Formulation and Evaluation of Fluconazole Loaded Liposomal Gel for Topical Drug Delivery

Fig no 01: Structure of Liposomes.

METHODS OF PREPARATION OF LIPOSOMES:

1. Thin Film Hydration Method

2. Sonication Method

3. French Pressure Method

4. Ether Infusion Method

5. Ethanol Injection Method

6. Reverse Phase Evaporation Method

1. Thin Film Hydration Method: The most used approach for MLV research. The process entails drying a lipid solution until a thin film form at the bottom of a round-bottom flask. The film is then hydrated by adding water buffer and briefly vortexing the dispersion. The temperature used for the hydration process is higher than the gel-liquid crystalline transition. temperature Tc of the lipid or higher than the Tc of the lipid mixture's maximal melting component. Depending on their solubilities, the chemicals to be encapsulated are added to either an organic solvent containing lipids or an aqueous buffer.This approach makes MLV easy to organize, and these liposomes can be used to encapsulate a wide range of compounds. limited internal volume, limited encapsulation efficiency, and a heterogeneous size distribution are the process's drawbacks

2. Sonication Method: Under an inert atmosphere, MLVs are solicited using either a probe sonicator or a bath type sonicator. This method's primary disadvantages are extremely low interior volume/encapsulation efficiency, potential degradation of phospholipids and compounds to be encapsulated, exclusion of big molecules, metal pollution from the probe tip, and the presence of MLV in addition to SUV.

3. French Pressure Method: MLV is extruded through a tiny aperture at 20,000 psi and 4°C. Compared to the sonication approach, the method has several advantages. The process is straightforward, quick, repeatable, and requires handling unstable materials gently. Compared to sonicated SUVs, the resultant liposomes are a little bigger.

4. Ethanol Injection Method: A large excess of buffer is quickly injected with a lipid ethanol solution. The MLVs are created right away. The method's disadvantages include the population's heterogeneity (30-110 nm), the liposomes' extreme dilution, the difficulty of eliminating all ethanol due to its azeotrope formation with water, and the potential for different biologically active macromolecules to become inactive in the presence of even minute amounts of ethanol.

5. Ether Infusion Method: An aqueous solution of the item to be encapsulated is slowly injected with a solution of lipids dissolved in diethyl ether or an ether/methanol combination at 55–65°C or under decreased pressure. Liposomes are created when ether is then removed under vacuum. The population's heterogeneity (70–190 nm) and the chemicals to be encapsulated's exposure to organic solvents or high temperatures are the method's primary disadvantages.

6. Reverse Phase Evaporation Method: First water in oil emulsion is formed by sonication of a two-phase system containing phospholipids in organic solvent and aqueous buffer. A viscous gel is created when the organic solvents are extracted under low pressure. When leftover solvent is eliminated through continuous rotating evaporation at lower pressure, liposomes are created. This technique can achieve high encapsulation competency of up to 65% in a low ionic strength media, such as 0.01 M NaCl. Small, big, and macromolecules have all been encapsulated using this technique. The exposure of the materials to be encapsulated to organic solvents and short sonication times is the method's primary drawback.

MATERIAL AND METHOD

MATERIAL –

Chemicals:

Flucanazole:

Vaginal thrush, oral thrush, esophageal candidiasis, and cryptococcal meningitis are among the fungal and yeast infections that can be treated and prevented using the prescription antifungal drug fluconazole.

It works by preventing the growth of fungus, and symptoms usually go away in 24 to 3 days.

Phospholipids:

The basic structural components of all biological cell membranes are phospholipids, a type of compound lipids.

They have two hydrophobic (water-repelling) "tails" and a hydrophilic (water-attracting) "head" because they are amphipathic molecules.

In aquatic settings, their special dual characteristic enables them to spontaneously self-assemble into a lipid bilayer, thereby regulating what enters and leaves a cell.

Chloroform:

Chloroform (systematic IUPAC name: trichloromethane) is a chemical compound that is transparent, colorless, and extremely volatile. It smells sweet and ether-like.

It was independently discovered in 1831 and became well-known for being the first inhaled medicinal anesthetic.

