Disulfide-Crosslinked Polymeric Nanosystems For Efficient Tumor Targeted Protein Delivery

Protein biotherapeutics have been widely used for cancer treatment due to their high biological activity,superb specificity and low toxicity.It should be noted,however,many proteins bear intracellular targets and are challenged with poor in vivo stability,fast degradation and inefficient cellular transportation,which largely hinder their clinical translation.Therefore,it is of critical importance to develop efficient intracellular protein delivery systems.In the past few years,various polymeric nanosystems have been engineered to improve the circulation stability,maintain the biological activity and realize effective intracellular delivery of protein drugs,thus achieving targeted tumor therapy.In Chapter 1,the progress and challenges of polymer based protein nanotherapeutics are introduced,accordingly different types of smart polymeric nanocarriers aiming at overcoming the challenges are summarized.To achieve targeted and high-efficiency intracellular protein delivery,herein,disulfide-crosslinked polymeric nanosystems integrating multi-functions including efficient protein loading,high stability,active cell targeting,endosomal escape and stimuli-sensitivity are developed from dithiolane containing polymers.In Chapter 2,smart polymersomes dually functionalized with cRGD and fusogenic GALA peptides(cRGD/GALA-Ps)were constructed to realize ?v?3-specific and high-efficiency cytosolic delivery of cytochrome C(CC)to A549 human lung cancer cells.cRGD/GALA-Ps was prepared with 20 mol%cRGD and varying GALA contents from 2,4 to 6 mol%via co-assembly of PEG-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-spermine(PEG-b-P(TMC-co-DTC)-spermine),cRGD-PEG-b-P(TMC-co-DTC)and maleimide-PEG-b-P(TMC-co-DTC)and post-michael addition with GALA-SH.cRGD/GALA-Ps loaded with?13 wt.%CC displayed a small size of ca.65 nm and fast glutathione-triggered protein release.Interestingly,cRGD/GALA-Ps maintained a similar targeting ability to cRGD-Ps in ?v?3-positive A549 lung cancer cells,while markedly enhanced cytosolic release of FITC-labelled CC,as revealed by confocal microscopy.MTT assays exhibited that CC-loaded cRGD/GALA-Ps was significantly more potent than CC-loaded cRGD-Ps,in which cell viabilities of 76.2,51.0,29.6 and 35.5%were discerned for cRGD/GALA-Ps with 0,2,4,and 6 mol%GALA,respectively,at 15.4 ?M CC.Apoptosis assays corroborated that cRGD/GALA-Ps-CC with 4 mol%GALA induced better apoptosis of A549 cells than cRGD-Ps-CC(cell apoptosis:36.4 versus 14.4%).In Chapter 3,disulfide-crosslinked hyaluronic acid nanoparticles(HA-NPs)based on natural components were prepared for CD44-targeted delivery of protein drugs(CC and saporin,SAP).Phenylalanine tetrapeptide-lipoic acid conjugates were coupled to the hydroxyl groups of HA via esterification,yielding HA-g-(Phe4-LA),which formed HA-NPs upon self-assembly and crosslinking.HA-NPs displayed a small size of ca.74 nm,a narrow size distribution(PDI=0.11)and high loading capability for CC and SAP with DLE of?100%at a theoretical protein loading content of 2.0 wt.%.In vitro cell experiments indicated that HA-NPs could actively target to CD44-positive A549 cells and were internalized via receptor-mediated endocytosis,for which the cellular uptake amount was about 3-fold higher than that in CD44-negative L929 cells and Anti-CD44 antibody pretreated A549 cells.In accordance,SAP-loaded HA-NPs induced 3-fold stronger cytotoxicity against A549 cells(IC50 of 1.6 ?g/mL)than L929 cells.