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h p://dx.doi.o g/10.1016/j.mic omeso.2016.05.004
h p://hdl.handle.ne /10251/78699
Else ie
Pé ez-Es e e, É.; Ruiz Rico, M.; To e, CDL.; Llo ca Ma ínez, ME.; Sancenón Gala za, F.;
Ma cos Ma ínez, MD.; Amo os Del To o, PJ.... (2016). S abili y o di e en mesopo ous
silica pa icles du ing an in i o diges ion. Mic opo ous and Mesopo ous Ma e ials. 230:196-
207. doi:10.1016/j.mic omeso.2016.05.004.
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
1
S abili y o di e en mesopo ous silica pa icles du ing an in i o
1
diges ion
2
3
Édga Pé ez-Es e ea*, Ma ía Ruiz-Ricoa, C is ina de la To eb,c, Empa Llo cad, Félix Sancenónb,c,
4
Ma ía D. Ma cosb,c, Ped o Amo óse, Ca men Guilleme, Ramón Ma ínez-Máñezb,c,, José Manuel
5
Ba a a
6
a G upo de In es igación e Inno ación Alimen a ia, Uni e si a Poli ècnica de València. Camino
7
de Ve a s/n, 46022, Spain
8
b Ins i u o In e uni e si a io de In es igación de Reconocimien o Molecula y Desa ollo
9
Tecnológico (IDM), Unidad Mix a Uni e si a Poli ècnica de València – Uni e sidad de Valencia.
10
Depa amen o de Química Uni e si a Poli ècnica de València, Camino de Ve a s/n, 46022,
11
Valencia, Spain
12
c CIBER de Bioingenie ía, Bioma e iales y Nanomedicina (CIBER-BBN)
13
d G upo de Mic oes uc u a y Química de Alimen os. Depa amen o de Tecnología de
14
Alimen os, Uni e si a Poli ècnica de València. Camino de Ve a s/n, 46022, Valencia, Spain
15
e Ins i u de Ciència dels Ma e ials (ICMUV), Uni e si a de València, P.O. Box 2085, 46071,
16
Valencia, Spain
17
18
*Co esponding au ho : edpees@up .es
19
20
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
2
Mesopo ous silica ma e ials ha e he abili y o en ap d ugs, nu ien s and unc ional
21
biomolecules and can be able o ac as sma deli e y sys ems capable o con ol and a ge
22
he elease o hei ca go in a pa icula pa o he gas oin es inal ac when adminis a ed
23
o ally. Howe e , he ap ness o hese encapsula ion suppo s in in i o o al con olled elease
24
elies on hei chemical s abili y h ough he diges i e ube. In his con ex , we ha e e alua ed
25
he s abili y o ou di e en mesopo ous silica pa icles, equen ly used as encapsula ing
26
suppo s, du ing an in i o diges ion p ocess comp ising buccal, s omach and in es inal
27
phases. Resul s showed ha a e 4 h o diges ion, he ex u al p ope ies o silica suppo s in
28
he o m o nanopa icles (MCM-41 and UVM-7 nanopa icles) we e los in a ying deg ees,
29
whe eas silica mic opa icles suppo s (MCM-41 and SBA-15 mic opa icles) endu es be e he
30
diges ion p ocess. Mo eo e , he unc ionaliza ion o he su ace wi h N1-(3-
31
ime hoxysilylp opyl)die hylene iamine, an o ganic moie y commonly used in he
32
p epa a ion o pH- esponsi e mesopo ous silica pa icles, esul ed in an imp o emen o he
33
s abili y o he suppo s.
