Drug Encapsulation and Controlled Release Via Stimuli Responsive Nano and Micro Structured Polymeric Materials

Drug Enc a psulation and Controlled Release Via Stimuli Responsi ve Nano an d M icro Structured Poly me ric Mate rial s Doc toral Thesis Nieves Igl esi as Blanco Su pervisors: Dra. M aría Vi o lan te de Paz Bá ñez Dra. Elsa Ga lbis Fus ter Sevill e , Feb ruar y, 2020 Universidad d e Sevill a Facul tad de Farmacia Depa rt amento de Q uímic a Or gánic a y Farmacéut i ca The Thesis en titled “Drug E n capsulatio n and C ontro lled R elease V i a Stimuli Resp onsive Nano and Micro Str uctu re d Polym eric Mater ials” , su bmit ted b y Nieves I glesias B la nco for th e degree of Do ctor in Ph ar macy (Dep artment of Organic an d Ph armac eu tical Chem ist ry, Fa cu lt y of Pha rm ac y, Unive rsity of S ev il le), meets th e requ irements to op t for the Internationa l Doctor men ti on . Fo r all in tents and pu rpo ses, the follow ing c erti ficat ion is signed : Universidad d e Sev il la Facul tad de Farmacia Depa rt amento de Q uímic a Or gánic a y Farmacéut i ca Dra . M. ª Violante de Paz Báñ ez Prof esora Titul ar de Universidad Department of Organi c an d Pharmaceu ti cal Chemistry Faculty of Phar macy University of Sev ille Dra . E lsa Galbis Fuster Prof esora Ayud a nte Doctor a I nterina Department of Organi c an d Pharmaceu ti cal Chemistry Faculty of Phar macy University of Sev ille Agradecimie ntos Sería imposi ble pod er nombr a r a todas l as pers on as que me han ayudad o en est e camino l leno de difíciles momentos y sinsabores, pero s í quiero dest acar a alg uno s que han si do fundamentales . Como no podía ser de otra forma, a mi t utor a, director a, guía, faro y AM IGA, l a Prof esora D ra. Viola. C on ell a no sól o he ap ren di do muc ho de cualquier ámbi to de la vi da, he aprendido a s er mejor persona, a crecer y a madurar . Si l os días s e hub ies en resumido a ir al laboratorio y hace r las reacciones o experimentos co rrespondientes… ¡qu é trist e hubiera s ido t odo ! Por s uerte, la tenía a el la, para poder c onversar de mi s días bueno s y no tan b uenos, para que me aconsejara y di era ánimos. Cada ensayo realiz ado, con result ados mejores o peores, suponía un escalón más supe rado y ell a si empre l o recibía con entusias mo y al egría, es a aleg ría que te con tag iaba y te hacía creer que el artículo estaba ya casi terminado (a unqu e hubiera acaba do de empezar). Gracias a el la he con seg uido i r superá ndo me día a día, y l ejos de mi famili a, era lo más cercan o a ést a par a mí. A la Prof esora Dra. Els a, mi o tra di recto ra que par a mí si empre fue algo más que eso. El verla tan joven y ce rcana, hizo q ue s iempre p udiera co ntarle las cosas más pr iv adas, nuestras muchas comidas y des ayunos en la fac u lt ad hici ero n de ell a una compa ñer a impresci ndible . Además, nos unen muc has más cosas , como nuestra pasi ón por ciertas comidas y todo eso, no puede ser casuali dad. Ellas, para mí Viola y Elsa, en especial, han s ido m i pilar cada mañana. SI EMPRE SERÉIS PARTE DE MÍ, GRACI AS. Un agradecimi ento especial al Profesor D r. D. Juan Antoni o Gal bis Pérez, el cual, dur ante mis estudios de docto rado , se jubil ó, pero tuve el placer de coincidir un br eve tiempo con él, nutriéndome de sus muc has cualidades y el cual, me hizo sentir querida nada más co menzar. Al Profesor Dr . Ricar do, ese pr ofesor q ue siempre lleg aba c on una sonrisa cada mañana y me h acía reír co n c ual quier cosa. Si empre c orr iend o, con sus l íos y aje treos. Mi compañer o de mesa durante añ os y mi comp añía en, a veces, e se fr ío labor atorio, si empre tenien do en l a boc a la siguiente frase: “y o te puedo ayudar si quieres, ¿eh?” Así qu e gracias, has sido más importante p ara mí d e lo qu e c rees. No p uedo ol vi dar me de l a Profesora D ra. Doña Marí a de Gracia, Pro fesora Dra. Elena y Dra. Lucía, que en es tos ú lti mo s mes e s hemos compartido más que artí culos y han hecho que me sienta aún más par te de esa famil ia. Al Dr. Isaac y a Miguel Ánge l, q ue siempre han r espondido a t odas mis petici ones co n una frase amab le, y no han p odido s er mejores compañeros en est os años. A Juan Moral y a Carmen de la Flor ( ¡qu é aleg ría has llev a do al departamento! ), por solucionar cada gesti ón de maner a impecable. También, dur ante mis es tudios, he tenido el placer de reali zar un a estancia predoctoral con l a Profesor a D ra. Ana María dos Santo s Rosa da Costa y todo s u equipo, en especial agradezco a M iguel , Jorge, Noel ia y Fili pa que hi cieron que una s ituación di fícil para mí , lejos de mi zona de co nfort, se convirtier a en una exper ienci a ú nica, l lena de risas y momentos buenos. Si empre ag radeceré s u acogi da y es fuer zo pa ra q ue me si ntier a có moda. En g eneral, gracias a t od os l os que, con s ólo una s onr is a han contribu ido a que mi est ancia , tanto en Sevi lla co mo en Portugal , haya si do mejor. Lógicamente, a mis padr es, pilares fun damentales. Sin el los no hubiera podido hacer nada de todo est o, si empre me decís que la mej o r her encia que me podíais d ej ar es la educación y vaya si l o habéis co nseg uido. Sé qu e han si do t iempos di fícil es y que, a veces, hemos es tado un poco más agobiados en d is tintos as p ectos de l a vi da, per o es o si empre os ha da do i gual , lo único que i mpor t aba era que y o me centrar a en mi s est udios y fuer a feliz . Sólo yo s é to do lo q ue hab éi s hecho y soi s capaces de hacer po r mí. Os tengo q ue agradecer tanto q ue no cabría ni en una tes is completa . Asi m is mo, a mi hermana, que como siempre dig o, es el mejor regal o que la v ida me pud o dar. Aún en la dist ancia, siempre a m i l ad o. Nun ca olvi daré lo much o que me ayudas en t odo y l a de horas que has ded icado a escuch arme, apoyar me y ayudar me. No me falta su mensaje de ánimo y apoyo cad a día, dis pon ibl e las 24 horas del día si y o la necesi to. A mi famil ia en general, en es pecial, a mi tí a C haro , p orque es ta tes is tambi én es gracias a el la. Tú sabes todo lo q ue te debo . A mi prima M ontse, sin ella esta tesis no t endr ía color. Se ha es merado en cada detal le de la por tada y tod o lo q ue ell o engl oba, como siempre hace, co n pasión p or s u trabajo . Hace t odo con muc ho amor y es o s e ha vi sto reflejado. Además , es fun damental en mi vida po rque ell a, mejor que n adie, me entiende y po rq ue mi día a día no sería ig ual si n su s palabras. A mi s amig os , los cuales considero famili a y que habéis si do mi mejor medicina. Des tacando a Raquel , por sus múlti ples consejos, ánimos, apoyos, visi ta s s or pr esas y audios interminables que h acían qu e la si ntiera más cerca de mí. A los qu e se fuero n demas iado p ronto, si n avis ar , p ero que estáis y estaréis pr esentes en mi vida para si empre. Y finalmente, pero no menos importante, a Fran, mi confidente, mi compañer o, mi pareja, la otra par te del equip o, po rq ue juntos s o mos uno . No te haces una idea de l o mucho que te debo. Esta t esis no es en parte tuya, es tuya también por que t ú me animas te a se guir, a luchar y a levantarme las tant as y tantas vece s que me he caído . Gracias a to dos, ¡ qué af o rtun ada soy de teneros! Resumen La es peran za de vi da en A ndalucí a y en Españ a ha c recido de ma ner a co nstante dur ante l os últi mos 20 años y , en 2017, p resentaron val ores cercanos a 8 6 y 80 a ños para mujer es y hombres, respectivamente. Esta tendencia es la m is ma que la observada en otr os p aís es des arr ollados. S in embargo, a pesar de las grandes inversi ones qu e se real izan en la lucha co nt ra muc has enfermedades, la cali dad de vida de n uestros mayores sig ue s in ser óp tima. La i nvestigaci ón biomédica se ha cent rado du rante much o tiempo en l a s íntesi s y el descubr imi ento de fármaco s innovadores para tratar div ersas enfermedad es. Si n embargo y s or pren de ntemente, l a s principales d i ficultades enco ntradas para lograr un tratamiento eficaz no es tán relaci onadas en mu cho s casos con l a disponibil idad d e fármaco s, si no con el hallazg o de vehículos adecuado s par a la l iberación de medicamentos en los ór ganos o teji dos d añados. La Tesis Doc toral que s e pr esenta aborda la preparación de si stemas po li m éricos p ara el desarro ll o de bi omateriales para terapias avanzadas. Entre ell os s e inc luyen nano partículas y mat eriales nano - y mi cro-est ru c turad os sensi bles a cambios de pH y temperatur a par a la inclusi ón de compuestos li pof ílicos o hidróf il os con uti lidad terapéutica, por ejemplo, en la terapia contra el cánce r y enfermedad es del tracto gas trointe sti na l . También s e proced erá al des arr ollo de materi ales i nteligentes y matrices deriv adas del quitosano y l a goma de s emi lla de al garrob o que presentarán comp ortamientos dinámicos en medios biológico s en l ínea co n s u uso fi nal. Con respecto a la prepar ación, se utilizará una batería de herr amientas sintéticas: desde métodos de polimeriz ación vi vo s como la polimerización por radicales de transferenc ia de át omo s , hast a l a química c li ck . Las pro pieda des de respuesta a es t í mulos serán pr oporcionadas por la presencia de enl aces covalent es d inámicos como los enl aces Diels -Alder y acetal , así como po r enlaces iónicos. La i nclusi ón de s us tancias de relevancia terap éutic a, y su liberación en condiciones con troladas también formará parte del pr esente e s tudio. Por último, s e op timi zarán u n método ecológi co y compatible con entornos fis io lógi cos para la síntesis de materiales poliméricos. V.2. Met hods .................................................................................................................... 102 SECOND PART ....................................................................................................... 109 Chapter VI: Core Cross-Linked Nanoparticles from Self-Assembling PolyFMA- Based Micelles . 113 VI.1. Abstract .................................................................................................................... 115 VI.2. Introduc tion .............................................................................................................. 116 VI.3. Resu lts and Discussion ................................................................ .............................. 118 VI.3.1. Prepar ation o f st abili zed nan opart icles based o n amphiphilic blo ck -copo lymer .. 118  Synthesis o f furan-c ont aining b lock -copolym ers and cross-l inkers ...................... 118  Nanopart icle formation in aqueo us media ......................................................... 122  Cri tical micel l e co ncent ration (CMC) .................................................................. 123  Stabilization o f nan opart icles via core cross-li nking rea c tions ............................. 124 VI.3.2. C harac terization of the self -assembl y nan opart ic les ........................................... 128 VI.4. Conclus ions ................................ ............................................................................... 134 VI.5. Bibliogr aphy ................................ .............................................................................. 135 VI.6. Annexes ................................................................................................ .................... 140 VI.6.1. Sp ecif ic M etho ds ................................................................ ................................ 140  Synthet ic p rocedu res and micell e format ion ...................................................... 140  Preparation of Cross-linkers ................................................................ ............... 141  Formation of micell ar nan opart icles thro ugh self -asse mbly p rocess ................... 142  Formation of stabilized co re c ross-l inking nanopart icle s .................................... 142  Stability of nanop articles ................................................................................... 143 VI.6. 2. Su pplement ary Informat ion ................................................................................ 144 Chapter VII: Validation of pH Responsive Polymeric Nanoparticles as Controlled Drug Delivery Systems ....................................................................................................................... 145 VII. 1 . Abstrac t ................................................................................................................... 147 VII. 2 . Intro duct ion ................................................................................................ ............. 149 VII. 3 . Results an d D iscussion ............................................................................................. 150 VII. 3 . 1. Lo ading of lipophil ic molecule /drug int o nan opart icles .......................................... 150  Load ing of pyrene .............................................................................................. 150  Load ing Ki netics of Pilocarpine ........................................................................... 154 VII. 3 . 2. St ruct ural st udies of NPs at acidic pH and p il ocarpine -l oaded NPs ......................... 158 VII. 3 . 3. St im ulus-respo nse release of pilocarpine .............................................................. 161 VII. 4 . Conclusions .............................................................................................................. 164 VII. 5 . Bibliography ............................................................................................................. 165 VII. 6 . Annexes ................................................................................................................... 168 VII. 6 . 1. Spec ifi c Met hods ............................................................................................... 168  Mat erial s ................................ ........................................................................... 168  Meth ods ............................................................................................................ 168 Chapter VIII: Optimization of the Loading Process of th e Anticancer Drug Camptothecin in to Dua l Stimuli-Sensitive Nanoparticles ...................................................................................... 171 VIII.1. Abstrac t .................................................................................................................. 173 VIII.2. Introduc tion ................................ ............................................................................ 175 VIII.3. Results and Discu ssion ............................................................................................. 176 VIII.3.1. Preparation of core crosslinked and non - cro ss li nked N Ps from the aut o-assem bly blo c k -cop olymer poly[(DMA 31% - ran dom -HEMA 19% )- bloc k -(DEA 45% - rand om -FMA 5% )] ....... 176 VIII.3.2. Mod elling and influence of sonication tim e and C PT/polymer ratio on th e encapsulat ion efficiency of t he NPs ................................................................ ............... 178 VIII.3.3. Model ling and inf l uence of t emperature and ti me on NP uptake ...................... 183 VIII.3.4. Size and shape morp hology of opt imi zed c amp to thec in -l oaded NPs ................. 188 VIII.3.5. Drug retent ion c apac ity modelization ............................................................... 189 VIII.4. Conclus ions ............................................................................................................. 193 VIII.5. Bib li ograph y ................................................................................................ ............ 194 VIII.6. Annex es .................................................................................................................. 198 VIII.6.1. Specif i c Metho ds .............................................................................................. 198  Mat erial s ................................ ........................................................................... 198  Synthet ic Proc edures an d Micell e Format ion ..................................................... 198  Preparation of Campto thec in-l oaded NPs .......................................................... 198  Opt imi zat ion of the Experi mental Para meters in th e Loading Assays .................. 199  Drug ret ention assays of CPT-l oaded nanop articles ............................................ 201  Experimental design t o stud y the effect of lo ading conditions on CP T encapsulat ion and t he effect o f ionic s trength and time in drug retent ion c apacity ....... 201 VIII.6.2. Supple mentary Information .............................................................................. 203 Chapter IX: Reversible pH -Sensitive Chitosan- Based Hydrogels. Drug Delivery Systems ......... 207 IX. 1. Abstrac t ................................................................................................ .................... 209 IX. 2. Intro duct ion ................................................................ .............................................. 211 IX. 3. Resu l ts and Disc ussion – First Part ................................................................ ............. 213 IX. 3.1 . Cross-linked chito san-tricarbally li c acid (CTS x -TCA y ) hydro gel s ............................ 213  Fourier t ransformed infrared spec tro scop y (F T-IR) ............................................. 216  Rheological charac terization of CTS x -TCA y h ydrogels .......................................... 217  Modu lated temperature di fferential scan ning calori met ry (MTDSC) stud ies of CTS - TCA hydrogel s ............................................................................................................... 221 IX. 3.2 . Diclofenac loaded (CTS x -TCA y -DCNa Z ) formu latio ns ............................................. 223  Scan ning El ectro n M icrosc opy (SEM) ................................ ................................ . 226  Spreadability ................................................................................................ ...... 227  Diclofenac So dium Release Studies .................................................................... 228 IX. 4. C onc lusi ons – First Part ................................ ............................................................. 232 IX. 6. Anne xe s – First Part ................................................................ ................................... 233 IX. 6.1 . Spec ific Met hods ................................................................ ................................ 233  Mat erial s ................................ ........................................................................... 233  Meth ods ............................................................................................................ 233 IX. 7. Resu l ts and D iscussion – Sec ond Part ........................................................................ 238 IX. 7.1 . Du al Ionic and Covalent Cross-linked Hydrog els as D DS ................................ ...... 238  Preparation of H ydrogels from Cross - li nked C h itosan (C TS), C itric A cid (CA) and Diiodinated T rehalose (ITrh) (CTS x - CA 10 -ITrh y ) ................................................................ 238  Rheological Charact erization of I Trh, C TS x - CA 10 -ITRh y Hydrogel s and C orre lations of Rheological Par ameters with CTS Conc entrar ion and Degree of Cross -linkin g ................ 239  Diclofenac Sodium Loaded Formulations from Cross-l inked Ch i tosan-Conjugat es and Studies o f Dru g Release ................................................................ .......................... 244  Scan ning El ectro n M icrosc opy (SEM) Stud ies ..................................................... 248 IX. 8. C onc lusi ons – Seco nd Part ................................................................ ......................... 250 IX. 9. Anne xe s – Sec ond Part .............................................................................................. 252 IX. 9.1 . Spec ific Met hods ................................................................ ................................ 252  Preparation of D oubly Cross-Linked CTS-Based Hydrogels ................................ .. 252  Synthesis of 2 ,3,4, 2',3',4'-Hexa- O -acetyl-6 , 6'-diiodo-6 ,6'-dideoxy- α -D- Glucopyrano syl- α -D-gl uco pyranoside ( I Trh ) ................................................................ ... 252  Spect ra of 2,3,4,2',3',4'-Hex a- O -ac etyl-6 ,6'-diiodo-6,6'-dideoxy- α -D- Glucopyrano syl- α -D-gl uco pyranoside ( I Trh ) ................................................................ ... 254 IX. 10. Bib l iogr aphy ............................................................................................................ 258 Chapter X: Chitosan -Nanoparticle Composites as Res veratrol Carriers: Sustained and Colonic Specific Drug Release ................................................................................................ .... 263 X.1. Abstract ................................ ..................................................................................... 265 X.2. Introduc tion ............................................................................................................... 266 X.3. Results and Discussion ................................................................................................ 268 X.3.1. Resverat rol-Loaded Nanopart icles ....................................................................... 268 X.3.2. Nanostruc tu red Chitosan-Based Composit es Con taining Resver atro l -Loaded NPs 274 X.3.3. Resverat rol Releas e Stu dies ................................................................................. 278 X.4. Conclusions ................................................................................................ ................ 280 X.5. Bibliography ................................................................................................ ............... 281 X.6. Annexes ..................................................................................................................... 288 X.6.1. Specific M ethods ................................................................................................ . 288  Preparation of Resverat rol-Loaded NPs .............................................................. 288  Experimental Des ign to Study t he E ffect of Load ing Conditions on Resveratrol Encapsulation ................................................................................................................ 288  Preparation of Nanostruc tured Chitosan-Based Hydrogels wi th Resveratrol-Loaded Nanopart icles ................................................................................................................ 289  Release Studies ................................ .................................................................. 289 Chapter XI: Locust Bean Gum Derivatives for the For mation of Nanoparticles Useful in Drug Delivery Systems .......................................................................................................... 291 XI. 1. Abstrac t ................................................................................................ .................... 293 XI. 2. Intro duct ion ................................................................ .............................................. 295 XI. 2. Resu l ts and D iscussion ............................................................................................... 297 XI. 2.1 . O xidation o f Lo cust Bean Gum ................................................................ ............ 298 XI. 2.2 . Fo rmation o f amine ............................................................................................ 302 XI. 2.3 . Fo rmation o f nano partic les based on the aminated derivativ e o f LBG ................. 306 XI. 3. C onc lusi ons ................................................................................................ ............... 312 XI. 4. B i bliograph y .............................................................................................................. 313 XI. 5. Annexes ................................................................ .................................................... 317 XI. 5.1 . Mat erials and General Metho ds ......................................................................... 317 - Mat erial s ................................................................ ............................................... 317 - General Met hods ................................................................................................... 317 XI. 5.2 . Spec ific Met hods ................................................................ ................................ 317 Chapter XII: Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Diels-Alder Click Reactions ............................................... 321 XII.1. Abstrac t .................................................................................................................... 323 XII.2. Introdu ct ion ............................................................................................................. 325 XII.3. Results and Disc ussion ................................................................ .............................. 327 XII.3.1. Preparation of mo nomers and small molecu les ................................................. 327 XII.3.2. Thermal stabili ty studies of the model DA adduct DMDOO -Fur (4). Ki netic parameters d etermination u nder mod el -based anal y se s. .............................................. 328 XII.3.3. Synthesis o f t he new funct ionalized polymers a s chemical scaffo lds .................. 332 XII.3.4. Determinat ion of mono mer co nversion during p olymerization by Infrared Spect rosc opy and a Second D eriv ative Proc edure ......................................................... 339 XII.3.5. Experim ental d esig n fo r t he op timization of polymerization c onditions ............. 342 XII.3.6. Thermal pro perties of th e new mat erial s ........................................................... 346 XII.4. Conclusions ................................................................................................ .............. 348 XII.5. Bibliography ................................................................................................ ............. 350 XII.6. Annexes ................................................................................................................... 355 XII.6.1. Specific Meth ods ............................................................................................... 355  Preparation of mon omers and small molecu les. E xperiment al proc edures ......... 355  Thermal stability studies of t he m ode l DA adduc t DM DOO-Furan (4). Kinetic parameters d etermination u nder mod el -based anal y se s ............................................... 357  Preparation of fu nct ionalized polymers as c hemical scaffo lds ............................ 358  Experimental desi gn fo r t he opt imization of polymerizat ion c onditions ............. 359  Determination of m ono mer con version i n polyme ri zation trial s by I nfrared Spect rosc opy and a Second D eriv ative Proc edure. ......................................................... 361 XII.6.2. Supplem entary Information ............................................................................... 362  Polymerization Trials .......................................................................................... 362  Determination of 𝑫𝑷 and 𝑴𝒏 from 1 H NMR ................................ ...................... 363  Determination of endo/exo ratios from 1 H NMR ................................................ 364  GPC chro matograms ................................................................ .......................... 365  1 H NMR an d 13 C NMR spec tra ............................................................................. 367 THIRD P ART ........................................................................................................... 379 Chapter XIII: Global Conclusions ................................................................ ................... 383 XIII.1. Conclus ions for Chapters VI, VII and VIII ................................................................... 385 XIII.2. Conclus ions for Chapters IX and X ............................................................................ 387 XIII.3. Conclus ions for Chapter XI ....................................................................................... 389 XIII.4. Conclus ions for Chapter XII ...................................................................................... 390 Chapter XIV: Annexes .................................................................................................. 391 XIV. 1. Pub li shed Papers ................................................................ ..................................... 393 XIV. 1.1 . E xperimental Model Design: E xploration and Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Ma terials by Cl ic k Die ls- Alder ................................ ............................................................................................. 394 XIV. 1.2 . Nanostruc tu red Chi tosan-Based Biomaterials for Sustained and Colon -Specific Resveratrol Rel ease ....................................................................................................... 4 22 XIV. 1.3 . Rev ers ible pH-Sensitive C h i tosan-Based Hydro gel s . Influence of Dispers ion Composition on Rheological Propert ies and Sustained Drug D eli ver y ............................. 439 XIV. 1.4 . L oading St udies of the Ant icancer Drug Camptot hec in Into Dual Stimul i -Sensitive Nanopart icles. St ability Scrutiny .................................................................................... 457 XIV. 1.5 . Validation of Smart N anopart icles as Contro lled Drug Deli ver y Systems: Loading and pH-Dependent Release of Pilocarpine ..................................................................... 500 XIV. 1.6 . pH-Responsiv e Poly meric Nanopart icles as D rug Delivery Systems ................... 515 XIV. 1.7 . Core C ross-L i nked Nanopartic les fro m Self-A s sembling PolyFM A -Based Mice ll es . Encapsulation of L ipophilic Mo lecules ........................................................................... 521 XIV. 2. C urriculum Vitae ................................................................................................ ..... 563 XIV. 2.1 . Su mmary .............................................................................................................. 563 XIV. 2.2 . University Education ................................ ......................................................... 564 XI V.2.3 . Investigation ................................................................ ..................................... 564  Scientific Articles ................................................................................................ 564  Science Chapter Books ....................................................................................... 565  Participation and Attend ance at Congresses ...................................................... 565  Published Books ................................................................ ................................ . 