Decorating hexahistidine-metal assemblies with tyrosine enhances the ability of proteins to pass through corneal biobarriers.

In recent decades, the use of protein drugs has increased dramatically for almost every clinical indication, including autoimmunity and cancer infection, given their high specificity and limited side effects. However, their easy deactivation by the surrounding microenvironment and limited ability to pass through biological barriers pose large challenges to the use of these agents for therapeutic effects; these deficits could be greatly improved by nanodelivery using platforms with suitable physicochemical properties. Here, to assess the effect of the hydrophobicity of nanoparticles on their ability to penetrate biological barriers, the hydrophobic amino acid tyrosine (Y) was decorated onto hexahistidine peptide, and two nanosized YHmA and HmA particles were generated, in which Avastin (Ava, a protein drug) was encapsulated by a coassembly strategy. In vitro and in vivo tests demonstrated that these nanoparticles effectively retained the bioactivity of Ava and protected Ava from proteinase K hydrolysis. Importantly, YHmA displayed a considerably higher affinity to the ocular surface than HmA, and YHmA also exhibited the ability to transfer proteins across the barriers of the anterior segment, which greatly improved the bioavailability of the encapsulated Ava and produced surprisingly good therapeutic outcomes in a model of corneal neovascularization. STATEMENT OF SIGNIFICANCE: Improving the ability to penetrate tissue barriers and averting inactivation caused by surrounding environments, are the keys to broaden the application of protein drugs. By decorating hydrophobic amino acid, tyrosine (Y), on hexahistidine peptide, YHmA encapsulated protein drug Ava with high efficiency by co-assembly strategy. YHmA displayed promising ability to maintain bioactivity of Ava during encapsulation and delivery, and protected Ava from proteinase K hydrolysis. Importantly, YHmA transferred Ava across the corneal epithelial barrier and greatly improved its bioavailability, producing surprisingly good therapeutic outcomes in a model of corneal neovascularization. Our results contributed to not only the strategy to overcome shortcomings of protein drugs, but also suggestion on hydrophobicity as a nonnegligible factor in nanodrug penetration through biobarriers.

Sheltering proteins from protease-mediated degradation and a de novo strategy for preventing acute liver injury.

Restoring protein functions or supplying proteins is considered one of the most powerful therapeutic strategies for many diseases, but it is mainly limited by the denaturation of proteins during encapsulation and degradation by proteases during in vivo delivery, and limits its delivery. Herein, by encapsulating a protein (catalase, an enzyme) in a hexahistidine-metal assembly (HmA) via a de novo strategy under mild conditions, we demonstrated that HmA could maintain the bioactivity of the enzyme, protect the enzyme from proteinase degradation, and deliver the encapsulated protein for the prevention of disease in an acute liver injury model.

Efficient delivery of cytosolic proteins by protein-hexahistidine-metal co-assemblies.

Proteins play key roles in most biological processes, and protein dysfunction can cause various diseases. Over the past few decades, tremendous development has occurred in the protein therapeutic market due to the high specificity, low side effects, and low risk of proteins. Currently, all protein drugs on the market are based on extracellular targeting; more than 70% of intracellular targets remain un-druggable. Efficient delivery of cytosolic proteins is of significance for protein drugs, advanced biotechnology and molecular cell biology. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for intracellular protein delivery. Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged protein encapsulation particles(Protein@HmA) through a co-assembly process with a high loading capacity and loading efficiency. Protein@HmA was able to deliver proteins with diverse physicochemical properties through multiple endocytosis pathways, and the protein could quickly escape from endosomes. In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells. Our results demonstrate that this strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics. STATEMENT OF SIGNIFICANCE: Intracellular targets with protein drugs may provide a new way for the treatment of many refractory disease. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for efficient intracellular protein delivery. Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged particles (Protein@HmA) with a high loading efficiency. Protein@HmA was able to deliver different proteins through multiple endocytosis pathways, and the protein could quickly escape from endosomes. In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells. This strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics.