Hierarchical structured and programmed vehicles deliver drugs locally to inflamed sites of intestine

Con en s lis s a ailable a ScienceDi ec Bioma e ials jou nal homepage: www.else ie .com/loca e/bioma e ials Hie a chical s uc u ed and p og ammed ehicles deli e d ugs locally o inflamed si es o in es ine Wei Li a,1 , Yunzhan Li b,1 , Zehua Liu a,1 , Na ha Ke dsakundee a,c , Ming Zhang b , Feng Zhang a , Xueyan Liu b , Tomás Baule h-Ramos a,d,e , Wenhua Lian b , E mei Mäkilä , Ma ianna Kemell g , Yaping Ding a , B uno Sa men o d,e , Ruedeeko n Wiwa anapa apee c , Ja no Salonen , Hongbo Zhang h , Jouni T. Hi onen a , Dong ei Liu a,i,∗∗∗ , Xianming Deng b,∗∗ , Hélde A. San os a,i,∗ a D ug Resea ch P og am, Di ision o Pha maceu ical Chemis y and Technology, Facul y o Pha macy, Uni e si y o Helsinki, Helsinki 00014, Finland b S a e Key Labo a o y o Cellula S ess Biology & Inno a ion Cen e o Cell Signaling Ne wo k and S a e-P o ince Join Enginee ing Labo a o y o Ta ge ed D ugs om Na u al P oduc s and School o Li e Sciences, Xiamen Uni e si y, Xiamen 361102, Fujian, China c Depa men o Pha maceu ical Technology, Facul y o Pha maceu ical Sciences, P ince o Songkla Uni e si y, 90110 Ha Yai, Thailand d Ins i u o de In es igação e Ino ação em Saúde (I3S), Ins i u o de Engenha ia Biomédica (INEB), Uni e si y o Po o, Rua Al edo Allen, 208, 4200-135 Po o, Po ugal e Ins i u o Ciências Biomédicas Abel Salaza (ICBAS), Uni e si y o Po o, Rua Jo ge Vi e bo 228, 4150-180 Po o, Po ugal Labo a o y o Indus ial Physics, Depa men o Physics, Uni e si y o Tu ku, Tu ku 20014, Finland g Depa men o Chemis y, Facul y o Science, Uni e si y o Helsinki, FI-00014, Helsinki, Finland h Depa men o Pha maceu ical Sciences Labo a o y & Tu ku Cen e o Bio echnology, Åbo Akademi Uni e si y, Tu ku 20520, Finland i Helsinki Ins i u e o Li e Science (HiLIFE), Uni e si y o Helsinki, Helsinki 00014, Finland ARTICLE INFO Keywo ds: Po ous silicon Hyb id nanopa icle S imuli esponsi e Ta ge ing D ug deli e y ABSTRACT O ally adminis able d ug deli e y ehicles a e de eloped o manage incu able inflamma o y bowel disease (IBD), howe e , hei he apeu ic ou comes a e comp omised by he side effec s o sys emic d ug exposu e. He ein, we use hyalu onic acid unc ionalized po ous silicon nanopa icle o b idge enzyme- esponsi e hyd ogel and pH- esponsi e polyme , gene a ing a hie a chical s uc u ed (nano-in-nano-in-mic o) ehicle wi h p o- g ammed p ope ies o ully and sequen ially o e come he mul iple obs acles o efficien ly deli e ing d ugs locally o inflamed si es o in es ine. A e o al adminis a ion, he pH- esponsi e ma ix p o ec s he embedded hyb id nanopa icles con aining d ug loaded hyd ogels agains he spa ially a iable physiological en i onmen s o he gas oin es inal ac un il hey each he inflamed si es o in es ine, p e en ing p ema u e d ug elease. The nega i ely cha ged hyb id nanopa icles selec i ely a ge he inflamed si es o in es ine, and g adually elease d ug in esponse o he mic oen i onmen o inflamed in es ine. O e all, he de eloped hie a chical s uc u ed and p og ammed ehicles load, p o ec , anspo and elease d ugs locally o inflamed si es o in- es ine, con ibu ing o supe io he apeu ic ou comes. Such s a egy could also inspi e he de elopmen o nume ous hie a chical s uc u ed ehicles by o he po ous nanopa icles and s imuli- esponsi e ma e ials o he local deli e y o a ious d ugs o ea plen y o inflamma o y gas oin es inal diseases, including IBD, gas o- in es inal cance s and i al in ec ions. 1. In oduc ion Inflamma o y bowel disease (IBD), including ulce a i e coli is and C ohn's disease, is ch onic, elapsing and medically incu able in- flamma o y diso de s o he in es ine [1]. Cu en a ailable he apies ail o adequa ely manage he IBD symp oms and a e equen ly h ps://doi.o g/10.1016/j.bioma e ials.2018.09.024 Recei ed 12 Augus 2018; Recei ed in e ised o m 13 Sep embe 2018; Accep ed 16 Sep embe 2018 ∗ Co esponding au ho . D ug Resea ch P og am, Di ision o Pha maceu ical Chemis y and Technology, Facul y o Pha macy, Uni e si y o Helsinki, Helsinki 00014, Finland. ∗∗ Co esponding au ho . S a e Key Labo a o y o Cellula S ess Biology & Inno a ion Cen e o Cell Signaling Ne wo k and S a e-P o ince Join Enginee ing Labo a o y o Ta ge ed D ugs om Na u al P oduc s and School o Li e Sciences, Xiamen Uni e si y, Xiamen 361102, Fujian, China. ∗∗∗ Co esponding au ho . D ug Resea ch P og am, Di ision o Pha maceu ical Chemis y and Technology, Facul y o Pha macy, Uni e si y o Helsinki, Helsinki 00014, Finland. 1 These au ho s con ibu ed equally o his wo k. E-mail add esses: dong ei.liu@helsinki.fi(D. Liu), [email p o ec ed] (X. Deng), helde .san os@helsinki.fi(H.A. San os). Bioma e ials 185 (2018) 322–332 A ailable online 19 Sep embe 2018 0142-9612/ © 2018 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/BY/4.0/). T associa ed wi h se e e side effec s esul ing om sys emic d ug ex- posu e [2]. One app oach o mo e efficacious and sa e IBD he apy could be nano/mic o- ehicles media ed local d ug deli e y o achie e high d ug concen a ion a he inflamed si es o in es ine wi h minimal exposu e o heal hy in es ine o dis an issues [3]. D ug deli e y ehicles can a he easily each inflamed in es ine o local d ug deli e y by ec al adminis a ion which howe e leads o poo pa ien compliance [4–6]. Fo IBD he apy, pa ien compliance can ne e be o e emphasized, which lies in he ac ha incu able IBD, wi h mos o he pa ien s diagnosed unde he age o 30, usually e- qui es li elong adminis a ion o d ugs o manage symp oms [1]. O al adminis a ion p o ides he g ea es deg ee o pa ien compliance, howe e i makes he local d ug deli e y o inflamed in es ine ex- emely challenging, as he co esponding d ug deli e y ehicles should ulfill mul iple asks du ing hei jou ney om o al ca i y o inflamed in es ine. Specifically, an op imal ehicle should p o ec he d ug and p e en p ema u e d ug elease in he