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doi:10.3748/wjg.14.4209

World J Gastroenterol 2008 July 14; 14(26): 4209-4215

World Journal of Gastroenterology ISSN 1007-9327
2008 The WJG Press. All rights reserved.

RAPID COMMUNICATION

Gene therapy for type 1 diabetes mellitus in rats by

gastrointestinal administration of chitosan nanoparticles
containing human insulin gene

Li Niu, Yan-Cheng Xu, Zhe Dai, Hui-Qin Tang

Li Niu, Yan-Cheng Xu, Zhe Dai, Hui-Qin Tang, Department
of Endocrinology, Zhongnan Hospital of Wuhan University,
Wuhan 430071, Hubei Province, China
Author contributions: Niu L and Xu YC contributed equally
to this work; Niu L and Xu YC designed the research; Niu L
performed the research; Dai Z and Tang HQ contributed to new
reagents/analytic tools; Niu L, Xu YC, Dai Z and Tang HQ
analyzed the data; Niu L and Xu YC wrote the paper.
Correspondence to: Yan-Cheng Xu, Professor, Department
of Endocrinology, Zhongnan Hospital of Wuhan University, 169
Donghu Road, Wuhan 430071, Hubei Province,
China. endocrine@mail.whu.edu.cn
Telephone: +86-27-62605165 Fax: +86-27-67812892
Received: May 1, 2008
Revised: June 2, 2008
Accepted: June 9, 2008
Published online: July 14, 2008

Abstract
AIM: To study the expression of human insulin gene in
gastrointestinal tracts of diabetic rats.
METHODS: pCMV.Ins, an expression plasmid of
the human insulin gene, wrapped with chitosan
nanoparticles, was transfected to the diabetic rats
through lavage and coloclysis, respectively. Fasting
blood glucose and plasma insulin levels were measured
for 7 d. Reverse transcription polymerase chain
reaction (RT-PCR) analysis and Western blot analysis
were performed to confirm the expression of human
insulin gene.
RESULTS: Compared with the control group, the
fasting blood glucose levels in the lavage and coloclysis
groups were decreased significantly in 4 d (5.63
0.48 mmol/L and 5.07 0.37 mmol/L vs 22.12 1.31
mmol/L, respectively, P < 0.01), while the plasma
insulin levels were much higher (32.26 1.81 IU/mL
and 32.79 1.84 IU/mL vs 14.23 1.38 IU/mL,
respectively, P < 0.01). The human insulin gene mRNA
and human insulin were only detected in the lavage
and coloclysis groups.
CONCLUSION: Human insulin gene wrapped with
chitosan nanoparticles can be successfully transfected
to rats through gastrointestinal tract, indicating that
chitosan is a promising non-viral vector.
2008 The WJG Press. All rights reserved.

Key words: Gastrointestinal tract; Human insulin

gene; Gene expression; Diabetes mellitus; Chitosan
nanoparticle
Peer reviewers: Kazuma Fujimoto, Professor, Department

of Internal Medicine, Saga Medical School, Nabeshima,

Saga, Saga 849-8501, Japan; Yasuji Arase, MD, Department
o f G a s t r o e n t e r o l o g y, To r a n o m o n H o s p i t a l , 2 - 2 - 2
Toranomonminato-ku, Tokyo 105-8470, Japan
Niu L, Xu YC, Dai Z, Tang HQ. Gene therapy for type 1
diabetes mellitus in rats by gastrointestinal administration of
chitosan nanoparticles containing human insulin gene. World J
Gastroenterol 2008; 14(26): 4209-4215 Available from: URL:
http://www.wjgnet.com/1007-9327/14/4209.asp DOI: http://
dx.doi.org/10.3748/wjg.14.4209

INTRODUCTION
Type 1 diabetes mellitus is the result of insulin deficiency
caused by the autoimmune destruction of insulinproducing pancreatic cells. Hyperglycemia would
cause a lot of long-term clinical problems, including
renal failure, retinopathy, neuropathy and heart disease[1].
Although intensive exogenous insulin therapy can
delay or prevent the onset of chronic complications,
it is rather cumbersome and sometimes would cause
hypoglycemia, which could be life-threatening. However,
the development of gene therapy has also generated a
greater hope and excitement for a possible cure of
diabetes since insulin gene was first cloned and expressed
in cultured cells in the late 1970s[2]. Many attempts have
been made, including islet transplantation [3-5], whole
pancreas transplantation[6,7], regeneration of cells[8-10]
and insulin gene therapy[11-13].
In general, whole organ transplants have more
sustained and durable function. Advances in islet
transplantation procedures now mean that patients
with the disease can be cured by transplantation of
primar y human islets of Langerhans. The major
drawbacks of these strategies are the insufficient
availability of donor islets, invasive procedure and
high cost. Due to the limited available number of
donor islet cells, researchers are looking for different

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sources of pancreatic islet progenitor or stem cells.

