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 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). Re- sults 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 age, ceramics, catalysis, automobiles, biosensor, water nanotechnology has provided a wide range of application in treatment and biomedicine[16–18]. The possible applications of fields of material, medical, computer, bio-sciences and in- these materials has not been fully explored especially in the formation technology. Formulation of nanomaterial based field of biomedical sciences. To the best of our knowledge, antimicrobial agents has received much attention due to its no studies have looked into the bacterial toxicity of lantha- unique and distinctive physical, chemical and biological num oxide bulk (La2O3 bulk) and lanthanum oxide nanopar- properties due to its size effect and large surface to volume ticle (La2O3 NP). ratio. A number of studies have been focused on the prepara- In this report, we have characterized the size, structure and tion of antimicrobial agents using nanomaterials and nano- chemical composition of La2O3 bulk and La2O3 NP’s using composites[1–5]. Interestingly, inorganic based antimicrobial scanning electron microscopy (SEM) and energy dispersive agent has shown better stability under higher temperature X-ray spectroscopy (EDS) techniques. Furthermore, these and pressure than organic based antimicrobial agents. Over materials were analyzed for their bactericidal property the past few years, numerous studies have been undertaken against some potential pathogenic clinical isolates like S. to investigate the antimicrobial activity of metal and metal aureus, E. coli and P. aeruginosa. oxide nanoparticles[6–8]. Among these, a number of reports on the antimicrobial activities of silver and zinc oxide 1 Experimental nanoparticles have been reported[9–12]. New nanoparticles are being investigated for their antimicrobial properties and to 1.1 Materials develop alternative antimicrobial agents to control bacterial infections. La2O3 bulk (Ottokemi, Mumbai, India), La2O3 NP Rare earth elements, characterized by their high density, (Nanoshel, Panchkula, India), nutrient broth (Himedia, high melting point, high thermal conductance and conduc- Mumbai, India), nutrient agar (Himedia, Mumbai, India), tivity, possess unique physical and chemical properties due chloroform (Rankem, Faridabad, India) and hydrochloric to their 4f orbital electron and this has been extensively ap- acid (Merck, Mumbai, India). plied in electronics, medical, biomedical and agronomic [13,14] 1.2 Characterization fields . Lanthanum oxide (La2O3) is a rare earth metal oxide, which has a band gap of 4.3 eV and the lowest lattice The surface topography of the sample was analyzed by energy with high electric constant[15]. Also, it has been in use scanning electron microscopy (SEM). The samples were as a p-type semiconductor and has several other applications spread on the carbon tap and analyzed with an accelerating in areas of electronics, fuel cells, optics, magnetic data stor- 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 1299 composition of the sample was obtained by energy disper- drochloric acid before and 24 h post treatment. The optical sive X-ray spectroscopy analysis (EDS). density of the extract was measured at 520 nm. The absorb- ance value of each sample was multiplied by 17.072 to ex- 1.3 Bacterial toxicity assessment assay press the concentration of pyocyanin production in micro- [19–21] Toxicity of La2O3 (bulk and NP) against S. aureus, E. coli grams per milliliter of culture supernatant . and P. aeruginosa bacteria was investigated using shake Three independent experiments were performed to check flask method. the reproducibility of the results.

La2O3 bulk and nanoparticles at a final concentration of 10 mg/ml were added to separate culture flasks containing the 2 Results and discussion pathogens S. aureus, E. coli and P. aeruginosa. The flasks were then incubated at 37 ºC for 24 h in a shaker water bath 2.1 SEM and EDS analysis set at 100 r/min. Prior to incubation and at the end of 24 h The size and surface morphology of both La O bulk and incubation, 1 ml samples from each culture flasks were taken, 2 3 NPs were examined using SEM. The SEM image of La O serially diluted twice, and 100 µL of this diluted sample was 2 3 bulk and NPs are given in Figs. 1(a) and (b). The sizes of spread on nutrient agar plates. These plates were incubated at La O bulk particles were in the region of 1 µm as seen in 37 ºC, to obtain 0 and 24 h colony counts. Untreated controls 2 3 Fig. 1(a). From Fig. 1(b) it is clear that these nanoparticles were also included in this study. are almost spherical in shape and are uniform in size of Colony counts at 0 and 24 h were used to determine the about 100 nm. The chemical composition of the La O bulk effect of the test items (La O bulk or NP) on bacterial vi- 2 3 2 3 and NP samples were analyzed by energy dispersive spec- ability. trum (EDS) and are shown in Figs. 1(c) and (d), respectively. 1.4 Quantitative evaluation of pyocyanin production The peaks reveal the presence of lanthanum (La) and oxygen (O). Preliminary experiments showed that La2O3 had some effect on the pyocyanin production by Pseudomonas. This was 2.2 Bacterial toxicity assays further investigated by evaluating the effects of La O bulk 2 3 2.2.1 Toxicity to S. aureus La O NPs showed signifi- and NP on pyocyanin production. Pyocyanin pigment was 2 3 cant toxicity against S. aureus. There were 2.6×108 cfu extracted in 6 ml of chloroform and 2 ml of 0.2 mol/L hy-

