Chitosan microparticles encapsulating PEDF plasmid demonstrate efficacy in an orthotopic metastatic model of osteosarcoma
Introduction
Tumorigenesis entails the sequential acquisition of numerous genetic defects that renders neoplastic cells capable of growing, invading and eventually metastasising [1]. Various specific events such as unregulated angiogenesis, aberrant cell-signalling pathways and disrupted cell–matrix interactions are rife in tumours. These events in turn form the foci for targeted molecular therapy. Therapy for non-superficial tumours is tricky due to the need for limiting effects of the transgene on neoplastic tissue and not affecting normal healthy tissue.
One tumour type that underscores the need for targeted treatment is osteosarcoma. Osteosarcoma is the second highest cause of cancer-related death in young people, essentially in the prime years of life. Although the current treatment of this aggressive tumour, which comprises of resection with pre- and post-operative multi-agent chemotherapy, has resulted in substantial improvements in survival, 1 in 3 patients still develop pulmonary metastases, and this remains the major cause of death from this condition [2], [3]. Therefore, it is the systemic spread of osteosarcoma which fails to be adequately eradicated with current regimes. Furthermore, dose intensification of chemotherapeutic agents, to the point of requiring hematopoietic stem cell reconstitution, has failed to consistently offer any added survival benefit to these patients [4]. One alternative source of potential osteosarcoma therapeutics is endogenous biologicals, which should preclude the problems faced with current cytotoxic agents such as drug resistance and toxicity. The progression of osteosarcoma adjacent to the growth plate cartilage in bone has revealed that this relatively avascular structure acts as a natural barrier to the progression of this tumour, which is attributable to the expression of anti-angiogenic factors such as pigment epithelium-derived factor (PEDF) in the growth plate cartilage [5].
PEDF is a widely expressed 50-kDa secreted glycoprotein that has been identified as one of the most potent endogenous inhibitors of angiogenesis, inducing endothelial cell apoptosis through the Fas/FasL death pathway, as well as decreasing the expression of important pro-angiogenic factors such as VEGF [6], [7], [8]. PEDF also plays a role in promoting cell differentiation and influencing cell proliferation by regulating the cell cycle [9] and inducing apoptosis [10]. Decreased levels of intratumoral PEDF have been correlated with higher microvessel density and a more metastatic phenotype and a more poorer outcome in numerous malignancies, highlighting the key role PEDF plays in neoplasia [11].
Previously, we have demonstrated the therapeutic potential of PEDF against osteosarcoma using two clinically relevant orthotopic models of osteosarcoma and a panel of cell-based assays relevant to osteosarcoma proliferation, apoptosis, differentiation, angiogenesis and metastasis [12]. The feasibility of PEDF as a biological drug candidate was also discussed with promising preliminary results for stability and toxicity, as well as bioactivity of short cost-effective peptides derived from the parent molecule (manuscript under consideration). However, the proof-of-principle studies used PEDF-overexpressing cells to demonstrate PEDF activity in vivo. We have extended the findings to include injection of cells with microparticles in an attempt to more closely emulate drug delivery that can be adapted in the clinic. Thus, what is needed is a realistic approach for delivery of genes to osteosarcoma primary tumours, one that can be translated into clinical usage.
An ideal delivery vehicle has to have the following features: be biocompatible, biodegradable, non-immunogenic, non-toxic, able to carry a variety of types of molecular agents without changing its own or their chemical constitution, able to release the ferried agent in a sustained (controlled) manner, be relatively easy and inexpensive to formulate, not require biohazardous chemicals or unsafe formulation procedures for manufacture, and be formulated from abundant natural raw materials. One such material is chitosan, a natural polysaccharide found in crustacean shells, which has been used for plasmid delivery in vivo via the oral route for vaccination against peanut allergy in mice [13]. Plasmid expression has been found to be higher in vivo than that achieved with naked plasmid or transfected with lipofectamine cationic liposomes when particles were injected intramuscularly [14]. Most importantly, expression in the muscle lasts up to 12 weeks post-transfection [15]. Apart from gene delivery, chitosan has also been used widely for sustained drug delivery mainly due to its biocompatible, low immunogenic, and biodegradable nature [13].
Here, we demonstrate the feasibility of using chitosan microparticles, prepared using two different methods of complex coercavation, for delivery of plasmids into a osteosarcoma cell line. One microparticle, performing better than the other in cell culture, was then chosen for in vivo analysis with PEDF. It was shown to be efficacious in an orthotopic model of osteosarcoma [16], reducing both primary and secondary tumour growth in mice.
Section snippets
PEDF plasmid
Human PEDF cDNA was generated from muscle mRNA and cloned into a pCR-II vector (Invitrogen) for transformation. A BamH1-Xba1 insert was then cloned into pcDNA3.1-his-myc(-)A vector (Invitrogen) for transfection. The plasmids expressing green fluorescent protein (pGFP), pPEDF and the pcDNA3.1 empty vectors were amplified using TOP10 bacterial cells (Invitrogen, Australia). Plasmids were extracted and purified using the Midi-prep kit (Invitrogen, Australia).
Formulation of particles
Low viscous chitosan
Microparticle characterisation
Microparticles formulated with stirring (SMPs) were 600 nm in diameter on average (∼95%) according to particle sizing due to DLS, while a small percentage (∼5%) were 2.5 μm in diameter (data not shown). However, EM picked up a large number of particles that were 50–100 nm in diameter, which were not detected by DLS, mainly due to instrument incapability (Fig. 1A). The charge potential was 5.0 mV. Microparticles formulated with vortexing (VMPs) were 440 nm in diameter although EM (Fig. 1B) revealed
Discussion
We have demonstrated the dramatic effects of PEDF on in vitro tumour cell invasion and adhesion when administered as a plasmid DNA-expressible form (pPEDF, [12]). In vivo, PEDF expression in osteosarcoma cells may play a key anti-metastatic role, possibly through regulation of the microenvironment or direct changes in tumour cell phenotype. This was evident in this study where pPEDF resulted in a 2-fold reduction in SaOS-2 spontaneous tumour formation in the lungs (secondary site of cancer cell
Conclusion
Osteosarcoma is a disease that is debilitating if not fatal mainly in adolescents. Due to the inadequate management of this disease with current surgery and chemotherapy, novel drugs are being tested, including the less toxic biologicals such as proteins. One such protein, PEDF, the most potent anti-angiogenic factor found endogenously in the body, was used in a plasmid-expressible form, and encapsulated within two different types of chitosan microparticles. The effects of transgene expression
Acknowledgements
This study was generously supported by grants from the Cancer Council of Victoria, the Australian Orthopaedic Association, and the Victorian Orthopaedic Research Trust Grant.
Competing interests statement: The authors declare that they have no competing financial interests.
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2018, Molecular and Cellular EndocrinologyCitation Excerpt :Bone pathologies such as osteogenesis imperfecta (OI) (Becker et al., 2011) and osteosarcoma (Ek et al., 2007) have absent or low levels of PEDF. Moreover, PEDF has shown potent anti-osteosarcoma efficacy as gene therapy (Dass et al., 2007) or protein therapy (Broadhead et al., 2011). Studies from our lab and others have demonstrated the potency of PEDF to regulate osteogenic genes and induce osteogenesis in mesenchymal stem cells (MSCs). (