34
35
36
Keywo ds: mesopo ous silica pa icles, in i o diges ion, s abili y, amine- unc ionaliza ion
37
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
3
1. In oduc ion
38
Mesopo ous Silica Pa icles (MSPs) a e ecei ing g ea a en ion in he ield o o al
39
con olled elease due o hei capabili y o imp o e d ug solubili y and s abili y in he
40
gas oin es inal ac (GIT), [1-2] as well as o dosage he ca go along ime (sus ained
41
con olled elease) in speci ic GIT places ( a ge ed con olled elease) [3-5]. These epo ed
42
ea u es, ha con e MSPs in unique sma deli e y sys ems, a e due o hei la ge loading
43
capaci y [6], low oxici y [7] and he ac ha hei su ace can be unc ionalized wi h
44
molecula /sup amolecula ensembles. This las ea u e allows he de elopmen o ga ed-MSPs
45
showing “ze o deli e y” and capable o elease hei ca go on-command in esponse o
46
speci ically designa ed ex e nal s imuli [8-10]. D ug deli e y/ o mula ion echnologies ha can
47
imp o e bioa ailabili y, d ug s abili y and subsequen ly inc ease d ug e ec i eness a e much
48
desi ed in he pha maceu ical sciences [11-12]. In ood echnology, encapsula ion o bioac i e
49
molecules (e.g. i amins, an ioxidan s, phy ochemicals, e c.) may imp o e hei biological
50
s abili y, acili a e componen s handling, mask unpleasan senso ial p ope ies and modula e
51
he bioaccessibili y o he molecule o in e es along he GIT [13].
52
Besides a high loading capaci y, con olled elease and biocompa ibili y, he sui abili y o
53
MSPs in o al con olled elease in in i o applica ions depend on he chemical s abili y o he
54
suppo s hough he whole diges i e ube. Howe e , i is known ha due o he me as abili y
55
o MSPs, silica can be biodeg aded in o silicic acids, including monome ic silicic acid and
56
a ious polysilicic acids wi h di e en polyme iza ion deg ees unde ha sh en i onmen s
57
p o oking a collapse o he po ous s uc u es [14]. In his line, Cauda, Schlossbaue & Bein
58
s udied he biodeg ada ion o colloidal mesopo ous silica nanopa icles (50 nm) in simula ed
59
body luid o ba e, globally unc ionalized, and su ace poly(e hylene glycol)-coa ed colloidal
60
mesopo ous silica nanopa icles in simula ed body luid (pH 7.4) o a pe iod o 1 mon h a 37
61
°C [15]. A e his pe iod o ime, he ex u al p ope ies o he mesopo ous sys em we e los
62
and po es we e blocked because he p ecipi a ion o ino ganic componen s om he
63
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
4
simula ed body solu ion. The s abili y o he pa icles inc eased by su ace unc ionaliza ion
64
wi h poly(e hylene glycol). The deg ada ion beha iou o su ac an -ex ac ed mesopo ous
65
silica in simula ed body luid was also e alua ed by He and co-wo ke s p oposing a h ee-s age
66
deg ada ion p ocess comp ising a as bulk deg ada ion on hou -scale, a silicon concen a ion
67
dec ease s age due o a deposi ion o a calcium/magnesium silica e laye , and a la e
68
con inuous sus ained di usion beyond days [16]. The same yea , Lin, Abadee & Haynes,
69
e alua ed he s abili y o small mesopo ous silica nanopa icles (<50 nm) unc ionalised wi h
70
poly(e hylene glycol) in H2O, phospha e bu e solu ion (PBS) (pH 7.5), and Dulbecco’s
71
modi ied Eagle’s medium (DMEM) wi h 10% e al bo ine se um (FBS) (pH ca. 7.5) [17]. These
72
pa icles exhibi ed long e m s abili y in all hese media a bo h, oom and physiological
73
empe a u e. In a di e en a emp , El Mou abi e al. s udied he s abili y o mesopo ous
74
silica unde acidic condi ions and a loss o ex u al p ope ies o he suppo s was obse ed
75
[18]. The au ho s also ound ha he deg ada ion a e was dependen on he na u e o he
76
acidic media (phospho ic acid ha e s onge impac han hyd ochlo ic o sulphu ic acids) and
77
he kind o mesopo ous silica used in he s udy. Mo e ecen ly, Choi e al. s udied he
78
biodeg ada ion o SBA-15 in bo h, simula ed body luid and in i o [19]. These au ho s ha e
79
shown ha he deg ada ion a e o SBA-15 was a ec ed by he p esence o su ace unc ional
80
g oups and syn hesis me hodologies. Fu he mo e, in i o expe imen showed ha SBA-15
81
deg ade in he animal and po e s uc u e de o ma ion occu s as a unc ion o ime.
