Efficient oral insulin delivery by dendronized chitosan: In vitro and in vivo studies

Article abstract of DOI:10.1039/c4ra07511k

The development of efficient and bio-safe polymeric carriers for oral insulin delivery is a major thrust in biomedical research. In this paper, dendronized chitosan (DCTS) is prepared using a Michael-type addition reaction by grafting polyamidoamine (PAMAM) onto chitosan to improve its water solubility, pH sensitivity, and insulin encapsulation efficiency for enhanced bioavailability of the oral insulin. The self-assembled dendronized chitosan nanoparticles are prepared using a mild coacervation method, in which almost sub-spherically shaped nanoparticles of 85-150 nm size are produced, with an insulin encapsulation of approx. 95%. In vitro release study confirms a pH-sensitive and self-sustained release of insulin, where the oral administration of these nanoparticles exhibits a pronounced hypoglycemic effect in diabetic mice, producing a relative bioavailability of ～9.19%. As no systemic toxicity is observed with its peroral delivery, these DCTS nanoparticles can effectively serve as a promising device in the efficient administration of oral insulin. This journal is

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PAPER
Efficient oral insulin delivery by dendronized
chitosan: in vitro and in vivo studies
Cite this: RSC Adv., 2014, 4, 43890
Piyasi Mukhopadhyay,a Kishor Sarkar,a Sourav Bhattacharya,b Roshnara Mishrab
a
*
and P. P. Kundu
The development of efficient and bio-safe polymeric carriers for oral insulin delivery is a major thrust in
biomedical research. In this paper, dendronized chitosan (DCTS) is prepared using a Michael-type
addition reaction by grafting polyamidoamine (PAMAM) onto chitosan to improve its water solubility, pH
sensitivity, and insulin encapsulation efficiency for enhanced bioavailability of the oral insulin. The self-
assembled dendronized chitosan nanoparticles are prepared using a mild coacervation method, in which
almost sub-spherically shaped nanoparticles of 85–150 nm size are produced, with an insulin
encapsulation of approx. 95%. In vitro release study confirms a pH-sensitive and self-sustained release of
insulin, where the oral administration of these nanoparticles exhibits a pronounced hypoglycemic effect
in diabetic mice, producing a relative bioavailability of ꢀ9.19%. As no systemic toxicity is observed with
its peroral delivery, these DCTS nanoparticles can effectively serve as a promising device in the efficient
administration of oral insulin.
Received 23rd July 2014
Accepted 29th August 2014
DOI: 10.1039/c4ra07511k
www.rsc.org/advances
Therefore, positively charged chitosan can easily encapsulate
negatively charged bio-macromolecules such as DNA or insulin
via electrostatic interaction.5,6 However, acidic solutions of
chitosan may not be desirable in many applications, such as
cosmetics, food and biomedicine.4 Hence, successive modi-
cations have been implemented to improve its water solubility,
physicochemical and biochemical properties.7–9
1. Introduction
Oral insulin delivery remains a great challenge in the biomed-
ical eld due to several problems, such as the harsh acidic
environment in the stomach, extensive enzymatic degradation
by different proteolytic enzymes of the GI tract, and barriers
presented by the mucosal surface and the tight junction in
between the intestinal epithelial cells.1 Despite remarkable
advancements in oral insulin delivery for combating diabetes
mellitus,2 an efficient and satisfactory oral insulin delivery
vehicle remains unavailable. Recently, biodegradable polymers
such as protein- and polysaccharide-based nanoparticles have
gained tremendous attention in pharmaceutical research as
efficient drug delivery vehicles for controlled and targeted drug
release, which aim to improve the therapeutic effects and also
reduce the side effects of the formulated drugs.3 Over the past
few decades, chitosan has received appreciable interest among
other polysaccharides for drug delivery due to its biodegrad-
able, biocompatible, non-toxic and non-immunogenic proper-
ties.1 Chitosan, isolated from chitin by alkaline hydrolysis, is a
cationic, linear and partly acetylated (1 / 4)-2-amino-2-deoxy-b-
D-glucan. Chitosan has a pKa value of 6.5, and therefore, it is
only soluble in acidic aqueous solutions such as formic, acetic,
pyruvic, 10% citric and lactic acids and becomes cationic due to
the protonation of its primary amine groups at pH < 6.5.4
On the other hand, a new class of synthetic polymers, the
polyamidoamine (PAMAM) dendrimer, has gained considerable
attention in biomedical research due to its water solubility, size
uniformity, and highly branched nano-spherical architecture
with precise molecular weight.10,11 Moreover, full-generation
cationic PAMAM dendrimers (amine terminated) can also
form complexes with negatively charged biomolecules such as
protein, DNA, RNA etc. by electrostatic interaction.12 However,
the severe toxicity of high-generation PAMAM dendrimers (with
a high density of primary amine groups) limits its widespread
application in the biomedical eld.13 Therefore, a chemical
combination of chitosan with PAMAM may provide a novel
biomaterial with improved water solubility, higher charge
density and lower toxicity for efficient oral insulin delivery.
In our previous studies, we prepared N-maleated, chitosan-
gra-PAMAM copolymer and PAMAM–chitosan conjugated
through the naphthalimide moiety for efficient gene delivery
with improved water solubility and minimal toxicity.5,14
However, it was observed during the preparation of N-(4-
bromonaphthalimide)-chitosan that a higher molar ratio of
naphthalimide to chitosan produced a water-insoluble product
that was not soluble even aer graing of PAMAM; the water-
insoluble product is not suitable for oral insulin delivery and
aDepartment of Polymer Science and Technology, University of Calcutta, 92, A.P.C.
