Formulation and Evaluation of Nanoemulsion Based Hydrogel Using Erythromycin for Bacterial Infectionss

Introduction

Drugs are transported from a topical product to a local target site via diffusion, metabolism, and dermal circulation before being eliminated from the body.¹ Numerous commonly used topical drugs, such as ointments, creams, and lotions, have several drawbacks, as they are often sticky and make patients uncomfortable when used. Within the main category of semisolid preparations, these formulations also have a lower spreading coefficient, require rubbing during application, and present stability issues. As a result, gels has become more popular in both cosmetic and pharmaceutical preparations.² Gels are semisolid preparations that contain one or more drugs in either a hydrophobic or hydrophilic base. Compared to other topical dose forms, gels have greater potential for administering drug topically because they require less energy for production.³

Nanoemulsions are a mixture of transparent or translucent oil globules dispersed in an aqueous phase and stabilised by an interfacial barrier created by extremely small droplet size surfactant or co-surfactant molecules. Nanoemulsions have great wettability due to their small particle size, larger specific surface area, and low surface tension, allowing for close contact with the skin.⁴ Incorporating nanoemulsion into a hydrogel base is an effective strategy to form a novel dosage form known as a nanoemulgel (NEG) for transdermal drug administration. NEG can improve drug penetration through the skin. In addition to its good rheological properties and the dual effects of nanoemulsion and hydrogel, it can also improve drug adherence to the skin, resulting in a larger concentration gradient towards the skin. Incorporating a drug into a NEG formulation may also improve its stability and assure sustained release.⁵

Erythromycin (ERY), a macrolide compound with a broad antibiotic spectrum, is currently being used to treat a variety of bacterial infections affecting the skin and has shown to be quite beneficial from a therapeutic perspective. Despite having significant benefits, ERY has a number of disadvantages that pose significant therapeutic challenges. Among them, their extremely low solubility in water and instability in acidic environments limit their efficacy and bioavailability. A wide range of ERY formulations, including nanoparticles, have been developed during the past few decades. Hence in this research, an attempt was made to formulate erythromycin nanoemulsion based hydrogel using tea tree oil to treat bacterial infections and provide sustained release of drug to maintain effective drug concentration.

Materials and Methods

Erythromycin was obtained as a gift sample from Alembic Pharmaceuticals, Vadodara, Gujarat. Tea tree oil (Multilabs, Bangalore), Carbopol 934 (HiMedia Laboratories), Methanol and Ethanol (CDH laboratory, New Delhi), Tween 80 (Sd fine-Chem Ltd, Mumbai).

Preformulation Studies⁶

Preformulation studies are a set of experiments that are conducted to understand the physical, chemical, and biopharmaceutical properties of a new drug substance.

Organoleptic characteristics⁶

The physical characteristics of the drug sample, such as appearance, colour, and odour, were evaluated.

Solubility studies⁶

The solubility of erythromycin was evaluated in solvents like ethanol, methanol, dimethyl sulfoxide, 0.05 M phosphate buffer pH 6.8, and distilled water.

Melting point⁷ Melting point of erythromycin was determined using the capillary tube method. A small amount of erythromycin was placed on a clean, flat surface, and a capillary tube sealed at one end was taken. The open end of the tube was pushed facilitating the drug entry into the tube. The capillary tube was then placed inside the Mel-Temp apparatus chamber. 

Estimation of Erythromycin by UV-spectrophotometric method⁶

a) Determination of λmax

The absorption spectra were determined over the wavelength range of 200-400 nm, with 0.05 M phosphate buffer pH 6.8, in quartz cuvettes of 1 cm width, using double beam UV-Visible spectrophotometer (Shimadzu UV-1900).

b) Standard calibration curve for erythromycin using 0.05 M phosphate buffer pH 6.8

c) Preparation of standard stock solution

1. Primary stock solution - 100 mg of accurately weighed erythromycin was dissolved in a 100 mL volumetric flask with 0.05 M phosphate buffer pH 6.8 as solvent and volume was made up to mark with the same to obtain a concentration of 1000 µg/mL.
2. Secondary stock solution - From the above solution, 10 mL was transferred into a 100 mL volumetric flask to obtain a concentration of 100 µg/mL.
3. Working standard solution - The aliquots of 1, 2, 3, 4, 5, 6, 7 µg/mL solutions were prepared in 10 mL volumetric flasks from the above solution and the final volume was made up with 0.05 M phosphate buffer as a solvent. The absorbance was measured at 234 nm using 0.05 M phosphate buffer pH 6.8 as a blank.

