Robust optimization of alginate-Carbopol 940 bead formulations

The Scien i ic Wo ld Jou nal Volume 2012, A icle ID 605610, 15 pages doi:10.1100/2012/605610 The cien i icWo ldJOURNA L Resea ch A icle Robus Op imiza ion o Algina e-Ca bopol 940 Bead Fo mula ions J. M. L ´ opez-Cacho,1Ped o L. Gonz´ alez-R,2B. Tale o,1 A. M. Rabasco,1andM.L.Gonz´ alez-Rod ´ ıguez1 1Depa men o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Se ille, C/ P o esso Ga c´ ıa Gonz´ alez 2, 41012 Se ille, Spain 2Depa men o Indus ial Managemen , School o Enginee ing, Uni e si y o Se ille, C/ Camino de los Descub imien os s/n, 41092 Se ille, Spain Co espondence should be add essed o M. L. Gonz´ alez-Rod ´ ıguez, [email p o ec ed] Recei ed 31 Oc obe 2011; Accep ed 8 Decembe 2011 Academic Edi o : Ali Nokhodchi Copy igh © 2012 J. M. L´ opez-Cacho e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo mula ion p ocess is a e y complex ac i i y which some imes implica es aking decisions abou pa ame e s o a iables o ob ain he bes esul s in a high a iabili y o unce ain y con ex . The e o e, obus op imiza ion ools can be e y use ul o ob aining high quali y o mula ions. This pape p oposes he op imiza ion o diffe en esponses h ough he obus Taguchi me hod. Each esponse was e alua ed like a noise a iable, allowing he applica ion o Taguchi echniques o ob ain a esponse unde he poin o iew o he signal o noise a io. A L18 Taguchio hogonala aydesignwasemployed oin es iga e he effec o eigh independen a iables in ol ed in he o mula ion o algina e-Ca bopol beads. Responses e alua ed we e ela ed o d ug elease p o ile om beads ( 50% and AUC), swelling pe o mance, encapsula ion efficiency, shape and size pa ame e s. Con i ma ion es s o e i y he p edic ion model we e ca ied ou and he ob ained esul s we e e y simila o hose p edic ed in e e y p o ile. Resul s e eal ha he obus op imiza ion is a e y use ul app oach ha allows g ea e p ecision and accu acy o he desi ed alue. 1. In oduc ion The use o na u al polyme s o he design o d ug deli e y sys ems has long been he subjec o g ea in e es du ing he pas decades. Sodium algina e is a sodium sal o alginic acid, a na u ally occu ing non oxic polysaccha ide ound in ma ine b own algae. Algina e has been widely used as ood and pha maceu ical addi i es, such as able disin eg an , hickening, and suspending agen . I con ains wo u onic acids, α-L-gulu onic (G) and β-D-mannu onic acids (M), andiscomposedo homopolyme icblocksandblocks wi h an al e na ing sequence [1]. This polyme can o m a e icula ed s uc u e when i con ac s wi h Ca2+ and hus i has been used o p oduce sus ained elease pa icula e sys ems o a ious d ugs, p o eins, and e en cells [2–4]. Gela ion occu s by an ionic in e ac ion be ween he calcium ions and he ca boxyla e anions o G-G blocks as calcium ions diffuse om he ex e nal sou ce in o he d ople o ming a polyanionic mic ocapsule. The addi ion o a polyca ion (poly-L-lysine o chi osan) o he gela ion medium induces he o ma ion o polyan- ionic-polyca ionic complexes, which s abilizes he ionic gel ne wo k and educes he algina e pe meabili y [5,6]. The main ad an age o using algina e o encapsula e d ugs is ha he algina e gela ion p ocess occu s unde e y mild condi ions wi hou using high empe a u es o chemi- cal c osslinking agen s. Ano he ad an age o using algina e is ha he algina e gel can also be con e ed o sol by adding chela ing agen s, such as Na+and EDTA. Howe e , he d ug eleasing p ope ies o Ca-sodium algina e ma ices suffe om some se ious p oblems. Fi s ly, he d ugs could be leaked du ing he gel o ma ion due o he long imme sion ime, which dec eased he encapsula ion efficiency. Secondly, he bu s elease o he d ugs om pu e Ca-sodium algina e beads is se e e due o he quick b eakdown o beads in he in i o elease p ocess. Cu en ly, much effo has been made o imp o ing he pe o mance o Ca-sodium algina e beads as d ug deli e y ca ie s. 2The Scien i ic Wo ld Jou nal Table 1: Some o he ac o s affec ing he encapsula ion p ocess o d ugs in o algina e beads. Fac o D ug Effec Re e ence Rela ed wi h he o mula ion Gliclazide Algina e concen a ion +EE + diame e −po osi y [7] − elease a e + swelling BSA + EE [5,8] Glucose Volume o he algina e solu ion Gliclazide −diame e [7] Di alen ca ion concen a ion BSA −EE [5,8,9] VEGF Glucose C osslinke /algina e a io Neu al (xan han gums, e hylcellulose, PVA) Diclo enac sodium +EE [10,11] + swelling Ca ionic (chi osan, poli-L-lysine) P o eins Oligonucleo ides +EE [3,5,12] Waxy compounds (Comp i ol 888 ATO, P eci ol ATO 5, Geluci es, glyce yl palmi os ea a e, magnesium aluminum silica e) Tamsulosin, diclo enac sodium +EE − elease a e [13–16] Anionic (CMC, Eud agi L100, dex an sulpha e) and ca ionic Insulin +EE + diame e − elease a e Su ac an p esence Gliclazide −diame e [7] +EE Rela ed wi h he p ocess Needle size BSA −diame e [5] Coaxial ai olume BSA −diame e [5] D ying p ocess BSA [5] S i ing a e Gliclazide −EE [7] −diame e The e o e, many ac o s a e in ol ed in he o mula ion o algina e mic osphe es. Some o hem a e summa ized in Table 1. D ug elease om calcium-algina e beads depends on he swelling o he beads and he diffusion o he d ug in he gel ma ix [17]. Al hough algina e beads do no swell app eciably in acidic luid [18], he beads swell and e ode/disin eg a e apidly in he in es inal luid, leading o a quick elease o he loaded d ug wi hin a ew minu es [6,19] and hence calcium algina e ma ix alone does no seem sui able as an o al con olled elease sys em [20]. Polyme ic ma e ials ha e been widely used in o de o con enien ly modi y and modula e he d ug elease om con olled- elease mic opa icles. Howe e , a la ge numbe o ac o s, including he chemical-physical p ope ies o he aw ma e ials (bo h d ug and excipien s), he composi ion and he ela i e amoun s o he componen s in he o mula- ions, as well as he manu ac u ing p ocess pa ame e s, can in luence he d ug elease beha io om he inal p oduc s [6,7,21,22]. In he p esen s udy, we a emp ed o ein o ce calcium- algina e beads con aining me hylene blue as a model d ug by inco po a ing Ca bopol 940 as hyd ophilic polyme . This is a poly ac ylic acid in anionic o m ha con ains many ee hyd oxyl g oups. Since i can o m a gel, i has been used in combina ion wi h o he polyme s o con ol he d ug elease. The e a e p e ious epo s abou he effec o ca bome s on cha ac e is ics o calcium-algina e beads [23,24]bu no o Ca bopol 940. The e o e, we in ended o in es iga e he The Scien i ic Wo ld Jou nal 3 effec o his hyd ophilic and swelling polyme on elease beha io o calcium-algina e beads. The p epa a ion o calcium-algina e beads o d ug en- apmen is a s epwise p ocedu e. The indus ial expe imen- a ion includes a g ea numbe o pa ame e s and a ull ac o ial design ha yield la ge numbe o expe imen s. Con- en ional op imiza ion p ocedu es in ol e al e ing one inde- penden ac o a a ime keeping all o he s emain cons an , which enables o assess he impac o hose pa icula pa am- e e s on he p ocess pe o mance. These p ocedu es a e ime consuming and cumbe some because equi e mo e expe - imen s and canno p o ide in o ma ion abou he mu ual in e ac ions o he pa ame e s [25]. To educe hese numbe s o expe imen s, a small se o ials wi h all possible combi- na ions is selec ed by using a ious s a is ical ools [26]. S a is ical expe imen al design me hods, such as he ac ional ac o ial design [8], he Placke -Bu man me hod [27], esponse su ace me hodology [28], he Box-Behnken design [29,30], and he Taguchi me hod [31], p o ide a sys ema ic and efficien plan o expe imen a ion o achie e ce ain goals so ha many con ol ac o s can be simul ane- ously s udied. In con as o he adi ional “one- ac o a a ime” app oach, hese me hods can be used o examine and op imize he ope a ional a iables while conside ing he in e ac i e effec s among he con ol ac o s. The Taguchi me hod ocuses on p oduc obus ness agains uncon ollable in luences, o noise. The me hod is designed o educe a iabili y, and op imize unc ion o pe - o mance wi h he lowes cos componen s. Taguchi me hod o o hogonal a ay design o expe imen (DOE) in ol es he s udy o any gi en sys em by a se o independen a iables ( ac o s) o e a speci ic egion o in e es (le els). This app oach also acili a es o iden