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

2. Screening of polymers for gel preparation:(13)

Preparation of Placebo batches of Gel by using different polymers.

Formulation of gel- the gel is form by adding different gelling agent in different concerntration in sufficient volume of water. propylene glycol is added 0.5 ml followed by methyl paraben then triethanolamine is added for neutralizing the gel. For that different polymers like hpmc, hpmc E5, hpmc E15, Carbopol 940 were used. From all cabopol 940 were used as it has proper gelling agent and viscocity.

3. Preparation of liposomal Gel using Carbapol 940 incorporate the optimized Liposomes (14-15)

The formulations was incorporated into the gel. The gel base Carbapol 940was developed separately with distilled water approximately 50 ml. The base was slowly added then stirred and added a neutralizing base TEA into the gel base, after a gel based was formed, liposomes suspension of levofloxacin was added and stirred until homogeneous mixture is formed, then methyl paraben is added which has been dissolved before in a portion of Propylene glycol and all stirred until homogeneous gel is formed.

Table No:6.1 Preparation of liposomal Gel

4. Evaluation (16-18)

7.1 Evaluation of liposomes 1. Microscopy

liposomes vesicles were observed under microscope by using 10 X magnification and 45 x magnification as shown in figure. Result are reported in 9.8.1

2. Particle size zeta potential analysis

Vesicle properties, particle size diameter and zeta potential, were determined at room temperature by Zeta Potential/ Particle Sizer analyzer. liposome formulations were diluted with phosphate buffered saline, pH 7.4, for Zeta potential and particle size determination, respectively. Result are reported in graph no. 9and 10

3. Drug entrapment efficiency

The entrapment efficiency of liposome was determined by calculating the amount of entrapped levofloxacin in the liposomes. To determine the entrapment efficiency of levofloxacin in liposome, an appropriate amount of dispersion was transferred in culture tube. The dispersion was centrifuge for 45 min at 1500 rpm. After centrifugation the supernatant was collected and Percentage Drug Entrapment amount of free levofloxacin was determined spectrophotometrically (?max= 287 nm). The entrapment efficiency has been determined according to the following equation:

EE % =        W (Added drug) −W (free drug) ×100

                         W (Added drug)

Where, W (added drug) is the amount of drug added during the preparation of liposomes, W (free drug) is the amount of free drug measured in the lower chamber of the culture tube after centrifugation. Result are reported in table 9.8.3.

4. % Drug content

Drug Content of liposome loaded can be determined by dissolving accurately weighed liposome loaded in 10ml methanol. After appropriate dilution absorbance may be determined by UV-Spectrophotometer (?max= 287 nm). The drug content was calculated. Result are reported in table 9.8.4.

8. Evaluation of liposomal gel (19-25)

1. Physical characteristic

After gel preparation the formulations were determined by visual examination for appearance, Homogeneity, consistency and phase separation. Result are reported in table 9.9.1 Physical characteristics

2. Measurement of pH

One gram of gel was dispersed in 50 mL of distilled water, and a digital pH meter was used to determine the pH value. Result are reported in table 9.9.2

3. Drug content

1 gm of gel was dissolved in a 100mL of phosphate buffer pH 7.4. The resultant solution was filtered, and drug content was analyzed spectrophotometrically. Result are reported in table 9 .9.3

4. Washability

The weighed quantity of gel (1gm) was taken and spread on the hand and washed under running water for 1 min. This test is crucial to assess how well the gel and its component remain on eye surface.

5 In vitro release study

The diffusion medium used was simulated tear 7.4 pH. The in vitro diffusion study was carried out using a Franz diffusion cell with egg membrane. The membrane with an effective diffusion area of 1.76 cm² was mounted between the donor and receptor compartments of the diffusion cell. The volume of the receptor medium was 20ml of simulated tear fluid, which was continuously stirred throughout the experiment. 2g of gel was applied on the membrane and placed between the compartments. At 1,2,3,4,5 6 and 7hr intervals 1ml of the solution in the reptor compartment was removed and replaced immediately with an equal volume of fresh solution. Samples were analyzed using a UV spectrophotometer and the data was recorded in table. 9.10.1

RESULT & DISCUSSION:

1. 0. Selection of Active Pharmaceutical Ingredient (API):

Levofloxacin was selected for incorporation into a liposomes for the treatment of ocular infections due to its antibacterial properties against many bacteria. In its ophthalmic formulation, levofloxacin is indicated for the treatment of bacterial conjunctivitis caused by susceptible organisms.