Stem cells with an extensive proliferative ability may
provide a valuable source of islet progenitor cells.
Several studies have demonstrated that progenitor/
stem cells can be expanded in vitro to generate a
large number of islet progenitor cells[14-16]. However,
efficient and direct differentiation of these cells to
an endocrine pancreatic lineage is difficult to achieve.
Insulin gene therapy including any approach involving
the introduction of a foreign gene into any cell
type in the body can produce insulin[17]. The gene(s)
introduced could be the insulin gene itself, perhaps
under control of a tissue specific promoter, allowing
for expression in a selected non--cell type, or in a
gene encoding for a factor that activates the insulin
gene, thereby allowing for ectopic insulin production.
In insulin gene therapy, one of the key issues is the
selection of carriers. Conventional viral vectors can
introduce exogenous genes into cells precisely and
effectively, but they can easily cause immune reactions
because of the existence of antiviral immune system.
More and more researchers are interested in non-viral
vectors.
In this study, we constructed an expression plasmid
pCMV.Ins expressing human insulin gene. Then, we
wrapped the pCMV.Ins with chitosan nanoparticles, a
non-viral vector, and transfected to diabetic rats through
gastrointestinal tract to explore the gene therapy for type
1 diabetes mellitus. At present, there is no report on
transfection of human insulin gene to gastrointestinal
tract and application of chitosan as a vector in gene
therapy for type 1 diabetes mellitus.

MATERIALS AND METHODS

Materials
The cDNA sequences of human insulin gene were cut
from the PBAT16, hInsG1.M2 (presented by Doctor
Michael German, Department of Hormone Research
Institute, University of San Francisco, USA.) using
Bgl/ Not enzyme inserted into the expression
vector of pCMV.eGFP in the cohesive end. Then
the pCMV.Ins was transfor med into E.coli. T he
top10F strains (Invitrogen) were screened. Plasmids
isolated with a large-scale alkaline lysis procedure
were purified, 0.1 g chitosan was dissolved in 100 mL
acetic acid, and the pH was adjusted to 5.5 with
sodium hydrate. T he solution was stored at 4
after determined with a micropore film, the aperture
of which was 0.22 m. Before it was used, the
chitosan solution was diluted with ddH 2O until the
concentration reached 0.02%. One hundred L
DNA solution at a concentration of 200 g/mL
was added into 100 L sodium sulfate solution at
a concentration of 25 mmol/L. After the chitosan
a n d D N A s o l u t i o n s we r e h e a t e d t o 5 5 i n a n
aqueous bath for 15 min, respectively, they were
mixed immediately and put into convolution for
1 min. The volume of reaction system should not
excess 500 L[18]. Experiments were carried out in 45
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7

250 bp

250 bp

100 bp

100 bp

Figure 1 PCR of recombinant plasmid pCMV.Ins. Lane 1: PCMV.eGFP, lanes 2

and 8: Molecular marker, lane 3: Negative control, lane 4: Recombinant plasmid
pCMV.Ins, lanes 5-7: Bacterial suspension.

male Wistar rats (weighing180-190 g) at the age of

8 wk. After an over night fasting, the rats were
given a single intraperitoneal injection of 60 mg/kg
streptozotocin (STZ, Upjohn, Kalamazoo, Ml, USA)
in a 0.01 mol/L citrate buffer. Forty rats with their
fasting blood glucose level > 16.7 mmol/L were used
in the study. The rats were randomly divided into 4
groups. Chitosan-DNA nanoparticles were transfected
to the diabetic rats through lavage and coloclysis,
respectively. The two control groups were treated with
naked chitosan and normal saline respectively. The
rats had free access to diets and water.
Identification of plasmid
pCMV.Ins was identified by PCR. The sequences of
primers used for human insulin specifically are 5'ATCACTGTCCTTCTGCCA-3' (forward) and 5'GGGTGTGTAGAAGAAGCC-3' (reverse). The
PCR products were analyzed by a 1.5% agarose gel
electrophoresis, and the expected amplification length
was 173 bp. The DNA sequencing of pCMV.Ins plasmid
was performed in the Shanghai Sangon Sequencing
Center.
Measurement of fasting blood glucose and
plasma insulin levels
The fasting blood glucose and plasma insulin levels
were measured in all groups. The glucose levels in blood
samples obtained from tail veins were measured with
the one touch meter and test strips (Lifescan Inc, USA)
every morning. The plasma human insulin was detected
with a commercial human insulin radioimmunoassay
(RIA) kit (Institute of Atomic Energy, China).
Reverse transcription polymerase chain
reaction (RT-PCR) analysis
RT-PCR was performed to verify the expression of human insulin gene in gastrointestinal tract. Half of the
rats in each group were killed on the 4th d after they
transfected with chitosan-DNA nanoparticles. Immediately upon death, stomachs, intestines and rectums
of the treated and control animals were flash-frozen
in liquid nitrogen, and stored at -70. Total RNA

