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Pharmaceutical Nanotechnology, 2020, 8, 239-254

RESEARCH ARTICLE
ISSN: 2211-7385
eISSN: 2211-7393

Eudragit L-100 Capsules Aromatize and Quaternerize Chitosan for

Insulin Nanoparticle Oral Delivery During Toxic Oxidative Stress in
Rat Liver and Kidney
BENTHAM
SCIENCE

Reza Mahjub1, Farzane K. Najafabadi2, Narges Dehkhodaei2, Nejat Kheiripour3, Amir N.

Ahmadabadi1, Sara S. Asl4, Masomeh Taheri-Azandariani2 and Akram Ranjbar2,5,*

1
Department of Pharmaceutics, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran;
2
Department of Pharmacology and Toxicology, School of Pharmacy, Medicinal Plants and Natural Products Re-
search Center, Hamadan University of Medical Sciences, Hamadan, Iran; 3Research Center for Biochemistry and
Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran; 4Anatomy Department,
School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; 5Research Center for Molecular
Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

Abstract: Background: Insulin, like most peptides, is classified as a hydrophilic and macromolecular
drug that is considered as a low permeable and unstable compound in the gastrointestinal (GI) tract.
The acidic condition of the stomach can degrade insulin molecules. Moreover, the presence of proteo-
Pharmaceutical Nanotechnology

ARTICLE HISTORY
Received: January 17, 2020
Revised: April 01, 2020
Accepted: June 02, 2020
DOI:
10.2174/2211738508666200628033442
lytic activities of some enzymes such as trypsin and chymotrypsin can hydrolyze amide-bonds be-
tween various amino-acids in the structures of peptides and proteins. However, due to its simplicity
and high patient compliance, oral administration is the most preferred route of systemic drug delivery,
and for the development of an oral delivery system, some obstacles in oral administration of peptides
and proteins including low permeability and low stability of the proteins in GI should be overcome.
Objective: In this study, the effects of orally insulin nanoparticles (INPs) prepared from quaternerized
N-aryl derivatives of chitosan on the biochemical factors of the liver in diabetic rats were studied.
Methods: INPs composed of methylated (amino benzyl) chitosan were prepared by the PEC method.
Lyophilized INPs were filled in pre-clinical capsules, and the capsules were enteric-coated with Eu-
dragit L100. Twenty Male Wistar rats were randomly divided into four groups: group1: normal control
rats, group 2: diabetic rats, group 3: diabetic rats received capsules INPs(30 U/kg/day, orally), group 4:
the diabetic rats received regular insulin (5 U/kg/day, subcutaneously). At the end of the treatment, se-
rum, liver and kidney tissues were collected. Biochemical parameters in serum were measured using
spectrophotometric methods. Also, oxidative stress was measured in plasma, liver and kidney. Histo-
logical studies were performed using H and E staining .
Results: Biochemical parameters, and liver and kidney injury markers in serum of the diabetic rats that
received INPs improved significantly compared with the diabetic group. INPs reduced oxidative toxic
stress biomarkers in serum, liver and kidney of the diabetic treated group. Furthermore, a histopatho-
logical change was developed in the treated groups.
Conclusion: Capsulated INPs can prevent diabetic liver and oxidative kidney damages (similar regu-
lar insulin). Therefore oral administration of INPs appears to be safe.
Lay Summary: Although oral route is the most preferred route of administration, but oral delivery of
peptides and proteins is still a challenging issue. Diabetes Mellitus may lead to severe complications,
which most of them are life-threatening. In this study, we are testing the toxicity of oral insulin na-
noparticles in kidney and liver of rats. For this investigation, we will prepare insulin nanoparticles
composed of a quaternized derivative of chitosan. The nanoparticles will be administered orally to
rats and the level of oxidative stress in their liver and kidney will be determined. The data will be
compared to the subcutaneous injection of insulin.

Keywords: Chitosan, diabetes, Eudragit L100, insulin nanoparticles, kidney, liver.

*Address correspondence to this author at the Research Center for Molecular Medicine, Hamadan University of Medical
Sciences, Hamadan, Iran; E-mail: akranjbar2015@gmail.com

2211-7393/20 $65.00+.00 © 2020 Bentham Science Publishers

240 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 Mahjub et al.

