Synthesis of dimethyl carbonate (DMC) based biodegradable nitrogen mustard ionic carbonate (NMIC) nanoparticles

Article abstract of DOI:10.1039/c4ra13290d

Chitosan-nitrogen mustard ionic carbonate nanoparticles (C-NMIC-Nps) were fabricated for the first time by an ionotropic gelation method. The preparation of C-NMIC-Nps involved four steps. In the first step, green reagent dimethyl carbonate (DMC) reacted with 2-dimethylethanolamine (DMEA) to form bis-(2-dimethyl amino-ethyl)carbonate (DAEC). In the second step, DAEC underwent cationization with (1-chloro-2,3 epoxy)propane to form NMIC with a stable carbonate backbone having a greater number of sorption sites. Later, NMIC was successfully cross linked with chitosan to form C-NMIC, which was later on used to form C-NMIC nanoparticles (C-NMIC-Nps) by ionotropic gelation. The chemical structure of NMIC and C-NMIC was identified by proton nuclear magnetic resonance (¹H-NMR), ¹³C nuclear magnetic resonance (¹³C-NMR) spectroscopy and elemental analysis. The spherical shaped, smooth and uniform distribution of the C-NMIC-Nps was observed by scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The average particle size distribution of the C-NMIC-Nps was found to be 14.4 ± 3.1 nm as observed using a particle size analyzer. The functional groups of DAEC, NMIC and C-NMIC were identified by Fourier transform infrared spectroscopy (FTIR). The conversion of the crystalline nature of chitosan to the amorphous state in C-NMIC-Nps was studied by X-ray diffraction (XRD). The thermal stability of the C-NMIC-Nps was confirmed by thermo gravimetric analysis (TGA). The good cell viability of C-NMIC-Nps was assessed by in vitro MTT assay using VERO cell lines. Overall, the results suggested that C-NMIC-Nps had been prepared successfully. The prepared C-NMIC-Nps will act as a nano carrier of growth factors for wound healing application.

Full text of DOI:10.1039/c4ra13290d

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Synthesis of dimethyl carbonate (DMC) based
biodegradable nitrogen mustard ionic carbonate
Cite this: RSC Adv., 2015, 5, 10560
(NMIC) nanoparticles†
*
P. Thyriyalakshmi and K. V. Radha
Chitosan-nitrogen mustard ionic carbonate nanoparticles (C-NMIC-Nps) were fabricated for the first time
by an ionotropic gelation method. The preparation of C-NMIC-Nps involved four steps. In the first step,
green reagent dimethyl carbonate (DMC) reacted with 2-dimethylethanolamine (DMEA) to form bis-(2-
dimethyl amino-ethyl)carbonate (DAEC). In the second step, DAEC underwent cationization with (1-
chloro-2,3 epoxy)propane to form NMIC with a stable carbonate backbone having a greater number of
sorption sites. Later, NMIC was successfully cross linked with chitosan to form C-NMIC, which was later
on used to form C-NMIC nanoparticles (C-NMIC-Nps) by ionotropic gelation. The chemical structure of
NMIC and C-NMIC was identified by proton nuclear magnetic resonance (1H-NMR), 13C nuclear
magnetic resonance (13C-NMR) spectroscopy and elemental analysis. The spherical shaped, smooth and
uniform distribution of the C-NMIC-Nps was observed by scanning electron microscopy (SEM) and high
resolution transmission electron microscopy (HRTEM). The average particle size distribution of the
C-NMIC-Nps was found to be 14.4 ꢀ 3.1 nm as observed using a particle size analyzer. The functional
groups of DAEC, NMIC and C-NMIC were identified by Fourier transform infrared spectroscopy (FTIR).
The conversion of the crystalline nature of chitosan to the amorphous state in C-NMIC-Nps was studied
by X-ray diffraction (XRD). The thermal stability of the C-NMIC-Nps was confirmed by thermo
gravimetric analysis (TGA). The good cell viability of C-NMIC-Nps was assessed by in vitro MTT assay
using VERO cell lines. Overall, the results suggested that C-NMIC-Nps had been prepared successfully.
