Materials Science and Engineering C 70 (2017) 278–286

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Preparation of chitosan-based multifunctional nanocarriers overcoming

multiple barriers for oral delivery of insulin
Lei Li a, Guohua Jiang a,b,c,⁎, Weijiang Yu a, Depeng Liu a, Hua Chen a, Yongkun Liu a, Zaizai Tong a,
Xiangdong Kong d, Juming Yao a,b,c
a
Department of Materials Engineering, Zhejiang Sci Tech University, Hangzhou 310018, China
b
National Engineering Laboratory for Textile Fiber Materials and Processing Technology (Zhejiang), Hangzhou 310018, China
c
Key Laboratory of Advanced Textile Materials and Manufacturing Technology (ATMT), Ministry of Education, Hangzhou 310018, China
d
College of Life Science, Zhejiang Sci Tech University, Hangzhou 310018, China

a r t i c l e i n f o a b s t r a c t

Article history: To overcome multiple barriers for oral delivery of insulin, the chitosan-based multifunctional nanocarriers mod-
Received 14 July 2016 ified by L-valine (LV, used as a target ligand to facilitate the absorption of the small intestine) and phenylboronic
Received in revised form 21 August 2016 acid (PBA, used as a glucose-responsive unit) have been designed and evaluated in this study. The resultant
Accepted 30 August 2016
nanocarriers exhibited low cytotoxicity against HT-29 cells and excellent stability against protein solution. The
Available online 3 September 2016
insulin release behaviors were evaluated triggered by pH and glucose in vitro. The chemical stability of loaded in-
Keywords:
sulin against digestive enzyme were established in presence of simulated gastric fluid (SGF) containing pepsin
Nanocarriers and simulated intestinal fluid (SIF) containing pancreatin, respectively. The uptake behavior of HT-29 cells was
Chitosan evaluated by confocal laser scanning microscope. After oral administration to the diabetic rats, an effective hypo-
Insulin glycemic effect was obtained compared with subcutaneous injection of insulin. This work suggests that L-valine
Oral delivery modified chitosan-based multifunctional nanocarriers may be a promising drug delivery carrier for oral admin-
Hypoglycemic effect istration of insulin.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction peptidase 4 inhibitor and thizalidinediones. For example, Glimepiride,

There are 415 million adults were living with diabetes in 2015 and
this number is expected to increase to around 642 million or one in
ten adults by 2040, according to a report from International Diabetes
Federation (IDF) [1]. It has become one of the most lethal diseases in
some counties, especially in the developing countries. Insulin is com-
monly used to treat diabetes [2]. Although the significant developments
in insulin therapy over the past few decades, subcutaneous injection of
insulin remains the preferred approach for the treatment of insulin-re-
quiring diabetic patients due to ease of administration [3]. However,
such injections must pass through the skin or mucosal barrier, resulting
in dermal trauma and pain [4]. Thus, focusing on the development new
route of administration (oral or pulmonary) or reducing the injection
doses are beneficial to reduce the inconvenience and drawbacks associ-
ated with this conventional method [5–7]. Oral diabetes drugs, pills you
take by mouth, are commonly prescribed to help treat diabetes. Cur-
rently, there are 6 groups of oral medicine, including biguanides,
sufonylureas, meglitinides, alpha-glucosidase inhibitors, dipeptidyl
⁎ Corresponding author at: Department of Materials Engineering, Zhejiang Sci Tech
University, Hangzhou 310018, China.
E-mail address: ghjiang_cn@zstu.edu.cn (G. Jiang).
common brand name as Amaryl, is a kind of sulfonylureas drug. Nausea
and upset stomach may occur after oral treatment. Acarbose (alpha glu-
cosidase inhibitor), common brand name as Percose, is often used to
control high blood sugar in people with diabetes. Diarrhea, gas, upset
stomach, constipation, or stomach pain may occur in the first few
weeks of treatment. At present, suitable oral formulations of proteins
are still under development, facing countless challenges despite all the
efforts, time and money spent on the research. Major obstructions in de-
veloping oral or pulmonary insulin formulations are either enzymatic
barriers or physical barriers (i.e. intestinal epithelium), which oral insu-
lin has to overcome [8]. Because insulin is a peptide that consisted of 51
amino acids, it can get deteriorated by gastric pH and intestinal en-
zymes, and even intestinal epithelial cell membranes serve as absorp-
tion barrier for intact peptide structure resulting in b 1% bioavailabity
of total insulin taken orally [9–12].
To overcome these problems mentioned above, various approaches
are tried which include micro and nanoparticles [13–15], liposomes
[16,17], gastrointestinal patches [18] and permeation enhancers [19,
20]. Among them, the use of biocompatible and biodegradable materials
has been described as a promising strategy toward oral administration
of proteins and peptides [21,22]. Due to favorable nature of chitosan
as a positively charged biocompatible, nontoxic, and mucoadhesive

http://dx.doi.org/10.1016/j.msec.2016.08.083
0928-4931/© 2016 Elsevier B.V. All rights reserved.
 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286
polymer, many researchers selected chitosan as an oral drug carrier
[23–29]. Chitosan is a mucoadhesive polycationic polymer composed
of N-acetyl-d-glucosamine and d-glucosamine. It, with an intrinsic pKa
of 5.5, will thus lose its charge and spontaneous assembly into nanopar-
ticles in an aqueous solution [30,31]. For example, together with
tripolyphosphate (TPP) or even just polyelectrolyte complexation
with insulin is the most common methods to produce chitosan nano-
particles. The interaction of chitosan and polyanions leads to a spon-
taneous formation of nanoparticles in an aqueous media and mild
conditions, with no need for using organic solvents or heat, avoiding
cytotoxicity concerns and threats to insulin stability, thus being the
main advantages of these carriers [32]. Another study conducted by
Mukhopadhyay et al. showed an average assimilation efficiency of
insulin with self-assembled chitosan nanoparticles of approximately
85% [33]. The nanoparticles retained insulin efficiently in simulated
gastric conditions with a significant amount of insulin released in
simulated intestinal conditions. These insulin-loaded nanoparticles
were effective for dropping the blood glucose level when adminis-
trated in vivo.
