Carbohydrate Polymers 236 (2020) 116101

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Chitosan-cellulose hydrogel conjugated with L-histidine and zinc oxide T

nanoparticles for sustained drug delivery: Kinetics and in-vitro biological
studies
Dhanya George, P. Uma Maheswari, K.M. Meera Sheriffa Begum*
Department of Chemical Engineering, National Institute of Technology, Tiruchirapalli, 620015, Tamilnadu, India

A R T I C LE I N FO A B S T R A C T

Keywords: Functionalised nanohybrid hydrogel using L-Histidine (HIS) conjugated chitosan, phyto-synthesised zinc oxide
L-Histidine - chitosan nanoparticles (ZNPs) and dialdehyde cellulose (DAC) was formulated as a sustained drug delivery carrier for the
Cellulose crosslinker polyphenol drugs – Naringenin (NRG), Quercetin (QE) and Curcumin (CUR). A maximum loading efficiency of
Nanohybrid hydrogel 90.55 %, 92.84 % and 89.89 %, respectively were optimised for NRG, QE and CUR in the hybrid hydrogel. The
Drug delivery
maximum drug release was favoured for the optimum drug loading and at pH-5. HIS-chitosan conjugation
Antimicrobial
Anticancer activity
stabilised the hydrogel and enabled a sustained drug delivery. Drug release kinetics predicted a non-Fickian
diffusion-based mechanism along with polymer erosion. Prominent antimicrobial activity against Staphylococcus
aureus and Trichophyton rubrum strains were predicted to evolve based on the synergic formulation. Significant
biocompatibility towards L929 cells revealed their support for normal cell survival. Anticancer studies towards
A431 cells exhibited excellent cytotoxicity with a 15 to 30-fold increase using the hybrid carrier, compared to
the free polyphenol drugs.

1. Introduction properties of the human tissues.

Novel nanohybrid hydrogels are highly researched for specific ap-
plications in the field of biomedical engineering. The nanohybrid
system consists of at least one constituent in nano-dimensions with
inorganic nature (Liu, Wang, & Wang, 2012). Based on the nature of
application, the organic/ inorganic constituents are further appro-
priately chosen and are suitably functionalised for the development of
materials towards the drug delivery and topical medical applications.
They possess superlative properties required for drug encapsulation,
release and sustainment including various stimuli responsive signalling
which resulted from the incorporation of selective functional groups in
the hybrid biomaterials.
The utilisation of natural polymers such as protein and carbohy-
drates over synthetic polymers as the primary precursors for the de-
velopment of nanohybrid hydrogels are of significant interest. The de-
velopment of stimuli sensitive hydrogels that can alter their
performance in tune to the biological stimuli such as change in pH,
enzyme, redox properties, temperature and small molecules are on a
high demand (Ananikov, 2019). When the natural precursors are used,
they exhibit self-imbibed biocompatibility, non-toxicity and eco-
friendliness with stimuli response and closely found to mimic the
Chitosan obtained from the shell source is highly appreciated in the
development of reinforced biomaterials with modifications (Pella,
Lima-tenorio, & Tenorio-neto, 2018; Sahariah & Masson, 2017). The
hydroxyl and amino groups on the polymer backbone provides broad
spectrum of scope for functional modifications. Amino acid based
chitosan derivatives ensure the presence of variety of side chain func-
tional groups that are acidic, basic and hydrophobic in nature. Their
presence in the hydrogel matrices is expected to improve the drug en-
capsulation, release and improve the therapeutic effects of the re-
spective drug. DAC, a cellulose derivative was found to effectively
crosslink the polymer matrices by the schiff base mechanism and hy-
drogel thus obtained was non-toxic and highly biodegradable (Kimura
et al., 2011; Munster, Capakova, Fisera, Kuritka, & Vicha, 2019). L-
Histidine (HIS) is an essential amino acid with a hydrophobic imidazole
group that is highly used in the nature for coordinating the metal ions
in the enzyme active sites (Zhang et al., 2019). They also participate in
stabilizing the quaternary folded structures of proteins through hy-
drogen bonding in the living cells. HIS is also vital for maintaining a
neutral pH in the body by shuttling proton to maintain acid/base bal-
ance in the tissues and blood. The imidazole group on HIS with a pKa
value of 6.1, protonates at acidic pH which is expected to improve the

⁎
Corresponding author.
E-mail address: meera.nitt.edu@gmail.com (K.M.M.S. Begum).

