A novel cyanoacrylate-based matrix excipient in HPMCP capsules forms a sustained intestinal delivery system for orally administered drugs with enhanced absorption efficiency

Article abstract of DOI:10.1039/d0tb02606a

Patients prefer oral drug delivery due to its convenience and noninvasiveness. Nevertheless, a multitude of potentially clinically important drugs will not reach the market or achieve their full potential, due to their low bioavailability and instability in gastric acid. In this study, a novel oral drug delivery system based on poly-cyanoacrylate [a polymer of 2-(2-methoxyethoxy)ethyl-2-cyanoacrylate (MECA)] and hydroxypropyl methylcellulose phthalate (HPMCP) was developed and shown to permit intestinal targeting and sustained drug release. Aspirin [acetylsalicylic acid (ASA)] was selected as a model drug for atherosclerosis treatment. It was physically dissolved in liquid MECA, and the ASA-MECA matrix was then polymerized into a solid drug-loading depot in an HPMCP shell. The delivery of the drug depot in the intestine was achieved with the HPMCP shell; then the polymerized MECA (polyMECA) provided sustained drug release. The polyMECA excipient was not absorbed by the intestine due to its high molecular weight; a fluorescein-labeled assay indicated that it was excreted completely in feces after drug release. The formulation, ASA-polyMECA-HPMCP, showed good intestinal targeting and sustained drug releasein vitroandin vivo. Pharmacokinetic studies indicated that this formulation improved the bioavailability of ASA relative to commercially available controls. ASA-polyMECA-HPMCP showed desirable anti-atherosclerosis efficacy in a rabbit model, with significant enhancement of atheromatous lesion stability. Biosafety tests proved the low toxicity of ASA-polyMECA-HPMCP and the polyMECA matrix. We believe that this work has provided a practical and biocompatible system for sustained intestinal drug delivery that can be applied broadly with various drugs for specific therapeutic aims.

Full text of DOI:10.1039/d0tb02606a

Journal of
Materials Chemistry B
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PAPER
A novel cyanoacrylate-based matrix excipient in
HPMCP capsules forms a sustained intestinal
delivery system for orally administered drugs with
enhanced absorption efficiency†
Cite this: J. Mater. Chem. B, 2021,
, 1288
9
ab
c
d
a
a
e
f
Liya Song, Pengfei Chen, Jin Yu, Xiaolu Han, Yabing Hua, Shan Liu, Bo Pang,
a
b
a
Jing Gao, Jiahua Ma* and Liang Xu
*
Patients prefer oral drug delivery due to its convenience and noninvasiveness. Nevertheless, a multitude
of potentially clinically important drugs will not reach the market or achieve their full potential, due to
their low bioavailability and instability in gastric acid. In this study, a novel oral drug delivery system
based on poly-cyanoacrylate [a polymer of 2-(2-methoxyethoxy)ethyl-2-cyanoacrylate (MECA)] and
hydroxypropyl methylcellulose phthalate (HPMCP) was developed and shown to permit intestinal
targeting and sustained drug release. Aspirin [acetylsalicylic acid (ASA)] was selected as a model drug for
atherosclerosis treatment. It was physically dissolved in liquid MECA, and the ASA–MECA matrix was then
polymerized into a solid drug-loading depot in an HPMCP shell. The delivery of the drug depot in the
intestine was achieved with the HPMCP shell; then the polymerized MECA (polyMECA) provided
sustained drug release. The polyMECA excipient was not absorbed by the intestine due to its high
molecular weight; a fluorescein-labeled assay indicated that it was excreted completely in feces after
drug release. The formulation, ASA–polyMECA–HPMCP, showed good intestinal targeting and sustained
drug release in vitro and in vivo. Pharmacokinetic studies indicated that this formulation improved the
bioavailability of ASA relative to commercially available controls. ASA–polyMECA–HPMCP showed desirable
anti-atherosclerosis efficacy in a rabbit model, with significant enhancement of atheromatous lesion stability.
Biosafety tests proved the low toxicity of ASA–polyMECA–HPMCP and the polyMECA matrix. We believe that
this work has provided a practical and biocompatible system for sustained intestinal drug delivery that can
be applied broadly with various drugs for specific therapeutic aims.
Received 4th November 2020,
Accepted 22nd December 2020
DOI: 10.1039/d0tb02606a
rsc.li/materials-b
are encountered with oral administration, such as the low
bioavailability of agents due to degradation in the stomach
Patients prefer the oral administration of pharmaceutical and gastrointestinal irritation, especially at high dosages.
Introduction
3
–6
agents because of its convenience and the avoidance of painful Intestine-targeting formulations for oral administration have
1
,2
procedures, among other reasons. However, many hurdles attracted increasing attention in recent years; they have been
developed with the aims of improving the treatment efficiency
a
for local intestinal diseases (e.g., colorectal infections and
colorectal cancer), providing an alternative route for the
systemic absorption of conventional and labile drugs, and
State Key Laboratory of Toxicology and Medical Countermeasures,
Beijing Institute of Pharmacology and Toxicology, Beijing, China.
