| Cancer is considered to be one of the most threatening diseases to human life and health in the world.So far,among the various methods of cancer treatment,chemotherapy is considered to be one of the ideal anti-cancer treatments due to its high curative effect.Some drugs have been found to have strong anti-cancer activity,but their own physical and chemical properties limit their clinical application.Dihydroartemisinin has excellent antimalarial activity and metabolic rate,and is also an important active metabolite of artemisinin,and recent research by domestic and foreign scholars has found that it contains the ability to inhibit 55 cancer cell lines.The DHA molecule contains a peroxy bridge structure,which generates active oxygen free radicals through Fenton reaction with transition metal ions to oxidize lipids,destroy cell membranes,proteins and DNA,and ultimately induce tumor cell apoptosis.However,DHA has poor water solubility and poor stability,which hinder its anti-tumor efficacy in clinical research.In order to solve the above problems,researchers in the past few years have designed drug delivery systems（DDSs）based on nanomaterials in order to improve the in vivo drug delivery methods and clinical efficacy of DHA.So far,scientists have successfully synthesized nanocarriers,including high molecular polymers,mesoporous silica,organic or metal compounds,and carbon nanomaterials.They show excellent spatial structure,controllable size,and passive tumor targeting ability with penetration and retention effect（EPR）,but also suffer from some biodegradability or toxicity issues.Current studies have shown that dihydroartemisinin can exert good efficacy in the mitochondria of tumor cells.Therefore,in view of the anti-cancer activity of DHA and the biodegradability and toxicity of nano-drug delivery systems,the subject of constructing a biodegradable mitochondrial targeted drug delivery system for cancer treatment and diagnosis has important research significance.In this thesis,we have cleverly designed and synthesized a biodegradable tumor cell mitochondrial targeted nano-drug delivery system,which delivers DHA through mitochondria targeting,so as to improve the chemotherapy efficacy of DHA on tumors from the organelle level.First,we synthesized the nano-sized metal-organic framework PCN-224 by a one-pot method,and further used triphenylphosphine（TPP）for surface modification,and finally loaded DHA to form DHA@PCN-224-TPP nano-drugs.DHA@PCN-224-TPP is enriched in tumor tissue through high permeability and retention effect（Enhanced Permeability and Retention Effect,EPR effect）.After using the positive charge and lipophilicity of TPP to further target into the mitochondria,the tumor mitochondria The high concentration of phosphate causes the disintegration of the nano-framework,thereby releasing DHA,increasing the cumulative concentration of the drug in the tumor mitochondria.At the same time,the increase of transferrin receptor in the tumor mitochondria leads to a higher level of ferrous ion,which triggers the DHA peroxy bridge Fenton reaction produces ROS and singlet oxygen in the fracture,and acts on apoptosis-related proteins to activate intracellular apoptosis pathways and improve the anti-tumor effect on tumor cells.In addition,PCN-224itself carries a fluorescent group,which is convenient for the quantitative and localization detection of nanomedicine in vivo and in vitro,which is convenient for tracking drug targets and explaining its anti-tumor internal mechanism,avoiding the false positive effect caused by the additional use of fluorescent dyes.This article mainly contains the following parts:This first chapter mainly covers the preparation and characterization of nanomaterials,mainly using transmission electron microscopy（TEM）,Fourier transform infrared spectrometer（FT-IR）,X-ray diffractometer（XRD）,ultraviolet-visible spectrophotometer（Uv-vis）,whole Automatic specific surface and porosity analysis（BET）and other instruments are used to analyze the morphology,particle size,potential,crystal characteristics,UV characteristic absorption band,surface atomic composition,specific surface area and mesoporous structure characteristics of the optimized nanomaterials.A comprehensive evaluation was carried out.The results show that the particle size and potential of the prepared nanoparticles meet the design requirements.According to the closed hysteresis loop characteristics of the adsorption-desorption curve,it is judged that the nanocarrier is a mesoporous material with a large specific surface area,and the pore structure and size are suitable for drug loading.The characteristics of TPP grafting amount and surface distribution can meet the needs of expected target design.In the second chapter,the drug loading evaluation of nanocarrier MOF-TPP was carried out.The designed and prepared MOF-TPP was evaluated for drug loading,and its drug loading process was optimized by measuring the drug loading rate and encapsulation rate of MOF-TPP.At the same time,by examining the in vitro release characteristics,phosphate-responsive degradation and stability of MOF-TPP under various conditions,the drug-loading characteristics,release principle and release behavior of MOF-TPP were comprehensively evaluated to verify the applicability and feasibility of nanocarriers.The results show that the prepared MOF-TPP can release the drug in response to the disintegration of the high-concentration phosphoric acid environment,and the release behavior is significantly different in the release system simulating the tumor cell mitochondrial microenvironment and the normal cell microenvironment.The carrier has the selective release effect of tumor cell mitochondria.The third chapter investigated the in vitro antitumor activity and mechanism of DHA@PCN-224-TPP.In this chapter,two cancer cell lines,4T1 and A549,were used to study the antitumor activity and mechanism in vitro,and the MTT assay,plate clone formation assay,cell cycle arrest assay,flow cytometric apoptosis assay,mitochondrial membrane potential assay and intracellular apoptosis-related protein expression assay were performed.The in vitro antitumor activity,cell cycle arrest,and apoptosis-related proteins in mitochondria were determined by the above experiments.Adjust the situation and confirm the position of nanocarriers and drugs entering mitochondria,so as to verify the effectiveness and therapeutic mechanism of tumor cell mitochondria-targeted delivery of DHA to inhibit cancer cells and its low toxicity to normal tissue cells.The results showed that the nanocarriers could improve the antitumor effect of dihydroartemisinin by reducing the mitochondrial membrane potential,regulating apoptosis-related proteins,and blocking the G2/M phase of the cell cycle after the drug was delivered to the mitochondria.The fourth chapter conducted the in vivo antitumor study of DHA@PCN-224-TPP.This chapter mainly selects BALB/c female mice-bearing 4T1 tumor（6-7 weeks）as the research object,and investigates the relative tumor volume coefficient changes and tumor weight of DHA@PCN-224-TPP during the treatment process to evaluate the in vivo efficacy of the animals.At the same time,the morphological changes of the heart,liver,spleen,lung,kidney,tumor and other tissues of nude mice after administration were observed to evaluate the anti-tumor activity and the targeting and safety of the drug.The results show that the nanocarriers have excellent biosafety,no obvious toxicity to the main organs of mice,and can significantly improve the inhibitory effect of dihydroartemisinin on tumor growth after drug loading.In summary,this project successfully designed and prepared a biodegradable tumor cell mitochondria-targeted nano-drug delivery system,and successfully delivered dihydro-artemisinin to the tumor cell mitochondria to exert its medicinal effect.In addition,the constructed nano-drug delivery system possesses biodegradability and good in vivo safety.At the same moment,it carries a fluorescent signal,which can realize the visual detection of the localization and quantification of the derivative dihydroartemisinin. |
