The Research Of Multi-target Anti-angiogenic Nanoparticles In The Treatment Of Ovarian Cancer

BackgroudOvarian cancer is one of the three malignant tumors of the female reproductive system,with a high incidence and high mortality,the incidence rate showed a rising trend in rescent years."2012 NCCN Ovarian Cancer Clinical Practice Guidelines (2nd Edition)" showed that the preferred method of treatmenting ovarian cancer is surgery,chemotherapy,biological therapy,hormone therapy,radiation therapy, etc. The chemotherapy drugs are paclitaxel,platinum, trabectedin,etc.Ovarian cancer tissue were rich in blood vessels,so it was sensitive to chemotherapy.However,the drug toxicity and long-term treatment of chemotherapy drugs greatly reduced the efficacy.the five-year survival rate is still low, hovering in 25-30%.Therefore there is an urgent need to find new ways to improve the five-year survival rate. People have focused on the research of ovarian cancer gene therapy in recent years,especially in the treatment of tumor vasculature targeting,which has become a cutting-edge topic of cancer treatment.Tumor growth, invasion and metastasis depend on the rich blood supply and angiogenesis,tumor angiogenesis not only transport nutrients and excrete metabolic waste out of the tumor tissue, but also is one of the main aspects of tumor metastasis. Inhibition of tumor angiogenesis, could significantly inhibit tumor growth and metastasis,which opened up a new way for the treatment of ovarian cancer.Ovarian cancer is a solid tumor, combined with two relatively independent parts: the tumor stroma and tumor parenchyma, there is a high density of microvessels in tumor stroma, the growth and metastasis of ovarian cancer has closed relationship with these microvessels.So, select a specific angiogenic gene as targeting, is a new method for the treatment of ovarian cancer.At present, the main anti-angiogenic therapy is to limit and prevent the formation of new blood vessels, mainly in the following three areas:(1) the role of blocking angiogenesis factors; (2) inhibition of EC proliferation and migration; (3) inhibition of vascular basement membrane and intercellular synthesis and degradation. There are three ways of target organs:angiogenic factors,EC, basement membrane or cytoplasm.Compared with conventional radiotherapy,taking the tumor angiogenesis as a therapeutic target, has higher direction,higher efficient,lower adverse reactions and lower resistance.The current angiogenesis inhibitors include inhibitors of endothelial cells, VEGF and VEGFR monoclonal antibodies or small molecule tyrosine kinase inhibitors, ECM degradation inhibitors, adhesion molecule inhibitors, and cell signaling inhibitors. In recent years, anti-vascular endothelial growth factor (VEGF) and VEGF receptor has made important progress in the treatment of a variety of tumors,recombinant humanized anti-VEGF monoclonal antibody bevacizumab is the first monoclonal antibody drugs to be approved for the inhibition of angiogenesis growth.The research of ovarian cancer treatment has been carried out to III clinical trials. Although bevacizumab in combination with chemotherapy drugs applied to recurrent ovarian cancer has achieved good results, it also caused serious adverse reactions during the treatment, such as hypertension, thrombosis, gastrointestinal perforation, so it is urgent to develop more anti-angiogenesis drugs for the treatment of ovarian cancer.However,most of the existing anti-angiogenic therapy programs for single target, no single drug can play a role throughout at different times of tumor progression. Angiogenesis at different growth stages have different vascular microenvironment, it can not make an effect all the time;Anti-angiogenesis inhibited tumor cells in the "sleep",prone to recur.If we can find the drug which can target variety of pro-angiogenic factors, or choose a variety of signaling pathway intersections or multiple targets,the effect may be better. In addition, a direct effect on proliferation vascular endothelial cells,tumor cells and angiogenesis against could play better anti-tumor effect. Therefore, this study proposed development drug which can target multiple tumor angiogenesis, and play the role of inhibiting tumor vascular endothelial cells and tumor cells.Biological macromolecules as the carrier of the self-assembly of nano drug has good biocompatibility, ease of modification of surface, which has a high permeability and retention in tumor tissue, the drug can prolong circulation time in the body, escape net macrophage phagocytosis, increased drug utilization.We can use biological macromolecules as a carrier to carry more of the active ingredient by physical or chemical means, the application of this strategy to achieve our target for the development of multi-purpose anti-angiogenic drugs, and applied to the treatment of ovarian cancer research. VEGF promote angiogenesis via paracrine mechanisms to promote tumor cell proliferation through an autocrine mechanism to participate in ovarian cancer cell proliferation invasion and metastasis.Take VEGF or its receptor as a target for anti-angiogenic therapy profound a new method of cancer. Angiogenesis is a matter of a variety of cell proliferation, apoptosis, migration, integration, extracellular matrix degradation and remodeling of complex processes. VEGF is the most specific endothelial cell division-promoting growth factor, capable of binding to its receptor VEGFR, promote