E-mail: xuliang24998@yahoo.com
b
c
d
e
f
7,8
School of Life Science and Engineering, Southwest University of Science and
Technology, Mianyang 621010, China
reducing gastric stimulation and side effects.
The use of polymeric materials is a valuable route to
Department of Cardiology, the 6th Medical Center, Chinese PLA General Hospital,
Beijing 100853, China
9
–11
meet the challenges of drug storage and delivery.
Several
natural and synthetic polymers, such as chitosan, pectin, and
Department of Neurology (the First Medical Center), Chinese PLA General
Hospital, Beijing 100853, China
ethylcellulose, have been explored as excipients in small
12–15
Pathology Department of PLA Rocket Force Characteristic Medical Center,
Beijing 100085, China
intestine- and colon-targeting formulations.
However,
several are limited by their restricted adaptivity, low bio-
availability, and/or complicated preparation, and commercially
available materials for intestine or colon targeting still lack
diversity. The development of new strategies and biomaterials
Clinical Laboratory, Guanganmen Hospital, China Academy of Chinese Medical
Science, Beijing 100053, China
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb02606a
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for intestine-targeting oral drug delivery (ITODD) systems Table 1 CA compositions of the ASA–polyCA–HPMCP formulations
remains essential.
Atherosclerosis is the leading cause for many fatal cardio-
vascular and cerebrovascular diseases.
CA monomer
composition
CA monomer
wt%
Sample
16,17
Accumulating evidence
ASA–polyBCA–HPMCP
ASA–polyMECA–HPMCP
BCA
MECA
—
—
shows that atherosclerosis is caused not only by the accumulation
of lipids in arterial walls, but also as a chronic inflammatory ASA–polyBCA/MECA–HPMCP
BCA/MECA
50/50
18
disease in response to vascular injury. Extensive research has
explored the targeting of inflammation to prevent the progression
19–22
Results and discussion
of atherosclerosis.
Aspirin [acetylsalicylic acid (ASA)], a con-
ventional anti-platelet and anti-inflammatory agent used widely in Monomers and ASA–polyCA–HPMCP formulations
clinical practice, plays therapeutic and preventive roles for ather-
osclerosis via the inhibition of platelet adhesion and aggregation,
Two CA monomers, BCA and MECA, were synthesized with
high purity, as confirmed by proton nuclear magnetic reso-
nance ( H-NMR; Fig. S1, ESI†) and gas chromatography (99.1%
for BCA, and 95.4% for MECA). MECA exhibited less polymer
film formation and a smaller contact angle, confirming that its
hydrophilicity was superior to that of BCA (Fig. S2 and Table S1,
ESI†). Samples containing BCA and MECA (50/50, wt%) yielded
intermediate results.
and the interruption of several steps in the inflammatory process
1
23–28
associated with atherogenesis.
However, the decomposition
of ASA in acidic environments and the rapid and extensive
absorption of this agent in the upper intestinal tract can lead to
severe side effects, such as gastrointestinal bleeding, ultimately
29–31
leading to ASA intolerance in some patients.
Thus, the
targeting of the intestinal region and extension of the drug’s
release time would improve the therapeutic effect of ASA for
atherosclerosis.
Three ASA–polyCA–HPMCP formulations with different CA
compositions were prepared (Table 1). ASA (5%) dissolved
readily, reaching a transparent liquid state, in the CA mono-
mers. After maintenance at 80 1C for 24 h, the liquid ASA–CA
In this study, a novel sustained ITODD system for the
treatment of atherosclerosis, with ASA loaded as the model
drug, was assessed. In particular, poly-cyanoacrylate (PCA) was
used as a novel drug matrix excipient, filled in a capsule shell
made of hydroxypropyl methyl cellulose phthalate (HPMCP).
The HPMCP coating is pH sensitive. It could withstand the low
pH in the stomach and will degrade in the neutral pH of the
1
complex had formed a solid depot. A H-NMR assay confirmed
the complete polymerization of the CA monomers, with the
shifting of peaks at 7.05 and 6.62 ppm, corresponding to the
olefinic double-bond protons, to 2.64 ppm (Fig. S3, ESI†). This
liquid–solid transformational property of the drug-loading
matrix leads to easy filling of shells molded into any shape
with individualized doses.
3
2,33
intestine,
which ensures that ASA can pass through the
stomach completely and be released from the ASA–PCA
complex depot in intestine. After drug release, the inert phar-
macological polymer PCA is meant to be excreted directly, as it
cannot be absorbed by the intestinal tract due to its high
molecular weight. To our knowledge, this report is the first to
describe the use of cyanoacrylate (CA) as a matrix excipient for
oral drug delivery.
