Carboxymethylated chitosan-stabilized Copper
nanoparticles: A promise to contribute a potent
antifungal and antibacterial agent
Sangeeta Tantubay1, Sourav K. Mukhopadhyay2, Himani Kalita1, Suraj Konar1,
Satyahari Dey2, Amita Pathak1* and Panchanan Pramanik1*
[1] Sangeeta Tantubay, Himani Kalita, Suraj Konar, Amita Pathak, Panchanan
Pramanik
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur
721302, West Bengal, India
[2] Sourav K. Mukhopadhyay, Satyahari Dey
Department of Biotechnology, Indian Institute of Technology Kharagpur,
Kharagpur 721302, West Bengal, India
*
Corresponding authors
Panchanan Pramanik (Corresponding author)
Email: pramanik1946@gmail.com
ppramanik1946@yahoo.in
Tel.: +91-9434016995
Amita Pathak (Corresponding author)
Email: ami@chem.iitkgp.ernet.in
Tel.: +91-3222-283312
Fax: +91-3222-282252
Sangeeta Tantubay (First author)
Email: sang.chem2@gmail.com
1
No.
Contents
Page No.
1
Comparison with some recent reports on CuNPs/nanocomposites synthesis (Section 1).
Required chemicals for synthesis of CMC-stabilized
Cu-NPs and chemical characterization of CMCstabilized Cu-NPs (Section 2).
MTT assay (Section 3).
3-4
4
Comparative table of antimicrobial activities of CuNPs/nanocomposites, copper salts, and commercially
available antibiotics (Table S1).
6
5
HD, PDI, ZP, and λmax values of CMC-stabilized CuNPs at various pHs (Table S2).
7
6
Hydrodynamic Size distribution curves of CMCstabilized Cu-NPs at various pHs (3-12) (Figure S1).
8
7
Zeta Potential distribution curves of CMC-stabilized
Cu-NPs in the pH range of 3 to7 (Figure S2).
9
8
Zeta Potential distribution curves of CMC-stabilized
Cu-NPs in the pH range of 8 to 12 (Figure S3).
10
9
References
11-12
2
3
4-5
5
2
Section 1. Comparison with some recent reports on Cu-NPs/nanocomposites
synthesis.
1a. “A simple robust method for synthesis of metallic copper nanoparticles of high
antibacterial potency against E. coli” (Chatterjee et al. 2012)
Summary: This work reports the synthesis of gelatin-stabilized Cu-NPs via aqueous based
chemical reduction of copper salt at room temperature in the synthesis period of ~ 6-8 h.
Particles’ average size range is ~ 60-80 nm & they have stability ~ one month. These Cu-NPs
exhibit good antibacterial activity.
Differences/Advantages: Microwave-assisted synthesis of CMC-Cu-NPs occurs within very
short time span (~ 2 minutes). About 4-15 nm sized CMC-Cu-NPs are stable up to two months.
This has been found that our synthesized NPs have good antibacterial and antifungal activities
without significant toxicity towards mammalian cells. However, neither antifungal property nor
the evaluation of cytotoxicity of Cu-NPs has been reported in the work of Chatterjee et al.
1b. “Synthesis, characterization, and antimicrobial properties of copper nanoparticles”
(Usman et al. 2013)
Summary: Usman et al. have synthesized chitosan-stabilized Cu-NPs through chemical means,
where use of antioxidant (ascorbic acid) and reaction under refluxing condition at 120 °C are
accepted. There is good control of particle size (2-350 nm) depending on concentration of
chitosan. They have studied antifungal and antibacterial activities of Cu-NPs.
Differences/Advantages: Our method is based on different synthetic approach. Chitosan, the
stabilizing agent, suffers from lack of solubility in the biological pH, i.e., 7.2-7.4. Usman et al.
have not demonstrated about the stability of dispersed form of NPs in aqueous medium at
different pHs including biological pH, which is very much important for studying biological
activities. There is no experiment related with cytotoxicity study which is important regarding
the potential health risks of Cu-NPs. In our method, surface stabilization of Cu-NPs with CMC
(a water soluble derivative of chitosan) overcomes the aqueous stability/dispersibility problem of
Cu-NPs. Besides, we have done the in vitro cytotoxicity study of CMC-Cu-NPs.
