CHITOSAN-BASED BIONANOCOMPOSITE FOR TRANSDERMAL THERAPY

Abstract

According to a general and official definition approved by the European Society for Biomaterials (Williams, 1987), "biocompatibility is the ability of a material to function within a particular medical application, producing an appropriate response in the host organism" [2]. According to the first attempts, biomedical researchers have suggested that hydrogels used as membranes meet all requirements for wound healing, appearing as an appropriate candidate for patients with burns, since the wounds are cured as soon as possible. [...]80 mg of CS (Sigma Aldrich) were added to 10 mL of 1% acetic acid (Sigma Aldrich) and kept under constant stirring till a clear viscous solution was obtained. [...]5 mL of AgNO3 were added to 10 mL chitosan solution under stirring for 1h at room temperature.

Headnote

Abstract

Chitosan has been widely used for tissue engineering of both epithelial and mesenchymal tissues. As major components in various organs, epithelial cells are used in the protection, secretion and maintenance of tissue homeostasis due to their sheet-like tight cellular arrangement and polarised nature. In the regeneration of damaged epithelial tissues, chitosan promotes proliferation of various embryonic and adult epithelial cells. In this study, chitosan-silver- hydroxyapatite (CS/nAg/HAp) hydrogels were prepared by a simple eco-friendly method.

Keywords: silver nanoparticles, biomaterials, chitosan, hydroxyapatite.

1. INTRODUCTION

The most important factor by which a biomaterial can be distinguished is its ability to exist in contact with tissues of the human body without causing unacceptable degrees of injury [1]. These issues are dealt with in the broad context of the biocompatibility category. According to a general and official definition approved by the European Society for Biomaterials (Williams, 1987), "biocompatibility is the ability of a material to function within a particular medical application, producing an appropriate response in the host organism" [2].

Hydrogels are highly hydrophilic macromolecular networks produced by chemical or physical crosslinking of soluble polymers. Due to their particular properties, such as very high physiological media, hydrophilic nature, soft tissue, as well as to their water content and adequate flexibility, they appear as excellent candidates for biomedical applications.

Winter [3] introduced the first generation of polymeric bandages and provided the optimal media for wound repair. In 1978, several chitinbased polymeric materials were used to cover wounds due to their biological activity, which recommended polymers as excellent candidates for biomaterial development [4]. The trend of research and development of hydrogels as polymeric transient bandaging membranes becomes the main commercial target. These hydrogels were synthesized by chemical crosslinking of acrylamide and methylene bisacrylamide, including polysaccharides [5,6]. According to the first attempts, biomedical researchers have suggested that hydrogels used as membranes meet all requirements for wound healing, appearing as an appropriate candidate for patients with burns, since the wounds are cured as soon as possible. In order to obtain better mechanical proprieties after the absorption of wound exudates, methods of incorporation of a biocompatible filler into the wound dressing material were developed. Particular focus was laid on hydroxyapatite as a filler material, which is a suitable biomineral for addition to wound dressing materials due to its biocompatibility, non-toxicity, and nonimmunogenicity [7,8]. It has been reported that addition of hydroxyapatite into biomaterials results in improved mechanical, barrier and thermal properties [9]. There are many methods that can be used to produce hydroxyapatite-containing composite materials, such as thermo-mechanical mixing, in situ precipitation, in situ polymerization and the sol-gel method [10].

Among metal ions, such as Ag+, Cu2+ and Zn2+, widely utilized in medicine as antimicrobial agents, Ag+ has effect with a broad spectrum of antimicrobial activities against bacteria, the reactivity of silver being even more efficient when used in nanometer-sized particles, due to their high surface-to-volume ratio that allows better contact with microorganisms [11]. Besides, silver nanoparticles (nAg) are less toxic at low concentrations [12]. An eco-friendly synthesis route for obtaining nAg uses the natural biopolymer chitosan [13]. Various forms of silver phosphates have already been used, from precipitation and coprecipitation in aqueous solution to ionic exchange [14-15], by co-sputtering [16], microwave-assisted processes [17], sol-gel [18] and thermal or cold spraying [19,20].

Chitin and chitosan are used to stimulate endogenous collagen production needed for skin regeneration [20]. Chitosan promotes fibroblast proliferation [21], enhances the migratory activity of human umbilical cord vein-derived endothelial cells [22] or interacts with the fibroblast growth factor, enhancing its bioavailability [23]. Chitosan conjugated with laminin is used to deliver keratinocytes in wounded skin [24], which could accelerate healing via reepithelialization and regeneration of nerves in the dermis. Due to such properties, chitosans have been used as scaffolds, hydrogels or tissue adhesives for skin repair [25,26].

