Design of folic acid decorated virus-mimicking nanoparticles for enhanced oral insulin delivery

Article abstract of DOI:10.1016/j.ijpharm.2021.120297

Mucus penetration and intestinal cells targeting are two main strategies to improve insulin oral delivery efficiency. However, few studies are available regarding the effectiveness of combining these two strategies into one nano-delivery system. For this

Full text of DOI:10.1016/j.ijpharm.2021.120297

International Journal of Pharmaceutics 596 (2021) 120297
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Design of folic acid decorated virus-mimicking nanoparticles for enhanced
oral insulin delivery
Hongbo Cheng, Shuang Guo, Zhixiang Cui, Xin Zhang, Yingnan Huo, Jian Guan, Shirui Mao*
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chitosan
Mucus penetration and intestinal cells targeting are two main strategies to improve insulin oral delivery effi-
ciency. However, few studies are available regarding the effectiveness of combining these two strategies into one
nano-delivery system. For this objective, the folic acid (FA) decorated virus-mimicking nanoparticles were
designed and influence of FA graft ratio on the in vitro and in vivo properties of insulin loaded nanoparticles was
studied systemically. Firstly, using folic acid as active ligand, different folic acid grafted chitosan copolymers
(FA-CS) were synthesized and characterized. Thereafter, using insulin-loaded poly(n-butylcyanoacrylate)
nanoparticles as the core, virus-mimicking nanoparticles were fabricated by coating of positively charged FA-CS
copolymer and negatively charged hyaluronic acid. Irrespective of the FA graft ratio, all the nanoparticles
showed good stability, similar insulin release in the gastrointestinal fluid, excellent and similar penetration in
mucus. The nanoparticles permeability in intestine was FA graft ratio and segment dependent, with FA graft ratio
at/over 12.51% presenting better effect in the order of duodenum > jejunum ≈ ileum. Both mechanism studies
and confocal microscopy observation demonstrated FA-mediated process was involved in the transport of FA
decorated nanoparticles. In vivo studies revealed hypoglycemic effect of the nanoparticles was FA graft ratio
dependent, a saturation phenomenon was observed when FA graft ratio was at/over 12.51%. In conclusion, folic
acid decorated virus-mimicking nanoparticles presented improved insulin absorption, implying combining
mucus penetration and active transcellular transport is an effective way to promote oral insulin absorption, while
the modification ratio of active ligand needs optimization.
Folate
Insulin
Nanoparticles
Mucus penetrating
Targeting
1. Introduction
and correct the defect of glucose metabolism caused by peripheral de-
livery (Arbit, 2004).
Biological drugs, such as peptides/proteins, are highly attractive in
modern medicine because of their high potency and specificity. Ac-
cording to the F&D report (2019), the global biomedical market reached
$261.8 billion in 2018 and will continue to grow at an annual -
growth rate of 11.8% over the next decade. However, due to the low
stability and poor absorption, biological drugs are mainly administrated
by injection in clinic. Therefore, in order to expand their application,
tremendous research and development have been carried out to develop
non-invasive routes for systemic delivery of biological drugs, including
oral, nasal, inhalation, buccal and transdermal routes (Anselmo et al.,
2019). Especially, as the most preferred and patient-friendly alternative
method to injection, oral route has attracted researchers interest for a
long time (Liu et al., 2017;Eilleia et al., 2018). More interestingly, oral
insulin therapy can not only improve patients compliance and reduce
the side effects, but also simulate the physiological secretion of insulin
It is known that mucus and the underlying intestinal cells are two
main obstacles for oral insulin delivery (Liu et al., 2017). Many viruses,
which are characterized by small particle size, densely coated with
negative and positive charges creating a net-neutral and highly hydro-
philic surface, can diffuse in mucus as fast as that in water (Lai et al.,
2009). Inspired by the features of viruses, the virus-mimicking nano-
particles were developed by our group (Zhang et al., 2018;Liu et al.,
2019b) to overcome the mucus barrier of oral insulin delivery, which
showed an excellent penetration in the porcine mucus. However, due to
lack of specific transport pathway, the uptake of virus-mimicking
nanoparticles in epithelial cells was restricted, limiting the oral insulin
delivery efficiency. It is reported that nanoparticles appended with
different targeting ligands such as lectin, CSK peptide and Vit B12 were a
promising strategy to improve the intestinal absorption of insulin by
interacting with the receptors on the intestinal cells membrane (Kaklotar
* Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, China.
E-mail address: maoshirui@syphu.edu.cn (S. Mao).
https://doi.org/10.1016/j.ijpharm.2021.120297
Received 30 September 2020; Received in revised form 30 December 2020; Accepted 15 January 2021
Available online 26 January 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
et al., 2016). Folic acid (FA), whose transport carrier is widely distrib-
uted across the gastrointestinal tract (GIT), was frequently used as a
target ligand to promote nanoparticles enterocyte endocytosis (El Leithy
et al., 2019; Xie et al., 2018; Xu et al., 2017). However, to the best of our
knowledge, there are few studies on the effect of FA graft ratio on the
intestinal absorption of drug-loaded nanoparticles. Moreover, the mucus
presented over the gastrointestinal tract might compromise the effi-
ciency of FA decorated nanoparticles. Therefore, in order to overcome
the mucus barrier and investigate the absorption enhancing effect of FA
targeting ligand on insulin loaded nanoparticles, virus-mimicking
nanoparticles with different FA graft ratios were designed herein. It is
assumed enhanced mucus-penetration in combination with ligand tar-
geting nanoparticles might be more effective in enhancing oral insulin
delivery.
Moreover, UV–Vis spectrophotometer method was used to measure
the content of FA in the FA-CS copolymers (Li et al., 2016). Firstly, FA-
CS was dissolved in 0.25% acetic acid, then its absorbance was measured
at 350 nm using UV–Vis spectrophotometer (UV 2000, Unico). And the
FA content in FA-CS was obtained by reference to the FA standard
calibration curve (A = 0.015C + 0.0034, R2 = 0.997). The coupling ratio
of FA was calculated by the following equation:
WFA
WFAꢀ CS ꢀ WFA
CR(%) =
× 100
(1)
where WFA represents the amount of FA in the FA-CS copolymer and
FA-CS represents the total amount of FA-CS copolymer.
