D-α-Tocopherol-Based Micelles for Successful Encapsulation of Retinoic Acid

Abstract

All-trans-retinoic acid (ATRA) represents the first-choice treatment for several skin diseases, including epithelial skin cancer and acne. However, ATRA's cutaneous side effects, like redness and peeling, and its high instability limit its efficacy. To address these drawbacks and to improve ATRA solubilization, we prepared ATRA-loaded micelles (ATRA-TPGSs), by its encapsulation in D-α-tocopheryl-polyethylene-glycol-succinate (TPGS). First, to explore the feasibility of the project, a solubility study based on the equilibrium method was performed; then, six ATRA-TPGS formulations were prepared by the solvent-casting method using different TPGS amounts. ATRA-TPGSs showed small sizes (11-20 nm), low polydispersity, slightly negative zeta potential, and proved good encapsulation efficiency, confirmed by a chemometric-assisted Fourier transform infrared spectroscopy (FTIR) investigation. ATRA-TPGS stability was also investigated to choose the most stable formulation. Using Carbopol^® 980 as gelling agent, ATRA-TPGS-loaded gels were obtained and analyzed for their rheological profiles. Ex vivo release studies from ATRA-TPGSs were performed by Franz cells, demonstrating a permeation after 24 h of 22 ± 4 µ cm^-2. ATRA-TPGSs showed enhanced cytotoxic effects on melanoma cells, suggesting that these formulations may represent a valid alternative to improve patient compliance and to achieve more efficacious therapeutic outcomes.

Keywords: TPGS; drug delivery systems; micelles; nanocarrier-loaded gels; nanocarriers; retinoic acid; topical application.

Figure 1

Chemical structures and schematic representation of micelles and their dispersion into the Carbopol^® network.

Figure 2

D-α-tocopheryl-polyethylene-glycol-succinate (TPGS) concentration-dependent solubility of all-trans-retinoic acid (ATRA). Total solubility of ATRA versus TPGS concentration measured by equilibrium method after 48 h at 37 °C in water (a). Linear regression obtained considering the values above TPGS critical micellar concentration (CMC) (b).

Figure 3

ATRA total solubility obtained by solvent-casting method as a function of ATRA:TPGS w/w ratio present in the starting mixture. Histogram summarizes quantitative data of the means ± S.D. of three independent experiments. * indicates statistical difference vs. sample mean (p < 0.05).

Figure 4

Fourier transform infrared spectroscopy (FTIR) spectra of ATRA (retinoic acid), TPGS (vitamin E) and of three ATRA-TPGS formulations.

Figure 5

Principle component analysis (PCA) results represented as a score plot (a) and a loading plot (b), performed on the matrix by collecting spectral data of all samples using R software.

Figure 6

Differential scanning calorimetry (DSC) thermograms of selected ATRA-TPGS formulations, ATRA:TPGS physical mixtures, TPGS, and ATRA (a). Overlapped thermograms with y axis reporting the real scale (b).

Figure 6

Differential scanning calorimetry (DSC) thermograms of selected ATRA-TPGS formulations, ATRA:TPGS physical mixtures, TPGS, and ATRA (a). Overlapped thermograms with y axis reporting the real scale (b).

Figure 7

Stability over time of aqueous micellar dispersions maintained at 25 °C and obtained using ATRA:TPGS 1:20, 1:30, 1:40, 1:50, 1:60, and 1:70 (w/w) ratios in the preparative mixture.

Figure 8

Carbopol ^® 980 gel loaded with the colloidal dispersion of ATRA-TPGSs prepared from ATRA:TPGS 1:50 (w/w) ratio.

Figure 9

Rheograms of ATRA-TPGS-loaded hydrogels at 0.5%, 1.0%, and 1.5% (w/w) Carbopol^® 980 concentrations, recorded at 25 °C.

Figure 10

Power Law rheograms of ATRA-TPGS-loaded gels at 0.5%, 1.0%, and 1.5% (w/w) Carbopol^® 980 concentrations.

Figure 11

Dose-dependent cytotoxic activity of ATRA on melanoma cells. The analysis was performed by CellTiter 96^® Aqueous One Solution Cell Proliferation Assay in melanoma cells exposed to ATRA (0.1–20 µM) for 72 h. The graph summarizes the quantitative data of the means ± S.E.M. of three independent experiments. ** p < 0.01 vs. Ctr cells; * p < 0.05 vs. Ctr cells.

Figure 12

Effect of ATRA-TPGSs on melanoma cell viability. Cell viability was evaluated by CellTiter 96^® Aqueous One Solution Cell Proliferation Assay. Cells were exposed for 72 h to free and encapsulated ATRA 5 µM (red columns) and 7.5 µM (light blue columns), and to TPGS at the same concentrations provided by the amounts of ATRA-TPGSs. Histograms summarize the quantitative data of the means ± S.E.M. of three independent experiments. * p < 0.05 vs. Ctr cells; ** p < 0.01 vs. Ctr cells; ° p < 0.05 vs. ATRA-treated cells.