Protective effect of mesoporous silica particles on encapsulated folates

Documen downloaded om: This pape mus be ci ed as: The inal publica ion is a ailable a Copy igh Addi ional In o ma ion h p://dx.doi.o g/ 10.1016/j.ejpb.2016.05.016 h p://hdl.handle.ne /10251/78363 Else ie Ruiz Rico, M.; Daubenschüz, H.; Pé ez Es e e, E.; Ma cos Ma ínez, MD.; Amo ós, P.; Ma ínez Mañez, R.; Ba a Ba ie a, JM. (2016). P o ec i e e ec o mesopo ous silica pa icles on encapsula ed ola es. Eu opean Jou nal o Pha maceu ics and Biopha maceu ics. 105:9-17. doi:10.1016/j.ejpb.2016.05.016. 1 P o ec i e e ec o mesopo ous silica pa icles on encapsula ed ola es 1 2 Ma ía Ruiz-Rico1,*, Hanna Daubenschüz1, Édga Pé ez-Es e e1, Ma ía D. Ma cos2,3, Ped o 3 Amo ós4, Ramón Ma ínez-Máñez2,3, José M. Ba a 1 4 5 1 G upo de In es igación e Inno ación Alimen a ia. Depa amen o de Tecnología de Alimen os, 6 Uni e si a Poli ècnica de València. Camino de Ve a s/n, 46022, Valencia, Spain 7 2 Cen o de Reconocimien o Molecula y Desa ollo Tecnológico (IDM), Unidad Mix a 8 Uni e si a Poli ècnica de València y Uni e si a de València. Depa amen o de Química, 9 Uni e si a Poli ècnica de València, Camino de Ve a s/n, 46022, Valencia, Spain 10 3 CIBER de Bioingenie ía, Bioma e iales y Nanomedicina (CIBER-BBN) 11 4 Ins i u de Ciència dels Ma e ials (ICMUV), Uni e si a de València, P.O. Box 22085, 46071 12 Valencia, Spain 13 *(M.R.R.) Phone: +34963877000 ex .83612. E-mail: ma ui i@e sia.up .es. 14 2 Abs ac 15 Mesopo ous silica pa icles (MSPs) a e conside ed sui able suppo s o design ga ed ma e ials 16 o he encapsula ion o bioac i e molecules. Fola es a e essen ial mic onu ien s which a e 17 sensi i e o ex e nal agen s ha p o oke nu i ional de iciencies. Fola es encapsula ion in MSPs 18 o p e en deg ada ion and o allow hei con olled deli e y is a p omising s a egy. 19 Ne e heless, no in o ma ion exis s abou he p o ec i e e ec o MSPs encapsula ion o p e en 20 hei deg ada ion. In his wo k, 5- o myl e ahyd o ola e (FO) and olic acid (FA) we e 21 en apped in MSPs unc ionalized wi h polyamines, which ac ed as pH-dependen molecula 22 ga es. The s abili y o ee and en apped i amins a e acidic pH, high empe a u e and ligh 23 exposu e was s udied. The esul s showed he deg ada ion o FO a e high empe a u e and 24 acidic pH, whe eas en apped FO displayed enhanced s abili y. F ee FA was deg aded by ligh , 25 bu MSPs s abilized he i amin. The ob ained esul s poin owa ds he po en ial use o MSPs 26 as candida es o enhance s abili y and o imp o e he bioa ailabili y o unc ional biomolecules. 27 28 Keywo ds: 5- o myl e ahyd o ola e; con olled elease; encapsula ion; olic acid; mesopo ous 29 silica pa icles; s abili y 30 31 32 33 Abb e ia ions used 34 5- o myl e ahyd o ola e (FO), asco bic acid (AA), encapsula ed 5- o myl e ahyd o ola e (E- 35 FO), encapsula ed olic acid (E-FA), Eu opean Food Sa e y Au ho i y (EFSA), olic acid (FA), 36 ee 5- o myl e ahyd o ola e (F-FO), ee olic acid (F-FA), leas signi ican di e ence (LSD), 37 mesopo ous silica pa icles (MSPs), N-(3- ime hoxysilylp opyl)die hylene iamine (N3), N- 38 ce yl ime hylammonium b omide (CTAB ), phospha e-bu e ed saline (PBS), powde X- ay 39 di ac ion (PXRD), e abu ylammonium hyd ogen sulpha e (TBAHS), e ae hylo hosilica e 40 (TEOS), ansmission elec on mic oscopy (TEM), ie hanolamine (TEAH3), ul a iole (UV). 