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.

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.

Contents
1 Introduction 10
1.1 Overview of brain tumors 10
1.1.1 Introduction of brain tumors 10
1.1.2 The role of blood-brain barrier 11
1.1.3 Current status of brain tumors treatment 12
1.1.4 Gene therapy in brain tumors 13
1.1.5 Research of ultrasound-mediated gene delivery in brain tumors 16
1.2 Antiangiogenic-targeting MBs in gene therapy 17
1.2.1 Properties of DNA-loaded MBs 17
1.2.2 Properties of antiangiogenic-targeting MBs 18
1.3 Mechanisms of ultrasound-mediated gene delivery 19
1.3.1 Properties of MBs combined with FUS 19
1.3.2 FUS-induced blood-brain barrier opening 19
1.3.3 Ultrasound-mediated gene delivery in vitro 20
1.3.4 Ultrasound-mediated gene delivery in vivo 21
1.4 Overview of dissertation 22
2 Material and Methods 24
2.1 DNA-loaded VCMBs 24
2.1.1 Plasmid preparation 24
2.1.2 Preparation of DNA-loaded VCMBs 24
2.1.3 Characterization of DNA-loaded VCMBs 26
2.1.4 Measurement of avidin-biotin binding efficiency 26
2.1.5 Measurement of DNA loading efficiency 27
2.1.6 Nuclease protection assay 27
2.1.7 Microscope and cryo-TEM image 28
2.1.8 Measurement of C6 glioma cells targeting efficiency 29
2.2 Acoustic properties of DNA-loaded VCMBs 29
2.2.1 Stability Analysis 29
2.3 Ultrasound-mediated gene delivery in C6 glioma cells 30
2.3.1 Cell membrane permeability test 30
2.3.1.1 Experiment setup for cell membrane permeability test 30
2.3.1.2 Experimental design 31
2.3.1.3 Analysis of cell membrane permeability 31
2.3.2 pLUC transfection in vitro 32
2.3.2.1 Experiment setup for pLUC transfection in vitro 32
2.3.2.2 Experimental design 33
2.3.2.3 Analysis of pLUC expression and cell viability 33
2.3.3 pHsv-TK transfection in vitro 33
2.3.3.1 Experiment setup for pHsv-TK transfection in vitro 33
2.4 Ultrasound-mediated gene delivery in vivo 34
2.4.1 Monitoring of pLUC delivery in subcutaneous tumors 34
2.4.1.1 Experiment setup for pLUC delivery in subcutaneous tumors 34
2.4.2 pLUC delivery in brain tumors 35
2.4.2.1 Brain tumor model 35
2.4.2.2 Confirmation and quantification of FUS-BBB opening 36
2.4.2.3 Experiment setup for pLUC delivery in brain tumors 36
2.4.2.4 Analysis of pLUC expression 38
2.4.3 pHsv-TK delivery in brain tumors 38
2.4.3.1 Experiment setup for pHsv-TK delivery in brain tumors 38
2.4.3.2 Magnetic resonance imaging (MRI) 39
3. Results and Discussion 40
3.1 DNA-loaded VCMBs 40
3.1.1 Characterization of DNA-loaded VCMBs 40
3.1.2 Measurement of avidin-biotin binding efficiency 41
3.1.3 Measurement of DNA loading efficiency 42
3.1.4 Nuclease protection assay 43
3.1.5 Microscope and cryo-TEM Images of VCMBs 44
3.1.6 Measurement of C6 glioma cells targeting efficiency 45
3.2 Acoustic properties of DNA-loaded VCMBs 46
3.2.1 MBs stability analysis 46
3.2.2 Measurement of inertial cavitation dose and subharmonic intensity 47
3.3 Ultrasound-mediated gene delivery in C6 glioma cells 48
3.3.1 Cell membrane permeability test 48
3.3.2 Ultrasound-mediated gene delivery of pLUC in vitro 53
3.3.3 Ultrasound-mediated gene delivery of pHsv-TK in vitro 61
3.4 Ultrasound-mediated gene delivery of pLUC in subcutaneous tumors 62
3.4.1 Assessment of pLUC Expression 62
3.5 Ultrasound-mediated gene delivery of pLUC in brain tumors 64
3.5.1 In vivo FUS-induced BBB opening and EB release 64
3.5.2 Analysis of pLUC Expression 65
3.6 Ultrasound-mediated gene delivery of pHsv-TK in brain tumors 66
4. Discussion 68
4.1 Safety in ultrasound-mediated gene delivery 68
4.2 CMBs for increase gene loading efficiency 68
4.3 Antiangiogenic-targeting MBs applied in ultrasound-mediated gene delivery 69
4.4 Limitation of pHsv-TK/GCV system 70
4.5 Overall conclusion 71
5. Future Work 71
Reference 72
Contents
