Formulation and Invitro Evaluation Azithromycin Liposomal Gel by Thin Film Hydration Technique

The aim of present research work is to formulate and evaluate azithromycin liposomal gel for topical application. In the present research, azithromycin liposomal suspension was formulated with different concentrations of phosphatidylcholine of which formulation F was optimized in terms of particle size, entrapment efficiency and invitro drug release study.   The Optimized formulation was incorporated into carbapol gel to form liposomal gel for topical application.  Physical drug and excipient interactions were evaluated by FT-IR and DSC studies. Azithromycin liposomal suspension was evaluated for particle size, entrapment efficiency and invitro drug release. Optimized formulation having a particle size 102.6nm, entrapment efficiency 99.44% and invitro drug release 99.89% for 14 hours.  The gel (F1) was evaluated for organoleptic properties, pH, viscosity, spreadability, extrudability, drug content, content uniformity and invitro diffusion studies. Azithromycin liposomal gel was found to be a white in colour with smooth texture, pH of 5.7, with excellent spreadability and extrudability, drug content was found to be 96.08% and invitro diffusion studies was exhibited with 98.45% for 16 hours. The present research study concluded that both liposomal suspension and gel formulations showed better invitro drug release for 14 and 16 hours respectively by following non-fickian diffusion (suspension) and fickian diffusion (gel).

Azithromycin is a broad-spectrum antibiotic belongs to macrolide class, act by blocking the protein synthesis by reversibly attaching to the P- site of the bacterial 50S ribosomal subunit [1]. It is used to treat skin and soft tissue infections, otitis media, sinusitis, respiratory tract infection in both children and adults. In the recent past, during pandemic azithromycin was extensively used [2].  Liposomes are the targeted drug delivery systems, which are popularly known for its localised action. Liposomes are lipid bilayer vesicles which are made up of lipids [3].  They contain natural non-toxic bio-lipids which can capable of encapsulation of both hydrophilic and lipophilic drugs which can be administered through oral, parenteral and topical routes. Liposomal gel was used for topical application. Liposomes can administer through topical route, but practically direct liposomal application on skin is less. So, these are generally formulated into semi-solid dosage forms for topical use [4]. In the present research, azithromycin a highly lipophilic drug was used for topical use to treat skin infection; therefore, azithromycin was loaded to in liposomes to enhance its effect. These liposomes were incorporated into carbopol for improved patient compliance. Azithromycin was purchased from Merck, L- a- phosphatidylcholine was purchased from Sigma Aldrich Chemicals, Cholesterol, Carbopol were purchased from Loba Chemie Pvt. Ltd. Mumbai.  Chloroform, Methyl paraben, Propyl paraben, Glycerine was purchased from Thermo Fisher Scientific Pvt. Ltd. Mumbai. Triethanolmine was purchased from Qualigens Fine chemicals Mumbai.

Preformulation Studies

DSC thermograms were obtained by using DSC, Q20 model of TA instruments with TA Q20 software. FTIR spectra were recorded on a BRUKER alpha model infrared spectrophotometer with OPUS software. Thermal analysis and spectral analysis were carried out for azithromycin, azithromycin and physical mixture of azithromycin, selected pharmaceutical excipients (phosphatidylcholine, cholesterol) in a 1:1 weight/weight ratio).

Preparation of azithromycin liposomal suspension

Liposomes were prepared by the thin film hydration technique. Accurately weighed phosphatidylcholine and azithromycin in specific ratios (5:1, 4:1, 3:1. 2:1) were dissolved in chloroform: methanol. The mixture was rotated until the solvent system was completely evaporated and a thin film was formed in the round bottom flask and was hydrated with distilled water. The suspension was sonicated for 15 minutes by Ultra sonicator.

Preparation of liposomal gel

In the present research liposomal gel was prepared with 1gm of carbapol-940 grade mixed with 88gms of distilled water which contained 10gm of glycerine previously added. The resultant mixture was stirred on a magnetic stirrer until it thickens and then by using triethanolmine drop wise addition and is neutralized. Optimised liposomal suspension (5:1) was incorporated into prepared carbapol gel.

Evaluation of Liposomal Suspension

Particle size analysis

The particle size of liposomes was determined by using a particle size analyzer HORIBA SZ-100 at 25⁰c.

Entrapment efficiency

Entrapment efficiency of drug was calculated by using centrifugation method. Liposomal suspension was centrifuged at 3500 rpm for 45 minutes. The clear supernatant obtained after centrifugation was used to determine amount of drug present by UV-Visible spectrophotometer at 208nm. The amount of azithromycin entrapped in liposomes was determined as follows: % Entrapment efficiency= (Wt/Wi) ×100 % where wt is the total amount of drug in the in liposomes and Wi is the total quantity of drug added initially during preparation.