Due to the gradual degradation kinetic profiles of the composite
nanoparticles in vivo, coupled with hydrophobic interactions between
the hydrophobic drug and hydrophobic segment of the polymer matrix,
the unfavorably slow drug release may be happen. The successful design
and synthesis of stimulus responsive chitosan-based intracellular deliv-
ery systems is in high demand to enhance the oral absorption while si-
multaneously preventing unwanted toxicities. In order to overcome
such issue, an alternative glucose sensor moiety can be introduced
from a synthetic component, phenylboronic acid as a potential candi-
date for such an objective, which is due to the fact that the boronic
acid binds reversibly to diols to form a cyclic boronic ester in aqueous
media [34–38]. Moreover, the boronic acid is a biocompatible functional
group with low cytotoxicity and low immunogenicity. In addition, the
oral bioavailability of insulin is always very poor because of the follow-
ing barriers: insulin is susceptible to digestion by proteases in the gas-
trointestinal (GI) tract; the GI tract is coated with a mucus layer that
may prevent macromolecules or drug delivery systems from reaching
beneath the epithelial cells [39–41]. Because of the poor cellular uptake
of a hydrophilic macromolecule in epithelial cells, insulin can hardly
permeate the epithelial cell layer in the intestine. Fortunately, it has
been reported that conjugating L-valine increases its transportation in
intestinal cells of rats [42]. For instance, L-valine modified chitosan in
the nanoparticles greatly contributed to high encapsulation efficiency
for protein drug, enhancement of drug absorption, prolonged drug res-
idence in the gut, and favorable enzymatic inhibition [43,44]. The L-va-
line-conjugated drug enhances oral bioavailability of drug, implicating
the great potential of L-valine-conjugated carrier in absorption of drug
[45].
The aim of this investigation is to prepare multifunctional
nanocarriers, in which chitosan used as nanocarriers backbone,
phenylboronic acid (PBA) used as hydrophobic and glucose-sensitive
unit, and L-valine (LV) used to facilitate the absorption of the small in-
testine. They can be easily obtained via graft reaction [46] and, thus,
spontaneously form nanocarriers in aqueous solution. Then, the novel
oral drug delivery system is further developed for overcoming multiple
barriers during the insulin delivery in GI tract. The insulin release
behaviors are evaluated triggered by pH and glucose in vitro. And the
hypoglycemic effect against diabetic Sprague Dawley (SD) rat models
is evaluated in vivo. Herein, using L-valine modified chitosan as
nanonarriers for oral delivery of insulin exhibit some advantages.
Firstly, chitosan is natural polymeric material. It exhibit higher bio-
compatible, nontoxic and biodegradable properties compared with
synthesized ones. Secondly, although chitosan is a mucoadhesive
polycationic polymer, the modification of L-valine to the chitosan is
beneficial to prolong their gastrointestinal retention time and pro-
mote the uptake of insulin before elimination from the intestinal
canal.
279
2. Experimental
2.1. Preparation of chitosan-based multifunctional nanocarriers
Firstly, carboxymethyl chitosan (CMCS) was prepared according to a
similar method described elsewhere [47]. Then, the terminal carboxyl
groups of CMCS were conjugated to the amino groups of 3-APBA and
L-Valine to synthesize CMCS-PBA-LV. Briefly, 300 mg CMCS was dis-
solved in 40 mL of deionized water containing EDC and NHS. The reac-
tion proceeded under stirring for 2 h at 0 °C to form active esters of
CMCS. 3-APBA (500 mg in DMF) and L-Valine (100 mg in deionized
water) were added to the above solution and stirred for 24 h to get
the reaction complete. The mixture was subjected to a 5-fold dilution
with deionized water and filtered. The filtrate was dialyzed against de-
ionized water using a dialysis bag (MWCO = 3500 Da) for 48 h and
freeze-dried to obtain CMCS-PBA-LV. The final yield of CMCS-PBA-LV
was around 87%.
2.2. Preparation of insulin loaded chitosan-based multifunctional
nanocarriers
The insulin-loaded CMCS-PBA-LV nanoparticles were prepared by
the similar route except the adding certain amount of insulin. Briefly, in-
sulin was dissolved in HCl solution (0.01 M, pH = 2.0) at a concentra-
tion of 1 mg mL−1, and the pH was adjusted to 8.0 using 1 M NaOH
solution. Then, the insulin solution was mixed with 1% CMCS-PBA-LV
solution. Then the sodium tripolyphosphate (0.5%) was added which
was continued stirring (1000 rpm) at room temperature for 30 min,
yielding an opalescent suspension. The resultant solution was centri-
fuged at 12,000 rpm for 30 min at 4 °C. The supernatant was removed
for the determination of entrapment efficiency and the formed insu-
lin-loaded multi-responsive nanocarriers were freeze-dried and stored
at 4 °C in the dark until further use. The FITC-insulin loaded CMCS-
PBA-LV nanocarriers were prepared by the same method.
2.3. Insulin release in vitro
The insulin release studies were carried out in pH = 1.2, 6.8 and 7.4
phosphate buffer solution (PBS) with temperature controlled at 37 °C.
The insulin loaded nanocarriers were suspended in the above buffer so-
lution (2.5 mg mL−1) containing different concentration of glucose. Al-
iquots of 200 μL were taken at one-hour intervals and the released
insulin was estimated by means of Lowry method [48]. Equivalent vol-
ume of the fresh buffer was replaced each time after the sampling. The
experiments were done in triplicates. The amount of insulin in the test
solution was calculated from the insulin standard maintained during
the assay.