41 3 G aphical abs ac 42 43 0 20 40 60 80 100 810 12 14 16 Vi amin eco e y (%) Time o exposu e (h) Encapsula ed i amin F ee i amin 4 In oduc ion 44 In he las yea s hyb id o ganic−ino ganic ma e ials ha e a ac ed conside able in e es due o 45 he combina ion o he bene icial cha ac e is ic o o ganic chemis y and ma e ial science in 46 o de o de elop sma nanode ices. Among di e en hyb id solids, mesopo ous silica pa icles 47 (MSPs) o e se e al unique ea u es ha allow he design o ga ed ma e ials o con olled 48 elease and sensing/ ecogni ion p o ocols [1]. The i s amily o MSPs called MCM-X was 49 desc ibed in he ea ly 1990s by he Mobil Co po a ion Labo a o ies. This amily o silica 50 suppo s include di e en po ous silica exhibi ing hexagonal (MCM-41), cubic (MCM-48) and 51 lamella (MCM-50) po e shapes. A e hese de elopmen s, new o de ed ma e ials (e. i. MSU, 52 KIT, FDU, AMS, SBA…) wi h a wide ange o ex u al p ope ies ha e been desc ibed by 53 di e en au ho s [2]. MSPs possess b oadly ad an ageous p ope ies such as biocompa ibili y, 54 he mal and chemical s abili y, huge loading capaci y, high su ace a eas, unable mo phologies 55 and po e sizes, as well as acile unc ionaliza ion o su aces and po es [3-7]. The su ace 56 unc ionaliza ion o MSPs o he de elopmen o ga ed ma e ials allows ha he deli e y o he 57 ca go s o ed in he ino ganic suppo can be igge ed by applying selec ed ex e nal s imulus [1]. 58 Fu he mo e, i is conside ed ha he ino ganic amewo k can e ec i ely p o ec he payload 59 molecules om enzyma ic deg ada ion o dena u a ion caused by en i onmen al changes [7]. 60 Howe e , he e a e ew s udies in he li e a u e abou he p o ec i e e ec o MSPs on he 61 s abili y o biomolecules in biological solu ions. 62 The unc ionalized MSPs ha e been used o encapsula e d ugs mainly o he biomedical ield, 63 bu also o encapsula e bioac i e molecules o o he sec o s such as ood echnology. Di e en 64 ood ing edien s and nu aceu icals including i amins [8-12], an ioxidan s [13,14], 65 an imic obials [15-17], a omas [18] o enzymes [19] ha e been en apped in ga ed mesopo ous 66 ma e ials. Mos s udies ha e been ocused on he de elopmen and op imiza ion o he 67 encapsula ion sys ems o con olled deli e y, bu i is expec ed ha he MPSs may be able o 68 enhance he s abili y o he en apped bioac i e compound. 69 5 Wa e -soluble i amins, like ola es, a e labile compounds in he p esence o en i onmen al 70 agen s, such as ex eme pH alues o high empe a u es [20]. As ola es a e essen ial o he 71 human body and canno be syn hesized de no o by he o ganism, his indispensable i amin 72 needs o be ob ained om ood o die a y supplemen s [21]. Thus he s abili y o his i amin 73 a e s o age and p ocessing in ood p oduc s o supplemen s should be aken in o accoun . Loss 74 o he biochemical ac i i y o na u al ola es can occu du ing ha es , s o age and ood 75 p ocessing [22,23]. In gene al, pH, empe a u e, p essu e, ligh and an ioxidan s, among o he s, 76 can a ec he s abili y o he na u al ola es and he syn he ic olic acid (FA) [24-30]. FA, wi h a 77 ully oxidized p e idine ing sys em, exhibi s g ea e s abili y han ola es. Among ola es, la ge 78 di e ences in s abili y exis in suscep ibili y o oxida i e deg ada ion, and 5- 79 o myl e ahyd o ola e (FO) is he mos s able [31]. Mo eo e , he s abili y o ola es is 80 in luenced by pH and oxygen, which p o okes hei oxida ion [30,32]. The inclusion o 81 an ioxidan compounds, such as asco bic acid (AA) o me cap oe hanol, is equi ed o p e en 82 he des uc ion o labile ola es om he mal exposu e and pho odeg ada ion du ing ood 83 p ocessing [20,23,33]. 84 Bea ing in mind hese ac o s, i is o in e es o c ea e ola es encapsula ion sys ems which can 85 ensu e he equi ed dose and ully gua an ee he s abili y and bioa ailabili y o his i amin. 86 The e o e, he objec i e o his s udy was he encapsula ion o FO and FA in a mesopo ous 87 silica suppo (MCM-41) unc ionalized wi h amines o c ea e a sys em o be used in o ally 88 deli e ed applica ions, and o s udy he s abili y o en apped i amins o es he e icacy o he 89 MCM-41 suppo as a p o ec o agains ex e nal agen s, such as acidic pH, high empe a u e and 90 ligh . 