Niu L et al . Gene therapy in gastrointestinal tracts by chitosan nanoparticles

30

Lavage group

Naked chitosan group

Coloclysis group

Normal saline group

4211

Fasting blood (mmol/L)

25
20
15
b

10

0
0

t /d

Figure 2 Fasting blood glucose levels in each group after the normal saline treatment. bP < 0.01 vs naked chitosan group and normal saline group ( n = 10).

(5 g), obtained using Trizol (Gibco BRL, Gaithersburg, MD, USA) according to the manufacturers instructions, was subjected to reverse transcription by
using an oligo-dT21 primer, recombinant RNAsin, and
AMV reverse transcriptase (all Promega, Madison, WI,
USA). AMV RT was inactivated at 95, and PCR was
performed by using a gene amp PCR system 9600 thermal cycler (Perkin Elmer, Norwalk, CT, USA), and Taq
DNA polymerase (Perkin Elmer). The cDNA-mixture
was allowed to react for 19 (GAPDH), or 22 (insulin) cycles. The sequences of primers used for human
insulin are 5'-ACCATGGCCCTGTGGATGCGC-3'
(forward), and 5'-CTAGTTGCA GTAGTTCTCCAG-3' (reverse). The sequences of primers for
GAPDH are 5'-ACCACAGTCCATGCCATCAC-3'
(forward) and 5'-TCCACCACCCTGTTG CTGTA-3'
(reverse). Insulin primers were designed to amplify
human insulin specifically. Primers for GAPDH were
used in control reactions. The RT-PCR products were
analyzed by a 1.5% agarose gel electrophoresis, and
the expected amplification lengths were 336 bp and
451 bp, respectively.
Western blot analysis
Four days after the transfection, stomachs and
intestines were harvested and resuspended in a lysis
buffer containing 1% nonidet P-40, 50 mmol/L TrisHCl (pH 7.4), 150 mmol/L NaCl, 200 U/mL aprotinin,
1 mmol/L phenylmethanesulfonyl fluoride. The tissue
lysates (50 mg of protein) were separated by 12%
polyacrylamide gel electrophoresis and blotted onto
poly-vinylidene difluoride membranes. Immunoblotting
was perfor med with the antibody against human
insulin (Sigma-Aldrich Corp, St Louis, MO, USA), and
the molecular weight of human insulin was known as
56 kDa.
Statistical analysis
Data were expressed as mean SD. The concentrations
of blood glucose and plasma insulin were evaluated by

one-way ANOVA (SPSS for Windows 11.5). P < 0.05

was considered statistically significant.

RESULTS
Identification of recombinant plasmid pCMV.Ins
The PCR products of recombinant plasmid pCMV.
Ins and bacterial suspension were amplified. One
specific band was obtained in lanes 4-7, respectively,
corresponding to the expected size of 173 bp. No
fragments in lanes 1 and 3 were amplified from pCMV.
eGFP and negative control (Figure 1), suggesting that
the human insulin gene was successfully inserted into
recombinant plasmid pCMV.Ins. The sequencing results
also revealed that the recombinant pCMV.Ins plasmid
was successfully constructed. The number of sequencing
reports was LE142.
Change in fasting blood glucose
The fasting blood glucose level was decreased in lavage
and coloclysis groups from 22.12 1.31 mmol/L to 5.63
0.48 mmol/L and 5.07 0.37 mmol/L, respectively,
after transfected (n = 10 each group). The levels of
fasting blood glucose were significantly lower in the
lavage group and coloclysis group transfected with
chitosan-DNA nanoparticles (P < 0.01) after the normal
saline treatment from the 1st to 4th d (Figure 2). The
blood glucose levels in the naked chitosan group also
decreased significantly, because chitosan had the effect
of decreasing blood glucose level. The blood glucose
levels in the lavage and coloclysis groups were much
lower than those in the naked chitosan group, and the
differences were significant (P < 0.01). There was no
difference in blood glucose levels between the lavage and
coloclysis groups.
Change in plasma insulin
The plasma insulin in the lavage and coloclysis groups
increased from 14.23 1.38 IU/mL to 32.26
1.81 IU/mL and 32.7 1.84 IU/mL after transfection
(n = 10 each group). The plasma insulin levels in the
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Lavage group

Naked chitosan group

Coloclysis group

Normal saline group

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Plasma insulin (IU/mL)

30

25

20
15
10
5
0
0

t /d

Figure 3 Plasma insulin levels in each group after transfection. bP < 0.01 vs naked chitosan group and normal saline group (n = 10).