1. INTRODUCTION fore, these nano-carriers are regarded as excellent

candidates for the oral delivery of peptides and
Type 1 and type 2 diabetes mellitus (T1DM and
proteins. Chitosan, which is a polymer with bio-
T2DM) are metabolic diseases characterized by a
compatible and biodegradable features, can be
progressive decrease in β-cell function and, when
used in the pharmaceutical industry as an absorp-
not or inappropriately treated, they may lead to
tion enhancer, dissolution improver, and sustained
severe complications, which are life-threatening
release factor in oral drug formulation. In this re-
and very costly to be controlled. Insulin is usually
gard, the mucoadhesive properties of chitosan are
administered by the subcutaneous route, which
introduced as the results of ionic interaction be-
significantly reduces morbidity and mortality
tween negatively charged functional groups in the
rates; however, approximately 60% of patients fail
surface of epithelial cells and positively charged
to achieve long-term glycemic control [1]. There-
amino groups in chitosan. Recently, aromatic de-
fore, the maintenance of blood glucose levels at
rivatives of chitosan received attention of many
near-normal levels reduces the risk of long-term
researchers, and the characterization of the synthe-
complications related to diabetes, such as cardio-
sized methylated chitosan has also been reported,
vascular diseases, non traumatic amputation, and
including an aromatic ring known as methylated
diabetic nephropathy. To overcome these prob-
N-(4-N,N-dimethyl aminobenzyl) chitosan. Ac-
lems, different routes of insulin administration
cordingly, it has been demonstrated that this deriv-
have been tested. The oral way still remains the
ative of chitosan can be applied as a capable gene
preferred choice for drug administration, because
carrier, which poses a greater transfection
of its noninvasive nature, simplicity, and high pa-
efficiency compared with trimethylated derivative
tient compliance [2]. Also, the significant obstruc-
and chitosan [5]. Also, the imbalance between the
tions for oral delivery of insulin are summarized as
production and the elimination of reactive oxy-
follows: hydrolytic enzymatic activity in the dif-
gen/nitrogen species (ROS/RNS) indicates Oxida-
ferent areas of gastrointestinal (GI) such as stom-
tive/Nitrosative stress and the decreased genera-
ach; intestinal lumen; and intestinal brush borders
tion of antioxidants leading to oxidative stress [6].
that cause decay of the peptides, unpleasant acidic
Under the hyperglycemic conditions, oxidative
environment of the stomach, the presence of tight
stress affects macromolecules in the kidney, liver,
junctions in the intestinal epithelium that act as a
and other tissues [7]. Although the relationship
hindrance for paracellular permeation, as well as
between oxidative stress inductions in diabetes has
the low mean residence time (MRT) of chemicals
been demonstrated, studying the effects of
through intestinal lumen that reduces the time of
nanoinsulin on various tissues in the body is still
exposure of insulin to the absorption site. There-
essential [8]. Therefore, the aim of this study was
fore, a wide variety of procedures have been eval-
to investigate the effects of oral administration of
uated to overcome these obstacles including the
Eudragit L-100 coated capsules containing insulin
application of permeation enhancers, niosomes,
nanoparticles (INPs) composed of a quaternized
chemical modification, mucoadhesive polymers,
aromatic derivative of chitosan on liver, kidney,
and development of lipid- based carriers including
and serum biomarkers oxidative damage compared
liposomes, enzyme inhibitors, microparticles, and
with subcutaneous insulin injection in male rats
solid lipid nanoparticles [3]. Entrapment of pro-
with type1 diabetes.
teins and peptides to mucoadhesive polymeric na-
noparticles is still regarded as the most useful ap- 2. MATERIALS AND METHODS
proach for the inhibition of drug instability against
degradation in GI as well as prolongation of pep- 2.1. Materials
tide permeability across the intestinal epithelium. In this study, Chitoclear™ chitosan [viscosity,
Notably, the preparation of a mucoadhesive sys- 1% (w/v) solution in acetic acid, 22 mPa s, with a
tem can improve the residence time of the thera- molecular weight of 120 KDa] was purchased
peutic compounds at the region of absorption with- from Primex (Siglufjordur, Iceland). Crystalline
in GI, and therefore, the bioavailability of an oral recombinant human insulin was provided by Exir
drug delivery system can be increased [4]. There- pharmaceutical company (Iran-Denmark). Manni-
Effect of Insulin Nanoparticles on Diabetes Pathogenesis
tol was obtained from Helm AG (Hamburg, Ger-
many).
Simulated intestinal fluid (SIF) was also
prepared using potassium dihydrogen phosphate
(0.05 M) and sodium hydroxide (0.2 M) was ad-
justed to a pH value of 6.8. Moreover, phosphate-
buffered saline (PBS) was prepared by mixing so-
dium chloride (137 mM), potassium chloride (2.7
mM), disodium hydrogen phosphate (10mM), and
potassium dihydrogen phosphate (2mM) (pH=7.4).
The degree of N-quaternization was determined
using 1H-NMR Spectroscopy, which was reported
to be 43%. All other reagents were of pharmaceu-
tical or analytical grade and were used as received.
2.2. Methods
2.2.1. Preparation and Characterization of
Nanoparticles
INPs were developed by polyelectrolyte com-
plexation (PEC) procedure. Previously, Mahjub

et al. [9] and Kheiripour et al. [10] in their studies
prepared insulin nanoparticles using the PEC

method. Firstly, a quaternized aromatic derivative
of chitosan (i.e., Methylated 4-N,N-dimethyl ami-
no benzyl chitosan) was synthesized and then
characterized according to previous reports [9].
Afterward, 5ml of insulin solution (1 mg/ml,
pH=8.0) was added to an equal volume of the so-
lution containing the newly synthesized polymer,
under gentle magnetic stirring via dropwise pro-
cess. For the improved curing of the particles, the
obtained opalescent colloidal nano-suspension was
continually stirred for 20 min., subsequently, the
obtained suspension was centrifuged at 17,000 rpm
for 20 min at 4°C. The separated nanoparticles
were then reconstituted in the freshly prepared dis-
tilled water, and the supernatant was discarded.
Finally, the obtained nanoparticles were character-
ized for their size, PDI, and zeta potential using a
Zeta sizer ZEN3600 (Malvern instrument, UK) at
25°C.
2.2.2. Insulin Content in Nanoparticles
The restructured NPs were submerged in HCl
(1N) aqueous solution for 7 min. The particles
were degraded, and their total contents of insulin
was released to the acidic aqueous media. The
whole insulin content of the particles was deter-
mined by the detection of insulin using HPLC.
Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 241
2.2.3. HPLC Analysis
For HPLC analysis, 20 microliters of the sam-
ples were injected into a Schimadzu™ LC-20AD
HPLC system (Kyoto, Japan) equipped with SPD-
10A vp UV-Visible detector set at the wavelength
of 214 nm. Also, the Shimadzu ODS C18 column
(250 mm*4.6 mm) was applied as the solid sta-
tionary phase. The mobile phase consisted of ace-
tonitrile/0.1% (v/v) trifluoroacetic acid (30:70),
and the pump flow was set at 1 ml/ min. The peaks
were automatically integrated using the lab solu-
tion® software.
2.2.4. Determination of Entrapment Efficiency
(EE%) and Loading Efficiency (LE%)
The direct procedure was used to determine the
EE% and LE%. In this method, the insulin value in
nanoparticles was calculated according to the fol-
lowing equations 1 and 2:















(Eq. 1)























(Eq. 2)