82
Mos o hese s udies e alua ed he s abili y o mesopo ous silica nanopa icles. Howe e ,
83
MSPs can be ab ica ed wi h a con olled size om 50 nm o a ew mic ons. When p epa ing
84
sma deli e y sys ems based on MSPs, pa icle size is e y impo an since i condi ions he
85
dis ibu ion and beha io o pa icles in li ing sys ems. In gene al, small MSPs can c oss
86
epi heliums, can be dis ibu ed in he body and be non-speci ically in e nalized by ce ain cells
87
[20]. In con a y, o e sized pa icles (mic opa icles) canno easily c oss physical memb anes in
88
he body, and hus la ge pa icle sizes a e p e e ed o de eloping o ally adminis a ed
89

Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
5
con olled elease de ices [4]. Ha ing in mind he impo ance o pa icle size in o al
90
adminis a ion, i may be o impo ance o s udy he s abili y o mesopo ous silica wi h mic o-
91
sized pa icles. Howe e , s abili y o la ge MSPs has been ba ely s udied. Mo eo e , as a as
92
we know, he e a e no s udies abou he e ec ha he consecu i e p esence o sali a (pH
93
7.5), gas ic (pH 1.2-2) and in es inal luids (pH 7.8-8) ha e on he s abili y o he small and
94
la ge MSPs. Thus, no wi hs anding he wo ks de ailed abo e, a lack o in o ma ion abou he
95
deg adabili y/s abili y o MSPs wi h di e en sizes du ing a whole diges ion is s ill una ailable.
96
The aim o his s udy was o e alua e he s abili y o di e en ba e and unc ionalised
97
mesopo ous silica pa icles di e ing in pa icle size, pa icle shape and po e s uc u e (po e
98
size and wall hickness) du ing a simula ed in i o diges ion. Wi h his pu pose a deep
99
e alua ion o he s abili y o ex u al p ope ies o MSPs du ing he in i o diges ion was
100
pe o med. Desc ip i e s udies we e comple ed wi h he assessmen o po en ial cy o oxici y
101
o diges ed pa icles o hei deg ada ion p oduc s.
102
2. Ma e ials and me hods
103
2.1 Chemicals
104
Te ae hylo hosilica e (TEOS), N-ce yl ime hylammonium b omide (CTAB ), Plu onic
105
P123 (P123), ie hanolamine (TEAH3), sodium hyd oxide (NaOH), hyd ochlo ic acid (HCl), N1-
106
(3- ime hoxysilylp opyl)die hylene iamine (N3), and all chemicals o he p epa a ion o he
107
simula ed diges i e luids we e p o ided by Sigma-Ald ich (Poole, Do se , UK). HPLC g ade
108
ace oni ile was p o ided by Scha lau (Ba celona, Spain). Rhodamine B was acqui ed om
109
Fluka (Missou i, USA).
110
Fo cell cul u e expe imen s, ypan blue solu ion (0.4%) cell cul u e g ade and dime hyl
111
sul oxide (DMSO), phospha e bu e ed saline (PBS) and Dulbecco's Modi ied Eagle's medium
112
(DMEM) wi h glucose, L-glu amine and py u a e o cell cul u e we e p o ided by Sigma-
113
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
6
Ald ich (Poole, Do se , UK). Mc Coy’s 5a Medium and Ke a inocy e Se um F ee Medium, Fe al
114
Bo ine Se um (FBS) and ypsin we e pu chased om Gibco (Li e Technologies, Mad id, Spain).
115
Cell p oli e a ion eagen WST-1 was pu chased om Roche Applied Science (Ba celona,
116
Spain).
117
2.2 Mesopo ous silica pa icles syn hesis
118
Syn hesis o he ou di e en silica pa icles was ca ied ou ollowing he p ocedu es
119
desc ibed p e iously [4].
120
MCM-41 (M) was syn hesized ollowing he so-called “a ane ou e”, using CTAB as he
121
s uc u e-di ec ing agen and a mola a io ixed o
122
7TEAH3:2TEOS:0.52CTAB :0.5NaOH:180H2O. The p ocedu e consis ed in adding CTAB o a
123
solu ion o TEAH3 and NaOH con aining TEOS a 118 °C. A e dissol ing CTAB in he liquo ,
124
wa e was slowly added wi h igo ous s i ing a 70 °C o o m a whi e suspension. This
125
mix u e was aged a oom empe a u e o e nigh .
