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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
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493
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494
ma e ials o on-command elease o gues molecules, Chem. Re . 116 (2016) 561−718.
495
[2] Choudha i, Y., Hoe e , H., Libana i, C., Monsuu , F., McCa hy, W., Mesopo ous silica
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d ug deli e y sys ems, in: Shah, N., Sandhu, H., Choi, D.S., Chokshi, H., Malick, A.W. (Eds.),
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Amo phous solid dispe sions, Sp inge , New Yo k, USA, 2014, pp. 665-693.
498
[3] Slowing, I.I., Vi e o-Esco o, J.L., Wu, C.W., Lin, V.S.Y., Mesopo ous silica nanopa icles
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as con olled elease d ug deli e y and gene ans ec ion ca ie s, Ad . D ug Deli . Re . 60
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