W
2.3. Preparation and characterization of folic acid decorated
nanoparticles
To test this hypothesis, first of all, the core of insulin-loaded PBCA
nanoparticles (Ins/PBCA NPs) was prepared by the self-polymerization
of butylcyanoacrylate (BCA) in aqueous system (Cheng et al., 2020),
which can not only provide good protection to its cargo, but also show
pH-responsive release characteristics in the gastrointestinal fluids (Graf
et al., 2009). Thereafter, folic acid-modified chitosan (FA-CS) with
different modification degrees were synthesized and coated on the NPs
surface to enhance ligand targeting, and negatively charged hyaluronic
acid (HA) was further added to achieve virus-mimicking shell for
enhanced mucus penetration. Influence of FA-CS graft ratio on the
properties and stability of the nanoparticles, in vitro insulin release,
mucus penetration and permeability of the nanoparticles in the intes-
tine, and in vivo therapeutic effect were investigated systemically.
Insulin-loaded PBCA nanoparticles (Ins/PBCA NPs) was prepared as
described previously (Cheng et al., 2020). Briefly, 20
μL BCA was
dispersed in 2.0 mL HCl solution (pH 2.0) containing F127 and SDS. And
the Ins/PBCA NPs were obtained by dropping insulin solution dissolved
in HCl/Tris solution (1 mg/mL, pH 7.6) to the BCA solution (2/1, v/v)
and stirring for 30 min at room temperature. Secondly, Ins/PBCA NPs
was dropped to FA-CS solution (0.25% acetic acid, 0.6 mg/mL) in a
volume ratio of 2:1 and stirred for 30 min to fabricate Ins/PBCA/FACS
NPs. At last, Ins/PBCA/FACS/HA NPs was prepared by adding Ins/
PBCA/FACS NPs to HA water solution (2.0 mg/mL) in a volume ratio of
2:1 and agitated for 30 min at room temperature.
The average particle size and zeta potential of the nanoparticles were
analyzed using dynamic light scattering (DLS, NANO ZS90, Malvern) at
25 ◦C with a scattering angel of 90◦. The entrapment efficiency (EE) of
insulin in the nanoparticles was determined by measuring the amount of
free insulin in the supernatant (15,000 rpm, 30 min) using high per-
formance liquid chromatography (HPLC, Agilent 1100) method (Cheng
et al., 2020), and EE was calculated by the following equation:
2. Material and methods
2.1. Material
Porcine insulin was purchased from Wanbang Biochemical Phar-
maceutical Company. N-butylcyanoacrylate was supplied by Beijing
Compant Medical Devices Co., Ltd. Poloxamer 407 (F127) was a gift
from BASF. Sodium dodecyl sulfate (SDS) was obtained from Beijing
Biotopped Technology Co., Ltd. Folic acid was purchased from Sigma.
Carbodiimide (EDC) and N-hydroxy-succinamide (NHS) were from
Shanghai Common Chemical Science and Technology Co., Ltd. Chitosan
(100 kDa, DD ≥ 85%) was from Jinan Haidebei marine bioengineering
Co., Ltd. Hyaluronic acid (200 kDa) was from Bloomage Biotechnology
Co., Ltd. Tris, Pepsin (>3000 U/mg), Trypsin (>250U/mg), Cy5-NHS,
and DAPI solution were all from Dalian Meilun Biotechnology Co., Ltd.
Total am ount of insulin added ꢀ free insulin in supernatant
EE(%) =
× 100
Total am ount of insulin added
(2)
Then, the precipitate (15,000 rpm, 30 min) was collected and freeze
dried for 24 h at ꢀ 40 ◦C, and the loading capacity of insulin was
calculated by the following equation:
LC(%) = WInsulin/WNPs*100
(3)
where WInsulin represents the amount of insulin wrapped in the nano-
particles and WNPs represents the weight of nanoparticles.
2.2. Synthesis and characterization of folic acid-chitosan (FA-CS)
copolymer
Moreover, in order to identify the interaction between Ins/PBCA NPs
and the coating layers, the prepared nanoparticles were freeze dried for
Firstly, FA, EDC and NHS (1:1.5:1.5, molar ratio) were dissolved in
◦
◦
24 h at ꢀ 40 C and the obtained powders were analyzed by FTIR as
DMSO and stirred at 30 C for 3 h in dark. Secondly, 100 mg CS was
described above.
dissolved in 0.25% acetic acid (2 mg/mL, pH 6.0), then the activated FA
solution was slowly dropped to the CS solution and stirred at 30 ◦C for
24 h in dark. Thereafter, the solution was centrifuged at 10,000 rpm for
10 min and the supernatant was adjusted to pH 9.0 with NaOH solution.
Then, the precipitate was collected, redissolved in acetic acid solution
and centrifuged at 10,000 rpm for 10 min. At last, the supernatant was
dialyzed against pure water for 72 h (MWCO 8–12 K), and FA-CS was
2.4. Stability of folic acid decorated nanoparticles
Firstly, dilution stability of the nanoparticles in simulated gastric
fluid (SGF, pH 1.2 HCl) and simulated intestinal fluid (SIF, pH 6.8 PBS)
was investigated using DLS method. In brief, 200 μL nanoparticles so-
◦
obtained after freeze-drying for 24 h at ꢀ 40 C (Li et al., 2016;Yang
lution was added to SGF and SIF respectively, and incubated at 37 ◦C/80
rpm for 4 h (SGF) and 9 h (SIF). And the particle size changes of
nanoparticles were monitored by DLS at 37 ◦C.
et al., 2014).
In order to identify the successful synthesis of FA-CS, its chemical
structure was analyzed by FTIR. Briefly, the powders were compressed
into disks with KBr, and scanned from 4000 to 400 cmꢀ 1 at resolution of
1 cmꢀ 1 using the spectrometer (IFS 55, Bruker). Moreover, the synthe-
sized FA-CS was characterized by 1H NMR. Firstly, FA was dissolved in
deuterated DMSO while CS and FA-CS were dissolved in D2O/TFA (2:1,
v/v). Then, the samples were analyzed by 1H NMR at 600 MHz (AV-600,
Bruker).
