Antimicrobial and antifungal potential of zinc oxide nanoparticles in comparison to conventional zinc oxide particles

Abstract

Nanotechnology has become increasingly internalized into pharmaceuticals & cosmetics and is of great significance as an approach to killing or reducing the activity of numerous microorganisms. Natural antibacterial materials, such as zinc and silver, are being claimed to possess good antibacterial properties. Nano-sized particles of ZnO have been claimed to have pronounced antimicrobial activities than large particles. Antimicrobial/antifungal potential of ZnO on five pathogens (Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160, Bacillus subtilis MTCC 441, Aspergillus niger MTCC 281, Candida albicans MTCC 227) and the influence of particles size of these inorganic powder on its antimicrobial /antifungal efficacy was considered in the present study. Results indicated that zinc oxide nanoparticles do have strong antibacterial and good antifungal activity against selected strains of bacteria and fungus as compared to that of conventional zinc oxide particles.

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Journal of Chemical and Pharmaceutical Research, 2013, 5(11):457-463
Research Article
ISSN : 0975-7384
CODEN(USA) : JCPRC5
457
Antimicrobial and antifungal potential of zinc oxide nanoparticles in
comparison to conventional zinc oxide particles
Priyanka Singh* and Arun Nanda
Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana
_____________________________________________________________________________________________
ABSTRACT
Nanotechnology has become increasingly internalized into pharmaceuticals & cosmetics and is of great significance
as an approach to killing or reducing the activity of numerous microorganisms. Natural antibacterial materials,
such as zinc and silver, are being claimed to possess good antibacterial properties. Nano-sized particles of ZnO
have been claimed to have pronounced antimicrobial activities than large particles. Antimicrobial/antifungal
potential of ZnO on five pathogens (Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160, Bacillus
subtilis MTCC 441, Aspergillus niger MTCC 281, Candida albicans MTCC 227) and the influence of particles size
of these inorganic powder on its antimicrobial /antifungal efficacy was considered in the present study. Results
indicated that zinc oxide nanoparticles do have strong antibacterial and good antifungal activity against selected
strains of bacteria and fungus as compared to that of conventional zinc oxide particles.
Keywords: Nanotechnology, antibacterial, antifungal, efficacy.
_____________________________________________________________________________________________
INTRODUCTION
Nanotechnology is being envisioned as a hurriedly developing field, it has potential to revolutionize pharmaceuticals
and cosmetics. Nanotechnology, or the use of materials with constituent dimensions on the atomic or molecular
scale, has become increasingly applied to pharmaceuticals & cosmetics and is of great interest as an approach to
killing or reducing the activity of numerous microorganisms. Some natural antibacterial materials, such as zinc and
silver, are being claimed to possess good antibacterial properties.
Microbial spoilage of cosmetic formulation has always been of special concern for cosmetic industry. The use of
authorized preservatives of the new regulation 1123/209 is required to prevent product damage caused by micro-
organisms and to protect the product from unwanted contamination by the consumer during its shelf –life. However
since few years, the cosmetic industry is facing some restrictions regarding the use of some preservatives. So, there
has been considerable interest in the development of new preservatives. Among raw materials exhibiting
antimicrobial/antifungal properties, inorganic powders such as zinc oxide (ZnO) represent a promising alternative to
these chemical preservatives [1, 2].
Zinc oxide is a non-toxic , II-VI semiconductor with wide band gap (3.37eV) and natural n-type electrical
conductivity [3, 4]. Zinc oxide because of its interesting properties, such as optical transparency, electrical
conductivity, piezoelectricity, near-UV emission [5, 6, 7, 8, 9, 10] and various morphologies, has become one of the
most attractive nanomaterials for research objectives. Its significant properties has made it applicable in UV-light
emitters, varistors, transparent high power electronics, surface acoustic wave devices, piezo-electric transducers, gas
sensors, etc.[11].
Introduction of zinc oxide in cosmetic creams and gels makes them sunlight-protective and antibacterial [12]. The
efficiency of their action largely depends not only on the concentration of the active substance, zinc oxide, but also

