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INTRODUCTION
Antimicrobial Resistance (AMR) has emerged as one of the most critical global health challenges of the 21st century. The World Health Organization (WHO) warns that AMR threatens to reverse decades of progress in the treatment of infectious diseases by rendering existing antibiotics less effective (World Health Organization, 2020). The increasing resistance of microbial pathogens to conventional antibiotics has intensified the need for innovative therapeutic approaches and advanced drug delivery systems to enhance the efficacy of existing antimicrobial agents.
Among available antibiotics, lincomycin hydrochloride, a member of the lincosamide class, exhibits potent activity against Gram-positive bacteria and certain anaerobes (Rusu et al., 2023). However, its therapeutic potential is limited by poor oral bioavailability, degradation within the gastrointestinal tract, and systemic side effects such as gastrointestinal irritation and diarrhea. Moreover, variable absorption and frequent dosing reduce patient adherence, thereby contributing to the risk of resistance development (Obana et al., 2023). These limitations necessitate the exploration of alternative delivery routes that can maintain therapeutic concentrations at the site of infection while minimizing systemic exposure.
Transdermal Drug Delivery Systems (TDDS) represent a promising strategy to overcome the drawbacks associated with conventional antibiotic delivery. TDDS can bypass gastrointestinal degradation and first-pass hepatic metabolism, offering sustained and controlled drug release with improved patient compliance (Ghaferi et al., 2024). Additionally, localized transdermal delivery can maintain adequate drug levels at the infection site, enhancing antimicrobial efficacy and reducing the probability of systemic toxicity and resistance development (Akombaetwa et al., 2023). Nevertheless, the major challenge of transdermal delivery lies in overcoming the skin’s outermost barrier the stratum corneum which restricts the permeation of many hydrophilic drugs. This challenge can be addressed by optimizing polymer composition, incorporating permeation enhancers, or employing formulation design strategies such as factorial optimization (Alkilani et al., 2024).
In this context, the present study focuses on the formulation and evaluation of lincomycin hydrochloride-loaded transdermal patches using a combination of Hydroxypropyl Methylcellulose (HPMC K100M) and Polyvinyl Alcohol (PVA) as film-forming polymers. Propylene glycol was used as a permeation enhancer, while glycerine acted as a plasticizer to improve flexibility and film integrity. A 2² full factorial design was employed to optimize the polymer ratios for maximum drug release and mechanical stability. The developed patches were subjected to comprehensive physicochemical evaluation, in vitro drug release studies, antimicrobial activity testing against Staphylococcus aureus, and compatibility analysis through Fourier-Transform Infrared (FTIR) spectroscopy.
This study aims to develop a stable, biocompatible, and effective transdermal system for lincomycin hydrochloride that offers sustained drug release, enhanced local antimicrobial action, and reduced systemic toxicity, thus presenting a potential strategy to combat antimicrobial resistance.
MATERIALS AND METHODS
Materials
Lincomycin Hydrochloride was obtained as a gift sample, while HPMC K100M, PVA, propylene glycol, and glycerine were procured from standard analytical-grade suppliers. All chemicals used were of analytical grade, and distilled water was employed throughout the study. Materials were stored at controlled room temperature (25±2ºC) and protected from light and moisture until use.
Methods
Preparation of Standard Stock Solutions
A primary stock solution of lincomycin hydrochloride (1 mg/mL) was prepared by dissolving 100 mg of drug in 100 mL of distilled water using a volumetric flask. From this, a secondary stock solution (100 µg/mL) was obtained by diluting 10 mL of the primary stock to 100 mL with distilled water (Naveed et al., 2014).
Determination of λmax
An aliquot of 1 mL from the secondary stock solution was diluted to 10 mL to obtain a 10 µg/mL working solution. The solution was scanned in the range of 200-400 nm using a UV-visible spectrophotometer (Shimadzu, Japan) with distilled water as blank. The maximum absorbance (λmax was observed at 204 nm, which was used for further analysis.
Calibration Curve of Lincomycin Hydrochloride
Aliquots of 1-8 mL from the secondary stock solution were separately diluted to 10 mL with distilled water to obtain concentrations ranging from 10-80 µg/mL. The absorbance of each solution was measured at 204 nm against distilled water as blank. A calibration curve was plotted between concentration (µg/mL) and absorbance to determine linearity and regression coefficient (Najim et al., 2014).