Secondly, enzymatic stability of insulin in the nanoparticles was
evaluated. Briefly, the nanoparticles solution (Insulin 118.5 μg) was
added to 800 μL pepsin solution (pH 1.2 HCl, 9 IU/mL) and trypsin
solution (pH 6.8 PBS, 375 IU/mL), respectively, and incubated at 37 ◦C/
80 rpm. At the set time points, the samples were withdrawn, cold 0.1 N
NaOH solution (pepsin) or HCl solution (trypsin) was added to stop the
enzymatic reaction, and the nanoparticles were dissolved by methanol.
2

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Then, the remaining insulin content was analyzed using HPLC after
centrifugation (15,000 rpm, 30 min).
2.8. Observation of folic acid decorated nanoparticles uptake in
enterocyte
In this study, uptake of the nanoparticles in enterocyte was intui-
tively observed by confocal laser scanning microscopy (CLSM, LSM 710,
Zeiss). Firstly, the fasted rats (6–8 weeks) were anesthetized with chloral
hydrate by intraperitoneal injection. Then, a small incision was made in
rats abdomen, both ends of the different intestinal segments (~4 cm)
2.5. In vitro release of insulin from folic acid decorated nanoparticles
The release amount of insulin from the nanoparticles was determined
by centrifugation method (Liu et al., 2019a). In brief, the nanoparticles
solution (Insulin: 118.5 μg) was added to 800 μL SGF, SIF and pH 7.4
PBS, respectively, and incubated at 37 ◦C/80 rpm. At set time points, the
samples were taken out and centrifuged for 30 min at 15,000 rpm. And
the content of insulin in the supernatant was analyzed by HPLC. Herein,
similar factor (f2) was used to compare the release difference between
nanoparticles:
were sealed after the Cy5 labeled nanoparticles solution (300 μL) being
injected. One hour later, the rats were sacrificed and their intestinal
segments were excised with gently rinsing by 5 mL saline. Then, the
intestinal segments were fixed for 2 h in 4% paraformaldehyde and
dehydrated for 12 h in 30% sucrose. Thereafter, the intestinal loops were
embedded into O.C.T compound and frozen for cutting with Cryotome
(SLEE medical GmbH, D-55136 Mainz). At last, the epithelial nucleus
∑
{
}
n
(Rtꢀ Tt)ꢀ 0.5
t=1
f2 = 50 × log 1 + (1/n)
× 100
(4)
was labeled by DAPI solution (10 μg/mL) and the intestinal slices were
observed by CLSM at EX/EM 405/497 nm for DAPI and EX/EM 633/
700 nm for Cy5-Insulin.
where n represents the number of time points, Tt is the release amount of
the sample at different time points and Rt is the release amount of the
reference at the corresponding time points. And the difference is
considered significant if f2 < 50.
2.9. Hypoglycemic effect
The hypoglycemic effect of FA decorated virus-mimicking nano-
particles was evaluated in diabetic rats. Firstly, the diabetic rats were
induced with alloxan in Wistar rats (200 ± 20 g) (Cheng et al., 2020).
Secondly, the diabetic rats were fasted overnight but with free access to
water and then administered by the following formulations (n = 4 for
each group): insulin solution by oral (50 IU/kg), insulin solution by
subcutaneous injection (5 IU/kg), Ins/PBCA/CS/HA and Ins/PBCA/
FACS/HA NPs solution by oral (50 IU/kg). At last, the blood glucose
level of diabetic rats was monitored with blood glucose meter (JPS-6,
Beijing Yicheng) as described previously (Liu et al., 2019a). And the
pharmacological availability (PA) was calculated according to the
following equation:
2.6. Penetration of folic acid decorated nanoparticles in mucus
In the study, all the animal experiments followed the principle of
Laboratory Animal Care and were approved by Ethics Committee of
Shenyang Pharmaceutical University. The penetration of nanoparticles
in mucus was evaluated as described previously (Zhang et al., 2018).
Firstly, the mucus was collected from porcine intestine and Cy5 labeled
insulin was synthesized. Secondly, the nanoparticles were prepared
using Cy5-insulin as described above, added to mucus, mixed evenly and
incubated for 10 min at 37 ◦C/80 rpm. At last the samples were
centrifuged for 10 min at 2000 rpm and fluorescence intensity of the
supernatant was measured by microplate reader (SpectraMax® M3,
Molecular Devices Corporation) at excitation: 600 nm, emission: 665
nm. And penetration percentage of the nanoparticles in mucus was
determined by the ratio of its fluorescence intensity in the supernatant
and original nanoparticles.
/
AACp.o. Dosep.o.
AACs.c./Doses.c.
PA(%) =
*100
(6)
where AAC is the area above the blood glucose level curve, s.c. means
subcutaneous injection, p.o. means oral administration.
2.7. Permeability of folic acid decorated nanoparticles across rat intestine
2.10. Toxicity studies
Herein, the permeability of nanoparticles across rat intestine was
evaluated using ex-vivo method (Zhang et al., 2018). Firstly, Wistar rats
(6–8 weeks) were executed by cervical vertebrae and their intestines
In order to investigate the in vivo safety of nano-delivery system, the
gastrointestinal irritation study was carried out in healthy Wistar rats
(200 ± 20 g). Firstly, the rats were divided into saline group and
nanoparticles group. Then, the saline and nanoparticles groups were
daily given physiological saline solution (3.0 mL/Kg) and Ins/PBCA/
FA12.51-CS/HA NPs solution (50 IU/Kg) by gavage, respectively. After 7
days, the rats were sacrificed and the different gastrointestinal segments
(stomach, duodenum, jejunum and ileum) were excised and gently
washed with physiological saline solution, fixed in paraformaldehyde
were excised. Then 300 μL Cy5-labeled nanoparticles were injected into
the intestinal segments with both ends sealed (~4 cm), and incubated in
10 mL Krebs-Ringer solution at 37 ◦C/80 rpm. At indicated time points,
200 μL sample was collected from the accept chamber and equivalent
Krebs-Ringer solution was compensated. Fluorescence intensity of the
collected samples was recorded by microplate reader at excitation 600
nm, emission 665 nm, and the apparent permeability coefficient (Papp)
of insulin across intestine was calculated as follows:
(4%, w/v) and cut into 5 μm thick paraffin sections. At last, the paraffin
sections were stained by H&E (hematoxylin and eosin) and observed
dQ
1
using stereomicroscope.