Its clinical use has been completely discontinued in contemporary medicine due to its extreme toxicity profile and tendency to cause organ damage.

It is now mostly used as a chemical solvent and heavy-duty industrial precursor.

Methylparaben:

A common preservative in food, cosmetic, and pharmaceutical items is methylparaben.

Methylparaben is a preservative used in emulgel compositions to stop bacteria and fungi from growing.

Triethanolamine:

pH adjuster Triethanolamine is used, especially in medicines and cosmetics, to change the pH of compositions.

Triethanolamine, an emulsifier, enhances the solubility of components and stabilizes emulsions.

Essential oils and perfumes are among the many compounds for which triethanolamine is employed as a solvent.

Ethanol:

The main purpose of ethanol in liposomes is to serve as a solvent for active substances, which aids in the development of a stable and uniform topical formulation.  It can also serve as a co-surfactant and a penetration enhancer, and at a certain concentration, it can protect the network structure of the liposomes, increasing their freeze-thaw stability.

Carbapol934:

A synthetic high molecular weight polymer of acrylic acid, carbopol 934 is frequently employed as a gelling agent in cosmetic and medicinal applications.

Gelling Agent: Carbopol 934 is a transparent, stable gel that swells in water. neutralized (such as sodium hydroxide or triethanolamine).

For topical applications, it has a smooth, non-greasy feel. By avoiding phase separation, it aids in stabilizing suspensions or emulsions in gel compositions.

Glycol Propylene:

The main role of propylene glycol in emulgel is to improve the solubility and skin delivery of hydrophobic medications by acting as a co-surfactant and penetration enhancer.

Additionally, it absorbs water and aids in moisture retention by acting as a humectant.

It is frequently utilized in liposomalgel formulation to assist generate a stable, effective product by dissolving additional ingredients like parabens.

EXPERIMENTAL WORK

FORMULA

Formula for liposomes:

Table no: 01 Formula for liposomes: (For 20 gm)

Formula for gel:

Table no: 02 Formula for gel: (For 20 gm)

How to Make Liposomal Gel:

Forming the liposomes carrying the active substance and then integrating them into a gel base for topical administration are two separate procedures in the manufacture of a liposomalgel.

METHODS:

Step 1: Liposome Preparation As indicated in Table No. 01, the various liposome formulations were made by dissolving flucanazole, soy lecithin, and cholesterol in ethanol and chloroform. This mixture was placed in a 250 ml round-bottom flask. To create a thin lipid coating on the flask wall, evaporate the organic solvent. A milky white suspension was produced by progressively hydrating the dry lipid film with 10 milliliters of phosphate buffer pH 6.8 and rotating the mixture for two hours. To extract the uncapsulated medication, the formulation is centrifuged for 30 minutes at 3000 rpm.

Fig no: 02 Liposomal Suspension

Step 2: Carbopol gel preparation the determined quantity of carbopol was added to the warm water while being continuously stirred at a moderate speed using a magnetic stirrer. Triethanolamine was used to change the pH of the Carbopol gel.

Fig no: 03 Carbopol gel

Step 3: Liopsomal gel formation to create the liposomal gel, the produced liposomal suspension and gel base were separately combined in the proper ratio while being continuously stirred. A container was filled with the prepared Liopsomal gel

Fig no: 04 Liosomal gel

EVALUATION PARAMETER:

1.Evaluation of Liposome:

1.Colour

2. Particle shape and size analysis

3.pH of Liposomes

4. Zeta potential

5. Entrapment efficiency

2.Evaluation for Gel:

1.Colour

2.pH of gel

3.Grittiness

4.Spreadability

3.Evalution for Liposomal gel

1.Physical Examination

1. Colour
2. Grittiness

2. pH of Liposomal gel

3. Spreadability

4. Washability

5. Drug content

6. Irritancy Test

7. Anti fungal Activity

1. Evaluation of Liposome:

1. Color: Visual inspection can also be used to evaluate it.

2. Particle size and shape analysis: An optical microscope is used to examine the shape of the particles.

Fig no. 05: Particle shape

3. Liposome pH: A digital pH meter was used to measure the formulations' pH. The formulation's pH was measured in triplicate, and average results were computed.

