Formulation and Evaluation of Levofloxacin Loaded Liposomal Gel for Ocular Infection

Despite the widespread use of traditional topical ocular treatments in modern times, there are still certain issues with their application, effectiveness, and safety. As a result, numerous methods have been developed and examined. Using nanotechnology in the ocular drug delivery system via nanoparticles and nanomicelles is one strategy. Liposomes are use in enhancing ocular delivery. Liposomes have an aqueous core containing medication which is encased by one or more phospholipid bilayers. Three forms of liposomes - small unilamellar vesicles (10–100 nm), big unilamellar vesicles (100–300 nm), and multilamellar vesicles (containing several bilayers) can encapsulate both hydrophobic and hydrophilic medicines. Because these liposomes have cell-like membranes, natural phospholipids, and exceptional biocompatibility, they hold promise for the delivery of ocular drugs. In addition, liposomes have the ability to adhere to the hydrophobic corneal epithelium, releasing the bound drug content continuously. This improves pharmacokinetics and lessens toxic side effects. Depending on the type of lipid composition chosen, multilamellar vesicles may be used to produce sustained release of the drug. Levofloxacin is also used as antibiotic eye drops to prevent bacterial infection. Levofloxacin, like other fluoroquinolone antibiotics, exerts its antimicrobial activity via the inhibition of two key bacterial enzymes: DNA gyrase and topoisomerase IV

Figure 1: Structure of levofloxacin

MATERIALS AND METHODS

2.0 A] Chemicals

Levofloxacin, Soy lecithin, Chloroform, Methanol, Carbapol 940, HPMC, HPMC E5, HPMC E5, Propylene glycol, Methyl paraben, Ethanol, Disodium Hydrogen Phosphate, Potassium Dihydrogen Phosphate, Sodium chloride

2. Instruments

Rotary Vacuum Evaporator, Digital pH Meter, Lab Stirrer, Franz Diffusion Cell Apparatus, Brookfield Viscometer, UV Spectrophotometer, Magnetic Stirrer.

1. 1. Preformulation study (8-9)

Preformulation study is defined as investigation of physical and chemical properties of the drug substance alone and combined with the excipients”. Preformulation studies are the first step in the rational development of dosage form of drugs. It involves the application of biopharmaceutical principles to the physicochemical parameters of the drug with the goal of designing an optimum delivery system that is stable, bioavailable and can be mass produced.

1. 1. Drug Identification Test

Identification of drug was done by organoleptic properties such as appearance, test, colour and odour. Result are reported.9.1.a

1. 2. Determination of Solubility

A semi-quantitative determination of solubility can be made by adding a solute in small amount to fixed volume of solvents. Solubility of drug was carried out in distilled water, Ethanol, Methanol, and chloroform. After each addition, the system is vigorously shaken and examined visually for any undissolved particles. Result are reported in 9.1b

1. 3. Melting Point Determination

Melting point determination of the obtained drug sample was done as it is a first indication of purity of the sample. It was determined by capillary tube method. Result is reported in section 9.1c

1. 2. Spectrophotometric method for the estimation of levofloxacin:

The Spectrophotometric method for the estimation of levofloxacin was carried out in Phosphate buffer pH 7.4 (10% Ethanol).

1. 1. 1. Determination of λmax of levofloxacin in:

100 mg of Levofloxacin was accurately weighed and transferred to 100 ml of volumetric flask. The drug was dissolved in 7.4 pH buffer (10 % Ethanol), and the volume was made up to 100 ml to obtain a stock solution of 1000 μg/ml. The resulting solution was scanned between 200 nm and 400 nm in a double beam UV-visible spectrophotometer. Result are reported in 9.2

1. 1. 2. Standard calibration curve of levofloxacin in 7.4 pH buffer (10% Ethanol):

Standard solution was prepared by dissolving accurately 100 mg of levofloxacin in 10% Ethanol and made up to the volume with 7.4 pH buffer100 ml stock solution was prepared. From this solution having concentration 1000 ug/ml. aliquots 0.2,0.4,0.6,0.8,0.8,1,1.2, 1.4,1.6, 1.8 and 2.0 ml were pipette out into 10ml volumetric flask. The volume was made up to Mark with phosphate buffer pH 7.4(10%Methanol) to get final concentration 2,4,6,8,20,12,14,16,18,20 ug/ml respectively. The absorbance of each concentration was measured at λmax 287 nm.A graph of absorbance vs concentration was plotted and its shown in graph no: 2.show straight line meaning the calibration curve obeys Beers Law. Result are reported in Table no:9.2.1

1. 3. Fourier Transform Infrared Spectroscopy for Analysis of Drug and Excipients:

FT-IR spectra for pure drug and polymer at room temperature using FT-IR spectrophotometer (FTIR-8400S, Shimadzu, Japan) in transmittance mode. The samples were ground in a mortar, mixed with Nujol and placed between two plates of KBr and compressed to form a thin film. The sandwiched plates were placed in the infrared spectrometer and the spectra were obtained. Scanning was performed between wave numbers 4000-400 cm^-1. Result are reported in 9.3.1, 9.3.2,9.3.3.

1. 4. Differential Scanning Calorimetery:

The compatibility between drug and polymers were determined by Differential Scanning Calorimetry (DSC). The thermal behavior of pure drug and polymer was studied at heating rate of 10°C/min from 25°C to 400°C in a thermetically sealed pan with a pinhole in the lid under a nitrogen purge of 20 ml min.The differential scanning calorimetry analysis gives an idea about the interaction of various materials at different temperature. Result are reported in graph no 6,7,8.

3. Formulations of liposomes (10-11)

Method of preparation

Liposomal formulation was prepared by Thin film hydration method. The specific amount of levofloxacin, cholesterol and soya lecithin were dissolved in chloroform and methanol (7:3) in a rotary round bottom flask followed by stirring for 1 hour at a temperature not exceed than 40^oC.Thin film of the sample was obtained to which phosphate buffer 7.4 added and precipitate obtained was collected and stored at amber-colored glass bottle in a desiccator for further used.

Table No: 3.1 Formulation of liposome by increasing soya lecithin concentrations.

In the above study, by increasing concentrations of soya lecithin and keeping cholesterol constant and processing with rotary evaporator method. The concentrations of soya lecithin was taken from 100 to 700 mg. Results are reported in table no.9.5.1

Table No 3.2: Formulation of liposomes by increasing cholesterol concentration

In the above study, 7 batches were prepared using different concentration of cholesterol from 100 to 700 mg. and processing with rotary evaporator method. Soya lecithin kept constant with 500 mg. Result are reported in the table no:9.5.2

2. Formulation Study (12)

In this study drug loaded batches were formulated.

Formulation of Levofloxacin Loaded liposomes

The next step after optimization of excipients (Soya Lecithin, cholesterol, choroform, Methanol) addition of levofloxacin was done. The optimized quantity of soya lecithin and cholesterol Were taken and levofloxacin added in different concentrations 0.5%,1%, and 1.5%,2 % The drug loaded liposomes are thus formulated by rotary evaporator method. During formulation of liposomes sterility is maintained. Sterilization is crucial for ophthalmic preparations to ensure safety and prevent infection. The choice of sterilization method depends on the specific formulation, the stability of the active ingredients, and the need to preserve the integrity of the product. Aseptic manufacturing and sterility testing are essential components of ophthalmic preparation production to guarantee the quality and safety of the final product Result are reported In the table no:9.6.1

Table No: 4.1 Formulation of levofloxacin Loaded liposomes