Papp =
*
(5)
dt A*C0
2.11. Statistical analysis
where dQ/dt means the permeability rate of Cy5-insulin, A means the
surface area of intestine and C0 means Cy5-insulin initial concentration
in the donor chamber.
The experimental data were expressed by mean value ± standard
deviation (n ≥ 3). And the group data were analyzed by two tail stu-
dent’s t-test and one-way ANOVA with GraphPad Prism 6 at the prob-
ability level of 0.05.
Furthermore, the uptake mechanisms of FA decorated nanoparticles
in intestine were studied. In brief, the labeled nanoparticles were loaded
into duodenum and incubated in Krebs-Ringer solution containing
different absorption inhibitors, including folic acid (40 mg/mL), indo-
methacin (20 μg/mL), chlorpromazine (10 μg/mL) and colchicine (5 μg/
mL) at 37 ◦C/80 rpm. In addition, the duodenum was also incubated at
◦
low temperature (4 C) without inhibitors while the other procedures
were the same as described above.
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H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 1. (A) Synthesis scheme of folic acid-chitosan (FACS) copolymers, (B) FTIR spectra of CS, FA and FA-CS copolymers, (C) 1H NMR spectrum of CS, FA and FA-CS
copolymers, (D) UV curve of FA, CS and FA12.51-CS copolymers.
3. Results and discussion
ester reacted with the primary amino groups of CS and the FA-CS co-
polymers were obtained. To investigate the influence of FA graft ratio on
the in vitro and in vivo properties of the nanoparticles, FA-CS copolymers
with different coupling ratios, which were 6.36%, 12.51% and 19.84%,
respectively, were synthesized herein by adjusting FA and chitosan
molar ratio. Firstly, FTIR was used to investigate the structural charac-
teristics of FA-CS copolymers (Fig. 1B). It is known the absorption peak
of FA at 1696 cmꢀ 1 and 1606 cmꢀ 1 belong to the vibration of carboxyl
and pethidine ring amino groups, respectively. The absorption peak of
3.1. Synthesis and characterization of folic acid-chitosan copolymers
The copolymers of folic acid grafted chitosan (FA-CS) was synthe-
sized by the reaction between the carboxyl group of FA and the amino
groups of CS via the formation of amide bond. As shown in Fig. 1(A), the
γ-COOH of FA, which has higher reactivity than α-COOH (Zhang et al.,
2007), was firstly activated by NHS/EDC reaction, then the activated FA
4

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 2. The preparation mechanism and structure of Ins/PBCA, Ins/PBCA/FACS and Ins/PBCA/FACS/HA NPs.
Fig. 3. (A) Influence of FA6.36-CS concentration on Ins/PBCA/FA6.36-CS NPs characteristics; (B) HA concentration on Ins/PBCA/FA6.36-CS/HA NPs characteristics.
Symbols indicate statistically significant differences (*p < 0.05).
CS at 1655 cmꢀ 1 and 1599 cmꢀ 1 correspond to the vibration of amide I
–
(C O stretch) and amide II (NH blending) groups. The spectrum of
Table 1
–
The characteristics of insulin-loaded PBCA nanoparticles.
synthesized FA-CS copolymers displayed that the peak of FA at 1696
cmꢀ 1 disappeared and a new peak corresponding to C O bond vibra-
–
–
Nanoparticles
Graft
Size/nm
Zeta/mV
EE/%
LC/%
tion of CONH appeared at 1640 cmꢀ 1. Moreover, the peaks of CS at
–
–
ratio/%
1655 cmꢀ 1 and 1599 cmꢀ 1 were shifted to 1640 cmꢀ 1 and 1607 cmꢀ 1
,
Ins/PBCA
/
120.90 ±
3.18
ꢀ 11.13 ±
1.00
99.76 ±
0.10
30.11 ±
1.65
respectively, indicating the successful synthesis of FA-CS copolymers (Li
et al., 2016). It can be found the intensity of FA-CS peak at 1607 cmꢀ 1
increased with the increase of FA amount, suggesting the increase of FA-
CS graft ratio. Then, the structural characteristics of FA-CS copolymers
were confirmed using 1H NMR. As shown in Fig. 1(C), the 1H NMR
spectrum of FA-CS copolymers contained a series of peaks originating
from both FA and CS. The signal at 7.8 and 6.9 ppm correspond to the
protons of H-13/15 and H-12/16 of FA, respectively, and the signal at
3.6, 2.7, 2.2 and 1.1 ppm correspond to the protons of H-4′, H-1′, H-2′
and H-a of CS, respectively (Wang et al., 2014), further confirming FA
was successfully grafted onto CS. Moreover, the increase of FA-CS co-
polymers peak intensity at H-13/15 and H-12/16 also indicated FA-CS
grafting ratio increased with the increase of FA amount. Moreover, UV
spectrum was used to identify the characteristic of FA-CS copolymers. As
shown in Fig. 1(D), in the range of 250–500 nm, CS didn’t show any UV
absorption, and FA showed maximum absorption at 281 and 350 nm
Ins/PBCA/FACS
CS
150.75 ±
4.32*
32.85 ±
1.35
99.87 ±
0.11
22.54 ±
1.29
FA6.36-CS
168.37 ±
32.00 ±
1.06
99.91 ±
0.09
21.52 ±
0.86
2.20#
FA12.51
CS
-
-
184.05 ±
6.55
31.15 ±
1.20
99.90 ±
0.06
21.95 ±
0.94
FA19.84
CS
200.25 ±
8.84
30.25 ±
1.89
99.89 ±
0.10
21.69 ±
0.59
Ins/PBCA/
FACS/HA
CS
197.13 ±
6.42
ꢀ 10.25 ±
0.20
99.91 ±
0.02
16.43 ±
0.42
FA6.36-CS
215.45 ±
7.22
ꢀ 10.37 ±
0.51
99.89 ±
0.01
16.67 ±
0.16
FA12.51
CS
-
-
241.15 ±
7.99
ꢀ 10.60 ±
0.21
99.87 ±
0.01
16.73 ±
0.33
FA19.84
CS
275.95 ±
7.00
ꢀ 11.70 ±
0.42
99.87 ±
0.02
16.46 ±
0.23
Note: Symbols indicate statistically significant differences (p < 0.05), (*)
compared to Ins/PBCA NPs, (#) compared to Ins/PBCA/CS NPs.