567  Teac hing Articles ................................................................ ................................ 567 XIV. 2.4 . Sc holarships, Award s and St ays ................................ ........................................ 567  Scho larships ................................................................ ....................................... 567  Stays .................................................................................................................. 568 XIV. 2.5 . Work Experienc e .............................................................................................. 568  Universidad de Sevil la. Departame nto de Q uímica Org áni ca y Fa rmacéut i ca ...... 568  Universidade do Alg arve. Depart amento de Química e Farmácia ....................... 569  St. M ary’s Scho ol. .............................................................................................. 569  Mont equinto Pharmac y. ................................ .................................................... 569 XIV. 2.6 . Certif icat ions .................................................................................................... 570 XIV. 2.7 . Courses ............................................................................................................ 570  Specialized trainin g c ourses ............................................................................... 570  Training, inno vation an d t eacher improvement c ourses ..................................... 570  Courses aimed at improv ing health c are fo r health care professionals ................ 571 Figures Inde x Figure III- 1. Number o f deaths est imated by the mai n t ypes of can cer i n the U nited States i n 2018. ...................................................................................................................................... 51 Figure III- 2. Two-d imensi onal and three -dimension al struc tu res of pil oc arpine hydrochloride. ................................................................................................................................ ............... 53 Figure III- 3. Two-dimensi onal an d t hree-dim ens i onal s tru ct ures of campt othecin. ................. 54 Figure III- 4. DALY s per year of i nflammatory bowel disease (I BD) in selected locations in 2 01 7, ordered by incidenc e and sex. ................................................................................................ . 56 Figure III- 5 . T wo-dimensional and t hree-dimensional s truct ures o f sod ium diclofenac. .......... 57 Figure III- 6. Two-dimensi onal an d t hree-dim ens i onal s truct ures o f resveratrol. ..................... 57 Figure III- 7. Examples of nan ocarr iers for c an cer detec tion an d t herapy. The main co mponents typically include a nanoc arrier, a targeting m oiety conjugat ed to the nano carrier and a cargo (the c hosen chemotherapeut ic drug). Reprinted from Natu re Publi shin g Group: (D. D. Peer, J.M. Karp, S. H ong , O.C. Farokhzad, R. Marga lit, R. Langer, Nanocarriers as an emergi ng platform for c anc er therapy, Nature Nano tec hnology. 2 (2 007 ) 75 1 – 760). .............................. 60 Figure III- 8. Nanoc arriers as promisi ng t ransport ers o f ant icanc er drugs to tumors by passive tissue targeting and ac tive cell ula r targeting. Reprint ed from N ature Publi shing Group: (D. Peer, J .M. Karp, S . H ong , O.C. Farokhzad, R. Marga li t, R. Langer, N anoc arriers as an e mer gi ng platform for c anc er therapy, Nature Nano tec hnology. 2 (2 007 ) 75 1 – 760). .............................. 62 Figure III- 9 . pH-responsive nanop articles prepa red fr om an amphiphilic d i -bloc k c opolymer in which the an ticancer drug is embedded i nto the l ipophilic core. (B) Drug rel ease of anticanc e r drug fro m pH-sensitive NPs t riggered by the acidic enviro nment fo und in solid tu mors. ......... 65 Figure III- 10. Formation of ag gregates or stable nano particles from cro ssli nking assays in t he micelles c ores or th e micelles shells, respect ively. ................................................................ ... 68 Figure III- 11 . Exampl es of biomed ical applicat ions of hydrogels . ............................................ 71 Figure III- 12. General st ructure of a cro ssli nked CTS-based h ydrogel. ..................................... 74 Figure VI- 1. Graphical abstract of the main objec tives in th is work. ...................................... 115 Figure VI- 2 . General struc ture of synthesized methacr yl ate-based block-co polymers and cross- li nkers . .................................................................................................................................. 118 Figure VI- 3. Mac r o- and mi cro- analyses of micell ar d i spersions (at a glance and by Dynamic Light Sc attering). ................................................................................................................... 122 Figure VI- 4. Absorb ance of aqueo us polym er s olution at 6 00 nm against p olymer con cent ra tion (i n red). Fl uoresc ence em i ssion of pyrene in aqueous solutions: spectral ratios II I I (384 nm)/II (37 2 nm) (in blue). ......................................................................................... 123 Figure VI- 5 . (A) Micel les from auto-assemb ly of the ami ne-cont aining amphiph il ic bloc k cop olyme rs in aqueous medi a (non-stabilized mi cel les); (B) sol ution of pro ton ated p oly mer chains, (C ) stabi lized NPs by core c ross -linking; and (D) stab le amine -con taining NPs wit h hydrophilic and hydrat ed c ore. ............................................................................................. 127 Figure VI- 6. SEM micrographs of the crosslinked N Ps at neutral pH : (a) sample M 2 -2; (b) sample M2-3 ; ( c) sample M 2-4; (d) sample M2-5. (e) Size di stribut ions extracted fro m stat istical anal ysis performed on v arious SE M images are also plot. The curves co rrespond to the fit of t he distributions using a log -normal funct ion. In the case of sample M2-5, analy sis of the size distributions has not been performed because the NPs were strongl y agglomerated . ................................................................................................................................ ............. 130 Figure VI- 7. SE M micrographs and si ze d istributi ons extrac ted from statistical analysis performed on vari ous SEM images fo r (a) sample M2 -1 at pH 7.0 and (b) sampl e M3-1 at pH 3.0. T he c urves co rrespond to t he fit of t he di stribut ions using a log -normal fun ct ion . .......... 131 Figure VI- 8. SEM mi crographs and size d i stribut io ns extracted from stat istical anal ysis performed o n various SEM images of c ore cross-li nked NPs in acidic media. a) sample M 3- 2; (b) sample M3-3 ; ( c) sample M 3-4; (d) sample M3-5. (e) Size di stribut ions extracted fro m stat istical analysis perform ed o n various SEM images are also p lot. ...................................... 132 Figure VI- 9 . C omparison of the mean NP si zes deter mi ned by DL S and SE M for samples M2- X (pH 7.0) and M 3-X (pH 3 .0). ................................................................ .................................. 133 Figure VI-S 1 . Zeta Pot ential distribution o f M3 -X samples at pH 3 .0. .................................... 144 Figure VII- 1 . Grap hical abst ra ct of the main objec tives in t his work. ..................................... 148 Figure VII- 2. Fluorescenc e-emi ssion data a t 3 80 nm (a rbitrary units) of pyrene loading tests a t selected p eriods of time. ................................ ....................................................................... 152 Figure VII- 3. Fluorescenc e microsc opy i mage of non -c ross-linked pyrene- loaded N Ps (LM-Pyr- 1) and the correspond ing size di stribut ion obtaine d from s tatistical analysi s. Th e curve corresp onds to the fit with the log -normal func tion. ............................................................. 153 Figure V II- 4. Comparison of t he size of t he un loaded and loaded p yrene nano particles. ...... 153 Figure VII- 5 . Ki netic studies at 25°C of pi locarpine loading i n NP s by U V−v is spect rosco py at 215 nm (ini tial pilocarpine c onc entrat ion = 0.1 mg/ mL; polymer co ncentration = 0.12 5 mg/ mL). (A) Absorbanc e of free pilocarpine i n NP dispersion vs time; (B) cumulative percent age of pilocarpine embedded int o t he NPs vs t ime, c alculated from ab sorbanc e values at 215 nm. . 155 Figure VII- 6 . Diels – A ld er adduc t formed b y t he reaction of two furan rings fro m furfu ryl methac rylate units present i n t he core of the NPs wi th the cross -l inker 1,8-dimale imide-3,6- dioxaoct ane (D MDOO). ................................................................ ......................................... 158 Figure VII- 7 . Sel ected SE M im ages of the NP syst e ms at pH 5.5 prepared from the block cop olymer (DMA 31% H EMA 19% )-block-(D EA 45% FMA 5% ) sy nthesized by A TRP: (a) non -cro ss-linked NP (M2-1 -ac); (b) core cross-linked NP with 20% of deg ree of cro ss-l inking (M 2-2-ac ). .......... 159 Figure VII- 8. Comparison of mean NP si zes determined by DLS and SEM at pHs 7.0 and 5. 5 (from l eft to ri ght: samples M2-1, M2-1-ac, M2-2 , M2-2-ac, M2-3 , M 2-3- ac; here “ac” denotes NPs dispersed in an acidic medi um at pH 5.5, “X L” d enotes cross - li nked N Ps and “20%” and “10 %” t he degree of cro ss -li nking). ................................ ....................................................... 159 Figure VI I- 9 . C umulative release of pi locarpine (in percent age, related to the entrappe d pilocarpine into the NPs) v s t ime ( in hours) from p il ocarp ine -l oaded NPs at pHs 7.0 and 5.5 at 37°C (final poly mer co ncent rat ion: 0 .125 mg/m L). ................................ ................................ 162 Figure VII-S 1 . Chemi cal struc ture of pil oc arpine. .................................................................. 169 Figure VII-S 2. Cal ibration c urves o f pilocarpine at 215 nm (U V-Vi s spec tro scop y) at 25°C . .... 170 Figure VII I- 1 . Graphical abst ract o f the main objec tives in t his work. .................................... 174 Figure VIII- 2 . St ruc ture of the am phiphilic block-copolymer and the cross-linker used i n the present work. ....................................................................................................................... 177 Figure VII I- 3 . S ch ematic representat ion of non cr oss-l inked NP (on the l eft), in which formulation the crosslinker has not been added, and core cro ss -li nked NPs (on t he right). In the latter samples, some of the furan rings hav e reacted wi th the bifunct ional c ross-linker DMD OO leading t o c ore cro ss-l inked NPs by means of D iel s-Ald er reac tion. ....................................... 178 Figure VIII - 4 . Response surfac e for CP T encapsulat ion efficiency percentage on both, non - and cro ss-li nked NPs . ................................................................................................................... 182 Figure VII I- 5. Response surfaces for encapsulation efficiency (EE in percentage) against temperatu re and l oading time on non- and cros s-li nked nanoc arriers for the second experiment al design. ............................................................................................................. 186 Figure VIII- 6 . Selected SE M im ages of the CPT -loaded NP sy stems at pH 7.0 prepare d acc ording to t he op timized con ditions found in the present study. (a) non - cross-linked NP (Non-T 32 -t 2 ); (b) c ore cross-li nked NP with 20 % of degree of cross-linki ng (Xr-T 32 -t 2 ). ............. 189 Figure VIII- 7. Response surface for drug retention capacity (DRC, i n percentag e) ag a inst sal t con cent ra tion (N aCl, in % w/v) and ti me on non - and cross-linked nanocarriers for the third experiment al design. ............................................................................................................. 192 Figure VII I-S 1. Chemi cal st ruc ture of campto thecin (CPT). .................................................... 199 Figure VII I-S 2. General sc heme of the loading p rocess of CPT into the select ed nan o part icles at 25°C. ..................................................................................................................................... 200 Figure VII I-S 3. Cali bration curve of CPT at 369 nm (UV - Vi s spec tro scop y) at 25°C. ................ 203 Figure IX- 1. Grap hical abst ract of the main objec tives in t his work. ...................................... 210 Figure IX- 2 . FT -I R spectra of C TS (blue); freeze -dried C TS 3 -TCA 10 hydrogel (green) and TCA (red). ..................................................................................................................................... 217 Figure IX- 3. Frequency dependenc e o f t he storage, G’ , and loss, G”, moduli and the l oss tangent for CTS x -TCA y hydrogels as fu nct ion of CTS (a, b). ..................................................... 218 Figure IX- 4. Frequency dependenc e o f t he storage, G’ , and loss, G”, moduli and the l oss tangent for CTS x -T CA y hydro gel s as funct ion o f degree of cro ss-li nking (c,d). ........................ 219 Figure IX- 5. Viscous fl ow curves for CTS x -TCA y hydrogel s st udied, at 25°C, as a function of (a) CTS c onc entration and (b) TCA conc ent ra tion. ................................................................ ...... 220 Figure IX- 6 . Reversi b l e heat-flow plot and phase transition temperatu re from the temperature at the peak of the transition endotherm ( °C ) of CTS 3 -TCA 10 (solid line, i n blue) , CTS 2 -TCA 15 (short dash li ne, i n red), and CTS 4 -TCA 0 (dash-dot li n e, in black) hydrogels established usi ng modulated temperatu re DSC (MTD SC) over l a id wi th the reversi b l e heat -flow versus temperatu re of CTS 3 -TCA 5 system (dash- dot line, in gre en). .................................................. 223 Figure IX- 7. SE M i mages showing m orpho logy of th e as-sy nt hesized unloaded chitosan-based hydrogel CTS 3 - TCA 10 (a -c ) and loaded h ydrogel CTS 3 -T C A 10 -DCNa 1 (d -f). ................................ 227 Figure IX- 8. I n vi tro release profiles of di clofenac so dium (DCN a) from chit osan hydrogels i n acet ate buffer solution at pH 5.5 at 37°C. Data were o btained from U V -vis spectro sco py at 280 nm and reported as m ean ± S.D from fi ve independen t experiments. E ffect of degree of cro ss - li nk i ng from non-cross-linked sam ples to 15% of cro ss -linked (CTS conc entrat i on 3%; DCNa con cent ra tion: 1%). ............................................................................................................... 229 Figure IX- 9. I n vi tro release profiles of di clofenac so dium (DCN a) from chit osan hydrogels i n acet ate buffer solution at pH 5.5 at 37°C. Data were o bt ained from U V -vis spectro sco py at 280 nm and reported as mean ± S.D from five ind ependent experi ments . E ffect of C TS Table VIII- 4. E quat ions y iel ded for the depende nt variabl e (E E) as a fun ct ion of the independent vari ables (temperature and loading ti me, norma lized values) for the secon d experiment al design. ............................................................................................................. 185 Table VII I- 5 . C ompari son of Z -avera ge, po lydi sp ersity i ndex (PdI), and hydro dyna m ic di am eter (D h , determined by DLS) of non-cr oss-linked N P (Non-Xr) and s tabili zed N P at 20% of cross- li nk i ng (Xr) (unloaded or loaded with CPT). ........................................................................... 188 Table VIII- 6. Experi mental drug retention capac ity v alues of CP T loaded NP s at 25°C an d different NaC l co ncent rations and times following t he experim ental design 1 . ....................... 190 Table VI II- 7. E quations yi elded for each dependent variable as a func tion of t he independent variables (no rmalized values) for t he t hird experiment al design . ........................................... 191 Table VII I-S 1. C PT-loaded NP s prepared at different C PT/polymer ratios and sonicat ion times. Final polymer co ncent ration: 0.1 mg/mL; t emperature: 25 °C . ............................................... 204 Table VII I-S 2. C PT-loaded NPs prepared at d i fferent temperatures and tim e s. Sonicat ion time: 14 min; CPT/polymer ratio: 1 .5. ............................................................................................ 205 Table VIII-S 3 . S tudies of drug retention capacity of CP T-l oaded NPs versus time (t = 1, 8, 1 5 days) and ionic strength (NaCl c onc entrat ion: 0.1, 0.5, 0.9% w/v). ........................................ 206 Table IX- 1 . Conc entrat ion and degree of cro ss -linki ng of the 12 CTS-based hydrogels prepared. Rheological para m eters. ....................................................................................................... 215 Table IX- 2. Thermal propert ies of select ed CTS-based hydrogels from modulated temperature differential scann ing calorimetry (MT DSC). ........................................................................... 222 Table IX - 3. Composition and evaluation o f dru g formul ations. ............................................. 225 Table IX- 4. Conc entration and degree of cro ss-l inking of th e 10 CTS-based hydro gels prepared. Rheological para m eters. ....................................................................................................... 239 Table IX- 5 . Composition and evaluation of drug fo rmulations. Drug releas e figures . ............ 245 Table IX-S 1. Concent ration and degree of cro ss-li nking of the 12 CTS-based hydrogels prepared. Rheo logical paramet ers. ................................ ....................................................... 234 Table IX-S 2 . Composition an d evaluation of d rug formulations. ........................................... 236 Table X- 1. Experi mental encapsulat ion efficiency v alues of resv eratro l l oaded N Ps for non- cro ss-li nked syst ems at different RES/polymer rat ios and t emperatures fol lowing the experiment al design 1 . ................................ ........................................................................... 269 Table X- 2. Experi mental encapsulat ion efficiency values of resv eratro l l oaded N Ps for non - cro ss-li nked syst ems at different RES/polymer rat ios and t emperatures fol lowing the experiment al design 1 . ................................ ........................................................................... 270 Table X- 3. E quat ions y ielded for the dependent vari a ble (EE ) as a func tion of th e independent variables (RES/pol ymer ratio and t emperature, no rmali zed values ) for the experimental design. ................................................................................................................................ ............. 271 Table X- 4. Comparison of Z -averag e, polyd ispersi ty index (PdI ), and hydrod ynami c diameter (D h , determined by DLS) of non-cross-li nked NP (N on-Xr) and s tabilized N P at 20% of cross- li nk i ng (Xr) (unloaded or loaded with RES). ................................................................ ............ 274 Table X- 5. C omparison of spreadability and TGA data of resveratrol -loaded hydrogel - NP compo sites (with cross-linked NP and non-cro ss-linked NP. ................................ .................. 276 Table XI- 1 . C omparison of Z -avera ge, polydispers i ty index (PdI), hydrodyna mic di ameter (D h ) and zeta potential (  ) determ ined by DLS of the nano particles formed at different LBG am /C RG ratios. ................................ ................................................................................................... 309 Table XI I- 1. Type-A Polymers: Ext ent of po lymeri zati ons of D iT -Fur ( 1 ) and DMDO O ( 3 ) und er different polymeri zation con ditions obtained fr om IR and NMR data at 24 h and 48 h, respect ivel y. ................................................................ ......................................................... 336 Table XI I- 2. Type-B Polymers: Extent of polymerizatio ns o f DTT-Fur ( 2 ) and DMDOO ( 3 ) un der different p olymerizat ion c onditions obtained from IR and NMR data a t 24 h and 48 h, respect ivel y. ................................................................ ......................................................... 337 Table XI I- 3 . Equation yielded for the dependent variable (M n ) as a function of the independent variables (wat er co ntent and t emperature, normalized values) fo r t he experimental desi gn. 343 Table XI I- 4. Thermal prop erties of selected DiT-Fur and DT T-Fur based polymers. ............... 347 Table XI I-S 1 . P olymeri zat io n trials at different water contents and temperatures of monomers DiT Fur ( 1 ) or DTT-Fur ( 2 ) with the bis-m aleimide mono mer DMD OO ( 3 ). .............................. 362 FIRST PART Chapter I: List of Publication s Chapter 1 ________________________________________________________ List of Publications 39 This Doctoral Thesi s wa s carr ied out fro m N ovember 2016 to Febr uary of 2 0 20 , in the Depa rtamen to d e Qu ímica Orgá n ic a y Fa r macéu tica, Univers id ad d e Sevilla and directed by the pr ofessors María V iolante de Paz Báñ ez and E l sa Galbis Fu st e r, an d in the Departam ento de Química e Fa rmácia, Uni vers idade do Algarve (Portugal) and directed by the professor Ana María dos Santos Rosa da Costa, during my predoc tor al s tay . The typology of thi s Thesis i s by comp endium of publi shed arti cles based on specific regulation s of the PhD pro gram of t he University of Sev ille. P ublications, for which, wi ll be detai led, next, impact factor and p osi tion accor ding to the las t upd ate o f th e Journ al Citati o n Report (J CR) . The order of the publicat ions s hown bel ow i s i n descending ord e r ac cording to the year o f pub li cati on :  Nieves Iglesi as , Elsa Gal b i s, Lucía Romero-Azogil , Elena Beni to, M. Je sús Díaz Bl anco, M . Gracia García Martín, M. Violant e de Paz. Experimental mod e l design: E xpl oration and optimiz ation of custom ized pol ymer iza t ion con ditions for the preparation of target e d smart m ater ials b y cl ic k Diel s-Al der. Poly m. Chem . 201 9; 1 0: 5473-5486. Polymer Chemistry Polymer Science: 9/ 87; Q1. Impact factor: 4.760  Nieves Iglesias , E ls a Galbis, M . Jesús D íaz B lanco, Ricardo L ucas, E lena Benito, M. Violante de Pa z. Nan ostructu re d Chito san-Based B i omater ia ls for S ustained and C olon -Specifi c Resveratrol Relea se. Int . J. Mol. Sci. 2019; 20 (2): 398. International Journ al of Molecul ar Sciences Biochemist ry & Molecular Biology: 7 8/2 99; T1 . Impact factor: 4 .1 83  Nieves Iglesias , Els a G albis, Concepción Val encia, M. Violante de Paz, Juan A. Galbis. Revers ib le pH-Sen sit ive Ch itosan-Based Hydrogels. I nfluence of Dispersion Compo si tion o n Rheol ogi cal Pro perties and Susta in ed Dr ug Deliver y. P olymers . 20 18; 10: 392. Polymers Polymer Science: 1 7/87; Q1. I mpact f actor : 3.164 Cha pter I ___________________________________________________________ L ist of Publ icat ions 40  Nieves Iglesi as , E ls a Galbis , M. Jesú s Díaz Blan co, M. Violante d e Paz, Juan A. Galbis . Load ing studies o f the anti cancer drug Campt othecin in t o dual s tim u li -sens itive nan opa rt icles. Sta b ilit y scrutin y. Int. J. Pharm . 20 18; 550: 429-43 8. International Journ al of Pharmaceutics Pharmaco logy & Pharmacy: 45/256; Q1. Impact fa ctor : 3.785  Elsa Gal bis, Nieves I glesias , Ri card o Lucas, E rnes to Ti najero-Díaz, M. Violante de Paz, Sebasti án Muñoz-Guerr a, Juan A. Galbis. Validatio n of s ma rt nan opa rt icles as cont ro ll ed d rug del ivery s ys tem s: Loadi ng and pH-dep endent relea se of pil oca r p ine . ACS Om e g a . 20 18 ; 3: 3 75-382 . ACS Omega Chemis try , Mul t idisciplinary: 76 /172; Q2. Impact f actor: 2. 584  Elsa Galbis, M. Violante de Paz, Nieves Iglesias , Ricardo Lucas, Juan A. Ga lbi s. pH - Responsive pol ymer ic nanoparticles as drug delive ry systems. J Drug Des. Res. 2017; 4 (4): 1047 . Jour nal Drug of Desig n and Research https:// www .js ci medcentral.com/Dr ugDesig n  Elsa G a lbis, M. Vi olante de Pa z, Nieves Iglesias , Bertrand Lacroix, Ana Al cudia, Juan A. Gal bi s . Core cross- li nked n a noparticles f rom s elf -ass emb ling pol yFM A-b ased micelles. Encapsul a ti o n of lipophi li c molecu l es. E ur. Polym. J. 20 17; 89: 406-4 18 . Eur opean Polymer Journal Polymer Science: 1 4/87; Q1. Impact f actor: 3. 62 1 Chapter II : Abbreviation s Chapter II ________________________________ ___________________________ Abbreviations 48 PVamine Poly(v in yl amine) RAFT Reversi ble addit ion−fr agmenta tion ch ain transfer polymerization RCS Refr ig erated co oling s ystem RES Resv eratr ol R f Rate factor RM -Pi l-X Pil ocar pine releas e tria ls fr om the NPs ROS Reactiv e oxy gen species S Sonication tr eatment SAOS Small ampl it ude oscillatory shear SEM Scanning electron microscop y SLN Solid li pid nan opar ticl es SNR Sig nal- to -n oise ratio Tan ẟ Loss tangent t BME tert -Butyl methy l et her TCA Tr icarbally li c acid TE MPO 2,2,6,6-tetramethyl p i peridine-1-o xy l T g Gl ass tran si t ion temperature TGA Ther mogravimet ric an aly ses THF Tetrah ydrofuran TLC Thin lay er c hromatography TNF- α Tumo r necrosi s factor UC Ulcerativ e col i tis UV -vis Ultravi ol et visi b l e spectroscop y Chapter I II : Introduct ion Chapter III ___________________________________________________________ Introduction 51 I II .1 . Pre val ent Worldwide D iseases: C ancer an d IBD Cancer i s a maj or public healt h pr oblem worldwide and i s the second lea ding cause o f death in devel op ed countries — only exceeded by cardiovas cular dis ea ses — , which accou nts for mo re than 8.8 mill ion d eaths per year. It is caused by damag e or mutations in the genetic material of the cells due to envi ro nmental or inherited facto rs; cancer is character ized by uncon t rolled cel l pr oli fer ati on and an absen ce of cell death that, except for hemat ological cancer s, generates an abno rmal cel l mass or tumor. T his primary tum or g rows thanks to new vas cularizati on and, in time, acquires meta static po tential and spreads to other b ody s it es, wh i ch causes metast a sis a nd finall y death. Th e lifeti me pr obabilit y of developing an invasi ve cancer is curren tl y abo ve 4 0% f or men and close to i t for w omen and, in 2018 , 1, 735,350 new cancer cas es and 609,640 can cer deaths were p rojected to o cc ur in the United Stat es ( Fi gure III -1 ) [1 ] . Figure III- 1. Nu mber of d eaths estimate d by t he main types of cancer i n t he United States in 2 018 . Tr eatment is di ctated by t he cancer ty pe, s tage at diag nosis, and the pati ent's tol eranc e to the pr escribed the rapy [ 2] . W hile s urgery and rad iotherapy are the primary treatment used f or local and no n-metastat ic cancer s, o ther treatments can be employed such as chemo thera py, hor mone and biologi cal t her apies. Th us, for ex ample, in the case of soli d tumor s, sur gery is the local treatment of c hoice as t he damag e i s confined i n a li mit ed area of the bod y. H owev er, most patients require the combination of t wo or mo re therapeutic treatments due to the potential Est ima t e d Deat hs Lu ng and bronchus Pr osta te Col on and r ectum Pa ncrea s Liv er Le ukem i a Esop hagus Urin ar y bla dder Non- Ho dkin g lym ph oma K idney and renal pe lvis Br east Ovar y Ute rine co rpus Br ain a nd ot her nervou s syst em Chapter III ___________________________________________________________ Introduction 52 spread of the dis ease as well as to effectively prevent t he ev olution of t he di seas e from early to advanced stages. Chemotherap y works inhibit ing the divisi on of rapi dly g ro win g cells and, combined wi th s urgery or radiother apy, the effecti veness of t hese treatme nt modaliti e s are increased [3] . The use of radiation and chemotherapy al so s ignificantly add s to the cost of cancer care due t o the high material costs and t he need for app ropriate s pecialists, appro p riate monitoring, and treatment of si de effects [2] . Al though chemothe rapy i s the mai n treatment for cancer pati ents, the acti ve phar maceutical ingredients (APIs ) used do not differentiat e between h eal thy cells and ca ncer cells . Both cancer and healt hy cel ls are exposed to the cytotoxi c effects of chemotherap e uti c drugs and, con sequently , the drugs i nterfer e the growing pat tern o f no rmal cell s wi th fast pr oli fer ati on rates, such as t he hai r f olli cles, bon e marr ow an d gas tro intes tinal tract (GIT)cel ls, and provoke l ong-ter m toxic effects on the heart, l ungs, and ki dneys [4] . Typical harmf ul s ide effects as sociated t o chemother apy s uch as nausea, v omiting, i mmu ne suppressi on , hepatotoxicit y, nephr otoxicit y , memory l oss, anem ia, and ev en death, are rooted in the us e of s uch nonspecific therapeutic sy s tems. One of the ap pro ac hes to dimi nis h the inconve niences menti oned above has been the us e of pr odrugs w ith reduced toxici ty that can be acti vated i nto the body to yield the active chemother apeutic molecule. This i s t he cas e of Capecitabine (Xeloda; Hoff man La Roc he), used f or the treatment of breast and colon can cers. C apecitabine is a prodr ug of the anti tumor ag ent 5-f luorour a cil that pr esents better efficacy and s afety pro fil es than t he l atter and can be adminis trated orally [5] [6 ]. Another exampl e is t he deve lopment of the p rodrug cyclopho s phamide (C ytoxan TM ), a cyt ostati c mo lecul e that has b een used t o s low the progressi on of s everal types of cance r, including ly mpho mas and l eukemias , mul t ipl e myeloma, neuro - and retinoblastoma and cancer of the b reast and ov ary. Cy cloph osphamide belongs to the fami ly of "nitrogen mustards". It i s a prodrug that is met abolized i n the organism and ev entuall y l ead to pho sphoramide mustard, its active met abol ite [5] . However, their cl ini cal app li cation throu gh con venti onal dru g-deli very appr oaches cau ses nonspecific bio -distribution and low sel ectivity. Ther efore, and regarding d ru g adminis tration, th e method s used h as been tr aditi onally l imited to maki ng t he drug acces s ibl e t o the bloodstream, relying on t he i rr ig ation and the dru g affinity for the tissues for the access to the target. In fact, b io avai lability is still measured fro m drug levels i n the b loodstream, not in the tar get surrounding s. In man y cases, o nly a small por tion o f the Chapter III ___________________________________________________________ Introduction 53 adminis tered drug reaches t he tumor s i te [7] . As a con sequence, cancer treatments generally involv e the administration of relati vely hig h doses of the drug i n t he h ope that a porti on , al though minor, wi ll go to damaged ti ss ues [8] . T herefore, there is a need to i ncr ease t he drug con centration in the cancerous tis sue w hile r educing t he s ide eff ects associated with chemother apeutic mol ecules. Th is req uirement is ev en more c om pell ing i n the case of hig hly toxic anticancer d rugs, whi ch may al so present to o -d eficient phys ico-chemical and s tabil ity features such as t he l abile camptothecin or pi locarp ine. Thu s, t he i ndiscriminate destruc ti on of nor mal cell s, t he toxi c ity of convent i onal chemotherapeu tic drugs, as