Cannabinoid derivate-loaded PLGA nanocarriers for oral administration: formulation, characterization, and cytotoxicity studies

© 2012 Ma ín-Bande as e al, publishe and licensee Do e Medical P ess L d. This is an Open Access a icle which pe mi s un es ic ed noncomme cial use, p o ided he o iginal wo k is p ope ly ci ed. In e na ional Jou nal o Nanomedicine 2012:7 5793–5806 In e na ional Jou nal o Nanomedicine Cannabinoid de i a e-loaded PLGA nanoca ie s o o al adminis a ion: o mula ion, cha ac e iza ion, and cy o oxici y s udies Lucía Ma ín-Bande as1 Jose a Ál a ez-Fuen es1 Ma ilde Du án-Loba o1 José P ados2 Consolación Melguizo2 Me cedes Fe nández- A é alo1 Mª Ángeles Holgado1 1Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain; 2Ins i u e o Biopa hology and Regene a i e Medicine (IBIMER), School o Medicine, Uni e si y o G anada, G anada, Spain Co espondence: Mª Ángeles Holgado Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Se ille c/ P o eso Ga cía González n° 2, 41012 Se ille, Spain Tel +34 954 551 624 Fax +34 954 556 085 Email [email p o ec ed] Abs ac : CB13 (1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone)-loaded poly(lac ic-co-glycolic acid) nanopa icles (NPs) we e p oduced by nanop ecipi a ion and es ed o hei in i o elease beha io and in i o cy o oxici y assays. The e ec s o se e al o mula ion pa ame e s such as polyme ype, su ac an concen a ion, and ini ial d ug amoun we e s udied. NPs had a pa icle size 90–300 nm in diame e . Resul s ob ained show ha he main in luence on pa icle size was he ype o polyme employed du ing he pa icle p oduc ion: he g ea e he hyd ophobici y, he smalle he pa icle size. In e ms o encapsula ion e iciency (%), high alues we e achie ed (∼68%–90%) o all o mula ions p epa ed due o he poo solubili y o CB13 in he ex e nal aqueous phase. Mo eo e , an in e se ela ionship be ween elease a e and NP size was ound. On he o he hand, low molecula weigh and low lac ide con en esul ed in a less hyd ophobic polyme wi h inc eased a es o wa e abso p ion, hyd olysis, and e osion. NPs showed no cy o oxici y and may be conside ed o be app op ia e o d ug-deli e y pu poses. Keywo ds: neu opa hic pain, CB13, nanop ecipi a ion In oduc ion Nano echnology has become one o he mos in ensi ely s udied esea ch a eas in ecen imes. Ma e ials a he scale o a nanome e ha e unique physicochemical p ope ies ha a e due o hei small size, su ace a ea, chemical composi ion, su ace s uc u e, solubili y, and shape. O e he las se e al decades, nume ous nanoca ie pla o ms ha e been s udied o hei use as he apeu ic agen s, wi h g ea en husiasm, in bo h academic and indus- ial applica ions. These nanoca ie pla o ms include liposomes, polyme he apeu ic conjuga es, polyme ic micelles, dend ime s, nanoshells, and nucleic acid-based nanopa icles (NPs). These polyme ic nanoca ie s bea g ea po en ial o biomedi- cal applica ions owing o hei biomolecula design and small size, and a e known o ha e shown exci ing esul s in p eclinical s udies demons a ing hei po en ial as he apeu ic ca ie s.1 Among he polyme ic ca ie s, poly(lac ic-co-glycolic acid) (PLGA) has a g ea po en ial in applica ions combining a ge ing, imaging, diagnos ics, and he apy.2 Fu he mo e, as is known, his polyme can easily be hyd olyzed in o indi idual monome s (lac ic acid o glycolic acid), which a e hen emo ed om he body ia no mal me abolic pa hways.3 Howe e , low wa e solubili y o hese d ugs limi s hei o al bioa ailabili y and abso p ion. The e is hus an u gen need o adequa e op ions o deli e hese d ugs o he pa ien . Al hough he e a e di e se s a egies (use o cosol en s, sal o ma ion, complexes wi h cyclodex ins, e c) o sol e his p oblem,4 se e al nano echnology-based Do ep ess submi you manusc ip | www.do ep ess.com Do ep ess 5793 ORIGINAL RESEARCH open access o scien i ic and medical esea ch Open Access Full Tex A icle h p://dx.doi.o g/10.2147/IJN.S34633 Numbe o imes his a icle has been iewed This a icle was published in he ollowing Do e P ess jou nal: In e na ional Jou nal o Nanomedicine 22 No embe 2012 In e na ional Jou nal o Nanomedicine 2012:7 d ug deli e y sys ems ha e eme ged o inc ease he bioa ail- abili y o nume ous d ugs ha a e poo ly soluble in wa e .5,6 Cu en ly, he e a e a conside able numbe o nanobased d ug-deli e y sys ems being