2.2.5. Lyophilization of the Nanoparticles
Before lyophilization, nanoparticles were fro-
zen at -20°C for a night. Lyophilization was pre-
ceded using Lyotrap Plus (LTE Scientific Ltd.,
Oldham, UK). Based on previous reports on
freeze-drying, the desired samples were dried for
48 h, at a working pressure of 0.07 mbar, and a
condenser temperature of -46°C. The process of
freeze-drying was carried out using mannitol 5%
as the cryoprotectant.
2.2.6. In Vitro Release Studies
In this study, to investigate the release profile
of nanoparticles in acidic and neutral media, both
of the simulated gastric fluid (SGF) and simulated
intestinal fluid (SIF) were used for in vitro release
studies. To prepare the simulated gastric fluid
(SGF), hydrochloric acid solution (0.2 N, 39 ml)
was added to sodium chloride solution (0.2 N, 250
ml), and the volume was then adjusted to 1000 ml
with deionized water, while the pH was main-
tained at 2.2. Besides, the simulated intestinal fluid
(SIF) was prepared by dissolving KH2PO4 (6.8 g)
in 250 ml of deionized water. The obtained solu-
tion was mixed with NaOH (0.2 N, 77 ml), and
242 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 Mahjub et al.

[+log]

7.1050

4.6924
4.5452
7.7399
7.5445

3.5709
3.4102
3.2386
3.0216
2.8457
2.7410
2.3376

1.2207
-N+(CH3)2
Ar-N(CH3)2
2

-N(CH3)2
Aromatic
1

Ar-N+(CH3)3
0

15.8236
0.2500
1.3551

1.5595

7.0671
7.1268
1.8566
4.7380
5.2231

9.7350

3.2931
10 8 6 4 2 [ppm]

Fig. (1). Proton nuclear magnetic resonance (H NMR) spectra of insulin nanoparticles. (A higher resolution / col-
our version of this figure is available in the electronic copy of the article).

finally, the volume was adjusted to 1000 ml using
deionized water, while the pH was maintained at
6.8. The proper amount of lyophilized powder (i.e.
414.75 mg) equivalent to 70 mg of insulin was
weighted and dispersed in the SGF by considering
the loading efficiency (LE %) and the amount of
cryoprotectant (i.e. 25 mg) for freeze-drying.
Throughout the study, the temperature was kept
constant at 37 ± 1.0°C, and the release medium
was gently agitated at 50 rpm using the USP bas-
ket apparatus I. Moreover, the experimental condi-
tion was adjusted in a manner to ensure that the
sink condition is established. At the predetermined
time intervals of 15, 30, 45, 60, 75, 90, and 120
min, one ml of the solution was sampled and re-
placed by a freshly prepared and preheated medi-
um. Afterward, the samples were centrifuged at
12,000 rpm for 20 min using a Beckman-Coulter
Optima™ XPN-100 ultracentrifuge (Georgia,
United States). The supernatant was also collected
for insulin determination using the HPLC method.
120 min post-incubation, the release medium was
changed to SIF by the addition of suitable amounts
of sodium hydroxide (NaOH) and potassium dihy-
drogen phosphate (KH2PO4). Subsequently, the
pH was ascertained to 6.8 using a Sartorious® pH-
meter, and the samples were taken at 150, 195,
240, 300, and 360 min (Fig. 1) [11].
2.2.7. Preparation of the Enteric-coated Capsules
Before performing in vivo studies, the nanopar-
ticles were filled in the Capsugle® preclinical cap-
sules, and the capsules were then enteric-coated
using Eudragit-L100. Firstly, Eudragit L-100® was
dissolved in double distilled water to produce a
concentration of 5 mg/ ml. After filling each cap-
sule with a proper amount of lyophilized powder,
the capsules were immersed in the Eudragit® solu-
tion and then remained there for 2 min. Afterward,
the capsules were removed and dried using hot air.
2.2.8. In Vivo Studies
In this study, 20 Male Wistar rats (220-250 g)
were housed under the controlled temperature
conditions and fed in terms of the laboratory ani-
mal standard diet with water provided ad libitum.
Effect of Insulin Nanoparticles on Diabetes Pathogenesis
These animals were then divided into four groups
as follows (n=5): group1: normal control rats,
group 2: diabetic rats, group 3: diabetic rats re-
ceived pre-clinical capsules INPs (30U/kg/day,
orally), group 4: the diabetic rats received regular
insulin (5U/kg/day, subcutaneously).
Before the study, the rats were rendered diabet-
ic by a single intraperitoneal (IP) injection of
60 mg/kg of streptozocin (STZ) in an isotonic cit-
rate buffer. The development of type 1 diabetes in
rats was confirmed by the estimation of fasting
blood glucose seven days after the STZ injection.
The animals with fasting blood glucose levels
above 250 mg/dl were considered diabetic [12].
This study design was approved by the Ethics
Committee Guidelines of Hamadan University of
Medical Sciences (IR.UMSHA.REC.1395.440).
Lyophilized nanoparticles were orally administered
to rats using pre-clinical capsules (Chitoclear™
Austria). Also, the capsules used had been previ-
ously enteric-coated using Eduragit L-100” aque-
ous enteric system (Colorcon, Idstein, Germany).
Subsequently, freeze-dried nanoparticles prepared
from amino benzyl chitosan were administrated at
an equivalent dose of insulin. After 28 days, the
fasted rats were anesthetized with ketamine (50
mg/kg), and serum samples for biochemical anal-
yses were collected and then stored at -20°C. Also,
the liver and kidney tissues were separated and
cleaned with ice-cold saline solution, then some of
their parts were transferred into cryotubes, kept in
liquid nitrogen for an hour, and finally stored at -
70°C until analysis. Notably, the remaining parts
of the tissues were fixed in 10% neutral buffer
formalin for histopathological evaluation.
2.2.9. Estimation of Oxidative Stress Parameters
To measure the rate of lipid peroxidation
(LPO), TBA was used, which reacts with lipid
peroxide molecules. The plasma samples were also
blended with TCA (20%), and the precipitate was
dispersed in H2SO4 (0.05 M). Subsequently, TBA
(0.2% in sodium sulphate 2M) was added and
heated for 30 min in a boiling water bath. TBARS
adducts were extracted using n-butanol, and the
absorbance was measured as 532 nm. This reac-
tion occurred in acidic pH on high temperature,
and the maximum absorption was observed for a
pink complex at 532 nm [13].
Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 243
Total antioxidant capacity (TAC) was measured
using ferric reducing ability of plasma (FRAP)
method. This procedure was performed based on
the strength of plasma in reducing Fe3+ to Fe2+ in
the presence of TPTZ. The reaction of Fe2+ and
TPTZ provided a complex with blue color and
maximum absorbance at 593 nm [14].
To evaluate the total thiol groups (TTG),
DTNB was used as a reagent. In this regard, it is
noteworthy that DTNB reacts with thiol molecules
and creates a yellow complex with a good absorb-
ance at 412 nm [15].
2.2.10. Estimation of Biochemical Parameters
Liver damage biomarkers (ALP, AST, ALT,
and total and direct bilirubin), glucose level, creat-
inine, gamma glutamine transferase (GGT), and
urea were measured in terms of the standard pro-
cedure of kits (Pars Azemon kit, Iran).
2.2.11. Liver and Kidney Histology
For histopathological assessments, a small
piece of tissues was fixed with 10% formalin and
then embedded in paraffin. Moreover, the serial
sections (thickness of 5 μm) were processed and
stained with hematoxylin and eosin (H&E), ac-
cording to the standard pathology laboratory pro-
tocols. Afterward, the stained liver and kidney
slices were evaluated under light microscopy.
2.2.12. Statistical Analysis
All values are expressed as Mean ± SE. Statisti-
cal analysis was done using one-way ANOVA,
and post hoc comparisons were carried out using
the Tukey test. P>0.05 was considered as non-
significant difference, while P<0.05 was consid-
ered as statistically significant.
3. RESULTS
The H-NMR spectra of the quaternized and
aromatized chitosan polymer synthesized are
shown in Fig. (1). Accordingly, the sharp peaks in
the regions of 2.3 and 2.7 ppm represent chitosan
methyl (-N (CH3)2) and (-N + (CH3)3) protons of
methyl aliphatic amines, respectively. Also, the
sharp peaks in the 3.4 and 3.6 ppm regions repre-
sent the methyl (-N (CH3) 2) and (-N + (CH3)3) pro-
tons of the aromatic amine attached to the benzyl
ring, respectively.
244 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 Mahjub et al.

