Dynamically Crosslinked Nanocapsules For Efficient Cytosolic Protein Delivery

Proteins are important components of living organisms,regulating majority of biological processes within cells.With the in-depth study of protein functions and mechanisms,protein therapies are rapidly developing in many fields of biomedical research and clinical applications.The clinical availability of protein therapies is currently limited to extracellular targets,as hydrophilic protein drugs with high molecular weights(MWs)have difficulty penetrating cell membranes.However,proteins targeting intracellular targets hold broader therapeutic potentials.Therefore,intracellular protein delivery is critical for the development of novel protein biopharmaceuticals and therapeutic approaches.However,current protein intracellular delivery vectors generally suffer from complex synthesis and low versatility.In addition,chemical modification of the protein is usually required before complexation with the carrier,and the biological activity of the natural protein is often compromised in the process.The most critical issue determining the in vivo application of protein therapies is the ability of nano delivery systems to maintain their own stability under serum conditions.Current intracellular protein delivery carriers tend to aggregate and lose their activity when administered into serum,and even cause immune reactions,posing a great challenge for protein therapy.Based on these understandings,in situ formed,protein-encapsulated nanocapsules(NCs),termed EPP-protein NCs,were herein designed for cytosolic protein delivery,achieved by dynamically crosslinked epigallocatechin gallate(EGCG),low molecular weight PEI(PEI 1.8k),and 2-acetylbenzeneboronic acid(2-APBA)via the formation of imine and boronate ester.Chapter 1 provided an overview of the development and applications of proteins and protein drugs,introduce the advantages and shortcomings of current intracellular protein delivery systems,and summarize the important roles of phenylboronic acid and polyphenols in protein delivery.In Chapter 2,a protein nano-delivery system by dynamically crosslinked EGCG PEI 1.8k,and 2-APB A was developed.First,the protein binds to EGCG by hydrophobic interaction and hydrogen bonding.Subsequently,the aldehyde group on 2-APBA reacts with the primary amino group on the PEI chain to form a Schiff base structure,while the phenylboronic acid group at the other end forms a boronic ester bond with the o-dihydroxy group of EGCG,thus cross-linking each other on the protein surface to form stable EPP-protein nanocapsules(NCs).The optimal reaction molar ratio of PEI,EGCG,and protein was obtained by dynamic light scattering and flow analysis screening to 80:10:1,at which the dynamically cross-linked EPP-protein NCs with a particle size of 120 nm had the highest intracellular delivery efficiency.In addition,the dynamically crosslinked NCs can effectively shield the positive charge of PEI 1.8k and have good cytocompatibility.Due to the pH sensitivity of the Schiff base structure and the borate ester structure,the protein NCs were able to dissociate under acidic conditions and effectively release the encapsulated protein.Subsequently,we used dynamically crosslinked NCs for efficient intracellular protein delivery and could dissociate in endolysosomal acidic environment to release the encapsulated active protein.The crosslinked EPP-protein NCs have excellent serum resistance.This delivery system enables efficient intracellular delivery of proteins of various molecular weights(1.6-430 kDa)and isoelectric points(pI,4.1-10.3),and maintains their natural activity after release.The strategy was also able to deliver proteins into various tumor cells.It is a cell-universal protein intracellular delivery platform.In Chapter 3,we investigated the anti-tumor efficacy mediated by EPP-protein NCs.Efficient delivery of EPP-RNase A with EPP-saporin NCs significantly inhibited HeLa and 4T1 tumor cell viability with semi-inhibitory concentrations(IC50)of 3.87 and 0.26 μg/mL,respectively.This system achieved efficient delivery of CRISPR-Cas9 ribonucleoprotein(RNP)in 293T-EGFP cells with a knockdown rate of 44%.The EPP-saporin NCs obtained by this strategy had good biocompatibility and can be retained in mice for a long time as well as enriched in tumor sites.In addition,EPP-saporin NCs exhibited excellent anti-tumor properties in 4T1 tumor-bearing mice.Chapter 4 provided a summary of the master dissertation and prospects for future work.In this thesis,we designed an intracellular protein delivery system with excellent serum resistance and versatility by dynamically crosslinked EGCG,PEI 1.8k,and 2-APBA.We explored the application of this strategy in gene editing and anti-tumor therapy,which provides new ideas for the development of anti-tumor protein drugs,gene editing and many other biomedical research and applications.