Dispersion of ASA in the polyCA depot
1
H-NMR, ultraviolet (UV), and high-performance liquid chromato-
graphy (HPLC) assays were used to characterize ASA dispersion in
the polyCA depot. Taking the ASA–polyMECA group as an
example, the typical spectral peaks of ASA were detected in
1
H-NMR spectra obtained before and after MECA polymeriza-
CA has well-known applications in the construction of
3
4–36
tion (Fig. S3, ESI†) and the UV absorption peak of ASA remained
at 276 nm, as for free ASA [Fig. 2(a)]. HPLC showed the
characteristic peaks of ASA in sample extracts [Fig. 2(b)], and
randomly sampled depots had similar integral areas. These
results indicate that ASA was physically and homogeneously
dispersed in the polyMECA depot, with no chemical reaction
occurring between these components. Homogeneous physical
medical adhesives and nanocarriers.
Its liquid monomers
polymerize into a solid under certain conditions, providing
opportunities for the direct dissolution of organic drugs by
liquid CA and the formation of drug-loading depots of any
desired shape and for any dose. However, n-butyl cyanoacrylate
[BCA; Fig. 1(a)], the most commonly used CA polymer, is
very hydrophobic and micromorphologically compact; thus,
polyBCA is not an optimal material for the construction of
3
7
drug-release depots. To overcome these limitations, 2-(2-
methoxyethoxy)-ethyl-2-cyanoacrylate [MECA; Fig. 1(b)], an
alkoxy-modified CA monomer with better hydrophilicity, was
used in this work for depot polymerization.
Fig. 1 Chemical structures of (a) n-butyl cyanoacrylate and (b) 2-(2-
Fig. 2 (a) UV assay results and (b) HPLC chromatograms of ASA alone and
methoxyethoxy)ethyl 2-cyanoacrylate.
ASA–polyMECA phosphate-buffered saline extract.
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dispersion is of great significance, as it permits the adjustment
of the drug dose via alteration of the volume of liquid used.
ASA release from the ASA–polyCA–HPMCP formulation in vitro
In an in vitro assay, the efficiency of ASA (5%, ASA in MECA)
release from the ASA–polyCA–HPMCP formulation was evalu-
ated under different pH conditions corresponding to the gastric
(HCl, pH 1.2) and intestinal media (PBS, pH 6.8) at 37 1C
(Fig. 3). As ASA partially and rapidly degrades to salicylic acid
(SA) in phosphate-buffered saline (PBS) solution, the amount of
ASA released was calculated by determining the total concen-
3
8,39
trations of free SA and ASA.
The ASA and SA peaks were well
separated under the test conditions (Fig. S4, ESI†).
All three groups showed a limited drug release at pH 1.2 for
the first 2 h, indicating that HPMCP provided reliable protec-
tion from gastric fluid. At pH 6.8, the outer layer of the capsule
Fig. 4 Release profiles of ASA–polyMECA–HPMCP, ASA-ETs, and ASA-
ECs in different media. The dashed line represents the change of medium
became degraded and corroded, and ASA–polyMECA–HPMCP pH from 1.2 to 6.8. Data are means Æ SDs (n = 3).
samples showed a good sustained drug release profile, with
approximately 90.65% of ASA released within 24 h. In contrast,
ETs) released little (5.61%) ASA at pH 1.2 and showed sustained
ASA–polyBCA–HPMCP samples released little ASA in simulated
intestinal fluid. ASA–polyBCA/MECA–HPMCP samples had a
slightly higher ASA release rate (9.64%) than did ASA–polyBCA–
HPMCP samples. Moreover, a control of ASA–HPMCP without
any polymer depot showed burst release within 1 h after placing
in pH 6.8. These results indicate that the CA formulation
affected the ASA release behavior. The higher drug release rate
in the ASA–polyMECA–HPMCP group may be attributable to the
superior hydrophilicity of polyMECA relative to polyBCA.
The increase in drug release with the addition of polyMECA
in the ASA–polyBCA/MECA–HPMCP group indicates that drug
release can be adjusted according to specific needs.
release at pH 6.8 (98.42% within 8 h). ASA–polyMECA–HPMCP
samples showed no ASA release in simulated gastric fluid and
had a slower drug release rate than did commercial ASA-ETs
(85.16% within 14 h, 90.65% within 24 h).
ASA–polyMECA–HPMCP was chosen as the preferred formu-
lation, given its appropriate drug release rate. Its drug release
properties under different drug loading rates and matrix
volumes were investigated further. Samples with the same
polyMECA volumes (200 mL) but different drug loading rates
(5%, 10%, and 15%) produced similar ASA release curves [Fig.
S5(a), ESI†]. Those with the same drug loading rate (5%) but
different polyMECA volumes (50, 100, and 200 mL) also showed
parallel tendencies, although those containing 200 mL poly-
MECA had a slightly slower release rate [Fig. S5(b), ESI†]. This
difference may be explained by the larger surface area of
samples with less vehicle than of those with a larger volume
contained in the same capsule shell. Moreover, Fig. S6 (ESI†)
indicated that the molecular weight of polyMECA had little
influence on ASA release.