<Chromatogram>
mAU

PDA Multi 1 214nm,4nm

4.021
12.5

10.0

7.5

2.691
5.0

2.287
2.031
2.165
2.5

2.488
0.0

0 1 2 3 4 5
min
Fig. (2). Chromatogram of insulin with 5 μgr/ml. Insulin eluted from the column with an approximate retention time
of 4 minutes. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Table 1. The loading efficiency (LE%) and entrapment efficiency (EE%) of HPLC injection.

LE% Mean ± EE% Mean ±

LE1% LE2% LE3% EE1% EE2% EE3%
SD SD

18% 18.40% 17.50% 17.96% ± 0.45 62% 62.28% 61.83% 62.03% ± 0.22

Table 2. Physico-chemical characteristics of nanoparticles, before and after lyophilization.

- Before Lyophilization After Lyophilization

Size (nm) 138.2 ± 31 324.3 ± 22.94

Zeta potential (mV) +9.31 ± 1.18 +13.01 ± 1.1
Polydispersity 0.243 ± 0.07 0.345 ± 0.05

The HPLC chromatogram of insulin is depicted
in Fig. (2). As shown in the figure, insulin was
eluted from the column with an approximate reten-
tion time of 4 minutes.
The LE and EE of INPs were 17.96% ± 0.45
and 62.03 ± 0.22, respectively. Also, the evalua-
tion of shape by TEM in different magnifications
confirmed the spherical shape of INPs (Table 1).
The size, zeta potential, and polydispersity of
INPs before and after lyophilization are presented
in Table 1. The results show that after lyophiliza-
tion, these parameters were increased further com-
pared to before lyophilization; however, this in-
crease was not significant (p>0.05) (Fig. 3) (Table 2).
The in vitro release of insulin from the enteric-
coated capsules was studied in the Simulated Gas-
tric Fluid (SGF) adjusted to pH value of 2.2 and
the Simulated Intestinal Fluid (SIF) adjusted to pH
value of 6.8. The release profile of different types
of prepared nanoparticles is shown in Fig. (4).
From minute 0 to 120, enteric-coated capsules
with Eudragit L100 and containing INPs in SGF
were released with deficiency and only 8.35% of
insulin was released into the SGF after 120
minutes. Then, the capsules were transferred to
SIF, and 84.81% of insulin was released into the
SIF after 240 minutes.
The results of the size of quaternized INPs,
their zeta characteristics, and the zeta potential
Effect of Insulin Nanoparticles on Diabetes Pathogenesis Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 245

100

80

Cumulative Release (%)

60

40

20

0
0 100 200 300 400

Time (min)
Fig. (3). Release of insulin from nanoparticles. The release of insulin in simulated gastric fluid (SGF) (0-120 min)
was 8.35% and this amount was 84.81% in simulated intestinal fluid (SIF) (120-360 min). (A higher resolution /
colour version of this figure is available in the electronic copy of the article).