Priyanka Singh and Arun Nanda J. Chem. Pharm. Res., 2013, 5(11): 457-463
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458
on the size of its particles, their modification, and the degree of polydispersity. Moreover, zinc oxide (ZnO) is listed
as “generally recognized as safe”(GRAS) by the U.S. Food and Drug Administration (21CFR182.8991). Nano-sized
particles of ZnO have been claimed to have pronounced antimicrobial activities than large particles, considering the
fact that the small size (less than 100 nm) and high surface-to-volume ratio of nanoparticles may allow for better
interaction with bacteria. Recent studies have shown that these nanoparticles have selective toxicity to bacteria but
exhibit minimal effects on human cells [1, 13, 14, 15, 16, 17, 18, 19].
Even if ZnO has been used for a long time in cosmetic or pharmaceutical ointments, its antimicrobial properties had
not been fully investigated in context of cosmetic preservation. A systematic and detailed study was designed, taking
into account above claims, to investigate the enhanced antimicrobial and antifungal properties of nano-zinc oxide
over conventional one.
Therefore, the purpose of the present paper is to demonstrate firstly the antimicrobial/antifungal potential of ZnO on
five pathogens (Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160, Bacillus subtilis MTCC 441,
Aspergillus niger MTCC 281, Candida albicans MTCC 227) that are used for challenge tests, and secondly to
determine the influence of particles size of these inorganic powder on its antimicrobial /antifungal efficacy.
EXPERIMENTAL SECTION
Materials
Nanoparticles of ZnO with a diameter of either ~65 nm were synthesized and used in this study. A representative
TEM image of the ~65 nm ZnO nanoparticles is shown in fig. 1. Conventional zinc oxide nanoparticles with a
diameter ~1000nm were used for the comparison.
Selection of Test Pathogens
Pathogenic microorganisms selected for the study include three bacteria, viz., Escherichia coli (MTCC 443),
Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441) and two fungus, viz., Aspergillus niger
(MTCC 281) and Candida albicans (MTCC 227).
Preparation of dilutions of synthesized compounds
10 mg of the each particle (nano and conventional zinc oxide) was weighed accurately and dissolved in 10 ml
DMSO giving a solution of 1mg/ml concentration. 1 ml of the above solution was again diluted to 10 ml with
DMSO giving a solution of 100µg/ml concentration.
Preparation of Agar nutrient broth (for bacteria)
5.6g of Agar was dissolved in 150ml distilled water and heated. The medium was the sterilized by autoclaving at
115
o
C for 30 mins.
Preparation of Sabouraud Dextrose Agar (for fungi)
9.20g of Sabouraud Dextrose Agar was dissolved in distilled water and heated. The medium was the sterilized by
autoclaving at 115
o
C for 30 mins.
Preparation of nutrient broth medium
0.75 g of media (Bacterial/fungal) was dissolved in 30 ml of distilled water and heated. The medium was then
sterilized by autoclaving at 115
o
C for 30 mins.
Preparation of bacterial and fungal slants
Five Nessler cylinders were sterilized by hot sterilization method in an oven at 160
o
C for 30 min. Laminar air flow
cabinet was wiped with cotton immersed in ethanol and UV was switched ON for 15mins. Sterlized bacterial and
fungal media were poured into 5 sterilized Nessler cylinders (3 bacterial, 2 fungal) and were allowed to stand in
slant position till the media in the cylinders was solidified. Sterlized loop wire was used to transfer bacterial and
fungal strains to nessler cylinders. The nessler cylinders were then labelled and cotton plugs were fitted into their
mouth and were incubated at 37
o
C except aspergillus niger (which was incubated at 25
o
C) for 24 hr. From each of
the strain, small portion was transferred to 3ml of nutrient broth media separately and incubated at 37
o
C for 24hrs.
0.1 ml of the above five medias were transferred to five different stoppered conical flasks containing 0.9% NaCl
solution.

