Formulation And Evaluation Of Benzocaine Transferosomes Containing Gel For Local Anesthetics Activity

NOVEL DRUG DELIVERY SYSTEM

Novel Drug delivery System (NDDS) refers to the approaches, formulations, technologies, and systems for transporting a pharmaceutical compound in the body as needed to safely achieve its desired therapeutic effects. It may involve scientific site-targeting within the body, or it might involve facilitating systemic pharmacokinetics; in any case, it is typically concerned with both quantity and duration of drug presence”.

• The conventional dosage forms provide drug release immediately and it causes fluctuation of drug level in blood depending upon dosage form.

•NDDS is advanced drug delivery system which improves drug potency, control drug release to give a sustained therapeutic effect, provide greater safety, finally it is to target a drug specifically to a desired tissue.

•NDDS is a combination of advance technique and new dosage forms which are far better than conventional dosage forms

Advantages of NDDS

• Decreased dosing frequency.
• Reduced rate of rise of drug concentration in blood.
• Sustained and consistent blood level within the therapeutic window.
• Enhanced bioavailability.
• To achieve a targeted drug release
• Reduced side effects
• Improved patient compliance.

IMPORTANCE OF NOVEL DRUG DELIVERY SYSTEM

• Enhanced drug efficacy: Novel drug delivery systems can improve the therapeutic effect of drugs by delivering them to the target site in a controlled and sustained manner.
• Reduced side effects: By targeting drug delivery, these systems can reduce the exposure of healthy tissues to the drug, minimizing side effects.
• Improved patient compliance: Systems that reduce dosing frequency or simplify drug administration can improve patient adherence to treatment regimens.
• Enhanced drug stability: Some delivery systems can protect drugs from degradation, improving their stability and shelf life.

       
           
       
Figure 1: Classification of NDDS

TRANSFEROSOMES: Transferosomes are advanced drug delivery systems that aim to transport therapeutic agents across biological membranes, such as the skin. Here’s a comprehensive overview of transferosomes:

Structure:

       
           
       
Figure 2: Structure of transferosomes

Transferosomes are ultra-deformable lipid vesicles, consisting mainly of phospholipids, surfactants, and water. They are characterized by their flexibility and ability to penetrate deep into the skin layers or even reach systemic circulation.

Components

1. Phospholipids: These form the lipid bilayer, similar to biological membranes, enhancing biocompatibility.

2. Surfactants: These provide the necessary flexibility to the vesicle, allowing it to squeeze through narrow pores without rupturing.

3. Water: Serves as the medium in which the lipids and surfactants are dispersed.

Composition of Transferosomes: Transferosomes are possessed majorly of phospholipids like phosphatidyl choline which assembles into lipid bilayer in an aqueous environment such that to form a vesicle upon closing. The second component in the composition of transferosomes is edge activator. It acts by increasing the lipid bilayer flexibility and permeability. An edge activator consists of single chain surfactant which destabilizes the lipid bilayer thus increasing its fluidity and elasticity.

By mixing suitable surfactants in the appropriate ratios, the flexibility of transferosomes membrane can be altered. The resulting transferosomes are flexibility and permeability optimized. Transferosomes can therefore acclimatize its shape to adjacent stress easily and rapidly, by adjusting local concentration of each bilayer component to the local stress experienced by the bilayer. The flexibility is of significant importance because it minimizes the threat of complete vesicle rupture in the skin and allows them to track the natural water gradient across the epidermis, when applied under non occlusive condition.(1,2)

Mechanism of Penetration of Transferosomes

When the formulation (lipid suspension (transferosomes)) is applied on to the skin the water gets evaporated and there is a formation of “osmotic gradient”, which is the major mechanism for transportation of transferosomes across the skin. Thus the transportation of these elastic vesicles is independent of concentration. When applied under suitable condition they can transfer 0.1mg of lipid per hour/ cm2 area across the intact skin. This value is considerably superior than which is typically driven by the transdermal concentration gradients8. Naturally occurring transdermal osmotic gradients i.e. “another much prominent gradient is available across the skin is the reason for this high flux rate(3).