Fig. 4 shows the drug release with ASA–polyMECA–HPMCP
(drug loading content is 5%, ASA in MECA) samples and two
commercial products. Aspirin enteric capsules (ASA-ECs) showed
little (2.79%) ASA release in simulated gastric fluid, and burst
when placed in simulated small-intestinal fluid (98.76% release
at 45 min). Aspirin enteric-coated sustained-release tablets (ASA-
The underlying interactions for drug releasing from depot
include drug diffusion, depot erosion and degradation. To deter-
mine the mechanism of ASA release from the ASA–polyMECA
complex in simulated intestinal fluid, the n parameter of the
Korsmeyer–Peppas model (ln M
t
/M
= 0.939). This value was 0.42, indicating that the release
pattern could be characterized as a Fickian transport mechanism
in which the diffusion flux is proportional to the concentration
N
= nln t + lnk) was computed
2
(R
(
40,41
gradient);
thus, diffusion was the principal release mecha-
nism. The moderate hydrophilicity of polyMECA could provide
water molecules gradually penetrating into the polymer, resulting
in a sustained drug release, neither too fast nor too slow.
In vivo pharmacokinetics of ASA
In an in vivo rabbit model, plasma concentrations of ASA–
Fig. 3 Drug release curves. The dashed line represents the change of
medium pH from 1.2 to 6.8. Data are means Æ SDs (n = 3).
polyMECA–HPMCP reached their maximum after
4 h
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À1
Table 2 Pharmacokinetic data for the in vivo oral administration of ASA–polyMECA–HPMCP (18 and 24 mg kg ASA), commercially available ASA-ECs
À
1
À
1
(
24 mg kg ASA), and free ASA solution (24 mg kg ASA)
À1
À1
Sample
Tmax (h)
C
max (mg mL
)
AUC(0–24) (h mg mL
)
Free ASA solution
ASA-EC (24 mg kg
ASA–polyMECA–HPMCP (24 mg kg
ASA–polyMECA–HPMCP (18 mg kg
0.25
0.005 Æ 0.002
0.019 Æ 0.006
0.021 Æ 0.002
0.016 Æ 0.003
0.015 Æ 0.001
0.123 Æ 0.061
0.188 Æ 0.025
0.136 Æ 0.016
À1
)
1
4
4
À1
À1
)
)
(Table 2). In contrast, the maximum drug concentration in Excretion of polyMECA
blood was reached within 1 h with ASA-ECs, and this concen-
tration was reached immediately but was very low with free ASA,
due to the instability of ASA in gastric acid (Fig. 5). Importantly,
Gel permeation chromatography (GPC) showed that the mole-
cular weight of polyMECA in ASA–polyMECA–HPMCP samples
exceeded 50 kDa (Fig. S7, ESI†), which exceeds the limit of
glomerular filtration.
the bioavailability of ASA–polyMECA–HPMCP samples with
À1
2
4 mg kg ASA (drug loading content is 20%) was superior
To intuitively observe its metabolism course in vivo, poly-
MECA was labeled with fluorescein isothiocyanate (FITC;
Scheme S3, ESI†) and administered orally in a mouse model.
Near-infrared fluorescence images showed that the polymer
was distributed mainly in the intestine (99.6%) at 1 h and in the
feces (90.1%) at 24 h after administration, as indicated by the
excised organs (Fig. 6). No distribution in the heart, liver,
spleen, lung, or kidney was observed at either time point.
Despite the weak adhesive capability of polyMECA, we observed
no long-term retention of this material (only a little residue
observed in the intestine at 24 h) or consequent intestinal
obstruction. Moreover, in an in vitro 24 h degradation test,
only 4.96 mg formaldehyde was generated (corresponding
to 0.2% polyMECA degradation, according to the reversed Knoe-
to that of ASA-ECs with the same ASA dosage. ASA–polyMECA–
À1
HPMCP had an area under curve (AUC) value of 0.188 h mg mL
,
which was 1.5-fold greater than that of commercial ASA-ECs and
1
2.5 times greater than that of free ASA solution. Moreover, the
À1
AUC value for ASA–polyMECA–HPMCP samples with 18 mg kg
ASA (drug loading content is 15%) was higher (1.1 times)
than that for ASA-ECs with 24 mg kg ASA. The improved
À1
bioavailability of the novel drug delivery system provides the
potential to reduce the drug dosage, and thereby drug-related
side effects. In our work, the ASA release and absorption in the
intestinal tract were related much more to the inner polyMECA
depot than to the outer HPMCP layer, due to the solubility of
3
3
HPMCP at pH 5.5 in the duodenum. PolyMECA showed a
weak, but definite, adhesive capability when smeared between
two pieces of intestine (Table S2, ESI†). This potential bioadhesive
property may be attributable mainly to alkoxy modification, and is
related to hydrogen bonding, van der Waals force, and mechanical
interaction. Thus, the ASA–polyMECA formulation could have a
prolonged retention time, which aids drug absorption and
transport in the intestinal tract, thereby improving drug
bioavailability.