Priyanka Singh and Arun Nanda J. Chem. Pharm. Res., 2013, 5(11): 457-463
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459
Antimicrobial activity: Determination of Minimum inhibitory concentration and Minimum Bactericidal/
Fungicidal concentration
Minimum inhibitory concentration (MIC) was determined for conventional and nano-sized zinc oxide nanoparticles
showing antimicrobial and antifungal activity against test pathogens by serial dilution method. Broth microdilution
method was followed for determination of MIC values. 1ml of media was taken in a test tube, to which, 1ml of test
solution (100µg/ml) was added. Thereafter, 0.1ml of the microbial strain (bacterial/ fungal) prepared in 0.9% NaCl
was added to the test tube containing media and test solution. Serial dilution were done five times giving
concentrations of 50, 25, 12.5, 6.25, 3.75, 1.5 µg/ml. The test tube were stoppered with cotton and incubated at
37
o
C. The time incubation time varied for different strains (bacteria/fungus), i.e., 24 hrs for bacteria and one week
for fungus.
The MIC values were taken as the lowest concentration of the particles in the test tube that showed no turbidity after
incubation. The turbidity of the contents in the test tube was interpreted as visible growth of microorganisms. The
minimum bacterial/fungicidal concentration (MBC/MFC) was determined by subculturing 50µl from each test tube
showing no apparent growth. Least concentration of test substance showing no visible growth on subculturing was
taken as MBC/MFC.
RESULTS AND DISCUSSION
Zinc oxide nanoparticles particles were fully characterized. A representative TEM image of the ZnO nanoparticles
(~65 nm) is shown in figure 1.
Secondly, the antimicrobial properties of conventional ZnO particles and ZnO nanoparticles were studied. Both, the
conventional particles and nanoparticles, showed antimicrobial activity against Escherichia coli MTCC 443,
Staphylococcus aureus MTCC 3160 and Bacillus subtilis MTCC 441 with a size dependent effect. Figure 2 & figure
3 portray the behaviour of bacterial populations following the incubation with conventional ZnO particles and ZnO
nanoparticles for 24 hrs. The Minimum inhibitory concentration of ZnO nanoparticles (as shown in table 1) against
the three bacterias, viz., Escherichia coli MTCC 443, Staphylococcus aureus MTCC 3160 and Bacillus subtilis
MTCC 441 was found to be 6.25µg/ml, 6.25µg/ml, 12.5µg/ml, respectively which is very less concentration as
compared to that of conventional zinc oxide particles (25µg/ml, 12.5µg/ml, 12.5µg/ml, respectively). Similarly the
Minimum bacterial count for ZnO nanoparticles was less in each case as compared to conventional ZnO particles
(table 1 & 2). Figure 4 & figure 5 portray the intense bacterial growth on the plate in the presence of conventional
zinc oxide particles and fewer bacterial growths on the plate in the presence of ZnO nanoparticles. The difference of
sensitivity to same test substance between these three strains can be attributed to structural and chemical differences
of their bacterial cell walls [20].
According to a study by Yamamoto et al., 2000 [21], the presence of reactive oxygen species (ROS) generated by
ZnO nanoparticles is responsible for their bactericidal activity. Zhang et al., 2010 [22], further proposed that the
antibacterial behaviour of ZnO nanoparticles could be due to chemical interactions between hydrogen peroxide and
membrane proteins, or between other chemical species produced in the presence of ZnO nanoparticles and the outer
lipid bilayer of bacteria. The hydrogen peroxide produced enters the cell membrane of bacteria and kills them. The
study also showed that nano-sized ZnO particles are responsible for inhibiting bacterial growth [22]. Further,
Padmavathy and Vijayaraghavan, 2008 [23], showed the bactericidal activity of ZnO nanoparticles. As per their
findings, once hydrogen peroxide is generated by ZnO nanoparticles, the nanoparticles remains in contact with the
dead bacteria to prevent further bacterial action and continue to generate and discharge hydrogen peroxide to the
medium. The results of the present study correspond with the results of the authors above, showing that ZnO
nanoparticles have an excellent antimicrobial activity.
Zin oxide also exhibited antifungal activity but in a minor extent than the antibacterial one since no fungicidal
activity is reported. The ZnO nanoparticles did show activity against Aspergilllus Niger and Cadida ablicans at a
concentration of 12.5µg/ml and 6.25µg/ml, respectively. Again these concentration were higher for conventional
ZnO particles, i.e., 25 and 12.5, respectively (table 1). The Minimum fungal count for zinc oxide nanoparticles was
found to be same as Minimum inhibitory concentration, i.e., 12.5µg/ml and 6.25µg/ml, respectively. Same was the
case with conventional ZnO particles in case of Aspergillus niger, where MFC (25µg/ml) was same as MIC
(25µg/ml). But the antifungal activity against candida albicans showed different pattern in view that MFC (25µg/ml)
was more than MIC (12.5µg/ml). Figure 6 & figure 7 represent the intense fungal growth on the plate in the
presence of conventional zinc oxide particles and fewer fungal growths on the plate in the presence of ZnO
nanoparticles.

