Bioactivity of gentamicin contained in novel transdermal drug delivery systems (TDDS) formulated with biodegradable polyesters

1 Department of Pharmaceutics, University of Nigeria, Nsukka 410001, Enugu State, Nigeria. 2 Department of Microbiology, University of Nigeria, Nsukka 410001, Enugu State, Nigeria. 3 Probiotics Research Unit, Microbiology Laboratory, School of Biosciences and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy. 4 Division of Epidemiology and Prevention, Institute of Human Virology, School of Medicine, University of Maryland, 725 West Lombard Street, Baltimore, MD 21201, USA.


INTRODUCTION
Transdermal drug delivery system is being extensively investigated as a viable alternative to drug delivery with improved bioavailability. Transdermal drug administration generally refers to topical application of agents to healthy *Corresponding author. E-mail: chimafrankduff@yahoo.com; frankline.kenechukwu@unn.edu.ng. Tel: +234-8038362638. Fax: +234-42-771709. intact skin either for localized treatment of tissues underlying the skin or for systemic therapy (Valenta and Auner, 2004). It offers many advantages over conventional administration such as enhanced efficacy, increased safety, and greater convenience and improved patient compliance (Valenta and Auner, 2004;Dnyanesh and Vavia, 2003;Chandak and Verma, 2008). Transdermal route permits the use of a relatively potent drug with minimal risk of system toxicity and avoids gastrointestinal degradation and hepatic first-pass metabolism (Mundargi et al., 2007;Mutalik and Udupa, 2004). In case of toxicity, the transdermal patch can easily be removed by the patient (Chang et al., 2006).
Gentamicin sulphate is an aminoglycoside antibiotic commonly used topically in the control of severe Gram positive and Gram negative microbial infections especially in burns and wounds as well as for treating bone and soft tissue infections (Nishijima and Kurokawa, 2002). Despite its benefits, bacterial barriers and adverse effects such as nephrotoxicity, ototoxicity and neurotoxicity upon prolonged use, limit gentamicin daily dosage (Drusano et al., 2007). In fact, many clinicians are reluctant to use it, even for a short term (Singh et al., 2003). Efforts have been made to determine its optimal therapeutic regimens in order to increase its overall efficacy while minimizing drug toxicity. These include various formulation techniques such as formulations for topical administration.
Though topical administration of gentamicin is not painful, the patient may not apply the ointment/cream as often as required. Therefore, there is a need for the preparation of a new dosage form of gentamicin. Owing to the advantages offered by transdermal drug delivery systems (TDDS) over conventional routes of administration (Mundargi et al., 2007;Mutalik and Udupa, 2004), this study was designed to evaluate the transdermal delivery system of gentamicin so as to develop nonparenteral and needle-less (non-invasive) gentamicin preparation that will not only reduce to the barest minimum the systemic toxicity associated with parenteral administration of gentamicin but also ensure patient's compliance. A transdermal patch is not painful and is cost effective (Chang et al., 2006). Technically, the patch is placed in a part of the body which releases the drug into the body for a long period of time (Valenta and Auner, 2004;Dnyanesh and Vavia, 2003;Chandak and Verma, 2008). Polymeric matrices are usually employed as carriers for transdermal delivery of drugs/actives (Dnyanesh and Vavia, 2003;Chandak and Verma, 2008;Mundargi et al., 2007;Mutalik and Udupa, 2004;Verma and Iyker, 2002;Gupta and Mukherjee, 2003;Lyman, 2007). The novelty embodied in this study lies in the formulation of gentamicin transdermal patches using PURASORB ® polymers, a well-established, safe, biocompatible and resorbable excipients commonly employed in the formulation of controlled release drug delivery systems. These biodegradable polyesters have wide applications, including as orthopedic implant devices, surgical sutures, cardiovascular products, tissue regeneration scaffolds, among others. PURASORB ® materials allow for maximum flexibility in formulation technologies, ranging from extrusion and solvent processing to spray drying. Moreover, they are the material of choice for the production of implants, microspheres, and depot systems (Yasukawa et al., 2001;Avitable et al., 2001;Arora and Mukherjee, 2002).
The objective of this study, therefore, was to design and formulate transdermal patches incorporating gentamicin using biodegradable polyesters for the purpose of enhancing the delivery of the drug, by providing controlled delivery of the drug. The suitability of four different biodegradable polyesters (PURASORB ® polymers: PLGA, PDL 05, PL 32 and PDL 04) for this purpose was assessed by evaluating some of the physicochemical properties of the patches formed, the efficiency of incorporation of the drug in the patches as well as the bioactivity of the incorporated drug.