https://doi.org/10.1016/j.carbpol.2020.116101
Received 15 February 2020; Received in revised form 24 February 2020; Accepted 28 February 2020
Available online 29 February 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
D. George, et al.
pH-responsive stimuli of the chitosan carrier, when suitably modified
with HIS (Wang et al., 2017). This is desirable for the targeted delivery
of anticancer drugs because tumor tissue has a strong acidic environ-
ment compared with normal tissues. Nanohybridisation of hydrogels
using metal/metal oxide nanoparticles such as gold, silver, zinc oxide,
titanium oxide, silica etc provides larger surface area and enhanced
porous structure which favours better drug binding and release prop-
erties (Utech & Boccaccini, 2016). Further, the incorporation of in-
organic nanoparticles results in the development of antioxidant hy-
drogels which synergistically contribute to the antimicrobial or
anticancer property of the loaded drug in the hydrogel carrier
(Senapati, Mahanta, Kumar, & Maiti, 2018).
Plant derived polyphenols are known as strong antioxidants which
exhibits UV protection, anti–inflammatory, antiaging, anticancer,
cardio-protective and antimicrobial properties (Piccolella, Crescente,
Candela, & Pacifico, 2019). However, formulation of polyphenolic
drugs in suitable carrier is required to improve their ADME profile,
stability, bioavailability and activity of the drug (Oliver, Vittorio, &
Boyer, 2016). A functionalised carrier mediated delivery of poly-
phenolic drugs is essential in improving the drug stability under phy-
siological conditions and to release the drug on tuning towards the
target site.
In the present study, chitosan modified by HIS conjugation and
embedded with green ZNPs was crosslinked with dialdehyde cellulose
to result in a highly functionalised nanohybrid hydrogel. This green
crosslinked hydrogel carrier was tested for the delivery three poly-
phenolic drugs with noticeable antimicrobial and anticancer properties,
namely NRG, QE and CUR. These drugs were optimally loaded in the
nanohybrid hydrogel carrier and its release properties were studied
under various conditions. The significant impact of the developed hy-
drogel carrier on improving the therapeutic activity of the polyphenolic
drugs such as antimicrobial and anticancer activities were compared in
the light of the participation of the hybrid carrier.
2. Materials and methods
2.1. Materials
Muskmelon seeds and SCB were collected from a juice stalls in the
nearby locality. Low molecular weight chitosan (85 % degree of dea-
cetylation), NRG, HIS and QE were procured from Sigma–Aldrich
Chemie, Steinheim, Germany. Sodium hydroxide, sodium chlorite,
glacial acetic acid and silicon oil were supplied by Loba Chemie Pvt.
Ltd., Mumbai, India. Hydrogen peroxide, sodium metaperiodate, trie-
thylamine, zinc acetate dihydrate, potassium chloride, hydrochloric
acid, acetone, potassium dihydrogen phosphate and Tween 80 were
purchased from Merck Life Science Private Limited, Mumbai. CUR and
EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)
was procured from Otto Chemie Private Limited, Mumbai. The micro-
bial strains Gram – positive bacteria, Staphylococcus aureus (MTCC 96)
was obtained from Microbial Type Culture Collection (MTCC) at the
Institute of Microbial Technology (IMTECH), Chandigarh, India. The
fungus strains of Trichophytonrubrum was collected from Rontgen di-
agnostic center, Thanjavur, India. L929 murine fibroblast cell lines and
A431 human skin carcinoma cell lines were obtained from National
Centre for Cell Science, Pune, India which were revived and kept under
constant culture. The cells were grown as monolayers in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10 % fetal calf
serum, penicillin (100 units/mL) and streptomycin (100 μg/mL).
Double distilled water was used for all the preparations.
2.2. Conjugation of chitosan with HIS
Chitosan (3% (w/v)) was dissolved in 1% acetic acid and pH of
2.0–3.0 was maintained by stirring overnight to ensure complete so-
lubility. 0.75 g of HIS was gradually added to the homogenised chitosan
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Carbohydrate Polymers 236 (2020) 116101
solution. EDC crosslinker weighing 0.6 g was added under continuous
stirring in the presence of the deprotonating base, trimethylamine and
stirred constantly at 40 °C for 3 days. The transformation of the dusty
white chitosan solution to a pale brown colour confirmed the amide
formation between the amino group (-NH2) of chitosan and the car-
boxyl group of HIS. The reaction mixture was neutralised using 3 N
sodium hydroxide solution. The resulted precipitate was repeatedly
washed with distilled water to obtain neutral condition. Finally, CH-HIS
was washed with acetone and lyophilised. The HIS modified chitosan
was further used as the polymer base for the preparation of nanohybrid
hydrogel.
2.3. Preparation of drug loaded nanohybrid hydrogel
The CH-HIS conjugated polymer precursor was embedded with the
phyto- synthesised ZNPs and crosslinked with biomass derived cross-
linker DAC to obtain the nanohybrid hydrogel. The experimental pro-
cedure adopted for the synthesis of ZNPs and DAC were briefly ex-
plained in our earlier reports (George, Maheswari, Arthanareeswaran, &
Begum, 2019). Nanohybrid hydrogels were prepared using Schiff base
crosslinking method by dissolving modified chitosan (CH-HIS) solution
of 2% (w/v) in 1% (v/v) acetic acid solution under stirring. ZNPs dis-
persion in water was added to the solution at varying ZNPs to CH-HIS
mass ratios (0.1–0.3). DAC was dissolved in water by stirring at 100 °C
and was added at varying crosslinker to CH-HIS mass ratios
(0.05–0.25), until an effective crosslinking leading to gelation was ob-
served. The nanohybrid hydrogel (HIS-CHGZ) obtained was repeatedly
washed with water and freeze-dried.
Drug loading in hydrogels was performed by swelling method where
dried hydrogel of 0.1 g was soaked in 10 mL of the drug solution for
24 h to obtain the drug loaded HIS-CHGZ and CHGZ nanohybrid hy-
drogels. The concentration of the drug solution was measured before
and after drug loading using UV–vis spectrophotometer at 290 nm for
NRG, 368 nm for QE and 421 nm for CUR respectively. The drug
loading efficiency (LE) was calculated using Eq. (1).
( Initial amount of drug used− unloaded drug)
LE (%) = × 100
(Initial amount of drug used) (1)
Drug loading efficiencies were optimised as a function of three
process parameters such as drugs to CH-HIS ratio, ZNPs to CH-HIS ratio
and DAC to CH-HIS ratio. Using Taguchi method of optimisation, nine
set of experiments were performed for three levels of the three process
parameters chosen (George, Maheswari, & Begum, 2020). Based on the
optimised drug loading conditions, the drugs were loaded on HIS-CHGZ
and used for further drug release and biological studies.
2.4. Material characterisation