The prepared C-NMIC-Nps will act as a nano carrier of growth factors for wound healing application.
Received 28th October 2014
Accepted 6th January 2015
DOI: 10.1039/c4ra13290d
www.rsc.org/advances
natural polymer aer cellulose. It is a linear co-polymer of
b-(1,4)-2-acetamido-2-deoxy-b-D-glucose and b-(1,4)-2-amine-
2-deoxy-b-D-glucose.4 Chitosan has both reactive amino and
hydroxyl groups, that can chemically alter its properties, under
mild conditions. It is generally regarded as biocompatible,
biodegradable and nontoxic biomaterial. Chitosan has been
proved to have antifungal property. It is an attractive candidate
for treating wounds by acting as a haemostat and initiates
broblast proliferation at the wound site.5
Chitosan shows high crystallinity and high degree of
hydrogen bonding leading to poor solubility in water and
common organic solvents.6 In recent years, there has been a
growing interest in the modication of chitosan and its utili-
zation as diversied agent in pharmaceutical, waste water
treatment, cosmetics, drug delivery, heavy metal chelation,
heterogeneous catalysis and many other applications.7 Ionic or
covalent crosslinking between the polymeric chains plays a vital
role in the formation of nanoparticles.8
1. Introduction
In modern polymer science a wide range of materials, such as
natural or synthetic polymers, lipids, surfactants and den-
drimers have been employed as drug carriers. Among these,
polysaccharides have received increasing attention because of
their outstanding physical and biological properties.1
In recent years, naturally occurring carbohydrate based
biopolymers chitin and chitosan have gained impetus because of
their excellent biocompatibility.2 Since, chitin and chitosan exist
with some unique properties like biodegradability and bioac-
tivity, and have a variety of potential applications in biomedical
and biotechnological elds. Chitosan is more efficient than
chitin in terms of adsorption capacity due to the presence of large
number of free amino groups on the chitosan chain.3
Chitosan is a poly cationic biopolymer obtained mainly by
deacetylation of chitin, which is the second most abundant
Crosslinkersaremoleculeswithtworeactivefunctionalgroups
thatallowtheformationofbridgesbetweenthepolymericchains.9
Cross linked chitosan is very stable and maintain their strength
even in acidic and basic solutions. It leads to the change in crys-
talline nature of chitosan and enhance sorption properties.10
Bio-Products Laboratory, Department of Chemical Engineering, A.C. Tech, Anna
University, Chennai-25, Tamil Nadu, India. E-mail: radhavel@yahoo.com; Tel:
+91-44-22359124
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra13290d
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Chitosan is able to form nanoparticles which were harvested C-NMIC-Nps was assessed by in vitro cytotoxicity test (MTT
spontaneously under mild acidic conditions in contact with assay).
negatively charged sodium tripolyphosphate (TPP) ions by
ionotropic gelation method. This type of ionic cross linking of
chitosan is usually a typical non-covalent interaction, which is
reversible and this may avoid the potential toxicity of the
reagent.11
2. Experimental methods
2.1 Materials
Analytical grade dimethyl carbonate (DMC), 2-dimethyletha-
The chemical modication of chitosan affords a wide range
of derivatives such as quaternary chitosan, carboxy alkyl chito-
san, thiolated chitosan, sugar bearing chitosan, bile acid
modied chitosan and cyclodextrin-linked chitosan. The
derivatives of chitosan impart amphiphilicity, which is an
important characteristic for the formation of self-assembled
nanoparticles, potentially suited for biomedical applications.12
Among the several chitosan derivatives, chitosan with
cationic moieties has been prepared by quaternization of the
amino group or by graing small molecules or polymer chains
on to chitosan backbone. By introducing the cationic nature of
chitosan several drug conjugate approaches have been devel-
oped towards therapeutic applications.13
nolamine (DMEA), triethyl amine (TEA) and epichlorohydrin
were purchased from Sigma Aldrich. Chitosan of low molecular
weight (85% deacetylation) with viscosity >200 cps, sodium
tripolyphosphate (TPP) with 85% purity and MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] with
98% purity were also obtained from Sigma Aldrich. All other
reagents were analytical grades and were used without any
further purication. Milli-Q grade (resistivity 18.2 MU cmꢁ1
)
water was used in all the experiments.
