Article
Journal of
Pharmaceutical Sciences
and Pharmacology
Copyright © 2014 American Scientific Publishers
All rights reserved
Printed in the United States of America
Vol. 1, 131–140, 2014
www.aspbs.com/jpsp
Structure-Toxicity Relationship of Chemically Modified
Chitosan as an Oral Protein Drug Delivery Carrier
Subrata Biswas1 , Mainak Chattopadhyay2 , Kalyan Kumar Sen3 ,
Malay Kumar Saha1 , and Himangshu Sekhar Maji2 ∗
1
National Institute of Cholera and Enteric Diseases, Indian Council of Medical Research, Beliaghata, Kolkata 700010, India
Bengal School of Technology, Sugandha, Delhi Road, Hooghly 712102, West Bengal, India
3
Gupta College of Technological Sciences, Ashram More, G.T. Road, Asansol 713301, West Bengal, India
2
N,N,N-Trimethylated chitosan has been extensively used as an absorption enhancer for macromolecules and as a
mucosal vaccine carrier. Both of these properties are molecular weight (MW ) and degree of quaternization (DQ) dependent. The aim of the present study was to evaluate the impact of some synthesized trimethylated chitosan with various
MW and DQ on biological systems in terms of biocompatibility and providing guidelines for the rational design of chitosan
derivatives for effective and safe drug delivery. For this purpose, cytotoxicity in HT29 cell line was monitored using the
MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observation was carried
out as indicators for blood cell compatibility. Furthermore, haemolysis was quantified spectrophotometrically for evaluation
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of haematotoxicity. Two polymer doses were used
for subby
acute
toxicity
in BALBc mice. After oral administration,
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On:Signs
Thu, of
15toxicity
Jun 2017
animals were monitored over 28 days
and necropised.
were15:09:52
evaluated via mortality and histopathology
Copyright:
American
Scientific
Publishers
of liver, kidney and spleen. The magnitude
of the cytotoxic
effects
all chitosan
derivatives were found to be concentration
dependent. Higher concentration of trimethylated chitosan with 22% DQ and native chitosan did not cause significant
abnormalities among experimental group of mice, whereas, trimethylated chitosan with higher DQ as 50% and 61% may
lead to concentration dependent cytotoxicity, hematotoxicity and increased renal and hepatotoxicity. All assays yielded
comparable results and concluded cationic charge density of the chitosan derivatives seems as key parameters for the
interaction with the cell membranes and consequently the cell damage. These results indicate that structure-toxicity
relationship is necessary to optimize the degree of modification of chitosan for the development of biocompatible and
biodegradable derivatives.
KEYWORDS: Biocompatible, Cytotoxicity, Haemolysis, Histopathology, Sub-Acute Toxicity, Trimethyl Chitosan.
INTRODUCTION
Chitosan (CS) is a natural linear polysaccharide consisting
of 1 → 4-D-glucosamine and 1→ 4-N -acetyl Dglucosamine units and is obtained by partial deacetylation of the natural polymer chitin [Aspden, 1996; van der
Lubben, 2003]. It can be found in a variety of forms differing in size (average molecular weight; MW ) and degree
of deacetylation (DD). It has gained increasing attention
in the field of drug delivery system due to its favourable
biological properties, such as non toxicity, biodegradability, mucoadhesive properties and biocompatibility [Kumar,
∗
Author to whom correspondence should be addressed.
Email: [email protected]
Received: 2 January 2014
Accepted: 28 February 2014
J. Pharm. Sci. Pharmacol. 2014, Vol. 1, No. 2
2004; Agnihotri, 2004]. However, solubility of CS only
restricted to acidic solution limits its applications to bioactive agents such as gene delivery carriers, peptide carriers and drug carriers [Kotze, 1999]. Partial quaternization
of CS’s primary amine groups has been used to obtain a
CS derivative that is soluble at physiological conditions.
N ,N ,N -Trimethylated chitosan (CS TM) has been shown
to have mucoadhesive properties, penetration enhancing
properties and therefore, can be applied to delivery of
hydrophilic macromolecules, such as peptides and proteins [Sandri, 2005]. This capability as enhancer is due
to opening the tight junctions between adjacent epithelial
cells through interactions between the protonated (positively charged) amino groups on the C-2 position and the
negatively charged sites on the cell membrane and/or in
the tight junctions [Hamman, 2003].