de eloped by a ious pha ma- ceu ical companies. A comp ehensi e e iew o s a egies o imp o e he o al bioa ailabili y o his ype o d ugs can be ound in Fasinu e al.7 The e ha e been con o e sial indica ions as o he ex en and mechanism o anspo o hese nanoca ie s, bu he e is now no dispu e o e he ac ha pa icula e up ake does ake place, especially ia he Peye ’s pa ches M-cells and isola ed ollicles in gu -associa ed lymphoid issue, and also ia he no mal en e ocy es.8 In his pape , he cannabinoid 1-Naph halenyl [4-(pen yloxy)-1-naph halenyl]me hanone (CB13), which ac s as a po en agonis a bo h he CB1 and CB2 ecep o s, is used as a model d ug. This compound, in beha io al animal models ( a and guinea pig) o ch onic pain (neu opa hic and nocicep i e), has been shown o e e se es ablished mechanical hype algesia a e bo h o al adminis a ion and local injec ion in o a hind-paw. In beha io al es s o cen al ne ous sys em (CNS) ac i i y in a , CB13 p oduced signi i- can CNS e ec s only a doses ha we e 20- old highe han he o al doses equi ed o e e se hype algesia. Thus, hese da a indica e ha CB13 p oduces an ihype algesic ac i i y p edominan ly ia an ac ion on pe iphe al senso y ne es.9 Howe e , CB13 is highly lipophilic and belongs o he class 2 compounds (low solubili y and a high pe meabili y) o he Biopha maceu ics Classi ica ion Sys em, showing a low wa e solubili y (∼0.001–0.002 mg/mL).9 As a consequence o i s poo solubili y and dissolu ion in he gas oin es inal luids, his compound is incomple ely abso bed.9 Hence, his d ug was selec ed on he basis o i s he apeu ic in e es and inadequa e physicochemical p ope ies. I is expec ed ha , by including CB13 in a PLGA nanoca ie , p oblems associa ed wi h low wa e solubili y will disappea . This way, CB13 o al iabili y would be imp o ed. Hyd ophobic d ugs a e usually encapsula ed in biodeg ad- able polyme s using one o h ee me hods: homogeniza ion, sonica ion, o nanop ecipi a ion. An ideal me hod would p oduce NPs wi h he ollowing cha ac e is ics: na ow size dis ibu ion; size in he ange 100–1000 nm (wi h unimodal popula ion); capaci y o high d ug inco po a ion; con olled d ug con en o e a wide ange; non oxic; and ela i ely easily p oduced.10 The selec ed p epa a ion me hod o NPs was he nanop ecipi a ion me hod, also known as sol en displacemen me hod. I is based on in e acial deposi ion o a polyme a e displacemen o a semipola sol en , miscible wi h wa e , om a lipophilic solu ion. Rapid di usion o he sol en in o he aqueous phase esul s in a dec ease in he in e acial ension be ween he wo phases, which inc eases he su ace a ea and leads o he o ma ion o small d ople s o o ganic sol en , e en wi hou any mechanical s i ing.11 Mo eo e , his me hod p o ides high encapsula ion e iciency (EE) o d ugs p esen ing low wa e solubili y.3 So, he main objec i es o he p esen s udy we e: (1) o p oduce CB13-PLGA loaded NPs using nanop ecipi a ion as he mos sui able echnique o syn hesis; (2) o ealize he in i o cha ac e iza ion o he NPs; and (3) o de e mine he in i o cy o oxici y o NPs and e alua e hei sa e y and po en ial use as nanoca ie s o o al d ug adminis a ion. Me hods Ma e ials CB13 was ob ained om Toc is Cookson L d (B is ol, UK). PLGA 50:50 was ob ained in di e en o mula ions, Resome ® RG 502 (molecula weigh [Mw]: 12,000; inhe - en iscosi y: 0.24 dL/g), Resome RG 502H (Mw:12,000; inhe en iscosi y: 0.19 dL/g), Resome RG 504 (Mw:48,000; inhe en iscosi y: 0.5 dL/g), Resome RG 504H (Mw: 48,000; inhe en iscosi y: 0.53 dL/g) and PLGA 75:25, and Resome RG 752S (Mw: 15.000; inhe en iscosi y: 0.24 dL/g), om Boeh inge Ingelheim Gmbh (Ingelheim, Ge many). The su ac an s employed, Span® 60 and Plu onic® F-68, we e ob ained om Sigma-Ald ich (S Louis, MO). Ace one PRS was pu chased om Pan eac Química (Ba celona, Spain); p opylene glycol (PPG) and glyce ol we e ob ained om Aco a ma Dis ibución SA (Ba celona, Spain). The e e sed phase high-pe o mance liquid ch oma- og aphy (RP-HPLC) analysis was ca ied ou on a Hi achi LaCh om® Se ies HPLC sys em (D-7000; Hi achi L d, Tokyo, Japan) equipped wi h an L-7200 au oma ic injec o , an in e phase D-7000 and a qua e na y pump (model L-7100), and DAD UV–VIS de ec o (model L-7455). A Wa e s Co p Sphe iso b ODS2 column (10 µm, 4.6 mm × 250 mm; Mil o d, MA), kep a 40.0°C ± 0.1°C (Eli e LaCh om L-2350 column o en; Hi achi L d), was used in his analysis. Da a collec ion and calcula ion we e done by using HSM D-7000 LaCh om® so wa e (Hi achi, L d). P epa a ion o CB13-loaded PLGA NPs The NPs we e p epa ed by he nanop ecipi a ion me hod wi h modi ica ion.11 B ie ly, a weighed amoun o PLGA was codissol ed wi h Span 60 in ace one o each a 1.5% w/ concen a ion. Then, 5 mL o such solu ion was added submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5794 Ma ín-Bande as e al In e na ional Jou nal o Nanomedicine 2012:7 d opwise a di e en a es (30, 15, and 5 mL/min) using a sy inge pump (Ha a d Appa a us L d, Edenb idge, UK) in o 15 mL Plu onic F68 aqueous solu ion (0.5% w/ ) unde magne ic s i ing. The ace one was hen e apo a ed a oom empe a u e (RT) o 4 hou s. The pa icle suspension was cen i uged o e a glyce ol bed (100 µL) a 10,000 pm o 15 minu es a 4°C o collec he NPs. A e washing wice, he NPs we e esuspended in he c yop o ec an solu ion and hen ozen in liquid ni ogen and lyophilized (C iodos-50; Tels a Indus ial SL, Te assa, Spain), a −80.0°C ± 0.5°C, and 0.057 mba , o ob ain a ine powde . All he o mula ions we e p epa ed in iplica e (n = 3). Fo CB13-PLGA loaded NPs, he polyme and d ug we e codissol ed in ace one a di e en concen a ions (6, 10, and 20% w/w). Cha ac e iza ion me hods The mean diame e and size dis ibu ion o CB13 loaded- PLGA NPs we e measu ed a 25.0°C ± 0.5°C by a lase sca e - ing echnique based on Mie heo y (Pa ica LA-950V2; Ho iba L d, Kyo o, Japan). An aliquo o 1 mL o ecen ly p epa ed pa icles was dilu ed in a 12 mL cell. Measu emen s we e ca - ied ou unde con inuous magne ic agi a ion. The in luences o (1) o ganic phase addi ion a e in o he aqueous phase, (2) he ype o polyme , and (3) d ug loading we e s udied. NP aspec and mo phology we e s udied by scanning and ansmission elec on mic oscopy (SEM and TEM). The shape and mo phology cha ac e is ics o he NPs we e de e mined by SEM (XL-30; Royal Philips Elec onics, Ams e dam, The Ne he lands) a e coa ing lyophilized samples wi h a gold hin ilm. Fo TEM analysis (CM-10; Philips), a sample o lyophilized pa icles was suspended in dis illed wa e . An aliquo o his suspension was d opped on a g id and d ied a RT. NPs su ace cha ge was de e mined by ze a po en ial (ZP) measu emen s. The ZP o he pa icles was de e mined by lase Dopple (Ze amas e 300; Mal e n Ins umen s L d, Mal e n, UK). ZP measu emen s we e ca ied ou in iplica e a e washing he NPs wi h dis illed wa e a RT. The e ec s o polyme ype and d ug loading we e s udied. Ch oma og aphic condi ions The in oduc ion o new HPLC me hods o ou ine qual- i y con ol o pha maceu ical p epa a ions s a s wi h he es ablishmen o he op imal measu emen condi ions and p o ides he maximum ele an in o ma ion by analyzing he expe imen al da a.12–14 The ch oma og aphic condi ions15,16 we e de e mined using a column C18 (Sphe iso b® 5 µm ODS2, 4.6 × 250 mm Analy ical Column; Wa e s Co p). The mobile phase consis ed o wo sol en s: • Sol en A: ace oni ile:wa e :ace ic acid (75:23.7:1.3 / ) • Sol en B: ace oni ile These sol en s we e main ained in a low a e o 1.000 mL/min: 70%:30% (A:B). The mobile phase was il e ed h ough a 0.22 µm ni ocellulose-memb ane il e (Me ck Mil- lipo e, Bille ica, MA) and degassed unde acuum p io o use. A calib a ion cu e was cons uc ed using s anda d CB13 solu ions in ace one. The calib a ion cu e was lin- ea be ween 2.5 and 750 µg/mL (y = 17,268 ⋅ x + 19,0397, R2 = 0.9900, F = 583.78, P = 3.25 ⋅ 1 0 −3). The limi s o de ec- ion and quan i ica ion we e 0.5 µg/mL and 1.25 µg/mL, espec i ely. The de ec ion wa eleng h UV was 230 nm and he injec- ion olume was 10 µL. The ope a ing empe a u es we e main ained a RT, bu he o en was hea ed a 40°C o a o he mobile phase low h ough he column. De e mina ion o Plu onic F68 esiduals The mos popula s abilize o he p oduc ion o PLGA- based NPs is poly( inyl alcohol), which is di icul o emo e om he NP su ace and has been epo ed o be po en ially oxic.17,18 In he p esen wo k, Plu onic F68 was employed o s abilize he NPs and could be conside ed as a aluable al e na i e o poly( inyl alcohol) and a p omising FDA- app o ed su ace ac i e agen o clinical use.19 To de e mine he esidual amoun s o Plu onic F68 on PLGA NPs, 1H-NMR (500 MHz) spec