2.4. Enzyme inhibition studies
To investigate the protective properties of the CMCS-PBA-LV against
enzymatic degradation of insulin, the insulin-loaded nanocarriers (1%)
were dispersed in (Simulated Gastric Fluid) SGF (USP) and (Simulated
Intestinal Fluid) SIF (USP). The SGF and SIF were consisted of 35 mM
NaCl, 80 mM HCl, 0.3% (w/v) pepsin, pH = 1.2 and 50 mM KH2PO4,
15 mM NaOH, 1.0% (w/v) pancreatin, pH = 6.8, respectively. Aliquots
of insulin solution (3 mL each, 98.6 μg L−1), insulin-loaded nanocarriers
(INCs) containing the same content insulin in 1 mL of 0.08 M HCl were
dispersed in SGF and SIF, respectively. The mixtures were incubated
under agitation at 100 rpm on an orbital shaker for 30 min and
60 min in the 37 °C water bath. The enzymatic reaction was stopped im-
mediately by the addition of 0.1 M NaOH. Samples (100 μL) were col-
lected at 30 min and 60 min, then followed by determination of
remained insulin concentration using the insulin ELISA kit [49].
280 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286
2.5. Cytotoxicity of chitosan-based nanocarriers
Cytotoxicity can be evaluated using the 3-(4,5-dimethyl-2-
thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay [50]. To
determine cell viability, the HT-29 cells were sowed in 96-well plates
at a density of 1 × 104 cells per well with 200 μL of growth medium con-
taining 10% FBS and allowed to grow overnight at 37 °C in 5% CO2 incu-
bator. After 24 h of cell attachment, the media were aspirated from the
wells and 200 μL of the blank nanocarriers and insulin-loaded
nanocarriers solutions at concentrations of 0, 20, 50, 100, 250, 500 and
1000 μg mL−1 were added to each well for 48 h (group 1) and at con-
centration of 500 μg mL−1 were added to each well for 0, 3, 6, 12, 24
and 48 h (group 2). Then 20 μL of MTT (5 mg mL− 1) solution were
added to each well and the cells were further incubated at 37 °C for
4 h. The medium containing unreacted MTT was removed carefully,
and the dark blue formazan crystals generated by the mitochondria de-
hydrogenase in the live cells were dissolved with 150 μL of dimethyl
sulfoxide (DMSO) to measure the absorbance at 490 nm by a Microplate
Spectrophotometer (MultiskanMK3, Thermo Electron Corporation). Ex-
periments were performed in quintic. Cell viability (%) = (Ni/Nc) × 100,
where Ni and Nc are the absorbance of surviving cells treated with or
without nanocarriers, respectively. The results are presented as the
mean ± the standard deviation (SD).
2.6. Cell uptake of multi-responsive nanocarriers
The cell uptake of multi-responsive nanocarriers was evaluated by
confocal laser scanning microscope (CLSM, C2, Nikon Corp., Japan).
Briefly, the HT-29 cells were inoculated in a glass bottom dish with a
density of 2 × 104 cells per dish. The prodrug (nanocarriers loaded
with FITC-labled insulin) with a final concentration of 10 μg mL−1 was
added after the wells were washed with PBS and replaced with fresh
media. Then, the cells were cultured for 1, 3 and 6 h and washed with
PBS three times. The cells were fixed with 4% paraformaldehyde. The
cells were permeabilized using 0.2% Triton X-100 in blocking solution
containing 1% bovine serum albumin for 10 min. Finally, the cells were
stained with 4,6-diamidino-2-phenylindole (DAPI). Images were ob-
tained using confocal laser scanning microscope.
2.7. Flow cytometry study
HT-29 cells were incubated into 24 well plates at a density of 2 × 105
cells per well in DMEM medium for 24 h. The prodrug with a final con-
centration of 10 μg mL−1 was added after the wells were washed with
PBS and replaced with fresh media. Then, the cells were cultured for 1,
3 and 6 h and washed three times with PBS. Cells were treated with
trypsin and centrifuged for 5 min at 1000 rpm. Then the cells were
suspended in 0.5 mL of PBS and analyzed using a BD FACScan flow
cytometer (Becton Dickinson, USA).
2.8. Evaluation on diabetic SD rats in vivo
The experiments on animals were carried out according to the guide
for the care and use of laboratory animals, provided by Experimental
Animal Center of Zhejiang Academy of Medical Sciences, China. The pro-
cedure was approved by the Animal Ethics Committee of Zhejiang Sci
Tech University. Rats were randomly divided into five groups (five
rats per group). Diabetes was induced in SD rats by an injection of
streptozotocin (STZ, 65 mg/kg) dissolved in a 10 mM citrate buffer
(pH 4.5) as previously described [51]. The blood glucose level was de-
termined using a glucose meter (JPS-6, Yicheng Biotech. Co. Ltd., Beijing,
China). For the analysis of plasma insulin levels, blood samples were
centrifuged (3000 rpm, 5 min) and subsequently quantified using an
appropriate insulin ELISA kit (DuMa Biotechnology Co., Ltd., China).
3. Results and discussion
3.1. Composition and structure of nanocarriers
Herein, the multifunctional nanocarriers are firstly synthesized via
graft reaction of chitosan. The synthetic strategy in the current report is
schematically illustrated in Fig. 1A. Therein, carboxymethyl chitosan
(CMCS) is firstly prepared using according to a described method [52], in
which the degree of substitution is 0.6. The multifunctional nanocarriers
(CMCS-PBA-LV) are endowed by introduction of phenylboronic acid
(PBA) unit into polymer backbone. The hydrodynamic radii of the
nanocarriers can be enlarged due to formation of hydrophilic PBA-glucose
complex with increasing of glucose concentration. L-valine is an attractive
targeting agent to many cell types that over express the L-valine receptors
[42,45]. In order to design the system with target specificity, L-valine is in-
troduced into the nanocarriers which facilitated the delivery of them via
receptor-mediated endocytosis. Insulin can be loaded into the nanocarreris
due to the hydrophoblic reactions between of insulin and PBA, and the
electrostatic reaction between of insulin and chitosan.