1. 1. Preformulation study:

Confirmation of Pure Drug:

1. Physical Properties:

Colour: pale yellow Odour: Odourless

Appearance: Crystalline Powder Taste: Slightly Bitter

Result: Identification of Levofloxacin has the same physical description properties as given in IP.

2. Determination of Solubility: The solubility of the pure drug 10mg /10ml of solvent was carried out and Results are given in Table

Table no. 9.1: Results of Solubility of Levofloxacin.

Result: Levofloxacin drug was showing the same Solubility as the pure drug as per the IP/BP.

3. Determination of Melting point:

The melting point of the obtained drug sample was determined. This was determined by using capillary tube method. The melting point of Levofloxacin was measured in the laboratory and found to be 227 °C

Result: Levofloxacin has the same melting point that of the monograph given in the IP/BP.

1. 2. Spectrophotometric method for the estimation of Levofloxacin: Determination of λmax of levofloxacin

After scanning 20 µg/ml solutions, highest peak at 287 nm was observed and considered as λ max. The UV spectrum is shown in Graph no.1, the confirmatory analytical test for the drug, which shows the UV spectrum as described in reference book.

Graph no. 1: Spectrum of Levofloxacin

Result: λ max of Levofloxacin was measured to be 287 nm. Standard calibration curve of levofloxacin in 7.4 pH buffer: From the standard curve, it was observed that the drug obeys Beer’s law in the concentration range of 2- 20 µg/ml in phosphate buffer of pH 7.4. The drug showed good linearity with regression of coefficient (R² = 0.999) and the equation for this line obtained was found to be y= 0.0824x +0.01

Graph no. 2: Standard Calibration curve of Levofloxacin

Result: From the standard curve, it was observed that the drug obeys Beer’s law

1. 0. Fourier Transform Infrared Spectroscopy for Analysis of Drug and Polymer:

1. IR Spectrum of Levofloxacin

Graph no. 3: IR Spectrum of Levofloxacin

2.: IR Spectrum soya lecithin

Graph no .4: IR Spectrum soya lecithin

IR Spectrum Levofloxacin +soya lecithin

Graph no .5. IR Spectrum Levofloxacin +soya lecithin

Result: FTIR spectra of the Drug, Polymer & Physical mixture of both were shown in Graph no..3, 4, & 5. It was observed that the principal peak was found in the FTIR spectra of a drug, & polymer, as well as the FTIR spectra of a physical mixture of drugs, and polymer.

1. 3. Differential Scanning Calorimetery (DSC):

The DSC of Levofloxacin (drug) shows a sharp endothermic peak a 226°C, confirming its crystalline nature and corresponding to its melting point. The Soyalecithin shows a broad endothermic peak at 256 °C with earlier thermal transition around 190°C indicating its semi crystalline nature and thermal degradation behaviour.

In the physical mixture (drug +polymer) the broader peak indicates a different crystalline structure or possibly an interaction or partial miscibility in molten state. The final thermal event may indicate decomposition, formation of new compound.

Graph no .6. DSC of Levofloxacin

Graph no .7. DSC of soya lecithin

Graph no .8. DSC of Levofloxacin soya lecithin

1. 4. Preliminary trials for formulation of liposome

Table no 9.5.1. Formulation of liposome by increasing soya lecithin concentrations.

In the above study, 7 batches were prepared using different concentration of Soyalecithin from 100 to 700 mg. and processing with rotary evaporator method. Cholesterol kept constant with 500 mg . Here keeping concentration of soya lecithin 500 mg and cholesterol 100 mg shows good result.

Table no 9.5.2 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 . Here also by keeping concentration of soya lecithin 500 mg and cholesterol 100 mg shows good result. So this concentration of liposomes were optimized.

9.6 Formulation of Levofloxacin Loaded liposomes

Table no 9.6.1 Formulation of Levofloxacin Loaded liposomes

The next step after optimization of excipients (Soya Lecithin, cholesterol, Ethanol, 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%, 1.5%, and 2% The drug loaded liposomes are thus formulated by rotary evaporator method. After optimization of soya lecithin 500 mg and cholesterol 100 mg, levofloxacin was loaded in placebo liposomes 1 to 4 batches were formulated with different concerntration of levofloxacin and all batches are in spherical form.