Formulation Studies

Development of pseudo-ternary phase diagram^8,9

The existence of nanoemulsion zones was determined by means of a pseudo-ternary phase diagram. Tea tree oil was selected as the oil phase. Tween 80 and ethanol were used as surfactant and co-surfactant, respectively. Water was used as an aqueous phase for the construction of phase diagram. The pseudo-ternary phase diagram was generated using titration method. In order to optimise and determine the ideal ratio based on the largest covering area of the nanoemulsion, a mixture of the surfactant-Tween 80 and the cosurfactant-ethanol was utilised in four different weight ratios (1:1, 2:1, 3:1, and 4:1). Different ratios, including 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, were selected for optimization and identification of the ideal concentration of oil to Smix. The phase diagram was constructed using ternaryplot.com.

Screening of formulation by central composite design¹⁰

The screening of oil and Smix was done using central composite design (Design Expert 13.0). Based on the central composite design, 11 formulations were prepared. The formulations were coded as E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11.

Independent variables - Oil concentration, Smix ratio.

Dependent variables (Responses) - Drug content, Globule size, Transmittance, Polydispersity index (PDI).

Preparation of nanoemulsion

Separate beakers containing varying quantities of oil and Smix were heated to 650C. Once they attained the required temperature, the S_mix was added to the oil phase and stirred on a magnetic stirrer for 15 minutes, maintaining the temperature at 100 rpm. 1% erythromycin was added to this mixture and stirred until the drug was fully dissolved. The aqueous phase was heated to 650C separately and then the oil phase was added to the aqueous phase dropwise with continuous stirring for 20 minutes till a yellow clear emulsion was formed. After developing the emulsion, it was sonicated using a probe sonicator for particle size reduction.

Central composite design for formulation of nano-emulsion

Evaluation parameters of nanoemulsion^6,11

1. Appearance

Visual examination of the generated emulsion formulations was done to assess colour, phase separation, and homogeneity.

2. pH

Using a calibrated digital pH metre, the pH of the developed nanoemulsion was determined.

3. Percentage transmittance

Transmittance was measured using a UV spectrophotometer. Each nanoemulsion formulation was diluted 100-fold with distilled water. Following that, using distilled water as a blank, the % transmittance was determined at 234 nm. 

4. Viscosity

Using spindle #62 at 100 rpm and torque varying from 20% to 90%, a Brookfield digital viscometer (DV-II+ Pro) was used to measure the viscosity of the formulations.

5. Particle size and Polydispersity index

The particle size and polydispersity index of the formulations were evaluated using a Malvern particle size analyzer.

6. Drug content

The drug content was determined by dissolving the form ulation equivalent to 10 mg of drug in methanol, followed by 0.05 M phosphate buffer pH 6.8. The solution was then filtered and diluted, and the resulting solution was analysed at 234 nm in the UV Visible spectrophotometer.

7. In vitro drug release studies

In vitro drug release of erythromycin loaded nanoemulsion was performed using Franz-diffusion cell. Dialysis membrane was immersed in phosphate buffer pH 6.8 for 24 hours after being rinsed with distilled water for 10 minutes. Then the dialysis membrane was placed between donor compartment and receptor compartment. Nanoemulsion equivalent to 10 mg of drug was taken in the donor compartment and the receptor compartment was filled with 0.05 M phosphate buffer 6.8. On a magnetic stirrer set to 50 RPM and 37±0.5°C, the media in the receptor compartment was stirred. At various time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 24th hour), 1 mL of sample was withdrawn from the receptor solution, and was replaced with fresh buffer solution. The resulting solution was diluted and analysed using UV-Visible spectrophotometer at 234 nm.

8. Zeta potential

The zeta potential of optimized formulation (E7) was measured using a Malven Zeta Sizer.

Formulation of nanoemulgel¹¹

 Two steps were used in the preparation of the nanoemulgel.

1. Step 1: The gel formulation was prepared by dissolving Carbopol 934 (1%, 1.5%, and 2%) in slightly warm distilled water, followed by stirring with a magnetic stirrer for 30 minutes. The mixture was then left to hydrate and swell for 24 hours.
2. Step 2: The optimized nanoemulsion and gel formulation were mixed in 1:1 ratio. Triethanolamine was used to adjust the pH levels of the formulation.

