synthesis, characterization and in vitro evaluation of cytotoxicity and...

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Synthesis, characterisation and in vitro evaluation of cytotoxicity and an-

timicrobial activity of chitosan-metal nanocomposites

Pawan Kaura, Rajesh Thakur

# a, Manju Barnela

a, Meenu Chopra

b, Anju Manuja

b and Ashok

Chaudhurya

aDepartment of Bio and Nano Technology, Guru Jambheshwar University of Science and

Technology, Hisar, India

bNational Research Centre on Equines, Sirsa road, Hisar, India

#Corresponding author. Tel: (+91) 1662-263514; E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which

may lead to differences between this version and the Version of Record. Please cite this

article as doi: 10.1002/jctb.4383

mailto:[email protected]


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Abstract

BACKGROUND: The present study explores the synthesis of chitosan-metal nanocompo-

sites in view of their increasing applications as antimicrobial material.

RESULTS: Chitosan nanoparticles were prepared by ionic gelation between chitosan and so-

dium tripolyphosphate. Copper sulphate hydrate (CuSO4.5H2O) and zinc acetate (Zn

(O2CCH3)2) were used as precursors for synthesis of chitosan-copper nanocomposites

(Cu/Ch) and chitosan-zinc nanocomposites (Zn/Ch), respectively. Synthesis of nanocompo-

sites was confirmed by Transmission Electron Microscope (TEM), Fourier Transform Infra-

red (FTIR) spectroscopy, Scanning Electron Microscopy with energy dispersive X-ray mi-

croanalyses (SEM-EDX) and Differential Scanning Calorimetry (DSC). Cytotoxicity of nano-

formulations was studied by Resazurin assay on Vero cell line (African green monkey kidney

cell line). Their antibacterial activities were assessed by zone of inhibition method and time

dependent growth curve against Micrococcus luteus MTCC 1809, Pseudomonas aeruginosa

MTCC 424 and Salmonella enterica MTCC 1253 in vitro. Antifungal activity also studied

against Alternaria alterneta, Rhizoctonia solani and Aspergillus flavus in vitro by mycelium

inhibition method.

CONCLUSIONS: It was observed that all nanoformulations show high antimicrobial activity

against all test microorganisms. So chitosan-metal complexes can be promising candidate for

novel antimicrobial agents that can be used in cosmetic, food and textile.

Keywords: Chitosan, nanocomposites, cytotoxicity, antimicrobial, percent inhibition.

Introduction

Utilization of metallic nanoparticles in various biotechnological and medical applications

represents one of the most extensively investigated areas of the current materials science.

These advanced applications require the appropriate chemical functionalization of the

nanoparticles with organic molecules or their incorporation in suitable polymer matrices i.e.


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nanocomposites. The polymer nanocomposites material is an innovative product having nano

fillers dispersed in the matrix of polymers that have gained much interest recently. Significant

efforts are underway to control the nano-structures via innovative synthetic approaches1-5

with excellent antimicrobial properties, increased biocompatibility and potential in wound

management.

Among polymers, chitosan (Ch) is one of the most abundant natural polymers, is non-

toxic, biodegradable and biocompatible. Its antimicrobial activity has been observed against a

wide variety of microorganisms including fungi, algae, and some bacteria6-7

. It has several

advantages as compared to some other disinfectants because it exhibits higher antibacterial

activity, broader spectrum of activity, higher killing rate, and lower toxicity toward mammal-

ian cells. Many attempts have been taken up to improve the antimicrobial activity of chitosan,

such as structural modification, adjustment of molecular factors, and forming complexes,

nanocomposites with other antimicrobial metal nanoparticles8-9

.

Chitosan is a powerful chelating agent, which easily forms complexes with transition

metals and heavy metals and shows antimicrobial activity10

. Du et al. (2004) found that an-

timicrobial properties of chitosan were enhanced by loading chitosan with various metals11

.

The composite was found to have significantly higher antimicrobial activity than its compo-

nent particles at their respective concentrations. Although antibacterial action of nanocompo-

sites has been reported12-13

but limited literature is available on the antifungal activity of

nanocomposites14

. In present work, we evaluated antifungal activity of chitosan-metal com-

plex by percent inhibition of mycelia growth method. Surprisingly, our data showed excellent

antifungal activity of all nanoformulations (chitosan, copper oxide nanoparticles (CuO NPs),

zinc oxide nanoparticles (ZnO NPs), chitosan-copper nanocomposites (Cu/Ch) and chitosan-

zinc nanocomposites (Zn/Ch), especially nanocomposites (Cu/Ch and Zn/Ch). The chitosan-

metal nanocomposites also showed significant antibacterial activity against Staphylococcus


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aureus MTCC 1809, Pseudomonas aeruginosa MTCC 424 and Salmonella enterica MTCC

1253 in vitro than component nanoparticles individually. Cytotoxicity studies conducted on

Vero cell line (African green monkey kidney cell line), revealed good biocompatibility of

nanocomposites as compared to metal nanoparticles. To date, we have not come across any

experimental report on cytotoxicity study of chitosan-copper oxide and chitosan-zinc oxide

nanocomposites.

