Preparation Of Stimulus-responsive Protein Nanoparticles For Cancer Therapy

Cancer has become the main disease causing human deaths worldwide.Compared with chemotherapy and gene therapy,protein therapy has unique advantages in anti-cancer applications,including high pharmacological efficacy,strong specificity,low toxicity,low drug resistance and less interference with normal biological processes.While directly treating cancer,there are disadvantages such as poor stability and cell membrane permeability for protein drug,which greatly limits its application.It is urgent to develope an efficient and good biocompatible carrier,which can promote the escape of endosomes and enhance the cellular uptake of protein,so as to promote the clinical research of protein drug for cancer.Nanoparticles have the following advantages for protein delivery: 1)Protecting the protein from degradation or denaturation under acidic and enzymatic conditions;2)Extending the half-life;3)Targeting diseased cancer cells and tissues to improve the safety and effectiveness of protein drugs in clinical applications.Stimulus-responsive nanocarrier can be designed based on the unique characteristics of the tumor microenvironment.The carrier can control the release of drugs only stimulated by endogenous or exogenous sources in the tumor site.In this way,the adverse toxicity to normal cells is minimized.In this study,we developed a stimulus-responsive nanocarrier through protein coupling that can efficiently deliver ribonuclease A(RNase A)to the nucleus with the purpose of enhancing its anti-tumor efficacy.This work also provides a new delivery idea for the intranuclear transport of other protein drugs.The main works are as follows:(1)Preparation of nanoparticles: The stimulus-responsive Linker containing the azide group was coupled to the surface of RNase A through covalent linkage.The benzocyclooctyne-containing lysine(DIBO-Lys)was further paired with the copper-free click chemistry,and a positively charged single protein nanoparticle RNL is obtained.The reduction-responsive disulfide bond will be broken in the tumor microenvironment with high concentration of GSH in the tumor,and then RNase A will be released in prototype form to exert enzyme catalysis and anti-tumor activity.(2)In vitro characterization of single protein nanoparticle RNL: The prepared RNL particles were characterized by DLS,Zeta potential and TEM.The results showed that the RNL nanoparticles are positively charged and the particle size is less than 50 nm,which is the effective particle size through the nuclear pore.Dithiothreitol(DTT)was used to simulate the reducing environment with glutathione in the tissues,and the results showed that RNase A can be released intactly in prototype form according to the agarose gel electrophoresis.(3)Cell experiment: Using mouse 4T1 breast cancer cells,we explored the anti-tumor activity of RNL in vitro.Fluorescence-labeled nanoparticles can be efficiently aggregated in the nucleus.Further exploration of the endocytosis mechanism revealed that RNL relies on Caveolin’s mediation to enter the cell,which belongs to energy-dependent active endocytosis.It escapes from the lysosome through the proton sponge effect and finally enters the nucleus by importin α/β.The strong lethality of RNL on mouse breast cancer cells was confirmed by performing MTT assays.(4)In vivo experiment: Through the establishment of mouse 4T1 breast tumor model,animal experiments were carried out to further study the anti-tumor activity of protein-loaded RNL in vivo.The results showed that RNL can effectively inhibit the growth of tumors,while the weight of mice tends to stabilize.H&E tissue sections revealed that RNL did not cause obvious damage,indicating that it has good biological safety while exerting anti-tumor activity.The nanocarrier induced the apoptosis of tumor cells by delivering RNase A in the nucleus.In vivo and in vitro experiments showed excellent anti-tumor efficacy,providing new ideas for the clinical treatment of protein drugs for cancer.