Size (d.nm): % Intensity: St Dev (d.nm):

Z-Average (d.nm): 159.0 Peak 1: 191.1 100.0 44.17

PdI: 0.221 Peak 2: 0.00 0.00 0.00

Intercept: 0.737 Peak 3: 0.00 0.00 0.00

Size Distribution by Intensity

20
Intensity (Percent)

15

10

0
0.1 1 10 100 1000 10000
Size (d.nm)

Record 6: 94/12/15 s2 1

Fig. (4). Size distribution of insulin nanoparticles. (A higher resolution / colour version of this figure is available in
the electronic copy of the article).

indicate the average size of the insulin nanoparti- chitosan nanoparticles (20.000×, 80,000×, and
cles to be 159 diameter nanometer (d.nm) and pol- 125,000×, respectively).
ydispersity index (PDI) equal to 0.221 (Fig. 4).
As summarized in Table 3, diabetes caused sig-
Moreover, as shown in Fig. (5), the zeta potential
nificant weight loss in this study. The final body
for the insulin nanoparticles was +8.31 mV.
weight significantly decreased in the diabetic
Fig. (6) indicates the Transmission electron mi- group compared to the control group (p<0.05).
croscopy (TEM) of the insulin-loaded trimethyl However, the oral administration of the coated
246 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 Mahjub et al.

Results
Mean (mV) Area (%) width (mV)
Zeta Potential (mV): 8.31 Peak 1: 8.31 100.0 4.27
Zeta Deviation (mV): 4.27 Peak 2: 0.00 0.0 0.00
Conductivity (ms/cm): 0.420 Peak 3: 0.00 0.0 0.00
Result quality: Good

Zeta Potential Distribution

500000

400000
Total Counts

300000

200000

100000

0
-200 -100 0 100 200
Zeta Potential (mV)

Record 85: 95/07/18 insu 1

Fig. (5). Monomodal graph of zeta potential of insulin nanoparticles. (A higher resolution / colour version of this
figure is available in the electronic copy of the article).

Fig. (6). Transmissionelectronmicroscopy (TEM) of insulin-loaded trimethyl chitosan nanoparticles. (20.000×,

80,000×, 125,000× respectively). (A higher resolution / colour version of this figure is available in the electronic
copy of the article).
Effect of Insulin Nanoparticles on Diabetes Pathogenesis Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 247

Table 3. Weight of Rat (g) in the studied groups.

Variable/Group C D D+N D+I

Initial body weight (g) 228.04 ± 9.3 230.3 ± 10.4 227.2 ± 9.2 224.2 ± 3.2
Final body weight (g) 274.8 ± 13.9 214.4 ± 11.32a 229.3 ± 6.7a,b 222.1 ± 8.3b
Mean weight level during treatment in different studied groups. Data are shown as Mean ± SE. C: healthy control. D: diabetic control. N: encapsulated insulin
nanoparticle (30U/kg). I: injectable insulin (5U/kg). a Significantly different compared to the control groups. b Significantly different compared to diabetic con-
trol groups. p<0.05.

compared to the healthy control rats (p<0.01).

Blood Glucose Level (mg/dl)

600 day1
a day7
a day28
400
b kidney p<0.01 and liver p<0.05) as well as the
200
a duce MDA (p<0.05). There were no substantial
0 groups treated by the non-coated form of insulin in
C D D+N D+I
Fig. (7). Fasting blood sugar level during treatment in
different studied groups. Data are shown as Mean ±
SE. C: healthy control. D: diabetic control. N: encapsu-
lated insulin nanoparticle (30U/kg). I: injectable insulin
(5U/kg). aSignificantly different compared to the con-
trol groups. bSignificantly different compared to diabet-
ic control groups. p<0.05. (A higher resolution / colour
version of this figure is available in the electronic copy
of the article).
insulin could significantly compensate for these
weight losses compared to the diabetic group
(p<0.05).
FBS of the control group did not change up to
the end of the treatment. On the other hand, after
the STZ injection, FBS in three diabetic groups
significantly increased compared to the healthy
control group (p<0.05). Also, a significant
decrease was observed in blood glucose levels of
diabetic groups receiving oral capsules containing
nanoparticles insulin and insulin-treated diabetic
group on day 28, compared to the control group
(Fig. 7). Notably, insulin treatment in a non-coated
way, similar to the coated form with a quaternized
INPs chitosan-based nanoparticle, could enhance
the blood glucose level (p<0.05). However, a sig-
nificant difference was observed between the in-
jectable insulin and the control rat (p<0.05).
The diabetic rats indicated an elevated lipid pe-
roxidation level in kidney, serum, and liver tissues
While STZ enhances the level of MDA, the oral
treatment with the coated form (in serum p<0.01,
subcutaneous treatment with the non-coated form
of insulin (in serum p<0.01) could remarkably re-
differences between diabetic groups and diabetic
the kidney and liver MDA levels (p>0.05)
(Fig. 8A).
The comparisons of the TAC levels among all
groups are summarized in Fig. (8B). As shown,
diabetes resulted in a notable decrease in the TAC
levels in serum, kidney, and liver tissues (p< 0.01).
The coated (in serum p<0.01, kidney p<0.01, and
liver p<0.05) and non-coated forms of insulin (in
serum p<0.01, kidney p<0.05, and liver p<0.05)
could relieve these oxidative markers with a sig-
nificant increase in the TAC level (p<0.05).
Furthermore, STZ-induced T1D significantly
increased TOS levels in the treatment groups com-
pared to the control group (p<0.01), but treatment
by encapsulated INP (in serum p<0.01, and kidney
p<0.05) and insulin (in serum p<0.05) remarkably
modulated the levels of TOS (Fig. 8C).
In addition, the levels of OSI were significantly
higher in diabetic groups compared to the control
group (p<0.01). Also, treatment with encapsulated
INP (in serum p<0.01, kidney p<0.01, and liver
p<0.01) and insulin (in serum p<0.01, kidney
p<0.01, and liver p<0.01) had a significant effect
on this level (Fig. 8D).
The levels of TTG decreased in diabetic group
compared to the control groups (p<0.01). How-
ever, encapsulated INPs significantly improved
TTG concentrations in serum, kidney, and liver
(p<0.05), and insulin also showed a significant
248 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 Mahjub et al.