The chemical composition of nanocarriers is confirmed by FTIR spec-
tra analysis. As shown in Fig. 1B, the FTIR spectra of CMCS and CMCS-
PBA-LV show a strong peak at 1000–1150 cm−1, which is the character-
istic peak of the saccharide structure. The very strong peak at around
3420 cm−1 can be assigned to the stretching vibration of O\\H, the ex-
tension vibration of N\\H and the intermolecular hydrogen bonds of the
polysaccharide. In the FTIR spectrum of CMCS, the strong peak at
1412 cm− 1 can be assigned to the symmetrical stretch vibration of
COOH. The asymmetrical stretch vibration of COOH at 1596 cm−1 ex-
hibits a very strong peak. Comparison the FTIR spectrum of physical
mixture of CMCS, 3-APBA and L-valine, some new peaks appeared in
range of 1800–1000 cm− 1 can be observed in CMCS-PBA-LV. The
peaks at 1633 cm− 1 and 1574 cm−1 are corresponds to the amide I
and II bands, respectively. The peak at 1347 cm−1 can be assigned to
the B\\O stretching [53]. The peak at 1238 cm− 1 represents C\\O
stretch of L-valine, indicating that PBA and L-valine functional units
have been successfully grafted onto the CMCS backbone.
The composition of resultant CMCS-PBA-LV is further verified by 1H
NMR analysis, as shown in Fig. 1C. The peaks in the range of 7.0–8.0 ppm
are attributed to the phenyl protons in the PBA segments [54]. The pro-
ton peaks that ranged in 3–4 ppm can be assigned to the sugar unit pro-
tons of CMCS. The characteristic peaks of the methyl protons of L-valine
are shown at about 0.89–0.93 ppm. The weak peaks at 2.26 and
2.77 ppm that corresponding to N C-H of L-valine can be founded. The
results verify the successful synthesis of CMCS-PBA-LV. Combined
with the integral value of L-valine segment protons (− CH3) at 0.89–
0.93 ppm, phenyl protons at 7–8 ppm and methylene (−CH2-) protons
of –OCH2COOH at 4.31 ppm, the calculated graft ratio of x: y: n in CMCS-
PBA-LV is 1.2: 2.0: 4.3.
Fig. 2A and B show the SEM and TEM images of insulin-loaded
chitosan-based multifunctional nanocarriers, respectively. These
nanocarriers exhibit a spherical morphology, which is benefit for the en-
docytosis of cells [55]. The diameter of resultant nanocarriers is ranged
from 100 to 300 nm. The average hydrodynamic diameter is 190 nm
that observed by Dynamic Light Scattering (DLS) measurement when
the concentration of glucose controlled at 0 mM in pH = 7.4 aqueous
solution, as shown in Fig. 2C. The average diameter determined by
SEM is slightly smaller than that determined by DLS. This discrepancy
is widely considered to be induced by the process of sample preparation
and the difference of investigation method between DLS and SEM. The
relatively smaller size of nanocarriers is beneficial to their applications
in biology, since particles with a size under 200 nm are more likely to
be taken up by cells [56]. To evaluate the stability of the nanocarriers
in protein solution, the nanocarriers is investigated in 10% FBS solution
by measuring the change of hydrodynamic diameter. No significant
changes in the mean particle size of the nanocarriers is found after incu-
bation in the 10% FBS solution for 24 h, suggesting that most of the
 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286
Fig. 1. Synthetic routine for preparation of chitosan-based multifunctional nanocarriers (A); FT-IR spectra (B) of carboxymethyl chitosan (CMCS, black), physical mixture of CMCA, 3-APBA
and LV (green) and CMCS-PBA-LV (red); 1H NMR spectrum of CMCS-PBA-LV (C).
nanocarriers remained their excellent stability against protein solution
(Fig. 2D).
3.2. Loading and release in vitro
The entrapment efficiency (EE%) of loaded insulin is calculated at
about 67% using the insulin ELISA kit. Then, the drug loading capacity
(DLC%) is calculated at about 9.8%. Insulin release behaviors from the in-
sulin-loaded chitosan-based multifunctional nanocarriers in the media
mimicking the gastrointestinal track were investigated in vitro. The mi-
metic physiological trigger are carried out in simulated gastric fluid
(SGF, pH = 1.2), simulated intestinal fluid (SIF, pH = 6.8) and in differ-
ent concentration of glucose (pH = 7.4) at 37 °C. As shown in Fig. 3A,
the insulin release percentage (only 16.6%) at SGF (pH = 1.2) is less
than that at SIF (pH = 6.8), which indicated that there is an inhibitory
effect on the release of insulin at pH 1.2. It may be due to the carboxyl
groups remain protonated at lower pH which restricts the swelling
and subsequent release of loaded drugs. When the pH is increased to
6.8, about 50.7% of loaded-insulin can be released from nanocarriers.
In SIF, the carboxyl group of the CMCS is deprotonated, thus creating
ionic repulsion between the polymer chains, allowing for release of in-
sulin. Therefore, these systems can be utilized as the pH shift between
the stomach and the small intestine (from ~pH 2 to 7) environmental
to trigger release of insulin [57]. To further confirm the controlled re-
lease behaviors of nanocarriers, the insulin release profiles against the
281
different concentration of glucose in PBS solution are investigated. As
shown in Fig. 3A, the higher the glucose concentration, the faster release
rate is observed. The insulin release percentage at the glucose concen-
tration of 20 mM is more effective than that at 5 and 10 mM. Only
about 55.4% of loaded-insulin can be released from nanocarriers with
the concentration of glucose at 5 mM. With increasing the concentra-
tion of glucose, the release rate of insulin is increased as well. N68%
and 92% of loaded-insulin can be released with concentration of glucose
at 10 and 20 mM, respectively. It is because that the glucose molecules are
permeated into the inner of nanocarriers, the negatively charged PBA can
combine with the 1, 2-diols of glucose and form stable and soluble phenyl
borates [58–60], which shifts the equilibrium to the side for forming more
combination and results in the swelling of the nanocarriers. This augur
swelling for the potential application of such nanocarriers as nanosized
drug delivery vehicles, since the abrupt change in hydrophilicity of the
cores is expected to allow “triggered release” of insulin drugs [56].
