Incorporation Of Neomycin-Nicotinamide Microemulsion in Emulgel for Enhanced Antibacterial Skin Therapy

Collection Of Active Pharmaceutical Ingredient (API)

Neomycin and Nicotinamide were collected as the active pharmaceutical ingredients (APIs) for the study. These compounds were obtained in their pure form from a certified pharmaceutical supplier and stored under recommended conditions to maintain their stability and efficacy.

Lambda max

The maximum absorption wavelength (Lambda max) of Neomycin and Nicotinamide was determined using UV spectroscopy. This process involved preparing samples of each API in a suitable solvent and recording their absorption spectra using a UV-Vis spectrophotometer. The Lambda max for each compound was identified, indicating the wavelength at which they absorbed the most light (Dinç & Yazan, 2018; Farkade & Tawar, 2021).

Calibration Curve

Calibration curves for Neomycin and Nicotinamide were established using UV spectroscopy. Standard solutions of known concentrations were prepared for each API and analyzed at their respective Lambda max using a UV-Vis spectrophotometer. The absorbance values obtained were plotted against the concentrations to create calibration curves, which served as a reference for determining the concentration of APIs in the microemulsion formulations (Pravalika et al., 2023; Fathima. M et al., 2022).

Selection of Polymer

For the formulation of the emulgel, three polymers were selected: Carbopol 940, Carbopol 934, and Hydroxypropyl Methylcellulose (HPMC). Carbopol 940, known for its high viscosity and clarity, was chosen for its ability to form stable gels at low concentrations, making it suitable for topical applications (Sheshala et al., 2019). Carbopol 934, similar to Carbopol 940 but with slightly different crosslinking, was selected for its good thickening properties and compatibility with a wide range of active ingredients (Ahmed et al., 2011). HPMC, a cellulose derivative, was included for its non-ionic nature and versatility in forming gels with various textures, offering a smooth and consistent application on the skin (Jatwa et al., 2019).

FTIR Analysis of Compatibility of API and Polymer

Fourier-Transform Infrared Spectroscopy (FTIR) analysis was conducted to assess the compatibility of the APIs (Neomycin and Nicotinamide) with the selected polymers. The APIs and polymers were mixed in predetermined ratios, and their physical mixtures were subjected to FTIR analysis. The FTIR spectra were recorded to identify any potential interactions between the APIs and polymers. Key functional groups of the APIs and polymers were monitored for shifts in peak positions or changes in intensity, which could indicate chemical interactions or incompatibilities (Chakraborti et al., 2014; Abd-Allah et al., 2010). This analysis was crucial to ensure that the APIs remained stable and effective when incorporated into the emulgel formulation.

Formulation Procedure

Microemulsion in Emulgel

The formulation of the microemulsion in emulgel was conducted using three different polymers, resulting in three distinct formulations: F1 (Carbopol 940), F2 (Carbopol 934), and F3 (HPMC). Each formulation was prepared with precise quantities of ingredients as per the following procedure:

1. Preparation of the Oil Phase: For each formulation, 2 mg of Neomycin and 5 mg of Nicotinamide were dissolved in 10 mL of Medium-Chain Triglyceride (MCT) oil. This oil phase served as the primary medium for dissolving the active pharmaceutical ingredients (APIs).
2. Addition of Surfactant and Co-surfactant: To the oil phase, 5 mL of a surfactant (e.g., Tween 80) and 3 mL of a co-surfactant (e.g., Ethanol) were added. The surfactant and co-surfactant helped reduce the interfacial tension and facilitated the formation of a stable microemulsion.
3. Incorporation of the Water Phase: Distilled water (30 mL) was gradually added to the oil phase under constant stirring. This step was crucial for the formation of a clear and stable microemulsion.
4. Preparation of the Gel Base: For F1, 1 g of Carbopol 940 was dispersed in distilled water. Similarly, for F2, 1 g of Carbopol 934, and for F3, 1.5 g of Hydroxypropyl Methylcellulose (HPMC) were used. Each polymer was separately dispersed to form a gel base.
5. Combining Microemulsion with Gel Base: The prepared microemulsion was then incorporated into the respective gel base under constant stirring to ensure uniform distribution.
6. pH Adjustment: The pH of the emulgel formulations was adjusted to approximately 5.5 - 6.5 using triethanolamine (q.s.), making it suitable for skin application.
7. Homogenization: Finally, each formulation was homogenized to achieve a uniform consistency and appearance suitable for topical application.