spa ially a iable physiological en i onmen s o he gas oin es inal (GI) ac , selec i ely a ge he discon inuously dis ibu ed inflamed si es o in es ine which is usually su ounded by heal hy in es ine, and g adually elease d ug in esponse o he mic oen i onmen o inflamed in es ine. To hese ends, di e se ehicles, such as polyme pa icles [7–11], ino ganic pa icles [12,13], liposomes [14], solid lipid pa icles [15], and hyd ogels [11,16], ha e been de eloped. These ehicles, al hough a e consis ed o no el ma e ials, ha e no demons a ed he abili y o ully and sequen ially o e come he mul iple obs acles o deli e ing d ugs locally o inflamed in es ine, and hei he apeu ic ou comes, hough encou aging, a e s ill subop imal and significan ly comp omised by he side effec s o sys emic d ug exposu e. Such us a ing si ua ion p omp s a e hink o he ehicle design s a egy solely dependen on ma e ial composi ion, and aises a en ion o he s uc u e design o ehicles o IBD he apy. A hie a chical s uc u ed ehicle, wi h each le el in i s hie a chy being endowed by a ional selec ed ma e ial wi h desi ed unc ion, holds p omise o sequen ially ackle each o he mul iple obs acles o local d ug deli e y o inflamed in es ine. Mesopo ous nanopa icles, such as po ous silicon (PSi), po ous si- lica, halloysi e, e c., a e flexible in he loading/co-loading o d ugs wi h diffe en physicochemical p ope ies [17–23], due o hei unable mesopo ous s uc u e, la ge po e olume and high specificsu ace a ea [24–26], and hei hie a chized mesopo es make hem p omising can- dida es o cons uc ing hie a chical s uc u ed ehicles [22]. Meso- po ous nanopa icles encapsula ed in pH- esponsi e polyme s ( wo le- els o hie a chy) can deli e d ugs o he desi ed segmen o in es ine, howe e he loaded d ugs a e usually apidly eleased om mesopo es nanopa icles in an uncon olled manne due o hei eely accessed po es [23,27,28]. Hyd ogels o med by small molecules such as i- glyce ol monos ea a e (507 g mol −1 ) and aco byl palmi a e (AP) (415 g mol −1 ) can encapsula e d ugs, and hei enzyme-labile bonds enable g adual elease o d ugs in esponse o deg ada i e enzymes, including ma ix me allop o einases (MMPs) and es e ases [5,29,30], ha a e up- egula ed and eleased in inflamed issue [5,29–34]. In- spi ed by hese ac s, we specula e ha AP (molecule size ∼2.60 nm [35]) can be co-loaded wi h d ugs and u he assembled wi h d ugs in o hyd ogels inside he mesopo es o PSi nanopa icles. The esul an AP loaded PSi nanopa icles a e hen expec ed o g adually elease d ugs in esponse o he mic oen i onmen o inflamed in es ine. Fu he mo e, he e sa ile su ace chemis y o PSi nanopa icles makes hem a p omising candida e o a ge ing he inflamed si es o in es ine. Specifically, inflamma ion o he colon mucosa is accom- panied by des uc ion and inc eased discon inui y o he mucus laye [36], and in si u accumula ion o posi i ely cha ged p o eins including ans e in [14], eosinophil ca ionic p o ein [37] and bac e icidal/ pe meabili y-inc easing p o ein [38]. Consequen ly, posi i e cha ges build up on he su ace o damaged epi helial offe a a ge o d ug deli e y ehicles wi h nega i e su ace cha ge [5,14,39]. Rega ding PSi nanopa icles, nega i e cha ge can be eadily ob ained on hei su ace h ough su ace modifica ions. He e, we epo a hie a chical s uc u ed ehicle (AP@PSi-HA@ HPMCAS) based on hyalu onic acid (HA) unc ionalized PSi nano- pa icles (PSi-HA), AP and hyd oxyp opyl me hylcellulose ace a e suc- cina e (HPMCAS) wi h diffe en solubili y p ofiles depending on pH (LF, MF and HF g ades) o efficien local d ug deli e y o inflamed in es ine ia o al adminis a ion. Budesonide (BUD), a glucoco icoid o IBD he apy [10], se es as a model d ug in his s udy. AP and BUD a e co-loaded and in si u gelled inside he mesopo es o PSi-HA hyb id nanopa icles, and hen delica ely encapsula ed in HPMCAS by mic o- fluidic echnique o o m BUD loaded AP@PSi-HA@HPMCAS. We hy- po hesize ha , a e o al adminis a ion, pH- esponsi e HPMCAS ma- ix will p e en p ema u e d ug elease om AP@PSi-HA nanopa icles in GI ac un il hey each o a e close o he inflamed si es o in es ine. A e he dissolu ion o HPMCAS, nega i ely cha ged AP@PSi-HA nanopa icles will hen selec i ely bind o he inflamed si es o in es ine by elec os a ic adhesion, and hen g adually elease d ug in esponse o inflamma ion due o he enzyme-labile bonds o AP (Fig. 1). O e all, he hie a chical s uc u ed and p og ammable e- sponsi e AP@PSi-HA@HPMCAS will load, p o ec , anspo and e- lease d ugs locally o inflamed si es o in es ine a e o al adminis a- ion, and con ibu e o no only supe io he apeu ic efficacy bu also conside ably educed sys emic d ug exposu e. 2. Resul s and discussion The p epa a ion p ocess o he hie a chical s uc u ed and p o- g ammable esponsi e BUDAP@PSi-HA@LF o deli e ing BUD is schema ically illus a ed in Fig. 2a. HA was conjuga ed on o he su ace o ca boxyl- e mina ed PSi nanopa icles (Fig. 2b, Fig. S1a) using Fig. 1. P oposed mechanism o he d ug deli e y by hie a chical s uc u ed and p og amed ehicles (AP@PSi-HA@HPMCAS) h ough GI ac wi h IBD. HPMCAS (LF, MF and HF g ades), wi h diffe en pH dissolu ion h esholds a ailable, dissol es in specific segmen o he GI ac . The eleased AP@PSi-HA nanopa icles wi h nega i e su ace cha ge do no adhe e o he in ac mucosa, whe eas only adhe e o he inflamed mucosa which is cha ac e ized by de- ple ion o mucus, accumula ion o posi i ely cha ged p o eins, and enhancemen o pe meabili y o he epi helial cell laye . The deg ada i e enzymes e- leased by he inflamed cells deg ade he AP hy- d ogel, which leads o he elease o d ug om AP@ PSi-HA. W. Li e al. Bioma e ials 185 (2018) 322–332 323 spe mine (SPM) as linke ia 1-e hyl-3-(3-dime hylaminop opyl) ca - bodiimide/N-hyd oxysuccinimide (EDC/NHS) eac ion o o m PSi-HA hyb id nanopa icles (Fig. 2c, Fig. S1b) wi h nega i e su ace cha ge (−30 ± 2 mV) o a ge ing inflamed in es inal mucosa and