Synthesis and anti-hepatitis B virus activity of acyclovir conjugated stearic acid-g-chitosan oligosaccharide micelle
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.
Section snippets
Materials
Chitosan with about 5.0 kDa average weight average molecular weight was obtained by enzymatic degradation from 95% deacetylated chitosan (Mw 450.0 kDa), which was supplied by Yuhuan Marine Biochemistry Co., Ltd., China. Stearic acid was purchased from Shanghai Chemical Reagent Co., Ltd., China. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co., Ltd., China. 2,4,6-trinitrobenzene sulfonic acid and
Synthesis of acyclovir-chitosan-g-stearate
The synthesis route was shown in Fig. 1A. O-succilylacyclovir was obtained with about 56% yield after recrystallization from methanol. The 1H NMR spectra were shown in Fig. 1B–E, and were in agreement with the previous report (Colla et al., 1983). In 1H NMR spectrum of acyclovir (Fig. 1B), the peak at 7.9 ppm was attributed to the proton of pyridine in acyclovir (NCH–N); in 1H NMR spectrum of O-succilylacyclovir (Fig. 1C), the peaks at 12.2 ppm was attributed to the proton of (CH2–COOH) in the
Conclusions
Antiviral agent such as acyclovir had low oral bioavailability, and high doses were usually administrated to reach the desired therapeutic effect. The structure modification of antiviral agent and the employing of effective drug delivery system are the main route to improve the drug bioavailability and reduce the drug dose. Here, acyclovir was successfully conjugated to chitosan-g-stearate micelles via a succinate linker. The acyclovir-chitosan-g-stearate micelles had nano-scaled size and kept
Acknowledgements
We are grateful for financial support of National Basic Research Program of China (973 Program) under contract 2009CB930300, National HighTech Research and Development Program (863) of China (2007AA03Z318), and Foundation of Science and Technology Department of Zhejiang Province (2008C23043).
References (24)
- et al.
Synthesis and characterization of a pentaerythritol-based amphiphilic star block copolymer and its application in controlled drug release
Reactive and Functional Polymers
(2009) - et al.
Stearic acid grafted chitosan oligosaccharide micelle as a promising vector for gene delivery system: Factors affecting the complexation
International Journal of Pharmaceutics
(2010) - et al.
Liver targeting and anti-HBV activity of reconstituted HDL–acyclovir palmitate complex
European Journal of Pharmaceutics and Biopharmaceutics
(2008) - et al.
Chemical stability and bioavailability of acyclovir coupled to α,β-poly(N-2-hydroxyethyl)-dl-aspartamide
Journal of Controlled Release
(1995) - et al.
Shell cross-linked stearic acid grafted chitosan oligosaccharide self-aggregated micelles for controlled release of paclitaxel
Colloids and Surfaces B-Biointerfaces
(2006) - et al.
Cellular uptake and cytotoxicity of shell crosslinked stearic acid grafted chitosan oligosaccharide micelles encapsulating doxorubicin
European Journal of Pharmaceutics and Biopharmaceutics
(2008) - et al.
Langmuir monolayers of the long-chain alkyl derivatives of a nucleoside analogue and the formation of self-assembled nanoparticles
Colloids and Surfaces B-Biointerfaces
(2005) - et al.
Inhibition of hepatitis B virus replication by shRNA in stably HBV expressing HEPG2 2.2.15 cell lines
Journal of Hepatology
(2006) - et al.
Macro molecular systems for chemotherapy and magnetic imaging
Advanced Drug Delivery Reviews
(1996) - et al.
Polymeric nanoparticulate system: A potential approach for ocular drug delivery
Journal of Controlled Release
(2009)