Efficient Polymerization And In Situ Assembly Of Intracellular Polypeptides With Diverse Biological Functions

Intracellular or in vivo construction of artificial nanostructures from small molecules with biofunctions has been recognized as a highly effective approach to enhance bioavailability of active components for disease detection and therapy.Enzyme as the fundamental and ubiquitous catalyst in biological system plays a crucial role in major life activities,which also have been widely utilized to regulate the assembly process in a certain region for drug release,bioimaging,tissue engineering and so on.Of course,when it comes to the construction of nanoassembles in living body,enzyme could be recognized as an efficient trigger factor that pushes the equilibrium from monomers to assemblies.Somehow similar with natural proteins,polypeptides represent a class of biocompatibility materials have been widely applied in living body.The elastic repeat peptide units can polymerize into ELPs with extensibility beyond natural elastin and are capable of undergoing an entropy-driven chain collapse process with temperature change.With enhanced hydrophobic interaction during collapsing process,the chains were inevitably entwined and formed regular structures spontaneously.Here,we firstly report an intracellular enzyme-catalyzed polymerization approach for high-efficient synthesis of polypeptides and in situ controlled construction of thermosensitive nanostructures with diverse biological functions.Transglutaminase(TGase)is one of the crosslinking protein candidates,which creates the covalent bond between amino group of lysine residue and carboxamide group of glutamine residue,exhibiting a high resistance to proteolysis.By de novo designing monomeric peptide unit,we are able to control their phase-transition and topology properties of ELPs.The modular monomeric peptide was composed by i)a functional molecule,ii)polymerization active sites(i.e.,H/H,Q/K or QK/QK)and iii)an elastin-based repeat unit(i.e.,AVHPGVGP,HHPGVG,HDPGVG,HPGVGH,RLGVGFP,RLGVGLP,RLGVGDP,VHPGVG,APGVG,VPHVG and APGVG).The polymerization was catalyzed by TGase via formation of isopeptide bond between the glutamine and lysine side chain.The encoded sequence of elastin-based repeat unit regulated the upper critical solution temperature(UCST)and lower critical solution temperature(LCST),phase transition temperature,polymerization degree and self-assembled property of the resulting polypeptides.The polymerization active sites commanded the self-assembled topological structure.Importantly,upon introducing monomeric units into cells,the intracellular dynamic polymerization process of elastin-like polypeptides and simultaneously in situ self-assemble behavior were investigated independently,revealing that polymerization was a slow chain growth process and self-assembly was a temperature-dependent fast process.Finally,a collection of polypeptides with variable topological structures were used for further biological function evaluation.The linear self-assembled ELPs enabled to significantly enhance their intracellular retention and accumulation,which was a promising candidate as a bioactive molecule carrier.In a sharp contrast,the 3D ELPs analog with volume phase transition property exhibited cell-apoptosis inducing capability,which was potential for drug-free therapeutic application.