2333-3715/2014/1/131/010
doi:10.1166/jpsp.2014.1016
131
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
Biswas et al.
Several studies have been performed to determine the
the reduction of NAD+ by LDH. The resulting reduced
optimal degree of quaternization (DQ) for either transepNADH/H+ is utilized in the stoichiometric conversion
ithelial delivery of low molecular weight drug molecules
of a tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5and/or proteins, or to increase transfection potential of
phenyltetrazolium chloride (INT) [Weyermann, 2005]. The
complexes of CS TM with plasmid DNA. It has been
LDH test provided information on the effects of polymers
shown that CS TM with higher DQ would display optiafter short incubation times. The HT 29 human epithelial
mum mucoadhesive and penetration enhancing properties
cell line was selected in the present study as a convenient
[Verheul, 2008]. These chemically modified polymers need
in vitro model to assess polymer toxicity. Changes in cell
to be biocompatible, non-toxic, non-immunogenic, and
morphology and erythrocyte aggregation were also used
biodegradable, with a high drug-carrying capability and
as indicators of cell survival by microscopic investigation.
controlled release of drugs at the target site [Sgouras,
Furthermore, the blood compatibility of the modified poly1999]. The term “biocompatibility” encompasses many
mers was quantified by spectrophotometric measurement
different properties of the materials, however, three difof haemoglobin release from erythrocyte after polymer
ferent aspects of the biomaterial screening refers to their
treatment. In vivo exposure to polymers is likely to have
in vitro cytotoxicity, blood-compatibility behaviour and
potential impact on the liver, spleen and kidney since expoin vivo sub-acute toxicity [Jintapattanakit, 2008; Kean,
sure to these particles is likely to occur through ingestion,
2010; Amidi, 2010].
distribution and clearance by the liver, spleen and kidney
In this study, two different MW of CS and its
and acute toxicity was investigated in Swiss albino mice
trimethyalted derivatives of different DQ considered as
via organ histopathology.
drug delivery systems were examined in terms of bioThe toxicity end points selected in the current study repcompatibility and biodegradation, especially for oral proresent vital biological functions of the mammalian system
tein delivery. These polymers are used to form neutral
as well as provide a general sense of toxicity in a relatively
or positively charged complexes with protein due to elecshort time. The results described in this paper provide
trostatic interactions [Leclercq, 2003]. Polycation-protein
information of in vitro and in vivo toxicity of two different
complexes were usually found to be less cytotoxic than
molecular weight of CS with different DQ, thereby propolymers [Cherng, 1996]. But administration of polymerviding guidelines for the rational design of CS derivatives
drug complexes to blood can result in their dissociafor effective and safe drug delivery.
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tion and aggregation due to interactions with erythrocytes
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and plasma proteins yielding non-complexed
polycations
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American Scientific
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MATERIALS
AND METHODS
[Fischer, 2003]. The free polymer then interacts with cell
Materials
membranes, extracellular matrix proteins and blood comHigh molecular weight CS (CS HMW) (MW 310–
ponents leading to undesired side effects [Kircheis, 2001].
375 kDa) and middle molecular weight CS (CS MMW)
CS TM administered orally can be absorbed through gastro
(MW 190–300 kDa) were purchased from Sigma
intestinal tract (GIT), then degraded gradually in blood and
Aldrich, USA. DMEM (Dulbecco’s Modified Eagle’s
liver, and finally discharged from the body through urine
Medium) and HEPES [N -(2-hydroxyethyl piperazine-N [Dong, 2010]. It is necessary for the acknowledgement of
(2-ethanosulphonic
acid)] were procured from Sigma
CS TM’s biological safety to study whether it has effects
(USA). 96-well flat bottom polystyrene plate was puron relevant systems and organs after being absorbed and
chased from Nunc (Denmark). Ultrapure water (MilliQ,
degraded gradually in vivo. CS degradation rate can be
Waters, USA) was used to prepare all solutions and freshly
affected by the polymer’s MW and degree of substitution
prepared solutions were used in all experiments.
[Kean, 2010].
