Arch Pharm Res Vol 31, No 7, 918-923, 2008

DOI 10.1007/s12272-001-1247-9 http://apr.psk.or.kr

Prolonged Antidiabetic Effect of Zinc-Crystallized Insulin Loaded

Glycol Chitosan Nanoparticles in Type 1 Diabetic Rats
Hyung Gon Jo, Kyung Hyun Min, Tae Hwan Nam, Seong Ju Na, Jae Hyung Park, and Seo Young Jeong
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Korea

(Received January 3, 2008/Revised June 3, 2008/Accepted June 10, 2008)

New basal insulin formulation was designed and their structural characteristics were investi-
gated in vitro and biological activities in type 1 diabetic rats. Zinc-crystallized insulin was physi-
cally loaded into hydrophobically modified glycol chitosan (HGC) nanoparticles by a dialysis
method. The series of insulin-HGC formulations were prepared with different feed weight ratio
of insulin to HGC from 0.5:1 to 4:1. The loading contents of insulin and size distribution of insu-
lin-HGCs were characterized, and blood glucose responses were investigated in streptozoto-
cin-induced diabetic rats after single subcutaneous injection of regular insulin and insulin-
HGCs. The highest loading efficiency and content were obtained in insulin-HGC when a 1:1
feed weight ratio of insulin to HGC was employed. The hydrodynamic diameter of insulin-HGC
nanoparticles were in the range of 200 to 500 nm with narrow size distribution. Insulin-HGC
effectively sustained insulin release up to 40% within 12 hours followed by a slower controlled
release. Insulin-HGC showed an extended blood glucose lowering effect up to 24 h and pro-
vided normal blood glucose levels after oral glucose (1.5 g/kg) load at 24 hours post-injection
while regular insulin showed severe hypoglycemia. The prolonged time action profiles and low
variability of insulin-HGC formulation resulted in improved blood glucose control in diabetic rats
and fulfilled a pattern desirable of a basal insulin.
Key words: Basal insulin, Blood glucose control, Nanoparticles, Type 1 diabetes

INTRODUCTION glucose (Skyler, 1986; Binder et al., 1984; Wearley, 1991;

Insulin is the primary hormone utilized to treat virtually
all types of diabetic patients. Monomeric forms of insulin
acts quickly whereas hexameric insulin has relatively a
longer half-life of approximately two hours after sub-
cutaneous injection in diabetic patients. Since this short
action of insulin in vivo is still insufficient to meet basal
patient needs, long-acting formulations of insulin have
been developed for many years (Owens, 2002; Frokjaer
and Otzen, 2005; Cefalu, 2004). The most commonly
used long-acting insulin formulations have been prepared
as suspensions of insulin complexes by addition of zinc or
protamine. Such basal insulins showed improved half-
lives, however, they still have some problems including
limited dosing accuracy or variation in the absorption
kinetics which introduces insulin induced low blood
Correspondence to: Seo Young Jeong, Department of Pharma-
ceutics, College of Pharmacy, Kyung Hee University, 1 Hoegi-
dong, Dongdaemoon-gu, Seoul 130-70,1 Korea
Tel: 82-2-961-9254, Fax: 82-2-962-0223
E-mail: syjeong@khu.ac.kr
Agu et al., 2001). To overcome these limitations, several
approaches have been proposed to improve its therapeu-
tic value. Various insulin formulations are being developed
up to now, these include, changing the iso-electric point of
insulin to crystallize insulin injection site (McKeage et al.,
2001; Brange and Volund, 1999), bioconjugation of small
or larger molecules to extend its half-life in vivo (Hamilton-
Wessler et al., 1999), or derivatization of insulin with
degradable moiety to show a time-dependent slow
release of insulin in vivo (Gershonov et al., 1999). The
described approaches showed a number of promising
results and some are now used in clinical applications.
Among diverse candidates, polymeric nanoparticles
have been widely investigated as carriers for drug delivery
including therapeutic peptide and protein drugs. Polymeric
nanoparticles provide massive advantages regarding im-
proved drug stability in biological environment, controlled
release of drug and with their additional potentials to
improve drug loading, targeting and transport (Takeuchi et
al., 2001; Romero-Cano and Vincent, 2002; Alonso and
Sanchez, 2003). Water-soluble glycol chitosans are