Invitro drug release studies

The release of azithromycin from liposomal formulation with different ratios was determined by using the membrane diffusion method. The dialysis membrane (Himedia laboratories, diameter- 21.5mm) was activated by heating it in distilled water at 100⁰C to remove impurities and for activation of dialysis membrane. Accurately measured amount of azithromycin liposomal formulation was placed in the dialysis membrane and ends were sealed. Then it is placed in beaker containing phosphate buffer pH 6.8 and placed in a magnetic stirrer with constant stirring. Aliquots of 5ml buffer were withdrawn from beaker at specific intervals up to 12 hours and to maintain sink conditions 5ml of fresh buffer was added after withdrawal of aliquot. The samples were analyzed by UV- Visible Spectrophotometer for drug release at 208nm (Table 1).

Evaluation of liposomal gel

Physical Evaluation

Formulated liposomal gel was evaluated for washability, occlusiveness and organoleptic properties such as colour, texture, pH, viscosity, spreadability, extrudability, drug content and content uniformity and invitro diffusion studies.

Viscosity

Viscosity of gel was determined by using Brookfield viscometer. The prepared gel was rotated at 1.5 rpm and viscosity was measured at centipoises.

Spreadability

The prepared liposomal gel sample (0.1gm) was placed between 2 slides and left for 5 minutes. After 5 minutes the diameter of the spreaded circles was measured in centimetres [5].

S=ML/T

M= weight on upper slide

L= Length of glass slide

T= Time

Extrudability

The formulated gel was determined by filling liposomal gel in the collapsible tubes. It was measured in terms of weight in grams that required extruding a 0.8cm ribbon of gel.

Drug content and content uniformity

Liposomal gel was withdrawn and drug content was examined by using a UV- Visible spectrophotometer at 208nm. The content uniformity was determined by analyzing azithromycin concentration in gel by taking from 3 to 4 different areas from the container.  The gel was mixed with sufficient of methanol to extract drug and then analysed at 208nm by using UV-Visible spectrophotometer.

Invitro diffusion studies

Invitro drug release was carried out utilising a Franz diffusion cell. A dialysis membrane was placed between receptor and donor compartments, whereas the donor compartment had gel and receptor compartment containing of buffer. Throughout the experiment to maintain skin conditions diffusion cell was kept at 37±0.5°C stirring at 200 rpm. Aliquot of 5ml was taken from the receiver compartment through a slide tube at predetermined time intervals for 16 hours. To maintain sink conditions 5ml of fresh buffer was added to diffusion cell. The samples were analysed at 208nm using a UV- Visible spectrophotometer. 

FT-IR and DSC studies concluded that there was no physico-chemical interaction between azithromycin and selected pharmaceutical excipients.  The particle size of liposome decreased as the concentration of lipid increased. In F1 the particle size was about 102.6nm. The F1 liposomal suspension showed more drug entrapment efficiency whereas F4 showed 93.93%.   The Invitro drug release was evaluated by dialysis membrane for 12 hours. From the result it was confirmed that for F1 the drug release was found to be 99.89 % and for F4 91.43%.  From the observation, concluded that F1 has excellent drug release and after plotting zero order, first order higuchi, Kosmayer peppas and Hixson graphs the liposomal suspension of F1 formulation follows non-fickian diffusion.

Evaluation of liposomal gel 

The liposomal gel has a white colour, smooth texture with better washable and occlusive property.  The pH value of F1 azithromycin gel was found to be 5.7. The normal skin pH value ranges between 5.0 – 6.0 and this pH value showed that F1 gel probably very safe for skin without any skin irritation. Hence, the gel is suitable for topical preparation.  The viscosity of F1 gel was found to be 3890 cps. The spreadability of gel is responsible for therapeutic efficacy of drug. Finally, F1 gel was a low spread time with high spreadability. Extrudability of F1 gel was found to be excellent. The drug content of F1 gel was found to be 96.08% ± 0.65. The method used to disperse the liposomal suspension into the gel was satisfactory because no significant difference was observed in % drug at various areas.  The Invitro diffusion studies of F1 gel was found to be 98.45% ± 0.52 16 hours of studies.  The gel follows fickian diffusion it was concluded based on the n value obtained after plotting zero order, first order, higuchi, Kosmayer Peppas and Hixson on graph.

Azithromycin loaded liposomal suspension was prepared by thin film hydration technique and the best formulation was incorporated into the carbopol gel. Liposomal gel was found to be smooth, white in colour and pH was found to be 5.7 which in the range of normal skin pH. The invitro drug release of gel showed better drug release of 98.45% ± 0.52 at 16 hours of diffusion studies which followed first order kinetics. The patient compliance was more for liposomal gel because they showed more localised effect and it is very easy for topical application without any issues. Spreadability and extrudability of liposomal gel was excellent which played a major role during dosage form application on to the skin.

Authors are grateful to Chalapathi drug testing laboratory, Chalapathi Institute of Pharmaceutical Sciences for their continuous motivation and providing the chemicals and instruments to complete my research work.  We also thank syndy Pharma, Hyderabad for financial support.

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