2.2 Characterization of DAEC, NMIC, C-NMIC and C-NMIC-
Nps
FTIR spectra of all the samples were recorded using Alpha
Recently, chitosan acetate with both hemostatic and anti-
microbial properties has been used for wound healing appli-
cation.14 A good wound healing material should maintain a
moist environment but allows the drainage of wound exudate
and prevents infection at the wound site. Hydroxypropyl chito-
san is used as a delivery vehicle for broblast growth factor that
accelerates wound healing.15 Biocompatible and water soluble
carboxymethyl chitosan nanoparticles has been studied for
delivery of antibiotics.16
Nowadays sulfur and nitrogen (half-) mustards are exten-
sively employed in inorganic synthesis, organic synthesis and in
the preparation of numerous pharmaceutical intermediates.
These compounds are toxic and the toxicity is strictly related to
their highly reactive chlorine atom. The replacement of chlorine
atom by a carbonate moiety with DMC17 an eco-friendly,
biodegradable and nontoxic reagent resulted in sulfur and
nitrogen half mustard carbonate analogues. These analogues
showed to maintain the chemical behaviour of the parent
chlorine compounds, while losing their toxic properties.18 Thus,
the current study mainly focussed on the synthesis of nitrogen
mustard ionic carbonate (NMIC).
Bruker spectrophotometer in attenuated total reectance (ATR)
mode in the range of 4000–400 cmꢁ1 1H-NMR and 13C-NMR
.
spectra of the samples were recorded on a Bruker D8 advance
500 spectrometer at 25 ꢂC using D2O and D2O/CF3COOD as
solvents. Mass spectra were recorded on a (EI) JEOL JMS-AX 500.
Elemental analysis was performed on a Heraeus CHN-O-Rapid
analyser. XRD Patterns were done with a Bruker D8 Advance
˚
XRD machine using Cu-Ka (l ¼ 1.54 A) source. The layer
spacing of the samples was calculated according to the Bragg's
equation. The diffraction angles 2q were set between 2ꢂ and 75ꢂ
incremented with a scanning angle of 1ꢂ minꢁ1
.
Thermal stability was examined by the TGA of TA Perkin
Elmer instrument at a heating rate of 20 K minꢁ1 in the
temperature range between 30–700 ꢂC under nitrogen atmo-
sphere. The average particle size distribution was determined
using dynamic light scattering (DLS) analyzer with a Malvern
Zetasizer Nano-S. For SEM analysis, the particles were sputter-
coated with gold lm using a sputter coater and imaged
under SEM (TESCAN-VEGA3 SBU). For HRTEM studies of the
nanoparticles, 5 mL of the sample was coated on a carbon-coated
copper HRTEM grid, which was subsequently air dried and was
analysed by a high resolution HRTEM (JEM 2100, Jeol, Peabody,
MA, USA) operating at a high voltage of 200 keV. The selected
area electron diffraction (SAED) pattern of the C-NMIC was also
recorded using the same instrument.
The objective of the study is to develop a novel, facile route
for the preparation of biodegradable chitosan-nitrogen mustard
ionic carbonate nanoparticles (C-NMIC-Nps) with a stable
carbonate backbone that offers a great promise as wound
healing platform. In the present work, a novel DMC based
NMIC was synthesized and cross linked with chitosan to
produce C-NMIC. Herein, DMC provides a stable carbonate
moiety to the newly synthesized NMIC. Then, a simple iono-
2.3 Preparation of NMIC and C-NMIC
2.3.1 Synthesis of bis-(2-dimethyl amino-ethyl)carbonate
tropic gelation method was used to prepare the C-NMIC-Nps. (DAEC). 0.1 M (8.4 mL) of DMC was allowed to reux with
The cationic and the hydrophilic property of NMIC, would 0.2 M (27 mL) of DMEA in the presence of TEA (13.9 mL) base as
enhanced the sorption properties of chitosan, making C-NMIC- a catalyst at 86–88 ꢂC for about 24 h. The crude compound was
Nps a promising material in therapeutics.
subjected to fractional distillation under vacuum at 60 ꢂC. The
The synthesized NMIC, and the prepared C-NMIC and yield of the coloured orange product was found to be 47.3%.