The following analytical techniques were used to characterize the
prepared functional materials. 1H NMR was performed using 500 MHz
Bruker Nuclear Magnetic Resonance (NMR) spectrometer. Samples
were prepared in HCl/D2O solution to ensure complete solubility. Solid-
state 13C NMR spectra were obtained on a Bruker 500 MHz FT-NMR
spectrometer instrument operating at 125.75 MHz. Spectra were ac-
quired using the technique of CP-MAS (cross polarised magic angle
spinning) for the samples spun at 8 kHz using a contact time of 3.3 ms
and an interscan delay of 5 s. FTIR (Fourier Transform Infrared
Spectroscopy) measurements were done in Thermo scientific FTIR
spectrophotometer (Nicolet iS5) in the range from 4000 to 400 cm−1
with 32 scans to identify the presence of functional groups in the re-
spective samples. The materials were homogenised and analysed by the
KBr pellet method. The XRD (X-Ray diffraction) data for the dry sam-
ples were collected in RIGAKU X-Ray diffractometer with Cu-kα mode
and the 2θ range was fixed between 10° and 80° for determining the
crystalline nature of the particles. The morphology and cross section of
D. George, et al.
Fig. 1. Mechanism of chitosan – histidine conjugation via amidation reaction.
the hydrogel were investigated by SEM (Scanning Electron Microscopy)
(VEGA3 TESCAN) analysis. The dried hydrogel was loaded on the
sample holder and sputtered with gold before the observation.
The swelling degree of the hydrogels were determined by soaking
0.1 g of dried hydrogel in 50 mL of the medium for 24 h at varying pH
and by using Eq. (2) (George et al., 2020).
Ws − Wd
% Q= × 100
Wd (2)
where Wd is the dry hydrogel weight (g) and Ws Ws is the swollen
hydrogel weight (g).
2.5. Drug release and kinetic studies
The drug release studies were performed with 0.1 g of dry hydrogel
at room temperature under constant slow stirring. Effect of pH on the
drug release was conducted for optimal drug loaded condition using
100 mL of buffer medium (PBS buffers with 2% Tween 80 at physio-
logical pH conditions of 5.0, 6.8 and 7.4). The drug release was quan-
tified periodically by analysing the buffer release medium using UV–vis
spectroscopy upto 12 h. Similarly, the effect of initial drug loading
concentration was studied at different drug concentrations at pH 5.0.
The in-vitro drugs release data were fitted in various kinetic models such
as zero order, first order, Korsmeyer – Peppas and Higuchi model to
understand the drug release mechanism and the rate controlling pro-
cesses for the various drug release (Purushothaman, Harsha,
Maheswari, & Begum, 2019). All release experiments were performed in
triplicates.
2.6. Biological studies
Antimicrobial studies were done by agar diffusion method (Anagha,
George, Maheswari, & Begum, 2019). Petri plates were prepared using
nutrient agar medium / potato dextrose agar for the bacteria / fungi
respectively. A sterile cotton swab was dipped into a standardised
bacterial / fungal test suspension of 106 CFU/mL and used to evenly
inoculate the entire surface of the nutrient agar plate / potato dextrose
agar plate respectively. Briefly, inoculums containing Staphylococcus
aureus (bacteria) and Trichophyton rubrum (fungi) were respectively
swabbed on the corresponding agar plate. The hydrogel samples were
arranged on the inoculated plate surface with the help of sterilised
forceps and incubated at 37○C for 24 h for the bacteria and 30○C for
24−48 h for fungal strains. The zone of inhibition (mean diameter)
measurement made around the disc in millimeters was used to evaluate
the antimicrobial activities of the samples. Each sample was analysed in
triplicates.
The biocompatibility and anticancer activity of the materials were
done through MTT assay and using the L929 (murine fibroblast) and
A431 (human skin carcinoma) cell lines respectively. Cells were plated
in 24-well plates at a seeding density of 1 × 105/well and incubated in
37 °C with 5% CO2 condition. The various concentrations of the samples
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Carbohydrate Polymers 236 (2020) 116101
(7.8–1000 μg/ml) were incubated in well for 24 h and further washed
with phosphate-buffered saline (pH 7.4) or DMEM without serum.
100 μl/well (5 mg/ml) of 0.5 % 3-(4,5-dimethyl-2-thiazolyl)-2,5-di-
phenyl–tetrazolium bromide (MTT) was added and incubated for 4 h
followed by the addition of 1 ml of DMSO. The % cell viability was
calculated from Eq. 3, absorbance at 570 nm was measured with UV-
Spectrophotometer using DMSO as the blank:
Absorbance at 570nm of treated cells
% Cell viability = × 100
Absorbance at 570nm of control cells (3)
Measurements were performed in triplicates and the concentration
of each sample that are required for 50 % inhibition of the cell growth
(IC50 value) was determined.
3. Results and discussions
3.1. Formulation of polyphenolic drugs loaded nanohybrid hydrogel
The bio-based precursors used in the development of the functio-
nalised nanohybrid hydrogel included the polymer base – chitosan,
amino acid conjugant – HIS, inorganic constituent – phytosynthesised
ZNPs and eco-friendly crosslinker – DAC. The amidation reaction be-
tween the amine groups of chitosan and carboxyl groups of HIS yielded
the conjugated CH-HIS as represented in Fig. 1. Triethylamine, a de-
protonating base activated the amine groups of chitosan. In addition,
EDC activated the carboxylic acid groups of HIS and together they
enhanced the conjugation of HIS on the backbone of chitosan (Xiao
et al., 2016). The modified chitosan was further embedded with ZNPs
and crosslinked to form nanohybrid hydrogel using DAC. Characteristic
elucidation of the synthesised ZNPs and DAC are presented in detail in
our previous work (George et al., 2019). The maximum loading
efficiency of 92.84 % for QE, 90.55 % for NRG and 89.89 % for CUR in
HIS-CHGZ were optimised using Taguchi method. Material character-
isations were employed to evaluate the presence of various functional
materials in the nanohybrid hydrogel are discussed in detail in the
following section. These analytical methods provide confirmation with
regard to the CH-HIS conjugation via amide bond formation, ZNPs
embedment in the polymer matrices, Schiff base mechanism based
hydrogel crosslinking and also the drug loading.
3.2. 1H and 13
C NMR spectroscopy
In Fig. 2(a–c), the 1H NMR spectra of chitosan, HIS and CH-HIS
revealed the effective conjugation of HIS with chitosan polymer. The
saccharide ring protons of chitosan resonating at approximately
2.5–5.0 ppm were also detected at δ 2.84 ppm for C2, δ3.5–3.8 ppm for
C3-C6 and δ4.81 ppm for C1 of chitosan and with δ1.91 ppm for the
solvent HCl/D2O.The signals observed at around δ 3.1 ppm and δ
6.5–8.0 ppm in HIS spectra corresponded to the methylene/amino
group and imidazole/carbonyl groups present in the HIS (Yang, Lee, &
Kim, 2006). In CH-HIS, in addition to the chitosan peaks, the strong
D. George, et al. Carbohydrate Polymers 236 (2020) 116101

