INTRODUCTION:

As polymer technology has advanced over the past two centuries, the peculiar characteristics of macromolecules synthetically available have also developed^[1]. The highly branched three dimensional man made polymeric macromolecules are known as dendrimers are the mainly known element of the polymer science. Dendrimers have often been referred to as the “Polymers of the 21^st century”. Dendrimers have developed a young set of polymers with a versatile architecture and unique chemical structures^[2]. The better encapsulating ability of dendrimers makes them excellent carriers for delivery of drugs. It has been proved that dendrimers may have potential in improving the solubility of low aqueous soluble drugs and as a carrier for bioactive materials^[3]. Among them, the polyamidoamine (PAMAM) dendrimer is one of the well known and is proven as a carrier to improve water solubility and rate of dissolution of drugs, such as ketoprofen^[4], ibuprofen^[5], aceclofenac^[6] and riboflavin ^[7]_.

Ketoconazole, an imidazole derivative with potent antileishmanial activity. Its clinical use is limited by the lipophilicity and poor aqueous solubility. Over the past several years, various efforts have been made to improve the aqueous solubility and in vitro dissolution profile of ketoconazole. The versatile architecture of dendrites, such as their size, branching length, shape and their surface functional groups permit to transform the dendrimers as per the requisites, constructs these macromolecules perfect carriers in drug delivery applications and enhancing the solubility of low aqueous soluble drugs^[8]. Here we made an attempt to incorporate ketoconazole in PAMAM dendrimers to explore the potential of PAMAM dendrimers to act as a carrier for ketoconazole and also to evaluate its cytotoxicity on mouse macrophage cell lines.

MATERIALS AND METHODS:

MATERIALS:

Ketoconazole was purchased from Micro Labs. (Bangalore, India). PAMAM dendrimer of different generations were received from Sigma Aldrich (USA). All other chemicals used were procured from S.D. Fine Chemicals (Mumbai, India)

Synthesis of PAMAM-KET Complexes:

Results of dendrimer – mediated solubility studies suggested that the solubility of ketoconazole was enhanced to increase in generation as well as dendrimer concentration^[9]. The optimized G3 dendrimer- based formulations were selected for advanced studies. The ketoconazole was dissolved in dendrimer solutions to a concentration of 2 mg/ml after dilution with distilled water. These were vortexed for 24 h at room temperature. The vortexed samples were spun at 14000 rpm for 10 min. These were then filtered through 0.22 µm membrane filter. These conjugates were used for in vitro release studies^[10].

In vitro release studies:

In vitro release studies of ketoconazole -PAMAM G3 dendrimer complex was performed by the dialysis method ^[11]. The ketoconazole was dissolved in dendrimer solutions to a concentration of 2 mg/ml after diluted with distilled water. Pure ketoconazole was dissolved in a small quantity of methanol, then diluted with distilled water and used as a control. Drug dendrimer solution of about 5 ml was filled in dialysis bags (M.W. cut off =1000 Daltons, Himedia, India), which was pretreated with 0.1 N HCl and the dialysis bags were suspended in 100 ml of 0.1 N HCl maintained at 37+ 0.5ᵒC under constant stirring^[12]. A perfect sink condition was achieved by withdrawing 1 ml of the sample from the outer phase, and it was again refilled with same volume of dissolution medium. The quantity of drug released at each time interval for 12h was estimated at 270 nm using UV spectrophotometer.