C-NMIC-Nps were characterized by various methods namely FTIR: nmax/cmꢁ1 1652 (ester C]O str), 1496 (N–CH3 assy def),
FTIR, 1H-NMR, 13C-NMR, elemental analysis, SEM, HR-TEM, 1386 (CH2 def CH2–O), 1254 (ester C–O assy str), 1094 (ester C–O
particle size analysis, XRD and TGA. The biocompatibility of sym str), 864 (C–N str), 660 (CH2 rock). 1H-NMR (500 MHz, D2O):
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dH ppm 3.06 (12H, s, N–CH3), 3.38 (4H, t, J ¼ 4.5, N–CH2), 3.91– diphenyl-tetrazolium bromide (MTT) assay. The VERO (kidney
3.95 (4H, m, O–CH2). 13C-NMR (500 MHz, D2O): dc 54 (N–CH3), epithelial from African monkey) cells were grown and main-
56 (N–CH2), 67 (O–CH2), 161 (C]O). EI-MS (m/z): 207 (M + 2, tained in Dulbecco's modied eagle medium (DMEM) con-
130%).
taining high glucose and pyruvate, with 10% fetal calf serum
2.3.2 Synthesis of bis-{2-[(3-chloro-2-hydroxy propyl)-N,N- (FCS) supplemented with penicillin (120 units per mL), strep-
dimethyl ammonium chloride]-ethyl}carbonate (CHPDAEC)/ tomycin (75 mg mLꢁ1), gentamycin (160 mg mLꢁ1) and
(NMIC). DAEC obtained in the rst step was mixed with excess amphotericin B (3 mg mLꢁ1) at 37 C in an incubator humidi-
ꢂ
amount 2 mL of 1-chloro-2,3-epoxy propane in methanol and ed with 5% CO2. Conuent cells were harvested by washing in
ꢂ
stirred at 50 C for 16 h in the presence of acid catalyst. The phosphate-buffered solution (PBS), followed by trypsinization
crude product NMIC was obtained at 65 ꢂC aer fractional for subculture. The cell proliferation and viability were
distillation under vacuum. Later, the pure product with a yield measured by MTT assay to determine the amount of the active
of 57.9% was subsequently obtained as a brown colour liquid mitochondrial enzymes present in the viable cells.
aer ether separation in diethyl ether. FTIR: nmax/cmꢁ1 3464
2.4.2 Experimental design for the evaluation of cytotoxicity
(alcoholic OH str), 1652 (ester C]O), 1386 (CH2 def CH2–O), effects. The cytotoxicity effects of C-NMIC-Nps were assessed
1336 (CO def of CHOH), 1162 (alcoholic C–O str), 1012 (CH2 def using VERO cell lines. The nanoparticles were sterilized by UV
of CH2OH), 1254 (ester C–O assy str), 960 (OH def of CHOH), treatment before subjecting them to the cell culture procedures.
