preliminary evaluation of the antifungal activity of samarium ...claudiu stefan turculet, alina...

Romanian Biotechnological Letters Vol. 23, No. 5, 2018 Copyright © 2018 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 23, No. 5, 2018 13927

Preliminary evaluation of the antifungal activity of samarium doped

hydroxyapatite thin films DOI 10.26327/RBL2018.220

Received for publication, May, 3, 2018 Accepted, August, 20, 2018

CLAUDIU STEFAN TURCULET1,2, ALINA MIHAELA PRODAN1,2, IONUT NEGOI1,2, GABRIEL TELEANU1,2, MARCELA POPA3, ECATERINA ANDRONESCU4,5, MIRCEA BEURAN 1,2, GEORGE A. STANCIU6, RADU HRISTU6, MONICA LUMINITA BADEA7, ALIN IOSIF8, STEFANIA MARIANA RAITA9, NICOLETA VINETICU10, ROXANA TRUSCA4, OLIVERA LUPESCU1,2,*

1Emergency Hospital Floreasca, 8 Calea Floresca, Bucharest, Romania, 2Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari, Bucharest, Romania, 3Earth, Environmental and Life Sciences Section, Research Institute of the University of Bucharest, 1–3 Portocalelor Lane, 77206 Bucharest, Romania 4UPB Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, Romania 5Academia Oamenilor de Știință din România, Romania 6Center for Microscopy-Microanalysis and Information Processing, University Politehnica of Bucharest, 313 Splaiul Independentei, 060042 Bucharest, Romania 7University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania 8BioSintex, 4 Vlădiceasca, Snagov, Ilfov 077168 9Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Bucharest, Romania 10Faculty of Physics, University of Bucharest, 405 Atomistilor Street, P.O. Box MG 1, 077125 Măgurele, Romania. *Address for correspondence to: [email protected]

Abstract

This study was focused on the obtaining of antifungal Sm:HAp (samarium doped hydroxyapatite xSm=0.07 and xSm=0.1) layers deposited on titanium substrate (Ti-Sm:HAp_7 and Ti-Sm:HAp_10) by spin coating technique. The Ti-Sm:HAp_7 and Ti-Sm:HAp_10 thin films were characterized using scanning electron microcopy (SEM) to obtain information on the surface morphology. Furthermore, the antifungal activity of the Ti-Sm:HAp_7 and Ti-Sm:HAp_10 thin films was investigated using Candida albicans ATCC 10231 fungal strain. The development of the C. albicans biofilm on the Sm:HAp layers was emphasize by confocal laser scanning microscopy (CLSM). The results of the antifungal studies demonstrated that the adherence of fungal strains and the biofilm development were inhibited by the Sm:HAp thin films.

Keywords: Candida albicans, samarium doped hydroxyapatite, antimicrobial layers.

1. Introduction

In the last years due to the high impact and rapid progress of nanotechnology a great deal of attention has been focused towards the design and fabrication of nanoscale materials with enhanced properties for biomedical applications (MIGLIARESI & NICOLAIS [1]; PREDOI [2]; LE MAY & al. [3]; PREDOI [4]; LOPEZ & al. [5]; WILLI & al. [6]; PREDOI [7]). One of the most used biomaterial in biomedical applications is hydroxyapatite (HAp) due to its similarity with the inorganic part from teeth and bones. HAp also exhibits good biological properties such as biocompatibility, bioactivity and osteoconductivity (LOPEZ &

CLAUDIU STEFAN TURCULET, ALINA MIHAELA PRODAN, IONUT NEGOI, GABRIEL TELEANU, MARCELA POPA, ECATERINA ANDRONESCU, MIRCEA BEURAN, GEORGE A. STANCIU, RADU HRISTU, MONICA LUMINITA BADEA, ALIN IOSIF, STEFANIA MARIANA RAITA, NICOLETA VINETICU, ROXANA TRUSCA, OLIVERA LUPESCU