i y he in luence o indi id- ual ac o s, and es ablish he ela ionship be ween a iables and ope a ional condi ions. In his me hodology, he desi ed design is sough by selec ing he bes pe o mance unde condi ions ha p oduce consis en pe o mance [32] and he esul s om small-scale expe imen s a e alid o e he scale up schedule [33]. Robus ness is essen ial in he p oduc ion o nea ly all p oduc s. The a ia ion in he quali y o he p oduc may ollow om en i onmen al ac o s and/o manu ac u ing a iables ha canno be easily con olled (noise ac o s). In addi ion o emphasizing ha he a iabili y in he quali y o he p oduc and ac o s in e ac ions should be educed [34], he Taguchi me hod uses he signal- o-noise a io (S/N ), which is di ec ly ans o med om he quad a ic quali y loss unc ion, as a measu e o de e mine he obus ness o a p ocess. Thus, op imizing p ocess pa ame e s by he Taguchi me hod is an a emp no only o b ing he a e age quali y nea o he a ge alue, bu also o simul aneously minimize he a ia ion in quali y [35]. The e o e, his me hod has been ex ensi ely applied in indus y and i s applica ion caused signi ican changes in se e al indus ies, in manu ac u ing p ocesses and o al quali y con ol [36–38]. This echnique is somehow no e y well known o op imiza ion o pha maceu ical p ocesses. This pape offe s a apid and efficien me hodology o s udy and op imize pha - maceu ical o mula ions, based on he Taguchi o hogonal a ay design. To do his, as an example, we used algina e- Ca bopol beads o mula ed by iono opic gela ion wi h me hylene blue (MB) as hyd ophilic d ug model. A e he selec ion o noise and con ol ac o s, we se- lec ed he p o iles ( esponses) o op imize wi h he aim o demons a e he applicabili y o his me hodology in he op imiza ion o diffe en p o iles such as d ug elease, swelling a e, o he mo phology o he beads. A e wa d, he expe imen al design and da a analysis p ocedu e, we selec ed he bes p o iles o Taguchi op imiza ion and hen we compa ed he esul s wi h p edic ed ones by lineal eg ession. Finally we compa ed he accu acy o he op i- miza ion. 2. Ma e ials and Me hods 2.1. Ma e ials. Sodium algina e ( iscosi y 2% w/ : 250 cP) and ie hanolamine (TEA) we e pu chased om Sigma Ald ich. Calcium chlo ide (Pan eac, Spain) was selec ed as ca ion dono sal . MB was used as hyd ophilic d ug model. Ca bopol 940 (Aco a ma, Spain) was added o con ol he d ug elease om he algina e beads. Sodium ipolyphos- pha e and T is(hyd oxyme hyl) aminome hane (TRIS) we e ob ained om Sigma (Spain). 2.2. P epa a ion o Beads. The beads we e p epa ed by ol- lowing he ex usion/p ecipi a ion me hod [39,40]. B ie ly, a sodium algina e aqueous solu ion con aining 0.01% (w/ ) MB and 2% (w/ ) o sodium algina e was p epa ed by a simple mixing s ep. Ca bopol 940 was dispe sed in pu i ied wa e and hen was added o he abo e solu ion. As a unc ion o he expe imen , TEA was added o his solu ion. The beads we e p epa ed by d opping he algina e-Ca bopol solu ion (50 mL) con aining MB om a sy inge using a pe usion pump (Pe uso m, B aun), h ough a 0.9 mm diame e needle in o a gen ly s i ed 0.25 M calcium chlo ide aqueous solu ion (IKA Eu os a ). Diffe en concen a ions o Ca bopol, a diffe en d opping a e and s i ing a es we e used, as was epo ed in Table 2. The ob ained hyd ogels we e main ained in o he calcium chlo ide solu ion o diffe en ime pe iods o comple e he chemical eac ion. The beads we e collec ed by decan ing he calcium chlo ide solu ion, washed wi h deionized wa e and d ied o a cons an weigh . A his s age, diffe en d ying me hods we e used: oom empe a u e (20◦C), o a y e apo a o ( educed p essu e, acuum and 80◦C), o o en (100◦C). 