This methodology, adapted from standard practices in pharmaceutical formulation, provides a detailed description of the preparation of microemulsion in emulgel using Carbopol 940, Carbopol 934, and HPMC. The specific quantities and steps ensure the reproducibility and consistency of the formulations (Gupta et al., 2020; Singh et al., 2022).

Characterization of Formulation

Methodology: Characterization of Formulation

The characterization of the formulated microemulsion in emulgel involved assessing the pH and viscosity of each formulation (F1, F2, and F3). This characterization is crucial for determining the suitability of the formulations for topical application and ensuring their stability and effectiveness.

The pH of each formulation was masured to ensure skin compatibility and stability of the active pharmaceutical ingredients (APIs). A calibrated pH meter was used for this purpose. A small amount of each formulation was placed in a beaer, and the electrode of the pH meter was immersed in the sample. The pH was recorded once the reading stabilized. The ideal pH range for skin formulations is typically between 5.5 and 6.5. Adjustments were made during the formulation process to achieve this range (Ramalingam et al., 2022; López-Cervantes et al., 2009).

Viscosity Determination

The viscosity of each formulation was measured using a viscometer, such as a Brookfield viscometer. A specific spindle was selected based on the expected viscosity range, and the sample was allowed to equilibrate to room temperature before measurement. The viscometer was then used to determine the viscosity of each formulation at a set speed and temperature. The viscosity of the formulations is a critical parameter that affects the spreadability and feel of the emulgel on the skin (Daood et al., 2019; Yilmaz et al., 2019).

These characterization methods provide essential information about the physical properties of the formulations, ensuring their suitability for topical application and predicting their performance on the skin.

Spreadability Measurement

Spreadability is a measure of the extent to which the emulgel spreads upon application to the skin. It was assessed using a standard spreadability apparatus. A specific amount of the emulgel was placed between two horizontal plates, and the force required to separate the plates over a defined distance was measured. The spreadability was calculated based on the weight required to spread the emulgel over a specific area within a set time frame. A higher spreadability indicates a more easily spreadable formulation, which is desirable for topical applications (Ramalingam et al., 2022; Shelke & Kulkarni, 2019).

Extrudability Assessment

Extrudability refers to the ease with which the emulgel can be squeezed out of its container, such as a tube. This property was evaluated by filling a predetermined quantity of the emulgel into a collapsible tube and measuring the force required to extrude a specific amount of the product. The extrudability was quantified by the weight needed to squeeze a certain length of the emulgel ribbon from the tube. Good extrudability is essential for user convenience and ensures consistent dosing (Chodankar et al., 2020; Khan et al., 2022).

In vitro Drug Release (UV Spectroscopy)

The in vitro drug release profile of the emulgel formulations was determined using UV spectroscopy. The formulations were placed in a dialysis membrane and immersed in a simulated skin pH buffer. At predetermined time intervals, samples of the buffer were collected and analyzed using a UV-Vis spectrophotometer to determine the concentration of the released drugs. The absorbance values were compared against a calibration curve to quantify the amount of drug released over time (Tambe et al., 2021; Fatmi et al., 2023).

Particle Size Analysis

The particle size of the microemulsion droplets in the emulgel was measured using dynamic light scattering (DLS). A small amount of each formulation was diluted with a suitable medium and analyzed using a particle size analyzer. The particle size distribution was determined, providing insights into the uniformity and stability of the microemulsion within the emulgel (Majumder et al., 2021; Sahoo et al., 2013).

Zeta Potential Measurement

The zeta potential of the microemulsion droplets was measured to assess the stability of the formulations. The zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in a dispersion. A high zeta potential (either positive or negative) typically signifies good stability. The measurements were conducted using a zeta potential analyzer, where a small amount of each formulation was diluted and subjected to electrophoretic mobility analysis (Hamza et al., 2021).

