Development of copper loaded nanoparticles hydrogel made from waste

biomass (sugarcane bagasse) for special medical application

Harish Kumar1,a, Avneesh Kumar Gehlaut1,b, Himanshu Gupta1,c, Ankur
Gaur1,d, Jin-Won Park2,e
1. Department of Chemical Engineering, Motilal Nehru National Institute of Technology,
Allahabad-211004, Uttar Pradesh, India
2. Department of Chemical and Biomolecular Engineering, Yonsei University, 262 Seoul 120-749,
Republic of Korea
a
nonu839@gmail.com, bakgehlaut@gmail.com, chimanshu9252@gmail.com, dankur@mnnit.ac.in,
e
jwpark@yonsie.ac.kr

Keywords: Carboxymethyl cellulose (CMC), copper oxide nanoparticles (CuO-NPs), hydrogel,

pH-sensitive, antibacterial activity.

Abstract. This research is based on the creation of copper oxide nanoparticles (CuO-NPs) hydrogel
through in situ method. The effective UV- Vis spectroscopy absorption peak of 610 nm indicates
the existence of copper oxide nanoparticles (CuO-NP) group because of surface Plasmon resonance
(SPR). Characterization of CuO-NPs was carried out by TEM, SEM, EDX and XRD. However, the
action of prepared nanocomposite hydrogels swelling was examined at different pH and saline
solutions. In addition, CuO-NPs were also tested using the disk plate diffusion method by means of
adverse effect of bacteria Gram-positive bacteria (Bacillus subtilis) as well as Gram-negative
bacteria (Escherichia coli). The CuO-NPs hydrogels isolated from lab-made CMC (made from
sugarcane bagasse) indicates that the Bacillus subtilis as well as Escherichia coli have greater
antibacterial activity compared with synthetic CMC purchase from the market. CuO-NPs can be
used effectively in biomedical applications with the production of hydrogels.

Introduction
Hydrogels are hydrophilic polymers that are cross connected in three dimensions linear and
branched. Compared to common absorbent materials, they are competent for significant amount of
water, salt and physiological alternatives [1]. Because of its high swelling level and not poisonous
to living tissue hydrogels made of this polymeric product, they can be used to encourage their
prevalent use in agriculture, in biomedical field as antibacterial materials, tissue engineering,
biosensors, drug delivery and used as sorbents for of heavily metal removal [2]. Cellulose is a
polymer substance that, due to its strong inter and intra molecular –OH bonds has linear and broad
molecular weight, but does not melt or dissolve readily, also in the typical organic solvents in hot
and cold water. The CMC is a water soluble imitation, formed from alkaline and mono chloro
acetic acid cellulose (MCA) reactions with organic solvents in heterogenous conditions. CMC is the
cellulose ether most widely used as an enviable element, including thickener and stabiliser for food
stuff manufacturing, food packaging, cosmetically fabric, tissue engineering, pharmaceuticals and
paper industry [3]. CMC is a class of hydrophilic polymer, which in contrast to cellulose, is easily
resolved in water and has a perfect swelling strength [4]. Due to strong antibiotic properties, Ag-
NPs and CuO-NPs became essential as new antimicrobials for a large number of microorganisms,
including multi-drug resistant bacteria. Silver is a costly metal that therefore has high Ag-NPs
synthesis costs. Copper is less costly than silver and easily accessible, ensuring that the synthesis of
CuO-NPs is cost efficient. Ag-NPs and CuO-NPs were used originally for antibacterial purposes
and are therefore now used in various antimicrobial formulations and devices such as food
packaging and surgical instruments as well as biomedical and water purification items. Several
prior studies have been conducted to produce CuO-NPs such as chemical, thermal, sono-chemical
reduction, vacuum vapour deposition, radiation methods, micro-emulsion techniques or laser
ablation. Copper (Cu) can become an admirable substitute for silver (Ag) in a variety of biomedical
purposes, for instance cellular analysis photo-thermal therapy, and used in pesticides etc [2]. This
work aims at summarizing and characterizing a sequence of Cu-NPs antibacterial CMC hydrogels.
However, this research is focused on comparing the synthetic CMC and lab made CMC using an in
situ method and thus the lab made CMC is more efficient than synthetic CMC bought from the
market.

Materials and methods

Materials
CMC which is acquired from market in the range (Mw = 10,000 Da, DS = 0.51) was acquired from
Molychem. The lab-made CMC was prepared from sugarcane bagasse (SCB) (DS = 0.65), and
Epichlorohydrin (98%) was acquired from Merck. Copper chloride (CuCl 2 2H2O) was purchased
from SRL Chemical.