Fig no. 06: pH of Liposomal suspension

4. Zeta potential: The partical size reveals the uniformity of the size distribution and displays the average diameter of the liposomal vesicles. The vesicles' surface charge is represented by their zeta potential, which aids in predicting their stability.

Fig no. 07: Particle size & zeta potential

5. Entrapment efficiency: The following formula can be used to calculate EE, which is the ratio of drug molecules encapsulated in liposomal particles to the total amount of medication used.

Entrapped drug divided by total drug times 100 is the entrapment efficiency.

Total medication = First medication added Free drug is the unentrapped drug found in the supernatant.

Entrapped drug is equal to total drug minus free drug. Following the preparation of liposomal solution, unentrapped drug is separated using centrifugation, gel filtering, and dialysis.

Centrifuge the sample for 60 minutes at 4°C at a high speed of 15,000 rpm. In order to separate the entrapped drug from the free (unentrapped) drug in the supernatant, carefully pipette or decant the supernatant without upsetting the pellet.

Evaluation for Gel:

1.Color: Visual inspection is another way to evaluate it.

2. Grit: Visual inspection and light finger rubbing were used to test the liposomal gel. A smooth texture and homogeneous dispersion of the medication and excipients were indicated by the formulation's lack of gritty particles. The formulation's fineness is confirmed by the lack of grittiness, which enables topical administration without irritating the skin.

3. pH of gel: A digital pH meter was used to measure the formulations' pH. The formulation's pH was measured in triplicate, and average results were computed.

Fig no. 08 pH of gel

4. Spreadability

Weigh out one gram of gel and put it in the middle of a pre-marked circle or the initial area of a glass plate. Put a second glass plate on top of the gel and apply weight. For five minutes, a 500-gram weight is applied to the upper plate in order to compress the gel into a homogenous film. Calculate the diameter. Once the weight has been applied for the predetermined amount of time, take it off and measure the spread gel circle's circumference. Determine spreadability: A formula can be used to determine spreadability.

S=Df-Di / Di

Were,

S= spredability

Df =Diameter after spreading

Di=Initial diameter

Fig no: 09 Spreadability of gel

1.Physical Assessment

Color: Visual inspection can also be used to evaluate it.

Grit: Visual inspection and light finger rubbing were used to test the liposomal gel. A smooth texture and homogeneous dispersion of the medication and excipients were indicated by the formulation's lack of gritty particles. The formulation's fineness is confirmed by the lack of grittiness, which enables topical administration without irritating the skin.

2. Liposomal gel pH: A digital pH meter was used to measure the formulations' pH. The formulation's pH was measured in triplicate, and average results were computed.

Fig no .10: pH of Liposomal gel

3. The ability to spread: Weigh out 1g of liposomal gel and put it in the middle of a pre-marked circle or the first spot on a glass plate. Put a second glass plate on top of the gel and apply weight. For five minutes, a 500-gram weight is applied on the upper plate in order to compress the liposomal gel into a homogenous layer. Calculate the diameter. Once the weight has been applied for the predetermined amount of time, take it off and measure the spread gel circle's circumference. Determine spreadability: A formula can be used to determine spreadability.

S=Df-Di / Di

Were,

S= spredability

Df =Diameter after spreading

Di=Initial diameter

Fig no. 11: Spreadability of liposomal gel

4. Washability: A gel washability test evaluates a formulation's ease of removal from the skin with water. This is often done by putting a predetermined quantity of gel to the skin, rinsing it under running water, and tracking the removal. Because gels shouldn't leave any oily residue, it guarantees patient compliance.

5. Drug content: 20 ml of phosphate buffer (pH 5.5) and 1 g of liposomal gel formulation were added to a 100 ml volumetric flask, and the mixture was swirled for 30 minutes. One milliliter of the aforementioned solution was further diluted to fifty milliliters using phosphate buffer (pH 5.5) after the volume was increased to one hundred milliliters of ethanol. Whatman paper was used to filter the final solution. Using a UV spectrophotometer set to 260 nm and phosphate buffer pH 5.5 as a blank, the absorbance was measured. The drug content was calculated using the fluconazole standard calibration curve.