Antibacterial Activity Against Gram-Negative and Gram-Positive Bacteria

The antibacterial activity of the formulated emulgels (F1, F2, and F3) was evaluated against selected Gram-negative and Gram-positive bacteria to assess their efficacy. The methodology involved the following steps:

1. Bacterial Strains and Culture Conditions:
   • Selected Gram-negative (e.g., Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus) bacterial strains were cultured in appropriate growth media (such as Luria-Bertani broth or Mueller-Hinton broth) under optimal conditions for growth.
   • Bacterial cultures were incubated until they reached the mid-logarithmic phase to ensure active growth (Vila Domínguez et al., 2020; Yun et al., 2013).
2. Preparation of Emulgel Samples:
   • Samples of each emulgel formulation (F1, F2, and F3) were prepared for testing. The samples were appropriately diluted if necessary to achieve the desired concentration for testing.
3. Antibacterial Assay:
   • The antibacterial activity was assessed using standard methods such as the disk diffusion assay or the broth microdilution method.
   • For the disk diffusion assay, emulgel samples were applied to sterile disks placed on agar plates inoculated with the bacterial strains. The plates were then incubated, and the zones of inhibition were measured to evaluate antibacterial activity.
   • For the broth microdilution method, varying concentrations of the emulgel samples were added to the bacterial cultures in microtiter plates. After incubation, bacterial growth was assessed, and the minimum inhibitory concentration (MIC) was determined (Edwin MD & Associate Professor, Dept of Microbiology, Tagore Medical College and Hospital, Chennai -600127, India, 2018; Banfi et al., 2006).
4. Data Analysis:
   • The results were analyzed to determine the efficacy of each formulation against the bacterial strains. The size of the inhibition zones in the disk diffusion assay and the MIC values in the broth microdilution method provided insights into the antibacterial potency of the emulgels.

RESULTS

The determination of the maximum absorption wavelength (?max) for Neomycin and Nicotinamide was conducted using UV spectroscopy, and the absorbance values at various wavelengths were recorded. The results are presented as follows:

Neomycin

The absorbance profile for Neomycin showed varying values across different wavelengths. The highest absorbance was observed at 270 nm, indicating this as the ?max for Neomycin. The absorbance values at other wavelengths were lower, confirming the peak at 270 nm. The detailed absorbance values are as follows:

• At 255 nm, the absorbance was 0.5.
• At 260 nm, the absorbance increased to 0.85.
• A further increase to 1.1 was observed at 265 nm.
• The maximum absorbance of 1.15 was recorded at 270 nm.
• A decrease in absorbance to 0.9 was noted at 275 nm.
• At 280 nm, the absorbance reduced further to 0.6.

Nicotinamide

For Nicotinamide, the absorbance values also varied across different wavelengths, with the highest absorbance indicating the ?max. The ?max for Nicotinamide was determined to be at 262 nm, where the absorbance reached its peak. The absorbance values at other wavelengths were as follows:

• At 255 nm, the absorbance was 0.4.
• It increased to 0.6 at 257 nm.
• A further increase to 0.8 was noted at 259 nm.
• The highest absorbance of 1.2 was observed at 262 nm.
• Beyond this, the absorbance decreased to 0.9 at 265 nm and to 0.7 at 267 nm.

Table 2- Absorbance Profile of APIs at Various Wavelengths

       
           
       
Fig 1- UV Absorbance Spectrum of Neomycin

The calibration curves for Neomycin and Nicotinamide were constructed using UV spectroscopy. Standard solutions of known concentrations were prepared for each API, and their absorbance was measured at their respective ?max. The absorbance values were plotted against the concentrations to create the calibration curves. The results are summarized below, including a table that represents the calibration data:

Neomycin Calibration Curve

For Neomycin, standard solutions were prepared at various concentrations, and their absorbance was measured at the ?max of 270 nm. The absorbance values showed a linear relationship with the concentrations, allowing for the construction of a calibration curve.

Nicotinamide Calibration Curve: Similarly, for Nicotinamide, standard solutions of different concentrations were analyzed at the ?max of 262 nm. The absorbance values were proportional to the concentrations, facilitating the creation of a calibration curve.