2.3. Cha ac e iza ion o Beads 2.3.1. En apmen Efficiency. The MB con en in he d y sam- ples was de e mined by dissol ing he beads, p e iously weighed, in a ipolyphospha e solu ion (0.03 M; 100 mL). The esul ing solu ion was il e ed and he d ug concen- a ion was analyzed by UV spec opho ome y, a 664 nm, using a UV- isible spec opho ome e (Hi achi U-2000). En apmen efficiency was calcula ed as he pe cen age (w/w) o he heo e ical d ug con en . Resul s we e based on ipli- ca e de e mina ions. 4The Scien i ic Wo ld Jou nal Table 2: Fac o s and hei co esponding le els implemen ed o he cons uc ion o L18 o hogonal a ay. Fac o Le els used, ac ual (coded) Low (−1) Medium (0) High (+1) Independen a iables A: addi ion o TEA No Yes B: concen a ion o Ca bopol (% w/ ) 1 2 4 C: lux a e o polyme ic solu ion (mL/min) 1 2 3 D: s i ing a e o s i e ( pm) 100 300 500 E: eac ion ime o beads in o CaCl2solu ion (min) 5 30 60 F: d ying me hod Room Ro a y e apo a o O en G: NaCl (% w/ ) 0 0.01 0.1 H: pH 7.2 7.6 8.0 2.3.2. In Vi o D ug Release S udies. The d ug elease p o iles o beads we e de e mined using he USP 28 paddle me hod. The dissolu ion medium was 900 mL (sink condi ions) o T is-buffe solu ion adjus ed a diffe en pH and NaCl concen a ion alues as a unc ion o he expe imen . The s i ing speed was 50 pm and he empe a u e was main- ained a 37 ±0.5◦C. Accu a ely weighed samples o 700 mg beads we e used o he es . A selec ed ime in e als o a pe iod o 420 min, aliquo s each o 3 mL we e wi hd awn om de dissolu ion medium h ough a 0.45 μmmemb ane il e and eplaced wi h an equi alen amoun o he esh dissolu ion medium. Concen a ions o MB we e hen spec- opho ome ically de e mined a 664 nm. Each expe imen was ca ied ou in iplica e and he esul s we e a e aged. T is-buffe solu ion was selec ed as he dissolu ion medium because in he o ma ion o algina e gel, algina e eac ion wi h calcium ions u ns e e sible wi h sodium ions, and a disagg ega ion o he beads occu s [6]. We selec ed his buffe o con ol swelling p ocess by modi ying NaCl concen a ion. 2.3.3. Swelling P ocess. The amoun o abso bed wa e was de e mined ollowing desc ibed me hods by many au ho s [7,41,42]. In a i s s age, a ce ain numbe o beads, accu a ely weighed (W0), we e placed in glass beake s con aining 80 mL o is-buffe and NaCl concen a ion se by DOE. Nex , he glass beake s we e placed in a he mos a ic ba h a 25◦C and 150 pm. A ime in e als o 1 h, beads we e il e ed and weighed (We). Swelling pe cen age (Esw) was calcula ed using he ol- lowing exp ession [43]: ESW =We−W0 W0×100, (1) whe e Weis he we weigh o beads and W0is he ini ial weigh o beads. 2.3.4. Mo phological P ope ies o Beads. Mo phological cha ac e is ics o beads we e obse ed by Scanning Elec on Mic oscopy, SEM (Philips XL30). The samples we e spu e - coa ed wi h Au/Pd using a acuum e apo a o (Edwa ds) and examined a 20 kV accele a ing ol age. The size and shape o he beads we e de e mined using image analyze so wa e (Image P o Plus). The leng h and b ead h we e measu ed om he digi al images o each bead and i s size calcula ed om he a e age o hese wo dimensions [44]. Fo each o mula ion, 30 beads we e andomly selec ed o measu emen ; he esul s we e hen a e aged. 2.4. Op imiza ion Me hodology. Op imiza ion me hodology adop ed in his s udy was di ided in o ou phases (wi h a - ious s eps): planning, pe o mance, analysis, and alida ion. The schema ic ep esen a ion o he designed me hodology was depic ed in Figu e 1 (modi ied om [34]). 2.4.1. Phase I. Planning (1) De ining he P oblem and he Goal. Fi s s ep in Phase I is o de e mine he ac o s o be op imized in he p oduc ion and cha ac e iza ion o algina e-Ca bopol beads ha ha e c i ical effec on he d ug elease, encapsula ion efficiency, and in he mo phological p ope ies o he beads. The goal is o e alua e he effec o diffe en ac o s on he elabo a ion o he beads and o ob ain he pe ec combina ion be ween he le els o he ac o s o op imize hei s p ope ies. (2) Iden i ying he Fac o s o Be Op imized. Six p ocess pa ame e s ( lux a e, s i ing a e, incuba ion ime, d ying mechanism, NaCl concen a ion, and pH o he dissolu ion medium) and wo o mula ion pa ame e s (p esence o TEA and Ca bopol concen a ion) we e e alua ed. The no mal p ac ice is o expe imen wi h he easible ange, so ha he a ia ion inhe en in he p ocess does no mask he ac o s effec s. The con ols ac o and hei s le els a e exposed in Table 2. (3) Iden i ying he Tes Condi ions o Op imize. The espons- es o be op imized om he cha ac e iza ion o algina e- Ca bopol beads a e hose ha ha e c i ical effec on he d ug