a we e eco ded a 303 K in CDCl3, on a B üke A ance 500 spec ome e (B üke AXS Inc, Madison, WI). Chemical shi s we e gi en in ppm, using he esidual p o ona ed sol en signal as e e ence. Di e en ial scanning calo ime y (DSC) The physical s a us o CB13 inside he d ug-loaded PLGA NPs was in es iga ed by DSC. The mog ams o CB13, Plu onic F68, PLGA 50:50, and NPs (6% and 20% w/w d ug/ polyme ) we e eco ded on a DSC (Se a am 131; Se a am Ins umen a ion, Calui e, F ance). Samples (5 mg weighed o a p ecision o 0.005 mg, Model CP 225D; Sa o ius AG, Goe ingen, Ge many) we e placed in aluminum pans, and he lids we e c imped using a Shimadzu c impe . The mal beha io o he samples was in es iga ed a a scanning a e o 10°C/minu e co e ing a empe a u e ange o 25°C–300°C. submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5795 Cannabinoid de i a e-loaded PLGA nanoca ie s o o al adminis a ion In e na ional Jou nal o Nanomedicine 2012:7 S abili y s udy o NPs I is well known ha NPs made o hyd oly ic deg adable polyme s will deg ade o e ime. The e ec s o pH and empe a u e ha e a c ucial e ec on long- e m s abili y.20 In o de o e alua e he s abili y o PLGA NPs, a b ie s udy was ca ied ou . Samples o blank and loaded NPs we e incuba ed in Milli-Q wa e , phospha e-bu e ed saline (PBS) (pH 7.4), and Dulbecco’s modi ied Eagle medium (DMEM) (Sigma- Ald ich), a 4°C and 37°C. Samples we e collec ed a e p e ixed incuba ion pe iods and cen i uged o 5 minu es a 4000 pm in o de o p ecipi a e he agg ega es. Finally, he mean pa icle size o he emaining nonagg ega ed NPs was de e mined by lase sca e ing.21 F eeze-d ying p ocess F eeze-d ying is a widely used p ocess o d ying and imp o ing he s abili y o a ious pha maceu ical agen s. As his p ocess is ela i ely slow and expensi e, i is usually only used o p oduc s wi h a high added alue, which is he case o mos ypes o NPs con aining expensi e ac i es, such as CB13. The esul ing eeze-d ied NPs should ha e ce ain desi able cha ac e is ics: he p ese a ion o he ini ial cha ac e is ics o he p oduc , sho econs i u ion ime and an accep able suspension, low o unmodi ied pa icle size dis ibu ion o he NPs suspensions, and unchanged ac i i y o encapsula ed d ug. The e a e di e en ac o s han can a ec he inal quali y o he p oduc : o mula ion, use o c yop o ec an , and he s o age condi ions. F eezing is he i s s ep o eeze-d ying and gene a es many s esses. Du ing his s ep, he liquid suspension is cooled, and ice c ys als o pu e wa e o m – he c ys alliza ion o ice may exe cise a mechanical s ess on NPs, leading o hei des abiliza ion. In he p esen wo k, he use o wo commonly employed c yop o ec an s a di e en concen a ions was e alua ed: p opylene glycol, a low molecula weigh alcohol; and manni ol, a suga . Bo h we e added di ec ly o he NPs suspension be o e eezing wi h liquid ni ogen. A e his, NPs we e de os ed a RT. To e alua e he e ec o each c yop o ec an , NP diame e s we e measu ed by lase sca e ing. De e mina ion o d ug inco po a ion CB13 con en o he NPs was assessed di ec ly om he ex ac ion o he d ug om NPs. The lyophilized NPs (abou 5 mg) we e accu a ely weighed using a high-p ecision analy ical balance (d = 0.01 mg; Model CP 225D; Sa o ius). Then, 1 mL ace oni ile was added, and he mix u e was accu a ely o exed o dissol e he pa icles in he o ganic phase. A e his, 10 µL o he solu ion was il e ed (Millex® GV sy inge il e , 0.22 µm; Me ck Millipo e) and injec ed in o he HPLC sys em o CB13 de ec ion. The d ug con en was exp essed as EE (%) and d ug loading (%) ollowing Equa ions 1 and 2: EE Ac ualamoun o CB loaded in NPs amoun o CB in %=13 13Theo e ical NNPs     ×100 (1) D ug loadingwwMasso CB in NPs Masso NPs ee ed (% /)=    × 13 co 1100 (2) In i o d ug elease NP samples we e suspended in phospha e bu e (pH 7.4), main ained a 37°C, and s i ed mechanically (100 pm) du ing he elease expe imen s (Uni onic Vai én; Selec a S.A., Ba celona, Spain). Aliquo s (500 µL) we e wi hd awn a ixed ime in e als and il e ed upon cen i uga ion a 8000 pm. The il e ed sample (Millex GV) (10 µL) was injec ed in o he HPLC appa a us o he e alua ion o CB13. Cell cul u e and in i o cy o oxici y Cy o oxici y assays we e ealized using human no mal colonic CCD-18Co cell lines and he human ca cinoma T-84 cell lines ob ained om Ame