Due to the stratum corneum, which acts as a skin penetration barrier this osmotic gradient is developed so that it prevents the water loss across the skin and maintains a water activity difference in the viable part of the epidermis (75% water content) & nearly completely dries the stratum corneum, near to the skin surface (15%water content) .The stability of osmotic gradient is very important because the ambient air acts as a perfect sink for the water molecules even when the transdermal water loss is un physiologically high(4). Due to the energetically favorable interaction between the hydrophilic lipid residues and their proximal water all polar lipids attract some water. Most lipid bilayers thus spontaneously resist an induced dehydration. As a result all lipid vesicles composed of polar lipid vesicles move from the rather dry location to the sites with sufficiently high water properties which is responsible for their greater deformability(5). Standard liposomes are confined to the skin surface, where they dehydrate completely and fuse, so they have less penetration power than transferosomes. Transfersomes attain maximum flexibility, so they can take full advantages of the transepidermal osmotic gradient (water concentration gradient) (5).  Transfersomes vesicle can therefore adapt its shape to ambient easily and rapidly, by adjusting local concentration of each bilayer component to the local stress experienced by the bilayer as shown in figure 2 in detail (6). So when lipid suspension is placed on the skin surface, that is partly dehydrated by the water evaporation loss and then the lipid vesicles feel this “osmotic gradient” and escape the complete drying by moving along this gradient. This can be achieved only when they are sufficiently deformable to pass through the narrow pores in the skin, because transferosomes composed of surfactants that have more suitable rheological anhydration.

Transferosomes work through a combination of osmotic gradient and elastic deformation:

1. Osmotic Gradient: Creates a driving force that pushes the transferosome into the deeper layers of the skin.

2. Elastic Deformation: The surfactants allow the vesicle to deform and pass through pores and skin barriers that are smaller than the vesicle itself.

       
           
Figure 3: MOA of Transferosomes

       
           
       
Table No.3 Materials for Transferosomes

Central Composite Design (CCD) is a statistical technique used for optimizing formulations by studying the effects of multiple factors and their interactions. When formulating transferosomes using soyalecithine and Span 80, CCD can help in determining the optimal concentrations of these components to achieve desirable characteristics such as particle size, encapsulation efficiency, and stability

       
           
       
Table No.4: Formulation Of Transferosomes

Thin film hydration technique is employed for the preparation of transfersomes which comprised of three steps

1. A thin film is prepared from the mixture of vesicles forming ingredients that is phospholipids and surfactant by dissolving in volatile organic solvent (chloroform methanol). Organic solvent is then evaporated above the lipid transition temperature (room temp. for pure PC vesicles, or 50°C for Souyalecithine) using rotary evaporator. Final traces of solvent were removed under vacuum for overnight.

2. A prepared thin film is hydrated with toluene by rotation at 60 rpm for 1 hr at the corresponding temperature. The resulting vesicles were swollen for 2 hr at room temperature.

3. To prepare small vesicles, resulting vesicles were sonicated at room temperature or 50°C for 30 min. using a bath sonicator or probe sonicated at 4°C for 30 min. The sonicated vesicles were homogenized by manual extrusion 10 times through a sandwich of 200 and 100 nm polycarbonate membranes.

C] CHARACTERIZATION:

1. PARTICLE SIZE:

The HORIBA HZ 100 particle size analyzer is a tool used to determine the particle size of benzocaine and other excipients. The process involves preparing the sample, selecting the appropriate dispersion medium, and dispersing the sample using an ultrasonic bath. The analyzer is set up, calibrated, and loaded into the sample cell. The sample is then measured using laser diffraction, and multiple measurements are recorded for accuracy and repeatability. The HORIBA HZ 100 software provides a particle size distribution curve, with key parameters such as mean particle size, standard deviation, and other percentile values. The data analysis is done using the HORIBA HZ 100 software, which provides a summary of the particle size distribution. The sample cell is then cleaned thoroughly between different samples to avoid cross-contamination and using appropriate solvents to remove residues. The process ensures accurate and repeatable results. The 1 ml of solution is taken and diluted upto 10 ml water after that is it sonicate for 10 min using ultrasonicator for 10 min and analyse .

2. ENTRAPMENT EFICIENCY:

To determine the entrapment efficiency (EE) for benzocaine transferosome, follow these steps:
1. Prepare transferosomes by dissolving phospholipids and surfactants in an organic solvent. Add benzocaine to the mixture, evaporate the solvent, and hydrate the film with an aqueous buffer.
2. Separate the encapsulated and free drug using ultracentrifugation or dialysis. Measure the total benzocaine content using techniques like UV-Vis spectrophotometry or HPLC. After separation, measure the free benzocaine content.
3. Calculate the entrapment efficiency by substituting the measured values into a formula. If specific data is needed, consult a professional. Otherwise, follow the steps outlined in the experimental setup to determine the entrapment efficiency.