Road, Kolkata-700009, India. E-mail: ppk923@yahoo.com; Fax: +91-2352-5106; Tel:
+91-2352-5106
bDepartment of Physiology, University of Calcutta, 92, A.P.C. Road, Kolkata-700009,
India
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may also cause systemic toxicity within the animal body. rst, chitosan was puried by re-precipitation method prior to
Therefore, in this work, we prepared a low-generation, conju- the reaction.6 Then, 1.0 g of puried chitosan was dissolved in
gated chitosan through the N-carboxyethyl chitosan methyl 50 mL of 1% acetic acid solution under constant stirring for
ester intermediate for oral insulin delivery. In comparison to 30 min and diluted with 200 mL of ethyl alcohol. To this solu-
previously reported materials,15,16 the gra copolymer in this tion, methyl acrylate (10 equiv./NH2 of chitosan) was added
article is unique with respect to its mode of preparation and slowly, and the reaction was further continued for 48 h at 25 ꢃC.
properties. Furthermore, the preparation of PAMAM (G 2.0)- Aer that, the reaction mixture was concentrated to approx.
conjugated chitosan through the N-carboxyethyl chitosan 100 mL under reduced pressure to remove the excess methyl
methyl ester intermediate is advantageous, because it allows the acrylate and solvent. The remaining mixture was dialyzed for 3
conjugation of high-generation dendrimer on chitosan, thereby days against deionised water and lyophilized for another 3 days
improving its water solubility. Finally, self-assembled nano- to obtain N-carboxyethylchitosan methyl ester.
particles were prepared with insulin, and physical character-
To conjugate PAMAM dendrimer with NCME, N-carboxy-
ization of the nanoparticles, together with their insulin loading ethylchitosan methyl ester (100 mg) was dispersed in methyl
capacity, insulin encapsulation efficiency, in vitro insulin alcohol (50 mL), and then, PAMAM (0.54 mmol: 1.0 equiv./
release, in vivo pharmacological responses, insulin bioavail- CO2Me) in MeOH (50 mL) was added to this suspension. The
ability and also their toxicity were investigated in detail in this reaction mixture was allowed to stir at room temperature. Aer
article.
3 days, the mixture was evaporated to dryness and dispersed in
0.2 M NaOH solution at room temperature for 2 h, dialyzed and
nally lyophilized to get the nal product, dendronized chito-
san (DCTS), as presented in Fig. 1.
2. Materials and methods
2.1. Materials
Chitosan (molecular weight, 222 kDa and degree of deacetyla-
tion, DDA 82%) was obtained from Himedia, India. Low-
molecular weight chitosan (25 kDa, DDA 82%) was prepared
by oxidative degradation using sodium nitrite (Merck, India) at
room temperature according to our previous method.6 Ethyl-
enediamine (EDA), methyl acrylate (MA), creatinine Merckotest
kit and methanol were purchased from Merck, India. PAMAM
dendrimer (G 2.0) was synthesized according to previous
reports.5,17 Insulin (bovine insulin, 27 USP units per mg) and
alloxan monohydrate were purchased from Sigma-Aldrich.
Bovine insulin ELISA kit, lactate dehydrogenase LDH (P-L) kit
and micro-protein kit were purchased from LILAC Medicare Pvt.
Ltd. and Crest Biosystems, India, respectively. Serum glutamate
pyruvate transaminase (SGPT) ALAT (GPT)-LS kit and serum
glutamate oxaloacetate transaminase (SGOT) AST (GOT)-LS kit
were obtained from Piramal Health Care Limited, India. Mul-
tistix reagent strips were purchased from Siemens, India. All
other chemicals were used as received.
2.3. Characterization of polymer
Fourier transform infrared (FT-IR) analysis was carried out with
ATR-FTIR (model-Alpha, Bruker, Germany) spectrometer,
scanning from 4000 to 500 cmꢂ1 for 42 consecutive scans at
room temperature. Samples were mixed separately with KBr and
pressed into pellets for measurements.
1
The H nuclear magnetic resonance (1H NMR) spectra were
measured on a Bruker AV 3000 Supercon NMR system (Ger-
many) at 500 MHz using D2O/DCl and D2O as solvent. Chemical
shis (d) were reported in ppm using tetramethylsilane (TMS) as
an internal reference.
The molecular weight of chitosan and its derivatives were
measured by gel permeation chromatography (GPC). The GPC
equipment consisted of ultrahydrogel 1000 (7.8 ꢄ 300 mm)
column, 515 HPLC pump and 2414 RI detector (Waters, USA).
The mobile phase was 0.1 M acetic acid/0.1 M sodium acetate
buffer. Mobile phase and chitosan solution were ltered
through a 0.45 mm lter (Millipore). The ow rate was main-
tained at 0.3 mL minꢂ1. The sample concentration was 0.3 mg
mLꢂ1. Polyethylene glycol (Sigma-Aldrich) standards were used
to calibrate the column. All data provided by the GPC system
were analyzed using the Empower 2 soware package.
Animals: Male Swiss albino mice (26 ꢁ 2 g) (M sꢂ1 Chakra-
borty Enterprise, Calcutta, India) were housed under
a
controlled environment (room temperature: 23 ꢁ 2 ꢃC, relative
humidity: 60 ꢁ 5%, 12 h day/night cycle) with a balanced diet
and water ad libitum. All the animal experiments were approved
by the animal ethical committee, Department of Physiology,
University of Calcutta, in accordance with the guideline of the
committee for the purpose of control and supervision of
experiments on animals (CPCSEA Ref no.: 820/04/ac/CPCSEA
dated 06.08.2004), Government of India.