ican Type Cul u e Collec ion (ATCC, Manassas, VA). CCD-18Co cells we e g own in Eagle’s minimal essen ial medium (MEM) (Sigma-Ald ich) wi h Ea le’s balanced sal solu ion (BSS) and supplemen ed wi h 10% e al bo ine se um (FBS), 2 mM L-glu amine, 1 mM sodium py u a e, and 14 mM NaHCO3. T-84 cells we e g own in DMEM, supplemen ed wi h 10% FBS, 2 mM L-glu amine, 15 mM HEPES, 14 mM NaHCO3. Gen amicin (40 µg/mL) and ampicillin (500 µg/mL) (An ibió icos SA, Mad id, Spain) we e used in bo h mediums. Cells we e main ained in monolaye cul u e a 37°C in an a mosphe e con aining 5% CO2. The cy o oxici y o CB13 loaded-PLGA NPs (Resome 502) was e alua ed using a p oli e a ion assay; a s udy o blank NPs and ee CB13 oxici y was included. Cells we e seeded in a 24-well pla e (25 × 103 cells pe well) and ea ed wi h CB13 loaded-PLGA NPs, blank NPs, and ee CB13 a a wide ange o concen a ions. A 24 and 48 hou s, cells we e ixed wi h 10% ichlo oace ic acid o 60 minu es a 4°C and s ained wi h 0.4% sul o hodamine B/1% ace ic acid by incuba ing o 10 minu es wi h cons an shaking. Cells p e iously washed wi h 0.1% submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5796 Ma ín-Bande as e al In e na ional Jou nal o Nanomedicine 2012:7 ace ic acid we e le in 10 mM T izma® (T6066-100G; Sigma-Ald ich) o 15 minu es a oom empe a u e wi h cons an shaking. The op ical densi y (OD) a 492 nm was de e mined using a Ti e ek Mul iscan MC (Flow Labo a o ies L d., I ine, Ay shi e, Sco land). To de e mine he pe cen age o ela i e cell iabili y (%RCV) we used he o mula %RCV = ( ea ed cells OD/un ea ed cells OD) × 100. Resul s and Discussion Cha ac e iza ion o he NPs The size and size dis ibu ion o he NPs was measu ed by lase sca e ing and he esul s a e epo ed in Table 1. The da a a e exp essed as he mean diame e alue and s anda d de ia ion (SD) ob ained om independen expe imen s. The analysis showed ha he samples had a pa icle size 90–300 nm in diame e ega dless o he ini ial condi ions in he p epa a ion, wi h a coe icien o a ia ion (CV) abou 30%. The e ec o polyme ype, su ac an concen a ion, and ini ial d ug amoun we e s udied. Resul s ob ained showed ha he main in luence on pa icle size was he ype o polyme employed du ing he pa icle p oduc ion. I can be seen ha o Resome 502 and 502H, pa icles we e 300 nm in diame e , and o 504, 504H, and 752S, pa icles we e a ound 100 nm in diame e . The pa icle size o NPs p epa ed wi h Resome s dec eased wi h inc ease in molecula weigh om 12,000 o 48,000 Da (502 s 504). These esul s can be explained by he hyd ophobici y o he polyme employed. A high molecula weigh , highe hyd ophobici y and smalle pa icle size we e ob ained due o longe alipha ic chains.22,23 No di e ences in size we e ound due o he p esence o e minal ca boxyl g oups in bo h cases.22 Compa ing esul s ob ained o R502 and R752S (simila molecula weigh ), smalle diame e s we e ob ained when R752S was used. In his case, he lac ic acid po ion inc eased up o 75%. The ep oducibili y o he p ocedu e was also e alua ed. In e lo CV% was in all cases less han 10%, which poin s o a eliable p ocedu e o NP p oduc ion. The ZP alues o he NPs shown in Table 1 indica e he nega i e cha ges on he NP ba e su ace, which is due o he o e all nega i e cha ges o unc ional g oups on PLGA in Milli-Q wa e . The high nega i e su ace change is an impo an indica ion o he s abili y o a colloidal sys em in medium. The epulsion among he highly nega i ely cha ged NPs p o ides ex a s abili y. The ZP o ou o he ypes o NPs was measu ed below –24 mV, p o ing he high s abili y o he NPs suspended in aqueous medium. When CB13- PLGA NPs we e e alua ed, a lowe ZP alue was ob ained (–20 mV; da a no shown). The use (o no ) o a su ac an on he o ganic phase also s ongly a ec ed he pa icle size o NPs elabo a ed wi h Resome 502 and 502H (see Table 2). I can be seen ha when no su ac an was employed o i s concen a ion was oo low, pa icle diame e inc eased up o 600 nm, and he e was a highe deg ee o polydispe si y. The ini ial amoun o CB13 was also e alua ed. No in lu- ence on pa icle size o pa icle size dis ibu ion was ound (see Table 3). As an example, Figu e 1 shows a SEM mic opho og aph o he PLGA NPs. The pic u e e ealed a egula and sym- me ical mo phology, and a e p ocessing he SEM image, he size o he esul