Elsevier

Polymer

Volume 51, Issue 15, 8 July 2010, Pages 3387-3393
Polymer

Water-soluble heparin–PTX conjugates for cancer targeting

https://doi.org/10.1016/j.polymer.2010.05.030Get rights and content

Abstract

Targeted drug delivery to cancer cells or tumor vasculature is an attractive approach to treating cancer. We here report the synthesis of an anticancer drug conjugate composed of paclitaxel (PTX) and polysaccharide heparin through the reaction of aminated PTX with the carboxyl group of heparin. The structure of the conjugates was identified by 1H NMR and FT-IR measurements. Heparin–PTX conjugates have high solubility in aqueous solutions. Unlike physically encapsulated drugs, heparin–PTX can self-assemble to form spherical nanoparticles in aqueous solution as characterized by Transmission Electron Microscopy (TEM). Size distribution of the nanoparticles as determined by Dynamic Light Scattering (DLS) was in the range of 200–400 nm depending on the coupling ratio of PTX to heparin molecules. The anticoagulant activity of heparin–PTX conjugates was decreased compared to that of heparin, thereby reducing hemorrhagic side effects. Cellular uptake of the nanoparticles was significantly enhanced compared to heparin as visualized by Confocal Laser Scanning Microscopy (CLSM). Furthermore, heparin–PTX conjugate nanoparticles exhibited higher cytotoxicity against KB cancer cells than did free PTX. The cytotoxicity of nanoparticles was found to depend on the amount of PTX conjugated to heparin as well as the conjugate concentration. Thus, conjugation of PTX to heparin may be useful for the solubilization and targeted delivery of PTX to solid tumors.

Introduction

Recent advances in tumor therapy have led to the development of new probes, drugs, and genes for tumor targeting [1], [2]. Among these new drug targeting systems, polymeric nanoparticles have been demonstrated to significantly improve the specificity of drug action. This effect is mainly attributed to changes in tissue distribution and pharmacokinetics [3], [4], [5]. Furthermore, it has been demonstrated that nanoparticles can escape from the vasculature through the leaky endothelial tissue surrounding the tumor and accumulate in certain solid tumors. Most polymeric nanoparticles display this enhanced permeability and retention effect (EPR effect). Several synthetic as well as natural polymer carriers such as poly(ethylene glycol) (PEG), N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA) and poly(l-glutamic acid) (PG) have been successfully used in improving cancer chemotherapy [6], [7], [8].

Heparin is a natural polysaccharide held together by glycosidic linkages. The covalently linked sulfate, hydroxyl and carboxyl groups of heparin are the main functional groups contributing to its anticoagulant activity [9], [10]. Other studies have shown that heparin is not just an anticoagulant, but actually a complex set of multifunctional glycosaminoglycan molecules with many other potential biological effects on cancer cells [11], [12]. Recently, many reports have shown that the effect of heparin on tumors is associated with the binding of growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [13], [14]. Heparin is also associated with the inhibition of heparanases enzymes that are thought to be required by tumor cells for invasion of the vascular basement membrane [15]. These features make heparin a promising candidate as a drug carrier for targeted drug delivery.

Paclitaxel (Taxol) is an effective anticancer drug that has shown significant antineoplastic activity against various human cancers, including breast and ovarian tumors [16], [17]. However, the clinical use of PTX is limited due to its poor solubility and toxicity. Currently, available PTX formulations include a combination of Cremophor EL and ethanol for solubilization. However, Cremophor EL is toxic and has side effects such as hypersensitivity, nephrotoxicity and neurotoxicity [17]. In order to overcome these limitations, polymeric carriers such as PG, polyethylene glycol (PEG) and albumin have been widely used to conjugate to or encapsulate PTX [18]. These carriers produced desirable pharmacokinetics, enhanced anti-tumor activity and low toxicity. To prepare a stable water-soluble conjugate exerting anti-tumor effects, we selected the natural polysaccharide heparin due to its following advantages: (a) it is highly hydrophilic due to the presence of sulfate, hydroxyl and carboxyl groups; (b) it is non-toxic in vivo and readily neutralized by antagonists; and (c) it acts as an anticancer agent.

Several strategies for the conjugation of drugs to polymer carriers have been developed to promote cancer cell targeting. In these approaches, drugs are directly conjugated to polymer carriers or to a linker for cleavage by lysosomal enzymes. This strategy can endow derivatives with enhanced water-solubility and chemical stability, an improved pharmacokinetic and distribution profile, and reduced side effects [4], [5], [18]. In this paper, we report the synthesis, characterization and in vitro experiment of heparin–PTX conjugate nanoparticles. We expect that our strategy will be applied to passive targeted delivery of therapeutic agents to tumor tissues.

Section snippets

Materials

Low molecular weight heparin (114.1 IU/mg, heparin) of average molecular weight ca. 5000 Da was purchased from Glaxosmithkline (Brentford, Middlesex, UK). Paclitaxel (PTX) was provided by Samyang Genex Co. (Daejeon, Korea). 4-nitrophenyl chloroformate (4-NPC), triethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarboimide hydrochloride (EDC), 4-methylmorpholine, N-hydroxysuccinimide (NHS), ethylenediamine, dimethyl sulfoxide (DMSO), methanol, and n-hexane were purchased from Sigma Chemical CO.

Synthesis and characterization of heparin–PTX conjugates

We synthesized heparin–PTX nanoparticles for cancer targeting by conjugating the polysaccharide heparin with PTX. To introduce an amine group to PTX, ethylenediamine (EDA) was covalently linked to the hydroxyl group of PTX in the presence of EDC and NHS as shown in Fig. 1. The presence of an amine group on the PTX structure was confirmed by characteristic peaks at 2.0 ppm in the NMR spectrum of NH2–PTX. However, the specific conjugation site in PTX could not be identified. Cavallaro et al. also

Conclusion

Paclitaxel was aminated to conjugate with heparin via an amide linkage. The conjugation of PTX with heparin significantly increased the water-solubility of PTX. A higher coupling ratio of PTX to the heparin backbone could be obtained by increasing the PTX feed ratio. Heparin–PTX conjugates readily self-assembled to form spherical nanoparticles (200 nm) in aqueous solution. Heparin–PTX conjugate nanoparticles demonstrated greater cytotoxicity to KB cancer cells than did free PTX. Furthermore,

Acknowledgement

This work was supported by a grant from the Fundamental R&D Program for Core Technology of Materials funded by the Ministry of Knowledge Economy, Republic of Korea, by the Korea Research Foundation Grant funded by the Korean Government (KRF-D00289), and by BioImaging Research center at GIST.

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    Park and Kim equally contributed to this article as co-first authors.

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