Double Drug Delivery Using Capped Mesoporous Silica Microparticles for the Effective Treatment of Inflammatory Bowel Disease

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00041

Silica mesoporous microparticles loaded with both rhodamine B fluorophore (S1) or hydrocortisone (S2), and capped with an olsalazine derivative, are prepared and fully characterized. Suspensions of S1 and S2 in water at an acidic and a neutral pH show negligible dye/drug release, yet a notable delivery took place when the reducing agent sodium dithionite is added because of hydrolysis of an azo bond in the capping ensemble. Additionally, olsalazine fragmentation induced 5-aminosalicylic acid (5-ASA) release. In vitro digestion models show that S1 and S2 solids are suitable systems to specifically release a pharmaceutical agent in the colon. In vivo pharmacokinetic studies in rats show a preferential rhodamine B release from S1 in the colon. Moreover, a model of ulcerative colitis is induced in rats by oral administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) solutions, which was also used to prove the efficacy of S2 for colitis treatment. The specific delivery of hydrocortisone and 5-ASA from S2 material to the colon tissue in injured rats markedly lowers the colon/body weight ratio and the clinical activity score. Histological studies showed a remarkable reduction in inflammation, as well as an intensive regeneration of the affected tissues.

Full text of DOI:10.1021/acs.molpharmaceut.9b00041

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Article
Double drug delivery using capped mesoporous silica microparticles
for the effective treatment of Inflammatory Bowel Disease
Adrián H Teruel, Édgar Pérez-Esteve, Isabel Gonzalez-Alvarez, Marta Gonzalez-Alvarez, Ana M.
Costero, Daniel Ferri, Pablo Gaviña, Virginia Merino, Ramon Martinez-Manez, and Felix Sancenón
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00041 • Publication Date (Web): 16 Apr 2019
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Double drug delivery using capped mesoporous
silica microparticles for the effective treatment
of Inflammatory Bowel Disease
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Adrián H Teruel,1,2 Édgar Pérez-Esteve,1 Isabel González-Álvarez,3 Marta González-Álvarez,3 Ana
M. Costero,1,2,4 Daniel Ferri,1,2,4 Pablo Gaviña,1,2,4 Virginia Merino,5 Ramón Martínez-
Máñez,1,2,6,7,* and Félix Sancenón1,2,6,7
1 Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo
Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Camino de
Vera s/n, 46022 Valencia, Spain.
2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN).
3 Departamento de Ingeniería, Sección de Farmacia y Tecnología Farmacéutica, Universidad
Miguel Hernandez, 03550, Alicante, Spain.
4 Departamento de Química Orgánica, Universitat de València, Doctor Moliner 50, Burjassot,
46100, Valencia, Spain.
5 Departamento de Farmacia y Tecnologia Farmacéutica, Universitat de València 46100,
Valencia, Spain.
6 Unidad Mixta de Investigación en Nanomedicina y Sensores. Universitat Politècnica de
València, Instituto de Investigación Sanitaria La Fe, Valencia, Spain.
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7 Unidad Mixta UPV-CIPF de Investigación en Mecanismos de Enfermedades y Nanomedicina,
València, Universitat Politècnica de València, Centro de Investigación Príncipe Felipe, Valencia,
Spain.
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KEYWORDS: Mesoporous silica microparticles, gated materials, smart drug delivery materials,
colon targeted release, inflammatory bowel disease
ABSTRACT
Silica mesoporous microparticles loaded with both rhodamine B fluorophore (S1) or
hydrocortisone (S2), and capped with an olsalazine derivative, are prepared and fully
characterized. Suspensions of S1 and S2 in water at an acidic and a neutral pH show negligible
dye/drug release, yet a notable delivery took place when the reducing agent sodium dithionite
is added due to a hydrolysis of an azo bond in the capping ensemble. Besides, olsalazine
fragmentation induced 5-aminosalicylic acid (5-ASA) release. In vitro digestion models show
that S1 and S2 solids are suitable systems to specifically release a pharmaceutical agent in the
colon. In vivo pharmacokinetic studies in rats show a preferential rhodamine B release from S1
in the colon. Moreover, a model of ulcerative colitis is induced in rats by oral administration of
2,4,6-trinitrobenzenesulfonic acid (TNBS) solutions, was also used to prove the efficacy of S2
for colitis treatment. The specific delivery of hydrocortisone and 5-ASA from S2 material to the
colon tissue in injured rats markedly lowers the colon/body weight ratio and the clinical
activity score. Histological studies showed a remarkable reduction in inflammation, as well as
an intensive regeneration of the affected tissues.
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1. INTRODUCTION
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Inflammatory bowel disease (IBD) is an idiopathic autoimmune, inflammatory and chronic
disease. There are essentially two main IBD subtypes: ulcerative colitis (UC) and Crohn’s
disease (CD). However this classification is not always obvious because clinical and pathologic
features substantially overlap.1-11 The IBD etiology is still poorly understood, but all the facts
indicate a multifactorial origin, where genetics, environmental, and intestinal microbiota are
the main factors. Since IBD are chronic diseases with a low mortality, continuous incidence or
newly diagnosed patients increase the number of prevalent cases. The whole costs of IBD are
quite overwhelming. More than 1,000,000 of people are estimated to suffer from IBD (just in
USA), and only the direct medical costs exceeded US$ 6 billion in 2004.12,13 In Canada over
200,000 people suffer IBD, with more than CDN$ 1.2 billion spent on direct healthcare costs
per year.14 Moreover, over 3,000,000 of European patients suffer from IBD, and the direct
healthcare spent is calculated to be over €5 billion per year.15 The biggest contributors in these
healthcare-related costs are pharmaceuticals, hospitalizations, surgery and outpatient care.16
Moreover, non-direct IBD costs (e.g. lost work productivity), exceed even direct costs. Besides,
in the last decades, developing countries (in Middle East, South America and Asia) have seen
an important emergence on IBD incidence and prevalence.17-21
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Considering together the above mentioned facts, plus the lack of a definitive solution
available on the market, the development of new strategies to treat IBD are of interest.