2.4. Preparation of polymer/insulin self-assembled
nanoparticles
Self-assembled nanoparticles were prepared by complex coac-
ervation method according to our previous report.6 Chitosan
and dendronized chitosan were dissolved in 5 mM sodium
acetate–acetic acid buffer solution at a pH of 5.5. Insulin was
2.2. Synthesis of dendronized chitosan
Dendronized chitosan was prepared by two consecutive steps; dissolved in 0.1 M HCl solution, and the pH of the solution was
the preparation of N-carboxyethyl chitosan methyl ester (NCME) adjusted to 8.4 by 0.1 N Tris (hydroxymethyl) aminomethane
followed by conjugation of PAMAM dendrimer (G 2.0) with the solution. Then, equal volumes of polymer and insulin solution
NCME. N-carboxyethyl chitosan methyl ester was prepared (1 mg mLꢂ1) were mixed at different weight ratios (0.5 : 1, 1 : 1
according to the previous report with slight modication.18 At and 2 : 1), immediately vortexed for 15–30 seconds and allowed
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Fig. 1 Synthesis of dendronized chitosan, preparation of dendronized chitosan and insulin self-assembled nanoparticles and their application in
in vivo models.
to incubate at room temperature for another 20–30 min to form ZS (Malvern Instrument, UK) digital autocorrelator at 37 ꢃC. The
the self-assembled nanocomplexes.
mean diameter was obtained by the Stokes–Einstein
relationship.
The size and morphology of the nanocomplexes were also
characterized by AFM (Nanoscope IV Bioscopet, Digital Instru-
ments, Veeco) and SEM (Carl Zeiss EVO 18, Germany). 1–2 mL of
samples containing complexes in acetate buffer was deposited
2.5. Determination of particle size and zeta potential and
morphological analysis of polymer/insulin self-assembled
nanoparticles
Particle size and zeta potential of polymer/insulin complexes at onto the centre of a freshly split, untreated mica disk and glass
different weight ratios were measured in DLS equipped with slide for AFM and SEM, respectively. AFM imaging was con-
Zetasizer. The solutions were ltered through a 0.45 mm lter ducted with silicon nitride tip in tapping mode and at a scan
(Millipore) prior to the experiment to avoid the inuence of dust speed of 1 Hz at ambient condition. Before SEM observation,
on the reliability of results. The intensity of autocorrelation was the samples were xed on an aluminium stub and coated with
measured at a scattering angle (q) of 173ꢃ with Zetasizer Nano gold by an ion sputter coater (Hitachi, Japan, Model-E1010) for
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6 min. An acceleration voltage of 20 kV was used during the an angle of 45ꢃ on a platform under a constant ow rate (10 mL
scanning.
minꢂ1) of phosphate buffer (pH 7.4). The percentage of nano-
particle adherence was calculated by comparing the weight of
adhered nanoparticles to the initial weight of nanoparticles
taken for the experiment.
2.6. Insulin loading capacity and insulin encapsulation
efficiency of self-assembled nanoparticles
Aer polymer/insulin complex formation at different weight
ratios, the solution mixture was centrifuged at 14 000 rpm
2.9. In vivo pharmacological response of polymer/insulin
self-assembled nanoparticles
minꢂ1 for 30 min at 4 C. The clear supernatant was analyzed
ꢃ
for insulin content using UV-vis spectrophotometer (OPTIGEN
POP BIO, Mecasys Co. Ltd., Korea) at 280 nm. All experiments
were done in triplicate to calculate insulin loading capacity (LC)
and encapsulation efficiency (EE) by the following formula:4,6
Diabetes was induced in mice (26 ꢁ 2 g) according to our
previous reports.6,21 To assess the in vivo pharmacological
response, the following formulations were orally administered
to diabetic mice (n ¼ 6 in each group): group I: oral insulin
solution (50 IU kgꢂ1 b.w.), group II: oral chitosan/insulin
nanoparticles (50 IU kgꢂ1 b.w.), group III: oral DCTS/insulin
self-assembled nanoparticles (50 IU kgꢂ1 b.w.) and group IV
(control): subcutaneous injection of insulin solution (5 IU kgꢂ1
b.w.). The blood glucose level was checked at regular intervals
(1 h) using a Bayer glucose meter.
LC ð%Þ ¼
total amount of insulin ꢂ free insulin in the supernatant
ꢄ 100
weight of the nanoparticles
(1)
EE ð%Þ ¼
total amount of insulin ꢂ free insulin in the supernatant
ꢄ 100
total amount of insulin
2.10. Measurement of serum insulin and relative
bioavailability
(2)
Again, the concentration of serum insulin was measured
following peroral treatment of insulin-loaded nanoparticles.
The following formulations were administered to diabetic mice
(n ¼ 6, each group): group I, oral insulin solution (50 IU kgꢂ1
b.w.); group II, oral chitosan/insulin nanoparticles (50 IU kgꢂ1
b.w.); group III, oral DCTS/insulin self-assembled nanoparticles
(50 IU kgꢂ1 b.w.); and group IV (control), SC injection of insulin
solution (5 IU kgꢂ1 b.w.). The blood serum was separated by
centrifugation for 10 min at 5000 rpm and at 4 ꢃC, and insulin
concentrations were quantied using enzyme-linked immuno-
sorbent assay (ELISA). The relative bioavailability (RA) of insulin
was calculated using the following formula:21,22
2.7. In vitro insulin release prole from the self-assembled
nanoparticles
To determine the insulin release prole and pattern, the
polymer/insulin complexes were immersed in simulated gastric
uid (SGF), pH 1.2, and simulated intestinal uid (SIF), pH 7.4,
with mild agitation. At specic time intervals, the samples were
centrifuged, and an aliquot from each sample was taken out.
The concentration of the released insulin was determined using
a UV-vis spectrophotometer at 280 nm. To determine the
insulin release mechanism, the in vitro insulin release data were
tted to standard zero-order, rst-order, Higuchi and Kors-
meyer–Peppas models,19–21
AUCðOralÞ ꢄ DOSE
Relative bioavailability ðRAÞ ¼
ðScÞ ꢄ 100%
Mt
AUCðScÞ ꢄ DOSE
¼ Ktn
(3)
ðOralÞ
MN
(4)
where Mt and MN are the absolute amounts of insulin released
at time (t); K is a constant showing the structural and geometric
characteristics of the device; n is the release exponent reecting
the diffusion mechanism. Values of the release exponent (n) ¼
where AUC is the total area under the curve of plasma insulin
concentration versus time.
