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Targeted Gene Therapy Using Targeted Cationic Microbubbles Conjugated With Cd105 Antibody Compared With Untargeted Cationic And Neutral Microbubbles

Posted on:2016-07-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y ZhouFull Text:PDF
GTID:1224330482453596Subject:Medical imaging and nuclear medicine
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PART I PREPARATION AND CHARACTERIZATION OF NEUTRAL MICROBUBBLES, CATIONIC MICROBUBBLES AND TARGETED CATIONIC MICROBUBBLES SECTION I PREPARATION AND IDENTIFICATION OF NEUTRAL MICROBUBBLES, CATIONIC MICROBUBBLES AND TARGETED CATIONIC MICROBUBBLESObjective To develop neutral microbubbles (NMB), cationic microbubbles (CMB) and targeted cationic microbubbles conjugated with a CD 105 antibody (CMB 105) for use in gene therapy.Methods Microbubbles were prepared by sonication of relevant lipid mixtures, DC-cholesterol was added to make the microbubbles charge positively, CD 105 antibody was conjugated to CMB via an "biotin-avidin" bridge to make it have the target ability, and isotype control antibody conjugated to CMB to serve as control (C-CMB105). The size distribution and zeta-potential were analyzed, the morphology was observed under the light microscope (LM), the success binding between antibody and microbubbles was assessed by confocal fluorescence microscopy. Conventional ultrasound imaging using NMB, untargeted CMB, C-CMB105 and CMB 105 were performed in healthy mice first to obtain their circulation characteristicsResults NMB, CMB, C-CMB105 and CMB 105 was constructed successfully, all of them had a good dispersion with uniform size under light microscope, and both the C-CMB105 and the CMB 105 shown green fluorescence under confocal laser scanning microscope. The zeta-potentials of CMB (26.44±2.13 mV) and CMB 105 (26.62±2.48 mV) were significantly higher than for NMB (-2.38±0.56 mV; P<0.001). The half-life of CMB, C-CMB105 and CMB105 was 8.35±1.27,7.63±1.61 and 7.27±1.33 min, respectively, while the half-life of NMB was longer than 20 mm.Conclusions NMB, CMB, C-CMB105 and CMB 105 was constructed successfully, the addition of CD 105 antibody to the CMB did not significantly affect the zeta-potential and diameter of CMB. The half-life of CMB, C-CMB105 and CMB 105 was shorter than that of NMB because of the surface of them charged positively.SECTION Ⅱ ASSESSMENT OF THE TARGET ABILITY OF NMB, CMB AND CMB105 BOTH IN VITRO AND IN VIVOObjective To detect the ability of NMB, CMB and CMB 105 to target to vascular endothelium cells which express CD 105 both in vitro and in vivo.Methods First, a special set up was developed by ourselves, which was used in our microbubbles target ability test and gene transfect experiments. A coverslip of HUVECs was placed on a platform with the cells facing the microbubbles to ensure that the cells were in contact with the microbubbles. After 15 min at RT, the coverslips were gently washed twice with PBS and examined under a microscope to evaluate the number of microbubbles which target binding to HUVECs. Second, a tumor model of subcutaneous breast cancer in nude mice was used for our experiments, the expression of CD 105 in neovascular endothelial cells in tumor was confirmed by immunohistochemistry. Different types of microbubbles were injected into the tail vein, the ability of target tumor neovascularization in vivo was measured through ultrasound imaging. C-CMB105 were measured as isotype control of CMB 105 both in vitro and in vivo, and competition tests were taken to confirm the special binding between CMB 105 and vascular endothelium cells which express CD 105.Results All of CMB, CMB 105 and C-CMB105 could target to HUVECs which express CD105 except for NMB, and more targeted bound CMB105 were observed compared with CMB, C-CMB105 (P<0.01). An obvious inhibition of CMB105 was observed when HUVECs were preincubated with anti-CD105 antibody (P<0.01), whereas no inhibition was found when preincubated with isotype control antibody (P>0.05). For in vivo experiments, all of NMB, CMB and C-CMB105 couldn’t target to tumor neovascularization cells which express CD 105 except for CMB 105. Preinjection of CD 105 antibody led to significantly decreased binding of CMB105 in vivo(P<0.001)Conclusions CMB, CMB 105 and C-CMB105 could bind to HUVECs in vitro, whereas only CMB 105 could target bind to HUVECs, the mechanism of CMB and C-CMB105 to target to HUVECs was electrostatic interaction, this advantage was disappeared in vivo, and further confirmed the target ability of CMB 105.PART Ⅱ ULTRASOUND MEDIATED GENE TRANSFER (UMGT) TO HUVECs WITH NMB, CMB AND CMB105SECTION I ASSMENT OF THE PLASMID LOADING CAPACITY AND HUVECs MEMBRANE PERMEABILITY AND VIABILITY AFTER UMGT WITH NMB, CMB or CMB105Objective To evaluate the endostatin (ES-GFP) plasmid loading capacity and the viability of HUVECs after transferred by ultrasound with NMB, CMB and CMB 105.Methods ES-GFP plasmid was amplified, extracted and identified first, 5×108 NMB, CMB or CMB105 were incubated with varying doses of ES-GFP plasmids (10,20,40, and 80 μg) in optiMEM for 15 min to allow spontaneous electrostatic charge-coupling between the DNA and microbubbles. Then, the samples were centrifuged and washed. The concentration of unbound DNA was detected through spectrophotometry. Then, the amount of bound DNA was calculated by subtracting the amount of unbound DNA from