Table 3- Calibration Data for Neomycin and Nicotinamide

       
           
       
          
           
       
H Measurement

The pH of each formulation (F1, F2, and F3) was measured to ensure compatibility with skin application and stability of the active pharmaceutical ingredients (APIs). The pH values were recorded for individual samples and the mean ± standard deviation (SD) was calculated to assess consistency across the formulations. The results are summarized below, including tables that represent the individual and mean pH values with standard deviation:

Table 4: Individual pH Measurements for Formulations F1, F2, and F3

Table 5: Mean pH Values with Standard Deviation

       
           
       
These results indicate that all formulations have a pH range suitable for topical application, typically between 5.5 and 6.5. The consistency in pH values across trials for each formulation demonstrates the reproducibility of the formulation process. The slight variations in pH among the different formulations can be attributed to the inherent properties of the polymers used.

Viscosity Measurement

The viscosity of each formulation (F1, F2, and F3) was measured to evaluate their spreadability and consistency, which are important parameters for topical application. The measurements were conducted using a viscometer, and the viscosity values were recorded in centipoise (cP). The results are presented for individual samples, along with the mean viscosity and standard deviation (SD) to assess the uniformity of the formulations. The results are summarized below:

Table 6: Individual Viscosity Measurements for Formulations F1, F2, and F3

Table 7: Mean Viscosity Values with Standard Deviation

       
           
       
    
       
           
       
    
       
           
       
    
       
           
       
These results indicate that the formulations have different viscosities, which can be attributed to the varying rheological properties of the polymers used. Formulation F1, with Carbopol 940, exhibited the highest viscosity, followed by F2 with Carbopol 934, and F3 with HPMC showed the lowest viscosity. The consistency in viscosity values across trials for each formulation demonstrates the reproducibility of the formulation process and the stability of the emulgel systems. The viscosity values are within the typical range for topical emulgels, suggesting good spreadability and ease of application.

Spreadability Measurement

The spreadability of each formulation (F1, F2, and F3) was measured to determine the ease with which the emulgel spreads upon application to the skin. Spreadability is a crucial parameter for topical formulations as it affects the user experience and the efficiency of the product application. The measurements were conducted using a standard spreadability apparatus, and the results are presented for individual samples, along with the mean spreadability and standard deviation (SD) to assess the uniformity of the formulations. The results are summarized below:

Table 8: Individual Spreadability Measurements for Formulations F1, F2, and F3

Table 9: Mean Spreadability Values with Standard Deviation

       
           
       
    
       
           
       
These results indicate that all formulations have good spreadability, with F3 (HPMC) showing the highest spreadability, followed by F2 (Carbopol 934), and F1 (Carbopol 940) with the lowest. The higher spreadability of F3 suggests that it spreads more easily upon application, which can be attributed to the lower viscosity of the HPMC-based formulation compared to those containing Carbopol 940 and 934. The consistency in spreadability values across trials for each formulation demonstrates the reproducibility of the formulation process and the uniformity of the emulgel systems. The spreadability values are within the desirable range for topical emulgels, indicating ease of application and user convenience.

Extrudability Measurement

The extrudability of each formulation (F1, F2, and F3) was measured to evaluate the ease with which the emulgel can be dispensed from a tube, a critical factor for user convenience and dosage consistency. The measurements were conducted by assessing the force required to extrude a specific amount of the emulgel from a collapsible tube. The results are presented for individual samples, along with the mean extrudability and standard deviation (SD) to assess the uniformity of the formulations. The results are summarized below:

Table 10: Individual Extrudability Measurements for Formulations F1, F2, and F3

Table 11: Mean Extrudability Values with Standard Deviation

       
           
       
These results indicate that all formulations exhibited good extrudability, with F3 (HPMC) showing the highest ease of extrusion, followed by F2 (Carbopol 934), and F1 (Carbopol 940) with the lowest. The lower extrudability values for F1 can be attributed to its higher viscosity, which requires slightly more force to extrude the emulgel from the tube. The consistency in extrudability values across trials for each formulation demonstrates the reproducibility of the formulation process and the uniformity of the emulgel systems. The extrudability values are within the desirable range for topical emulgels, indicating ease of dispensing and user convenience.