ing pa icles was be ween 170 and 230 nm (by lase sca e ing analysis). D ug loading The capaci y o d ug loading o he NPs is an impo an ac o in hei o mula ion since high loading implies smalle amoun s o he NPs a e needed o a gi en dose o he ea men . The CB13 loading (%w/w) as well as he EE% a e epo ed in Table 4. In e ms o EE%, high alues we e achie ed o all NPs p epa ed due o he poo solubili y o CB13 in he ex e nal aqueous phase,24 and highes alues we e achie ed using Resome 502. Panyam e al25 demons a ed ha when hyd ophobic d ugs a e used, NPs d ug loading is closely Table 1 Pa icle mean diame e , size dis ibu ion, and ZP alues ob ained as a unc ion o polyme used (n = 6) Polyme Dmean (nm) SD (nm) CV (%) in alo CV (%) in e lo ZP ± SD (mV) R502 311.02 12.94 34.2782 4.16 −24.5 ± 2.3 R502-H 281.37 11.30 34.628 4.02 −42.6 ± 0.6 R504 89.33 0.96 35.0231 1.08 −28.4 ± 1.1 R504-H 91.97 10.38 34.8708 2.29 −44.8 ± 0.9 R752S 131.06 6.02 38.1749 4.60 −33.2 ± 0.7 Abb e ia ions: R, Resome ®; Dmean, mean diame e ; SD, s anda d de ia ion; CV (%), coe icien o a ia ion pe cen age; ZP, ze a po en ial. Table 2 Pa icle mean diame e and size dis ibu ion o he nano- pa icles as a unc ion o su ac an concen a ion assayed (n = 3) Polyme Csu (%w/ ) Dmean (nm) SD (nm) CV (%) R502 0 592.60 296.10 49.97 0.1 462.26 533.70 115.45 0.5 304.22 111.50 36.65 R502-H 0 600.56 219.89 36.61 0.1 269.40 96.70 35.89 0.5 287.35 116.38 52.22 Abb e ia ions: R, Resome ; Csu , su ac an concen a ion; Dmean, mean diame e ; SD, s anda d de ia ion; CV (%), coe icien o a ia ion pe cen age. submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5797 Cannabinoid de i a e-loaded PLGA nanoca ie s o o al adminis a ion In e na ional Jou nal o Nanomedicine 2012:7 Table 3 Size and size dis ibu ion o he nanopa icles as a unc ion o ini ial amoun o CB13 employed (polyme used: Resome 502) (n = 3) CB13 (%w/ ) Dmean (nm) SD (nm) CV (%) 0 291.28 99.8 34.2782 10 321.73 108.9 33.857 20 315.25 108.3 34.360 Abb e ia ions: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone; Dmean, mean diame e ; SD, s anda d de ia ion; CV%, coe icien o a ia ion pe cen age. 18 15 10 5 0 0.010 0.100 1.000 10.00 Diame e (µm) q (%) 100.0 1000 3000 AB Figu e 1 (A) Typical size dis ibu ion o CB13-PLGA NPs (polyme used: Resome 502, loading: 10% w/w). (B) SEM pho og aph o he same o mula ion. Abb e ia ions: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone; PLGA, poly(lac ic-co-glycolic acid); NPs, nanopa icles; SEM, scanning elec on mic oscope. ma ched wi h he espec i e solid-s a e d ug–polyme solubili y, ha is, he abili y o he polyme ic ma ix o en ap d ug in he dispe sed s a e. The solid-s a e solubili y o he d ug in he polyme inc eased wi h an inc ease in he lac ide con en in he polyme and wi h a dec ease in he molecula weigh . Mo eo e , he p esence o ee acid end g oups in he polyme esul ed in a dec ease in he d ug’s solubili y in he polyme . This can be explained on he basis o he inc easing hyd ophobici y o he polyme wi h inc easing lac ide con en o wi h es e end g oups, esul ing in be e solid-s a e solubili y o he hyd ophobic d ug in he hyd ophobic polyme ma ix. So, in ou s udy his hypo hesis is ue. NPs elabo a ed wi h Resome 502 showed he highes alues in d ug load- ing due o hei lowe molecula weigh and he p esence o he es e i ied end g oups. On he o he hand, NPs elabo a ed wi h Resome 504H showed he lowes alues due o hei highe molecula weigh and he p esence o ee acid end g oups. Finally, NPs elabo a ed wi h Resome 752S showed simila d ug loading alues o hose ob ained using Resome 502, whe e he e ec s o molecula weigh and lac ide con en would be balanced. In ela ion o he ini ial amoun o CB13 employed (6, 10, o 20%w/w), EE dec eases when CB13 ini ial con- cen a ion inc eases. This means ha he e is a maximum quan i y o d ug ha can be en apped in he polyme ma ix in he dispe sed s a e; ha is, he e is a limi o d ug miscibili y in he polyme .26 The emainde o he d ug can mig a e o he su ounding emulsi ie aqueous phase. As was indica ed by Panyam e al,25 i he su ac an u ilized o s abilize he emulsion is p esen a a concen a ion g ea e han i s c i ical micella concen a ion, su ac an micelles can solubilize he d ug, esul ing in a loss o encapsula ion by he NPs. In ou case, he concen a ion o su ac an - used, Plu