w e ll as t he development of multi dru g resistance, suppo rt the need to find new effective targeted treatments based on t he changes in the molecular biology o f the tu mo r cell s. Pil ocar pine i s an al ka loid that is obtained fro m the leaves of tro pical shrubs of the g enus Pil ocar pus. It i s a par asympathomimetic dr ug, a no n -s electi ve a gonis t of the mus car ini c receptors of the parasympatheti c nervou s s yst em (Fi gure III -2). The e ffect of pilocarpine of the sw eat and saliv ary glands, compar ed to other cholinergic drugs , i s mor e potent, because pi locarp ine has no quatern ary amine in its s truc ture, which allows i t to c ros s the bloo d brain barrier. Figure III- 2. Two-dimensi onal and t hree - di mens iona l s tructures of p ilocarpine hydrochlor ide. Pil ocar pine is a therap eutic molecule wit h l ow wat er sol ubili t y, clinically used as co -dru g i n gl auco ma and xerostomia as we ll as i n the treatme nt of head and neck can cer; it is also pr escript agai ns t Sjogren's s yndr ome. Sal iv ary gl and h ypofunction, commo nly devel oped during radiati on therapy to the head a nd neck cancer and i n pati ents wit h Sjögren s yndro m e [9], leads to dimi ni shed secretions and the aci dificati on of sal i va. The later character is tic would not only affect the regular homeo st asi s of the o ral cavi ty, l ead ing to speci fic changes in the s ali vary bacterial pr ofiles , but also the demineralization of tooth en a mel , wi th t he consequent i ncrement i n the risk for caries [10]. I n t his context, pilocar pine is us ed to redu ce t he s everity of xerostomia and saliv ary Chapter III ___________________________________________________________ Introduction 54 dysfunction si nce this drug can stimulate s a livary t issues. Hence, t he i ncor po ration of pi locarp ine into pH-sens itive NP s allows the release of the drug under those acidic envi ro nments to ex ert its therapeu ti c activity. ( S )-c amptothecin (CPT) is a s tron gl y cyt otoxic mol ecule w ith ex ce llent antitumo r activi ty over a wide spectrum of human cancers l ik e l ung, colon and breast [ 11] (Fi gure II I -3 ). CPT i s a wat er insol uble , natural pentacycli c alkaloid isolated fro m the oriental t ree Camp t otheca accumin ata and s everal researcher s have reported that camptothecin i nhibits a cell ular enzyme DNA topo is omerase I and induces apoptosis in various can cer cell s [12] . Figure III- 3. Two-dimensi onal and t hree - di mens iona l s tructures of camptot hecin. However, i t s cl inical use is curr ently li m ited because of it s poor solubil ity in wat er, l ow plasma stabili t y l inked to the cleavag e of i ts l actone ring at physiol ogica l pH and s evere toxi ci ty . D ue t o CPT insol ubilit y in biocompatible s ol vents, i t is very difficult t o apply conventional drug adminis trat ion routes, including or al, intraveno us o r intramuscular injecti on, to distr ibute thi s comp ound th roughout the body [13]. F urther m ore, there a re other negat iv e s as pects t hat li m it the use of CPT in cl inical t ria ls: prono unced loss of acti vi ty due to l actone -r ing hydro ly si s, reversibility of dr ug-target i nteraction and s evere toxici ty, including hemo rr hagic cystitis and myel otoxic ity [14]. Nowadays, one of t he m ost ef fective research s trategi es t o achiev e a safe an d efficient release of camptothecin t o target cells i s the us e of na no -vehicl es and a r ecently pub li shed review i ncludes the mo st innovati ve ap pr oach es [15]. Alt ho ugh inflamma tion is part of t he no rmal host res po nse t o i nfection and i njur y, exces sive or inappr opriat e i nflammation contr ibutes to a range of acute an d ch ron ic h uman dis eases w hich are char acterized by the p ro d uction of i nflammatory cytokines, arach i do nic acid – de rived ei cosanoids, other i nflammatory agent s and adhesi on molecules [16] . Thus, high con centrations o f tumo r necr osis factor (TNF - α ), i nterleukin - 12 (I L-12), an d i nterleukin-6 (IL-6) are particularly des truc ti ve Chapter III ___________________________________________________________ Introduction 55 and are i mpli cated in s ome of the pathologi c respon ses that oc cur i n end otoxic s hock, i n acute respirator y dis tress sy ndr ome, and in chro nic i nflammatory di seases such as rheumatoid arthritis and inflammat or y bowel dis ease (IBD ) [17] . IBD is a g ro up of dis or de rs characterized by a chro nic and relapsi ng i nflammation of the gas trointe sti na l tract frequent i n W est ern coun tri es [18] . The two m ost common forms of I BD are Croh n’s diseas e (C D) and ulcerativ e colitis (UC) . They l ead to long -term and someti mes i rr ev ersible impai rment of g ast ro- intesti na l structur e and functi on [19] . In UC, a diffuse mucosal inflammation of the co lon i s mai nly fou nd as wel l a s the coincident pr oducti on of a complex mix ture of inflammat or y mediators and extensi ve superf ici al mucosal ulceration. Conversely , in CD, any part of the g ast ro- intes tinal tract (GIT) from t he mo uth to the anus can be affected, al though it is usuall y the i leum and c olon. Unli ke UC , C D may be patchy and seg mental . I n addit ion, inflammation c an be typi cally tran smural [16] [17]. It has been hypothesi zed that IBD r esul ts fro m an i napp ro p riate and exag gerated muco sal immune respon se to n or mal co nsti tuents of the mucosal mi cro flora that i s, i n par t, g enetically determined [17] . It i s now wel l establ is hed that mi cro bial componen ts of the r esi dent mi cro biota can regul ate gut inflammat ion [20] . I n CD , in particular, the int es tinal mi cro biota is s tron gl y sus pected to play a ro le i n i nitiat ing and t riggering t he i mmune syst em , leading t o character is tic i nflamma tion [21] [22]. Howev er, the inher itabl e co mp onen t als o pl ays a ro le an d seems st ro nger in C D than in ulcerativ e coli tis [23] . M oreover, it is w or thy to h ig hlight that in sev eral coun tries w ith his torica l ly low rates of IBD, a pattern of ris ing inci denc e in t he pas t on e to tw o deca des, par ticularly fo r CD , has occ urred, sugg esting that environmental factor s are als o inv olved [19] . IBD has an e no rm ou s i mpact on peop le’ s l iv e s. Acc or ding to the Insti tute for Health Met rics and Evaluati on (GBD 2017 , Univ ersity of Was hingt on ) IBD was respon si ble , globally, for the 0.07% of deaths that occurr e d i n t he wo rld i n 20 17, i .e. , the death o f 3. 86 mi llion people was directl y cor related with some of these path ologi es. Th ey al so have a mar ked influence on the qualit y of li fe of il l people. Thus, the g lobal disabi lit y -adjusted l ife years (DALYs) co nnected wi th these dis or ders was of 18 4.95 mil li on i n 201 7. The hig hest DALYs v alues were foun d f or pe ople fro m E ngland, The United States of Amer ica and N or way , as c an be seen in Figur e III - 4. Ther efor e, finding n ew therapies aimed at the overall redu ction of the inci dence o f IBD is a necessi ty. Chapter III ___________________________________________________________ Introduction 56 Figure I II - 4. D AL Ys per year of infl ammator y bowel d isease (IBD) in selected locati ons in 20 17 , or de red by incidence and sex . Conventional treatment of IBD is based on the dail y adm inis trat ion of hig h doses of i mmune - suppr ess ant or anti -inflammatory drugs, often c ompli cated by seri ou s adverse effects. Thu s, a carr ier sy stem that de li v ers the drug s pecifically t o the i nflamed intest inal reg ions and s hows pr olonged drug r el ease would be desi rab le [2 4] . Sodium dicl ofen ac (DC Na) (Fig ure II I -5), i s a nonsteroidal anti -inflammator y dru g (NSAID) used t o treat pai n and i nflammatory dis eases [4] . The primary mechanism respon si ble for i ts anti - inflammat or y, antipyretic , and analg e si c action is t ho ught to be i nhibition of pro stag landin sy nthes is by i nhibit ion of the cycloxyg enase -2 (COX-2). I nhibition of pr ostagl andin synthesis occu rs sy s temi ca lly resulti n g i n undesi rab le s ymptoms such as irritati on of the gas tric epithel ium, this is the main si de effect of diclofenac. Serious si de eff ects may include heart dis ease, st ro ke, kidney pr oblems , and s tomach ulceration [25]. It is bel iev ed to work by dec reasi ng t he pro ducti on o f Chapter III ___________________________________________________________ Introduction 57 pr ostag landin [26] . Dicl ofenac is used commonly to treat mi ld to moderate po st op e rativ e or post - traumatic pain, in par ti cular when inflammati on is also pr esent. Figure III- 5 . T wo-d imensio nal and three -d imensional s tructures of sodium diclofe na c. Resv eratr ol (R ES) (Figure II I-6), a natural o ccu rr i ng p olyphenol, is the main biological ly activ e comp onent in red wi ne. Hundreds of publicat ions have demonstrated that RES can prevent or delay the pr ogression of a wi de variet y of dis ea ses [27] du e to i ts antipl at el et acti vi ty [28] as well as antit umor [29] , neuroprotecti ve [30] [31] and a nti -inflammat or y [32] [3 3] properti es. Resveratrol has been repor ted to decrease i nflammati on by inhibi t ing the induced pro ducti on of pro - inflammat or y cytok ines, such as T NF - α, IL - 1β , IL -6, and IL-8, and matrix metallopro tei nases M MP - 2, MMP -3 , M MP-9 and MMP -13 , i n in vitro and i n vivo models [34]. Th e main problem as sociated wit h the therapeutic us e of R ES are due to i ts rapid met abolis m in vivo [35] i ts l ow w ater s olubility and it s chemical i nstability. These dr awbacks has been ad d ressed by the prepar ati on of resveratrol derivatives so t hat their i ntrinsi c bi ological activit y and bioavai labili t y would be i mpro ved [34] [36][37]. Figure III- 6. Two-dimensi onal and t hree - di mens iona l s tructures of res veratro l. Due to t he aforementioned d rawbacks relat ed to s ome of the cu rr ent the rapeutic t reatments, new target ed therapies are of g rowing i nteres t. I n addit ion, bl ock i ng the bi o l ogical transduction Chapter III ___________________________________________________________ Introduction 64 I II .3 . Towar ds the Desi gn of Stable Smart Polyme r Nano par ticles To date, rema rkable init iatives hav e been dedicated to the advancement of st imuli -responsive NP. They have been devel op ed to achi eve the controll ed releas e of pa yl oads at the target s ites i n respon se t o a s pecific sti mulu s. They are call ed in tel ligent, smart , or envi ron mentall y -respon si ve polymers [2] [8][43][45]. When a smart sys tem is involved, the release of a dru g encapsulated i nto the s mart NP can be trig g ered by means of a c hange i n a particular pr o per ty (en dogenou s or exogenous). Once the NPs mee t the harmed t is sue, they respond to the v ariati on of a specifi c pr operty, releasi ng the dr ug and i mproving both, speci ficity t oward s the harm ti ss ue and therapeutic outcomes [46]. As a res ult, t h i s appro ach not onl y improves contr ol of d rug relea se, i t als o b oo st s its therap eutic features and the dr ug adv erse effects ar e di minis hed [8] [47] . Ther apies based on st imuli -sen si t iv e nan opar ticl es dis play some interest ing pro perties, such as the fact that t hey can successfully lower t he d osage f req uency since the drug r elea se is co ncentr ated in targeted or gans/tiss ues and retained for a much l onger period; s mart s yst em s are al so respon si ble for t he re duction in d ru g fluctuati on and a re capable of enhancing the anticancer therapeu ti c efficacy [48]. Common s timul i exp lored by s timul i -respon si ve polymer s i nclude endo genous [e.g ., r eactiv e oxygen speci e s (ROS), r edox, pH, and enzymes ] and exogenous (e.g ., light, temperatur e, magnetic field , and u ltrasound) sti muli [49] . I II.3.1. pH -sensitive NPs for DDS in Anticancer Therapy The l ower ex tracellul ar pH of tumo r mi cro e nvironments (wi th pH as low as 5.7) dis ti nguishes it fr om normal t iss ue , and the difference is attribu ted to t h e hig her rate of ae robic and anae robic gl ycol ysis in cancer cell s. This gradient has been used to desi gn pH -responsiv e t umor- targeted dr ug release s ystems based on pH-sensi ti ve poly m ers [40] [46] . The es senti a l part of a pH -sensi tiv e DDS is a pH-trig ger ing g ro up (an i onizable wea k ac idic or basi c moiety ) that is attached to the backbone (Fi gure III -9). T hus, most of the pH-sensi tiv e polymers are polyel ectro lytes whose coi led ch ains expand upo n ionizati on , in respon se to chan ges in environmental pH, and their hydro dynamic vol ume i ncreases dramatically due to el ectro st at ic repu ls ion f ro m the ch arges generated (anion s o r cations) along th e b ackbone [ 48] . However, two Chapter III ___________________________________________________________ Introduction 65 main strateg ies ex ist in t he desi gn of pH -sensi tiv e dr ug deliv ery s ystems: firstly, the us e of polymeric s y st ems wi th i on izable g ro ups a ttache d t o t he backbo ne that thei r conformational or solubility proper ties changes in response to cha ng es i n pH v alues . Second ly , the use of polymers bearing acid-s ensitive bo nds whose cl eavag e triggers t he release of d rug m olecules att ached to the polymer backbon e, modificat ion of the polymer charge or the ex po sure of target ing lig ands. The first st rategy is preferred in t he development of pH -respon si ve sys tem s for drug releas e at tumor sit es since a sharp r esponse is obtained to changes in pH in the t umo r extracell ular microenviron ment. Figure III- 9 . pH-responsive nanopartic les prepa red fro m an amph i philic di - b lock cop olymer in w h ich the anticancer d rug i s embed ded into t he lipoph ilic core. (B ) D rug rele ase of antica ncer drug fr o m pH-sensitive NPs triggere d by t he ac idic env ironment fo und in so lid t umors. Alt ho ugh current research an d revi ews have exp lored numerous and novel as pects for s timul i - respon si ve dru g deli very nanoplatfor ms [50], t here are s till i mpo rtant features that must be addr ess ed fo r the future devel opment of s uch s ystems, s o that they can achiev e a rapid and Chapter III ___________________________________________________________ Introduction 66 effective cli n i cal transl ation . Table II I-2 displ ays the most s ig nificant prop erties to pursue when desi gn ing polymer NPs as D DS. Tho se features are ma rked by some NPs p roperties s uch as comp osit ion, si ze, charge and target ing l ig and functi on ali zation, properties that can s ubstantially and p osi tively af fect their biod is tribution and NP blood circ ulati on hal f -li fe, r educing rates o f non -specific uptake, thus del ayi ng opsonizati on, and increasing the ex tent of t is sue specific accumu lat ion [5 1] . Table II I- 2 . Ratio nal des ign o f po ly mer N P s as DD S: pr oper ti es t hat the na n ocarriers sh ould meet for a ra pid and effect ive cl i ni c a l tra nslation. Rational design of polymer N Ps as DDS: pr operti es t hat the na noc arriers should meet • Nanocarriers sho uld be m ade from a material that is bioco mpatib le, well characterized, and easily functio nalized • Nanocarriers sho uld be either solub le or collo idal under aqueo us cond itions for increased eff ectivene ss • Nanocarriers sho uld have an extended circu lating half -lif e, a low rate of a ggregation, and a lo ng shelf lif e • Nanocarriers sho uld exhibit high differ ential uptak e efficiency in the ta rget cell s over normal cells (or tiss ue ) The fo rmation of polymer na noparticl es can be achiev ed not only by the s elf -assembly of two mix ed compo nents of op posed hydrophili c/hydr op h obic nature [52] but als o by the use of amphiphili c block -co po ly mers, leading to nano si z ed micelles [53] . By ensuring the homo genei t y of the g enerated nanoparti cles, t he lat t er method all ows t he re l iabl e prepar ati on of formulati ons widely used in varied fiel ds, fro m drug deli very, bios ensor s and g ene therapy to cosmetics , among other s [ 8][54]. To attai n t he p reparation of s uch bl ock -cop olymer s, li v ing poly merization procedur es, which have been ex tensi vely devel oped fr om mi d -90 s t o now , are the proce dures of ch oice. The attributes of these poly meriza ti on methods include l ow mol ar mas s polydi spersity , hi gh end g ro up fi delit y, capacity for c ontinued chai n gr owth and access to com plex architectures. These att ributes are cru cial to many appl icati ons in ensuring a repr odu cible, rel iable and un iform response [5 5]. Amon g the livi ng polymer ization t echniques can be hig hli ghted oxyan ionic polymeriz a tions [56] , atom transfer r adical poly merizati ons (ATRP ) [57],[58], radical additi on-f ragmentati on ch ain -tran sfer Chapter III ___________________________________________________________ Introduction 67 polymerizations (RAFT) [59] and polymeriz ation-induced sel f-ass embly (PI SA) [60] . Al l t ho se techniques lead t o the produ ction of poly mers with low po ly dispers it ies and well -co ntro ll ed comp osit ions and molecul ar w eig hts fr om a w ide v ariety of mono mers, in contrast t o cla ss ical radical po ly merization. Micelles fro m di- block - co polymers are one of the most popu lar DDS l eading to cor e−shell m icelles si nce they are able to transpor t l ipophili c molecules i nto their cores [43][61]. Regarding the comp osit ion of the polymeric material, the presence of a highly hydro phili c cor ona l ayer makes the micel les v ery s table i n aqueous medi a [62]. A mong the most comm only chosen bioc ompatible hydro phili c b locks i n biomedical f iel d, poly(ethyl ene gl ycol) (PEG) [39][63] , c hitosan [64], and some polymers obtained fro m methacrylate est ers suc h as 2 -hydro xy ethyl methacrylate [HE MA [65] ], and N,N - dimet hylaminoethyl met hacr ylat e [DMAE MA, [66]] can be found. In t he hydr o phobic blocks, the presence of t he biocom patible polycaprolactone [(PCL, [39]] , poly(l actic acid) [PLA, [43] ], poly(lacti c glycolic acid) [PLGA, [ 67]] , p oly(prop yl ene o xi de) [ PPO, [ 68]], polydimet hy lsiloxane [PDMS, [66]], cicl od ext ri ns [8] [ 63] , and other polymers based on methacrylate derivatives s uch as N,N -d iet hy laminoethyl met hacr yl a te [DEAEMA, [69] ] a re t he most commo n options. However, the dynamic and revers ible na ture of mi celle for mation i s a sourc e of inst abi li ty. As micelles can dis sociate at l ow concentr ations (below their cr it ical mi celle con centrations , CMC , for exampl e , in the blo odstream), the pr emature drug releas e in nor mal t issues or organs may t ake place and consequentl y cause serious side effects [39] . The disinteg ration o f N Ps by dil uti on can be pr evented i f t hey are pro perly cro ss -li nked, either in the she ll or i n the core [70] , l eading to st able unimolecular N Ps able to keep their co ntents intact until a tr ig gered st imul us oc cur s [ 71][72]. Alt ho ugh s hell -cro ss linkin g has been o ne o f the pr eferred met hods to st abili ze NP upo n di luti on , this proced ure must be c onducted at hi gh di luti on s s o th at the c ro ss -l inki ng reacti on s take pl ace among functi onal g ro ups present w ithin the s a me NP an d hence, avoidi ng the formati on of agg regates (Fi gure III - 10) [ 7 3]. T he core cro ss -linking appr oach circu mvents inter -micell ar reactions, both at hig h and low NP concen t rations, since the h ydro phili c segments fr om t he shel l behave as steric st ab i lizer blocks pr eventi ng int er -micelle cro ss -li nk ing [74] . Chapter III ___________________________________________________________ Introduction 68 Figure III- 10 . Formatio n of aggreg ates or sta ble nan oparticles from crossli nking assa ys in t he mice l les c ores or the micel les shells , respect iv e ly. To effecti vely cro ss -li nk the micelles, the Diels – A ld er (DA) reaction, that is encomp ass ed withi n th e cli ck reacti on s, bec omes an i deal cand idate for its use i n the pr eparation of f unctional and respon si ve biomateria ls. C li ck Chemistry (CC ) ha s emerged as a wide -spr ead appro ach t hat uses only t he most -pr actical and -r el iab le chemica l transfor mations, with an explosive growth in pub li cati on s i n recent year s [75] [7 8]. The t erm CC w as coined by Sharpless and col. [75] . According to the authors, a r eaction sho uld meet a s et of s t ringent cr it eria to be defined as a click reaction. Thu s, “ the react ion must be modula r , wid e in scope, give very hig h yield s, genera te onl y in offensive byprodu cts that can b e removed by n onchro ma t ograph ic m e thod s , a nd be stereospecific (but not necessaril y enantio-selective). The requi re d process ch aracteri st ics i nclu de sim ple reaction con d ition s (ideall y, th e process shoul d be insens i ti ve to oxygen and w ater), readi ly avail a bl e startin g mat er i al s and reagents, t he use o f no sol v en t or a solvent that is benig n (such as wa ter) or easil y removed, and simple p ro duct isol a tio n. Purif ica t ion — i f requi re d — m ust be b y non chr om a tog ra phic m e tho d s , such as crystallizati o n o r disti ll ation, and the p ro duct must be stabl e un der physiological con di t io ns” [75] . Alt ho ugh fitti ng the r equirements of a click reaction is a tall or der , sev eral p ro cess es have don e so. Thu s, t he DA -coupli ng reacti on (Scheme II I- 1) i s a wel l -established cli ck reaction t hat combines a diene moiet y, such as f uran rings and a dienoph il e g ro up, such as malei mide rings, i n a robu st, Chapter III ___________________________________________________________ Introduction 69 efficient, and or thogonal method f or t he fun ctio nali zation of differen t c ompounds [79][80] . T he chemoselectivi t y of DA reactions is narr owly defined, that i s, i t is ortho gonal to an unusually br oad range o f r eagents , sol vents, and other functional group s such as the – C OOH, – OH and – NH 2 grou ps fou nd i n biomolecules. I t is als o moisture and oxy gen to lerant and, under mi ld conditi on s, does not generate si de pr oducts. Scheme III- 1. Typical scheme of a Die ls Alder re action between fu r an rin g (F) and ma leim id e r i ng (M ) . Endo and ex o adducts format ion (AendoFM , AexoFM , respect iv ely) As the cli ck reaction that i t is, the yields are usually very hig h , and the reaction can be ca rried o ut und er particularly mi ld conditions (aqueous soluti on , neutral pHs and moderate temperatures). Moreo ver, that DA reactions can proceed i n the a bsence of met al catal ysts is an ex tra bo nus [8 1] . One of the prefe rr ed DA reactions i s the reacti on between furan and malei mi de moie ties. This comb inati on has been w idely us ed since its DA adducts are for med at mod erate temperatur es and can be reverted to t he s tarting mal e imide a nd fur a n pai r at el evated temperatures [20] . Temper ature ca n accelerate the D A and the retro -DA reactions si multaneously, affectin g the overall results. Additi onal ly, water h as been demo nstrated to ex ert a unique eff ect in this ty pe of reaction, not o nly i n its kinetics but als o i n its ster eosel ectivity [25] as was demonst rated i n t he early 198 0s by t he resea rc h teams headed b y B resl ow [26] and G rieco [2 7]. These findings can b e con si dered the “Bi g Bang” in aqueou s sy nthes is and have trigg ered gener al i ntere s t in the u s e of water as a solvent in or gani c chemis try [25] . C onsequently, t h i s react ion all ows c lean, reliable and sequential transfo rmations of wide scope [ 7 5] . Chapter III ___________________________________________________________ Introduction 70 III .4 . Hydro gels. A Br ief Over v iew Hydro gel s consti tut e a group of p olymeric materials t hat exh ibit t he ability to swell and retain a si gn ificant fraction o f water wi thin their thr ee-di mens ional (3D) networks b ut do not diss olve i n water o r biological f lui ds. Hydr ogels h ave been defined as two - or m ulti -compo nent sys tems con si st in g of a three-dimens ional networ k o f polymer chains and water that fills the s pace between macromolecules. Many materials, bo th naturally occur ri ng and s yntheti c, fi t the definiti on of hydrogel s [82] . Their abil ity to absorb water de pend s o n the polymer (po ly mers) com positi on, i.e., the hydro phili c fun ctional gro ups attached to the p olymeri c back bone s uch as -OH, -CONH – , – CONH 2 and – SO 3 H, as well as on the natur e and densit y of the net wor k joints. Water co ntent o n t hese 3D s tructur es can vary over a broad val ue range; ty pically, i n the sw ollen stat e , the ma ss fraction of wat er i n a hydro gel is much h ig her than t he po ly mer mass fraction (someti mes, more than 90% wt. ). The resis tance of t hese mat erials to diss olut ion arises fro m the cross -li nk s between netw or k chains which are pro vi ded by covalent bond s, hydrogen binding, v an der W aa ls i nt eractions, or physi ca l entangl ement s [38] . In g eneral terms, hydro gel s can be cl ass if ied based o n a v ariety o f cha racter is tics, i ncluding the natur e o f s ide g ro ups ( neutral o r i onic, i. e., basic , acidic, ampho teric el ectro ly te or zw itterion ic), or ig in of t he polymer (n atural o r sy nthetic), method of p repar ation (h omo - o r co-poly mer), physical st ru cture (amorphous, semi crys talli n e, hydrogen b ond ed, s uper molecular, and hydro collodial), and res po nsi veness to physi ol ogic envi ron ment s t imuli (pH, i onic strength, temperatur e, el ectro- mag netic radi ati on , etc. ) [38] . They can als o be divi ded i nto two categ or ies based on the chemi cal o r p hysi cal nature of t he cross -link junctions. Chemi ca lly cross -li nked networks have per manent junctions, whil e physical netw or ks have transient junctions t hat ari se fr om ei ther p ol ymer chain entangleme nts or p hys ical i nteractions s uch as ionic interact ions, hydro gen bonds, or hydrophobic interactions. More than a ny other clas s of sy nthetic bi omat erials, hydrogels have s ome commo n physi cal pr operti es resembling those of the li vi ng ti ssues , which is at tributed to their high -wat er content, their sof t and r ob bery consi st ency , and low inter facial tension with wa ter or biologi cal f lui ds [38] [83]. Likewis e , the e lasti c nature of hydrated hydrogels mi n imi ze s irritati on of surr ound ing tissues after impl antat ion. Als o, the low interfacial tensi on between the h yd rogel surface an d body Chapter III ___________________________________________________________ Introduction 71 fluid l ess en s protein adsor ption and cell adhesi on, whi ch red uces t he chances of a neg ati ve immune reaction [84] . Havi ng s aid t hat, i t i s no s ur prise that hydrogels have foun d their most relevant appli cations in the b iomedical and pharm aceutical fields where it is necessary that ge ls be degradable und er physi ologi ca l cond iti ons a nd l e ad t o the dis integ rat ion of t he t hr ee - dimens ional structur e, prefer abl y i n har mles s pr od ucts, to ensure the good biocompatibili ty of the hydrogel [85] . T hus, the ever-growing hydrogel technolog y has l ed t o advances in biomedica l appli cat ions (Fig ur e III-11) as div erse as dr ug delivery sy stems [84] [86] , ocular dru g deli very [87] , cel l pr oli fer ati on and di ffer e ntiat ion [88] , tis sue eng ineering and regenerative medici ne s [89] [90] , wound dress ing [91] [92], anti-biofouli ng pr oper ties [93] , and biosensors [82][94]. Figure III- 11 . Exa mples of biomedic al app li c a tions of h y drogels. As menti oned above, t heir unique physi coc hemical and biologi cal characteris ti cs wit h t he i r huge diversity, collectively , have l ed the hydr ogels to co nsiderable att ention as exce ll ent candidate s for deli very systems of therapeutic agents. A cla ssification b ased on the ro ute of adminis tration of the hydro gel drug deli very s y st em s, seems to i nclud e the v ast area of thes e therapeutic mat eri als. Accor dingly , pharmaceutical hydro gel s can be cl assi f ied as : (a) oral hyd rogel s ys tems; (b) transdermal an d implantable hydro gel sys tems; (c) t opical and transdermal h ydrogel sys tems ; (d) Chapter III ___________________________________________________________ Introduction 72 hydro gel devices for gas tro-intes tinal tract (GI T) drug deli very; and (e) hyd ro gel -based o cular deli very sy stems. Fur thermor e, hydrogel -based for mulati ons applied via other r ou tes are als o notewor thy. In this regard, no vel app ro aches to i mpro ve bioavai lability thr ough nasal and vaginal ro utes usi ng hydr ogels have been presented [ 9 5] . The poly mers common ly used in prepar ati on of hydro gel s with pharmaceu tical and biomedical applicati ons are fro m natural or s yntheti c or ig ins. Typical example s of natur al, s ynthetic and comb inati onal, i.e., s em is ynthe tic polymers us e d in hydrogel prep a ratio ns are summariz ed in Table III -3 . Table II I- 3 . Hydro phi li c p o lymers use d in prep aration o f hyd r og e ls . Nat ur al polymers and their derivatives Anionic polymers HA, alginic acid, pectin , carrageenan , chon droitin sulfate, dextran sulfate Cationic po lymers Chitosan, polylysine Amphip athic po lymers Collagen ( and g elatin), carbox ymethyl chiti n, fibrin Neutral polymers Dex tran, a garose, pullulan Synthetic polymers Polyesters PEG – PLA – PEG , PEG – PLGA – PEG, PE G – PCL – PEG, PLA – PEG – PLA, PHB, P(PF- co - EG)6acrylat e end groups, P(PEG/ PBO terephthalate) Other polymers PEG-bis-(P LA -acrylate), PEG 6CDs , PEG-g - P(AAm- co -Vamine), PAAm, P(NIPAAm- co - AAc ), P(NIPAAm- co -EMA), P V Ac/PVA, PNVP, P( MM A - co - HEMA), P(AN- co -allyl sulfon ate), P(biscarboxy- phenoxy - phosph azene), P(GEMA-sulfate) Combinations of natural an d synth et ic polymers P(P EG- co -peptides), alginate- g-(PEO – PPO – PEO), P(P LGA- co - serine) , collag en -acrylate, alginate- acr ylate, P(HPM A- g - peptide), P(HEMA/Matri gel®), HA-g-NIPAA m Abbreviations : HA, hyaluronic acid; PEG, poly (ethylene glycol); PLA, poly(lactic acid); PLGA, poly(lactic- co -glycolic acid); PCL, polyc aprolacto ne; PHB, poly(hydroxy butyrate ); P F, propylene fumarate; EG, ethylene glycol; PBO, poly(butylene oxide); C D, cyclodextrin; PAAm, polyacrylamid e PNIPAAm, po ly(N-iso propyl acrylamide ); PVA, poly (vinyl alc ohol); PVamine, poly(vinyl amine) PVAc, poly(vinyl acetate); PNVP, poly ( N -vinyl pyrrolid one); P A Ac, poly(acrylic acid); HEMA, hydroxyeth yl methac rylate; PAN, polyacrylonitrile; PGEMA, poly (g luco syleth yl methacrylate ); PEO, poly(ethyl ene oxide); PPO, poly(propyleneoxide ) ; PHP MA, poly(hyd roxypropyl methacrylamid e); PEMA, poly (ethyl methac rylate) ; PMM A, poly(methyl methacryl ate) . Chapter III ___________________________________________________________ Introduction 73 III .5 . Chito s an Alt ho ugh hydrogels of natural o rigi n may s how mechanicall y sub-optimal characteristi cs, they do off er v arious advantageous proper ti es s uch as being usually non -toxi c, biocompatible and biodegradab le, apart f ro m other rema rkable properties that make them sui table for diffe rent applicati ons in d rug del i very systems [38] . A relev ant example is the po lys acchar ide c hitosan (CTS). The accumulated i nfo rmation ab out t he phys icochemical and biologi ca l prop erties of C TS led to the r ecogniti on of this po ly mer as a p ro mis ing mater ial fo r d rug deli very [38] . The p olys acchar ide CTS, a weak cati onic p oly sacc har ide com po sed of ran do mly di st ributed β - (1 – 4) -li nked D -glucosamine and N - acetyl - D -g lucosamine repeating units, is a copo lymer p rep ared from renewable r esources. It can be obtai ned f ro m the partial deacety lation of the seco nd most important natural po ly mer i n the world: chiti n or poly( N -acetyl- β - D -g lucosami ne). Its abundan ce i n marine crustacean, s uch as shrimp and c rabs, makes i t a commer cial prod uct w ith gl obal i mpact in polymer s cience. Thus, f rom a t echnical p oint of v iew, it is ex tremely important that chit osan i s hydro soluble and posi ti vely char ged. These pro pe rties enable this polymer to i nteract with neg a tively char ged poly mers, macro molecules , and ev en wi th certain polyani ons upon contact i n aq ueous envi ron ment. T hese interactiv e forc es and the resulti n g s ol – g e l tra nsiti on st ages have been exploited for nano - encapsulati on pur po ses [9 6] . On the other hand, chitosan has the special possibility of adher ing t o the mucosal s ur faces wit hin the bod y, a property leadi ng to the att ention t o thi s polymer i n muco sal dru g deli very [87] . 