high col- loidal s abili y (Fig. 2d and e) o mic ofluidic encapsula ion. The success ul conjuga ion o HA on o PSi was indica ed by he pa icle size and su ace cha ge a ia ions a e each eac ion s ep (Fig. 2 ), and confi med by he appea ance and enhancemen o amide I band a 1642 cm −1 in he Fou ie - ans o m in a ed spec oscopy (FTIR) spec a (Fig. 2g) a e each eac ion s ep in addi ion o he inhe i ance o cha ac e is ic bands o PSi, SPM and HA in PSi-HA (Fig. 2g). The amoun o HA in PSi-HA was de e mined o be 12.5 ± 1.3 w % by he mog a ime ic analysis (Fig. S2). Small molecula BUD and AP we e co-loaded in o PSi-HA a 65 °C, and hen we e cooled down o oom empe a u e o he in si u gela ion o AP and BUD inside PSi-HA o o m BUDAP@PSi-HA, making use o he eadily empe a u e con- olled gela ion beha io o AP (Fig. S3). BUDAP@PSi-HA was subse- quen ly encapsula ed in LF o o m he final hie a chical s uc u ed BUDAP@PSi-HA@LF pa icles (35 ± 1 μm) (Fig. 2h) using d ople mic ofluidics. The ob ained uni o m pa icle size is beneficial o achie ing con olled and ep oducible d ug elease p ofiles [40]. The success ul co-loading o BUD and AP in PSi-HA and encapsula ion o BUDAP@PSi-HA in LF we e confi med by FTIR (Fig. S4). The loading deg ee o BUD in BUDAP@PSi-HA and BUDAP@PSi-HA@LF was 24.5 ± 0.1% and 3.8 ± 0.3%, espec i ely. The dis ibu ion o BUDAP@PSi-HA nanopa icles in he LF ma ix was e alua ed by con ocal mic oscopy. To enable he isualiza ion, PSi- HA was conjuga ed wi h fluo escein iso hiocyana e (FITC, g een) and LF ma ix was labelled wi h e ame hyl hodamine (TRITC, ed). The con ocal images indica ed ha PSi-HA was e enly dis ibu ed in he LF ma ix (Fig. 2h), and scanning elec on mic oscopy (SEM) image showed ha BUDAP@PSi-HA@LF had smoo h su ace and well defined sphe ical shape (Fig. 2h). The de eloped hie a chical s uc u ed AP@PSi-HA@LF was hen in es iga ed o i s p og ammable esponsi e p ope ies. The pH- e- sponsi e dissolu ion beha io o p o ec i e ma ix was fi s ly e alua ed by imme sing PSi-HA@LF in aqueous buffe solu ions a a ious pH alues, s a ing om pH 1.2 o 5.5. PSi-HA@LF was in ac a pH < 5.5, and immedia ely began o dissol e a pH 5.5 (Fig. 3a), while pa icles made by MF and HF only s a ed o dissol e when pH eached 6.0 and Fig. 2. a) Fab ica ion p ocess o hie a chical s uc u ed and p og ammable esponsi e ehicles (BUDAP@PSi-HA@LF). b–c) T ansmission elec on mic oscopy (TEM) image o b) PSi and c) PSi-HA. d) Size and e) polydispe si y index (PDI) o PSi and PSi-HA dispe sing in Milli-Q wa e du ing 2 h, demons a ing he high colloidal s abili y o PSi-HA o e ime. Da a ep esen mean ± SD. (n= 3). ) Size, PDI, and su ace ze a po en ial o PSi, PSi-SPM and PSi-HA. g) FTIR o PSi, SPM, PSi-SPM, HA and PSi-HA. h) The monodispe si y o he ab ica ed BUDAP@PSi-HA@LF was s udied by con ocal mic oscopy and SEM. PSi-HA was conjuga ed wi h FITC andLF laye was labelled wi h TRITC. The con ocal images showed he FITC (g een), TRITC ( ed) and he o e lay (yellow) channels. The su ace o he pa icles was obse ed by SEM. (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he Web e sion o his a icle.) W. Li e al. Bioma e ials 185 (2018) 322–332 324 6.8, espec i ely [23,41]. To u he e alua e he pH- esponsi e p op- e ies o PSi-HA@HPMCAS (LF, MF o HF), BUD was solely loaded in PSi-HA@HPMCAS (LF, MF o HF). BUD@PSi-HA@LF, BUD@PSi-HA@ MF and BUD@PSi-HA@HF only began o elease BUD when he pH eached 5.5, 6.0 and 6.8, espec i ely, and he elease was nea ly comple e wi hin 30 min (Fig. S5) due o he sho disin eg a ion ime (∼10 min acco ding o he manu ac u e ) o HPMCAS (LF, MF and HF). The p ecisely pH- igge ed d ug elease beha io s o PSi-HA@HPMCAS sugges i s po en ial o elease BUD a specific segmen o he in es ine by he flexible selec ion and combina ion o LF, MF and HF. The in- es inal pH o mice is lowe han ha o human [42], he e o e, LF a he han MF o HF was used o he animal expe imen s. The enzyme- esponsi e p ope ies o d ug loaded AP@PSi-HA@LF we e fi s ly in es iga ed in buffe solu ions. In con as o he apid elease o BUD om PSi-HA@LF, AP@PSi-HA@LF only sligh ly e- leased BUD (6.5% a 6 h) in he absence o lipase e en hough pH eached 5.5. Wi h inc easing concen a ion o lipase, g adual elease o BUD om AP@PSi-HA@LF was accele a ed, i.e., 8.5%, 75.6% and 88.4% a 6 h o 0.01, 1 and 100 U/mL lipase, espec i ely (Fig. 3b), sugges ing AP@PSi-HA@LF can adjus d ug elease o ma ch he dis- ease ac i i y a inflamed in es ine. IBD is cha ac e ized by a iable disease ac i i y o e ime wi h fla es and pe iods o low disease ac- i i y. A disease ac i i y-dependen d ug deli e y ehicle is likely o a oid sub- o sup a- he apeu ic d ug concen a ions locally du ing pe iods o high o low disease ac i i y, espec i ely, esul ing in max- imized he apeu ic efficacy and minimized sys emic d ug exposu e [30]. O e all, BUDAP@PSi-HA@LF exhibi ed pH and enzyme p og am- mable esponsi e d ug elease beha io s. To simula e d ug elease om AP@PSi-HA@LF unde inflamma o y condi ions, BUDAP@PSi-HA@LF was incuba ed wi h supe na an collec ed om human mac ophages cul u ed wi h o wi hou lipopolysaccha ide (LPS). In compa ison o uns imula ed mac ophages, mac ophages ac i a ed by LPS enhanced he enzyma ic ac i i ies in he supe na an [5,30,32,33], which esul ed in significan ly inc eased BUD elease (Fig. 3c). The abo e enzyme- e- sponsi e d ug elease beha io s sugges ha AP@PSi-HA@LF would p e e en ially elease BUD a he si es o inflamed in es ine a he han heal hy in es ine o dis an heal hy issues, and he e o e educe sys- emic d ug exposu e which o en causes side effec s. Since AP@PSi-HA@LF is designed o o al d ug deli e y, wo human in es inal epi helial cell lines, Caco-2 clone C2Bbe1 and HT29- MTX, we e used o assess i s sa e y. A e 24 h incuba ion wi h bo h cell lines, he e was no e idence o cy o oxici y o PSi-HA@LF and BUDAP@PSi-HA@LF as well as hei componen s, i.e., PSi-HA, AP and BUD (Fig. S6, Fig. S7 