Fabrication of chitosan-bis (4-formyl-2 methoxy phenyl carbonate) Schiff base nanoparticles and evaluation of their antioxidant and anticancer properties

Article abstract of DOI:10.1007/s11033-019-04887-4

Abstract: The present study details on the mechanism of synthesis of bis (4-formyl-2 methoxy phenyl carbonate), using two green reagents dimethyl carbonate and vanillin for application as therapeutic agent. The synthesized FMPC was identified from the ¹³C nuclear magnetic resonance spectra. The novel modified Schiff base nanoparticles resulted from the crosslinking of FMPC with chitosan were confirmed by cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance spectroscopy. The incorporation of the FMPC was identified from the amorphous X-ray diffraction patterns of C-FMPC-Nps. The thermal stability of the formed nanoparticles was predicted using thermogravimetric analysis. The morphology of the nanoparticles as observed from HRTEM was found to be smooth and spherical in nature. Both FMPC and C-FMPC-Nps showed significant radical scavenging potential and anticancer property. The carbonate ester backbone and the moiety present in chitosan-FMPC-nanoparticles, underwent hydrolysis at the targeted cancer causing microenvironment to release vanillin and chitosan and enhance the anticancer activity. Both FMPC and C-FMPC-Nps exhibits a dose dependent cytotoxicity towards the different cell lines and it was tested with a commercial drug for application studies. Graphical abstract: Effective synthesis of FMPC, successful incorporation onto chitosan nanoparticles for the formation of C-FMPC-Nps. The formed Schiff base compound proves to have enhanced antioxidant and anticancer efficacy.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11033-019-04887-4

Molecular Biology Reports
https://doi.org/10.1007/s11033-019-04887-4
ORIGINAL ARTICLE
Fabrication of chitosan‑bis (4‑formyl‑2 methoxy phenyl carbonate)
Schif base nanoparticles and evaluation of their antioxidant
and anticancer properties
P. Thyriyalakshmi1 · K. V. Radha1
Received: 26 September 2018 / Accepted: 15 May 2019
© Springer Nature B.V. 2019
Abstract
The present study details on the mechanism of synthesis of bis (4-formyl-2 methoxy phenyl carbonate), using two green
reagents dimethyl carbonate and vanillin for application as therapeutic agent. The synthesized FMPC was identifed from
the 13C nuclear magnetic resonance spectra. The novel modifed Schif base nanoparticles resulted from the crosslinking
of FMPC with chitosan were confrmed by cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance
spectroscopy. The incorporation of the FMPC was identifed from the amorphous X-ray difraction patterns of C-FMPC-Nps.
The thermal stability of the formed nanoparticles was predicted using thermogravimetric analysis. The morphology of the
nanoparticles as observed from HRTEM was found to be smooth and spherical in nature. Both FMPC and C-FMPC-Nps
showed signifcant radical scavenging potential and anticancer property. The carbonate ester backbone and the moiety present
in chitosan-FMPC-nanoparticles, underwent hydrolysis at the targeted cancer causing microenvironment to release vanillin
and chitosan and enhance the anticancer activity. Both FMPC and C-FMPC-Nps exhibits a dose dependent cytotoxicity
towards the diferent cell lines and it was tested with a commercial drug for application studies.
Graphical abstract
Efective synthesis of FMPC, successful incorporation onto chitosan nanoparticles for the formation of C-FMPC-Nps. The
formed Schif base compound proves to have enhanced antioxidant and anticancer efcacy.
Keywords FMPC · DMC · Vanillin · Chitosan · Anticancer · Oxaliplatin
Abbreviations
* K. V. Radha
FMPC
Bis (4-formyl-2 methoxy phenyl carbonate)
radha@annauniv.edu
DMC
Dimethyl carbonate
1
13C-NMR 13C nuclear magnetic resonance
Bioproducts Laboratory, Department of Chemical
Engineering, A.C. Tech, Anna University, Chennai,
Tamil Nadu 600025, India
Nps
Nanoparticles
Vol.:(0123456789)
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C
Chitosan
easily and enhance the availability of the drug at the target
site [10].
