Construction Of A Novel Nano-composite Vector And Its Therapy For Breast Cancer By Mediating NK4 Gene

With the in-depth study of medical molecular biology and application, gene therapy for cancer has become an important biological therapeutic method and showed good application prospects in the treatment of breast cancer. At present, gene therapy for breast cancer contains the following issues, at first, there have been several gene transfer methods for research or clinical, but the transfection efficiency is still not satisfactory. The retrovirus carriers have high transfection efficiency and a stable expression, but it is only active in proliferating cells, and it can be integrated into the host cell and occurr the insertion mutation and oncogene activation in the process of transfection, so it is unfit for human-mediated gene transfer in vivo. The gene mediated by Adeno-virus carriers can not be integrated into the host cell's chromosomes, although it has a high transfection efficiency but can not transfect the target cells with a long-term stable expression, induce immune response also. Meanwhile, lack of carrier with specificity and efficiency, if the tumor cells are only partially transfected, the treatment will be affected, while the vector with low specificity can lead to victimize cancer tissues and bone marrow and other major organizations. Therefore, gene therapy is necessary to enhance the target, safety, developing a new and efficient vector system has become a focus research.Cationic polymer polyethyleneimine (PEI) has become the most successful example among the non-viral cationic polymer vector and has been the standards to design a new cationic polymer transfection carrier, because it can condense DNA molecules effectively to form stable nano-particles and protect the DNA molecule's biological activity, avoid hydrolysis, effective endocytosis, and dissociation to achieve the release of DNA molecules. Current research mainly focus on the lower cytotoxicity, high transfection efficiency and cell targeting to transform PEL One of the most attractive strategy is applying receptor-mediated endocytosis to replace the non-specific electrostatic interaction between PEI and the cell-cell, it is essential to use targeted ligand binding parts of the targeted receptors on cell surface with high affinity recognition and specificity. Hyaluronic acid (HA), as a targeted vector for anti-cancer drugs, can stick small drug molecules to its network structure or graft drug molecules to the carrier of hyaluronic acid-type drugs, the formed particles or complexes combined with the receptor-targeted tumor cell surface, so that more drug molecules arrive the tumor tissue, increase tumor uptake and retention time in tumor and lymph nodes, thereby enhance the efficacy of drugs and reduce the toxic or side effects. In this study, HA as a targeting ligand was to modify the PEI/DNA complexes for the first time, construct HA/PEI/DNA ternary nanocomposites, and investigate its characterization and performance.Currently gene therapy of breast cancer need a more effective targeted gene, NK4 as an HGF antagonist, has two roles of angiogenesis inhibition and tumor invasion suppression, which has been confirmed in transplant trials of breast cancer in nude mice, it prompted that NK4 could be a new anti-cancer drug with both of the unique features.Therefore,this experiment choose NK4 gene to build HA/PEI/PCAGGS-NK4 nanocomposites for the first time, and its effects on breast cancer cells were studied in vitro, established the breast cancer xenograft model in nude mice andobserved the antitumor effect of HA/PEI/PCAGGS-NK4 ternary complex in vivo. 1. Construction and performance test of the HA/PEI/DNA ternary nanocomposites(1) Construction and characterization of the HA/PEI/DNA ternary complexesTo prepare HA/PEI/DNA ternary complex with different charge ratios of HA and PEI, agarose gel electrophoresis, DNA gel retardation experiments, particle size distribution of nanoparticles and Zeta Potential was analyzed by Malvern, compared the particle size in BSA and in water.(2) Cell transfection experiments of HA/PEI/DNA ternary complex in vitro.Breast cancer cells MDA-MB-231, MCF-7, MDA-MB-435 were added a certain amount of nano-composites with different charge ratios, transfection efficiency was detected in the flow cytometer.(3) CD44 expression was detected in breast cancer cell lines by western blotThree kinds of breast cancer cells were cultured in serum-free culture medium. Collection of cell supernatants, total protein of cell extracts used BCA Protein Assay Kit quantitatively. After SDS-PAGE electrophoresis, transferred to NC film, plused CD44s monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled by horseradish peroxidase, color, (3-actin was used as the internal standard.(4) COS-1 cells toxicity of the ternary complexes was determined by MTTCOS-1 cells in Logarithmic phase were managed by ternary complex with different concentrations and different charge ratio of HA and PEI, continue to culture 48 h, plused MTT solution, shaked slowly, incubated 4h, added DMSO, Selected wavelength 570nm, measured the absorbed value by UV spectrophotometry.2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticles Preparation of nanoparticles HA/PEI/PCAGGS-NK4 with HA/PEI/ PCAGGS-LacZ, DNA gel retardation experiments, characterized by particle size and Zeta potential, the methods were same with the HA/PEI/DNA ternary complex in section 1.