Study Of The Growth Inhibition And The Mechanism Of Ovarian Cancer By Gold Nanoflower Modified By 15 Peptide-mediated Polypyrrole (15P-PPy-NPS)

Research BackgroundThe ovarian cancer is a common malignant tumor of female genital tract, and ranks second in the female reproductive tract cancer, which is only after cervical cancer. It shows by the recent five years’survey that the incidence rate increased by 6%, which compare with 2005-2010 period, today the incidence rate is 64%. The early diagnose of ovarian cancer is difficult, and easy transfer, once the diagnosis is often intermediate or terminal stage, so the postoperative rehabilitation is difficult, really a threat to the patients’ health. Low sensitivity for ovarian cancer radiotherapy, and the chemotherapy primarily through intravenous injection or intraperitonealh injection drug puncture method. Due to the great side effects, resistance to drugs or other properties, we have difficulty getting the result we really want. Even if combine with mutual adjuvant therapy, the patients with ovarian cancer for 5 years survival rate was 32%. To reduce side effects and improve treatment effects, which use biological methods and physical methods at the same time. We get the biologically active polypeptide molecules attached to biological materials with chemical connection method, and apply the characteristics of the combination between the polypeptide molecules and some tumor cell membrane or the cell-specific expression of the molecule, which can more effectively act on the tumor cell specificity, and can be used to block tumor cell growth, metastasis, inhibit or kill tumor cells within the cell-specific expression. With the target of elimination of tumor, expecting to improve cancer cure rates.The ovarian cancer transfered by Lymphatic, some papers have reported the lymphangiogenesis promotes cancer metastasis. So inhibition of tumor lymphangiogenesis will become a new possibility method for the cancer diagnosis and treatment. Studies have shown that VEGF-C and VEGFR-3 can induce the new lymphangiogenesis inside the tumor and lymphangiectasia around the tumor tissue. Lifang Zhu had experimental study on the molecular imaging and therapy for VEGFR-3 binding peptide targeting Lymphangiogenesisa in nude mouse with ovarian cancer, the results shown inhibit the generation of VEGFR-3 could inhibit the ovarian cancer cell growth. Some papers report the VEGFR-3 mainly express in the new lymphatic endothelial cells of the tumor, but not express in normal lymphatic endothelial cells, which make VEGFR-3 as a target to do ovarian cancer lymphatic generate therapy become possible. In 2006, lifeng Shi in 2006 stand VEGF receptor extracellular region of 3 analysis of the obtained 15 peptide (SHSWHWLPNLRHYAS) in by ELISA and immunofluorescence assays in vitro, and VEGFR-3 extracellular domain strong affinity and capacity, affinity constants (2.37±1.10) ×106M-1, the study also found 15 peptide binding VEGFR-3 inhibits later lymphangiogenesis, which will ovarian provide new theoretical basis of anti-cancer cells.The gold nanoparticles are really small that their diameters arel-250nm,due to they have small particle size, quantum effect, surface effect and macroscopic thermal effects, nanotechnology research has been through medical, life sciences and other fields. Apply some special advantage of nanoparticles, after the modification of surface, can carry biological molecules, drug missile carriers, drug parcels and so on, which will upgrade the existing diagnostic medical level, to achieve the treatment of localized lesions, reducing damage to health organ, and greatly reducing the treatment side effects of cancer and other diseases. These features provide the possibility for the development of new drugs.In 2003, Hirara used beta-catenin (ctnnb1) regulatory proteins to hinder the generation of VEGF-C in human bladder cancer, inhibit cancer cell amplification, effectively delaying the patient’s condition, we use biological polypeptide in coordination with surgery, radiotherapy and chemotherapy to the patients, from biology, chemistry, physics etc. to make the patients feel better and live longer, prolong the survival time of patients.Apply the nanomaterial as photosensitizer in the photothermal therapy, which base on the macro-thermal effects of nanomaterials. Under the action of light, the cancer cell of the body will deform or the biological molecules will change, which will lead to cell damage and necrosis, reaction involved in oxygen. So called photosensitizing-oxidation or photodynamic therapy (photodynamic therapy, PDT). By photochemical reactions convert light energy to molecular internal