Fig. 2. Spectroscopic images of 1H NMR spectra for (a) Chitosan, (b) Histidine, (c) CH-HIS conjugation and (d) 13C NMR spectra for HIS-CHGZ nanohybrid hydrogel.

signal observed between δ 6.5–7.0 ppm could be attributed to the amide

bond formation between the carbonyl group of HIS and amino group of
chitosan (Berger et al., 2004). Also, the existence of imidazole group
immobilised on chitosan backbones was confirmed from the signal at
around δ 6.5–8.0 ppm assigned to imidazole groups adjacent to me-
thylene protons.
The 13C CP-MAS solid-state NMR spectra for HIS-CHGZ is displayed
in Fig. 2d. The characteristic peaks present for 50–110 ppm, in general,
conforms to the carbohydrate region. In addition, the chemical shift
values at 23.75 ppm and 120−140 ppm corresponding to (−CH3) ali-
phatic carbon and imidizole group were also observed (Li, Zhou, Su,
Han, & Deng, 2013). The signals that indicate the amine bonds of
chitosan are generally reported in region of 30−65 ppm and in the
present case could have overlapped with the carbohydrate peaks. The
strong signal at 187.29 ppm confirmed the formation of the schiff base’s
azomethine linkage (-C = N) between the aldehyde of DAC and amine Fig. 3. Swelling degree plot.
of chitosan in addition to the amidation (R-CO-NR1) product of HIS –
chitosan conjugation (De La Rosa, Miller, & Knicker, 2018; Sreenivas, nanohybrid hydrogels compared to pure chitosan – cellulose hydrogel.
Aruna, & Ravinder, 2014). Thus the conjugation of HIS with chitosan ZNPs in the hydrogel matrices aids in the hydration process by holding
and formation for hydrogel crosslinking with DAC was confirmed using more water in its lattice structure. Also the osmotic pressure gradient in
1
H and 13C NMR spectroscopies, respectively. the hydrogel created by the surface electric charge of the ZNPs was
balanced by the penetration of more amount of water. Additionally,
3.3. Swelling characteristics swelling degree in general was found to be higher at acidic pH due to
the electrostatic attraction between the protonated amine groups and
The swelling degree was measured for varying pH and compared the H2O molecules. The protonation of imidazole group of HIS present
between pure chitosan – cellulose hydrogel (CHG), ZNPs embedded HIS-CHGZ resulted in an enhanced swelling in the acidic region of
CHGZ and HIS-CHGZ nanohybrid hydrogel as shown in Fig. 3. The pH > 3 (Tang, Zhao, Yang, Wang, & Zhang, 2019). However, on fur-
presence of ZNPs has significantly improved the swelling ability of the ther decrease in pH, swelling was lowered which may be due to the

