Elsevier

Biomaterials

Volume 30, Issue 27, September 2009, Pages 4815-4823
Biomaterials

A chitosan hydrogel delivery system for osteosarcoma gene therapy with pigment epithelium-derived factor combined with chemotherapy

https://doi.org/10.1016/j.biomaterials.2009.05.035Get rights and content

Abstract

Osteosarcoma (OS) is the most common type of malignant bone cancer, and the sixth most common type of cancer in children and young adults. Currently, gene therapy is being evaluated as a novel method for OS treatment. Here we report on an in situ gelling chitosan-based hydrogel system that sustains the release of a potential anti-cancer gene (pigment epithelium-derived factor) to the tumor site. A significant reduction of the primary OS in a clinically relevant orthotopic model was measured. The combination of plasmid treatment and chemotherapy together with the use of this delivery system led to the highest suppression of tumor growth without side effects. The results obtained from this study demonstrate the potential application of a biodegradable hydrogel technology as an anti-cancer drug delivery system for successful chemo-gene therapy.

Introduction

OS is the most common primary solid tumor of bone in children and young adults [1], [2], comprising about 20% of primary bone sarcomas [3], and represents the second highest cause of cancer-related death in this age group [4]. Currently, the primary treatments of this disease include chemotherapy and surgery. Chemotherapy drugs can be administered both before and after surgery. Contemporary chemotherapy is normally the intravenous or oral administration of different chemotherapeutic agents [5], [6]. However, most chemotherapeutics carry the risk of both short-term and long-term toxic effects. In addition, despite improved aggressive treatment comprising of multi-agent chemotherapy and surgical resection, approximately 30–40% of all patients still develop lung metastasis which is the major cause of death from this disease [4], [7]. Since the current regimes used to treat OS have failed to eradicate the systemic spread of OS, alternative treatment such as gene therapy which should exclude the problems faced with current cytotoxic agents such as drug resistance and systemic toxicity has attracted significant research interest.

The study of chronological and spatial patterns of OS growth and invasion of local tissue structures including growth plate cartilage in bone has revealed that this relatively avascular structure acts as a natural barrier to the progression of this tumor [2]. This corresponded with the growth plate layers with high expression of the most potent endogenous anti-angiogenic factor, pigment epithelium-derived factor (PEDF). Therefore, PEDF may have potential therapeutic applications in OS, by improving the local control of tumor growth and increasing the potential for limb-sparing surgery, as well as reducing the risk of metastatic spread in this disease. Ek et al. have demonstrated the therapeutic potential of PEDF against OS using two clinically relevant orthotopic models of OS and a set of cell-based assays relevant to OS proliferation, apoptosis, collagen-1 adhesion, differentiation, angiogenesis and metastasis [7].

The delivery of foreign DNA into host cells has been accomplished by non-viral vectors such as liposomes and DNA–polymeric complexes [8], and by viral vectors such as retroviral and adeno-associated viral vectors [9]. However, the efficiency of non-viral vectors is very low and they are rapidly inactivated in the presence of serum. On the other hand, viral vectors have provided high transduction efficiency but faced serious safety problems with some clinical deaths reported [9]. Recently, PEDF plasmid (pPEDF) has been encapsulated in chitosan microparticles as a delivery vehicle for OS treatment [10], though proper efficacy was not determined.

In the previous study, we have developed a thermal and pH responsive chitosan in situ gelling system using low concentrations of an inorganic phosphate salt, dipotassium orthophosphate (DPO) as the gelling agent [11]. We have also demonstrated the sustained releasing properties of Chi/DPO hydrogels which are liquid at low or room temperature but gel at body temperature (37 °C). The successful application of this biodegradable and biocompatible Chi/DPO hydrogel in the delivery of Doxorubicin (Dox), a small anti-tumor molecule, for the treatment of OS in a clinically relevant orthotopic mouse model has been also reported [12]. In this paper, a further application of Chi/DPO technology in delivering pPEDF, a large bioactive molecule, in OS treatment was presented. The same clinically relevant metastatic mouse model of OS [4] was employed in this study. The dual-pronged regime for OS treatment provided by the combination of chemotherapy and plasmid treatment was discussed.

Section snippets

Materials

Low molecular weight chitosan from crab shells (∼120 kDa) with deacetylation degree between 75% and 85% was obtained from Fluka BioChemika (Switzerland). Dipotassium phosphate (K2HPO4) (DPO) and acetic acid (CH3COOH) were purchased from Ajax Finechem (Australia). Doxorubicin was obtained from Sigma–Aldrich, Australia. PEDF plasmid was obtained as previously described [10]. Briefly, PEDF DNA insert was cloned into pcDNA3.1-his-myc(−)A vector (Invitrogen) for transfection. The plasmids expressing

Characterization of Chi/DPO solutions

Five different Chi/DPO formulations were characterized and tested in an in vitro release study to figure out the most suitable formulation for delivery of pPEDF. They were prepared by varying chitosan concentration, orthophosphate concentration and drug loading parameters (Table 1).

Discussion

PEDF, a widely expressed 50 kDa secreted glycoprotein is a member of the serine protease inhibitor (serpin) family, although it does not inhibit proteases [18]. It has been identified as one of the most potent endogenous inhibitors of angiogenesis. It induces endothelial cell apoptosis through the Fas/FasL death pathway and decreases the expression of important pro-angiogenic factors such as VEGF [19], [20], [21]. It also promotes cell differentiation and influences cell proliferation by

Conclusion

The potential application of Chi/DPO hydrogel DDS for OS treatment has been demonstrated again in this paper. pPEDF, a potential anti-tumor bio-agent was successfully released from this DDS in a sustained manner with its functionality preserved. The release of PEDF plasmid was mainly governed by the degradation of Chi/DPO hydrogel. The incorporation of pPEDF in Chi/DPO significantly inhibited the growth of OS and osteolysis. However, due to the slow release of pPEDF from the hydrogel matrix,

Acknowledgement

This project was supported by the University of Melbourne and St Vincent's Hospital (Australia). We would like to thank Mr. Bruce Abaloz (Department of Zoology, the University of Melbourne) for his help with the histology analysis.

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