CP/MAS 13C-NMR cross-polarization magic angle
spinning carbon-13 nuclear magnetic
resonance
Recently, N-(2-hydroxyl) propyl-3-trimethylammonium
chloride [11], N-trimethyl chitosan [12] and glucomannan-
chitosan [13] nanoparticles were fabricated for the delivery
of proteins by ionic gelation method. Among wide variety
of crosslinked chitosan nanoparticles, vanillin crosslinked
chitosan nanoparticles and microparticles have received
considerable attention as vanillin is regarded as a safe mate-
rial and exert potent antioxidant activity, antibacterial and
anti-infammatory activity [14]. There is a great need to
address the challenge of preparing stable vanillin and it can
be achieved by crosslinking it with chitosan to form chitosan
particles with prolonged functionality that fnds application
in food and drug administration [15]. It has recently been
reported, that vanillin cross linked chitosan microspheres
was explored as bioactive microcarrier of drugs such as the
control release of resveratrol [16]. In addition, chitosan van-
illin nanoparticles were fabricated, that can act as a promis-
ing vehicle for the delivery of 5-furouracil against HT-29
cells [17]. Novel polymeric prodrug of vanillin termed
poly(vanillin oxalate) nanoparticles, have been developed
with great potential that fnds application as novel antioxi-
dant therapeutics and drug delivery systems [18].
XRD
X-ray difraction
TGA
Thermogravimetric analysis
High resolution transmission electron
microscopy
HRTEM
TPP
Sodium tripolyphosphate
Attenuated total refectance
Deuterium oxide
ATR
D2O
EI-MS
ZrO
Electron ionization mass spectrometry
Zirconium oxide
KHZ
TMS
TEA
MTT
Kilohertz
Tetramethyl silane
Triethyl amine
[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide]
DPPH
MEM
FBS
(1,1-Diphenyl-2-picryl hydrazyl)
Minimal essential medium
Fetal bovine serum
Introduction
In this current framework, eforts were made to synthe-
size FMPC, using green reagents vanillin and DMC. Herein,
the carbonate linkages present in FMPC which links the van-
illin moieties together would provide enhanced stability to
FMPC. The objective of the present study is to develop a
novel, facile route to fabricate C-FMPC-Nps, which would
act as a potential biomaterial in therapeutics. The incorpo-
rated FMPC will undergo hydrolysis at the site of carbonate
linkages producing hydroxyl groups. This would act as a
radical scavenger at the targeted site making the C-FMPC-
Nps a better antioxidant and anticancer agent.
Chitosan is a linear poly-amino saccharide obtained by
N-deacetylation of chitin, the second most abundant natu-
ral bio-polymer [1]. While, chitin occurs naturally in cell
walls of fungus and crustacean shells, chitosan is a fully
biodegradable and biocompatible natural polymer, and can
be used as an adhesive and as an antibacterial and antifun-
gal agent. Chitosan ofers many advantageous properties,
including hydrophilicity [2], non toxicity [3], biodegradabil-
ity [4] and biocompatibility [5] and it has been well docu-
mented for the preparation of value added products such as
food additives, cosmetics and drugs [6].
Chitosan, due to its biocompatible properties, has been
extensively studied for making it as prospective drug car-
rier. Chitosan can be modifed accordingly to its uses as the
elemental composition of the chitosan polymer is carbon
(44.11%), hydrogen (6.84%) and nitrogen (7.97%) with an
average molecular weight of ~5.3×105 Daltons [7]. Even
though search to form chitosan as micro and nano system for
biomedical application is common, majority of the attempts
are for preparation and synthesis of chitosan nanoparticles
[8]. Researchers have suggested using chitosan to coat nano-
particles made of other materials, in order to reduce their
impact on the body and increase their bioavailability. Nano
sizing of chitosan and its derivatives were found to have
high saturation solubility and dissolution rate due to large
surface area and small difusion layer thickness compared to
larger particles [9]. Polysaccharide nanoparticles with ultra-
tiny volume can penetrate through the cells and tissue gap
Materials and methods
Instrumentation
FTIR spectra of the samples were analyzed using Alpha
Bruker spectrophotometer in attenuated total refectance
(ATR) mode in the range of 4000–400 cm−1.
1H-NMR and 13C-NMR spectra of the samples were
recorded on a Bruker D8 Avance 500 spectrometer at 25 °C
using Deuterium oxide (D2O) as solvent.
Mass spectrum was recorded on an electron ionization
mass spectrometry (EI-MS) JEOL JMS-AX 500. Solid state
NMR spectroscopy was conducted using a Bruker Avance
600 spectrometer (14.10 T). Solid powder samples were
packed in a zirconium oxide (ZrO) ceramic cylindrical rotor
with 3.2 mm diameter and spun at 4.5 kHz. The chemical
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shift of the 13C nuclei was estimated using tetramethyl silane
(TMS), with δ=0 ppm as external reference. Contact time
of cross-polarization was 2.5 ms with accumulation of
1400–4000 scans.
tubes were kept in dark room for 20 min at ambient
temperature and measured for absorbance at 517 nm.