elease, encapsula ion efficiency, and mo phological p ope ies o he beads. Selec ed esponses a e exposed in Table 3. The Scien i ic Wo ld Jou nal 5 Valida ion expe imen s Phase I. Planning Indi idual ac o con ibu ion Rela i e ac o in e ac ion De e mina ion o op imum le els o ac o s Es ablishmen o bes op ional condi ions o p ocess op imiza ion Pe o mance unde op ional condi ions Analyze he da a using DOE pack 2000 so wa e P edic he pe o mance a hese le els Iden i y he con ol ac o s and hei le els Design he expe imen al ma ix De ine he da a analysis p ocedu e Pe o m he designed expe imen s Iden i y he ac o s o be op imized Iden i y es condi ions De ining he p oblem and he objec i e Phase II. Pe o mance Phase IV. Valida ion Phase III. Analysis Figu e 1: Schema ic diag am o he s eps in ol ed in he Taguchi me hodology. Table 3: Tes condi ions o op imize. P o ile analyzed Selec ed a iable D ug elease Time o elease 50 pe cen o en ap- men d ug ( 50%) A ea unde cu e (AUC) En apmen efficiency En apmen efficiency (EE) Swelling pe o mance Swelling a e (SR) Shape o he pa icles Shape ac o (SF) Size o he pa icles Mean diame e (Dm) (4) Expe imen al Design and Da a Analysis P ocedu e. The undamen al p inciple o he Taguchi me hod is o imp o e he quali y o a p oduc by minimizing he effec o he causes o a ia ion wi hou elimina ing he causes [45]. Th ee majo ools used in he Taguchi me hod a e he o hogonal a ays, analysis o a iance (ANOVA), and he signal o noise a io (S/N). The Taguchi app oach p o ides an oppo uni y o selec a sui able o hogonal a ay depending on he numbe o con- ol ac o s and hei s le els [46]. The o hogonal a ays gi e equal c edence o all he pa ame e s being in es iga ed while ANOVA de e mines he con ibu ion o each pa ame e in he design o he expe imen . This is a ac ional ac o ial app oach and educes he numbe o expe imen s. O hogonal a ay is a ma ix o numbe s a anged in ows and columns. Each ow ep esen s he le el o ac o s in each un and each column ep esen s a speci ic le el o a ac o ha can be changed o each un. S/N a io is an indica i e o quali y, and he pu pose o he Taguchi expe imen is o ind he bes le el o each ope a ing pa ame e o maximize he a io [47]. Analysis o he expe imen al da a using he ANOVA and effec s o he ac o s gi es he ou pu ha is s a is ically signi ican o inding he op imum le els. In he Taguchi me hod, quali y is measu ed by he de ia ion o a cha ac e is ic om i s a ge alue, and a loss unc ion [L(y)] is es ima ed o he de ia ion as L(y)= k×(y−m)2,whe ekdeno es he p opo ionali y cons an , m ep esen s he a ge alue and yis he expe imen al alue ob ained o each ail. In case o bigge and be e quali y cha ac e is ics, he loss unc ion can be w i en as L(y)= k×(1/y2) and he expec ed loss unc ion can be ep esen ed by: ELy=k·E1 y2,(2) whe e E(1/y2)canbees ima ed omnnumbe o samples as: n i=11/y2 i n.(3) 6The Scien i ic Wo ld Jou nal In o de o measu e he quad a ic loss unc ion, he Taguchi in oduces he signal- o-noise (S/N ) a io o mea- su ing he quali y h ough o hogonal a ay based expe i- men s. S/N a ios ake in o accoun bo h he a iabili y in he esponse da a and he closeness o he a e age esponse o a a ge alue. The S/N a io cha ac e is ics can be di ided in o h ee ca ego ies when he a iable is con inuous [48]: (i) nominal he bes : S N=−10 logy2 i νyn,(4) (ii) smalle he be e : S N=−10 logiy2 i n,(5) (iii) bigge he be e : S N=−10 log1/iy2 i n,(6) whe e yis he a e age o obse ed da a, nis he numbe o obse a ions, and yi he obse ed da a o he i h un. In he nominal bes s udy, yand na e nominal alues hose a e o be achie ed. Fo each ype o cha ac e is ics, wi h he abo e S/N a io ans o ma ion, he highe he S/N a io, he be e is he esul . The uncon ollable ac o s which cause he unc ional cha ac e is ics o a p oduc o de ia e om hei a ge alues a e called noise ac o s. The o e all aim o quali y is o make p oduc s ha a e obus wi h espec o all noise ac o s [49]. The design was pe o med by an L18 a ay. In he p esen s udy, h ee le els o se en ac o s and wo le els o one ac o (Table 2) we e conside ed and he size o expe imen a ion was ep esen ed by symbolic a ay o L18 (which indica ed 18 expe imen al ials) (Table 2). The h ee le els o all he ac o s ha e been assigned (excep TEA (21)), wi h a layou o L18 (21×35). 