Angiogenesis-targeting microbubbles combined with ultrasound-mediated gene therapy in brain tumors.

The major challenges in gene therapy for brain cancer are poor transgene expression due to the blood-brain barrier (BBB) and neurologic damage caused by conventional intracerebral injection. Non-viral gene delivery using ultrasound-targeted microbubbles (MBs) oscillation via the systematic transvascular route is attractive, but there is currently no high-yielding and targeted gene expression method. In this study, we developed a non-viral and angiogenesis-targeting gene delivery approach for efficient brain tumor gene therapy without brain damage. We developed a VEGFR2-targeted and cationic microbubbles (VCMBs) gene vector for use with transcranial focused ultrasound (FUS) exposure to allow transient gene delivery. The system was tested in a brain tumor model using the firefly luciferase gene and herpes simplex virus type 1 thymidine kinase/ganciclovir (pHSV-TK/GCV) with VCMBs under FUS exposure for transgene expression and anti-tumor effect. In vitro data showed that VCMBs have a high DNA-loading efficiency and high affinity for cancer cells. In vivo data confirmed that this technique enhanced gene delivery into tumor tissues without affecting normal brain tissues. The VCMBs group resulted in higher luciferase expression (3.8 fold) relative to the CMBs group (1.9 fold), and the direct injection group. The tumor volume on day 25 was significantly smaller in rats treated with the pHSV-TK/GCV system using VCMBs under FUS (9.7±5.2mm3) than in the direct injection group (40.1±4.3mm3). We demonstrated the successful use of DNA-loaded VCMBs and FUS for non-viral, non-invasive and targeted gene delivery to brain tumors.

An epirubicin-conjugated nanocarrier with MRI function to overcome lethal multidrug-resistant bladder cancer.

Multidrug resistance (MDR) presents a major obstacle to curing cancer. Chemotherapy failure can occur through both cell membrane drug resistance (CMDR) and nuclear drug resistance (NDR), and anticancer effectiveness of chemotherapeutic agents is especially reduced by their nuclear export. Here we report an exciting magnetically-targeted nanomedicine formed by conjugation of epirubicin (EPI) to non-toxic and high-magnetization nanocarrier (HMNC). Strikingly, HMNC-EPI overcomes both CMDR and NDR in human bladder cancer cell models, without using P-glycoprotein (P-gp) and nuclear pore inhibitors. Besides, the half-life of drug is prolonged ~1.8-fold (from 45 h to 81 h) at 37 °C, with a ~10-fold increase in concentration at the tumor site through magnetic targeting (MT). Moreover, malignant NDR bladder cancer can be effectively inhibited after 14 days in mice by just two injections and MT. We are the first to demonstrate the nanomedical strategy that can overcome the CMDR and NDR bladder cancers simultaneously, and proceed to the excellent MT therapy, significantly reducing the dosage and cardiotoxicity and holding great promise for incurable human MDR bladder cancer.

In vivo assessment of macrophage CNS infiltration during disruption of the blood-brain barrier with focused ultrasound: a magnetic resonance imaging study.

Focused ultrasound has been discovered to locally and reversibly increase permeability of the blood-brain barrier (BBB). However, inappropriate sonication of the BBB may cause complications, such as hemorrhage and brain tissue damage. Tissue damage may be controlled by selecting optimal sonication parameters. In this study, we sought to investigate the feasibility of labeling cells with superparamagnetic iron oxide particles to assess the inflammatory response during focused-ultrasound-induced BBB opening. We show that infiltration of phagocytes does not occur using optimal parameters of sonication. Taken together, the results of our study support the usefulness and safety of focused-ultrasound-induced BBB opening for enhancing drug delivery to the brain. These findings may have implications for the optimization of sonication parameters.

Quantitative micro-SPECT/CT for detecting focused ultrasound-induced blood-brain barrier opening in the rat.

