Acyclovir-Loaded Chitosan Nanospheres from Nano-Emulsion Templating for the Topical Treatment of Herpesviruses Infections

Abstract

Acyclovir is not a good candidate for passive permeation since its polarity and solubility limit is partitioning into the stratum corneum. This work aims to develop a new topical formulation for the acyclovir delivery. New chitosan nanospheres (NS) were prepared by a modified nano-emulsion template method. Chitosan NS were characterized by Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and an in vitro release study. The in vitro skin permeation experiment was carried out using Franz cells and was equipped with porcine skin. Biological studies were performed on the Vero cell line infected by HSV-1 and HSV-2 strains. The acyclovir loaded chitosan NS appeared with a spherical shape, a size of about 200 nm, and a negative zeta potential of about 40.0 mV. The loading capacity of the drug was about 8.5%. In vitro release demonstrated that the percentage of acyclovir delivered from the nanospheres was approximately 30% after six hours. The in vitro skin permeation studies confirmed an improved amount of permeated acyclovir. The acyclovir-NS complex displayed a higher antiviral activity than that of free acyclovir against both the HSV-1 and the HSV-2 strain. The acyclovir-loaded NS showed no anti-proliferative activity and no signs of cytotoxicity induced by NS was detected. Confocal laser scanning microscopy confirmed that the NS are taken up by the cells.

Keywords: acyclovir; antiviral activity; chitosan nanospheres; topical infections.

Figure 1

Transmission electron microscopy (TEM) image of acyclovir-loaded chitosan nanoparticles.

Figure 2

In vitro release kinetics of acyclovir from the drug aqueous solution and from the chitosan acyclovir NS.

Figure 3

Comparison of Acyclovir permeation through porcine skin from the chitosan NS gel and commercial cream.

Figure 4

Antiviral activity of free acyclovir, acyclovir-loaded NS, and plain carrier (NS) against HSV-1 (A) and HSV-2 (B). Vero cells were infected at a multiplicity of infection (MOI) of 0.01 and then exposed to different drug concentrations. Virus titers in the supernatants of cell cultures were determined by a standard plaque assay at 48 h or 24 h post-infection for HSV-1 or HSV-2, respectively. Values are the means of three separate determinations.

Figure 5

Effect of acyclovir, acyclovir-loaded NS, and only NS on the viability of non-infected Vero cells as a function of the drug concentration at 24 h (A) and 48 h (B). X axis: NS concentration, Y axis: cell viability (% of untreated control). Each point represents the mean ± SD (n = 3).

Figure 6

Cytotoxic effect of acyclovir, acyclovir-loaded NS, and NS alone on non-infected Vero cells by measuring the release of lactate dehydrogenase in culture medium at 24 h (A) and 48 h (B). X axis: nanospheres concentration, Y axis: release of lactate dehydrogenase (% of untreated control). Each point represents the mean ± SD (n = 3).

Figure 7

Cell uptake of fluorescent NS. Vero cells were incubated with the formulation for the times indicated and then analyzed by confocal laser scanning microscopy without fixation. The upper panels show the fluorescence images while the lower panels show fluorescence images merged with phase-contrast images. The first column on the left shows the control cells, which were not incubated with the formulation (NT = no treated).