Journal of Drug Delivery Science and Technology 57 (2020) 101738

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Journal of Drug Delivery Science and Technology

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

Formulation and characterisation of insulin-loaded chitosan nanoparticles T

capable of inducing glucose uptake in skeletal muscle cells in vitro
Chun Y. Wonga,b, Hani Al-Salamia,b,c, Crispin R. Dassa,b,∗
a
School of Pharmacy and Biomedical Sciences, Curtin University, Bentley, 6102, Australia
b
Curtin Health Innovation Research Institute, Bentley, 6102, Australia
c
Biotechnology and Drug Development Research Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University,
Bentley, 6102, Australia

ARTICLE INFO ABSTRACT

Keywords: A successful oral protein nanocarrier needs to exhibit the following characteristics: stability in the gastro-
Chitosan intestinal (GI) tract, drug release at specific site, overcoming high mucus turnover, mucoadhesive properties and
Dz13Scr enhanced epithelial absorption. The purpose of this research was to examine the potential of complex coa-
Glucose uptake cervation technique for manufacturing of insulin-loaded chitosan-Dz13Scr nanoparticles. The nanoparticles were
Insulin
characterized for size, polydispersity index, entrapment efficiency, drug loading capacity, zeta potential, mor-
Nanoparticle
Oral
phology, stability and consistency of physiochemical properties. Additionally, toxicity, GI permeation, en-
docytosis absorption pathway, and glucose consumption capacity, were examined in cellular assays. The opti-
mized formulation variables [0.5% of natural polymer (chitosan) and 10 μg of an oligonucleotide (Dz13Scr)]
could control the physiochemical properties of nanoparticles. The complex coacervation technique used in the
study could tighten the size uniformity and generate nanoparticles with high encapsulation efficiency (88%). In
vitro drug release model also confirmed that the nanoparticles could withstand the acidic environment with only
13% of insulin released. The formulation could maintain its stability upon storage, promote the permeation of
encapsulated insulin via paracellular absorption and endocytic pathway, and induced glucose uptake in the
skeletal muscle cells. Therefore, this insulin-loaded chitosan-Dz13Scr nanoparticle is a potential drug delivery
system for management of diabetes mellitus.

1. Introduction injections can lead to unwanted side-effects such as lipoatrophy, skin

Diabetes mellitus is one of the most prevalent metabolic diseases
and it is anticipated that 642 million people will be living with diabetes
by 2040 [1]. During the last few decades, a variety of protein or pep-
tide-based nanocarriers have been developed, which could enhance the
stability of formulations (against extreme pH and enzymatic degrada-
tion) and improve the oral bioavailability of therapeutic agents. To
date, insulin injection remains a common therapy injected for patients
with type 1 diabetes mellitus. As there is no treatment available to cure
diabetes mellitus, multiple daily injections of exogenous insulin via
parental injection becomes essential to manage blood sugar level in
conjunction with adherence to strict healthy lifestyle. Insulin injections
have been proven to minimize the occurrence of long-term complica-
tions such as limb amputation, renal dysfunction, and retinopathy.
However, daily injections of immediate-acting insulin and multiple
long-acting insulin are inconvenient and painful. Furthermore, insulin
necrosis, nerve pain, peripheral hyperinsulinemia, atherosclerosis, and
hypertension [2]. In contrast, oral administration of insulin, which is a
non-invasive and convenient route, could mimics the physiological
behaviour of endogenous insulin that is directed to the liver to regulate
the glucose homeostasis [3]. Therefore, this route is favourable for
diabetes patients to improve their compliance to therapy and manage
blood glucose level.
Oral administration of insulin encounters challenges such as in-
stability under extreme pH variation, degradation by gastrointestinal
(GI) enzymes and impermeable across the GI tract [4]. Various drug
delivery systems such as nanoparticles [5], microparticles [6] and li-
posomes [7] have been investigated to encapsulate insulin and increase
its bioavailability for diabetes treatment. Amongst the above sub-mi-
cron sized drug delivery systems, polymeric nanoparticle is one of the
drug delivery systems with promising characteristics, which include
size in nano-scaled range and optimized surface characteristics, that can

∗
Corresponding author. School of Pharmacy and Biomedical Sciences, Curtin University, GPO Box U1987, Perth, 6845, Australia.
E-mail address: Crispin.Dass@curtin.edu.au (C.R. Dass).

https://doi.org/10.1016/j.jddst.2020.101738
Received 30 January 2020; Received in revised form 23 March 2020; Accepted 5 April 2020
Available online 10 April 2020
1773-2247/ © 2020 Elsevier B.V. All rights reserved.
C.Y. Wong, et al. Journal of Drug Delivery Science and Technology 57 (2020) 101738

Table 1
Insulin-loaded nanoparticles prepared by complex coacervation/polyelectrolyte complexation.
Insulin-loaded NPs Characteristics Reference

1. Chitosan-g-polyethylene glycol monomethyl ether NPs Modification of nanoparticle surface by using optimal mPEG graft ratios to improve oral 2019: [1]
bioavailability of insulin
2. Polyurethane-chitosan NPs with alginate protective shell Incorporation of polyurethane, chitosan and alginate to encapsulate insulin within the core–shell 2018: [44]
and improve insulin oral bioavailability
3. Poly(ethylene glycol)-carboxymethyl chitosan NPs Improvement of heart ischemia/reperfusion injury in diabetes mellitus due to the antioxidative, 2018: [41]
antiapoptotic, and anti-inflammatory features of NPs
4. N,N-Dimethyl-N-Octyl chitosan NPs Enhancement of insulin permeation by using octyl chitosan instead of trimethyl chitosan 2018: [12]
5. Trimethylated poly(allylamine) NPs Complexation of thiomers and insulin to improve the stability against enzymatic degradation 2018: [2]
6. Polyethylene imine NPs Coating with three-layer films to improve colonic release of insulin 2016: [3]
7. Modified-chitosan NPs Addition of quaternary ammonium groups and sodium tripolyphosphate to improve the stability of 2015: [43]
the formulation
8. Quaternized chitosan NPs Modification of chitosan to improve the transport efficiency of the formulation 2014: [33]
9. Chitosan/alginate NPs Encapsulation of insulin by using two natural, hydrophilic and biodegradable polymers 2013: [4]
10. Poly(methylaminophosphazene)/dextran sulfate NPs Formation of ternary interpolyelectrolyte complexes to protect insulin against proteolysis by pepsin 2013: [10]
11. Chitosan NPs Formation of self-assembled formulations between therapeutic proteins and natural polymers 2013: [13]
12. Chitosan/hydroxypropyl methylcellulose phthalate NPs Ionic cross-linking of chitosan nanoparticles with hydroxypropyl methylcellulose phthalate to 2011: [42]
improve stability against pepsin, intestinal mucoadhesion and hypoglycaemic effect
13. Modified chitosan NPs Optimization of physiochemical properties by using N-(4-N,N-dimethylaminobenzyl), methylated 2011: [38]
N-(4-pyridinyl), and methylated N-(benzyl) chitosan
14. Chitosan/Eudragit L100-55 NPs Utilization of Eudragit L100-55 to develop a pH sensitive drug delivery system 2010: [46]
15. Water soluble chitosan NPs Utilization of low molecular weight chitosan to encapsulate insulin 2010: [35]
16. PEGylated trimethyl chitosan NPs Formation of PEGylated trimethyl chitosan nanocomplexes to improve the mucoadhesiveness of 2009: [47]
formulations
17. Insulin-chitosan complex Combination with oily-based system to enhance oral bioavailability of insulin 2009: [40]
18. Chitosan NPs, triethyl-chitosan NPs, dimethylethyl- Utilization of quaternized-modified chitosan to formulate colon-targeting nanoparticles 2008: [37]
chitosan NPs
19. Chitosan/dextran sulfate NPs Development of a pH-sensitive and mucoadhesive delivery system with two natural polymeric 2007: [45]
polysaccharides 2006: [36]
21. Polyethylenimine-dextran sulfate NPs Preservation of insulin secondary structure and biological activity 2003: [39]