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Chapter IV : Objectives Chapter IV ______________________________________________________________ Objectives 96  Pre paration of nanoparti cl es a s potential DDS by m eans of polyelectrol yte co m plex ati on ( Un iversid ad e d o Al garve ) The general g oal of t he resear ch cond ucted i n the Dep art am en to de Qu ímica e Farm á cia , Universida d e do A lg arve (Portugal) i s the develop ment of po ly cationic derivatives from the natur al occu rring locust bean g um (LBG) w ith a degree of aminati on cl ose to that fo und i n most of t he commer cial ch it osan mater ial s. Th e use of t he amine d e rivat ives in the p reparati on of nano particles as potential D DS by means o f polye lect ro lyt e co mplexation will be i nvestigated (Tab le I V-3 ). The formation of new poly cationic LBG derivat iv e s will be i nv estigated thro ugh a two -st ep pr ocedure: oxidati on of LBG raw material and sub sequent reductiv e aminati on. T h e intermediates and final products wi ll be studied by infr ared and ultravi ole t -visi b l e spectrosco pies . The f ormation of NPs based on the ani on ic k appa-carr agee nan and the s ynthesi zed poly cationic LBG derivat ive (LGB am ) w ill be ex p l or ed by means of poly electrolyt e complexa ti on. Comp lexes pr epared wi th sev eral LBG am -carr ageenan mass ra t ios will be te sted. D ynamic light scat tering data and zeta potential st udies will b e the too ls to exami ne and und e rstand the f inal result s. Table IV- 3. Summary of the m ai n tar gets for t he prepa ration of NPs as DDS by m eans of po ly e lectro ly te complexation Nanopa rticle s as potent ial DDS by means of poly ele ctrolyte comp lexati on (Ch apter XI) Objectives How / What Com ments Controlle d oxidatio n of LBG Sodium periodat e, sodium hypoc hlorite and ammonium persu l fate * T ransformations followed by second derivative of FT IR absorb ance spectra Formation of p o lycatio n ic LBG materi als Reduct ive amination of oxidized LBG derivatives * T ransformations followed by second derivative of FT IR absorb ance spectra and UV -vis spec tro scop y Nanoparti cle formation. Characteri zation Polyelect rolyte complexation * K-carr ageenan used as po lyani onic cou nterpart * Several LBG am -c arrageenan mass ratios will be t ested * St udy o f influence o f +/- charge ratios * Size, polydispersit y , z-potent ial Chapter IV ______________________________________________________________ Objectives 97  O ptimizati on of Cus to m iz ed Pol yme rizati on Con dit ions for the Preparati on of Target ed Smart M at er ial s by the Di els -Alder Click Reactions ( Un iversi da d d e Sevi ll a ) This thesis will also s tudy t he op timi zation of t he DA polymerizat ion conditi on s to synthesize two famil ies of thermo-sensit ive poly mers , either wi th reduction-respon si ve pro perties, or wi th enhanc ed hydrophil icity w ith po tenti al use i n biomedici ne (Table IV-4). To fi nd the optimal polymerization conditi on s, a Box – Behnken experimental de sign will be used s o that the r elati ve influence o f the chosen variables (temperature an d water content) can be determined. The influence of temperature on t he s tabil ity of furan − mal ei mide adduc ts will be explor ed by thermo gravi metric analyses i n or der to have a first approach to t he t hermo -degradab il it y of the new materials. Addit ionally, the scope an d limi tati on s of the use of IR spectroscop y, via the second derivative of the a b sorption spectra, w il l be investigat ed as a tool to t rack the deg ree o f con versi on of the monomers in the polymer synthes es. Table IV- 4. Summary of the m ai n tar gets for t he Opti mization of Customize d Po ly mer i zati on C o nditi ons f or the Preparati on of Target ed Sma rt M aterials by the Di els -Alder Click Re a ctio ns. Optimization of C ustomized Polymeriza ti on Cond it ions for the Prepa ration of Targeted Smart Materials by t he Diels-Alder Click Reacti ons ( Cha pter XII ) Objectives How / What Com ments Preparation of monom ers for DA reacti ons and DA ad duct * Difuran monomers * D imal e imide- prot ect ed model compo und * Synt hesis and characterizat ion by con ventional metho dology (FT IR, M S, NMR) Preparation of functio nal thermo-re sponsive polym ers DA reac tion * Polymers w ith respo nsive propert ies to reduc tive environments wi ll be ai med * Mu ltihydroxyl DA polym ers wi ll be aimed Thermal degradati on studies of DA adduct Thermogravi metric analyses * Kinetics d ata wil l be ob tained from experiment s c onducted at fixed temperatu res Optimal polymeri zation conditions Box – Behnken experiment al design *Determination of t he relat ive i nfluence of the independent variables: temperatu re and water content Degree of monomer conversi on in polyme r s yntheses IR spect rosc opy * Quant itative analyses v ia the second derivative of IR abso rbanc e spec tra Chapter V: General Met hods Chapter V _________________________________________________________ General Methods 101 V.1. Mater ials All chemica ls used were pur chased from Aldrich C hemical C o (Madrid, Spain) apart fr om Jeffamine D- 230 polyetheramine, which was pur chased from Huntsman ( Huntsman Corporation), Camptothecin which w as pur chased from TCI Europe (Tokyo Chemi cals I ndu stry) and Res veratro l which was pr ovided by Se gu ra’s Center of Edap hology and App li ed Biology (CEBAS -CSI C) (Mur cia, Spain). A commerc ial chitosan (CTS) fro m Sig ma-Aldrich (Sai nt Louis, MO , USA) has been cho sen wit h a deacety lation degree of 7 5% and molecular wei ght 299 kDa . Locust bean g um (L BG) was a kind gi ft from Industrial Farense (Faro, Portugal). Potas sium bromide (KBr) was obtained fro m Riedel -del-Haën (Germany). Ul trapur e water (Mi li -Q Plus, Mili por e Iberica, Madrid, Spain) was used throughout. The 1kDa cut -off mi ni-dial ysis tub es used were purchased f rom G E Heal thcare (Wauwatosa, WI, USA). In addit ion, t he acetate buf fe r at pH 5.5 (25 °C ) f or release as says was pr epared i n-house, w ith pH variations vs. t emperatur e o f 0.1 from 5 to 50 °C . Voltaren emul gel ® (No vartis Farmaceuti ca, Barcelona, Spain) was purch ased from a li censed drugstore (Sevill e , Spain). 2-H ydroxyethyl methacrylate (HEMA), N ,N -diethyl am inoethyl methacrylate (DEA), N,N -dimethyl am inoethyl methacr y lat e (DMA), and furfur yl me thacr ylat e (FMA ) were passed thro ugh a basi c alumina column and dis til led befo re u se. Chapter V _________________________________________________________ General Methods 102 V. 2. Meth od s IR spectra were recorded o n a Jasco FT/IR 4200 spectro meter (G reat Du nmow, Essex , UK) equipped wit h att enuated total s ingl e reflection (ATR) ac ces sory i n t he range betwee n 4000 and 60 0 cm -1 . Nuclear magnetic reson ance (NMR) and mass s pectra w ere recorded at t he CI TIUS Se rvice (Universi t y of Sevill e) . 1 H and 13 C NMR spectra wer e recorded at 300 K wi th a Bruker AMX - 500 a nd a Br uker A dvance AV- 50 0 f or soluti on s in CDC l 3 a nd DM SO- d 6 . Chemical shift s ( δ ) are rep or ted as parts per mil li on do wnfiel d fr om Me 4 Si and J i n Hz. J i s as signed and not repeated. Al l the ass ignments were co nfirmed by COSY an d H SQC ex periments. Mass spectra were ob tained using a Kratos M S80 RFA i nstrument. Hig h resoluti on mas s spectra w ere recor ded o n a Q -Exacti ve spectromete r. Meas ur ements of Ultraviolet a nd visible light absor bance (UV- Vis) were carried out at a n Ag il ent 84 53 UV – vi si ble s pectro pho tometer (Pal o Alto, C A, USA), which presents diode a rray detecti on (DAD). UV-vis measur ements of resveratrol -loaded hydrogels were perf or med wi th a Shimadzu UV -2 102 PC UV – vi si ble spectroph otometer (Kyot o, Japan). In b oth cases , the data were the result of, at least, three measurements. The c hosen s amples w ere exami ned by thermog rav imetri c analysis (TGA), an d the decomposit ion temperatur es of t he different sampl es could be observed. Thermo grav imet ric anal y zer w as TA Inst ru ments Q-600 SDT (New Cas tle, D E , USA). Plati num pans co ntaining appr oximately 5 mg of each s ample were us ed. Trial s were con d ucted u nder i nert atmos phere ( ni trogen, fl ow rate: 1 0 0 mL/min, heating r ate: 10 °C/min), fro m 0°C to 7 00 ° C . The thermal behavi or of the p oly mers was examined by Differential S canning Calorimetry (DSC), using a TA DS C Q-200 Ins trument (calibrated wi th indium, Cerdanyola del V alles, Spain). DSC data were o btained from 1 – 5 mg of sampl es at heati ng/co oling r a tes of 10°C/min u nder a nit ro gen flow (flow rate: 50 mL/min). The gl ass transition temp eratures were determined at a heati ng rate o f 10 °C/mi n from rapidly quenched p olymer sampl es. The ph ase t rans iti ons exhibited by the new pr epared cros s-l inked hydro gel s were ex amined by mod ulated temperature differential sca nning calorimetry (MTDSC), and a refr ig erated coo li n g sy stem (RCS) to ens ur e pr oper temperature cycli ng . Accurately weighed 3 – 6 mg samples wer e then hermeticall y s ea led i nto alum inum DSC Chapter V _________________________________________________________ General Methods 103 pans, and the e quipment was o pe rated i n mo dulati on mode. Cal or imet ric s cans were car ried o u t at a scanning rate of 2°C/min under nitro gen atmosphere o ver an app ropriate temperature range (fr om 0 °C to 140°C). Modulation ampli tude was ±0.159°C every 3 0 s . Gel permeation c hr omat ogra p hy (GPC) analyses w ere perfo rmed using a W ater s appar atus equipped wit h a W aters 24 1 4 refractive index de tector and t wo St yragel ® HR c olumns (7.8× 30 0 mm 2 ) l inked in series , thermostatt ed at 40°C, and us ing N,N -dimethyl fo rmamide (DMF) contai ning 5.8 mM Li Br as t he mobil e phase at a flow rate of 0.5 mL/min. Molecular wei ghts were es timat ed agai ns t methyl methacr ylate standards. The morphology and di stribution o f the NPs were char acterized by sca nning elect ron mi croscopy (SEM) usi ng a HITACHI S5200 fi eld-emi ssion mi cro scope o perating at 5 kV, al so us ed at the C ITIUS Service (University of Sev ille). Before SEM observat ions, the dispers ions were deposit ed and all owed to dry on a monocrystal line s il icon s upp ort treat ed wit h oxyg en pl a sma for 80 s t o make i t mor e hydrophili c o r on a carbon c oated grid in sampl es loaded with camptothecin. In the cas e of hydr ogel s loaded or not l oad ed w ith sodium diclofenac, the samples s caffo ld were directly frozen at - 20 °C for 3 h, t hen at - 80 °C for 2 4 h. The s amples were then lyophilized by freeze dr yi ng for 2 4 h. Fi nall y, the dry hydro gel was fi xed on alumi num s tubs, coated wit h a thi ckness of abou t 25 nm of gold, and analy zed at the Electro n M icroscop y Di vision of the Scienti fic Int e grated Services (SC -ICYT) of the Univ ersity of Cádiz (S pain) usi ng a fiel d emi ss ion scanning el ectro n microscop e FEI Nova Nano S EM 450 (Hil lsboro , Or egon, US) o per ated at 5 kV . In the case of hydrogels l oaded or not loade d wi th resveratrol, the method of l yophiliz ation is t he same. Finally, the dry hydrogel was fi xed on al uminum s tubs, coated wit h a thi ckness of a bo ut 10 nm of platinum-iridi um (Pt -Ir), and imag ed by sca nning elect ro n mi cro scopy using a fiel d emissi on FEI T ENEO micro scope (Hil ls bor o, OR , US A) operati ng at 5 kV at the G eneral Research Services of the Universi t y of Sevi lle (CITIUS). In or de r to determine the NP si ze dis tribution and t o c ompare the result ing data wi th D LS results, statis t ical ana lysis was also conduc ted fr om SEM and fl uor escence imag es. For t hat pur pose, an alg or it hm based on an adaptiv e thresholdin g meth od was used 1 . This appro ach, which makes use of local t hr eshold eval uati on , allows par ti cle s t o be detected aut omati cally and/o r ma nuall y and 1 Y. Li, K. Xiao, J. Luo , W. Xi ao , J.S. Lee, A.M. Gonik , J . K ato, T. A. Don g, K.S. Lam , We ll-defined , rev ersib le disul fid e cross- linked micel les for on- de mand pa cli tax el delive ry , Bio materi als. 32 (2011) 663 3 – 664 5. doi:10 .1016/ j.biomate r ia ls.2011 .05.050. Chapter V _________________________________________________________ General Methods 104 char acterized w ith greater accuracy than when usi ng more -conventional met hod s in w h i ch a gl obal t hr eshold is used. The det ected particl e s’ b oun da ries were then fitted by ellipses def ined by their major and minor ax is 𝑎 an d 𝑏 , respectively, and the size of each NP was determined u si ng the mean geometric d iame ter of t he fitted el li pse 𝑑 = √ 𝑎𝑏 . Final ly, the mea n particl e si ze 𝑑𝑚 was obtained b y fitti ng the size distribut ion with a log -no rmal function d efined as ( Equation V- 1) : where 𝛽 represents the r elat iv e po lydi spers it y 2 . It i s worth mentioning that the use of the adapti ve thresholding al gorithm to detect the particl es was appli ed wi th s pecia l car e i n or der t o rem ove possibl e clusters of sev eral NPs detected f rom th e stati stica l analysis, and to keep only indivi dual particles that ar e the mo st r epresentativ e of the size d is tribution wi thin the samples. Fluorescence int ensity was mea sured on a Va rian C ary -Ec li pse Fluorescence Spect rometer (Varian Iberica, Madrid, Spai n) equipped wi th a x enon di scharge l amp, two Czerny -Tur ner mono chromators, and an R - 298 p hotomultipl ier t ube detector. Al l the meas ur ements to ok place in a standard 10 mm p ath-l ength qu a rtz cell, and excitat ion an d emiss ion slits were bo th set at 5 nm. Fluorescence emi ss ion was measured at 380 nm w ith exci tati on wavel en gth of 360 nm o r, f or CMC calcula tions, at 3 83 nm and 372 n m with exci tati on wavelength of 31 9 nm . Fluorescent ima ges were obtained wi th a ZEISS ApoT ome epi fluorescence equipment w ith motor ized XY stag e (maximum scann ing area 130×85 cm), epifluo rescence i ll uminati on Exci te 12 0HXP wi th mercury -vapor lamp of 1 20 W and high -s ensit ivi ty mon ochrome camer a Axi ocam- 50 6 to capture fluor escent imag es [of 1936×1460 px sensor (3 Megapix el)]. The av erage diameter, si ze distribution (polydis p ersi ty index, PdI), and Z -potenti al of the samples were determi ned wi th a Mal vern Zetas izer Nano ZS (Mal vern Ins truments, Mal ver n, UK) at 25°C, wit h a particle si ze analysis ran ge of 0.6 nm to 6 µ m. Th e intensi ty of the scattered li ght (expressed in ki lo cou nts per s eco nd) was mea sured by dyna mic light sca tteri ng (DLS). The ins trument was pr ovided wi th 4 mW He –Ne l aser (λ = 63 3 nm), digi tal cor relator ZEN3600, and n on -invasi ve 2 H. S. Obero i , F.C . Laque r , L.A. Ma rky, A. V Kab anov, T .K. B ro nich , Co r e cross -linked blo ck i ono m e r micel les as pH - responsive ca rriers for cis -dia mmin edichlo r oplat inu m(II). , Jo urnal of Co ntro lled Releas e . 153 (2011 ) 64 – 72. doi:10 .1016/ j.jconrel .2011 .03.028 . 𝑓 ( 𝑑 ) = 1 𝑑𝛽 √ 2𝜋 exp [ − ( ln ( 𝑑 𝑑 𝑚 ) 𝛽 √ 2 ) 2 ] ( Eq . V -1) Chapter V _________________________________________________________ General Methods 105 backscatter (NIBS®) technology. M easurements were carr ied out at a scatt ering ang le of 173 ° to the i ncident beam, and data analyzed using CON TIN alg or it hms (Malvern I nstruments) . D ata for each di spersion we re collected from at leas t t hree runs. T he Z -Averag e si ze o r Z-Average mean obtained fr om D LS is a parameter also kno wn as the cu mulants mean. I t is the primary and m ost stabl e par ameter pro duced by the technique. Thi s mean i s cal culated fro m t he i ntensi ty weighted dist ribution, l ead ing to the s tateme nt that the Z -Av erage size i s the har monic i ntensi ty -we ighted arithmeti c averag e particl e diame t er. The hy drod ynami c si ze measured by Dy namic Li ght Scattering (DLS) i s defined as the s iz e of a hypothetical hard sphe re that diffuses in the s ame fashion as that of the pa rticle being mea sured. I n practice th ough, pa rticles or macromolecules in soluti on are non -spher ical , dynamic (tumb li ng), and s olvated. Because of this , the di ameter calculated fro m the diffusional properties of the particle will be i ndicative o f t he ap pa rent s ize of the dynami c hydrated/solvat ed partic le and hence, the term “Hydr odynamic diame ter”. Th e hydro dynamic diamet er, or Stokes diameter, t herefo re i s that of a s pher e t hat has the same translat ional diffu si on co efficient as the particle bei ng meas ur ed, assuming a hydration l a yer surr ounding the pa rticle or molecul e. The hyd rodynamic diamet er i s measured usi ng the Stokes - Einstei n (E quation V - 2 ): 𝐷 𝐻 = 𝑘𝑇 3𝜋η𝐷 Eq. V-2 where D H = hydrod ynamic di ameter, k = Bol tzmann’s constant , T= abs olute temperature, η = vis cos i ty, and D = diffu si on c oeff ici ent. The zet a potenti al (  ) was calculated fr om the el ect ro phoretic mob il it y (  ) a nd then a pply ing the Henr y equation. The Smoluchows ki a ppro xi mation  =   where  >>1 (wh ere  is the solution vi scos it y ,  is the dielectric constant of the medium, and  and  are the Debye – Hückel parameter and the pa rticle radius, respecti vely) wa s used. The el ectroph oreti c mobil ity (  ) was obtai ned by perf orming an electropho resis ex periment on t he s ample and meas uring t he velocity of t he particles us ing Laser Doppler V elocime try (LDV). Dat a acquisi tion s were performed using ZetaSi zer Nano software. Chapter VI: Core Cr oss-Lin ked Nanoparticles f rom Self - Assembling Pol y FMA- Based Mi celles Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 115 VI.1. Abstr act The present w or k describes the prep aration o f s table organic nanopar ti cles with potential use as “smart” dr ug deliv ery systems. A we ll -defined s elf-ass emb ly hydr oph il ic-hydro phobic di- blo ck cop olymer has been synthes ized vi a atom transfer r a dical polymerizat ion (AT RP). The hydrophili c block i s based on a ra ndo m copo lymer with DMA and HEMA units , while the hydro phobic comp onent i s a l inear rando m cop olymer of DEA and FMA. Stabil ization of the nan opar ticl es by means of core cross -li nk ing is add ressed by Diels -Alder r eactions (DA) betwee n t he f uran rings of the hydr ophobic blocks and the sy nthe siz ed b is dien oph il es; the influence of both the cro ss -li nk ing agent and the degree of cross -li nk ing i n the p ro ce ss i s dis cussed . Figure VI- 1. Grap hi c a l abstract of t he main objecti ves in t his work. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 116 VI.2. Intro duc tion The so-call ed amp hiphil ic block -co polymers cons tit ut e a s pecific t ype of copoly mers of s pecial interest b ecause of thei r potential abili t y to s el f -ass emble i nto nanoparticl es (NPs). M icelles fr om diblock copo ly mers are one of t he most p opular dr ug deli very system s (DD S) which are a ble to transpor t l ipophili c molecul e s into its core [1] [2] . The presence of a highly hydr oph il ic cor ona lay er makes them very stable in aqu eous media [3][4] . M ethacrylate esters [5] , poly e thylene g lyco l (PEG) [3 ][6] , and chitosan [ 7] , amo ng othe rs ar e the most c ommon hydrophili c s egments and regarding t he hydrop hob ic blocks poly capr olact one (PCL) [3] , po ly pr opylene oxide (PPO) [8] , polydimet hy lsiloxane (PDMS) [5] , ci clodextrin s [6] [9] , and other methacr yl ate derivati ves [10] can be f ound. Moreo ver, smart N Ps can be desi gned so that they respond to changes to specifi c sti mulus, boo sti ng the drug concentr ati on wher e it is required. On e highly useful trigger stimul us i s a redu ction in pH, which i s co mmonly encoun tere d between healt h cells and cancer ous ti ssues in soli d tumor s. The presence of basi c tertiary amine groups in the bl ock -co polymers could impart pH -respo nsi ve pr o per ti es to the f inal N Ps . However, the dynamic and revers ible nature of mi celle for mation is a s our ce of ins tability. Their disi nte grati on by dil ution (at concentr ati ons bel ow its CMC) can be avoided if they a re co rr ectly cro ss li nked [11] , which leads to s table unimolecu lar N Ps able to keep t heir contents intact until a trigg ered stimulus occur s [12][13] . The core crosslinki ng appr oach cir cumvents the potential i nter - micellar reaction found i n s hell -cr oss -linki n g meth od si nce t he hydrophil ic shell blocks can behave as a s teric stabilizer m ini mizing the overlap o f core blocks between adjacent mi ce ll e s and con sequently , preventing i nter-micelle crosslinks [ 14]. Of interest for this pur po se i s the use of the highl y re li abl e Diels Al der reaction in whi ch furan-malei mide units [15] [20] are i nvolved. Such reaction is categorized as a cli ck chemi st ry (CC ) reaction [21] [2 4] and di splay s all the benefit s ass ociated w ith such p ro ce dur es. For the prepar ati on of t ail or-made block-copoly mers, si nce t he mid -90 s, techniqu es of li ving polymerizations have been extens iv e ly developed , i ncluding oxyani onic p olymeriz a tions [25] , atom transfer radi cal polymerizations (ATRP) [20] [2 6], a nd radi cal additi on-fr agmentati on chain-transfer polymerizations (RAFT) [2 7]. All those techniques l ead to t he pro ducti on o f polymers wi th l ow Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 117 polydis per siti e s and we ll -co ntro ll ed compo si tion s and molecular weights fr om a wi de varie ty of mono mers, in contr ast to clas si ca l rad ical po ly mer i zation. The overall ai m of t he present c hapter is t he p repar ati on of new smart polymer NPs as d rug deli very sys tems (DDS) for anti cancer the rapy an d other dis eases by mea ns of a t andem s ynthetic method capable of g enerating stabl e nanopar t i cles wi th v aried prop erties . Th is g oal w ill be addr ess ed by combining highl y efficient syntheti c tools: Atom T ransfer Radical Polymeriz ation (ATRP) for the sy nthesis of amph iphil ic diblock -c opolymers; phys ical interactions (sup ramolecular sel f-co mplexation and mi cell e for mation) and d y namic coval en t l inkages (D iels -Alder reactions, DA) for the st abilization of the nano particles b y cro ss linking in the cor e. Both ATRP and DA reactions have pr oven to be very versatil e tools f or t he preparati on of advanced material s [28] [2 0 ] [28] [1] [20] . The NPs are aimed to be s table upon diluti on and s ensit ive t o acidic pH — such as thus fou nd in soli d tumors — . Th e stabil izat ion of the NPs wi ll be ad dressed in the core by the formation of dynami c DA comp lex es and the s en sitivity to acidi c pH will be imparted by the presence of tertiary ami ne g ro ups in both hydro p hili c and hydro phobic blocks. Mor eover, the NPs wi ll po ss e ss fr ee hydroxy l g ro ups fo r v ectorizati on ex -profeso , when requ ired. T he pre pared NPs w ill be fully char acterized so as to determine their s iz e, polyd is persity , z -potential , stabi li ty under dil ution and und er acidic pH and the CMC of the NPs will be determined. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 118 VI.3. Results and D iscussi on VI.3.1. Pr eparatio n of stabil ize d nanoparti cl es based on amphi philic block -copolymer  Synth e sis of f u ran-contai ning bl ock -c opolymer s and cr os s-li nkers First, the synthesis of amphiphil ic met hacr ylate -based b loc k -copo lymers (Figure VI -2 ) ca pable o f sel f-assembly into stimul u s -r esponse NPs was addressed. For the fur ther st abilizati on of the NPs, the cross -linking of either the micell ar c ore or shell would n eed the presence of reactive functi onal group s i n ei ther t he hydrophobic or hyd ro phi li c blocks , respective ly . One major drawback i n the sy nthes is o f co nventional shel l cro ss -li nk ed micelles is that the r eaction must be c arr ied out at h ig h diluti on i n or der t o av oid extens iv e inter -mi cella r cross-li nking . In t he repo rted cas es w here the shel l was successfull y cro ss -li nk ed, a st eric stabilizer was present, and the i nter-micellar reacti ons were prevented [26]. These drawbacks can be avoided i f t he co re i s cross - lin k ed i nstead , si nce t he hydro phili c segment will beh ave a s a steri c stabi lizer in the p ro cess [14][ 2 8] [29]. Figure VI- 2 . General st ructure of syn the size d met ha crylate-b ased b lock -c o pol ymers and cros s-linkers. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 119 Als o, of interes t i s the presence of fu nctional g ro ups i n the NPs, avai l ab l e for the i nclusion o f reco gnit ion l ig ands , useful for acti ve vectorizat ion toward specific ti ss ue s and cel ls, if required . As a r el iable option, the hydr oxyl g ro ups ar e biological ly co mpatib le and o f us e in many r eactions. The hyd ro phobic block w as mai nly constit uted by the pH sens itive poly( N,N -d iet hy lami noethy l methacrylate) (pDEA), which tu rns into hyd ro p hili c polymer at aci d ic pH; the seco nd monomer incor porated in this block, furfur yl methacrylate (FMA), is a reactiv e mo n omer in Diel s - Alder reactions with a key ro le i n the crossli nk ing of the NPs . This lipoph il ic block will be responsible for the encap sul ation o f the h yd rophob ic d ru gs . The hyd rophil ic block i s desi gned to ensure the f uture s tabil ity o f t he N P in an a queou s medium. The choice of the hydrophili c compon ents was made to guarantee t he biocompatibility of the resulti ng NPs as wel l as to ensure the a bil ity to res pond to pH ch a nges useful in the d rug releas e in acidic envi ron ments s uch as s olid tumors [3 0] . T hus, i t i s well known that pH EMA is a non -toxic and biocompatible hydro phi li c material and is particularly attractive for biomedical engi neering applicati ons [27] . Moreover, because of i ts abundan t hy droxyl functional g ro ups co ntent, poly(2-h ydroxymethyl methacr yl ate) (pHEMA) can be easil y func ti on ali zed by co val ent co nj ugati on wit h, for exampl e , targeting l igands for v ector iz ation w ith the approp riate marker ag ents , and fluor escent molecules [20] . The pH sens itive poly( N,N -dimethyl am inoethyl methacr yl ate) (pDMA ) is another biocomp ati ble polymer used i n t he co -deliv ery of pacli t axel and D NA [22] wi th pH respon si ve behavi or [9] . This polymer forms part of graft co p olymers or b loc k -copo lymers wit h other biocompatibl e blocks such as polycapro lactone (PCL ) o r pol y(ethyl ene glycol) (PEG). For the preparation of the b lo ck -copo ly mers, t he well - known “l iving” polymer ization technique atom transfer radical p olymerizations ( ATRP) [ 25 ][31] was used (S cheme VI- 1) , and ex cell ent con trol over t he molar comp osit ions and polydi spersit ies of the material s w as achiev ed . Several polymers wi th differ ent molar composit ions were sy nthesized i n order to find t he o pti mal block comp osit ion so that the material would d is play sel f -assembl e pr oper ties in aqu eous media. Th e synthes is of t he polymers was con ducted at r oom temperature using ethyl 2- bro mo-2 - -methylprop ionate (EB iB) as a radical i nitiat i or . The constit uent mon ome rs of b oth blocks were added i n t wo sel ected times of the proc ess , so t hat the hydroph il ic bl ock w ou ld be s ynthesi zed first, and the add ition of the monomer mi xture, DEA and FMA, was don e when ca. 95% of the hydro phili c mo nomers was co nsumed (d ata obtained f rom 1 H NMR). Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 120 Scheme VI- 1 . General scheme fo r t he synthesis of am phiphilic met hacrylate -based b lock -c o pol ymers. The com po si ti on of the hydr o phili c block was determined by ex amining an al iquot ex tracted from the r eacti on medium befo re th e additi on of the h ydrophobic monomer s. Thus, the composi tion (in mole percentage) of each repeating unit was ca lculat ed by 1 H NMR. S imilarly, and us in g those val ues a s refer ence, the o verall cop olymer co m pos ition was revealed. The peaks from the 1 H NMR s pectra sel ected to determi ne t he copolymer compositi on were t hose cor responding to the methyl ene g rou p adjacent t o the est er group ( -COOCH 2 -) : f or the first block, the i ntegrals of t he peaks at 4.44 ppm and 3.59 ppm, correspon ding to DM A and HE MA respectiv e ly, were compar ed. For the second bl ock and, hence, fo r the final copolymer comp osit ion, the combined i nformation ex tracted from t he anal ys es of t he fi rst block, and from the integ ra l s of the peak s at 4.51 – 4.32 ppm and 4.98 ppm, corresponding t o DM A + DEA and FMA respectiv e ly, r endered the f inal co poly mer mol e co mpositi on. It was fo und that the only po ly mer able to spo ntaneou sl y sel f -assemble i nto mi cell ar NP in aqueous media w as the p olymer wi th t he experimental c ompo si t ion [ (DMA 31% - HEMA 19% )] - blo ck -(DEA 45% -FMA 5% )]. Th e values of molecul ar wei ght s and p oly dispersity were cal culated by gel permeati on chromatograph y a nd w ere f ound t o be M n = 34 ,7 00; M w = 4 5,1 00 ; and M w /M n = 1.3. A maj or chall enge i n the design of smart NPs is to avoid the inst abi l i ty associated to the mi celle - unimers equili brium due t o dil ution phe nomena i n the huma n fluids an d the serious si de effects con comitant wi th the prematur e release of t he d r ug i n normal tis sues [32] . That i s the reason why various nano -sized samples were cor e cr oss -linked to circumvent n ot only i ts potential disi nte grati on but al so some i nter -mice ll ar cr oss -linki ng [33] . The presence of fu rfu ryl Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 121 methacrylate (FMA ) moi et ies in the polymer structure would ena ble the st abilization of t he NPs for med by cross -li nk ing reaction when requ ired. Wi th the future ai m o f core c ross -li nking t he micell e s once formed i n order to o btain s table NP dispersi ons, a simpl e , fas t, and reliable react ion (i.e. , with high yields and abs ence of side pr oducts ) i s needed. The reacti on s that meet such attributes are grou ped under the denominati on of cli ck reactions, the Diels−Al der reaction being one of them [22][24][34][35]. One of the most widely used Diels−Alder reactions is that involving a malei mi de r ing and a fur an r ing [ 20]. The c ro ss -li nking agents pr epared were two di malei mi de s capable o f per forming DA r eactions wi th the fu ran rings present i n t he hydrop hobic bl ock co polymer. For t hat purpose, two s tarting diami nes were u sed: 1, 8-diamino-3,6- dioxaoctane and Jeffamine D-230, leading to their respecti ve bisdi enop hil e s, DMDOO and D MJF ( Fig ur e VI -2) b y a g ram-scale reaction i n g ood t o mo dest yiel ds (72 % and 3 5%, respectiv e ly ). Commerc ial Jeffa mine D -23 0 is a mix ture of s everal d iami ne s differing from each other i n the numbe r of prop ylene oxi de units present i n the spacer . DM DOO has been described i n a previous w or k [20] , and DMJF was sy nthesized foll ow i ng the s ame pr ocedure (a det ail ed characterizati on of t he n ew cr oss -li nker is i ncluded i n the ex perim ental part). Al though i n the case of DMJF , a mi x t ure of bis dienoph il es is present a s a lo gi cal con sequence of t he natu re of t he s tarting materi a l, DMDOO and DMJF are quite s imilar i n size and structur e; h owever, DM JF is l e ss polar t han DMD OO due to the mo re hyd ro p ho bic natu re o f the spacer (o lig opr opylene oxide) present in the former than that ( olig oethy lene oxide) in DMDOO. The formation of DA ad duc ts f ro m D MDOO w ith fu ran rings has p reviousl y been confirmed by NMR s tudies and mas s s pectro metry, either by fas t at om bo mbard ment (FAB) or chemi cal ionization (CI ). The l att er technique, together w ith calorimetric st udies, confirmed the reversibi lit y of t he DA furan- malei mi de adduc t. Phas e chang es (g e l t o sol) of D A cro ssli nked hydr ogels took place at temperatur es close to 85 °C [20] . Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 128 of cor e c ro ss -li nked NPs . Moreover, i n acidic med ia, micellar s truc tures w it h hydrated cores mig ht be o btained, wi th the c onsequent b oo st i n h ydrop hili c ity. The ex per imenta l DLS data first confirmed the pres ence of NPs in the acidic soluti ons, in al l cases , even i n the non-cro ss -linked sys t ems, whi ch remained stabl e under those experimental con diti ons. Unexpectedl y , proto nation of t he pol y mer in non-crossl inked NPs was not capable of br eaki ng up t he mi cell e s, which kept the ir integ r i ty. This behavior may be due to the difficulti es encou ntered by the acidic water soluti on to e nter the c or e of the micel les and then pr oton ate the basi c resi dues of the DEA units, leadi ng to a polymer soluti on . Besi des, it i s hypothesized t hat t he high number of hyd ro xyl g ro ups fro m H EMA mo iet ies present in the hy dr oph il ic bl ocks partiall y pr evented t he electrostati c repu ls ion between the cati onic char ge s in the s hell of t he micel les, keeping them intact a t acidi c pH. At pH 3.0 these dis persions pos sess ed positive zet a-potentials ranging fr om +49 mV to + 55 mV (Fig ure VI -S1, Sup plementary Infor mation), confirming pr otonation of the weakl y basi c am i ne r esi dues [27] . To es tabli sh the stabi li ty of the NP upon diss oluti on phen omena, another s et of as say s were con ducted (M4-X, Table VI -1 ). Di spersi ons were dil uted wi th N,N -dimethyl for mamide (DMF), a good sol vent for the two blocks of (PDM A- ra ndom -PHEMA)- blo c k -(PDEA- rando m -PFMA), to con centrations below CM C . A liquo ts of s amples M2-X were centrifugated at 35,000 rpm for 30 min. The supernatants were discarded and the NPs s uspended in DM F (final p olymer con centration = 0.25 mg /mL) at 2 5°C and g entl y sti rr ed for 7 2 h. Interes tingly, D LS studies of sampl es M4 -X (Table VI-1) i n D MF s howed that t he cr oss-li nked mi cell e s can s till mai nta ined their nano structures wher eas the no n-cross-li nked s ystems l ost their s upr amolecular as semb ly , demon st rating t hat t he coval ent cr oss - li nk ing of th e hydro phobic block have a definitive i mpact i n the final NP s tabil ity [27] . In addition, an increa se in t he hydro dynamic v olume of the NP w as observed, sugg e sti n g the incor poration o f DM F in the cor e of the NPs . VI.3.2. Characterizati on o f the se lf -asse mbly nan oparticles The data o f the first NP -stabil iz at ion trial s (M1-X) are rec or de d in (Ta ble VI-S1, Supplementary Infor mation ). In general terms, when the polymer con centration s tood at 0.4 5 mg /mL or hig her, a high degree of cross -linki ng wa s targeted (50% or 25%), t he presence o f a precipitate was observed, and the filt ered samples were analyze d by DLS. Po lydisperse s ystem s w ith 2 or 3 NP pop ulati ons were obtained ( one of av erage si ze l ower than 100 nm (60 – 83 n m) an d anothe r Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 129 aro und 340 nm (3 17 – 438 nm), heralding a certain tendency to ag gregat e formation. Fu rthermore , the presence of a p opu lat ion of mi cr on -sized particles i n ev ery cross -li nked s amp le was respon si ble for the large polydis persitie s. This trend al so became evi dent i n sampl es M1-3 and M1 - 5, where the cross -li nking degree s tood at 25%. The NPs for med at l ower polymer concentr ati on (fr om M2 - 1 to M2-5, ai med degree of cro ss - li nk ing 20% , 10% , 0%), s howed a higher particle s ize t han those from the more conc entrated dispersi ons M1-X. Thus, the av erage partic le size fou nd by DLS fo r t he non-c ro ss -li nked sample was about 2 00 nm (M2-1) wher eas this siz e decr e ased when the core was cro ss -li nked (with val ue s ranging fr om 95 nm to 1 50 nm) . Con versel y, s a mple M2 -5 dis played a d ifferent trend than the other s, wi th an av erage mai n population of 400 nm, correspond ing to s ome clust ers, as can be observed i n the SEM i mages , w h i ch mak es the extr action of NP s ize dis tribut ion unreliable . (Fi gure VI -6 ). The av erage par ticl e siz e s were al s o cal culated fr om SEM imag es (Fig ur es VI - 6 and VI-7). Sampl es M2 - 3, M2-4 , an d — especial ly — M2 - 2, co ntain smal ler N Ps than tho se corresponding to non -cross - li nked micellar s tructu re (M2 -1). A si mi lar trend is observed by D LS . However, av erage siz e s (determined fr om S EM imag es) of the NPs found in samples M2 -1 , M2-2 and M2- 3 — 95, 49, and 65 nm, respectively. On the oth er han d, a sig nifican t redu ction in size (between 31% an d 48% from SEM d ata) was observed for th e cro ss -linked NPs, in co mparison with sample M2 -1 . It is noteworthy that the prepared NPs (sampl e s M2-X) remained wit ho ut change for a leng thy period (> 5 m onths) and at a bro a d range of t emperatur es (f ro m 5°C to 40 °C), w hich v erifies t heir stabili t y in a nu mber of en viron mental conditi ons. Next, the nanosized sampl e s were acidified in o rd er to determine their s tability in the medi a and to o bserve i f any change in si ze and/or s hape to ok pl ace. T he freshly aci dified suspensi ons w ere studied by DLS and SEM. The expe rime ntal DLS d ata of the NPs dis played av erage D h ranging fro m 19 9 nm to 2 62 nm — values hi gher than those fr o m t he s ame s amples at neutral pH (Tab le VI -1). This is in agreement with the hypothesis on the change fro m amphi phili c NPs , at aci d ic pH, i nto fully water-soluble str uctur es wi th hydr ated cor es. At pH 3.0, the aggregates observed at neutral pH i n s ample M2-5 (s ee data fo r s ample M3 -5 , Ta ble VI -1 ) were not present a ny mo re a nd a sl ig ht redu ction in PdI, probably due to the r epul si on between char ged micell e s, wa s observed. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 130 Figure VI- 6. SE M microg r ap h s of the crossli nked N Ps at neutral pH : (a) sample M2-2; (b) sample M2-3; (c) sampl e M 2-4; (d) sample M2-5 . (e ) Size d istributions ex tract ed from statistica l anal ysis perf or med o n va r ious SEM im a ges are also p lot. The cur ves corresp ond to the fit of the distributi ons using a l og -norm al function. In t he case of sam p le M2-5, analysis of the siz e di stri bu ti o ns has n ot been per forme d because t he NPs were st r on g ly a gglome rated. Fig ure VI-7 s hows t wo SEM micr ograph s represent ative of the non -cross-l inked micellar s tructur es of the samples M2 -1 and M3- 1 (at ne utral and acidic pH, respecti vely) . The i mag e s clearl y evi dence t he almost spherical s hape of the na no si zed, n on- cro ss -li nked micellar structures at neutral and acid ic media. According t o the s ize distributions obtained from s tati stical analysis of a set o f SEM imag es, the mean size of the NPs in s ample M3 - 1 w as hig her ( d m = 127 nm) than t hat fou nd i n sample M2 - 1 ( d m = 95 n m). Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 131 The SEM imag es representativ e of the cross -linked structur es u nder a cidic pH (samples fro m M3 - 2 to M 3-5 ) a re shown i n Fi g ure VI -8. The m or phol ogy of the NPs observed in the i mages con firms that nano si zed cr oss -linked structur es remain sta ble. The NP siz es i n s amples M3-4 and M3-5 ( d m =123 nm and d m = 122 nm, r especti vely) are v ery si mil ar to those of the n on -cross-li nked micel lar structur e M3-1, while the NPs observed in sampl e M3-3 are smal ler ( d m = 6 7 nm). It is impor tant to mention that s ample M3 -2 contains struc tures su bstanti a ll y larg er t han ( d m = 484 nm), and has a mor pholog y significantly differ ent fr om, the rest of the samples. Figure VI- 7. SE M microg r ap h s and size distri butions ex tracted from statistica l analysis performed o n va r ious SEM im a ges for (a) sample M 2-1 at pH 7 .0 and ( b) samp le M3-1 at pH 3 .0. The curves corresp ond to the fit of the distributi ons using a log -norma l function. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 132 Figure VI- 8. SE M microg r ap h s and size distri butions ex tracted from statistica l analysis performed o n va r ious SEM im a ges of core cross-l i nked NPs i n acidic medi a . a) sample M3-2; (b) sample M3-3 ; (c) sample M3- 4; (d) sampl e M3 -5. (e) Size distri bu ti ons extr acted fr om st atistical an alysis perfo rmed o n various S EM images are al so plot. The ev olution of the mean NP size determined by DLS (Z -average, main po pulation) an d SE M are comp ared in Fig ur e VI -9. As a general trend, t he siz e evolution is similar us ing the different measurement methods, especiall y fo r s amples M2-X. Ho wev er, the v alues obtained by SEM are generally lower than those o bt ai ned by DLS. This differ ence can be at tributed t o the fact that DLS tends to overest imate the mean si z e due t o t he p resence o f pa rti cle agglomerates (ev en at a low perc entage ), whi le diame ters determined by SE M must be c onsidered as t he lower l imi ts of th e particle size [38] . Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 133 Figure VI- 9 . Compari son of t he mean N P size s determ ined b y DLS an d S EM for samp le s M2 -X (pH 7.0) an d M3 -X (pH 3.0). Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 134 VI.4. Conclusions This work set s out a method to pro vi de s table sti mulus -respon se NPs by total s ynthesis w it h potential use as smart dru g deli ver y systems (DDS ) u seful in biomedicine and pharmacy. Of the amphi phili c block -co polymers synthesized by ATRP, the polymer w ith the best sel f-assemblable prop erties in aque ous medi a w as the o ne wi th the rel ativ e molar composi ti on poly[(DMA 31% - HEMA 19% )- block -(DEA 45% - FMA 5% )] . The criti cal mi ce lle concentration ( CMC ) was determined by pyrene fluorescence i ntensi ty and vi sible spectroscop y and the v alues rendered by both methods were comparable (0.08 5 mg/mL an d 0 .078 mg/mL). Stable core c ross-l inked NPs have been s uccessfully pr epared by mea ns of a Diel s -Alder cou pling reaction betwee n the fu ran rings in the c o re an d the malei mi de g ro ups present in ei ther of the two freshly prepar ed bisdi enop hil es, th e hydrop hil ic DMDOO or the l ipophilic DM JF . The best results were ach iev ed w i th degrees of cr oss -linki n g of 10 % a nd 20%. At pH 7.0, NPs in the range fr om 100 nm t o 200 nm and with low polydis persities were fou nd. T he trial s condu cted at hig h di luti on and in aci dic m edi a (pH 3.0) confirmed the a ccompli shment of cro ss li nks since th e i ntegrit y of the NP s was pres erved . Al though non -crossl inked NPs were st able at aci dic pH, the need for cross li nk ing treatmen t w a s veri fied t o s tabiliz e their s upr amolecular structur es in high diluti on ass ays . I ncr eases in NP siz es of f reshly aci dified sampl es were observed , as ascertai ned by means of DLS and S EM studies. Al though the trends in siz e observed by DLS or SEM were si m il ar fo r al l the s a mples , D LS overestimat ed the average s ize of the NPs and henc e, the diameters determined by SE M should be consi der ed the lower li mits of t he par ticl e siz e . 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Tuzar, Synthesis of Shel l Cross -Link ed Mi ce lles a t Hi gh Solids in Aqueou s Media., Macro molecules . 33 (2000) 1 – 3. doi:10.1021/ ma 9 91 4669. Chapter VI ______________ Core Cross-Linked Nanoparticles f rom Self -Assembling PolyFMA- Based Micelles 144 VI.6.2. S upplementary Information Figure VI- S 1 . Zeta Potentia l d istr ibution of M 3-X samp les at pH 3.0. Table VI- S 1. Values of Pd I and avera ge size (D h , nm) of t he NPs prep ared at polymer co ncentrat ions = 0 .45 mg/mL. Sampl e Bisdienophile Z-av era ge (nm) PdI Peak No. Pop u lation 1 Pop u lation 2 Size (± S D) (D h .nm) % Size (± S D) (D h .nm) % M1 -1 … 107 0,40 2 79 (± 30) 52 317 (± 150) 48 M1 -2 DMD O O 50% 125 0,44 3 73 (± 28) 48 341 (± 154) 45 M1 -3 DMD O O 25% 142 0,71 3 61 (± 22) 30 335 (± 202) 57 M1 -4 DMJF 50% 149 0,48 3 83 (± 31) 45 438 (± 200) 50 M1 -5 DMJF 25% 151 0,77 3 60 (± 19) 31 334 (± 142) 52 Chapter VII: Validation of pH Responsive Pol ymeric Nanoparticles as Controlled D rug Delivery Systems Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 147 VII.1. Abstr act In the pr esent wo rk , s elected mi celles designed in C hapter VI are tes ted as drug deli very sy stems. The NPs were prepared f rom t he am phiphil ic b lo c k -copo lymer, poly[(HE MA 19% - DMA 31% ) — bl o ck — -(FMA 5% -DEA 45% )]. For the pr esent research, three sy stems wer e validated, two of them w ith cro ss li nked cor es and the other wit hou t ch emical stab i lization. A co mpar is on of th ei r loading kinet ic s and releas e pr o files is discussed, with the support o f addi tional data obtained by s cann ing elect ro n mi cro scopy a nd dynamic li ght scatt er i ng. It was eval uated not only t he i ncorporation i nto the NPs of a li pop hilic fluorescent molecule ( pyr ene) and a therapeutic agent (pil ocarp ine) , but als o the pH - depend ent release of the latter in hyd ro phili c environ ments. When pyrene was imme rsed i nto t he mi cell es, th ere w as a si gni ficant boo st of the flu or escence emissi on (fr om 10 to 40 -f old), due to the hydrophobic environment of the inner pa rt of t he NPs . Pil ocar pine was also able to be encapsul ated b y t he prepared sys te ms. The d rug was loaded into the NPs wi thin t he first minute s and the dr ug l oading w as dependent on the deg ree of cross - li nk ing. To s tudy t he releas e of pil ocar pine, t hr ee s et of trials a different pH v alues were con si dered. All the systems experienced a b oost i n drug releas e at acidic pH, ranging from 5 0 % t o 80 % wi thin the fi rst 48 h. NPs with the hig he s t deg reeo f cor e c ro ss -li nking (20 %) delivered the highes t per centage of d rug at f ix ed times. The st udied NPs pr oved t o behave as smart controlled drug del ivery s y st em s capable of respon ding to chan ges in pH. They may pro vi de a continuou s deli very of li poph il ic dru g s at speci fic acidic locati on s. Th is featur e makes them ideal can didates in the treatment of certain tum or s si nce the aci dic pH foun d i n damag ed ti ssues in so li d tumors can trig ger t he drug releas e fro m the desi gned N Ps. Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 148 Figure VII- 1 . Graphi ca l ab str a ct of the m ain o bjectives in t hi s w o rk. Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 149 VII.2 . Intr odu ction A number of dis eases are curr ently a t hr eat to the heal th of t he pop ulati on and, in paral lel, a bun ch of di ffer ent t he rap eutic st rategi e s have come i nto play to combat them. T he adminis tration of li pop hil ic drugs i s as sociated wit h a num ber of d rawbacks depen ding on the rou te of adminis trat ion [1] , bes ides some i nher ent dis ad vantages, such as the need for reit erative dose adminis trat ion at certain intervals; and monitoring drug con centrations s o that they are wit hin t he therapeu ti c range ( ratio betw een the d ose of a d ru g that p rod uces a t arget ed therapeutic ef fect and that capable of causing a toxi c eff ect); a furth er c hall enge is the ad mini strati on of drugs wi th a marked variabi lity in t heir ki netic behavior. Hence, new met hod s f or the adminis tration of li pop hil ic dr ugs are need ed. As on numerous occasions, nature has inspi red t he desi gn of therapeutic al ternativ es, such as those sys tems t hat display t he ability to respond t o a s timul us, wh ich is t he basis of l iving systems. Thu s, poly mer materials that respo nd to ex ogenous or endogenous sti mu li — i. e . , changes i n temperatur e, pH, l ig ht, or el ectric/magneti c field, the presence or absence of certai n chemical pr oducts , and i onic strength — have been synthesi zed . The fas cinating "stimu lus - respon se" behavior of thes e "s mart" syst em s has sparked off th e dev elopment of new appli cat ions in fields as div erse as bi omed i cine, pai nt s, tissue engineer ing, sens or s, and more [2] [5] . In particular, the use and devel opment of pol ymer mi cro /nanostru ctures i n smart dr ug/gene deli very systems have caught t he att ention of the research c ommunity for the las t two decades: they can s uppress th e si de-eff ects of t oxic drugs — es pecially anti cance r molecules — and they can al so overcome the dr awbacks ass ociated to insoluble dr ugs. Some interest ing reviews h ave summarized the last trends in th is field [6][8] . The ai m of the p resent chapter i s the v ali dat ion o f the sy nthesiz ed smart, pH-respo nsi ve cor e cro ss -linked NPs as drug carriers abl e to control the release of an activ e li pop hilic molecule up on a trigg er stimulus. As far as the authors are awar e, this is the fi rst ti me that the influence of the degree of core c ro ss -l ink in the uptake an d rel ease of hydr op h obic d rug s has been inves tigated in NPs used as DDS. Pi locarpine, a h ydrophob ic dru g, is the molecule of cho ice. T he up take ki netics of the dr ug by cr oss -li nked and no n – cross -li nked s ys tems are co mpar ed, as wel l a s its r elea s e und er various cond iti ons. The fi ndings are suppo rted by data collect ed from s cann ing el ectronic microscop y (SEM), dynamic li ght scat ter i ng (DLS), UV-Visi ble spectroscopy, and other techni ques. Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 150 VII.3. Resu lts and Discussion The sy nthesis of the s e lf -ass embly poly mer and the prepa ration of the micel lar dispersi ons t o be used in the present chapter a re described in Chapter VI. T he properties of the generated N Ps, such as s iz e , poly dispersity, shape, and criti cal mi cell e concentration (CMC ) [9][10] w ere explored by means of SEM, DLS, and fluores cence spectro scopy . The CMC value (0.078 mg /mL) demonst rate d t he g reat t endenc y of the copoly mer used to sel f-assemble i nto mi celle s in aqueou s media. Accordingly , well -defined mi celles, st abi lized or n ot, were formed at p H 7.0 w hen the p olymer concent ration st oo d at 0.25 mg / mL (s ample M2 -X , Chapter VI ). The hydrophobic blocks, formed by DEA/FMA moieti es, constit ut ed the dehyd rated micelle cor es [ 11] , l eavi ng the hydro phi li c blocks (formed by DMA an d HEMA m oiet ies ) in the outer par t of the NPs in co ntact with the aq ueou s medium. Th e NPs were s table for months, in a wide range of pH ( from pH 3.0 to pH 8.0) an d un der d il ution ass ays in the case of t he cross -li nked NPs (fr om M2-2 to M2- 5) [12]. Pyrene loading st udies was one of the ai ms of the cur rent Chapter and they were conducted in the five syst em s prepar ed i n Cha pter VI (M2- 1 – M2 -5): the non-c rossli nked and the four c ro ss li nked by DMDOO and DMJF (degree of cro ss li nked: 20% and 10%). In t he cas e of pi locarp ine loading and release i nvestigat ions, systems M 2 -1, M 2-2 an d M2-3 (non-cross li nked or s tabil ized by DM DOO) were empl oyed. The encapsulation trials of th e fl uor escent mol ecule, pyrene [13] , and the therapeu ti c mol ecule, pil ocar pine, were foll owed by means of UV-vi s s pectro scopy, fluor escence spectroscopy, and fluorescence microscop y . VII.3.1. Load ing of lipo phi li c mole cu le/d rug into nanop arti cl es  L oading of pyren e Pyrene is a poly aro mati c hydr ocar b on that presents an ensembl e of fluor escence emiss ion peaks in the range from 375 nm to 4 05 nm. I t is exquis itel y sensitive to polarity of the probe’ s microenviron ment, and c onsequently could be us ed for the anal ys is of i ts s ur roundings [14]. Thus, onc e the fluor escent molecule i s i mmersed i nto a hydropho bic envi ro nment, such as t he core of the prep a red NPs , a boos t in the fluorescence e mis sion is expected. This phenomenon was al so fou nd i n other fluorescent molecules s uch as gatiflo x acin , an anti b iotic of the four th -generation Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 151 fluor oquinolone family ; Ocaña et a l. reported a fi nal fluorescence i ntensit y enhanc ement of a bout 75 % by this method [15] . To carry o ut t he incorp orati on of pyrene, the s amples were prepar ed by t he additi on of an aqueo us soluti on of pyre ne ( final pyrene concent ration = 1.2 μ g/mL) into t he mi cell e di spersi on s pr evi ously pr epar ed (Section “Nanoparticle for ma ti on in aqueous media” , Chapter VI) to get a f inal polymer con centration o f 0.225 mg/mL . The mi xtu res were g ently stirr ed on a rotor for 6 days and the fluor escence emiss ion was measured (λ absor ptio n = 3 38 nm; λ emissi on = 380 nm) at f ou r es tablished times to track t he ev olution of data . T o si mplify the termi no logy , s ample s l oaded wit h pyrene wil l be cal led LM-Py r- X (LM-Pyr-1 to LM -Pyr-5 , Ta ble VII -1 an d Fi gure VII -2 ). Sample LM -Pyr - 0 cor responds to a dilute s olut i on of pyrene (1.2 μg /mL); even though sample LM-P yr- 0 contained the maxi mum con centration of free pyrene, it s h owed a weak nat ive -emi ssion s ignal i n aqueo us medium, i n agreement wi th the polar aq ue ous envi ron ment. Table VII - 1 . Fluorescence emissio n data of pyre ne emb edded in to N Ps (samp les L M-Pyr -1 to L M-Pyr - 5) compared with a m odel a queous pyr e ne soluti on (LM- P yr-0, co n c. = 1.2 𝝻 g/m L) at 38 0 nm. Sampl e Bisdienophile Time (days) Enhancement of fluorescen t emission compared with LM - Pyr-0 (fold ) 6 9 12 46 Average values (± SD) LM -Py r-0 (Aq. pyrene solutio n) 14 13 11 9 -- LM -Py r-1 … 420 390 477 369 36.1 (± 7.1) LM -Py r-2 DMD O O 20% 189 198 192 164 16.1 (± 2.2) LM -Py r-3 DMD O O 10% 142 176 149 122 12.7 (± 1.7) LM -Py r-4 DMJF 20% 96 134 115 94 9.5 (± 1.8 ) LM -Py r-5 DMJF 10% 215 190 228 178 17.6 (± 3. 1) Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 152 Figure VII- 2. Fluo rescence-em ission da ta at 38 0 nm (arbi tr a ry u n its) of p yrene l oading t ests at selected periods of time. Fro m the fi gures recorded i n Ta ble VII -1, it was inf err ed t hat pyrene was s ucc ess fully embedded i n the N Ps [ 15] in all the sampl es; the fl uo rescen t emission ro se to val ues far ab ove tho se for aqueo us pyrene soluti ons (from 9 .5 t o 3 6.1 times h ig her), confirming the non - polar surroundings of the l ipoph il ic mo lecul e i n the NPs. Some addit ional infor mati on was revea led: mor e -ap olar surr oundings were pr ovided by the non-c ross-li nked mi celles, with emission val ues that do ubled those fr om cross-li nked NPs. Fluorescence mi cro scope i mages fro m pyrene-l oaded sampl es were taken. I t was found that the micellar size of n on -cross -linked micelle s ro se substanti ally co mpar ed with th at of non -cross- li nked, pyrene-fr ee micel les (Figure VII -3). A prio ri , the only NPs able to substanti ally change their si ze are the no n-cross -li nked s amp l es (samples 1 in each ass ay) as the dynami c equil ibrium between unstabiliz ed NPs and the po ly mer chai ns (unimers) all ows the reorganizati on of those structures. Chapter VII ______ Validation of pH Responsive Poly meric Nanoparticles as Controlled Drug Delivery Systems 153 Figure VII- 3. Fluo rescence micr osc op y image of non -cr oss-linked py rene - loaded NPs (LM-P yr -1) and t he correspond ing s ize distr ibution obta ined from statistic a l analysis. T he curve corr es ponds to t he fit with t he log - norm al fu nctio n. Fro m the analys es of t he i mages, t hi s pheno men on was fou nd o nly i n s ample LM -Pyr-1 ( d m = 1.27 μm) , w it h an incr ease in size clo se to 10-fold compared with M2 -1 ( d m = 95 nm) (Figure VII - 4) . Sample M 2-1 s eemed to ev olve into ther modynami cally more-st able micro particles with pyrene embedded in the core (LM-Pyr-1). Figure VII- 4. Compari s on of t he size of the unl oaded and loade d pyrene n anopartic l es. Chapter IX __________________ Reversible pH -Sensitive Chitosan- Based Hydrogels. Drug Delivery Systems 256 Figur e IX- S 4 . 13 C NM R sp ect rum of the d ielec tr ophile IT rh us ed as c ovalent c ro ss-li nker i n the form ation of CT S-base d h ydr ogels. Chapter IX __________________ Reversible pH -Sensitive Chitosan- Based Hydrogels. Drug Delivery Systems 257 Figur e IX- S 5. Hi gh res o l utio n ESI m a ss s p ect rum o f t he die lect rop hile use d as c ovale n t cr oss- linke r in t h e f ormat i on of CTS- Chapter IX __________________ Reversible pH -Sensitive Chitosan- Based Hydrogels. Drug Delivery Systems 258 IX. 10 . Bibl iography [1] M. Hamidi, A. Az ad i , P. Rafiei, Hydrogel nanopar ticl es i n drug deli very, Adv anced Drug Del ivery Rev i ews. 60 (2008 ) 16 38 – 1649. doi:10 .10 16 /j.addr .2 008 .0 8.002 . [2] W. E. 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De -Paz , Nano structur e d C hitosan -based biomaterial s for s ustai ned and colon -specifi c resveratrol release, I ntern ati onal Jour nal of Molecul ar Sciences. 20 (20 19) 3 98 (1 – 16 ). doi:1 0.3390/ ij ms20020398. Chapter X: Chitosan-Nan op articl e Composites as Resverat rol Carriers: S usta ined and Colonic Specific Drug Release Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 265 X.1. Abstr act In the pr esent work, we demonstrate the prepar ation of chitosan -based comp osites as v eh i cles of the natural occ ur ring mul ti -drug resv eratro l (R ES). Such sy stems are endowed wi th p otential therapeu ti c effects on inflamma tor y bowel diseases (IBD) , s uch as C roh n’s dis ease (CD) and ulcerativ e coli t is, t hr ough the s ustai ned colonic releas e of RES from l ong -las ti ng mucoadhesive dr ug depots . Th e loading of R ES into nanoparticles (NPs) was op ti m ized regard ing two independen t variables : RE S/po ly mer ratio, a nd temperatur e. Twenty experiments were carr ied out and a Box-Behnken ex perimental design was us ed to ev aluate the signi ficance of thes e independen t variables related to e ncapsulat ion effici ency (E E). T he enh an ced R ES EE values were achieved i n 24 h at 39°C a nd at R ES/polymer ratio of 0.75:1 w /w. Sizes and polydi spersities of t he optimiz ed NPs were st udied by dynamic lig h t scatt ering (DLS). C hitosan (CTS) dis persions con tai ning the RES-loaded NPs were i onically gelled w ith tricarballylic ac id to yield C TS -NPs comp osit es. M acro and microscopic features ( morp hol ogy and porosity s tudied by SEM and spreadabili t y), thermal s tability (st udied by TGA), and release ki net ics of t he R ES - loaded C TS-NPs were inves tigated. Release patterns in s imulated colon co nditi on s f o r 48 h dis played signi f i cant differen ces between the NPs (fi nal cumu lat iv e dr ug r el ease: 79 – 8 1%), and the CTS -NPs com posites (29 – 34%). Figure X- 1 . Graph ical abst ract of t he main object ives i n this work. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 272 Figure X- 2 . Rela tive infl uen ce of t he stu d ied va riables on enca psulat ion efficiency ( E E%) in n on -cr o ss l inked and cr o ss-linked n anoparticle s. The Pareto cha rts (Fi gure X - 2) s how t hat the mo st important v ariable on non -cross-l inked EE evoluti on is temperatur e (8 3%); i t hap pens, l ikew ise, on campto thecin loaded NPs [32] . However, si m il ar relative statistical i nfluence (48 % and 52% for temperature and drug/polymer rati o respectiv e ly ) has been found for 20% c ross -l inked treatment. In g eneral terms, as observed pr evi ously for other s yst ems, the hi gher t he drug/po ly mer ratio, t he g reater the EE. T he EE val ues are also i n agreement wi th those obtained for camptothecin at si milar dr ug/po l ymer ratios. To help the interpretation of the obtai ned equations, the response s ur face f or eac h depen dent variable is s ho wn (Figure X-3). Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 273 Figure X- 3 . Res pon se s urface for RES e nca psul ation eff i cienc y percentag e on both, n o n - a n d cr oss -l in k ed NPs. The RES -loaded NPs wit h optimi zed EE (Table X -4 ) were st udied by DLS. The Z -average and D h of the non-cross-l inked s ystem ex perienced a s light r eduction compar ed t o t he unloaded cou nterpart. This could be d ue t o the  -  tacki n g of the dru g molecules and the f ur furyl rings of the cor e of t he NP, causi ng mor e comp act micelles. In the cas e of cro ss -link ed NP loaded w ith RES, the si ze became slightly hig her t han t he unloaded cor responding NP, as has been ob served for other sy stems [31] [32]. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 274 Table X- 4. Compar ison of Z-ave rage, pol ydispersit y ind ex (PdI), an d hydr odyna m i c di ameter (D h , determined by DL S) of n on -cross- linked NP (Non-Xr) an d stabilize d NP at 2 0% of cross - linking (Xr) ( u nloaded or loaded with R E S). Unloaded s a mples [31] Resveratrol-loaded NPs Degree of crosslinking Sampl e Z- av (± SD) PdI (± SD) Size (± SD) (D h .nm) Sampl e Z- av (± SD) PdI (±SD) Size (± SD) (D h .nm) (nm) (nm) Non- Xr S- 01 177 (± 1) 0.1 4 (± 0.02) 210 (± 80) RES-Non- Xr 115 (± 1) 0.4 6 (± 0.01) 170 (± 90) Xr 20% S- 02 108 (± 1) 0.3 3 (± 0.01) 130 (± 70) RES- Xr 121 (± 1) 0.2 7 (± 0.01) 170 (± 90) The RE S-loaded NPs were prepared at pH 7.0 acco rding to the op timized co nditio ns found in th e present study: Sample RES-Non-Xr = Non- Xr -Res 0.75 -T 39; RES-Xr = Xr -Res 0.75 -T 39 Temp erature = 39  C; RES/polym er ratio = 0 .75 :1; loadin g time = 24 h. X. 3.2. Nanostructured Chi t osan -Based Composites Con ta in ing Resve rat rol-Loaded N Ps Hydro gel s are curr ently being studied as matr ices for the c ontro ll ed release of bioacti ve molecules, and f or the encapsulation of li vi ng cells. For th es e appli cati on s, it i s often required that the hydr ogel s degrade u nder physi ologi cal cond it ions, i.e., the or ig inal ly t hr ee -d imens ional structur es have to d isinteg rate pr efer ably i n harmless pr odu cts to ensur e a g ood biocompatibi li t y of the biomaterial. Recently, much attenti on has been pai d to chito san (CTS) because o f i ts advantag eous bi ological pr operti es such as biodegradabi lity, b iocomp ati bility and non -toxi city as well as it s physicochemical pro per ti es [43]. T h us, CTS and i ts der iv atives have been us ed as an abs orption enhanc er [44], drug ca rrier [45][46], mucoad hesiv e and permeation enhancing poly mer i n for mulati on s for b uccal/subling ual, nasal , g a stroin test ina l, vaginal, colonic drug deliv ery [47] and fo r gene deli very [48] . Chit osan has been fo und to be degradable b y the colon mi cro flora. Taking advantag e of this pr operty, CTS has t ur ned out t o be a useful mat erial to guarantee t he colonic dr ug deli v ery as long Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 275 as it is part of the cont rolled drug releas e s yste ms [49] . T hese material s can adher e t ightl y to muco sal surf aces such as gastro intestinal t ract (GI T) walls and transiently op ening the t ight junction between epitheli al cells , enhancing drug abs or ptio n ac ross intest ina l epithelial ce lls wit hou t injuring them [50] . Thu s, for ex ample, sust a ined i ntest ina l del ivery of dr ugs , such as 5-f luo ro uracil and i nsuli n, seems to b e a f easi ble alt ern ative to i nject ion ther apy [51] . In add iti on, the bi oadh esi veness pr oper ty of chitosan-based DDS has made i ts formulatio ns useful as local dru g-depots [52]. The above-mentioned properties make CTS an i deal p olymer fo r colon -s pecific dr ug releas e and it w as the bioco mpatible polymer cho sen in the pr esent work. In general terms, polymeric networ k formation can be ac complis hed ei ther by no n -covalent physical as sociations, such as s econd ary f orces (hydrogen, ionic, or hydrophobic bond s) and physical entan gleme nt s, or by covalent cro ss -link s. To for m s tabil izing li nka ges i n chitosan -based materials, t he amine moiet ies present in their str uctur e all ow t he l inkag e between the chains thro ugh select ed c ross-l inkers to pr event gel dissociation. In a “C hapter I X” , we have carr ied out the sy nt hesis of mi cro- st ructur ed biomaterials based on ionically cross -linked C TS fo r t heir applicati ons as biocompatible carriers of drugs and bioactiv e comp ounds. The influence of the dis persion compo si ti on on i ts rheological proper ties was eval uated. Th e rel ea se pr ofiles of a model dr ug, diclofenac sodium (DCNa) as well as thei r relati onships w ith po ly mer concentration, dr ug loading and degree o f cross -linki ng were est abl is hed [53] . Based on t his s tudy, we have carried out the p rep aration o f CTS -based bioadhesiv e hydr ogels with RES -loaded NP embe dded into their structur e. In thi s case, par t of the dist il led water necess ary for the hyd ro gel for m ati on has been r eplaced by a n o ptimi zed RES - lo aded N P suspensi on. The sprea dabili ty of the sys tems as well as t heir morphologi es and thermo gravi metric analys es of the freeze-dried s amples w ere investigated. Su rpr isi ngl y , the macro scopic prop erties of t he comp osit es w ere simi lar to those wi thout the i nclusion of the nanoparticl es. Table X -5 dis plays some relevant data fr om the sy stems studied . Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 276 Table X- 5. Compar ison of sp reada bi l ity and T GA dat a of resveratr ol -loaded h ydro gel -NP comp o sites (w ith cross -l inked N P and non -cross-li nked NP. Sampl e Spreadability ( diameter, cm) a TGA b t 1 min t 30 min  diameter (%) o T d (  C) max T d (  C) Δ W (%) Mass residue at 650  C (%) CTS -- 110 72/29 7 9/58 30 Xr -C TS-RES 5 8.2 64 98 62/28 1 24/43 32 Non- Xr -C TS-RES 5.6 8.9 59 99 111 /281 24/42 32 a: Spreadability measured in cm ;  diam eter = ch ange of diameter ( in pe r centage ) after 30 min; b: Onset deco mpositio n te mperature c orresponding to 10% of weight loss ( ° T d ); maximu m rate decom positio n temperatures ( max T d ) and weight los s at the respective decom positio n step [Δ W ( %)] determined by TG A. The mechanical p rop e rties of formulati ons desi gn ed fo r G IT a dmi nis tration are fundamen tal i n comp osit e perf ormance. The sp readabili ty of t he pr odu ct is one of them and c ontributes to t he final cli nica l efficacy of t he p roduct [54]. On the other hand, the composi tes need t he dis play ing muco adhesion properties to act as a RES depot i n the G IT. M uco adhesion i s controlled by the affinity of the material fo r t he mucin g lycopro teins of the mucus and CTS has demonstrated the exhibi t ion o f excel lent muco adhesiv e pr operties due to its amine and hy dr oxyl gro ups , which ar e involv ed in its pr olonged res idence t ime i n t he g astrointes t inal t ract [49] . Th e comp osit e for mulated i n the in the present work display ed excel len t s pr eadability pr oper ti es as expected , which en sure the satis factor y applicati on o n th e d amaged tis sue. Fro m d ata obtained in the cur rent and previ ous works, i t can be concluded that the hig h er the CTS concentr ati on in the for mulati on s, the mo re struc tu red hyd ro gel s are obtai ned, and the lower the spread ibi li ty foun d. A thermogravimetric s tudy of t he commer cial CTS and the two freeze -dried CTS-NP conjugat es was condu cted i n order t o reveal t he v ariation s in thermal degradability between the s tarting material and the composit es. Their t races ar e superimposed i n Fi gure X - 4. For il lust rat i ve pur poses , the c urves that rep resent the der iv ativ e weig ht los s vs . temperature are also included. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 277 Figure X- 4 . TGA t races of commercial ch i tosan (so lid li ne) and t he two RES - loaded CT S-NP com posites . The new c hitosan -based conj ugates d is play al mo st identical ther mogravimet ric p ro fil es and s how a max imum degradat ion temperature at 281°C. Al though this v alue i s slig ht ly shi fted to l ower temperatur es compared wit h the st arting material (297 ° C), t he peak p ro fil e i s s imilar to CTS’s one, as can clea rly be seen in t he i r derivat ive t her mogravi metric analysis (DT G ) curves. The new for mulati on s experience a re duc ti on i n mas s of 24% at l ow temperatures i n contrast wi th the commer cial CTS (9 %), which co uld be mostl y due to water co ntent in the CTS sample. As SEM has proved to be a pa rticularly relev ant te chn ique to determine th e scaffo ld c har acteris tics (po re s iz e and morphology) of hydrogel s and biol ogical systems, some SEM image s were obtained in or der to s tudy the mi crostructure o f the c omposi tes formed. To preserve the skel eton s tructur e of o ur sy stems prior to S EM observati ons, the freeze dried method p revi ou sl y repor ted f or cel ls has been used [53] [ 55 ] [5 6] . Fi gure X -5 compares SEM micrograph s of the chitosan -based hydro gel s with RES-loaded Xr and no n- Xr NPs . The two i mages taken at the same magnif icat ion show t hat both s amples have a po ro us mi cros truc ture. I t shou ld be noted that, unli ke the macro scopic pr oper ties , the mi cro structur e of t he tw o prepar ed c onjugated di ffer i n thei r 3D - micro-stru cture and the scaffold of the Xr sample dis plays larger po res. -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Deriv. Weight (%/°C) 20 40 60 80 100 120 Weight (%) 0 100 200 300 400 500 600 700 Temp eratur e (°C) CT S PM.00 1 ––––––– CT S- RES- N XRL .001 – – – – CT S- RES- XR L.00 1 ––––– · U n i ve r sa l V 4 . 5 A T A I n st r u m e n t s Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 278 Figure X- 5 . SE M images of t he loaded chit o san-based hy d rogels: (A) X r an d (B) Non - Xr . X. 3.3. Resv eratrol Release Stu dies The l ast part o f the present work foc used on eva l uating the r elea se of R ES from the NPs and the CTS-NP comp osit es in simul at ed colonic envi ro nments. Due t o the highl y - structur ed hydrogel scaffolds, the mechanisms of drug releas e from them are markedly diff ere nt from those of other DDSs such as mice lles and dend rimers. Previ ous modeli ng st udi es predicted that t he release of an activ e agen t fro m a hydrogel i s determined by the rate -limi ting s tep of the p rocess and, therefore, categorized as diffusion-contro ll ed, sw e lling -co ntrolled, or chemical ly-con trolled, bei ng the former the pr imary mechanism that governs the r el ease of d ru gs fr om hydrogel s [49] . Both the NPs and the CTS-NPs c ompo si tes were immersed in a s imul ated co lonic e nvironment at 37 °C and t he dr ug rel ease was eval uated by UV- V is mea surements at 30 7 nm. T he c umulat iv e RES release prof il es of NPs and CTS- NPs comp osit es are sho wn in Figure X- 6A a nd X-6B, respectiv ely. The percentag es of dru g releas e were d etermined using Equation X- 1: Cumu lative RES releas e (%) = 𝑚 𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒 𝑑 ( 0 ) − 𝑚 𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 ( 𝑡 ) 𝑚 𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒 𝑑 ( 𝑜 ) × 100 ( Eq. X- 1) where m entrap p ed (0) i s the weig ht of init ial entrapped RES i nto NPs; m r esidual(t) i s the weig ht of residual RES at time “t ” into the nano -microc arriers. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 279 Figure X- 6 . In vitro releas e profiles of res ver atr ol in si mulated colo n conditi on s fr o m resv e ratr ol - loaded NPs (A) and resveratr o l - loaded CTS-NPs compos ites (B). For these t rials , the use of normali zed data was chosen in order to compa re the capacity of the sy s tems s tudied to retain o r not the drug into t he NPs (non- or cross -linked) on their own (Figure X-6a) o r imme rsed i nto a C TS net wor k (Fig ure X - 6b). The most si gni ficant fact observed was the si m il ar behavior of b oth NPs s tudied, wi th a posi t ive s ustained releas e of RES over t ime. When t he CTS-NPs compo si tes were st udied , i t was be taken into accoun t that cross -linkag e s i n hydro gel s cou ld s ufficiently restrain t he hydrogel 3D -networ ks and the water fl ow wi thin the s ys tem s. On the other ha nd, p hysi cal as sociations would rise to reversi ble bonds, labi le over ti me and wi th definitive effects on the drug release ki net ics of hydrogel -based DDS. As expected, once t he RES res ervoir was i mmersed into a CTS network, a substanti al decrement i n RES relea se rates was observed, and hence mos t of t he dr ug remained i n the composi te (final cumulativ e relea se (%) after 48 h: 29 – 34%), highlighting the marked influence of network environments o n the dr ug r el ease rates. Thes e facts s upp or t the hypothesi s that t her e ar e si gn ificant benefits associa ted w ith t he desi gn of drug depo t s ys tems based o n nano - microstruc tured CTS biomaterials . Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 280 X. 4. Conclusions Resv eratr ol (RES), a natural oc curring multi -therap eutic drug, h as demo nstrated i ts cap abil ity of redu cing i nflammation as sociated wi th g a st ro intestina l tract pathologies. Our efforts have foc used on the des ig n and devel opment of new smart micro - and nano-structur ed gel -li ke dr ug delivery sy s tems as colon-specific RE S depo ts. The d rug w as s uccessfull y encapsulated i n freshly -prepar ed bi oco m patibl e no n- and cross -li nked NPs, which, in t ur n, were integrated into a chitos an g el matrix . Th e influence o f two pa rameters, the temperatur e and the drug/poly mer r atio, on the encapsulati on efficiency (EE ) in the non - cross- li nked NP was co mpared wit h that fou nd on the cross -li nked systems, disco vering that the temperatur e exerted the pr evai li ng influence. R ES -loaded CTS- NP formulati ons were efficientl y pr epared by imme rsing RES-loaded NPs i n 4% CTS s uspensions with s ubsequent g elation by ionic cro ss -linki ng . The s tudy of the two drug -depo ts by TGA an d S EM dis closed porous s caffo lds wi th great si m il ari ties in their th erm al st abi lity patterns. RES release fro m NPs was next inv e stigated fin ding t hat non - and c ro ss -li nked NPs behaved si m il arl y over the period of ti me studied (48 h). W hile t hey displayed a sus tained RES releas e (final cumulativ e dr ug releas e after 48 h: 7 9 – 8 1%), t he RES-loaded C TS -NP hydrogels , prepar ed at 3 7°C , 4% C TS and 10% of cross -li nk ing , showed a mark ed reduction i n RES release rates (red uctions in RES release after 48 h: 57 % a nd 64%). Ther efore, i t has been pro ved that, i n the prep ared R ES depots, th e chit osan netw or k is respon si ble for s ubstantially reduc ing the rele ase rate of dru gs , conforming such sy st e ms into a versatile t oo l t hat cou ld potential ly endow therapeuti c benefits in the treatment of IBD throu gh pr olonged retenti on and deli very. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 281 X. 5. Bibli o graph y [1] P. C. Cal der, n-3 Pol yunsaturated fatt y ac ids , i nfla mmation, and i nflamm atory dis ea ses, The American Journal of C li nical Nutrition. 83 (2 00 6) 1505S -1519S. doi:h ttps://doi. or g/10.1093 /ajc n/83.6. 1505S. [2] G. Bouma, W. Strober , The immunologi cal and g eneti c basi s of inflammatory b owel disea se, N ature Reviews Immuno logy . 3 (2 00 3) 521 – 533 . d oi:10 .1 03 8 /nr i1132. [3] E. V Loftus, Cl ini cal epi demiology of inflammat ory bowel disea se: incidence, p revalence, and environmental i nfluences, Gas troent erology. 126 (2004) 1 504 – 1517. doi:h ttps://doi. or g/10.1053 /j.gas tro.20 04.01.063. [4] R.J. Xavier, D. K . Podolsky, Unr avelli ng the patho g enesis of i nflammatory bowel di sease , Natur e. 448 (2007 ) 427 – 434. doi:10.1038/nature0 60 05. [5] S. Rakoff-N a houm, J. Pa gl ino, F. Esl ami -Varzaneh, S. Ed berg, R. Medzhitov, Recogniti on of Commensal M icrof lora by Toll -Like Receptors Is Required for Intes ti nal Homeosta si s , C ell. 11 8 (2004) 229 – 2 41. doi:https:// d oi.org/1 0.1 01 6/j. cell.200 4 .07.00 2. [6] P. Seksik, H. Soko l, P. Lepage, N . Vasquez, C. Manichanh, I. Mangi n, P. Pochar t, J. Do ré, P. Marteau, Review article: the ro le of bacteria in onset and per petuation of i nflammatory bowel d is ease, Al imentary Phar macology & Thera peutics. 24 (2006) 1 1 – 18. doi:1 0.1111/ j.1 365-2036.2006.03053 .x . [7] H. Sokol, B. Pig neur , L. Watterlot, O. Lakhdar i, L .G. Bermud ez -Humaran, J.-J. Gratadou x, S. Blugeon, C . Bridonn eau, J. -P. Fu ret, G. Corthier, C. G ran get te , N. Vas quez, P. Pochar t, G. Tr ugnan, G. T homas, H.M. Blot ti ere, J . Dore, P. Marteau, P. Sek s ik, P . Lan gella, Faecali bacterium prausnitzii is an anti -inflammator y commensal bacterium i dentified by gut mi cr obiota anal ysis of Croh n di seas e patien ts, Proc eedings of the National Academy of Sciences. 10 5 (2008) 16731 – 16736. doi:10.10 7 3/ p nas.0804 812105. [8] M. Orho lm, P. Munkholm, E. Langholz, O.H. Nielsen, T. I. A . S φ r ensen , V. Binder, Familial Occurren ce of Inflammatory B owel Dis ea se, N ew England Journal of Medicine. 324 (1991) 84 – 88. doi:10 .1 056/NE JM199101103240203. [9] E. -M. Col lnot, H. Al i, C . -M. Lehr, Nano- an d mi cropar ti culate dru g carriers fo r target ing of the inflamed intestinal mucosa, Journ al of C on trolled Releas e . 161 (2012) 23 5 – 246. doi:h ttps://doi. or g/10.1016 /j.jco nr el. 201 2.01.028. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 288 X. 6. Annexes X. 6.1. Specific Me tho ds The spreadabili ty of the nanostructur ed hydrogels w as a ttained by measuring the extension area (diameter in cm, after 1 mi n and 3 0 min) of t he sampl e when it i s s ituated between two gl a ss plates (2 0 cm x 20 cm) and exposed to a co nstant wei ght.  Pre paration o f R esverat rol -Load ed N Ps The g eneral l oading procedu re w as conducted as follows : t he sel ected mi cellar dispersi on was introdu ced i nto a mi ni -dialys is tube (1 kDa cut -o ff, GE Healt hcar e). The l atter was placed i nto a seal ed tub e con taini ng a freshly pr epar ed aqueous-based RES s olution (1 :4 v/v DMSO -water) at a pr edetermined RES/poly mer rati o an d gently sti r red f or 24 h at 2 5, 32 o r 39 ° C. T o determine the encapsulati on efficiency in the l oading proces ses, t he remaining RES co ncentr ati on s w ere measured at p redetermined times b y UV spectr os copy at 3 07 nm.  Exp erim ental De s ign to Stu dy t he Effect of L oadin g Conditi ons on Resve ratrol Enca psulation The enca psulat ion s tudies of resveratrol by the f reshly pr epared n on - and co re c ross-l inked NPs were cond ucted by means of UV s pectro scopy varyi ng sev eral experimental parameters s uch as RES/ polymer r atio and t emperature. The R ES -loa ded NPs with the optimi zed EE w ere al so studied by D LS. Th ese sy stems were the RE S -loaded NP of c hoice to be embe dded i nto CTS - based hydro gel s. I n ord er to obtain optimi zed conditi ons for t he loa ding st ep, a Box – Behn ken experimental desi gn (CSS St at isti ca , StatSoft Inc ., Tuls a , OK, USA) was u sed to ev a luate the si gn ificance of the independen t variabl e s (tempe ratur e and resveratrol/poly mer ratio) , as well as the i nteractions among t hem i n the n on - and c or e c ro ss -li nked NPs . This experimental des ign [60] [61] enabl ed t he co nstruction of s econd -orde r polynomial s for each i ndepen dent v ariable and the i dentifi ca tion of s tatistical signifi cance in th e v ariables. For two v ariables , 10 experimental points are est ablished. Independ ent variables wer e nor mali zed by usi ng Eq uation (VIII -S5) . Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 289 To st udy t he i nfluence o f RES/polymer ratio and t emperatur e in the s yst ems , 20 RES-loaded NP sy s tems, 10 of t hem named NonX r - RES/POL x - T y a nd t he other 1 0 s ys tems named X r -RE S/POL x -T y were prepar ed from non -cross li nked NPs and cr oss -li nked NPs , re s pectively. The final polymer con centration was 0.1 mg/mL, t he final targeted RES/ polymer rati os w ere 0.25, 0.50 and 0.75, and the l oad ing temperatures were 2 5° C, 32 °C an d 39°C. E ncapsulati on efficiency of R ES embed ded into the n anoparticl es were calculated accor ding to Eq uation (V II I- S2 ).  Preparat ion of Nanost ructu red Chi tosan -Bas ed Hydrog els with Resve ratrol-Loa ded Na noparticles Two sy stems were prepar e d w ith CTS of mole cular wei ght 199 k Da, one of t hem wi th the incor porati on of non-cross-li nked RES-loaded NPs and the other w ith the cro ss -linked coun terpart. The final CTS concentration was set at 4% w/w and the degree o f c ross li nk ing 10%. Firstly, t he NPs (cross -l inked and non -cross -linked) w ere loaded w ith RES under the optimiz ed con diti ons di scl osed i n t he ex perimenta l desi gn described above: RES/polymer ratio: 0.75; temperatur e: 39°C, loading time: 24 h. Secondly, the h ydrogel preparation was conducted si milarly to t he procedure recentl y described by us [53] th at can be su mmarized as f ollows : CTS (0.4 g, 1.74 mmol of free amine g ro ups) was char ged in a round-bottom flas k pro vided wi th a stirrer bar; then, an aqueous s olution of tricarb all yl ic acid (1.02 mL , 10 mg/mL, 0.05 mmol ), a s olution of acetic acid (0.1 mL, 52% w/v), a dis persion o f resveratrol -loa ded NPs (5.5 mL, RES 0.93 7 5 mg/mL) and doub le - dist il led water [up to a fi nal wei ght of 10 g (f inal p olymer conc e ntration: 4% w/w)] were a dded i n sequence. The mi xture was s ti rr ed t o homogenization at 40 °C during 1.5 h. The so l ution w as coo led at 25°C and the stirring proceeded overnight at 25°C .  Releas e Stu dies In or de r to c heck the release of t he R ES -loaded N Ps , two samp les, crossli nk ed and non -c rossl inked NPs, wer e pr epared und e r o pti mum uploading c onditions (RE S/poly mer r ati o: 0.75; t empe ratur e: 39 °C, l oad ing ti me: 24 h). Next, t he RES rel eased du ring t he trial s were determined by UV -Vis spectroscop y at 307 nm, similarly to the general pr ocedure d escribed abo ve f or the loading ass ays. In thes e cas es, a min i -dial ysi s tube containi ng t he NP dis persion w as immersed into s imulat ed colon fluids (pH = 6.0 ± 0.25 [6 2]) an d the temperat ure was s et at 37°C. Chapter X _______________________________ Chitosan -Nanoparticle Composite as Resveratrol Carriers: Sustained and Colonic Specific Dr ug Release 290 When the release of R ES fr om hydrogel -NP composi tes w as cond ucted, the sel ected RES -loaded comp osit e was t ran sferred to a dial ysis ba g (m olecular w eight cut -of f: 8 00 0 – 14, 00 0 D a), then immersed in simulated colon fluids and g ently s tirred at 37 °C i n a s haker incubator (Hei dolph Unimax1 010 -Heidolph Inkubator 1000, Schwaba ch, Al emania). At pre -desi gned ti me i nterva ls, ali quo ts were taken fro m the release medium and the amou nt of RES released was determi ned by UV – Vis spectroscop y at 307 n m. R ES r eleas e experiments were perf or med in triplic at e. Chapter XI: Locust Bean Gum De rivatives for t he Formation of Nanoparticles Us e fu l in Drug D elivery Systems Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 293 XI.1. Abstract Polys acchar ides constitute a central grou p of nat ur al -occurr ing mat erials t hat are of g reat i ntere st in the p repar ation and der iv atiz at ion o f bi oco m p atibl e nan opar ticl es . Many o f them are ob tai ned fr om pl ants or marine or ganis ms, whi ch are g reen and re newab le s our ces. Fi nding other uses t o renewable materials has a h ig h impact o n the pres ervati on of the p lanet . Locu st bean gum (LBG ) i s a neutral poly saccharide produc ed from the see d o f the l ocu st bean tree which grows in cou ntries bord ering the Medit erranea n. The present work deals wi th the chemical modification of LBG i n or der t o o btain a cationic polyel ectrolyte that, in turn, by pol yel ectro ly t e comp lex ati on wit h the anionic po ly electr olyt e k -carr agee nan (CRG), w ou ld afford NPs wi th potential appli ca tions in the development of DD S. Cationic deriva tives of LBG have been prep ared thro ugh a tw o-step procedur e : a first oxidati on reacti on and a s ubsequen t redu ctive aminatio n. The outcomes were veri fied by FTIR and UV -vis s pectrosco pies . When the oxi dat ion was con ducted by the oxidant sodium pe riodate, the bes t results were achieved. The f or mati on of NPs wi th low polydis persi ti e s was achiev ed w hen one of t he cha rged poly mers used (ei ther mul t iami ne-LBG or C RG) w as i n hi gh er ratio than the other , s tabilizing the NP by surface cha rge repulsion and hence, preventi ng their floccu lati on f rom the medi um . For mulati ons wit h multiamine-LBG/CRG ratios equal t o 3/1 and 4/1 dis played the l owe st polydispers it ies (va lues close to 0.2 and 0.3) and Z-av erage figures (below 2 50 nm). Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 294 Figure XI- 1. Grap hical a bstract of t he main objectives i n this work. Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 295 XI.2. Intr odu ction Polymeri c nanop articl es (NP s ) are one of the most s tudi ed organic strategies i n advanced therapies s ince polymers are amo ng t he mo s t versat ile buil ding units, av ailable for the straig htfor ward modificati on of their pr operties . Organic polymeric nano particles can be prod uced using either s y nthetic or natural po ly mers. In fact, natural materials may disp lay bet ter cell adhesion pro pe rties as well a s enhanced mechanical pro perties si m ilar to those pro vided by natur al t is sues [1][4] . Protei ns and p oly saccharides are among t he mos t commonly us ed natural polymers for the for mati on of NPs . Polys acchar ides have fou nd a wi de range of appl icat ions as s tabilizing a gents, thi ckeners , gelling agents and emul si fi ers, commonly used in food and pha rmaceutical products [5]. Many of them are obtai ned fr om pl ants or mari ne o rganis ms, w hich are g reen and renewable s our ces. Due t o their highly functionalized nature, they can be transfor med i nto a wi de vari e ty of new mater ials by means of conventional chemical methods an d the new materi als g eneral ly preserve the degradabili t y and biocompatibil ity prop erties of t he origi nal polymers . These features allow t he ir biomedical use due to the absence o f side effects that co uld be associated wi th other new syst ems [5] . That is why poly saccharides co nsti tute a centr al gro up of natu ral -o curring material s that are of great interest in the prep aration an d deriv atiz at i o n of biocompatible nano pa rticles [7] [ 11]. Ther e a re many metho ds t o produce p oly meric nanoparticl es and the ch oice of a pa rti cular methodo logy w ill ma inly depen d on s pecific character is t ics of t he drug t o be encapsul ated and the mat erial t o be used a s matrix [12 ]. M ethods based on the es tablishment of i ntermolecular elect ro static i nteractions are one of the m ost repo rted and are appli ed when the mat rix of nano particles is composed by at l east on e polyel e ctro ly te, t hat i s, a poly mer that ex h i bits char ged group s when is in s olution [13]. Polyel ectrolyte complexati on i s the name o f the technique when the group s medi ati ng the interaction are pr ovided by tw o oppo si tel y charged poly e lectrolytes [10] . N on -toxic hydrophili c material s with opposi te charges are needed for t he fo rmation of nano particles by this thecnique. The mai n adv antage o f the met ho d is that it usual ly i nvolv e s a comp let e hydrophil ic env ironment and mil d pr epar ati on conditi ons [14] , avoiding t he use of or gani c s olvents and al lowi n g the encapsulat ion of l abile dr ugs [13][15]. On t he othe r hand, t he pr esence of sugar unit s on ca rbohydrate p olymer backb one s makes them go od candidates f or Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 296 targeted dru g deli very by carbohydr ate reco gni zing receptors t hat ca n be found on the su rface of sev eral cel ls [6] . Locu st bean gum (LBG ) i s a neutral poly saccharide produc ed from the see d o f the l ocu st bean tree (car ob tree), Ceraton ia siliqua , which grows in coun tries bord ering the Medit err anean, es pecial ly Spain, Portugal, Moroc c o, Al geria, Cyprus and C rete. LBG i s a g alact omann an c onsis ting of D -mann ose unit s joined by  - 1,4 -linkag e s to f o rm long s traight chains. D -Gal actose units are attached to mannose un its by  -1,6-gly cos i dic linkages. LBG has an average rat io of 1 :4 gal actose to mann ose units, with the galactose un its attach ed in blocks as dis played in Figure XI- 2. Figure XI- 2. Genera l structur e of locust bean gum, h ighlighti ng t he structu ral unit present i n LBG. M = D -mannose u nits , G = D -galactose u nits. The mai n goal o f this work is to expl or e di ffer ent strateg ies t o c hemically modify LBG i n order to obtain a cationic polyel ectrolyte that, in turn, b y poly electrolyte complexation, wit h an anionic polyel ectro lyte, would afford NPs w ith p otential application s in the devel opment of D DS. Kappa-c arragee nan (CRG), a poly sacc har ide carryi ng negatively charged sul fate g roups at neutral pH, will be the o ther polysaccharide of use. Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 297 XI.2. Results an d Discu ssion Recently, there has been a g rowi ng interest i n the chemical fun ctionali zati on of polys acchar ides, particularly those of natural or ig in, making use of t he free hydro xyl grou ps present in the chain to create d erivat iv e s with custom pr operties for the desi red applicati ons [16] . In thi s Chapter, chemi ca l modifications of LBG were carried out t o obtained positi vely char ged derivatives for the for mati on of NPs by polyelect ro ly te complexati on. Cationic derivatives were pr epared th ro ugh a tw o-step proc e dur e, that con si st ed on a fi rst oxidation reaction and a subsequent reducti ve ami nation. For the first step , a variety of o xi dativ e methods were test ed: sodium per iodate, sodium hypoch lorite in the pr esence of sodium bro mide and a cataly tic amount of 2,2,6,6-tetramethyl piperidine -1 -oxyl (TEMPO) and ammonium persulfate wi th iron(II) s ulfate heptahydr ate as catalys t w ere ch osen as oxidizi ng agents (Sch eme XI -1). Scheme XI- 1 . Scheme of the chemica l modificati ons int r oduced i n LBG. Once the corresponding oxi dized der iv atives were obtained, the imi ne d erivat ives were formed by an adaptati on o f t he procedure des cr ibed by Berg man et al . [17] . The reducti on of t he i mine wi th sodium bo rohydride, method bro a dly described in the literat ure, was accomp lished next [18] [19]. Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 304 Figure XI- 6 . The positive seco nd deri va ti v e spectr um fo r t he samples LBG ox (blue line), LBG im (green l i ne) and LBG (ora nge li ne). A vertical l ine ind icates the positi on of the unique b and due to N-H bo nd s i n ami no derivative at wavenumber ν ma x of 15 80 cm -1 . The reduc ti on of i mine group s by s od ium bor ohy dr ide was acco mpli shed next . This react ion co uld be foll owed at a gl ance s ince the LBG im soluti ons evolved fro m a dark b rownish color t o the li ghter yellow color of the amine s olutions . Apart from t his change i n color, the reduction of the imi ne group s was verifi ed by ul trav iolet -visible s pectro scopy (UV-vis ). The IR s pectr oscopy w as not the spectroscop ic technique of choice in t his case be cause the reactions were ca rr ied out i n aq ueous media , w it h the consequen t str ong di sturbanc e caused by the infrar ed abs or ption bands fr om water. Th e dis appear ance of the band at 2 30- 260 nm in UV-vis s pectr oscopy, resulti ng fro m the n - > п* transi tion in the C=N group s [2 9] could b e cor related wi th t he ami ne for mati on . For t his reason, qu ick scans of the reaction mixture at d iff erent timepoints were perf or med (Figure XI-7). Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 305 Figure XI- 7. Kin etics of imin e reduct ion f o l lo we d by UV-v is spect roscopy at t = 0 m in (li ght blu e l ine), t = 3 0 min (pink l ine), t = 6 0 min (gr ee n line) an d t = 90 min (red line). To follow the kinet ics of the ami ne fo rmation by UV -vi s spectroscop y, a n ini tial meas ur ement of the s tarting i mine was made, and then al i quo tes were wi thdr awn from t he reduction mi xt ur e at sel ected time s t imes (30, 60 and 90 mi n). It w as observed that the i ntense ban d pr esent in the imi ne spectrum (250-300 nm ) reduc ed wi th ti m e , as expected, w hich was an unequ iv ocal signa l that the reaction had taken place. It w as also obvi ous t hat , regarding time, the reaction pr ocess was al mos t complet e in 30 minutes and totally complete after 60 minutes, since no changes were observed betwee n the s uper imposed green and red li nes (correspond ing to 6 0 an d 9 0 mi nutes of reaction, r especti vely). When the reaction finis hed, the corr esp onding a mine der iv ative (LBG am ) was dial yzed agai n st acid water (pH=4) and l yophili zed and its i nfr ared s pectru m was adquired (Fig ur e XI -8). As expected, the band correlated wit h t he s tretching of the C=N bon ds at 1675 cm -1 dis appear ed, and the 1090 cm -1 band correspon ding to t he stretching of the C N ami ne b onds i s present, thus co nfirming the success o f the r eduction process . Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 306 Figure XI- 8. FTIR spectra of L BG, LBG ox and L BG am . Arrows in d icate the car b ony l group at 17 25 cm -1 in LBG ox and t ha t of C-N at 1 090 cm -1 in L BG am . XI.2.3. Fo rmat ion of nanopartic les based on t he amin ate d derivative of LBG The production o f the aminated derivati ve de scribed above endowed t hem wit h basic am i ne group s — is olated in the form of i ts ammon ia salts — , which al lows t he p repar ation o f nano particles b y poly electrolyte complexati on (Fi gure XI-9). This N P formation technique i s a gentl e me thod that occur s in hydrophili c medi um , devoid of agg res sive conditions such as or ganic solv ent s or high shear forces, and that i nvolve s electrostatic i nteractions betwee n polymer s with opp osit e ch arges [3 0][31] . The LBG am sel ected fo r the NP for mati on were tho se sy nthesiz ed from the LBG ox derivati ves prep ared by t he oxidati on o f LBG wit h t he max imum s odium pe riodate con centration. Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 307 Figure XI- 9. Repr ese ntation of t he prepar ation of t he NPs by po ly e lectro ly te complexati on m etho d. Carrageenan (CRG) was used as a negati vely char ged poly saccharide. Of natur al orig in, t hi s polysaccharide s tands ou t for i ts ability t o for m g els . CRG is a mixture of s everal anionic polysaccharides for med by s ulphated g alactose derivat ives and i s fou nd i n the cell w alls of some alg ae . Ther e are three important types of carr ageenan: kappa (k), i ota (I) and lambd a ( λ ) , (Fig ur e XI -10). k -carrageenan, which w as used i n this work, i s a polygalact an consi st in g of l ong li near chains of alternating α -(1,3)- D -gal actose 4- sulphate est er and β -(1 ,4)-anhydr o- D -gal actose [32] . Carrageenan i s curr ently a versat ile and promisi ng biomaterial for a variety of bioengi neering applicati ons [32] . There are s everal s tudies tha t r eport the appli cations of carrageenan-based sy s tems not only related to i ts use in s ustained rel ease of drugs, but a ls o their application s i n bone and ca rtil age tiss ue eng ineering, wound s heali ng and in a nti microb ial for mulati on s [32][33] . Thus , the polys acchar ides chosen f or the formati on o f nan op articl es by polyel ectrolyt e complexation were the ami nated, posi tively charged deriv ativ e of l oc ust bean gum (LBG am ) a nd, o n the other hand , the negati vely char ged carr ageenan (CRG). In other studies conducted by this resea rc h group, sul fated and carboxyl ated derivatives of locust bean gum were used wi th c hi tosan. In these investigati on s , C TS w as the posi tiv ely charged polyel ectro lyte and t he LBG deriva tives wer e neg atively charged . It was concluded that the Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 308 physi coc hemical ch aracteristi cs of the na nopa rticles were hig hly dependen t on their co m positi on and the charge ratios applied in each co mplexat ion that w as perform ed [10][20][34][35]. Figure XI- 10 . General st ructure of the three mo st important types of carra geenan: ka ppa, iota and la mb da. For the formation of nanoparticl es by po ly elect ro lyt e complexation (Figure XI-9), seven mass ratios of LBG am /CRG were used: 4/1 , 3/ 1, 2/1, 1/1, 1 / 2, 1/3 , and 1/ 4. The ratio s of charges/g ram for LBG am and C RG were taken into acc oun t. LBG am was expected t o hav e f our basic grou ps per structur al unit (Scheme XI -2), whi le car rageenan has one negati ve charge per structu ral unit. For each polymer, the cha rge per mas s rati o can b e obtained by di vidi ng the number o f ionizabl e group s p resent in the repetiti ve unit by it s molar mas s. Thus, LBG am i s as sumed to hav e 0.00 41 8 positive charges per g ram, and i n the cas e of t he CRG, t he val ue foun d was 0.00245 negati ve char ges per g ram. Z -average, polydis pers ity i ndex (PdI), hydrod ynami c di ameter (D h ) and zet a potential ( ) studies were carried out, and data result in g fro m al l formu lat ions a re coll ected be l ow (Tab le XI -1). Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 309 Table XI- 1 . Comparison of Z -a verage, po lydispers ity in dex (PdI), h ydrody namic diameter (D h ) and zet a potential (  ) determined b y DL S of t he na nopa rticles f ormed at diff erent LBG am /CRG rati os. Sampl es LBG am / CRG Ratio of Charges* (+/- ) [LBG am ] (mg/mL) [CRG] (mg/mL) Z-av era ge (± SD) (nm) PdI (± SD) Size (± SD)  (± SD) (mV) (D h .nm) 4/1 6.8 3 0.7 1 0.1 8 115 (± 1) 0.2 0 (± 0.01) 138 (± 72) +8.8 (± 0.982 ) 3/1 5.1 2 0.7 1 0.2 4 245 (± 3) 0.2 9 (± 0.04) 338 (± 213) +10.7 (± 0.153 ) 2/1 3.4 1 0.7 1 0.3 6 1774 (± 412) 0.8 4 (± 0.14) 870 (± 118) +5.7 (± 1.42) 1/1 1.7 1 0.7 1 0.7 1 2080 (± 13 20) 0.7 8 (± 0.20) 406 (± 23) -14.5 (± 3.40) 1/2 0.8 5 0.3 6 0.7 1 354 (± 18) 0.4 4 (± 0.05) 602 (± 431) -42.2 (± 0.451 ) 1/3 0.57 0.2 4 0.7 1 313 (± 8) 0.3 6 ( ± 0.0 4) 478 (± 299) -43.7 (± 0.987 ) 1/4 0.43 0.1 8 0.7 1 271 (± 13) 0.4 7 (± 0.06) 325 (± 173) -44.4 (± 4.82) *Theoretic al ratio of positive vs neg ative char ges found for nanopartic led samples. Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 310 The ev olution of the zet a-potenti al as a function of the ch a rge ratios inv olved in each formulation of nanoparticles is depicted in Figure XI- 11. Figure XI- 11 . Effect of charge ratio (+/-) in t he zet a po t ential of the na nopartic les. The f or mati on of NPs wi th l ow polydi spersities w as achiev ed when one of the cha rged polymers used (either LBGam or C RG) was in higher ratio ( LBGam/CRG r atios: from 3:1 to 4:1 and f rom 1/2 to 1/4 ) than t he othe r. The fo rmation of i onic co mplex a t ion t oo k place and the stabi li ty of t he NP was reinfo rced by the final s urf ace charge of the complex that enable d the repulsion between the NPs, preventi ng their flocculation in the media. Thu s, i n s amples 1/ 2, 1/3 a nd 1/4, i n w hich the negative charged polymer ( carrageenan) was i n hi g her rates, charge ratios (+/ -) l ower than 1 were antici pated. This was confirmed by thei r hig hly negati v e z eta potential val ue s. The repulsi on between the neg atively char ged NPs result ed in s table NP s uspens ions over ti me. These suspensi ons a lso displayed the typ ical b luis h appe arance of nan op articl es sus pens ions . In the case of f or mulati ons wi th LBG am/CRG ratios of 3/ 1 and 4/1, the ami nated derivati ve was the pr edominant p olyel ectrolite, which transl at e d i nto positi ve potential zeta v a lues . These sys tem s displ ayed the bes t morphologi cal parameters, i .e. , the lowest polydispersiti e s were fo und (values close to 0.2 and 0.3) and the figures of Z -average were below 2 50 nm. Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 311 When the propor ti on s o f LBG am a nd ca rrageen an approach to ea ch othe r, t he formati on of agg regates w a s observed . This is w hat was observed i n samples 1/1 and 2/1, findings that w ere cor roborated with their h ig h s iz es and polydi spersities d etermined b y DLS. In additi on, pr ecipi tati on was fou nd to occ ur fo r th ese for mulati ons, b eing coincident w ith z eta po tential val ues close to zero . Another fi nding that can be inferr ed f rom the data is that, for a ratio of posi tive /negat iv e theor eti cal charges equal to 1, t he z eta potential was ex pected to be cl ose to 0. H owev er, and based on the zeta po tenti al fig ur es of the trial s perfor me d, the number of amine group s present in LBGam has been overest imated. The degree of a mi ne g ro ups in LBGam is curren tl y being unde r study. All in all, the po ly electrolyte co mplexi t i on met ho d has proved t o be a successful pathway for the for mation of nanopar ti cles based o n an a nionic polys acchar ide (CRG) and the cationic de rivative of LBG (LBG am ), with reasonab ly good polydi spers it ies and si ze val ue s. Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 312 XI.3. Con clusions LBG has p roved t o be a s uitabl e renewable raw mat erial for t he for mati on o f new ami no polysaccharides and their subsequent use in N P m anuf acture. The oxidati ve cleavage of LGB has been condu cte d by s od ium periodate, sodium hypoch lorite and ammonium persulfate, dis playing the most pro m ising r esul t s t he materials obtained by the fi rst method (sodium periodate). T he ami dati on of t he oxidi zed LBG mat eri al s was addressed by a redu ctiv e ami nation pro cedure — th rough imi ne formation — and the i ntermediates and final pr oducts were studied by infrar ed and ultraviolet - vi sible spectro scopies. NPs based o n t he an ionic carr ageenan an d the cati on ic aminated LBG w er e s ucc ess ful ly p repar ed by means o f polyel ectrolyte complexat ion. NP s iz es as good as tho se ranged from 115 nm t o 300 nm wi th polydis persity val ue s close to 0.3 were obtained. Zeta p otential data w ere compr ised between +11 m V to -44 mV, depending o n the p ol ymer ratios chosen. C omplexes prep ared wi th LBG am -carrageenan mass ratios of 1/3 , 1/ 4, 3/1 and 4/1 ca n be s uitable NPs for contro ll ed dru g deli very appl icati ons . Additional ex periments are bei ng con ducted t o: (a) optimi ze the s ynthesis of amino -based LBG polymers, (b) characterize LBG derivati ves by N MR and (c) inves ti gate NP formation at di ffer ent pH . The u se o f the NPs fo rmed as DDS is als o under study. Chapter XI _ Locust Bean Gum Derivatives for the Formation of Nanoparticles Useful in Drug Delivery S ystems 313 XI.4. Bibli o grap hy [1] B.L. Banik, P. Fattahi , J. L . Brown, Pol ymer ic nano p arti cles : the futur e of nanomedicine, W IREs Nano medicine and N a nobiotechnology. 8 (2016 ) 2 71 -299. doi: 10 .1 00 2 /wnan .1364 [2] H. C ou rrier, N. B utz, T. Van damme, Pul monar y dr ug deli very s ys tem s: recent devel opments and pr ospects, Critical Rev iews™ in Therapeuti c Dru g Carrier Sy stems , 19 (200 2) 425 -498. doi:1 0.1615/critrevtherdr ugcarriersy st.v19.i45.40 [3] T. C aon, L. J in, C.M.O. Simões, R. S . Norto n, J.A. Nicolazz o , Enhan cing the buccal mucosal deli very of peptide and protein therapeuti cs, Pharmaceutical Research. 32 ( 2014) 1 -21. doi: 10 .1007/s11095 -0 1 4- 1485-1 [4] D.A. Canelas, K.P. Herlihy, J. M. D e Si mone, To p -d own particle fab ricati on : Contr ol of s iz e and shape for diag nostic imag i ng and dr ug deli very, Wil e y Interdisci plinar y Revi ew s. Nano medi cine and Nanobiotechnology. 1 (2009) 391- 404 . doi : 10 . 10 02/ wnan.40 [5] T.C. Yih, M. Al -Fandi, Engineered nanoparticles as precise dru g deli very s ystems, Journ al of Cellular Biochemistry. 9 7 (2 00 6) 1 18 4- 11 90 . doi: 1 0.10 02/jcb.20796 [6] P. Calvo, C. Remuñán-Lóp ez, J.L. Vila-Jato, M.J. Alonso, Novel hydroph yli c chitosan polyethyl ene oxide nan opar ticl es as protein ca rr iers, Journ al of A ppli ed Pol ymer Science. 63 (19 97) 125- 13 2. doi: 10.1002/(SICI )10 97-4628 ( 19970 1 03 ) 63 : 1%3C1 25::AID - APP13%3E 3.0.CO;2-4 [7] M. de la Fuente, N. Csaba, M. G arc ia -Fuentes, M.J. Alonso, N anop articl es as pr otein and g ene carr iers to mucosal sur faces, Nanomedici ne. 3 (2 00 8) 84 5 -857. do i: 10 .2217/17435889.3.6.845 . [8] L. Zhao, A. Seth, N. W ibowo, C -X. Zhao, N. Mitt er, C. Yu, et al ., Nanopar ticl e vaccines, Vaccine. 32 (2014) 327- 33 7. doi: 10.1016/j.vaccine. 20 13.11.069. [9] J. das Neves , R. Nunes, A. Machado, B. Sarmento, Pol ymer -based nan ocarriers fo r vag i nal dr ug deli very , Advanced Dru g Del ivery Revie ws. 92 (20 15 ) 53 -7 0. d oi: 10.1016/j.addr.2014.12.004. [10] N. Bhattarai , J. Gunn, M. Zhang, Chitosan-based hydro gel s for controlled, l ocalized drug deli very , Advanced Dru g Del ivery Revie ws. 62 (20 10 ) 83 -9 9. d oi: 10.1016/j.addr.2009.07.019. [11] D. Poncelet, M icroenc apsulati on: fundamentals, met hod s and applicati ons. In: Bli t z J, Gun’ko V, edi tor s. Surf ace Chemi stry in Bi omedical and En vi ro nmental Sci ence. Dordr echt: Springer; (2005). p. 23- 4. Chapter XI __ Locust Bean Gum Derivates for T he Formation of Nanoparticles Useful in Drug Delivery Sys tems 320 Final ly, to purify t he final produ ct, sampl es were dial y si z ed ag ai ns t acidic water at pH 4 (5 L) for 72 h. Th e samples were f rozen at -80°C for 24 h and l y oph il ization was con ducted f or 3 days. A slig ht ly yellowis h po wder was collected (LBG am ) (0 .16 8 g ). - Forma tio n of nano particles ba sed on the aminate d derivat ive o f LBG All nanopar ticl es were prepar ed by p oly electrol yte complexation met ho d whi ch c onsi sts in t he elect ro static interaction betw een the p os itive a nd neg ativ e ch arges of th e diff erent polymers [23] [36]. Seven LBG am /CRG mass ratio s we re used to prep are the nano pa rticles by pol yel ec tro ly te comp lex ati on. Thes e were di vi ded i nto two sys tems: sy stem 1 i n w hich LBG am p redominated (4 :1, 3: 1 and 2:1) and system 2 ( 1: 1, 1:2, 1:3 and 1:4) in which CRG pr edominated. For system 1 , a s tock soluti on of C RG was prepared w ith ultrapu re water at a con centration of 2 mg of C RG/mL. I n ad dit ion, a st ock soluti on of L B G am in ultrapure water was p repared wi th a fi nal con centration of 1 mg of LBG am /m L. For system 2 , the solutions were prep ared i n t he s ame manner, but t hi s time the CRG s tock soluti on ha d a c oncen tration of 1 mg /mL and the LBG am st ock solu tion a concentration of 2.5 mg /mL. Al l soluti ons were filtered thro ugh a 0.45 μ m filter befo re use. The fo rmulations were prepar ed by the s lowly additi on of the ch osen LBG am s olution over the sel ected CRG solution under gentl e mag netic s tir ring at ro om t emperatur e, as s hown i n Fig ur e XII -8. The s uspensions of na nop a rticles were mi xed by magneti c s ti rr ing for 10 min and then the si ze , zet a potentia l (ζ) and p olydi spersion i ndex (PdI) determinati on of the nanoparticles were perf ormed on freshl y pr epared sa mples (T able XI -1). Chapter XII: Optimization of Customize d Polymerizatio n Conditions for the Preparation of Targeted Smart M ateria ls by the Diels-Alder Cli ck Reactions Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 323 XI I.1. Abstrac t Usi ng an experimental de si gn provides sign ifi c ant benefit i n the prep a ration of tai lor - made respon si ve materi als for biomedical appli cations. The abi li ty to rap idly and ea si ly dis cover the optimal po ly merization cond it ions f or t he p rep a ration of f unctional p oly mers, makes it an i deal tool i n t he devel opment of personaliz ed bi omaterials. The use of reversibl e covalent bon ds for the sy nthes is of new mat er i als pr ovi des the polymer structur es wi th novel func ti onal ities and applicati ons. Th e Diels -Alder (DA) reacti on is an i deal s ynthe ti c tool for the prepar ati on of fun ctional and responsiv e biomaterials un der mil d and undemanding conditi ons. The present work detail s t he optim ization of t he polymeriz ation cond iti ons to sy nthes ize two famil ies of thermo-sensit ive poly mers , either wi th red uction -respon si ve pro perties, or wi th enhanc ed hydr oph il ici ty. These polymers ha ve been s uccessfully prep ared from two functional bisdi ene mono mers: a di furfur yl dithi od iet hano l derivati ve (D iT -Fur, 1 ) and a di furfur yl - dithiothreitol derivative (DTT-Fur, 2 ), respectiv ely, using the same dimaleimide (DM DOO , 3 ) derived fr om triet hylene glycol in bo th sy stems. Twent y experiments were co ndu cted and a Box – Behnken experimental design used t o ev a luate th e importance of temperature an d water content in t erms of the polymer mol ecu l ar weights ( M n , determined by 1 H NMR). Higher t emperatur es were dis card ed s ince p revious ther mo -labil it y s tu dies demonstrated that t he retro -DA reaction cou ld occur at temperatu res as low as 50°C. Temperature p roved to be the m os t influential variable i n fi nal M n , ex ertin g opp osit e influences in bo th syst ems. In additi on, the progress of the polymeriz at ions at sel ec ted times w as accur ately monit or ed via IR spectroscop y. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 324 Figure XII - 1. Graph ical ab stract of the m ai n o bjectives in this w ork. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 325 XI I.2. Intr od uction Over t he l ast few years, increasing att ention has been dedicated t o the use of new polymeriz ation techniques that meet the thr ee main criteria of an ideal synthesis: effici ency , versa tili t y, and sel ect ivity [1] . There is als o a need fo r the construction of functional and respon si ve tai lor -made materials [2] f or their appli cati on in a wide ran ge of fiel ds, especiall y in biomedicine [3]. T he use of reversibl e co val ent bo nds in the prep a ration of po ly meric materia ls i s an attractive tool that all ow s changes in their arr angement and bond st ru ctu re sparked of f by an ex ternal st imulus such as heating, light or pH , whil e retaining their stab ilit y in the absence of trigg er. In recent years, more and more researc h has been dev oted to the uti li zation of reversibl e covalent bon ds in synthesizing new material s [4] , which not only overc omes dis advantages of permanent coval ent po ly mers, but als o brings in new func ti on ali ties such as self-heali ng, smart dr ug del ivery and contro ll able degradation, b road ening vast ly the scop e o f use o f these material s [4] . Among th e gro u p of reactions cap able of for ming reversi ble covalent linkages, Diels -A l der (DA) has emerged as a cli ck reaction t hat p ro vi des an i mmerse n umber of o ppo rtunit ies and adva n tag es, making i t an i deal candidate fo r it s use in the pr epar ati on of functio nal and r espo nsiv e biomateria ls. DA reacti on i s a robu st, effici ent, and or thogonal met hod for t he functi on ali zation of diffe rent comp ounds [ 1][9]. Th e c hemoselecti vit y of D A reactions is narrowly defined, that is , it i s or thogonal to a n unusually broad range o f reagents, s olvents , and other fu nctional groups s uch as the – COOH, – OH and – NH 2 grou ps found i n b iom olecules . It i s also moi sture and oxy gen tolerant and, und e r mi l d co nditions, does not generate s ide products. As the cli ck reaction that i t is , t he yi e lds are usually very high, and the reaction can be carried o ut und e r particularl y mi ld con diti ons (aqueo us s olution, ne utral pHs and moderate t emperatures). M or eover, t hat DA reactions can pr oceed i n t he a bsence of metal catal ysts is an extra bonu s [10]. C onsequentl y, this reaction all ows c lean, reli ab l e and sequential transfo rmatio ns of wide scope [5]. DA cycloaddit ion reacti on s have demon strated t o pro vi de benefits in a v arie ty of fields and sy s tems such as in 3D l aser mi cro- and nanoprinting [11] , i n bioconju gati on procedur es, immobilization of oli gonucleotides, pr ote i n, peptides, carb ohydrates and antibodies [1 ][12], i n t he pr eparation of s mart d rug deli very systems (DD S) [13] , i n t he core-stabi li zation of nanoparticles to pr event thei r disruption by dil ution i n the organi sm [14] [15] , in the g elat ion proc ess es of Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 326 hydro phili c polymers [16] , and in the p repar ati on of brus h polymers [10], sel f-heali ng materials [17] [18] and functional polymers [19] amo ng others. One of the p refer red DA sy stems is t he reactio n betwee n fur an an d malei mi de moieties. This comb inati on has been w idely us ed since its D A a dduc ts are for med at moderate temperatures and can be reverted back t o t he starting maleimide and furan pair at el evated temperatures [20] . A wide v ariety of reaction cond iti ons have been fo und in the s cienti fic literatur e among fur an an d malei m ide derivatives. Th us, f or example, it has been ca rried out in many s olvents : f rom toluene [10] , wat er an d chlo ro f or m [1 6 ][19] , and tetrahydro furan [21] up to i n bulk [1 ] . Depending on the con diti ons ch osen, co u pling r eactions pro ceed from a few h ours to days , at temperatures as varied as 30 °C, 40 °C, 60 °C or ev en at 120 °C in a re tro -DA procedur e [ 21] . M onomer co ncentrati on usuall y range s fro m 7.0 t o 9.0 mM [10][19] and the reacti on can be s peeded up by t he us e of a numb er of catalys ts [22] [24]. Taking i nto accoun t the findings from publis hed s cienti fic literatur e [9] [20], t emperatur e seems to be a key parameter am ong t he reaction c onditi ons s ince, toget her wi th t he accelerati on of the DA reaction, t he retro-DA reaction can occur s imul ta neously, affecti n g the l atter t o the overall results. Additionally, w ater has demonstrated to ex ert a u nique effect i n thi s type of reactions, not onl y in its ki ne tics but als o in i ts stereoselecti vi ty [25] a s was demonstrated i n the ea rly 1980s by the research teams headed by Bresl ow [26] an d G rieco [2 7] . These findings can be con si dered the “Bi g Bang” in aqueo us sy nthesis and trigg ered the gener al interes t in the us e of w ater as a s ol vent i n or gani c chemi stry [25] . Surp risi ngly, and as far as the authors are aware, ther e i s no systematic study on the influence of both independent var iabl es (temperature and wat er c ontent) on DA polymerizations of furan-malei mide mono mers and thei r impact in the final material s. The main goal s of the present wo rk are, on the one ha nd, to find the o ptimal conditi on s for the pr eparation of functi onal and r esponsiv e polymer s wi th potential use i n biomedicine throu gh a Box – Behn ken experimental d esi gn; on the other hand, to determine the relat ive influence o f the cho sen variables (te mperatur e and water co ntent) and to v erify w hether the general tre nds observed fo r one sy stem can be g eneralized or , on t he co nt rar y, depen ds on the syst em st udied. Additionally, the s cop e and li mi tati on s of the use of IR spectro sco py, vi a the s econd deriv ative of the absorption s pectra, w il l be inves tigated as a tool to t rack the deg ree of c onversion of t he mono mers in the polymer synthes es. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 327 XI I. 3. Resu lts and Discu ssion XI I.3 .1. Pr eparatio n of mon omer s and small molecules For the pr esent s tudy, the functionalized bisdifur fur yl mono mers Di T-Fur ( 1 ) and DT T-Fur ( 2 ) we re fr eshl y prepared by the reacti on of furfuryl is ocyanate wi th either 2,2’ -dithiodiethanol (Di T) or D,L -di thiothreitol (DTT), respecti vely. In both cas es, the p resence of difu rfu ryl ur ea as a si de pr oduct was found . T he i nterest in the use of bot h monome rs i n bi omedical for mulati on s i s due t o the fact t hat monomer 1 i mparts degrada bili ty u nder hypoxic envi ro nments character is ti c i n s olid tumor ti ss ues; [26] mo nomer 2 , wit h two free hydroxyl group s per m olecule, will increase the hydro phili c nature of the final poly mer. The bis di enoph il e chosen for the current work , DMD OO ( 3 ), was pr epa red by an al ready pub li shed method [ 12] . This dimalei mide al so displ a ys a hydro phili c char acter du e to the short olig oethylene g ly co l chain that conn ects both male imi de rings (Fi gure XI I-2). In order t o anti cipate the t h ermal lability of the polymers to be prepar ed, a model compound bas ed on the bis malei mide monomer was s ynthesi zed. The DM DOO monomer was made to r eact with fur an to rend er mo lecul e DMDOO -Fur ( 4 ) i n almost quantitative yi e lds. Figure XII - 2 . Chemical structu re of t he mon omers and small mo lecules use d in the p resent stud y. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 328 XI I.3.2. Thermal stability studies of the mo del D A adduct DMDOO- Fu r (4). Kineti c par ame ters determin atio n under model-b ased anal yses. The total reversi bility of furan-malei mi de-based adducts s eems t o occur f rom 100 °C to 12 0°C, mainl y at t he upp er val ue s of the range [1][7] [1 3][ 27][28]. This effect has been used to pro vi de wit h s elf-healing pr op e rties t o th erm o-labile materials at t emperatur es close t o 120 °C [13] . Moreo ver, at lower temper atures, t he retro -DA r eaction was observed. For i nstance, at 60°C , thi s reaction w as s ig nif icant when l ong reaction t imes (f or exampl e, 24 h ) were used [27] . Oth e r author s have s tated that at 70°C, [28] fu ran−malei mide adducts w ere partially rev ersible and, at higher temperatures, s hor t period s of ti me w ere needed (at 80°C in 2 ho ur s [ 1]) to i solat e the pr oducts f rom the retro-DA reaction. Some s tudies related to the ther mo -r eversi bility of fur a n−malei mide DA adducts have been pub li shed. T hus, for ex ample, Sanyal and cowor kers prepar ed polymethacrylates with protected malei m ide functionalization t o deblock the malei mide units upon heating s o t hat t hey would l a ter participate in the prep aratio n of hyd rogel micro-patterns by Michael addition. They followed the reversibility of the Di els -Alder reacti on by NMR at 80°C and f ound that, upon heati ng for 30, 60 and 120 minutes , the conversion of t he cy cl oaddu ct to malei mide was 18 , 36 and 56%, respectiv e ly, whi le heati n g for 8 h ens u res compl ete conversion w ith no trace of the bicy clic moiety [29] . The particular nature of each DA r eaction has already been demons t rated by the extensiv e work car ried out by the group o f P ro f . Konovalov from Kazan Federal U ni versi t y and sev eral pred ictive models hav e been assessed for calculati ng ki net ic parameters of cycloaddition reactions [30] . Among the cycloadditi on reactions s tudied (1849 ), most of them correspond ed to DA reactions. Each DA r eacti on (a nd c on sequently , it s cou nterpar t retr o - DA) is , therefo re, uniqu e. Gi ven t he above and t hat the po ly mer mater ials to be synthes ized are based on DM DOO mono mer, it i s nece ssary to car ry out a deep st udy of the influence of temperatur e on the stabi li t y of furan−malei mide adduc t based on DMDOO. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 329 To study the eff ect o f temperatu re on the reversi bility of t he Diels -Alder add uct, a series o f thermo gravi metric trials were conduc ted on the model comp oun d DM DOO-Fur ( 4 ) (Sc heme XII -1). Compou nd 4 provided some interesti ng featur es: first, DMD OO w as the bis malei m ide to be used in the p olymeriz ation experiment s; secon d, the d eg rad ati on of ad duct 4 would relea se the highly volat ile fur an (bo il ing point: 32°C/75 8 mmHg; vapor p ress ur e: 493 mmHg at 2 0°C ; data from www.sigmaal dr ich.com) and, consequently, t he k ineti cs of the p rocedure c ould be u nequivocall y li nked to the w e ig ht l oss o bserved du ring the tria l s; third , thermogravimetr ic analyse s ar e able to detect minute loss o f weig ht (c hanges of a few thou sandths of a mil ligram can be meas ur ed). Fig ure XI I-3 dis plays the thermograms obtai ned when c ompound 4 w as heated u nder inert atmosphere at 50°C, 60 °C an d 80°C for 100 min. c am Scheme XII- 1 . Scheme of thermally -induced retr o -Diels -Ald e r reacti on of add uct 4 . Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 336 Table XI I- 1 . Type-A Polymers: E xtent of p olymer ization s of DiT-Fur ( 1 ) an d DM DOO ( 3 ) und er d ifferent polymerizati on conditi ons obta in e d from IR a n d NMR d ata at 24 h and 4 8 h, respecti vely. Pol ymeriz ation tr ials Pol ymeriz ation conditions FTIR-AT R data Pol ymeriz . Time: 24 h 1 H NMR Data Pol ymeriz ation Time: 48 h Formulation code Sampl e Wa ter content (%) Temperatu re (°C ) Functi o nal groups consumed (%) Molar ratio endo/exo M n DiT-W 0 -T 20 A1 0 20 50 68/32 13 , 200 DiT-W 0 -T 30 A2 0 30 57 62/38 22 , 000 DiT-W 0 -T 40 A3 0 40 62 56/44 25 , 300 DiT-W 10 -T 20 A4 10 20 71 62/38 4, 900 DiT-W 10 -T 30 A5 10 30 80 66/34 10 , 700 DiT-W 10 -T 30 A6 10 30 78 66/34 11 , 300 DiT-W 10 -T 40 A7 10 40 86 62/38 11 , 400 DiT-W 20 -T 20 A8 20 20 65 66/34 8, 900 DiT-W 20 -T 30 A9 20 30 * 60/40 15 , 800 DiT-W 20 -T 40 A10 20 40 * 55/45 18,2 00 [Bis-maleimide mon omer] = [Difurfuryl mo nom er] = 65 mM; polymerization time: 48 h. Solvent: THF with variable water content (in %v / v): 0%, 1 0% o r 20%. Temperature: 20° C, 30°C, 40 °C. * Signal- to -noise ratio (SNR)< 10. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 337 Table XI I- 2 . Type-B Polymers: Extent of p olymeriz ation s of DT T-Fur ( 2 ) and D MDOO ( 3 ) under di ffer ent polymerizati on conditi ons obta in e d from IR a n d NMR d ata at 24 h and 4 8 h, respecti vely. Pol ymeriz ation tr ials Pol ymeriz ation co nditions FTIR-AT R data Pol ymeriz . Time: 24 h 1 H NMR Data Pol ymeriz ation Time: 48 h Formulation code Sampl e Wa ter content (%) Temperatu re (°C ) Functi o nal groups consumed (%) Molar ratio endo/exo M n DTT-W 0 - T 20 B1 0 20 55 74/26 22 , 500 DTT-W 0 - T 30 B2 0 30 46 53/47 9, 000 DTT-W 0 - T 40 B3 0 40 65 59/41 6, 700 DTT-W 10 - T 20 B4 10 20 67 70/30 16 , 900 DTT-W 1 0 -T 30 B5 10 30 80 66/34 12 , 500 DTT-W 1 0 -T 30 B6 10 30 * 56/44 13 , 000 DTT-W 10 - T 40 B7 10 40 * 45/55 7, 600 DTT-W 20 - T 20 B8 20 20 * 71/29 16 , 800 DTT-W 2 0 -T 30 B9 20 30 * 64/36 15 , 400 DTT-W 20 - T 40 B10 20 40 * 56/44 14 , 600 [Bis-maleimide mon omer] = [Difurfuryl mo nom er] = 65 mM; polymerization time: 48 h. Solvent: THF with variable water content (in %v / v): 0%, 1 0% o r 20%. Temperature: 20° C, 30°C, 40 °C. * Signal- to -noise ratio (SNR)< 10 Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 338 Select ed s a mples were also analyzed by GPC. It was observed that, i n general t erms , the M n values obtained by G PC were magni fied comp a red to th ose val ues cal culated fr om quantit ativ e 1 H NMR. Thu s, fo r ex ample, the t rial that render ed the hi ghest M n for the Type-A poly mers acco rding to 1 H NMR ( M n = 25,300, sample A3 , DiT-W 0 -T 40 ) showe d the followi ng values in the GPC chr omatogr am: M n = 42 ,200; M w = 65,300; M w /M n = 1.6 (Fig ure X II -S1 , Supplementary I nfo rmation). A s imilar behavior was o bserved f or DTT -based polymers and t he v alues accessed fro m GPC were overesti mat ed. Thus, fo r ex ample, sampl e B4 (DTT-W 10 -T 20 ) wi th a cal culated M n = 16 ,900 f rom NMR data, di splay ed t he foll owing results from i t s G PC chr omatogram: M n = 6 4,60 0; M w = 95,500; M w /M n = 1.5 (Fig ur e XI I-S2, Supplem entary Info rmation). This effect may be due to the fo rmation of hyd rogen bonds among the urethan e, hydroxy l and /or the thiour etha ne groups when the sampl es are diss ol ved i n the G PC s olvent (DMF) causi ng some reversi ble chain connections and, therefo re, an inc rease in the hydr odynami c volume of the polymer samples . The es timat ion of the en do/exo ratios i n the p oly meric material s by 1 H NMR was straig htfor ward. In the cas e of Type-A polymers and Type -B polymers, the mol ar end o/exo rati os w ere calcul ated accor ding to E quation XI I-S3a an d E quation XI I-S3b (S upplementary Inf ormation ), r espectiv e ly . One of the benefits l inked to the use o f water as a (co )solvent i n DA reactions i s i ts capacity to boo st reaction s electi vity towards the end o iso mer. Thus, en do/exo ratios have been al most invariably higher in water than in o rganic solvent s [34] . This fact has been observed ev en when t he rea ctants are s paring ly so luble or i nsoluble i n this medium [32]. Dens ity fun ctional theory studies c onfirm these finding s si nce the computed end o pr eference is increased in aq ueou s media up to 2. 4 Kcal ·mol -1 [35] . It needs highlig ht ing that t he for mation of the end o adducts prevail ed over the exo addu cts i n almost all t he polymerization trial s (Table XI I -1 and XI I-2). This ag rees wi th the Alder r ule [3 6 ] which predicts the pr edominance of the end o stereois omer when the fur an/maleimi de D A reaction i s carr ied out at low to moderate reac tion temperatur es [37] . W hen hig her temperatures were set for the polymeri zati on experiments, a ge neral tren d towards a sli ght but steady redu ction in the end o/ex o ratios was conf irmed. This concu rs wit h the f indings that the end o configuration is kinet ica lly favored over the ex o conf ig uration whe reas the latter is ther modynamically mor e stabl e [38] . The excepti on was ob served in sample B7 ( DTT-W 10 -T 40 ) wi th an en do/exo ratio close to 1:1 (45 :55). Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 339 XI I.3.4. Determinati on of mono mer co nver sio n dur in g pol ymerization by Infrared Sp ectroscopy and a Se co n d Derivative Pr oc edure Another target of the present work was to explo re the poss ibili t y of m onitoring the polymeriz ati on trial s with a s tra ig htfo rward and af fordable IR -bas ed pr ocedure t hat takes advantage of the fact that the required eq uipment is pr esent i n most research l abo rator ies . I n additi on, the pro cedure for tes ting polymeriz a tion reactions is mini ma ll y i nvasive (r emovi ng a small a li quo t from the medium for anal ysi s is sufficient) , and i t is not necessary to be extremely caut ious when t ak ing out the ali quo t since the r e action is not sensitive neit her to oxyg en no r to water. Consequently, IR spectroscop y was us ed as a suppo rting tool to track the polymerization pr ocesses after 2 4 h of reaction. These studies were co nduc ted using at tenuated total reflection (ATR) — Fourier Transform (FT) IR spectro sco py. To maximi ze the br oad co nvergence between the desi gned polymer s y st ems and thus, enable compar is on betw een them, polymeriz at ion trial s shared the s ame bis male imide mono mer (DMD OO, 3 ) and the difurfuryl mono mers have identi cal molecular wei g ht s (4 00 Da fo r DiT -Fur ( 1 ) an d D TT- Fu r ( 2 ); Fi g ur e XII -2). Lik ewise , identical mono mer concentrations an d reaction cond iti ons were used for the p repar ati on of type -A and type-B materials. The deg ree o f p olymeriz at ion was calculat ed by means of quantifyi ng the un reacted mal ei mide fun ctional g ro ups fr om mono me r D MDOO ( 3 ). Th e fo cus was placed on the stretching band at ν max 30 98 cm -1 associat ed wi th its C sp 2 — H bonds (Fig ure XI I-6). T he enhanced sens iti vity of the negative s econd der ivat iv e s of the abso rp ti on IR s pectra was used to det ermine the unreacted malei m ide g rou ps present i n the reaction mi xt ure. Th us, fo r ex ample, the peak height found at polymerization time = 0 w as set a s the refer ence val ue for the p resence of 10 0% u nreacted malei m ide mono mer. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 340 Figure XII - 6. The ab sorbance spectr um (bottom) and its corr es ponding ne ga ti ve seco nd d er ivati ve spect rum (top) for the b ismaleimi de mon omer DMD OO ( 3 ). A vert ical l ine ind ica tes the po sition of the u n ique b and due to =C-H b on d s in ma lei mi de rings at wave number ν max of 3 098 cm -1 . Polymeri za tion t rials w ere analy zed at 0 h, 7 h, 24 h and 48 h. In all samples, the peak at ν max 3098 cm -1 was i ndist inguishable from the baseli ne of t he IR s pectru m after 48 hours. F or ill ustrative pur pose s, Figure XI I-7 s hows the seco nd deri vative of the I R absorban ce s pectra of t he polymerization sampl e A1 at 0 h and at 48 h. H owever, thi s s tretching band w as sti ll evi dent in most experiments after 24 h of p olymerizat ion. To es tabli sh t he range of mono mer c onsumption that can be acc urately meas ur ed wi th thi s technique, a mi nimum signal - to - noise rati o (SNR) of 10/1 w as set . Data fro m polymerization trial A7 (Di T-W 10 -T 40 ) at 24 h w ere us ed. The bas eli ne noise fo r t he second der ivat iv e spectrum was fou nd to be 0.00 2 mi ll iabsorban ce unit pe r cm -2 , whil e the negative second de rivat ive band hei ght observed for t he p eak at ν max 30 98 cm -1 was 0.022 mAU cm -2 (sig nal- to -n oise r atio = 11 /1 ). The perc entage of malei mide g ro ups consumed was then quanti fied by 1 H NMR and f oun d to be 86%. Th ese data w ere correlated with the sig nific ant lower heig ht of the selected IR b and. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 341 Co nsequently , for t he experiments conduc ted, mono mer consumption rang ing from 0 % to 86% cou ld be provided by th is metho d. Figure XII - 7. Expa nded spectr a l re gi o n that exh ibi ts th e negative seco nd deri va ti ves of the v inylic C - H stretching band from mal eimide ri ngs at ri ngs at ν max = 309 8 c m -1 (Sol i d green li ne: DM DOO monomer; blu e dash l ine: po l ymerizati on trial A1 (DiT- W0 -T2 0) at time = 0. All the pol ymerization tes ts of the curr ent w or k ( A1 -A10 an d B1 -B 10 ) were anal yzed by I R at 24 hou rs. Th e heights of the second deriv ative o f thei r absorption band s were mea sured. The con versi on degrees were calculat ed by means of the reduc ti on in intensi ty observed of t he band mentioned above (data reco rd ed i n Tables XII -1 and XI I-2). This s ignal tended to v anish gradually as the po ly merization pro ceede d. Type-A poly mers exhibi ted mo nomer c onsumptions larger than 50% in al l cases (Tab le XII-1). Temper ature play ed a signi ficant role speeding t he polymeriz ation reactions l ikewise the increment of water co ntent in the polymerizat ion medi a. Thus, f or samples A9 and A10 , the peak Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 342 at ν max 3098 cm -1 was un detectable after 24 h of reacti on . Regarding T ype B polymers, they displ ayed the same trend that Type -A polymers when t he solv ent was TH F. However, th e tria ls wit h wat er as co solv ent w ere quicker tha n t hei r counterparts, wi th mon omer co nsumption greater than 80% in 24 hours. This may be due to the presence of hydroxyl g ro ups i n the bi sdiene mono mer 2 that may enhance the solubil it y of the olig omers in the reaction media. Overall, considering the affor dable nature of t he required equipment and t his t echnique bei ng mini mally invasive (the removal of a smal l aliquo t for analys is i s sufficient), quantitative IR analys e s are highly co nvenient to deter mine to what extent DA po ly merizations h ave tak en p lace. XI I.3.5. Exp erimental design for the opti mizati on of pol ymerization c on d iti ons One of the main g oals of t he cu rren t work is to draw the opti mal conditions for conducting the polymerization of the chosen bis fur a nyl monomers 1 and 2 wit h bis malei mide 3 under mi ld con diti ons. To dis close optimi zed polymerization con diti ons for the diene mo nomers, 1 and 2 , a Box – Behn ken ex perimental design was utilized and the s elect ion of t wo independent v ariables was required . The i ndepen de nt variables ch osen were temperatur e and solvent, and they r anged fr om 20°C to 40°C and from 0% to 20 % of w ater content, respecti vel y. As dependent v ariable, M n , calculated by 1 H N MR, was sel ected. T able X II -1 an d XI I-2 displ ay the val ues o f independen t variables and the ex perimental v alue s of M n obtained for the polymeriz ation of D MDOO ( 3 ) wi th both bisfuranyl-based mon omers: DiT -Fur ( 1 ) a nd DTT-Fur ( 2 ), r especti vel y. Both tabl e s also con tai n i nfor mation a bout endo /exo rati os fou nd in each t rial . In Tab le X I I-3, the equati ons obtained u si ng po ly nomial regression and statis tic al parameters ( R 2 , d f an d F) ar e sh own. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 343 Table XI I- 3 . Eq uation yiel ded f or t he depende nt variab l e (M n ) as a functi on of the i ndepen den t variab les (water conte nt and tem perature , normal ized values) fo r t he experi me n tal de sign. Equation R 2 d f F 𝐷𝑖𝑇 − 𝑀 𝑛 = 10 , 900 − 2 ,933. 33 𝑊 + 4, 650 𝑇 + 8, 100 𝑊 2 − 2650 𝑇 2 0.9 8 4.5 51.7 7 𝐷𝑇𝑇 − 𝑀 𝑛 = 13 ,5 00 − 1, 433. 33 𝑊 − 4 ,5 5 0 𝑇 + 3, 400 𝑊𝑇 0.9 5 5.4 0 29.3 3 W = water content, no rmalized value; T = Temp eratu re, normalized value; M n = Numb er Ave rage Molecu lar Weig ht determined by end -g roup an alyse s (from 1 H NMR) for the po lymerization of DMD O O with (a ) D iT-Fur (DiT- M n ) and (b ) D TT-Fur (DTT- M n ). Concer ning the response equation, i n most case s acceptable R 2 (>0.90 ) an d F (> 29) v alues were fou nd. Both eq uations con tai n complex terms t hat invol ve interaction s between the indepen dent variables. I dentify ing t he i nfluence of t he relat iv e independ ent stat istical variables on the depend ent variable i n t he di splay ed equati on s i s not s traightforwar d, nor are the calculati ons t o obtain the tw o v alues of i ndep endent variables at which the max imum M n c ould b e achieved. To overco me this drawback, the respon se s ur faces for t he de pendent vari able in each sy st em studied are s ho wn in Fig ur e XI I-8A as we ll as the compar ati ve influence of the i ndepend ent v ariables i n M n in DTT- based sys tems (Fi gure XII -8B ) and DiT sys tems (Figure XII -8C). The M n values for T ype-A Poly mers were c alcu lat ed and were fou nd t o r a nge f rom 4,90 0 t o 25 ,300, d epending o n the polymeriz ation conditions (Tab le XI I-1). T he experimental endo/ exo seg ment ratios were calculated and were found to range from 68/3 2 to 55/45. In the case of Type - B Polymers , M n v alues were w ithi n 6,700 and 2 2,5 00, fig ures als o depending on the polymerization con diti ons (Table XI I-2). Likewis e, experimental endo/exo s e gment rat ios were determined and they vari ed between 74 / 26 and 45/55. It could be observed that both polymerization s ys tem s (Type -A polymers an d Ty pe -B polymers ) showed s imilar r anges o f M n but wit h d ifferent b ehavi or s when the p olymeriz at ion co nditions var ied as is described in detail below. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 344 Figure XII - 8. Left column: (A) Response su rface fo r Num ber Av e rage M olecul ar Weight (M n ) (calculate d by 1 H NMR) ag ainst t emperature and w a ter content fo r the experime ntal desi gn of the po ly me r izat ion of DMD OO and DT T-Fur (green res p onse surf ace) DM DOO an d DiT-Fur (b lue re spon se s u rface). Ri g ht col umn: Rela ti ve i nfl u ence (in perce n tage s) of indepe nd e nt va r iables in Die l s A ld er P olymeriz ation of (B) DT T -Fur with DMD OO (green) and (C) DiT-Fur w i th D MDOO (blue). The most relev ant fi ndings were that the correl ations of M n wi th the i ndepend ent v ariables (temperatur e and wat er con tent) w ere dis si mila r fo r b oth p olymeri zation s ys tem s (Type -A and Type-B pol ymerizat ions) , w ith temperature being, the most influential parameter as can be seen in Fig ure XII-8A, XI I-8B an d XI I-8 C. Surp risi ngly, the influence of t emperatur e w as divergent i n the two sys tems studied. Thus, for D iT - Fur based p oly mers (f rom A1 to A1 0 ), t emperat ur e had a p osit ive i nfluence (increments in molecular wei ghts) over the entire temperatur e range, whereas a con s is tent decrease l inked to temperatur e i n the final M n was observed i n DT T-Fur polymers ( B1 - B1 0 ). T his fact can be eas ily br ought t o t he fo refront whe n c ompar ing trial s c ondu cted at eq ual w ater co ntent, i. e., tria ls B1 , B2 an d B3 . The re duction in t he degree o f po ly merizati ons l inked to m od erate temperatures were partially comp ensated when water was added to the sys tems (f or example, B9 a nd B6 compa red to B2 ). On t he other hand, i n Type -A trial s, the wat er content s howed a no n-lineal behav ior, leadi ng to t he hig hest M n in the absence of water . However, t he mi nimum M n fig ures w ere found Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 345 wit h a medium water content (10% i n volume). From the response s urf aces fo r the depen de nt variable i n the two s ystem s studied (Fig ur e XII -8 A), si milar degrees of polymeriz ations were achieved at intermediat e te mper atures and water con tents (cross ing l ine between cur ves). It is clear that there is n ot a direct cor r elat ion betw een r eaction r ates and M n . Other facto rs such as the reversibi lity of the reaction at mode rate temperatures and access ibi lity of the final polymer chain t o kee p t he polymerizati on pro cess active need to be considered. Thus, for example, fro m our own res ults described i n Secti on XI I.2.4. and al so suppor ted by ot her studies [16] [37] temperatur e increased th e DA rates in all cases . However, thi s trend di d n ot necess arily l ead to polymers wit h hig h molecular wei ghts . Based on the the rmal labi li t y of D MDOO -Fur ( 4 ) demon st rated in Secti on XI I. 2.2. the new poly meric mat erials are expected to be sens itive t o intermediate t emperatur es, experiencing retro-DA reactions to some degree. T herefore, although high temperatur es can s peed up D A reacti on s, retr o -DA process es occur, an d need to be introdu ced in the final equati on. This could be the case of Type B po lymers, whi ch di splayed a trend towards low M n at moderate temperatures. Regarding the water content, it was assumed that t he access to terminal functional g ro ups i n polymer chains was an essenti a l requisite for keepi n g the polymeriz at ion process acti ve . The use of water as a c osolv ent mig ht i nterfer e wit h t he g row th of the p olymer chains. This effect may be respon si ble fo r t he redu ction in M n of D iT-based polymers, d ue to the p oor solv at ion o f the olig omers g enerated d uring the polymerization p rocess. These observati ons are al so respon si ble for a peculiar behavior o f s ome polymeric s ur factants [39] . The lack of w ater s olubility of the hydro phobic block f rom the menti oned polymeric surfactants w as c rucial for t heir i rr eversi ble thermal deacti vation in w hich the po lar a nd non-p olar seg ments were initially joined by a fur an/maleimi de DA adduct. Consequentl y, the solv ation o f the final mater ial in the r eaction medium plays a key ro le i n keepin g the DA p olymeriz ation active . To sum up, fo r Type-A p olymers the best p oly me ric con diti ons w ere fo und at 40 °C when THF was used as the solvent , w hereas for t he Ty pe -B polymeri zation s, l ow temperature was cri tical to rend er hig her M n . 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[45] J.R. M cElhano n, T. Zifer, S.R. Kl ine, D .R. Wheel er, D.A. Loy, G.M. Jamison, T.M. Lon g, K. Rahimian, B.A. Simmons, Ther mall y cleavab le surfactants based o n f uran -mal eimi de diel s-a lder add ucts, Langmui r. 2 1 (200 5) 3 25 9 – 32 66. d oi:10.1021/la04707 4z. [46] E. Blas co, M .B. Sims, A. S. Gol dmann , B.S. Su merl in, C. Barn er -Kowo ll ik , 50th Anniversary Perspective: Polymer Functionali zat i on, Macromolecules. 50 ( 2017) 5215 – 52 52. doi:10 .1 021/acs.macromol.7b00465 . Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the D iels-Alder Click Reactions 355 XI I. 6 . Ann exes XI I.6.1. Specific Method s Thin lay er chromatograph y ( TLC) were pe rformed on s il ica gel 60 F 254 (Me rc k), wi th detection by UV l ig ht charring with p -anisal dehyde, phosphomolybdic acid o r ninhydr in. Sil ica gel 6 0 (Me rck, 40 -60 and 63- 2 00 μm) was used for preparative chro matography. Melt ing point s were determined in a Bi bby Steril ing LTD devi ce, in open capil lary t ubes and are uncorrected. Ther m ogravimet ric analys e s (TGAs ) were perf or med under a nit ro gen atmos pher e (flow rate 100 mL·mi n − 1 ) wi th a Universal V4.3A TA Inst ru ment at a heati ng rate of 1 0°C·mi n − 1 . For st abil ity st udie s of the Di el s - Alder adduct bo nds, is otherms at 50°C, 60°C a nd 80°C were c onducted. In ad diti on , ramps at sev eral heat ing rates were car ried o ut for the mo del comp ound 4 .  Pre paration of mono mers and s mall m ole cules. Expe rim ental procedure s - Disulf a ned iyl bis(et hane- 2,1-d iyl) bis[(f uran -2-ylmet hyl)carbamat e]. Furfury l deriv at ive o f 2,2’ - Dith iodiethanol (DiT -F ur (1)) A ro und bo ttom flas k l oaded with 2 -hydro xyet hyl disul fide (D iT, 616 mg , 4 mmo l ) w as subjected t o three cycl es of vacuum-argon an d dry T HF (2 mL) w as added to g et a soluti on. Then, fur fur yl is ocyanate (0.85 mL, 984 mg , 8 mm ol) was added foll owed by one d r op o f t he ca taly s t dibutyl tin dilaur a te. After s ti r ring 1.5 h ours at roo m temperatu re, a soli d w as fo r med. The s tirr ing w as kept for further 5 hou rs and the n the r eacti on mix tur e wa s concent rated to dr ynes s . The res idue w as pur i fied by column c hromatography us i ng ethyl a cetate -hexa ne 1: 3 as eluent and the titl e compound was obta ined as a white s olid (6 72 mg, 42%). M.p. 96 - 98° C. FTIR-ATR: ν (cm -1 ) 3 30 7 N- H ; 3060 =C -H; 16 8 1 C=O ; 1540 δ N-H; 1505 C=C; 1256 δ C- N. 1 H NMR (DMSO-d 6 , 500 MHz): δ ( ppm) 7.68 (t, 2H, H NH, J NH,e = 5.5 Hz), 7.54 (d, 2 H, H a , J a,b = 2.0 Hz, ), 6.37 (dd, 2H, H b , J b,c = 3.0 Hz), 6.21 (d, 2H, H c ), 4 .20 (t, 4H, H f , J = 6.5 Hz), 4.16 (d , 4H, H e ), 2.95 (t, 4H, H g ). 13 C NMR (DMSO-d 6 , 125 MHz): δ (ppm ) 15 5.7 (C=O), 15 2.2 (C d ), 1 41 .5 (C a ), 110.2 (C b ), 10 6.3 (C c ), 61.6 (C f ), 37,3 37,1 (C e, g ) (see Sup pleme ntary Infor mati on). Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 356 ESI-Mass Spectr ometry: m /z 423.06 [M + Na] + . HR -E SI-MS m/z: calculated from C 16 H 20 O 6 N 2 S 2 Na = 42 3.0655; f ound = 4 2 3.06 48 (differ ence bet ween t he t heor etical and experimenta l val ues: 1.64 98 ppm). - S,S' - (2,3 -d ihydroxy butane -1, 4-diyl) bis[( f uran -2-ylmethy l)carbam othioate]. Furfury l derivati ve of D,L - Dith ioth r eito l (D TT -F ur (2)) To a s olution of di thiothreitol (DTT, 3 08 mg , 2 mmol ) in THF ( 7 mL), triethyl amine (0.14 mL, 1 mmol) was added a nd s ti rr ed for 5 mi n. T he s oluti on was cooled at 0°C an d t hen, a s econ d soluti on of fu rfu ryl i socyanate (0.58 mL, 5.44 mmo l) i n THF (6 mL ) w as added d ropwise. The reaction mi xture was all owed to warm up sl owly to rt a nd pr oceeded for 6 h ou rs. The s olvent was removed under vacuum an d t he resi due dis solved i n et hyl acetate (100 mL). The o rganic l ayer was washed wit h an aqueous s olution of sulfuric aci d ( 0.3 M , 2 x 20 mL), dried wit h s od ium sul fate and the s olvent evapor ated under reduc e d press ur e l ea ding to a colored soli d. The soli d w as washed wit h w arm dichloromethane l eadin g to the t itle compo u nd as a pure w hite s ol id (35 8 mg , 45%). M.p.: 12 9°C-1 31 °C. FTIR-ATR: ν (cm -1 ) 3265 N- H, O -H; 3018 =C-H; 1631 C=O; 1520 δ N-H; 1505 C=C; 1211 δ C-N. 1 H N MR (DMSO-d 6 , 500 M Hz): δ (ppm) 8.60 (bs, 2H, NH ), 7.58 ( bs, 2 H, H a ), 6.40 ( bs, 2 H, H b ), 6 .25 (bs, 2H, H c ), 4.85 (d, 2H, OH , J OH ,g = 6.0Hz), 4.30 (d, 4H, H e , J e ,NH = 3.0Hz), 3.49 (bd , 2H, H f ), 2.98 (dd, 2H, H g’ , J g’, g ’’ = 13.5 Hz; J g’,f = 5 .0 Hz;), 2 .89 (dd, 2H, H g’’ , J g’’,f = 7 .5 Hz;). 13 C NMR (DMSO-d 6 , 125 MHz): δ (ppm) 166 .2 (C=O), 151 .8 (C d ), 142.2 (C a ), 110 ,4 (C b ), 107.1(C c ), 71 .6(C f ), 3 7.1(C e ), 32.3(C g ) ( see Sup plementary Inf or mati on ). ESI-Mass Spectr ometry: m/z 42 3.0 6 [M + Na ] + . HR-ESI-MS m/z : calcul ated from C 16 H 20 O 6 N 2 S 2 Na = 42 3.0655; f oun d = 42 3.0651 ( difference between the theor eti cal and ex perimental v alues: 0 .83 47 ppm). - 2,2'- (( E thane- 1,2- diylbis(oxy))b is(ethane -2,1-diyl))bi s(3a,4,7, 7a-tetra hydro- 1 H - 4,7 -e poxyiso indole- 1,3(2 H )-dion e) (DMDO O -Fur an (4)) A mixture of 1 ,8 -dimalei mide - 3,6-dioxaoctane (D MDOO ( 3 ), 125 mg , 0.4 1 mmol) and fur a n (0.12 mL, 1.62 mmol ) w as dis solv ed in dichloro metha ne (1.25 mL) and w as st irred at 20°C for 7 day s. The solvent and the ex ce s s of fur an were remo ved under v acuum and the ti tle compo und was is olated as an u ncolored o il in qu a ntitat iv e y iel ds . FTIR-ATR: ν (cm -1 ) 3006 =C-H; 1690 C=O. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the D iels-Alder Click Reactions 357 1 H NMR (CD Cl 3 , 500 MHz) δ (ppm) 6.5 0 (bs, 4H, H b exo ), 6.39 (bs, 4H, H b endo ), 5.31 (bs, 4H, H a endo ), 5.25 (bs, 4H, H a exo ), 3.70-3.40 (m, 28H, H h , H i endo ), 2.85 (bs, 4H, H i exo ). Rat io end o/exo : 43 :57. 13 C NMR (CD Cl 3 , 12 5 M Hz): δ (ppm) 1 76.1, 174. 8, (C=O amide), 13 6.5 (C b exo ), 134 .4 (C b, endo ), 80 .9, (C a exo ), 79 .5 (C a, endo ), 70 .1, 69.9, 67 .1, 67.0, 3 8,3, 37.7 (C h ), 4 7.5 (C i exo ), 4 6.0 (C i endo ) (see Supp leme ntary Info rmation). HR -E SI -MS m /z : calculat ed from C 22 H 24 O 8 N 2 Na = 467 .1425; found = 467.1417 (difference bet ween the theor etical and experimental value s: -1.67 87 ppm).  Ther m al stabil it y s tudi es of the m o del DA add uct DMDOO- Furan (4). Kin eti c para met ers deter minati o n under m o del-bas e d anal y ses A model-based analysis was used t o find the activation energy of t he thermally i ndu ced retro - Diels -Alder reaction o f compo u nd 4 under the acc eptance of a ki netic model [25] . Thr ee as sumptions hav e been made f or the reacti on und e r st udy. Fi rstly, it was assumed that reactions are the result of sev eral eleme ntary rea ction s teps; the reacti on rate of each step can be described by its own kineti c equation f( α) , which i n turn de pend s o n mas s conversion ( α) . T he kinet ic equation of each elementary step f(α ) is li nked to the initi al co ncentr ation o f the reactant and the concentration of the product [ Equation XII -S4 ]: 𝑑𝛼 𝑑𝑡 = 𝑘 𝑓 ( 𝛼 ) (E q. XII-S4) The rate constant “k ” ob eys to th e Arrhenius law [Eq uation (XII-S5)]: 𝑘 = 𝐴 𝑒 − 𝐸𝑎 𝑅𝑇 (Eq . XII -S5) where “k” is the rate constant, “T” is the temper a ture (in kel vins ), “A” is the p re-expon ential factor — a constant for ea ch c hemical reaction — , “E a ” i s the activ ation energy fo r the reaction, and “R” i s the un iv ersal g a s con stant. Second ly , i t was presumed that the k ineti c par am eters kept constant dur ing t he reacti on pr ogress for each indivi dual r eaction step and thirdly, i t w as ass umed that t her mo -chemical signal wa s the Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 358 sum of the si gnals of t he s ingle reaction st eps. NE TZSCH Ki netics Neo® h as been the software us ed to analyze thermo-ch emical pr ocesses data.  Pre paration o f functi onal iz ed poly mers as che m ical scaffol ds In a t ypical Diels -Alder polymeriz at ion reaction, ei ther the difur f ur yl monome r 1 or 2 an d the dimal e imi de DMDOO ( 3 ) were di ss olved i n the p olymerization sol vent (m ixt ur es of THF -H 2 O wit h variable w a ter content: 0%, 10% or 20% v/v ) so that the maxi mum monomers concen trations were reach ed (concentrati on for mon ome r 1 or 2 and monomer 3 : 65 mM). The temperatur e was set at 20°C, 30°C or 40°C by means of a s il icon e oil bath and the poly merizati on reacti on was sti rr ed for 48 hou rs. The reaction was quenched by freezing the reacti on mi xture at -20°C. The solv ent s were removed u nder vacuum at low temperature a nd the bulk pol ymers fur ther d ried at high vacuum for 7 days , leadi n g to t he polymer ic mat erial in y ields above 95% i n al l cases. Purification steps were unnecessary since unr eacted mon omers w ere not found, and t he reactions pr oceeded w ithout the fo rmation o f si de product s. Al l the s amples were st udied by 1 H NMR t o determine not only the nu mbe r molecul ar wei g ht but als o t he seg ments’ ratios endo -ex o . In all cases , the pr esence of endo and exo adducts s egments w ere found w ith prevalence of the for me r. Fro m every experiment and after 2 4 -h our poly merizati on ti me , an aliquot was r emoved fr om t he reaction mix ture an d an aly zed by ATR-FTIR to det ermine the p ercentage of mono me r conversion. - Prepar ation of DiT -Fur -base d Po lymers: Type -A Materials In the preparation of a batch o f Type - A p olymeric materi als, ten polymerizat ion reacti on s devi sed to optimi ze the polymeri c conditions were con ducted, as recor ded in Table X I I-S1 (Sup plementary Infor mation), followi ng the general met ho d desc ribed above. Al l the polymers derived form Di T - Fur ( 1 ) were white soli ds. FTIR-ATR: ν (cm -1 ) 3309 N- H, 1684 C=O; 125 7 δ C- N. 1 H NMR (CDCl 3 , 500 MHz): δ ( ppm) 7.35 (bs, 1H, H a f ro m terminal furfuryl gro up), 6.5 3 (b s, 4H, H b and H c ex o ), 6.42 and 6.33 ( 2 bs, 4H, H b and H c end o ), 5.71 (bs, 2H, NH), 5.29 -5.26 (m, 2H, H a en do ), 5.22 ( bs, 2H, H a ex o ), 4.34 ( bs, 4H, H f ), 4.04-3.80 (m, 4H, H e en do/exo ), 3.70-3.40 (m, 1 4H, H h , H i endo ), 3.35-3 .20 (m, 2H, H i’ endo ), 3.10- 2.8 5 (m, 6 H, H g , H i e xo , H i’ exo ) . 13 C NMR (CD Cl 3 , 12 5 MHz): δ ( ppm) 175.9, 175.1, 174 ,7, 170.7 (C=O amide), 156.4 (C=O urethane), 13 8.5, 137 .3 (C b, C c exo ), 135.8, 13 5.1 (C b, C c en do ), 90.2 (C d ), (80 .8 (C a ex o ), 79 .4 (C a en d o ), 70.1, Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the D iels-Alder Click Reactions 359 69 .9, 68.0, 67.8, 6 7.1, 67.0, 38.3, 37.2 (C h ), 62.9 ( C f ), 50 .2 (C i ’ exo ), 48.2 (C i endo , C i ex o ), 43,8 (C i’ endo ), 42.0 (C e exo ), 40 .3 (C e end o ), 3 7.9(C g ) (see Supp leme ntary Inf ormation ). - Prepar ation of DTT - Fur- based Polym er s: T ype- B Ma terials Wi th a s imilar s trategy for the mat eria ls pr epar ed fr om m onomer 1 , ten po lymeri zation reac ti ons between D TT- Fu r ( 2 ) an d DMD OO ( 3 ) w ere ca rr ied out wit h suc cess i n agreement w ith the experimental p arameters shown in Tab le XII -S1, Su pplementary Infor mation. In this case, the polymers derived fo rm DT T-Fu r ( 2 ) were sy ru p -li k e material s . FTIR-ATR: ν (cm -1 ) 3339 O-H , N -H, 1694 C=O; 1530 N-C=O st sy (amide II), 124 4 δ C-N. 1 H NMR (DMSO-d 6 , 5 00 MHz) : δ (pp m) 8.34 and 8 .28 (2 bs, 2H, NH), 7.5 8 ( bs, 1H, H a from termi nal fur furyl g rou p), 6.57, 6.41 and 6.2 7 (3 bs, 4H, H b and H c en do and ex o i somers), 5.25 (bs, 2H, H a endo ), 5.10 (bs, 2H, H a exo ), 4.84 (b s, 2H , OH), 4.42 -2.71 (m, 26 H, H e , H f , H g , H h , H i , H i ’ ). 13 C NMR (DMSO-d 6 , 125 MHz): δ (ppm) 1 76.5, 17 5.5, 17 5.2, 175 .0 (C =O amide), 167.3, 16 6.7 (C=O thiourethan e), 138.1, 135.9, 135.7 (C b, C c end o and exo is omer s), 91.2, 90.9 (C d endo and exo is omers), 80.6 (C a exo ), 78.9 (C a en do ), 70 .1, 69.8, 69.7, 67.4, 66.8, 38.1,37.6 (C h ), 7 2.0 (C f ), 5 0.5, 48. 7, 48.2, 47 .6 (C i , C i ’ en do and exo is omers) , 4 1.4, (C e exo ), 4 0.3 (C e en do ), 32.8 (C g ) (s ee Supp leme ntary Info rmation). Fro m quan ti tati ve 1 H-N MR anal yses, the values of M n were cal culated and ranged 6,700 to 22 ,5 00, depend ing on the p olymerization conditi ons. Li kewise, exper imental endo /exo segment r atios were determined and they varied between 74/26 and 45/55.  Exp erim ental design fo r the opt imizati on of poly meri zation condit ions To discl ose op ti mized polymer iz a tion conditi on s for the diene monome rs, 1 and 2 , a Box – Beh nken experimental des ign (CSS Statis ti ca , StatSoft Inc., Tulsa, UK) was used t o ev a luate the significance of the independen t variables (wa ter content and temperatur e), as we ll a s the i nteractions among them in the p olymeriz ation with the bisd ienophile 3 . The number of experiments “N” necessary was defined b y t he E quati on (XII-S5): 𝑁 = 𝑘 2 + 𝑘 + 𝑐𝑝 (Eq . XII -S5) Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 360 where “k” rep resents t he number of f actors (v ariables ) i nvolved i n t he s tudy and “cp ” i s the numb er of replicates of t he central point . Box – Behnken cou ld be seen as a cub e, consisting of a central po int and the middle points of the edges. Thu s, t he i nfluence of wa ter co ntent and temperatur e in t he final molecular wei ght of the polymers was s tudied by means of 20 p olymeri z ati on sys tems , 10 of them named D iT- W x -T y and the o ther 10 sys tems named D TT-W x -T y . T hey we re prepa red from mon omers DiT -Fur ( 1 ) or DT T - Fur ( 2 ), respecti vel y. In Table X I I-S1, Supplementar y I nfo rmation and al ong the text “x ” denotes the water content in percentage (v/v) i n the polymerizat ion s olvent and “y” de no tes t he polymerization temperature (Cel si u s degrees). The bisdienophile used was D MDOO ( 3 ) i n all polymerization sy stems, t he i n i tial mono mer concentrati on was s et at 65 mM led to proceed for 48 h. The factorial desi gn was used for monomer DiT -Fur ( 1 ) and DT T-Fur ( 2 ) i n which number average molecular weight ( M n ) was stat ed as dep endent variabl e. The total number of experiments requ ire d f or e ach s ys tem considered i ndependent variables at three lev e ls was 10. The values of the s elected pai r of i ndepen dent variabl es were normalized fr om -1 to +1 by usi ng E quation (XI I-S6) to facil itat e direct comparison of the c oeff ici ents and vi sua li z ati on of the eff ects o f the ind iv idual independent variables on th e respon se variabl e. 𝑋 𝑛 = 𝑋 – 𝑋  ( 𝑋 𝑚𝑎𝑥 −𝑋 𝑚𝑖𝑛 ) 2 ⁄ (E q. XII-S6) where “X n ” i s the normaliz ed val ue of independen t v ariables ; “X” is the abs olute exper imental val ue of the variable con cerned ; “ 𝑋  ” is the mean of all fixed val ue s for the variabl e in questi on; and “X ma x ” and “X min ” are the maxi mum and minimum val ues of th e variable, r espectively. The data, analy zed by multipl e r egress ion analys e s fo ll owing polynomial equation, were derived to repr esent M n as a function of the indep en dent vari ables te sted [Eq uation (XII -S7 )] 𝑦 = ∑ 𝛽 𝑖 𝑥 𝑖 𝑖 = 1 𝑡𝑜 3 + ∑ ∑ 𝛽 𝑖𝑗 𝑥 𝑖 𝑥 𝑗 𝑖 =1 𝑡𝑜 3 𝑖 < 𝑗 + ∑ 𝛽 𝑖 𝑥 𝑖 2 𝑖 =1 𝑡𝑜 3 (Eq. XI I-S7) where “y” is the predicted M n , “  ”, “  i " , and “  ij ” , deno tes the regress ion coefficients and “x i ” , “x j ” are the normalized val ues betw een pairs of independent variables (wa ter con tent and temperatur e). Only the es timat ed coefficients w ith si gn ifi cant l evels hig her than 95% (p<0.05) Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the D iels-Alder Click Reactions 361 were included in the final models. The di ffer ences betwee n the experimenta l val ue s and those that were calculated using the pr evi ous equ ati ons never exceeded 5 % of the forme r.  D eterminat ion of m ono mer conv ersi on in polym erization trial s by Infrar ed Spectro scopy and a Second Derivative P roc edu re. Infrar ed spectra were recorded wi th a Jas co FT/ IR 420 0 spectrometer eq ui pped wi th ATR. FTIR spectra w ere coll ected in a wa venumber range of 4,000 cm -1 t o 600 cm -1 at a res olution of 4 cm -1 . To enhance the s ig nal - to -no is e ratio, 64 s ca ns w ere co -add ed and si gnal averaged. The backgrou nd reference mat erial used was air. To improve the s pectral features, t he second derivatives of the absor ption spectra were g enerated, and multi plied by -1 w ith the so le objectiv e that the ban ds point upwards for c onvenience. To st udy t he percentage of monomer conversi on at reacti on ti m e of 24 hours, the hei ght of t he band at 3 098 cm -1 — ass ociated w ith the s tre tching band of =C - H mal eimi de bonds — was measured i n the negative secon d -d erivat iv e absor ption s pectra. These values di m inis hed unp arallel l y with the monomer con versi on. Th e band hei ght was monitored in each polymerization t rial and compared to s elected reference s tandard s. Among the l atter , the v a lue fou nd at 0 hour for the p olymeri zation medi a were l inked to 0% of conversion. The fig ur es ass ociated with 10 0 % con versi on were the heig hts of t he peaks fou nd when polymeric s yst em s wit h number av erage mol ecular w eights ( M n ) abo ve 17,500 determined by 1 H NMR (conversi on degree ≥ 98) w ere uti li zed . T he reduc tion of t he se data in percen tag e were corr elat e d w ith the degree o f conversion at select ed time s. Chapter XII _______________ Optimization of Customized Polymerization Conditions for the Preparation of Targeted Smart Materials by the Di els-Alder Click Reactions 368 Figur e X II- S 4 . 13 C NMR spectr a of DiT-F u r. [Document text truncated for crawler view.]