834 (C–N str), 660 (CH2 rock), 602 (C–Cl str). 1H-NMR (500 MHz, C-NMIC-Nps of various concentration (0.5, 1, 1.5, 2.0 and
D2O): dH ppm 3.2 (12H, s, N–CH3) 3.5–3.67 (8H, m, NCH2), 3.9– 2.5 mg mLꢁ1) were placed in the 96 wells of a polystyrene tissue
4.1 (4H, m, OCH2), 4.8–4.9 (2H, m, CH2–CH–CH2), 3.4–3.48 (4H, culture plate. The cellular behaviour of the nanoparticles was
m, CH2Cl). 13C-NMR (500 MHz, D2O): dc ppm 54 (N–CH3), 55 (N– observed aer 12 h, 24 h, 48 h and 72 h respectively. The
CH2–CH2) and (O–CH2), 63 (N–CH2–CH), 67 (CH2–CH–OH), 53 polystyrene surface (wells without C-NMIC-Nps) was treated as
(C–Cl), 165 (C]O). EI-MS (m/z): 391.2 (M+ ꢁ Cl, 100%).
control. In all the culture conditions, the medium was renewed
2.3.3 Modication of chitosan with NMIC. 1.6 g of chitosan every day and at the end of incubation, the supernatant of each
was being dissolved in 1% acetic acid. The pH of the solution well was replaced with MTT diluted in a serum-free medium.
was adjusted to 4 by the addition of 0.1 M Na2CO3 solution and The plates were then incubated at 37 ꢂC for 4 h. Aer aspirating
kept for continuous stirring. Solution of 0.1 M NMIC in meth- the MTT solution, DMSO was added to each well, and pipetted
anol was added drop wise to chitosan. To the resultant mixture, up and down to dissolve all of the dark blue crystals of for-
0.1 M Na2CO3 was added until the solution attained the pH 7–8 mazan, and then le at room temperature for few minutes.
and stirring was continued for 24 h. The precipitated NMIC was Finally, the absorbance at 570 nm was measured, using the UV
subjected to repeated centrifugation and washed with 1 : 1 spectrophotometer. Experiments were repeated as triplicate per
CH3OH/H2O, till the pH of the solution becomes neutral. The sample. The absorbance of the control group was taken as 100%
formed C-NMIC was dried in a vacuum oven at 55 ꢂC overnight and the percentage of cell viability was recorded by the
and stored in a desiccator. FTIR: nmax/cmꢁ1 3280 (OH str and following equation.
NH str of primary amine), 1642 (C]O str of amide), 1564 (NH
OD ðtestÞ ꢁ OD ðblankÞ
Cell viability ð%Þ ¼
ꢃ 100% (1)
bends of amide), 1374 (sym angular deformation of CH3), 1318
(CO def of CH–OH), 1258 (ester assy C–O str), 1150, 1062, 1030
and 896 (sym str of C–O–C), 822 (C–N str), 798 (OH bend).
1H-NMR (500 MHz, D2O/CF3COOD): dH ppm 1.3 (br, 3H, s,
NHCOCH3), 2.4–2.5 (br, 4H, d, (NH–CH2)), 2.9–3.2 (br, 4H, d,
(N–CH2–CH(OH))), 3.3 (br, 12H, s, (N–CH3)), 3.5–4.9 (br, m, (2H,
N–CH2–CH2–O), (H1, H2, H3, H4, H5, H6, H60), (4H, OCH2),
OD ðcontrolÞ ꢁ OD ðblankÞ
3. Results and discussion
3.1 Mechanism
(1H, CHOH)). 13C-NMR (500 MHz, D2O/CF3COOD): dc ppm 22 DAEC was synthesized by the reaction of DMC with DMAE at
(CH3CO), 52 (N–CH3), 54 (NH–CH2), 56 (N–CH2), 60 (C6), 61 88 ꢂC through BAC2 mechanism as given in Scheme 1. When
(O–CH2), 63 (C2), 67 (CH2–CH–OH), 73 (C3), 74 (C5), 78 (C4), 105 DMEA attacks the carbonyl carbon of DMC, carboxy methyla-
(C1), 167 (C]O).
tion reaction takes place resulting in the formation of the
2.3.4 Preparation of C-NMIC nanoparticles. To about 0.1% expected DAEC and by-product methanol.17 The second step in
w/v of C-NMIC in aqueous acetic acid, 0.01% w/v TPP was gently Scheme 1 represents the synthesis of NMIC by nucleophilic
dripped and the solution was allowed to stand for 10 min at addition reaction. Herein, DAEC in the presence of acid catalyst
room temperature to get an opalescent suspension. The attack the C3 carbon of epoxy ring of 1-chloro(2,3 epoxy)
resulting nanoparticles were centrifuged at 10 000 rpm for propane, resulting in the three member ring opening reaction.