(A) 4
(B) (C) Serum
Kidney

MDA (nmol / L serum or mg protein)

400 80 Liver
a#

TOS (nmol / L serum or mg protein)

TAC (nmol / L serum or mg protein)
a#
3 60
300
b* b* a#
b# b# b* a#
2 b# a# 40 b*
a# 200
a# b#
a# b* b#
b# b#
b#
1 100 20
a#

0 0 0

(D) (E) (F)

I
C
1.0

+
+

D
80

D
TTM (nmol / L serum or mg protein)
a#
0.8 b* 40 a#
60

Serum 8-oxo-dG (ng/ml)

OST (TOS/TAC)

0.6 30
a# b# b#
40 b*
0.4 a# b*
b# b* b* 20
a# b#
20 a#a#
0.2 b# b#
b# b# 10

0.0 0
0

I
N
D
D

+
I

C
C

I
N
+

D
+

C
D
+

+
D

+
D
D

D
D
Fig. (8). A: Lipid peroxidation, B: Total antioxidant capacity, C: Total oxidant status, D: oxidative stress index,
E: Total thiol groups in serum, kidney and liver in rats and F: Serum DNA damage. (A higher resolution / colour
version of this figure is available in the electronic copy of the article).
Data are presented as Mean ± SE.C: healthy control. D: diabetic control. N: encapsulated insulin nanoparticle
(30U/kg). I: injectable insulin (5U/kg),a Significant differences with the control group, b Significant differences with
the diabetic group. *p < 0.05, #p < 0.01.

effect on TTG level (in serum p<0.05 and kidney amelioration of these markers. The benefits of
p<0.05) (Fig. 8E). ALP and total and direct bilirubin reached the val-
ues of the healthy control group by subcutaneous
The results of DNA damage in serum are pre-
treatment with non-coated insulin.
sented in Fig. (8F). Induction of diabetes by STZ
caused a significant increase in DNA damage As shown in Fig. (10), the levels of creatinine,
(p<0.01), and treatment of diabetic group with in- urea, and GGT activity were significantly higher
sulin as well as encapsulated INPs significantly in the diabetic non-treatment group compared to
improved the serum DNA damage (p<0.01). the control group (p<0.05). Besides, it was found
that the treatment with insulin-coated trimethyl
As depicted in Fig. (9), the levels of ALP, AST,
chitosan-based nanoparticles and injectable insulin
ALT, and total and direct bilirubin were signifi-
could better ameliorate creatinine, urea, and GGT
cantly higher in the STZ non-treatment group
activity compared to the diabetic group (p<0.05).
compared to the healthy control group (p<0.05).
Moreover, it was found that the treatment with in- The diabetic rats showed a decreased urine cre-
sulin-coated trimethyl chitosan-based nanoparti- atinine level compared to the healthy control ani-
cles could significantly ameliorate ALT, ALP, and mals (p<0.05). The oral treatment with coated
total and direct bilirubin (p<0.05). However, no form and the subcutaneous treatment with a non-
remarkable differences were observed between the coated form of insulin could more increase urine
coated and the non-coated forms of insulin in the creatinine compared to the diabetic animals
Effect of Insulin Nanoparticles on Diabetes Pathogenesis Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 249

(A) (B) 250

(C) 3000 a
150
200 a

serum AST (U/L)

serum ALP (U/L)

a
serum ALT (U/L)
2000
100 150
b
b 100
1000 b
50
50

0 0
0

D
.I

.N
.N
C

.I
D

D
.N
C

.I
D

D
D
D
D
(D) 1.5
(E)
0.20
a
Total bilirubin mg/dl

Direct bilirubin mg/dl

1.0 a
0.15
b
b b
b 0.10
0.5
0.05

0.0 0.00
C

.I
.N

.I
.N
D

D
D

D
Fig. (9). Liver function factors in different studied groups. Data are shown as Mean ± SE. (A) Serum Alanine ami-
notransferase level, (B) Aspartate aminotransferase level, (C) Alkaline phosphatase level, (D) Total bilirubin, (D)
Direct bilirubin. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
C: healthy control. D: diabetic control. N: encapsulated insulin nanoparticle (30 U/kg). I: injectable insulin (5 U/kg),
a
Significantly different compared to the control groups. bSignificantly different compared to diabetic control groups.
p < 0.05.
(A) (B) (C)
1.0 40
5
a a a
0.8 b
CGT activity (unit/L)
Cratinine(mg/dl)

30 4
b
urea (mg/dl)

0.6
b b 3
20
0.4 2
b b
10 1
0.2

0
0.0 0
C D D+N D+I C D D+N D+I C D D+N D+I
(D) 80 (E)
400
b Healthy Control
Urin cratinin (mg/dl)

60 a
a 300 Diabetic
b
Alb (mg/dl)

