Gene-Loaded Fluorescent AuMBs Combined With US/NIR Bi-Mode Imaging,Double Irradiation-Photothermal-Gene Treatment For Hepatoma And Its Mechanism Study

Part I Preparation, characterization and biocompatibility research of gold nanorods, fluorescent AuMBs and gene loaded fluorescent AuMBs[Objective] To provide the theoretical and experimental basis for further clinical application in dual-modal imaging and hepatoma treatment, we aim to prepare, characterize and evaluate the biocompability and photo-thermal dynamics characteristics of gold nanorods (AuNRs), AuNRs-loaded fluorescent microbubbles (fluorescent AuMBs) and gene loaded fluorescent AuMBs.[Methods]①AuNRs were prepared using the improved one-pot method with phenols as the reducing agent. Transmission electron microscopy (TEM), Malvin laser particle size analyzer and UV-visible near-infrared spectro photometer were used to investigate the characteristics of AuNRs, including size, shape, dispersion, hydrate particle size, zeta potential and ultraviolet absorption spectrum.② NIR797-labled fluorescent AuMBs were fabricated through the method of lipid thin-film, hydration and mechanical agitations. TEM and optical microscope were used to observe the morphological characteristics, distribution and homogeneity. Malvin laser particle size analyzer was used to measure average hydration diameter and zeta potential. The UV-visible near-infrared spectro photometer was used to detect the NIR797-conjugated. Then gene loaded fluorescent AuMBs were further developed through conjugating plasmid DNA.③The cytotoxicity and hemolysis in vitro of the gene loaded fluorescent AuMBs were evaluated using MTT assay and hemolysis test respectively. ④Thermodynamic test was used to observe the temperature changes of various concentrations of AuNRs and gene loaded fluorescent AuMBs under near-infrared laser irradiation. [Results]①AuNRs were observed approximately rod-like, uniform in size and monodispersed under TEM. The mean hydrodynamic size of them were-110 nm in length and ～22nm in width, with an average aspect ratio of 5. And the zeta potential was-+25.6 mV. UV-vis spectra displayed that AuNRs had two SPR bands, longitudinal SPR band at 880 nm and the transverse plasmon band at 520 nm.② Under TEM, the fluorescent AuMBs were approximately spherical with a thin layer of phospholipid membrane filled with SF6 gas. AuNRs were observed at both sides and inside the membrane. The average particle size of AuMBs was about ～1.8 um and zeta potential value was～-17.7mV. The NIR797-conjugated was detected by the UV-visible near-infrared spectro photometer. ③ The result of MTT assay indicated that the gene loaded fluorescent AuMBs had no toxicity on mouse fibroblast cell line (L-929) and no hemolysis activity occurred. ④ When exposed to NIR light (output power= 2.5W; wavelength=808nm), different concentrations of AuNRs and gene loaded fluorescent AuMBs could rise to a steady temperature ranging from 38℃-53℃ and 39.5℃-51℃ respectively. In addition, the temperature raised rapidly within 15 minutes and stabilized up to a certain level after 30 minutes.[Conclusion] ①AuNRs were successfully fabricated using the improved one-pot approach with phenols as the reducing agent. ②NIR797-labled fluorescent AuMBs were successfully synthesised by the approach of lipid thin-film, hydration and mechanical agitations. And gene loaded fluorescent AuMBs with good biocompatibility were further developed.③ Self-prepared AuNRs and gene loaded fluorescent AuMBs exhibited good characteristic of photo-thermal absorption and the ability to absorb light energy to heat up and reach the thermostat (44℃), meaning they could be used as photoabsorbers for subsequent photothermal ablation of tumors.Part Ⅱ Construction and identification of pDoubleEx-EGFP-COX2p-yCDglyTK[Objective] ①Using COX2p as a promoter and yCDglyTK as therapeutic gene, we aim to construct eukaryotic recombinant expression plasmid pDoubleEx-EGFP-COX2p-yCDglyTK for subsequent targeted gene therapy on hepatoma.② To evaluate the feasibility of AuMBs as gene transfer vector in combination with ultrasound-targeted microbubble destruction (UTMD).[Methods]① COX2 promoter was synthesized and pUC19-Kan-COX2p was cut using Mlul+EcoRI enzymes to obstain COX2p fragment. COX2p fragment was inserted into the pCDNA3.1-EGFP vector to replace the CMV promoter to generatepCDNA3.1-COX2p-EGFP plasmids. Then, yCDglyTK gene was inserted intopDoubleEx-EGFP vector to obtain pDoubleEx-EGFP-yCDglyTK. After that, CMV promoter was replaced by COX2p and pDoubleEx-EGFP-COX2p-yCDglyTK plasmids were constructed. All the recombinant plasmids were confirmed by enzyme digestion and sequencing. ③ Gene-loaded AuMBs combined with UTMD were transfected into hepatoma Bel-7402 cells (bear plasmid, Lipofectamine 2000 and bear plasmid combined with UTMD as control). Fluorescence microscope and flow cytometry (FCM) instrument were applied to observe the EGFR reporter gene expression representing exogenous gene transfecting tumor cells and to detect transfection efficiency.