1 Introduction 10
1.1 Overview of brain tumors 10
1.1.1 Introduction of brain tumors 10
1.1.2 The role of blood-brain barrier 11
1.1.3 Current status of brain tumors treatment 12
1.1.4 Gene therapy in brain tumors 13
1.1.5 Research of ultrasound-mediated gene delivery in brain tumors 16
1.2 Antiangiogenic-targeting MBs in gene therapy 17
1.2.1 Properties of DNA-loaded MBs 17
1.2.2 Properties of antiangiogenic-targeting MBs 18
1.3 Mechanisms of ultrasound-mediated gene delivery 19
1.3.1 Properties of MBs combined with FUS 19
1.3.2 FUS-induced blood-brain barrier opening 19
1.3.3 Ultrasound-mediated gene delivery in vitro 20
1.3.4 Ultrasound-mediated gene delivery in vivo 21
1.4 Overview of dissertation 22
2 Material and Methods 24
2.1 DNA-loaded VCMBs 24
2.1.1 Plasmid preparation 24
2.1.2 Preparation of DNA-loaded VCMBs 24
2.1.3 Characterization of DNA-loaded VCMBs 26
2.1.4 Measurement of avidin-biotin binding efficiency 26
2.1.5 Measurement of DNA loading efficiency 27
2.1.6 Nuclease protection assay 27
2.1.7 Microscope and cryo-TEM image 28
2.1.8 Measurement of C6 glioma cells targeting efficiency 29
2.2 Acoustic properties of DNA-loaded VCMBs 29
2.2.1 Stability Analysis 29
2.3 Ultrasound-mediated gene delivery in C6 glioma cells 30
2.3.1 Cell membrane permeability test 30
2.3.1.1 Experiment setup for cell membrane permeability test 30
2.3.1.2 Experimental design 31
2.3.1.3 Analysis of cell membrane permeability 31
2.3.2 pLUC transfection in vitro 32
2.3.2.1 Experiment setup for pLUC transfection in vitro 32
2.3.2.2 Experimental design 33
2.3.2.3 Analysis of pLUC expression and cell viability 33
2.3.3 pHsv-TK transfection in vitro 33
2.3.3.1 Experiment setup for pHsv-TK transfection in vitro 33
2.4 Ultrasound-mediated gene delivery in vivo 34
2.4.1 Monitoring of pLUC delivery in subcutaneous tumors 34
2.4.1.1 Experiment setup for pLUC delivery in subcutaneous tumors 34
2.4.2 pLUC delivery in brain tumors 35
2.4.2.1 Brain tumor model 35
2.4.2.2 Confirmation and quantification of FUS-BBB opening 36
2.4.2.3 Experiment setup for pLUC delivery in brain tumors 36
2.4.2.4 Analysis of pLUC expression 38
2.4.3 pHsv-TK delivery in brain tumors 38
2.4.3.1 Experiment setup for pHsv-TK delivery in brain tumors 38
2.4.3.2 Magnetic resonance imaging (MRI) 39
3. Results and Discussion 40
3.1 DNA-loaded VCMBs 40
3.1.1 Characterization of DNA-loaded VCMBs 40
3.1.2 Measurement of avidin-biotin binding efficiency 41
3.1.3 Measurement of DNA loading efficiency 42
3.1.4 Nuclease protection assay 43
3.1.5 Microscope and cryo-TEM Images of VCMBs 44
3.1.6 Measurement of C6 glioma cells targeting efficiency 45
3.2 Acoustic properties of DNA-loaded VCMBs 46
3.2.1 MBs stability analysis 46
3.2.2 Measurement of inertial cavitation dose and subharmonic intensity 47
3.3 Ultrasound-mediated gene delivery in C6 glioma cells 48
3.3.1 Cell membrane permeability test 48
3.3.2 Ultrasound-mediated gene delivery of pLUC in vitro 53
3.3.3 Ultrasound-mediated gene delivery of pHsv-TK in vitro 61
3.4 Ultrasound-mediated gene delivery of pLUC in subcutaneous tumors 62
3.4.1 Assessment of pLUC Expression 62
3.5 Ultrasound-mediated gene delivery of pLUC in brain tumors 64
3.5.1 In vivo FUS-induced BBB opening and EB release 64
3.5.2 Analysis of pLUC Expression 65
3.6 Ultrasound-mediated gene delivery of pHsv-TK in brain tumors 66
4. Discussion 68
4.1 Safety in ultrasound-mediated gene delivery 68
4.2 CMBs for increase gene loading efficiency 68
4.3 Antiangiogenic-targeting MBs applied in ultrasound-mediated gene delivery 69
4.4 Limitation of pHsv-TK/GCV system 70
4.5 Overall conclusion 71
5. Future Work 71
Reference 72

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03/23/accepted 2015.
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