126
Nanopa icula ed MCM-41 (N) was syn hesized using he ollowing p ocedu e: NaOH was
127
added o he CTAB solu ion, ollowed by adjus ing he solu ion empe a u e o 95 °C. TEOS
128
was hen added d opwise o he su ac an solu ion. The mix u e was allowed o s i o 3 h o
129
gi e a whi e p ecipi a e.
130
UVM-7 (U) was syn hesised using, once again, he “a ane ou e”. The mola a io o he
131
eagen s in he mo he liquo was ixed a 7TEAH3:2TEOS:0.52CTAB :180H2O. The TEOS/TEAH3
132
mix u e was hea ed o 120 °C un il no elimina ion o e hanol was obse ed. The mix u e was
133
cooled o 90 °C and he CTAB was added g adually in small po ions, ollowed by wa e . The
134
mix u e was aged o 24 h.
135
The SBA-15 (S) sample was syn hesized using P123 as he s uc u e-di ec ing agen wi h
136
he eac an mola a ios: 0.017P123:1.0TEOS:6HCl:196H2O. The p epa a ion was ca ied
137
mixing an aqueous solu ion o P123 wi h HCl solu ion, and s i ing o 2 h, a e which he silica
138
sou ce, TEOS, was added. This inal mix u e was s i ed o a u he 20 h.
139
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
7
A e he syn hesis, he di e en solids we e eco e ed, washed wi h deionised wa e ,
140
and ai -d ied a oom empe a u e. The as-syn hesized solids we e calcined a 550 °C using an
141
oxidan a mosphe e o 5 h in o de o emo e he empla e phase.
142
The pa icles we e also unc ionalised wi h N1-(3- ime hoxysilylp opyl)die hylene iamine
143
(N3). In pa icula , 1 g o he di e en MSPs we e suspended in 40 mL o ace oni ile and an
144
excess o N3 (4.3 mL, 15.0 moll g-1) was hen added. Final mix u es we e s i ed o 5.5 h a
145
oom empe a u e. Finally, he solids we e il e ed o , washed wi h 30 mL o deionised wa e ,
146
and d ied a oom empe a u e.
147
2.3 Simula ed diges ion p ocedu e
148
An in i o diges ion model consis ing o mou h, gas ic and in es inal phases desc ibed by
149
Ve san oo e al. was used o simula e he ypical chemical composi ion, pH and esidence
150
ime pe iods o each o he h ee main compa men s o he GIT [21]. A schema ic
151
ep esen a ion o he in i o diges ion model is p esen ed in Figu e 1. The pH alues o he
152
diges i e juices we e checked and, i necessa y, adjus ed o he app op ia e in e al wi h
153
NaOH (1 M) o HCl (37% w/w).
154
155
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
8
156
157
Figu e 1. Schema ic ep esen a ion o he in i o diges ion p ocess. The in i o diges ion model desc ibes a h ee-
158
s ep p ocedu e simula ing he diges i e p ocesses in mou h, s omach and small in es ine. In each compa men , he
159
ma ix is incuba ed a 37 °C o a ime ele an o he compa men . The diges ion is ini ia ed by addi ion o
160
a i icial sali a o he ma e ial. Subsequen ly, gas ic juices and in es inal luids a e added o simula e he diges i e
161
p ocesses in s omach and small in es ine, espec i ely. A e each o hese s eps, samples we e aken o conduc
162
cha ac e iza ion p ocedu es. Cha ac e iza ion ypically in ol es mic oscopy, PXRD, size dis ibu ion, ze a po en ial,
163
N2 adso p ion-deso p ion iso he ms, 29Si RMN, silicon analysis and biocompa ibili y using WST-1 es .