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H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 4. FTIR spectra of Ins/PBCA NPs, folic acid-chitosan (FA12.51-CS) copolymers, Ins/PBCA/FACS NPs, hyaluronic acid (HA) and Ins/PBCA/FACS/HA NPs.
while the spectrum of FA-CS copolymers shifted to higher wavelength
and the second absorption peak was less obvious relative to the FA
spectrum, suggesting the successful synthesis of FA-CS copolymers.
were introduced into the system to improve the dispersion of BCA and
physical stability of the nanoparticles (Dong et al., 2018). As shown in
Fig. 3(A), the Ins/PBCA NPs were slightly negatively charged. After
adding to FACS solution, surface charge of the nanoparticles changed
from negative to positive and the particle size increased significantly (p
< 0.05), indicating the successful coating of FACS on Ins/PBCA NPs. And
particle size and surface charge of the nanoparticles increased gradually
with the increase of FACS concentration, indicating the increase of
coating thickness. As it is generally recognized that the nanoparticles
3.2. Influence of FA-CS graft ratio on the properties of nanoparticles
Herein, insulin was firstly wrapped in PBCA nanoparticles (Ins/PBCA
NPs) with BCA self-polymerization by the stimulation of Tris (Cheng
et al., 2020) (Fig. 2). And in the process of preparation, SDS and F127
Fig. 5. The size change of Ins/PBCA/FACS/HA NPs versus time (A) in pH 1.2 HCl, (*) compared to initial, (#) compared to CS group, (&) compared to FA12.51-CS
group; (B) in pH 6.8 PBS (0.05 M), (*) compared to initial, (#) compared to CS group. Symbols indicate statistically significant differences (*p < 0.05). Enzyme
stability of Ins/PBCA/FACS/HA NPs (C) in pepsin, (D) in trypsin. (*) compared to insulin.
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H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 6. Insulin release from Ins/PBCA/FACS/HA NPs in pH1.2 HCl, pH6.8 PBS and pH7.4 PBS (0.05 M).
with surface charge greater than 25 mV are more stable, 0.6 mg/mL
FACS was used to prepare the Ins/PBCA/FACS NPs. As shown in Table 1,
with the increase of FA-CS graft ratio, the particle size of Ins/PBCA/
FACS NPs increased while its surface charge decreased slightly. Since the
primary amino groups of CS decreased with the increase of FA graft
ratio, the electrostatic interaction between FACS and Ins/PBCA NPs
might be weakened, probably resulting in a relatively loose structure of
Ins/PBCA/FACS NPs (Zhang et al., 2018) and thus a slightly decrease of
its surface charge (Agrawal et al., 2015).
its symmetric stretching peak (1412 cmꢀ 1) disappeared, implying HA
coating layer was also formed by electrostatic interaction (Gilli et al.,
1994; Li et al., 2013).
3.3. Influence of FA-CS graft ratio on the stability of nanoparticles
It is known that stability of the nanoparticles was greatly affected by
the pH and enzymes in the GIT (Zhang et al., 2018). Herein, dilution
stability of the Ins/PBCA/FACS/HA NPs in different pH was firstly
investigated by DLS. As revealed in Fig. 5(A), in pH 1.2 HCl, particle size
of the nanoparticles with different FA-CS graft ratios showed a decrease
at first and remained constant for the next few hours. Compared with the
unmodified nanoparticles, FA decorated nanoparticles all showed a
bigger particle size in pH 1.2 medium, and the FA19.84-CS decorated
nanoparticles had the biggest particle size while no significant differ-
ence was found between the FA6.36-CS and FA12.51-CS groups. As
revealed in Fig. 5(B), in pH 6.8 PBS, particle size of the CS group showed
a slight increase, and no significant change in particle size was found for
FA6.36-CS and FA12.51-CS groups while a slight decrease in particle size
was observed in FA19.84-CS group. Overall, the FA19.84-CS decorated
nanoparticles showed the largest particle size in pH 6.8 PBS while no
significant difference was found among the other nanoparticles with
lower FA-CS graft ratios. The particle size of Ins/PBCA/FACS/HA NPs
with different FA-CS graft ratios ranged from 100 to 300 nm in pH 1.2
HCl and pH 6.8 PBS, indicating their good dilution stability in the
gastrointestinal fluids.
It is known mucus present on the intestinal epithelial cells greatly
impeded the transport of nanoparticles (Pearson et al., 2016), which
might discount the contribution of ligand based epithelial cells target-
ing. Cone et al. found many viruses with dense coating of positively and
negatively charged groups could diffuse in mucus as quick as that in
water (Olmsted et al., 2001). Thus, in order to overcome the mucus
barrier, the virus-mimicking nanoparticles were fabricated with the
coating of hyaluronic acid (HA) on Ins/PBCA/FACS NPs (Fig. 2). Firstly,
taking FA6.36-CS coated nanoparticles as an example, influence of HA
concentration on the properties of the nanoparticles was investigated. As
shown in Fig. 3(B), with the increase of HA concentration, surface
charge of the nanoparticles decreased, which changed from positive to
negative when HA concentration was increased to 1 mg/mL. Moreover,
particle size of the nanoparticles increased significantly (p < 0.05) with
HA addition, indicating successful coating of HA on Ins/PBCA/FACS
NPs. It was noted particle size of the nanoparticles increased sharply
with 0.25 mg/mL HA addition, indicating structure of the nanoparticles
was not compacted contributed to the weak electrostatic interaction
(Zhang et al., 2018). And no particle size difference was found in the HA
concentration range of 0.5–2 mg/ml while it increased significantly
when HA concentration was increased to 4.0 mg/ml, indicating increase
of coating thickness. Moreover, probably due to the nearly net-neutral
surface charge and thin coating layer, aggregation of nanoparticles
was observed after 48 h when HA concentration was less than 1.0 mg/
mL. Therefore, in order to simulate the surface properties of virus and
meanwhile guarantee stability of the nanoparticles, 2.0 mg/mL HA was
selected for coating in further study.
