(T1230-07-45) Double RSM Design QbD Optimization of Emulsomal Gel

Purpose: β-Carotene (β-C) is an anti skin-cancer drug, proven to induce apoptosis in melanoma cells and inhibit tumor angiogenesis [1]. However, it has poor bioavailability and is prone to rapid thermal and photodegradation which limits its use as a therapeutic drug. Emulsomes, a novel lipid nanoparticle, have been proven to enhance transdermal delivery and stabilize encapsulated drugs, however they are typically created with a trial and error approach which can lead to significantly higher costs and experimentation times, and poor translatability of the DDS into the industry due to the lack of knowledge of the relationship between the CMAs and CPPs with the CQAs. Therefore, to solve these problems and efficiently stabilize β-C and improve its transdermal delivery, an optimized biodegradable emulsomal gel was formulated with a thin film hydration method, utilizing a streamlined QbD method with 2 RSM experimental designs.

Methods: 5mL batches of emulsomes were formulated with a thin film hydration method utilizing ethyl acetate (474 mbar, 50 ± 1^oC, 600rpm), and downsized with homogenization and sonication (15,000rpm, 40kHz), before being introduced into a gel matrix. Different phospholipids (PC, PE, PS), glycerides (C18:0, GMS, GML, C888ATO), organic solvents (EtOAc, Hx, EtOh, MeOH), and CPPs such as hydration volume and temperature, and vacuum pressure were screened. A Box Behnken design was used to first optimize the CQAs: % Entrapment Efficiency (%EE) and % Transmittance (%T) of an emulsomal suspension, with the inputs being the CMA molar ratios: PC:SL, CH:PC, and API:TL (TL=SL+PC). %EE was measured with a validated standard curve (y=0.043x+0.0195, R² = 0.999, 1-20 ug/mL, LOQ = 2.39ug/mL), by adding a nonionic surfactant (TX-100) and inducing lysis in the emulsomes which allowed for precipitation, centrifugation (6000 rpm), filtration (0.45µm) and isolation of the drug. A Wolfe reduced gradient approach was used to find the optimal suspension from the Box Behnken design. Then, a second I-optimal RSM design was developed to optimize the cumulative drug permeated over 24h (Q₂₄) and dynamic viscosity of the emulsomal gel. Q₂₄ was measured in vitro with a franz diffusion cell (32 ± 1^oC, PBS pH 7.4, 400 rpm, CA membranes), with samples taken periodically for analysis. Dynamic viscosity was measured with a spindle viscometer (12rpm). Using prediction and contour profilers, an optimal emulsomal gel was found. This gel was then characterized for rheological properties, in vitro drug permeation with release kinetics, drug assay and retention, pH, and short term stability.

Results: The results revealed that the optimal formulation (1 PC:SL, 2 CH:PC, 30 TL:API, 1.05% XG, Figure 3E) successfully stabilized β-C, increasing drug retention nearly ninefold (89.13 ± 0.81%, p≤0.0001), and improving its in vitro transdermal delivery and flux by a factor of four (0.507 ± 0.01ug/cm ²/h, p≤0.0001, Figure 3B) when compared to the gelled control. The optimal gel also had favorable rheological properties with shear thinning (3756.67 ± 192.96 mPas, Figure 3G), high stability, and allowed controlled release (73.55 ± 1.86ug/cm², p≤0.0001, Figure 3A). The gel’s release profile was seen to be a rapid burst release in the first 6 hours, followed by controlled release, and had a significant release rate for all time points (p≤0.05) except 12 and 24h. Following storage for a week at 4 and 25 C, the optimal suspension’s %EE and %T stayed above 80% of their original measurements (Figure 3C). In addition, The response surface designs used in the study generated predictive models that elucidated the relationship between the CMAs and CPPs with the CQAs (R²≥0.9, p≤0.05, Figure 2B), and contour and prediction profilers were highly accurate in predicting the composition of an optimal formulation (Figure 2A, 3E, 3F). The residuals and studentized residuals of both models were within the set boundaries and had no patterns, validating the models. Furthermore, both models' effects were mostly significant, demonstrating that modulation of the emulsomal gel’s CMAs and CPPs had significant effects on its CQAs.

Conclusion: In conclusion, emulsomes were successful in stabilizing β-Carotene and enhancing its transdermal delivery in vitro. The established QbD double RSM method can also likely be used to incorporate almost any water-insoluble drug into an optimized emulsome gel.

References: [1] Chinembiri TN, du Plessis LH, Gerber M, Hamman JH, du Plessis J. Review of natural compounds for potential skin cancer treatment. Molecules. 2014 Aug 6;19(8):11679-721 PMID: 25102117; PMCID: PMC6271439.

Acknowledgements: Gratitude is expressed to Instilling Goodness Developing Virtue Schools and Hiep X. Nguyen, Ph.D., DTM for providing the lab equipment used in this study.


Graphical representation of emulsomal gel design, formation, and characterization.


Box Behnken design formulation list; F0 is the optimized emulsomal suspension. (A) Contour profilers and response surface profilers for the Box Behnken design, showing possible CQA combinations. (B) Predicted vs Actual plots for Box Behnken and RSM designs. All data is expressed in mean ± SD, and all experiments were repeated 3 times (n=3) unless otherwise stated. p≤0.05 was considered to be statistically significant.


(A) Drug permeation graph of optimized emulsomal gel and control over 48h. (B) Drug retention and Flux plot of emulsomal gel and control. (C) Short term stability of %EE and %T of emulsomal suspension at 4oC and 25oC. (D) Formulation list of RSM design, with plotted dynamic viscosity and Q24. (E) (F) Prediction profilers for Box Behnken and RSM design. (G) Regression models for RSM design and gel rheology plot for optimized gel. All data is expressed in mean ± SD, and all experiments were repeated 3 times (n=3) unless otherwise stated. p≤0.05 (*) was considered to be statistically significant, p≤0.0001 (****) was considered highly significant.