al. [5]; WILLI & al. [6]; PREDOI [7]; CIOBANU & al. [8]; CIOBANU & al. [9]; DOROZHHIN [10]; YIN & al. [11]). Moreover, it is well known that HAp possess a great affinity for substitutions with various ions such as: Ag+ (CIOBANU & al. [8]; CIOBANU & al. [9]), Eu3+ (ICONARU & al. [12]; YANG & al. [13]; CIOBANU & al.14]), Zn+ (BHATTACHARJEE & al. [15]; POPA & al.16]), Sm3+ (MORAIS & al. [17]; CIOBANU & al. [18]; CIOBANU & al. [19]), etc. Usually these ions replace calcium in the HAp structure which improves its physico-chemical and biological properties such as biocompatibility and antimicrobial activity. In the last decade the use of hydroxyapatite coatings in implantology have demonstrated that this type of coatings promote the formation of bonds between the implant and the surrounding living bone (AIZAWA & al. [20]; KOKUBO [21]). Furthermore, in the literature have been reported studies that have shown that HAp is able to guide the bone formation along the coating surface of the implant (JOKANOVIC & USKOKVIC [22]). Recent studies (CIOBANU & al. [18]; CIOBANU & al. [19]) have shown that the substitution of HAp with lanthanides (samarium) improve its antimicrobial activity against Gram-negative and Gram-positive bacterial strains.

Also, has been reported that the use of Sm isotopes and samarium doped hydroxyapatite in cancer therapy could reduce the pain intensity (TURNER & al. [23]; TURNER & CLARINGBOLD [24]; EAT & al. [25]; PODOLOFF & al. [26]). On the other hand, another biological role of samarium is represented by the fact that it can stimulate the metabolism (http://www.lenntech.com/periodic/elements/sm.htm [27]). If we consider this encouraging results that show that samarium doped hydroxyapatite could be used in a series of medical applications, contributing to the improvement of medical care for patients suffering from painful illness. The goal of this research was to investigate that the Sm:HAp thin films were deposited successfully by sol-gel spin coating method as a homogeneous and uniform coating on the Ti substrates forming a homogenous and uniform composite layer. Another objective of this study was to show that the adherence of fungal strains and the biofilm development of C. albicans cells were inhibited by the Sm:HAp thin films. 2. Materials and Methods

2.1. Sample preparation Nanoscale samarium doped hydroxyapatite particles (Sm:HAp) were obtained as

previously described by Ciobanu et al. (CIOBANU & al. [18]; CIOBANU & al. [19]). The composition ratios in the Sm:HAp (Ca10-xSmx(PO4)6(OH)2, xSm=0.07 and xSm=0.1) sols were adjusted in order to obtain a [Ca+Sm]/P ratio equal with 1.67 (FRUMOSU & al. [28]). The Ti substrates used for the deposition were cleaned and rinsed with acetone and deionized water. The Sm:HAp thin films (Ti-Sm:HAp_7 and Ti-Sm:HAp_10) deposited on a Ti substrate were obtained by spin coating technique. For this, a proper amount from the obtained sol (Sm:HAp) was dropwise added onto the substrate (commercially pure Ti), and the substrate was spun at 1000 rpm for 60 s. The obtained films were thermally treated in air at 70°C for 1 h. This procedure was repeated for 5 times. Finally, after the last layer deposition, the coatings were treated at 300°C for 2 h.

2.2. Scanning Electron Microscopy (SEM) The morphology of the Sm:HAp layers prepared by spin coating technique was

investigated by Scanning Electron Microscopy (SEM) with an Inspect F50 microscope.

A system for in vivo endothelial-specific and conditional expression of apolipoprotein E3


2.3. Confocal Laser Scanning Microscopy (CLSM) The adherence and development of C. albicans cells on the Ti-Sm:HAp_7 and

Ti-Sm:HAp_10 layers were investigated by Confocal laser scanning microscopy (CLSM) technique with a Leica TCS SP (Leica Microsystems, Germany) microscope. Prior to the CLSM examination the substrates were stained in the dark with ethidium bromide (5µg/mL) for approximately 5 minutes. For image acquisition a Leica HCX PL FLUORITE 40X/0.75 NA equipped with an Ar laser with an excitation of 488 nm and an emission between 580 and 660 nm, with a dry objective (Leica Microsystems, Germany) system was used.

2.4. Antimicrobial assay The antifungal activity of the Ti-Sm:HAp_7 and Ti-Sm:HAp_10 was investigated using

Candida albicans ATCC 10231 fungal strain as previously reported (CHIFIRIUC & al. [29]; MARUTESCU & al. [30]; PREDOI & al. [31]). 3. Results and discussion

The surface morphology of the Sm:HAp (Ti-Sm:HAp_7 and Ti-Sm:HAp_10) thin films deposited on a Ti substrate by spin coating technique investigated by scanning electron microscopy (SEM) are presented in Figure 1A-B. The SEM micrographs presented in Figure 1A-B depict that Sm:HAp layers deposited on a Ti substrate are homogenous without any cracks.