enhance the oral absorption of insulin. These polymeric nanoparticles
can be prepared by a mild technique called complex coacervation
(polyelectrolyte complexation) [8,9]. As shown in Table 1, this tech-
nique has been employed extensively to prepare insulin-loaded nano-
particles with promising results, such as protection of therapeutic
proteins against enzymatic degradation [1,2,10], release of drug in a
controlled or active targeting manner [3], prolonged retention time in
the GI tract, and enhanced permeation of therapeutic proteins across GI
mucus layer and epithelial barriers [1].
The objective of the present research was to examine the potential
of chitosan-Dz13Scr nanoparticles using complex coacervation for oral
delivery of insulin. Chitosan is a natural polymeric polysaccharide,
which is biocompatible, biodegradable, non-immunogenic, non-toxic
and mucoadhesive [11–13]. It is the second most abundant polymer
and can be derived from chitin deacetylation [4]. Its positive charge
enables the formation of a self-assembled polyelectrolyte complex with
negatively charged molecules such as the 34-mer oligonucleotide,
Dz13Scr. Dz13Scr is an anionic oligonucleotide exhibiting excellent
biocompatibility and minimal cytotoxicity. To understand the influence
of the scaling up process, comparisons between the pre-scaled up na-
noparticles and scaled-up nanoparticles were made in terms of phy-
siochemical properties and cellular responses. Fig. 1 illustrates nano-
particle preparation, which involves a pre-screening stage (to
understand the encapsulation potential of the novel drug delivery
system), an optimization stage (to refine the amount of encapsulated
insulin and explore the physiochemical properties of pre-scaled up
nanoparticles), a scaled-up stage (to explore the physiochemical prop-
erties of scaled-up nanoparticles), a physical characterization stage (to
visualise the stability and drug release kinetic), and lastly a cellular
characterization stage (to visualise the cytotoxicity, glucose consump-
tion capacity, permeation efficiency and endocytic absorption me-
chanism). Overall, the developed nanoparticles prepared by complex
coacervation method could produce nanoparticles with acceptable size
range, uniform size distribution and high encapsulation efficiency.
2. Materials and methods
Low molecular weight (LMW) chitosan (95% deacetylated; MW
150 kDa), sodium sulfate (SS), sodium acetate (SA) and Dz13Scr were
purchased from Sigma-Aldrich (St Louis, MO, USA). Insulin was ob-
tained from Gibco (Fort Worth, TX, USA). Trifluoroacetic acid (Sigma-
aldrich), acetonitrile (Thermofisher, VIC, Australia) and all chemical
agents were of pharmaceutical and analytical grade.
2.1. Pre-screening stage
2.1.1. Preparation of insulin-loaded nanoparticles
Complex coacervation [14] was used to manufacture insulin-loaded
chitosan-Dz13Scr nanoparticles. In brief, the first step was to dissolve
insulin at a concentration of 20.9 μM in Milli-Q water. The second step
was a gentle mixing process of 50 μL insulin (20.9 μM) and 100 μL of
Dz13Scr (5, 10 or 15 μg; in 50 mM SS buffer). In this study, the effect of
nine concentrations of chitosan (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4,
0.45, 0.5%; in 25 mM SA buffer) on encapsulation efficiency of nano-
particles were examined. The mixture was vortexed for 30 s at
2500 rpm in a 600 μL microfuge tube. Due to its cationic nature,
chitosan is readily complexed with negatively charged Dz13Scr via
electrostatic interactions to form insulin-loaded chitosan-Dz13Scr na-
noparticles. The nanoparticles can be collected by ultracentrifugation at
18200 g for 15 min. The nanoparticles were stored at 4 °C in the fridge
for further experimentation.
2.1.2. Insulin quantification
Chromatographic quantification of insulin was conducted at room
temperature using a Shimadzu HPLC LC-20AT system (Kyoto, Japan)
coupled with a UV–Vis detector. Separation was carried out using
Apollo C18 column (150 mm × 4.6 mm, 5 μm, Maryland, USA)
[15,16]. The mobile phase consisted of solution A (Mill-Q water) and
solution B (0.1% TFA in acetonitrile). The validated HPLC method