500
400
300
200

bp
bp
bp
bp

100 bp

Figure 4 Amplification
results of GAPDH (A)
and human insulin gene
mRNA (B) in each group.
Lane 1: Lavage group,
lane 2: Coloclysis group,
lane 3: Naked chitosan
group, lane 4: Normal
saline group, lane 5:
Molecular marker.

kDa

119
79
Human
Insulin
46

31

Figure 5 Expression of human insulin in each group. Lane 1: Lavage group,

lane 2: Coloclysis group, lane 3: Naked chitosan group, lane 4: Normal saline
group, lane 5: Molecular marker.

1000 bp
800 bp
600 bp
500 bp
400 bp
300 bp
200 bp
100 bp

lavage, coloclysis and naked chitosan groups (Figure 3)

were higher than those in the normal saline group
from the Day 1 to the Day 4 (P < 0.01). There were
remarkable differences among the lavage, coloclysis
and naked chitosan groups (P < 0.01). The differences
between the lavage and coloclysis groups did not show
any statistical significance. These results were consistent
with reduction in the fasting blood glucose levels.
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Verification of human insulin gene expression

To analyze the expression of the human insulin gene,
RT-PCR and Western blot analysis were carried out.
Total RNA from the gastrointestinal tract was amplified
using primers specific for GAPDH, revealing a
451 bp fragment in all groups (Figure 4A). However,
human insulin specific primers produced a fragment,
corresponding to the expected size of 336-bp in lanes
1 and 2, only in reactions containing RNA from the
lavage and coloclysis groups, respectively (Figure 4B).
No products were amplified from cDNA in the naked
chitosan group (lane 3) and normal saline group (lane 4).
T he molecular weight of human insulin was
56 kDa (Figure 5). One specific band was only
obtained in the lavage and coloclysis groups in lanes
1 and 2, respectively. No products were obtained
in the naked chitosan group (lane 3) and nor mal
saline group (lane 4). These results suggest that the
exogenous transferred pCMV.Ins plasmid but not the
endogenous insulin gene expressed the mRNA and
human insulin.

Niu L et al . Gene therapy in gastrointestinal tracts by chitosan nanoparticles

DISCUSSION
One factor critical to successful gene therapy is the
development of efficient delivery systems. Despite the
advances in gene transfer technology including viral
and non-viral vectors, no ideal vector system is available
at present [19] . Although viral vectors can introduce
exogenous genes into cells precisely and effectively,
they can easily cause immune reactions because of
the existence of antiviral immune system. Due to the
growing concerns over the toxicity and immunogenicity
of viral DNA delivery systems, DNA delivery via
improving viral routes has become more desirable and
advantageous[20].
A perfect vector should also be biocompatible,
efficient, and modular so that it can be applied both in
research and in clinical settings[21]. Taking into account
this point, we selected chitosan nanoparticle, a kind of
non-viral vector, in the study. We found that human
insulin gene wrapped with chitosan nanoparticles could
decrease the fasting blood glucose level and increase
the insulin level in STZ diabetic rats. The mRNA in
human insulin gene and human insulin was detectable
in the gastrointestinal tract. These results demonstrate
that chitosan nanoparticles can mediate the transfection
of human insulin gene and that chitosan nanoparticles
can be used as a good vector in gene therapy of type 1
diabetes mellitus. Kping-Hggrd et al[22] reported that
aerosol delivery formulated with chitosan oligomers
can improve the distribution of pDNA polyplexes in
the lungs and increase 6-fold of the efficiency of gene
delivery in vivo over the commonly used intratracheal
instillation method.
Chitosan nanoparticles are a kind of non-viral vector.
Non-viral vector includes liposome[23-25], composite[26],
microsphere, and nanopar ticles, etc [27,28] , but the
cytotoxicity of the bangosome limits its application in vivo.
Owing to its loose constitution, the constancy of
composites is poor. The diameter of microsphere is
bigger than that of nanoparticles. Chitosan nanoparticles
are a comparatively promising non-viral vector. Chitosan
nanoparticles[29] have a good biocompatibility and no
toxicity, and are economically available. The transfection
efficiency of chitosan can be regulated by changing
its molecular weight, plasmid concentration, and the
chitosan/plasmid ratio. After the plasmid is embedded
in chitosan, it can resist the degradation of nucleases.
It also exhibits an antibacterial activity by inhibiting the
bacterial metabolism.
In our study, the fasting blood glucose level was
decreased during the first 4 d, due to the regeneration of
gastrointestinal tract epithelial cells, which is consistent
with the reported data[30].
Further study should be performed to detect the
cells intaking the plasmid. We speculate that gut K-cells
may intake the expression plasmid. The gut K-cells
in the epithelium mucosa of gastrointestinal tract
secrete glucose-dependent insulinotropic polypeptide
(GIP)[31], an incretin hormone secreted by endocrine
K-cells in response to nutrient absorption. GIP can