The batch no .1 is selected as a optimized batch.

a) 3D graph

Figure 4: B The figure 8 B shows entrapment efficiency is dependable on the variables. The entrapment efficiency increases with the concentration of variables.

b) Counter Plot:

Figure 5: B The figure 9:B shows that entrapment efficiency is affected by the concentration of variable X that is soyalecithine are not affected  by variable Y

c)Interaction:

Figure 6: B  The line of both the variables shows 4 designs of points of interaction and shows the initial entrapment and dependency in lower concentration.

d) Predicted VS Actual:

       
           
       
Figure 7: A                                                     Figure 7: B

Figure 7: B The figure 11 B shows 9 points and all of are into the diagonal line. So predicted values are match with actual values

e) ANOVA:

ANOVA for Quadratic model

Response 1: PS

ANOVA for Quadratic model

Response 1:EE

3. IN VITRO DRUG RELEASE:

The transdiffusion procedure for benzocaine transferosome gel involves evaluating the drug's permeation through a biological membrane. The procedure requires the use of benzocaine transferosome gel, Franz diffusion cells, egg skin, phosphate buffer solution (PBS) 7.4 ph, magnetic stirrer, analytical balance, syringe, sampling vials, and a UV-Vis spectrophotometer for drug quantification. The membrane is prepared by removing any subcutaneous fat and hydrating it in PBS for at least an hour before the experiment. The Franz diffusion cell is assembled, consisting of a donor compartment and a receptor compartment, filled with PBS and maintained at a constant temperature. A magnetic stirrer is placed in the receptor compartment to ensure uniform drug distribution. The benzocaine transferosome gel is applied onto the membrane between the donor and receptor compartments. Samples are withdrawn at predetermined intervals and replaced with fresh PBS to maintain sink conditions. The concentration of benzocaine in the receptor compartment is determined using UV-Vis spectrophotometer, and the cumulative amount of benzocaine permeated per unit area is plotted against time to obtain the permeation profile.

4. PREPARATION OF GEL:

Preparing benzocaine transferosomes gel involves several key steps: preparation of transferosomes, incorporation of benzocaine into these transferosomes, and formulation of the gel. Here's a step-by-step guide:

1.Preparation of Transferosomes

       
           
       
Table No. 3: Preparation of Transferosomes

Materials Needed:

1. Phosphatidylcholine (PC)
2. Edge activators (Span 80)
3. Benzocaine
4. Ethanol
5. Distilled water
6. Sonicator
7. Rotary evaporator (optional)

Steps:

1. Dissolve Lipids: Dissolve phosphatidylcholine and the edge activator in ethanol. The typical ratio might be PC: edge activator at 85:15 by weight.

2. Hydration: Hydrate the lipid mixture with distilled water. This can be done by slowly adding water to the lipid mixture while stirring.

3. Formation of Vesicles: Sonicate the mixture using a probe sonicator to form small vesicles. This step can also be done using a rotary evaporator to remove the solvent, leading to the formation of a thin lipid film which is then hydrated and sonicated.

4. Sizing: If needed, extrude the vesicles through a polycarbonate membrane to achieve the desired size.

2. Incorporation of Benzocaine

Steps:

1. Loading Benzocaine: Add benzocaine to the hydrated lipid mixture before sonication. Benzocaine can be dissolved in a small amount of ethanol if needed to aid in its incorporation.

2. Sonication: Continue sonication until benzocaine is uniformly distributed within the transferosomes.

3. Preparation of Benzocaine Transferosome Gel

Materials Needed:

- Carbopol 934 or another suitable gelling agent

- Distilled water

- Benzocaine-loaded transferosomes

Steps:

1. Prepare Gel Base: Disperse Carbopol 934 in distilled water (usually around 1-2% w/w). Allow it to hydrate completely, usually requiring a few hours with occasional stirring.

2. Neutralize: Slowly Carbopol to adjust the pH and form a clear gel. The pH should be around 6-7.

3. Incorporate Transferosomes: Add the benzocaine-loaded transferosomes to the gel base. Mix gently but thoroughly to ensure even distribution of the transferosomes within the gel.