(b) Antioxidant property of C-FMPC-Nps suspension was
determined, by slight modifcation [20]. 0.2–2.2 mg/
mL of C-FMPC-Nps were mixed with 0.5 mL of
100 μM DPPH which was subjected to stirring for 2 h
under dark condition at ambient temperature and meas-
ured for absorbance at 517 nm. Chitosan was used to
compare the antioxidant activity of the prepared nano-
particles.
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 (λ=1.54 Å) source. The layer spac-
ing of the samples was calculated according to the Bragg’s
equation. The difraction angles 2θ were set between 2° and
75° incremented with a scanning angle of 1° min−1.
Thermal stability was examined by the TGA of TA Per-
kin Elmer instrument at a heating rate of 20 K min−1 in the
temperature range between 30 and 700 °C under nitrogen
atmosphere.
All the determinations were performed in triplicate and
the activity was measured using the following equation.
Ac − As
DPPH radical scavenging activity (%) =
× 100%
Ac
For high resolution transmission electron microscopy
(HRTEM) studies of nanoparticles, 5 mL of the sample was
coated on a carbon-coated copper HRTEM grid, which was
subsequently air dried and was analysed by HRTEM (JEM
2100, Jeol, Peabody, MA, USA) operating at a high voltage
of 200 keV.
Ac is the absorbance of control (all reagents except stand-
ard/sample), As is the absorbance of standard/sample.
Evaluation of cytotoxicity against cell lines (cell culture
preparation and experimental design)
The bioassay of the nanoparticles was done using Shi-
madzu UV-3101 spectrophotometer.
Human cancer cell line MCF-7 and A 2780 used in this
study was procured from National Centre for Cell Science,
Pune. HT-29 (ATCC HTB-38) cell lines were procured from
American Type Culture Collection (Rockville, MD, USA).
MCF-7 and A 2780 cells were grown in Minimal essen-
tial medium (MEM) supplemented with 4.5 g/L glucose,
2 mM l-glutamine and 5% fetal bovine serum (FBS) (growth
medium) at 37 °C in 5% CO2 incubator. HT-29 cells were
grown on E-MEM Minimum Essential Medium (MEM) with
Earle’s salt and nonessential amino acids, supplemented with
5% heat-inactivated fetal calf serum (FCS), 1 mM sodium
pyruvate, and 2 mM l-glutamate. The MTT assay developed
by Mosman [21] was modifed and used to determine the
inhibitory efects of test compounds on cell growth in vitro.
In brief, the trypsinized cells from T-25 fask were seeded
in each well of 96-well fat-bottomed tissue culture plate at
a density of 5 × 103 cells/well in growth medium and cul-
tured at 37 °C in 5% CO2 to adhere. After 48 h incubation,
the supernatant was discarded and the cells were pre-treated
with growth medium and were subsequently mixed with dif-
ferent concentrations of test compounds (12.5, 25, 50, 100,
200 μg/mL) to achieve a fnal volume of 100 μL and then
cultured for 48 h. All experiments were carried in triplicates.
The compound was prepared as 1.0 mg/mL concentration
stock solutions in DMSO. Culture medium and solvent were
used as controls. Each well then received 5 μL of fresh MTT
(5 mg/mL in PBS) followed by incubation for 2 h at 37 °C.
The supernatant growth medium was removed from the wells
and replaced with 100 μL of DMSO to solubilize the colored
formazan product. After 30 min incubation, the absorbance
Chemicals
Analytical grade dimethyl carbonate (DMC), 4-hydoxy-
3-methoxy benzaldehyde (vanillin) and triethyl amine (TEA)
were purchased from Sigma Aldrich. Chitosan of low molec-
ular 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 bro-
mide] with 98% purity, drug oxaliplatin obtained from local
drug store and DPPH (1,1-diphenyl-2-picryl hydrazyl) were
also obtained from Sigma Aldrich. All other reagents were
of analytical grades and were used without any further puri-
fcation. Milli-Q grade (resistivity of 18.2 MU cm−1) water
was used in all the experiments.
In vitro bio assays
Antioxidant activity by DPPH method
The free radical scavenging activity was measured by DPPH
assay as described below.