42
venagel approach ). The contribution of the formaldehyde
pathway to the degradation of polyMECA was surprisingly poor,
due to its limitation in water and at physiological pH. Hydrolysis
À1
Fig. 5 Pharmacokinetics of 24 and 18 mg kg ASA loaded with the
À1
polyMECA–HPMCP formulation, 24 mg kg ASA in ECs, and free ASA
Fig. 6 Representative ex vivo fluorescence images of the dissected
organs of mice given polyMECA by oral gavage and sacrificed 1 and 24 h
thereafter.
À1
solution (24 mg kg ) in New Zealand white rabbits. Data are means Æ SDs
(n = 9).
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of the ester function is another degradation route (Fig. S8, ESI†). from the heart to the iliac bifurcation were stained with Oil-
This was confirmed by the estimation of the acid produced by Red-O (ORO). En face aorta images and the quantification of
the ester hydrolysis. The consumption of sodium hydroxide lesion area revealed atherosclerotic lesions with typical post-
corresponded to the limited hydrolysis of the ester bond modeling features in the control group, and significantly
(1.07%). These results, which are consistent with the GPC delayed plaque development in the aortic arch and thoracic
analysis, not only indicate the hurdle of the degradation of the aorta in the ASA–polyMECA–HPMCP group relative to the
polymer, but also suggest the restricted absorption of orally controls [Fig. 7(b) and (c)]. Average plaque areas in ORO-
administered polyMECA.
stained samples were 21.47% Æ 2.12% in the ASA–polyMECA–
HPMCP group and 47.76% Æ 4.15% in the control group.
As the rupture of vulnerable plaques may lead to fatal
Anti-atherosclerosis treatment effect of ASA–polyMECA–HPMCP
4
3
complications, we histopathologically examined the stability
of atherosclerotic plaques after treatment. Masson’s trichrome
staining revealed that the content of collagen surrounding
plaques was enhanced considerably, resulting in thicker
fibrous caps, after treatment with ASA–polyMECA–HPMCP
The anti-atherosclerosis effect of ASA–polyMECA–HPMCP was
evaluated in a rabbit model [Fig. 7(a)]. After treatment, aortas
[Fig. 7(d) and (e)]. Immunohistochemical analysis revealed a
reduced level of matrix metalloproteinase (MMP)-9, which can
4
4
contribute to the development of vulnerable plaques, in
aortas [Fig. 7(f) and (g)]. Taken together, these results suggest
that ASA–polyMECA–HPMCP exerted good anti-atherogenic
effects without increasing plaque vulnerability.
ASA–polyMECA–HPMCP prevents ASA irritation of the gastric
mucosa
In our formulations, the HPMCP coating and polyMECA depot
were meant to prevent ASA degradation and burst release in the
stomach, thereby preventing irritation of the gastric mucosa. In
the rabbit study of atherosclerosis treatment, rabbits given
ASA–polyMECA–HPMCP showed no gastric bleeding or histo-
morphological abnormality, indicating good biocompatibility,
Fig. 7 Treatment of atherosclerosis with ASA–polyMECA–HPMCP in New
Zealand white rabbits. (a) Time course of the experiment. Saline, ASA–poly-
À1
MECA–HPMCP (18 mg kg ASA) was administered orally once a day after
1
week of ND. (b) Representative photographs of en face ORO-stained aortas
from each group. (c) Areas of lesions in (b). (d) Representative histological
images of aortic root sections after Masson staining. Â100 magnification. The
scale bar is 200 mm. (e) Quantification of the collagen content in (d). (f) Sections
stained with antibodies to MMP-9. Â100 magnification. The scale bar is
Fig. 8 Protective effect of ASA–polyMECA–HPMCP against ASA-induced
bleeding in the stomach. (a) Representative images of the gastric mucosa,
showing damage in rabbits given free ASA solution. (b) Representative
histomorphological images of the gastric mucosal layer, showing ulcera-
tion induced by ASA. Â100 magnification. The scale bar is 200 mm.
2
00 mm. (g) MMP-9 levels. Data are means Æ SDs. **p o 0.01.
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Fig. 11 Histopathological sections of major organs after the 90 day
repeated dose toxicity assessment. Â200 magnification. The scale bar is
100 mm.
Fig. 9 H&E-stained histological sections of the heart, liver, spleen, and
kidneys of (A) normal group and (B) ASA–polyMECA–HPMCP group at 9
weeks after treatment initiation. The images were obtained using a Leica
microscope. Â200 magnification. The scale bar is 100 mm.
Table
3
Effects of the subchronic 12 week oral administration of
whereas those given free ASA solution (control) showed mucosal polyMECA or 0.5% CMC on hematological parameters in SD rats
damage and even perforation in the glandular region of the
Hematological parameter 0.5% CMC
PolyMECA
p values
stomach, resulting in the death of all animals within 6 days after
treatment initiation [Fig. 8(a)]. Microscopic examination of the
gastric mucosa of rabbits in the control group revealed the
occurrence of epithelial erosion, the separation and exfoliation PLT (10 L
of cells lining the gastric pit, the presence of inflammatory
infiltrates, and the suffusion of blood into the mucosa [Fig. 8(b)].
