Introduction

Liposomes are spherical, self-enclosed vesicles in which aqueous part is entirely enclosed by phospholipid bilayer and liposomes is amphiphilic in nature, means it possess both lipophilic and hydrophilic character¹. Liposomes are prepared from a variety of natural and synthetic phospholipids, and considered as drug-carrying structures or vesicles².

Liposomes, which are versatile, biodegradable, nontoxic phospholipid vesicles, are known to reduce toxicity associated with many compounds including antitumor³, antibiotics⁴, and immunosuppressive agents⁵.The reasons for using liposomes can be summarized as follows: liposomes can enhance cellular internalization of the drug; they can generally decrease unwanted systemic toxic effects; and they may increase drug solubility in biological fluids, modulating at the same time the drug release profile. Furthermore, in some case, the use of phospholipid vesicles to carry the drug to target cells could possess specific release by passive or active targeting strategies⁶.

Controlled drug release can be achieved by transdermal drug delivery systems (TDDS) which can deliver medicines via the skin portal to systemic circulation at a predetermined rate over a prolonged period of time. TDDS has gained a lot of interest during the last decade as it offers many advantages over the conventional dosage forms and oral controlled release delivery systems notably avoidance of hepatic first pass metabolism, less frequency of administration, reduction in gastrointestinal side effects and improves patient compliance^7,8.

Drug was chosen because of its low meting point, low partition coefficients and optimum range of molecular weight which is prerequisite for development of transdermal drug delivery system⁸.

Transdermal therapeutic systems are defined as a self-contained, discrete dosage forms which, when applied to the intact skin, deliver the drug, through the skin at control rate to the systemic circulation. Transdermal formulation maintain drug concentration within the therapeutic window for prolong period of time ensuring that drug levels neither fall below the minimum effective concentration nor exceed the maximum effective concentration⁹.

Baclofen (BCF) is a direct agonist at GABA_B receptors. Baclofen used to relax the muscle during the condition of muscle contraction¹⁰. The purpose of the current study was to investigate the feasibility of liposomes to formulate the transdermal administration of baclofen.

Materials and Methods

Baclofen was a provided as gift sample from Sun Pharmaceutical Industries Limited, India. Soya lecithin was purchased from Acros Organics, New Delhi, India. Cholesterol was purchased from Sigma, USA. Stearic acid purchased from Research Lab, Mumbai India. Ethanol (AR), Methanol and Chloroform purchased from Merck, India. The all other materials used in this study were of analytical grade.

The baclofen liposomes prepared as per method reported A. Kumar, et al., (2010) with slight modifications. For the preparation of liposome various formulations were prepared using different ratio of lecithin, cholesterol and ethanol but drug and stearic acid ratio kept constant (Table 1). The lecithin, cholesterol, stearic acid and drug were dissolving in organic solvents (ethanol) and injected in to 10 ml preheated PBS water at 55-65^oC with continuous stirring at 500 rpm using magnetic stirrer. The solvent was evaporated by heating so as to obtain drug loaded liposomes¹¹.

Characterization

Vesicle size:

The size of liposomes was determined by using a film of vesicle dispersion, fixed with 10% w/v gelatin solution, was observed under a light microscope (BEM-21, Besto Microscope, India) fitted with an ocular micrometer and stage micrometer at a magnificationof 100X¹².

Vesicle shape:

Vesicles morphology of baclofen liposomes obtained from ethanol evaporation was determined using a photomicroscope (Nikon Model UFX-II, Japan) at 1000 x magnification¹².

Entrapment efficiency:

The lipoosomal dispersions were centrifuged (90 XL Ultracentrifuge, Beckman, USA) at 10,000 × g for 20 min to separate unentrapped drug and washed with phosphate buffered saline (pH 7.4). The clear supernatant was analyzed for baclofen by UV-spectrophotometer (Shimadzu UV-1700, Japan) at 226 nm. The amount of entrapped drug was calculated using the following equation¹³.

Results and Discussion

Vesicle size:

Particle size determination of liposomes was performed by light microscope after storage. The size of different formulations of baclofen liposome was found to be in the range of 3.98±0.45-4.24±0.65 µm.

Vesicle shape:

The microscopic study of drug loaded liposomes shows that small unilamellar vesicles were formed by ethanol injection method. Microscopic observations of the prepared liposome formulation confirmed the formation of spherical particles as shown in Figure 1.