2
C.Y. Wong, et al.
Fig. 1. Schematic presentation of nanoparticle preparation.
comprised flow rate (1 mL/min), UV–Vis detection wavelength
(214 nm), run time (15 min) and sample injection volume (50 μL). The
gradient elution of solution B was pumped from 10 to 90% in 9 min. A
stock insulin solution of 1.2 mg/mL was freshly prepared using Milli-Q
water. Insulin stock solutions of known concentrations (2.97, 6.06,
12.13, 24.25, 48.5, 80.81, 121.25, and 202.06 μg/mL) were used to plot
a calibration curve with high linearity (r > 0.99).
2.1.3. Encapsulation efficiency and loading content
The insulin-loaded chitosan-Dz13Scr nanoparticles were ultra-
centrifuged at 18200 g for 15 min to separate the free or encapsulated
insulin. After ultracentrifugation, the amount of non-encapsulated in-
sulin in the clear supernatant was determined by RP-HPLC to evaluate
the encapsulation efficiency and loading capacity (section 2.1.2). The
percentages of insulin encapsulation efficiency (Eq. (1)) and drug
loading capacity (Eq. (2)) were calculated as follows [17–19]:
Drug encapsulation efficiency (%)
Initial amount of insulin added Free insulin
= x100%
Initial amount of insulin
Loading capacity (%)
Amount of encapsulated insulin
= x 100%
Initial amount of insulin + Amount of chitosan + Amount of Dz13Scr
Journal of Drug Delivery Science and Technology 57 (2020) 101738
2.2. Optimization stage
2.2.1. Small scale production of nanoparticles
In the pre-screening stage, the potential amount of insulin that
could be encapsulated in the drug delivery system was determined. To
refine the amount of insulin that is added in the preparation of nano-
particles, the calculated encapsulation efficiency in section 2.1.3 was
taken into account. The same operating protocol (section 2.1.1) was
carried out to prepare nanoparticles, but only 8.61 μM of insulin (50 μL)
was added to the mixture.
2.2.2. Particle size, polydispersity and zeta potential
The particle size, polydispersity index and zeta potential of the
nanoparticles could influence the GI cellular uptake and efficacy of
therapeutic agents. Therefore, these parameters were examined in the
optimization stage by dynamic light scattering using a Zetasizer Nano
ZSP (Malvern Instruments Ltd., UK) at room temperature according to
the established method [20,21]. In this study, the effect of Dz13Scr (5,
10 or 15 μg) and chitosan concentration (0.5%) on the size, poly-
dispersity index and zeta-potential of the nanoparticles were de-
(1)
termined. In brief, the samples in Milli-Q water were analysed using a
folded capillary electrophoresis cell [22–24]. Dynamic light scattering
was used to determine the mean particle size and zeta-potential. Zeta-
sizer software (Version 7.11) was used to obtain the data. All samples
were characterized in triplicates within 3 days of preparation.
(2)
3
C.Y. Wong, et al.
2.3. Scaled-up stage
2.3.1. Scaled-up production and characterization of nanoparticles
The production of nanoparticles was scaled-up 6-fold at the same
ratio of chitosan, insulin and Dz13Scr. The measurement of size,
polydispersity index, zeta-potential and encapsulation efficiency was
carried out as described above. To examine the consistency of physio-
chemical properties, these parameters were compared between pre-
scaled up nanoparticles and scaled up nanoparticles. The morphology of
the nanoparticles was also examined by scanning electron microscopy
at 5 kV (Zeiss Neon 40EsB, Germany). An aliquot of the nanosuspension
(10 μL) was mounted on aluminium stubs, dried overnight at room
temperature and coated with platinum. The nanoparticle formulations
that contain 10 μg Dz13Scr and 0.5% chitosan were subject to further
physical (drug release, stability) and cellular characterization (cyto-
toxicity, glucose consumption, permeation capacity and endocytic ab-
sorption).
2.4. Physical characterization stage
2.4.1. In vitro drug release of nanoparticles
The nanosuspension was centrifuged at 18200 g for 15 min
(Eppendorf 5702 Centrifuge, Beckman Coulter, Hauppauge, NY, USA),
followed by withdrawal of supernatants. The in vitro insulin release
profile of nanoparticles was examined at pH 2 (hydrochloric acid so-
lution; 250 μL) and pH 6.8 (phosphate buffer solution; 250 μL), simu-
lating the pH environments in fasting stomach and upper small intestine
respectively. The samples were placed on orbital shaker (PSU-20i,
Biosan, Riga, Latvia). At predetermined time points (0.5 h, 1 h, 2 h, 3 h,
4 h, 5 h, 6 h, 8 h and 10 h), the release medium (55 μL) was removed
and analysed by RP-HPLC as previously described. The same amount of
fresh medium (55 μL) was added to maintain the samples at sink con-
dition.
2.4.2. Insulin release kinetic analysis
In order to evaluate the kinetic of insulin release from chitosan-
Dz13Scr nanoparticles, the data obtained from in vitro release study was
analysed by fitting the data in the following release models: zero order,
first order, Higuchi model, and Korsemayer-Peppas model [25,26]. The
best-fitting model was selected for the model with the highest R2 value.
2.4.3. Stability studies
Taking into account the storage stability of insulin-loaded nano-
particles, the freshly prepared formulations were stored at 4 °C for 2
months. In Fourier transform infrared (FTIR) spectroscopic analysis, the
characteristic peaks of insulin, fresh pre-scaled up nanoparticles, pre-
scaled up nanoparticles (2 months storage at 4 °C) and scaled up na-
noparticles (2 months storage at 4 °C) were analysed using ATR-FTIR
spectrometer (PerkinElmer, Buckinghamshire, England) at resolution of
4 cm−1 scanning from 4000 to 400 cm−1. The effect of preparation
condition (complex coacervation) and storage condition on the in-
tegrity of insulin secondary structure was evaluated by the character-
istic peaks. Double subtraction [8] and ATR correction were carried out
with the in-built software (Spectrum, PerkinElmer). Meanwhile, the
changes in physiochemical properties (size, pdi, zeta potential) of the
nanoparticles were also analysed after 2 months of storage.
2.5. Cellular characterization stage
2.5.1. Cell culture
C2C12 cells (mouse myoblasts) and HT29 (human epithelial color-
ectal adenocarcinoma cells) were purchased from the American Type
Culture Collection (ATCC, USA). The cell culture medium consisted of
Dulbecco's Modified Eagle Medium, supplemented with 10% fetal bo-
vine serum, 1.5 g/L sodium bicarbonate, and 1% penicillin-strepto-
mycin solution. The medium was changed every second day. The cells
4
Journal of Drug Delivery Science and Technology 57 (2020) 101738
were cultured on T-75 flasks at 37 °C under 5% carbon dioxide and 95%
relative humidity (CO2CELL 170 incubator, MMM, Germany).
C2C12 cells and HT29 cells were used within passage 14 and passage 15
respectively.
2.5.2. Cytotoxicity analyses
The metabolic effect of blank medium (negative control), insulin
solution (10, 55, 100 nM), pre-scaled up insulin-loaded nanoparticles
(10, 55, 100 nM), scaled up insulin-loaded nanoparticles (10, 55,
100 nM) was investigated with C2C12 cells. In 96-well plates (Nunc,
Denmark), the cells were seeded at 1 × 105 cells/well in DMEM
medium for 12 h at 37 °C under 5% CO2 to ascertain cell attachment.
After 24 h of treatment with samples, the cells were washed with PBS.
The Cell-Titre Blue assay (Promega, NSW, Australia) was employed to
investigate the toxicity of samples on C2C12 cells. The analysis was
conducted using a microplate reader (PerkinElmer, Germany) at ex-
citation/emission wavelength at 560/590 nm. The cell viability was
expressed as percentage in relative to untreated cells.
HT29 cells were also used to evaluate the cytotoxicity of insulin
solution, pre-scaled up insulin-loaded nanoparticles, and scaled up in-
sulin-loaded nanoparticles. The cells were grown in 96-well plates at
1 × 105 cells/well until a monolayer is formed. In DMEM medium
(without FBS), the cells were treated with free insulin or nanoparticle
samples (containing 5, 12.5, 25, 50, 100 μg/mL insulin) for 24 h. The
Cell-Titre Blue assay was carried out according to the instruction from
manufacturer.
2.6. Glucose consumption analyses
The efficacy of glucose consumption in C2C12 cells treated with the
above three conditions was confirmed by glucose consumption assay kit
[27]. The samples (containing 10, 55, 100 nM insulin) were re-dis-
persed in DMEM that consisted of 25 mM glucose. At 12 h post-treat-
ment, Amplex® Red reagent (Thermofisher) was used according to the
manufacturer's instructions to determine the changes in glucose levels
of the medium. The percentage change of glucose level can be obtained
by the detection of red fluorescence using a microplate reader.
2.6.1. Mucus permeation measurements
The in vitro permeation capacity of pre-scaled up insulin-loaded
nanoparticles and scaled up insulin-loaded nanoparticles across
HT29 cells was evaluated [28–31]. The cells were seeded at
2 × 105 cells/mL on a polyester membrane (0.4 μm in pore size, dia-
meter 12 mm, 1.12 cm2 of cell growth area) and incubated in a
Transwell 24-well plate for 2–3 weeks. In the donor (0.5 mL) and re-
ceiver (1 mL) chambers, the DMEM medium was changed every day.
Upon reaching confluence, the DMEM medium was discarded in both
chambers, followed by the addition of pre-warmed HBSS. After 30 min
of equilibration at 37 °C, the cells were washed twice with pre-warmed
HBSS. Then, the HT29 cells in donor chamber were treated with 200 μL
pre-scaled up or scaled up nanoparticles (containing 200 μg/mL insulin
and dispersed in pre-warmed HBSS). In the pre-determined time in-
tervals (1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h and 9 h), an aliquot of test
samples (160 μL) in receiver chamber was collected. Subsequently,
same volume of fresh pre-warmed HBSS medium (160 μL) was added to
the donor chamber. The collected test samples were centrifuged at
18200 g for 15 min. The amount of insulin permeating from the donor
chamber to receiver chamber was measured by RP-HPLC (section
2.1.2).
2.6.2. Endocytic absorption mechanistic studies
The endocytic absorption pathways of pre-scaled up insulin-loaded
nanoparticles and scaled up insulin-loaded nanoparticles were in-
vestigated in the HT29 cell line [32]. The cells (5 × 104 cells/mL) were
seeded in 48 well-plates and incubated at 37 °C for 5 days. Prior to the
initiation of experiment, the cells were equilibrated with GI inhibitors
C.Y. Wong, et al.
in HBSS for 1 h at 37 °C, including chlorpromazine (clathrin vesicles
inhibitor; 10 μg/mL), genistin (caveolae pinching inhibitor; 0.2 mM),
amiloride (macropinocytos inhibitor; 12 μg/mL), and sodium azide
(active transport inhibitor; 1 mg/mL). Then, the above pharmacological
membrane entry inhibitors were co-incubated with cells and tested
samples (containing 200 μg/mL insulin) for 3 h at 37 °C [32]. The
HT29 cells that were treated with pre-scaled up and scaled up nano-
particles in the absence of GI inhibitors were used as negative controls.
After sample treatment, the cells were washed thrice with ice-cold PBS,
and subjected to the BCA protein assay. The data was presented as the
relative changes of cellular protein amount in relative to the negative
control.
2.7. Statistical analysis
The results are expressed as mean ± SD of triplicate data obtained
from two experimental repeats. Comparisons among groups were per-
formed using Analysis of Variance (ANOVA) and Dunnett multiple
comparison t-test by GraphPad Prism (GraphPad Software, USA). A
p < 0.05 is denoted as statistically significant.
3. Results
3.1. Pre-screening stage: examination of insulin encapsulation potential
In the pre-screening stage, we examined the encapsulation potential
of the self-assembled nanoparticles by addition of excessive insulin in
the manufacturing process (20.9 μM; 50 μL). The encapsulation effi-
ciency and loading capacity of insulin-loaded nanoparticles are illu-
strated in Fig. 2a and Fig. 2b respectively. While the concentration of
insulin was kept constant, it was found that the concentration of chit-
osan (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5%) and amount of
Dz13Scr (5, 10 or 15 μg) could influence the encapsulation efficiency of
insulin. For instance, when Dz13SCr (5 μg) were kept constant, an in-
crease in chitosan concentration from 0.1 to 0.5% could lead to sig-
nificant changes in encapsulation efficiency (p < 0.0001). When
Dz13SCr (10 μg) was used in the formulation, the addition of 0.5%
chitosan could significantly improve the encapsulation efficiency as
compared to formulations with lower chitosan concentrations
(0.1–0.45%) (p < 0.005). However, when Dz13SCr (15 μg) was added,
an increase in chitosan concentrations (0.1–0.45%) did not lead to any
significant changes of encapsulation efficiency (p > 0.05). Amongst all
the formulation, nanoparticles that were fabricated at the highest
concentration of chitosan (0.5%) and Dz13SCr (10 μg) demonstrated
the highest encapsulation efficiency (32.98 ± 1.64%) and loading
capacity (28.29 ± 1.41%). This phenomenon could be related to the
fact that 0.5% chitosan was sufficient in stabilising the polyelectrolyte
nanocomplex, thereby generating particles with the highest entrapment
Fig. 2. Encapsulation efficiency and loading capacity of insulin nanoparticles in pre-screening stage. (a) Effect of chitosan concentration and Dz13Scr amount
on encapsulation efficiency (%) of insulin-loaded NPs in pre-screening stage. (b). Effect of chitosan concentration and Dz13Scr amount on loading capacity (%) of
insulin-loaded NPs at the pre-screening stage.
5
Journal of Drug Delivery Science and Technology 57 (2020) 101738
efficiency. Below the optimal chitosan concentration, an excessive
amount of Dz13Scr could destabilize the nanoparticles.
3.2. Optimization stage: characterization of pre-scaled up insulin
nanoparticles
After determining the encapsulation potential in the pre-screening
stage, the obtained result was used to refine the amount of insulin
(8.61 μM; 50 μL) that was added in the preparation of nanoparticles. In
the optimization stage, the effect of Dz13Scr (5, 10 or 15 μg) and
chitosan concentration (0.5%) on the size, polydispersity index and
zeta-potential of the nanoparticles were determined. As shown in
Fig. 3a, as the amount of Dz13Scr increased from 5 μg to 10 μg, the
average size of nanoparticles increased from 244 ± 106 nm to
885 ± 50 nm. It was also revealed that the polydispersity index varied
between 0.31 ± 0.06 and 0.86 ± 0.23 for all formulations (Fig. 3a).
High polydispersity index and uneven particle size could be attributed
to an imbalance in the ratio of positively charged polymers and nega-
tively charged oligonucleotides. As shown in Fig. 3b, the zeta potential
of nanoparticles decreased slightly from 14.6 ± 1.3 mV to
13 ± 1.73 mV with higher concentration of Dz13Scr, however the
difference in zeta potential amongst all formulations was not statisti-
cally significant (p > 0.05). Therefore, negatively charged Dz13Scr
had minimal impact on the zeta potential of the formulations. The re-
sultant positive zeta potential could be attributed to the presence of
chitosan over the outermost layer, which could improve the mu-
coadhesiveness of the drug delivery system to the negatively charged
mucin in the GI tract. Amongst the tested conditions, the combination
of Dz13Scr (10 μg) and chitosan (0.5%) could produce nanoparticles
with desirable particle size (534 ± 24 nm), polydispersity index
(0.31 ± 0.06), positive zeta potential (14.57 ± 1.1 mV), loading
capacity (56.31 ± 2.79%) and high encapsulation efficiency
(79.96 ± 3.96%). Therefore, these preparation parameters were se-
lected as the optimal conditions to generate nanoparticles for further
examination in the scaled-up stage.
3.3. Scaled-up stage: characterization of scaled-up of nanoparticles
The physical properties of the scaled-up nanoparticles, which were
prepared by escalating the quantity of excipients 6-fold, were char-
acterized and compared with the pre-scaled up nanoparticles. As shown
in Fig. 3c and d, it was observed that the scaled-up nanoparticles de-
monstrated slightly smaller particle size (479 ± 24 nm), comparable
polydispersity index (pdi 0.34 ± 0.06), and similar zeta potential
(14.47 ± 2.2 mV) when compared to pre-scaled up nanoparticles.
Meanwhile, the scaled-up nanoparticles demonstrated slightly higher
loading capacity (58.9 ± 0.2%) and encapsulation efficiency
(88.71 ± 0.3%) (Fig. 3e). However, statistical analysis revealed that
C.Y. Wong, et al. Journal of Drug Delivery Science and Technology 57 (2020) 101738