Conventional IBD therapy includes 5-aminosalicylic acid (5-ASA), corticosteroids and
immunosuppressants.22 Steroids were the cornerstone of ulcerative colitis therapy for several
years due to their proved anti-inflammatory efficacy. However, the therapeutic efficacy of
steroids in IBD is controversially affected by serious secondary effects. As an alternative, in the
last decade, biological agents (especially anti-TNF (tumor necrosis factor) therapies) have been
introduced to be used in UC/CD patients who failed conventional therapy.23,24 As an alternative
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to these therapies, nano- and microparticle as drug carriers for oral administration have been
tested in experimental colitis models to increase drug efficacy.25-27 However, among different
nano- and microparticles, mesoporous silica particles (MSPs) have still been barely studied as
carries for IBD treatment.28,29 As containers of selected cargo molecules, mesoporous silica
nano- or microparticles have been broadly studied in recent decades for their promising
characteristics, such as their biocompatibility, large specific surface areas (up to 1200 m2 g-1),
customizable pore size (within the 2-10 nm diameter range), large load capacity,
homogeneous porosity, tunable particle, inertness and good thermal stability. Compared to
organic nanocarriers, mesoporous silica materials are generally more resistant to pH,
variations in temperature and mechanical stress, which renders them improved capability to
protect molecules and biomolecules when they come into contact with body fluids. Besides,
MSPs can be decorated with selected molecular or supramolecular entities to either enhance
selectivity to cells and tissues or control the delivery of certain entrapped cargos at will using
specific external stimuli.30-38
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Considering the urge to find a better solution for the treatment of IBD, we report herein the
synthesis and characterization of mesoporous silica microparticles loaded with rhodamine B or
hydrocortisone and capped with an olsalazine derivative (see Scheme 1). As a consequence of
the grafting of bulky olsalazine groups on the external surface of the loaded inorganic
framework, the prepared microparticles are unable to release the entrapped cargo. However,
when a reducing agent such as sodium dithionite (which mimic the azoreductase enzyme
activity of the colon microbiota) is present the olsalazine derivative is hydrolyzed and yields 5-
aminosalicylic acid (5-ASA). This hydrolysis process induces pore opening and delivery of the
entrapped cargo (i.e. rhodamine B or hydrocortisone). As a novelty, one of the prepared
mesoporous microparticles is able to deliver two drugs at the same time; i.e. hydrocortisone
(located in the pores of the inorganic silica scaffold) and 5-ASA (product of the hydrolysis of
the gating olsalazine). The effect of the co-delivery of both drugs is studied in an in vivo IBD rat
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model. The information obtained by parameters such as the clinical activity score, the
colon/body weight ratio and the histological evaluation, confirms the usefulness of these
microparticles system to treat IBD.
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OH
HOOC
COOH
NH2
HO
OH
HO
O
O
OH HO
H2N
N
N
N
N
H2N
NH2
HO
OH
OH
HO
NH
Si
HN
Si
HN
Si
NH
Si
O
O
O
O
O
O O
O
O O
O
O O
O
O O
azoreductor
rhodamine B (S1) / hydrocortisone (S2)
Scheme 1. Representation of rhodamine B (S1) or hydrocortisone (S2) loaded silica
mesoporous particles capped with an olsalazine derivative. Delivery of both cargos (rhodamine
B or hydrocortisone) is induced by an azoreductor.
2. EXPERIMENTAL SECTION
2.1. General Techniques. Transmission electron microscopy (TEM), scanning transmission
electron microscopy (STEM), thermogravimetric analysis (TGA), N2 adsorption-desorption
isotherms, powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR),
nuclear magnetic resonance (NMR), fluorescence spectroscopy and HPLC techniques were
used in order to characterize the prepared materials. The instruments used were: JEOL JEM-
1010 microscope for TEM images acquisitions. Bruker D8 Advance diffractometer (CuK
radiation) for PXRD measurements. TGA/SDTA 851e Mettler Toledo balance for TGA, using air
(80 mL/ min) as an oxidant atmosphere with the following heating program: heating ramp of
10 °C/min from 393 to 1273 K, then an isothermal heating step at 1273 K for 30 minutes.
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Micromeritics ASAP 2010 automated analyzer for N2 adsorption-desorption isotherms
recording, samples were degassed at 120 °C in a vacuum overnight. The specific surface areas
were calculated from the adsorption data within the low-pressure range using the BET
(Brunauer–Emmett–Teller) model. Pore size was determined following the BJH (Barrett-
Joyner-Halenda) method. Bruker AV400 spectrometer to measure 1H NMR spectra. Bruker
Tensor 27 equipment to record infrared spectra. PerkinElmer EnSpire Multimode Plate Reader
for fluorescence measurements. Hitachi LaChrom Elite HPLC with a UV detector (L-2400) and
an auto-sampler (L-2200) for HPLC analysis, using Kromaphase 100 C18 column (150 mm × 4.6
mm i.d., 5 μm particle size) for separations.
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2.2. Chemicals. The chemicals tetraethylorthosilicate (TEOS), 1-hexadecyltrimethylammonium
bromide (CTABr), sodium hydroxide (NaOH), triethanolamine (TEAH3), N-hydroxysuccinimide
(NHS), N,N′-dicyclohexylcarbodiimide (DCC), (3-aminopropyl)triethoxysilane, rhodamine B,
triethylamine, hydrocortisone, sodium dithionite and 2,4,6-trinitrobenzene sulfonic acid
solution (TNBS) were purchased from Sigma-Aldrich. Sodium acetate anhydrous, potassium
chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen
phosphate, hydrochloric acid and all the solvents were provided by Scharlab. Olsalazine
sodium salt (Mordant yellow 5) was purchased from Molekula. The animal facilities supplied
isoflurane pentobarbital (dolethal®). All the chemicals were used as received.