Human intestinal epithelial cell (HT 29) were cultured
Different techniques were used to measure changes
and maintained in DMEM with supplemented with 10%
in cell viability and interactions with erythrocyte.
fetal bovine serum and 1% antibiotic-antimycotic solution
The MTT (3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetra(Invitrogen, Carlsbad, CA). Cells were maintained in 5%
zolium bromide) assay was selected to determine detriCO2 at 37 C humidified incubator for 10 days.
mental intracellular effects on mitochondria and metabolic
Specific pathogen-free male BALB/c mice (6 weeks old,
activity and is frequently used for screening of cytotoxicity
22–25 g) and male New Zealand rabbits weighing 2.0–
[Mosmann, 1983]. MTT is oxidized by mitochondrial
2.5 kg were obtained from the animal house of Gupta
dehydrogenases in living cells and gives a dark purple
College of Technological Sciences (GCTS), India were
formazan product. Damaged or dead cells show little or
used. They were kept into individual cages with autoclaved
no dehydrogenase activity [Klajnert, 2006]. Membrane
food and water ad libitum. All animals were housed in
damage effects caused by polymer were analyzed by the
an aseptic room with 12 h light/dark cycle and temperarelease of the cytosolic enzyme lactate dehydrogenase
ture of 20 ± 2 C. Animals were acclimated to the new
(LDH) which is a potential site of interaction of cationic
environments for 1 week before being used for immunizamacromolecules [Chokakulnimitr, 1995]. The LDH activtion. Animal study protocol was reviewed and approved by
ity is determined in an enzymatic test and based on
132
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
Biswas et al.
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
the Institutional Animal Care and Use Committee, GCTS,
India.
Synthesis and Determination of DQ of CS TM
The two-step reaction pathway to synthesize CS TM with
a various DQ is based on the method published by Verheul
et al., with some modifications [Verheul, 2008]. In the first
step N -alkyl CS was prepared by introducing a methyl
group into the amine group of CS via Schiff’s base followed by reducing the C N bond, in the next step
methyl iodide was used to produce CS-TM. CS-TM products synthesized were analyzed by 1 H-Nuclear Magnetic
Resonance (1 H-NMR) spectroscopy (Bruker, Germany).
The NMR spectrum of CS TM was obtained by dissolving 5 mg of each sample in 750 l of D2 O at 80 C.
The DQ was calculated using data obtained from the
1
H-NMR spectra using the following equations [Sieval,
1998; Polnok, 2004; Amidi 2006]
DQ = CH3 3 /H × 1/9 × 100
In Situ Cytotoxicity (Lactate Dehydrogenase
(LDH) Assay)
The LDH assay was performed according to the method
described by Baba et al. [Baba, 2005]. HT-29 cells were
seeded into 96-well microplates (Nunc) at a density of
3000 cells/cm2 . After 24 h cell culture, medium was
removed and replaced by fresh medium containing CS
and its derivatives at concentration of 0.25, 1, 4 mg/ml
were added. The cells were incubated for 1 h at 37 C.
After incubation the culture supernatants were harvested
to carry out the LDH assay. Fifty l of the culture supernatant from each well were transferred to the corresponding well in another microtitre plate. The LDH activity
of the supernatant was measured utilizing a commercial
kit (Sigma Aldrich, USA) according to the manufacturer’s
instruction. The absorptions were measured in triplicate at
490 nm, with a background correction at 690 nm, using
a microplate ELISA reader (Multiscan). Controls were
performed with 0.1% (w/v) Triton X-100 (Sigma) and
set as 100% LDH release. The relative LDH release is
defined by the ratio of LDH released over total LDH in
the intact cells. The percent cell viability was calculated
after incubation with polymers from the release of LDH
in the supernatants, measured spectrophotometrically by
absorbance at 490 nm:
Here CH3 3 is the integral of the chemical shift of
the N -trimethyl amino group at 3.3 ppm attributed to the
nine hydrogen atoms of the methyl groups pertaining to
trimethylated amino groups. [H] is the integral of the H-1
peaks between 4.7 and 5.7 ppm; the signal related to the
protons attached to the carbon of the glucosamine unit of
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− c
the glucopyranose ring.
× 100%
Cell
viability
% = 100 −
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15:09:52
100% − C
Copyright: American Scientific Publishers
In Vitro Cytotoxicity (MTT Assay)
where is the absorbance of the sample incubated with a
The MTT assay was performed according to the method
polymer, C is the absorbance of the culture supernatant
described by Mosmann [Mosmann, 1983]. HT 29 cells
without
polymer, and 100% is the absorbance of the samwere seeded into 96-well microplates (Nunc) at a density
2
of 3000 cells/cm . The medium was removed 24 h after
ple treated with Triton X-100. Thus, the percentage cell
plating and fresh media containing CS and its derivatives
viability equals 100% if there is no release of LDH.
at concentrations of 0.25, 1.0 and 4.0 mg/ml were added.