[Results] ①The recombinant eukaryotic expression vectorpDoubleEx-EGFP-COX2p-yCDglyTK was analyzed by restriction endonuclease digestion and sequencing. The segaments (COX2p and yCDglyTK) were all inserted into the correct sites, which were confirmed by digestion gel electrophoresis, and their sequences were identical with the corresponding published data. Thus, thepDoubleEx-EGFP-COX2p-yCDglyTK plasmids were successfully constructed. ② Gene-loaded AuMBs combined with UTMD as gene vectors could induce gene delivery into Bel-7402 cell. Green fluorescence could be observed in the cells 6 hours after transfection and became the strongest at 48 hours post-transfection. Such combination had the ability to enhance gene transfection and its transfection efficiency could reach 79.73%.[Conclusion] ① pDoubleEx-EGFP-COX2p-yCDglyTK recombinant eukaryotic expression plasmids were successfully constructed and confirmed, which lays the foundation for subsequent targeted gene therapy research in combination with UTMD. ②Gene-loaded AuMBs combined with ultrasound (US) exposure could deliver exogenous gene intoBel-7402 cell lines and lead to high-efficiency expression, thus could be used as an efficient gene transfer vector.Part III Ultrasound and near-infrared fluorescencebi-mode imaging study of gene loaded fluorescent AuMBs[Objective] ①To observe ultrasound contrast enhancement imaging and UTMD experiment in vitro. ②To observe ultrasound contrast enhancement imaging in kidneys of normal rabbits and in hepatoma xenografts of nude mice. ③To observe near-infrared fluorescence (NIRF) imaging in vitro. ④To observe NIRF imaging in hepatoma xenografts of nude mice using gene loaded fluorescent AuMBs combined with UTMD.[Methods] ①A self-made set-up was applied for in vitro US imaging study. A plastic pipe with an inner diameter of-2 mm was fixed in a water tank filled with 1L degassed distilled water and a US transducer was vertically positioned on the surface of the pipe. The B-mode images were first collected from degassed distilled water in the tube. Then different concentrations of gene-loaded fluorescent AuMBs and commercially available SonoVue(?) were randomly injected into the tube from one end. At the same time, contrast specific mode (Contrast-Tuned Imaging, CnTI TM) was opened for ultrasound contrast imaging of flowing microbubble. When the images reached maximum contrast enhancement, an extra therapeutic US probe was applied to irradiate the tube. After that, the tube was observed by contrast-enhanced US. ②In vivo US imaging:Firstly, contrast-enhanced US images of normal rabbit kidneys after injecting the gene loaded fluorescent AuMBs through ear margin vein were acquired. SonoVue(?) was used as control. Then, contrast-enhanced US images of xenografts after injecting the gene loaded fluorescent AuMBs via tail vein of tumor-bearing mice were captured. In vitro and in vivo US imaging were carried out using a clinically available ultrasonic diagnostic instrument MyLab Twice (Esaote, Italy) with a LA332 linear-array transducer. All US images were collected and stored. Image J software was used to quantitatively analyze and compare the mean gray values in region of interest (ROI) of images. Time-intensity curve was also described. Statistical analysis was done using SPSS 18.0 software. ③ For in vitro NIRF imaging study, different concentrations of gene loaded fluorescent AuMBs were placed into 10-mm-diameter Eppendorf tubes for NIRF scan. SonoVue(?) and physiological saline were used as control. ④ For in vivo NIRF imaging study, tumor-bearing mice were randomly assigned to UTMD group and non UTMD group. US irradiation was performed in UTMD group when the gene loaded fluorescent AuMBs were injected. Both groups of mice underwent NIRF scan at 0 hour,1 hour,2 hours,4 hours,6 hours,24 hours and 48 hours after injecting contrast agent. At the end of in vivo imaging, all nude mice were dislocated executed and hearts, brains, livers, spleens, kidneys, intestines and tumors were immediately removed for ex vivo NIRF imaging. All NIRF imaging were performed using Near-infrared multispectroscopy scan (CRi, Woburn, MA, USA) and NIR sequence excitation (670 nm-900 nm or 740 nm-950 nm,10 nm interval). MaestroTM EX 2.10 software was applied for spectral separation of the specific signaling.[Results] ①In vitro US imaging and UTMD experiment:compared with anechoic degassed distilled water, both SonoVue(?) and the gene loaded fluorescent AuMBs exhibited strong echo in the tube. Moreover, they caused similar intensity of contrast enhancement which was concentration dependent. The UTMD experiment involving the gene loaded fluorescent AuMBs demonstrated that strong echo in the tube became echoless after US irradiation. ②In vivo US imaging indicated that the gene loaded fluorescent AuMBs could lead to effective and ongoing US contrast-enhancement of normal rabbits kidneys and xenografts. After injection of the gene loaded fluorescent AuMBs, the echo of xenografts began to enhance at 1 seconds, reached the strongest at about 12 seconds, and gradually weakened after 1 minute.