30 min at 4 ꢂC. The precipitated nanoparticles were once again Covalent attachment of NMIC to chitosan through N-alkylation
re-suspended in water, sonicated, centrifuged and freeze dried. was shown in Scheme 2. Wherein, N-alkylation of chitosan with
NMIC takes place through the liberation of HCl at pH 7–8.19
The successful attachment of NMIC to chitosan was
conrmed by elemental analysis (Table 1). The presence of
2.4 Cytotoxicity studies
2.4.1 Cell culture. The cytotoxicity of the pure C-NMIC-Nps NMIC in the modied chitosan caused a decrease in the carbon
on VERO cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- and hydrogen content in the resulting C-NMIC material.
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Scheme 1 Formation of DAEC and NMIC.
pendent chain in C-NMIC forms co-valent cross linking between
its chloride atoms and amino groups present on the chitosan
polymer backbone. In addition the decrease in the C/N ratio
suggests that C-NMIC cross linking occurred successfully on the
addition of NMIC. Thus chitosan was chemically modied by
the successful incorporation of NMIC as specied in Scheme 2.
3.2 Characterization studies
3.2.1 FTIR for DAEC, NMIC and C-NMIC. The FTIR spectra
in Fig. 1(a), indicates the formation of DAEC by the observation
of basic characteristic peaks at 1652 cmꢁ1 corresponding to the
C]O stretch of ester.21,33 The peaks due to asymmetric defor-
mation of N–CH3 group and C–O asymmetric stretch of ester
appeared at 1496 cmꢁ1 and 1254 cmꢁ1 respectively.22,26 The
presence of alcoholic OH conrms the successful cationization
of DAEC by the appearance of peak at 3464 cmꢁ1 (Fig. S1(b)†).23
The peak at 602 was assigned to C–Cl stretching of the so
formed CHPDAEC (NMIC).21,24
Scheme 2 Schematic structure newly synthesised C-NMIC.
However, the percentage of nitrogen increases with the addition
of NMIC to the chitosan sequence of the modied polymer.20
From the results it can be inferred that the presence of NMIC as
Table 1 Elemental analysis of chitosan and C-NMIC
Element (%)
Sample
C
H
N
C/N
Chitosan
C-NMIC
40.43
40.22
6.57
5.71
7.17
7.62
5.63
5.27
Fig. 1 FTIR spectra of (a) DAEC and (b) NMIC.
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The unmodied chitosan spectrum (Fig. 2(a)) was similar to
13C-NMR spectra of C-NMIC exhibited the chitosan signals,
that of previously reported.25–28 In comparison to the supporting that was consistent with that as reported earlier.9,19,32 In addi-
biomaterial chitosan, the FTIR spectra (Fig. 2(b)) of the tion signals at 167, 67, 61, 56, 54 and 52 ppm, were assigned to
prepared C-NMIC conrmed the successful addition of NMIC to the NMIC carbonyl carbon, methine carbon of (CH–OH),
chitosan through N-linkage by the appearance of new peak at methylene carbon of (O–CH2), (N–CH2), (NH–CH2) and methyl
1546 cmꢁ1
.
It should be noted that unlike chitosan, a char- carbon of (N–CH3) respectively, which were an indication of
28
acteristic band could be observed at approximately 1470 cmꢁ1 neat formation of C-NIMC (Fig. S4(c)†).19,31,32
in the C-NMIC sample, which was a clear evidence for the
The EI mass spectra (Fig. S5(a) and (b)†) of the synthesized
chemical modication of chitosan.25,29 Further, conrmation DAEC and NMIC showed molecular ion peak at (m/z) 207 and
was done from the absence of peak at 602 cmꢁ1, that arose from 391.2 respectively which was in good agreement with the
C–Cl stretching of NMIC.24
3.2.2 NMR (1H and 13C) and mass spectroscopy. The H-
calculated theoretical values.