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Table 1 Determination of MIC and MBC for conventional zinc oxide particles
Pathogen Concentration
(µg/ml) Observation Minimum Inhibitory
concentration
(µg/ml)
Minimum bacterial
concentration
(µg/ml)
Escherichia coli
(MTCC 443)
50
No turbidity
12.5
12.5
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Staphylococcus aureus
(MTCC 3160)
50
No turbidity
12.5
12.5
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Bacillus subtilis
(MTCC 441)
50
No turbidity
25
25
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Aspergillus niger
(MTCC 281)
50
No turbidity
25
25
25
No turbidity
12.5
Turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Candida albicans
(MTCC 227)
50
No turbidity
12.5
25
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Table 2 Determination of MIC and MBC for zinc oxide nanoparticles
Pathogen Concentration
(µg/ml) Observation Minimum Inhibitory
concentration
(µg/ml)
Minimum bacterial
concentration
(µg/ml)
Escherichia coli
(MTCC 443)
50
No turbidity
6.25 6.25
25
No turbidity
12.5
No turbidity
6.25
No turbidity
3.75
Turbidity
1.5
Turbidity
Staphylococcus
aureus
(MTCC 3160)
50
No turbidity
6.25 6.25
25
No turbidity
12.5
No turbidity
6.25
No turbidity
3.75
Turbidity
1.5
Turbidity
Bacillus subtilis
(MTCC 441)
50
No turbidity
12.5 12.5
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Aspergillus niger
(MTCC 281)
50
No turbidity
12.5 12.5
25
No turbidity
12.5
No turbidity
6.25
Turbidity
3.75
Turbidity
1.5
Turbidity
Candida albicans
(MTCC 227)
50
No turbidity
6.25 6.25
25
No turbidity
12.5
No turbidity
6.25
No turbidity
3.75
Turbidity
1.5
Turbidity

Priyanka Singh and Arun Nanda J. Chem. Pharm. Res., 2013, 5(11): 457-463
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461
Figure 1 TEM image of zinc oxide nanoparticles
Figure 2 Determination of MIC for conventional zinc oxide particles against bacterial strain
Figure 3 Determination of MIC for zinc oxide nanoparticles against bacterial strain

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Figure 4 Bacterial growth in the presence of conventional zinc oxide particles
Figure 5 Bacterial growth in the presence of zinc oxide nanoparticles
Figure 6 Fungal growth in the presence of conventional zinc oxide particles
Figure 7 Fungal growth in the presence of zinc oxide nanoparticles
CONCLUSION
Results in present study indicate that zinc oxide nanoparticles had strong antibacterial and good antifungal activity
against selected strains of bacteria and fungus as compared to that of conventional zinc oxide particles. In summary,