Bioactivity of protein decreases during preparation of drug-loaded
nanocarriers due to protein adsorption and denaturation at the water/or-
ganic solvent interface. The circular dichroism (CD) spectroscopy is an ef-
fective technique to evaluate the conformational stability of proteins. The
secondary structure of the insulin after release in pH 7.4 phosphate buffer
is further examined by circular dichroism CD spectroscopy, which is an ef-
fective technique to evaluate the conformational stability of proteins.
Compared with the CD spectra of pristine and released insulin, no signif-
icant difference in their secondary structure can be observed, as shown in
282 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286

Fig. 2. SEM image (A), TEM image (B) and DLS distribution in water (C) of insulin-loaded chitosan-based multifunctional nanocarriers, stability of as-prepared nanocarriers in 10% fetal
bovine serum (FBS) solution at 37 °C against incubating time (D).

Fig. 3B. Moreover, native insulin shows two negative bands in the far-UV
region at 208 and 223 nm, respectively. The bands at 208 and 223 nm cor-
respond to the α-helix structure and β-pleated sheet structure, respective-
ly [49]. The ratio of intensity of 208 and 223 bands ([Ф]208/[Ф]223)
quantitatively indicates the overall conformation of insulin. The [Ф]208/
[Ф]223 for native and released insulin are 1.03 and 1.08, respectively. It in-
dicates an undistorted secondary structure of insulin after release.
3.3. Cell cytotoxicity and cellular uptake
It is necessary to verify the innocuous nature of the drug carrier ma-
terials in the present work. Fig. 4A shows the cell viability of blank
CMCS-PBA-LV and insulin-loaded CMCS-PBA-LV against HT-29 cells
using a MTT assay with the different concentrations after incubating
24 h. The cells maintained viability of N98% when the concentrations
ranged from 20 μg mL−1 to 500 μg mL−1. Even with high concentration
of nanocarriers reached 1000 μg/mL, the viability of cells is still kept
above 96%, indicating low toxicity of the as-prepared nanocarriers.
After the insulin encapsulated into CMCS-PBA-LV, no significant differ-
ence of the viability of cells can be founded. The blank CMCS-PBA-LV
and insulin-loaded CMCS-PBA-LV against different incubation time
also reveals its low cytotoxicity (Fig. 4B). These indicate that resultant
chitosan-based nanocarriers possess excellent cytocompatibility and
therefore having great potential for in vivo application.

Fig. 3. Insulin release profiles of insulin-loaded nanocarriers against SGF (pH = 1.2) and SIF (pH = 6.8) in vitro, and PBS solution with different concentration of glucose (A: T = 37 °C); CD
spectra of free and released insulin (B).
 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286 283

Fig. 4. In vitro cell viability of HT-29 cells against the blank nanopcarriers (NCs) and insulin-loaded nanocarriers (INCs) with different concentration (A) and culture time (B) by MTT assay.
Data are presented as average ± standard deviation (n = 5).

To investigate the cellular uptake of the insulin-loaded CMCS-PBA-
LV, the cell adhesive and penetrating properties of the FITC-insulin-
loaded nanocarriers are evaluated by confocal laser scanning microsco-
py (CLSM) after 1, 3, 6 h of incubation using the HT-29 cells as model.
Usually, most epithelium-targeting systems are performed on the
Caco-2 cell model which possesses no mucus [61]. However, the fact,
being often neglected, is that the real effect of epithelial cell-targeting
may be greatly affected by the mucus layer presenting on epithelium.
Therefore, it is important to exploit more effective targeting ligands
which possess high specificity for recognition, so as to provide sufficient
targeting effectiveness while at the same time avoid being blocked by
mucus. Meanwhile, evaluation on the targeting efficiency and mecha-
nism of the targeting system in the presence of mucus is considered as
another important issue [62]. Therefore, in our study, HT-29 cells are ap-
plied to evaluate the transport of the obtained nanocarriers due to their
having mucus layer similar with that of intestinal epithelial cell.
As shown in Fig. 5, the first column shows the red fluorescence from
the cell cytoskeleton stained by rhodamine phalloidin. The second col-
umn shows the blue fluorescence from the cell nuclei that stained by
DAPI (4, 6-diamidino-2-phenylindole). The third column shows the
green fluorescence from the FITC-insulin from insulin-loaded CMCS-
PBA-LV nanocarriers which have been internalized in the HT-29 cells.

Fig. 5. CLSM images of HT-29 cells treated with FITC-insulin loaded chitosan-based nanocarriers. The red signal represents cytoskeleton stained with rhodamine-phalloidin, the blue signal
represents nuclei stained with DAPI and the green signal represents FITC-insulin. Scale bar = 25 μm.
284 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286
The images in the fourth column are merged images corresponding
from the first, second, and third images. Only the weak green fluores-
cence can be observed after 1 h incubation and following stronger
with increasing the incubation time. Moreover, it also can be seen
from CLSM that most cyan fluorescene (overlay of blue and green) can
be clearly observed in intercellular spaces, suggesting that the insulin-
loaded CMCS-PBA-LV nanocarriers can facilitate insulin across the cell
membrane via the paracellular route. Moreover, after incubation of
6 h, some stronger green dots are observed inside of cells, indicating
the insulin-loaded CMCS-PBA-LV nanocarriers can internalize into the
cell and release insulin within the cells.
Flow cytometer analysis is a potent way to further reveal the cellular
uptake efficiency of FITC-labeled insulin. From the data displayed in Fig.
