| The incidence of ovarian cancer is the fifth most common malignant tumor in women,and the mortality rate is the highest.In the early stage of ovarian cancer,there is a lack of typical signs&symptoms,so most of the patients with ovarian cancer have been diagnosed at the late stage(FIGO ?-?).The five-year survival rate in the late stages is less than 40%.Surgery is the first line of treatment of patients in the early stage of Ovarian cancer:Standard first-line therapy for advanced epithelial ovarian cancer is Cytoreductive surgery and postoperative chemotherapy of paclitaxel combined with carboplatin,more than 70%of patients will relapse;there is still no effective treatment for relapse and drug resistance.Chemotherapy is the main adjuvant therapy for ovarian cancer.However,although new chemotherapeutic drugs and chemotherapy regimens have been put into clinical use,the prognosis of the patients has not been significantly improved in the past few decades.Therefore,it is urgent to improve the prognosis of patients with new and effective therapeutic methods.Nanotechnology can be used to achieve targeted therapy of malignant tumors,which provides a new method for the treatment of ovarian cancer.Nano technology has experienced rapid development from the early 1990s to date,and has achieved remarkable results.At present,nanotechnology has been widely used in many fields,such as aerospace,biomedicine,materials and so on.In the field of medicine,the emergence and development of drug loaded nanoparticles provide a new way for the development of anticancer drugs.The use of nano carriers to encapsulate drugs can increase the solubility of insoluble drugs,enhance the stability of water-soluble drugs,improve the bioavailability of drugs and reduce the toxic side effects of drugs.When the tumor tissue grows rapidly,a large number of new blood vessels are produced,and the permeability of blood vessels is increased,therefore,nanoparticles have enhanced permeability and retention effect(EPR effect),So as to achieve passive targeting and sustained release effect on tumor tissue;Anaerobic glycolysis increases in tumor tissue,tumor local environment becomes acidic,Nano carrier biodegradable materials are prepared using PH sensitivity,which can be used to selectively release the drug in the tumor site,and to maintain the stability in the normal tissue so as to fulfill the physical targeting of tumor;The corresponding ligand or the specific monoclonal antibody is coupled with the nano carrier by physical or chemical bond,The active targeting of nano carriers can be achieved by targeting the tumor cells specifically to enhance the selectivity of tumor tissues and reducing the aggregation of drug loaded nanoparticles in the surrounding tissues.Based on physical and chemical targeting,passive targeting and active targeting,nano carriers can be widely used in the diagnosis and treatment of gynecological tumors.The main contents of this thesis include two amphiphilic copolymers PEG(Polyethylene glycol,PEG)&PLA(Polylactic acid,PLA)which were connected with active targeting group Folic acid molecules,The preparation of active targeting nanoparticles of micelles,was done with nano micelles been encapsulated with domestic second generation photosensitizer hypocrellin B(HB)and ovarian cancer common chemotherapy drug paclitaxel,the preparation process and the physical and chemical properties of the nanoparticles were investigated by measuring the size of the nanoparticles and the change of the potential and the morphology of the transmission electron microscope,In this paper,the toxicity and the uptake experiments of nano micelles were carried out to verify the low toxicity and tumor targeting characteristics of the nanoparticles,The distribution of drug in vivo and pharmacokinetics showed that the nano carrier has a slow release effect:The Objective of discussing the pharmacokinetics and pharmacodynamics of the drug was in order to investigate the active targeting of nano carriers on ovarian cancer cells and tissues.At the same time,the corresponding effect of pH was proved by the cytotoxicity of cells and the uptake of nanoparticles with different amino acids.In this experiment,the passive targeting of nano carriers,Physical pH targeting as well as the active targeting components after the folic acid were studied,and it proved that the drug loaded nano carrier has the characteristics of slow release and tumor targeting.PART 1:Preparation of folate receptor nanoparticles and amino protective nanoparticles and determination of physical and chemical propertiesObjective:Preparation of two amphiphilic copolymers polylactic acid-polyethylene glycol(PEG-PLA),was on the basis of nano block in which the folic acid molecule is connected or the nanometer amino terminal is closed to prepare polylactic acid polyethylene glycol(FA-PEG-PLA)and(HOOC--PEG-PLA).To explore the preparation process of various nano carriers,and to examine the physical and chemical properties,and to evaluate the feasibility of the follow-up studyMethods:Using the purified lactide under the action of initiator and ethylene oxide lucifugal isothermal reaction after 24h