After incubation for 1 h the medium was discarded, the
Haemolysis Test
cells were washed twice with phosphate-buffered saline
The haemolytic activity of the polymers was investigated
and 50 l of 5 mg/ml MTT (Sigma Aldrich, USA) solution
according to Parnham and Wetzig [Parnham, 1993]. Whole
in PBS were added to each well. The plates were incublood from rabbit was diluted with 3% sodium citrate solubated in a humidified atmosphere of 5% CO2 at 37 C for
tion. Erythrocytes were separated from the plasma and leu6 h and the formazan crystals were dissolved by adding
cocytes by centrifugation (5000× g, 5 min) (Sorval Legend
100 l dimethyl sulfoxide (DMSO) to each well. The
X1R, Germany) at 4 C, washed three times with phosabsorptions were measured in triplicate at 570 nm, with
phate buffered saline (PBS) and resuspended in saline to
a background correction at 630 nm, using a microplate
achieve 2% (v/v) erythrocyte dispersion. They were used
ELISA reader (Multiscan). Results were recorded as perimmediately after isolation.
centage absorbance relative to untreated control cells. The
To study the effects of CS and its derivatives on haemolcytotoxicity assay results were used to calculate cell viaysis,
the red blood cells were suspended at three differbility after incubation with polymers as follows:
ent concentrations of polymer 0.25, 1.0, 4.0 mg/ml in
x
PBS at a haematocrit of 1% and incubated for 30 minutes
Viability % = × 100%
xc
at room temperature (20 C). The incubated suspensions
were centrifuged at 1000× g, 5 minutes. For reference,
Where x is the absorbance in a well containing a parred blood cells were treated with double-distilled water,
ticular polymer concentration and xC is the absorbance
which effects 100% haemolysis. The haemolytic index
for untreated control cells. All experiments were run four
was determined from the released haemoglobin in the
times.
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
133
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
supernatants, measured spectrophotometrically (Mecasys
Optizen, Korea) by absorbance at 540 nm:
haemolytic index % = 100 −
− c
× 100%
100% − C
where is the absorbance of the sample incubated with a
polymer, C is the absorbance of the sample treated with
phosphate-buffered saline, and 100% is the absorbance of
the sample treated with Triton X-100. Thus, the haemolytic
index equals 100% if there is no release of haemoglobin.
The polymers themselves contributed no more than 1% of
the absorbance at 540 nm. The experiments were run in
triplicate and were repeated twice.
Biswas et al.
objective of X40 magnification. Histopathology changes
were observed and grouped based on vascular changes and
necrotic changes.
Statistical Analysis
Data were expressed as mean values ± standard deviation
(SD). The effect of the degree of quaternization and molecular weight on the cell viability compared to native CS was
assessed with the aid of SPSS 16.0 (SPSS Inc., Chicago,
USA) with the significance level set at p < 0
05. Paired
sample t-test for pair-wise comparison was performed at
each concentration of the CS HMW, CS LMW and their
various DQ. Samples that were not normally distributed
were tested using the Mann Whitney test.
Erythrocyte Aggregation Assay
The erythrocyte aggregation potential of CS derivatives
RESULTS
was investigated according to the reported procedure of
Synthesis and Determination of the DQ of CS TM
Zhang et al. [Zhang, 2008]. A 2% (v/v) stock suspension
CS TM was synthesized based on reaction of CS with
of erythrocytes was prepared for the study. CS and its difCH3 I. The successful incorporation and analysis of the
ferent derivatives at concentrations of 0.25, 1 and 4 mg/ml
spectra of quaternized derivatives of CS was based on the
1
of 1 ml were added into 1 ml of erythrocyte dispersion
H NMR assay and was used to determine the DQ (percent
and incubated for 1 h at room temperature. A 20 l samof trimethylated amine groups) of synthesized CS TM.
ples was placed on a glass plate and agglutination was
The hydrogen (H1) peak bonded to the anomeric carbon
observed using a Carl Zeiss phase contrast microscope
at 5
3 < < 5
5 ppm, the hydrogen (H3, H4, H5, H6)
(Carl Zeiss, Germany) equipped with an objective of X40
peaks bonded to the 2nd carbon of the glucosamine unit
magnification.