onic F68 (0.06 mM), was sligh ly abo e i s c i ical micella concen a ion (0.04 mM), which may explain his phenomenon. In ela ion o he CB13 loading (%w/w), his seems o inc ease wi h he ini ial amoun o d ug employed du ing he p epa a ion o he NPs. Se e al s udies showed ha d ug loading can be enhanced by inc eased ini ial amoun o d ug. Howe e , EE does no necessa ily inc ease wi h highe ini ial amoun o d ug. Once he maximum loading capaci y o NPs is eached, u he inc ease in ini ial amoun o d ug can e en dec ease he EE.27 Di e en ial scanning calo ime y (DSC) The physical s a es o he d ug in he NPs ha could in luence he in i o and in i o elease o he d ug om he sys em was in es iga ed by DSC.28 Figu e 2 shows he DSC he - mog ams o pu e CB13 powde , CB13-loaded PLGA NPs, and blank PLGA NPs. Plu onic F68 and PPG he mog ams we e also analyzed. The mel ing endo he mic peak o pu e CB13 appea ed a 69.55°C (∆H = 64,651 J/g). Howe e , no mel ing peak was de ec ed o bo h NP o mula ions. Resome 502 and Plu onic F68 p esen ed endo he mic peaks a 45.90°C and 53.07°C, espec i ely. A Plu onic F68 peak was no de ec ed o NPs. Thus, i can be concluded ha CB13 inside he NPs submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5798 Ma ín-Bande as e al In e na ional Jou nal o Nanomedicine 2012:7 Table 4 D ug loading and encapsula ion e iciency o NPs (n = 6) Polyme CB13 (%w/w)ini ial EE ± SD (%) CB13 loading ± SD (%w/w) R502 6 89.61 ± 0.25 6.16 ± 1.12 10 82.26 ± 3.73 12.22 ± 6.69 20 70.75 ± 4.82 16.26 ± 0.97 R502H 6 80.74 ± 2.95 4.84 ± 1.12 10 77.094 ± 3.01 6.71 ± 2.30 20 67.709 ± 1.98 15.64 ± 3.01 R504 6 75.44 ± 2.79 16.90 ± 1.10 R504H 6 71.53 ± 3.06 16.11 ± 2.24 R752S 6 83.21 ± 1.57 18.75 ± 3.05 Abb e ia ions: NPs, nanopa icles; R, Resome ; CB13, 1-Naph halenyl[4-(pen yloxy)- 1-naph halenyl]me hanone; EE, encapsula ion e iciency; SD, s anda d de ia ion. −30 −25 −20 −15 −10 −5 0 5 10 15 20 25 30 29.3 29.9 38.4 50.3 61.9 73 83.8 94.4 105 116 126 137 147 158 168 179 189 200 210 221 231 242 252 263 273 284 294 305 315 T (ºC) Hea low Resome 502 F ee CB13 Loaded NPs Plu onic F68 PPG Blank NPs Figu e 2 DSC cu es o Resome 502, ee CB13, loaded-PLGA NPs (20% w/w), Plu onic® F68, PPG, and blank-PLGA NPs. Abb e ia ions: DSC, di e en ial scanning calo ime y; CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone; PLGA, poly(lac ic-co-glycolic acid); NPs, nanopa icles; PPG, p opylene glycol. was ei he in an amo phous, diso de ed c ys alline phase o in he solid solu e.19,29 Residual amoun o Plu onic F68 The e a e se e al me hods o de ec esidual amoun s o su ac- an s. One o he mos popula is a colo ime ic me hod de el- oped by Childs,30 employed also o quan i y esidual amoun s o Plu onic F68. The colo ime ic me hod is based on he o - ma ion o a colo ed complex be ween wo hyd oxyl g oups o Plu onic F68, Ba2+, and an iodine molecule. Then, he complex is e alua ed by measu ing i spec opho ome ically a 540 nm. In he p esen wo k, he de e mina ion o he esidual amoun s o Plu onic F68 was ca ied ou by 1H NMR spec- oscopy.31 The 1H NMR spec um o CB13, Plu onicF68, Resome 502, and blank and loaded PLGA NPs a e shown in Figu es 3 and 4. The cleaned NPs we e collec ed by cen i uga ion and lyophilized. The powde ob ained was dissol ed in CDCl3 o 1H NMR analysis. The esidual Plu onic F68 adso bed on o pa icles was expe imen ally es ima ed acco ding o he ollowing equa ion:31 Nmgg M M I I PF PLGA PF PLGA (/)(.) (..) =    × = =− 68 68 117 535515 87 δ δ 11000, (3) in which IPLGA was he peak in eg al o he – CH g oups o he lac ide uni om Resome 502 (δ = 5.35–5.15 ppm) co esponding o 1 p o on; IPF68 was he peak in eg al o he – CH3 g oups o polye hylene oxide uni om Plu onic F68 (δ = 1.17 ppm) co esponding o 87 p o ons; MPLGA was he sum o he molecula weigh s o he lac ide and he gly- colic uni s in PLGA (130 g/mol); and MPF68 was he molecula weigh o he Plu onic F68 (8350 g/mol). Addi ionally, 1H-NMR spec a we e analyzed o quan i y he amoun s o CB13 p esen in NPs, ollowing Equa ion 4. Resul s we e exp essed as Q (w/w%): QM M I I CB PLGA CB PLGA (%) / () () =    × = = 13 13 12 52 3100 δ δ . . (4) whe e MCB13 was he molecula weigh o CB13 (368.47 g/mol) and MPLGA was he sum o he molecula weigh s o he lac ide and he glycolic uni s in PLGA (130 g/mol). ICB13 was he