CMC hydrogel preparation (in situ)
The CMC were made according to the technique described [5]. 3 g of CMC (synthetic and lab-
made) were mixed individually in 100 ml of 3% w/v sodium hydroxide solution by consistently
homogeneous blending with mechanical agitation. Subsequently epichlorohydrin (ECH) (5 ml) was
wisely mixed with the solutions of CMC for an hour before uniform mixer was obtained. The
formed mixture was then placed 2 hours at 80 ºC in a hot water bath. In Millipore water, the
developed insoluble CMC adhesive was collected and washed to remove the remaining sodium
hydroxide (NaOH) and ECH and then dried overnight in an oven at 65 ºC.

CMC nanocomposite preparation hydrogels consisting of CuO-NPs
The CuO-NPs hydrogel were made according to the technique described [2]. For a whole day, 0.6 g
of CMC hydrogels was combined with the solution of 0.030 M copper chloride (CuCl 2). Cu2+ was
cleaned with Millipore water to eliminate the Cu2 + charged ion linked to copper hydrogel. During
washing of these hydrogels they were coated with 0.2 M sodium hydroxide (NaOH) for 24 hour in
100 ml solution and after oxidation these hydrogel were then washed with Millipore water after that
dried whole day at 60 ºC in an oven.

Characterization and analysis

The dried specimens samples were then compressed in disks to measure through an X-ray
diffractometer in 2q ranges from 5º to 65º operated at 40 mA and 40 kV and 10º min -1 scanning
speed were confirmed with X-ray diffractometer system using (Malvern Panalytical, XPERT-PRO)
diffractrometer using (1¼ 1.5406 Å) as a X-ray source. TEM samples were produced by casting the
drop of suspension on the carbon (C) copper grid, discarding extra water solution with the tissue
paper allowing air to dry for whole night at room temperature. The TEM research was carried out
on a 200 kV operating HITACHI H-800. UV-Vis spectrum were shown to be very prone to copper
colloid formation, as CuO-NPs exhibit an extreme peak absorption due to SPR. UV-Vis
spectroscopy (Perkin Elmer, Shimadzu, Japan) is an essential technique used to ratify CuO-NPs
growth.

Swelling ratio
100 ml of aqueous solutions in 1 g of CuO-NPs dried hydrogels, with a neutral pH were mixed for
2 hours to attain highest swelling equilibrium [3].
Antibacterial action
Antibacterial action made according to the technique described [3].

Results and Discussion

The cross-linking agent ECH was used in aqueous basic conditions to synthesize the CuO-NPs
hydrogels. The CMC (-OH) group is converted into alcoholic anions under basic conditions.
Alcoholic anion strikes the ECH form chloro-propanediol monoether, and after rearranging
chloride a fresh group of epoxy chloro-propanediol was created. The relation between CMC and
ECH occurs after the new epoxide group interacts with the –OH groups of one more CMC [3].

CuO-NPs hydrogel in situ formation

The CuO-NPs in the CMC hydrogel system. CMC networks, including such as Al 3+, Cu2+, Co2+,
Mo6+ and Zn2+ have several metal cations [6].

XRD analysis
(Fig. 1a, b) shows the XRD pattern of synthetic CMC and lab made CMC/CuO nanocomposite
hydrogel in the range of 5-65ᵒ. The CMC/CuO nanocompsoite hydrogel diffractogram at 2θ values
of about 35ᵒ, 39ᵒ, 49ᵒ, respectively allocated to the (110), (002) and (112) diffractions of CuO
crystals. To identify the creation of CuO-NPs within the hydrogel arrangement of the CMC, each
peak matches the CuO monoclinic section crystal. The peak at 17.8, 22.8 in the CMC X-ray
diffraction which are the distinctive diffraction of cellulose [2].

Fig. 1 XRD patterns of of (a) Synthetic CMC and lab made CMC (b) Synthetic CMC/CuO and lab
made CMC/CuO hydrogel...

Uv-Vis spectrophotometry
With their SPR, CuO-NPs have distinctive and variable optical characteristics based on the
distribution of the nanoparticles by size, and size distribution. For both the synthetic CMC and lab
made created CMC, an absorption band at pH 7 occurred at 610 nm. As pH increases the maximum
absorbent band strength to pH 7 at 610 nm, the decrease Cu2+ ions to CuO atoms increases, the
reduction of Cu2+ ions as well as CMC’s dual role as a competent stabiliser and reducing agent.
[2,3].