Drug-Excipient Compatibility Study (FTIR)
Compatibility between lincomycin hydrochloride and polymers was evaluated using Fourier Transform Infrared (FTIR) spectroscopy (PerkinElmer, USA). Spectra were recorded for pure drug, individual excipients (HPMC K100M, PVA, propylene glycol, and glycerin), and their physical mixtures in the range of 4000-400 cm⁻¹ using the KBr pellet method. Any significant shifts or disappearance of characteristic peaks were noted to assess possible chemical interactions (Ganjoo et al., 2016).
Preparation of Lincomycin Hydrochloride Transdermal Patches
Preparation of Backing Membrane
Polyvinyl alcohol (0.8 g) was dissolved in 15 mL of distilled water under constant stirring at 300 rpm with gentle heating. Glycerin (0.3 mL) was added as a plasticizer. The homogeneous solution was poured into a clean glass Petri dish and allowed to dry at ambient temperature for 24 hr to form a flexible backing membrane (Alkilani et al., 2023).
Preparation of Drug-Loaded Layer
Required quantities of HPMC K100M and PVA (as per factorial design) were dissolved in 15 mL of distilled water with continuous stirring until a clear solution was obtained. Lincomycin hydrochloride, propylene glycol, and glycerin were then added and mixed thoroughly to obtain a uniform viscous dispersion. The prepared mixture was carefully cast over the pre-formed PVA backing membrane and dried at room temperature (25±2ºC) for 24 hr. After complete drying, patches were cut into 2 × 2 cm squares and stored in airtight containers until further use (Park et al., 2018) (Table 1).
| Ingredients | F1 | F2 | F3 | F4 |
|---|---|---|---|---|
| Lincomycin HCl (mg) | 1500 | 1500 | 1500 | 1500 |
| HPMC (mg) | 750 | 750 | 500 | 500 |
| PVA (mg) | 800 | 1200 | 800 | 1200 |
| PG (mL) | 1 | 1 | 1 | 1 |
| Glycerine (mL) | 0.4 | 0.4 | 0.4 | 0.4 |
| Water (mL) | 15 mL | 15 mL | 15 mL | 15 mL |
Application of Adhesive and Release Liner
A thin layer of hot-melt pressure-sensitive adhesive was applied to one surface of the dried patch. The patch was then covered with a siliconized release liner to protect the adhesive layer. Finished patches were stored in moisture-proof containers to prevent degradation and maintain integrity (Wokovich et al., 2010).
Experimental Design (2² Full Factorial Design)
A two-factor, two-level (2²) full factorial design was used to optimize the formulation variables. The independent variables were: X₁: Concentration of HPMC K100M (mg), X₂: Concentration of PVA (mg). Each variable was studied at two levels (low and high). The dependent response variable was the cumulative percentage drug release at 6 hr. Four experimental batches (F1-F4) were formulated as per the factorial combinations, and evaluated for physicochemical and in vitro parameters (Gunst et al., 2009).
Evaluation of Transdermal Patches
Physical Appearance
The prepared patches were visually examined for color, transparency, surface smoothness, flexibility, and homogeneity (Arora et al., 2002).
Thickness
The thickness of each patch (2 × 2 cm) was measured at three random positions using a digital micrometer screw gauge. The mean±Standard Deviation (SD) was calculated to assess uniformity.
Weight Uniformity
Three patches from each batch were individually weighed using an analytical balance. The mean weight and standard deviation were determined to ensure uniform distribution of polymer and drug.
Folding Endurance
A strip of each patch (2 × 2 cm) was repeatedly folded at the same point until it broke. The number of folds required to break the patch was recorded as the folding endurance value, which indicates flexibility and mechanical strength (Prabhakara et al., 2010).
Surface pH
Each patch (2 × 2 cm) was placed in contact with 2 mL of distilled water for 1 hr. The surface pH was measured using a digital pH meter by placing the electrode in contact with the surface. This test ensures compatibility with skin pH and minimizes the risk of irritation.
Moisture Content
Patches were initially weighed and kept in a desiccator containing activated silica gel for 24 hr. They were re-weighed until a constant weight was obtained. The percentage moisture content was calculated from the initial and final weights.
Adhesive Strength (Tack Test)
Adhesive strength was determined by lightly pressing the patch between thumb and forefinger and assessing its tackiness upon removal. A good patch should exhibit moderate tackiness, ensuring firm adhesion without leaving residue on the skin.