EXPERIMENTAL

Reagents

Chitosan, tri-sodium citrate purified LR and acetic acid were obtained from S.d fine-chem.

Limited, India. Copper sulphate hydrate and zinc acetate were purchased from Sisco Re-

search Laboratories, India. Resazurin sodium dye was purchased from Sigma-Aldrich

Chemicals Private Ltd. (Bangalore, India). The test strains Staphylococcus aureus MTCC

1809, Pseudomonas aeruginosa MTCC 424 and Salmonella enterica MTCC 1253 were pro-

cured from Institute of Microbial Technology, Chandigarh. All fungal strains were available

in our department. Vero cell lines (African green monkey kidney cell line) maintained at Na-

tional Research Centre on Equines, Hisar (India).

Preparation of chitosan-metal nanocomposites

Copper sulphate hydrate (5mM) and zinc acetate (5mM) were used as precursors for synthe-

sis of chitosan-metal nanocomposites according to reported method11

with some modifica-

tions. Reduction of copper ions and zinc ions into nanoparticles was achieved in acidic solu-

tion (acetic acid in distilled water (1% v/v)) of chitosan; the mixture was stirred for 12h, then,

treated with sonication at 1.5 kW for 30 min. The suspension was subsequently centrifuged at

12,000 rpm for 20 min at 4°C. The pellet was resuspended in distilled water, centrifuged

again, and freeze dried (Lyophilized). The freeze-dried chitosan-metal nanocomposites were

either used directly or suspended in distilled water for characterization and other experiments.


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Characterization of chitosan-metal nanocomposites

The morphology & size of nanocomposites were observed using Transmission Electron Mi-

croscope (TEM) (JEOL 2100F TEM instrument) operating at an accelerating voltage of 100

kV. Chitosan-metal nanocomposites were suspended in double distilled water, homogenized

using an ultrasonic cleaner, a drop was placed on a copper grid with a lacey carbon film,

which was then air dried and observed in TEM.

IR spectra of the lyophilized nanocomposites were obtained using FTIR spectrophotometer

(Spectrum BX11, Perkin–Elmer) in the range 4600 – 400 cm-1

and resolution ± 4 cm-1

. The

tested samples were pelletized with KBr in the weight ratio 1/100 for observations.

The differential scanning calorimetry (DSC) thermogram of nanoformulations was obtained

using DSC (Q10V9.0 Build 275, TA Instruments). Samples were heated under nitrogen at-

mosphere in an aluminium pan at the rate of 10°C per min over a temperature range of 0-

300°C.

The elemental analysis of the nanoformulations were carried out by means of a Scanning

Electron Microscope (SEM-Leo 435VP) working with EDX Link 300 ISIS from Oxford In-

struments. The samples were prepared by fixing the powder particles to microscope holder,

using a conducting carbon strip.

Cytotoxicity studies

In vitro cytotoxicity study of chitosan and their nanocomposites was determined by Resazurin

assay as described previously15

. The cytotoxic activity was assessed by colorimetric assay

using resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) which is blue in colour and

nonfluorescent until it is reduced to the highly fluorescent pink coloured resorufin. Vero cell

lines at a density of 1 × 104 cells per well were cultured in a 100μL volume of cell culture

medium (EMEM supplemented with 10% fetal bovine serum 10 mM HEPES, 2 mM L-


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glutamine, 25 mM sodium bicarbonate, penicillin 10000 units/ml, streptomycin 10 mgml-1

,

amphotericin B 25 mgml-1

) in a 96-well cell culture plate. After 24h of incubation at 37 °C

with 5% CO2, cultured cells were treated with different concentrations of chitosan-metal

nanocomposites, chitosan nanoparticles and metal nanoparticles (10µgml-1

to 200µgml-1

),

well dispersed in deionized water (1 mgml-1

) and incubated again for 24h. After incubation,

the samples were treated with 10 µl of the resazurin solution prepared in distilled water (1

mgml-1

) and incubated for 4h under conditions as described above. After 4h the pink coloured

resorufin was formed, which was estimated by taking absorbance at 590 nm in a spectropho-

tometer (ELISA plate reader, PowerWave™ XS2; Bio-tek, VT, USA). The reduction from

blue to pink color is directly proportional to metabolic activity of cells. The data was normal-

ized with reference to the background absorbance of media. Cytotoxicity percentage was

calculated with reference to untreated cells15

.