Priyanka Singh and Arun Nanda J. Chem. Pharm. Res., 2013, 5(11): 457-463
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463
the present study reveals that zinc oxide nanoparticles could potentially be an antibacterial and antifungal agent to
treat infections caused by bacteria and fungus. In future, these nanoparticles might replace conventional
preservatives in cosmetics. However, antibacterial/antifungal effects, safety, and detailed mechanisms of zinc oxide
nanoparticles should be further studied in vitro and in vivo.
Acknowledgement
The authors are thankful to CSIR for providing funding in the form of SRF and encouragement to carry out this
research work.
REFERENCES
[1] J Sawai, J. Microbiol. Methods, 2003, 54, 177–182.
[1a] SB Prasad; Yashwant; V Aeri, Journal of Chemical and Pharmaceutical Research, 2013, 5(9), 194-197.
[2] JS Wellings; NB Chaure; SN Heavens; IM Dharmadasa, Thin Solid Films, 2007, 516, 3893-3898.
[3a] S Udayakumar; V Renuka; K Kavitha, J. Chem. Pharm. Res., 2012, 4(2), 1271-1280.
[3] DC Look, Mater. Sci. Engg: B, 2001,80,383-387.
[4] P Yang; H Yan; S Mao; R Russo; J Johnson; R Saykally, Adv. Func. Mater. 2002;12:323-331.
[5] MH Huang; S Mao; H Feick; H Yan; Y Wu, Science, 2001, 292, 1897-1899.
[6] T Shiono; H Yamamoto; S Nishino, Jap. J. Appl. Phys., 2004,43, 4941-4944.
[7] T Sato; T Tanigaki; H Suzuki; Y Saito; O Kido; Y Kimura, J. Cryst. Growth., 2003,255, 313-316.
[8] JF Hochepied; AP Almeida De Oliveira, Prog. Colloid Polym Sci., 2004,125, 68-73.
[9] SJ Pearton; DP Norton; K Ip; YW Heo; T Steiner. Superlattices Microstruct., 2005, 50, 3-32.
[10] AV Gudkova; KI Kienskaya; VV Nazarov; V Kim; SE Mukhtarova. Russ. J. Appl. Chem., 2005, 78, 1757-
1760.
[11] KM Reddy. Appl. Phys. Lett., 2007, 90, 2139021–2139023.
[12] O Yamamoto. Int. J. Inorg. Mater. 2001, 3,643–68.
[13] JT Seil; TJ Webster, Nanotech. , 2012 , 23, 495101.
[14] AC Dodd; AJ McKinley; M Saunders; T Tsuzuki, J. Nanopart. Res., 2006, 8, 43–51.
[15] L Zhang; Y Jiang; Y Ding; M Povey; D York, J. Nanopart. Res., 2007, 9, 479–89.
[15a] S. Dugal; N. Mamajiwala, J. Chem. Pharm. Res., 2011, 3(1), 584-589.
[15b] N. haque; R. R. Khalel; N Parvez; S Yadav; N Hwisa; MS Al-Sharif; BZ Awen; K Molvi, J. Chem. Pharm.
Res., 2010, 2(5), 161-168.
[16] RK Dutta; PK Sharma; R Bhargava; AC Pandey, J. Phys. Chem. B, 2010, 114, 5594-5599.
[17] O Yamamoto; J Sawai; T Sasamoto, Int. J. Inorg. Mats., 2000, 2, 451-454.
[18] L Zhang; Y Jiang; Y Ding; N Daskalakis; L Jeuken; M Povey; A O’Neill; D York, Springer Netherlands
2010.
[19] N Padmavathy; R Vijayaraghavan, Sci. and Tec. of Adv. Mats., 2008, 9.