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Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

ABSTRACT: The purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs).Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the HRECs was measured using the 3-(4,5-dimthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. The transcription and expression of endostatin, VEGF, Bcl-2, and Bcl-xl were measured via quantitative real-time PCR (qPCR) and western blotting, respectively.CMBs differed significantly from NMBs in terms of the zeta potential, but no differences in size were detected. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group (p<0.05), while the transcription and expression of VEGF, Bcl-2, and Bcl-xl were lowest compared with the other groups. Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).This study demonstrated that ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of ES-GFP, which had obvious impacts on the inhibition of the growth process of HRECs in vitro. These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.

SUBMITTER: Xu Y

PROVIDER: S-EPMC4548789 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

Xu Yan Y Xie Zongyuan Z Zhou Yu Y Zhou Xiyuan X Li Pan P Wang Zhigang Z Zhang Qunxia Q

Molecular vision 20150825

<h4>Purpose</h4>The purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs).<h4>Methods</h4>Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the H ...[more]

PMID: 26321867

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Project description:ObjectivesUltrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection.BackgroundContrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound.MethodsCationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging.ResultsCharge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles.ConclusionsWe conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Project description:Hypoxia-inducible factor 1α (HIF-1α) plays a critical protective role in ischemic heart disease. Under normoxic conditions, HIF-1α was degraded by oxygen-dependent prolyl hydroxylase-2 (PHD2). Gene therapy has become a promising strategy to inhibit the degradation of HIF-1α and to improve cardiac function after ischemic injury. However, conventional gene delivery systems are difficult to achieve a targeted and localized gene delivery into the ischemic myocardia. Here, we report the localized myocardial delivery of shRNA against PHD2 through ultrasound-targeted microbubble destruction (UTMD) for protection the heart from acute myocardial infarction. In this study, a novel cationic microbubble was fabricated by using of the thin-film hydration and sonication method. The resulting microbubbles had a 28.2 ± 2.21 mV surface zeta potential and could greatly improve DNA binding performance, achieving 17.81 ± 1.46 μg of DNA loading capacity per 5 × 108 microbubbles. Combined with these cationic microbubbles, UTMD-mediated gene delivery was evaluated and the gene transfection efficiency was optimized in the H9C2 cardiac cells. Knockdown of PHD2 gene was successfully realized by UTMD-mediated shPHD2 transfection, resulting in HIF-1α-dependent protective effects on H9C2 cells through increasing the expression of HIF-1α, VEGF and bFGF. We further employed UTMD-mediated shPHD2 transfection into the localized ischemic myocardia in a rat ischemia model, demonstrating significantly reduced infarct size and greatly improved the heart function. The silencing of PHD2 and the up-regulation of its downstream genes in the treated myocardia were confirmed. Histological analysis further revealed numbers of HIF-1α- and VEGF-, and CD31-positive cells/mm2 in the shPHD2-treated group were significantly greater than those in the sham or control vector groups (P < 0.05). In conclusion, our study provides a promising strategy to realize ultrasound-mediated localized myocardial shRNA delivery to protect the heart from acute myocardial infarction via cationic microbubbles.

Project description:ObjectiveTo investigate the efficacy and mechanism of ultrasound-targeted microbubble destruction (UTMD) combined with radiotherapy (XRT) on glioblastoma.MethodsThe enhanced radiosensitization by UTMD was assessed through colony formation and cell apoptosis in Human glioblastoma cells (U87MG). Subcutaneous transplantation tumors in 24 nude mice implanted with U87MG cells were randomly assigned to 4 different treatment groups (Control, UTMD, XRT, and UTMD + XRT) based on tumor sizes (100-300 mm3). Tumor growth was observed for 10 days after treatment, and then histopathology stains (HE, CD34, and γH2AX) were applied to the tumor samples. A TUNEL staining experiment was applied to detect the apoptosis rate of mice tumor samples. Meanwhile, tissue proteins were extracted from animal specimens, and the expressions of dsDNA break repair-related proteins from animal specimens were examined by the western blot.ResultsWhen the radiotherapy dose was 4 Gy, the colony formation rate of U87MG cells in the UTMD + XRT group was 32 ± 8%, lower than the XRT group (54 ± 14%, p < 0.01). The early apoptotic rate of the UTMD + XRT group was 21.1 ± 3% at 48 h, higher than that of the XRT group (15.2 ± 4%). The tumor growth curve indicated that the tumor growth was inhibited in the UTMD + XRT group compared with other groups during 10 days of observation. In TUNEL experiment, the apoptotic cells of the UTMD + XRT group were higher than that of the XRT group (p < 0.05). The UTMD + XRT group had the lowest MVD value, but was not significantly different from other groups (p > 0.05). In addition, γH2AX increased due to the addition of UTMD to radiotherapy compared to XRT in immunohistochemistry (p < 0.05).ConclusionsOur study clearly demonstrated the enhanced destructive effect of UTMD combined with 4 Gy radiotherapy on glioblastoma. This could be partly achieved by the increased ability of DNA damage of tumor cells.