Fig. 3. Physical characteristics of pre-scaled up and scaled-up insulin nanoparticles in optimization stage and scaled-up stage. (a) Effect of chitosan
concentration and Dz13Scr amount on particle size and polydispersity index of pre-scaled up insulin nanoparticles in optimization stage. (b) Effect of chitosan
concentration and Dz13Scr amount on zeta potential of pre-scaled up insulin nanoparticles in optimization stage. (c) Comparison of particle size between pre-scaled
up and scaled-up insulin nanoparticles in scaled-up stage. (d) Comparison of zeta potential in scaled-up stage. (d) Comparison of loading capacity and encapsulation
efficiency in scaled-up stage.

the difference in mean values (i.e. size, pdi, zeta potential, loading
capacity and encapsulation efficiency) between the above two for-
mulations were not statistically significant (p > 0.05), hence the im-
pact of scaling up on the physical properties of formulations were not
significant. In Fig. 1, SEM images illustrated the spherical pre-scaled up
and scaled up nanoparticles that were prepared by Dz13Scr (10 μg) and
chitosan (0.5%). These two formulations were subject to further phy-
sical and cellular examination.
3.4. Physical characterization of pre-scaled up and scaled-up nanoparticles
3.4.1. pH-dependent drug release
The insulin-loaded nanoparticles demonstrated pH-dependent drug
release characteristics in simulated GI fluids at 37 °C (Fig. 4a). At pH 2
hydrochloric acid solution, the maximum cumulative drug release of
pre-scaled up nanoparticles was 13.49 ± 9.27% within 10 h. Similarly,
scaled-up nanoparticles had only 14.03 ± 9.27% of cumulative drug
release at pH 2. Therefore, the formulations presented minimal pre-
mature drug release and demonstrated protective effect against acidic
pH. In contrast, at pH 6.8 phosphate buffer solution, an approximately
88 ± 13% and 85 ± 13% of insulin was release from the pre-scaled
up and scaled-up nanoparticles respectively after 10 h. At both pH 2
and pH 6.8, statistical analysis showed that the mean differences in the
percentage of insulin release between pre-scaled up and scaled-up in-
sulin nanoparticles were not statistically significant (p > 0.05)
throughout the experiment. Also, the obtained results showed that both
pre-scaled up and scaled-up insulin nanoparticles exhibited biphasic
release pattern with an initial rapid burst release during the first hour,
followed by a sustained release profiles within 10 h. For instance, it was
found that the pre-scaled up nanoparticles had a rapid burst release of
insulin (49.49%) at pH 6.8, followed by a sustained release pattern
(88%) in 10 h. This phenomenon could be attributed to insulin ad-
sorption on the surface of particles as well as their high encapsulation
efficiency, which were in accordance with previous studies [33,34].
3.4.2. Mathematical modelling for insulin release kinetics
Mathematical models were applied to examine the insulin release
mechanism from the optimized pre-scaled up and scaled-up formula-
tions. The best-fitting models with the highest R2 were tabulated in
Table 2. According to R2 values, the release mechanism of optimized
formulations can be elucidated by either the first-order or Higuchi
model. At pH 2, insulin release from both pre-scaled up and scaled-up
nanoparticles were found to be best fitted by Higuchi square root
model. On the other hand, first-order was the best-fitting model for
both formulations at pH 6.8. The obtained results suggested that insulin
release from nanoparticle matrix was directly proportional to the initial
insulin concentration (first-order model) in alkali condition, while the
Higuchi model implies the insulin release in acidic medium occurred via
diffusion from the porous matrix based on Fickian diffusion.
3.4.3. Stability
FTIR study was conducted to examine the interaction between
components (insulin, chitosan, Dz13Scr) of the nanoparticulate sys-
tems. Fig. 4b illustrates the FTIR pattern of native insulin, fresh pre-

6
C.Y. Wong, et al. Journal of Drug Delivery Science and Technology 57 (2020) 101738

Fig. 4. Drug release profiles and FTIR spectra of insulin nanoparticles. (a) Effect of pH on the cumulative release (%) of insulin release from both pre-scaled up
and scaled-up nanoparticles at pH 2 and pH 6.8. (b) FTIR spectra of insulin, pre-scaled up insulin NPs, pre-scaled up insulin-loaded NPs (2 months storage) and
scaled-up insulin-loaded NPs (2 months storage). (c) Storage stability of particle size for both pre-scaled up and scaled-up insulin nanoparticles. (d) Storage stability
of polydispersity index for both pre-scaled up and scaled-up insulin nanoparticles. (e) Storage stability of zeta potential for both pre-scaled up and scaled-up insulin
nanoparticles.