91 92 6 Ma e ials and me hods 93 Chemicals 94 Te ae hylo hosilica e (TEOS), N-ce yl ime hylammonium b omide (CTAB ), sodium 95 hyd oxide (NaOH), ie hanolamine (TEAH3), N-(3- ime hoxysilylp opyl)die hylene iamine 96 (N3), sodium phospha e monobasic (NaH2PO4), sodium phospha e dibasic (Na2HPO4) and 97 e abu ylammonium hyd ogen sulpha e (TBAHS) we e p o ided by Sigma-Ald ich (Mad id, 98 Spain). 5- o myl e ahyd o ola e (FO) and olic acid (FA) we e pu chased om Schi cks 99 Labo a o ies (Jona, Swi ze land). Ace oni ile HPLC g ade was p o ided by Scha lab 100 (Ba celona, Spain). 101 102 Mesopo ous silica pa icles syn hesis 103 Syn hesis o mic opa icula ed MCM-41 pa icles was ca ied ou ollowing he so-called 104 “a ane ou e”, whe e CTAB was used as he s uc u e-di ec ing agen . A mola a io, ixed a 7 105 TEAH3: 2 TEOS:0.52 CTAB :0.5 NaOH:180 H2O. CTAB , was added o a TEAH3 and NaOH 106 solu ion, which con ained TEOS a 118 ºC. A e dissol ing CTAB in he solu ion, wa e was 107 slowly added along wi h igo ous s i ing a 70 ºC o o m a whi e suspension. This mix u e was 108 aged a 100 ºC o 24 h. Following syn hesis, he solid was eco e ed, washed wi h deionized 109 wa e and d ied a 70 ºC. The as-syn hesized mic opa icles we e calcined a 550 ºC in an 110 oxidan a mosphe e o 5 h o emo e he empla e phase [16]. 111 112 Syn hesis o encapsula ed ola es 113 The design o he encapsula ion sys em was based on a p e ious wo k, in which FA was 114 en apped in a MSP unc ionalized wi h amines o deli e FA du ing a simula ed diges ion 115 p ocess [12]. Dissolu ions o FO and FA (10 mg/mL) we e p epa ed in dis illed wa e and 116 phospha e-bu e ed saline (PBS), espec i ely. Solu ions we e added o 300 mg o MCM-41 in 117 3 addi ion cycles (1.5 mL pe cycle). A e each addi ion cycle, solids we e d ied a 37 ºC o 118 7 emo e wa e con en . A e loading and d ying, solids we e collec ed and unc ionalized wi h 119 1.29 mL o N3 using di e en media; i.e. ace oni ile (E-FO) o ace a e bu e a pH 2 (E-FA). 120 The inal mix u es we e s i ed o 5.5 h a oom empe a u e, isola ed by acuum il a ion, 121 washed wi h 300 mL o wa e adjus ed o pH 2, and d ied a oom empe a u e o 24 h. 122 123 Cha ac e iza ion o solids 124 Powde X- ay di ac ion (PXRD), ansmission elec on mic oscopy (TEM), N2 adso p ion- 125 deso p ion iso he ms and ze a po en ial we e used o cha ac e ize he syn hesized ma e ials. 126 PXRD was pe o med in a B uke D8 Ad ance di ac ome e using CuKα adia ion (B uke , 127 Co en y, UK). Fo he TEM analysis, pa icles we e dispe sed in dichlo ome hane and 128 sonica ed o 2 min o p eclude agg ega es. The suspension was hen deposi ed on o coppe 129 g ids coa ed wi h a ca bon ilm (Aname SL, Mad id, Spain). The MSPs samples we e imaged 130 by JEOL JEM-1010 (JEOL Eu ope SAS, C oissy-su -Seine, F ance) a an accele a ion ol age 131 o 80 kV. The single-pa icle size was es ima ed by a e aging he measu ed size alues o 50 132 pa icles. The N2 adso p ion-deso p ion iso he ms we e eco ded wi h a Mic ome ics 133 ASAP2010 au oma ed so p ion analyze (Mic ome i ics Ins umen Co po a ion, No c oss, 134 USA). Samples we e degassed a 90 ºC in acuum o e nigh . Speci ic su ace a eas we e 135 calcula ed om he adso p ion da a wi hin he low p essu e ange by he BET model. Po e size 136 was de e mined ollowing he BJH me hod. To de e mine he ze a po en ial o he ma e ials, a 137 Ze asize Nano ZS (Mal e n Ins umen s, UK) was employed. Samples we e dispe sed in wa e 138 a a concen a ion o 1 mg/mL. Be o e aking each measu emen , samples we e sonica ed o 2 139 min o p eclude agg ega ion. The ze a po en ial was calcula ed om he pa icle mobili y alues 140 by applying he Smoluchowski model. The a e age o i e eco dings was