0.45, 0.45 < n < 0.89 and 0.89 indicate Fickian (Case I) diffusion, 2.11. In vivo toxicity assay of the polymers
non-Fickian (anomalous) transport, and diffusion and zero-
Acute toxicity studies were conducted with peroral treatment of
order (Case II) transport, respectively.
chitosan and dendronized chitosan (DCTS) nanoparticles at a
dose of 300 mg kgꢂ1 b.w. per day in mice. Experimental animals
were divided into the following three groups (n ¼ 6): Group I
(control), only 0.5 mL 0.9% saline perorally; group II, chitosan
2.8. Mucoadhesion studies of self-assembled nanoparticles
in animal model
Mucoadhesion studies were carried out on small intestine nanoparticles (300 mg kgꢂ1 b.w. per day) orally; group III, DCTS
tissue freshly excised from mice according to our previously nanoparticles (300 mg kgꢂ1 b.w. per day) orally.
described method.21 Saline was ushed through freshly excised
On the next day, urine was collected, and the animals were
intestinal tissue to remove luminal contents, and the sample anesthetized to collect blood from the retro-orbital vein. Serum
was placed in a glass support with the help of adhesive. The was separated and stored at ꢂ20 ꢃC for assessment of different
polymer/insulin self-assembled nanoparticles (freeze dried) biochemical parameters.
were uniformly spread and allowed to interact with the intes-
2.11.1. Liver function test. The serum glutamate pyruvate
tinal mucosal lining for 10–15 min and then were mounted at transaminase (SGPT), serum glutamate oxaloacetate
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transaminase (SGOT) and lactate dehydrogenase activity (LDH) stretch).4 Fig. 2b shows the FTIR spectrum of N-carboxy-
were estimated to analyse hepato-toxicity in the treated mice. ethylchitosan methyl ester. A new strong absorption peak at
2.11.2. Nephro-toxicity test. Urine samples were analyzed 1728 cmꢂ1 appears due to the stretching vibration of the
for quantitative measurement of creatinine (serum and urine), carbonyl bond (C]O) of the ester group. The FTIR spectrum of
micro-protein and urea to evaluate the nephro-toxicity in the PAMAM dendrimer (G 2.0, Fig. 2c) shows the strong absorption
treated animals. Again, urine samples were qualitatively peaks at 3281 cmꢂ1 and 1545 cmꢂ1 corresponding to the N–H
analyzed using multistix reagent strips.
bond of primary amine (–NH2) groups. The strong absorption
2.11.3. Pathohistological diagnosis. For pathohistological peak at 1636 cmꢂ1 is associated with the amide N–H bond in the
diagnosis, liver and kidney were xed in 10% phosphate buff- –CONH group. From Fig. 2d, it is found that the intensity of
ered formalin, and tissue sections were stained with haema- absorption peaks at 1651 cmꢂ1 (NH CO(I) stretch), 3282 cmꢂ1
toxylin and eosin (H&E) and observed under microscope.
,
and 1557 cmꢂ1 (N–H stretching and bending, respectively)
increased aer reaction of the PAMAM dendrimer (G 2.0) with
the N-carboxyethylchitosan methyl ester, and the peak for ester
group at 1728 cmꢂ1 disappeared, indicating the formation of
dendronized chitosan.
2.12. Statistical analysis
The acute toxicity results are expressed as mean ꢁ SE, n ¼ 6. The
signicance level was determined by one-way ANOVA following
Tukey's post hoc test. p < 0.05 was considered as signicant.
The 1H NMR spectra of chitosan, N-carboxyethylchitosan
methyl ester and DCTS are shown in Fig. 3. In Fig. 3a, typical
peaks at 3.4–4.0 ppm are assigned to the glucosamine unit (H3,
H4, H5, H6) of chitosan; the peak at 3.1 ppm is responsible for
H2; the peak at 2.1 ppm is assigned to the methyl protons of the
N-acetyl group. Aer the reaction between chitosan and methyl
acrylate, a new triplet peak appeared at 2.2–2.7 ppm in the NMR
spectrum of N-carboxyethylchitosan methyl ester (Fig. 3b); this
peak is attributed to the methylene protons of methyl ester.
Aer further reaction of PAMAM dendrimer (G 2.0) with N-car-
boxyethyl chitosan methyl ester, the peak for the methylene
proton of the methyl ester disappeared, and another new broad
3. Results and discussion
3.1. Characterization of polymers
FTIR spectra of the polymers are shown in Fig. 2. The FTIR
spectrum of chitosan (Fig. 2a) shows the basic characteristic
peaks at 3427 cmꢂ1 (O–H stretch and N–H stretch overlap), 2922
cmꢂ1 and 2859 cmꢂ1 (asymmetric and symmetric stretching of
C–H, respectively), 1652 cmꢂ1 (NH–CO(I) stretch), 1597 cmꢂ1(N–
H bend), 1154 cmꢂ1(bridge –O stretch), and 1092 cmꢂ1 (C–O
Fig. 2 The FT-IR spectra of (a) chitosan, (b) N-carboxyethylchitosan methyl ester, (c) PAMAM (G 2.0) and (d) DCTS.