Figure 1:  Photomicrograph of baclofen liposome formulation (RLP5) 

Stability studies:

For stability studies liposome formulations were stored at different temperature such as 4±2 °C, 25±2°C and 37±2°C. The maximum % entrapment was observed in 4±2 °C (refrigerated) temperature compared to 25±2°C and 37±2°C temperature (Table 4). The magnitude of drug retention within the vesicles, on storage under defined conditions, ultimately governs the shelf life of the developed formulation. Acceleration in drug leakage at higher temperatures, as observed in storage-stability studies, suggested keeping the liposomal product in the refrigeration conditions, to minimize the drug leakage from liposomal-systems. The optimized formulation RLP5 shows better storage stability at refrigerated condition than other formulations.

Table 4:  Stability study of different formulations of baclofen liposomes 

Formulation Code	Time (month)
	1	3	6
	Temperature (°C)
	4±2
	% Entrapment
RLP1	57.61±0.91	56.82±0.34	56.72±0.43
RLP2	57.65±0.82	58.68±0.56	58.62±0.51
RLP3	58.31±0.45	59.98±0.42	60.83±0.67
RLP4	62.22±0.54	61.65±0.31	61.62±0.74
RLP5	64.19±0.26	63.54±0.68	63.45±0.23
25±2
RLP1	46.48±1.36	45.78±0.71	46.56±0.28
RLP2	46.78±0.86	47.67±0.15	48.59±0.61
RLP3	49.67±0.81	50.62±0.43	49.23±0.13
RLP4	55.38±0.46	52.56±0.71	51.34±0.35
RLP5	58.84±0.91	55.52±0.87	53.69±0.41
37±2
RLP1	38.77±0.18	37.89±0.12	37.89±0.71
RLP2	40.88±0.35	39.67±0.24	38.26±0.18
RLP3	41.36±0.78	40.57±0.45	41.91±0.40
RLP4	44.37±0.71	42.51±0.69	43.59±0.51
RLP5	47.74±0.21	46.48±0.58	46.73±0.30

In vivo study:

In vivo study was carried out to check the potential activity of formulation, and it was determined by rota-rod apparatus at CT Institute of Pharmaceutical Sciences, Jalandhar (Punjab), India. For the in vivo study 45 mice (Mus musculus) were selected. The dosage regimen was 1 to 4 mg/kg for optimized formulation RLP5 and plane drug as per literature. The plane drug treated (1, 2, 3 and 4 mg/kg) mice showed lesser number of falls from rota-rod apparatus in comparison to optimized formulation RLP5 (1, 2, 3 and 4 mg/kg) and diazepam (2 mg/kg), results are given in Table 3.1. Similarly optimized formulation RLP5 treated mice showed increased number of falls than plane drug treated mice but lesser number of falls was observed in case of diazepam treated mice. When the dose of optimized formulation was increased (1, 2, 3 and 4 mg/kg) there was increase in number of falls from rota-rod apparatus, because liposomal formulation permeated well through the skin and absorbed in systemic circulation, and may completely relax the muscle. The mice treated with formulations have shown improved muscle relaxant activity which was evident by increased number of falls in rota-rod test as compared to plane drug treated mice. The data pertaining to number of falls were analyzed by one way ANOVA followed by Student-Newman-Keuls test. The effect of formulation was dose dependent (p values are 0.001 for 1, 2, 3 and 4 mg/kg, respectively). However, diazepam treated mice may resulted higher muscle relaxation than any dose of formulation tested (Figure 2).

Figure 2:  Comparison of plane drug, optimized formulation RLP5 and diazepam on albino mice for skeletal muscle relaxant activity (n=6). NOTE: M.D.= Mean deviation. S.D.= Standard deviation. ***=P<0.001. 

Conclusions

Liposomal formulation containing baclofen possessed particle size, particle shape, entrapment efficiency, in vitro drug release, stability and in vivo studies and on the basis of above these studies revealed that due to small size and spherical shape of liposomes gaining access from the epidermal layer of the skin to effectively deliver the baclofen. Liposomal formulation releases the baclofen for longer period of time in a sustained release manner. Liposomal formulations stable at 4±2°C temperature because in accelerated temperature more drug leakage occurs. Baclofen loaded liposomal formulation shows skeletal muscle relaxant activity performed by rota rod method in mice which could deliver the baclofen from liposomes in the therapeutic range. For transdermal drug delivery of baclofen via liposomes could deliver and entrap lesser quantity of drug for the treatment of spasticity. Finally concluded, the formulation RLP5 of baclofen liposome shows better performance than those of respective formulations.