1. 7. Result of prepared Placebo Batches of gelling agents

The measurement of viscosity of the sample was done using digital viscometer Model). The required quantity of Gel was placed in a small volume holder and the spindle used was L4 30 (rpm) rotations speed per minute. The corresponding viscosity value in cp (centipoises) was noted.

Table No 9.7.1. Result of prepared Placebo Batches of CARBAPOL 940

From the above study, batches of different concentration of Carbapol 940 gel were prepared Triethanolamine added to adjust the pH of the gel and after preparation of gel. Carbapol 940 showed proper gelling capacity, viscosity increases as the conc of Carbapol 940 increases. having proper gelling consistency. thus, Carbapol 940 optimized for the preparation of gel. It having proper gelling consistency. at concertation of 0.5 gm because at concerntion of 1gm lumps were formed So 0.5 gm optimized for the preparation of gel.

Table No 9.7.2 liposomal Gel using Carbapol 940 incorporate the optimized Liposomes

1. 7. Evaluation of liposomes 9.8.1. Microscopy

liposomes vesicles were observed under microscope by using 10 X magnification

Fig 10. no Microscopy of liposomes

(10 X) 

Fig no 11 Microscopy of lipososome

( 45 X )

9.8.2 Particle size and zeta potential analysis

Zeta potential liposomes was determined by ZETASIZER. Figure illustrate Zeta potential for optimized batch of liposome was -36.2 mV indicating presence of optimum charge on the surface of formulations to prevent aggregation during their shelf life.

Graph no.9. Zeta potential of liposomes

This graph illustrate the distribution of particle sizes within a sample. The x-axis represents the size of particles, typically ranging from nanometers to micrometers, while the y-axis shows the frequency or proportion of particles within each size range. The average particle size of optimize formulation was found to be 510.5 nm.

Graph no .10 Particle size of liposomes

9.8.3. % Drug entrapment efficiency

Table no 9.8.3. Result of % Drug entrapment efficiency

Table shows the drug entrapment efficacy of different formulation. Batch F4 batch shows the maximum drug entrapment efficacy which was 94.57 %.

6.8.4 % Drug content

Table no 9.8.4 Result of % Drug content

Drug Content of liposome loaded can be determined by dissolving accurately weighed liposome loaded in 10ml methanol. After appropriate dilution absorbance may be determined by UV- Spectrophotometer (?max= 287 nm). The drug content was calculated. Table shows the drug content efficacy of different formulation. Batch F4 batch shows the maximum drug entrapment efficacy which was 93.34 %

1. 9. Evaluation of liposomal gel

Table no 9.9.1 Physical characteristics

9.9.2 Measurement of pH

Table no 9.9.2 Measurement of pH

One gram of gel was dispersed in 50 mL of distilled water, and a digital pH meter was used to determine the pH value.

9.9.3% Drug content

Table no 9.9.3% Drug content

1 gm of gel was dissolved in a 100mL of phosphate buffer pH 7.4. The resultant solution was filtered, and drug content was analyzed spectrophotometrically. Drug content of F4 batch was found to be highest.

9.9.4 Washability

The weighed quantity of gel (1gm) was taken and spread on the hand and washed under running water for 1 min. Carbapol gel remain intact for long time.

1. 10. In vitro release study

The Drug Release of formulation F1 =7, F2=7.5, F3 = 8.2, F4=8.24. Batch 4 shows high drug release as compared to other batches. Thus, batch F4 is optimized.

Graph no. 11: Drug Release (mg) Vs Time (hr)

1. 1. 1. % Cumulative Drug Release for F1, F2, F3 and F4 Batches

The% Cumulative Drug Release of formulation F1 =64%, F2=66%, F3% = 67, F4=73%. Batch 4 shows high % Cumulative drug release as compared to other batches. Thus, batch F4 is optimized.

Graph no. 12: Percent Cumulative Drug Release (%) Vs time (hr)

Table no. 9.10.2: Kineics of drug release of F4 Formulation

Graph no. 13: Zero order release kinetic

Graph no. 14: First order release kinetic

Graph no. 15: Higuchi Matrix release kinetic

Graph no. 16: Korsmeyer peppas release kinetic

Graph no. 17: Hixson crowell release kinetic

Drug release study shows that the release of drug from the Liposomes shows by first order followed by higuchi model and hixson model.

1. 11. Stability and Sterility studies:

The stability studies of Optimised formulation revealed that there is no significant reduction in drug content and Physical appearance was observed over period of 10 days. The results are shown in Table.

Table No 9.11.1. Stability Study of Liposomes