and Fig. S8). The he apeu ic efficacy and sys emic abso p ion o BUD deli e ed by AP@PSi-HA@LF we e fi s ly s udied using in i o IBD model. The in i o IBD model consis ed o iple co-cul u ed cells including in es inal epi helial cells, human blood-de i ed mac ophages and dend i ic cells ha we e s imula ed by p o-inflamma o y cy okine in e leukin-1β(IL- 1β) in combina ion wi h lipopolysaccha ide (LPS) (Fig. S9). The ex- pe imen al imeline o se ing up he in i o IBD model and subsequen es ing o BUD o mula ions is shown in Fig. S10. As an indica ion o he in eg i y and pe meabili y o he monolaye , ansepi helial elec ical Fig. 3. a) SEM images o PSi-HA@LF a e imme sion in buffe solu ions a pH 1.2 and 5.0 o 2 h, and a pH 5.5 o 5 min and 2 h b) pH and enzyme sequen ially esponsi e BUD elease om AP@PSi-HA@LF in a se ies o pH buffe s a 37 °C. Enzyme (lipase; 0.01, 1, and 100 U/mL) was added a 3 h. Da a a e means ± SD (n= 3). c) BUD elease om AP@PSi-HA@LF upon incuba ion o 24 h a 37 °C wi h cul u e supe na an om LPS ac i a ed human mac ophages. Da a a e means ± SD (n= 3). d) No malized IL-8 elease o Caco-2 clone, mac ophages and dend i ic cells iple co-cul u e IBD model a e induc ion o inflamma ion and subsequen ea men wi h a ious BUD o mula ions and blank ehicle con ol (wi hou BUD). Da a a e means ± SD (n= 4). e) Pe mea ion p ofiles o BUD ac oss he Caco-2 clone monolaye s co-cul u ed wi h mac ophages and dend i ic cells in bo h heal hy and inflamed condi ions. ) Appa en pe meabili y coefficien s (P app ) o BUD calcula ed om he pe mea ion p ofiles o BUD. Da a a e means ± SD (n= 3). W. Li e al. Bioma e ials 185 (2018) 322–332 325 esis ance (TEER) alue was moni o ed du ing he expe imen pe iod. Compa ed o ee BUD and BUD@PSi-HA@LF, BUDAP@PSi-HA@LF ea men eco e ed he TEER o he highes le el, and was significan ly highe a day 1 (Fig. S11). Fu he mo e, as a ma ke o inflamma ion, IL-8 elease om he cells unde he ea men o diffe en BUD o - mula ions was measu ed. BUDAP@PSi-HA@LF ea men significan ly educed he IL-8 p oduc ion o he lowes le el (Fig. 3d), which in- dica es a p olong he apeu ic effec o p og ammable esponsi e BUDAP@PSi-HA@LF han ee BUD and BUD@PSi-HA@LF, mainly owing o he sus ained d ug elease p o ided by he emaining enzyme- esponsi e BUDAP@PSi-HA on monolaye s. Since AP@PSi-HA@LF is designed o local deli e y o BUD o p ecise he apy o IBD ia o al adminis a ion, i s effec s on sys emic d ug abso p ion is ano he impo an e alua ion index. AP@PSi-HA@ LF significan ly educed he pe meabili y o BUD ac oss he monolaye s in compa ison o ee BUD and PSi-HA@LF in bo h inflamed and heal hy condi ions (Fig. 3e and ), which is a ibu ed o he much slowe elease o BUD om AP@PSi-HA@LF (Fig. 3b and c). Thus, deli e ing d ug by hie a chical s uc u ed and p og ammable e- sponsi e AP@PSi-HA@LF will esul in a majo educ ion in sys emic d ug abso p ion and consequen ly educe he possibili y o side effec s. To maximize he he apeu ic efficacy and minimize he sys emic exposu e o d ug deli e ed by AP@PSi-HA@LF in i o, AP@PSi-HA@LF should selec i ely adhe e o he inflamed si es o in es ine. We hy- po hesize ha he nega i e su ace cha ge o AP@PSi-HA (−32 ± 1 mV) will acili a e i s a ge ing o he inflamed in es inal epi helium, whe e posi i ely cha ged p o eins a e accumula ed [14,37,38]. To e i y his hypo hesis, he adhesion p ope ies o AP@ PSi-HA we e fi s ly es ed in i o using syn he ic su aces. BUDAP@PSi- FITC/HA (−33 ± 2 mV) was incuba ed on glass slides o polys y ene pla es coa ed wi h human ans e in (posi i ely cha ged) o po cine mucin (nega i ely cha ged), simula ing inflamed and heal hy epi he- lium, espec i ely [5]. Compa ed o mucin coa ed su aces, ans e in coa ed su aces e ained much mo e pa icles (Fig. 4a) and showed a 14.9- old highe fluo escence signal (Fig. 4b) a e ex ensi e washing wi h phospha e buffe ed saline (PBS). In ano he con ol expe imen , BUDAP@PSi-FITC/HA was incuba ed wi h a ca ionic polye hylenimine (PEI) solu ion o con e i s su ace cha ge om nega i e o posi i e (35 ± 2 mV). As p edic ed, his cha ge con e sion abolished he p e- e en ial adhesion o BUDAP@PSi-HA o ans e in coa ed su aces and enhanced i s adhesion o he mucin coa ed su aces (Fig. S12). Following he p omising esul s in i o, we u he examined he a ge ing o AP@PSi-HA o inflamed mucosa using in i o IBD model, i.e., dex an sul a e sodium (DSS)-induced mouse coli is model. Mice wi h coli is and no mal mice ecei ed a single dose o BUDAP@PSi- Cy7/HA@LF (−31 ± 1 mV o BUDAP@PSi-Cy7/HA) ( o IVIS ima- ging) o BUDAP@PSi-FITC/HA@LF ( o con ocal imaging) by o al ga- age. Mice wi h coli is showed significan ly g ea e e en ion o fluo - escence han no mal mice a e he adminis a ion o BUDAP@PSi-Cy7/ HA@LF o 2 h and 5 h (Fig. 4c and d). Con ocal mic oscopy images o dis al end o colon sec ions (Fig. 4e) showed he a ge ing o BUDAP@ PSi-FITC/HA o he inflamed si es o colon. Toge he wi h he pH and enzyme esponsi e p ope ies o AP@PSi-HA@LF, we would expec ha hie a chical s uc u ed AP@PSi-HA@LF can efficien ly deli e BUD locally o inflamed si es o in es ine by execu ing i s p og ammed unc ions in he GI ac suffe ed wi h IBD. We hen es ed he in i o he apeu ic efficacy o BUD deli e ed by hie a chical s uc u ed and p og ammable esponsi e AP@PSi-HA@LF in DSS-induced mouse coli is model. BUD was s udied a low dose (L, 0.5 mg/kg) and high dose (H, 1.5 mg/kg), espec i ely. F ee BUD (FBUDL and FBUDH), blank ehicles wi hou BUD, i.e., AP@PSi-HA@ LF (PL and PH) and pH- esponsi e BUD@PSi-HA@LF (BUDL and BUDH) we e employed as con ols o demons a e he he apeu ic benefi s o hie a chical s uc u ed and p og ammable esponsi e BUDAP@PSi-HA@LF (BUDAPL and BUDAPH). A e induc ion o he coli is o 7 d, he ea men was conduc ed by daily o al ga age o 7 d, du ing which he body weigh o coli is mice g adually dec eased (Fig. 5a). Whe eas ea men wi h BUDAP@PSi-HA@LF (BUDAPL and BUDAPH) significan