4
D. George, et al.
shielding effect of excess H+ ions. Also, a moderate increase in swelling
degree at higher pH was observed in HIS-CHGZ which could be asso-
ciated with the electrostatic repulsion offered by the deprotonated
COO− and imidazole groups of HIS in the basic medium (Casolaro
et al., 2008). A one-way ANOVA test was performed using MINITAB 16
to determine the statistical significance of the differences in the swel-
ling degree obtained by the hydrogels. A p-value < 0.05 was observed
at all the conditions which confirmed that the differences in swelling
degrees were statistically significant
3.4. FTIR spectroscopy
Conjugation of chitosan with HIS was confirmed using FTIR analysis
(Yanming, Congyi, & Jianwei, 2001). The degree of substitution of HIS
in chitosan was calculated to be 70.5 %, from the absorption ratio of
A1560/A2920obtained from the FTIR bands of modified and unmodified
chitosan and fitting them in the calibration curve. The spectra of chit-
osan depicted in Fig. S1a showed the characteristic peaks of poly-
saccharides at 1060 cm−1, 1420 cm-1 and 2920 cm−1 which indicated
CeOeC stretching of saccharide structure, CH3 assymetric deformation
and C–H stretching, repectively. The peak at 3350 cm−1 was related to
the OeH stretching which overlapped with the NeH stretching of
chitosan with broadening. The characteristic peaks shown in Fig. S1b at
1510 cm−1, 1432 cm−1, 808 cm-1 and 712 cm-1 corresponded to the
NeH in plane bending, CeN stretching, imidizole ring deformation,
NH3 bending and C–H out of plane bending of HIS. An increase in the
peak at 1645 cm−1corresponding to C]O stretching and amide peak in
amide I region was observed confirmed HIS conjugation onto the
polymer chain in Fig. S1c. The increase in band intensity related to CH2
vibrations at 1432 cm−1 and 2920 cm−1, quantitatively confirmed the
amidation of chitosan (Badhe et al., 2015).
The FTIR spectra in Fig. 4. illustrates the characteristic peaks cor-
responding to the embedment of ZNPs, hydrogel crosslinking and drug
loading in the nanohybrid hydrogel. The functional peak at 535 cm−1
corresponded to the metal – oxide stretching vibration in ZNPs. The
presence of this characteristic peak in Fig. 4(a, c, e and g) confirmed the
embedment of ZNPs in hydrogel matrices (Elumalai & Velmurugan,
2015). The peak at 1645 cm-1 also confirmed the C]O stretching in
amide and NeH adsorption in NH2 due to partially acetylated amino
groups. In addition, the confirmation for crosslinking of chitosan by
DAC via a schiff base reaction and reduction was provided by C]N
stretching vibration also coincided with amide bonds of chitosan at
1645 cm-1as shown in Fig. 4a (Kim et al., 2017). The characteristic
peaks of QE at 3283 cm-1 and 1379 cm-1 confirmed the OeH stretching
of phenol groups. The peaks at 1666 cm−1, 1560 cm−1, 1510 cm−1,
1200 cm-1 and 1165 cm−1corresponded to the C]C aromatic ring
Fig. 4. FTIR spectra of (a) HIS-CHGZ, (b) QE, (c) HIS-CHGZ-QE, (d) NRG, (e)
HIS-CHGZ-NRG, (f) CUR and (g) HIS-CHGZ-CUR.
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Carbohydrate Polymers 236 (2020) 116101
vibrations, in plane bending of aromatic C–H, CeO stretching of
phenol, CeCOeC stretching and bending in ketone respectively in QE
as shown in Fig. 4b. The characteristic peaks of NRG observed in Fig. 4d
at 3340 cm−1 was due to the NeH/O–H stretching vibrations, while the
peak observed at 2900 cm−1 was due to CH2 asymmetric stretching
vibrations. Bands at 1645 cm-1 and 823 cm−1 corresponded to −CONH
amide I and CeOeC stretching vibrations. The characteristic peaks of
curcumin at 3508 cm-1 and 1544 cm-1 conformed to OeH stretching of
phenol groups and C = O- stretching and carbonyl group bending vi-
brations as shown in Fig. 4f. The presence of characteristic peaks of the
drugs QE, NRG and CUR were also observed in the corresponding drug
loaded nanohybrid hydrogel as shown in Fig. 4(c, e and g).
3.5. XRD studies
Although chitosan biopolymer is of amorphous nature, the con-
stituents – HIS, ZNPs and the drugs QE, NRG and CUR exhibited sig-
nificant crystallinity which were validated from the displayed char-
acteristic crystallite peaks. The conjugation of chitosan with HIS and
nanohybrid hydrogel formation with the addition of the green synthe-
sised ZNPs contributed to the crystalline nature of the carrier as shown
in Figs. S2 and S3a. Highly crystalline QE exhibited diffraction peaks as
displayed in Fig. S3b at the following diffraction angles: 10.11°, 11.34°,
12.7°, 14.21°, 16.87°, 22.33°, 26.57°and 28.54°. Also, NRG displayed
sharp characteristic crystalline peaks at different intensities of 2θ at
10.42°, 11.06°, 15.4°, 17.74°, 18.07°, 20.88°,23.34°, 25.4° and 27.3°,
while CUR exhibited at 28.9°, 27.549°, 25.44 9° and 18.329° as shown
in the Fig. S3(d and f). The presence of these characteristic crystalline
peaks were also observed in the drug loaded nanohybrid hydrogel as in
Fig. S3(c, e and g).
3.6. SEM analysis
The surface morphology of the nanohybrid hydrogel (HIS-CHGZ) in
different combinations the three drugs, QE, NRG and CUR are depicted
in Fig. 5. A highly porous three dimensional crosslinked structures were
exhibited by the nanohybrid hydrogels as a result of the electrostatic
interaction between the conjugated polymeric backbone and the ZnO
NPs as shown in Fig. 5a. This open porous macrostructure aids in im-
proved swelling of hydrogel with better structural stability to aid the
drug delivery applications. Fig. 5(b–d) showed the morphological
images of the drug loaded nanohybrid hydrogels. The surface char-
acteristics of the drug loaded systems showed a non-porous morphology
indicating that the porous hydrogel matrices were completely occupied
by the drug molecules. Additionally, better drug-carrier interaction in
the presence of HIS resulted in an improved surface homogeneity of
HIS-CHGZ hydrogel with smoother drug loaded surface compared to
non-conjugated hydrogel (Hu, Sun, Li, Sui, & Li, 2018). The extend of
hydrophobic interactions between the different drugs and the HIS-
CHGZ hydrogel were revealed from the variation in surface homo-
geneity (Li & Yang, 2015). The highly hydrophobic CUR exhibited a
comparatively low degree of surface smoothness followed by NRG and
QE.
3.7. Release studies of QE, NRG and CUR from HIS-CHGZ with varying pH
The developed nanohybrid hydrogel carriers were tested for the in-
vitro release studies of QE, NRG and CUR under the conditions of
varying pH at room temperature for 12 h. A relatively higher release
rate was observed commonly for all the drugs at acidic pH of 5.0 when
compared to pH at 6.8 and 7.4 from both HIS-CHGZ and CHGZ hy-
drogels as shown in Fig. 6(a–c). These release characteristics could be
attributed to an increased swelling of the nanohybrid carriers in acidic
medium which in turn lead to the enhanced release of the drug. Also,
the protonation of imidazole group of histidine under acidic conditions