40 200 b Diabetic + Insulin Nanoparticle

b
a
Diabetic + Injectable Insulin
20 100

0 0
C D D+N D+I C D D+N D+I

Fig. (10). Kidney function factors in different studied groups. Data are shown as Mean ± SE. (A) Urea level, (B)
Creatinine level, (C) γ-Glutamyl transferase (GGT) activity in serum and (D) Creatinine, and (E) Albumin (Alb) in
the urine. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
C: healthy control. D: diabetic control. N: encapsulated insulin nanoparticle (30 U/kg). I: injectable insulin (5 U/kg),
a
Significantly different compared to the control groups. bSignificantly different compared to diabetic control groups.
p < 0.05.
250 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3
(p<0.05). However, significant differences were
seen in the urine creatinine levels between the in-
jectable insulin and the control rats (p<0.05).
Also, the levels of urine albumin showed an el-
evation in the diabetic animals compared to the
healthy control animals (p<0.05). It was indicated
that the treatment with INPs and injectable insulin
could decrease urine albumin compared to diabetic
animals (p<0.05).
Liver tissues of all the control rats showed typi-
cal lobular architecture with central veins, radiat-
ing hepatic cords, the portal triads containing por-
tal vein, hepatic artery, and bile duct. Notably, the
sinusoids are naturally in the rows of hepatocytes
with no dilatation, and the Kupffer cells are at-
tached to the sinusoidal wall.
In the diabetic control group, dilated sinusoids
were grown. Hepatocytes have been removed from
the radial cord, and the number and size of hepato-
cytes decreased. Besides, the number of macro-
phages and lymphocytes has significantly in-
creased compared to the healthy control group. In
the diabetic group receiving capsules containing
insulin nanoparticles, dilated sinusoids have in-
creased; however, hepatocytes maintained the ra-
dial cord state. Furthermore, there was a signifi-
cant decrease in the number of macrophages in
this group compared to the diabetic control group.
Moreover, there was a substantial increase in
the number of lymphocytes in this group compared
to the healthy control group. In the diabetic group
receiving injectable insulin, dilated sinusoids have
increased. Also, hepatocytes were released from
the radial cord. The number of macrophages and
lymphocytes counted in this group has decreased
compared to the diabetic control group and in-
creased compared to the healthy control group.
In the kidney tissues of the control group, the
proximal and distal convoluted tubules, renal cor-
puscles, glomerulus, and glomerular capsule
showed their typical structures. Additionally, the
urinary pole had a rational size. In addition, the
urinary pole significantly decreased in the diabetic
group compared to the healthy control group. It
should also be noted that there was no considera-
ble difference between the other study groups and
the healthy control group; however, there was a
Mahjub et al.
significant increase compared to the diabetic con-
trol group. There was no significant difference in
glomerular and proximal convoluted sizes among
the diabetic group and the healthy controls and the
other groups.
In the diabetic group, distal tubule wide and
length have significantly decreased compared to
the healthy control group. It should also be noted
that there was no substantial difference between
the other study groups and the healthy control
group; however, there was a significant increase in
the distal tubule wide and length compared to the
diabetic control group (Figs. 11 and 12).
4. DISCUSSION
Diabetes mellitus is one of the main challenges
in the world that burdens society. Currently, in-
jectable insulin is the best choice for the treatment
of diabetes mellitus. However, in addition to sev-
eral side effects, this method of therapy has many
psychological consequences for diabetic patients.
Nowadays, nanotechnology plays an essential role
in the development of pharmaceutical strategies.
The current study showed that encapsulated insu-
lin nanoparticles, as well as insulin, have a consid-
erable effect on the maintenance of the body
weight, glucose homeostasis, liver and kidney tox-
icity and oxidative stress. However, encapsulated
insulin nanoparticles were more effective in the
correction of diabetes pathogenesis.
In recent years, ionic gelation and polyelectro-
lyte complexation (PEC) has been extensively
used to prepare the nanoparticles entrapping sensi-
tive molecules such as peptides and proteins. In
these methods, procedures such as sonication and
the presence of organic solvents can be avoided,
thus reducing the possible damage to peptides and
proteins. The PEC method can prepare nanoparti-
cles with a high EE%, zeta potential, and ionic ge-
lation that can provide drug retention and eventu-
ally, higher bioavailability. Methylated N-(4-N,N-
dimethyl aminobenzyl) chitosan are considered a
polycation and a polyanion, respectively, which
can form a polyelectrolyte complex and take part
in peptide and protein delivery [9]. Traditional in-
jectable insulin is the current treatment of diabetes;
this procedure has caused a heavy burden on the
patient. Today, attempts are being made to use
Effect of Insulin Nanoparticles on Diabetes Pathogenesis Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 251

(A) (B) hp

hp Dilated
sinosoid

cv sn
cv kup

sn

kup

lym
20 pixels 20 pixels
lym
(C) (D)

hp

cv
sn
sn kup

cv
kup

hp

lym
20 pixels 20 pixels

Fig. (11). Histopathologic changes of the liver. Normal control rats (A), diabetic rats (B), diabetic rats that received
encapsulated insulin nanoparticle (30U/kg) (C), diabetic rats that received injectable insulin (5U/kg) (D) (40×).
(A higher resolution / colour version of this figure is available in the electronic copy of the article).

(A) (B) Distal

convoluted
tubules
Distal
Proximal convolated
convoluted Urinary pole
tubule
tubule

Glomerulus

Glomerular Urinary pole Progximal

convoluted
tubule

2.5 inches 2.5 inches

Progximal
(C) convoluted (D)
tubule
Necrotic Urinary pole

cell debris

Glomerular

Distal urinary pole

convoluted
Distal
tubule
Proximal convoluted
Necrotic
convoluted tubule
cell debris
tubule