9
À1
WBC (10 L
RBC (10
)
7.63 Æ 1.76
7.91 Æ 0.79
8.73 Æ 1.27
8.72 Æ 1.50
0.43
0.46
0.61
12
À1
L )
À1
HGB (g L
)
141.00 Æ 10.82 145.33 Æ 8.50
9
À1
À1
)
909.00 Æ 119.15 951.67 Æ 150.13 0.72
9
GRAN (10 L
)
0.93 Æ 0.25
6.53 Æ 1.70
0.17 Æ 0.06
1.10 Æ 0.26
7.43 Æ 0.92
0.20 Æ 0.10
0.47
0.47
0.64
9
À1
À1
LYM (10 L
)
)
9
MID (10 L
ASA–polyMECA–HPMCP showed low systemic toxicity
Compared with the normal group, rabbits treated with ASA–
polyMECA–HPMCP in the in vivo atherosclerosis experiment
showed no pathological alteration or toxicity in the liver,
Table 4 Effects of the subchronic 12 week oral administration of
polyMECA or 0.5% CMC on biochemical parameters in SD rats
spleen, kidneys, heart, or lungs (Fig. 9). Thus, the ASA–poly- Biochemical parameter 0.5% CMC
PolyMECA
p values
À1
À1
MECA–HPMCP sample and the novel CA-based matrix excipi-
ent did not induce inflammation in tissues and was very safe at AST (U L
the dosage used.
ALT (U L
)
)
77.38 Æ 13.22
87.92 Æ 10.90 0.43
148.90 Æ 36.81 146.35 Æ 9.99
0.93
0.59
À1
BUN (mg dL
)
23.02 Æ 1.61
53.87 Æ 5.44
21.40 Æ 3.55
À1
Cr (mmol L
)
50.73 Æ 11.59 0.75
Safety of polyMECA
pathological examination revealed no abnormality in any major
organ (Fig. S10, ESI†).
The safety of polyMECA, a novel drug-loading depot ingredient,
was further evaluated in acute and subchronic toxicity studies.
À1
In the subchronic toxicity test, daily oral administration of
No acute toxicity was observed for polyMECA, even at 2.5 g kg
À1
0
.25 g kg polyMECA for 12 weeks resulted in no treatment-
(orally administered), in a mouse model. No death or hazardous
related mortality or clinical sign of general toxicity. Body
weight, the external physical structure of the organs, hemato-
logical parameters, and serum biochemistry profiles did not
differ between polyMECA-treated and control rats (Fig. 10, 11
and Tables 3, 4).
sign of toxicity was recorded during the 14 day observation period.
Increases in body weight over time were similar in polyMECA-
treated and control [0.5% (wt%) hypromellose aqueous solution
(CMC)-fed] mice (Fig. S9, ESI†). Upon sacrifice at 14 days,
Taken together, these results demonstrate the good biologi-
cal safety of orally administered polyMECA and ASA–poly-
MECA–HPMCP in vivo.
Conclusions
A novel sustained intestinal drug delivery system featuring
polyMECA excipient filling in an HPMCP capsule was devel-
oped in this work. The HPMCP acted as the protective shell for
intestinal targeting. The liquid MECA can directly dissolve ASA,
and the liquid–solid transformational property endows easy
molding into desired shape and doses. Alkoxy modification
of the CA matrix provided a suitable drug release rate and
appropriate bioadhesive capability, resulting in improved
Fig. 10 Body weights of rats treated with polyMECA and CMC. Data are
means Æ SDs (n = 5).
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bioavailability of ASA loaded in an ASA–polyMECA–HPMCP Estimation of drug release from ASA–polyCA–HPMCP capsules
formulation. ASA–polyMECA–HPMCP had desirable anti- in vitro
atherosclerotic effects in a rabbit model. The novel medicinal
adjuvant polyMECA was excreted completely in feces after drug
complex was investigated using an HPLC device (C-10AT;
release, and showed good biological safety. Thus, this work
The profile of ASA release from the ASA–polyCA–HPMCP
Shimadzu, Japan) equipped with a Diamonsil C-18 column
resulted in the development of a novel ITODD system with
(4.6 Â 250 mm, 5 mm particle size; Dikma, China). The ASA–
improved drug absorption efficiency. In addition to ASA
delivery, it has potential broad applicability, such as in the
treatment of enteritis and intestinal cancer.
polyCA–HPMCP complexes were immersed successively in buf-
fer solutions simulating gastric fluid (pH 1.2) and intestinal
fluid (pH 6.8). The dissolution media were maintained at 37 1C
and stirred at 100 rpm. Samples (500 mL) were withdrawn at
predetermined time points (n = 3) and the media were replaced.