1c. “Synthesis and antimicrobial activity of monodisperse copper nanoparticles” (Kruk et
al. 2015)
Summary: This work describes the synthesis of sodium dodecyl sulfate-stabilized Cu-NPs
having particle size range of ~ 50-70 nm via chemical reduction of copper salt in aqu. medium at
room temperature in the synthesis period of ~ 2 h. Synthesized Cu-NPs exhibit good
antibacterial & antifungal activities.
3
Differences/Advantages: Very less time-consuming preparation, smaller particle size, use of
more bio-compatible stabilizing agent (SDS is more toxic than CMC) distinguish our work.
1d. “Nanomaterial with High Antimicrobial Efficacy-Copper/Polyaniline Nanocomposite”
(Bogdanovic et al. 2015)
Summary: The authors report a new route to preparation of Copper/Polyaniline Nanocomposite
through in situ polymerization at room temperature in the synthesis period of ~ 20 h. These
nanocomposites have potent antibacterial and antifungal activities.
Differences/Advantages: Apart from notable procedural differences, the above method does not
control surface oxidation of Cu-NPs. In addition, Bogdanovic et al. have not discussed about the
pH/pH range where Cu-NPs form stable colloidal form(s). Toxicity of Cu-NPs to mammalian
cells has not been studied also.
Section 2. Required chemicals for synthesis of CMC-stabilized Cu-NPs and
chemical characterization of CMC-stabilized Cu-NPs.
Chemicals: The chemicals used in the experiments were copper(II)sulfate pentahydrate
(CuSO4∙5H2O, 98.5 %; Qualigens Fine Chemicals, Mumbai), chitosan (low molecular weight,
75‒85 % de-acetylated; Sigma-Aldrich), sodium hydroxide pellets (97 %; Merck, Mumbai),
chloroacetic acid (minimum acidimetric assay: 99 %; Loba Chemie Pvt. Ltd. Mumbai), 2propanol (GC assay: 99 %; Merck, Mumbai), hydrazine (N2H4, 99‒100 %; Merck, Mumbai), and
ethanol (100 %; Merck, Mumbai ). All the chemicals were used as received without further
purification. Water used throughout the experiments was Milli-Q water.
Chemical Characterization of CMC-stabilized Cu-NPs: Phase analysis and crystallinity of the
prepared CMC-stabilized Cu-NPs were investigated by X-Ray Diffractometer (XRD) (Phillips:
XPERT-PRO) employing CuKα radiation (λ = 1.5425 Å) over 2θ range of 5°‒95° at a scan rate
of 1.00266 °/min and with a sampling interval of 0.016711 (30 mA and 40 kV). The functional
group analyses of CMC and CMC-stabilized Cu-NPs were executed through Fourier
Transformed Infrared (FT-IR) spectroscopy (Perkin-Elmer Spectrum: RXI instrument) within the
scan range of 4000‒400 cm-1. Particle size and shape of CMC-stabilized Cu-NPs were obtained
using High-Resolution Transmission Electron Microscope (HRTEM) (JEOL, Japan: JEM-2100)
at an accelerating voltage of 200 kV. For HRTEM observation, the sample was prepared by
dispersing CMC-stabilized Cu-NPs in water (40 µg/mL) and dropping 5 μL of the dispersion on
the surface of a 300-mesh carbon coated copper grid (Allied Scientific Product, USA). Surface
morphology of the powder sample was characterized by Field Emission Scanning Electron
Microscope (FESEM) (Carl Zeiss SMT AG, Germany: SUPRA 40) with an accelerating voltage
4
of 5 kV. Thermal properties of the nanoparticles in the temperature range of 35‒800 ºC under a
heating rate of 5 °C/min in both argon and oxygen environments were investigated by
Thermogravimetric-Differential Thermal Analysis (TG-DTA) instrument (Perkin-Elmer: Pyris
Diamond) using alumina crucibles. Hydrodynamic radii of CMC-stabilized Cu-NPs were
obtained through Dynamic Light Scattering (DLS) measurements in a particle size analyzer
(BROOKHAVEN INSTRUMENTS CORPORATION: 90 Plus Particle Size Analyzer) using a
laser light (λ = 660 nm) scattered at 90°. Zeta potentials were measured using Zetasizer
instrument (Malvern, UK: Zetasizer-4) by taking the aqueous dispersions in disposable folded
capillary cells. UV-Vis absorption spectra were recorded on an UV/Vis spectrophotometer
(Shimadzu: UV-2450) by placing the dispersions in 1 cm quartz cuvettes. X-ray Photoelectron
Spectroscopy (XPS) analysis of CMC-stabilized Cu-NPs was performed at 1 × 10-10 Torr
pressure on a PHI 5000 Versa Probe II (ULVAC-PHI, INC, Japan) system using a micro focused
(100 µm, 25 W, 15 kV) monochromatic Al Kα source (hν = 1486.6 eV), a hemispherical
analyzer, and a multichannel detector.