2.4.2. Phase II. Pe o mance o Expe imen s. A e phase I, in which he ac o s o be op imized, hei s le els and he expe imen al ma ix we e selec ed, he expe imen s we e ca ied ou by iplica e (Table 4) and he selec ed a iables we e de e mined. 2.4.3. Phase III. Analysis o he Da a and Op imiza ion (1) Analysis o Da a. A his s age, a s a is ical analysis o a iance was pe o med o de e mine which es pa ame e s we e s a is ically signi ican o e e y a iable. Mo eo e , he in luence o each ac o on he diffe en esponse a iables selec ed was de e mined. (2) Op imiza ion. A e he analysis o he da a, we p o- ceeded o de e mine he op imal se pa ame e con igu a- ion. Op imiza ion p ocess was made applying he ma ginal means me hodology (also called a e age a e o main effec s analysis). In o de o do ha , we ob ained he a e age alue o S/N a io o e e y a iable in e e y le el o esponse and hen he esul s we e plo ed in a g aphic o ma ginal means. In he Taguchi me hod, we always ied o maximize he S/N a io, so we selec he le els wi h he highe alues o S/N a io in e e y ac o ha gi e us he maximum S/N inal a io. The diffe ence in e e y esponse is he me hod ollowed o he consecu ion o he S/N a io, as i was explained be o e. S/N a ios o e e y a iable we e ob ained as Table 3 ex- poses. 2.4.4. Phase IV. Valida ion Expe imen s. Wi h S/N a io and ANOVA analysis, he op imal combina ion o he es ing pa ame e s could be p edic ed o a 95% con idence le el. Also we can p edic he u u e esul s ha e e y combina ion is going o ob ain by eg ession o he main in luencing ac o s acco ding o he ANOVA. In o de o alida e his me hodology, alida ion expe - imen s we e u he pe o med using he op imized condi- ions ob ained o each esponse and hen he esul s will be compa ed o he p edic ed by eg ession. I inal esul s a e simila o he p edic ed, we could alida e he p oposed me hodology. 3. Resul s and Discussion 3.1. ANOVA. The applica ion o he expe imen al design me hodology has he objec i e o iden i ying he mos signi ican ac o s in luencing he o mula ion o algina e- Ca bopol beads con aining a wa e -soluble d ug, and es ab- lishing he bes le els o op imizing diffe en esponse a iables. Resul s ob ained o hese pa ame e s, exp essed as mean alues, a e p esen ed in Table 4. ANOVA could be used as a ool o hede ec iono maineffec s. Da a epo ed in Table 5 e ealed ha he elease pa ame e s (i.e., 50% and AUC o elease p o iles) we e signi ican ly affec ed by Ca bopol concen a ion and addi ion o TEA in o he polyme ic solu ion. Also, he p esence o TEA signi ican ly in luenced on he en apmen efficiency and he pa icle size, whe eas he swelling pe cen age was also affec ed by he NaCl concen a ion in o he dissolu ion medium. Finally, he mo phological pa ame e s, exp essed as shape ac o , we e mainly affec ed by he d ying p ocedu e and he s i ing a e applied om he s i e appa a us. Con ibu ion deg ees o each ac o o e e y esponse ha e been g aphically showed in Figu e 2. These indings will be discussed in nex sec ions. 3.1.1. Release P o iles: Time o Release 50%o En apmen D ug ( 50%) and A ea unde he Cu e (AUC). As shown in Table 5, all ac o s excep he NaCl concen a ion exe signi ican in luence on 50%. In he case o AUC, NaCl con- cen a ion nei he pH had in luence on his esponse. F om Figu es 2(a) and 2(b), we can conclude ha he Ca bopol concen a ion ( ac o B) had he mos signi ican effec on The Scien i ic Wo ld Jou nal 7 Table 4: Expe imen al layou using a L18 o hogonal a ay and esul s ob ained in e e y un. Run 50% AUC EE SR SF Dm Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N 1A−1B−1C−1D−173.00 28.82 268.68 49.11 48.43 37.04 2863.78 69.14 0.52 −5.74 1.34 −2.71 E−1F−1G−1H−1 2A−1B−1CoDo50.00 27.96 421.78 49.29 54.88 38.26 1937.37 65.74 0.20 −13.88 1.47 −3.43 EoFoGoH0 3A−1B−1C1D169.33 35.57 117.23 48.60 53.63 38.08 1320.67 62.42 0.25 −12.06 1.53 −3.70 E1F1G1H1 4A−1BoC−1D−191.33 31.94 461.89 48.23 54.88 38.17 1681.62 64.51 0.58 −4.77 1.72 −2.96 EoFoG1H1 5A−1BoCoDo102.33 