Mitochondria-targeting And Carrier-free Delivery Of Proteins And Its Application

Proteins are the main executor of life activities.Loss of activity and dysfunction of intracellular proteins are closely linked to the occurrence and development of multiple diseases.The direct and effective manner for interfering and treating these diseases is delivering the functional proteins into cells.In the past decade,protein drugs have gradually become the dominant player in the pharmaceutical market due to their high activity,well-defined target and function,little side effects and good biocompatibility.However,natural proteins can not cross cell membrane autonomously,which restricts the application of functional proteins as drugs.The current protein delivery systems have different disadvantages,such as poor universality,carrier dependence and difficulties in preparation.Therefore,we spare no efforts to develop a simple and universal carrier-independent protein delivery system.By introducing the light stimulation-response mechanism,the controllability of the protein delivery system on time and space will be further improved.Reactive oxygen species(ROS)is a kind of oxygenates with strong oxidation ability and active chemical properties.ROS is main produced from mitochondria and NADPH oxidase system.The effects of ROS depends on its level.The balanced level of ROS is closely related to cell growth,metabolism and proliferation.Low level of ROS causes imbalance of cell metabolism,while higher level of ROS contributes to cell canceration and development of tumor and over high level of ROS results in cell oxidative damage,apoptosis and necrosis.The intracellular ROS level can be regulated directly and efficiently by the delivery of antioxidases,which will interfere the progress of disease,such as anti-cancer.In addition,mitochondria are not only the main site of ROS production,but also the important site of ROS action.Targeting delivery of antioxidases to mitochondria will effectively regulate mitochondria function,and lay the foundation for the treatment of mitochondria-related diseases.In conclusion,the development of functional protein delivery system and the use of antioxidases to interfere disease processes are of great significance for the application and development of protein drugs.Based on this,we mainly carried out the following achievements:1.We developed a general carrier-free and mitochondria-targeting delivery system of functional proteins by the Rhodamine B specific conjugation to the protein N-termini,which could mediate the endocytosis-independent cellular delivery of ten proteins into cells via organic cation transporters.The specifically modified antioxidases(copper zinc superoxide dismutase,catalase)remained activities and regulated the level of intracellular ROS within A549 cells.In this way,functional proteins can be delivered into cells and target mitochondria without any physical tools,chemical or biological carriers.In addition,by introducing the coumarin photocages into the protein delivery system,we achieved light-controlled release of proteins in cytoplasm and mitochondria.2.Through the mitochondria-targeting protein delivery system to deliver antioxidases into cells,it was found that the RhB-modified antioxidases could selectively kill cancer cells.This is because the rapid growth and proliferation of cancer cells depending on the high ROS level and the activation of related redox signaling pathway.The RhB-modified antioxidase had low toxicity in mice and accumulated in OCTs-overexpresed organs,such as liver and kidney.These organs are more likely to be damaged by ROS.Furthermore,the RhB-modified antioxidase was able to penetrate BBB and enter into brain.Compared with N-RhB,the modified antioxidase had lower renal clearance rate and longer retention time.The enrichment of RhB-modified catalase in tumor tissue inhibited the proliferation of tumor cells and played a role in inhibiting tumor growth.3.Mitochondria dysfunction is closely related to many diseases such as neurodegenerative diseases,tumors,type 2 diabetes,obesity and aging.The delivery of catalase into cancer cells(A549)resulted in the disorder of ATP synthesis and tricarboxylic acid cycle,decrease of mitochondrial membrane potential,opening of MPTP and outflow of Cytochrome C.Moreover,we ptotected the COS-7 normal cells from oxidative damage via the mitochondria-targeting delivery of catalase.These works establish a relationship between antioxidant enzymes,ROS level and mitochondria function and lay a foundation for the prevention,interference and treatment of mitochondria diseases caused by ROS.