918
Prolonged Antidiabetic Effects of Insulin-LGC Nanoparticles in Type 1 Diabetic Rats
emerging as novel carriers of drugs because of their
solubility and biocompatibility in vivo (Sinha et al., 2004;
Calvo et al., 1996; Park et al., 2003). In our previous study,
hydrophobically modified biocompatible glycol chitosan
(HGC) formed polymeric nanoparticles in aqueous solu-
tion and showed highly sustainable release profiles of
loaded small molecule anticancer drugs in vitro and in
vivo (Kwon et al., 2003; Kim et al., 2006). Based on sub-
stantiated observations that HGC associated physically
loaded drug could enhance controlled delivery, we have
designed prolong-acting zinc-crystallized insulin loaded
HGC nanoparticle formulation. We hypothesized that
amphiphilic crystallized insulin can be loaded into HGC by
i) hydrophobic interaction between hydrophobic inner core
of HGC and hydrophobic side of zinc-crystallized of the
peptide and ii) electrostatic interaction between positively
charged chitosan and negatively ionized insulin.
In this study, we investigated the potential of insulin-
HGC formulation by examining the impact of insulin
loading on the size distribution of nanoparticles, insulin
release in vitro, and its in vivo glucose lowering effect in
type 1 diabetic rats.
MATERIALS AND METHODS
Materials
Human zinc-crystalline insulin (Zn2+ insulin) was pur-
chased from Serologicals Corp. (Norcross, GA) and used
without purification. Glycol chitosan (MW = 2.5 × 105 : degree
of deacetylation: 82.7%) and streptozotocin (STZ) were
obtained from Sigma (St. Louis, MO). The HGC (degree
of substitution = 12 %) was synthesized in the presence of
1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) and N-hydroxysuccinimid (NHS), as described
previously (Kwon et al., 2003). All reagents and organic
solvents were of analytical grade.
Preparation of insulin-HGC nanoparticles
Insulin-HGC nanoparticles were prepared by a dialysis
method. Briefly, HGC and zinc-crystallized insulin were
dissolved in dimethylsulfoxide (DMSO). The solution was
stirred for 12 h at room temperature and then dialyzed
against 0.01% NH4HCO3 using a membrane with a
molecular weight cutoff (MWCO) of 50 kDa (Spectrapor,
Rancho Dominquez, CA). After dialyzed for 2 days, the
solution was lyophilized. The amount of insulin in the HGC
nanoparticles was measured by using reverse-phased
high-performance liquid chromatography (RP-HPLC, Agilent
1200 series HPLC system with a Zorbax SB-C18 column,
Agilent Technologies, Wilmington, DE). The mobile phases
used were 0.1% trifluoroacetic acid (TFA) in distilled water
(eluent A) and acetonitrile (ACN) containing 0.1% TFA
(eluent B). The column was run using a linear gradient
919
from 20 to 75% of eluent B for 30 min at a flow rate of 1
mL/min and UV absorbance of the eluent was monitored
at 230 nm.
Characterization of insulin-HGC nanoparticles
The mean hydrodynamic diameter of HGC and insulin-
HGC nanoparticles was measured by dynamic light scat-
tering (DLS) measurements using the helium ion laser
system (Spectra Physics Laser Model 127-35) operated
at 633 nm and 25 ± 0.1oC. The intensity autocorrelation of
the sample (1 mg/mL in PBS) was measured at a scat-
tering angle of 90o with a BI-9000AT digital autocorrelator
(Brookhaven, NY, U.S.A.).
In vitro insulin releases profiles
Insulin-HGC nanoparticles (3 mg/mL) were dispersed
in PBS (pH 7.4). Solutions were placed into a cellulose
membrane (MWCO 50 kDa) and incubated in PBS while
gently shaken in a water bath with a speed of 100 rpm at
37oC. At various determined time points, the insulin
concentrations were determined by micro-BCA assay.
In vivo biological potencies
The National Institute of Health (NIH) guidelines for the
care and use of laboratory animals were followed throughout
the experiments (NIH publication 85-23 Rev. 1985). For
the glucodynamic studies, STZ-induced type 1 diabetic
rats were used. Male Sprague-Dawley rats weighing from
250 to 270 g were fasted for 12 h before inducing diabetes
mellitus. Diabetes was induced by a single intra-peritoneal
injection of STZ (in a citrate buffer at pH 4.5) at 70 mg/kg.
Two weeks after the STZ treatment, the rats whose blood
glucose level in the non-fasting condition was above 450
mg/dl were selected as diabetic rats for further investiga-
tions. Glycemic responses were monitored after a single
subcutaneous injection of regular insulin or insulin-HGC
(insulin, 2 and 5 IU/kg, 1 mL/kg and the equivalent amount
of the nanoparticles) in fasted diabetic rats. During the
experiments, all rats were kept in metabolic cages with
free access to water only. Blood samples for the analysis
of blood glucose were taken from tail veins and deter-
mined immediately on fresh samples by using an one
touch blood monitoring system (Glucocard II, Arkray,
Kyoto, Japan).
RESULTS AND DISCUSSION
Insulin-HGC nanoparticles
HGC is an amphiphilic polymer which consists of
hydrophphilic and hydrophobic segments, thereby can
form self-assembled nanoparticles in aqueous condition
(Fig. 1). In the aqueous phase, the hydrophobic cores,
induced by 5β-cholanic acid, of polymeric nanoparticles are
920 H. G. Jo et al.