at 3.7 ppm and the methyl hydrogen peak of acetamido
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? ppm were determined (Fig. 1). According to
group at
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Sub-Acute Toxicity
theJun
peak2017
assignment
and intensity, the DQ of synthesized
Copyright: American Scientific Publishers
CS TM was calculated. The NMR findings pertaining to
Fifty four male mice were exposed to CS and CS TM
CS are close to that of Britto et al. [Britto, 2004]. Dimethywith two different doses by oral gavages in 0, 7, 14,
lated amine peak at 2.5 ppm and trimethylated amine
21 days interval to examine the morphological and patho(quaternized amine) peak at 3.7 ppm were observed in
logical changes which were labelled as M1A, M1B,
NMR spectrum of CS TM. The NMR data of CS TM are
M2A M4B; N1A, N1B, N2A N4B. After 28 days
in accordance with the results of the other work [Sieval,
(Table I), they were anesthetized by i.p. phenobarbitol at
1998; Britto, 2007].
48 h and autopsied. The organs such as liver, kidney and
spleen were stripped out. All of the organs were immeCytotoxicity (MTT Assay)
diately fixed in 10% formalin. The tissues of organ samples were embedded in paraffin blocks, then sliced and
The in vitro cell metabolic activity of different molecuplaced onto glass slides. After histological haematoxylin
lar weight of CS and its derivatives was analyzed in HT
and eosin (H–E) staining, the slides were observed and
29 cells by MTT assay. The cell viability percentage was
the pictures were taken using a Carl Zeiss phase condetermined after 6 h of incubation with a series of concentrast microscope (Carl Zeiss, Germany) equipped with an
tration (0.25, 1, 4 mg/ml). The results indicated that both
Table I.
Exposed doses for CS and CS TM used in the animal experiment.
Experimental group
M1A
M1B
M2A
M2B
M3A
M3B
M4A
M4B
Polymer used
Dose (mg/kg)
Experimental group
Polymer used
Dose (mg/kg)
CS-HMW
CS-HMW
CS-HMW-TM-24
CS-HMW-TM-24
CS-HMW-TM-48
CS-HMW-TM-48
CS-HMW-TM-61
CS-HMW-TM-61
108
540
108
540
108
540
108
540
N1A
N1B
N2A
N2B
N3A
N3B
N4A
N4B
CS-MMW
CS-MMW
CS-MMW-TM-22
CS-MMW-TM-22
CS-MMW-TM-50
CS-MMW-TM-50
CS-MMW-TM-61
CS-MMW-TM-61
108
540
108
540
108
540
108
540
Notes: Abbreviations: CS: Chitosan; HMW: High molecular weight; MMW: Middle molecular weight; TM: Tri methylated.
134
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
Biswas et al.
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
Figure 1. 1 H NMR spectra of (A) CS HMW TM 24 DQ%, (B) CS
HMW TM 61 DQ%, (C) CS MMW TM 22 DQ%, (D) CS MMW TM
61 DQ%. The solvent proton resonates at 4.7 ppm.
CS HMW (% cell viability 83
03 ± 5
40) and CS MMW
(% cell viability 85
18 ± 1
80) had minimal effects on HT
29 cells even at higher concentration of 5 mg/ml, suggesting the relatively non toxic nature of native CS (Table II).
No significant difference in cytotoxicity was observed
between CS HMW and MMW-derivatives (p = 0
298).
Interestingly, the interaction between cell metabolic activity and polymers were found to be influenced by the degree
of quaternization (p < 0
05). Compared with native CS
(83
03 ± 5
41) at higher concentration, it is observed CS
with 20% DQ did not exhibit significant toxicity (76
76 ±
5
35) while CS with 50% DQ (42
78 ± 5
94) and CS with
60% DQ (42
61 ± 4
87) showed significant toxicity. Such
result appears that the cytotoxicity of CS is related to
the charge density of the molecule, toxicity increases with
increasing positive charge density of quaternized polymer.
The result also showed that, all CS derivatives affected
the metabolic activity in a concentration dependent manner
(p < 0
01). The viability of the cells treated with CS HMW
with 61% DQ at 0.25 mg/ml was significantly decreased
when treated with the same at 4 mg/ml (76
89 ± 9
02 vs.
41
2 ± 5
53, p < 0
01).