Antibacterial assay

Antibacterial activity of different nanoformulations was screened by agar well diffusion

method16

. Stock solutions of nanoformulations were prepared by adding 1mg of nanopowder

in 10ml distilled water at room temperature and at neutral pH.

For antibacterial activity tests, 1.0 ml of 24h old bacterial broth culture was homoge-

neously spread on solidified agar plates. 5-10 mm wells were made by using gel puncture and

filled with 500 μl of stock solution (homogenized using an ultrasonic cleaner). The plates

were incubated at 37°C for 24 h. The inhibition zone size was determined by measuring its

radius with the help of a divider and a scale.

Growth curve of the bacterial cells under treatment with different nanoformulations

was studied at varying time intervals (1h, 2h, 4h, 8h and 12h, 24h, 36h, 48h and 54h) by tak-

ing OD of the culture broth at 600nm in a nanodrop spectrophotometer. For this 2ml of nano-


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formulations (50µgml-1

) was added to 10 ml bacterial broth culture (OD 1.0) in 50 ml conical

flasks and incubated on rotary shaker (180 rpm) at 37°C. Untreated culture and culture

treated with antibiotic were used as negative and positive controls, respectively.

Antifungal activity assay

Nanoformulations of different nanoparticles were screened for antifungal activity by observ-

ing percentage inhibition of mycelial growth under their effect. Stock solutions of nanofor-

mulations were prepared by adding 1mg of nanopowder in 10ml distilled water at room tem-

perature and at neutral pH. For this, about 15 ml of the Potato Dextrose Agar (PDA) me-

dium was poured into petri plates and allowed to solidify. After solidification, 500 μl of stock

solution was spread on them with the help of sterilised swab. Five mm discs of 7-day-old

culture of the test fungi were placed at the centre of the above petri plates and incubated at

25±2 ºC for seven days. After incubation the colony diameter was measured in millimetre.

For each treatment three replicates were maintained. PDA medium without the nanoformula-

tions served as control. The toxicity of nanoformulations to fungi in terms of percentage inhi-

bition of mycelial growth was calculated by using the formula

% inhibition = (dc – dt)/dc x 100

Where dc = Average increase in mycelial growth in control, dt = Average increase in myce-

lial growth in treatment17

.

Synthetic fungicides i.e. amphotericin B were also tested at their recommended dosage (2gm

l-1

) for antifungal activity.

Statistical calculations

For statistical analysis, SPSS Statistics 17.0 software was used and all tests were performed

in triplicate, and the results were expressed as the mean ± the standard errors of the mean. P

values lower than 0.05 were considered significant.


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RESULTS AND DISCUSSION

Synthesis and characterisation of nanocomposites

Dispersion of CuO and ZnO metal nanoparticles in chitosan matrix as observed in TEM im-

ages in Fig. 1, indicates synthesis of chitosan-metal nanocomposites. All nanofiller metal par-

ticles were 10-15 nm in size, approximately spherical and well capped. However, there is a

need to improve distribution of nanoparticles uniformly in the chitosan matrix. In Fig. 1(c),

inset represents that nanoparticles are polydispersed and less than 15nm in size, imparting

long term stability and redispersibility to the nanocomposites without affecting their physical

and chemical characteristics10

.

Chitosan nanoparticles and chitosan nanocomposites were characterized by FTIR

spectra, as shown in Fig. 2. In this figure, the spectra of chitosan show a strong broad vibra-

tion at around 3391, 1458, 1089 cm-1,

which can be assigned to the stretching vibration of O-

H and N-H, C-N and C-O-H group. In addition, some obvious shifts are shown in the spectra

of its corresponding complexes. Comparing with the vibrations of the above given groups

copper and zinc complexes show a little red shift at wave numbers 3304, 1419, 1142 cm-1

and

3386, 1418, 1080 cm-1

. Almost, a similar FTIR spectrum has been discussed previously18

.

Figure 3 shows DSC spectra for chitosan and its complexes. It is clear from the ther-

mal analysis data (Table 1), that the dehydration endotherms moved at 41 - 349 °C. The

thermograms of chitosan exhibited endothermic peaks at 167.82 °C with enthalpy of fusion

248.1Jg-1

corresponding to its melting point, indicating its crystalline nature; on the other

hand peaks at the 72.97°C with enthalpy fusion 91.88Jg-1

& 88.74°C with enthalpy fusion

174.9 Jg-1

for Zn/Ch and Cu/Ch, respectively. Thermal decomposition temperature of chito-

san moved to lower temperature in chitosan-metal complexes which indicates that chitosan


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chain of complexes can be broken more easily. The last endotherm of complexes belongs to

metal salt18

.