is expected to improve the hydrogel swelling leading to the release of
D. George, et al.
Fig. 5. SEM images of (a) HIS-CHGZ, (b) HIS-CHGZ-QE, (c) HIS-CHGZ-NRG and (d) HIS-CHGZ-CUR.
drugs (Wang et al., 2017). Whereas, the deprotonation of the amino
groups of chitosan in neutral to basic medium improved the binding of
the drugs to the carrier and thus hindered the drug release. The im-
proved release rate of the drugs in the acidic environment was found to
be appropriate for cancer-targeting applications.
On comparing the release pattern of HIS-CHGZ and CHGZ, it was
observed that the non-conjugated nanohybrid hydrogel showed a burst
release of the surface bound drug owing to the rapid swelling of the
hydrogel and was followed by a moderate release of the remaining
drug. The maximum release of 72.41 %, 63.65 % and 58.68 % for QE,
82.19 %, 70.06 % and 63.68 % for NRG and 70.05 %, 67.34 % and
66.48 % for CUR, respectively was obtained using CHGZ at pH 5.0,6.8
and 7.4. The functionalised HIS-CHGZ hydrogel, on the other hand,
exhibited a minimal burst release followed by a sustained release of
drugs. The increased stability of the carrier as a result of the HIS con-
jugation protected the drug against burst release and aided in a sus-
tained release leading to better bioavailability of the loaded drugs. This
constructively prolonged the saturation time of the drug release by an
average of 4 h which could ensure improved biocompatibility and low
associated drug toxicity. The better hydrophobic interactions between
the histidine functionalised hydrogel carrier and the hydrophobic drugs
could have aided in the stabilisation of the drugs in HIS-CHGZ nano-
hybrid hydrogel (Mishra et al., 2015). Thus, a sustained delivery of
drugs with a maximum release of 70.32 %, 64.54 % and 60.44 % for
QE, 77.54 %, 69.23 % and 64.63 % for NRG and 65.19 %, 61.27 % and
59.98 % for CUR respectively were obtained over a period of 12 h at pH
5.0, 6.8 and 7.4, respectively.
3.8. Release studies of QE, NRG and CUR from HIS-CHGZ for varying drug
concentration
The effect of drug loading concentrations on the release profiles of
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Carbohydrate Polymers 236 (2020) 116101
NRG and QE is represented in Fig. 6(d and e). Both the hydrogels, CHGZ
and HIS-CHGZ gave the maximum release for the optimal conditions of
1.0 mg/mL initial drug concentration. With increase in initial drug
loading concentration, drug release was enhanced due to the increased
osmotic potential leading to better QE release from the hydrogel into
the release medium (Kamaraj et al., 2018). The release was reduced at
higher concentration of loaded QE due to the reduced osmotic pressure
gradient. As observed in the previous case, an initial burst release was
followed by a gradual release of drug giving a maximum release of
82.19 %, 70.45 % and 55.65 % for NRG and 72.41 %, 66.94 % and
48.90 % for QE respectively, for 0.5, 1.0 and 2 mg/mL of initial drug
loading at pH 5.0. The improved stability of the carrier with HIS con-
jugation and the resultant sustained release of drugs from HIS-CHGZ
compared to CHGZ at a particular concentration was clearly observed
from the release profiles. Thus, a sustained delivery of drugs with a
maximum release of 72.23 %, 64.60 % and 52.76 % for NRG and 68.22
%, 62.33 % and 44.98 % for QE were obtained for 0.5, 1.0 and 2 mg/mL
of initial drug loading at pH 5.0 respectively. The release studies for
CUR performed at the optimum loading condition of 5 mg/mL gave
maximum release of 62.11 % and 66.65 % with CHGZ and HIS-CHGZ,
respectively.
3.9. Kinetic modelling of the drugs release
The experimental release data for the three different drugs at
varying pH and initial drug concentrations were fitted to different ki-
netic models such as zero order, first order, Korsmeyer – Peppas and
Higuchi models to determine the suitable control mechanism of the
drug release. The best fitting model was determined by comparing the
R2 value (close to 1) obtained for different models as listed in Tables S1
and S2. It was inferred that the Korsmeyer – Peppas model was the best
fitting model, in general to all the different release experiments. The
D. George, et al.
Fig. 6. Drug release profiles of (a) QE, (b) NRG and (c) CUR for varying pH and (d) QE and (e) NRG for varying initial drug concentrations.
release mechanism of the drugs from the carriers were determined
using the release exponent, n values calculated from the model as (i)
n < 0.5 for Fickian diffusion; (ii) 0.5 < n < 1.0 for non-Fickian dif-
fusion/ anomalous transport and (iii) n > 1.0 for Case II transport
(polymer erosion).
A Fickian release was observed in all the three drug release from
CHGZ with n values of 0.30, 0.29 and 0.27 for QE, 0.26, 0.24 and 0.29
for NRG and 0.50, 0.35 and 0.43 for CUR respectively at pH 5.0, 6.8 and
7.4. The deduced kinetic parameters indicated that the rate of diffusion
was much slower than the rate of hydrogel swelling and hence the re-
lease was controlled by molecular diffusion mechanism. While the re-
lease from HIS-CHGZ hydrogel provided n values of 0.96, 0.92 and 1.05
for QE, 0.85, 0.97 and 1.12 for NRG and 0.83, 0.89 and 1.19 for CUR,
respectively at pH 5.0, 6.8 and 7.4 which confirmed a non-Fickian
diffusion/polymer erosion controlled mechanism of drug release. The
gradual rate of swelling of the hydrogel matrices in addition to mole-
cular diffusion of the drugs aided towards a slow and sustained release
of the drugs. The improved stability of the hydrogel carrier as a result of
HIS conjugation and the pH effect mediated steady swelling rate was
7
Carbohydrate Polymers 236 (2020) 116101
thus identified as the factors that controlled drug release.
Further analysing the kinetics for the effect of initial drug con-
centrations, Korsmeyer – Peppas model satisfactorily fitted the release
data and found suitable to understand the drug release mechanism. The
drug release rate constants, kkp were determined as 51.25, 59.12 and
41.02 for QE and 55.12, 63.29 and 47.01 for NRG usingHIS-CHGZ and
57.6.0, 68.72 and 46.67 for QE and 61.91, 73.27 and 51.29 for NRG
using CHGZ at different concentrations of 0.5 mg/mL, 1.0 mg/mL and
2.0 mg/mL respectively. The rate of QE release was determined to be
directly proportional to the amount of drug loaded at varying con-
centrations. Also, the n values obtained for drug release from HIS-CHGZ
were 0.83, 0.86 and 0.99 for QE and 0.93, 0.97 and 1.08 for NRG and
that from CHGZ were 0.36, 0.42 and 0.47 for QE and 0.39, 0.34 and
0.41 for NRG, respectively, at different concentrations of 0.5 mg/mL,
1.0 mg/mL and 2.0 mg/mL, respectively. The curcumin release kinetics
gave n value of 0.49 and 1.09 for CHGZ and HIS-CHGZ at optimum
loading of 5 mg/mL.From the listed n values, a polymer erosion along
with molecular diffusion controlled drug release from the HIS-CHGZ
hydrogel and Fickian diffusion controlled drug release from CHGZ were
D. George, et al. Carbohydrate Polymers 236 (2020) 116101