Cytotoxicity (LDH Assay)
The damage of cell membranes, as the initial point of
interaction with polymers, was analyzed in HT 29 cells
by LDH assay. The results demonstrated that exposure
to different molecular weight of CS and its derivatives
for 1 h resulted in concentration-dependent increase in
LDH leakage (p < 0
05) and did not produce significant cytotoxicity up to the concentration of 0.25 mg/ml
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Table II. Percentage cell viability byIP:
MTT
assay and LDH
assay,15
percentage
5.10.31.211
On: Thu,
Jun 2017haemolytic
15:09:52index after incubation with different
concentrations of chitosan and their trimethylated
derivatives
(Mean
± SD, nPublishers
= 6).
Copyright:
American
Scientific
Concentration
(mg/ml)
% Cell viability
(MTT assay)
% Cell viability
(LDH assay)
% Haemolytic
index
0.25
1.00
4.00
0.25
1.00
4.00
0.25
1.00
4.00
96.95 ± 2.45
88.91 ± 4.91
83.03 ± 5.41
97.96 ± 1.80
94.17 ± 1.39
85.18 ± 4.02
91.36 ± 2.38+
83.58 ± 5.61
76.76 ± 5.35
94.30 ± 2.90
90.04 ± 4.11
88.72 ± 5.38
97.41 ± 2.07
94.43 ± 5.27
84.60 ± 6.42
95.15 ± 2.03
92.76 ± 3.55
88.09 ± 4.13
96.30 ± 1.51
94.19 ± 3.13
88.06 ± 5.66
91.32 ± 6.71
82.30 ± 4.97+
77.87 ± 6.52+
91.01 ± 5.89
90.31 ± 3.10
79.10 ± 5.16+
CS-HMW-TM-48
0.25
1.00
4.00
82.42 ± 6.58+
62.10 ± 7.43+
48.78 ± 5.94+
CS-HMW-TM-61
0.25
1.00
4.00
76.76 ± 5.35+
60.65 ± 3.67+
42.61 ± 4.87+
81.98 ± 7.53+
63.11 ± 6.91+
47.94 ± 5.45+
76.89 ± 9.02+
59.31 ± 9.46+
41.20 ± 5.53+
85.59 ± 4.63+
77.88 ± 4.18+
71.09 ± 6.67+
81.57 ± 7.42+
74.18 ± 3.05+
66.82 ± 6.97+
CS-MMW-TM-22
0.25
1.00
4.00
93.69 ± 4.01
86.23 ± 3.40∗
80.60 ± 5.60#
CS-MMW-TM-50
0.25
1.00
4.00
89.24 ± 2.91∗
77.97 ± 5.36∗ #
65.92 ± 6.85∗ #
93.49 ± 4.16∗
86.89 ± 6.11∗
80.47 ± 5.49
90.03 ± 6.12
79.22 ± 6.01∗ #
65.28 ± 5.00∗ #
92.04 ± 2.96∗
88.61 ± 4.30
81.26 ± 4.93∗
84.28 ± 8.01∗
78.39 ± 3.42∗
73.22 ± 3.36∗
CS-MMW-TM-61
0.25
1.00
4.00
82.67 ± 4.70∗
68.22 ± 7.66∗
52.35 ± 7.16∗ #
82.57 ± 6.95∗
69.79 ± 7.24∗
51.60 ± 5.97∗ #
79.63 ± 2.91∗
71.32 ± 2.98∗ #
66.38 ± 5.39∗
Group
CS-HMW
CS-MMW
CS-HMW-TM-24
Notes: +p < 005 in comparison with group A (HMW), ∗ p < 005 in comparison with group B (MMW); # p < 005 in comparison with group C (HMW-CS-TM).
Abbreviations: CS: Chitosan; HMW: High molecular weight; MMW: Middle molecular weight; TM: Tri methylated.
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
135
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
(Table II). An appreciable increase of membrane leakage was observed with degree of quaternization. For CS
TM-23, CS TM-49 and CS TM-61 when treated with
5 mg/ml, the cell viability decreased by 8.6% (79
17 ±
5
90, p = 0
686), 34.6% (56
61 ± 10
34, p < 0
05) and
46.5% (46
40 ± 7
72, p < 0
001), compared to native CS
(86
66 ± 6
05).