epo ed as he ze a 141 po en ial. Measu emen s we e aken a 25 ºC in iplica e. 142 143 8 Release s udies 144 Deli e y s udies we e conduc ed o es he elease capaci y o he encapsula ion sys em and o 145 con i m he unc ionali y o he ga es o modula e he elease o i amins acco ding o he pH o 146 he medium (closed ga es a pH 2, opened ga es a pH 7.5). To de e mine he elease o FO and 147 FA om he amine-ga ed mesopo ous suppo (E-FO and E-FA), 10 mg o he solids we e 148 placed in 25 mL o PBS a pH 2 and pH 7.5. A ce ain ime poin s (0, 2, 5, 15, 30, 60, 120, 180 149 min), aliquo s we e sepa a ed, he suspension was il e ed and he solu ion was analyzed by 150 HPLC. 151 152 S abili y assays 153 The in luence o di e se ex e nal agen s, such as acidic pH, high empe a u e and ligh , on he 154 s abili y o he ee and en apped i amin was s udied. F ee FO and FA we e ea ed, whene e 155 possible, as he encapsula ed i amin in o de o ensu e ep oducibili y. In o de o simula e no 156 only he 3 day-loading (72 h d ying a 37°C) o he pa icles wi h he i amin, bu also a u he 157 24-hou d ying pe iod a e unc ionaliza ion, compounds in hei ee o m we e incuba ed o 158 96 h a 37°C. The s abili y assays wi h he ee i amin we e all conduc ed wi h hese incuba ed 159 samples (F-FO and F-FA). 160 Fo he s abili y assays, 4 mg o he en apped i amins (E-FO and E-FA) and he 161 co esponden amoun s o he ee o ms (ca. 0.02 mg o FO and ca. 0.3 mg o FA) we e 162 dissol ed in 10 mL o PBS (pH 2 o pH 7.5). All he s abili y expe imen s we e pe o med in 163 iplica e. The i amin eco e ies we e p esen ed by assuming he pe cen age eco e ed unde 164 op imal condi ions o be 100% (pH 7.5, no ea men ). 165 166 pH s abili y 167 These expe imen s we e ca ied ou o s udy he s abili y and solubili y o i amins a di e en 168 pH alues; e.g., he acidic pH a which FO and FA exhibi ed e y low solubili y [30,32]. These 169 15 285 Func ionaliza ion e iciency was e i ied by he ze a po en ial de e mina ions o ba e MCM-41, 286 MCM-41 loaded wi h FO/FA, and MCM-41 loaded and unc ionalized wi h amines. Ba e 287 pa icles e ealed an a e age nega i e ze a po en ial o -31 mV. A e loading pa icles wi h 288 FO/FA, he ze a po en ial changed sligh ly o alues o ca. -30 mV. Ye a e unc ionaliza ion 289 wi h N3, he ze a po en ial changed posi i ely o alues o ca. 50 mV o E-FO and E-FA, 290 which con i med he a achmen o amines o he pa icle su ace. 291 292 Release s udies 293 The elease s udies con i med he mechanism o he amine-ga ed MSPs o modula e i amin 294 elease acco ding o he pH o he medium. The pH-dependen eleases o he encapsula ed FO 295 and FA a e shown in Figu e 4. Ga es we e la gely closed a pH 2 and he i amin was ba ely 296 de ec ed, which con i med ha i amin deli e y was hinde ed by he combina ion o he low 297 solubili y o he i amins unde acidic condi ions, he e ec o he amines ancho ed o he 298 su ace o MSPs and he polyammonium g oups-anionic species in e ac ion. A an acidic pH, 299 polyamines we e ans o med in o polyammonium g oups, which adop ed a igid-like 300 con o ma ion due o Coulombic epulsions and coo dina e anions (phospha es p esen in 301 solu ion), which blocked po es and a oided i amin elease [8,34]. 302 In con as , FO and FA showed a p og essi e elease among ime a pH 7.5. A e 2 h, 303 maximum i amin eleases we e ob ained a pH 7.5, wi h 41.9±7.2 mg FO/g solid o E-FO and 304 84.3±7.8 mg FA/g solid o E-FA. The maximum eleased amoun s we e used o calcula e he 305 equi alen amoun o solids needed in he s abili y assays o make a compa ison be ween he 306 ee and encapsula ed FO and FA. A sus ained elease was p oduced because polyamines we e 307 less p o ona ed a a neu al pH, and he Coulombic epulsion be ween hem and he a ini y o 308 anions signi ican ly educed. These e ec s, along wi h inc eased i amin solubili y, allowed he 309 deli e y o FO and FA om po es. This pH- esponsi e deli e y e ec has been sugges ed o be 310 16 sui able o eleasing i amins in he gas oin es inal ac (closed ga es in he s omach, opened 311 ga es in he in es ine) [12]. Encapsula ion was also expec ed o p o ec i amins om 312 deg ada ion a e exposu e o en i onmen al agen s ( ide in a). 