SEM-EDX spectra recorded on the examined samples are shown in Fig. 4 and 5. Figures 4(a)

and 5(a) show morphology of nanocomposites which is consistent with TEM micrograph.

The spectra obtained during EDX studies were used for carrying out the quantitative analysis.

In the left part of the presented spectrum one can clearly see three peaks located between 0.2

keV and 2 keV (Fig. 4(b) and 5 (b)). The maxima located on the left part of the spectrum at

0.2 keV and 0.5keV confirms the presence of carbon and oxygen, respectively. The hardly

visible maxima located near 1 keV indicates presence of copper (2.5%) and zinc (6.6%) as

can be seen in Fig. 4(b) and 5(b), respectively. The carbon (26%) and oxygen (67-70%) peaks

in the examined samples confirms the presence of chitosan matrix. Besides, presence of oxy-

gen in the examined samples also confirms the formation of CuO and ZnO nanoparticles in

chitosan matrix18, 19

.

Cytotoxic study

Cytotoxicity studies (Fig. 6) clearly revealed the relation between cytotoxicity and type and

concentration of the nanoformulations used in treatments. Cytotoxicity decreased signifi-

cantly with the decrease in their concentrations. Chitosan was found to be highly biocompati-

ble up to 200 μgml-1

concentration. Zn/Ch nanocomposites were less toxic i.e., more biocom-

patible up to 100 μgml-1

as compared ZnO NPs which were more toxic i.e. 97% even at conc.

60 μgml-1

. Cu/Ch nanocomposites and CuO NPs were more toxic i.e. 100% at 60 μgml-1

as

compared to any other formulation used. Chitosan was found to be highly biocompatible

similar to that of the controls at all the studied ranges. Nanocomposites showed less toxicity

i.e. more biocompatibility than CuO NPs and ZnO NPs nanoparticles at all of the studied

concentrations. Many recent studies on chitosan are based on its good biocompatibility and

non-toxicity20-21

. Therefore, it could be deduced that, the presence of chitosan in all nano-


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composites somehow compensates the negative impact of the nanoparticles to the biocom-

patibilities. Cao et al. (2010) reported similar cell behaviour when L-929 was exposed to chi-

tosan and silver/chitosan nanocomposites22

.

Antibacterial activity assay

It is well known that all nanoformulations exhibit antibacterial activity due to their well-

developed surface which provides maximum contact with the environment. Fig. 7 shows that

biocompatible concentrations of the nanoformulations (200 μgml-1

concentration of chitosan,

100 μgml-1

concentration of Zn/Ch and 60 μgml-1

concentrations of CuO NPs, ZnO NPs and

Cu/Ch) were able to inhibit bacterial growth and create a zone of inhibition against all bacte-

rial strains. It was observed that Cu/Ch nanocomposites exhibit size of zone i.e. 17.16 ± 2.36

mm, 17.5 ± 0.5 mm, 18 ± 1.82 mm that are higher than CuO NPs (15.5 ± 2.64 mm, 13.83 ±

1.5 mm, 14.8 ± 1.52 mm) and chitosan NPs (13 ± 0.5 mm, 11 ± 1.73 mm, 11.5 ± 1.5 mm),

against P. aeruginosa, S. enterica and M. luteus, respectively. Zn/Ch nanocomposites also

showed 17.5 ± 1.5 mm, 19 ± 1.0mm, 19 ± 0.5mm zone of inhibition as compared to ZnO NPs

(7.5 ± 0.5 mm, 7.83± 0.29mm and 8.5 ± 0.87 mm) and chitosan, against P. aeruginosa, S.

enterica and M. luteus, respectively. So nanocomposites showed higher antibacterial activity

against all test bacteria. Similar results have been reported earlier11

. They also found that an-

tibacterial activity was significantly enhanced by metal ion loaded nanoparticles, especially

for Cu2+

, and Zn2+

, compared to those of chitosan nanoparticles and related metal ions inde-

pendently.

The dynamics of bacterial growth curve was monitored in liquid nutrient broth. Here, anti-

bacterial effects of nanoformulations were studied by studying optical density as function of

time for upto 54 hours with 50µgml-1

concentration. Fig. 8 (a), (b) and (c), show growth

curve of P. aeruginosa, S. enteric and M. luteus, respectively, when treated with 50µgml-1

concentration of nanoformulations. We can conclude that in the absence of nanoformulations


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there is increase in optical intensity showing bacterial growth but in cultures of cells treated

with concentration of nanoformulations, there is reduction in the bacterial growth curve of all

bacteria. Among these nanoformulations, Cu/Ch nanocomposites and Zn/Ch nanocomposites

show higher antibacterial activity than chitosan, CuO NPs and ZnO NPs.