Evaluation of nanoemulgel

1. Physical appearance¹² The colour, phase separation, homogeneity, and consistency of the prepared emulgel formulations were visually assessed. 

2. pH¹³ The pH was assessed after dissolving one gram of nanoemulgel in 100 mL of distilled water and storing the mixture for two hours.

3. Viscosity¹³ The viscosity of the formulation was measured using a Brookfield digital viscometer (DV-II+ Pro) with spindle #64 at 100 rpm and torque range of 20% to 90%.

4. Drug content¹⁴ The drug content was determined by dissolving the nanoemulgel equivalent to 10 mg of drug in methanol, followed by 0.05 M phosphate buffer pH 6.8. The solution was then filtered and diluted with 0.05 M phosphate buffer pH 6.8 and analysed under UV spectrophotometer at 234 nm.

5. In vitro drug release study¹¹

Franz diffusion cells were used to measure the in vitro drug release study. Dialysis membrane was immersed in 0.05 M phosphate buffer pH 6.8 for 24 hours after being rinsed with distilled water for 10 minutes and it was mounted between donor and receptor compartments. The entire assembly was set up on a magnetic stirrer spinning at 50 rpm after the receptor chamber was filled with 0.05 M phosphate buffer pH 6.8 as a diffusion medium. In the donor chamber, 1 g of nanoemulgel was evenly spread across the membrane. The receptor chamber was filled with fresh buffer solution, and samples were withdrawn at predetermined intervals: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 hours. After filtration and appropriate dilution with buffer, the samples were examined using a UV spectrophotometer set to λ_max 234 nm.

Antimicrobial Study¹⁵

The antimicrobial activity of the optimized nanoemulsion (E7) was evaluated using the cup and plate method. In this method, a cylinder containing antibiotic is diffused into the agar layer containing the microorganisms. The activity of the nanoemulsion against the microorganisms was measured and compared to the activity of the standard reference drug erythromycin.

The microorganisms used were: Gram negative - Escherichia coli (E. coli) and Gram positive - Staphylococcus aureus (S. aureus).

Preparation of agar media

Fourteen grams of nutrient agar was taken in a conical flask and dissolved in 500 mL of water by heating for 20 minutes. The media was then sterilized in an autoclave for 15 minutes at 15 psi pressure and 121°C.

Preparation of inoculum

The bacterial stock cultures were sub cultured onto fresh nutrient media, and were then incubated for 24 hours at 37°C.

Preparation of standard solution of erythromycin

Five milligrams of eythromycin was dissolved in 1 mL dimethylformamide to obtain a concentration of 5 mg/ mL and was used for the antimicrobial study.

Procedure for antimicrobial study

• The petri plates and agar media were autoclaved for 15 minutes at 15 psi pressure and 121°C . The working area was disinfected using ethanol and the sterilized agar media and petri plates were stored in a sterile environment. Once cooled to room temperature, agar media was poured into petri plates and allowed to solidify.
• A swab of pure bacterial culture was evenly spread over agar plates using a sterile non-absorbent cotton. Wells were then created with a borer and the nanoemulsion was poured into the bore at different concentrations using a micropipette.
• The standard solution of erythromycin was also introduced into the well as a reference to compare the activity of microorganisms.
• The plates were incubated at 37°C for 24 hours, after which the zones of inhibition around the samples were observed and recorded.

Stability Studies^16,17 

Stability studies were conducted in accordance with the International Council for Harmonization (ICH) regulations. The formulations were stored for three months in a stability chamber at 40±20C and 75±5% RH. The samples were withdrawn at the end of storage period and analyzed. The change in pH of the gel or drug deterioration were used to measure the stability.

Results

Preformulation Studies

Organoleptic properties

Organoleptic properties of erythromycin pure drug were found to be as per I.P. monograph. Erythromycin is white in colour, odourless, and appears as a crystalline powder.

Solubility studies

Erythromycin exhibits the following solubility profile in various solvents. It is freely soluble in ethanol, soluble in methanol and dimethyl sulfoxide, sparingly soluble in 0.05 M phosphate buffer at pH 6.8, and slightly soluble in distilled water.

Melting point

The reference melting point of erythromycin is 135°C to 140°C, while the observed melting point was 137 ± 2.64°C.