313 314 315 Figu e 4. Release p o iles o i amin om he po es o E-FO (A) and E-FA (B) in PBS a pH 316 2.0 (do ed lines) and pH 7.5 (solid lines). Values a e Means ± SD, n = 3. 317 318 S abili y assays 319 The in luence o di e se ex e nal agen s ela ed o ood p ocessing o s o age, such as pH, 320 empe a u e and ligh , on he s abili y o ee 5- o myl e ahyd o ola e and olic acid (F-FO and 321 F-FA) and he co esponding en apped i amins (E-FO and E-FA) was s udied. 322 323 pH 324 The s udy o he e ec o pH on he s abili y o FO and FA a di e en pH alues was 325 conduc ed in wo s eps. In he i s s ep, wa e solu ions o ee FA and FO we e adjus ed o 326 di e en pHs and s i ed o 1 h be o e being analyzed by HPLC. Figu e 5 shows he de ec ed 327 concen a ions o FA and FO (in e ms o eco e y) in he aqueous solu ions unde all he s udy 328 condi ions (i.e. pH 1-10). As obse ed, eco e ies eached alues o ca. 100% om pH 5 o 10, 329 which con i ms he s abili y o bo h molecules a hese pH alues. Below his pH ange, he 330 0 10 20 30 40 50 60 70 80 90 100 060 120 FA elease (%) Time (min) 0 10 20 30 40 50 60 70 80 90 100 060 120 FO elesase (%) Time (min) A B 17 concen a ions o bo h molecules lowe ed. The FO concen a ion in wa e g adually lowe ed 331 om ca. pH 4 o pH 1, whe eas his e ec was obse ed o FA below ca. pH 3. 332 333 334 Figu e 5. F-FO and F-FA eco e ies a di e en pH alues. Di e en le e s in he ba s indica e 335 s a is ically signi ican di e ences (p<0.05) om le els o pH. Values a e Means ± SD, n = 3. 336 337 The d op in he eco e y o FO and FA a an acidic pH can be explained by h ee phenomena: 338 (a) loss o solubili y; (b) in e con e sion in o o he de i a i es; (c) oxida i e deg ada ion. 339 Fola es a e sligh ly soluble a an acidic pH, and a e highly soluble unde neu al/basic 340 condi ions due o he p o ona ion and dep o ona ion o molecules in aqueous en i onmen s [32]. 341 In addi ion o oxida i e deg ada ion, FO can nonenzyma ically in e con e wi h 5,10- 342 me henyl e ahyd o ola e h ough changes in pH, empe a u e and oxygen [35]. 5,10- 343 me henyl e ahyd o ola e is o med by he acidi ica ion o 5- o myl e ahyd o ola e because one 344 molecule o wa e is los (dehyd a ion), which leads o he cycliza ion o he molecule in a 345 e e sible manne . The equilib ium g adually shi s owa d 5,10-me henyl e ahyd o ola e, and 346 i s o ma ion becomes as e he lowe pH becomes [36]. 347 Bea ing all hese ac o s in mind, which could explain loss o eco e y a an acidic pH, in a 348 second s ep, expe imen s we e un o de e mine he amoun o i amins los a an acidic pH. In 349 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 9 10 Reco e y (%) pH F-FO F-FA a a a a b ccc c c a a b c d dd d d d 18 hem aqueous solu ions o ee FA and FO we e adjus ed o pH 2, s i ed o 1 h and hen pH 350 was adjus ed o 7.5 be o e he HPLC analysis. The pe cen age o i amins de e mined a pH 2 351 and a e neu aliza ion o pH 7.5 is shown in Fig. 6A, whe e almos no eco e y o i amins is 352 de ec ed a e s i ing hem o 1 h a pH 2 (which ag ees wi h Fig. 5). Howe e , he i amins 353 eappea ed wi h a pe cen age o ca. 40% o F-FO and o ca. 72% o F-FA a e neu alizing 354 he pH. F-FA ga e highe alues a e adjus ing o he neu al alue han ee FO, bu none o 355 hem achie ed comple e eco e y. Some s udies ha e e ealed ha FA migh no be soluble a a 356 low pH [12], bu o he au ho s ha e epo ed i s deg ada ion a an acidic pH [29]. The na u al 357 ola e showed less s abili y a an acidic pH han FA, p obably due o deg ada ion [36]. 