ly mi iga ed weigh loss as compa ed o ee BUD (FBUDL and FBUDH) and BUD@PSi-HA@LF (BUDL and BUDH) (Fig. 5a). The se e i y o coli is was assessed by disease ac i i y index (DAI). In e es ingly, all ea ed g oups showed significan ly educed DAI a e ea men o 7 d as compa ed o coli is g oup, and he minimal DAI was ound o BUDAP@PSi-HA@LF (BUDAPL and BU- DAPH) (Fig. 5b). Besides, BUDAPH no ably p ese ed colon leng h when compa ed o FBUDH and BUDH (Fig. S13). Fu he mo e, he le- els o ypical p oinflamma o y cy okines including IL-1βand IL-6 we e quan ified. Coli is mice exhibi ed ele a ed le els o bo h IL-1βand IL-6 han no mal mice (Fig. 5c and d). O e all, he le els o IL-1βand IL-6 dec eased a e ea men wi h BUD o mula ions, and he g oup o mice ea ed wi h BUDAP@PSi-HA@LF showed he lowes concen a- ion o IL-1βand IL-6 in compa ison o he o he ea ed g oups. Addi ional in o ma ion was ob ained by examining and sco ing hema oxylin and eosin (H&E) s ained his ological sec ions o dis al end (Fig. 5e and ) o colon issues. The coli is was cha ac e ized by su- pe ficial e osions, deple ion o goble cells, infil a ion o he colon lamina p op ia wi h inflamma o y cells, and c yp hype ophy [5,9]. Disease se e i y was significan ly educed in mice ea ed wi h BUD@ PSi-HA@LF (BUDL and BUDH) and BUDAP@PSi-HA@LF (BUDAPL and BUDAPH) (Fig. 5e), and ep esen a i e images (Fig. 5 ) demons a e ha ea men wi h BUDAP@PSi-HA@LF offe ed he bes imp o emen in pa hological inju ies, which was also confi med by TUNEL s aining (Fig. S14). O e all, BUD deli e ed by hie a chical s uc u ed and p o- g ammable esponsi e AP@PSi-HA@LF exhibi s supe io he apeu ic efficacy han ee BUD and BUD deli e ed by pH- esponsi e PSi-HA@ LF, which was a ibu ed o he p o ec ion o BUDAP@PSi-HA in GI ac by LF, a ge ing o BUDAP@PSi-HA o he inflamed si es o colon and subsequen p olonged elease o BUD om AP@PSi-HA in esponse o he mic oen i onmen o inflamed in es ine. 3. Conclusions In summa y, we ha e de eloped a hie a chical s uc u ed and p o- g ammable esponsi e AP@PSi-HA@HPMCAS ehicle o efficien local d ug deli e y o inflamed si es o in es ine in IBD he apy ia o al ad- minis a ion. The p epa ed ehicles showed well-defined sphe ical shape and uni o m pa icle size. The pH- esponsi e ma ix p o ec s he d ug and AP co-loaded PSi nanopa icles agains he complex en- i onmen o he GI ac be o e eaching he desi ed segmen o in- es ine, and p e en s p ema u e d ug elease. The nega i ely cha ged AP@PSi-HA nanopa icles selec i ely a ge he inflamed si es o in- es ine, and locally elease he d ug in esponse o he inflamma ion g adually o e a p olonged du a ion. Compa ed o ee d ug and con- en ional pH- esponsi e pa icles, d ug deli e ed by hie a chical s uc u ed and p og ammable esponsi e AP@PSi-HA@HPMCAS ex- hibi supe io he apeu ic efficacy and significan ly educed sys emic d ug exposu e, subs an ia ing he impo ance o enginee ing hie - a chical s uc u ed and p og ammed ehicles o deli e ing d ugs lo- cally o inflamed si es o in es ine. 4. Ma e ials and me hods 4.1. P epa a ion o PSi nanopa icles Elec ochemical anodiza ion me hod was used o p epa e un- decylenic acid modified he mally hyd oca bonized PSi (UnTHCPSi), as desc ibed in de ail elsewhe e [43]. B iefly, monoc ys alline p + - ype Si (100) wa e s (0.01–0.02 Ωcm esis i i y) we e elec ochemically anodized in a 1:1 ( / ) aqueous hyd ofluo ic acid (38%)–e hanol elec oly e by applying epea ed low and high cu en densi y pulsed e ching p ofile. The esul ing PSi wi h high po osi y ac u e planes and hyd ogen- e mina ed su ace was subsequen ly de ached om he W. Li e al. Bioma e ials 185 (2018) 322–332 326 subs a e as ee-s anding mul ilaye films by ab up ly inc easing he cu en densi y o elec opolishing egion. The mal hyd oca boniza ion o he PSi films was pe o med by exposing hem o a 1:1 ( / ) N 2 / ace ylene (C 2 H 2 )flow (1 L min −1 ) o 15 min a oom empe a u e, ollowed by a hea ea men o 15 min a 500 °C. The ob ained THCPSi films we e hen ea ed wi h 10-undecenoic acid o 16 h a 120 °C. The UnTHCPSi films we e finally we ball milled o p oduce nanopa icles and sepa a ed using cen i uga ion o ob ain he final PSi nanopa icles wi h desi ed pa icle size. Ni ogen adso p ion–deso p ion iso he ms o PSi nanopa icles we e measu ed a 77 K on a gas adso p ion analyze (Mic ome i ics T iS a 3000, USA). The specific su ace a ea was de- e mined om he adso p ion b anch o he ni ogen iso he m using he B unaue -Emme -Telle (BET) heo y [44], and he po e olume was de e mined as he o al adso bed amoun a a ela i e p essu e (p/p 0 )o 0.97. The a e age diame e o he po es was 4 × po e olume/specific su ace a ea. The specific su ace a ea and po e olume o PSi nano- pa icles used in his s udy we e 305 ± 10 m 2 /g and 0.89 ± 0.01 cm 3 /g, espec i ely. The a e age diame e o he po es was calcula ed o be 11.6 ± 0.4 nm. 4.2. P epa a ion o HA The aqueous solu ion o HA sodium sal (Sigma-Ald ich, USA) was dialyzed agains 0.01 M HCl o 24 h and hen agains Milli-Q wa e o an addi ional 24 h o con e HA om i s sodium sal o m o acid o m. The acid o m o HA was eco e ed by lyophiliza ion [45]. Fig. 4. a) Con ocal mic oscopy images and b) fluo escence in ensi ies (quan ified by mic opla e eade ) o BUDAP@PSi-FITC/HA a e incuba ion wi h uncoa ed, mucin coa ed (simula ing heal hy epi helium) o ans e in coa ed (simula ing inflamed epi helium) su aces o 1 h a 37 °C, ollowed by washing wi h PBS o 3 imes. Da a a e means ± SD (n = 6). c–d) Mice wi h DSS-induced coli is and no mal mice we e o ally adminis a ed wi h BUDAP@PSi-Cy7/HA@LF. c) The li ing mice we e imaged using IVIS fluo escence imaging sys em a e 2 h and 5 h, espec i ely. d) The mice we e sac ificed 5 h a e adminis a ion, and he in es ines we e dissec ed and imaged using IVIS fluo escence imaging sys em. The o al fluo escence in ensi y was de e mined in a s anda d-size egion o in e es (ROI) d awn a ound he whole in es ine (c) o colon (d). (c) and (d) sha e he same adiance scale ba s. Da a a e means ± SD (n = 3). e) Immunofluo escence images o dis al end o colon dissec ed om no mal mice and mice wi h coli is 5 h a e o ally adminis a ed wi h BUDAP@PSi-FITC/HA@LF. Nucleus and mac ophages we e s ained by DAPI and F4/80, espec i ely. All he images ha e he same scale ba . W. Li e al. Bioma e ials 185 (2018) 322–332 327 4.3. P epa a ion o PSi-HA nanopa icles PSi nanopa icles we e co alen ly conjuga ed wi h SPM using he EDC/NHS eac ion. Typically, 1 mg PSi nanopa icles we e dispe sed in 1 mL o anhyd ous dime hyl o mamide (DMF) (Sigma-Ald ich, USA). Then 4 μL (22.6 μmol) EDC (Sigma-Ald ich, USA) and 3 mg (26.1 μmol) NHS (Sigma-Ald ich, USA) we e added in o he abo e suspension and s i ed o e nigh . Subsequen ly, 20 mg (98.8 μmol) SPM (ACROS O ganic, UK) we e added and s i ed o e nigh . The p epa ed PSi-SPM nanopa icles we e collec ed by cen i uga ion and ex ensi ely washed wi h DMF o 3 imes. 4 mg HA we e dissol ed in 1 mL anhyd ous di- me hyl sul oxide (DMSO) (Sigma-Ald ich, USA). Then 2.8 μL EDC and 1.8 mg NHS we e added and s i ed o 2 h. A e wa ds, 1 mg PSi-SPM nanopa icles we e dispe sed in o he abo e solu ion and s i ed o e - nigh . The ob ained PSi-HA nanopa icles we e collec ed by cen- i uga ion and ex ensi ely washed wi h Milli-Q wa e o 5 imes. The amoun o HA on he su ace o PSi nanopa icles was es ima ed using TGA 7 he mog a ime e (Pe kinElme , USA). The samples we e hea ed om oom empe a u e o 850 °C a a hea ing a e o 20 °C/min unde a 200 mL/min N 2 flush. The measu emen s we e done in ipli- ca es. 4.4. Loading o BUD and AP in PSi-HA nanopa icles BUD and AP we e co-loaded in o PSi-HA nanopa icles using an imme sion me hod. BUD and AP we e dissol ed oge he in a mix u e o e hanol–H 2 O (1:1 / ) a a concen a ion o 25 and 75 mg mL −1 , e- spec i ely, a 65 °C. 10 mg PSi-HA we e added in o 2 mL o he abo e solu ion and s i ed o 3 h a 65 °C. A e wa ds, he BUD and AP loaded PSi-HA (BUDAP@PSi-HA) nanopa icles we e collec ed by cen i uga- ion o 3 min using p e-wa med o o , and hen we e allowed o cool down o oom empe a u e o he gelling o AP be o e washing by Milli-Q wa e . The p ocedu es o p epa e PSi-HA loaded wi h BUD only (BUD@PSi-HA) and PSi-HA loaded wi h AP only (AP@PSi-HA) we e he same as BUDAP@PSi-HA, and he concen a ion o BUD and AP in e hanol–H 2 O (1:1 / ) was 25 and 75 mg mL −1 , espec i ely. 4.5. Fab ica ion o flow- ocusing de ice o mic ofluidics Bo osilica e glass cylind ical capilla ies (Wo ld P ecision Ins umen s, USA) and glass slides we e used o assemble he mic o- fluidic flow- ocusing de ice [23,27]. The inne capilla y had an ou e diame e o 1000 μm. Fi s ly, he inne capilla y was ape ed using a mic opipe e pulle (P-97, Su e Ins umen , USA), and hen enla ged o 350 μm. Subsequen ly, he ape ed capilla y was inse ed in o he igh end o he ou e capilla y wi h an inne diame e o 1120 μm. Two Fig. 5. The apeu ic efficacy o a ious BUD o mula ions on IBD model o DSS-induced coli is in mice. a) Body weigh and b) DAI o mice du ing 7 d o induc ion o coli is model and 7 d o ea men . Da a we e no malized as a pe cen age o he body weigh a day 0. c–d) The le els o c) IL-1βand d) IL-6 a e 7 d o ea men . e) His opa hology sco e o he dis al end o colon a e 7 d o ea men . ) Rep esen a i e H&E his ology images o he dis al end o colon a e 7 d o ea men . All he images ha e he same scale ba . Da a a e means ± SD (n= 5). *p< 0.05, **p< 0.01 and ***p< 0.001 e sus he coli is g oup. W. Li e al. Bioma e ials 185 (2018) 322–332 328 sy inges we e linked o he mic ofluidic de ice using polye hylene ubes o allow he independen injec ion o he inne and ou e fluids con olled by pumps (PHD 2000, Ha a d Appa a us, USA). A ans- pa en epoxy esin (5 min ® Epoxi, De con, USA) was used o seal he mic ofluidic de ice whe e equi ed. 4.6. Fab ica ion o pH- esponsi e mic opa icles PSi-HA nanopa icles we e encapsula ed in pH-sensi i e HPMCAS (ShinE su, Japan) o o m PSi-HA@HPMCAS mic opa icles using wa e -in-oil-in-wa e (W/O/W) double emulsion h ough a mic ofluidic flow- ocusing de ice. Th ee g ades (LF, MF and HF) o HPMCAS, which a y in hei pH-dependen solubili y, we e used. The LF, MF and HF g ades o HPMCAS dissol e a pH ≥5.5, 6.0 and 6.8, espec i ely. Fo he mic ofluidic encapsula ion, HPMCAS (LF, MF o HF) was dissol ed in e hyl ace a e (EA) a a concen a ion o 10 mg mL −1 2 mg PSi-HA nanopa icles we e dispe sed in 100 μL Milli-Q wa e and added d op- wise in o 1 mL HPMCAS (LF, MF o HF) solu ion unde sonica ion (Sonics, Sonics & Ma e ials Inc, USA) o p oduce he p ima y emulsion (W/O) which se ed as he inne dispe sed fluid. 2% w/ P-407 (BASF, Ge many) aqueous solu ion (pH 5) was used as he ou e con inuous fluid o ob ain a s able W/O/W emulsion. The inne fluid (3.5 mL h −1 ) was ocused by he ou e fluid (15 mL h −1 ), and he o med d ople s we e collec ed in 1% w/ P-407 aqueous solu ion (pH 5). A e wa ds, he p oduced PSi-HA@HPMCAS (LF, MF o HF) mic opa icles we e collec ed by cen i uga ion. 4.7. Fab ica ion o BUD and/o AP loaded mic opa icles BUDAP@PSi-HA@LF, BUD@PSi-HA@HPMCAS (LF, MF o HF) and AP@PSi-HA@LF mic opa icles we e p epa ed using BUDAP@PSi-HA, BUD@PSi-HA and AP@PSi-HA nanopa icles, espec i ely, unde he same condi ion as he PSi-HA@LF mic opa icles. The loading deg ees o BUD in pa icles we e de e mined by s i ing he pa icles in a mix u e o PBS and ace oni ile (1:1 / ) o dissol e he HPMCAS ma ix and AP hyd ogel, and o elease all he loaded BUD. The amoun o BUD was quan ified by Agilen 1100 high pe o mance liquid ch o- ma og aphy (HPLC) (Agilen Technologies, USA) wi h a mobile phase composed o phospho ic acid (0.1%, pH 3.2) and ace oni ile ( olume a io 52:48) a a flow a e o 1.4 mL min −1 a 25 °C. The wa eleng h used o BUD quan ifica ion was 244 nm. A Disco e y ® C18 column (4.6 × 150 mm, 5 μm, Supelco Analy ical, USA) was used as s a iona y phase and