Abstract
Glioblastoma multiforme (GBM) is a malignant brain tumor with poor prognosis and high recurrence rate despite traditional chemotherapy. Ultrasound-targeted microbubbles destruction (UTMD) has been approved to achieve local blood-brain barrier disruption (BBBD), enhancing therapeutic agents into the brain. Besides, UTMD has been employed to deliver tumor-killing gene for cancer therapy. Tumor vessels in GBM are highly rich in VEGF-A, which could bind VEGFR2 on endothelial cells, is widely used in targeted therapy. In this study, we fabricated DNA-loaded cationic microbubbles with anti-VEGFR2 antibody (VCMBs) for transient BBBD and targeted therapy in brain tumors. We used VCMBs for improving gene delivery by loading DNA on MBs shell and actively attaching on cancer cell. Expression of reporter gene, luciferase (pLUC, 6.5 kb), was used for monitoring gene transfer and optimization ultrasound parameters. Male Sprague-Dawley rats were injected 5x105 C6 glioma cells in left hemispheres of the brain in vivo. At 7 days post injection, 2x109 DNA-loaded VCMBs were injected via jugular vein, then ultrasound- mediated gene delivery was actuated by insonation of the brain tumor xenografts. Comparisons of treatment conditions across all time points revealed that the use of VCMBs in brain tumors resulted in significantly higher luciferase expression measured by IVIS ([5.5±1.5] to [11.6±4.4] x 103 photons/sec/cm2/sr,) relative to the use of CMBs in brain tumors ([4.4±1.4] to [7.3±1.1] x 103 photons/sec/cm2/sr, p*<0.5). Herpes simplex virus type 1 thymidine kinase (pHsv-TK, 7.2 kb) in combination with ganciclovir (GCV) has been shown as one of the most promising suicide gene systems for brain tumors treatment. For in vitro studies, 24 hours post transfection under FUS, the pHsv-TK transfected C6 glioma cells were incubated in the presence of 0-10 μg/ml GCV in medium. At day 4, cell viability of pHsv-TK transfected C6 glioma cells were decrease (100.0±10.5%, 90.2±21.4%, 63.6±8.9% and 57.6±4.9%) when GCV concentrations increase (0, 0.1, 1 and 50 μg/ml), respectively, showing significant cell death compared with cell viability of C6 glioma cells without treatment at different GCV concentrations (p*<0.5). For in vivo studies, rats were transfected with pHsv-TK with VCMBs under FUS after 6 days of tumor growth. Each rat was intraperitoneally injected with 0.2 ml (100 mg/kg/day) GCV every 24 h lasting for 8 days. The tumor volume measured by MRI on Day 18 was significantly smaller in the rats treated with pHSV-TK/GCV system with VCMBs under FUS (3.8 mm3) than in the rats without treatment (19.75 ± 8.3 mm3). Additionally, rats with treatment were significantly prolonged survival time compared to the rats without treatment. Overall, this study aimed to develop the DNA-loaded VCMBs, using UTMD for achieving local BBBD, as a non-viral, noninvasive and targeted gene delivery approach in brain tumors.