Section 3. MTT assay.
Cell line: L929 (normal mouse fibroblast cell line) were obtained from National Centre for Cell
Science (NCCS) Pune, India and was maintained in DMEM medium supplemented with 10%
fetal bovine serum at 37 ºC in a 5% CO2 incubator.
Cytotoxicity assay: Cytotoxicity of CMC-Cu-NPs in vitro was determined by conventional
MTT assay (Mosmann 1983) using normal cell line, L929. Briefly, cells in exponential growth
phase were trypsinised and seeded in 96 well culture plates at a density of 1 × 105 cells/well.
After 12h of cell seeding, cells were treated with CMC-Cu-NPs in different concentrations (3.90.24 µg mL-1).Then the plate was further incubated for 24 h at 37 ºC in CO2 incubator. Cell
viability was measured spectrophotometrically by measuring absorbance at 570 nm using a
micro plate reader (Bio-Rad 550). Cell viability was calculated using the formula;
% Viability = (𝑇𝑒𝑠𝑡 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒/𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒) × 100
5
Table S1. Comparative table of antimicrobial activities of
NPs/nanocomposites, copper salts, and commercially available antibiotics.
Microorganisms
E.coli
ATCC 25922
E.coli
ATCC 25922
E.coli
ATCC 25922
E.coli
ATCC 25922
E.coli
ATCC 25922
E.coli
ATCC 25922
E.coli
K12
E.coli
E.coli
E.coli
KACC 10005
E.coli
ATCC 25922
E.coli
KACC 10005
E.coli
F220
E.coli
ATCC 25922
C. tropicalis
ATCC 750
C. tropicalis
NCIM 3110
C. albicans
ATCC 10231
C. tropicalis
NCIM 3110
Type of Antimicrobial agent
Cu-NPs adsorbed mullite powder
(Particle Size range ~ 2-5 nm &10-20 nm) (Bagchi et al. 2012)
Cu-NPs (Size range: 6.86–16.53 nm) (Ruparelia et al. 2008)
Alginate-stabilized Cu-NPs (Size range: 5.4–7.5 nm)
(Diaz-Visurraga et al. 2012)
Cu-NPs (Size ~ 60 nm)
(Effenberger et al. 2014)
Poly-l-lysine-modified reduced graphene oxide stabilized Cu-NPs
(Size range: 10–50 nm) (Ouyang et al. 2013)
Bovine serum albumin– Copper Nanocomposites (Average diameters
of embedded Cu-NPs: 28 ± 12 nm )
(Rastogi and Arunachalam 2013)
Gelatin–stabilized Cu-NPs (Average size range: 60–80 nm)
(Chatterjee et al. 2012)
Iodine–stabilized Cu-NPs Chitosan composite (Average size: 8±4 nm)
(Mallick et al. 2012)
Commercial Cu-NPs (Size ~ 100 nm)
(Yoon et al. 2007)
Copper Salt [Cu(NO3)2 2.5 H2O] (Veerapandian et al. 2012)
Copper Salt[CuSO4. 5 H2O] (Effenberger et al. 2014)
Kanamycin (Standard antibiotic) (Veerapandian et al. 2012)
Amoxicillin (Known antibiotic) (Li et al. 2005)
CMC-stabilized Cu-NPs (in this study)
Fluconazole (Known antifungal drug) (Law et al. 1996)
Voriconazole, Anidulafungin, Amphotericin B (Known antifungal
drugs) (Mandal et al. 2014)
Cu-NPs (Size range: 50–70 nm) (Kruk et al. 2015)
CMC-stabilized Cu-NPs (in this study)