2.3. Synthetic protocols.
2.3.1. Synthesis of mesoporous silica microparticles (S0). We used the “atrane route” to
synthesize the micro-sized mesoporous silica particles.39 In a typical synthesis, TEAH3 (25.8 g,
0.17 mol) and sodium hydroxide (2 mL, 6 M) were mixed and the obtained solution was heated
to 120 oC and then cooled down to 70 oC. Afterward, TEOS (11 mL, 0.045 mol) was added to
the initial solution that was then heated to 120 oC. Next, solution was left to cool down and
the surfactant CTABr (4.7 g, 0.013 mol) was added at 118 °C. At this time, distilled water (80
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mL) was slowly added under vigorous stirring at 70 °C. The resultant mixture was aged at 100
oC in an autoclave for 24 h. The obtained powder was filtrated and washed with abundant
water. In the last step of the protocol, the solid is dried at 70 °C. To obtain the solid (S0), a
calcination process at 550 ºC is performed in an oxidant atmosphere during 5 h in order to
remove the surfactant template from the as-synthesized microparticles.
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2.3.2. Synthesis of 1a. Olsalazine sodium salt (0.5 g, 1.65 mmol) was dissolved in an acidic
solution of pH≈0 (28.5 mL of distilled water and 1.5 mL 37% HCl solution). The resultant
solution was stirred for 5 min at room temperature and then centrifuged. The supernatant was
discarded and the resulting protonated product (1a, 0.45 g, 1.49 mmol, 90% yield) was
recovered and dried at 70 oC for 24 h. This protocol was repeated 4 times.
2.3.3. Synthesis of 1. DCC (1.04 g, 5 mmol) and NHS (0.59 g, 5 mmol) were dissolved in
anhydrous THF (25 mL). Afterward, this mixture was then poured over a solution of compound
1a (1.53 g, 5 mmol) in anhydrous THF (25 mL). The crude reaction was stirred during 5 h at
room temperature in an argon atmosphere. A white-yellowish precipitate of dicyclohexylurea
(DCU) was discarded by centrifugation. The supernatant was left stirring for 15 h in an argon
atmosphere at room temperature. After 15 h the crude reaction was recentrifuged to remove
the newly formed DCU. Then, (3-aminopropyl)triethoxysilane (1b, 1.2 mL, 5 mmol) was slowly
added to the crude reaction and was stirred for a further 24 h period (room temperature,
argon atmosphere). The solvent was removed using a rotary evaporator to isolate the final
product 1 (yellow-orange oil, 2.05 gr, 4.05 mmol, 80% yield). 1H NMR (400 MHz, DMSO-D6):  =
8.31 (d, 2H), 7.98 (dd, 2H), 7.05 (d, 2H), 3.65 (m, 6H), 3.36 (t, 2H), 1.67 (m, 2H), 1.17 (m, 9H),
0.68 (t, 2H) ppm. HRMS-EI m/z: calcd for C23H31N3O8Si: 505.1880; found: 436.3410 (M-
2(CH3CH2OH)+Na) and 391.2828 (M-3(CH3CH2OH)+Na).
2.3.4. Synthesis of S1. Solid S0 (1 g) was suspended in a THF (40 mL) solution containing
rhodamine B (400 mg, 0.8 mmol/g of solid) under argon. Suspension was stirred at room
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temperature overnight. Afterward, compound 1 (2.05 g, 4.05 mmol/g of solid) was dissolved in
anhydrous THF (25 mL) and this mixture was added to the solid/rhodamine B suspension and
then stirred for 6 h at room temperature in an argon atmosphere. S1 particles were isolated as
a red solid, washed with H2O and EtOH, and finally dried in a vacuum for 24 h.
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2.3.5. Synthesis of S2. Solid S0 (1 g) was suspended in 40 mL of THF/hydrocortisone solution
(296 mg, 0.8 mmol/g of solid) under argon. Suspension was stirred at room temperature
overnight. Next, compound 1 (2.05 g, 4.05 mmol/g of solid) was dissolved in anhydrous THF
(25 mL) and was then added to the solid/hydrocortisone suspension. The obtained suspension
was stirred under argon and at room temperature during 6 h. S2 particles were isolated as a
yellow solid, washed with EtOH and H2O, and finally dried for 24 h in a vacuum.
2.4. Controlled release studies. The controlled release behavior of solids S1 and S2 at pH
values found along the gastrointestinal tract (GIT) (2.0, 4.5 and 7.4) and in the presence of a
chemical reducing agent (sodium dithionite), to simulate the azo-reductive environment, were
assessed. For this purpose, corresponding solid (2 mg) was suspended in 2 mL of the aqueous
solution at the selected pH. Aliquots were taken at scheduled times up to 24 h and centrifuged
to discard the solid before analyzing. The controlled release performances of both solids were
tested under in vitro digestion conditions using simulated solutions for GIT fluids including
saliva, gastric juice, duodenal juice, bile and colon (in which azoreductor agent sodium
dithionite was added for colon reducing environment simulation). In vitro digestion assays
were performed with 2 mg of solid S1 or S2 at 37 oC (simulating the body temperature in
humans) and shaken (100 rpm) to obtain homogeneous suspensions of microparticles.