164
165
2.4 Cha ac e iza ion o he mesopo ous silica pa icles
166
All ma e ials, as syn he ized and a e a simula ed diges ion p ocess, we e cha ac e ized
167
by s anda d p ocedu es: i.e. X- ay di ac ion (XRD), N2 adso p ion-deso p ion iso he ms,
168
ansmission elec on mic oscopy (TEM), ield emission scanning elec on mic oscopy (FESEM),
169
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
15
he o de N (wall hickness = 1.91), closely ollowed by M (wall hickness = 2.05) and U (wall
294
hickness = 2.22). The di e ences in wall hickness among N (less han 2 nm) and M and U
295
(mo e han 2 nm) migh be due o he syn hesis ou e. N and U ollow he a ane ou e o
296
syn hesis, a p ocedu e ha p o ides wide amewo k walls.
297
Fo i s pa , S wi h a wall hickness o 2.56 nm seemed o be he pa icle ha be e conse e
298
he hexagonal s uc u e o he pa icle. In ac he di ac og am o diges ed S was e y simila
299
han ha o he undiges ed pa icle meaning ha mesopo ous a angemen is p ese ed
300
du ing he in i o diges ion p ocedu e. In sho , he po e wall hickness seems o be he key
301
ac o o p ese e he mesos uc u e in eg i y, being he silica s abili y highly a ou ed when
302
he po e wall hickness inc eases.
303
Ni ogen so p ion da a (Fig 3) show a educ ion o he adso bed N2 olume in all solids,
304
which sugges ed ha he diges ion p ocedu e p o okes a loss o speci ic su ace a ea and po e
305
olume. This educ ion is mo e ma ked in he case o N (177 m2 g-1; 0.29 cm3 g-1) and U (372 m2
306
g-1; 0.78 cm3 g-1) ha in M (321 m2 g-1; 0.46 cm3 g-1) and S (368 m2 g-1; 0.62 cm3 g-1) in consonance
307
wi h XRD pa e ns. This loss o su ace a ea and po e olume as a consequence o he con ac
308
wi h biological media has also been obse ed by o he au ho s [15,18]. In hese wo ks, he
309
loss o ex u al p ope ies o silica suppo s was associa ed wi h a p og essi e elimina ion o
310
he po osi y by dissolu ion o he silica o by po e blockage due o p ecipi a ion o ino ganic
311
compounds on o he su ace o he po ous silica. The combined esul o silica edisolu ion and
312
sal p ecipi a ion has a ma ked e ec on he o m o he iso he ms. In ac , only SBA-15 shows
313
a e diges ion a N2 adso p ion-deso p ion iso he ms quali a i ely simila o he o iginal one,
314
wi h a well-de ined adso p ion s ep a ela i e p essu e alues in he 0.6-0.8 ange. In he case
315
o he emaining silica suppo s wi h hicke walls (M and U), he loss o su ace a ea and
316
olume seems o be mo e p onounced han he mesos uc u al diso de e idenced h ough
317
XRD. A his poin , p obably he exis ence o small mesopo es o samples M, N and U (ca. 2.5
318
nm) leads o an easie po e blocking h ough sal ep ecipi a ion wi h he subsequen
319

Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
16
signi ican loss o su ace and po e olume. In con as , he la ge alues o he oids and he
320
walls in he S suppo a ou s he p ese a ion o he mesos uc u e and hinde po e blocking.
321
322
323
Figu e 3. Ni ogen adso p ion-deso p ion iso he ms o mic opa icula ed MCM-41 (M) (a), nanopa icula ed MCM-
324
41 (N) (b), SBA-15 (S) (c) and UVM-7 (U) (d) be o e (i) and a e (ii) he in i o diges ion p ocedu e.
325
326
To u he unde s and which o hese mechanisms a e in ol ed in he diges ion-induced
327
silica deg ada ion, in pa allel o XRD and N2 abso p ion-deso p ion iso he ms expe imen s,
328
TEM and FESEM obse a ions o he ou silica suppo s we e ca ied ou . Figu e 4 shows
329
FESEM and TEM pic u es o di e en ba e pa icles be o e and a e he in i o diges ion
330
p ocess. This igu e allows obse ing no only he pa icle size and shape o he single pa icle,
331
bu also pa icle po osi y.