6A, it can be concluded that the relative geometrical mean fluorescence
intensities is increased as prolonging the incubation time from 1 to 6 h
compared with blank cells. This increase in fluorescence signals can be
attributed to the internalization of FITC-labeled insulin by HT-29 cells.
Another important feature of nanocarriers is protection the loaded
drugs from being destroyed or degradated by enzymes in the body
[63,64]. Fig. 6B shows the percent insulin remaining after incubation
in SGF containing pepsin and SIF containing pancreatin. In the case of
free insulin, only about 10% of insulin is remained after 30 min. And al-
most all free insulin is completely degraded after 60 min (b 3%). Howev-
er, the insulin stability is strikingly different appearance after
encapsulation of insulin into nanocarriers. N71% of insulin is remained
with same incubation time (30 min). The insulin-loaded nanocarriers
exhibit more effective to protect the insulin from degradation. Especial-
ly, the better stability (62.3% and 58.6% for SGF containing pepsin and
SIF containing pancreatin, respectively) is achieved for insulin that load-
ed into nanocarriers after incubation for 60 min. From these data, we
can conclude that the L-valine modified chitosan-based nanocarriers
can delay the degradation of insulin during the drug delivery process.
The small intestine has been shown to be able to transport the L-forms
of amino acids against a concentration gradient and that they compete
for the mechanism concerned [65]. So, L-valine is used as a ligand for
carrier-mediated transport of insulin-loaded chitosan nanoparticles.
3.4. Hypoglycemic effect
Streptozotocin-induced diabetic SD rats are further used to evaluate
the oral pharmacological effects of the insulin-loaded nanocarriers [50,
51]. Fig. 7A shows the blood glucose levels at different time intervals
after subcutaneous injection (SC) of insulin solution or oral administra-
tion of different formulations to diabetic rats. As expected, almost no
any influence of blood glucose level is observed after oral administration
of either free insulin solution or the blank saline solution. For the group
treated with a subcutaneous injection of insulin solution, the blood
Fig. 6. Flow cytometric profiles of HT-29 cells incubated with FITC-insulin-loaded nanocarriers for 0, 1, 3 and 6 h (A) and chemical stability of insulin in SGF containing pepsin and SIF
containing pancreatin (B).
glucose level is decreased rapidly that the minimum glucose level is ob-
served at 2 h after the injection of insulin solution. However, the blood
glucose level is returned to initial level in short time due to the short cir-
culating half-life of insulin in blood. In comparison, after oral adminis-
tration of insulin-loaded nanocarriers with the same insulin level, a
better hypoglycemic effect with the maximal blood glucose depression
of 60.2% at 5 h can be observed. Furthermore, the hypoglycemic effect
can be kept longer time compared with the subcutaneous injection of
insulin solution. Using blood glucose at 60% initial level as base line
with remarkable hypoglycemic effect, the hypoglycemic time is only
3.4 h for subcutaneous injection, much less than that of 8.4 h for oral ad-
ministration. It indicates the obtained insulin-loaded nanocarriers can
facilitate the prolonging the time of pharmacological effect. The relative
pharmacological availability of the oral administration of the insulin-
loaded nanocarriers is found to be 7.55 ± 1.32%, which is much higher
than that of the oral administration of free insulin solution (b0.8%).
The efficiency of facilitating the absorption in the small intestine be-
tween normal chitosan and L-valine modified chitosan has been com-
pared. Almost no insulin can be transferred into cells using normal
chitosan as carriers. It suggests that a carrier-mediated mechanism is in-
volved in its intestinal absorption [66]. In this study, L-valine modified
chitosan, consisting of L-valine as a targeting ligand and mucoadhesive
chitosan as backbone of carriers, is a mucoadhesive system that causes
bioadhesion to gastrointestinal mucosa and limits emptying of the dos-
age forms. Although the release and cell uptake of insulin is relative
slow, the insulin still can be uptaken before it is eliminated from the in-
testinal canal.
Furthermore, a pharmacokinetic analysis is further investigated to
determine the relative bioavailability of insulin released from
nanocarriers. Fig. 7B shows the plasma insulin levels following
intragastric administration (i.g.) of insulin-loaded nanocarriers (INCs)
and free insulin solution, as well as subcutaneous injection (s.c.) of insu-
lin solution. It is found that subcutaneous injection of insulin solution
resulted in a sharp increase of the plasma insulin concentration with a
highest value at 129.3 ± 4.9 μIU/mL in a short time (~ 1 h) and then
decreased rapidly to a lower level within 4 h. In contrast, after oral ad-
ministration of insulin-loaded nanocarriers, the plasma insulin concen-
tration is increased in a low rate before 4 h, while then declined slowly.
The highest plasma insulin level is 61.1 ± 7.6 μIU/mL after oral admin-
istration for 4 h. Although the value of highest plasma insulin level of
oral administration is less than that of subcutaneous injection, the
higher plasma insulin level can be maintained for longer time. Especial-
ly, a higher plasma insulin level can be obtained after post-administra-
tion for 2.8 h compared with subcutaneous injection.
The aforementioned studies are conformed that chitosan-based
multifunctional nanocarriers prepared in this work may be a promising
drug delivery carrier for oral administration of insulin. This citosan-
 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286
Fig. 7. Blood glucose levels in diabetic rats following oral administration of insulin-loaded nanocarriers, saline and insulin solution (A); Plasma insulin level in diabetic rats following oral
administration of insulin-loaded nanocarriers and insulin solution (B); Data are presented as the average ± standard deviation (n = 5) (*P b 0.05, **P b 0.01); Schematically illustrated for
fabrication of chitosan-based multifunctional nanocarriers overcoming multiple barriers for oral delivery of insulin (C).
based nanocarriers are glucose-sensitive and capable of withstanding
prolonged contact with acidic gastric media and digestive enzymes in
GI tract [67], and improving their transportation in intestinal cells due
to the targeted induction of L-valine conjugation. This approach can pre-
vent the rapid release of insulin from nanocarriers while in the stomach
and enhance their absorption on the surface of the small intestine, thus
further increasing their bioavailability (Fig. 7C) [67,68].