Table 1 Liang, 2012). Additionally, ZNPs were also reported to induce sig-
Zone of inhibition for the hydrogels with / without loaded drugs. nificant morphological changes, measurable membrane leakage, and
Sample Antibacterial zone diameter (mm) Antifungal zone substantial inhibition of cell growth eventually leading to cell death
diameter (mm) (Raghupathi, Koodali, & Manna, 2011). The cumulative effect of both
the additives was observed from the enhanced zone diameters obtained
CHG 7.20 ± 0.20 5.40 ± 0.30
for HIS-CHGZ.
CHGZ 13.75 ± 1.50 12.00 ± 1.82
HIS-CHGZ 16.00 ± 1.63 14.25 ± 1.89
The antimicrobial activity of QE, NRG and CUR are established and
HIS-CHGZ-QE 20.75 ± 2.06 18.85 ± 0.81 were also prominently observed from the improved zone diameters
HIS-CHGZ-NRG 17.35 ± 1.60 17.55 ± 1.21 obtained for HIS-CHGZ-QE. Zone of inhibition was found to be
HIS-CHGZ-CUR 22.40 ± 1.91 20.50 ± 1.50 20.75 mm for QE loaded in nanohybrid hydrogel compared to 16 mm
for nanohybrid hydrogel without drug and 8 mm for QE extract re-
ported by Pahal et al. respectively (Pahal, Devi, & Dadhich, 2018). Si-
evident. This was in agreement with our findings that increased stabi-
milarly, HIS-CHGZ-NRG and HIS-CHGZ-CUR displayed 17.35 mm and
lity of the drug loaded HIS-CHGZ hydrogel resulted in a sustained drug
22.45 mm of antibacterial zone inhibition. This revealed that the drugs
release.
possessed significant antimicrobial activity against Staphylococcus
aureus which further increased when loaded in nanohybrid hydrogel
3.10. In-vitro biological characterisations system due to the collective effect offered by the carrier and the drug.
With regard to the antifungal activity, as in the previous case, the
3.10.1. Antimicrobial studies zone of inhibition of Trichophytonrubrum showed a constructive in-
The antimicrobial activity of the samples was studied using agar crease with the addition of ZNPs, HIS and the drugs. The low isoelectric
diffusion test and the zone of inhibition diameters exhibited by the point of HIS due to the basic imidazole group would contribute to the
samples are reported in Table 1. Common skin infection causing mi- cell selectivity between normal cells and Trichophytonrubrum around
crobes (Staphylococcus aureus and Trychophyton rubrum) were chosen as infected tissues and inhibits the growth of fungal cells alone (Park et al.,
microbial targets. The influence of HIS, ZNPs and the drugs on the 2018). It was inferred that HIS conjugation was beneficial towards the
antibacterial and antifungal activity of the developed nanohybrid hy- antimicrobial potential of the carrier and the antimicrobial activity of
drogel based drug delivery systems were explored. The zone of in- all the drugs were significantly preserved in the loaded form. Thus HIS,
hibition diameters obtained for all modified hydrogels such as HIS- ZNPs and drugs showed a synergistic effect towards the antimicrobial
CHG, CHGZ and HIS-CHGZ against both the microbial strains were activity of drug loaded HIS-CHGZ and the differences in the anti-
remarkably greater than the unmodified hydrogel CHG. The positive microbial potential obtained by hydrogels were determined as statisti-
charge and electrostatic cation-π interactions in HIS were crucial for its cally significant (p < 0.01). Amongst the three drugs loaded carriers,
biological properties and were likely to play an important role in the CUR loaded hydrogel showed larger zones and thus better antimicrobial
expression of the antimicrobial activity (Mao et al., 2013). Kharidia potential followed by QE and lastly NRG.
et al. had reported the enhanced bacterial cell lysis activity with re-
duced cytotoxicity towards normal human cells exhibited by the in-
clusion of histidine in the modified peptides (Kharidia, Tu, Chen, &

