(549d) Application of DNA Vector-Based RNA Interference (RNAi) Technology in Controlled Release Gene/Drug Delivery Systems for Synergetic Therapy of Malignant Glioma

Tumors of the central nervous system (CNS) represent one of the most devastating forms of human disease. Malignant gliomas are characterized by three major physiological processes: proliferation, angiogenesis, and invasion. Traditional cytotoxic chemotherapies control malignant gliomas by blocking growth and proliferation mechanisms (e.g. Paclitaxel), but leave angiogenesis and invasion unchecked. Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix, disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Angiogenesis is absolutely essential for the growth and spread of tumor because tumors must acquire the ability to stimulate capillary formation to progress from a small localized growth with a limited oxygen and nutrient supply to a well-vascularized enlarged tumor. Therefore, synergistic therapy simultaneously suppressing glioma proliferation, angiogenesis and invasion becomes our pursuit to contribute a higher glioma therapeutic effect.

Matrix metalloproteinase-2 (MMP-2), a member of the MMP family that commonly overexpressed in glioma, can degrades most of the components of basement membranes and extracellular matrix, thus be proposed as an essential role in both glioma invasion and angiogenesis signal pathway. In an effort to achieve specific MMP-2 suppression, we have developed the designer RNAi plasmid by applying the recent RNA interference (RNAi) technology. RNAi can induce highly sequence-specific degradation of homologous mRNA by small interfering RNA (siRNA, a double-stranded RNA molecule of 21?23 bp), thus provided a powerful tool for silencing a target gene in gene therapy. Comparing with short half-life, poor penetration into the cells and high cost of synthetic siRNA, DNA vector-based RNA interference (RNAi) technology offers a unique advantage over conventional drugs, thus development of efficient RNAi plasmid delivery systems poses a huge challenge and offers great potential.

Biodegradable controlled gene/drug release devices encapsulating the MMP-2 RNAi plasmid and anticancer drug (Paclitaxel), have been fabricated by using electrohydrodynamic methods. Designed MMP-2 RNAi plasmid with a gene knockdown efficiency larger than 70% was complexed with polyethylenimine (PEI, a gene carrier), to achieve a high gene transfection efficiency and serve as the gene delivery unit. Poly (D,L-lactide-co-glycolide) (PLGA) based microfiber implants bearing both gene and drug was fabricated by electrospinning, while core/shell microparticles incorporating the two agents in different compartments was fabricated by coaxial Electrohydrodynamic Atomization (EHDA) process. The performance of these formulations in sustaining gene/drug release, inhibiting tumor cell viability, angiogenesis and invasion, were also evaluated based on C6 glioma cells.

The fabricated microfibers with a diameter of about 1 ìm was proved to achieve very high encapsulation efficiency (EE) for hydrophobic drug paclitaxel (>90%), while a moderate EE for the hydrophilic PEI/DNA complex (~50%) by forming emulsion right before electrospinning. The fibrous matrices not only provide greater surface area to volume ratio for effective drug release rates but also offer the ease for implantation in post-surgical brain tumor resected cavity. They are able to release the encapsulated agents in a sustained manner for more than 30 days. The core/shell microparticles incorporating paclitaxel in the hydrophobic PLGA phase and PEI/DNA in the hydrophilic poly(ethylene glycol) (PEG) phase, aim to entrap different agents in different compartment of the device, thus achieving tunable release kinetics relationship. Core(PEG)/shell(PLGA) and core(PLGA)/shell(PEG) microparticles were both fabricated with high encapsulation efficiency. All the dosage forms showed the advantage of sustained release of paclitaxel compared to acute Taxol® administration in prohibiting C6 glioma cell proliferation in vitro. The MMP-2 RNAi effects was induced in C6 glioma cells resulting suppressed angiogenesis and invasion. In addition, therapeutic effects of these formulations against intracranial C6 glioma tumors in BALB/c nude mice are also under investigation.