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from 222 kDa to 25 kDa. However, its molecular weight was
slightly increased from 25 kDa to 37.4 kDa and 41.86 kDa due to
the incorporation of methyl acrylate and PAMAM dendrimer
(G 2.0), respectively, on the chitosan backbone. On one hand,
the water solubility of chitosan was increased aer conjugation
of PAMAM dendrimer with chitosan because of the augmenta-
tion of primary amine groups of dendronized chitosan. On the
other hand, we got insoluble products (data not shown) when
we used an equivalent or lower amount of PAMAM dendrimer
during the conjugation of N-carboxyethyl chitosan methyl ester
(NCME) and PAMAM dendrimer. This is possible due to inter-
molecular crosslinking. To avoid this, we used an excess
amount of PAMAM dendrimer compared to the methyl ester of
chitosan. When the lower or equivalent amount of PAMAM was
used, the free primary amine groups of the chitosan methyl
ester might have reacted with other chitosan methyl esters and
resulted in an insoluble crosslinked product. However, when
the PAMAM dendrimer is in excess, its reaction with the chi-
tosan methyl ester is preferred due to the smaller size of
PAMAM dendrimer in comparison to the size of the chitosan
methyl ester, and this reaction results in water-soluble
dendronized chitosan. Similar results have been observed in
earlier reports.5
3.3. Particle size, zeta potential and morphology of polymer/
insulin self-assembled nanoparticles
An appropriate particle size of polycation/insulin nano-
complexes is a very important parameter for oral insulin
delivery, as particles below 1000 nm in diameter are reported
to be more desirable for better absorption through the
intestinal tract.23 The diameters of the self-assembled nano-
particles at various polycation/insulin ratios were measured
by dynamic light scattering (DLS), and the average particle
size is shown in Fig. 4a. It is observed that the average
particle size of DCTS/insulin nanocomplexes at different
weight ratios is 80–175 nm, whereas the average particle size
of unmodied chitosan/insulin nanoparticles at similar
weight ratios are comparatively larger and in the range of
150–475 nm.6 This may be attributed to the availability of
more positive charges on the surface of DCTS (due to graing
Fig. 3 The 1H NMR spectra of (a) chitosan, (b) N-carboxyethylchitosan
methyl ester, and (c) DCTS.
peak with multiplicity appeared at 2.2–3.0 ppm, indicating the
formation of dendronized chitosan (Fig. 3c).
3.2. Molecular weight and solubility
The molecular weight and solubility of chitosan and its deriv- of PAMAM) compared to the native chitosan, thereby tightly
atives are shown in Table 1. To improve the water solubility of entrapping the excess amount of insulin (negatively charged)
chitosan, CTS was depolymerised by oxidation using nitrous to form smaller nanoparticles. Again, a slight increase in
acid, and the molecular weight of native chitosan was decreased particle size is documented with further augmentation in
Table 1 Molecular weight and solubility of chitosan and modified chitosana
Solubility
Average molecular
Polymeric compound
weight (Mw) (kDa)
H2O
0.1 M HCI
0.1 M NaOH
Chitosan
Depolymerized chitosan
N-Carboxyethylchitosan methyl ester
PAMAM (G 2.0)
222
25
37.4
1.321
41.86
I
I
P
S
S
S
S
I
S
S
I
I
P
I
I
N-Carboxyethylchitosan methyl ester graed PAMAM (G 2.0) or dendronized chitosan
a
I ¼ insoluble, P ¼ partly soluble, S ¼ soluble.
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polymer concentration; this may be attributed to the strong
The zeta potential values of the self-assembled nanoparticles
repulsion between excess positive charges available on the are shown in Fig. 4b. The zeta value of insulin is found to be
surface of polymeric molecules aer complete electrostatic ꢂ10.06 mV, whereas a sharp increase in zeta potential of the
interaction with the negatively charged insulin. Similar chitosan/insulin and DCTS/insulin nanoparticles at 0.5 : 1
results are also found in our previous study.6 In previous weight ratio is observed, at +2.83 mV and +6.82 mV, respectively.
studies, chitosan nanoparticles (265–387 nm) were used for Again, with an increase in weight ratio to 1 : 1 and 2 : 1, the zeta
oral insulin delivery,24 and other polymeric systems with a value is found to further increase to +8.9 mV and +18.44 mV for
particle size of 200 nm have also been reported for insulin chitosan/insulin and +16.46 mV and +25.71 mV for DCTS/
delivery.22 In the present article, the particle size is compa- insulin nanoparticles, respectively (Fig. 4b). The zeta potential
rable to earlier reports.
of the nanoparticles is positive, although insulin is negatively
Fig. 4 (a) Particle size, (b) zeta potential, (c) AFM images of chitosan/insulin and (d) AFM images of DCTS/insulin nanoparticles, (e) SEM images of
chitosan/insulin and (f) SEM images of DCTS/insulin nanoparticles.
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charged; this has happened due to the neutralization of all the (22–27%) as well as insulin encapsulation efficiency of 74%,
negative charges on the insulin molecules by the cationic 89% and 95%. As DCTS carries more cationic charges over
charges on polymers (chitosan and DCTS) via electrostatic unmodied chitosan, stronger ionic interaction between the
interaction (Fig. 4b). As DCTS carries comparatively more negative charges of insulin and the positive charges of DCTS
surface cationic charges than native chitosan, the zeta potential may induce better loading capacity and improved insulin
of DCTS/insulin nanoparticles is found to be more positive as encapsulation.
compared to native chitosan/insulin nanocomplexes. There-
fore, the positive zeta potential value of nanoparticles can
provide prolonged attachment with the negatively charged
intestinal mucus layer, facilitating sustained release of encap-
3.5. In vitro insulin release prole in simulated gastric uid
(SGF) and simulated intestinal uid (SIF)
sulated insulin by increasing their stability with effective The cumulative release proles of insulin from DCTS/insulin
protection against self-aggregation.
