Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles

JOURNAL OF RARE EARTHS, Vol. 30, No. 12, Dec. 2012, P. 1298 Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles Brabu B...
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JOURNAL OF RARE EARTHS, Vol. 30, No. 12, Dec. 2012, P. 1298

Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles Brabu Balusamy1,2, Yamuna Gowri Kandhasamy2, Anitha Senthamizhan3, Gopalakrishnan Chandrasekaran1, Murugan Siva Subramanian2,4, Kumaravel Tirukalikundram S2,4 (1. Nanotechnology Research Center, SRM University, Chennai-603203, India; 2. GLR Laboratories Private Limited, Chennai-600 060, India; 3. Department of Physics, Indian Institute of Technology-Madras, Chennai-600 036, India; 4. GLR Laboratories Private Limited, Willingham, Cambridge, CB24 5GX, United Kingdom) Received 30 May 2012; revised 30 October 2012

Abstract: This study evaluated the bacterial toxicity of lanthanum oxide micron and nano sized particles using shake flask method against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Particle size, morphology and chemical composition were determined using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Results indicated that lanthanum oxide nanoparticles showed antimicrobial activity against Staphylococcus aureus, but not against Escherichia coli and Pseudomonas aeruginosa. It was speculated that lanthanum oxide produced this effect by interacting with the gram-positive bacterial cell wall. Furthermore, lanthanum oxide bulk particles were found to enhance the pyocyanin pigment production in Pseudomonas aeruginosa. Keywords: lanthanum oxide: nanoparticles: bacterial toxicity: pyocyanin; rare earths

In recent years, the rapid development in the field of nanotechnology has provided a wide range of application in fields of material, medical, computer, bio-sciences and information technology. Formulation of nanomaterial based antimicrobial agents has received much attention due to its unique and distinctive physical, chemical and biological properties due to its size effect and large surface to volume ratio. A number of studies have been focused on the preparation of antimicrobial agents using nanomaterials and nanocomposites[1–5]. Interestingly, inorganic based antimicrobial agent has shown better stability under higher temperature and pressure than organic based antimicrobial agents. Over the past few years, numerous studies have been undertaken to investigate the antimicrobial activity of metal and metal oxide nanoparticles[6–8]. Among these, a number of reports on the antimicrobial activities of silver and zinc oxide nanoparticles have been reported[9–12]. New nanoparticles are being investigated for their antimicrobial properties and to develop alternative antimicrobial agents to control bacterial infections. Rare earth elements, characterized by their high density, high melting point, high thermal conductance and conductivity, possess unique physical and chemical properties due to their 4f orbital electron and this has been extensively applied in electronics, medical, biomedical and agronomic fields[13,14]. Lanthanum oxide (La2O3) is a rare earth metal oxide, which has a band gap of 4.3 eV and the lowest lattice energy with high electric constant[15]. Also, it has been in use as a p-type semiconductor and has several other applications in areas of electronics, fuel cells, optics, magnetic data stor-

age, ceramics, catalysis, automobiles, biosensor, water treatment and biomedicine[16–18]. The possible applications of these materials has not been fully explored especially in the field of biomedical sciences. To the best of our knowledge, no studies have looked into the bacterial toxicity of lanthanum oxide bulk (La2O3 bulk) and lanthanum oxide nanoparticle (La2O3 NP). In this report, we have characterized the size, structure and chemical composition of La2O3 bulk and La2O3 NP’s using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) techniques. Furthermore, these materials were analyzed for their bactericidal property against some potential pathogenic clinical isolates like S. aureus, E. coli and P. aeruginosa.

1 Experimental 1.1 Materials La2O3 bulk (Ottokemi, Mumbai, India), La2O3 NP (Nanoshel, Panchkula, India), nutrient broth (Himedia, Mumbai, India), nutrient agar (Himedia, Mumbai, India), chloroform (Rankem, Faridabad, India) and hydrochloric acid (Merck, Mumbai, India). 1.2 Characterization The surface topography of the sample was analyzed by scanning electron microscopy (SEM). The samples were spread on the carbon tap and analyzed with an accelerating voltage of 20 kV using Quanta 200 FEG SEM. The chemical

Corresponding author: Kumaravel Tirukalikundram S (E-mail: [email protected]; Tel.: +91 9500064248) DOI: 10.1016/S1002-0721(12)60224-5