4. Conclusions
In summary, chitosan-based multifunctional nanocarriers modified
by L-valine target ligands and glucose-responsive units have been pre-
pared for overcoming multiple barriers for oral delivery of insulin. The
resultant nanocarriers exhibited relative lower cytotoxicity and excel-
lent stability against protein solution. The insulin release behaviors
from nanocarriers were evaluated triggered by pH and glucose in vitro.
The chemical stability of loaded insulin against digestive enzyme were
established in presence of simulated gastric fluid (SGF) containing pep-
sin and simulated intestinal fluid (SIF) containing pancreatin, respec-
tively. The obtained chitosan-based multifunctional nanocarriers
exhibited excellent protective properties for insulin against enzymatic
degradation. The hypoglycemic effect for oral delivery of insulin was
studied in vivo using STZ-induced diabetic SD rats as models. After
oral administration to the diabetic rats, the insulin-loaded nanocarriers
exhibit an obvious hypoglycemic effect. And higher serum insulin level
can be obtained after post-administration and maintained higher level
for a longer time. These results suggest that the L-valine modified chito-
san-based multifunctional nanocarriers have a potential application in
diabetes treatment via oral ingestion.
285
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (51373155) and “521 Talents Training Plan” in
Zhejiang Sci-Tech University. L. L. thanks the Cultivation Fund Program
for Excellent Dissertation in Zhejiang Sci-Tech University.
References
[1] IDF, Diabetes Atlas, 7th edition, 2015www.idf.org/diabetesatlas.
[2] G. Sharma, A.R. Sharma, J.-S. Nam, G.P.C. Doss, S.-S. Lee, C. Chakraborty, J.
Nanobiotechnol. 13 (2015) 74.
[3] M. Lepore, S. Pampanelli, C. Fanelli, F. Porcellati, L. Bartocci, A.D. Vincenzo, C.
Cordoni, E. Costa, P. Brunetti, G.B. Bolli, Diabetes 49 (2000) 2142.
[4] X. Du, G. Jiang, L. Li, Y. Liu, H. Chen, Q. Huang, Colloid Polym. Sci. 293 (2015) 2129.
[5] M. George, T.E. Abraham, J. Control.Release 114 (2006) 1.
[6] K.Y. Lee, S.H. Yuk, Prog. Polym. Sci. 32 (2007) 669.
[7] K. Kawakami, M. Ebara, H. Izawa, N.M. Sanchez-Ballester, J.P. Hil, K. Ariga, Curr. Med.
Chem. 19 (2012) 2388.
[8] E. Roger, F. Lagarce, E. Garcion, J.-P. Benoit, Nanomedicine 5 (2010) 287.
[9] P. He, Z. Tang, L. Lin, M. Deng, X. Pang, X. Zhuang, X. Chen, Macromol. Biosci. 12
(2012) 547.
[10] K. Park, I.C. Kwon, K. Park, React. Funct. Polym. 71 (2011) 280.
[11] T. Yamagata, M. Morishita, N.J. Kavimandan, K. Nakamura, Y. Fukuoka, K. Takayama,
N.A. Peppas, J. Control. Release 112 (2006) 343.
[12] P. Mukhopadhyay, K. Sarkar, S. Bhattacharya, A. Bhattacharyya, R. Mishra, P.P.
Kundu, Carbohydr. Polym. 112 (2014) 627.
[13] G. Jiang, T. Jiang, H. Chen, L. Li, Y. Liu, H. Zhou, Y. Feng, J. Zhou, Colloid Polym. Sci. 293
(2015) 209.
[14] C. He, L. Yin, C. Tang, C. Yin, Biomaterials 33 (2012) 8569.
[15] X. Wang, X. Sun, G. Jiang, R. Wang, R. Hu, X. Xi, Y. Zhou, S. Wang, T. Wang, J. Appl.
Polym. Sci. 128 (2013) 3289.
[16] N.A. Peppas, D.A. Carr, Chem. Eng. Sci. 64 (2009) 4553.
[17] L. Wu, M. Liu, X. Zhu, W. Shan, Y. Huang, Curr. Pharma. Design 21 (2015) 5198.
[18] Z. Antosova, M. Mackova, V. Kral, T. Macek, Trends Biotechnol. 27 (2009) 628.
286 L. Li et al. / Materials Science and Engineering C 70 (2017) 278–286

[19] J. Hombach, A. Bernkop-Schnürch, Int. J. Pharm. 376 (2009) 104. [44] M. Thanou, M. Nihot, M. Jansen, J. Verhoef, H. Junginger, J. Pharm. Sci. 90 (2001) 38.
[20] E. Khafagy, M. Morishita, Y. Onuki, K. Takayama, Adv. Drug Deliv. Rev. 59 (2009) [45] A. Jain, S.K. Jain, Acta Diabetol. 52 (2015) 663.
1521. [46] K.V. Harish Prashanth, R.N. Tharanathan, Carbohydr. Polym. 54 (2003) 343.
[21] M. Amidi, E. Mastrobattista, W. Jiskoot, W.E. Hennink, Adv. Drug Deliv. Rev. 62 [47] T. Sun, P. Xu, Q. Liu, J. Xue, W. Xie, Eur. Polym. J. 39 (2003) 189.
(2010) 59. [48] B. Singh, N. Chauhan, Int. J. Diabetes Mellitus 2 (2010) 32.
[22] M.P. Patel, R.R. Patel, J.K. Patel, J. Pharm. Sci. 13 (2010) 536. [49] A. Makhlof, Y. Tozuka, H. Takeuchia, Eur. J. Pharm. Sci. 42 (2011) 445.
[23] V. Lassman-Vague, D. Raccah, Diabete Metab. 32 (2007) 513. [50] L. Li, G. Jiang, W. Yu, D. Liu, H. Chen, Y. Liu, Q. Huang, Z. Tong, J. Yao, X. Kong, Mater.
