Synthesis and anti-hepatitis B virus activity of acyclovir conjugated stearic acid-g-chitosan oligosaccharide micelle

Abstract

The controlled release of chemotherapeutical reagent with high water solubility was a challenge for targeting drug delivery. In this article, an antiviral agent, acyclovir was conjugated to chitosan-g-stearate via a succinate linker. Chitosan-g-stearate was synthesized by the reaction between the amino group of chitosan oligosaccharide and the carboxyl group of stearic acid. Both chitosan-g-stearate and acyclovir-chitosan-g-stearate could self aggregate to form micelles in aqueous solution. Acyclovir-chitosan-g-stearate micelle had smaller size (24.9 ± 1.1 nm), lower positive zeta potential (24.4 mV) and higher critical micelle concentration (123.23 mg mL⁻¹) in distilled water, compared with those of chitosan-g-stearate (34.2 ± 3.8 nm, 46.9 ± 6.2 mV and 90.07 mg mL⁻¹, respectively). Acyclovir release from acyclovir-chitosan-g-stearate micelles could prolong to 24 h in vitro. For the free acyclovir and acyclovir-chitosan-g-stearate micelle with acyclovir concentration of 0.044 μM mL⁻¹, the inhibition of acyclovir on hepatitis B surface antigen was increased from 12.7% to 22.3% from 5 d to 9 d, while the inhibition of acyclovir-chitosan-g-stearate was increased from 58.2% to 80.3% from 5 d to 9 d. The cellular uptake and antiviral activity of acyclovir was successfully increased and improved through chemical conjugation of acyclovir to chitosan-g-stearate.

Introduction

Antiviral agent had low drug specificity towards the affected organs, thus high doses are frequently needed for clinic. Excessive or insufficient water-solubility and low cellular internalization also reduce their bioavailability (Park, Saravanakumar, Kim, & Kwon, 2010). To overcome these problems, two main approaches have been used: the employment of new therapeutic substances with higher bioavailability or more efficient drug delivery systems (Torchilin, 2001).

Acyclovir, a synthetic purine nucleoside analogue, is the prototype antiviral agent, which is activated by viral thymidine kinase. Acyclovir triphosphate inhibits DNA synthesis by acting as a chain terminator and the inhibition activity of acyclovir is highly selective due to its special affinity for the thymidine kinase enzyme encoded by herpes simplex virus and varicella zoster virus (Suzuki, Okuda, & Shiraki, 2006), and its anti-hepatitis B virus activity has been reported (Feng, Cai, Huang, & Zhou, 2008). However, the average oral bioavailability of acyclovir is fairly low (only 10–20%) and its plasma elimination half-life is 2.5–3.3 h (Tao et al., 2009), which means that frequent administrations are needed to maintain therapeutic drug concentration. The low oral bioavailability of acyclovir can be explained mainly by its low diffusivity, because plasma membrane is the primary barrier to entering the cytosolic space (Saito, Swanson, & Lee, 2003).

The drug delivery system could change the in vivo distribution of drug. The distribution and elimination patterns depended mainly on its physicochemical properties, such as size, charge, hydrophilic/lipophilic balance, shape, flexibility and deformability and the anatomical characteristics of endothelial capillaries (Taylor & Granger, 1983).

Ideal drug carriers should be easily synthesized with low cost, freely water-soluble, non-toxic, non-immunogenic and well characterized from the physicochemical point of view (Matthews et al., 1996, Nagarwal et al., 2009). Chitosan is a natural polysaccharide derived from chitin by alkaline deacetylation, which is regarded as non-toxic, low immunogenicity and biodegradable. Chitosan with low molecular weight was obtained by enzymatic degradation from chitosan, which indicated good water solubility in physiological pH condition (Ye et al., 2008). The chitosan-g-stearate was synthesized via coupling reaction between the amino groups of chitosan with low molecular weight and carboxyl group of stearic acid (Hu, Ren, Yuan, Du, & Zeng, 2006). The chitosan-g-stearate could form micelles spontaneously in the aqueous medium, and could be rapidly internalized into cells (You, Hu, Du, Yuan, & Ye, 2007). Chitosan-g-stearate micelle had a hydrophobic core and hydrophilic shell, and it has been used as a potential carrier for antitumor drug in order to improve its antitumor activity (Hu et al., 2006, Hu et al., 2008, You et al., 2007, You et al., 2008). However, it was proved that chitosan-g-stearate micelle was not suitable for physical entrapment of hydrophilic antiviral agent, such as acyclovir.

In this research, acyclovir was used as a model hydrophilic antiviral drug to conjugate with Chitosan-g-stearate micelle via succinic spacer (Colla, De Clercq, Busson, & Vanderhaeghe, 1983). The toxicity and antiviral activity of acyclovir and acyclovir-chitosan-g-stearate were carefully examined on HepG₂ cells and hepatitis B virus-transfected human hepatoma cells, respectively.