Preparation Of Nanoscale Ultrasound Microbubbles And It-Mediated PNP/Fludarabine Suicide Gene Therapy For Hepatocellular Carcinoma In Vitro

Gene vector is one of the keys to gene therapy. Gene vector is divided into two categories, viral vector and non-viral vector. Gene transfer rate of viral vector is relatively high, but its safety and overcoming body's immune response against virus are difficult problems. Lower transfection efficiency of non-viral vector as liposome affects the effectiveness of gene therapy to a certain extent. Ultrasound microbubbles contrast agent-mediated gene transfer system is a newly developing hot spot in recent years. And its unique advantages and development potential have been confirmed by more and more researchers. The average diameter of current ultrasound contrast agents is several microns. However, research has shown that the vascular endothelial gap in disease state only allows the particle whose diameter less than 700nm across, so that micron microbubbles can not pass through the vascular endothelial gap. Therefore it limits its gene transfer capabilities greatly. The rapid development of nanotechnology provides a theoretical basis for nanoscale ultrasound microbubbles mediated gene therapy. In recent years, gene therapy for liver cancer, of which suicide gene therapy is direct, reasonable and effective for concern, is becoming one of hot spots in life sciences and clinical medicine. PNP/Fludarabine is a novel suicide gene system, and its killing effect is the strongest of the systems by far. Its anti-tumor mechanism mainly is that killing tumor cells in many steps, and having a powerful killing effect both for division and resting stage of tumor cells. And toxicity product after the effects of prodrug can pass through the lipid membrane, kill nearby cells, and have a strong bystander effect.[Objective] The research aims to develop a nanoscale ultrasound microbubbles which diameter less than 700nm. To verify the feasibility of that it as a novel gene vector by detection of gene transfer efficiency and relative expression levels in vitro using nanoscale ultrasound microbubbles as media and GFP as target gene. Suicide gene expression plasmid pcDNA3.1(+)/PNP was built, and nanoscale ultrasound microbubbles as gene vector mediated PNP/Fludarabine suicide gene system. To explore its therapeutic effect and the mechanism on therapy for liver cancer, and to provide an experimental basis for nanoscale ultrasound microbubbles-mediated system as clinical gene therapy method in the future.[Methods] 1. Mixture phospholipid of DPPC and DPPA, perfluoropropane gas and other materials were selected, high shear dispersion method was used for nanoscale ultrasound microbubbles development, and the best preparation method was chosen through the uniform design.2. The cytotoxicity of different concentrations of nanoscale ultrasound microbubbles to HepG2 hepatoma cells was tested by MTT assays. And safe concentration range was chosen. The combining efficiency of nanoscale ultrasound microbubbles with the DNA plasmid and gene transfer efficiency in vitro were tested by fluorescence microscopy, flow cytometry and real-time fluorescence PCR methods, with GFP as the target gene. And results were compared to the gene transfer efficiency of liposomes under the same conditions, as well as gene transfection efficiency changes after ultrasound irradiation.3. Eukaryotic gene expression system pcDNA3.1 (+) and the PNP were selected to build the suicide gene expression plasmid pcDNA3.1 (+) /PNP, Gene cloning was identified by restriction enzymes SalI and EcoRV double-enzyme digestion, PCR, and recombinant plasmid nucleic acid sequencing. 4. Self-made nanoscale ultrasound microbubbles after ultrasound irradiation was used as gene transfer system; self-constructed suicide gene expression plasmid pcDNA3.1(+)/PNP was used as the therapeutic gene, and Fludarabine was processed as prodrug, in gene therapy research for liver cancer. RT-PCR method was used for detection of the expression of PNP in two cell lines. HepG2 cells of AFP high expression and SMMC7721 cells of AFP low expression were used as experimental materials, and the recombinant plasmids pcDNA3.1(+)/PNP were transfected into each set of cells. To investigate the killing effect of two kinds of human liver cancer cells, MTT assays were used for testing the effect of the PNP/Fludarabine system on two kinds of liver cancer cell growth. Annexin V-FITC apoptosis kits for cell apoptosis test were used after the effects of the suicide gene system in order to understand apoptosis effects of the PNP gene. After treatment of 0.75μg/ml Fludarabine, MTT assays were tested for cell survival rates of two kinds of liver cancer cells, idling PNP and positive transfection PNP cells, mixing in different proportions. Bystander effect was observed.