Brabu Balusamy et al., Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles

composition of the sample was obtained by energy dispersive X-ray spectroscopy analysis (EDS). 1.3 Bacterial toxicity assessment assay Toxicity of La2O3 (bulk and NP) against S. aureus, E. coli and P. aeruginosa bacteria was investigated using shake flask method. La2O3 bulk and nanoparticles at a final concentration of 10 mg/ml were added to separate culture flasks containing the pathogens S. aureus, E. coli and P. aeruginosa. The flasks were then incubated at 37 ºC for 24 h in a shaker water bath set at 100 r/min. Prior to incubation and at the end of 24 h incubation, 1 ml samples from each culture flasks were taken, serially diluted twice, and 100 µL of this diluted sample was spread on nutrient agar plates. These plates were incubated at 37 ºC, to obtain 0 and 24 h colony counts. Untreated controls were also included in this study. Colony counts at 0 and 24 h were used to determine the effect of the test items (La2O3 bulk or NP) on bacterial viability. 1.4 Quantitative evaluation of pyocyanin production Preliminary experiments showed that La2O3 had some effect on the pyocyanin production by Pseudomonas. This was further investigated by evaluating the effects of La2O3 bulk and NP on pyocyanin production. Pyocyanin pigment was extracted in 6 ml of chloroform and 2 ml of 0.2 mol/L hy-

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drochloric acid before and 24 h post treatment. The optical density of the extract was measured at 520 nm. The absorbance value of each sample was multiplied by 17.072 to express the concentration of pyocyanin production in micrograms per milliliter of culture supernatant[19–21]. Three independent experiments were performed to check the reproducibility of the results.

2 Results and discussion 2.1 SEM and EDS analysis The size and surface morphology of both La2O3 bulk and NPs were examined using SEM. The SEM image of La2O3 bulk and NPs are given in Figs. 1(a) and (b). The sizes of La2O3 bulk particles were in the region of 1 µm as seen in Fig. 1(a). From Fig. 1(b) it is clear that these nanoparticles are almost spherical in shape and are uniform in size of about 100 nm. The chemical composition of the La2O3 bulk and NP samples were analyzed by energy dispersive spectrum (EDS) and are shown in Figs. 1(c) and (d), respectively. The peaks reveal the presence of lanthanum (La) and oxygen (O). 2.2 Bacterial toxicity assays 2.2.1 Toxicity to S. aureus La2O3 NPs showed significant toxicity against S. aureus. There were 2.6×108 cfu

Fig. 1 SEM images and their corresponding EDS analysis of La2O3 bulk material (a, c) and La2O3 NP (b, d)

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JOURNAL OF RARE EARTHS, Vol. 30, No. 12, Dec. 2012

Table 1 Quantitative evaluations of S. aureus bacteria growth at 0 and 24 h of treatment with La2O3 bulk, La2O3 NP and control (cfu/ml) 0 h untreated

