Atorvastatin-loaded emulsomes foam as a topical antifungal formulation

Abstract

Dermal fungal infection faces many challenges, especially for immunocompromised patients. Recently, the repositioning of atorvastatin (ATO) as a promising anti-mycoses therapy is used to overcome some issues of conventional therapeutic agents such as microbial resistance. The goal of this study was to develop a suitable formula for dermal fungal infection. Wherefore, ATO was entrapped into emulsomes and then incorporated in a foam system for topical convenient application. The D-optimal design was used for the optimization of ATO-emulsome and foam to achieve suitable responses. Regarding emulsomes, cholesterol weight and sonication time were independent variables that impact emulsome size, polydispersity index, surface charge, and entrapment efficiency. The optimum formula showed a size of 359.4 ± 8.97 nm, PDI of 0.4752 ± 0.012, a zeta potential of -21.27 ± 0.53 mV, and a drug entrapment of 95 ± 2.38%. Transmission electron microscope and Fourier-transform infrared spectroscopy (FT-IR) proved the assembly of ATO-emulsome. Foam composition was optimized to achieve good expansion, stability, and viscosity using a surfactant triple mixture and hydroxypropyl methylcellulose. The selected ATO-emulsome foam which consisted of 1% HPMC, 1.249% SDS, and 4% pluronic showed prolonged drug release. Efficient permeation through skin layers was asserted by using a confocal laser scanning microscope. Moreover, the homogenous distribution of the foam bubbles upholds stability and conserves the system from rapid collapse. The antifungal activity was confirmed by an in-vitro and in-vivo microbiology study beside in-vivo biocompatibility. In conclusion, ATO-emulsome and incorporation in foam have demonstrated good antifungal activity which presented a unique aspect for potential clinical applications.

Keywords: Atorvastatin; Emulsomes; Foam; Fungal infection; Repurposing; Topical.

Graphical abstract

Scheme 1

Study design: Phase 1 depicted the formulation and evaluation of atorvastatin-loaded emulsome (ATO-EMLs), Phase 2 represents formulation and evaluation of blank foam systems, Phase 3 shows further characterization of optimized ATO-EMLs foam system, Phase 4 demonstrates skin tissue permeation by in-vivo visualization study using the confocal laser scanning microscope, Phase 5 represents the microbiology studies of atorvastatin against C.albicans and A.fumigatus, Phase 6 revealed the in-vivo studies.

Fig. 1

3D plot of cholesterol weight and sonication time impact as independent variables on (a) Particle size (PS), (b) Polydispersity index (PDI), (c) Zeta potential (ZP), and (d) Entrapment efficiency (EE), Graphically illustrated of variables interaction upon studying (e) Particle size (PS), (f) Polydispersity index (PDI), (g) Zeta potential (ZP), and (h) Entrapment efficiency (EE);results represent the significant influence of cholesterol on all variables, otherwise, sonication time affect PS and PDI only.

Fig. 2

(a) Interaction plot showing independent variables desirability for selected ATO-EMLs optimum formula, Further characterization on the optimum formula: (b) TEM image of emulsome vesicle system; observed uniform spherical shape around 300 nm in size, (c) Fourier transforms infrared spectra for free atorvastatin, cholesterol, compritol, phosphatidylcholine, and optimum emulsome formula; prove vesicle formation and efficient drug loading.

Fig. 3

3D plots of foam expansion (%) response corresponding to different variable levels; pluronic low level: 0% (w/v) with different concentration (% w/v) of Tween 20 (a) 0, (b) 2, (c) 4, pluronic medium level: 2% (w/v) with different concentration (% w/v) of Tween 20 (d) 0, (e) 2, (f) 4, pluronic high level: 4% (w/v) with different concentration (% w/v) of Tween 20 (g) 0, (h) 2, (i) 4. (Measured responses color change from blue to red indicate increase response value). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

3D plots of foam volume stability (%) response corresponding to different variable levels; pluronic low level: 0% (w/v) with different concentration (% w/v) of Tween 20 (a) 0, (b) 2, (c) 4, pluronic medium level: 2% (w/v) with different concentration (% w/v) of Tween 20 (d) 0, (e) 2, (f) 4, pluronic high level: 4% (w/v) with different concentration (% w/v) of Tween 20 (g) 0, (h) 2, (i) 4.