formulations, the physiochemical properties of scaled-up nanoparticles,

Table 2
including particle size (473 ± 5.85 nm), polydispersity index
Mathematical model for insulin release kinetics.
(0.35 ± 0.03) and zeta potential (15.1 ± 0.49 mV), had negligible
Formulations Conditions Release R2 difference. ANOVA analysis showed that formulations with 2 months of
kinetic storage did not have significant statistical difference in terms of changes
1. Pre-scaled up insulin NPs pH 2 HCl; 37oC Higuchi model 0.9619
in physiochemical properties as compared to fresh formulations
2. Scaled-up insulin NPs Higuchi model 0.9208 (p > 0.05). Under this storage condition, the stability of both for-
3. Pre-scaled up NPs (pH 6.8 PBS; o
pH 6.8 PBS; 37 C First-order 0.9507 mulations was maintained.
37 °C)
4. Scaled-up insulin NPs First-order 0.9161
3.5. Cellular characterization

scaled up nanoparticles, pre-scaled up nanoparticles (2 months storage 3.5.1. Cytotoxicity assay

at 4 °C) and scaled up nanoparticles (2 months storage at 4 °C). FTIR
spectrum of native insulin shows two characteristic peaks at 1656 and
1554 cm−1 that correspond to amide I region (C]O stretching vibra-
tions) and amide II region (N–H bending), respectively. For the fresh
formulations and formulations that were stored for 2 months, FTIR
spectra also revealed the characteristic peaks of native insulin at nearly
same wavenumber, confirming the compatibility between components
of nanoparticles and encapsulation of insulin within the core of nano-
particles. A slight shift of wavelength was attributed to the electrostatic
interactions between insulin and the ionizable excipients during the
manufacturing process of nanoparticles [35,36]. Similar observations
were also noted by other research groups, who reported interactions
between polymers and proteins [25]. In terms of the storage stability,
the changes in physiochemical properties (size, pdi, zeta potential) of
the nanoparticles were measured after 2 months of storage at 4 °C
(Fig. 4c, d and e). For pre-scaled up nanoparticles that were stored for 2
months, the measured properties had minimal changes including par-
ticle size (493 ± 9.09 nm), polydispersity index (0.31 ± 0.03) and
zeta potential (15.1 ± 1.5 mV). Similarly, as compared to fresh
The cytotoxicity of pre-scaled up formulation and scaled-up for-
mulation on C2C12 was examined. The obtained data suggested that all
tested samples (containing 10, 55, 100 nM insulin) did not introduce
any cytotoxic effect to C2C12 cells after 12 h of treatment. As illustrated
in Fig. 5a, all samples displayed at least 80% C2C12 cell viability.
Statistical analysis revealed that pre-scaled up nanoparticles and scaled-
up nanoparticles (containing 55 nM insulin) had significant protective
effect to C2C12 cells (p < 0.002). Meanwhile, the differences in mean
cell viabilities between non-treated cells and the remaining samples
were not statistically significant (p > 0.05). Therefore, the developed
formulations were biocompatible and did not induce any cytotoxicity.
The cytotoxicity of free insulin, pre-scaled up formulation and scaled-up
formulation were also examined in HT29 colon cancer cells. When
HT29 cells were incubated with free insulin or nanoparticles, the used
experimental doses (containing 5, 12.5, 25, 50, 100 μg/mL insulin) did
not induce any cytotoxicity effect in the cells. As shown in Fig. 5b, when
compared to non-treated cells, statistical analysis indicated that pre-
scaled up nanoparticles (containing 12.5, 50 and 100 μg/mL insulin)
and scaled-up nanoparticles (containing 5 and 25 μg/mL insulin)