nanocomplexes were investigated in pH gradients correspond-
Both atomic force microscopy and scanning electron ing to the gastrointestinal tract, i.e., pH 2.0 (simulated gastric
microscopy were utilized to observe the morphology and size of uid, SGF) and pH 7.4 (simulated intestinal uid, SIF) at 37 ꢁ
the prepared nanocomplexes. Fig. 4c and d show representative 0.5 ꢃC. The amount of released insulin is measured by UV
tapping mode AFM, and Fig. 4e and f show SEM micrographs of spectrophotometer at 280 nm, as shown in Fig. 5c. It is observed
the chitosan/insulin nanoparticles and DCTS/insulin self- that in SGF, at pH 1.2, chitosan/insulin self-assembled nano-
assembled nanoparticles, respectively. Almost all of the nano- particles retard the immediate release of insulin, releasing only
particles are of compact structure, having spherical or sub- ꢀ16% of the encapsulated insulin in the initial 2 h, while DCTS/
spherical shape with smooth surface. The diameter of the insulin self-assembled nanoparticles show better protection of
DCTS/insulin self-assembled nanoparticles is roughly within insulin, releasing only ꢀ10% for the same period of time. As
the range of 65–100 nm (Fig. 4d and f), much smaller than insulin is more strongly bound to DCTS due to the presence of
unmodied chitosan insulin nanoparticles of 90–120 nm size more positively charged amines compared to chitosan, there is
(Fig. 4c and e) at the same weight ratio, which is quite consis- less possibility of penetration of acidic media and thereby less
tent with the DLS measurement. Furthermore, the higher release of insulin from DCTS/insulin nanoparticles. The
number of cationic charges offered by DCTS (due to graing of residual protonated amines in both chitosan and DCTS do not
PAMAM) is able to tightly condense higher amounts of nega- allow them to interact with the protonated medium, as charges
tively charged insulin to form smaller particles than native of similar nature repulse. On the contrary, in SIF (pH 7.4), the
chitosan. The micrographs also demonstrate that the nano- amount of released insulin from the native chitosan/insulin
complexes are scattered independently over the eld, suggest- self-assembled nanocomplexes is about 82% of the encapsu-
ing possible stabilization against self-aggregation. Our previous lated amount. Aer the rst hour, only ꢀ28% insulin release is
reports also showed similar results.6 SEM images of self- observed, which reaches >74% on the 5th h. On the other hand,
assembled nanocomplexes also show that the polyplexes are DCTS/insulin self-assembled nanoparticles show more sus-
spherical in shape, and the particle size is in the range of 65–100 tained insulin release, where initially (1–2 h), 40–50% of insulin
nm, which is also in good agreement with the AFM micro- is released, but above 90% of insulin is released aer 6 h of
graphs. These results are very similar to the previously reported incubation. The presence of residual positive charges in both
ones.23 The above results conrm that the dendronized chitosan chitosan and DCTS (these are available even aer their partial
is more capable of condensing signicant amounts of insulin neutralization with insulin) allow them to interact with the
into nanoparticles of sufficiently small sizes for uptake by moderately alkaline medium, which thereby penetrates these
intestinal epithelial cells.
nanoparticles, leading to more release of insulin in SIF
compared to SGF (Fig. 5c). Thus, the pH-responsive, prolonged
release pattern of insulin can facilitate its oral administration in
the animal model, minimising the chances of hypoglycemia.
The mathematical analysis also shows the diffusion-controlled
3.4. Insulin loading capacity and insulin encapsulation
efficiency
Encapsulation and loading capacity of insulin is another and the swelling-controlled insulin release pattern from the
important aspect in oral delivery. Poor encapsulation efficiency nanoparticles. Results related to experiments in the pH gradient
restricts the wide use of polymeric carriers, as they would be medium are shown in Table 2. It is noted from the obtained
unable to pursue the desired function aer administration. correlation coefficient values (R $ 0.99) that the release data t
Therefore, the percentage of insulin loading capacity and well to the empirical equation. The “n” release exponent ranged
insulin encapsulation of the nanoparticles with different weight from 0.53 to 0.78 (Table 2), indicating a non-Fickian (anoma-
ratios (0.5 : 1, 1 : 1 and 2 : 1) was investigated, as shown in lous) transport (0.45 < n < 0.89) for all the tested nanoparticle
Fig. 5a and b. Insulin loading capacity of chitosan/insulin samples.19,20 So, the results indicate both diffusion-controlled as
nanoparticles at different weight ratios varies between 15– well as swelling-controlled insulin release (anomalous transport
22%, and the encapsulation efficiency of chitosan/insulin or non-Fickian diffusion mechanism) from the self-assembled
nanoparticles at 0.5 : 1, 1 : 1 and 2 : 1 weight ratios are 63%, nanoparticles.
78% and 86%, respectively; whereas DCTS/insulin nano-
Fig. 5d demonstrates the signicant mucoadhesive property
particles at these weight ratios showed better insulin loading of the DCTS/insulin self-assembled nanoparticles, which
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Fig. 5 (a) Percentage of insulin encapsulation, (b) insulin loading capacity, (c) cumulative insulin release profile, (d) mucoadhesion study in
excised animal tissue, (e) in vivo pharmacological response and (f) serum insulin concentration after peroral treatment of self-assembled
nanoparticles.
remained attached to the mouse intestinal lumen aer contin- electrostatic interaction with the negative charged mucous layer,
uous washing with buffer (pH 7.4) for 30 min. The mucoadhesive offering prolonged resident time and helping sustained release of
features of the nanoparticles could be attributed to an interaction the encapsulated insulin. Moreover, increased paracellular
between the positive charged amino groups of dendronized chi- permeability could be achieved by redistribution of F-actin and
tosan and the negatively charged sialic acid groups of mucin, the tight junction's protein through the interaction between the
which provides a prolonged interaction within the intestinal positively charged nanoparticles with the negatively charged cell
mucosa. Hence, the mucoadhesive nanoparticles have strong surfaces and tight junctions.
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Table 2 Estimated parameters and drug release mechanism of self-assembled nanoparticles
Korsmeyer–Peppas
Drug transport
mechanism
Sample
Zero-order r2
First-order r2
Higuchi r2
r2
n
pH 1.2
CS/insulin nanoparticles
DCTS/insulin nanoparticles
0.8968
0.9295
0.9215
0.9503
0.9891
0.9983
0.9921
0.998
0.5651
0.5396
Non-Fickian
Non-Fickian
pH 7.4
CS/insulin nanoparticles
DCTS/insulin nanoparticles
0.893
0.7912
0.9785
0.9662
0.9644
0.9515
0.9895
0.9665
0.7826
0.7822
Non-Fickian
Non-Fickian
shock in diabetic mice. However, the administration of free oral
insulin solution does not have any pronounced effect on gly-
cemia. The relative availability (RA) of insulin was found to be
ꢀ9.19% with DCTS/insulin nanoparticles, which is almost
double that of native chitosan/insulin nanoparticles (ꢀ5.28%),
suggesting signicant absorption of insulin in an active form.