Thereafter, pepsin and trypsin, which are two important digestive
enzymes in GIT, were used to evaluate the enzyme stability of insulin in
Ins/PBCA/FACS/HA NPs. As shown in Fig. 5(C), free insulin was quickly
degraded by pepsin and no insulin could be detected after 1 h incubation
in pepsin solution. In contrast, the stability of insulin encapsulated in
nanoparticles was dramatically improved. The remained insulin content
was still above 60% after 2 h incubation in pepsin solution and no sig-
nificant difference was found among the different FA-CS graft ratio
nanoparticles (p > 0.05). Similarly, as revealed in Fig. 5(D), the trypsin
stability of insulin was also greatly improved by nanoparticles, and the
content of insulin in nanoparticles was about 50% after 2 h incubation in
trypsin solution while the content of free insulin dropped to 30% within
half an hour and could not be detected after 2 h. No significant differ-
ence in insulin content was found among the different FA-CS graft ratio
nanoparticles in trypsin solution (p > 0.05). Overall, the Ins/PBCA/
FACS/HA NPs with different FA-CS graft ratios all provided good pro-
tection for insulin in GIT, which would be beneficial for clarifying the
effect of FA-CS graft ratio on insulin intestinal absorption.
As revealed in Table 1, after HA coating, surface charge of all the Ins/
PBCA/FACS/HA NPs was reversed and their particle size increased
significantly in a FA-CS graft ratio dependent manner, indicating suc-
cessful coating of HA on the Ins/PBCA/FACS NPs. The entrapment ef-
ficiency (EE) of insulin in all the nanoparticles was more than 99%,
indicating the coating process and FA-CS graft ratio had no effect on the
EE of insulin.
Moreover, the coating mechanism was analyzed by FTIR. As shown
–
in Fig. 4, after coating of FACS on the Ins/PBCA NPs, the amide I (C
O
–
stretch, 1640 cmꢀ 1) absorption peak of FACS was shifted to 1658 cmꢀ 1
and its amide II (NH blending) absorption peak at 1607 cmꢀ 1 dis-
appeared, indicating electrostatic interaction was involved in the
coating process of Ins/PBCA/FACS NPs (Jin et al., 2011). For the Ins/
PBCA/FACS/HA NPs, after HA coating, the asymmetric stretching peak
(1622 cmꢀ 1) of carboxylate anion of HA was shifted to 1659 cmꢀ 1 and
3.4. Influence of FA-CS graft ratio on the in vitro release of insulin
Herein, the in vitro release of insulin from Ins/PBCA/FACS/HA NPs
was investigated in different pH media. As shown in Fig. 6(A), in pH 1.2
HCl, no insulin was released from nanoparticles with different FA-CS
graft ratio, indicating their good stability in acid fluid. In pH 6.8 PBS,
7

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 7. (A) Penetration percentage of Ins/PBCA/FACS/HA NPs in original mucus. The accumulative amount of insulin transported through different rats’ intestinal
segments (B) Duodenum; (C) Jejunum; (D) Ileum; (E) Apparent permeability coefficient (Papp) of insulin in different rat intestine segments (*versus CS group,
#versus FA6.36-CS group); (F) Papp of insulin from Ins/PBCA/FA12.51-CS/HA NPs in duodenum in presences of different uptake inhibitors (*versus control, *p < 0.05,
**p < 0.01).
insulin was continuously released from nanoparticles and no significant
difference was found among the nanoparticles with different FA-CS graft
ratio (f2 > 50). It is known that the release behavior of drug from
nanoparticles is closely related to the properties of carrier materials and
their combination mode. It is reported that insulin was encapsulated in
PBCA nanoparticles by the non-covalent bond (Sullivan and Birkinshaw,
2004) and the degradation rate of PBCA in alkaline fluid was very slow
(Sulheim et al., 2016). Therefore, it can be speculated that the main
release mechanism of insulin from Ins/PBCA/FACS/HA NPs was passive
diffusion.
penetration in porcine mucus was investigated. As shown in Fig. 7(A),
the permeation percentage of Ins/PBCA/FACS/HA NPs with different
FA-CS graft ratios were all more than 60% after 10 min of incubation in
mucus, and no significant difference was found among them (p > 0.05),
which was much larger than the positively charged Insulin/Chitosan
NPs (5%) or negatively charged Insulin/Chitosan/Alginate NPs (22%)
previously designed by our group (Zhang et al., 2018). It is known the
gastrointestinal mucus are composed of water, mucins, lipids and pro-
teins, etc (Lai et al., 2009). And the negatively charged mucins are the
main component of mucus, which are easy to capture the positively
charged nanoparticles by electrostatic interaction. Meanwhile, the
network formed by the long chain of mucins tend to trap the particles
larger than 500 nm (Roger et al., 2010). Moreover, the hydrophobic
interaction offered by mucins and lipids can hinder the penetration of
hydrophobic substances. The mucus-penetrating nanoparticles designed
herein was composed of hydrophobic core (Ins/PBCA) and hydrophilic
coating layer (FACS/HA) with a slight negative surface charge and small
particle size (<300 nm). Furthermore, the nanoparticles showed good
stability and little insulin release (10 min) in the gastrointestinal fluids
(Fig. 6). Therefore, probably due to the virus-mimicking surface prop-
erties and good stability in GIT (Malhaire et al., 2016), the Ins/PBCA/
FACS/HA NPs were less entrapped by mucus, resulting in their excellent
penetration in mucus. Overall, the Ins/PBCA/FACS/HA NPs with
different FA-CS graft ratios showed excellent and similar permeability in
mucus, which is beneficial for clarifying the effect of FA-CS graft ratio on
insulin intestinal absorption.
Moreover, the release of insulin from Ins/PBCA/FACS/HA NPs was
also evaluated in physiological fluid (pH 7.4 PBS) to test the influence of
pH on drug release. As shown in Fig. 6(B), consistent with that in pH 6.8
PBS, insulin was released from the nanoparticles in a sustained manner
in pH 7.4 PBS and the FA-CS graft ratio had no significant effect on its
release rate (f2 > 50). However, a higher insulin release rate was found
in pH 7.4 PBS relative to that in pH 6.8 PBS. Overall, the release of in-
sulin from Ins/PBCA/FACS/HA NPs was pH-dependent, and insulin
release was inhibited in acidic medium, and increased with the increase
of pH. To the best of our knowledge, this pH respond release mode is an
ideal nano-delivery system for oral biomacromolecule, which can not
only improve insulin stability in stomach, but also promote its absorp-
tion in intestine and physiological fluid (Wong et al., 2017). Moreover,
the similar insulin release behavior of Ins/PBCA/FACS/HA NPs with
different FA-CS graft ratios in GIT provides a prerequisite for the study of
their intestinal absorption.