7
C.Y. Wong, et al.
Fig. 5. Cellular assays. (a) C2C12 cell viability after 12 h exposure to insulin, pre-scaled up and scaled-up insulin nanoparticles. (b) HT29 cell viability after 24 h
exposure to insulin, pre-scaled up and scaled-up insulin nanoparticles. (c) Glucose consumption in C2C12 cells. (d) Cumulative insulin transport through HT29 cell
monolayer. (e) Endocytic pathways of lyophilized nanoparticles.
introduce significant protective effect (p < 0.05). All other tested
samples had no cytotoxic effect to HT29 cells. Therefore, the used ex-
cipients (chitosan and Dz13Scr) and nanoparticles were safe within the
tested insulin dosage range (from 5 to 100 μg/mL).
3.5.2. Glucose uptake study
In medium containing 25 mM glucose, free insulin, pre-scaled up
formulation and scaled-up formulation (containing 10, 55 and 100 nM
insulin) elicited higher glucose uptake in C2C12 cells, as compared to
non-treated cells (Fig. 5c). It was found that non-treated cells demon-
strated 6.87% of glucose uptake, whereas samples containing 100 nM
insulin could result in 16.98–20.79% of glucose uptake at 12 h post-
treatment. When compared to free insulin (10, 55, 100 nM), both pre-
scaled up and scaled-up nanoparticles (containing 10, 55 and 100 nM
insulin) demonstrated comparable effects in C2C12 glucose uptake
(p > 0.05). Hence, the bioactivity of insulin could be preserved in both
pre-scaled up and scaled-up nanoparticles, leading to hypoglycaemic
effect in the medium, C2C12 cells glucose uptake, and a reduction in
glucose concentration.
3.5.3. Permeation across HT29 cells
Mucus layer and tight junctions can limit the permeation capability
of therapeutic proteins in the GI tract [28]. To understand the per-
meation capacity of pre-scaled up insulin-loaded nanoparticles and
scaled up insulin-loaded nanoparticles across HT29 cells, an in vitro
transwell system was used to examine the amount of insulin that can
permeate from the apical chamber to basolateral chamber. As illu-
strated in Fig. 5d, the cumulative percentage of permeated insulin for
both pre-scaled up and scaled up formulations had no significant dif-
ference throughout the whole experiment (p > 0.05). An
8
Journal of Drug Delivery Science and Technology 57 (2020) 101738
approximately 68% of insulin translocated to the receiver chambers for
both formulations within 1 h. Meanwhile, almost all encapsulated in-
sulin could be detected in the receiver chamber within 24 h. The ob-
tained results can be elucidated by the comparable physiochemical
properties (particle size, polydispersity, zeta potential) between pre-
scaled up and scaled up nanoparticles, thereby the variation in cellular
performance of both formulations was negligible after the scaling up
process.
3.5.4. Endocytic absorption mechanism
The endocytic absorption pathways of pre-scaled up and scaled up
insulin-loaded nanoparticles were assessed. As illustrated in Fig. 5e, the
cellular uptake of pre-scaled up nanoparticles reduced significantly by
39%, 28% and 35% in the presence of chlorpromazine, amiloride, and
sodium azide. Similarly, as compared to control (that is, HT29 cells
treated with nanoparticles in the absence of GI inhibitors), the cellular
uptake of scaled up nanoparticles reduced by 38%, 27% and 34% in the
presence of chlorpromazine, amiloride, and sodium azide. ANOVA
analysis revealed that pre-scaled up and scaled up insulin-loaded na-
noparticles did not have any significant difference in terms of changes
in protein uptake (p > 0.05). Therefore, the endocytic absorption
mechanisms and GI uptake efficiencies of both formulations were
comparable.
4. Discussion
4.1. Complex caocervation for preparation of insulin-loaded nanoparticles
Complex coacervation (polyelectrolyte complexation) is a mild
technique that has been adopted widely for the preparation of
C.Y. Wong, et al.
nanoparticle to encapsulate labile therapeutic agents such as proteins,
peptides and nucleic acids [36,37]. In this technique, the use of harsh
conditions such as organic solvents, sonication, mechanical stress and
heat can be eliminated, thereby the structure and therapeutic effect of
labile drug can be maintained during nanoparticle formation
[6,7,12,24,38–40]. In contrast, the above harsh procedures can be
present in the other nanoparticle preparation techniques, such as mi-
croemulsion method, solvent evaporation and emulsion polymerization
[12,13], hence limiting their use in encapsulation of sensitive drugs
because the drug compounds can be prone to structural changes [2,13].
In recent years, studies revealed that nanoparticles prepared by com-
plex coacervation could improve the colloidal stability and bioactivity
of therapeutic protein [2,13], promote sustained and controlled de-
livery of drugs [10,12], and protect peptides from enzymatic degrada-
tion [40]. Other favourable characteristics, including reasonable par-
ticle size [37], spherical shape [35], homogeneous particle size [37],
high encapsulation efficiency and enhanced cellular uptake [38], were
also reported for nanoparticles that were prepared by complex coa-
cervation. For example, polyethylenimine-dextran sulfate nanoparticles
could improve the stability of formulations and recover at least 95% of
the insulin potency [39]. Also, it was reported that quaternized chitosan
nanoparticles that were prepared by complex coacervation demon-
strated small particle size (170–270 nm), spherical morphology, high
encapsulation efficiency (80%), improved formulation permeation
across the colon membrane ex vivo, and more efficient GI absorption as
compared to native insulin [37]. When insulin nanoparticles were
prepared by complex coacervation, the formulation could elicit cardi-
oprotective effects against heart ischemia in diabetes mellitus, in-
cluding a reduction in myocardial infarction size, enhancement in left
ventricular filling pressures, amelioration of cell apoptosis, and reg-
ulation of biomarkers expression (lactate dehydrogenase, substance P,
ornithine decarboxylase, polyamine pools, spermidine/spermine N′-
acetyltransferase), as compared to free insulin [41]. Chitosan is a po-
tential biodegradable and biocompatible candidate for the encapsula-
tion of orally administered drugs [10]. For chitosan-based nanocarriers,
numerous studies revealed that they are capable of adhering to the
negatively charged mucin (mucoadhesion), reducing transepithelial
electrical resistance, and opening the tight junctions reversibly due to
cytoskeletal F-actin and tight junction protein ZO-1 redistribution
[4,33,36,38]. All of these favourable characteristics could contribute to
improvement in paracellular absorption and GI uptake of therapeutic
proteins [38]. Owing to the above promising attributes, the insulin-
loaded chitosan-Dz13SCr nanoparticles were prepared through complex
coacervation (Fig. 1). This technique, which is driven by the electro-
static interaction between oppositely charged ionizable polymer and
oligonucleotide, is a well-acknowledged technique for the preparation
of multifunctional nanomedicine [2,4,13,36,37,42]. In the present
study, the negatively charged Dz13Scr was mixed with insulin solution
to avoid aggregation, followed by the gradual addition of low molecular
weight chitosan at ambient temperature. Upon mixing of the oppositely
charged solvents, the self-assembled polyelectrolyte complex can be
fabricated in an aqueous environment. This gentle preparation proce-
dure ensures the perseverance of the insulin stability and bioactivity.
4.2. Physical characteristics of nanoparticles
4.2.1. Encapsulation efficiency and loading capacity
A successful nanocarrier should possess high drug encapsulation
efficiency to minimize wastage in the preparation stage and maximize
therapeutic effect in clinical settings. Previous studies reported that
chitosan-based nanoparticles that were prepared by complex coa-
cervation had at least 82% of insulin encapsulation efficiency, which
can be further increased by using modified-chitosan such as triethyl-
chitosan (88%) and dimethyl-ethylchitosan (87%) [37]. However,
when polyethylene glycol monomethyl ether-grafted chitosan was used,
the generated nanoparticles demonstrated reduced encapsulation
9
Journal of Drug Delivery Science and Technology 57 (2020) 101738
efficiency [1]. This phenomenon was attributed to steric hindrance and
a reduction in the amino groups on chitosan, which weakened the
electrostatic interactions between polycationic and polyanionic ex-
cipients. One of the studies optimized the insulin-loaded N,N-dimethyl-
N-octyl chitosan nanoparticles statistically by using the Box-Behnken
response surface methodology, which reported that the concentrations
of polymer and insulin had significant impact on the encapsulation
efficiency of insulin [12]. Similarly, the amount of incorporated insulin
could have an impact on the encapsulation efficiency of modified-
chitosan nanoparticles [43]. In brief, the encapsulation efficiency of the
nanoparticles increased with the amount of incorporated insulin, but
the value decreased when a maximal amount of insulin was used
[4,35]. Therefore, we examined the encapsulation potential of the
formulations with various amount of chitosan (0.1, 0.15, 0.2, 0.25, 0.3,
0.35, 0.4, 0.45, 0.5%) and Dz13Scr (5, 10 or 15 μg) in the pre-screening
stage. When an excessive amount of insulin was incorporated to the
mixture, the excess proteins could not interact with the charged ex-
cipients [4]. This enables the estimation of the saturated level of insulin
that can be encapsulated within the nanoparticles. In this study, it
should be noted that the oppositely charged chitosan (0.5%) and
Dz13Scr (10 μg) could introduce the optimal strength of electrostatic
interaction, resulting in the generation of nanoparticles with the highest
insulin encapsulation efficiency [3]. Below the optimum ratio of chit-
osan and Dz13Scr, the unreacted positively charged polymer or nega-
tively charged oligonucleotide could destabilize the formulation. Pre-
vious studies also demonstrated that excess concentrations the
interacting excipients could increase nanoparticle size and form ag-
gregates [2]. After determining the encapsulation potential of nano-
particles in the pre-screening stage, the refined amount of insulin was
used to prepare pre-scaled up nanoparticles and scaled-up nanoparticles
in the optimization stage and scaled-up stage respectively. Under the
optimal conditions, pre-scaled up nanoparticles (79.96) and scaled-up
nanoparticles (88.71%) could achieve high encapsulation efficiencies.