24 h untreated

0 h treatment

24 h treatment

0 h treatment

control

control

with La2O3 bulk

with La2O3 bulk

with La2O3 NP

with La2O3 NP

Experiment 1

2.1×108

TNTC*

2.2×108

TNTC

2.3×108

1.0×107

Experiment 2

2.8×108

TNTC

2.8×108

TNTC

2.7×108

2.8×107

Experiment 3

8

TNTC

8

TNTC

8

1.7×107

8

2.9×10

Mean

8

2.6×10

Standard Deviation

0.4×108

**

2.8×10

2.8×10

24 h treatment

NA

8

2.6×10

NA

2.6×10

1.8×107

NA

0.3×108

NA

0.3×108

0.9×107

* TNTC—too numerous to count; ** NA—not applicable

before and only 1.8×107 cfu following treatment with La2O3 NPs. This activity was not observed with La2O3 bulk material (see Table 1). The specific antibacterial activity of La2O3 NP against S. aureus may be attributed to lanthanide ions suppressing the activities of Ca2+ ions. Isomorphic capabilities of lanthanide ions replace the Ca2+ in the binding sites of staphylococcal nucleases and interrupt the activation and prevent the growth metabolism of the S. aureus.[22] If the above mechanism holds good, the toxicity against S. aureus should be observed following treatment with both bulk material as well. But our results clearly suggest that La2O3 bulk did not have toxicity against S. aureus. Therefore, some other mechanisms, other than calcium ion replacements may be involved in the bactericidal activity against S. aureus. Another possible explanation for of bacterial toxicity is via the induction of free radicals. Generations of free radicals like super oxide and hydroxyl ions mainly affect the macromolecules (DNA, lipids and proteins). Generally lanthanide oxides are known for their strong production of OH radicals. The generation of free radical in rare earth elements are ranked in the order of La2O3>Nd2O3>Sm2O3>Yb2O3>> CeO2[23]. Furthermore, another study reported the reactive oxygen species produced by the lanthanum, damaged the hepatic nuclei and mitochondria[13]. In this study, the bactericidal properties against S. aureus may not be fully attributable to the generation of free radicals, because other bacteria tested were not affected. Furthermore, it is also possible that bactericidal effect against S. aureus induced by La2O3 NP may be due to the interaction between the positively charged NP and negatively charged cell wall[24–28]. It therefore appears that the activity of lanthanum NP against S. aureus is multi-factorial and further research is necessary. A brief postulated mechanism is depicted in Fig. 2, which hypothesize that the bacterial toxicity may be due to the electrostatic interaction between the positively charged NP and negatively charged S. aureus. Overall charge of the S. aureus is negative due to the presence of excessive carboxylic acid in the cell wall. Therefore, the positively charged NPs are easily attracted towards the bacteria and are attached to the bacterial cells. Attached NPs mechanically damaged the bacterial cell wall and penetrated into the cell. Once the cell wall is damaged all cell constituents leak and cause cell death. 2.2.2 Toxicity to E. coli and P. aeruginosa With regard to the activities of both La2O3 NP and bulk materials, no sig-

nificant reduction in the growth of E. coli and P. aeruginosa were observed and the results are not presented here. Bactericidal properties of La2O3 NPs were compared with positive control silver nanoparticles (Ag NP) and found to be nil growth. The results suggested that La2O3 NPs did not show the same effectiveness and spectrum of antibacterial properties as seen with positive control Ag NP. Ag NP exhibited strong antibacterial activity against all pathogens tested. There are a number of studies reporting the antimicrobial activities of Ag NP. Some hypothesis that were proposed antibacterial mechanism of Ag NP, are free radicals released by the Ag NP that could attack the cell wall of bacteria and penetration of Ag NP into the cell and causing cell death[9,29]. 2.2.3 Quantitative evaluation of pyocyanin production Interestingly, treatment of P. aeruginosa cultures with La2O3 bulk material resulted in a significant increase in the

Fig. 2 Mechanism representing bacterial toxicity induced by La2O3 NP

Brabu Balusamy et al., Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles

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Table 2 Quantitative evaluations of P. aeruginosa bacteria pyocyanin production and growth at 0 and 24 h of treatment with La2O3 bulk, La2O3 NP and control (µg/ml) 0 h untreated

24 h untreated

0 h treatment

24 h treatment

0 h treatment

24 h treatment

control

control

with La2O3 bulk

with La2O3 bulk

with La2O3 NP

with La2O3 NP

Experiment 1

1.20

1.20

1.37

14.47

1.37

1.02

Experiment 2

1.20

1.37

1.02

14.00

1.20

1.02

Experiment 3

1.37

1.20

1.37

13.49

1.20

1.20

Mean

1.26

1.26

1.25

13.99

1.26

1.08

Standard Division

0.10

0.10

0.20

0.49

0.10

0.10

production of pyocyanin pigment. This effect was not seen with La2O3 NP. The pigment produced by the P. aeruginosa in the presence of La2O3 bulk material was confirmed as pyocyanin by treating it with chloroform and hydrochloric acid, which gave its characteristic colour. La2O3 bulk treated Pseudomonas produced comparatively much higher concentrations of the pyocyanin compared to both untreated control and La2O3 NP treated for 24 h of treatment. The amount of pyocyanin production at 0 and 24 h of treatments are given in Table 2. The exact biological significance of this phenomenon is not fully understood.

3 Conclusions In conclusion, we showed that La2O3 NPs had antibacterial activity against S. aureus and possibly against other gram-positive bacteria. Further research needed to identify the exact mechanism of bactericidal effect is still underway.

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