(a) The determination of the antioxidant activity of the
FMPC, was done by a slight modifcation of the litera-
ture protocol [19]. 2 mL of 100 μM FMPC prepared
in distilled ethanol was added to ethanol solution of
0.5 mL of 100 μM DPPH in test tube. Ascorbic acid
(100 μM) was used as a standard antioxidant. The test
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(OD) of the culture plate was read at a wavelength of 570 nm
on an ELISA reader, Anthos 2020 spectrophotometer. Per-
centage inhibition of compounds against the cell lines were
calculated using the following formula [22].
of FMPC was added to the same and refuxed for 48 h at
60 °C with continuous stirring. FMPC crosslinked chitosan
nanoparticles were collected, after washing the particles with
methanol several times by centrifugation at 10,000 rpm for
20 min. The C-FMPC-Nps were fnally dried in a vacuum
oven at 40 °C for 12 h. FTIR: νmax/cm−1 1646 (–CH=N– str),
1600 (C=C aromatic str), 1374 (sym angular def of CH3),
1254 (ester C=O assy str), 1152 (anti sym C–O–C), 887
(sym str of C–O–C). 13C-CP MAS (150.32 MHz,): δc
ppm 171 [O–(C=O)–O/(Ci)], 167 [–HC=N–/(Cg)],
148 [O–C=C– and CH3O–C–Ar/(Ca and Cb)], 126
[–N=CH–C–(Ar)/(Cd)], 116 [–C=CH–CH–(Ar)/(Ce and
Cf)], 103 [CH3O–C=CH–C–(Ar)/(C1 and Cc)], 82 (C4),
74 (C5 and C3), 56 [CH3O–(Ar)/(Ch, C6 and C2)], 22
(CH3CO– of chitosan).
At − Ab
% cell survival =
× 100
Ac − Ab
where At is the absorbance of test, Ab is the absorbance of
the blank (media), Ac is the absorbance of control (cells)
% cell inhibition = 100 − % cell survival
IC50 determination
The drug concentration that reduced the viability of cells by
50% (IC50) was determined by plotting triplicate data points
determined by plotting a graph of log (concentration of com-
pound) versus % cell inhibition.
Results and discussion
Mechanism of FMPC synthesis and its reaction
with chitosan for efective Cytotoxicity
Synthesis of FMPC
8.4 mL of 0.1 M DMC was allowed to react with 30.4 g of
0.2 M vanillin in the presence of 13.9 mL of triethyl amine
base and refuxed at 86–88 °C for about 48 h. The crude com-
pound obtained was subjected to purifcation using dry diethyl
ether to get a fnal brown coloured product FMPC with 40%
yield. FTIR: The peaks obtained (max/cm−1) are as follows:
1668 (ester and aldehyde C=O str), 1590 (C=C aromatic
str), 1087 (ester C–O sym str), 1039 (ether C–O str), 878 and
798 (phenyl C–H out-of-plane bends). 1H-NMR (500 MHz,
D2O): δΗ ppm 9.4 (2H, s, Ar–CHO, H-g), 7.4 (2H, d, J=2 Hz,
–O–C–C=CH–, H-e), 7.3 (2H, s, O–C–CH=C–CHO, H-c),
6.7 (2H, d, J = 8 Hz, –O–C–CH=CH–, H-f), 3.7 (6H, s,
–OCH3, Ar-OCH3, H-h). 13C-NMR (500 MHz, D2O): δχ ppm
193 (Ar–CHO), 171 (–O–C=O–O–(Ar)), 148 ((–O–C–Ar) and
(CH3O–C(Ar)), 126 (CHO–C(Ar)), 116 (–O–CH=CH(Ar))
and (–O–CH=CH–CH(Ar)), 103 (CH3O–C=CH(Ar)), 56
(Ar-OCH3).
Studies were carried out to check the detailed mechanism
undergone on the formation of C-FMPC-Nps. If the back-
bone of the compound is stable then the anticancer activity
was enhanced. For this initial steps were carefully optimized
for efective synthesis of FMPC and incorporating into chi-
tosan nanoparticles. On study it was found that the DMC hits
the hydroxyl group of vanillin and carboxy methylation [23]
reaction takes place through BAC2 mechanism (Scheme 1).
This resulted in the formation of FMPC that was carried
out at 88 °C and methanol was formed as byproduct [24].