Preparation of transdermal patches
The patches were prepared by solvent evaporation technique (Amunuaikit et al., 2005), using gentamicin, plasticizers and other film forming polymers. Gentamicin (5 g) was dissolved with distilled water (5 ml) in a beaker followed by addition of 10 g of propylene glycol. The mixture was stirred continuously until a solution (drug reservior) was formed. The backing membrane was cast by weighing approximately 10 g of the film forming polymer (PLGA) into a separate beaker, adding about 70 ml of ethyl acetate and . This dispersion was properly stirred and poured into an aluminium foil-lined petri dish of defined area (10 cm 2 ). A funnel of suitable size was inverted over the petri dish to minimize solvent evaporation. Casting solvent was then allowed to evaporate 48 h to obtain dry films. The above procedure was repeated using PDL 05, PL 32 and PDL 04 as the film forming polymers as well as appropriate quantities of either or both ethylacetate, chloroform and sodium hydroxide (0.1N NaOH ), as depicted in Table 1. The patches were stored between sheets of wax paper in a desiccator until further analysis.

Differential scanning calorimetry (DSC)
The stability and compatibility of gentamicin and different polymers to be used for the development of gentamicin transdermal film formulations was studied using a differential scanning calorimeter (Netzsch DSC 204 F1, Germany). Sample (2.5 -5 mg), placed in an aluminum crucible cell was firmly crimped with the lid to provide an adequate seal. The thermal properties such as melting temperature, enthalpy and glass transition of the drug and transdermal patches were deteremined in the range of 35 -190°C under a 20 ml/min nitrogen flux at a heating rate of 10°C/min. The baselines were determined using an empty pan, and all the thermograms were baseline corrected.

Bioevaluation of transdemal patches
The antimicrobial activity of the transdermal patches was tested against each isolates using the agar diffusion technique (Umeyor et al., 2011). This method depends on the diffusion of antibiotics from holes on the surface of the microbial seeded agar. This test was carried out for the prepared gentamicin patches as well as the commercially available gentamicin sulphate cream. Molten nutrient agar (20 ml) was inoculated with 0.1 ml of S. aureus broth culture. It was mixed thoroughly, poured into sterile Petri dishes and rotated for even distribution of the organism. The agar plates were allowed to set and a sterile cork borer (8 mm diameter) was used to bore holes in the seeded agar medium. For the tested patch formulae as well as the commercially available cream, a definite quantity containing equivalent amount of gentamicin was accurately weighed and inserted in a corresponding hole. The plates were allowed to stand at room temperature for 15 min to enable prediffusion before incubating at 37 ± 0.5°C for 24 h. The experiment was repeated for E. coli, P. aeruginosa, S. typhi and K. pneumoniae. Three replicate tests were performed in each case. Growth was examined after incubation and the diameter of each inhibition zone was measured and the average determined.

Stability study of transdermal patches
Time resolved stability studies were carried out on the various batches of the formulations following the ICH guidelines (Verma and Chandak, 2009). The transdermal patches were stored at 40 ± 0.5°C in a humidity chamber having a RH of 75 ± 5%. After four weeks, six months and one year of storage, the patches were withdrawn and evaluated for the drug content following the method stated above.

Statistical analysis
All experiments were performed in replicates for validity of statistical analysis. Results were expressed as mean ± SD. ANOVA and Student's t-test were performed on the data sets generated using Statistical Package for the Social Sciences (SPSS). Differences were considered significant for p-values < 0.05.

Differential scanning calorimetry (DSC)
Table 2 presents the thermal properties of the transdermal patches. The physicochemical compatibility of the drug and the polymers studied by differential scanning calorimetry suggested absence of any incompatibility. The results revealed the compatibility of gentamicin and the polymers as well as the stability of the drug in the polymeric matrices (formulations). This is because the formulations gave lower endotherms than gentamicin,  implying that gentamicin exists in amorphous state in the formulations and also is properly solubilized in the matrix systems (Nnamani et al., 2010). Moreover, there was general disappearance of drug peak in all patches but PURASORB ® PL 32 patch had the least enthalpy (-1.981 mW/mg) reflecting the degree of disorder in the matrix.