Project description:BackgroundThe present study aimed to determine whether ultrasound-targeted microbubble destruction (UTMD)-mediated angiopoietin 1 (Ang1) gene transfection can improve angiogenesis and potentially reverse left ventricular (LV) structural and sympathetic nerve remodeling in canines with myocardial infarction (MI).MethodsThirty dogs were randomly divided into groups (n=10/group) as follows: the MI group (MI dogs without UTMD treatment), the UTMD group (MI dogs with UTMD-mediated negative control plasmid treatment), and the UTMD-Ang1 group (MI dogs with UTMD-mediated Ang1 plasmid treatment). LV dimensions, systolic function, and synchrony were used to reflect the structural remodeling. The density of tyrosine hydroxylase (TH)- and growth-associated protein 43 (GAP43)-positive nerve fibers were calculated to assess the sympathetic nerve remodeling.ResultsOne month after treatment, the UTMD-Ang1 group showed lower LV end-diastolic dimension (LVEDD: 31.2±2.3 mm) and higher LV ejection fraction (LVEF: 44.6%±4.3%) than the MI group (LVEDD: 34.5±2.2 mm, t=2.282, P=0.014; LVEF: 37.3%±3.1%, t=3.718, P=0.003) and the UTMD group (LVEDD: 34.1±2.8 mm, t=2.264, P=0.040; LVEF: 39.3%±4.5%, t=2.408, P=0.030). LV synchrony was higher in the UTMD-Ang1 group compared with the MI group by 2-dimensional speckle-tracking echocardiography. Angiogenic density was higher in the UTMD group than the MI group but was highest in the UTMD-Ang1 group according to immunohistochemistry of CD31 and α-smooth muscle actin staining. The density of TH- and GAP43-positive nerve fibers were decreased in the UTMD-Ang1 group (TH: 1,928.2±376.6 μm2/mm2; GAP43: 2,090.8±329.2 μm2/mm2) compared with the MI group (TH: 2916.5±558.4 μm2/mm2, t=4.069, P=0.001; GAP43: 3,275.4±548.6 μm2/mm2, t=5.153, P=0.000) and the UTMD group (TH: 2,552.7±408.1 μm2/mm2, t=3.181, P=0.007; GAP43: 2,630.5±419.3 μm2/mm2, t=2.863, P=0.013). The relative Ang1 and sarcoplasmic reticulum Ca2+-ATPase 2a protein levels were significantly higher in the UTMD-Ang1 group than the UTMD and MI groups by Western blot, while the phospholamban levels exhibited the opposite trend. Plasma norepinephrine and N-terminal pro-B-type-natriuretic peptide were significantly reduced in the UTMD-Ang1 group from day 1 to 1 month after MI.ConclusionsUTMD-mediated Ang1 transfection can promote angiogenesis, reverse LV structural and sympathetic nerve remodeling, and improve LV synchrony after MI.

Project description:BackgroundContrast ultrasound-mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver.MethodsCharge coupling of fluorescently labeled cDNA to either neutral MBs (MBN) or cationic MBs (MB+) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MBN or MB+ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 μg/10(8) MBs).ResultsCharge-coupling of cDNA was detected for MB+ but not MBN. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB+ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB+, which was approximately fivefold higher (P < .05) than that for MBN. There was no effect of DNA supersaturation. The same pattern was seen for liver except that supersaturation further increased transfection with MBN equal to that of MB+.ConclusionsEfficient charge-coupling of cDNA to MB+ but not MBN occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake.

Dataset Information

Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

Publications

Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

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