Formulation and Evaluation of Anti-Bacterial Microemulsion Based Gel Containing Tetracycline Hydrochloride for Topical Drug Delivery

Fig no.1. Structure of the skin

Advantages and Disadvantages of Topical drug delivery system:

Topical drug delivery system have many advantages like local treatment and direct effectiveness of medications like anti- bacterial, antifungal, acne preparation and other medications that used for different purposes. It is characterized by avoidance of gastrointestinal incompatibility and avoidance of first pass metabolism. Also more selective to a specific site, improve patient and suitability for self medication. local drug administration has some disadvantages like skin irritation or possibility of allergic reactions. Other disadvantages is low penetration of drugs of large particle size, which means not easily to be absorbed through the skin.

Factors Affecting Topical Absorption of Medications:

• Physiological factor of skin
• Skin thickness 
• Lipid content & Part of Skin
• Density of sweat glands
• Skin pH
• Blood flow
• Hydration of skin

Skin penetration:

Skin penetration refers to the process where substances, like chemicals, drugs or ingredients from skincare products, pass through the skin's outermost layer (the epidermis) and into the deeper layers. it's a crucial aspect of various applications, including skincare, drug delivery, and understanding environmental exposures.

Fig no.2 Representation of skin showing different routes of penetration.

Mechanism of Skin Penetration:

Skin penetration enhancers are the molecules which reversible to remove the barrier resistance of the stratum carenum. They allow drugs to penetrate more readily to the viable tissues and hence enter the systemic circulation. The intercellular routes accelerants may interact at the polar head groups of the lipid, within aqueous region between lipid head group and between the hydrophobic tails of the barrier. The common mechanism is to protect the body for unwanted particles from the environment. The main barrier of the skin is located in the outermost layer of skin that is epidermis. Since the lipid regions in the stratum corneum forms the only continuous structure, substance applied on to the skin always have to pass these region. The major obstacle for topical drug delivery is the low diffusion rate of drug across the stratum corneum. Several methods have been increased the permeation assessed to rate of drugs temporarily.

Microemulsion development: -

A micro emulsion is a thermodynamically stable, transparent or translucent system consisting of:

Oil phase 

Water phase (dissolved API)

Surfactant and co- surfactant (Smix) 

Drug profile – Tetracycline Hydrochloride

Fig.no.3 Tetracycline hydrochloride drug and it’s structure

Name: Tetracycline hydrochloride

Molecular formula: CoH24NO: ·HCI

Class: Broad spectrum antibiotic

Mechanism of action:

Tetracycline hydrochloride bind to to the 30s ribosomal subunit of bacteria

                                       ↓

Prevent the aminoacyl- tRNA from binding to the A-site of ribosome

                                       ↓

Inhibition of protein synthesis (Inhibition stop the amino acid)                         

Dosage forms:

Tablet 

Capsule

Topical formulations (ointment, Solution)

Adverse effects –

Nausea

Vomiting

Diarrhoea

Abdominal discomfort

Rashes

Itching

Advantages of Microemulsion system:

1. Because of their superior thermodynamic stability, microemulsions are simple to make and require minimal energy input.

2. Microemulsion creation is reversible.

3. Microemulsions allow the system to self - emulsify and are thermodynamically stable.

4. In contrast to emulsions, Microemulsion are less viscous.

5. Being able to transport both hydrophilic and lipophilic medications.

6. The hydrophilic or lipophilic dispersion phase (O/W or W/O) may serve as possible reservoir for hydrophilic or lipophilic medications, respectively.

Disadvantages of Microemulsion system:

1. Having a limited ability to dissolve compounds that melt easily.

2. A lot of surfactants are needed to stabilize the droplet.

MATERIALS AND METHODS: -

Chemicals:

1. Tetracycline hydrochloride
2. Olive oil
3. Tween80
4. Poly-ethylene glycol
5. Methyl paraben
6. Carbopol 934
7. Triethanolamine
8. Distilled water.

Tetracycline HCl solubility research -

Tetracycline hydrochloride is a water-soluble broad-spectrum antibiotic, exhibiting high solubility in aqueous media due to its ionizable hydrochloride salt form. It is freely soluble in water.