Drug Content Uniformity
An accurately weighed patch (4 cm²) was dissolved in 100 mL of phosphate buffer (pH 7.4) and stirred overnight. The solution was filtered, suitably diluted, and analyzed spectrophotometrically at 204 nm. Drug content (%) was calculated in relation to the theoretical amount of lincomycin hydrochloride.
In vitro Drug Release Study
In vitro drug release was performed using a Franz diffusion cell equipped with a dialysis membrane (molecular weight cutoff 12,000-14,000 Da). The receptor compartment contained 50 mL of phosphate buffer (pH 7.4) maintained at 37±0.5ºC and stirred continuously at 100 rpm. A patch of 4 cm² was mounted on the donor compartment with the drug-loaded side facing the membrane. At predetermined intervals, 1 mL of the receptor medium was withdrawn and replaced with an equal volume of fresh buffer to maintain sink conditions. Samples were analyzed spectrophotometrically at 204 nm to determine cumulative percentage drug release (Shivalingam et al., 2021; Hardainiyan et al., 2017)
RESULTS
Determination of Absorption maxima
The Absorption Maximum (λmax) of Lincomycin Hydrochloride was determined using a UV-visible spectrophotometer with distilled water as solvent. The maximum absorbance was observed at 204 nm as shown in Figure 1, confirming the characteristic absorption peak of the drug.
Standard Calibration Curve of Lincomycin Hydrochloride
A standard calibration curve was constructed for Lincomycin Hydrochloride in the concentration range of 10-60 µg/mL. The absorbance values were recorded at 204 nm, and the mean of triplicate readings was analyzed using a linear regression model:
The calibration curve exhibited excellent linearity (Figure 2), indicating compliance with Beer-Lambert’s law.
Drug-Excipient Compatibility Study (FTIR Analysis)
Fourier Transform Infrared (FTIR) spectroscopy was employed to assess potential interactions between Lincomycin Hydrochloride and excipients (HPMC K100M, PVA, propylene glycol, and glycerin). The spectra were recorded over the 4000-400 cm⁻¹ range.
The FTIR spectrum of pure Lincomycin Hydrochloride (Figure 3) showed characteristic peaks corresponding to functional groups such as O-H, N-H, and C-H stretching. The mixture of drug and excipients (Figure 4, Table 2) exhibited all the prominent peaks of the pure drug with no significant shifts, indicating absence of chemical interactions and excellent compatibility. These characteristic peaks confirmed the structural integrity of the drug and its compatibility with selected excipients.
| Observed Peak (cm⁻¹) | Reported Range (cm⁻¹) | Functional Group |
|---|---|---|
| 3448.77 | 3400-3500 | O-H / N-H stretching (hydroxyl or amine) |
| 3275.9 | 3200-3300 | N-H stretching (secondary amine) |
| 3291.61 | 3200-3500 | O-H stretching (alcohol) |
| 3021.6 | 3000-3100 | =C-H stretching (aromatic/aliphatic) |
| 2955.88 | 2850-2960 | C-H stretching (alkane) |
Formulation of Lincomycin Hydrochloride Transdermal Patches
Transdermal patches (F1-F4) were successfully prepared using the solvent casting technique based on a 2² full factorial design. The patches were smooth, flexible, and transparent, as shown in Figure 5.
Evaluation Parameters
Physical Appearance
All patches were smooth, translucent, and flexible, indicating successful film formation and uniform polymer distribution.
Folding Endurance
The folding endurance values of the patches ranged between 217±1.23 to 291±1.52, suggesting excellent flexibility and mechanical strength required for application on skin. Evaluation parameters of formulated patches are shown in Table 3.
| Formulation Code | Folding Endurance | Thickness (mm) | Weight uniformity (g) | % Moisture Content | Surface pH | % Drug Content |
|---|---|---|---|---|---|---|
| F1 | 217±1.23 | 0.345±0.03 | 0.28±0.05 | 0.481±0.01 | 5.6 | 80.62±0.01 |
| F2 | 245±0.09 | 0.398±0.09 | 0.34±0.07 | 1.334±0.04 | 6.2 | 78.98±0.03 |
| F3 | 291±1.52 | 0.312±0.08 | 0.37±0.07 | 2.312±0.01 | 6.4 | 82.87±0.01 |
| F4 | 234±1.32 | 0.361±0.04 | 0.32±0.03 | 0.830±0.01 | 5.8 | 86.65±0.08 |
Thickness
The patch thickness ranged from 0.312±0.08 mm to 0.398±0.09 mm, demonstrating uniformity across formulations. Increased polymer concentration resulted in slightly thicker patches.