Cu-
Antimicrobial
Activity (µg/mL)
63.5
(Antibacterial)
140
( Antibacterial)
4
( Antibacterial)
> 600
( Antibacterial)
48.9
( Antibacterial)
50
( Antibacterial)
3
( Antibacterial)
21.5
( Antibacterial)
> 60
( Antibacterial)
128
( Antibacterial)
200
( Antibacterial)
128
( Antibacterial)
~ 525
( Antibacterial)
3.9
( Antibacterial)
4.0
( Antifungal)
1.562, 0.39, 0.19
( Antifungal)
3.75
( Antifungal)
3.9
( Antifungal)
6
Table S2. HD, PDI, ZP, and λmax values of CMC-stabilized Cu-NPs at various
pHs.
pH
Hydrodynamic
Diameter (HD)
(nm)
Polydispersity
Index
(PDI)
Zeta Potential
(ZP)
(mV)
λmax
(nm)
3
4
5
6
7
8
9
10
11
12
159.2
189.4
191.6
206.5
212.6
222.2
187
175.5
166.8
131.9
0.241
0.162
0.183
0.226
0.220
0.240
0.106
0.23
0.267
0.226
21.2
12.5
11.9
10.2
8.28
7.20
-2.91
-4.8
-13.7
-14.1
565
570
573
575
578
583
572
568
566
563
7
Figures
(A)
(B)
(C)
(D)
(E)
(F)
(H)
(G)
(I)
(J)
Figure S1. Hydrodynamic Size distribution curves of CMC-stabilized Cu-NPs at various
pH: (A) pH = 3, (B) pH = 4, (C) pH = 5, (D) pH = 6, (E) pH = 7, (F) pH = 8, (G) pH = 9, (H)
pH = 10, (I) pH = 11, and (J) pH = 12.
8
(A)
(B)
(C)
(D)
(E)
Figure S2. Zeta Potential distribution curves of CMC-stabilized Cu-NPs in the pH range of
3 to7: (A) pH = 3, (B) pH = 4, (C) pH = 5, (D) pH = 6, and (E) pH = 7.
9
(F)
(G)
(H)
(I)
(J)
Figure S3. Zeta Potential distribution curves of CMC-stabilized Cu-NPs in the pH range of
8 to 12: (F) pH = 8, (G) pH = 9, (H) pH = 10, (I) pH = 11, and (J) pH = 12.
10
References
Bagchi B, Dey S, Bhandary S, Das S, Bhattacharya A, Basu R, Nandy P (2012) Antimicrobial
efficacy and biocompatibility study of copper nanoparticle adsorbed mullite aggregates.
Mat Sci Eng C-Mater 32:1897-1905. doi:10.1016/j.msec.2012.05.011
Bogdanovic U, Vodnik V, Mitric M, Dimitrijevic S, Skapin SD, Zunic V, Budimir M,
Stoiljkovic M (2015) Nanomaterial with high antimicrobial efficacy-copper/polyaniline
nanocomposite. ACS Appl Mater Interfaces 7:1955-1966. doi:10.1021/am507746m
Chatterjee AK, Sarkar RK, Chattopadhyay AP, Aich P, Chakraborty R, Basu T (2012) A simple
robust method for synthesis of metallic copper nanoparticles of high antibacterial potency
against E. coli. Nanotechnology 23. doi:10.1088/0957-4484/23/8/085103
Diaz-Visurraga J, Daza C, Pozo C, Becerra A, von Plessing C, Garcia A (2012) Study on
antibacterial alginate-stabilized copper nanoparticles by FT-IR and 2D-IR correlation
spectroscopy. Int J Nanomed 7:3597-3612. doi:10.2147/Ijn.S32648
Effenberger FB, Sulca MA, Machini MT, Couto RA, Kiyohara PK, Machado G, Rossi LM
(2014) Copper nanoparticles synthesized by thermal decomposition in liquid phase: the
influence of capping ligands on the synthesis and bactericidal activity. J Nanopart Res 16.