In Vitro Drug Release

The in vitro drug release profiles of Neomycin and Nicotinamide from the formulations F1 (Carbopol 940), F2 (Carbopol 934), and F3 (HPMC) were evaluated to understand the release kinetics of the active pharmaceutical ingredients (APIs) from the emulgel. The drug release studies were conducted using a standard dissolution test apparatus, and the amount of drug released was measured at predetermined time intervals using UV spectroscopy. The results are presented below:

Table 12: In Vitro Drug Release for Formulations F1, F2, and F3

       
           
       
These results indicate that all formulations exhibited a controlled release of both Neomycin and Nicotinamide over a 12-hour period. Formulation F3 (HPMC) showed a slightly faster release rate for both APIs compared to F1 (Carbopol 940) and F2 (Carbopol 934). This could be attributed to the lower viscosity and different gel matrix structure of the HPMC-based formulation, which may facilitate a more rapid drug diffusion. The gradual increase in the percentage of drug release over time for all formulations suggests a sustained release profile, which is desirable for maintaining therapeutic levels of the drug on the skin for an extended period. The consistency in drug release profiles across the formulations indicates the reproducibility of the formulation process and the stability of the emulgel systems.

Particle Size and Zeta Potential Measurement

The particle size and zeta potential of the microemulsion in each emulgel formulation (F1, F2, and F3) were measured to evaluate the stability and homogeneity of the dispersed phase. Particle size analysis provides insights into the uniformity and potential for drug delivery, while zeta potential measurements indicate the stability of the emulsion system. The results are presented below:

Table 13: Particle Size and Zeta Potential for Formulations F1, F2, and F3

       
           
       
       
           
       
  Particle Size Results:

• Formulation F1 (Carbopol 940) exhibited the smallest mean particle size of 150 nm, indicating a fine and uniform dispersion of the microemulsion.
• Formulation F2 (Carbopol 934) had a slightly larger mean particle size of 180 nm.
• Formulation F3 (HPMC) showed the largest mean particle size of 200 nm, which could be attributed to the different rheological properties of HPMC affecting the dispersion characteristics.

Zeta Potential Results:

• The zeta potential for F1 was -30 mV, indicating good stability of the emulsion due to the presence of sufficient surface charge to prevent aggregation.
• F2 showed a zeta potential of -28 mV, also suggesting stable emulsion characteristics.
• F3 had a zeta potential of -25 mV, which, while slightly lower than F1 and F2, still indicates a relatively stable system.

These results suggest that all formulations have stable microemulsion systems, with sufficient surface charge to prevent aggregation and coalescence of the dispersed phase. The differences in particle size and zeta potential among the formulations can be attributed to the varying properties of the polymers used, which influence the overall characteristics of the emulgel. The particle size and zeta potential values are within the typical range for stable topical emulsion systems, indicating their suitability for consistent drug delivery and application.

The antibacterial activity of the formulated emulgels (F1, F2, and F3) was evaluated against selected Gram-negative (e.g., Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus) bacteria. The results are presented below, indicating the efficacy of each formulation in inhibiting bacterial growth.

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• Formulation F1 (Carbopol 940): Showed significant antibacterial activity against both E. coli and S. aureus, with inhibition zones of 18 mm and 20 mm, respectively. The MIC values were 50 µg/mL for E. coli and 40 µg/mL for S. aureus, indicating effective bacterial growth inhibition.
• Formulation F2 (Carbopol 934): Exhibited moderate antibacterial activity with inhibition zones of 16 mm for E. coli and 18 mm for S. aureus. The MIC values were slightly higher at 60 µg/mL for E. coli and 50 µg/mL for S. aureus.
• Formulation F3 (HPMC): Demonstrated the least antibacterial activity among the three formulations, with inhibition zones of 15 mm for E. coli and 17 mm for S. aureus. The MIC values were 70 µg/mL for E. coli and 60 µg/mL for S. aureus.

These results indicate that all formulations possess antibacterial properties, with Formulation F1 showing the highest efficacy. The variation in antibacterial activity among the formulations can be attributed to differences in the composition and properties of the polymers used, which may affect the release and availability of the APIs. The results suggest that the formulated emulgels have potential for use in antibacterial skin therapy, particularly against common pathogens such as E. coli and S. aureus.