peak in eg al o he – CH3 g oups (δ = 1.2 ppm) co esponding o 3 p o ons and IPLA was he peak in eg al o he – CH g oups o he lac ide uni (PLA [polylac ic acid]) om Resome 502 (δ = 5.35–5.15 ppm) co esponding o 1 p o on. These esul s acco ded wi h HPLC esul s o CB13 loading in Resome 502 NPs (see Table 4). submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5799 Cannabinoid de i a e-loaded PLGA nanoca ie s o o al adminis a ion In e na ional Jou nal o Nanomedicine 2012:7 10 987654321 ppm 3.01 2.03 6.13 1.99 2.03 1.00 3.03 3.01 1.03 1.00 1.01 1.00 1.01 1.00 A B 8.07.5 7.06.5 6.05.5 5.04.5 4.03.5 3.02.5 2.01.5 1.00.5 0.0ppm 3.81 1.92 1.00 Lac i O O O O xy O ** CH3 O Glico Figu e 3 (Con inued) F eeze-d ying NP in eg i y a e he eezing p ocess in liquid ni ogen was s udied unde di e en expe imen al condi ions in o de o de e mine he op imal concen a ion o c yop o ec an . C yop o ec an s a e ypically small, highly hyd oxyla ed molecules, such as monosaccha ides o low molecula weigh alcohols, ha can be added o he suspension p io o lyophiliza ion o p ese e he mo phological in eg i y o a nanoma e ial du ing eeze-d ying. We he e o e wen on o explo e an op imal concen a ion o wo o hese subs ances, p opylene glycol and manni ol. The amoun s o he c yop o- ec i e agen we e a ied om 0 o 50 w/w%, by dissol ing he agen di ec ly in o he NP suspension immedia ely p io o eeze-d ying. As can be seen in Figu e 5, he suspension wi hou c yo- p o ec an o med s icky gels du ing eeze d ying. These submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5800 Ma ín-Bande as e al In e na ional Jou nal o Nanomedicine 2012:7 C 9.08.5 8.07.5 7.06.5 6.05.5 5.04.5 4.03.5 3.02.5 2.01.5 1.00.5 ppm 3.00 1.03 23.47 0.20 0.11 —CH2 HOOOOH CH3 aab Figu e 3 1H NMR spec um o he CB13 (A), Resome 502 (B), and Plu onic F68 (C). Abb e ia ion: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone. lyophilized gels could no be esuspended in Milli-Q wa e , PBS, no DMEM unde any o he condi ions ied (including ul asonic agi a ion). The pa icle size a e he eezing p ocess was e alu- a ed measu ing mean pa icle diame e by lase sca e ing. Figu e 6 shows he e ec o he addi ion o c yop o ec an on NPs size and size dis ibu ion. Resul s ob ained o manni ol, sugges ed ha he op imal concen a ion was no eached and ha NP diame e s we e dec easing wi h inc easing manni ol concen a ion. When p opylene glycol was used, he amoun o 100 µL o p o- pylene glycol was ound o be op imal (NPs diame e and CV% we e close o ini ial pa icle diame e ). NP s abili y Fo a be e unde s anding o he in i o elease p o iles, NP s abili y in di e en incuba ion media a 37°C was analyzed. Con e sely, o de e mine he op imal s o age condi ions, s abili y s udies a 4°C we e also ca ied ou . Figu e 7 shows he NP size dis ibu ion a di e en imes in Milli-Q wa e , PBS, and DMEM a 4°C o 37°C. As e e ence, he g een (and ed) line indica es he ini ial pa icle size (300 ± 102 nm; = 0 hou s). A e incuba ion in PBS a 37°C o 6 hou s (black line), NP size inc eased o up o 100 µm in diame e . In PBS a 4°C o 6 hou s (blue line), a bimodal pa icle size dis ibu ion was ob ained. A small ac ion o NPs we e up o 100 µm in diame e , bu he majo % was a ound 700 nm in diame e . These esul s poin ou a slowe deg ada ion o NPs in PBS a 4°C s 37°C. Ne e heless, compa ing his esul wi h s abili y in Milli-Q wa e a 4°C, he esul is exac ly he con a y. A bimodal pa icle size dis ibu ion was ob ained, wi h a small ac ion o NPs a a ound 1 µm. In DMEM a 37°C incuba ion, esul s we e simila o hose ob ained in PBS a 37°C (da a no shown). In i o elease The wo main elease mechanisms associa ed wi h d ug elease om PLGA-based deli e y sys ems a e di usion and deg ada ion/e osion. The elease a e is o en said o be di usion con olled ini ially and deg ada ion/e osion con- olled du ing he inal s age o he elease pe iod. Howe e , many ac o s in luence he a e o d ug di usion and he deg ada ion kine ics, o example, polyme –d ug in e ac- ions, d ug–d ug in e ac ions, wa e abso p ion, and po e closu e. An ex ensi e e iew ocusing on he mechanisms o d ug elease om PLGA-based deli e y sys ems has been ealized by F edenbe g e al.32 submi you manusc ip | www.do ep ess.com Do ep ess Do ep ess 5801 Cannabinoid de i a e-loaded PLGA nanoca ie s o o al adminis a ion