1
3.2.3 X-ray diffraction (XRD) study for C-NMIC-Nps. The
NMR spectrum as given in Fig. S3(a)† conrmed the forma- XRD prole of original chitosan and C-NMIC-Nps were given in
tion of DAEC from the presence of characteristic resonances at Fig. S6(a).† The diffractograms of chitosan showed two peaks
3.06 ppm and 3.91–3.95 ppm that were attributed to N–CH3 (ref. one at 2q of 11.6ꢂ corresponding to anhydrous chitosan crystals
26) and O–CH2 group protons respectively.18,29–31 In Fig. S3(b),† and the other peak at 20.3ꢂ with (100) plane of orthorhombic
the peaks at 3.4–3.48 ppm were assigned to CH2–Cl protons24 of crystals.5 The diffractograms of C-NMIC-Nps (Fig. S6(b)†)
NMIC that exhibited the neat cationization of DAEC with showed large broad amorphous halo centred at 2q ¼ 24ꢂ 4,39 that
1-chloro-2,3-epoxy propane.
exhibits amorphous or disordered crystalline phase of chitosan.
1
As shown in Fig. S3(c),† H-NMR of C-NMIC was similar to The decrease in chitosan crystallinity might be due to the weak
the one of chitosan except for the additional signals of N–CH3, ionic interaction of TPP which, diminishes the regularity of the
and (NH–CH2) group protons at 3.3 ppm, 2.4–2.5 ppm respec- extended C-NMIC crystal structure.20,35 But, the results implied
tively. These signals appeared as a singlet and doublet due to that the crystalline domains of chitosan were not disrupted
the presence of methyl and methylene protons.18,26 The doublet completely due to the presence of the active and exible NMIC
at 2.4–2.5 ppm showed the N-crosslinking of original chitosan groups present in C-NMIC-Nps.7 The poly crystalline nature of
with NMIC. Since a pH of 7–8 was maintained, N-alkylation the nanoparticles were further conrmed from the SAED
takes place predominantly leading to 80–90% N-crosslinked pattern obtained from HRTEM analysis.
C-NMIC.32
3.2.4 Thermo gravimetric analysis (TGA). The thermal
The 13C-NMR spectra (Fig. S4(a)†) indicated the formation of stability of chitosan, C-NMIC and C-NMIC-Nps were studied by
DAEC by the appearance of signals at dc 161, 67, 56 and 54 ppm TGA analysis as shown in Fig. S7.† The initial weight loss of all
for (C]O), (O–CH2), (N–CH2) and (N–CH3) groups respec- the samples around 100 ꢂC might be ascribed to the evaporation
tively.18,32–34 In addition to the signals found in DAEC, reso- of absorbed water. It was noticed that the two TGA curves (a)
nances at dc 67 and 53 ppm for the newly formed NMIC were and (b) showed similar trends at both low and high tempera-
attributed to the methine carbon of CH–OH32 and chloro carbon ture. Rapid decomposition of the chitosan back bone occurred
of C–Cl (Fig. S4(b)†).24
in the temperature range between 270 ꢂC to 340 ꢂC for both
original chitosan and C-NMIC.27,36,39 This indicated the good
thermal stability of C-NMIC. TGA curve of C-NMIC-Nps curve (c)
showed a lower shi in degradation temperature at 240 ꢂC. This
was mainly attributed to the ionic interaction of TPP with C-
NMIC favouring the formation of nanoparticles.28,36 The
decrease in thermal stability conrmed the decrease in the
crystalline nature of C-NMIC-Nps.19
From differential thermo gravimetric (DTG) curves (Fig. S8†),
it was observed that the degradation temperature of chitosan in
ꢂ
C-NMIC was lowered by 10 C compared to original chitosan.
