Design,Construction And Application Of Genetically Engineered Multi-functional Nano-proteins For Orthotopic Hepatocellular Carcinoma Theranostics

Because of the insidiousness,high heterogeneity and insensitivity to radiotherapy and chemotherapy,hepatocellular carcinoma(HCC)is difficult to be treated by traditional methods effectively.Magnetic hyperthermia therapy(MHT)and sonodynamic therapy(SDT)are new approaches for minimally invasive treatment of cancer with infinite tissue penetration depth,which could overcome the bottleneck of optical therapy and have great potential to treat orthotopic HCC.However,both of them lack safe and efficient agents for further application.Protein-based nanomaterials have the characteristics of good biocompatibility,high bioactivity,easy modification and low cost,and could avoid the side effects caused by radiotherapy and chemotherapy effectively.According to the advantages of nanotechnology in tumor theranostics,we designed protein based MHT agent and sonosensitizer,and investigated their applications in the diagnosis and treatment of orthotopic HCC respectively,which also given new ideas for the development of protein-based nanomaterials.The main content is summarized as follows:In chapter 1,we summarized the current research status of protein drugs in cancer,and introduced different types of protein drugs in detail.And systematically introduced the development of molecular imaging technology,and the advantages and bottleneck of new therapeutic methods in tumor therapy.In consideration of the attributes of orthotopic HCC,we investigated the characteristics and functions of different protein materials,and determined to develop protein-based nanomaterials to solve the bottleneck of magneto-thermal therapy and sonodynamic therapy,thus realizing efficient treatment of orthotopic HCC.In chapter 2,we genetically engineered encapsulin protein nanocage,a type of iron-stored nanocage,and obtained magnetic protein nanocages(eMIONs)by bionic mineralization technique,and investigated its magnetic properties.It is found that the crystal nucleus of eMIONs is about 22 nm in diameter,is FeO@Fe3O4 multi-crystalline structure,and the degree of crystallization is nearly 100%.The magnetic field specific absorption rate(SAR)of eMIONs is as high as 2 390 W/g,which is 20 times higher than that of Feridex(115 W/g).Therefore,eMIONs is an excellent MHT agent.eMIONs also has T2 imaging capability to guide precise MHT.Furthermore,we found that eMIONs is a type of catalase nanoenzyme,and increased its catalytic efficiency by 44%in the presence of alternative magnetic fields.Therefore,eMIONs is not only an excellent MHT agent,but also a magnetic responsive nano-enzyme,which is expected to realize MRI-guided efficient magnetic catalytic therapy for orthotopic HCC.In Chapter 3,we explored the efficacy of eMIONs in the diagnosis and treatment of hepatocellular carcinoma.On the one hand,eMIONs promoted apoptosis of tumor cells through magnetothermal effect,and on the other hand,it significantly reduced the expression of hypoxic factor HIF-1α and growth factor VEGF in tumor cells through magnetic field enhanced catalase-like catalysis,thereby inhibiting the growth of liver cancer through magneto-catalysis therapy,and nearly doubling the survival time of orthotopic HCC mice models.It is proved that eMIONs prepared by genetic engineering technology and biomimetic strategy has successfully broken through the bottleneck of MHT,and expanded MHT application to realize the effective treatment of orthotopic HCC,bringing a new way for the follow-up research.In Chapter 4,based on the advantages and bottlenecks of sonodynamic therapy,we innovatively developed a highly specific HCC and mitochondria dual-targeted nano-protein sonosensitizer(NGR@mNPs).NGR@mNPs are based NGR peptide modified red blood cell membrane vesicle,and encapsulated SPIO carried mitoSupernova(mSN),an mitochondria targeted,catalase-like and sono-responsive protein,inside the cavity(NGR@mNPs).The NGR@mNPs exhibit an excellent targeting ability in both subcutaneous xenograft and orthotopic settings,and then mSN specifically targets mitochondria via the assist of SPIO-PEI and takes the efficient catalytic features to convert tumor over-expressed H2O2 into O2,thereby significantly promoting ROS production and augment SDT efficacy subsequently.