Synthesis of amphiphilic miktoarm star copolymers for preparation of biohybrid polymersomes

Amphiphilic block copolymers, composed of hydrophilic and hydrophobic blocks can spontaneously self-organize into ordered structures in aqueous solutions. The morphology of the structures formed depends primarily on the chemical composition and block lengths of the block copolymer. With a suitable hydrophilic weight fraction, polymer vesicles (polymersomes) can be formed that are hollow, water-filled membrane structures, with the hydrophobic blocks forming the core of the membrane. Increasing the molecular weight of the hydrophobic block increases the membrane thickness, which determines the permeability and stability of the polymersome. By modifying polymersomes with biological nanopores (biopores), selective permeability of their membrane can be achieved, enabling new technological applications in biomedicine, such as nanopore sensors capable of single molecule analysis. The main challenge is the design of the macromolecular parameters (molecular weight, chemistry of the blocks, architecture, etc.) of the block copolymers to achieve the required fluidity, flexibility, stability and thickness of the polymersome membrane to allow the reconstitution of the pore-forming proteins. In the case of a large hydrophobic mismatch due to the high thickness of the polymersome membrane, the block copolymer must be able to adapt to the size of the pore-forming protein to maintain its functionality, which currently limits the incorporation of biopores almost exclusively to linear poly(dimethylsiloxane)-based block copolymers due to their high flexibility. The goal of the proposed project is to design and synthesize a new generation of bioinspired amphiphilic miktoarm star block copolymers that can form stable polymersomes suitable for the reconstitution of pore-forming proteins. Inspired by natural lipids, we will focus on miktoarm star block copolymers instead of linear copolymers to mimic the architecture of natural phospolipids. Miktoarm polymers are star-shaped polymers with at least three arms that differ in chemical composition and are bound to the same core. By using two shorter hydrophobic blocks instead of a single long block in the linear block copolymers, we aim to reduce membrane thickness without compromising membrane stability, thus facilitating biopore reconstitution. The miktoarm stars will consist of biocompatible and biodegradable blocks prepared by ring-opening polymerizations. Sarcosine N-carboxyanhydride will be used to prepare the hydrophilic block, while the nature of the hydrophobic block will be varied by preparing polycarbonate, polyether and polypeptide blocks. The structure-property profile of polymersomes prepared from miktoarm star copolymers will be studied with the aim of achieving a membrane thickness similar to that of cell membranes while being sufficiently fluid to allow successful reconstitution of the biopores. The synthetic routes also include modifications to introduce specific functionalities, such as receptor molecules at the ends of the hydrophilic block, to support the attachment of pore-forming proteins and/or the incorporation of the UV cross-linkable moieties. It is expected that cross-linking of the polymersome membrane after reconstitution of the biopores will further increase the structural stability of the biohibrid polymersomes, similar to the spectrin network in red blood cells. The results obtained in the study of the relationship between the miktoarm star structure and the properties of the polymersome will provide a solid foundation for the design of self-assembled membrane structures capable of incorporating transmembrane proteins and biopores suitable for advanced applications such as biosensors, nanoreactors, drug delivery, artificial organelles, etc.