③ In vitro NIRF imaging revealed that bright fluorescent signal could be detected in gene loaded fluorescent AuMBs group. And as the concentration of gene-loaded AuMBs increased, the intensity of detectable NIRF signal gradually enhanced. In contrast, no fluorescent signal could be detected in SonoVue④ group or in physiological saline group. ④n vivo NIRF imaging revealed that fluorescence signal could be detected in tumor tissues of UTMD group from injection of contrast agent till the end of observation. However, the fluorescence signal in tumors of non UTMD group only appeared for a short period of time after MBs injection and became undetectable after 1 hour.[Conclusion] ① It is proved that the gene loaded fluorescent AuMBs have the ability of ultrasound contrast enhancement as well as NIRF imaging, thus have the potential of serving as US/NIR double modal imaging contrast agent for tumor detection.② It is proved that US irradiation could effectively destruct the gene loaded fluorescent AuMBs and deliver the loaded agents to targeted sites. As a result, UTMD in combination with gene loaded fluorescent AuMBs could provide the basis for subsequent experiment of targeted treatment of liver cancer.Part IV The gene loaded fluorescent AuMBs combined with US/NIR double irradiation-photothermal-gene treatment for Hepatoma and its mechanism study[Objective] To investigate in vitro and in vivo therapeutic effect of gene loaded fluorescent AuMBs combined with US/NIR double irradiation-photothermal-gene therapy on HCC, and to explore the molecular mechanism.[Methods] ①Human hepatoma Bel-7402 cells were seeded in 6-well plates and randomly assigned to one of the following five groups:(1) blank control group; (2) gene loaded fluorescent AuMBs group (referred to as gene therapy group); (3) gene loaded fluorescent AuMBs combined with NIR irradiation group (referred to as photothermal-gene therapy group); (4) gene loaded fluorescent AuMBs combined with US irradiation group (referred to as US irradiation-gene therapy group); (5) gene loaded fluorescent AuMBs combined with US/NIR double irradiation (referred to as double irradiation-photothermal-gene therapy group). The five groups of cells were treated respectively. Forty-eight hours post-treatment, CCK-8 and FCM assay were applied to evaluate therapeutic effects.②Twenty five tumor-bearing nude mice were randomly assigned to five groups as above. Treatment were carried out accordingly in each group after the gene loaded fluorescent AuMBs were injected via tail veins. Body weight of nude mice were measured every 5 days and tumors’growth was observed. And the treatment was repeated once a week. Thirty days later, nude mice were killed and tumoricidal effect were evaluated using the tumor volume inhibition rate, body weight of nude mice, histopathological examination of tumor tissue and internal organs. ③ The indicators associated with apoptosis such as Bax, Bcl-2, P53 and Survivin were detected using immunohistochemistry, quantitative real-time PCR and western blot assay.[Results] ① CCK-8 assay showed that the average cell viability of the control group, gene therapy group, photothermal-gene therapy group, US irradiation-gene therapy group, double irradiation-photothermal-gene therapy group were 100%,73.19%,45.97%,31.24% and 18.56%, respectively. And the corresponding average apoptotic indices in the five groups were 4.19%,14.2%,26.04%,38.94% and 50.6%, respectively. Although electron microscope showed that all therapy groups could induce liver cancer cell apoptosis, the most typical morphological changes of cell apoptosis were seen in the gene loaded fluorescent AuMBs in combination with US/NIR double irradiation/photothermal/gene triple targeted therapy group. ② In vivo tumor suppression experiment suggested that the average tumor volume inhibition rate were 97.03% in US/NIR double irradiation-photothermal-gene group, which were significantly higher compared with other groups (P< 0.05). HE staining showed that the degree of necrosis of tumors was the most serious in US/NIR double irradiation-photothermal-gene therapy group among all groups. Immunohistochemical staining showed the protein expression of Bcl-2, P53 and Survivin was significantly decreased in treated groups compared to the control group. While the opposite result was observed in Bax expression. The results of quantitative real-time PCR and western blot showed the strongest Bax expression but the weakest Bcl-2, P53 and Survivin expression in double irradiation-photothermal-gene groups.[Conclusion] Gene loaded fluorescent AuMBs in combination with US/NIR double irradiation/photothermal/gene triple targeted therapy had obvious complementary and synergistic effects and can effectively inhibit the growth of hepatoma.