Digestions started by the addition of 320 µL of simulated saliva fluid (pH = 6.8  0.1) and
incubating during 5 min. Next, 630 µL of simulated gastric juice was added and the mixture
was stirred 2 h (total volume of 950 µL and pH = 1.3  0.2). Then, 630 µL of simulated
duodenal juice, 320 µL of bile and 100 µL of a bicarbonate solution (1 M) were added
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simultaneously (total volume of 2 mL and pH = 6.0  0.5). The resulting solution was stirred
during 2 h. Finally, colon fluid was simulated with the addition of 2 mg/mL of sodium dithionite
to the last mixture (total volume of 2 mL pH = 6.0  0.5). Aliquots were collected at scheduled
times up to 28 h (4 h approximately to reach the colon, plus 24 h of colon residence time),
centrifuged, and analyzed. In order to determine the amount of cargo delivered from S1
microparticles, supernatants were loaded into 96-well plate and then the rhodamine B
emission at 572 nm (upon excitation at 555 nm) was measured. The amount of hydrocortisone
released from S2 microparticles was assessed from the supernatants using an HPLC. The
released 5-ASA was also analyzed by HPLC (vide infra).
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2.5. 5-ASA and hydrocortisone quantification. HPLC (with a reverse-phase column) was used
for 5-ASA and hydrocortisone determination using the methods described by Lunn40 and
Navarro.41 The HPLC equipment used was a Hitachi LaChrom Elite liquid chromatograph
connected to an auto-sampler and a UV detector (L-2200 and L-2400, respectively). For both
analytes, a C18 column (Kromaphase 100 of 150 mm × 4.6 mm i.d., 5 μm particle size) was
used for separations. For the 5-ASA determinations, mobile phase: (A) 50 mM sodium
phosphate / (B) methanol, isocratic program: 90% A / 10% B for 15 min. The wavelength of the
UV detector was set at 365 nm. For hydrocortisone determinations, mobile phase: (A) water /
(B) acetonitrile, isocratic program: 70% A / 30% B for 15 min. The UV wavelength of the UV
detector was set at 245 nm. Both analytes were quantified using a calibration curve (peak area
vs. analyte concentration). The used method was validated using a recovery study. At this
respect, simulated GIT fluids were spiked with both analytes (5-ASA or hydrocortisone) at five
different concentrations. The recovery values were estimated from the determined
concentration vs. the amounts of 5-ASA or hydrocortisone added. All the recovery values were
near to 100%.
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2.6. In vivo studies. These studies followed the Principles of Laboratory Animal Care and were
approved by the Institutional Ethics Committee of the University of Valencia (according to RD
1201/2005). For the in vivo studies male Wistar rats (aged 8-12 weeks and with an weight in
the 270-330 g interval) were used. Animals were housed in an air-conditioned room (12 h
light/dark cycles, 22 ± 3 ºC, 55 ± 5% humidity) and were allowed free access to laboratory
chow and water during all the time of the studies.
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2.6.1. In vivo pharmacokinetic studies. In a first step, male Wistar rats (with an average weight
of 300±30 g) were anesthetized to facilitate the cannula implantation 24 h before the
experiment. Then, in a second step, a previously reported method for jugular vein permanent
cannulation was followed.42 The implanted cannula allowed blood sampling. The animals were
randomly ascribed (n= 8-10) to different groups: Group 1: 1 mL of rhodamine B solution (750
µg/mL in saline solution) was given orally. Group 2: 1.75 mL of a S1 suspension (containing 30
mg of solid which should deliver about 750 µg of rhodamine B) was given orally. Blood samples
(0.6-0.7 mL) were drawn with heparinized syringes and replaced by heparinized saline (10
IU/mL), at previously established sampling time (up to 4 h). Plasma samples were centrifuged
(10,000 rpm, 10 min) and supernatant was stored at -20 °C until analyzed by HPLC. A
fluorescence detector, with excitation and emission wavelength fixed at 225 and 535 nm
respectively, was used. The solvent delivery system (Alliance System, Waters 2695) was used
to provide a mobile phase 70:30 v/v of trifluoroacetic acid aqueous solution (pH 3)-
acetonitrile. The stationary phase was a Waters model Nova Pak C-18 (150 mm length, 3.9 mm
in diameter and particle size of 4 μm) column preceded by a Teknocroma TCR-C130-B pre-
column. Flow rate was set at 1 mL/min and the injection volume was 90 µL for in vivo samples.
Retention time of rhodamine B was 2.5 min. Analytical method was validated in terms of
selectivity, specificity, precision, linearity and accuracy. After 4 h, rat subjects were euthanized
and the cecum, colon tissues and feces were extracted and treated in order to determine
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rhodamine B amount. The analyzed rhodamine B corresponded to that released from S1
particles in the GIT of subjects (S1 release was not forced in vitro).
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2.6.2.1. Inducing colonic inflammation. Several slight modifications were introduced in the
method described by C. Mura et al. to induce the chronic inflammation model in colon rat.43
For this purpose, rats were indistinctly separated in different treatment groups (having free
access to water) and were then anaesthetized with isoflurane. TNBS solution in 59% v/v
ethanol (0.6 mL, 78 mg/kg body weight) was instilled into the lumen of the colon using a
graduated rubber cannula inserted rectally into the colon (taking into account that the tip was
8 cm proximal to the anus). After the induction procedure, the development of inflammation
was monitored daily for the next 10 days that the assays lasted. Rats were sacrificed using an
overdose of isoflurane at the end of the 10th day (after induction with TNBS). Inflammation
development was evaluated through determination of the clinical activity score, colon/body
weight ratio and also studying changes in tissue histology.