332
A e he diges ion, wo pa icles did no modi y hei appea ance (FESEM) and po e
333
in eg i y (TEM). These pa icles a e M and S which a e pa icle wi h size in he mic oscale. This
334
implies ha o hese pa icles he loss o o de obse ed in XRD was no p o oked by a
335
collapse o he mesos uc u e, bu p obably by he o ma ion o a small olume ac ion o
336
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
17
“gel” consis ing mos ly o (SiOH2)n18 o he adso p ion o calcium and phospha e ions p esen
337
in he diges ion luids on he silica su ace o ming a hyd oxyapa i e phase [15, 23-25].
338
In con as , suppo s based on nanopa icula ed ma e ials, N and U biodeg aded wi h a
339
modi ica ion o he appea ance o bo h, su ace and po e s uc u e as a consequence o he
340
diges ion p ocedu e. Conc e ely, he mos a ec ed suppo was N. A e he whole diges ion
341
p ocess, N and U nanopa icles los he uni o mi y o po e s uc u e (see TEM images) and
342
despi e keeping i s pa icle size and shape, he e is a clea al e a ion o he su ace (FESEM). In
343
hose cases, besides he po e closu e p o oked by he appa i ion o new phases, he dec ease
344
o he mesos uc u e obse ed by XRD and N2 adso p ion-deso p ion iso he ms can also be
345
o igina ed, a leas pa ially, by po e collapse.
346
347
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
18
348
Figu e 4. Cha ac e iza ion o pa icle size, pa icle shape and po e sys em o ba e MSPs be o e and a e he in i o
349
diges ion p ocedu e (IDP). MCM-41 (M), MCM-41 nano (N) SBA-15 (S) and UVM-7 (U).
350
351
Silica deg ada ion necessa ily implies he b eak o siloxane bonds wi h he subsequen
352
gene a ion o silanol g oups and his was co obo a ed h ough he e olu ion o he 29Si NMR
353
spec a be o e and a e diges ion. Fo his s udy we selec ed wo samples (S and N) ha can
354
be conside ed as ep esen a i e o he wo obse ed beha iou s wi h low (solid S) and high
355
(solid N) biodeg ada ion acco ding o TEM images. The 29Si NMR spec a a e shown in Figu e 5.
356
While in he case o sample S, he diges ion does no a ec he p opo ion o Q4:Q3:Q2
357
(68:30:2) si es, a dec ease o he Q4 si es om 65% o 60% is obse ed in he case o he N
358
sample (Q4:Q3:Q2 om 65:31:4 o 60:33:7).
359
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
19
360
361
Figu e 5. 29Si NMR spec a o SBA-15 (a) and nanopa icula e MCM-41 (b), be o e (i) and a e (ii) he in i o
362
diges ion p ocedu e.
363
364
Ha ing in mind hese esul s, i is appa en ha all s udied MSPs a e al e ed as a
365
consequence o he in i o diges ion p ocess. Howe e , he deg ada ion deg ee depends on
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he ype and size o he pa icles. In his line, El Mou abi e al. s udied he s uc u e al e a ion
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o se e al po ous silica suppo s di e ing in pa icle size, pa icle shape, po e-size dis ibu ion,
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speci ic su ace a ea, po e olume and a e age o po e diame e caused by imme sion in acid
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solu ions and ound ha he deg ada ion o he suppo s was no ob iously in luenced by
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ex u al p ope ies o he pa icles [18]. Ne e heless, in ou s udy, i seems o be clea ha
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pa icle size and wall hickness seem o be essen ial pa ame e s ha condi ion deg ada ion.
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In o de o co ela e he impac o each o he phases o he diges ion wi h he pa icle’s
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deg ada ion, a u he expe imen was done. Fo his MCM-41 nanopa icles (solid N) we e
374
selec ed gi en ha his was he mos a ec ed suppo by he whole diges ion p ocess. Fo his
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pu pose, N was pu in con ac wi h wa e o 4 h. In pa allel, a ypical in i o diges ion p ocess
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(4 h) was pe o med. A e each o hese s eps, samples we e washed and obse ed by TEM.