Bioavailability for oral insulin trials has been reported to be low,
as only <2.5% insulin bioavailability has been documented with
pH-responsive poly(methacrylic-g-ethylene glycol) hydrogel
microparticles in oral delivery;25 ꢀ4% bioavailability has been
reported with chitosan nanoparticles;26 7% of bioavailability is
observed in Alg/Chit nanoparticles;23 ꢀ4.43% insulin bioavail-
ability was found in our previous study using N-succinyl chito-
san graed polyacrylamide hydrogels;21 and ꢀ8.11% of
bioavailability was found using core shell nanoparticles.27 In the
present investigation, the encapsulated insulin is well protected
from enzymatic degradation, as it is sheltered within the
nanoparticles. As enzymes (pepsin and trypsin) in the GI tract
are positively charged species, they cannot bind to the positively
charged nanoparticles. Therefore, an intimate contact of the
nanoparticles with the intestinal wall is established, allowing a
signicant amount of insulin absorption, although some
amount of the released insulin is probably damaged by the
classical degradation phenomena during its transit through the
GI tract. While the zwitterionic chitosan/PAMAM complex has
been reported as a potential drug carrier to solid tumors,28 in
the present investigation, self-assembled nanoparticles of
PAMAM-graed chitosan for oral insulin delivery showed
excellent hypoglycemic effects in in vivo model.
3.6. In vivo pharmacological response
In vivo pharmacological responses of DCTS/insulin self-
assembled nanoparticles aer peroral treatment in diabetic
mice are illustrated in Fig. 5e. Both chitosan/insulin and DCTS/
insulin self-assembled nanoparticles show hypoglycemic effects
aer oral delivery, as compared to the control (subcutaneous
injection of insulin 5 IU kgꢂ1 b.w.). But DCTS/insulin self-
assembled nanoparticles exhibit more pronounced effects in
the lowering of blood glucose level compared to native chitosan
nanoparticles. Aer subcutaneous injection, the blood glucose
level starts decreasing signicantly within 30–45 min and rea-
ches 85 mg dLꢂ1 at the 2nd h post-administration. However, the
hypoglycemic effect is further sustained for only 2 h and
subsequently returns to its basal level at the 5th h of adminis-
tration (Fig. 5e). On the other hand, oral administration of
chitosan/insulin self-assembled nanoparticles and DCTS/
insulin self-assembled nanoparticles (both 50 IU kgꢂ1 b.w.)
result in the signicant reduction of glycemia. In the case of
these nanoparticles, the reduction in the glucose level in dia-
betic mice is initiated aer 2–3 h of administration. However,
DCTS/insulin self-assembled nanoparticles show sustained
hypoglycemic effects for 8 h, reducing the blood glucose level up
to 119 mg dLꢂ1. On the other hand, chitosan/insulin nano-
particles lower the glucose level down to 165 mg dLꢂ1 at the 5th
h, and the effects sustain only for another 30 min to an hour.
The corresponding serum insulin concentrations at different
time proles were studied and are shown in Fig. 5f. It is
observed that the subcutaneous injection of insulin solution (5
IU kgꢂ1 b.w.) results in the maximum serum insulin concen-
tration at the 3rd h post-injection, whereas the oral adminis-
tration of chitosan/insulin self-assembled nanoparticles at a
dose of 50 IU kgꢂ1 b.w. shows the maximum concentration of
serum insulin at the 5th h of administration. Again, DCTS/
3.7. Evaluation of in vivo toxicity aer oral administration of
dendronized chitosan (DCTS)
3.7.1. Minimum lethal dose (MLD). No lethality is observed
insulin self-assembled nanoparticles exhibit the maximum up to 300 mg kgꢂ1 b.w. of DCTS nanoparticles at peroral dose.
serum insulin at the 6th h post-administration. In contrast, a
3.7.2. Liver function test analysis. The liver-specic
negligible amount of serum insulin (bovine insulin) is detected enzymes ASAT (asparate aminotransferase) and ALAT (alanine
in animals treated perorally with insulin solution. From the aminotransferase) are signicantly elevated in hepatobilliary
AUC(0–10h) data of the DCTS/insulin nanoparticle-orally treated diseases with a direct correlation with liver parenchyma
mice, the relative bioavailability (RA) is found to be ꢀ9.19%, damage. As shown in Fig. 6a, the SGOT value is 64.8 U Lꢂ1 for
where the RA of chitosan/insulin nanoparticles is only ꢀ5.28%. the control animal and 94.8 U Lꢂ1 and 114.75 U Lꢂ1 for the
Orally administered DCTS/insulin nanoparticles remain animals treated with chitosan nanoparticles and DCTS nano-
biologically active and show prolonged hypoglycemic effects for particles (300 mg kgꢂ1 b.w.), respectively. These values are
2–8 h (Fig. 5e) without producing any sudden hypoglycemic within the reference range of SGOT (55–251 U Lꢂ1) for mouse.
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Fig. 6 Acute toxicity study of DCTS nanoparticle-treated animals, (a) serum SGOT, (b) serum SGPT, (c) serum creatinine, (d) serum LDH, (e) urine
creatinine, (f) urine microprotein and (g) urine urea.