Formula –

Table no. 1 Role of ingredients

Instruments used -

1. Magnetic stirrer 

2. UV visible spectrophotometry 

3. Weighing balance 

4. Zeta sizer

5. pH meter

6. Incubator

Formula for Microemulsion [ 20 ml] –

Table no. 2 Composition of microemulsion formula

Formula for Gel base [ 20 gm] –

Table no. 3 Composition of gel base

Method –

1. Phase Titration method –

1. Firstly weigh accurate quantity of Tetracycline hydrochloride in a beaker and solubilized in distilled water.

                                       ⬇️

2. In another beaker, prepare mixture of surfactant and cosurfactant (S/CoS) and add in oil phase. Mix properly. Then dissolve drug add in (S/Cos) mixture.

                                       ⬇️

3. Titration with aqueous phase: Add water gradually drop by drop, while stirring constantly, untill the mixture becomes translucent and clear, signifying the creation of a microemulsion.

2. The alternative phase Inversion method –

1. Combine surfactant, cosurfactant and oil phase.

                                     ⬇️

2. Slowly add water while stirring until a clear microemulsion forms and inversion takes place.

B. Preparation of Microemulsion based gel-

Procedure:

1. Disperse the gelling agent (Carbopol 934) in distilled water. Allow it to hydrate.

                                     ⬇️

2. Then add preservative (methyl paraben). Adjust pH to 6-7 using triethanolamine to form a clear gel.

C. Formulation of Microemulsion based gel-

1. Slowly add the microemulsion to the gel base with gentle stirring to avoid air entrapment

                                     ⬇️

2. Check pH, Viscosity

Experimental Work-

Components Required:

Oil phase: Olive oil 

Aqueous phase: Distilled water or buffer solution

Surfactant: Tween 80

Co-surfactant: polyethylene glycol

Procedure - (By phase titration method)

A. Preparation of Microemulsion -

1. Firstly weigh accurate quantity of Tetracycline hydrochloride in a beaker and solubilized in distilled water

                                         ↓ 

2. In second beaker prepare the mixture for Tween 80 and poly- ethylene glycol and mix them together. Add oil phase. Then dissolve drug added in S/ Cos mixture.

                                         ↓                                 

3. Titration with aqueous phase: Add water gradually drop by drop while stirring constantly, untill the mixture becomes translucent and clear.

                                          ↓                

4. When the clear, stable microemulsion was formed then stirr this whole mixture on magnetic stirrer for 1 hour.

Fig no. 4 Preparation of Microemulsion

B. Preparation of Microemulsion based gel-

1. Prepare gel base:

Disperse the gelling agent (Carbopol 934) in distilled water, allow it to hydrate.

2. Neutralize:

Add in preservative, and adjust pH to 6 – 7 using triethanolamine to form a clear gel.

Fig no.5 Gel base

C. Formulation of Microemulsion based gel-

Slowly add the microemulsion to the gel base with gentle stirring to avoid air entrapment.

Check pH and viscosity.

Fig no.6 Microemulsion based gel

Evaluation of Microemulsion: -

Evaluation of microemulsion involves assessing their physical and chemical properties. Here's a structured overview of key evaluation parameters:

1. Physical Evaluation -

1. Appearance:

Microemulsion are typically light yellow coloured or slightly opalescent. Visual inspection can reveal phase separation of turbidity.

 1.2. Determination of pH:

Fig no. 5 pH of the Microemulsion

Accurately measure 1ml of prepared microemulsion and dispersed in 100ml of distilled water. The pH was measured by digital pH meter. In order to ensure that formulation can be used without the harm of kin irritancy, the pH (5.9) of the preparation has been determined.

1.3. Droplet size & Distribution :

Measured using Zeta size. Microemulsion usually have droplet sizes between 50 - 500 nm.

1.4. Zeta potential:

It indicates surface charge and stability of the formulation.

General zeta potential ranges:

+0 to +10 mV: Low stability (particles likely to aggregate)

+10 to +30 mV: Moderate stability 

+30 mV or more: High stability (good electrostatic repulsion).

The zeta potential (29 mV) has been determined.

1.5. Viscosity:

The viscosity (1.613cp) of the preparation has been determined.