4213

stimulate cells to release insulin, and promote the

regeneration of cells[32]. GLP accounts for about 60%
of the stimulation of insulin by oral glucose, but the
determinants of their secretion from the small intestine
are poorly understood[33]. There are many similarities
between the release of GIP and the secretion of insulin.
The concentration of GIP will obviously increase several
minutes after glucose ingestion, and return to its basal
level after 2 h. It was reported that the antagonist of
GIP can remarkably degrade the secretion of insulin[34].
Furthermore, the promoter of GIP can only be activated
in K-cells.
One of the key points in gene therapy for diabetes
is the modification (processing) of proinsulin to
insulin. Most non- cells functioning as target cells
in g ene therapy for diabetes are lack of typical
prohormone convertases which are essential to the
processing of proinsulin, whereas K-cells can produce
PC2 and PC3, which can help process the proinsulin
correctly [35]. Palizban et al have transfected rat small
intestine K-cells with pGIP/Ins plasmid by DOTAP
liposome [36] . As mentioned above, K-cells might
be the best target cells in gene therapy for type 1
diabetes mellitus due to their response to glucose and
resistance to destruction mediated by cytokines and
free radicals.
In conclusion, the human insulin gene can be
transfected successfully by chitosan-DNA nanoparticles
and expressed efficiently in the gastrointestinal tract
of diabetic rats, and chitosan is a promising non-viral
vector. If it is applied in clinic practice, it would be
accepted by patients. Although much work remains to be
done, the rapid progress in insulin gene therapy provides
an optimistic outlook for its clinical applications in the
treatment of type 1 diabetes mellitus.

ACKNOWLEDGMENTS
The authors thank Dr. Michael German, Department
of Hormone Research Institute, University of San
Francisco (San Francisco, USA) for donation of
pBAT16, hInsG1.M2 plasmid.

COMMENTS
Background

Gastrointestinal K-cells might be the best target cells in gene therapy for type
1 diabetes mellitus due to their response to glucose and the resistance to the
destruction mediated by cytokines and free radicals. A perfect vector should
also be biocompatible, efficient, and modular so that it can be applied both in
research and in clinical settings. Chitosan is an ideal non-viral vector and has
drawn wide attention.

Research frontiers

There are considerable endocrine cells in the gastrointestinal tract.

Gastrointestinal K-cells are the potential and ideal target cells in gene therapy
for diabetes. Progress in gene therapy has produced promising results that
translate experimental research into clinical treatment. The main barrier in gene
transfer is a safe and effective gene delivery system.

Innovations and breakthroughs

The exogenous insulin genes can be transfected by chitosan nanoparticles

and expressed efficiently in the gastrointestinal tract of diabetic rats, indicating
that chitosan is a promising non-viral vector. At present, there is no report on

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the transfection of human insulin gene to the gastrointestinal tract and on the
application of chitosan as a vector in gene therapy for type 1 diabetes mellitus.

Applications

12

Terminology

13

The superiority of human insulin gene expression in gastrointestinal tract by

chitosan nanoparticles is its safety without any wound. If it can be applied in
clinic practice, it would be accepted by patients. Chitosan is an ideal non-viral
vector and can be widely used in gene transfer.
Gastrointestinal K-cells exist in the epithelium mucosa of gastrointestinal
tract and can secrete glucose-dependent insulinotropic polypeptide (GIP),
which can stimulate cells to release insulin and promote the regeneration
of cells. Chitosan nanoparticles are a kind of non-viral vector and have a
good biocompatibility without any toxicity, and are economically available. The
transfection efficiency of chitosan can be regulated easily. After the plasmid is
embedded in chitosan, it can resist the degradation of nucleases.

14

Peer review

This paper reports the expression of human insulin gene in the gastrointestinal
tract by chitosan nanoparticles, thus providing new technologies of gene
transfer to endocrine cells in the gastrointestinal tract. This study is well
designed and interesting.

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