Apart from using the optimal concentration of ionizable excipients, one
of the studies suggested that nanoparticles with a higher value of mean
diameter could lead to higher encapsulation efficiency [43]. For in-
stance, insulin-loaded modified-chitosan nanoparticles with a mean
diameter of 154 nm had only 59% of encapsulation efficiency, but
formulations with a mean diameter of 1312 nm could encapsulate 95%
of insulin. In this study, pre-scaled up nanoparticles (534 ± 24 nm)
and scaled-up nanoparticles (479 ± 24 nm) achieved reasonable
particle size, which further maximised the amount of encapsulated in-
sulin within the nanocarrier. Similarly, quaternized chitosan nano-
particles (85%) [38], chitosan/dextran sulfate nanoparticles (85%)
[36], self-assembled chitosan/insulin nanoparticles (91.2%) [13] and
chitosan/polyurethane nanoparticles coated with alginate (98.5%) [44]
could encapsulate more insulin, when both excipient concentration and
excipient ratio were optimized.
4.2.2. Particle size and polydispersity index
The particle size is a critical determinant for the GI uptake efficiency
of nanoparticles [2,43]. In general, the GI internalization and para-
cellular absorption of therapeutic proteins could be facilitated with
particle size in nano-scaled range when compared to microparticles
[13,33,43]. The size of nanoparticles can be influenced by the polymer
to protein ratio [2,38], polymer to polymer ratio [36,42] and con-
centration of polymer [12]. For instance, the size of the self-assembled
chitosan/insulin nanoparticles increased from 210 nm to 465 nm when
the ratio of chitosan to insulin changed from 1:1 to 2:1 [13]. With re-
spect to the polymer to polymer ratio, the nanoparticles prepared with
dextran sulfate and chitosan at ratio of 1:2 demonstrated larger size
than formulations with a polymers ratio of 1:1, 1.5:1, 2:1 and 2.5:1
[36]. In terms of the concentration of polymer, previous study also
revealed that high concentration of chitosan and N-(2-hydroxy)propyl-
3-trimethyl ammonium chitosan chloride (> 2.5 mg/mL) could result
in the formation of aggregate with size over 1 μm in complex
C.Y. Wong, et al.
coacervation [43]. In the current study, low molecular weight chitosan
was manipulated because it could produce nanoparticles with smaller
size in complex coacervation or polyelectrolyte complexation [45]. It
was noted that as the amount of Dz13Scr increased from 5 μg to 15 μg,
the mean diameter of the pre-scaled up nanoparticles increased from
244 nm to 885 nm. Under the optimal combination of Dz13Scr (10 μg)
and chitosan (0.5%), insulin-loaded pre-scaled up nanoparticles and
scaled-up nanoparticles had particle size of 534 nm and 479 nm re-
spectively. Similarly, insulin-loaded chitosan/alginate nanoparticles [4]
and chitosan/dextran sulfate nanoparticles [45] prepared by complex
coacervation demonstrated average mean particle size of 552 nm and
500 nm respectively. The strong electrostatic interactions between
chitosan and Dz13Scr could produce compact nanoparticles with small
size, high encapsulation efficiency and improved stability [12,36].
Polydispersity index, with a numerical value ranging from 0 to 1, is an
index that indicate the size homogeneity of particles [37,38]. The
polydispersity index of nanoparticles that were prepared by complex
coacervation could be also influenced by the concentration of polymer
and of insulin [12,38]. In the study, pre-scaled up nanoparticles (0.31)
and scaled-up nanoparticles (0.34) demonstrated comparable poly-
dispersity index, indicating the uniformity of particle size.
4.2.3. Zeta potential
Zeta potential, an indicator of the surface charge, can have impact
on the colloidal stability [12,38,46], cytotoxicity and absorption effi-
ciency of the protein-based nanoparticles [34,37]. Zeta potential of the
nanoparticles is predominantly influenced by the concentration as well
as ratio of polymer and proteins in the formulation [13,38,47]. For
instance, a change in ratio of chitosan:insulin from 1:1 to 2:1 could
increase the zeta potential of nanoparticles from 2.83 mV to 24.69 mV
[13]. Previous studies revealed that the zeta potential of free chitosan
was 56.83 mV [40], whereas insulin had a zeta potential of
−10.06 mV. In the present study, the obtained zeta potentials for pre-
scaled up and scaled-up nanoparticles were 14.57 mV and 14.47 mV
respectively, which can serve as evidence for the formation of nano-
particles between chitosan and Dz13Scr. The positive zeta potential was
contributed by the unoccupied amine groups of the chitosan over the
outermost layer [44]. In one of the studies, insulin-loaded nanoparticles
that were prepared by complex coacervation, such as chitosan nano-
particles (17.6 mV), triethyl-chitosan nanoparticles (25.1 mV) and di-
methylethyl-chitosan nanoparticles (26.2), demonstrated similar zeta
potential [37]. The positive surface charge could contribute to the
mucoadhesion of nanoparticles to the negatively charged intestinal
mucosa via electrostatic interactions [2,43]. Consequently, the reten-
tion time of the formulation in the tract can be prolonged, and a drug
concentration gradient between the intestine and systemic circulation
can be created, resulting in an elevation of cellular uptake of ther-
apeutic proteins by the enterocytes [3]. Meanwhile, the capability to
escape from endolysosomes was another favourable characteristic re-
ported for cationic nanoparticles [43,48]. Last but not least, it was
stated that nanoparticles with zeta potential higher than 30 mV could
be cytotoxic to epithelial cells [38]. As the obtained nanoparticles in
this study had zeta potential of approximately 14.5 mV, the developed
formulations are expected to pose limited cytotoxicity and preserve the
cell viability.
4.3. Nanoparticle drug release
In order to determine the drug release behaviour of nanoparticles in
vitro, the dissolution study was conducted in simulated gastric fluid (pH
2 hydrochloric acid) and simulated intestinal fluid (pH 6.8 phosphate
buffer) [12]. As mentioned, the drug release profiles of both pre-scaled
up and scaled-up nanoparticles were similar. At pH 2, approximately
14% of insulin released from the formulations within 10 h, suggesting
the stability of the developed nanocarriers against acidic environment.
Such protective effect was also observed in chitosan/insulin self-
10
Journal of Drug Delivery Science and Technology 57 (2020) 101738
assembled nanoparticles, which release only 15% of insulin at pH 1.2
throughout the experiment [13]. It was suggested that more than 95%
of amino groups over chitosan are protonated in an acidic condition,
hence it could maintain the strong ionic interaction with Dz13Scr and
entrap insulin. At pH 6.8, the nanoparticles were capable to dissociate
and release around 85–88% of insulin in a sustained manner after 10 h,
suggesting the pH-sensitive nature of the formulations. The obtained
results showed that both pre-scaled up and scaled-up insulin nano-
particles exhibited biphasic release pattern with an initial rapid burst
release during the first hour (46%), followed by a sustained release
profile within 10 h. Similarly, it was reported that N,N-dimethyl-N-
octyl chitosan nanoparticles (38%) [12] and methylated-chitosan na-
noparticles (36.1) [38], which were prepared by complex coacervation,
demonstrated burst release of insulin at pH 6.8 within 30 min. The
initial phase of insulin release was attributed to the insulin located at
the surface of the nanoparticles, and the subsequent sustained release
phase could be attributed to the diffusion of entrapped insulin from
nanoparticle core to the surface [36]. In order to alleviate the effect of
burst release, one of the studies manipulated chitosan-polyethylene
glycol monomethyl ether conjugates to formulate nanoparticles via
complex coacervation, which could reduce the release of insulin to 10%
within 30 min, followed by a cumulative release of 60% insulin within
6 h at pH 6.8 [1]. Another study utilized chitosan derivatives including
triethyl-chitosan and dimethylethyl-chitosan to prepare insulin-loaded
nanoparticles, which could exhibit a lessened burst release effect and
release at most 47.3% of insulin over 5 h at pH 6.8 [37]. In the present
study, the Higuchi square root model was the drug release mechanism
of both pre-scaled and scaled-up nanoparticles at pH 2, and both for-
mulations demonstrated the first-order drug release kinetic at pH 6.8. In
acidic condition, as the obtained n values of nanoparticles were be-
tween 0.96 and 1.13, it could be inferred that the drug release me-
chanism was mass transfer, followed by a Fickian diffusion and erosion
of nanoparticle matrix [46]. In contrast, the percentage of insulin re-
lease from a swelled porous nanocarrier is directly proportional to the
initial insulin concentration within the core in an alkali condition [33].
4.4. Nanoparticle cytotoxicity
Polymeric nanoparticles, which were prepared by complex coa-
cervation, have demonstrated promising safety profile [12,33,41,44].
For instance, it was reported that insulin-loaded poly(ethylene glycol)-
carboxymethyl chitosan nanoparticles (up to 200 μg/mL) demonstrated
low cellular toxicity in H9C2 myoblast cells [41]. Although insulin-
loaded quaternized chitosan nanoparticles exhibited a concentration
dependent toxicity to Caco-2 epithelial colorectal adenocarcinoma
cells, their overall cytotoxicity was not significant in MTT and lactate
dehydrogenase assay [33]. Reduced cell viability could be associated
with the permeation of nanoparticle across the GI cells. In the current
study, the cytotoxicity of pre-scaled up formulation and scaled-up for-
mulation were assessed to evaluate the safety profile and biocompat-
ibility of drug delivery system. The developed nanoparticles displayed
negligible toxicity to C2C12 cells (containing 10, 55, 100 nM insulin)
and HT29 cells (5, 12.5, 25, 50, 100 μg/mL insulin). The obtained data
could be linked to the resultant positive surface charge (14.5 mV),
which was less than the toxic level (30 mV). Therefore, the used ex-
cipients (chitosan and Dz13Scr) and nanoparticles were safe and non-
toxic within the tested insulin dosage range.
4.5. Glucose consumption analyses
In the current study, the in vitro glucose uptake study confirmed that
the bioactivity of encapsulated insulin within both pre-scaled up and
scaled-up formulations was maintained. In the presence of insulin,
previous studies stated that the expression of GLUT-4 transporters in-
creases, hence glucose uptake can be induced [49]. When compared to
