METRONIDAZOLE- LOADED CHITOSAN/ POLY(MALEIC ANHYDRIDE-ALT-VINYL ACETATE) HYDROGELS FOR DENTAL TREATMENTS

Headnote

Abstract

A new approach to prepare polymeric hydrogels was developed to obtain an efficient controlled release system for dental treatments. Hydrogels were prepared by a method of condensation through amidation, using a natural (chitosan) and a synthetic polymer - poly(maleic anhydride-alt-vinyl acetate). The influence of different reaction parameters on hydrogels properties was investigated. Infrared spectroscopy studies confirmed the crosslinking by the presence of amide bonds, formed between the two polymers chains. In addition, these hydrogels presented a good release capacity, which was influenced by the reaction parameters used in the study (molar ratio between the two polymers). The cytotoxicity of these materials was investigated on human dermal fibroblasts.

Keywords: hydrogels, natural polymers, controlled release, metronidazole.

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1. INTRODUCTION

Carrier-based drug delivery, already with enormous impact on medical technology, represents an alternative approach to improve drug bioavailability[1]; the use of carriers, such as hydrogels, has been reported as a promising solution in this respect. Hydrogels have generated great interest as biomaterial vehicles for drug delivery, because many of them present excellent biocompatibility, with minimal inflammatory responses. [2,3]. Hydrogels are three-dimensional polymeric networks with a high capacity to absorb aqueous solutions or biological fluids, and a high degree of flexibility, similar to that of natural tissues [4]. The networks, composed of either homopolymers or copolymers, are insoluble, due to the presence of chemical (tie-points, junctions) or physical crosslinks [5,6]. These hydrogels also exhibit thermodynamic compatibility with water, which allows them to swell in aqueous media. Accordingly, they can be used for a wide range of applications, such as drug delivery devices, in making artificial muscles, immobilization of enzymes and cells, contact lenses, membranes for biosensors, materials for artificial skin, linings for artificial hearts [7,8]. Biocompatibility is the basic requirement which makes possible the application of a biomaterial for a therapeutic treatment. A large variety of synthetic [poly(hydroxyethyl methacrylate), poly(ethylene glycol), poly(vinyl alcohol)] and natural (proteins, polysaccharides, deoxyrhibonucleic acids) polymers can be used for the preparation of these release systems [7,9]. Natural polymers are widely used for the preparation of drug delivery systems, due to their good biocompatibility, biodegradability and nontoxicity [10-12]. On the other hand, the use of synthetic polymers has the advantage of high purity and good reproducibility over natural polymers. Also, they have an important advantage, that of permitting a sustained release of the therapeutic agent over a longer period of time, in comparison with the natural ones, with a relatively short drug release duration [13]. Considering this previous information, the main purpose of the present work was to explore the possibilities to obtain hydrogels based on a new concept, for improving the biological properties and the drug loading capacity and also for avoiding the direct contact of the drug with the tissues, which should reduce the side effects at the administration site. Tor this end, the preparation method preferred was that of condensation through amidation, as it offers the possibility of a rapid formation of the hydrogels, at ambient temperature and atmospheric pressure, without using additional cross-linking agents which are frequently toxic and difficult to remove from the synthesis products. For the preparation of these hydrogels, a combination between a natural (chitosan) and a synthetic {poly(maleic anhydride-alt-vinyl acetate) [poly(MAVA)]} polymer was realized. The reaction took place through opening of the anhydride cycles from the copolymer, under the action of the -NH2 groups from chitosan, a thin polymer membrane being finally formed. Chitosan was preferred as a natural polymer due to its good biocompatibility, biodegradability, and capacity to form membranes and to inhibit the growth of a wide variety of bacteria [14]. On the other hand, poly(MAVA) is an alternative copolymer with functional role in neoplastic processes in immunology and high resistance to viruses [15]. The prepared hydrogels were loaded with metronidazole, used as model drug. The objective of the investigation was to improve the bioavailability of metronidazole through the inclusion of the drug in the obtained hydrogels. This work also provides a very important study on the in vitro cytotoxicity of hydrogels on human dermal fibroblasts (HDF).

2. MATERIALS AND METHOD

2.1. Materials

Chitosan MMW (degree of deacetylation: 91%) and Metronidazole were purchased from Sigma Aldrich. Poly(maleic anhydride-alt-vinyl acetate)-poly(MAVA) was synthesized by radical copolymerization in our laboratory. Glycerol and lactic acid were purchased from Merck. Distilled water was used to prepare all solutions. Cryopreserved primary human dermal fibroblasts (HDF) cells (CO135C), Dulbecco's Modified Eagle's Medium, Fetal Bovine Serum (FBS) were purchased from Life Technologies. Penicillin, streptomycin, trypsin, non-essentials amino acids have been acquired from Antisel.

2.2. Hydrogels preparation

The hydrogels based on CS and poly(MAVA) were prepared by condensation through amidation. Opening of the poly(MAVA) anhydride cycles by the amino groups of CS led to the formation of new amide bonds. After this reaction, a polymer membrane with hydrogel character has been obtained. The experiment began with the preparation of the polymer solutions. A certain amount of CS was added in a lactic acid solution (1 wt%), under magnetic stirring. Copolymer solutions with specific concentrations were prepared by dissolving poly(MAVA) in DMSO. The copolymer solution has been added dropwise into the aqueous solution of CS, under continuous stirring. Finally, the hydrogels were poured into polycarbonate rectangular templates. The films were obtained after water evaporation in an oven at 37 ± 0.5°C for 24 h. In order to remove any possible unreacted products, the films were purified by repeated washings with distilled water and then dried at room temperature for 24 h.