358 Simila s udies o hose shown abo e ha e been conduc ed in he p esence o asco bic acid 359 (AA), and hei esul s a e shown in Fig. 6B. This an ioxidan was included because p e ious 360 s udies ha e demons a ed ha i s inco po a ion inc eases ola e s abili y as oxida ion eac ions 361 a e p e en ed [35]. As o he i amins supplemen ed wi h AA, he F-FO concen a ion only 362 sligh ly inc eased ( om 40% in he absence o AA o 50%). Howe e , AA ema kably 363 in luenced he s abili y o F-FA, and e ealed a eco e y o almos 90%. 364 Las ly, he e ec o pH changes on he encapsula ed i amins was s udied in o de o p o e he 365 p o ec i e unc ion o he suppo . As seen in Fig. 6C, mino i amin eco e ies ook place a 366 pH 2. When pH was adjus ed o 7.5, E-FO and E-FA we e almos ully de ec able wi h a 367 eco e y o 94% and 99%, espec i ely. Bo h encapsula ed i amins we e highly p ese ed in 368 he acidic en i onmen by he pH- esponsi e ga ed ma e ial. As a esul , he highly p o ec i e 369 unc ion o he MSPs unc ionalized wi h amines a a low pH was e idenced by bo h i amins. 370 This app oach be e imp o ed he s abili y o i amins han he s a egy epo ed o enhance he 371 s abili y o na u al ola es (addi ion o an ioxidan s). Nei he he ee o m no he i amins 372 supplemen ed wi h AA we e as s able a an acidic pH as hey we e inside he po es o MSPs. 373 374 19 375 Figu e 6. FO and FA eco e ies a e pH changes o ee i amins (A), ee i amins in 376 p esence o asco bic acid (B) and encapsula ed i amins (C). Di e en le e s in he ba s 377 indica e s a is ically signi ican di e ences (p<0.05) om le els o pH. Values a e Means ± SD, 378 n = 3. 379 380 Tempe a u e 381 P e ious expe imen s conduc ed wi h i amins dissol ed in PBS a empe a u es below 100 °C 382 had no impac on hei s abili y (da a no shown). In o de o in es iga e he impac o highe 383 empe a u es on he i amins, a s udy was ca ied ou by simula ing s e iliza ion condi ions (121 384 °C, 1 ba ) a di e en imes. The empe a u e assays pe o med in he au ocla e a e p esen ed in 385 Figu e 7. The esul s showed ha encapsula ed ola e did no signi ican ly educe i amin 386 con en a a ious exposu e imes. In con as , F-FO e ealed a signi ican loss o ca. 27% a e 387 15 min, p obably due o he o ma ion o in e con e sion p oduc s [30]. Wi h FA, no signi ican 388 di e ences we e ob ained o bo h he E-FA and F-FA esul s. These esul s a e in acco dance 389 wi h p e ious s udies ha ha e epo ed good FA s abili y a e he mal exposu e in he solid 390 s a e and wi h dissolu ion [24,26]. Syn he ic i amin has been sugges ed o be he mos s able 391 ype in he ola e g oup because o i s oxidized p- e idin ing [37]. The he mos abili y o FA 392 and FO has been p e iously epo ed as being simila a a neu al pH [23]. The esul s ob ained 393 wi h he encapsula ed i amins e ealed ha en apped FO could bea up unde he mal p essu e 394 exposu e and g ea e s abili y a e p o ing FO encapsula ion. Howe e , encapsula ed FA could 395 also esis he bu den o he mal p essu e as well as i s ee o m. 396 0 10 20 30 40 50 60 70 80 90 100 2 7.5 Reco e y (%) pH E-FO E-FA 0 10 20 30 40 50 60 70 80 90 100 2 7.5 Reco e y (%) pH F-FO+AA F-FA+AA aa 0 10 20 30 40 50 60 70 80 90 100 2 7.5 Reco e y (%) pH F-FO F-FA BA aa b b C aa b b b b 20 397 Figu e 7. In luence o empe a u e exposu e on he s abili y o encapsula ed (E-) and ee (F-) 398 FO (A) and FA (B) i amins. Di e en le e s in he ba s indica e s a is ically signi ican 399 di e ences (p<0.05) om le els o ime exposu e (small le e s) and di e ences be ween he 400 encapsula ed FO/FA o in hei ee o m (capi al le e s). Values a e Means ± SD, n = 3. 