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Figu e 6 shows TEM mic og aphs o N a e 4 h in con ac wi h wa e (a) and a e each o he
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phases o he in i o diges ion p ocess: buccal (b), gas ic (c) and in es inal (d). As obse ed,
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he pa icle size (ca. 100 nm) did no a y along he diges ion sugges ing ha pa icle s uc u e
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emains unal e ed a e he whole diges ion p ocess. Mo eo e , su ace and po osi y o MCM-
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41 emained unchanged a e 4 h in wa e , meaning ha pa icles do no collapse easily in
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wa e solu ion. Pa icles a e also in ac a e he 5 min o con ac wi h simula ed sali a.
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Howe e , pa icles change d ama ically a e he 2 h o gas ic phase. In pa icula , a e his
384
diges ion s ep, pa icles loss clea ly hei sphe ical shape and o de ed po ous con o ma ion
385
and become i egula shaped sphe es wi h diso de ed po osi y. Li le di e ences among
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pa icles obse ed a e gas ic and a e bo h, gas ic and in es inal phases we e obse ed
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sugges ing ha once he diges i e solu ion is neu alized by he addi ion o in es inal juices,
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he deg ada ion p ocess s opped.
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390
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Figu e 6. TEM images showing pa icle size, pa icle shape and po e sys em o ba e MCM-41 nanopa icles
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(solid N) a e 4 h in wa e , and buccal, gas ic and in es inal phases o he in i o diges ion p ocedu e.
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These indings con i m ha gas ic phase (pH 2) is he esponsi e o pa icle’s deg ada ion. The
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ole o acids in po ous silica deg ada ion has p e iously been desc ibed [18]. These au ho s
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ealised ha p o ons play a ole in he acidic al e a ion p ocess o silica. Mo eo e , hey
397
poin ed ou ha in acidic condi ions, anions (i.e. SO42-, Cl-, PO43-) p esen in he media can ac
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as nucleophilic ca alys s accele a ing he deg ada ion eac ions. Ha ing in mind he
399

Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
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composi ion o he wo luids comp ising gas ic phase o he diges ion (i.e. sali a and gas ic
400
juice) i can be say ha all hese deg ada i e species (i.e. NaSO4, NaH2PO4, HCl, NaCl, KCl, CaCl2
401
and NH4Cl) a e p esen in ou s udies.
402
403
3.3 E ec o in i o diges ion in mic os uc u e o amine- unc ionalised pa icles
404
To in es iga e i he unc ionaliza ion o he su ace o he pa icles wi h ce ain o ganic
405
molecules ha e any in luence in he p ese a ion o he s uc u e du ing he in i o diges ion,
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he ou pa icles objec o s udy we e unc ionalised wi h N1-(3-
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ime hoxysilylp opyl)die hylene iamine. This polyamine is one common o ganic molecule
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used o p epa e capped mesopo ous silica pa icles able o modula e payload elease in
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esponse o pH changes [26]. A e unc ionaliza ion, pa icles we e subjec ed o he diges ion
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p ocess desc ibed in Figu e 1. Diges ed pa icles we e washed wi h wa e , d ied and
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cha ac e ized by XRD, TEM and FESEM.
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Figu e 2 shows XRD pa e ns o amine- unc ionalized pa icles be o e (ii) and a e (i ) he
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in i o diges ion p ocess. In con as o ba e pa icles, unc ionalised pa icles showed he
414
same di ac ion peaks be o e and a e he diges ion, which indica e a p ese a ion o he
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po ous s uc u e a e he diges ion p ocedu e. The s uc u e p ese a ion was con i med by
416
mic oscopic analysis. As obse ed in Figu e 7, mo phology, pa icle size and po ous s uc u e
417
o he di e en amine- unc ionalized suppo s is e y simila be o e and a e he in i o
418
diges ion p ocess. These s udies poin ou he ole played by amines in he p o ec ion o
419
po ous silica, especially in he nanopa icles (solids N and U), agains he a ack by acids and
420
chemical species p esen in he diges i e juices. The p e en ion o silica deg ada ion a e
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o ganic unc ionaliza ion has also been obse ed by o he au ho s. Lin e al. ound ha
422
deg aded Si amoun s om 42 nm diame e silica nanopa icles we e g ea e han ha om
423
he equi alen pegyla ed nanopa icles a e bo h 10 days in deionized wa e and PBS a oom
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empe a u e and 37 °C [17]. Cauda e al. also obse ed ha he a achmen o a poly(e hylene
425
Pé ez-Es e e e al. 2016. Mic opo Mesopo Ma e
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glycol)-laye on he ou e su ace o colloidal mesopo ous silica s abilized he pa icles by
426
educing he a e o deg ada ion in simula ed body luid a 37 °C o 1 mon h [15]. The
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p e en i e e ec o unc ional g oups a ached o he su ace o he silica suppo s on he
428
deg ada ion o po ous silica could be a ibu ed o he capabili y o hese molecules o inhibi
429
he a ack caused by acids and ca aly ic anions as well as o p e en he adso p ion o
430
calcium/phospha e ca ions and he ea angemen o silicon species on a new “gel” ac ion on
431
he walls o he pa icles.