Uniformity of Weight
Patch weights were consistent, ranging between 0.28±0.05 g and 0.37±0.07 g, ensuring reproducibility.
Moisture Content
All formulations exhibited acceptable moisture content values between 0.481±0.01% and 2.312±0.01%, which help maintain patch flexibility and prevent brittleness.
Surface pH
The surface pH of all formulations ranged from 5.6 to 6.4, which is within the skin’s physiological range (4.5-6.5). This ensures that the patches are non-irritant and skin-compatible.
Drug Content Uniformity
Drug content among all formulations ranged between 78.98±0.03% and 86.65±0.08%, indicating uniform drug distribution throughout the matrix and compliance with pharmacopeial limits.
Antibacterial Activity (Zone of Inhibition)
Antibacterial activity was evaluated using the disc diffusion method against Staphylococcus aureus (ATCC 25923). The inhibition zone increased proportionally with drug concentration, indicating dose-dependent efficacy (Figure 6).
1 mg disc: 26±0.4 mm.
3 mg disc: 29±0.5 mm.
5 mg disc: 32±0.6 mm.
The Lincomycin HCl transdermal patch (50 mg) exhibited a significantly larger zone of inhibition (39±0.5 mm), confirming sustained and potent antibacterial activity due to prolonged drug release.
In vitro Drug Release Study
In vitro release studies were performed in phosphate buffer (pH 7.4) using the Franz diffusion cell method. The results demonstrated that drug release was inversely proportional to polymer concentration. Formulation F3 exhibited the highest cumulative drug release (78.44%), whereas F1 showed the lowest (67.92%) after 6 hr (Figure 7).
Statistical Analysis and Design of Experiment (DOE)
A 2² full factorial design was employed to evaluate the influence of polymer concentrations on the cumulative drug release of Lincomycin HCl transdermal patches. The independent factors were HPMC K100M (X₁) and PVA (X₂), each studied at two levels (low and high), while the dependent variable was the percentage cumulative drug release (Y) after 6 hr. Four formulations (F1-F4) were prepared to cover all combinations of the factor levels.
The cumulative drug release values demonstrated that drug diffusion decreases with increasing polymer concentrations, consistent with the expected behavior of hydrophilic polymer matrices, where higher polymer content forms a denser network that slows drug release.
ANOVA Analysis
The factorial model was statistically analyzed using ANOVA to determine the significance of the factors. The model exhibited a high F-value (518.44) and a p-value of 0.0310, indicating statistical significance (p<0.05). Among the individual factors, PVA (A) showed a stronger effect on drug release than HPMC K100M (B) (Table 4).
| Source | Sum of Squares | df | Mean Square | F-value | p-value | |
|---|---|---|---|---|---|---|
| Model | 64.8 | 2 | 32.4 | 518.44 | 0.031 | Significant |
| PVA (A) | 55.5 | 1 | 55.5 | 888.04 | 0.0214 | |
| HPMC K100M (B) | 9.3 | 1 | 9.3 | 148.84 | 0.0521 | |
| Residual | 0.0625 | 1 | 0.0625 | — | — | |
| Total | 64.87 | 3 | — | — | — |
Model Summary Statistics
The model exhibited excellent predictability with R²=0.9990, Adjusted R²=0.9971, and Predicted R²=0.9846, while the adequate precision (48.49) indicated a strong signal-to-noise ratio, confirming the suitability of the model for optimization.
Regression Equation
The regression equation in terms of coded factors is:
Where:
Y = % cumulative drug release.
A = PVA concentration.
B = HPMC K100M concentration.
The negative coefficients indicate that increasing either polymer reduces drug release, confirming the anticipated impact of matrix density on diffusion kinetics.
Graphical Analysis
Contour plots (Figure 8) and 3D surface plots (Figure 9) illustrate the interaction between HPMC K100M and PVA on drug release. Maximum drug release occurred at lower polymer concentrations, while increasing either factor progressively decreased drug diffusion. These plots effectively demonstrate the combined effect of both polymers on cumulative release.
Optimization of Formulation
Optimization using the desirability function targeted maximum drug release with minimal polymer content. The overlay plot (Figure 10) indicated the optimal formulation at:
The predicted drug release for this optimized combination was 73.03%, while the experimentally observed value was 75.87%, confirming model reliability.