Next step is the formation of C-FMPC-Nps which was done
through condensation reaction of aldehyde functionality
of vanillin with the free amine groups on chitosan. This
resulted in formation of Schif base chitosan exhibiting the
presence of azomethine (–CH=N–) linkage in C-FMPC-Nps
(Scheme 2) [25].
The successful formation of schif base was confrmed
by elemental analysis (Table 1). From the results it could be
inferred that the C-FMPC-Nps showed an increase in nitro-
gen percentage but a decrease in carbon and hydrogen per-
centage compared to chitosan. This behaviour would lead to
a conclusion, that FMPC has been successfully incorporated
onto chitosan backbone. In addition, the decrease in the C/N
ratio is an indicative of the successful crosslinking of chi-
tosan with the pendent FMPC to form Schif base [26, 27].
Preparation of chitosan nanoparticles
Initially, with 1 g of chitosan prepared in 100 mL of 1% aque-
ous acetic acid, 0.1 g of TPP in 100 mL of water was gently
dripped and kept at room temperature for 10 min to get an
opalescent suspension. The obtained suspension was subjected
to centrifugation at 10,000 rpm for 30 min at 4 °C. The formed
nanoparticles were again re-suspended in distilled water, soni-
cated, centrifuged and freeze dried.
Spectral studies for the formation of (FTIR, NMR
and Mass) of FMPC and C‑FMPC‑Nps
Fabrication of C‑FMPC‑Nps
Various studies were performed to analyze and predict the
formation of C-FMPC-Nps through FTIR, NMR and Mass
spectroscopy.
100 mg of chitosan nanoparticles dispersed in methanol
was taken in a 100 mL round bottom fask. Excess amount
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Scheme 1 Synthetic route for the preparation of FMPC
Scheme 2 Schematic structure of newly synthesized C-FMPC-Nps Schif base
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Table 1 Elemental analysis of C-FMPC-Nps
Element %
Sample
Chitosan
C-FMPC-Nps
C
40.43
33.63
N
6.57
5.63
H
7.17
7.31
C/N
5.63
4.60
FTIR confrms the presence of (C–O) phenolic stretch
at 1299 cm−1 present in vanillin (Fig. 1), was found to be
absent in the synthesized FMPC which is a clear indica-
tion of the successful formation of FMPC. In the formed
C-FMPC-Nps (Fig. 2b), the peak at 1668 cm−1 corresponds
to free aldehyde group of vanillin (Fig. 2a) which was found
to be absent and a new peak appeared at 1646 cm−1 that indi-
cates the formation of schif base [28]. The absence of peak
at 1559 cm−1, in C-FMPC-Nps corresponding to the amide
II band of chitosan, was a clear evidence for the covalent
attachment of FMPC with chitosan. In addition, the identi-
fcation of peaks at 1600 cm−1 and 1152 cm−1 are due to the
presence of C=C aromatic stretching vibration of vanillin
and C–O–C anti symmetric bridging vibration of chitosan.
This confrms the successful incorporation of FMPC with
chitosan [29].
Fig. 2 FTIR of (a) chitosan and (b) C-FMPC-Nps
1
The H-NMR spectrum of FMPC (Fig. 3), exhibits the
same characteristic resonances of vanillin [30]. Here, the
formation of FMPC was confrmed by the reappearance of
proton signal which corresponds to aldehyde functionality
of vanillin at 9.4 ppm [31]. Therefore, it proves that vanil-
lin –OH is involved in reaction with the carbonyl group of
DMC through BAC2 mechanism.
Further confrmation was done through 13C-NMR spec-
trum (Fig. 4). The characteristic resonance at 193 ppm indi-
cates the presence of free (–C=O) group in FMPC [32].
In addition to the signals found in FMPC, the most pre-
dominant resonances at 171 and 148 ppm corresponds to
(–C=O–) of ester and (–O–C–(Ar)) of carbonate linkage
that exhibited the formation of FMPC with a stable carbon-
ate backbone [33].
The EI mass spectra (Fig. 5) of the synthesized FMPC
showed a molecular ion peak at (m/z) 328.18 that was in
good agreement with the calculated theoretical values.
In the CP/MAS 13C-NMR spectrum of C-FMPC-Nps
(Fig. 6), the peak of aldehyde carbon at 195 ppm [34] disap-
peared and a new signal at 167 ppm appeared. This has been
attributed to the formation of schif base chitosan, resulted
from the condensation reaction between chitosan and FMPC
[35].