2.6.2.2. Treatment study design. Rats were distributed into four groups: Group 1 (positive
control group, 3 rats) with saline solution as treatment; Group 2 (8 rats) treated with a
suspension of S0 (empty mesoporous silica microparticles); Group 3 (8 rats) treated with
hydrocortisone solution; Group 4 (8 rats) treated with an aqueous suspension of S2
microparticles. The hydrocortisone dose that groups 3 and 4 received (5.58 mg/kg/day) was
calculated from the dose normally used for humans.44 All the treatments were carried out
using an oral gavage once a day during 3 days in the most intensive inflammation period (days
3, 4 and 5 after TNBS administration). Groups and treatment are summarized in Table 1.
Table 1. Treatment groups.
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Group 1
Group 2
Group 3
Group 4
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7
saline solution
(1.5 mL)
60 mg of S0 in
1.5 mL
1.5 mg hydrocortisone in 1.5 mL
30 mg S2 in 1.5 mL
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2.6.2.3. Clinical activity score system. A clinical score that assessed weight loss, stool
consistency and rectal bleeding (see Table 2) was used to quantify colitis activity.27,45
Table 2. Clinical activity score system.
CONTROL
RANGE
SCORE
<1%
0
1
2
3
4
0
2
4
0
2
4
1-5%
% WEIGHT LOSS
5-10%
10-20%
>20%
Well-formed pellets
Pasty and semi-formed stools
Liquid stools or absence
No blood
STOOL CONSISTENCY
BLEEDING
Positive finding
Gross bleeding
The sum of these scores resulted in the clinical activity which ranged from 0 (healthy animal)
to 12 (higher colitis activity). An activity score of 12 was assigned to rats that died or had to be
sacrificed before day 10.
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2.6.2.4. Assessing colonic injury and inflammation. Rats were monitored during the 10 days
period of the assay and, finally, the clinical activity score of colitis was evaluated. On day 10
after administering TNBS, rats were sacrificed with isoflurane to obtain the distal colon for
subsequent processing and analyzing. The samples from inflamed tissue were excised to
calculate C/B ratio, being C the distal colon weight, and B the body weight. Besides, an
histological evaluation was also carried out.
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2.6.2.5. Determining the colon/body weight ratio. After animal scarification, the distal colon
was extracted and longitudinally opened along the mesenteric edge. Then, colon was washed
with a 0.9% (w/v) saline solution, weighted and placed on a glass plate with the mucosal
surface lying upward.46 The 8 cm segment distal colon weight ratio was calculated as an index
of colonic tissue edema.
2.6.2.6. Histological evaluation. A 3 cm sample from distal colon (and a similar portion from
proximal part) were excised from each subject and maintained in 4% (v/v) p-formaldehyde for
24 h. Afterward, samples were changed to a 0.4% (v/v) p-formaldehyde medium in order to
perform the microscopic studies. These colonic tissue samples were routinely processed and
then embedded in paraffin. Then, longitudinal sections of 5 µm were stained with eosin and
hematoxylin. Microscopic assessment by light microscope was performed blind on coded
slices.
2.6.2.7. Statistical analysis. Non-parametric U-Mann-Whitney and Kruskal-Wallis tests were
performed to assess the differences among groups using SPSS v.22.0 (SPSS Inc., USA) on
Windows.
3. RESULTS AND DISCUSSION
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3.1. Synthesis of mesoporous silica microparticles functionalized with molecular gates. The
mesoporous silica carrier was synthesized following previously reported procedures with
minor adjustments.39 In this synthetic protocol, tetraethylorthosilicate (TEOS) was employed as
the source of silica and 1-hexadecyltrimethylammonium bromide (CTABr) as a structure
directing agent. The resulting white powder was rinsed and then calcined (in order to remove
the CTABr template). This protocol yielded the mesoporous silica microparticles S0. Pores in S0
where loaded with a selected dye (rhodamine B) or hydrocortisone and then the external
surface of both microparticles was functionalized with the olsalazine derivative 1. This
procedure yielded the final solids S1 (loaded with rhodamine B) and S2 (containing
hydrocortisone) as depicted in Scheme 1. The pores of S1 and S2 were expected to be capped
due to the presence of bulky azoderivative 1 anchored to the external surface of
microparticles. Besides, rhodamine B or hydrocortisone release should be observed in the
presence of azoreductor agents such as azoreductase enzymes, produced by colon bacteria, or
chemical reducing species.
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The synthesis of azo derivative containing trialkoxysilane moieties (1) was carried out using a
two-step sequence (see Scheme 2). First olsalazine sodium salt was protonated to yield the
free acid (1a), which was then reacted with (3-aminopropyl) triethoxysilane (1b). The final azo
compound containing alkoxysilane groups (1) was fully characterized using 1H and 13C-NMR
and HRMS.
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Scheme 2. Synthetic route used to prepare 1.
3.2. Characterization of the final materials. Solids S0, S1 and S2 were characterized using
powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), thermogravimetric
measurements (TGA), and N2 adsorption-desorption isotherms. The PXRD pattern of the as-
synthesized microparticles showed four reflections indexed as (100), (110), (200) and (210)
Bragg peaks typical of a mesoporous material with a hexagonal order. A marked shift of the
(100) reflexion in calcined material S0 was observed. This shift was ascribed to silanol groups
condensation during the calcination step. Moreover, the preservation of the (100) reflection in
the PXRD patterns of S1 and S2 microparticles showed that the mesoporous structure was
preserved during the loading process and functionalization with 1. TEM images of S0 and S2
also showed the typical porosity of mesoporous silica materials, which confirmed that there
was no framework modification (data not shown).
