Reversibly Crosslinked Temperature-responsive Nano-sized Polymersomes For Efficient Protein Delivery

Polymersomes have attracted significant attentions for biomedical applications and drug delivery systems. In this thesis, we have synthesized a series of well defined block copolymer of PEG-PAA-PNIPAM, studied their thermal sensitivity during the formation of the polymersomes and the reversible crosslinking, and explored the encapsulation of the proteins into the polymersomes and the intracellular release of the proteins.(1) Water-soluble temperature responsive triblock copolymers, poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-PAA-PNIPAM), were prepared in one pot by sequential reversible addition–fragmentation chain-transfer (RAFT) polymerization using a PEO-trithiocarbonate (PEO-S-1-dodecyl-S-(R,R- dimethyl-R-aceticacid) trithiocarbonate) as a macro chain transfer agent. The block copolymers have Mn PEO of 5 kDa, Mn PAA of 0.35-1.45 kDa, and Mn PNIPAM varying from 11-39 kDa. They were freely soluble in water as unimers at room temperature, but quickly self-assembled into nano-sized vesicles (about 220 nm) when raising the solution temperature to 37 oC. The vesicular structure was confirmed by confocal scanning laser microscope (CSLM) and static light scattering (SLS) measurements. The size and size distribution of the polymersomes depended on solution concentration, molecular weight of PNIPAM, the equilibrium time and shaking. Interestingly, thus formed vesicles could be readily cross-linked at the interface using cysteamine via the carbodiimide chemistry. The crosslinked polymersomes, while showed remarkable stability against dilution, organic solvent, high salt conditions and change of temperature in water, were otherwise rapidly dissociated under reductive conditions mimicking intracellular environment. Notably, FITC-dextran were shown to be encapsulated into the polymersomes with an unprecedently high loading efficiency. The release studies showed that most FITC-dextran was retained within the crosslinked polymersomes after lowering the temperature to 20°C. However, in the presence of 10 mM dithiothreitol (DTT), fast release of FITC-dextran was achieved. These reversibly crosslinked temperature responsive nano-sized polymersomes are highly promising as smart carriers for triggered intracellular delivery of biopharmaceutics such as pDNA, siRNA, pharmaceutical proteins and peptides.(2) The LCST of the above mentioned triblock copolymer PEO-PAA-PNIPAM can be adjusted from 28 oC to 50 oC by changing the block ratio, salt concentration, and pH. We designed copolymers with suitable proportion, in order to have their LCST in between 38 oC and 43 oC, so that after de-crosslinking the copolymer can dissolve in PBS molecularly. Notably, FITC-BSA was efficiently encapsulated into the polymersomes. For other proteins studied here, they all were encapsulated into the polymersomes with loading efficiency of 35-50%, and loading content of 4-50 wt.%. In vitro release studies showed that (37 o C in PB), in the presence of 10 mM dithiothreitol (DTT), fast release of FITC-BSA (80 % released within 7-8 hr) was achieved. However, without 10 mM DTT, most of the protein (80-85%) remains within the polymersomes after 8 hr. Cell experiments demonstrated that the proteins can effectively be transported into the cells. Thus these reversible cross-linked temperature-sensitive polymersomes can overcome the shortcomings of existing technologies, which can enhance loading efficiency of small molecule drugs, macromolecular drugs and probe molecule, enhance stability of polymersomes in the blood circulation, and enhance the endocytosis efficiency, so it can increase the bioavailability.