energy, molecular internal energy can produce a variety of reactive oxygen molecules within the cell, disrupting the sub-cells structure, the cell apoptosis or death. Currently this technology has been successfully used in alleviative treatment of lung cancer, esophageal cancer, and achieved gratifying results.Because nanomaterials can selective accumulation in the abnormal proliferation of tissue tumor, and have a light selection of wavelength, intensity and location in the clinical, has reached a minimum treatment of diseased tissue, reduce side effects of treatment. Its specificity is better, trauma is small, low toxicity, well selectivity, strong applicability, treatment can be repeated, can improve the efficacy of co-operation, can be palliative treatment, can eliminate the hidden lesions, can protect vital organs function, reducing the patient pain. Kelly et.al applied photosensitizing effects combined with targeting biomolecules in liver tumors have been reported. In this paper, the synthesis of polypyrrole modified gold nanoflower, combined 15P targeting molecules, study the inhibition of tumor cell and the mechanism of killing effect.Research purposesBy chemical synthesis of polypyrrole modified gold nanoflower as photosensitizer, and binding with target polypeptides, which have VEGFR-3 inhibitory effect as mediated to synthesis new materials (Gold nanorods of 15-pentadecapeptide 15p-PPy-NPS). Investigate the killing effect on ovarian cancer cell with different doses photosensitizers, the time of different laser illumination, different light frequencies. Ovarian cancer cell culture, build nude mice tumor models, and the biological safety evaluation of nanomaterials, to study the photodynamic tumor inhibition rate and destruction mechanisms of new nanomatrial. Desirable have a new ideas for clinical studies of ovarian cancer, which apply the new targeting gold nanoflowers.Research Methods1. Synthesis the gold nanoflower modified by 15 peptide-mediated polypyrrole (15P-PPy-NPS) then characterization and identification.The preparation of gold nanoparticles by reduction of gold chloride acid, apply polypyrrole modified to increase the surface area, and then screen the flower-like gold nanoparticles. By comparison particle filter, PH value stability, laser irradiation frequency photothermal conversion efficiency and other aspects to evaluate the chemical, physical, biological activity of the gold nanoparticle targeting peptide. Through UV-VIS-NIR absorption spectra, electron microscopy to characterization and identification.15P affinity targeting peptide is present laboratory through to the 12-mer random peptide library 4 affinity selection, analyzed the obtained 15 peptide (SHSWHWLPNLRHYAS) and the extracellular domain of VEGFR3 protein have strong affinity and capacity, which combine with gold nanoparticle via an amide bond to synthesised gold nanoflower modified by 15 peptide-mediated polypyrrole (15P-PPy-NPS).2. Screening the15P-PPy-NPS tumor cell lines in vitro.By MTT assay, Cell scratch test, Observation of 15P-PPy-NPS into cell experiments with fluorescence microscope.Transwell invasion assay in vitro compared the active, infection, the sensitivity to 15P-PP.y-NPS and the cell growth inhibition rate of the various cancer cell lines, which screened specific cell from HO-8910 human ovarian cancer cell line, SW626 human ovarian cancer cell lines, within RL95-2 human uterine adenocarcinoma cell line, SKOV-3 human ovarian adenocarcinoma cell line, HeLa-S3 cervical cancer cell lines, HeLa229 human cervical carcinoma cell lines etc. To detect the gold nano flower,15p, different laser irradiation frequencies and time of apoptosis in cancer cells.3.15P-PPy-NPS photothermal to inhibit tumor growth in animal modelsSKOV-3 tumor cells in culture, cell suspension through the armpit injection establish nude mice to build the bearing human ovarian cancer nude mice model, until the tumor grew to a certain size, through the tail vein injection of the photosensitive material 15p-PPy-NPS, NPS,15P, saline, combined laser irradiation with a certain frequency, recorded the tumor growth situation, activity status, food intake and the treatment adaptability of the nude mice daily. With 0.1% pentobarbital anesthesia, removed parts of patients to do the immunochemical analysis, detected the tumor cell apoptosis situation.4. Detection of NPS and 15P-PPy-NPS visceral tissues damageTake the nude mice treated with 15P-PPy-NPS, after laser irradiation, obtained the liver, spleen, kidney and internal organs paraffin sections, through fluorescence scanning electron microscope to observe the apoptosis or death situation.Research results1. Complete reduction method