Figure 1. SEM micrographs of Ti-Sm:HAp_7 (A) and Ti-Sm:HAp_10 (B).

The SEM results presented in Figure 1 confirm that Sm:HAp thin films were deposited

successfully by sol–gel spin coating method. The SEM images show a homogeneous and uniform coating on the Ti substrates forming a homogenous and uniform composite layer. The antifungal activity of the Ti-Sm:HAp_7 and Ti-Sm:HAp_10 thin films at different time intervals (24 h, 48 h and 72 h) was investigated using the most common fungal strain which is responsible for severe post operatory infections that usually led to dangerous complications and the need for reopening the patients. The quantitative results of the antifungal activity of Ti-Sm:HAp_7 and Ti-Sm:HAp_10 layers are presented in Figure 2. The results have revealed that both Ti-Sm:HAp_7 and Ti-Sm:HAp_10 layers successfully inhibited the development of C. albicans ATCC 10231 fungal strain. The number of colony forming units (CFU) showed a substantial decrease for Ti-Sm:HAp_7 and Ti-Sm:HAp_10 samples comparative to Ti substrate, which was used as control. Moreover, the results of the colony forming unit count (CFUc) assay highlighted that the antifungal activity of Ti-Sm:HAp_10 layers was greater than that of Ti-Sm:HAp_7. Furthermore, confocal laser scanning microscopy (CLSM) was used for obtaining information regarding C. albicans biofilm morphology. CLSM images of



C. albicans biofilm growth on Ti-Sm:HAp_7, Ti-Sm:HAp_10 layers and Ti substrate investigated at different time intervals are presented in Figure 3A-I.The CLSM observations revealed that the cell growth and biofilm development of C. albicans fungal strain was greatly dependent on the layer type and somewhat dependent on the incubation time. More than that, the images pointed out that the morphology of fungal cells developed on the Ti-Sm:HAp_7, Ti-Sm:HAp_10 layers and Ti substrate is distinctive to C. albicans cells.

Figure 2. Graphical representation of C. albicans ATCC 10231 colony forming units on Ti,

Ti-Sm:HAp_7 and Ti-Sm:HAp_10 at different time intervals (24 h, 48 h and 72 h).

Figure 3. CLSM images of C. albicans biofilm development on different substrates

at various time intervals. (A-I).

A system for in vivo endothelial-specific and conditional expression of apolipoprotein E3


The results obtained from the CLSM investigations were in good accord with the quantitative antifungal assay. In the case of Ti substrate, no antifungal activity was observed for all the tested time intervals (Figure 3G-I). The results highlighted that the antifungal activity of the tested layers was dependent on the incubation time interval and Sm concentration (Figure 3). Therefore, the antifungal properties of the tested layers were attributed to the samarium antimicrobial properties. The use of propidium iodide staining has revealed an antifungal activity of the tested samples characterized by a decrease of fungal cells development and biofilm formation in the following order: Ti-Sm:HAp_10<Ti-Sm:HAp_7<Ti. 4. Conclusion

Homogenous Ti-Sm:HAp_7 and Ti-Sm:HAp_10 thin films were obtained by spin coating technique. The SEM micrographs showed that the Sm:HAp layers deposited on a Ti substrate were homogenous with no cracks or other impurities. The antifungal activity of the composite layers was investigated using a Candida albicans ATCC 10231 fungal strain. The results of the quantitative assay of the antifungal activity revealed that both Ti-Sm:HAp_7 and Ti-Sm:HAp_10 inhibited the growth of C. albicans fungal strain. The results obtained by CLSM visualization of the biofilm development on the composite layers confirmed the quantitative results and revealed that the C. albicans biofilm development on the composite layers was time dependent. The results obtained in this study highlighted the potential of these composite layers of being used in medical applications as efficient antifungal agents. Acknowledgements This research was financially supported by the Ministry of Education of Romania through Project PN II Contract Number 259/2014. References

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preliminary evaluation of the antifungal activity of samarium ...claudiu stefan turculet, alina...

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