[Results] 1. Using mixed phospholipid and perfluoropropane gas as a basic raw material, perfluoropropane lipid ultrasound could be successfully prepared through high-shear distributed processing. By uniform design and multiple regression analysis, the optimal preparation conditions of preparation of nanoscale ultrasound microbubbles were:6 files of high-shear rate,8min of high-shear time,20mg/50ml of DPPC concentration,1 of T80/S60 ratio. Nanoscale ultrasound microbubbles that prepared under optimal conditions was small in diameter, at high concentrations and good uniformity. Particle size distributed in 208-416nm, with an average particle size of only (335±5) nm.2. It had no significant cell toxicity that nanoscale ultrasound microbubbles to HepG2 liver cancer cells in the concentration of≤5.0%. In that concentration nanoscale microbubbles combined with the DNA plasmid with high efficiency; in vitro gene dilivery, the nanoscale microbubbles could effectively transfer GFP gene expression plasmid into HepG2, its transfer efficiency up to 32%. It had no significant difference from the liposome transfection efficiency-34% under the same conditions. After ultrasound irradiation, gene transfection efficiency of nanoscale microbubbles significantly increased, reaching 79.9%.3. Restriction enzyme digestion and electrophoresis results showed that there was a specific band about 750bp, which was nearly the length of the target gene. Then the recombinant plasmid was amplified using specific primers by PCR method. Electrophoresis results showed that a specific band about 750bp and proved that the target gene had been correctly inserted pcDNA3.1(+), and the eukaryotic expression vector pcDNA 3.1(+)/PNP had been constructed. Sequencing results comparing to the sequences in Genbank was entirely correct.4. RT-PCR results showed that there was a clear band about 750bp, indicating pcDNA 3.1(+)/PNP plasmid had been successfully transfected into liver cancer cells and highly expressed through the nanoscale ultrasound microbubbles. In MTT assays it was found when the concentration of Fludarabine was only 0.75g/ml, PNP/Fludarabine system had obvious cytotoxic effects on HepG2 cells and SMMC7721 cells; and compared to SMMC 7721 cells of AFP low expression HepG2 cells of AFP high expression that only needed lower concentrations of Fludarabine (0.5 g/ml) could achieve the same killing effect. In Annexin V-FITC apoptosis test, after treatment of the prodrug,0.75μg/ml Fludarabine, two groups of transfected human hepatoma cells by pcDNA 3.1(+)/PNP were apparent apoptosis. Apoptosis rates were 41.34% and 53.15% respectively. After the 0.75g/ml of Fludarabine treatment, cell survival rates were observed by MTT assays of two kinds of liver cancer cells, idling PNP and positive transfection PNP cells, mixing in different proportions. The results showed that only 5%-10% of the PNP-positive transfection cells caused survival rate significantly to reduce 10%-20%, indicating that PNP/Fludarabine system had obviously bystander effects on the killing effect of two kinds of human liver cancer cells.[Conclusion] 1. Nanoscale ultrasound microbubbles that prepared under optimal conditions was small in size and distribution, uniformly dispersed, and good stability. It was a relatively small nanoscale ultrasound microbubbles in international and domestic literature, and it provided a good basis for follow-up study.2. Ultrasound-mediated self-made nanoscale ultrasound microbubbles system was non-toxic, stable, and highly efficient in gene transfer. It was a very promising, safe, efficient and novel gene transfer system. And it was expected to be a new method of clinical gene therapy.3. The self-constructed eukaryotic expression vector pcDNA3.1(+) /PNP was sequenced. Sequencing results comparing to the sequences in Genbank was entirely correct, that indicated successful amplification of the E.coli purine nucleoside phosphorylase gene DNA sequences with no mutation.4. Ultrasound-mediated self-made nanoscale ultrasound microbubbles could effectively transfected plasmid into the liver cells; at low concentration the suicide gene PNP/Fludarabine system had inhibited tumor cell growth successfully and induced apoptosis significantly for two kinds of human liver cancer cells. It was proved that apoptosis and bystander effect were important mechanisms for liver cancer cell killing effect induced by the PNP/Fludarabine suicide gene system.Through these experiments, we believed that self-made nanoscale ultrasound microbubbles could highly transfect exogenous genes efficiently into cells and obtain a valid expression. Ultrasonic irradiation could significantly increase efficiency and specificity of expression of exogenous genes in cells. The PNP/Fludarabine system mediated by the nanoscale ultrasound microbubbles system, could kill tumor cells effectively by direct cytotoxicity and good bystander effects. This novel nanoscale ultrasound microbubbles-mediated suicide gene targeting therapy strategies provided new ideas and experimental evidence for liver cancer and related tumors.