Fig. 1 SEM images and their corresponding EDS analysis of La2O3 bulk material (a, c) and La2O3 NP (b, d) 1300 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 24 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 2.9×108 TNTC 2.8×108 TNTC 2.8×108 1.7×107 Mean 2.6×108 NA** 2.6×108 NA 2.6×108 1.8×107 Standard Deviation 0.4×108 NA 0.3×108 NA 0.3×108 0.9×107 * TNTC—too numerous to count; ** NA—not applicable

7 before and only 1.8×10 cfu following treatment with La2O3 nificant reduction in the growth of E. coli and P. aeruginosa

NPs. This activity was not observed with La2O3 bulk mate- were observed and the results are not presented here. rial (see Table 1). The specific antibacterial activity of La2O3 Bactericidal properties of La2O3 NPs were compared with NP against S. aureus may be attributed to lanthanide ions positive control silver nanoparticles (Ag NP) and found to be 2+ suppressing the activities of Ca ions. Isomorphic capabili- nil growth. The results suggested that La2O3 NPs did not ties of lanthanide ions replace the Ca2+ in the binding sites of show the same effectiveness and spectrum of antibacterial staphylococcal nucleases and interrupt the activation and properties as seen with positive control Ag NP. Ag NP ex- prevent the growth metabolism of the S. aureus.[22] If the hibited strong antibacterial activity against all pathogens above mechanism holds good, the toxicity against S. aureus tested. There are a number of studies reporting the antim- should be observed following treatment with both bulk ma- icrobial activities of Ag NP. Some hypothesis that were terial as well. But our results clearly suggest that La2O3 bulk proposed antibacterial mechanism of Ag NP, are free radi- did not have toxicity against S. aureus. Therefore, some cals released by the Ag NP that could attack the cell wall of other mechanisms, other than calcium ion replacements may bacteria and penetration of Ag NP into the cell and causing be involved in the bactericidal activity against S. aureus. cell death[9,29]. Another possible explanation for of bacterial toxicity is via 2.2.3 Quantitative evaluation of pyocyanin production the induction of free radicals. Generations of free radicals Interestingly, treatment of P. aeruginosa cultures with like super oxide and hydroxyl ions mainly affect the mac- La2O3 bulk material resulted in a significant increase in the romolecules (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>> [23] CeO2 . 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 attribut- able 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 elec- trostatic 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 Fig. 2 Mechanism representing bacterial toxicity induced by La2O3 NP to the activities of both La2O3 NP and bulk materials, no sig- Brabu Balusamy et al., Characterization and bacterial toxicity of lanthanum oxide bulk and nanoparticles 1301

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 implications. Water Res., 2008, 42: 4591. with La2O3 NP. The pigment produced by the P. aeruginosa [8] Diao M, Yao M. Use of zero-valent iron nanoparticles in inac- tivating microbes. Water Res., 2009, 43: 5243. in the presence of La2O3 bulk material was confirmed as pyocyanin by treating it with chloroform and hydrochloric [9] Kim J S, Kuk E, Yu K N, Kim J H, Park S J, Lee H J, Kim S H, Park Y K, Park Y H, Hwang C Y, Kim Y K, Lee Y S, acid, which gave its characteristic colour. La2O3 bulk treated Jeong D H, Cho M H. Antimicrobial effects of silver nanopar- Pseudomonas produced comparatively much higher concen- ticles. Nanomed. Nanotechnol. 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