doi:10.1007/S11051-014-2588-7
Kruk T, Szczepanowicz K, Stefanska J, Socha RP, Warszynski P (2015) Synthesis and
antimicrobial activity of monodisperse copper nanoparticles. Colloids and surfaces B,
Biointerfaces 128C:17-22. doi:10.1016/j.colsurfb.2015.02.009
Law D, Moore CB, Joseph LA, Keaney MGL, Denning DW (1996) High incidence of antifungal
drug resistance in Candida tropicalis. Int J Antimicrob Ag 7:241-245.
doi:10.1016/S0924-8579(96)00328-7
Li P, Li J, Wu CZ, Wu QS, Li J (2005) Synergistic antibacterial effects of beta-lactam antibiotic
combined with silver nanoparticles. Nanotechnology 16:1912-1917.
doi:10.1088/0957-4484/16/9/082
Mallick S, Sharma S, Banerjee M, Ghosh SS, Chattopadhyay A, Paul A (2012) Iodine-Stabilized
Cu Nanoparticle Chitosan Composite for Antibacterial Applications. Acs Appl Mater
Inter 4:1313-1323. doi:10.1021/Am201586w
Mandal SM, Mahata D, Migliolo L, Parekh A, Addy PS, Mandal M, Basak A (2014) Glucose
Directly Promotes Antifungal Resistance in the Fungal Pathogen, Candida spp. The
Journal of biological chemistry 289:25468-25473. doi:10.1074/jbc.C114.571778
Mosmann T (1983) Rapid Colorimetric Assay for Cellular Growth and Survival - Application to
Proliferation and Cyto-Toxicity Assays. J Immunol Methods 65:55-63.
doi:10.1016/0022-1759(83)90303-4
Ouyang Y, Cai X, Shi QS, Liu LL, Wan DL, Tan SZ, Ouyang YS (2013) Poly-L-lysine-modified
reduced graphene oxide stabilizes the copper nanoparticles with higher water-solubility
and long-term additively antibacterial activity. Colloid Surface B 107:107-114.
doi:10.1016/j.colsurfb.2013.01.073
Rastogi L, Arunachalam J (2013) Synthesis and characterization of bovine serum albumincopper nanocomposites for antibacterial applications. Colloid Surface B 108:134-141.
doi:10.1016/j.colsurfb.2013.02.031
Ruparelia JP, Chatteriee AK, Duttagupta SP, Mukherji S (2008) Strain specificity in
antimicrobial activity of silver and copper nanoparticles. Acta Biomater 4:707-716.
doi: 10.1016/j.actbio.2007.11.006
11
Usman MS, El Zowalaty ME, Shameli K, Zainuddin N, Salama M, Ibrahim NA (2013)
Synthesis, characterization, and antimicrobial properties of copper nanoparticles. Int J
Nanomed 8:4467-4478. doi:10.2147/Ijn.S50837
Veerapandian M, Sadhasivam S, Choi J, Yun K (2012) Glucosamine functionalized copper
nanoparticles: Preparation, characterization and enhancement of anti-bacterial activity by
ultraviolet irradiation. Chem Eng J 209:558-567. doi:10.1016/j.cej.2012.08.054
Yoon KY, Byeon JH, Park JH, Hwang J (2007) Susceptibility constants of Escherichia coli and
Bacillus subtilis to silver and copper nanoparticles. Sci Total Environ 373:572-575.
doi:10.1016/j.scitotenv.2006.11.007
12