32.18 405.74 48.66 59.00 38.92 2177.52 66.76 0.27 −11.32 1.64 −4.50 E1F1G−1H−1 6A−1BoC1D1198.33 25.60 348.62 47.04 53.51 38.08 1119.52 60.98 0.65 −3.83 1.69 −4.61 E−1F−1GoH0 7A−1B1C−1Do118.67 19.17 354.48 48.22 54.34 38.21 1610.31 64.14 0.30 −10.47 1.51 −3.65 E−1F1GoH1 8A−1B1CoD1293.33 28.10 257.57 46.46 54.46 38.24 1082.83 60.69 0.25 −12.01 1.54 −3.73 EoF−1G1H−1 9A−1B1C1D−1106.00 18.80 427.85 47.90 57.14 38.65 1459.06 63.28 0.55 −5.26 1.66 −4.42 E1FoG−1H0 10 A1B−1C−1D160.00 24.77 448.50 48.89 59.73 39.04 2786.18 68.90 0.19 −14.68 2.07 −6.33 E1FoGoH−1 11 A1B−1CoD−182.67 22.18 634.32 48.71 59.33 38.97 1151.14 61.22 0.26 −11.95 1.83 −5.27 E−1F1G1H0 12 A1B−1C1Do119.33 40.29 401.70 47.93 56.07 38.49 3685.23 71.32 0.55 −5.20 1.57 −3.97 EoF−1G−1H1 13 A1BoC−1Do111.67 23.30 1326.03 47.92 58.83 38.91 1868.79 65.39 0.40 −9.30 1.73 −5.12 E1F−1G1H0 14 A1BoCoD1148.67 36.17 225.15 47.45 59.88 39.06 3003.21 69.55 0.20 −13.88 1.72 −4.70 E−1FoG−1H1 15 A1BoC1D−1209.33 36.72 469.47 47.14 59.49 39.00 2017.31 66.10 0.38 −8.50 1.56 −4.01 EoF1GoH−1 16 A1B1C−1D1238.67 40.29 1052.68 46.04 53.97 38.15 3094.41 69.81 0.35 −9.26 1.67 −4.54 EoF1G−1H0 17 A1B1CoD−1142.00 32.25 536.70 48.08 56.39 38.52 1554.57 63.83 0.34 −9.40 1.41 −3.04 E1F−1GoH1 18 A1B1C1Do138.33 33.61 841.84 47.79 60.02 39.08 1420.16 63.05 0.26 −11.71 2.81 −8.99 E−1FoG1H−1 elease cha ac e is ics. Howe e , his effec is posi i e in case o 50% (97.11) and nega i e in case o AUC (−6316). The in luence o he Ca bopol pe cen age on d ug elease p o ile is due o he effec o his polyme on he s uc u e o he beads, as was discussed by o he au ho s [50]. The densi y o he beads inc eases as he concen a ion o his polyme inc eases, sugges ing ha he beads o med a highe con- cen a ions a e mo e compac and less po ous han hose p epa ed a low polyme concen a ion [7].Thep esenceo a highe ne wo k densi y in he s uc u e has been demon- s a ed by o he au ho s in he p epa a ion o Ca bopol and PVP mic osphe es [16,50]. Mo eo e , aking in o accoun he swelling p ope ies o his hyd ophilic polyme , i is con i med ha he p esence o he gel laye hickness ac s as a ba ie o he pene a ion medium he eby e a ding he diffusion o MB [7,11]. All hese con ibu e o p oduce a slowing in MB elease a e (inc easing he 50%), making difficul he ou pu o he d ug o he dissolu ion medium (diminishing he AUC). 3.1.2. En apmen Efficiency. Acco ding o ANOVA analysis, he ac o wi h highe con ibu ion and s a is ical signi i- cance on he en apmen capaci y o he d ug was he inco - po a ion o TEA in o o mula ions. TEA is a weak base ha ionizes ca boxylic g oups in he chains o ca bome s. As a esul , he s uc u e o he gel 8The Scien i ic Wo ld Jou nal Table 5: ANOVA esul s: effec o he a iables on 50%, AUC, % en apmen (EE), swelling pe cen age (SR), shape ac o (SF), and pa icle size (DM). Fac o s 50% AUC EE F- a io PI-ha F- a io PI-ha F- a io PI-ha A 89.52 <0.001 16.48 144.1 <0.001 −1892 11.2 0.004 5.57 B 2072 <0.001 97.11 1070 <0.001 −6316 0.27 0.607 1.07 C 132.5 <0.001 24.56 136 <0.001 −2252 1.41 0.25 2.42 D 564 <0.001 50.67 251.7 <0.001 −3063 0.003 0.955 −0.12 E 172.9 <0.001 −28.06 52.87 <0.001 1404 1.27 0.274 2.30 F 83.07 <0.001 −19.44 21.36 <0.001 892.2 2.18 0.157 3.01 G 0.01 0.918 −0.22 0.01 0.921 −19.4 0.67 0.425 1.66 H 213.4 <0.001 −31.17 3.20 0.082 345.2 0.53 0.474 −1.49 Fac o s SR SF DM F- a io PI-ha F- a io PI-ha F- a io PI-ha A 1842 <0.001 592 34.79 <0.001 −0.072 12.78 <0.001 0.255 B 1208 <0.001 −587.2 0.78 0.383 0.013 2.19 0.148 0.128 C 808.8 <0.001 −480.5 11.9 <0.001 0.051 2.19 0.148 0.130 D 274.4 <0.001 279.9 69.35 <0.001 −0.124 1.69 0.201 0.115 E 0.00 0.99 −0.22 4.49 0.041 −0.032 2.73 0.108 −0.142 F 62.8 <0.001 −133.9 100.4 <0.001 −0.149 0.80 0.377 0.077 G 5856 <0.001 −1293 23.4 <0.001 −0.072 8.78 0.005 0.261 H 25.09 <0.001 84.64 15.63 <0.001 0.059 8.15 0.007 −0.251 changes i s con o ma ion, going om a elaxed s a e in spi al o m o a igid o m, which p o ides iscosi y and consis- ency o he beads. This s uc u al con o ma ion inc eases he en apmen efficiency. The same esul s ha e been ob- ained by o he au ho s when hey added chi osan o he algina e ma ix o p epa e beads [5]. Once he base was added, he inc ease o en apmen efficiency is as consequence o he c ea ion o a highe ne wo k densi y and he elec os a ic in e ac ion be ween he d ug and he polyme s [16]. A he end o he p ocess, he esul ing beads become ha de and hicke , a o ing en ap- men efficiency inside he beads bu making difficul i s la e elease. 