The N2 adsorption-desorption measurements on the calcined solid (S0) showed a type IV
isotherm, typical of the mesoporous silica materials. This isotherm presented at intermediate
P/P0 values (0.2-0.4) an adsorption step. From the adsorption branch of the isotherm the
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values of pore volume (0.98 cm3 g–1) and pore diameter (2.55 nm) were calculated. Besides,
the pore diameter estimated from the TEM images agreed with this calculated value. The total
specific surface of S0 microparticles (1193 m2 g–1) was evaluated using the Brunauer–Emmett–
Teller (BET) model. On the other hand, the N2 adsorption-desorption isotherms of S1 and S2
microparticles were typical of mesoporous systems with partially filled mesopores. When
compared with S0, both, the specific surface area and adsorbed N2 volume were clearly
reduced (see Table 3). The loading of pores with rhodamine B (for S1) or hydrocortisone (for
S2), and the grafting of olsalazine derivative 1 onto the external surface accounted for the
reduction in the BET surface observed for both solids.
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Table 3. Structural parameters for S0, S1 and S2 microparticles.
SBET
(m2 g-1)
1193
200
pore volumea
(cm3 g-1)
0.98
pore sizea,b
(nm)
S0
S1
S2
2.55
0.06
-
-
770
0.35
a Total pore volume estimated using the BJH model.
b Pore size according to the BJH model applied to the adsorption branch of the isotherm (for
P/P0 < 0.6) which can be assigned to the mesopores generated by the surfactant.
Table 4. Total organic matter (in g) content per gram of SiO2, olsalazine derivative 1 (in g)
content per gram of SiO2, dye or drug (hydrocortisone: HC) released (in µg) per mg of solid and
drug (5-ASA) released per mg of solid for S1 and S2.
organic content
(g/g SiO2)
0.24
1
dye/HC release
(µg/mg solid)
21.50
5-ASA release
(µg/mg solid)
107
solid
S1
(g/g SiO2)
0.16
S2
0.19
0.11
24.36
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Thermogravimetric analysis and 1H NMR measurements were employed to assess the
organic content in S1 and S2 (see Table 4). The amount of total organic matter was larger in S1
than in S2, which agrees with the larger specific surface found for S2 (see Table 3), indicating
that the loading of the mesoporous support with rhodamine B in S1 was more efficient than
for hydrocortisone in S2. The amounts of olsalazine derivative grafted onto the outer surface
of S1 and S2 were determined by 1H NMR measurements. For this purpose, the corresponding
solid was dissolved in NaOD/D2O using (CH3CH2)4NBr as internal standard. Moreover, HPLC was
used to quantify the amount of 5-ASA released. In the presence of a reducing agent (vide infra)
S1 and S2 were able to deliver 107 µg/mg and 93 µg/mg of 5-ASA, respectively. FT-IR analysis
on S2 revealed the presence of typical amide signals at 1633 cm-1 (carbonyl), 1594 cm-1 (N-H
torsion) and 1486 cm-1 (C-N stretching), and azo signal at 1558 cm-1 (data not shown).
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3.3. Payload delivery from S1 and S2. The cargo release (rhodamine B or hydrocortisone) from
solids S1 and S2 was evaluated at acidic pH (2.0 and 4.5) and at neutral pH (7.4) in the absence
of in the presence of sodium dithionite (an azoreductor agent). These pH values were selected
bearing in mind typical pH values for gastric juices (pH 2.0), transition from stomach to the
intestine (pH 4.5) and intestine (pH 7.4). Cargo release profiles for S1 and S2 microparticles are
shown in Figure 1a and 1b, respectively. Aqueous suspensions of S1 and S2 showed poor
rhodamine B (S1) or hydrocortisone (S2) release at acidic and neutral pHs. However, a marked
delivery of the entrapped payload (rhodamine B from S1 and hydrocortisone from S2) was
seen at pH 7.4 when sodium dithionite was present, which showed a sustained release profile
that reached ca. 90% (for S1) and 80% (for S2) of the maximum dye/drug delivered after 6 h.
The observed cargo release was ascribed to the presence of the azoreductor agent (sodium
dithionite), which is able to reduce the azo groups in the capping molecule 1. This azo group
reduction resulted in pore opening and rhodamine B (S1) or hydrocortisone (S2) release. The
maximum rhodamine B release from S1 was ca. 21.50 µg/mg solid, in the presence of an
azoreductor (pH = 7.4). Under the same experimental conditions, the maximum
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hydrocortisone release from S2 was ca. 24.36 µg/mg solid. The release of 5-ASA in the absence
of the azoreductor agent was negligible (at pH of 2.0, 4.5 and 7.4), although a clear cargo
release was attained after 1 h for S1 and S2, respectively, when sodium dithionite was present.
To conclude, both pharmaceutical active compounds (i.e. hydrocortisone and 5-ASA) were
released at a neutral pH when the reducing agent sodium dithionite was present, mimicking
the colon environment.
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a)
b)
Figure 1. (a) Release profiles of rhodamine B from S1 microparticles in water at pH ≈ 2
(square), pH ≈ 4.5 (circle), pH ≈ 7.4 (triangle) and pH ≈ 7.4 in the presence of sodium dithionite
(2 mg/mL) (cross). (b) Release kinetics of hydrocortisone (black) and 5-ASA (red) from S2
microparticles in water at pH ≈ 2 (square), pH ≈ 4.5 (circle), pH ≈ 7.4 (triangle) and pH ≈ 7.4 in
the presence of sodium dithionite (2 mg/mL) (cross: hydrocortisone, star: 5-ASA).
3.4. In vitro digestion model assay. In a further step the delivery of olsalazine and 5-ASA from
S2 was thoroughly evaluated in a digestion model using simulated solutions. The used model
was inspired in that reported by Oomen, and consisted in a procedure of three steps
simulating the digestive process (from the mouth, through the stomach, and to the small
intestine).47,48 Moreover, we added an additional step after the small intestine, in order to
simulate digestion at colonic area. In a typical experiment, S2 (2 mg) was suspended for 5 min
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in the simulated saliva at 37.5 oC, afterwards simulated gastric juice (stomach) was added and
the suspension was stirred during 2 h. Next, the simulated small intestine fluid (duodenal
simulated juice, bile and bicarbonate solution) was added and the mixture was stirred for 2 h.