the total amount of DNA added initially. Second, after target bound to HUVECs, NMB, CMB and CMB 105 was destructed by ultrasound, blank group was used as control. The permeabilization was detected by staining with FDA and PI, FDA+/PI+meant that the cell membrane was reversibly open. The effects of microbubbles and ultrasound on cell proliferation were evaluated by an MTT assay.Results the DNA loading capacities of NMB, CMB and CMB 105 were 16.76±1.75 μg,18.21±1.22μg, and 0.48±0.04μg per 5×108 microbubbles, respectively, difference between CMB and CMB 105 wasn’t significant (P>0.05). The positive rate of FDA+/PI+ was highest in CMB 105 group (36.89±4.28%) compared with control group (0.121±0.013%), NMB (6.19±2.35%) group and CMB group (28.12±3.58%, P<0.01). The cell viabilities for each group were 92.45±2.74%,90.63±2.52%,84.29± 5.07%,77.38±5.33%, respectively. The proliferation activity in CMB and CMB105 group was decreased, and the difference between them was significant (P<0.01).Conclusions The plasmid loading capacity of CMB and CMB105 was increased significantly because of the surface of them charged positively, and the plasmid loading capacity wasn’t affected after conjugation with antibody. With the increasing of target bound microbubbles, the permeability of cell membrane increased also, while the proliferation activity decreased.SECTION Ⅱ ASSESSMENT OF THERAPY EFFECT AFTER ES PLASMID TRANSFERRED BY ULTRASOUND WITH NMB, CMB AND CMB105Objective To detect the transfection efficiency and cell cycle, to evaluate the therapy effect on angiogenesis and tumor cell invasion in vitro.Methods The HUVECs were divided into four groups as follows:① control, ② NMB,③ CMB,④ CMB 105, ultrasound was applied at 1 MHz,1 W/cm2, and 50% duty cycle (DC) for 30 s, cells were collected for transfection efficiency and cell cycle detection by flow cytometry, the mRNA and protein level of endostatin, VEGF and caspase-3 were detected by qPCR and Western blot, respectively. The therapy effects were evaluated through vascular tube formation and MDA-MB-231 cells invasion in vitro.Results The transfection rates for each group 24 h post-transfection were 0.22±0.02%,1.36±0.13%,22.87±1.64 and 33.60±2.02%, respectively, the differences among each group were significant (P<0.01). The cell cycle was inhibited in Gl phase 24 h post-transfection both in CMB and CMB 105 group, the inhibition was more obvious 48 h post-transfection, and the difference between CMB and CMB 105 group was significant (P<0.01). The mRNA and protein level of endostatin and caspase-3 were increased both in CMB and CMB 105 group, while for VEGF decreased, the difference between CMB and CMB105 group was significant (P<0.05). For vascular tube formation experiments, the number of tube for each group were 22.33±3.082,21.05±3.120,14.33±2.150 and 6.33±2.520, respectively. For tumor cells invasion tests, the number of invasive tumor cells were 67.33±5.06,64.72±5.38,34.33±3.22 and 25.17±2.84, respectively.Conclusions The transfection efficiency for CMB and CMB 105 group was higher than that of NMB group, and transfected with CMB 105 could led to the highest transfection efficiency and best therapy effect, an obvious inhibition on vascular tube formation and tumor cells invasion could be found.PART Ⅲ STUDY ABOUT THE THERAPY EFFECT ON BREAST CANCER IN NUDE MICE BY UMTRASOUND-TRAGETED MICROBUBBLE DESTRUCTION (UTMD) WITH NMB, CMB AND CMB105Objective Study about the therapy effects on breast cancer in nude mice by UTMD with NMB, CMB and CMB 105 carrying ES plasmid.Methods For inoculation, MDA-MB-231 tumor cell suspensions were injected subcutaneously in both hind limbs of nude mice. Xenograft tumors were allowed to grow for 14 days before the experimental studies began, NMB, CMB and CMB 105 were injected into the tail vein, and 10 min after injection, ultrasound-mediated gene transfer was performed. Ultrasound was applied at 1 MHz,2 W/cm2, and 50%duty cycle (DC) for 30 seconds, tumor xenografts on the left hind limb received the ultrasound mediated gene therapy and the right hind limb tumor xenografts were used as control. The size of tumor xenografts was recorded every 3 days. Fifteen days after transfer, the mice were sacrificed, the tumor growth inhibition rate was calculated and the tumor growth curve was delineated. TUNEL and PCNA were used to evaluate the apoptosis and proliferation of tumors, respectively.Results UTMD-mediated endostatin gene delivery both with CMB and CMB 105 significantly inhibited tumor growth, The inhibition rate of the CMB105 group was highest (53.18±5.69%) compared with CMB (27.57±3.02%) and NMB (10.63±1.47%). UTMD-mediated endostatin gene delivery with CMB105 could inhibit the tumor growth successfully. The apoptosis index (AI) for each group was 8.217±3.628%,25.06± 5.721% and 56.643 ±8.534%, respectively, and the proliferation index (PI) for each group was 78.353±8.726%,50.98±6.142% and 21.873±4.309%, respectively.Conclusions UTMD-mediated endostatin gene delivery with CMB105 could inhibit the tumor growth successfully, the therapy effects were better than with CMB and NMB.
Keywords/Search Tags:Neutral microbubbles, Cationic microbubbles, Targetedcationic microbubbles, Biotin-avidin, Targeted cationic microbubbles, Ultrasound imaging, Ultrasound, Permeability, Proliferation, Gene therapy, Endostatin, Angiogenesis, Invasion, Genetherapy
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