Construction Of Proteinosomes Based Hybrid Hierarchical Structure And The Controlled Transportation Of Biomacromolecule

Recently,artificial cells for mimicking the structures and functions of eukaryotic cell have become a focus,with promising prospects in the fields of biomimetic materials,biocatalysis,drugs and gene delivery.However,the structures cannot fully represent the structural complexity and functional diversity of cells as the basic unit of life.Moreover,the key of “live” property of an artifical cell is mimicking the molecule transportation among cells and between cells and external environment.This thesis designed and constructed single/multi-compartmental structures based on multiple building blocks to mimic cellular membrane,cytoskeleton and intracellular organelles,and investigated the mimicking behaviors of artificial cells in cell fusion,self-healing,stimulus response and molecules transportation.The main research contents are as follows:Inspired by the supporting function of cytoskeleton in cells,protein hydrogel based proteinosomes as protocells were prepared based on the building blocks of BSAPNIPAAm bioconjugates,bovine serum albumin and dextran.The dynamic Schiff base covalent binding of protein hydrogel endows the single-compartment structure with a certain p H response and self-healing ability,and the adjacent proteinosomes will be fused due to the reversible rupture and recombination of chemical bonds,mimicking the cell fusion behavior successfully.Utilizing the fusion behavior of this protein hydrogel based proteinosomes,the transportation and exchange of biological macromolecules,inorganic particles,and Saccharomyces cerevisiae among the hydrogel based proteinosomes or between the hydrogel based proteinosomes and the external environment can be realized.And the fusion process can be accelerated by the local p H decrease which was triggered by the fusion of the two different proteinosomes loading glucose oxidase(GOx)and glucose respectively and the following enzymatic reaction.Moreover,the fusion process can be also inhibited by forming a protective layer of cinnamyl aldehyde on the surface of proteinosomes.Thus,the molecules transportation driven by proteinosome fusion was further regulated.The fusion behavior of protein hydrogel based proteinosomes realized the molecule transportation among artificial cells.For further mimicking of molecule transportation between cells and external environment,a hierarchical polymersomes-in-proteinosome multicompartment structure(CMMS)was constructed via integrating PEGylated insulin(PEG-Ins)-loaded p H-responsive polymersomes(Psomes)and GOx into the hydrogel based proteinosomes,realizing the transportation of PEG-Ins under biological stimuli for mimicking pancreatic β cells.The porous protein hydrogel based cytoskeleton in CMMS plays a key role for the even distribution of Psomes and GOx as well as the enhanced structural stability of CMMS.Moreover,the p H-responsive characteristics of both the protein hydrogel based cytoskeleton and Psomes,emphasize synergistic acid triggered swelling behaviors of the whole CMMS,influencing the release of PEG-Ins.Significantly,a glucose-triggered release of PEG-Ins from the constructed CMMS is available at glucose concentrations simulating a diabetic blood level regardless of the outer neutral aqueous environment.The diffusion of glucose from the external environment into CMMS can trigger the reaction of GOx to produce gluconic acid.Thus,at low glucose concentrations,the p H decrease was not enough to completely protonate the Psomes,resulting in extremely slow release of PEG-Ins.By contrast,at high glucose concentrations,PEG-Ins would be quickly transported into the external environment in a free state from CMMS with complete protonation of Psomes and cytoskeleton.Thus,the molecules transportation between cells and external enviornment was smartly regulated due to the stimuli-responsive property of inner compartments.For artificial cells,the simple stimuli-responsive molecules transportation cannot meet the requirements as new drug carriers.Thus,it was necessary to increase the regulation in a spatiotemporal scale.Accordingly,a dual-responsive and p H selfmonitoring multi-compartment(microgel in PEO micro-chamber in hydrogel loaded proteinosome(MPHP)structure)based on twice ATPS and a Pickering emulsion method was constructed.The adoption of glucose-like diol as specific binding site for anchoring phenylboronic group modified PEGylated insulin(Ins-PEG-PBA),as well as the electrostatic interaction between the modified insulins and p H-sensitive dynamic network of hydrogel,showed the possibility that MPHP structure could load Ins-PEG-PBA with high-efficiency.In the presence of low concentration glucose,the boronic ester exchange between the diol and glucose resulted in the release of insulin.However,in the presence of high concentration glucose,glucose triggered the reaction of GOx and followed a obvious p H decrease.Except the boronic ester exchange,boronic ester hydrolysis and the swelling of the hydrogel at low p H were the main driving forces for the long-term release of insulin.Moreover,by incorporating a p H indicator BSA-FITC/RBITC into the MPHP structure,the release process of insulins at different concentrations of glucose can be selfmonitored via confocal fluorescence microscope.In conclusion,in this thesis three new single/multi-compartmental structures were constructed and the controllable molecules transportation in artificial cells was realized according to different requirements,taken together the advantages of multicompartment,dynamic covalent and electrostatic interaction,p H-and glucose-responsive properties and enzymatic reaction modulated process.Thus,these artificial cells are expected to not only inspire more designs towards eukaryotic cell biomimetics in structure and functions,but also provide promising carriers for further therapeutic protein,gene and drug delivery.