Biotin receptor-mediated intracellular delivery of synthetic polypeptide-protein complexes

Pulmonary delivery offers a non-invasive route for the administration of biotherapeutics. In this context, understanding and control of a transport into, and across cellular barriers is central to the design of delivery systems. Here, we report our study on receptor mediated delivery of protein cargo by a formulation comprising sub-300 nm sized non-covalent protein complexes with biotin-conjugated PEG-poly(glutamic acid) (biotin-PEG_2k-b-GA₁₀) and PEG_2k-b-GA₃₀ copolymers blend as targeting and complexing functionalities. Designed complexes achieve intracellular delivery of the cargo in lung derived A549 epithelial cells in vitro via sodium-dependent multivitamin transporter (biotin receptor). We further show that biotin receptor driven endocytosis preferentially involves dynamin- and caveolae-dependent vesicular internalization, switching the transport pathway away from predominantly clathrin-dependent entry of free protein. Significantly for a protective intracellular delivery of biotherapeutics based on non-covalent complexation with polymeric excipients, the study provides evidence of intracellular presence of the complexing copolymer; demonstrated exploiting biotin in biotin-PEG_2k-b-GA₁₀ copolymer as a tag for binding with fluorescently labelled avidin. Moreover, analysis of intracellular localization of constitutive species shortly following cellular internalization suggests a co-localization of biotin-PEG_2k-b-GA₁₀ copolymer and protein constitutive species. The study demonstrates intracellular delivery of biotin targeted non-covalent complexes with a protein cargo, the result with important implications in a design of enabling technology platforms for protective, receptor mediated intracellular delivery of biotherapeutics.