(2) NK4 protein expression in breast cancer cells was measuered by western blottingMDA-MB-231, MDA-MB-435, MCF-7 cells, added with 100μg/ml PCAGGS-NK4 plasmid serum-free medium Nanocomposite,4h plused serum medium to culture, recovered the cell, cell lysate were scraped to conduct SDS-PAGE separation of protein bands, transfer film, add NK4 an anti-(human HGF monoclonal antibody), then add horseradish peroxidase labeled secondary antibody,β-actin as controls.(3) Cell proliferation was determined by MTTThree kinds of breast cancer cells were exchanged the medium containing different concentrations of nano-composite material with serum-free medium diluted PCAGGS-LacZ, PCAGGS-NK4 plasmid. Drew cell growth curve. Compared the cell proliferation of each group.(4) Invasive ability of cancer cells was investigated by TranswellAdherent cells were exchanged medium containing different concentrations of PCAGGS-LacZ, PCAGGS-NK4 plasmid nanocomposites, after 12h, were digested with trypsin and seeded in the upper Transwell chamber, joined the culture medium with 10%newborn calf serum to the lower room, after 12h, removed the small rooms, fixed, stained, count the number of the cells through the membrane under the microscope in five different point of view.(5) Apoptosis rate was analyzed by flow cytometryThe pre-staining cells were washed twice with cold PBS, re-suspended in staining buffer. Took 100ul cells (1x105) to a test tube. Adding an appropriate amount of fluorescent labeled Annexin V reagents and PI, mixed in dark, after incubating for 15 minutes at room temperature, added 400ul staining buffer and immediately analyzed on a flow cytometer. Took small drops to films and observed the morphology of apoptosis under fluorescent microscope, caculated the apoptosis, necrosis percentage.3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice(1) Establish the xenografts of human breast cancer cell in nude mice and administrationBALB/c (nu/nu) nude mice 30, each was vaccined 0.2ml (1×107/ml MDA-MB-231) cells under the second nipple on the right chest fat pad. Afer 8d, they were randomly divided into 3 groups, named respectively as followed:(①control group, the HA/PEI/PCAGGS-LacZ injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-LacZ at tumor and tumor-surrounding);②treatment group, namely, HA/PEI/PCAGGS-NK4 injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-NK4 at tumor and tumor-surrounding);③positive control group, namely, adriamycin group,0.2ml (including adriamycin 100μg) injected intraperitoneally. Drawing tumor growth curve. Inhibition rate= (1-mean tumor weight of treatment group/mean tumor weight of control group)×100%.(2) NK4 protein expression was detected in xenografts cells by western blotPrestored in liquid nitrogen, the organization (0.2g) were take out to wash by pre-cooling PBS for three times, Shredded with scissors, milled into homogenate rapidly in 10 times of its volume lysis buffer,4℃in centrifuge 10min, aspirated supernatant of total cellular protein, the protein concentration measured by Bradford method. DS-PAGE electrophoresis, transferred to NC film, and then closed with 5% skim milk TBST solution, and then added HGF monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled horseradish peroxidase, detected NK4 protein expression in different samples with ECL chemiluminescence.4.Statistical analysisApplied the statistical software SPSS 11.0, the measured data showed as mean± standard deviation (x±s), one-way ANOVA analysis and LSD test were used to compare between groups, P<0.05 for the statistically significant difference.Results1.Construction and Performance Test of the HA/PEI/DNA ternary nanocomposites(1) Construction and characterization of the HA/PEI/DNA ternary complexesThe prepared HA/PEI/DNA ternary complexes were managed by agarose gel electrophoresis, the results show that no DNA bands appear, the complexes can effectively combine DNA molecules. With the increasing amount of HA, the size of the composite increased gradually in the double distilled water and decreased in the BSA, Zeta potential gradually decreased, when the HA and PEI charge ratio to 100%, Zeta potential was negative.(2) Cell transfection experimentsWith adding the HA, the transfection efficiency of the ternary complexes in MDA-MA-231 and MDA-MB-435 was significantly higher than that of binary complexes PD (P<0.05), has a 2-13 times increased, when the HA and PEI charge ratio was 7.5%or 100%, the transfection rate of the ternary complexes in MCF-7, MDA-MA-231 and MDA-MB-435 was significantly higher than that of PD (P<0.05). When the HA and PEI charge ratio of 7.5%, the ternary complex had the highest transfected efficiency in the three kinds of cells compared with the other groups. Difference was significant (P<0.05).