3.5. Influence of FA-CS graft ratio on the mucus penetration of
nanoparticles
3.6. Influence of FA-CS graft ratio on the permeability of nanoparticles in
intestine
Mucus, which distributes along the GIT and protects the underlying
epithelial cells, is also a natural barrier for oral nano-delivery system.
Nowadays, mucus-penetrating nanoparticles, which could penetrate the
mucus layers deeply even to the absorption surface of intestinal cells,
have been widely used to overcome the mucus barrier (Liu et al., 2015).
Herein, the mucus-penetrating nanoparticles (Ins/PBCA/FACS/HA NPs)
was designed by mimicking the surface properties of virus and their
In order to investigate the influence of FA-CS graft ratios on insulin
intestinal absorption, the permeability of Ins/PBCA/FACS/HA NPs in
different intestine segments were studied by ex-vivo method and shown
in Fig. 7(B-D). It was demonstrated the absorption of insulin increased
over time in duodenum, and compared with the unmodified nano-
particles (CS group), the FA-CS decorated nanoparticles showed signif-
icantly enhanced insulin absorption in 75 min and 120 min (p < 0.05)
8

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 8. Observation of Ins/PBCA/CS/HA and Ins/PBCA/FA12.51-CS/HA NPs on microvilli absorption (1 h). (A) Confocal images; (B) Average fluorescence intensity.
The cells were stained by DAPI (Blue). Symbols indicate statistically significant differences (p < 0.05, *versus CS group, #versus indicated group).
while no significant difference was found in 30 min, indicating FA car-
riers participated in the intestinal transport of FA-CS decorated nano-
particles and the process was time dependent. Moreover, in duodenum,
no significant difference was found in insulin absorption among the
FA6.36-CS, FA12.51-CS and FA19.84-CS groups (p > 0.05). Compared to
that in duodenum, insulin absorption in jejunum was remarkably
decreased and the influence of FA graft ratio was more apparent. It was
noted that in jejunum, no significant difference in insulin absorption was
found between the CS and FA6.36-CS groups (p > 0.05), while statisti-
cally enhanced insulin absorption was observed in FA12.51-CS and
FA19.84-CS groups, and no difference was found between these two
groups (p > 0.05). And in ileum, the FA19.84-CS group showed more
insulin absorption than that of the CS or FA6.36-CS group (p < 0.05)
while no significant difference between the FA12.51-CS and FA19.84-CS
groups was found. Moreover, the apparent permeability coefficient
(Papp) of insulin was further calculated to evaluate the effect of FA-CS
graft ratio on the intestinal absorption of insulin. As revealed in Fig. 7
(E), the FA-CS decorated nanoparticles showed the best insulin ab-
sorption in duodenum, which was 2.6- to 3.3-fold higher than that of
unmodified nanoparticles. And in jejunum/ileum, the nanoparticles
with higher FA-CS graft ratios (FA12.51-CS, FA19.84-CS) showed more
insulin absorption than that of CS group while no significant difference
was found between the FA12.51-CS and FA19.84-CS groups (p > 0.05). It is
reported that the reduced folate carrier (RFC) and proton-coupled folate
transporter/hem carrier protein 1 (PCFT/HCP1) are two main trans-
porters mediating folic acid intestinal absorption, and the RFC is widely
distributed across the GIT while PCFT/HCP1 mRNA is particularly
expressed in the duodenum and to a less extent in jejunum (Qiu et al.,
2006). Furthermore, it is known the duodenum is characterized by a thin
mucus layer, which is beneficial for nanoparticles penetration (Ensign
et al., 2012). Therefore, probably due to the thin mucus and specialized
carrier, the FA-CS decorated nanoparticles were easier to pass through
the mucus layer to intestinal epithelial cells and interacted with the FA
receptors, resulting in their better absorption in duodenum.
Moreover, FA-CS graft ratio dependent drug absorption enhancing
effect can probably be explained by the fact that herein hyaluronic acid
(HA) was used to improve nanoparticles penetration in mucus by
coating on the surface of Ins/PBCA/FACS NPs. The HA coating layer
might produce a shielding effect to the FA targeting ligand. In addition,
the expression of PCFT/HCP1 mRNA was less in jejunum. Therefore,
probably due to the shielding effect of HA and reducing of FA receptors,
few ligands of the FA6.36-CS decorated nanoparticles interacted with the
FA receptors, resulting in limited insulin absorption improvement in
jejunum. However, with the increase of FA-CS graft ratio, more FA li-
gands might be exposed to the surface of nanoparticles, and the FA
ligand-receptor interaction might occur between the nanoparticles and
intestinal cells, leading to the improvement of insulin absorption for the
FA12.51-CS and FA19.84-CS groups in jejunum. In ileum, probably due to
the limited transport efficiency of RFC (Qiu et al., 2006), the nano-
particles with high FA-CS graft ratio (FA19.84-CS) was more likely to be
absorbed by the FA ligand-receptor transport pathway, resulting in its
best absorption in ileum. Overall, the study revealed that FA decorated
nanoparticles could improve insulin intestinal uptake and the FA-CS
graft ratio had a certain effect on its absorption.
To further understand the transport mechanisms of FA decorated
nanoparticles in intestine, taking FA12.51-CS as the targeting ligand and
duodenum as the research segment, the intestinal uptake of Ins/PBCA/
FACS/HA NPs in Krebs-Ringer (KR) solution containing different ab-
sorption inhibitors was studied. As shown in Fig. 7(F), in the KR solution
containing oversupplied FA, the intestinal absorption of insulin was
reduced by 60.57%, further verifying FA carrier-mediated process was
involved in the transport of Ins/PBCA/FACS/HA NPs. And the signifi-
cant decrease of insulin absorption in the colchicine and indomethacin
solution (p < 0.05) indicated that the macropinocytosis and caveolin-
9

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 9. Blood glucose level vs. time profiles following administration of nanoparticles in diabetic rats. Date are means ± SD (n = 4). Symbols indicate statistically
significant differences (*p < 0.05), (*) FA6.36-CS group versus FA12.51-CS group, (#) CS group versus FA19.84-CS group. Note: the results of saline group and insulin
solution groups were adapted from our previous study (Cheng et al., 2020).
mediated endocytosis also participated in the transport of Ins/PBCA/
Table 2
FACS/HA NPs in intestine. Moreover, insulin absorption was greatly
Pharmacological availability in diabetic rats given an oral insulin dose of 50 IU/
reduced in low temperature (4 ◦C), suggesting the intestinal transport of
kg. Data are means ± SD (n = 4).