Characterization of gentamicin transdermal films
The results of the physicochemical characterization of the patches are shown in Table 3. All the patches of the different polymers were transparent, colourless, smooth and uniform but the PDL 05 polymer had the greatest clarity. The results indicated that the formulated gentamicin-loaded patches exhibited good organoleptic and physicochemical properties (Valenta and Auner, 2004;Dnynesh and Vavia, 2003;Chandak and Verma, 2008;Mundargi et al., 2007;Mutalik and Udupa, 2004).

Bioevaluation of transdemal patches
The results of drug release studies using the agar plate diffusion method are presented in Figure 1. Compared with the commercially available gentamicin sulphate cream, all batches of the transdermal patches gave great zone of inhibition against S. aureus, E. coli, S. typhi and P. aeruginosa, while K. pneumoniae was mildly susceptible to the formulations. From the results presented in Figure 1, the patch formulations gave these zones of inhibition in decreasing order of magnitude: PL 32>PLGA>PDL 04>PDL 05 against E. coli, PL 32>PDL 05>PLGA>PDL 04 against S. typhi, PL 32>PDL 04>PLGA>PDL 05 against S. aureus, PL 32>PDL 05>PDL 04>PLGA against K. pneumonia, and PL 32> PDL 04 > PDL 05 > PLGA against P. aeruginosa. Compared with the rest of the batches of the formulations, PURASORB ® PL 32 gave the greatest zone of inhibition against all the transdermal patches employed in the study. While PURASORB ® PDL 04 gave the least zone of inhibition against S. typhi, PURASORB ® PDL 05 showed the least inhibition zone diameter against S. aureus and E. coli whereas PLGA exhibited the smallest zone of inhibition against K. pneumoniae and P. aeruginosa, respectively.
The microbiological test was performed to establish that gentamicin sulphate did not lose activity during formulation and after short-term storage, and was done one month after preparation. The determination of inhibition zone diameter (IZD) using agar plate method was based on the diffusion of an antibiotic agent or formulation thereof through a solidified nutrient agar (Umeyor et al., 2011). From the results presented in Figure 1, the transdermal patches released gentamicin which showed higher IZD compared with the commercially available gentamicin sulphate cream against E. coli, S. typhi, S. aureus and P. aeruginosa, while K. pneumoniae was mildly susceptible to the formulations. Patches of PURASORB ® PL 32 showed the best bioactivity. The results indicate that gentamicin loaded into transdermal patches produced very significant zones of inhibition against the Gram positive organism (S. aureus) and Gram negative organisms (S. typhi, E. coli, S. typhi, and P. aeruginosa) used in the study. The release of gentamicin from the patches depended on the type of polymer since the results showed that batches formulated with PURASORB ® PL 32 gave the greatest zones of inhibition against most of the isolates compared with the rest of the formulations. It is discernible from Figure 1 that the biodegradable polyesters, especially PURASORB ® PL 32, enhanced the release of gentamicin and therefore are good carrier for its transdermal delivery (Valenta and Auner, 2004). Figure 2 shows the drug content of the formulations after storage for one year. It is always very important to assess the stability of novel formulations. Stability could be viewed from the degradation of the active ingredients or physical property of the formulation (Panigrahi et al., 2005;Umeyor et al., 2012). In order to determine the change in drug content on storage, stability study was carried out. The results of the stability studies showed that the content of gentamicin in the formulations was not significantly changed on storage, as is evident from Figure 2. The result indicates that the formulations were stable on the required storage condition. In other words, there was a non significant change in the content of gentamicin in the formulations based on the required

Conclusion
Polymeric and/or lipid carriers could be exploited to improve the delivery of therapeutic molecules. In this study, gentamicin-loaded transdermal patches based on biodegradable polyesters were successfully prepared by the solvent evaporation technique, which is simple, cheap and reproducible. All the formulations showed good stability, tolerability on rat skin and physicochemical properties. The isolates of E. coli, S. aureus, P. aeruginosa, S. typhi and K. pneumonia were susceptible to gentamicin incorporated into the transdermal patches. In other words, the antibacterial properties of the drug was retained, with PURASORB ® PL 32 patches exhibiting the best bioactivity compared with the commercially available gentamicin sulphate cream that gave the least. It follows that this delivery system (PURASORB ® PL 32 patches) could offer a better and more promising approach for the treatment of topical infections caused by gentamicin-susceptible micro-organisms than the commercially available topical gentamicin sulphate cream.