Haemolysis Test
The interactions of the CS and their derivatives with negatively charged membranes have also be studied by haemolysis experiments. The release of haemoglobin was used
to quantify the erythrocyte membrane-damaging properties of the polymers. As 100% and 0% values we used
Triton X-100 and phosphate-buffered erythrocytes, respectively. Erythrocytes were incubated with three different
Biswas et al.
polymer concentrations in the range of 0.25–4 mg/ml for
30 min. Under these conditions, CS and their derivatives
showed no haemolytic effects up to 0.25 mg/ml indicating
no detectable disturbance of the red blood cell membranes
(88
20 ± 7
51). Consistent with the MTT assay and LDH
assay, the interaction between erythrocyte cell membrane
and polymers were found to be influenced by the degree
of quaternization (p < 0
001) and concentration (p < 0
05)
(Table II). For CS TM-23, CS TM-49 and CS TM-61at
5 mg/ml, the haemolytic index decreased by 9.0% (80
18±
4
94, p = 0
232), 18.1% (72
15 ± 3
53, p < 0
001) and
24.4% (66
60 ± 5
95, p < 0
001), compared to native CS
(88
07 ± 4
73).
Erythrocyte Aggregation Assay
In order to investigate the interaction of the CS and their
derivatives with erythrocytes, an erythrocyte aggregation
assay was performed. As shown in Figure 2, there was no
aggregation between the cells in the sample treated with
phosphate buffered saline. Furthermore, when cells were
incubated in the presence of either CS or CS with 20%
DQ, no aggregation was observed (Figs. 2(C), (D), (F)).
However, the addition of CS with 51% DQ (Fig. 2(H)),
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Figure 2. Agglutinations of CS HMW and CS MMW with various DQ at different concentrations with erythrocytes and
agglutinations were observed by microscopy. (A) Phosphate
buffered saline, (B) Triton X-100, (C) CS HMW at 4 mg/ml, (D)
CS HMW with 22% DQ at 1 mg/ml, (E) CS HMW with 61% DQ
at 4 mg/ml, (F) CS MMW with 22% DQ at 0.25 mg/ml, (G) CS
MMW with 51% DQ at 1 mg/ml, (H) CS MMW with 61% DQ at
4 mg/ml.
136
Figure 3. The microscopic pictures (H and E magnification
×100) show the pathological changes in liver tissues of experimental mice treated with (A) sterilized saline, (B) CS HMW at
higher dose, (C) CS MMW at higher dose, (D) CS HMW with
24% DQ at lower dose, (E) CS HMW with 61% DQ at higher
dose, (F) CS MMW with 61% DQ at higher dose.
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
Biswas et al.
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
the aggregation level was drastically increased and incubation with CS of 61% DQ caused severe aggregation and
lysis due to high positive charge density of the polymer
(Figs. 2(E), (G)).
Histopathological Examination
In mice exposed to saline, necropsy and histopathological
examinations of the mice showed well defined histological
structure without any signs of vascular and inflammatory
changes. The histopathology analysis of the liver revealed
signs of toxicity after administration of CS with DQ 50%
or CS with DQ 61% resulted in mild vascular and inflammatory changes with congested sinusoids and centrilobular necrosis (Fig. 3). However, even at higher doses of
CS or CS with DQ 22%, there was no significant finding
observed in liver histology.
Normal histology of the glomerulus and tubules was
found in kidney tissue of mice that received saline only
(Fig. 4(A)). In mice treated with CS or CS with 22%
DQ; there were no vascular or inflammatory changes after
28 days of administration. Treatment with higher doses
of CS with 61% DQ resulted mild vascular and inflammatory changes with signs of vascular congestion, tubular
Figure 5. The microscopic pictures (H and E magnification
×100) show
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experimental
treated with (A) sterilized saline, (B) CS
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HMW at higher
dose, (C) CS MMW at higher dose, (D) CS HMW
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with 24% DQ at lower dose, (E) CS HMW with 61% DQ at higher
dose, (F) CS MMW with 61% DQ at higher dose.
necrosis and glomerular atrophy, which is a degenerative
phenomenon. The histology assessment in the spleen did
not reveal any vascular changes in the saline treated or CS
treated mice (Fig. 5).
DISCUSSION
Figure 4. The microscopic pictures (H and E magnification
×100) show the pathological changes in kidney tissues of
experimental mice treated with (A) sterilized saline, (B) CS
HMW at higher dose, (C) CS MMW at higher dose, (D) CS HMW
with 24% DQ at lower dose, (E) CS HMW with 61% DQ at higher
dose, (F) CS MMW with 61% DQ at higher dose.