Scanning electron microscopy and Energy-dispersive X-ray spectrum

The SEM and EDX analysis of CMC hydrogels, which included the CuO-NPs was acquired in situ
method. The porous structure within the hydrogels is shown by SEM images (Fig. 2a, b). There are
several carboxylate (COO-) anions in the hydrogel network. The pores within the hydrogel
increases in size as a result of electrostatic repulsions around carboxyl anion (COO -) [2,3].The
quantitative analysis of EDX confines the nanostructure current during in situ method which
includes around 46.52 wt % Cu, 15.61 wt % oxygen, 14.59 wt % Na, 10.7 wt % Cl and about 12.58
wt % carbon for synthetic CMC and about 59.16 wt % Cu, 16.82 wt % oxygen, 10.38 wt % Na, 4.2
wt % Cl and about 9.44 wt % carbon for lab-made CMC (Fig. 3a, b).
Fig. 2 SEM images of (a) Synthetic CMC/CuO nanoparticles hydrogel (b) Lab made CMC/CuO
nanoparticles hydrogel

Fig. 3 EDX images of (a) Synthetic CMC/CuO nanoparticles hydrogel (b) Lab made CMC/CuO
nanoparticles hydrogel

The swelling effect of the pH
Hydrogel swellings were tested for the pH range from 2 to 10 to determine the pH tolerance of the
CuO-NPs. As illustrate in the Fig. 4 (a). The highest swelling ratio for synthetic CMC and lab-
made CMC were obtained at pH 7. The lab-made CMC demonstrates greater swelling capacity as
compare to the synthetic CMC can be certified to the existence of CuO-NPs with variance in sizes,
morphology, and external charges[2,6].

In saline solutions swelling behaviour

Hydrogels are the subjects of major concerns due to various practical applications. The
environment affects the hydrogel swelling ability because of ionic strength. The effect of salt
absorption and the cation power of swelling were thus investigated in the current scenario. The
charge source of the extra cations contributing to incomplete anion-anion repulsion that lower the
difference in osmotic pressure between the outer solution and hydrogel. The Fig. 4 (b) reveals that
the equilibrium swelling data collected from (NaCl), (MgCl2) and (AlCl3) the same amount of salt
solutions. As illustrated in Fig. 4 (b) multivalent cation significantly reduces the swelling strength
due to dramatically reduced water intake in solutions. [2,6].
Fig. 4 (a) Swelling behavior of the hydrogels at different pHs and (b) Swelling behavior for CuO
NPs hydrogel in saline solutions

Transmission electron microscope

TEM results indicate that the CuO-NPs are spherical and distributed smoothly within a 50–100 nm
size range and are shown in (Fig. 5a, b) [3].

Fig. 5 TEM images of (a) Synthetic CMC/CuO nanoparticles hydrogel (b) Lab made CMC/CuO
nanoparticles hydrogel

Antibacterial action
A disc plate diffusion analysis is conducted to demonstrate adverse CuO-NPs bacterial activity
against Gram-positive (B. Subtilis) are shown in (Fig. 6 A) and Gram-negative (E. coli) are
displayed in (Fig. 6 B). Higher concentration of CuO-NPs improves the inhibition area and the
findings are shown in Table 1. Lab-made CMC confirm the larger effect towards Gram-positive
bacteria due to the smaller particles size and having large surface area and more reactive oxygen
species (ROS) as compared to the synthetic CMC. Gram-negative bacteria, demonstrate less
sensitivity to ROS. The Gram-negative bacteria have a cell wall consisting of an inner thin
peptidoglycan structure and lipopolysaccharides exterior layer (LPS). Moreover there is a
lipopolysaccharides (LPS) surface and several peptidoglycan layers of Gram-positive bacteria. The
outer layer of Gram-negative bacteria may interfere and block the positive charges of CuO
nanoparticles to reach the cytoplasmic membrane [2,3].

Table 1. The antibacterial activity against ZnO nanoparticles hydrogel

Inhibition Zone (mm)
Gram positive Gram negative
B. Subtilis E. coli
18 mm For Lab made CMC 16 mm For Lab made CMC
16 mm For Synthetic CMC 15 mm For Synthetic CMC

Fig. 6 A) Antibacterial activity of Gram-positive bacteria (a) Synthetic CMC (b) lab made CMC ,
B) Antibacterial activity of Gram-negative bacteria (a) Synthetic CMC (b) lab made CMC

Conclusion
In situ method was used to develop CuO-NPs by copper chloride oxidation at room temperature
from synthetic CMC and lab-made CMC with epichlorohydrin in an alkaline medium. TEM
micrographs reveal the configuration of CuO-NPs with a size range of 50-100 nm for lab-made and
synthetic CMC in the hydrogel matrix. In salt solutions with the similar concentration, swelling
capability for these hydrogels is in order of (NaCl)>(MgCl2)>(AlCl3). Antibacterial action against
Gram-positive and Gram negative bactericides has been shown by CuO-NPs hydrogels. The
hydrogels developed CuO-NPs are useful for various medical areas for instance drug delivery and
wound healing.

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