acid hydrolysis,filtration and drying of polyethylene glycol polylactic acid(H2N-PEG-b-PLA-MMA)copolymer molecule.Synthesis of(FA-PEG-b-PLA-MMA)with folic acid coupling,PEG-PLA has been synthesized successfully by Nano block and folic acid under DCC and NHS mediated reactions in 24h,Dialysis drying to obtain brick red containing nano block of folic acid,With the above mentioned H2N-PEG-b-PLA-MMA,two methyl maleic anhydride was added to a certain proportion to obtain HOOC-PEG-PLA.The structural changes of different nano carriers were detected by nuclear magnetic resonance and ultraviolet spectroscopy.The particle size and the potential change of different nano blocks were measured by particle size scattering instrument,Nuclear magnetic resonance and infrared detection of the molecular weight of the nanoparticles has been successfully linked to the PEG-PLA block.Results:Three kinds of nano block copolymers were successfully prepared,and the properties of the three kinds of nano block carriers were stable and the particle size was uniform.At about 120nm,the nanoparticles were spherical.Raw materials for the subsequent package of different drugs.PART 2:Study on the preparation and antitumor effect of HB(ethylene glycol)nano carrierObjective:Preparation of hypocrellin nano carrier and folic acid containing hypocrellin B nano carrier micelle and its physicochemical properties were detected by in vitro and in vivo experimental verification of folate receptor targeted nanoparticles on differential expression of folate receptor in ovarian cancer cells or tissue targeting for in vitro release experiment the distribution of drugs,organizations have experimental verification of slow-release nanoparticlesMethods:HB-PEG-PLA and HB-FA-PEG-PLA nano carrier micelles were successfully prepared by rotating evaporation dialysis.The morphology,size and size of the micelles were measured by transmission electron microscopy and dynamic light scattering.Determination of entrapment efficiency and drug loading of HB nanoparticles with high performance liquid chromatography.In vitro release of micelles by dialysis method,The expression of folate receptor in ovarian cancer cell line A2780,HO8910 and SKOV3 was detected by quantitative PCR technique,and the high expression of folate was used to carry out the experimental study.MTT was used to detect the cytotoxicity of different nano carriers,and the cell uptake experiment confirmed that the carriers with folic acid could be absorbed more by tumor cells To establish an animal model and to inject different dosage forms of different drugs,The mice were sacrificed at different time points,and the HB content in the tissue was detected by HPLC technique,so as to verify the sustained-release effect and tumor targeting effect.Results:The preparation of nano micelles was successfully carried out.The nanoparticles were spherical in size and dispersed evenly in the samples.The size of HB-PEG-PLA nanoparticles was 137.8nm,the Zate potential was 17.34mV,the size of HB-FA-PEG-PLA nanoparticles was 145.9nm,and the Zate potential was 22.34mV.1.The expression of folate receptor in SKOV3 cells was significantly higher than that of HO8910.by quantitative PCR2.The cytotoxicity of HB solution was stronger than that of the same drug solution in the cytotoxicity test,It is proved that the nano carrier can reduce the toxic effect of the drug on the cells of the body,and it has a clinical application value.Cellular uptake experiments demonstrated that folate containing nano carriers have a targeting effect on tumor cells.Folic acid nanoparticles are more likely to be absorbed by cancer cells.3.The in vitro release test showed that the nanoparticles had sustained release effect in vitro and the pharmacokinetics of the drug in vivo showed that the nanoparticles had sustained release effect in vivo,In vitro and in vivo distribution proved the sustained release effect of nano carrier.4.The distribution of drug in vivo has further proved the slow release effect of nano carrier and the targeting effect of tumorDiscussion:The nanoparticles have uniform particle size,good stability and low toxicity,which can be used in vivo.Folic acid modified block copolymer has the targeting ability of ovarian cancer cells and tissues with high folate expression,Mediated by the folate receptor that nanoparticles more carrying photosensitizer HB into tumor cells and ovarian cancer tissues,so as to promote the production of photosensitizer HB cytotoxic effect on tumor cells and tissues in a certain wavelength of stimulation.PEG-PLA has low toxicity and slow release effect,and can be used as a drug carrier for clinical applicationPART 3:Preparation of paclitaxel loaded nanoparticles and determination of physical and chemical propertiesObjective:To prepare paclitaxel loaded micelles(PTX-PEG-PLA-NP and PTX-FA-PEG-PLA-NP)and to test its physical and chemical properties,MTT detection of toxic effects of different paclitaxel micelles and paclitaxel solution on ovarian cancer cells,Thus,it can be concluded that the effect of the nano block is an effective carrier which can improve the solubility of paclitaxel,improve the antitumor