Again, Fig. 6b shows that the SGPT value is 35.02 U Lꢂ1 for the DCTS nanoparticles. The values are within the reference range
control animals, 47.5 U Lꢂ1 for the animals treated with chito- of 28–184 U Lꢂ1 in mouse. The serum creatinine level is 0.42 mg
san nanoparticles, and 69.45 U Lꢂ1 for the animals treated with dLꢂ1 for the control animal, 0.61 mg dLꢂ1 for the animals
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treated with chitosan nanoparticles and 0.67 mg dLꢂ1 for the Furthermore, proteins (albumin and globulin fractions) are
animals treated with DCTS nanoparticles (Fig. 6c), where the known to be involved in the maintenance of the normal
normal reference range is 0.7–1.1 mg dLꢂ1. Again, the LDH distribution of water between the blood and the tissues. Mainly,
value (Fig. 6d) is 241.2 U Lꢂ1 for the control animal, 358.1 U Lꢂ1 proteinuria occurs with increased glomerular permeability or
for animals treated with chitosan nanoparticles and 379.7 U Lꢂ1 defective tubular reabsorption. Therefore, the concentration of
for the animals treated with DCTS nanoparticles; these values urine microprotein could be an indicator of renal disease or any
are within the normal reference range of 230–460 U Lꢂ1
.
glomerular damage, and increased urea is generally accompa-
Although signicant changes in SGPT, SGOT and LDH values nied by renal damage. Fig. 6f shows that the concentration of
are found in comparison to the control, all these values are urine microprotein is 93 mg per 24 h for the control animal,
within the reference range, suggesting no damage of the liver, 115.7 mg per 24 h for the animals treated with chitosan nano-
and no toxicological and functional disorder in the animal by particles and 125.1 mg per 24 h for those treated with DCTS
the treatment of DCTS nanoparticles.
nanoparticles. A signicant change is found in comparison to
3.7.3. Assessment of nephro-toxicity. Urine creatinine is an the control group, but the microprotein values are within
indicator of the urinary tract obstruction, kidney failure, dehy- normal reference range of 28–140 mg per 24 h. The concentra-
dration, severe kidney disease, shock, renal outow obstruction tion of urea in urine was also measured and is presented in
and acute tubular necrosis. Therefore, the concentration of Fig. 6g. The urea concentration is 491.8 mmol in 24 h for the
urine creatinine was measured and is reported in Fig. 6e. The animals treated with DCTS nanoparticles (300 mg kgꢂ1 b.w.)
creatinine value in control mice is 9.4 mg kgꢂ1 b.w., it is 14.79 and 458.6 mmol in 24 h for those treated with chitosan nano-
and 11.95 mg kgꢂ1 b.w. in mice treated with DCTS and chitosan particles. A signicant change is observed as compared to the
nanoparticles (300 mg kgꢂ1 b.w.), respectively. The values fall control animal group (335 mmol in 24 h), although these values
within the reference range of 8.4–24.6 mg kgꢂ1 b.w. are within the reference range of 333–583 mmol per 24 h. Thus,
Fig. 7 Sections of liver and kidney (H&E staining, magnification 40ꢄ) of control and treated (peroral chitosan and DCTS nanoparticles) mice.
Table 3 Qualitative analysis of different biochemical parameters of urine in polymeric nanoparticle-treated animals
Peroral treatment with dendronized
Control animal
(treated with 0.9% NaCI)
Peroral treatment with chitosan
nanoparticles (300 mg Kgꢂ1 b.w)
chitosan nanoparticles
Parameters
(300 mg Kgꢂ1 b.w)
Urobilinogen
Protein
pH
0.2
Negligible
7.5
1
0.2
Negligible
8.0
Trace
7.5
Blood
Specic gravity
Ketone
Bilirubin
Glucose
Moderate (non-haemolysed)
1.015
15–30
Negligible
Negligible
Moderate (haemolysed)
1.020
40
+
Trace (haemolysed)
1.005
Negligible
Negligible
Negligible
Negligible
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all these studies indicate that DCTS can be a safe polymer for
oral insulin delivery, indicating no renal dysfunction.
5 K. Sarkar and P. P. Kundu, Int. J. Biol. Macromol., 2012, 51,
859.
6 P.
Mukhopadhyay,
K.
Sarkar,
M.
Chakraborty,
3.8. Pathohistological diagnosis
S. Bhattacharya, R. Mishra and P. P. Kundu, Mater. Sci.
Eng., C, 2013, 33, 376.
No gross histopathological changes in liver and kidney sections
(Fig. 7) were observed. The liver sections (H&E staining) of the
treated animal group exhibit a central vein with the radiating
hepatic cells, similar to the control tissue. The sections of the
kidney also show the renal corpuscles surrounded by Bowman's
capsule and kidney tubules, lined by the simple cuboidal
epithelium. A urinary space (appearing as a clear space) is also
visible on the histological slides. The glomerulus (tu of
capillaries) appears as a large cellular mass, implying no hepatic
or renal toxicity in the animal model. Furthermore, qualitative
analyses of different biochemical parameters (Table 3) also
indicate its safety measures.
7 E. A. Stepnova, V. E. Tikhonov, T. A. Babushkina,
T. P. Klimova, E. V. Vorontsov, V. G. Babak, S. A. Lopatin
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A low-toxicity, pH-sensitive, water-soluble chitosan derivative
was successfully prepared for oral insulin delivery. The self-
assembled nanoparticles with ꢀ27% insulin loading and
ꢀ95% insulin encapsulation efficiency are prepared through a
mild process, avoiding harmful chemicals. The pH-responsive
nanoparticles are able to establish a prolonged hypoglycemic
effect compared to those obtained from oral insulin and native
chitosan nanoparticles, revealing an almost doubly-enhanced
insulin bioavailability (ꢀ9.19%) compared to native chitosan
nanoparticles (ꢀ5.28%). Since no systemic toxicity is observed
in experimental trials, one is assured that the dendronized
chitosan can be a promising polymeric vehicle in oral insulin or
other therapeutic drugs due to its efficient and safe
administration.
Acknowledgements
21 P.
Mukhopadhyay,
K.
Sarkar,
S.
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We are highly grateful to the Department of Science and Tech-
nology, Government of West Bengal for their nancial support
for this work and the project entitled ‘Synthesis of derivatives of
chitosan and their IPNs for oral insulin delivery’; Sanction no. 428
(Sanc./ST/P/S & T/2G-7/2011).
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