Construction Of Small And Smart Nanogels By Combining Microfluidics And Photo-Click Chemistry For CD44-Targeted Cytoplasmic Delivery Of Therapeutic Proteins And SiRNA

Malignant tumors continue to pose a serious threat to human health.Protein and nucleic acid drugs possessing a high specificity,strong potency,and low toxicity to healthy tissues,have exhibited great potential on cancer treatment.However,their practical application generally suffers from several disadvantages including easy degradation by enzymes,short circulation time,and poor cell uptake.Thus,a variety of nanocarriers including liposomes,polymeric nanoparticles,polymersomes,and nanogels have been developed to achieve efficient delivery of protein and nucleic acid drugs.In chapter 1,we have introduced the current status of protein and nucleic acid drugs on cancer treatment,summaried various strategies on nanogel construction,described the applications of smart nanogels on protein delivery,and reviewed different nanoplatforms used for siRNA delivery.In Chapter 2,small,traceable,endosome-disrupting and bioresponsive nanogels(NG)were developed for CD44-targeted cytoplasmic delivery of therapeutic proteins.NG was formed from hyaluronic acid derivatives,i.e.,HA-lysine-tetrazole(HA-Lys-Tet)and HA-cystamine-methacrylate(HA-Cys-MA),by combining microfluidics and catalyst-free photoclick reaction.By adjusting the microfluidics flow rates,polymer concentration and chain length of HA,we can readily obtain NG with controllable size and uniform structure.NG exhibits strong green fluorescence and efficient loading of various proteins including saporin(Sap),cytochrome C,Herceptin,immunoglobulin G(IgG),and bovine serum albumin(BSA).Interestingly,80 nm-sized NG revealed clearly better cellular uptake than 150 nm counterparts in both CD44 negative U87 cancer cells,and CD44 positive 4T1 and MDA-MB-231 cells.Moreover,NG modified with GALA,a pH-sensitive fusogenic peptide,exhibited accelerated endosomal escape.Accordingly,Sap-loaded small and GALA-functionalized HA-NG showed the highest cytotoxicity in CD44 positive MDA-MB-231,4T1,A549 and SMMC-7721 cancer cells.In vivo experiments showed that the NG-150 group had longer circulation time,while NG-80 revealed a higher accumulation at the 4T1 tumor site,which may stem from the deeper tumor penetration of the small-sized NG.Therefore,small,uniform and bioresponsive NG with easy and controlled fabrication holds a great potential for targeted protein therapy.In Chapter 3,zinc-dipicolylamine(Zn-DPA)was introduced into the NG developed in chapter 2 for efficient entrapment,endosome escape,targeting delivery and superb gene silencing of small interfering RNA(siRNA).Zn-DPA functionalized nanogel(NG/Zn-DPA)was formed from hyaluronic acid derivatives,i.e.,HA-Cys-MA and HA-lysine-tetrazole/Zn-DPA(HA-Lys-Tet/Zn-DPA),by combining microfluidics and catalyst-free photoclick reaction.NG/Zn-DPA could efficiently encapsulate siRNA,in which the loading content was up to 8.9 wt.%when the theoretical loading content was 10 wt.%.The superior encapsulation efficiency of siRNA in NG/Zn-DPA mainly results from the strong coordination bond between Zn-DPA in NG/Zn-DPA and the phospholipid group of encapsulated siRNA.siRNA-loaded NG/Zn-DPA exhibits small size(125 nm),uniform structure,and high stability.Flow cytometry results manifested that the cellular uptake level of NG/Zn-DPA group in MDA-MB-231 breast cancer cells was 5.1-fold higher than that of NG group.Meanwhile,NG/DPA exhibited a more effective escape from endosome The significantly enhanced endocytosis and endosomal escape is mainly due to the interaction of Zn-DPA with the phospholipid groups on the cell membrane and endosomal membrane,thereby promoting the cytoplasmic delivery of siRNA.siGL3-encapsulated NG/Zn-DPA(NG/Zn-DPA-siGL3)caused 71%silencing of luciferase reporter gene in HeLa-luc cells within 48 hours at a concentration of 200 nM siRNA,and similarly NG/Zn-DPA-siPLK1 significantly down-regulated 73%PLK1 mRNA in MDA-MB-231 cell at the same concentration.Importantly,NG/Zn-DPA displayed good biocompatibility,in which MDA-MB-231 cells following the treatment with NG/Zn-DPA at at a concentration up to 600μg/mL for 48 hours revealed nealy 100%cell viability.Therefore,NG/Zn-DPA holds great promising to serve as an innovate nanoplatform for efficient targeted delivery of siRNA due to its high stability,high loading efficiency,low toxicity and high silencing efficiency.The fourth chapter summarizes the whole work and presents perspectives on our future research direction.