(T1230-03-13) Enhancement of Transdermal Delivery for β-Carotene as a Treatment of Skin Cancer: Quality by Design Optimization of Microemulsion Formulation

Purpose: This QbD study was conducted through in vitro testing and JMP 17.0.0 software to determine the optimal microemulsion for efficient transdermal delivery of water insoluble β-Carotene as an anticancer agent used to treat skin cancer.¹

Methods: Preformulation Studies: The analytical method was first validated through several calibration curves with a visible light spectrophotometer, and intra-day and inter-day results were collected. β-Carotene was then added in excess to a range of oil, surfactant, and co-surfactant, and the samples were shaken for 96 hours to screen excipients for each phase. To measure solubility and stability, the samples were centrifuged at 24-hour intervals. Subsequently, 100µL of the supernatant was filtered through a 45µm filter and diluted for measurement with a spectrophotometer. To choose the optimum Surfactant: Cosurfactant (Smix) ratio, three pseudo ternary phase diagrams were constructed. The process involved combining them in the ratios (1:1), (1:3), and (3:1), and then mixing each of these with oil in the ratios: Smix:oil (1:9), (2:8), (3:7), (4:6), (5:5), (6:4), (7:3), (8:2), and (9:1). Water was then added incrementally to make up 0-95% of the solution while mixing at 21 ± 0.5 °C. The transparency and turbidity of the mixture was observed to determine microemulsion and emulsion phases, respectively. DOE Mixture

Design: Afterwards, a DOE mixture design was run on JMP to limit the study design and to calculate the values that would be used for in vitro testing. The microemulsions were loaded so that each 520mg of formulation would have 20mg β-Carotene. Three different responses were optimized, namely: % Transmittance, % Drug release, and drug loading. For the % Transmittance, the microemulsions were diluted with acetone at a ratio of (1:99) (w/w), and measured using spectrophotometry. In order to measure the in vitro % Drug release, 500uL of each microemulsion was put into a dialysis cassette, and was dialyzed in 200mL of water under 37±1°C, to mimic in vivo conditions with constant stirring at 150 rpm. 5 mL samples were taken out on the following intervals: 5, 10, 15, 20, 25, 30, 45 minutes and drug content was measured with a spectrophotometer. In measuring the drug loading, 0.52g of the formulations were diluted with 5mL of acetone, and the absorption was measured. JPM software was then used to fit the responses, and the design space and optimal formulation was found. Optimal Microemulsion Characterization: The optimal microemulsion was then characterized for stability, Viscosity, pH, % Transmittance, % Drug release, and Drug loading.

Results: The calibration curves demonstrated excellent linearity (r²=0.999±0.001, n=3), and the intra day and inter day results showed minimal deviation from each other. Transcutol CG (Solubility: 18.16±0.43µg/mL), Tween 20 (28.11±0.32µg/mL), Labrafac CCMB (28.56±0.54µg/mL), and water were determined to be separate phases from the solubility and stability experiments (n=3), as β-Carotene had the highest overall solubility and stability in them (Image 1). Smix was selected at a ratio (1:1 w/w), as the largest design space of microemulsion formation was found at this ratio (Image 2, n=3). The DOE mixture design provided 10 formulations, and the responses were most desirable in formulation 7 (Image 3). The in vitro % drug release, drug loading, and %Transmittance of formulation 7 were found to be 92.83±1.6%, 76.03±3.2µg/mL, and 92.5±0.7% respectively (n=3). JMP software was then used to calculate the prediction formulas of each response, and the resulting mixture profiler and contour profiler design space were found (Image 3), with white space showing the viable design space. The optimal formulation had a pH of 7.6±0.4 (n=3), a viscosity measured at 21±0.5°C to be 98.2±1.2 cP (n=3), and maintained stability at room temperature for 120 hours (n=1). Minimal phase separation was observed when the formulation was subject to centrifugation for 15 minutes (n=3).

Conclusion: Microemulsions are necessary for enhancing delivery of water-insoluble drugs, such as β-Carotene. In the present study, the microemulsion with 24% Labrafac CCMB, 1% Water, and 75% Smix was found to be the optimal formulation. This is due to its highest overall drug release, high drug loading, high formation of globules in the nano range due to its high transmittance, and optimal stability for long term storage.

References: 1. Azhar F, Naureen H, Shahnaz G, Hamdani SDA, Kiani MH, Khattak S, Manna MK, Babar MM, Rajadas J, Rahdar A, Díez-Pascual AM. Development of chitosan based β-carotene mucoadhesive formulation for skin cancer treatment. Int J Biol Macromol. 2023 Dec 31;253(Pt 1):126659. doi: 10.1016/j.ijbiomac.2023.126659. Epub 2023 Sep 1. PMID: 37660856.

Acknowledgements: We are grateful to Instilling Goodness Developing Virtue Schools for funding this project, and Gattefosse for providing us with free samples of Transcutol CG and Labrafac CCMB.


From top right clockwise: Mixture profiler depicting optimal design space, Zoomed in mixture profiler of optimal design space, Prediction profiler for optimal formulation, and Contour profiler for optimal formulation


Smix (1:1) Ternary diagram, Black portion indicates microemulsion formation


β-Carotene Solubility in different excipients, and it's drug release in the optimal formulation