INTRODUCTION: Focused ultrasound has been discovered to be able to locally and reversibly increase the permeability of the blood-brain barrier (BBB). The purpose of this study was to investigate the feasibility of micro-single photon emission computed tomography/ computed tomography (micro-SPECT/CT) and 99mTc diethylenetriamine pentaacetate (99mTc-DTPA) for identifying disruption of the BBB induced by focused ultrasound in a rat model. We also assessed the amount of radiotracer that had crossed the BBB using various intensity levels of ultrasound energy. METHODS: Immediately after sonication, three Sprague-Dawley rats were scanned for 2 h to determine the optimum time for data acquisition. Static SPECT with 1.5-h acquisition time was then performed in 12 rats sonicated with focused ultrasound pressure amplitudes of 0.78-2.45 MPa. Radiotracer and blue dye were used for lesion delineation. SPECT images were evaluated quantitatively and compared to results of histology and autoradiography. Terminal deoxynucleotidyl transferase biotin-desoxyuridine 5'-triphosphate (dUTP) nick end labeling staining was used to examine hemorrhage and tissue damage. RESULTS: The disruption to nondisruption radioactivity ratio showed a gradual increase from dynamic SPECT images, reaching a peak at 1.5 h post injection. The extent and intensity of radioactivity showed a good correlation with autoradiographic distribution and blue dye staining. SPECT measures correlated significantly with quantitative autoradiographic results (r(2)=0.90). According to SPECT findings, high acoustic powers allowed the delivery of larger amounts of radiotracer [0.001+/-0.002%ID (percent injected dose) under 0.78 MPa vs. 0.036+/-0.022%ID under 2.45 MPa]. Brain hemorrhage and tissue damage occurred at pressure amplitudes higher than 1.9 MPa. CONCLUSIONS: Our data demonstrate the usefulness of (99m)Tc-DTPA micro-SPECT/CT for detecting focused ultrasound-induced BBB disruption in the rat. This method may be used in vivo in combination with quantitative analysis for evaluating the amount of BBB opening.

Magnetic resonance imaging enhanced by superparamagnetic iron oxide particles: usefulness for distinguishing between focused ultrasound-induced blood-brain barrier disruption and brain hemorrhage.

PURPOSE: To investigate the usefulness of a fully flow-compensated heavy T2*-weighted imaging enhanced by superparamagnetic iron oxide (SPIO) particles for distinguishing between focused ultrasound-induced disruption of blood-brain barrier (BBB) and brain hemorrhage. MATERIALS AND METHODS: Focused ultrasound (frequency: 1.5 MHz) was used to induce disruption of the BBB in 39 rats. Two T2*-weighted images were obtained before and after SPIO administration. Preenhanced T2*-weighted images were used to detect hemorrhage. Detection of BBB disruption was performed on SPIO-enhanced images. Thirty-four rats were sacrificed after magnetic resonance (MR) scanning for histological confirmation of brain lesions. The remaining five animals were followed up for 35 days. Prussian blue staining was performed on histological sections to detect SPIO particles in the brain. RESULTS: After SPIO injection the areas of BBB disruption in rat brain were significantly enlarged. The area of mismatch between the T2*-weighted images indicated a safe region where BBB opening occurred without hemorrhagic complications. In the longitudinal study, removal of SPIO occurred at a faster rate in hemorrhagic areas, albeit being closer to that occurring in the liver. The presence of SPIO was confirmed by Prussian blue staining in brain parenchyma and capillary endothelial cells in areas of BBB disruption. CONCLUSION: T2*-weighted images-either with and without SPIO enhancement-may differentiate focused ultrasound-induced BBB disruption from brain hemorrhage.

Hemorrhage detection during focused-ultrasound induced blood-brain-barrier opening by using susceptibility-weighted magnetic resonance imaging.

High-intensity focused ultrasound has been discovered to be able to locally and reversibly increase the permeability of the blood-brain barrier (BBB), which can be detected using magnetic resonance imaging (MRI). However, side effects such as microhemorrhage, erythrocyte extravasations or even extensive hemorrhage may also occur. Although current contrast-enhanced T1-weighted MRI can be used to detect the changes in BBB permeability, its efficacy in detecting tissue hemorrhage after focused-ultrasound sonication remains limited. The purpose of this study is to investigate the feasibility of magnetic resonance susceptibility-weighted imaging (MR-SWI) for identifying possible tissue hemorrhage associated with disruption of the BBB induced by focused ultrasound in a rat model. The brains of 42 Sprague-Dawley rats were subjected to 107 sonications, either unilaterally or bilaterally. Localized BBB opening was achieved by delivering burst-mode focused ultrasound energy into brain tissue in the presence of microbubbles. Rats were studied by T2-weighted and contrast-enhanced T1-weighted MRI techniques, as well as by SWI. Tissue changes were analyzed histologically and the extent of apoptosis was investigated with the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling method. The results demonstrated that SWI is more sensitive than standard T2-weighted and contrast-enhanced T1-weighted MRI techniques in detecting hemorrhages after brain sonication. Longitudinal study showed that SWI is sensitive to the recovery process of the damage and, therefore, could provide important and complementary information to the conventional MR images. Potential applications such as drug delivery in the brain might be benefited.