of gold chloride acid to synthesis the gold nanoparticle, modified the gold nanoparticles with different thickness of polypyrrole package. Within the scope pH 1-9, the particle morphology obviously, and under 808nm laser irradiation at 3W/ cm2 stable. Synthesis 15P successfully. Through an amide bond covalently linked with gold nanoparticle, analyzed by spectrometry, conditions screening, spectral test results show that, 15P peptide polypyrrole Modified gold nanoparticle successfully.2. In the microscope, the cells were observed to be in the vicinity of the cell membrane, and the 30min was less than that in the cells. The 60min was obviously observed in the cytoplasm.A variety of gynecological cancer cell lines after treated with 15P-PPy-NPS material the MTT sensitivity indicates:HO-8910 human ovarian cancer cell inhibition rate of 53%, SW626 human ovarian adenocarcinoma cell line inhibition rate of 53%, RL95-2 human uterus adenocarcinoma cell line inhibition rate of 48%, SKOV-3 human ovarian adenocarcinoma cell line inhibition rate of 75%, HeLa-S3 cervical cancer cell lines inhibition rate of 63%, HeLa229 human cervical carcinoma inhibition rate of 51%. The treatment of 15P-PPy-NPS+irradiation group by Hochest33342 staining showed obvious apoptosis, apoptosis 15P-PPy-NPS+irradiation group rate of 84.93%, NPS+ irradiation group reached 71.83%,15P+irradiation Group reached 52.74%,15P-PPy-NPS group reached 15.72%,15P group reached 16.49%, NPS group reached 17.36%, laser irradiation group reached 24.17%; control group reached 12.0%.3.15P-PPy-NPS photothermal treatment of nude mice results:Inhibition rate of tumor (%):15P-PPy-NPS+irradiation group reached 23.57±2.626, NPS+irradiation group reached 16.61±0.7915,15P+irradiation Group reached 8.926± 1.651,15P-PPy-NPS group reached 5.883±1.625, NPS group reached 1.584±0.5264,15P group reached 6.159±1.403, laser irradiation group reached 1.430±1.788, the control group reached-18.65±3.901. There were no significant dfferences between before treatment and after treatment.(P>0.05).Tunel staining and PI, Hoechst staining showed that,15P-PPy-NPS+irradiation group apoptosis rate of 78.51%, NPS+irradiation group reached 62.10%,15P+irradiation Group reached 37.48%,15P+PPy-NPS group reached 22.5%, NPS group reached 11.69%,15P group reached 19.67%, laser irradiation group reached 10.02%, the control group reached 4.09%. The results showed that there were significant dfferences between group one, group two and other groups (P<0.001;P<0.05).4. Take organs from the 15P-PPy-NPS nude, paraffin tissue sections showed no difference 15P-PPy-NPS+ irradiation group and the control group in liver, kidney, spleen cell apoptosis. The results show the 15P-PPy-NPS has a better tissue compatibility for the mice, and have little damage for organ tissue. We can get it is safty for the experimental animal.ConclusionIn this thesis, through the synthesis of polypyrrole gold nanoparticle, binding polypeptide molecules, provide a efficient and stable photosensitizers for Photodynamic therapy of tumor cells, laser irradiation nude mouse tumors as a means to achieve the purpose of inhibiting or killing cancer cells, cancer tumor. The treatment provides a new idea.1. The successful synthesis of polypyrrole-coated gold nanoparticles at 6nm, synthetic 15 peptides, and connected the 15 peptide with Au NPs, through mass spectrometry, infrared, ultraviolet, chemical analysis to analysis structure, stability, photothermal conversion efficiency and other aspects of the gold NPS,15P,15P-PPy-NPS. Providing the material basis for subsequent experiments.2. Successful screened from six associated gynecologic cancer cells, which can efficiently kill cancer cells under the 15-PPy-NPS as photosensitizer, SKOV-3 cell model constructed. Animal model provides a foundation to build.3. Successfully constructed gland cells in human ovarian SKOV-3 tumor-bearing nude mice,15P-PPy-NPS inhibited tumor growth under the laser irradiation, a large number of apoptotic cells and dead cells were appeared in the tumor tissue sections, which provide new photosensitizer for clinical photothermal therapy, in combination infrared laser effectively kill cancer cells provides an experimental basis.4. After monitored the body weight, food intake, activity levels, response to external stimuli and anatomical visceral tissues liver, spleen, kidneys of the 15P-PPy-NPS experiment mice, then compared with the control groups; organ tissue sections apoptosis shown littel damage to internal organs; long with 15P-PPy-NPS+laser irradiation group compared with the control group of biological treatment of the material and photothermal therapy has a good safety and reliability.