Glomerular

2.5 inches 2.5 inches

Fig. (12). Histopathological changes in the kidney. A: Healthy control, B: Diabetic + encapsulated insulin nanopar-
ticles (30 U/kg), C: Diabetic group, D: Diabetic + injectable insulin (5 U/kg). (A higher resolution / colour version
of this figure is available in the electronic copy of the article).
252 Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3
alternatives for the treatment. One of these ways is
the oral administration of Eudragit 100 insulin
[16]. In this work, Eudragit-coated quaternized
aromatic derivatives of chitosan used for INPs
preparation. The Eudragit 100 INPs, which were
applied in the present study, were designed on the
basis of biocompatibility, insulin stability, and in-
testinal epithelial permeability. Therefore, to
achieve this purpose and to increase its intestinal
absorption, the oral administration of insulin ne-
cessitated the design of the nanoparticles, which
would protect and facilitate its intestinal absorp-
tion, and mimic its physiological rate [17]. It
seems that by the increased intestinal mucosal ab-
sorption of Eudragit 100 insulin, the hepatic glu-
coneogenesis, and the glycogenolysis effect of this
hormone in the liver could notably progress [18].
In our study, the efficacy of the insulin ab-
sorbed from the GI was evaluated by its effect on
the body weight, the FBS level, and the biochemi-
cal and histopathological changes in the liver and
kidney tissues, and the oxidative biomarkers. Stud-
ies have shown that STZ decreased weight due to
an impact on the pancreas and suppresses the pro-
duction of insulin. Insulin promotes glycogen and
lipid synthesis in muscle cells. Therefore; reduc-
tion in insulin production causes reduced entry of
glucose into muscle and increasing lipolysis and
gluconeogenesis can be causing muscle wasting
and weight loss [19]. The results of our study dem-
onstrate that treatment with INPS, induced weight
gain in diabetic rats compared to the untreated di-
abetic rat. Given that injectable insulin keeps the
concentration of insulin up for a short time, it has a
much lower effect on weight gain than oral INPs.
In line with our results, Heidarisasan et al.,
showed that INPs remarkably induced weight gain
more effectively than injectable insulin [20].
STZ penetrates the pancreas and induces diabe-
tes moderately by destroying the pancreatic beta
cells, inducing DNA alkylation, reducing the pro-
duction of insulin and increasing concentration of
plasma glucose level. Our results showed that the
fasting blood sugar of diabetic control rats was
significantly increased, but in diabetic treatment
with INPs, it was significantly decreased. Our result
validated the previous study that used insulin-incor-
porated nanoparticles of chitosan and alginates to
decrease the glucose level [21]. Jamshidi et al.
Mahjub et al.
indicated that INPs directly affect glucose metabo-
lism, and increase the expression of glucokinase
and pyruvate kinase genes. These enzymes are the
key rate-limiting enzymes mediating the oxidation
of glucose and generating ATP [8].
In diabetic condition, hyperglycemia can also
induce toxic oxidative stress through various
mechanisms and decline antioxidant capacity. A
high level of glucose inhibits mitochondrial respir-
atory complex III and leads to the production of
reactive oxygen species in the liver and other or-
gans. Oxidative stress also has a vital role in the
pathology of diabetes [22]. In the present study,
the oxidative stress due to diabetic rats was ap-
proved via the significant increase in oxidative
markers such as LPO, TOS, OSI and DNA dam-
age, and decrease in the antioxidant factors such as
TTG and TAC level. Similar results were obtained
in the studies on animal models and human studies
[8, 20]. These results show that Eudragit L-100
capsules aromatize and quaternerize chitosan for
insulin nanoparticle coating that can stimulate the
bioavailability of insulin and reduce oxidative
stress toxicity.
Hyperglycemia in the STZ groups induced liver
injury causing changes in liver enzyme levels and
secretion of soluble cytosolic enzymes into the cir-
culation. Aminotransferase (ALT, AST and ALP),
direct and total bilirubin, are the most sensitive
and the most widely used enzymes to detect any
liver injuries. INPs inhibit liver injury by its effect
on the improvement in liver histopathology. Our
result validated the previous study that used INPs
for improving liver injury [8].
Diabetes mellitus is generally accompanied by
significant changes in kidney function, including
increased creatinine, urea and GGT in serum and
decreasing the renal filtration of creatinine, urea in
urine. In this study, INPs improved renal histo-
pathology in diabetic rats. The results reveal that
creatinine and urea and GGT serum in the diabetic
group have shown significant changes and in-
creased the values of diabetic groups who received
insulin (oral and SC) at normal levels. Also, in the
diabetic group, the level of albumin is increased
and they show a degree of proteinuria. On the oth-
er hand, a few studies strongly support the out-
come of our study. In line with these findings, an-
other study showed that treatment with insulin
Effect of Insulin Nanoparticles on Diabetes Pathogenesis
could reduce urea and creatinine concentration in
the serum of STZ-diabetic rats. To support this
notion, it has been reported that the use of Eu-
dragit® RS 100 microparticles (ERS) containing
insulin (ILNP) in the alloxan-induced diabetic
rabbits and sheep model [23] and the biopolymer-
ic- based delivery nanoparticulate system im-
proved disorders caused by type 1 and type 2 dia-
betes mellitus [24].
CONCLUSION
Diabetes type 1 and type 2 cause kidney and
liver complications. The results of this study
showed that Eudragit L-100 capsules aromatize
and quaternerize chitosan for insulin nanoparticle
oral delivery that can be effective in kidney and
liver toxicity in diabetic rats. Results showed that
INPs reduced the liver and kidney injury and ame-
liorated oxidative toxic stress in these tissues.
However, we need further studies to examine and
verify the molecular pathways underlying the ef-
fects of insulin nanoparticles (INPs) in the liver
and kidney tissues of diabetic animal models. The-
se data may contribute to the management and
treatment strategies of diabetic patients.
AUTHORS’ CONTRIBUTIONS
In this study, all authors have contributed: Reza
Mahjub: analysis & revising of the manuscript,
Farzane Kazemi Najafabadi: design & performance,
Narges Dehkhodaei: design & performance Nejat
Kheiripour: analysis & performance, Amir Nili
Ahmadabadi analysis & drafting of manuscript,
Sara Soleymani Asl: report of work & histology
analysis, Masomeh Taheri -Azandariani, perfor-
mance of animal treatment, Akram Ranjbar de-
sign, analysis, report work and drafting and revis-
ing of the manuscript, must also be acknowledged.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
This research was approved by Animal Ethics
Committee Guidelines of Hamadan University of
Medical Sciences, Hamadan, Iran.
HUMAN AND ANIMAL RIGHTS
No human were used in this study. Research
work on animals was carried out in accordance
Pharmaceutical Nanotechnology, 2020, Vol. 8, No. 3 253
with the animal Ethics Committee Guidelines of
Hamadan University of Medical Sciences, Ap-
proval no. IR.UMSHA.REC.1395.440.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERI-
ALS
The authors confirm that the data supporting
the findings of this research are available within
the article.
FUNDING
This study was financially supported by the
Deputy of Research, Hamadan University of Med-
ical Sciences (No. 9510146103).
CONFLICT OF INTEREST
The authors declare no conflict of interest, fi-
nancial or otherwise.
ACKNOWLEDGEMENTS
Declared none.
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