Biomimetic Mineralization And Genipin-mediated Crosslinking Strategies For The Intracellular Delivery Of RNase A

In the past two decades,the incidence of cancer and mortality has shown an increasing trend year by year,making it a threat to human health and an urgent need to resolve the malignant disease.A series of therapeutic proteins or peptides including growth factors,cytokines,antibodies and enzymes have been successfully developed due to the rapid progress of biotechnological techniques.Meanwhile,protein-based therapy has exhibited great potential in the treatment of various diseases owing to the characteristics of high pharmacological potency and low toxicity.In order to solve these problems,more and more researches focused on overcoming the stability limitation of therapeutic proteins and improving their intracellular delivery efficiency.Among them,the cytotoxic ribonuclease A(RNase A)could achieve the cleavage of the intracellular RNA molecules and induce the cell apoptosis,which has been demonstrated to possess favorable killing ability against tumor cells.In this paper,two efficient RNase A systems are designed to achieve its intracellular delivery and rapid release.The contents are summarized as follows:ZIF-8 was utilized as an efficient platform for intracellularly delivering RNase A,an anti-tumor protein.The ZIF-8 crystal could not only efficiently encapsulate RNase A molecules through a facile method but also maintain its enzymatic activity.Moreover,RNase A@ZIF-8 nanoparticles could control the release of RNase A in a p H-dependent manner,and RNase A could be rapidly released from the nanoparticles in an acidic condition.Further,RNase A@ZIF-8 could realize an efficient cellular uptake by cancer cells,leading to an anti-proliferative effect through the induction of cell apoptosis.Finally,RNase A@ZIF-8 exhibited an excellent biocompatibility through hemolysis assay.Therapeutic protein RNase A and cationic polymer PEI25 K were covalently crosslinked to construct a protein-polymer hybrid nanoparticle RGP using genipin as a crosslinker.The characteristics of RGP nanoparticles showed that RGP nanoparticles were positively charged and uniform in size,which could achieve efficient intracellular delivery.The enzyme activity analysis showed that the genipin crosslinking did not significantly reduce the catalytic activity of RNase A.The protein-polymer hybrid system RGP could realize an efficient intracellular delivery of therapeutic protein RNase A and could escape from endosome.The RGP nanoparticle could induce the apoptosis by cleaving the RNA molecules in the cytosol,thereby achieving inhibiting the proliferation of cancer cells.In summary,we successfully constructed two RNase A delivery systems,through biomimetic mineralization and genipin-mediated cross-linking strategies,respectively.Both systems achieved efficient intracellular deliver,facilitated the endosomal escape of protein cargo in tumor cells,and greatly improved the killing capability of therapeutic proteins to tumor cells.The above studies provide good guidance for solving the difficulty in transmembrane transport and stability defects of therapeutic protein drugs,and have great significance for promoting the clinical application of therapeutic protein delivery.