Hydrophobic Polymeric Nanocapsules For Protein Delivery With Enhanced Transport Barriers Penetrating Abilities

Recently,protein therapy has been widely used in the field of disease treatment,such as cancer therapy,gene editing,autoimmune disease treatment and so on.Efficient and controlled delivery of protein to the target sites in the organism is the key of protein therapy.However,achieving efficient protein delivery in living organisms remains a challenge.There are a variety of delivery barriers in the organism,such as blood-brain barrier,cell membrane,vesicle capture,non-specific adsorption,etc.These biological barriers hinder the effective delivery of protein and are one of the main reasons for the low delivery efficiency of protein.Therefore,it is of great practical significance to design a protein delivery platform that can efficiently penetrate the delivery barriers for effective protein delivery and therapy.Here,we show a novel protein delivery platform based on hydrophobic polymeric nanocapsules with enhanced delivery barriers penetrating ability.This platform can significantly improve the efficiency of protein delivery at the cellular level.The main research contents of this thesis are as follows:(1)Design and preparation of hydrophobic polymeric nanocapsules coated with proteins.With bovine serum albumin(BSA)as the template protein,hydrophobic monomers and acid degradable crosslinkers were in situ polymerized on the proteins,resulting in nanocapsules consisting of protein core and hydrophobic polymer shell.Then a few biological functional molecules were coupled on the surface of nanocapsules.The final products were dispersed in aqueous environment with the aid of a small proportion of organic cosolvent.We named these hydrophobic polymeric nanocapsules coated protein as Cs-n(protein)-BM.Different from traditional hydrophilic protein delivery platforms,Cs-n(protein)-BM has a large proportion of hydrophobic surface and a small proportion of organic cosolvents to disperse in aqueous environments.Then,we investigated the physicochemical properties of Csn(protein)-BM and the release mechanism of proteins under acid environment.At the cellular level,it was confirmed that hydrophobic polymeric nanocapsules could also be used for the delivery of protein and nucleic acid complexes into cells for release and expression.In addition,Cs-n(protein)-BM showed low toxicity.(2)Research on the ability of Cs-n(protein)-BM to penetrate biological delivery barriers and its targeted delivery in cells.Cell experiments results showed that Csn(protein)-BM exhibited enhanced biological transport barriers penetrating efficiency,which was demonstrated as follows: Cs-n(protein)-BM showed high cell membrane penetrating efficiency,in the meantime,when biological functional molecule Tat with nucleus targeting ability was used,Cs-n(protein)-Tat showed enhanced nucleus targeting,strong vesicle escape ability and transcytosis efficiency.In addition,the Csn(protein)-BM system could remarkably enhance the selectivity of biological functional molecules to cells.Further,the effects of the types and concentrations of cosolvents,the proportion of hydrophobic surface of nanocapsules and the non-specific adsorption of proteins on the ability of Cs-n(protein)-BM to penetrate delivery barriers were explored.The cosolvent could significantly improve the penetration ability of the Cs-n(protein)-BM system.And with the increase of the proportion of hydrophobic surface of Cs-n(protein)-BM,the ability of Cs-n(protein)-BM to penetrate the biological delivery barriers and the selectivity of biological functional molecules to cells first increased,then decreased and then increased,and the optimal proportion may be related to the types of cells and biological functional molecules.The large proportion of hydrophobic surface of the Cs-n(protein)-BM system showed low non-specific adsorption effect,and the barrier penetrating ability of the Cs-n(protein)-BM system was not undermined after incubating with serum for 3 h in advance.Finally,based on the ability of Cs-n(protein)-BM to efficiently penetrate the delivery barriers,we selected the anticancer drug doxorubicin(DOX)to study the drug loading of the novel nanocapsules system and its drug delivery efficiency at the cellular level.Compared with free DOX and conventional hydrophilic polymeric nanocapsules loaded with DOX,DOX@Cs-n(protein)-BM exhibited a stronger killing effect on cancer cell.Finally,Csn(protein)-Tat could also penetrate multiple barriers at the animal level.Compared with conventional hydrophilic polymer nanocapsules,Cs-n(protein)-Tat could penetrate deeper skin tissues and were expected to be used in the field of transdermal therapy.