Fig. 7. MTT assay (a) biocompatibility studies towards L929 normal cells and (b) cytotoxicity studies towards A431 cancer cells based on cell viability after 24 h.

8
D. George, et al.
3.10.2. Biocompatibility studies
In vitro biocompatibility is an important aspect of a novel drug
carrier and L929 fibroblast epidermal cells (normal cells) is re-
commended by many standard institutions as reference cell line for the
cytotoxicity testing of polymeric carriers. The cell viabilities of the
samples were tested for varying sample concentrations (7.8–1000 μg/
mL) against the L929 cells for 24 h and represented in Fig. 7a. Among
them, HIS-CHGZ-QE, HIS-CHGZ-NRG and HIS-CHGZ-CUR had shown
cell viability greater than 100 % which indicated the cell proliferating
ability of the drug - hydrogel carrier system. The improved cell viability
of HIS-CHGZ when compared to CHGZ could be related to the presence
of HIS which was reported to possess cyto-protective activity against
normal cell death. Eso et al. reported that the invivo studies confirmed
the improved biocompatibility of the developed dentrimer carrier
through HIS association (Aso et al., 2019). Although pure drugs QE and
NRG did not exhibit significant proliferation characteristics and de-
creasing cell viability compared to CUR, all the drugs in their respective
carrier loaded forms showed a significant proliferation and hence good
biocompatibility upto 250 μg/mL. However, with increase in sample
concentration, the cell viability, in general, showed a decreasing trend
at higher concentrations. Nevertheless, with IC50 concentrations greater
than 1000 μg/mL, good biocompatibility characteristics were ensured.
3.10.3. Anticancer activity
In-vitro cytotoxicity assay is crucial as a pre-clinical screening test in
the development of a drug delivery system. The anticancer activity was
established for the samples with lower cell viability (IC50 concentra-
tions), obtained for varying sample concentrations of 7.8–1000 μg/mL.
The ability of the samples to inhibit cancerous cell growth which
thereby promotes a reduction in the cell viability was observed.
Abnormal activation of signalling G proteins such as Rho GTPases
caused the cancer proliferation, survival, mitigation and metastasis in
A431 cells (Kalailingam, Tan, Pan, Tan, & Thanabalu, 2019).
In the present study, cell viability of A431 cells was studied for
different concentrations of ZNPs, CHGZ, HIS, HIS-CHGZ, QE, HIS-
CHGZ-QE, NRG, HIS-CHGZ-NRG, CUR AND HIS-CHGZ-CUR as shown
in Fig. 7b. The IC50 values obtained for the listed samples along with the
corresponding concentrations of pure drug in the loaded forms are
presented in Table 2. The non-cytotoxic chitosan polymer with IC50
concentrations > 500 μg/mL was transformed to HIS-CHGZ hydrogel
with significantly improved cytotoxicity by the addition of ZNPs and
HIS. In addition to the ROS mediated cell apoptosis, the enhanced
permeation and retention effect of ZNPs selectively in the cancer cells
helped nanoparticles to be localised specifically in the tumour cell.
Also, the existence of ZNPs as ZnOH2+ at low pH (acidic conditions)
and high concentration of (negatively charged) anionic phospholipids
on the outer membrane of the cancerous cells resulted in their elec-
trostatic attraction. This thereby promoted selective localization, cel-
lular uptake, phagocytosis and finally cytotoxicity (Bisht & Rayamajhi,
2016). Similarly, the positively charged imidazole group of HIS could
serve towards cytotoxicity through the electrostatic attraction with the
Table 2
IC50 concentrations of the samples against A431 human skin carcinoma cells.
Sample IC50 (μg/mL) Amount of pure drug (μg)
ZNPs 58 – conjugated chitosan-cellulose nanohybrid hydrogel embedded green
CHGZ 121 –
HIS 61 –
HIS-CHGZ 52 – treatment against microbial infections and cancer therapy.
QE 81 40.5
HIS-CHGZ-QE 25 1.33
NRG 83 41.5
HIS-CHGZ-NRG 35 2.57
CUR 23 11.5
HIS-CHGZ-CUR 12 0.80
9
Carbohydrate Polymers 236 (2020) 116101
anionic phospholipid on the cancerous cells membrane (Chu et al.,
2015). HIS is also expected to cause inhibition of Autotaxin (ATX)
plasma lysophospholipase D (LPLD) activity in tumour growth with all
the three major functional groups of histidine: α-amino group, α-car-
boxyl group, and the metal-binding imidazole side chain equally con-
tributing for the inhibition (Clair et al., 2005).
In the context of the drug induced cytotoxicity, it was revealed that
higher cell death was obtained when the drug was administered in
loaded form than by the direct application. The enhanced cytotoxicity
of drugs when loaded in HIS-CHGZ could related to the better release of
the active drug moieties from the hydrogel carrier. This consequently
increased the cellular uptake of the drug in the cells and thus act upon
it. Amongst the drugs, CUR in loaded HIS-CHGZ hydrogel showed su-
perior cytotoxicity with 0.80 μg required for attaining 50 % cell death
compared to pure CUR of 11.50 μg. Similar improvement in cytotoxicity
was exhibited by QE and NRG with IC50 values in terms of pure drug
requirement as 1.33 μg and 2.57 μg in loaded form and 40.50 μg and
41.5 μg in pure forms, respectively. Thus, the present formulation
possesses remarkable anticancer potential with the synergic effect ex-
hibited by ZNPs, HIS and drugs towards the A431 cell death.
The phase inverted microscopic images of the A431 cells tested for
7.8 μg/mL, IC50concentration and 1000 μg/mL of the listed samples are
displayed in Fig. 8. The untreated A431 cells showed the highest cell
viability which then consequently decreased with increase in sample
concentrations. The cell death was significantly observed for the lowest
measured concentration of 7.8 μg/mL. Thus, the promising anticancer
activity of QE, NRG, CUR, ZNPs and HIS and the remarkable en-
hancement in cytotoxicity of all the drugs when delivered using HIS-
CHGZ against A431 cells were established.
4. Conclusion
The prospective application of a bio-organic/inorganic functiona-
lised drug carrier system was studied. While ZNPs based nanohy-
bridisation improved the drug loading and enhanced release properties,
the HIS conjugation stabilised the drug- carrier system enabling sus-
tained release of the loaded drugs, QE, NRG and CUR. The imidazole
group of HIS conjugation played the key role in improving the overall
functionality of the developed carrier. An optimal QE loading efficiency
of 92.84 % for QE, 90.55 % for NRG and 89.89 % for CUR in the na-
nohybrid hydrogel was obtained. Maximum sustained drug release with
prolonged saturation time was obtained in the initial 12 h at optimal
loading condition and pH 5 which makes it a promising drug carrier
towards targeted anti-cancer applications. Kinetic modelling of the drug
release at varying pH and initial drug concentrations revealed that the
release mechanism was controlled by both diffusion and polymer ero-
sion. Remarkable antimicrobial potential with synergically enhancing
zone of inhibition diameters was observed with a maximum of
22.40 mm and 20.50 mm for HIS-CHGZ-CUR against Staphylococcus
aureus and Trichophyton rubrum, respectively. L929 (Normal) cells
showed improved proliferation rate for all the drug loaded nanohybrid
hydrogels upto 250 μg/mL and found highly biocompatible with IC50
concentrations > 1000 μg/mL. In addition, the developed nanohybrid
based delivery system showed approximately a 15-fold increase for QE
and NRG and a 30-fold increase for CUR in cell cytotoxicity towards
A431 human skin carcinoma cells when compared to the direct ad-
ministration of the drug. Thus, we propose the dual functionalised HIS
ZNPs is a therapeutic choice for the delivery of polyphenol drugs for
CRediT authorship contribution statement
Dhanya George: Writing - original draft, Investigation, Data cura-
tion, Methodology. P. Uma Maheswari: Conceptualization, Writing -
review & editing. K.M. Meera Sheriffa Begum: Conceptualization,
D. George, et al.
Fig. 8. Phase inverted microscopy images of A431 cells at various sample concentrations.
Methodology, Investigation, Supervision.
Acknowledgments
Dr. P. Uma Maheswari acknowledges DST-WOS-A (SR/WOS-A/CS-
75/2016), India for financial support.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116101.
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