432
433
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Figu e 7. Cha ac e iza ion o pa icle size, pa icle shape and po e sys em o amine- unc ionalised MSPs be o e and
435
a e he in i o diges ion p ocedu e. MCM-41 (M), MCM-41 nano (N) SBA-15 (S) and UVM-7 (U).
436
437
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In ou s udy, acco ding o CNH elemen al analysis da a, he pos - unc ionaliza ion deg ee
438
o ou samples was comp ised in he 0.14-0.15 mol o N1-(3- ime hoxysilylp opyl) g oups pe
439
100 g o sample. This alue suppose a densi y o unc ional g oups o ca. 1-1.5 g oups/nm2
440
assuming ha hei inco po a ion is e ec i e along he whole su ace (ex e nal and in e nal).
441
As i is well known, he leng h o his o ganic g oup cons i u es a se ious d awback o achie e a
442
good di usion and dispe sion along he mesopo es. Then, a ce ain accumula ion and
443
condensa ion o he o ganic g oups on he ex e nal su ace and in he en ance o he
444
mesopo es is expec ed. This ela i ely hick shell seems o be he esponsible o he s abili y
445
o he silica suppo s a e diges ion. Mo eo e , in ou case, amine g oups a ached o
446
pa icle’s su ace can also able o locally neu alize he acidic en i onmen c ea ed by HCl.
447
448
3.4 E ec o in i o diges ion in mac oscopic s uc u e
449
To de e mine he changes o MSPs s uc u es and agg ega ion s a e along he whole in i o
450
diges ion p ocedu e, con ocal lase scanning mic oscopy (CLSM) mic og aphs we e aken upon
451
s aining M, N, S and U suppo s a di e en s age o he diges ion wi h hodamine B (Fig 8).
452
This s udy e ealed ha all he pa icles ended o o m agg ega es in wa e . This end o
453
o m la ge agg ega es, especially obse ed in N and U, is in acco dance wi h esul s p e iously
454
epo ed [27,28]. Figu e 8 also allows obse ing ha o all pa icles he gas ic phase end o
455
p o oke an enla gemen o pa icles agg ega es, and ha in none o he cases diges ion
456
igge ed he loss o mac oscopic pa icle s uc u e.
457
458
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459
Figu e 8. Cha ac e iza ion o pa icle size and pa icle shape o ba e MSPs be o e and a e he in i o diges ion
460
p ocedu e. MCM-41 mic o (M), MCM-41 nano (N), SBA-15 (S) and UVM-7 (U).
461
462
Agg ega ion endency obse ed in gas ic phase o all pa icles was con i med by pa icle
463
size dis ibu ion measu emen s using ligh di ac ion. As shown in Figu e 9, acco ding o
464
pa icles’ g ain size in di e en diges i e media, S and M mic opa icles, exhibi ed a size
465
dis ibu ion in he ange 0.5-2 m, while size dis ibu ion o N and U anged om 5 up o 40
466
m. The inse g aphs o he same igu e shows size dis ibu ion o nano-sized silica p esen in
467
he diges ion luids. As p esumable, only wo samples (N and U) also shown pa icles in he
468
100-200 nm ange, co esponding o hose pa icles no pa icipa ing in he pa icle’s clus e s.
469
470