Fig. No. 12 Drug Content by UV Spectroscopy

6. Irritation Test: A shaved section of skin was treated with the liposomal gel. Any indications of redness, irritation, or itching were noted at the test location.

7. Anti-fungal Test: Fungal cultures were used to prepare the microorganism inoculum. Clean, sterile Petri dishes were filled with 15 milliliters of Saubroad agar (Hi media) medium, which was then left to cool and harden. Using a spreading rod, 100 µl of the fungal strain's broth was pipetted out and evenly distributed over the medium until it dried completely. A sterile cork borer was used to drill wells with a diameter of 6 mm. Compound solutions (100µl/ml) were made in DMSO, and the wells were filled with 100µl of the prepared test solutions and standard. The petri dishes were incubated for twenty-four hours at 37°C. DMSO was used as the negative control, and miconazole (1 mg/ml) was created as the positive control. The diameters of the zone of inhibitions (ZI) were measured to assess antifungal activity; all measurements were made in triplicate.

Fig no. 13: Atifungal Activity of test samples against C. albicans

RESULT AND DISCUSSION

Liposomal Suspension :

1. Particle shape analysis:

The Liposome prepared using flucanzole drug was studied under microscope to observe the formation of Liposomal vesicles. The Liposomal vesicles were found to be uniform in size and shape. The shape of the Liposomes was observed spherical.

2. pH :

The standard range of the pH of the Liposomal solution was 5.0- 7.4 and the pH of the Liposomal solution was found to be 5.22 which is ideal pH.

Table No. 03 pH of Liposomal suspension

3. Zeta potential & Particle size:

The prepared Flucanazole -loaded Liposomes  showed an average particle size of 182 nm, indicating larger vesicle formation. The zeta potential was found to be 30 mV, suggesting acceptable stability of the Liposomal suspension.

4. Entrapment efficiency :

Entrapped drug /Total drug× 100

=10 -1.373 / 10 × 100

=86.27 %

The entrapment efficiency of the fluconazole loaded Liposomal vesicles was found to be 86.27 %. For topical or transdermal delivery, an EE of 70-90% is desirable.

Gel :

1. pH : The standard range of the pH of the gel was 5.5 - 7.5  and the pH of the gel solution was found to be 5.83 which is ideal pH.

Table No. 04 pH of gel

2. Spreadability:

S=Df-Di / Di

= 6-1 / 1

= 5 gm.cm/sec

Liposomal gel:

1. Physical Examination: The Liposomal gel was observed for colour, odour and appearance, which is shown in below table

Table no: 05 Observation of physical Examination

2. pH : The standard range of the pH of the Liposomal gel was 5.1- 7.4 and the pH of the Liposomal gel solution was found to be 6.18 which is ideal pH.

Table No. 06 pH of Liposomal  gel

3. Spreadability::

S=Df-Di / Di

=6.8-1/1

=5.8 gm.cm/sec

4. Washability:

The Liposomal gel was Easily Washable

5. Irritancy Test:

No any irritation or rashes seen on skin.

6. Antifungal Activity : The antifungal profile of Fluconazole Loaded Liposomal Gel was evaluated by measuring the zone of inhibition against fungal strains C albicans  Via well diffusion method. The compounds Fluconazole Loaded Liposomal Gel exhibited good activity as compared to the standard Miconazole

Table no: 07: Antifungal Activity of test samples against C. albicans

7. Drug Content:

Drug Content (%) = Sample absorbance / Standard absorbance × 100

Average absorbance

=0.512+0.518+0.526+0.535 /  4

=2.091 / 4

=0.52275 ≈ 0.522

If standard absorbance = 0.614, then

Drug Content (%) =0.522 /  0.614 × 100

=85.01%

The drug content was studied for the formulation prepared by Thin film hydration method. The drug content was found to be  85.01 %

Table no: 07: Evaluation Parameters and Standard Ranges