surrounded by hydrophilic outer shells, glycol chitosans.

Thus, the inner core can serve as a nano-container for
hydrophobic drugs. Since zinc-crystallized insulin is amphi-
philic, we hypothesized that the hydrophobic side of insulin
crystals can be incorporated into the hydrophobic inner
core of self-assembled HGC nanoparticles. Furthermore,
negatively charged insulin can be compacted by wrapping
around positively charged chitosan’s side chains. Thus,
crystallized insulin incorporated stable HGC nanoparticles
may associate through hydrophobic and ionic interactions.
Therefore insulin in HGC nanoparticles would be expected
to be released in a sustained manner. Fig. 1. Chemical structures of HGC conjugate

Fig. 2. Size distribution of HGC and insulin-HGC nanoparticles. (a) HGC only and insulin-HGC nanoparticles with feed weight ratio between insulin
and HGC at (b) 0.5:1, (c) 1:1, (d) 2:1, (e) 4:1.
Prolonged Antidiabetic Effects of Insulin-LGC Nanoparticles in Type 1 Diabetic Rats 921

Table I. Characterization of insulin-HGC nanoparticles

Sample Loading efficiency Loading content of
(weight ratio) (%) insulin (wt.%)
HGC-insulin (0.5:1) 58.15 ± 26.84 36.34 ± 4.28
HGC-insulin (1:1) 83.03 ± 13.39 41.51 ± 6.69
HGC-insulin (2:1) 20.50 ± 25.11 15.38 ± 3.83
HGC-insulin (4:1) 29.35 ± 22.47 11.69 ± 3.09