Fig. 5

3D plots of foam liquid stability (%) response corresponding to different variable levels; pluronic low level: 0% (w/v) with different concentration (% w/v) of Tween 20 (a) 0, (b) 2, (c) 4, pluronic medium level: 2% (w/v) with different concentration (% w/v) of Tween 20 (d) 0, (e) 2, (f) 4, pluronic high level: 4% (w/v) with different concentration (% w/v) of Tween 20 (g) 0, (h) 2, (i) 4.

Fig. 6

3D plots of half-life response corresponding to different variable levels; pluronic low level: 0% (w/v) with different concentration (% w/v) of Tween 20 (a) 0, (b) 2, (c) 4, pluronic medium level: 2% (w/v) with different concentration (% w/v) of Tween 20 (d) 0, (e) 2, (f) 4, pluronic high level: 4% (w/v) with different concentration (% w/v) of Tween 20 (g) 0, (h) 2, (i) 4.

Fig. 7

3D plots of viscosity response corresponding to different variable levels; pluronic low level: 0% (w/v) with different concentration (% w/v) of Tween 20 (a) 0, (b) 2, (c) 4, pluronic medium level: 2% (w/v) with different concentration (% w/v) of Tween 20 (d) 0, (e) 2, (f) 4, pluronic high level: 4% (w/v) with different concentration (% w/v) of Tween 20 (g) 0, (h) 2, (i) 4.

Fig. 8

Design optimization model concerning variables and targeted responses; (a) desirability contour plot, (b) graphical optimization contour plot, Comparative study of ATO-EMLs foam with vesicle dispersion and blank foam regarding (c) Particle size, (d) Polydispersity index, (e) Foam calculated parameters, (f) and foam half-life; revealed insignificant differences as loaded foam conserve the optimum specifications.

Fig. 9

(a) Depicted foam collapse time from the side view after actuating; showing good stable structure with a long collapse time, (b) measured and analyzed bubble size and distribution using ImageJ software.

Fig. 10

(a) Release study illustrated as cumulative percentage of atorvastatin free drug in comparison to optimized ATO-EMLs before and after incorporation in foam system, Stability study for 6 months, evaluated sample storage at 4 °C represented by (b) Particle size, (c), polydispersity index, and (d) Zeta potential; reflect stable attributes with no significant difference in comparison to fresh sample.

Fig. 11

Confocal laser scanning microscopy images of EMLs-foam system (a) tile x-y image of skin treated with FDA-loaded emulsome foam system, (b) 3D plot using z-stack images of the skin section from 0 μm to 300 μm, and (c) sequential images from 0 to 300 μm with 15 μm increments showed that penetration occurred across different skin layers revealed with high fluorescence intensity, (d) quantitative measurement of fluorescence intensity profile across skin layers.

Fig. 12

In-vitro microbiology studies: In way to determine MIC; optical density values of two fungal species against the diluted concentration of the evaluated samples were determined (a) C.albicans, (b) A.fumigatus, the susceptibility of isolated fungal species to atorvastatin, blank and ATO-loaded EMLs foam in addition to commercial control was estimated through measured (c) MIC values, (d) diameters of inhibition zone, inhibition zones on C. albicans were visualized for (e) Atorvastatin, (f) ATO-EMLs foam, (g) blank-EMLs foam, (h) Commercial control, inhibition zones on A.fumigatus were visualized for (i) Atorvastatin, (j) ATO-EMLs foam, (k) Blank-EMLs foam, (l) Commercial control.

Fig. 13

In-vivo biocompatibility study: (a) Normal skin, (b) placebo foam after 1 week, (c) placebo foam after 3 weeks, (d) ATO-EMLs foam after 1 week, (e) ATO-EMLs after 3 weeks.

Fig. 14

In-vivo microbiological study: photographic images of the infected skin area of rat dorsal (a) positive control group, (b) standard drug group, and (c) ATO-EMLs foam group. (d) C. albicans concentration in CFU/mL of different groups (**** p < 0.0001). Histopathological evaluation: (e) Positive group, (f) Standard drug group, (g) ATO-EMLs foam.