(3) CD44 expressionThe results of western blotting showed that the expression of CD44s was significantly higher in MDA-MB-435, MDA-MB-231 than in MCF-7. The average expression levels were 1.15±0.036,0.92±0.072 and 0.16±0.028.(4) COS-1 cells toxicityThe results showed that the non-modified PEI/DNA complexes (N/P,10) will cause COS-1-cell toxicity, so that cell viability decreased to 58%. HA was added to construct the ternary complex HA/PEI/DNA which had significantly reduced toxicity, cell survival rates were 95%, almost non-toxic.2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticlesThe prepared HA/PEI/PCAGGS-NK4, HA/PEI/PCAGGS-LacZ nanoparticles could effectively integrated DNA molecules. With the increasing amount of HA, Zeta potential decreased gradually in the BSA, nanoparticles did not cause aggregation, when the HA and PEI charge ratio was 100%, Zeta potential was negative.(2) NK4 protein expression in breast cancer cellsThe average expressive levels of endogenous HGF in MDA-MB-231, MDA-MB-435, and MCF-7 cells respectively were 0.91±0.062,0.83±0.029 and 0.33±0.017. after 100μg/ml HA/PEI/PCAGGS-NK4 was transfected in three kinds of cell lines, the average exogenous expression of NK4 protein respectively were:2.23±0.039,1.91±0.062,1.30±0.018.(3) Cell proliferationThe results showed that:the blank plasmid does not affect cell proliferation, with the increasingconcentration of PCAGGS-NK4, cell proliferation was inhibited gradually, when the concentration of PCAGGS-NK4 arrive the dose of 100μg/ml, 200μg/ml,500μg/ml, cell proliferation was significantly inhibited compared with that of the control group(P<0.05). The HA/PEI/PCAGGS-NK4 with various concentration had the largest inhibited effect on MDA-MB-231 cells (P<0.05), and the inhibited effect in MDA-MB-435 cells was significantly higher than MCF-7 cells (P<0.05).(4) Invasive ability of cancer cellsThe invasion capacity comparison of the cells without treatment by transfection were:MDA-MB-231>MDA-MB-435>MCF-7. Invasive ability had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, three kinds of cells had relative lower invasion capacity to a certain degree, the differences were statistically significant (P<0.05).(5) Apoptosis rateApoptosis rate of the MCF-7, MDA-MB-231, MDA-MB-435 cell line without treatment by transfection were 2.41±0.05%,2.13±0.06%,2.32±0.09%. Apoptosis rate had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). after Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, MCF-7, MDA-MB-231, MDA-MB-435 cells had relative higher percentage of apoptosis.The difference was statistically signifiant (P<0.05).3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice(1) Establish the xenografts of human breast cancer cell in nude mice and administrationThe xenografts in each group nude mice were generally good. After inoculated MDA-MB-231 cells for 8d, small bulge appeared at the inoculation site on three groups of nude mice. Tumorigenicity rate was 100%. The individual positive control group of nude mice died for apparent discomfort after administration. The tumor growth were significant and rapid in control group of nude mice, reach to the final volume of 445.32±26.65 after 40 days, while the tumor growth in positive control group and treatment groupof nude mice were significantly inhibited (P<0.01), final volume were respectively 272.96±24.57 and 225.64±17.36 after 40 days.Tumor inhibition rates of positive control group and treatment group were 47.75%,55.81%(compared with control group, P<0.05).(2) NK4 protein expression in xenograftsThe blank control group and the positive control group have a small expressive amount of HGF, expressive levels were 0.89±0.03 and 0.97±0.02, protein expression in treatment group was significantly increased compared with the blank control group (P<0.05), the expressive level of treatment group was up to 3.33±0.05.Conclusions1.HA/PEI/DNA could combined DNA molecules effectively and could not cause aggregation in the BSA. HA was added to construct the nano-composite, could significantly increase the transfection efficiency in MDA-MB-231, MDA-MB-435 (high-CD44S expression), produced cell targeting effect, when the HA and PEI charge ratio was7.5%, HA/PEI/DNA had the highest transfection efficiency in three kinds of cells.The toxicity of ternary complex on COS-1 cells was greatly reduced compared with the PEI/DNA binary complexes, showed almost non-toxic.2.HA/PEI/PCAGGS-LacZ and HA/PEI/PCAGGS-NK4 ternary complex could not cause aggregation in the BSA, it showed that the structure of plasmid could not affect the size and Zeta potential such as physical properties of nanoparticles. Three kinds of breast cancer cell lines transfected by HA/PEI/PCAGGS-NK4 had expressed exogenous NK4 protein; cell proliferation was inhibited; cell invasive capacity was declined; apoptosis rate was increased. The differences were all statistically signifiant (P<0.05).3.Successful established the xenografts of human breast cancer cell in nude mice, the tumor growth were significantly inhibited in positive control group (doxorubicin group) and treatment group (HA/PEI/PCAGGS-NK4 ternary complex group) nude mice,and treatment group nude mice had showed a certain level of NK4 protein expression in xenografts cells.