The samples were injected into the injector port of the HPLC
device and analyzed at 266 nm; the mobile phase used was
Experimental
Chemicals
acetonitrile:acetic acid:tetrahydrofuran:water at a ratio of
ASA and SA were obtained from InnoChem Chemical Reagent
Beijing Co., Ltd (Beijing, China). All other chemicals, reagents,
and solvents used in this work were purchased from Sinopharm
À1
20 : 5 : 5 : 70 and a flow rate of 1 mL min
.
Chemical Reagent Beijing Co., Ltd (Beijing, China). All Drug distribution in the ASA–polyMECA complex
solutions were prepared with deionized water produced by
a Milli-Q Advantage water purification system (Millipore,
USA). Soluble ECs made of HPMCP were obtained from
Shaoxing Kangke Capsule Co., Ltd (Zhejiang, China). ASA-ECs
were obtained from Yung Shin Pharm. Ind. (Kunshan) Co.,
Ltd (Jiangsu, China). ASA-ETs were obtained from Bayer
1
3
CDCl was used as the solvent for H-NMR analysis. The UV
absorbance of ASA–polyMECA at 200–350 nm was measured in
methyl alcohol using a Cary-100 Bio UV-visible spectrophoto-
meter (Varian) and a standard scanning program. HPLC was
performed as described in the previous section.
(China).
In vitro release of ASA from ASA–polyMECA–HPMCP
Animals and ethical approval
The in vitro release of ASA from ASA–polyMECA–HPMCP was
measured in triplicate using the method described in the
Estimation of drug release from ASA–polyCA–HPMCP capsules
in vitro section. Commercially available ASA-ECs and ASA-ETs
served as positive controls. In addition, the release profiles of
ASA–polyMECA–HPMCP with different drug loading rates (5%,
Animals were purchased from the Beijing Experimental Animal
Center (Beijing, China). All animal care and experiments
were performed according to the Care and Use of Laboratory
Animals guidelines of the Institute of Laboratory Animal
Resources of the National Research Council (USA). The
in vivo experiment was performed at the State Key Laboratory
of Toxicology and Medical Countermeasures, Beijing
Institute of Pharmacology and Toxicology, and approved by
the institute’s Animal Care and Use Committee. Best efforts
were made to minimize the number of animals used and their
suffering.
10%, and 15%, determined by quantificationally dissolving ASA
in liquid MECA) and matrix volumes (50, 100, and 200 mL) in
different media were investigated by the same method as in
previous section.
In vivo analysis of ASA–polyMECA–HPMCP pharmacokinetics
For the pharmacokinetic analysis, male New Zealand white
rabbits with a mean body weight of 2 Æ 0.2 kg (8 weeks) were
divided randomly into four groups (n = 9/group) orally admi-
Monomer synthesis
BCA and MECA were synthesized using conventional
methods (Schemes S1 and S2, ESI†). The total productivity
was 52.3% for BCA and 46.5% for MECA. Their chemical
structures were confirmed by H-NMR (JNM-ECA-400; JEOL,
3
Japan); CDCl was used as the solvent. The purity was con-
À1
nistered ASA–polyMECA–HPMCP with 18 mg kg ASA, ASA–
À1
polyMECA–HPMCP with 24 mg kg ASA, free ASA solution
1
À1
À1
with 24 mg kg ASA, and ASA-ECs with 24 mg kg ASA,
respectively. Before administration, the rabbits were fasted for
firmed by gas chromatography (GC2010 PLUS; Shimadzu,
Japan).
1
2 h, but had access to water. Blood samples were collected into
heparinized tubes at different time points and centrifuged at
000 rpm for 5 min. Plasma samples were stored at À20 1C
before analysis. Salicylate was extracted from the plasma
4
ASA–polyCA–HPMCP complex preparation
The ASA and liquid CA monomers were mixed at a certain samples by vortexing 50 mL of plasma and 125 mL of 0.5%
weight ratio in a vortex mixer to yield a clear and transparent formic acid with two 500 mL aliquots of methyl tert-butyl ether
liquid state at room temperature. An appropriate amount (5%, (MTBE). The MTBE layers were combined, evaporated to
ASA in CA) of drug-loading liquid was poured into the HPMCP dryness, and reconstituted in 200 mL of the HPLC mobile phase
capsule, which was then stored at 80 1C for 24 h until the CA for analysis. The HPLC conditions were the same as in the
matrix excipients had polymerized completely and solidified, Estimation of drug release from ASA–polyCA–HPMCP capsules
yielding the final ASA–CA–HPMCP complex.
in vitro section.
1294 | J. Mater. Chem. B, 2021, 9, 1288À1296
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Journal of Materials Chemistry B
À1
GPC analysis
excised and opened along the greater curvature. 18 mg kg
of free ASA solution is used as the control. Gastric lesions were
photographed with a camera. Histological examination of the
stomach samples was also performed. The photographs of the
gastric lesions were taken with a photomicroscope.
GPC was performed with a Waters 1515 system (Waters, USA) to
determine the molecular weight of polyMECA. The polymers
(25 mg) were dissolved in tetrahydrofuran (THF, 0.5 mL), and
then refiltered through syringe filters with 0.22 mm aperture
before analysis. The experiments were repeated at least
three times.