This decreasing trend could be due to the random cross linking
of NMIC having more number of polar groups with chitosan
polymer backbone.43 C-NMIC-Nps (Fig. S8(c)†) showed two
decomposition peaks at 260 ꢂC and 370 ꢂC respectively. This
corresponds to the degradation temperature of chitosan and
TPP.35 Once again herein, a further decrease in chitosan
decomposition temperature would be due to the decrease in
chitosan crystallinity that arises as a result of ionic cross linking
of TPP with C-NMIC.19 On the basis of the results obtained from
TGA and DTG, it can be stated that increase of polar groups and
decrease in the crystalline domains caused the reduction in
thermal stability.
Fig. 2 FTIR spectra of (a) chitosan and (b) C-NMIC.
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3.2.5 Particle size analysis using DLS, SEM and HRTEM
analysis. The particle size data of C-NMIC-Nps by Dynamic light
scattering (DLS) (Fig. 3(a)) showed a size range of 6.5 to 24 nm
with a mean diameter of 14.4 ꢀ 3.1 nm. SEM (Fig. 3(b)) and
HRTEM (Fig. 3(c)) images showed that the average size of
C-NMIC-Nps was around 10 nm with smooth surface and
spherical morphology. The observed lattice fringes (Fig. 3(d))
RSC Advances
with different concentrations of pure C-NMIC-Nps along with
control (100%) was assessed by MTT assay. MTT was used to
assess the cell viability as a function of redox potential. Actively
respiring cells convert the water soluble yellow tetrazolium MTT
to an insoluble purple formazan.
It has been claimed that, the MTT formazan gives rise to
extracellular deposits of needle-shaped crystals by exocytosis.38
These crystals were dissolved in DMSO. The resulting purple
solution was spectrophotometrically measured. The effect of
the nanoparticles on the cell proliferation was expressed as (%)
cell viability.39–42
The results (Fig. S9†) exposed that the C-NMIC-Nps at 0.5 mg
mLꢁ1 dilution aer 72 h showed remarkable biocompatibility to
VERO cell lines with a maximum cell viability of 93.5%. A slight
decrease in cell viability was observed as the concentration of
the C-NMIC-Nps increased from (0.5–2.5 mg mLꢁ1). This indi-
cates the concentration dependent cytotoxicity of the nano-
particles.43 The signicant increase in the cell viability of
C-NMIC-Nps at all given dilutions with increasing time interval
(12 h to 72 h) suggested a good development of cells within.
Thus the positive time dependence property was a neat indi-
cation of the cell adherence and proliferation over the C-NMIC-
Nps. The phase-contrast images of the viable cells in VERO cell
lines seeded on control and C-NMIC-Nps aer 72 h of incuba-
tion were shown in Fig. S10.† The results suggested that the
C-NMIC-Nps has negligible cytotoxicity. Hence, the nano-
particles were biocompatible with the living cells and act as a
potential material for wound healing application.
˚
with 3.8 A inter lamellar spacing indicated that the crystalline
structure was not destructed completely. Also, the selected area
electron diffraction (SAED) pattern (Fig. 3(e)) of the nano-
particles supported the formation of polycrystalline C-NMIC
nanoparticles.4,37
3.3 Biocompatibility study
Biocompatible studies were done to evaluate the suitability of
any polymer based nanoparticles used for biomedical applica-
tions. Biocompatibility of the C-NMIC-Nps was carried out by
MTT assay using VERO cell lines. Cell viability of VERO cells
4. Conclusion
In conclusion, synthesis of novel NMIC, preparation of C-NMIC
and C-NMIC-Nps and their characterization has been reported.
Cross linking of NMIC with biodegradable carbonate moiety
through N-alkylation with chitosan was veried using instru-
mental analysis. The morphology of the nanoparticles as
observed by SEM and TEM images were found to be smooth and
spherical in shape. The size of the C-NMIC-Nps was found to be
in 10 nm range. The C-NMIC-Nps were proved to be non-toxic
and showed high cell viability 93.5% at a concentration of
0.5 mg mLꢁ1 highlighting their potential application in
biomedical eld as growth factor nano carrier for wound heal-
ing application.
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