Finally, addition of sodium dithionite simulated the azoreductor colon environment. This final
suspension was left for 24 h. The release of hydrocortisone and 5-ASA from S2 was quantified
by HPLC under the conditions described above. Figure 2 shows the obtained results.
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Figure 2. Release kinetics of the hydrocortisone (HC) and 5-ASA from S2 in simulated GIT fluids.
As seen in Figure 2, the released hydrocortisone or 5-ASA from S2 was less than 5% in the
simulated mouth, stomach and small intestine juices, which indicates that acidic media over a
period shorter than 2 h does not induce hydrocortisone or 5-ASA delivery. In contrast, a clear
release of both drugs (maximum delivery was 30.44 and 81.25 µg/mg solid for hydrocortisone
and 5-ASA respectively) was observed when S2 came into contact with the simulated colon
juice due to the reduction of the azo linkages in the gated material.
3.5. In vivo pharmacokinetic studies. To test both the specific colon release and systemic
absorption prevention, in vivo pharmacokinetic studies with S1 microparticles were
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performed. Rats in group 1, which were administered with a rhodamine B solution, showed
plasma levels of rhodamine B with a maximum of 0.45 µg/mL 1 h after administration (Figure
3). Group 2, which received formulation S1, showed negligible rhodamine B systemic
absorption and no rhodamine B was found in plasma (Figure 3). The presence of rhodamine B
in the cecum, colon and feces was also evaluated. Figure 4 shows significant larger presence of
rhodamine B in the colon and cecum in rats fed with S1 (Group 2) compared with those
treated with rhodamine B (Group 1). These results evidence the specific dye/drug delivery to
the colon from the systems designed in this work, and also demonstrated that systemic
absorption was avoided when rhodamine B was encapsulated in S1. Besides, Cmax and Tmax
values for rats in groups 1 (administered with rhodamine B solution) and 2 (treated with S1)
are shown in Table 5.
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Figure 3. The rhodamine B concentration (µg/mL) in plasma. For the subjects which received
rhodamine B solution (Group 1) and those which receiving suspension S1 (Group 2).
Table 5. Cmax and Tmax of both formulations assayed. (n = 4).
AUC t0-t∞/Dose (mL-1 h-1)
Cmax (µg/mL)
0.45*
Tmax (min)
50*
Group 1
Group 2
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9.21
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Cmax (maximal drug concentration), Tmax (time to reach Cmax) of rhodamine B plasma profiles
were calculated and compared to determine if the prepared suspension modify the dye
solution parameters. Presented results are the average of four animals per group.
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Figure 4. Rhodamine B concentration (µg/mL) in the cecum, colon and feces. For the subjects
that received rhodamine B solution (Group 1) and those administered S1 suspension (Group
2).
3.6. In vivo efficacy studies
3.6.1. Inducing colonic inflammation. TNBS was used to induce the IBD model in rats. In this
model, enema administration of TNBS (50% V/V in ethanol) induced IBD-like injuries. The
reproducibility, simplicity dose- and time-related development of inflammation are the most
remarkable advantages of this model.49,50 Inflammation development was monitored daily.
During the period of study, individuals suffered weight loss and diarrhoea, but no rectal
bleeding.
Figure 5A shows the typical appearance of a healthy colon of rats. Figure 5B shows the
opened colon of the rats that received saline serum (placebo treatment, Group 1), whereas
Figures 5C to E show the opened colons of rats after inducing colitis with TNBS and treated
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with S0, hydrocortisone and S2, Groups 2, 3 and 4 respectively. All rats were sacrificed 10 days
after treatment with TNBS. Colons from Groups 1 (Figure 5B) and 2 (Figure 5C) were strikingly
similar and both presented a thick rigid bowel and showed necrotic tissue. In colons from
individuals treated with hydrocortisone (Group 3, Figure 5D) some tissue started to heal, but
although thickened tissue and necrotic zone had reduced, they were still present. In contrast,
colons of rats from Group 4 treated with S2 (Figure 5E) generally appeared healthy, and only a
few presented very small mild injuries. In addition, after the administration of S2, subjects
from Group 4 gained some weight and presented normal stools, similar to some subjects of
Group 3 (which received hydrocortisone solution). On the other hand, after the administration
of saline solution or S0, rats from Groups 1 and 2 had diarrhea and lost weight throughout the
experiment. Figures 6 and 7 showed the effects after oral administration of the different
formulations on the clinical activity score and the colon/body weight, respectively.
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Figure 5. Pictures of rat colon 10 days after inducing colitis with TNBS: A healthy control
(without TNBS administration), B positive control (Group 1, treated with saline serum), C
(Group 2, treated with S0), D (Group 3, administered with hydrocortisone) and E (Group 4,
administered with S2).
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The colon/body weight ratio decreased considerably in rats of Group 4 (treated with S2)
compared to the control, which received saline solution (Group 1). In contrast no accentuated
effect was observed on the colon/body weight ratio for subjects treated with S0 particles
(Group 2). Rats in Group 3 (treated with the hydrocortisone solution) had a wide variability,
with several cases of improvement and several cases without improvement at all (Figure 6).
Similar results (i.e. wide variability) were found for Group 3 in the clinical activity score (Figure
7). This large variation observed for Group 3 can be due to one of the following reasons: a)
orally taken hydrocortisone is almost absorbed completely before reaching its site of action; b)
the dose of an administered drug in not enough to allow recovery. In contrast, the S2
formulation allowed the release of two drugs, i.e. hydrocortisone (carried inside the
mesoporous silica microparticles) and 5-ASA (externally anchored to the surface of
microparticles), which results in a high concentration of both active ingredients in the injured
area having a remarkable therapeutic effect.