2.3. Hydrogels characterization Structural characterization

The hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR) using a Digilab Scimitar FTS 2000 FTIR spectrometer (absorbance mode; 400 - 4000 cm-1).

Morphological characterization

The surface morphology of hydrogels was investigated on a Scanning Electron Microscopy (SEM) type TESCAN Vega. This analysis provided information regarding the pores size and distribution, and allowed evaluation of hydrogels thickness.

Metronidazole release from hydrogels

Metronidazole release experiments were performed in phosphate buffer solutions (pH=7.2). The amount of released drug has been determined by UV-VIS spectrophotometric measurements. The dried sample of drug loaded hydrogels was placed in 100 ml phosphate buffer solution and maintained at 37°C. At specific time intervals, a certain volume of PBS solution was taken over and the metronidazole concentration was measured spectrophotometrically. Also, the drug release efficiency, , was calculated as follows:

...

where: m = amount of released metronidazole (mg); ml = amount of metronidazole loaded into the hydrogels (mg).

In vitro cytotoxicity assays

Hydrogels cytotoxicity test was performed according to EN ISO 10993-5:2009 (Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:2009)). The tests were performed in the laboratory of cell cultures at "Apollonia" University, equipped at international standards. Cryopreserved primary HDF cells were cultured in DMEM supplemented with: 10% Fetal Bovine Serum (FBS), an antibiotic cocktail consisting of penicillin and streptomycin (1% v/v) and non-essential amino acids 1% (v/v). Incubation took place at 37°C in a humidified atmosphere of 5% CO2 in air (air/ CO2 incubator; MCO-5AC, Sanyo). When the cell monolayer reached a 80% confluence, the cells were trypsinized with a 0.25% trypsin solution at 37°C for 3 min, followed by addition of fresh medium, at room temperature, to neutralize trypsin.

After centrifugation (Hettich ROTOFIX-32A centrifuge) and re-suspension in fresh medium (in flasks - 25 cm2), the cells were exposed to hydrogels (50µg hydrogel/1ml fresh medium) or unexposed - the control sample. The cells were allowed to attach to the substratum for 24 h prior to the treatment. Cell viability was determined using an inverted optical microscope (CKX41, Olympus) and the number of cells was counted with a Neubauer hemocytometer. All procedures were performed under laboratory hood with laminar flow (LAMIL PLUS 13, Kartusalan Metally Oy) using sterile instruments.

3. RESULTS AND DISCUSSION

3.1. FTIR Spectroscopy

The structure of the starting polymers and of CS-A-1 hydrogels was characterized by FTIR spectroscopy. The FTIR spectrum obtained is presented in Figure 1. The FTIR spectra of poly(MAVA) had an absorption band at 1782.42 cm-1, corresponding to the C=O stretching vibration of the anhydride groups [16]. CS exhibited characteristic bands of carbonyl (C=O - from amidic groups) at 1651 cm-1 and of free amine group (-NH2) at 1588 cm-1 [17,18]. The hydrogels presented an absorption band at 1655 cm-1, which corresponded to the carbonyl bond from the newly formed amide groups. In addition, the peak at 1726 cm-1 corresponded to the absorption band of specific -C=O bonds from the carboxylic group, suggesting that most of the anhydride groups participated either in the condensation reaction with amino groups or were hydrolyzed.

3.2. Hydrogels morphology

Hydrogels morphology was evidenced by scanning electron microscopy (Fig. 2). Microscopy images (in cross-section) showed that the hydrogels present a porous structure, pores' dimension being non-uniform, with a diameter between 200-400 µm. Hydrogels thickness was around 500 µm.

3.3 Metronidazole release profile

The release profiles of metronidazole in phosphate buffer solution (pH = 7.2) are represented in Figure 3. Analysis of results showed that the drug release efficiency varyed between 80% and 92%. Metronidazole release from hydrogels took place by a diffusional mechanism of the drug through the polymeric membranes. Another interesting observation was that, within the first two hours, a rapid release of metronidazole from hydrogels took place (the so-called "burst" effect). After this time interval, a slow drug release was observed and, after approximately 4 hours, the system reached equilibrium. It is obvious that the molar ratio between the two polymers (which determines membrane thickness and density of the polymer network) has an important role in the release of metronidazole from hydrogels.

Cell viability assays

Cell viability in contact with CS-A-1 hydrogels was approximately 87%, which means that the material has a non-cytotoxic behaviour. A slight decrease in the number of cells from the experimental cell cultures may be caused by the local mechanical damage, as a result of the considerable consistency of the material (Fig. 4).

No significant differences were recorded in cell morphology between the control cultures and the cells incubated with the CS-A-1 sample, as evidenced by the microscopic images presented in Figure 5.

4. CONCLUSIONS

A new type of hydrogels based on both natural and synthetic polymers was prepared by a condensation method through amidation. These hydrogels offer important advantages, such as simplicity of preparation and the possibility of adjusting their properties as a function of the desired application. Microscopic images showed that the hydrogels present a porous structure, pores' dimension being non-uniform, with a diameter between 200-400 µm. Release of the model drug is controlled by the diffusion rate through the polymeric membrane and the release capacity is high in alkaline medium. The obtained results showed that cell viability of the CS-A-1 hydrogels was approximately 87%, which means that the material was non-cytotoxic.

Acknowledgements: This paper was supported by the Sectoral Operational Programme Human Resources Development (SOP HRD), financed by the European Social Fund and the Romanian Government under the contract number POSDRU/144/6.3/S/127928.

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