the proliferation of vascular endothelial cells, generation of tumor blood vessels in tumor tissue. Tumor cells can simultaneously autocrine and paracrine VEGF, the VEGF and VEGFR secreted by the vascular endothelial cells can combine to achieve the purpose of promoting tumor angiogenesis. Thus, antagonism of VEGF and VEGFR binding can inhibit tumor angiogenesis. Integrin is an important member of the family of cell adhesion molecules, mainly mediated cell-cell, cell-cell adhesion each other extracellular matrix (ECM) between two-way and mediates signaling between cells and ECM.Integrins is highly expressed in tumor angiogenesis,which plays an important role in angiogenesis, especially αvβ3. Integrin αvβ3 as a target for many anti-angiogenesis drug,plays an important role in the process of angiogenesis by inhibiting endothelial cell apoptosis.Thus,VEGF,VEGFR,integrin αvβ3 are several important targets in anti-angiogenesis.Heparin is a mucopolysaccharide, with glucosamine and glucuronic acid crosslinked glycosaminoglycan ester, contains large amounts of sulfuric acid group and a carboxyl group, with a large number of negative charges. Study found that heparin can inhibit stimulating the formation of vascular endothelial cell proliferation and angiogenesis by VEGF and FGFs, as well as the impact of fibrin aggregation structure, change the sensitivity of plasm inogen activator.Thus, heparin has a good effect in anti-angiogenesis, anti-tumor growth, reducing cell adhesion, tumor invasion, metastasis.Thus, heparin can be a carrier,also nano drug to inhibit tumor angiogenesis. Studies have shown, RGD peptide is a class of arginine-glycine-aspartate (Arg-Gly-Asp) of short peptides,and it can be recognited by αvβ3 integrin receptor.Due to the multivalent,RGD peptide have higher receptor binding specificity than linear RGD peptides. K237 peptide is a kind of small peptides,which is isolated from phage. It has a high affinity with VEGFR2.By interfering the binding between VEGF and VEGFR2, K237 peptide can inhibit the proliferation of primary cultured human umbilical vein endothelial cells (HUVEC). Peptide RGD and K237 are short peptides, which can be chemically modified easily, capable of binding to heparin, and self-assemble into nano-drug.However, normal vascular endothelial cells also express VEGFR2, which can weaken the specificity to tumor cells. To solve this problem, we introduce a group that can target tumor cells.Biotin,also known as vitamin H, as an enzyme cofactor, involved in the body carbohydrates, lipids, proteins and nucleic acid metabolism, is also involved in signal transduction and gene expression.Biotin plays a physiological role via cell surface (sodium dependent multivitamin transporter, SMVT).the proliferation of tumor cells requires a large amount of biotin, therefore biotin can increase its specific targeting to tumor cells.In this study we take heparin as a carrier, combined with biotin, K237 peptide, RGD cyclic peptides chemically, to prepare the multi-target drugs targeted vascular endothelial cells and ovarian cancer cells.Chapter 1 Synthesis Multi-target Anti-angiogenic NanoparticlesObjective:Synthesis of stable multi-target drug chemically nanoparticles, for further study of its anti-angiogenic and anti-tumor effect.Methods:1. Synthesis of multi-target anti-angiogenic nanoparticles and its physicochemical properties.Succinylated heparin (40 mg) was dissolved in 2 mL of anhydrous DMSO and stirred at room temperature until fully dissolved, followed by adding biotin, cyclic peptide RGD, K237, EDC, NHS.the reaction was stirred under room temperature, after 48 h, dialysis in the water to remove DMSO. 2. The laser confocal microscope Qualitative analysis HUVEC cells and ovarian cancer cells the difference of uptaking nanoparticles.We content with the same amount nanoparticles in HUVEC cells and ovarian cancer cells were incubated for 4 h, after transfection Hochest33342, laser confocal microscopy observe the distribution of nanoparticles. 3. The flow cytometry analysis of human HUVEC cells and ovarian cancer cells to different targets content ratio of anti-angiogenesis drug uptake nano:After the same amount of different target content ratio of Oregon green green fluorescent labeled anti-angiogenic drug for HUVEC, OVCAR-3 and SKOV-3 cells were incubated for 4 h, flow cytometry three cells to target point content ratio of different anti-angiogenic drug uptake nanometers.Results:1. Preparation of the resulting multi-targeted nanoparticles in TEM spherical nanoparticles were no significant aggregation. Dynamic light scattering instrument display multi-targeted nanoparticles of particle size of about 90±7 nm, potential of about -27±4 mV.2. Qualitative analysis by confocal cell content of different proportions of multi-target uptake of nanoparticles under the microscope:the umbilical vein endothelial cells and ovarian cancer, multiple targets with a green fluorescent nanoparticles are mainly distributed in the cytoplasm, Description multi-targeted nanoparticles capable of targeting human umbilical vein endothelial cells and ovarian cancer cells and into the cells.3. The flow cytometry analysis of cell uptake:With the increase in the target, the fluorescence intensity of intracellular nanoparticles gradually increased, and in multi-target nanoparticles, the ratio of (40:6:4:4) the fluorescence intensity of the strongest, followed by (50:6:4:4), and finally (60:6:4:4) a combination of these