In Vitro Enhanced Skin Permeation and Retention of Imiquimod Loaded in β-Cyclodextrin Nanosponge Hydrogel

Abstract

Imiquimod (IMQ) is an immune response modifier clinically used for the treatment of various topical diseases. However, its poor aqueous solubility and skin penetration capability make the topical delivery of IMQ a challenging task. This work aims at developing a nanomedicine-based topical formulation, carrying IMQ to control the scarring process for the treatment of aberrant wounds. For this purpose, IMQ was loaded in β-cyclodextrin-based nanosponges and dispersed in a hydrogel suitable for dermal application. The formulation was characterized in vitro and compared with IMQ inclusion complexes, with (2-hydroxy)propyl β-cyclodextrin(HPβCD) and carboxymethyl β-cyclodextrin (CMβCD) showing enhanced penetration properties. The hydrogel containing IMQ-loaded nanosponges could act as a drug reservoir and guarantee the sustained release of IMQ through the skin. A greater inhibitory effect on fibroblast proliferation was observed for IMQ loaded in nanosponges compared to the other formulations.

Keywords: controlled release; cyclodextrins; imiquimod; inclusion complex; nanosponges.

Scheme 1

Imiquimod chemical structure.

Figure 1

Phase solubility tests of the imiquimod (IMQ) inclusion complexes of (2-hydroxy)propyl β-cyclodextrin (HPβCD) (A) and carboxymethyl β-cyclodextrin (CMβCD) (B).

Figure 2

(A) Differential scanning calorimetry (DSC) thermograms of imiquimod (IMQ) and IMQ inclusion complexes: (A) IMQ, (B) IMQ–HPβCD, (C) IMQ–HPβCD physical mixture, (D) IMQ–CMβCD, (E) IMQ–CMβCD physical mixture; (B) Attenuated total reflection–Fourier transformed infrared (ATR–FTIR) spectra of IMQ and HPβCD complex; (C) Attenuated total reflection–Fourier transformed infrared (ATR–FTIR) spectra of IMQ and CMβCD complex.

Figure 3

(A) DSC thermograms of IMQ (A), IMQ–NS physical mixture (B) and IMQ-loaded NS (C); (B)ATR–FTIR spectra of IMQ and IMQ-loaded NS.

Figure 4

In vitro release profile of IMQ from the NS in comparison with the cyclodextrin (CD) inclusion complexes and free IMQ.

Figure 5

In vitro permeation profiles of IMQ-loaded NS and IMQ complexes through pig skin.

Figure 6

In vitro accumulation of IMQ in the stratum corneum (SC), epidermis, and dermis following application on the pig skin of a hydrogel containing IMQ-loaded NS for 24 and 48 h.

Figure 7

Effect of free IMQ, IMQ–NS and IMQ–CD complexes on the proliferation of normal skin-derived fibroblasts. Fibroblasts were left untreated or treated with 5 and 25 µg/mL of free IMQ, blank NS (NS) and IMQ–NS (A,B) or with NS, IMQ–NS, IMQ–CMβCD and IMQ–HPβCD at 25 µg/mL (C) for the indicated times. Data are shown as the optical density (O.D.). Cell proliferation is presented as mean ± SEM of three independent experiments. # p < 0.05 vs. untreated cells, * p < 0.05 vs. NS as appropriate, by two-way ANOVA followed by Bonferroni post tests.

Figure 8

Inhibitory effect of IMQ–NS in a scratch wound assay. The figure shows micrographs of the extent of closure obtained under control conditions compared to those with free IMQ (5 µg/mL), NS or IMQ–NS (5 and 25 µg/mL) after 24- and 48-h treatment. The fibroblasts were synchronized for 2 h, wounded and treated as indicated, and phase-contrast microscopy pictures were taken of the wounded area (scale bar 100 µm). One representative experiment out of three is shown.