native insulin, both formulations (containing 10, 55, 100 nM insulin)
C.Y. Wong, et al.
demonstrated comparable effect in promoting 16.98–20.79% of glucose
uptake in C2C12 cells after 12 h of treatment. The hypoglycaemic effect
was also observed in other formulations that were prepared by complex
coacervation, including insulin-loaded chitosan/dextran sulfate nano-
particles (50 and 100 IU/kg) [45] and insulin-loaded chitosan/alginate
nanoparticles (10 IU/Kg) [4]. The above formulations could effectively
promote glucose reduction in diabetic rats. For instance, the orally
administered insulin-loaded chitosan/dextran sulfate nanoparticles re-
duced the blood sugar level by 35%, and the hypoglycaemic effect
could sustain over 24 h [45]. Therefore, complex coacervation is
deemed to be a mild preparation technique that can conserve the
conformational structure of encapsulated insulin and maintain its
therapeutic effect.
4.6. Nanoparticle permeation across GI cells
Mucus layer is one of the GI barriers that can hinder the permeation
of therapeutic proteins such as insulin across the GI tract
[28–30,50–53]. Previous studies demonstrated that the GI absorption of
nanoparticle could be dependent on zeta potential and surface property.
Although cationic polymers (chitosan, alginate, poly(acrylic acid),
carbomer) could promote mucoadhesiveness and adhere to the anionic
mucus layer [34], the effectiveness of the nanoparticles to permeate
across GI cells can also be affected by other factors such as mucus
clearance and stability of the nanocarriers [1]. One of the studies de-
veloped insulin-loaded chitosan-g-polyethylene glycol monomethyl
ether (mPEG) nanoparticles by complex coacervation, which could
improve the hydrophilicity of the formulation and enhance the muco-
penetrating capacity of formulations across the mucus layer by using
10% of mPEG [1]. In this study, the muco-penetrating capacity of pre-
scaled up and scaled-up nanoparticle was evaluated. It was found that
both formulations had approximately 68% of encapsulated insulin
translocated to the basolateral chambers within 1 h. The obtained data
can be elucidated by the comparable small particle size and hydrophilic
nature of both formulations. The efficient permeation capability of the
nanoparticles could be caused by the cationic chitosan [24,54] and
hydrophilic Dz13Scr, which result in a balance between the mu-
coadhesiveness strength and muco-penetrating capacity. Firstly, the
cationic component of the nanoparticles (chitosan) supports the mu-
coadhesiveness and prolong the residence time of the drug delivery
system in the intestine [4,12,13,37], which creates a drug concentra-
tion gradient between the apical surface and basolateral side of the GI
tract [2]. A number of insulin-loaded positively charged chitosan-based
formulations, such as polyurethane-chitosan nanoparticles with algi-
nate protective shell [44], triethyl-chitosan nanoparticles [37], di-
methylethyl-chitosan nanoparticles [37] and quaternized chitosan na-
noparticles [33], enhanced the adhesion of the drug delivery systems in
the rat intestine due to strong electrostatic interaction with mucosal
surface. The use of chitosan could open the tight junctions of GI epi-
thelial cells transiently [11,33,55], hence the total amount of per-
meated insulin that can reach the systemic circulation could be further
increased via paracellular absorption [28,33]. On the other hand, the
hydrophilic component (Dz13Scr) promotes the muco-penetration of
the nanoparticle [56]. Previous studies showed that hydrophilic surface
of the nanoparticle could improve the permeation amount of insulin-
loaded chitosan-g-polyethylene glycol monomethyl ether nanoparticles,
which were prepared by complex coacervation, in duodenum and je-
junum of rats as compared to free insulin [1]. Other studies also ob-
served the following characteristics, including small particle size and
hydrophilic surface nature, could improve the muco-penetrating capa-
city and minimize excessive mucoadhesiveness [51,57,58].
4.7. In vitro endocytic absorption mechanism
Apart from paracellular absorption, polymeric nanoparticles, such
as alginate-C18 conjugate nanoparticles, can also be absorbed by
11
Journal of Drug Delivery Science and Technology 57 (2020) 101738
endocytotic pathway in the GI tract [37,58]. With regards to endocytic
absorption mechanism, the nanoparticles could be adsorbed by ad-
sorptive endocytosis, but not receptor-mediated endocytosis (active
targeting) due to the absence of ligand conjugation in the developed
formulations [37]. In the current study, both pre-scaled up and scaled-
up formulations presented the following adsorptive endocytosis me-
chanisms including clathrin-mediated endocytosis, macropinocytosis
and comprehensive active transport. A previous study also demon-
strated that insulin-loaded alginate-based nanoparticles could facilitate
the endocytic absorption of therapeutic proteins via mixed mechanisms
[58]. Having taken the nanoparticle component and zeta potential into
consideration, it can be proposed that both paracellular and endocytosis
absorption mechanisms are involved in the developed formulations,
hence the permeation of therapeutic proteins across the GI cells can be
improved.
4.8. Stability of insulin nanoparticles
Insulin is a liable therapeutic protein that is prone to degradation
during the manufacturing process and storage of nanoparticles [59]. In
contrast, nanoparticles that were prepared by complex coacervation
could protect insulin against high temperature and GI enzyme [40]. In
terms of the stability against GI enzymes, a number of studies reported
that the encapsulation of insulin within nanoparticles by complex
coacervation was an effective strategy [1,2,10,40,42]. For example, as
compared to free insulin, chitosan-g-polyethylene glycol monomethyl
ether nanoparticles could significantly enhance the amount of intact
insulin when incubated in trypsin medium [1], while chitosan complex
[40] and chitosan/hydroxypropyl methylcellulose phthalate nano-
particles [42] could protect insulin against pepsin. It is essential to
verify the stability of insulin-loaded nanocarrier to ensure the intact-
ness of the encapsulated protein and preservation of its pharmacolo-
gical activity [2]. As shown in the glucose uptake study, native pre-
scaled up and scaled-up formulations promoted similar glucose uptake
efficacy in C2C12 cells, as compared to native insulin. Hence, complex
coacervation or polyelectrolyte complexation retained the bioactivity of
insulin and did not result in degradation [12]. The beneficial effect of
complex coacervation on the secondary structure and biological activity
of encapsulated insulin within nanoparticles has been confirmed by
circular dichroism in a few studies [33,39,41,46]. As shown in the
obtained data, upon storage for 2 months at 4 °C, FTIR spectral study
revealed that pre-scaled up and scaled-up formulations demonstrated
characteristic peaks of native insulin at nearly same wavenumber, in-
dicating the encapsulated insulin could preserve its conformation
structure and interact with ionizable groups within the core of nano-
particles [4,35,36,38,59]. The storage stability was confirmed by the
assessment of physiochemical properties (size, pdi, zeta potential) after
2 months of storage at 4 °C. In the present study, ANOVA analysis re-
vealed that both pre-scaled up and scaled-up formulations with 2
months of storage did not have significant difference in physiochemical
properties as compared to fresh formulations. Similar results were ob-
served in several insulin-loaded nanoparticles. For example, the size
and zeta potential of the chitosan/dextran sulfate nanoparticles were
maintained for 28 days [36]. Also, the chitosan complex could maintain
the chemical stability of insulin after 30 days storage at 4 and at 25 °C,
which was confirmed by HPLC due to the absence of degraded insulin
peak. Overall, the present study demonstrated that the bioactivity of
insulin could be preserved during the manufacturing process, and the
stability of formulations in terms of physiochemical properties could be
preserved after 2 months of storage.
5. Conclusions
In this study, the self-assembled chitosan-Dz13Scr nanoparticles for
oral administration of insulin were synthesized. The physicochemical
properties (including particle size, zeta potential encapsulation,
C.Y. Wong, et al.
morphology, drug release, stability) and cellular response (cytotoxicity,
glucose consumption, GI cell permeation, endocytosis absorption
pathways) were characterized. The developed nanoparticles demon-
strated improved stability against acid conditions mimicking those in
the stomach. In alkaline condition, a biphasic release pattern, including
initial burst release and subsequent controlled drug release pattern, was
observed in vitro drug dissolution study. We demonstrated that phy-
siochemical properties, such as size, polydispersity index and en-
capsulation efficiency, of insulin-loaded chitosan-Dz13Scr nano-
particles could be controlled effectively by optimising formulation
variables including chitosan concentration and Dz13Scr amount. Using
0.5% chitosan and 10 μg Dz13Scr, the optimal formulation with the
maximum encapsulation efficiency could be achieved. FTIR study and
stability study revealed the storage stability of nanoparticles in terms of
the consistency of physiochemical properties. The prepared formulation
could maintain the bioactivity of insulin in glucose uptake study. Lastly,
the developed nanoparticles could achieve a balance between the mu-
coadhesiveness strength and muco-penetrating capacity, hence the en-
capsulated insulin could permeate across the GI cells in vitro and induce
glucose consumption.
CRediT authorship contribution statement
Chun Y. Wong: Conceptualization, Methodology, Formal analysis,
Investigation, Writing - original draft. Hani Al-Salami: Writing - review
& editing, Supervision. Crispin R. Dass: Writing - review & editing,
Supervision, Project administration.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This paper was not prepared with a specific grant from any funding
agency in the public, commercial, or not-for-profit sectors. Wong ac-
knowledges the support from Australian Government through
Australian Government Research Training Program Scholarship. Al-
Salami is partially supported by the European Union's Horizon 2020
SALSETH research and innovation programme under the Marie
Skłodowska-Curie grant agreement No 872370.
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