[24] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, J. Control. Release 100 (2004) 5. Sci. Eng. C 69 (2016) 37.
[25] S.M. Alex, M.R. Rekha, C.P. Sharma, Int. J. Pharm. 410 (2011) 125. [51] Y. Kawashima, H. Yamamoto, H. Takeuchi, S. Fujioka, T. Hino, J. Control. Release 62
[26] S. Sajeesha, C.P. Sharma, Drug Deliv. 18 (2011) 227. (1999) 279.
[27] V. Dodane, M.A. Khan, J.R. Merwin, Int. J. Pharm. 182 (1999) 21. [52] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, J.Control. Release 186 (2014) 54.
[28] M. Thanou, M.T. Nihot, M. Jansen, J.C. Verhoef, H.E. Junginger, J. Pharm.Sci. 90 (2001) [53] X. Du, G. Jiang, L. Li, W. Yang, H. Chen, Y. Liu, Qin Huang, J. Appl. Polym. Sci. 133
38. (2016) 43026.
[29] F. Chen, Z.-R. Zhang, F. Yuan, X. Qin, M. Wang, Y. Huang, Int. J. Pharm. 349 (2008) [54] Q. Guo, Z. Wu, X. Zhang, L. Suna, C. Li, Soft Matter 10 (2014) 911.
226. [55] J. Li, Y. Qu, J. Ren, W. Yuan, D. Shi, Nanotechnology 23 (2012) 505706.
[30] Y. Yang, S.-X. Yuan, L.-H. Zhao, C. Wang, J.-S. Ni, Z.-G. Wang, C. Lin, M.-C. Wu, W.-P. [56] L. Li, G. Jiang, X. Du, H. Chen, Y. Liu, Q. Huang, X. Kong, J. Yao, RSC Adv. 5 (2015)
Zhou, Mol. Pharm. 12 (2015) 644. 75766.
[31] M. Wu, K. Guo, H. Dong, R. Zeng, M. Tu, J. Zhao, Mater. Sci. Eng. C 45 (2014) 162. [57] K.M. Wood, G.M. Stone, N.A. Peppas, Biomacromolecules 9 (2008) 1293.
[32] A. Makhlof, Y. Tozuka, H. Takeuchi, Eur. J. Pharm. Sci. 42 (2011) 445. [58] S.S.N. Ling, K.H. Yuen, E. Magosso, S.A. Barker, J. Pharm. Pharmacol. 61 (2009) 445.
[33] J. Emami, M. Rezazadeh, M. Rostami, F. Hassanzadeh, H. Sadeghi, A. Mostafavi, M. [59] A. Matuszewska, M. Uchman, A. Adamczyk-Woźniak, A. Sporzyński, S. Pispas, L.
Minaiyan, A. Lavasanifar, Drug Dev. Ind. Pharm. 41 (2015) 1137. Kováčik, M. Štěpánek, Biomacromolecules 16 (2015) 3731.
[34] K. Kataoka, H. Miyazaki, M. Bunya, T. Okano, Y. Sakurai, J. Am. Chem. Soc. 120 (1998) [60] W. Zhai, X. Sun, T.D. James, J.S. Fossey, Chem. Asian. J. 10 (2015) 1836.
12694. [61] I. Kadiyala, Y. Loo, K. Roy, J. Rice, K.W. Leong, Eur. J. Pharm. Sci. 39 (2010) 103.
[35] J. Yan, H. Fang, B. Wang, Med. Res. Rev. 25 (2005) 490–520. [62] Y. Jin, Y. Song, X. Zhu, D. Zhou, C. Chen, Z. Zhang, Y. Huang, Biomaterials 33 (2012)
[36] G. Jiang, T. Jiang, X. Li, Z. Wei, X. Du, X. Wang, Mater. Res. Express 1 (2014) 025708. 1573.
[37] G. Jiang, T. Jiang, Y. Wang, X. Du, Z. We, H. Zhou, RSC Adv. 4 (2014) 33658. [63] Y. Daimon, H. Izawa, K. Kawakami, P. Zywicki, H. Sakai, M. Abe, J.P. Hill, K. Ariga, J.
[38] G. Jiang, X. Du, Z. Wei, T. Jiang, H. Zhou, Z. Qiu, Eur. Polym. J. 60 (2014) 33. Mater. Chem. B 2 (2014) 1802.
[39] L.M. Ensign, R. Cone, J. Hanes, Adv. Drug Deliv. Rev. 64 (2012) 557. [64] A. Silva-Cunha, J.L. Grossiord, F. Puisieux, M. Seiller, Int. J. Pharm. 158 (1997) 79.
[40] A.-C. Groo, P. Saulnier, J.-C. Gimel, J. Gravier, C. Ailhas, J.-P. Benoit, F. Lagarce, Int. J. [65] A. Jain, S.K. Jain, Acta Diabetol. 52 (2015) 663.
Nanomedicine 8 (2013) 4291. [66] R.L.A. De Vrueh, P.L. Smith, C. Lee, J. Pharm. Experim. Therap. 286 (1998) 1166.
[41] J. Sheng, L. Han, J. Qin, G. Ru, R. Li, L. Wu, D. Cui, P. Yang, Y.H.J. Wang, ACS Appl. [67] H.-W. Sung, K. Sonaje, Z.-X. Liao, L.-W. Hsu, E.-Y. Chuang, Acc. Chem. Res. 45 (2012)
Mater. Interfaces 7 (2015) 15430. 619.
[42] R.L.A. de Vrueh, P.L. Smith, C.-P. Lee, J. Pharm. Exp. Ther. 286 (1998) 1166. [68] W. Shan, X. Zhu, M. Liu, L. Li, J. Zhong, W. Sun, Z. Zhang, Y. Huang, ACS Nano 9 (2015)
[43] N. Schipper, S. Olsson, J. Hoogstraate, A. Boer, K. Varum, P. Artursson, Pharm. Res. 14 2345.
(1997) 923.