401 402 Ligh 403 P e ious a icles, which ha e epo ed he in luence o a ious ligh sou ces on he deg ada ion 404 o syn he ic FA, ha e in es iga ed he impac o isible and UV ligh on bo h i amins [25]. 405 P elimina y expe imen s e ealed no o e y li le deg ada ion a e 6 h (da a no shown). 406 The e o e, assays we e ca ied ou om 8 h o 16 h. 407 Visible ligh assays we e conduc ed wi h a lamp, which gene a ed isible ligh wi h an in ensi y 408 ca. 8 mW/cm². The esul s ob ained om isible ligh expe imen s a e p esen ed in Figu e 8. 409 The good s abili y o FO (Fig. 8A) a e ligh exposu e was e idenced. Nei he F-FO no E-FO 410 showed deg ada ion du ing isible ligh exposu e. 411 Wi h FA (Fig. 8B), F-FA showed conside able loss a e 8 h o isible ligh exposu e, wi h a 412 emaining a e aged amoun o 40%. G adual educ ion o F-FA was de ec ed up o 12 h 413 i adia ion, wi h a low alue o 12%, and o al deg ada ion occu ed a e 16 h wi h an a e age 414 emaining amoun o 3%. Con e sely, E-FA was well-p o ec ed by he unc ionalized suppo 415 and a non-signi ican dec ease was de ec ed. 416 0 10 20 30 40 50 60 70 80 90 100 510 15 Reco e y (%) Time (min) E-FA F-FA 0 10 20 30 40 50 60 70 80 90 100 510 15 Reco e y (%) Time (min) E-FO F-FO aA aA aA aA aA bA aA aA aA aA aA aA BA 21 Gi en F-FA’s endency o be deg aded by ligh in solu ion, he e ec o an ioxidan AA was 417 also e alua ed as a s a egy o imp o e s abili y and o con i m he mechanism o deg ada ion 418 (i.e. oxida ion). F ee FA dissolu ion supplemen ed wi h AA showed ma ginal luc ua ion a e 419 16 h o isible ligh exposu e, bu appea ed o s abilize FA subs an ially in he same way as he 420 encapsula ion sys em. 421 422 423 Figu e 8. In luence o isible ligh exposu e on he s abili y o encapsula ed (E-) and ee (F-) 424 FO (A) and FA (B) in p esence o no o asco bic acid. Di e en le e s in he ba s indica e 425 s a is ically signi ican di e ences (p<0.05) om le els o isible ligh exposu e (small le e s) 426 and di e ences be ween he encapsula ed FO/FA o in hei ee o m (capi al le e s). Values 427 a e Means ± SD, n = 3. 428 429 Figu e 9 shows he ee and encapsula ed FO and FA eco e ies a e UV ligh exposu e wi h an 430 es ima ed in ensi y o 4 mW/cm². The esul s e ealed ha E-FO and F-FO exhibi ed good 431 s abili y unde UV ligh (Fig. 9A). Hence na u al ola e was highly s able in bo h he ee and 432 encapsula ed o ms. FA s abili y was a ec ed by UV ligh (Fig. 9B) and F-FA showed losses 433 a e 8 h o UV exposu e. As in he isible ligh assays, he impac o AA enhanced he good 434 s abili y o he syn he ic i amin and demons a ed mo e e ec i e p o ec ion han he 435 mesopo ous sys em a e 16 h o ligh exposu e. 436 437 0 10 20 30 40 50 60 70 80 90 100 810 12 14 16 Reco e y (%) Time (h) E-FO F-FO aAaA aA aA aA aAaA aA aA aA A 0 10 20 30 40 50 60 70 80 90 100 810 12 14 16 Reco e y (%) Time (h) E-FA F-FA F-FA+AA aB aB aB aB aB abB aA bA cA cA cA aB abcB cB bcB B 22 438 Figu e 9. In luence o UV ligh exposu e on he s abili y o encapsula ed (E-) and ee (F-) FO 439 (A) and FA (B) in p esence o no o asco bic acid. Di e en le e s in he ba s indica e 440 s a is ically signi ican di e ences (p<0.05) om le els o isible ligh exposu e (small le e s) 441 and di e ences be ween he encapsula ed FO/FA o in hei ee o m (capi al le e s). Values 442 a e Means ± SD, n = 3. 443 444 Summing up he wo ligh expe imen s, i was no ed ha FO did no deg ade unde ei he isible 445 ligh exposu e o UV ligh s ess, bu FA was suscep ible o isible and UV ligh exposu e. 446 Howe e , he mesopo ous sys em was able o e icien ly p o ec he i amin and s abili y was 447 g ea ly enhanced. 