Construction Of Multifunctional Biomimetic Nanoparticles Based On Improving Hypoxia Combined With Enhancing Tumor Infiltration Strategy And Study Of Anti-breast Cancer Effect

The World Health Organization’s International Agency for Research on Cancer（IARC）released the latest cancer research data,showing that the number of women with breast cancer has surpassed lung cancer to become the world’s most common cancer.Chemotherapy remains one of the most effective therapies for breast cancer,and anthracyclines remain an important part of chemotherapy.However,the drug resistance phenomenon of chemotherapy is increasingly prominent,and the mechanism is relatively complex,which is closely related to the overexpression of drug-resistant protein,changes of tumor microenvironment（TME）,and complex signaling pathways.Hypoxia,as one of the characteristics of TME,exists in the center of most solid tumors and is closely related to chemotherapy resistance.Although the nano-drug delivery technology for hypoxia and chemotherapy has shown beneficial therapeutic effects,the median value of tumor site accumulation in nanoparticle is only 0.7%of the injected dose due to the presence of dense extracellular matrix（ECM）in TME.This may also be one of the key causes of drug resistance.Based on the above problems,this paper intends to enhance the treatment effect of Dox for breast cancer through tumor oxygen supply intervention therapy and enhanced infiltration of nanoparticles.In this study,hollow mesoporous manganese dioxide（H-MnO₂）was used as the nanodrug delivery core to load Dox,and H-MnO₂can catalyze excessive H₂O₂in tumor cells to produce oxygen to alleviate the hypoxia of tumor sites.In addition,H-MnO₂surface was modified with collagenase（Col）to degrade ECM and further improve the infiltration of nanoparticles at tumor sites.In order to protect the stability of Col during in vivo transport and to improve the active targeting of nanoparticles,this paper further coated the Col-modified H-MnO₂-Dox（H-MnO₂-Dox-Col）surface with p H-sensitive liposomes（P）and macrophage membrane（M）fusion membrane（MP）.Finally,we constructed a biomimetic nanoparticles MP@H-MnO₂-Dox-Col and we systematically evaluated the preparation process,oxygen production,ECM degradation and anti-breast cancer effects of the biomimetic nanoparticles in vitro and in vivo.This research is mainly divided into three chapters.The first chapter is the construction and characterization of multifunctional biomimetic nanoparticles.This chapter is mainly divided into three parts.The first part is the preparation of carrier H-MnO₂:using solid silicon dioxide as template,H-MnO₂was synthesized by alkali etching method.The mesoporous structure and components of H-MnO₂were detected and identified.The results showed that the mesoporous structure of H-MnO₂was uniform.The specific surface area was 175.696 m²/g,and the pore size was 4.5nm,which had a large specific surface area,and the pore size was suitable for loading small molecules such as Dox.The identification result of component elements is that the sample contains Mn element and O element,and Mn in the nanoparticle is+4 valence state.The above results indicate successful synthesis of H-MnO₂.In addition,the drug loading rate and encapsulation rate were（73.02±2.2）%and（90.72±9.5）%,respectively.The second part is the preparation of MP:firstly,RAW264.7 macrophages（RAW for short）were collected in vitro cell culture,and M was extracted by repeated freeze-thaw method,then P was prepared by thin-film hydration,and finally MP was prepared by water bath ultrasound.In addition,F?rster resonance energy transfer（FRET）,fluorescence co-location and fourier transform infrared spectroscopy were used to investigate the fusion of M with P.The third part is the grafting of Col and the encapsulation of MP to construct the final multifunctional biomimetic nanoparticles MP@H-MnO₂-Dox-Col.The particle size was about（222.3±8.5）nm,and the potential was about（-35.6±1.3）m V,which was close to the potential of MP（-33.5±1.36）m V.In addition,the"core-shell"structure of MP@H-MnO₂-Dox-Col was observed by transmission electron microscopy（TEM）as well as SDS-PAGE and western blot results also verified the successful coating of MP on H-MnO₂surface.In the second chapter,we preliminarily evaluated the in vitro penetration enhancing effect,oxygen production capacity and anti-breast cancer effect of MP@H-MnO₂-Dox-Col.Firstly,we detected the oxygen production capacity and the activity of Col of the biomimetic nanoparticles in vitro.The preliminary results showed that the biomimetic nanoparticles had a good catalytic capacity for oxygen production,and remained the enzyme activity of Col modified on the H-MnO₂surface,and the enzyme activity of Col was protected after MP coating.Secondly,we observed the in vitro drug release of the biomimetic nanoparticles under H⁺and H₂O₂conditions.The results showed that MP@H-MnO₂-Dox-Col had good triggering response release characteristics of H⁺and H₂O₂,which could achieve the successful release of Dox in tumor microenvironment.In addition,the intracellular distribution results showed that the encapsulation of MP could mediate more distribution of Dox in tumor cells.The results of blank carrier toxicity experiment also show that the biomimetic nanoparticles have good biocompatibility.Finally,the anti-breast cancer effect of the biomimetic nanoparticles was evaluated by observing the growth of tumor sphere and cell death after different treatment.The results showed that the biomimetic nanoparticles had a strong inhibitory effect on breast cancer cells.In the third chapter,we investigated the in vivo safety and anti-tumor effects of multifunctional biomimetic nanoparticles,as well as the functions of relieving hypoxia and degrading collagen at tumor sites.We established the orthotopic transplantation tumor model of breast cancer in mice loaded with 4T1 cells.In vivo imaging experiments of small animals were conducted to investigate the distribution of biomimetic nanoparticles.The results showed that compared with the free Di R,biomimetic nanoparticles loaded with Di R（MP@H-MnO₂-Di R-Col）could be more enriched in tumor sites.Secondly,the changes of tumor volume growth curve,tumor weight and survival time in mice experiments confirmed that MP@H-MnO₂-Dox-Col had a good inhibitory effect on tumor.HE staining and body weight change in mice showed the biocompatibility of MP@H-MnO₂-Dox-Col.Finally,Masson trichromatic staining and immunofluorescence staining of tumor tissues were used to investigate the degradation of collagen,infiltration of nanoparticles and improvement of hypoxia state of tumor tissues with different treatment.The results showed that MP@H-MnO₂-Dox-Col had a good collagen decomposition ability and could promote the infiltration of Dox into the deep tumor,meanwhile,the catalytic oxygen production capacity of MnO₂can be utilized to alleviate the hypoxia of tumor tissues and further enhance the anti-tumor effect of Dox.In summary,we constructed multi-functional biomimetic nanoparticles MP@H-MnO₂-Dox-Col.On the one hand,active targeted drug delivery and tumor microenvironment responsive drug release could be achieved by MP,on the other hand,H-MnO₂carrier had excellent drug delivery and catalytic oxygen production characteristics,and Col penetration promoting characteristics of surface modification on H-MnO₂,which could improve the tumor hypoxic microenvironment and enhance the infiltration of nanoparticles,and ultimately promoted the better anti-breast cancer effect of Dox.This paper hopes to provide a new treatment strategy for the comprehensive treatment of cancer and to provide a new design ideas and research methods for tumor targeted therapy.