The insulin-HGC was prepared by conventional dialysis

methods. The loading efficiency and the loading content
of insulin were determined by varying the feed weight
ratio of insulin to HGC. After lyophilization, insulin-HGC
samples were dissolved in organic solvent, DMSO, and
content of incorporated insulin was analyzed by using RP-
HPLC. Table I shows that the loading efficiency decrease Fig. 3. In vitro release profiles of insulin from insulin-HGC nanoparticles
with increasing feed weight ratio of insulin to HGC from (3 mg/mL) with feed weight ratio between insulin and HGC at 1:1 (in
1:1 to 4:1. As the feed ratio increased, the aggregation of PBS, pH 7.4, at 37oC). Results are presented at mean ± SD (n=4).
insulin-HGC in the dialysis membrane tube was more pro-
nounced and consequently resulted in the lower loading single subcutaneous injection of PBS and insulin with or
efficiency. From the several variables for insulin incorpora- without nanoparticle association are illustrated in Fig. 4.
tion into HGC, the highest loading efficiency and content After PBS administration, a blood glucose level of animals
were obtained in insulin-HGC when a 1:1 feed weight ratio was not appreciably lowered. As expected, insulin (5 U/
of insulin to HGC was employed. The size distribution HGC kg) reduced blood glucose levels rapidly in 4 hours after
nanoparticle alone and insulin-HGCs were illustrated in administration and blood glucose slowly increased. In
Fig. 2. Insulin-HGC with feed weight ratio of 1:1 showed contrast, the insulin-HGC had similar blood glucose nadir
narrow and decreased size distribution compared to that concentrations; however, the initial rate of blood glucose
of HGC nanoparticle alone. elimination was less than that caused by regular insulin.
Although, insulin-HGCs had lesser effects than regular
In vitro release of insulin insulin on initial blood glucose, their effects were markedly
After determination of an optimized dose ratio between improved. Two different doses of insulin-HGC were
insulin and HGC, we fixed the ratio and evaluated in vitro evaluated (2 and 5 U/kg based on insulin content). Injec-
insulin release profiles. The in vitro release of insulin-HGC
was evaluated at 37oC in PBS, pH 7.4. Sink conditions
were maintained in the dialysis bag method during the
experiment. Fig. 3 illustrates the percentage of insulin
released from insulin-HGC nanoparticles with feed weight
ratio between insulin and HGC at 1:1. The insulin release
profile from HGC is typically constituted of two different
phases: an initial period, characterized by a relatively fast
and linear phase, followed by a second one, charac-
terized by a slower release of the insulin. HGC effectively
sustained insulin release up to 20 % within 12 h followed
by a slower controlled release over the next hours. The
release profile of insulin in self-assembled HGC nanopar-
ticles is consistent with the hypothesis that incorporated
insulin in HGC by hydrophobic and oppositely charged
interaction confers a decrease in release rates.
Fig. 4. Blood glucose concentrations following a single subcutaneous
In vivo hypoglycemic effect injection of PBS (●), insulin 5 U/kg (○) and insulin-HGC nanoparticles,
The pharmacodynamic potencies of the regular insulin 2 IU/kg (▼), 5 IU/kg (△) in STZ-induced diabetic rats. At 24 h post-
and insulin-HGC were investigated in STZ-induced type 1 injection, 1.5 g/kg of oral glucose was loaded to each group. Results
diabetic rat. Changes in blood glucose levels following a are presented on mean ± SE (n=3, 4).
922
Fig. 5. The glucose AUC for the blood glucose responses following a
single subcutaneous injection of insulin and insulin-HGCs. The glucose
AUC (a) from 0 to 24 h and (b) after oral glucose (1.5 g/kg) treatment.
(*, p<0.05 ; **, p<0.005).
tion of insulin-HGC (2 U/kg) showed similar blood glucose
reducing effect compared to that of insulin alone (5 U/kg).
Insulin-HGC with 5 U/kg of insulin showed significantly
reduced blood glucose and maintained normal glucose
levels up to 24 h experimental times. As shown in Fig. 5a,
the blood glucose AUC0-24h value of insulin-HGC (2 U/kg)
were comparable to that seen at insulin 5 U/kg, however,
insulin-HGC (5 U/kg) induced significant reduction in glu-
cose level (p < 0.05) compared to insulin alone (glucose
AUC0-24h, for PBS, insulin, insulin-HGC, 2 and 5 U/kg, 1649
± 19, 1459 ± 248, 1397 ± 81, 603 ± 109 h•%, respectively).
In addition, oral glucose tolerance test performed to
confirm its effectiveness. When glucose (1.5 g/kg) was
orally administered to insulin treated rats at 24 h post-
injection, only 5 U/kg of insulin-HGC formulation markedly
reduced blood glucose excursion and stabilized blood
glucose level compared to insulin alone (p < 0.005); as
evident by significantly reduced glucose AUC24-28h (Fig. 5b,
glucose AUC24-28h, for PBS, insulin, insulin-HGC, 2 and 5
U/kg, 298 ± 19, 396 ± 33, 289 ± 19, 116 ± 26 h•%, respec-
H. G. Jo et al.
tively). Taken together, insulin-HGC formulation showed
consistent blood glucose levels time dependently with low
animal to animal variability compared to that of zinc-
insulin which meets criteria for once-a-day insulin formula-
tion. Furthermore, stable distributed forms of nanoparticle-
based formulations could provide more accurate dose
controls.
The hypothesis of a mechanistic reason for prolong
action of insulin-HGC in vivo can be explained by the
physico-chemical structures of HGC nanoparticles and
crystallized insulin. The rate of diffusion of a subcutane-
ously administered peptide drug is generally proportional
to its in vivo half-life. In this respect, physically incorporat-
ed zinc-crystallized hexameric form of insulin in self-
assembled HGC nanoparticles would be expected to be
released more slowly into blood stream, and therefore, to
develop activity after being dissociated into the monomer
form in blood. Taken with our prolonged glucose lowering
effects in diabetic rats, insulin-HGC fulfilled all the features
expected of a basal insulin formulation. However, to
further investigate the clinical relevancy of insulin-HGC,
the prolonged mechanism and chronic study will be
carried out.
In conclusion, we found that the described insulin-HGC
demonstrated longer duration of action in vivo than the
native form. These enhanced biologic potencies highlight
their potentials as a novel prolonged activity insulin-based
antidiabetic agents.
ACKNOWLEDGMENTS
This research was financially supported by BK21 BNT
Scientist Renovating for the Drug Development Coping
with Aged Society, and by Seoul R&BD Program.
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