1.6. Conductivity: 

Helps differentiate between oil- in- water ( O/W) (10 to1000uS/cm) and water- in- oil (W/O) systems (<10uS/cm). The conductivity (595us/cm) of the preparation has been determined.

Evaluation of Gel –

1. Determination of pH-           

Fig no 7. pH of Carbopol gel

Accurately measure 1gm of prepared gel dispersed in 10ml of distilled water. The pH was measured by digital pH meter. In order to ensure that formulation can be used without the harm of skin irritancy, the pH (5.5) of the preparation has been determined.

2. Appearance – Gel are typically transparent. Highly viscous in nature.

Evaluation of microemulsion based gel –

1. Physical Evaluation –

1.1 Appearance – Should be homogenous, smooth and slightly opalescent.

1.2 Determination of pH – Accurately measured 1gm of prepared microemulsion based gel and dispersed in 10ml distilled water. The pH was measured by digital pH meter. In order to ensure that formulation can be used without the harm of skin irritancy, the pH (6.3) of the preparation has been determined.

Fig no 8. pH of Microemulsion based gel

1.3 Colour –

The formulation were viewed for their visual appearance and for no colour change. White colour is obtained.

1.4. Consistency of microemulsion –

To determine the consistency of the prepared gel, a small amount of gel was squeezed between the thumb and the index finger and the consistency of the gel was observed and determined.

1.5 Homogeneity of Microemulsion based gel-

The formulated gel was visually inspected for Homogeneity after they were stored for 7 days in the container and examined for availability of any aggregates and appearance.

1.6 Spreadability –

The spreadability of the microemulsion gel was tested using two glass slides. The formulation whose spreadability was to be evaluated was spread across one slide, and the gel was sandwiched between the two by placing the other slide on top of it. The slides were pressed together to eliminate any potential air before the adhering gel was removed. It was recorded how long it took the upper slide to fully separate from the lower slide. Where T is the time it takes to separate the slide, 1 is the length of glass slide, and M is the weight attached to the upper slide.

 Formula: S= M*L/T

Where:

S= Spreadability

M= Weight tied to upper slide (g)

L= Length of glass slide (cm)

T= Time taken to separate slides (sec)

M=20g

L=7.5cm

T=20sec

Then, S= 20×7.5÷20 = 7.5g.cm/see

The spreadability of microemulsion based gel was found to be 7.5cm

2.Chemical Evaluation –

2.1 Drug Content -Amount of active pharmaceutical ingredient ( API) in the formulation, measured using UV – Visible spectrophotometer.

Procedure

1. Preparation of Standard Solution

o Accurately weigh 10 mg of tetracycline hydrochloride.

o Dissolve in a small amount of methanol or buffer.

o Transfer to a 100 mL volumetric flask and make up the volume - 100 μg/mL stock solution.

o Prepare suitable dilutions (e.g, 2-10 μg/ml) to construct – calibration curve.

2. Preparation of Sample Solution

o Weigh accurately 1 g of microemulsion gel. Transfer into a 100 mL volumetric flask.

o Add about 50 mL of methanol or phosphate buffer (pH 6.8).

o Sonicate or stir for 20-30 minutes to extract the drug completely.

o Make up the volume to 100 mL.

o Filter the solution using Whatman filter paper.

o Dilute appropriately if required.

3. Measurement

o Measure the absorbance of the sample solution at  kmax (= 275 nm)using a UV spectrophotometer.

o Use the blank (solvent) as reference.

Fig no. 9 Result of UV- Visible spectrophotometer

Table no 5. Reading of calibration curve

Drug Content (%) = Actual amount of drug

                                                                                     ×100

                                 Theoretical amount of drug

Drug Content (%) =      492

                                                                   × 100

                                       500

=0.984×100

=98.4%

Drug Content was found to be 98.4%

Biological Evaluation –

Antibacterial Test –

Procedure -

1. Test organism (E-coli, and streptococcus) spread on the muller hinter agar.

2. Then, wells are made on it with the help of cork borer.

3. Then inoculated the sample into these wells.

4. Keep it in freeze for 10 min

5. For diffusion of sample, then incubated the plates at 37’ C in incubator for 24hrs.

Fig no .10 Antibacterial Activity

RESULT AND DISCUSSION:

Table no. 5 Evaluation parameter of microemulsion, Gel, Microemulsion based gel