J. Pharm. Sci. Pharmacol. 1, 131–140, 2014
A primary objective of oral delivery systems is to protect
protein and peptide drugs from acid and luminal proteases
in the GIT [Park, 2011]. Facile chemical modification is
one of CS’s great strength, enabling its optimization to be
effectively used as carrier in oral drug delivery [Aranaz,
2010]. Amphiphilic CS derivatives are widely proposed
for their use in pharmacy and biomedicine, but neither CS
nor its derivatives have yet been approved by the U.S.
FDA. Studies regarding the biocompatibility and toxicity
of chemically modified CS derivatives become now one of
the important key factors for the rational design as desired
pharmaceutical excipients.
HT-29 cells, an intestinal epithelial cell line, have been
widely used for cytotoxicity study [Watson, 1994; Sergent,
2012]. Although different assay techniques were used,
the results of the polymers with regard to their cytotoxicity were correlated [Fischer, 2003; Baba, 2005]. MTT
137
Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier
Biswas et al.
and LDH assay gave similar results, since loss of memdose as 540 mg/kg. The MW and hydrophilicity of CS
brane integrity after 1 h (LDH assay) was always folcan be considered as a critical factor for the absorption
lowed by a decrease of mitochondrial function (MTT
of the CS in mice after oral administration [Chae, 2005].
assay) after exposure of the polymer [White, 1998].
Absorbed CS was distributed to all organs such as liver,
The severe membrane damage caused by CS with 61%
kidney, blood, spleen and so on [Zeng, 2008]. All aniDQ at 1 h resulted a loss of the metabolic activity at
mals survived the duration of the study, with no signif6 h. Similarly, exposure to native CS of both molecular
icant changes of body weight and food consumptions.
weight and their DQ with 22% even at higher concenPathological changes on kidney and liver were observed
tration (4 mg/ml) did not change the release of LDH or
in mice exposed to CS with higher DQ (e.g., mild vascumetabolic activity demonstrating their good cell compatilar and inflammatory changes with signs of vascular conbility. Charge density resulting from the number and the
gestion, tubular necrosis etc.) but they were not found in
three-dimensional arrangement of the cationic residues is
mice exposed to native CS or CS with lower DQ. There
an important factor for cytotoxicity [Ryser, 1967; Singh,
were no signs of toxicity in the spleen in all groups of
1992]. Although it has been demonstrated that increase in
mice.
cytotoxicity is a function of molecular weight [Morgan,
1988; Haensler, 1993; Fischer, 1999], no significant difCONCLUSION
ference was observed between CS HMW and CS LMW
The native CS of two different MW and their trimethyin our experiment. Concentration of the polymer signiflated derivatives with various degree of quaternization
icantly affects cell viability (p < 0
01) and therefore is
an important bio safety consideration used as an excipient
show a linear correlation between DQ and concentra[Do, 2008].
tion with toxicity. Although, CS with 61% DQ seems to
Aggregation, crenation, haemolysis and coagulation of
show much more effective physicochemical properties than
red blood cells induced by polymer treatment would
the lower DQ as a drug carrier, it has higher toxicity;
largely restrict the safety application of polymers as
therefore its use as an excipient in a biological matrix
drug carriers [Cui, 2012]. Quaternized CS derivatives
should be considered carefully and it should be replaced by
possess positive charge density and can easily interact
lower DQ derivatives with appropriate concentration when
with negatively charged erythrocyte membrane, resulting
possible.
haemolysis and toxicity. Therefore analysis forDelivered
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patibility of synthesized CS derivatives
are highly
neces- Scientific
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American
ConflictPublishers
of Interests
sary. Most in vitro studies of polymer-induced haemolysis
The authors declare that they have no competing
evaluate the percent haemolytic index by spectrophotointerests.
metrically detecting plasma free haemoglobin derivatives
after incubating the particles with blood [Dutta, 2007;
Acknowledgments: The authors thank Professor
Dobrovolskaia,2008]. The results of the haemolysis assays,
Debesh Chandra Majumder and Professor Pranabesh
presented as haemolytic indices, showed CS with higher
Chakraborty for the encouragement and support provided.
DQ have an effect on haemolysis. Native CS and their
trimethylated derivatives with 22% DQ have very less
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