Acute oral toxicity assessment
Ten healthy male Kunming mice (20 Æ 2 g) were divided
randomly into two groups (n = 5 each). The animals were fasted
overnight, but had free access to water. They were weighed before
oral administration of 0.5% CMC aqueous solution (control) or a
limit dose of the 0.5% CMC suspension containing 20% MECA
Assessment of the metabolism and distribution of orally
administered polyMECA in mice
An FITC-labeled CA monomer 6-amino-1-hexanol cyanoacrylate
was synthesized using conventional methods (Scheme S3,
ESI†), then mixed and copolymerized with MECA (97%, wt%).
A suspension of the copolymer powder (20%) in 0.5% carbox-
ymethyl cellulose (CMC) solution was prepared for intragastric
À1
prepolymer powder (2.5 g kg ). Food was withheld for an
additional 3–4 h after treatment. The animals were observed
individually to detect changes in general behavior and body
weight, toxic symptoms, and mortality periodically for the first
À1
administration (2.5 g kg ) in Kunming mice (male, 20 Æ 2 g,
24 h and daily thereafter for 14 days. At the end of the experi-
n = 3). Normal saline is used as the control. The animals were
placed in individual metabolism chambers immediately after
dosing with the FITC-labeled copolymer, then sacrificed at
mental period, all animals were weighed and sacrificed by cervical
dislocation, and the organs were removed for necropsy.
1
and 24 h, respectively. The stomach, small intestine, colon,
Twelve-week subchronic toxicity study
heart, liver, spleen, lungs, kidneys, and feces were collected.
Fluorescent images of the excreta and major organs were
acquired using an IVIS SPECTRUM in vivo imaging system
According to the acute toxicity study results, the subchronic
À1
toxicity dose was set at 0.25 g kg . Twenty rats of both sexes
(
5
180 Æ 10 g) were divided into two groups [n = 10 (5 males and
(PerkinElmer, UK) for the observation of polyMECA metabo-
females)/group] and their weights were recorded. The control
lism and distribution.
group was given 0.5% CMC aqueous solution and the polyMECA
group was given 0.5% CMC suspension containing 20% MECA
prepolymer powder (0.25 g per kg body weight per day) by oral
gavage for 12 weeks. The body weight and food and water intake
were recorded weekly. At the end of the study period, the rats
were sacrificed by cervical dislocation and blood samples were
taken by retro-orbital puncture using EDTA capillary tubes for
hematological and biochemical studies. The liver, kidneys,
spleen, lungs, and heart were collected, washed immediately in
NaCl (0.9%), and examined macroscopically. Hematological
analysis of blood samples was performed using an automated
hematology analyzer (Mindrary BC-2800vet, China). Biochemical
parameters were estimated using a Rayto Chemray 800 auto-
mated biochemical analyzer (China). Histopathological analysis
of the preserved organs and tissues was performed. Photomicro-
graphs were taken using a NIKON DS-F12 digital sight imaging
system (Nikon Corporation, Tokyo, Japan).
Rabbit model of atherosclerosis treatment
Male white New Zealand rabbits (n = 16) weighing 2.0 Æ 0.2 kg
(8 weeks) were used for the atherosclerosis treatment model.
The rabbits were fed with ND for 1 week, and then HFD
containing 0.5% cholesterol, 5% sucrose, and 5% lard for
8
weeks. They were divided randomly into two groups (n = 8
each) given normal saline (model control) and ASA–polyMECA–
HPMCP (drug efficiency is 15%) orally daily from week 2.
At the end of the treatment period, the rabbits were eutha-
nized. The degree of pathological changes was evaluated by
measuring the lesion (plaque) area on the aorta from the heart
to the iliac bifurcation. The aorta was fixed with 10% neutral
formaldehyde, the periadventitial tissue was cleaned, and the
aorta was opened longitudinally and stained with ORO. To
determine the stability of atherosclerotic plaques at the aortic
root, the aortic sinus was fixed in 10% formalin for 50 min,
then embedded in paraffin and cut into 6 mm sections. The
sections were stained with Masson stain to quantify the content
of collagen. In addition, overnight incubation with antibodies
to matrix metalloproteinase-9 (MMP-9) was performed for
MMP-9 quantification. The histological and immunohisto-
chemical analyses were performed with a Panoramic 250 device
Statistical analysis
The data were analyzed using two-tailed Student’s t tests.
P values o0.05 were considered to be significant.
Conflicts of interest
(3D HISTECH, Hungary).
The authors have no conflict of interest to declare.
Side effect evaluation
Following 2 months of atherosclerosis treatment in vivo, the
liver, spleen, lungs, kidneys, and heart of rabbits were sampled
Acknowledgements
to study the histotoxicity of ASA–polyCA–HPMCP. For the Financial support from the Beijing Natural Science Foundation
examination of gastric mucosal damage, the stomach was (7202146) is gratefully appreciated.
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