The factorial design confirmed that both HPMC K100M and PVA significantly influence cumulative drug release. Higher polymer concentrations reduce drug release due to matrix densification, whereas lower concentrations allow faster diffusion. The optimized patch achieved a balance between mechanical integrity and drug release, providing sustained delivery suitable for antimicrobial applications. The DOE approach demonstrated robust predictive capability, ensuring formulation reproducibility and efficiency.
DISCUSSION
The present study focused on the formulation and evaluation of Lincomycin HCl transdermal patches to improve antimicrobial efficacy and overcome the limitations of oral delivery. Preformulation studies confirmed the λmax of Lincomycin HCl at 204 nm, and the calibration curve exhibited linear absorbance in the 10-60 μg/mL range, validating the method for quantitative analysis. FTIR spectroscopy indicated no significant drug-excipient interactions, confirming chemical compatibility of HPMC K100M, PVA, propylene glycol, and glycerine with Lincomycin HCl, consistent with previous reports on polymer-based transdermal systems.
All prepared patches were smooth, flexible, and translucent, with folding endurance ranging from 217±1.23 to 291±1.52 folds, demonstrating excellent mechanical integrity suitable for handling and application. Patch thickness and weight were uniform across formulations, while moisture content (0.48-2.31%) and surface pH (5.6-6.4) were within acceptable ranges, ensuring skin compatibility and stability. Drug content analysis revealed uniform dispersion (78.98-86.65%), fulfilling pharmacopoeial standards and indicating successful incorporation of Lincomycin HCl into the polymer matrix.
In vitro release studies demonstrated that cumulative drug release ranged from 67.92% to 78.44% over 6 hr, with F3 (lower polymer concentration) showing the highest release. This trend correlates with the hydrophilic nature and network density of the polymers, where higher polymer content creates a denser matrix, slowing drug diffusion. The 2² factorial design and DOE analysis confirmed the significant effect of both PVA and HPMC K100M on drug release, with PVA exerting a more prominent influence. The regression equation:
3D response plots illustrated that drug release decreases as polymer concentrations increase, in agreement with similar studies on HPMC and PVA-based transdermal systems.
Antimicrobial evaluation against Staphylococcus aureus demonstrated a clear dose-dependent zone of inhibition. The optimized transdermal patch (50 mg drug loading) produced a zone of 39±0.5 mm, exceeding the inhibition observed with equivalent drug-loaded discs, indicating enhanced localized antibacterial efficacy. This improvement is likely due to sustained release from the polymeric matrix, ensuring prolonged exposure of bacteria to the drug while reducing systemic absorption and side effects.
The optimization process identified the ideal formulation (HPMC K100M 1032 mg, PVA 663 mg) that balanced mechanical stability with maximal drug release. The observed cumulative release (75.87%) closely matched the predicted value (73.03%), validating the factorial model and demonstrating the reliability of DOE for transdermal formulation development. Overall, these results suggest that Lincomycin HCl transdermal patches provide a viable alternative to oral therapy, offering sustained local delivery, reduced systemic toxicity, and potential to mitigate antimicrobial resistance.
In summary, the study highlights the successful formulation of stable, effective transdermal patches with desirable physicochemical properties, optimized drug release, and significant antibacterial activity. The integration of DOE allowed systematic optimization, providing a reproducible and efficient platform for antimicrobial patch development.
CONCLUSION
The present study successfully developed and evaluated Lincomycin HCl transdermal patches using HPMC K100M and PVA as polymeric matrices, with propylene glycol as a permeation enhancer and glycerine as a plasticizer. Preformulation studies confirmed drug stability and compatibility with excipients, while physicochemical evaluation demonstrated uniformity in thickness, weight, folding endurance, moisture content, surface pH, and drug content, ensuring mechanical integrity and skin compatibility. In vitro drug release studies revealed that polymer concentration significantly influenced cumulative drug release, with lower polymer levels promoting faster diffusion. Factorial design and statistical optimization identified an optimized formulation (HPMC K100M 1032 mg, PVA 663 mg), achieving a cumulative release of 75.87%, closely matching predicted values, confirming model reliability. Antimicrobial studies showed that the patches exhibited significant dose-dependent antibacterial activity against Staphylococcus aureus, demonstrating their potential for localized, sustained antimicrobial therapy. Overall, Lincomycin HCl transdermal patches represent a promising alternative to conventional oral therapy, offering improved patient compliance, sustained drug release, reduced systemic side effects, and potential to mitigate antimicrobial resistance. This study provides a robust formulation and evaluation strategy that can be applied to other antibiotics and therapeutic agents for enhanced transdermal delivery.