experiments and streaming cell uptake measurement results, we selected the best NP1 targeted research its anti-angiogenic and anti-tumor effect.Chapter 2 multi-target anti-angiogenic drug nano anti-angiogenic and anti-tumor effectsChapter 2 The role of multi-target anti-angiogenic nanoparticleson HUVEC cells and ovarian cancer cellsObjective:Study the role of multi-target anti-angiogenic nanoparticles on HUVEC cells and ovarian cancer cells.Methods:1.MTT assay multi-targeted nano toxic effects of drugs on human umbilical vein endothelial cells and ovarian cancer.LMWH and NP1 respectively different concentrations in human umbilical vein endothelial cells and ovarian cancer cells after 48 h, MTT assay relative survival rates for each group of cells.2.Scratch test multi-targeted nano-scratch test the impact of drugs on ovarian cancer cell migration in vitro testingRespectively, the same concentration of LMWH and NP1 role in human umbilical vein endothelial cells and ovarian cancer cells 0 h,6 h,12 h,24 h later, three randomly selected microscopic vision camera observation.3.Transwell assay nanometer multi-target drugs on ovarian cancer cells in vitroRespectively, the same concentration of LMWH and NP1 role in human umbilical vein endothelial cells and ovarian cancer cells after 12 h, the producer received a small room, five randomly selected field of view with a microscope camera and statistical processing of data.4.Multi-targeted nano-assay in vitro angiogenesis drugs on tubule formationUsing CHEMICON in vitro angiogenesis assay (ECM625) kit to detect the impact of multi-targeted nanoparticles HUVEC tubule formation. Respectively LMWH and NP1 role in vitro tubule formation,10 h after inversion observed in vitro multi-targeted nanoparticles on angiogenesis under an optical microscope.5.the body of multi-targeted nano-angiogenesis drugs on Matrigel experimental determinationAfter subcutaneous Matrigel and drug mix in nude mice with bilateral back,14 days out Matrigel plugs, camera observation, and doing immunohistochemical expression of CD34 in Matrigel plugs.6.Matrigel plugs in CD34 expression was measured by immunohistochemistryTo further qualitative analysis of nano drug in vivo inhibition of tumor angiogenesis, the Matrigel plugs into the subcutaneous tissue and tumor tissue immunohistochemical staining of vascular markers CD34 antibody,the fixed, paraffin-embedded,dewaxing,antigen retrieval blocking of endogenous peroxidase, serum closed, antibody incubation, DAB color, stained, transparent, were mounted after the other steps, staining was observed under an optical microscope and the upright three groups of vascular markers CD34 antibody expression and analyze whether the difference was statistically significant difference in the presence or absence.Results:1.MTT assay multi-targeted nano toxic effects of drugs on human umbilical vein endothelial cells and ovarian cancerLMWH and NP1 respectively different concentrations in human umbilical vein endothelial cells and ovarian cancer cells after 48 h, MTT assay relative survival of cells in each group, the results indicate that multi-targeted nanomedicine could inhibit HUVEC cells and ovarian cancer cells proliferation, and increases as the concentration, effect enhancement.2.Multi-targeted nano-scratch test the impact of drugs on human umbilical vein endothelial cells and ovarian cancer cells in vitro migration testingRespectively, the same concentration of LMWH and NP1 role in human umbilical vein endothelial cells and ovarian cancer cells 0 h,6 h,12 h,24 h later, the microscope camera observation. The results show that multi-targeted nano drugs can inhibit the migration of HUVEC cells and ovarian cancer cells.3.Transwell assay nanometer multi-target effects of drugs on human umbilical vein endothelial cells and ovarian cancer cells in vitro migrationRespectively, the same concentration of LMWH and NP1 role in human umbilical vein endothelial cells and ovarian cancer cells after 12h, five randomly selected using a microscope field of view camera and statistical processing of data, the results show the drug can inhibit multiple targets nano HUVEC cells and ovarian cancer cells migration.4.Multi-targeted nanomedicine assay of angiogenesis in vitro tube formationUsing CHEMICON in vitro angiogenesis assay (ECM625) kit to detect the role of multi-target nanoparticles to HUVEC tubule formation. Respectively LMWH and NP1 role in vitro tubule formation,10 h after the inversion observed angiogenic nanoparticles vitro against multiple targets under an optical microscope. The results show that, in vitro, multi-targeted nano-drugs can inhibit tube formation, and LMWH substantially no effect.5.Matrigel experimental determination of multi-target nanoparticles on angiogenesis in vivoAfter mixing the drug and the Matrigel subcutaneously into nude mice with bilateral back,7 days out Matrigel, camera observation, the results show that in vivo, multi-targeted nano-drugs can inhibit blood vessel growth.Determination the expression of CD34 in Matrigel plugs6.ImmunohistochemicalMatrigel plugs removed from the nude mice, embedded in paraffin, sliced, select vascular marker CD34 immunohistochemical staining analysis showed:CD34 expression of LMWH group and the control group was significantly more than the nano-drug group, indicating that the LMWH group compared to the nano-angiogenesis drug was significantly reduced, further evidence of nano-drugs can inhibit angiogenesis in vivo.