Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation

Abstract

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of -11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

Keywords: antibacterial activity; dermal drug delivery; ex-vivo studies; fusidic acid; lyophilization; nanocrystals; rat excision wound infection model.

Figure 1

Line plots for the main effects of the type of stabilizer, drug to stabilizer ratio and homogenization time on (A) particle size (PS) and (B) span value (SV) of the prepared FA-NC.

Figure 2

Saturated solubility of FA coarse powder (FA), FA coarse powder dispersed in water containing PVA 4-88 (FA-PVA), FA-NC-F12, and FA-NC-F12/L in pure water at 37 °C.

Figure 3

In-vitro dissolution profiles of FA coarse powder, FA-NC-F12 and FA-NC-F12/L in phosphate buffer (pH 7.4) at 37 °C.

Figure 4

DSC thermograms of FA coarse powder (1); FA: mannitol PM (2); FA: PVA PM (3) and FA-NC-F12/L (4).

Figure 5

XRD spectra of FA coarse powder, FA-NC-F12/L, and physical mixture (PM) of FA with PVA and mannitol.

Figure 6

TEM micrographs of (A) optimized FA-NC-F12 suspension showing spherical particles with clear distinct thick layer of adsorbed stabilizer molecules around the particle and (B) lyophilized FA-NC-F12/L suspension showing no sign of particle aggregation.

Figure 7

Ex-vivo permeation profiles of FA from FA-NC-F12 and FA-NC-F2/L compared to coarse FA suspension through rat skin in PBS (pH 7.4) at 32 °C.

Figure 8

Cumulative amount of FA retained and permeated through rat skin from optimized FA-NC-F12, FA-NC-F12/L or coarse FA in PBS (pH 7.4) in 24 h. SC, stratum corneum; Tissue, epidermis + dermis; RC, receiver compartment.

Figure 9

In-vitro release profiles of FA from FA-NC-F12, FA-NC-F12/L and Fucidin creams in PB (pH = 7.4) at 32 °C.

Figure 10

Percentage (% ± SD) wound contraction in the four treatment groups during 10-day treatment with respective formulations.

Figure 11

Macroscopic changes in skin wound site in the different groups during the 10-day treatment with respective formulations.

Figure 12

CFU/swab (±SD) collected from infected area of the skin of rats in the four treatment groups at day-2, day-6 and day-8 from treatment with respective formulations (n = 6).

Figure 13

Micrographs of histological sections of healthy skin of normal rat (A) and wounded infected skin of rats treated for 10 days with normal saline (B), plain cream (C), Fucidin cream (D) and FA-NC-F12/L cream (E). All sections stained with hematoxylin and eosin are displayed at 200× magnification. (EP) epidermal layer, (De) dermal layer, (IF) inflammatory cells infiltration, (GT) granulation tissue, (NT) necrotic tissue, (BA) bacterial aggregate, (HG) hemorrhage, (BV) blood vessel, (CD) cellular debris, (K) keratinization and (Co) collagen.