Antifungal activity assay

The results of studies on effect of nanoformulations on the growth of test fungi (Fig. 9) re-

vealed a significant inhibitory activity against all test fungi. Aspergillus fungal strain showed

susceptibility to the tested nanoformulations in the order i.e. Amphotericine B = Ch/Zn >

CuO NPs > Cu/Ch > ZnO NPs > Ch. Zn/Ch caused 93% inhibition, against Aspergillus sp.

Similarly, strains of Rhizoctonia sp. and Alternaria sp. also showed highest susceptibility to-

ward Zn/Ch nanocomposites followed by CuO NPs > Cu/Ch > ZnO NPs > Ch and Cu/Ch >

CuO NPs > Ch > ZnO NPs, respectively. The control plate did not exhibit inhibition on the

tested fungi whereas standard antifungal drug amphotericin B caused growth inhibition even

at 5μg/well (not shown). The findings indicate that the nanoformulations can be used as po-

tent biocide to treat diseases in plants caused by A. alternata, R. solani and A. flavus as they

showed maximum activity nearly equal to the standard antifungal agent.

CONCLUSION

Nanobiotechnology is an important area of research that deserves our attention owing to its

various potential applications such as fighting against bacteria and fungi in more efficient

manner. It was observed that, antimicrobial activity was significantly enhanced when chito-

san nanoparticles were loaded with metals, especially for Cu and Zn, as compared to

nanoparticles of chitosan or metal nanoparticles alone. It is concluded that the chitosan-metal

complexes showed higher antimicrobial activities because of the stronger positive charge af-

ter complexation. All the results showed that chitosan-metal complexes can be promising

candidate for novel antimicrobial agents that can be used in cosmetic, food and textile.


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ACKNOWLEDGMENTS

We gratefully acknowledge Advanced Instrumentation Research Facility (AIRF), Jawaharlal

Nehru University, New Delhi for TEM characterisation. We are thankful to Department of

Science and Technology (DST), New Delhi, India for funding. Ms. Pawan acknowledges the

fellowship received from Guru Jambheshwar University of Science and Technology.

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Table1 Data of TG-DSC analysis

DSC spectra

Samples *TR (°C)

#DT (°C) Area (J/g)

Chitosan 142.56-335.51 152.65, 167.82, 269.48,

312.97

15.15, 248.1, 281.3,

75.91

Cu/Ch 42.20-300.18 88.74,166.17, 288.58 174.9, 2.970, 4.843

Zn/Ch 41.19-349.73 72.97, 165.71, 322.78 91.88, 172.2, 231.9

*Temperature range,

#Decomposition Temperature


FIGURE CAPTIONS

Figure 1. TEM micrograph of Cu/Ch and Zn/Ch nanocomposites. (A) Copper oxide nanopar-

ticles embedded in chitosan matrix, scale bar = 5nm (B) Single copper nanoparticle at magni-

fication 800000X, (C) ZnO nanoparticles in chitosan matrix, Inset shows size of ZnO

nanoparticles.


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Figure 2. FTIR spectra of Chitosan, Cu/Ch, Zn/Ch nanoformulations.

Figure 3. Differential scanning calorimetric thermograms of Chitosan, Cu/Ch and Zn/Ch

nanocomposites.


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Figure 4. (a) SEM image and (b) EDX-spectra of Cu/Ch nanocomposites.


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Figure 5. (a) SEM image and (b) EDX-spectra of Zn/Ch nanocomposites.


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Figure 6. Cytotoxic effect of nanoformulations i.e. Chitosan, Cu/Ch, CuO NPs, Zn/Ch and

ZnO NPs, on Vero cell lines in EMEM culture medium.


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Figure 7. Antibacterial activity of nanoformulations, I, II, III - Cu/Ch (a), CuO NPs (b) and

IV,V,VI - Zn/Ch (a), ZnO NPs (b) against P. aeruginosa (I, IV), S. enteric (II, V) and M. lu-

teus (III, VI).

Figure 8. Growth curve of a) P. aeruginosa, b) S. enteric and c) M. luteus when treated with

nanoformulations.


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Figure 9. Percent inhibition of various fungal strains i.e. A. flavus, R. solani and A. alternate

by nanoformulations.

synthesis, characterization and in vitro evaluation of cytotoxicity and...

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