Standard calibration curve of erythromycin by UV-Visible spectroscopy

Medium : 0.05 M phosphate buffer pH 6.

λmax : 234 nm

Beer’s range : 1-7 µg/mL

Formulation and Evaluation of Nanoemulsion

Development of pseudo-ternary phase diagram

Using the previously discussed method, pseudo-ternary phase diagrams were developed. The isotropic nanoemulsion region was presented as shadowed area. The maximum isotropic region was observed at 3:1 ratio of Tween 80 and ethanol as the Smix, and nanoemulsions were formulated using this ratio.

Evaluation of nanoemulsions

All the formulations were transparent and clear with the particles fully dispersed in the emulsion. Among 11 formulations, E7 demonstrated 74.17±1.18% drug release at the end of 24 hours. The zeta potential of E7 was found to be +45.6mV, indicating the stability of nanoemulsion.

Formulation & Evaluation of Nanoemulgel

Based on the results of particle size, PDI, pH, viscosity, drug content, % drug entrapment and % drug release, formulation E7 was selected as the optimized emulsion. It was then converted into a nanoemulgel using different concentrations of Carbopol gel base (1%, 1.5%, 2%) to produce erythromycin nanoemulgels (E7F1, E7F2, E7F3).

Evaluation of nanoemulgel

Among the three formulations, E7F1 showed better drug release profile of 72.02±0.33% at the end of 24 hours. Based on the results of pH, viscosity, spreadability, drug content and % drug release formulation, E7F1 was selected as the best formulation.

Stability Studies

Formulation E7F1 was selected for accelerated short stability studies. After three months, no significant changes were observed in homogeneity, pH, viscosity, spreadability, extrudability, % drug content and in vitro drug release, compared to the initial values. These results indicate good stability of the prepared nanoemulgel.

Discussion

Erythromycin (ERY), a macrolide compound with extensive antibiotic properties, offers significant therapeutic benefits in treating diverse bacterial skin infections. Despite having significant benefits, ERY has several disadvantages such as extremely low solubility in water and instability in acidic environments. Compared to other formulations like alcohol-based solutions, the gel base is an appropriate vehicle for topical applications to prolong the residence time of drug on the surface layer of skin. Hence in this research, an attempt was made to formulate an erythromycin nanoemulsion based hydrogel using tea tree oil, for treating bacterial infections and providing sustained release of drug to maintain effective therapeutic concentrations.

The drug was subjected to preformulation studies, followed by formulation and evaluation. Using a pseudoternary phase diagram, the existence of nanoemulsion regions was determined. Water, tea tree oil, and Smix (Tween 80 and ethanol) were used to create the pseudoternary diagrams. Central composite design was used to optimize the nanoemulsion (CCD) and was evaluated for pH, viscosity, globule size, PDI, drug content, and zeta potential. The nanoemulsion with the lowest concentration of oil (8.32% w/w) showed a desired globule size of 13.61, a PDI of 0.168 with 98.8±0.75% drug content and 74.17±1.18% drug release at the end of 24 hours and a zeta potential of +45.6mV. Based on these results, formulation E7 was selected as the optimized formulation and was then incorporated into different concentrations of Carbopol gel base (1%, 1.5%, 2%) to obtain erythromycin nanoemulgel (E7F1, E7F2, E7F3).

Among the three formulations, E7F1 showed better results with a drug content of 98.6±0.63% and a drug release of 72.02±0.33%, at the end of 24 hours. The antimicrobial tests demonstrated that formulation E7 had the ability to inhibit the growth of both E. coli and S. aureus. The zone of inhibition produced against E. coli was more clear and well-defined compared to S. aureus.

Conclusion

The goal of the present research was to formulate an erythromycin nanoemulsion based hydrogel using tea tree oil for wound healing. Erythromycin is a macrolide antibiotic used to treat a variety of bacterial infections, belonging to BCS class III. However, it has several disadvantages, including gastrointestinal upset, drug interactions, and allergic reactions. Additionally, oral erythromycin exhibits relatively low bioavailability. To address these disadvantages, topical erythromycin nanoemulgel was developed. Furthermore, the incorporation of tea tree oil into erythromycin enhances its antimicrobial activity against a wide range of bacteria. Thus, it can be concluded that the objective of this research was achieved by developing erythromycin nanoemulgel with improved therapeutic efficacy and reduced side effects.

Conflict of Interest

The author declares that there is no conflict of interest