448 This s udy simula ed indi ec ligh -induced s ess and, he e o e, in ensi ies un il deg ada ion 449 occu ed. We we e unable o compa e hese esul s wi h p e ious s udies as hey all used di ec 450 exposu es [25,27,28]. E en hough i has been sugges ed o be conside ably mo e s able a pH 451 7.5 han in acidic media [29], we de ec ed comple e oxida i e deg ada ion a e 16 h o isible 452 ligh and UV exposu e. Akh a e al. [25] sugges ed he mechanism o his oxida i e 453 deg ada ion as hey hin ed a deg ada ion being p oduced in he C9-N10 posi ion. Aqueous 454 solu ion can o m a ious adioly ic p oduc s, which ini ia e oxida i e dehyd ogena ion and lead 455 o an enamine compound. This in e media e o m is highly suscep ible in acidic media and can 456 unde go as deg ada ion. In alkaline media, his decomposi ion p ocess has been p oposed o 457 ake place mo e slowly, bu i also led o i e e sible clea age be ween C9-N10 bonding a e 16 458 0 10 20 30 40 50 60 70 80 90 100 810 12 14 16 Reco e y (%) Time (h) E-FO F-FO 0 10 20 30 40 50 60 70 80 90 100 810 12 14 16 Reco e y (%) Time (h) E-FA F-FA F-FA+AA aA aAaAaA aAaA aA aA aA aA aA aB aB bB bB aB aA aA bA bA aB aB aB aB aC A B 23 h o ligh exposu e. This caused inal decay in he sepa a ion o he p- e idin moie y om p- 459 aminobenzoylglu ama e [25,27]. 460 Encapsula ed FA, which was sol ed in PBS (pH 2) in he s abili y assays, showed good 461 s abili y. No e en he deg ada ion- a o able acidic su ounding could ha dly a ec E-FA 462 compa ed o he ee o m. Apa om success ul FA imp o emen h ough encapsula ion, 463 an ioxidan AA enhanced s abili y in he same way. Compa ed o he well-known p o ec i e 464 mechanism o an ioxidan s [38], he p o ec i e unc ion o he mesopo ous suppo has no been 465 epo ed o da e, and he s abilizing mechanism emains unclea . Al hough i is ully accep ed 466 ha MSPs show e y li le abso p ion wi hin he isible and ul a iole ange [39,40], enhanced 467 encapsula ed i amin eco e y was con i med he ein. The possible ole o MSPs as a s abili y 468 enhance could hinde access o weak poin s (C9-N10 bonding) by en apping he i amin in 469 mesopo es in such a way ha con o ma ional ans o ma ions o he molecule a e a oided. 470 471 Conclusions 472 The success ul en apmen o na u al FO and syn he ic FA in pH- esponsi e MSPs and he 473 con olled elease o he compounds ha mimic he gas oin es inal ac we e accomplished 474 he ein. The abili y o MSPs o p o ec i amins a e en i onmen al deg ada ion was clea ly 475 e idenced. The s abili y assays e ealed ha encapsula ed FO and FA we e e ec i ely p o ec ed 476 agains deg ada ion a an acidic pH compa ed o hei ee om. The s e iliza ion s udies 477 showed ha encapsula ion allowed i amins o wi hs and he mal exposu e and enhanced hei 478 s abili y. The esul s ob ained a e exposu e o isible and UV ligh displayed good s abili y o 479 ee FO, which was no in luenced by encapsula ion, bu imp o ed FA s abili y a e en apmen 480 in MSPs. When conside ing he p o ec i e e ec o MSPs agains ex e nal agen s and acidic 481 s omach condi ions, and p og essi e deli e y wi h ime unde he in es inal condi ions, he FO- 482 and FA-loaded suppo s p oposed he ein can be conside ed p omising po en ial sys ems as 483 supplemen s o ood sys ems. 484 485 24 Acknowledgemen s 486 Au ho s g a e ully acknowledge he inancial suppo om he Minis e io de Economía y 487 Compe i i idad (P ojec s AGL2012-39597-C02-01, AGL2012-39597-C02-02 and MAT2012- 488 38429-C04-01), FEDER ounding and he Gene ali a Valenciana (P ojec 489 PROMETEOII/2014/047). M.R.R. and E.P.E. a e g a e ul o he Minis e io de Ciencia e 490 Inno ación o hei g an s (AP2010-4369 and AP2008-0620). 491 492 Re e ences 493 [1] Azna , E., O o al, M., Pascual, L., - , Ga ed 494 ma e ials o on-command elease o gues molecules, Chem. 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