effect of paclitaxel,and reduce the toxic and side effects of paclitaxel.Cellular uptake further verifies that folate has a tumor targeting effect on the coupling of active targeting substances to the nano block.Methods:1.The nano micelles of paclitaxel were prepared successfully.The nanoparticles in the two kinds of nano micelles were spherical,the size distribution was uniform and the dispersion was better.The size of PTX-PEG-PLA nanoparticles was 128.8nm,the Zate potential was 35.4mV,the size of PTX-FA-PEG-PLA nanoparticles was 143.6nm,and the Zate potential was 42.53mV.2.The cytotoxicity of PTX solution was stronger than that of the same drug solution in the cytotoxicity test,this proved that the nano carrier can reduce the toxic effect of the drug on the cells of the body,and it has a clinical application value.Cellular uptake experiments demonstrated that folate containing nano carriers have a targeting effect on tumor cells.Folic acid nanoparticles are more likely to be absorbed by cancer cells3.The in vitro release test showed that the nanoparticles had sustained release effect in vitro and the pharmacokinetics of the drug in vivo showed that the nanoparticles had sustained release effect in vivo,In vitro and in vivo distribution proved the sustained release effect of nano carrier.4.The experimental results of drug distribution in vivo and the efficacy of the drug showed that the nano carrier conjugated folic acid had a tumor targeting effectDiscussion:The nanoparticles have uniform particle size,good stability and low toxicity,which can be used in vivo.Folic acid modified block copolymer has the targeting ability of ovarian cancer cells and tissues with high folate expression,By means of folate receptor mediated delivery,more nano carriers were carried into the tumor cells and ovarian cancer tissues,Paclitaxel nano carriers can be used to slow the release of taxol in vivo,which can prolong the time of taxol carrier,reduce the dosage of taxol and reduce the toxic side effects of paclitaxel.PEG-PLA has low toxicity and slow release effect,and can be used as a drug carrier for clinical application.Folate conjugated nanoparticles can be used as a tumor targeting agent,and have a highly effective killing effect.In normal cells,the uptake of HOOC-PEG-PLA nanoparticles was less than that of tumor cells,and the toxicity was the same as that in the normal tissues,Because of the acidic environment of the tumor cells and the potential change of the nano carrier,a series of reactions can be absorbed by the tumor cells.PART 4:Antitumor effect of paclitaxel loaded nanoparticlesObjective:Paclitaxel not easily soluble in water,but the application of nanometer carrier paclitaxel could solve this problem,drug-loading nanoparticle carrier can be taxol wrapped in block,improve the anti-tumor effect,in order to further detect taxol content in different organizations in drug group,established the taxol high performance liquid(HPLC)method,and the establishment of the subcutaneous tumor nude mice model.Through different intravenous drug,observe the subcutaneous tumor size,weight and nude mice from in vitro animal level further confirmed drug-loading nanoparticle carrier has antitumor effect,the experiment offers a new way for the drug for the treatment of ovarian cancer.Methods:1.The determination of physical and chemical properties of taxol(absorbance,the preparation of standard curve,precision experiment,the HPLC method for the establishment of)2.The standard curve of taxol drug content in different organizations 3.Three different kinds of intravenous drugs,executed a tumor-burdened mice atdifferent time points,respectively to detect the contents of different organization in different time points of paclitaxel,thus draws the drug distribution organization.4.Venous injection of different group of drugs,the size of the subcutaneous tumors were measured and a tumor-burdened mice body weight,different drug group of antitumor effect.Conclusion:Paclitaxel concentrations in 10-70 ug/ml absorbance had good linear relationship,according to the days of taxol and the precision of the result in the day,and that paclitaxel properties are stable.Paclitaxel in the mobile phase(acetonitrile:water =1:1)column temperature is 25? sample volume of 1 ml/min under the conditions of the HPLC peak is better,different concentration of the peak time is relatively fixed.Concentration in the 2.5-35 ug/ml,a linear distribution.Concentrations of paclitaxel-peak area of standard curve for Y = 180.085 + 93.195 X(r = 0.997).Organizations of taxol standard curve conforms to the linear regression,better detection of peak type,can be separated with other impurity peak very good,the peak time is relatively fixed.Folic acid modified or not,drug-loading nanoparticle carrier drug taxol content in the body are the highest content at 6 h,and pure taxol group of taxol drug was achieved at 2 h peak in the body,thus can be concluded that the nanometer carrier has sustained release effect in the body.Folic acid modification of drug-loading nanoparticle carrier can improve the active target of tumor tissue. |
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