he injec ion olume o he samples was 20 μL. 4.8. Physicochemical p ope ies o nanopa icles and mic opa icles The pa icle size, PDI and su ace cha ge o nanopa icles we e de e mined by Ze asize Nano ZS (Mal e n Ins umen s L d, UK) in Milli-Q wa e wi h pH adjus ed o 7.4 by HCl/NaOH. The s abili y o nanopa icles was e alua ed by ollowing hei changes on size and PDI a e incuba ion o 200 μgmL −1 o nano- pa icles wi h Milli-Q wa e (pH 7.4) du ing 2 h. Samples we e wi h- d awn a 15, 30, 60, 90 and 120 min, and dilu ed in Milli-Q wa e (pH 7.4) o measu ing he size and PDI. Milli-Q wa e was used o s abili y es as i was used o dispe sing he PSi-HA nanopa icles du ing he mic ofluidic ab ica ion o PSi-HA@HPMCAS. The s uc u e o nanopa icles and mo phology o mic opa icles we e obse ed by TEM (JEOL 1400, Japan) and SEM (Hi achi S-4800, Japan), espec i ely. The mic opa icles we e pla inum spu e ed be- o e imaging. The pa icle size o mic opa icles was de e mined using Image J (NIH, USA), and a leas 100 pa icles we e analyzed. The chemical composi ion o he nanopa icles and mic opa icles was cha ac e ized by FTIR (Ve ex 70, B uke , USA). The samples we e mixed wi h KB (FTIR g ade, Sigma-Ald ich, USA) and p essed in o pelle s. The pelle s consis ed o 1 mg o sample and 200 mg o KB . The FTIR spec a we e eco ded in he ange o 4000–400 cm −1 wi h a esolu ion o 4 cm −1 using OPUS 5.5 so wa e. Fo s udying he dis ibu ion o PSi-HA nanopa icles wi hin he LF mic opa icles, PSi-HA was labelled wi h FITC (The mo Fishe Scien ific, USA) as desc ibed in ollowing sec ions, and he LF polyme was mixed wi h TRITC (The mo Fishe Scien ific, USA) a a mass a io o 100 o 1. A e mic ofluidic ab ica ion as desc ibed abo e, he p oduced PSi-HA@LF mic opa icles we e placed in o 35 mm Pe i dish (Ma Tek, USA), and imaged using con ocal mic oscope (Leica SP5 II HCS A, Ge many). 4.9. pH- esponsi e dissolu ion beha io o mic opa icles To in es iga e he pH- esponsi e dissolu ion beha io o he p e- pa ed PSi-HA@LF mic opa icles, hey we e moun ed on o s ubs using ca bon ape, and hen ea ed wi h buffe solu ions a diffe en pH alues (1.2 and 5.0) o 2 h and a pH 5.5 o 5 min and 2 h. The excess solu ions we e emo ed and he samples we e d ied a oom empe a- u e o 24 h. The mic opa icles wi hou any ea men we e used as a con ol. All samples we e pla inum spu e ed be o e imaging. 4.10. pH- esponsi e d ug elease s udy The pH- esponsi e d ug elease p ofiles o BUD@PSi-HA@HPMCAS (LF, MF and HF) we e s udied in con inuous g adien pH media a 37 °C wi h shaking a 150 pm. F ee BUD was used as con ol g oup. Samples con aining 50 μg BUD we e added in o 50 mL media a pH 1.2, and he pH was hen g adually inc eased o 5.0, 5.5, 6.0 and 6.8. A p e- de e mined ime in e als, 200 μL o sample we e wi hd awn om each sample and he elease media was eplaced wi h 200 μL o esh media. Samples we e fi s ly cen i uged (16100 g, 3 min), and hen he d ug concen a ions in he supe na an we e quan ified by HPLC. 4.11. Enzyme- esponsi e d ug elease s udy The enzyme- esponsi e d ug elease p ofiles o BUDAP@PSi-HA@ LF we e s udied in con inuous g adien pH media wi hou and wi h 0.01, 1 o 100 U/mL o The momyces lanuginosus lipase (Sigma-Ald ich, USA) a 37 °C wi h shaking a 150 pm. BUD@PSi-HA@LF and ee BUD we e used as con ol g oups. Samples con aining 50 μg BUD we e added in o 50 mL media a pH 1.2, and he pH was hen g adually inc eased o 5.0, 5.5, 6.0 and 6.8. Lipase was added when pH was inc eased o 5.0. A p ede e mined ime in e als, 200 μL o sample we e wi hd awn om each sample and he elease media was eplaced wi h 200 μLo esh media. Samples we e fi s ly cen i uged (16100 g, 3 min), and hen he d ug concen a ions in he supe na an we e quan ified by HPLC. BUD elease was also s udied in cul u e supe na an om human mac ophages. The me hod o ob aining mac ophages was desc ibed in de ail in la e sec ions. Mac ophages we e cul u ed wi hou and wi h 0.1 μgmL −1 lipopolysaccha ide (LPS) (Sigma-Ald ich, USA), and su- pe na an was collec ed a e 4 and 24 h. 10 mg BUDAP@PSi-HA@LF we e suspended in 1 mL o Milli-Q wa e (pH 5.0), and 20 μL o his suspension we e added o 1 mL o supe na an om uns imula ed o LPS-s imula ed mac ophages. A e incuba ion a 37 °C o 24 h, sam- ples we e cen i uged a 16100 g o 5 min, and BUD concen a ion in he supe na an was measu ed by HPLC. 4.12. Cell line cul u e Human colon ca cinoma (Caco-2), Caco-2 clone C2Bbe1 and human colon adenoca cinoma (HT29-MTX) cells we e sepa a ely cul u ed in Dulbecco's modified Eagle medium (DMEM) (HyClone, USA) wi h 4.5 g L −1 glucose, supplemen ed wi h 10% e al bo ine se um (Gibco, In i ogen, USA), 1% nonessen ial amino acids, 1% L-glu amine, peni- cillin (100 IU mL −1 ) and s ep omycin (100 mg mL −1 ) (all om HyClone, Logan, UT). The cells we e main ained a 37 °C wi h an W. Li e al. Bioma e ials 185 (2018) 322–332 329 a mosphe e o 5% CO 2 and 95% ela i e humidi y, and he cul u e media we e changed e e y o he day. P io o each es , he cells we e ha es ed using 0.25% ( / ) ypsin–e hylenediamine e aace ic acid (EDTA)–phospha e buffe solu ion. 4.13. Mac ophages and dend i ic cells cul u e Buffy coa s we e p ocessed by Ficoll-Paque g adien cen i uga ion o ob ain he pe iphe al blood mononuclea cells (PBMCs). Mac ophages and dend i ic cells we e diffe en ia ed om CD14 + monocy es ha we e isola ed om PBMCs using CD14 Mic oBeads (Mil enyi Bio ec). These cells we e cul u ed wi h mac ophage medium (RPMI 1640 medium wi h 1% L-glu amine (HyClone, USA), supple- men ed wi h 10% human se um AB (BioWes , F ance), 1% nonessen ial amino acids, penicillin (100 IU mL −1 ) and s ep omycin (100 mg mL −1 ), and 1% sodium py u a e (Gibco, UK)). Mac ophages we e ob ained a e cul i a ion o 6 d wi h mac ophage medium, while dend i ic cells we e ob ained a e cul i a ion o 6 d wi h mac ophage medium supplemen ed wi h 80 ng/mL ecombinan human GM-CSF (Pep o ec, USA) and 80 ng/mL ecombinan human IL-4 (Pep o ec, USA) [46]. 