XRD of C‑FMPC‑Nps
Figure 7 indicates the XRD profile of pristine chitosan,
vanillin and C-FMPC-Nps respectively. The high degree
of crystallinity of chitosan (Fig. 7a) is indicated by the
presence of sharp diffraction peak at 2θ = 20.3° and a
weak diffraction peak at 2θ = 11.6° [36]. The presence of
a sharp diffraction peak of vanillin at 2θ= 13.3° (Fig. 7b),
appeared as semi crystalline peak at 2θ = 13.7° in
Fig. 1 FTIR of (a) vanillin and (b) FMPC
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Fig. 3 1H-NMR of FMPC
Fig. 4 13C-NMR of FMPC
C-FMPC-Nps (Fig. 7c) [37]. Herein, for C-FMPC-Nps the
observed peak at 2θ = 13.7° with weak reflection exhibits
the existence of interdigited hydrogen bond between chi-
tosan and vanillin unit present in FMPC [38]. In addition,
C-FMPC-Nps exhibits a broad peak at 2θ = 20.6° with
decrease in magnitude that is an indicative of molecular
miscibility between the chitosan, TPP and FMPC. Thus,
the successful formation of C-FMPC-NPs has been con-
firmed by the XRD patterns.
TGA of C‑FMPC‑Nps
The thermal stability of chitosan, vanillin and the prepared
C-FMPC-Nps was studied from TGA results and are indi-
cated in Fig. 8. For chitosan, vanillin and C-FMPC-Nps,
the initial weight loss around 50–150 °C is due to the mois-
ture vaporization and bound water of hydration. Chitosan
(Tmax at 298 °C) (Fig. 8a) was an indicative of disruption of
strong inter and intra molecular hydrogen bonds, due to the
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Fig. 5 Mass spectra of FMPC
Fig. 7 XRD of (a) chitosan, (b) vanillin and (c) C-FMPC-Nps
depolymerisation and thermal decomposition of the acety-
lated and deacetylated units of polymeric chitosan [39]. For
vanillin (Fig. 8b) Tmax value has been observed at 230 °C
that corresponds to the degradation or thermal evaporation
of vanillin [40].
It is evidenced from Fig. 8c, for C-FMPC-Nps
(Tmax =269) the thermal stability, was comparatively higher
than vanillin. This could be due to the incorporation of sta-
ble carbonate backbone present in FMPC moiety. But, rather
the decrease in the stability of C-FMPC-Nps compared
Fig. 6 (CP/MAS) 13C-NMR of FMPC
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repulsion, hydrophobic interaction and hydrogen bonding
exists between diferent functionalities present in C-FMPC-
Nps that results in transparent colloidal aggregates,as clearly
observed from TEM analysis [42].
DPPH radical scavenging activity of FMPC
and C‑FMPC‑Nps
DPPH is a relatively stable nitrogen centred free radical. It
has the ability to react with electron/hydrogen atom donat-
ing reducing agents and converting it into a nonradical
1,1-diphenyl-2-picryl hydrazine (yellow) diamagnetic mol-
ecule which can be measured spectrophotometrically [43].
The IC50 values of the newly synthesized FMPC and
standard ascorbic acid to scavenge the DPPH radicals were
found to be 48.7% and 89.6% respectively. FMPC was found
to be almost equally potent as compared to ascorbic acid by
DPPH assay. The functional groups present in the targeted
compound are highly responsible for the free radical scav-
enging activity. It could be understood from the structure
that the antioxidant activity of the FMPC might be attributed
to the electron donating nature of the substituent –OCH3
[44]. The presence of –OCH3 on FMPC increases both the
electron density and electron mobility in the core structure
[45] and stabilizes the generated radical during oxidation
[46] thus, exhibiting predominant DPPH radical scaveng-
ing activity.
Fig. 8 TGA of (a) chitosan, (b) vanillin and (c) C-FMPC-Nps
to chitosan was because of the introduction of the FMPC
moiety in chitosan. FMPC decreases the number of amine
groups on chitosan by obstructing the chain packing of chi-
tosan resulting in the decreased stability of C-FMPC-Nps
[41].
TEM observation of C‑FMPC‑Nps
The visualization of the particles as performed by TEM
analysis indicates the size and morphology of the formed
C-FMPC-Nps (Fig. 9). TEM image revealed that the size of
the nanoparticles were around 25–30 nm in diameter with
smooth surface and spherical morphology. The electrostatic
Figure 10 represents the antioxidant activity of the
C-FMPC-Np. The IC50 values of the chitosan and C-FMPC-
Nps were found to be 1.859 and 0.9739 mg/mL respectively.