4. Adjust Consistency: If needed, adjust the final consistency of the gel by adding more distilled water or additional Carbopol.

Storage: Store the gel in airtight containers at a cool temperature to maintain stability.

EVALUATION TESTS FOR GEL

Evaluating benzocaine transferosomes-containing gel involves various analytical techniques to ensure its quality, efficacy, and safety. Below are detailed descriptions of the essential tests:

1. Physical Appearance:

- Objective: To assess the visual and tactile properties of the gel.

- Procedure: Observe and document the gel’s color, homogeneity, presence of any aggregates, phase separation, and any other visible inconsistencies.

2. pH Measurement:

- Objective: To ensure the gel's pH is suitable for topical application.

- Procedure:

- Prepare a 1% gel solution in distilled water.

- Measure the pH using a calibrated pH meter.

3. Viscosity:

- Objective: To determine the gel’s spreadability and consistency.

- Procedure:

- Use a viscometer.

- Measure the viscosity at different shear rates.

4. Drug Content Uniformity:

- Objective: To ensure even distribution of benzocaine within the gel.

- Procedure:

- Accurately weigh several samples of the gel.

- Dissolve each sample in a suitable solvent.

- Analyze benzocaine concentration using UV spectroscopy.

- Calculate the drug content uniformity across samples.

5. In Vitro Release Studies

- Objective: To study the release profile of benzocaine from the gel.

- Procedure:

- Use Franz diffusion cells with synthetic membranes.

- Fill the receptor compartment with phosphate buffer (pH 7.4).

- Place a known amount of gel in the donor compartment.

- Maintain the system at 37°C.

- Collect samples from the receptor compartment at predetermined intervals.

- Analyze the samples for benzocaine content using UV spectroscopy.

6.Skin Irritation Studies

-Objective: Ensures safety for human skin.

- Procedure:

Apply the gel to human skin and observe for any signs of irritation.

RESULT AND DISCUSSION

A] PREFORMULATION STUDY

1. Organoleptic Properties:

           
       

Table No :4 Organoleptic Properties

2.Solubility of Benzocaine:

Benzocaine is having low solubility in water, but it is moderately soluble in organic solvents like ethanol, methanol.

       
           
       
Table No :5 Solubility of Benzocaine

3. Melting Point:

For Benzocaine the experimentally determined melting point typically falls within a specific range. The melting point of Benzocaine is generally reported to be 88-92°C.

4. Characterization by UV Spectrophotometer:

The ?max (wavelength of maximum absorbance) for benzocaine typically falls around 254 nm in

UV-Vis spectroscopy. However, it's essential to verify this value in your specific experimental

conditions, as factors like solvent, concentration, and pH can influence it. Conducting a calibration

curve with known concentrations of benzocaine can help determine the exact ?max for your setup.

       
           
       
Figure:8 Calibration of Benzocaine ? max=292 nm

a. Drug FTIR

The sample is prepared by grinding benzocaine with potassium bromide powder and pressing into a pellet, or by placing a thin film of the liquid on a KBr or NaCl window. The FTIR spectrometer is set up, calibrated, and the sample is placed in the sample holder. The spectrometer is then run to identify peaks in the spectrum, such as the ester C=O stretch (1735-1750 cm??1;), aromatic C-H stretch (3050-3100 cm??1;), aromatic ring vibrations (1400-1600 cm??1;), and ester C-O stretch (1050-1300 cm??1;). The data is then compared with reference spectra for confirmation.

       
           
       
Figure 10: Drug FTIR

       
           
       
Table No. 7: Functional groups present in drug FTIR

The FTIR procedure for benzocaine with soy lecithin and Span 80 involves combining benzocaine, soy lecithin, and Span 80 in appropriate proportions. The mixture is ground with potassium bromide powder and pressed into a transparent KBr pellet. If the mixture is liquid or needs to be dissolved, a thin film is placed on a KBr or NaCl window. The FTIR spectrometer is set up, and a background scan is run with nosample or a blank KBr pellet for calibration. The sample is placed in the sample holder and scanned over the typical wavenumber range (4000 to 400 cm??1;). Data collection involves recording the absorption spectrum produced by the spectrometer. The absorption peaks identified include benzocaine, soy lecithin, and Span 80, with benzocaine having the most significant absorption peaks.

       
           
       
Table No.8: Functional groups present in drug and polymer FTIR