As to the importance of wound dressing biomaterials for biomedical applications, the main goal in this work was to develop a simple method for the synthesis of chitosan-silver nanoparticles containing hydroxyapatite composites [27].

2. METHODS AND CHARACTERIZATION

2.1. CS/ nAg/HAp composite fabrication

The CS/nAg /HAp (chitosan/nano-silver/ hydroxyapatite) bio-composite scaffold was obtained by the following procedure. Firstly, 80 mg of CS (Sigma Aldrich) were added to 10 mL of 1% acetic acid (Sigma Aldrich) and kept under constant stirring till a clear viscous solution was obtained. Secondly, 5 mL of AgNO3 were added to 10 mL chitosan solution under stirring for 1h at room temperature. The mixture was transferred to glass tubes and kept at 90 °C for 12 h in a temperature-controlled bath. After cooling at room temperature, 20 mg of HAp (Sigma Aldrich) were added, while stirring contined. Thereafter, the film-forming solution was poured into plastic Petri dishes and dried at room temperature for 3 days, at dark. Films' thickness was measured using a Tencor Alpha-Step D 500 stylus profiler and a value of 400 pm was obtained.

2.2. Characterization of CS/nAg /HAp scaffold

Silver ion reduction to metal silver nanoparticles is due to the fact that silver ions are easily reduced by the lone pair electrons of the nitrogen and oxygen atoms. Each of the nitrogen and oxygen atoms of the functional groups in chitosan has lone pair electrons for producing complexation, as well as reduction of silver ions. Thus reduction was achieved because of the intrinsic property of chitosan, without the usage of any external chemical reducing agent.

Scanning electron microscopy (SEM) was used to analyze surface morphology and topography. The electron micrographs samples were registered with a Quanta 200 microscope at an accelerating voltage of 15 kV and with an EDAX system of elemental analysis.

Raman spectra and images were recorded on a Renishaw inVia Qontor confocal Raman microscope, using a laser beam with an excitation wavelength of 633 nm, 1,000 spectra/ sec.

Fig. 1 plots the Raman (a) and SEM (b) images of the CS/ nAg/ HAp sample. The surface of the composite scaffold has patterns of different size and shape, due to the presence of nAg and HAp (Fig 1a). SEM images at 5 pm scale revealed the presence of microspheres, with round-like welldispersed morphology. EDAX measurements (Table 1) show the presence of AgNp on the film in low quantities, the nitrogen content being due to chitosan, while the P and Ca contents are due to hydroxyapatite's presence on the studied sample.

In Fig. 3 is shown the Raman spectra of CS/ nAg/Hap sample while in Table 2 and Table 3 are presented the characteristic vibration bands of chitosan and HAp and from this spectra.

The characteristic vibration bands of the PO4 groups in HAP crystals are: v2 bending of POP at 444 cm-1, v4 bending of POP at 619 cm-1, v1 stretching of PO at 960-962 cm-1, and v3 stretching of PO stretching at 1030 cm-1.

Fig. 4 presents the measurements at the surface of the sample. The roughness of the sample was calculated as Ra=12.3 nm.

The cytotoxicity of samples was evaluated on the primary fibroblast cell line at passage 5, obtained from Albino Rabbit dermis. The cells were plated 24 h prior the test, on 48 well culture plates, using 1 x 104 cells/well cell density and DMEM culture media supplemented with 10% BFS and 1% P/S/N.

In our study, in vitro cytotoxicity tests were performed according to the ISO 10993-5:2009 standard recommendations. The investigated sample proved to be non-cytotoxic (Fig. 5).

3. CONCLUSIONS

Concerning the importance of wound dressing biomaterials for transdermal therapy, the main goal in this work was to develop a simple method for the synthesis of chitosan-silver nanoparticlescontaining hydroxyapatite composites, to replace the existing wound dressing materials. The casting solution used in this study is simple, easy and inexpensive, requiring no specialized equipment and toxic solvents.

Acnowledgement. The authors acknowledge the financial support of this research through the Project "Partnerships for knowledge transfer in the field of polymer materials used in biomedical engineering" ID P_40_443, Contract no. 86/8.09.2016, SMIS 105689, cofinanced by the European Regional Development Fund by the Competitiveness Operational Programme 2014-2020, Axis 1 Research, Technological Development and Innovation in support of economic competitiveness and business development, Action 1.2.3 Knowledge Transfer Partnerships.

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