Nanoparticles
Ins/PBCA/FACS/HA NPs also relayed on energy. In summary, multiple
Administration
Dosage
(IU/kg)
AAC (%*h)
PA (%)
mechanisms were involved in the transport of Ins/PBCA/FACS/HA NPs
in intestine and insulin absorption could be improved by the FA ligand-
receptor targeting effect.
route
Insulin solution
Insulin solution
Ins/PBCA/CS/HA
Injection
Oral
5
422.40 ±
62.22
100
50
50
50
50
50
50.33 ±
43.25
1.20 ±
1.03
3.7. Observation of folic acid decorated nanoparticles uptake in
enterocyte
Oral
296.68 ±
54.82
7.02 ±
1.30
Ins/PBCA/FA6.36
-
Oral
332.90 ±
65.05
7.88 ±
1.54
The intestinal permeability study revealed the absorption of Ins/
PBCA/FACS/HA NPs was FA graft ratio and intestinal site dependent.
Herein, taking Ins/PBCA/ FA12.51-CS/HA NPs as an example, its uptake
and penetration in intestinal microvilli was further observed by CLSM
technique. As revealed in Fig. 8, compared with the unmodified nano-
particles (CS), the FA decorated nanoparticles (FACS) showed stronger
fluorescence intensity in duodenum (p < 0.05), further demonstrated
that the FA carriers participated in the transcellular transport of Ins/
PBCA/FACS/HA NPs and promoted insulin intestinal uptake. Moreover,
the Ins/PBCA/FACS/HA NPs showed more insulin uptake in duodenum
relative to that in jejunum/ileum (p < 0.05), and more nanoparticles
could be found in the middle and bottom of duodenum (A-C), indicating
FA receptors, especially PCFT/HCP1 transporter, promoted the uptake
and penetration of Ins/PBCA/FACS/HA NPs in duodenal microvilli. In
jejunum, the FACS group also showed a better insulin absorption than
the CS group (p < 0.05), and probably due to the limited expression of
PCFT/HCP1 mRNA (Qiu et al., 2006), the Ins/PBCA/FACS/HA NPs had
a less uptake in jejunum than that in duodenum (p < 0.05). Compared
with the CS group, the FACS group promoted insulin uptake in ileum (p
< 0.05). However, probably due to the thick mucus (Ensign et al., 2012)
and limited RFC transport efficiency (Qiu et al., 2006), compared with
the duodenum, the uptake of Ins/PBCA/FACS/HA NPs in ileum was
poor (p < 0.05) and mainly distributed in the border of ileal microvilli.
Thus, CLSM studies further verified that the intestinal absorption of
insulin could be improved by the FA receptor mediated transport of Ins/
PBCA/FACS/HA NPs, with the best absorption in duodenum.
CS/HA
Ins/PBCA/FA12.51
-
-
Oral
414.05 ±
65.67
9.80 ±
1.55*
9.67 ±
1.73*
CS/HA
Ins/PBCA/FA19.84
CS/HA
Oral
408.60 ±
73.07
Symbols indicate statistically significant differences (p < 0.05, * versus CS
group).
Note: the results of insulin solution groups were adapted from our previous study
(Cheng et al., 2020).
loaded nanoparticles all showed obvious hypoglycemic effect while the
insulin solution failed to reduce the blood glucose level in diabetic rats,
indicating the nanoparticles designed herein could improve insulin oral
delivery efficiency. And compared with subcutaneous injection of in-
sulin, oral insulin nanoparticles showed a mild and sustained hypogly-
cemic effect, and this might be able to avoid the side effect of
hypoglycemia caused by subcutaneous injection. Probably due to the
limited FA carriers-mediated transport, the FA6.36-CS decorated nano-
particles showed comparable hypoglycemic effect relative to the CS
group and no significant difference in pharmacological availability (PA)
was found between them (Table 2) (p > 0.05). In contrast, a more
obvious hypoglycemic effect was observed when FA graft ratio was
increased to 12.51% (FA12.51-CS) (p < 0.05), indicating FA carriers-
mediated transport contributed to improved insulin in vivo absorption.
However, further increasing of FA graft ratio to 19.84% led to no further
improvement in insulin absorption, and no significant difference in PA
was found between the Ins/PBCA/FA19.84-CS/HA NPs and Ins/PBCA/
FA12.51-CS/HA NPs (p > 0.05), indicating FA-mediated transport was
saturated at the graft ratio 12.51% studied here. And this phenomenon is
in good agreement with the in ex-vivo permeation results.
3.8. Hypoglycemic effect
In vitro studies revealed the Ins/PBCA/FACS/HA NPs could improve
insulin intestinal uptake by promoting its mucus penetration and
transcellular transport mediated by FA carriers. And in order to evaluate
the influence of FA-CS graft ratio on insulin in vivo absorption, the hy-
poglycemic effect of Ins/PBCA/FACS/HA NPs in diabetic rats was
further studied. As shown in Fig. 9, after oral administration, the insulin-
3.9. Toxicity studies
Moreover, the in vivo safety of nano-delivery system was evaluated
by histopathology examination. As revealed in Fig. 10, similar with that
10

H. Cheng et al.
International Journal of Pharmaceutics 596 (2021) 120297
Fig. 10. Gastrointestinal irritation evaluation after the oral administration of Ins/PBCA/FA12.51-CS/HA NPs at dose of 50 IU/Kg for 7 days.
of saline group, no histopathological lesions and hyperemia were
observed in the different gastrointestinal segments after 7 days orally
administration of Ins/PBCA/FA12.51-CS/HA NPs, indicating the good
tissue compatibility and safety of this nano carrier.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.120297.
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