Cytotoxicity Studies:

The capability of the cells to stay alive a toxic response has been the basis of most of the cytotoxicity assays. MTT [(3-(4, 5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide] assay was performed to assess viability or cell proliferation ^[13]. This study is based on the principle that the tetrazolium compound is bioreduced by cells into a colored formazan product by mitochondrial enzyme succinate dehydrogenase. Dead cells or their products do not reduce tetrazolium compound. Cytotoxicity studies were carried in J774A, mouse macrophage cell lines. The cells were cultured in Dublecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2mM L- glutamine. Cells were placed in a 96-well sterile Nunc Cell –culture plates and each well containing 1× 106cells. The culture plates were incubated overnight in a CO₂ incubator at 37°C. 100 µl of different concentrations of drug, dendrimers and drug dendrimer conjugates were added to the wells. For each control, drug, dendrimers and drug- dendrimer conjugate triplicate wells were made. The plates were incubated for 72h. MTT reagent of 50 µl was added to each of these wells. After addition of MTT reagent, the plate was incubated for 4h. After 4h incubation, the plates were gently shaken and were read at 540 nm in a microplate reader to determine the formation of soluble formazan by live cells^[14].

RESULTS AND DISCUSSIONS:

Synthesis of PAMAM-KET Complexes:

To examine the influence of PAMAM dendrimer generation with EDA as core (G1, G2, and G3) and concentration on the solubility of KET a sequence of solubility studies were conducted. In association with PAMAM dendrimers aqueous solubility of KET has been significantly improved than in distilled water and drug loading was found maximum in the case of third generation of PAMAM dendrimers ^[15,16]. The enhancement of solubility of KET was due to the internal architecture that are offered to incorporate KET molecules (host- guest interaction) and theses versatile features make them excellent carrier for drug delivery applications^[17].The optimized G3 dendrimer- based formulation was selected for further studies.

In vitro release studies:

The In vitro release studies of ketoconazole -PAMAM G3 complex carried out in 0.1 N HCl of pH 1.2 shows the potency of the drug – dendrimer complex. Pure ketoconazole was released (59.11%) in 6 h, whereas KET- dendrimer complex exhibited the delayed release of the drug (Fig. 1). After 10h, 71.63 % release was accomplished for the pure KET while 40.07 % release was shown by KET-G3 PAMAM complex. In comparison with the pure ketoconazole the release profile of KET from the drug dendrimer complexes was in a sustained manner. This is probably due to hydrophobicity of KET which permit them to reside little longer in the hydrophobic pockets of the dendrimers. It is clear from these results that electrostatic interaction plays an inevitable role in release of drugs from dendrimer complexes^[18].

Figure 1: Comparative In vitro release of KET in G3 PAMAM dendrimer solution compared with the pure KET(ketoconazole) release behavior

Cytotoxicity Studies:

Cytotoxicity studies of, drug solution and drug dendrimer solution of different concentration were conducted using MTT assay and the results are shown in the Fig.2. Based on the results, it is concluded that drug dendrimer complexes exhibited higher percentage cell viability than the free drug at same concentration. Results of the cytotoxicity studies also showed that the complexation of drugs with PAMAM dendrimer leads to the reduction in cytotoxicity of drugs. High entrapment efficiency and sustained release profile of the drug dendrimer complex are the probable reasons for the reduced cytotoxicity of drug dendrimer complex.

Figure 2: Comparative cytotoxicity of the free drug and drug dendrimer complex (P-KET- PAMAM G3-Ketoconazole, KET- Ketoconazole)

CONCLUSION:

The PAMAM dendrimers with terminal amine functional groups have the ability to enhance the solubility of low aqueous soluble drugs, for instance ketoconazole. Our study presented that the conjugation of ketoconazole with PAMAM dendrimers led to sustained release of drug in vitro. Results of the cytotoxicity studies also showed that the complexation of ketoconazole with PAMAM dendrimer leads to the reduction in cytotoxicity of drugs. In vivo studies toxicity studies should be conducted for assessing the safety and efficacy of such formulations in prior to the clinical studies. Even if the drug delivery based on dendrimer is in its initial phase, it tenders numerous features in the delivery of drugs.

ACKNOWLEDGEMENTS:

Supports from Nitte University, Mangalore and National Institute of Immunology, Delhi were highly valued.

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Received on 06.11.2015 Modified on 21.11.2015

Research J. Pharm. and Tech. 9(1): Jan., 2016; Page 17-19

DOI: 10.5958/0974-360X.2016.00004.4