3.1.3. Swelling Pe cen age. A e he ANOVA analysis, we can de e mine ha p ac ically all ac o s ha e s a is ical in luence on his dependen a iable. The ac o p esen ing he highe in luence was NaCl concen a ion in he dissolu ion medium ollowed by TEA addi ion and Ca bopol concen a ion. Acco ding o ANOM es o he ac o NaCl concen- a ion (da a no showed), he p esence o sodium ions ac s hinde ing he swelling p ocess. Swelling pe cen age will inc easeasNaClconcen a iondec eases(I-ha =−1293). This esul can be explained on he basis o he swelling mechanism o hese polyme ic s uc u es algina e-Ca bopol. I is well accep ed ha he algina e beads in an aqueous medium wi hou sodium ions do no swell well, bu when hese ions a e p esen in he medium, he p e o med complex be ween he algina e and calcium u ns uns able [51,52]. This could be explained by he iono opic phenomenon ha occu s be ween he Ca2+ ions o he algina e beads and he Na+ions p esen in elease medium ha inally p oduces a cap u e o he Ca2+ by he chlo ine ions. This ac p oduces a disagg ega ion o he bead s uc u e leading o e osion and dissolu ion o he swollen beads and inc easing he d ug elease a e [5,9,51]. This can be app ecia ed in Figu e 3, whe e beads co esponding o ba ch 11 wi h highe NaCl concen a ion ha e lowe swelling pe cen age a he same ime han o he s. In addi ion, TEA inco po a ion p oduces a igid s uc- u e inside he beads and inc ease ca boxylic g oups wi h nega i e cha ge. These g oups ha e a high affini y o wa e molecules so mo e quan i y o wa e can ge inside he bead, inc easing he swelling o he beads in ime and in ensi y. These esul s we e isualized by pho og aphing he samples du ing he swelling p ocess es s. We can no ice a highe swelling in ba ches wi h TEA addi ion (Figu e 4) han in o he lo s wi hou TEA a he same ime. 3.1.4. Shape Fac o . Acco ding o ANOVA analysis, he s i - ing a e and he d ying me hod o beads a e he ac o s ha ha e he highe in luence on his mo phological pa ame e . S i ing a e has an impo an s a is ical signi icance con- ce ning he shape and size pa ame e s [7]. When algina e- Ca bopol d ops all in o CaCl2solu ion, s i ing a e p oduce a o ce ha molds he inal mo phological aspec o he beads, in ela ion o hei shape and su ace. As he s i ing a e inc eases, beads will be less sphe ical and mo e i egula in i s su ace, which p oduces an inc ease o shape ac o and a dec ease in i s ci cula i y (in e se o SF), as we can demons a e in Figu e 5. F om he e alua ion o he d ying p ocess, we mus keep in mind he cha ac e is ics o e e y ea men , because hey can affec many p ope ies o he beads [40]. D ying The Scien i ic Wo ld Jou nal 9 B∗ D∗ H∗E∗C∗A∗F∗G ×103 −5 5 15 25 35 45 55 65 75 85 95 SS o 50% (a) B∗ D∗ A∗C∗ E∗F∗H∗G −2.5E + 07 2.5E + 07 7.5E + 07 1.25E + 08 1.75E + 08 2.25E + 08 2.75E + 08 3.25E + 08 3.75E + 08 4.25E + 08 SS o AUC (b) A∗ FCEGHBD −100 0 100 200 300 400 500 SS o EE (c) G∗ A∗ B∗ C∗ D∗ F∗H∗E (d) F∗ D∗ A∗ G∗ H∗C∗E∗B −0.1 −0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 SS o SF (e) A∗ G∗ H∗ EBCDF −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 SS o Dm ( ) Figu e 2: Sc ee plo ob ained o he a iables using he esul s o he ANOVA analysis. (a) Time o elease 50% o en apmen d ug, (b) a ea unde he cu e, (c) en apmen efficiency, (d) swelling a e, (e) shape ac o , and ( ) Diame e medium. ∗Values wi h s a is ical signi icance (α<0, 01). A: TEA; B: Ca bopol (% w/ ); C: lux a e (mL/min); D: s i ing a e ( pm); E: e en ion ime (min.); F: d ying me hod; G: NaCl (% w/ ); H: pH. (a) (b) (c) Figu e 3: Digi al images co esponding o samples du ing swelling es s (a e 180 minu es). (a) ba ch 11 (0.1 M NaCl), (b) ba ch 15 (0.01 M NaCl) and (c) ba ch 16 (wi hou NaCl). 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