The antioxidant activity of C-FMPC-Nps indicates that the
nanoparticles act as a potent antioxidant in scavenging
DPPH free radicals.
Fig. 10 DPPH radical scavenging property (a) chitosan (b) C-FMPC-
Fig. 9 HRTEM image of C-FMPC-Nps
Nps. Each value represents the mean SD (n=3)
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The antioxidant property is far higher than the synthe-
sized FMPC. The reason behind this high activity might be
due to the synergistic efect of both FMPC and chitosan [47].
Notably, the electron donating –OCH3 present in vanillin
moiety, the hydroxyl groups present at C3 and C6 position of
chitosan and the presence of (–C=N–) linkage enhanced the
radical scavenging activity of the C-FMPC-Nps. Wherein,
the high electron density over nitrogen present in the schif
base (–C=N–), overlaps with the neighbouring (–O·) radi-
cal electrons formed at the C3 carbon of chitosan would
stabilize the free radical formed [48]. Further, the increased
electron fuidity of the prepared nanoparticles upon forma-
tion of the schif base [49] would resulted in the enhanced
antioxidant property of the schif base C-FMPC-Nps [50].
Inhibitory efect of FMPC and C‑FMPC‑Nps
against MCF‑7, HT‑29 and A 2780 cell line
As the antioxidant result of FMPC is encouraging, further
screening of FMPC for anticancer activity against MCF-7,
HT-29 and A 2780 cell lines were undertaken. The in vitro
cytotoxic activity was accessed and graphically represented
for Chitosan and FMPC in Fig. 11 and C-FMPC-Np’s in
Fig. 12. The IC50 value was determined to be 0.501 μg/mL,
23.098 μg/mL and 25.189 μg/mL respectively for MCF-7
cell line and all other cell line values are tabulated. (Table 2).
From the result it could be inferred that the electron with-
drawing –OCH3 group is highly responsible for the posi-
tive efect against stress related H2O2 scavenging anticancer
activity of the synthesized compound [51].
Fig. 12 Anticancer efect of C-FMPC-Nps
cancer induced acidic environment the carbonate back bone
might be cleaved [52] to release vanillin, which suppress
the generation of ROS, such as H2O2 and exerts, excellent
activity against oxidative stress [53]. Thus FMPC exhibits
dose dependent cytostatic/cytolysis behaviour [54] towards
all cell lines. The high levels of H2O2 found in cancer cell
line induce the hydrolytic cleavage of carbonate ester bonds
present in FMPC [55]. Thus the release of vanillin from
FMPC would result in the anticancer activity of the syn-
thesized FMPC. The percentage inhibition of C-FMPC-Nps
on MCF-7 cancer cell line is indicated and the activity of
the nanoparticles was compared with chitosan. The inhibi-
tory efect of C-FMPC-Nps was higher on comparison with
chitosan (Fig. 13). The increase in the anticancer activ-
ity of C-FMPC-Nps arises from the schif base formation
between vanillin and chitosan. Here, the reactive oxygen
species produced at the targeted site were quenched by
the localized electron dense nitrogen atom present in the
schif base. In addition to the aforementioned property, the
hydrolytic cleavage of the carbonate backbone [56] and the
Synthesized FMPC with carbonate ester backbone
structure has high response towards H2O2. Thus under the
Table 2 IC50 values of the FMPC and C-FMPC-Nps against MCF-7,
HT-29 and A 2780 cell line
Anticancer activity cell lines IC50 (µg/mL) S.D
MCF-7
HT-29
A 2780
Chitosan
FMPC
C-FMPC-Nps
0.501 1.34
35.39 1.16
25.189 2.05
12.45 1.71
65.21 2.43
59.32 1.18
13.67 3.12
47.28 1.87
67.55 2.21
Fig. 11 Anticancer efect of synthesized (a) chitosan and (b) FMPC
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Molecular Biology Reports
Fig. 13 Images of (a) control
and (b) FMPC treated MCF-7
cells
C=N linkage [57], would release vanillin and chitosan from
C-FMPC-Nps.
method is by employing drug response assays in vitro.