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Figure 6. The colon/body weight ratio of rats with TNBS-induced colitis after receiving
subsequent treatments: saline solution (Group 1, positive control), S0 (Group 2),
hydrocortisone (Group 3) and S2 (Group 4). In each box represents the median value, the 25%
and 75% percentiles, the minimal and maximal values (a,b statistical significant difference (P <
0.05) compared to the saline and S0 solid, respectively).
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Figure 7. The clinical activity score of rats with TNBS-induced colitis after receiving subsequent
treatments: saline solution (Group 1, positive control), S0 (Group 2), hydrocortisone (Group 3)
and S2 (Group 4). In each box represents the median value, the 25% and 75% percentiles, the
minimal and maximal values (a,b statistical significant difference (P < 0.05) compared to the
saline and S0 solid, respectively).
3.6.2. Histological evaluation. Histological tissue examinations in a healthy control rats, and in
rats treated with TNBS of different groups: i.e. 1 (saline solution, positive control group); 2
(S0); 3 (hydrocortisone solution); 4 (S2 formulation) were carried out. Samples were always
taken from the sacrificed subjects on day 10 after TNBS treatment. The healthy control
showed a usual colon structure: i.e. connective tissue and goblet cells (lamina propria, Figure
8A), healthy mucosa with enterocytes, muscularis mucosae, normal submucosa and muscularis
externa. On the other hand, tissues from rats of Groups 1 and 2 showed loss of necrotic
mucosa and substitution for granulation tissue (see Figure 8B and 8C). Besides, a strong
inflammatory process was present in the lamina propria, submucosa and muscularis extern in
the individuals of both groups. Figure 8B and 8C also show the ulceration process with the
fibrinoid necrosis of the mucosal surface and granulation tissue below necrotic tissue. Rats of
Group 3 (treated with hydrocortisone) only showed superficial erosion, thinning mucosa
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(complemented by a thickening of muscularis mucosae), and a chronic inflammatory process
which affected the mucosa and submucosa with an early development of lymphoid follicle.
Besides, Figure 8D also shows a small number of parts with a normal mucosa structure.
However, the presence of strong follicular hyperplasia (in the muscularis externa and parts
with necrosis), loss of mucosa and substitution for granulation tissue, and the inflammation
process is also observed in Figure 8D. The individuals treated with S2 microparticles (Group 4)
showed a normal mucosal structure with the presence of minor chronic inflammation in
muscularis propria (see Figure 8E).
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Figure 8. Histology of a representative colon specimen of healthy rats (A) and rats after
inducing colitis with TNBS, treated and sacrificed on day 10: (B) administered with saline
solution (positive control, Group 1), (C) treated with S0 (Group 2), (D) administered with
hydrocortisone (Group 3) and (E) treated with S2 (Group 4).
The histological findings indicated that Group 1 individuals (the saline-treated TNBS group)
showed necrosis, strong inflammation and mucosa loss. Similar results were observed with the
rats treated with microparticles S0 (Group 2). In contrast, the histological findings of the rats
treated with S2 (Group 4) showed an intensive regeneration, softer inflammation, and an
almost normal mucosal structure. The information obtained by the colon/body weight ratio,
the clinical activity score and the histological evaluation, confirming the usefulness of S2
system to treat IBD. Moreover, it can be stated that our formulation improved the results
reported in previous studies by C.Mura et al., M. Naeem et al. or A. H. Teruel et al., with the
added benefit of not involving any systemic intervention.29,43,51
4. CONCLUSIONS
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A novel oral colon drug delivery system is designed and prepared, and its efficiency to treat
IBD was evaluated in vivo in a TNBS-induced colitis rat model. The prepared system consists of
mesoporous silica microparticles loaded with either, rhodamine B (S1) or hydrocortisone (S2),
and decorated on the external surface with an olsalazine derivative. This olsalazine derivative
is a bulky azo compound, covalently grafted onto the external surface of the loaded support
through amide linkages, which yielded drug 5-ASA upon hydrolysis of the azo bonds. Both
materials (i.e. S1 and S2) remained capped at neutral an acidic pH, yet a remarkable payload
delivery is evident when sodium dithionite (a reducing agent that mimics the azo-reductase
enzymes present in colon) was present. Cargo release is a consequence of the sodium
dithionite-induced reduction of azo bonds in the bulky capping ensemble, whereas 5-ASA is
also delivered from both microdevices upon the hydrolysis of the olsalazine derivative. In vivo
pharmacokinetic studies performed with S1 showed specific rhodamine B delivery at colonic
area. The clinical activity score, the colon/body weight ratio and the histological colon tissue
evaluation evidenced that rats with chronic colon inflammation and treated with
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microparticles S2 show an improvement in the pathology due to the simultaneous
hydrocortisone and 5-ASA release. In conclusion, we demonstrate that microparticles reported
here can be used to release drugs specifically in the last intestine part, while diminishing
systemic absorption. Specifically S2 is a formulation able to treat IBD due to the double drug
delivery (i.e. hydrocortisone and 5-ASA release) at the colon.
5. ACKNOWLEDGEMENTS
We thank the Generalitat Valenciana (Project PROMETEO2018/024) and the Spanish
Government (Projects AGL2015-70235-C2-2-R and MAT2015-64139-C4-1-R (MINECO/FEDER))
for support. AHT thanks the Spanish MEC for his FPU fellowship. The authors also thank the
support of the Electron Microscopy Service at the UPV. The SCSIE (of the Universitat de
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València) is also gratefully acknowledged for all the equipment used. NMR spectra were
measured at the U26 facility of ICTS “NANBIOSIS” at the Universitat de València.
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