4.14. Cy o oxici y s udies 100 μL Caco-2 clone and HT29-MTX cells we e sepa a ely seeded in 96-well pla es a a densi y o 2 × 10 5 cells mL −1 . A e he cells we e le o a ach o 24 h, he cul u e medium was disca ded and he cells we e washed wi h 100 μL esh 1 × Hank's Balanced Sal Solu ion (HBSS)−HEPES (pH 7.4). Then PSi, PSi-HA and PSi-HA@LF a con- cen a ions o 25, 50, 100, 250 and 500 μgmL −1 (PSi o equi alen ) in HBSS−HEPES (pH 7.4), AP a concen a ions o 5, 10, 25, 50 and 100 μgmL −1 in HBSS−HEPES (pH 7.4), and ee BUD, BUDAP@PSi- HA and BUDAP@PSi-HA@LF a concen a ions o 5, 10, 20, 50 and 100 μgmL −1 (BUD o equi alen ) in HBSS−HEPES (pH 7.4) we e added o each well. A e incuba ion o 24 h, he cells we e washed wice wi h HBSS−HEPES (pH 7.4) and he numbe o iable cells was quan ified using CellTi e -Glo ® (P omega Co po a ion, USA). The lu- minescence was measu ed using Va ioskan Flash pla e eade (The mo Fishe Scien ific, USA). HBSS−HEPES (pH 7.4) and 1% o T i on X-100 we e used as nega i e con ol and posi i e con ol, espec i ely. All he expe imen s we e pe o med a leas in iplica e. 4.15. In i o IBD model In i o IBD model was modified om p e ious s udies [47–49]. 6×10 4 Caco-2 clone cells we e seeded in o he apical compa men o 12-T answell ® pla e, 1 × 10 4 mac ophages and 1 × 10 4 dend i ic cells we e seeded in o he basola e al compa men . The co-cul u e was kep wi h Caco-2 medium (0.5 mL) in he apical compa men and mac o- phage medium (1.5 mL) in he basola e al compa men , and he medium was changed e e y o he day in bo h he apical and basola e al compa men s. The co-cul u e was cul i a ed o 21 d o o m cell monolaye s. Fo he inflamed model, 0.5 mL Caco-2 medium wi h 20 ng/mL IL-1β(Pep o ec, USA) and 0.1 μgmL −1 LPS was added o he apical compa men and incuba ed o 2 d. 4.16. D ug pe meabili y ac oss in es inal cell monolaye s The pe meabili y expe imen s we e pe o med using in i o IBD model a 37 °C wi h shaking a 100 pm, in compa ison wi h heal hy in es inal cell monolaye s. A e 2 d o cul i a ion wi h o wi hou s i- mula ion, he medium om he apical compa men was collec ed o dispe sing he samples. 500 μL ee BUD, BUD@PSi-HA@LF and BUDAP@PSi-HA@LF in apical medium we e added in o he apical compa men a a BUD (o equi alen ) concen a ion o 50 μgmL −1 .A specific ime poin s (5, 15, 30, 60, 90, 120 and 180 min), 100 μL sample we e collec ed om he basola e al compa men and eplaced wi h he same olume o esh medium. The amoun o pe mea ed BUD in he basola e al compa men was quan ified by HPLC, and he P app was calcula ed, as desc ibed elsewhe e [20]. The expe imen s we e ca ied ou in iplica e. 4.17. In i o ea men es s A e 2 d o s imula ion, bo h apical and basola e al medium we e emo ed, and cells we e washed 3 imes wi h PBS. F ee BUD, BUD@ PSi-HA@LF and BUDAP@PSi-HA@LF we e added in o he apical compa men , p o iding an equi alen BUD dose o 25 μg pe well. A e 4 h o incuba ion, which app oxima es d ug e en ion ime in he in es ine o IBD pa ien s [50], he apical medium was emo ed and eplaced wi h esh Caco-2 medium. The monolaye s we e used bo h o ansepi helial elec ical esis ance (TEER) and IL-8 measu emen s. IL-8 was quan ified by enzyme-linked immunoso ben assay (ELISA) ki s (BD Biosciences) acco ding o he manu ac u e 's p o ocol. 4.18. Fluo escence labelling o nanopa icles FITC labelled PSi-SPM (PSi-FITC) and Cy7 labelled PSi-SPM (PSi- Cy7) we e p epa ed by o e nigh s i ing o 10 mg o PSi-SPM wi h 0.1 mg o FITC-NHS (The mo Fishe Scien ific, USA) and 0.1 mg o Cy7- NHS (Lumip obe, Ge many) in DMF, espec i ely. The ob ained PSi- FITC and PSi-Cy7 we e conjuga ed wi h HA using he EDC/NHS eac- ion as desc ibed abo e o p oduce FITC labelled PSi-HA (PSi-FITC/HA) and Cy7 labelled PSi-HA (PSi-Cy7/HA), espec i ely. 4.19. In i o adhesion o nanopa icles FITC labelled PSi-HA we e used o imaging in all in i o adhesion expe imen s. Heal hy and inflamed mucosa we e simula ed by coa ing Lab-Tek™8-chambe slides (The mo Fishe Scien ific, USA) o 96-well polys y ene pla es (Co ning, USA) wi h mucin om po cine s omach (Sigma-Ald ich, USA) and human ans e in (Sigma-Ald ich, USA), espec i ely [5]. B iefly, mucin solu ion (30 mg mL −1 in HBSS) was added o he chambe slides/pla es and incuba ed a oom empe a u e o 2 h wi h gen le shaking. T ans e in solu ion (500 μgml −1 in PBS) was added o he chambe slides/pla es and incuba ed o e nigh a 37 °C. Chambe slides/pla es incuba ed wi h PBS o e nigh a 37 °C we e used as uncoa ed chambe slides/pla es. A e incuba ion, chambe slides/pla es we e washed wi h PBS o 3 imes. FITC labelled BUDAP@PSi-HA we e dispe sed in PBS a a concen a ion o 50 μgml −1 , and hen added o each well o he chambe slides/pla es. The chambe slides/pla es we e gen ly shaken a oom empe a u e o 1 h. Wells we e hen washed wi h PBS o 3 imes. The chambe slides we e imaged using con ocal mic oscope (Leica TCS SP5 II HCS A, Ge many), and he fluo escence in ensi ies o he pla es we e quan ified using Va ioskan™LUX (The mo Fishe Scien ific, USA). In a second se o expe imen s, ca ionic b anched polye hylenimine (PEI) (∼25,000 g mol −1 , Sigma-Ald ich, USA) (10 mM, pH 4.0) was added o he FITC labelled BUDAP@PSi-HA o con e i s su ace cha ge om nega i e o posi i e, and he adhesion expe imen was pe o med using uncoa ed-, mucin-coa ed, and ans e in-coa ed chambe slides/pla es as desc ibed abo e. 4.20. DSS-induced coli is model Male C57BL/6 mice (6–8 weeks) we e pu chased om Beijing Vi al Ri e Labo a o y Animal Technology Co., L d. Mice we e ed o - mula ed d inking wa e con aining 3% DSS (MW 36–50 kDa; MP Biochemical, Hlki ch, F ance) ad libi um h oughou he expe imen (day 0 o day 14). The body weigh s o mice we e moni o ed and he weigh s we e eco ded as pe cen age o ini ial bodyweigh . Clinical e alua ions including he assessmen o s ool consis ency and he W. Li e al. Bioma e ials 185 (2018) 322–332 330