The MTT assay is one of the methods used to predict the
drug response in malignancies [59]. Incorporation of cell
culture studies ofers good possibility to assess the drug in
controlled conditions. The cytotoxic response of diferent
cell lines is evaluated using high-throughput MTT assay. It
measures the cell respiration and the amount of formazan
produced is proportional to the number of living cells pre-
sent in culture. An increase or decrease in cell number
results in a concomitant change in the amount of formazan
formed, indicating the degree of cytotoxicity caused by the
drug. MTT was used to assess the cell viability as a func-
tion of redox potential. Actively respiring cells convert the
water soluble yellow tetrazolium MTT to an insoluble purple
formazan.
The synergistic effect of both chitosan and vanillin
moieties released at the targeted carcinogenic site would
enhanced the anticancer activity thus curtailing the growth
of cancer cells (Scheme 3). The inhibitory efect led us to
speculate, that there is a steady and linear increase in the
cytotoxic efect with increase in concentration of the pre-
pared nanoparticles [58].
Cytotoxic response of diferent cell lines
to the commercial drug oxaliplatin
One of the goals of oncology is to predict the response
of patients with cancer to chemotherapeutic agents. One
Scheme 3 Hydrolytic cleavage
of C-FMPC-Nps
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Molecular Biology Reports
It has been claimed that, the MTT formazan gives rise to
extracellular deposits of needle-shaped crystals by exocy-
tosis. These crystals were dissolved in DMSO. The result-
ing purple solution was spectrophotometrically measured
and the percentage of viability was calculated. The results
(Fig. 14) exposed that with increasing time period from 12 to
72 h, the vero cells adhere and proliferate on C-FMPC-Nps
facilitating cell attachment [18]. The signifcant increase in
the optical density (OD) value of C-NMIC-Nps suggested a
good development of cells within. The images of the viable
cells in vero cell lines treated using control and C-FMPC-
NPs after 72 h were shown in Fig. 15. The test performed on
vero cell lines, showed the cell viability of 93.5% at a maxi-
mum concentration of 0.5 mg/mL after 72 h of incubation.
This suggested that the prepared C-FMPC-Nps was found
to be less cytotoxic in vero cell lines.
IC50 is the concentration of the tested drug able to cause
the death of 50% of the cells and can be predictive of the
degree of cytotoxic efect. The lower the value, the more
cytotoxic is the substance. Figure 16, shows the IC50 of
drug oxaliplatin against human cancer cell lines. IC50 val-
ues resulted were 1.65, 0.98 and 0.44 for MCF-7, HT-29 and
A 2780 cell lines indicating the cytotoxicity of the drug to
that specifc cancer cell type. The premise of in vitro drug
response testing is that it can provide the knowledge of
the relative efcacy of the various agents used in standard
therapy before an empiric in vivo trial. Cell-based assays
may help in the selection of chemotherapeutic drugs with
the greatest likelihood for clinical efectiveness, and in the
exclusion of inefective therapy. This can lead to improved
disease management, response, survival and use of fnancial
resources. The IC50 was estimated from the curve generated.
The lower the IC50, the more cytotoxic the drug is to that
specifc cancer cell type.
Conclusion
In the present study, synthesis, characterization and mecha-
nism of novel FMPC, C-FMPC and C-FMPC-NPs using
DMC has been reported. Successful formation of FMPC
and its incorporation in chitosan nanoparticles through
cross linking by N-alkylation of chitosan was verified
using instrumental analysis. C-FMPC-NPs were proved to
be non-toxic and showed high cell viability even when the
concentration of the C-FMPC-NPs was 0.5 mg/mL high-
lighting their potential application in biomedical feld. The
prepared nanoparticles were found to be more efective
than FMPC in the screening of anticancer activity against
cell lines. Both FMPC and C-FMPC-Nps exhibits a dose
dependent cytotoxicity towards various cell lines. The struc-
ture activity relationship revealed that in addition to –OCH3
moiety the –CH=N– imine moiety plays a vital role for the
Fig. 14 Cell viability test performed on vero cell lines after cultivat-
ing with diferent concentrations of C-FMPC-NPs
Fig. 15 MTT assay of (a) con-
trol and (b) C-FMPC-Nps
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Molecular Biology Reports
IC50 = 1.65 +/- 0.1
(a) MCF-7 cells
IC50 = 0.98 +/- 1.3
(b) HT -29
IC50 = 0.44 +/- 4.8
(c) A 2780
IC50 = 1.272
Combined effect
Fig. 16 IC50 of drug oxaliplatin against human cancer cell lines (a) MCF-7 cells, (b) HT-29, (c) A 2780, (d) total
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Compliance with ethical standards
Conflict of interest The authors have no confict of interest.
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