Reversibly Crosslinked Chimaeric Polymersomes For Targeted Therapy Of Non-Small Cell Lung Cancer In Mice

Lung cancer with the highest morbidity and mortality among all the cancers has urged researchers to devote to efficient therapy.Many anticancer nanomedicines have been developed for lung cancer therapy.However,their clinical efficacy is not satisfactory.The insufficient in vivo instability,short circulation time,low tumor tissue accumulation and tumor cell uptake as well as sluggish intracellular drug release all lead to low intracellular drug concentration thus less antitumor efficacy.Moreover,for highly efficient and low toxicity hydrophilic drugs including small molecular anticancer drugs?e.g.methotrexate sodium?MTX?and pemetrexed disodium?PEM??and biomacromolecules?e.g.proteins and peptides?,no suitable nanocarriers have been developed.To deal with these problems,in this thesis we have designed a series of reversibly crosslinked chimaeric polymersomes which can realize highly efficient loading of these hydrophilic drugs and targeted therapy of non-small cell lung cancer?NSCLC?in mice.The thesis mainly consists of two parts:biocompatible polymersome nanomedicines based on poly?ethylene glycol?-b-poly?N-2-hydroxypropyl methacrylamide-g-lipoic acid??PEG-P?HPMA-LA??and biodegradable polymersome nanomedicines based on copolymers polyethylene glycol-b-poly?trimethylene carbonate-co-dithiolane trimethylene carbonate?-b-polyethyleneimine?PEG-P?TMC-DTC?-PEI?.In chapter 2,we designed and synthesized poly?ethylene glycol?-b-poly?N-2-hydroxypropyl methacrylamide-g-lipoic acid?-b-poly?2-?dimethylamino?ethyl methacrylate??PEG-P?HPMA-LA?-PDMA?and anisamide coupled polymer Anis-PEG-P?HPMA-LA?-PDMA.The co-selfassembly of the two copolymers in the presence of MTX yielded MTX loaded disulfide-crosslinked polymersomes?MTX-Anis-RCCPs?,which showed excellent efficacy in treating NSCLC.In this design,Anis groups on the PEG outer shell can specifically bind to sigma receptors on H460 cells improving cellular uptake.PHPMA is FDA approved biocompatible water soluble polymer.LA is a natural antioxidant and its grafting onto HPMA renders hydrophobicity of P?HPMA-LA?.The side dithiolane rings of P?HPMA-LA?facilitate disulfide crosslinking in the polymersome membrane supplying the in vivo stability and reduction sensitive decrosslinking.PDMA in the polymersome inner shell becomes positively charged and can efficiently encapsulate negatively charged drugs via electrostatic interaction.MTX-Anis-RCCPs?160 nm?with MTX loading content of 22.0 wt%have been obtained,which show reduction sensitivity.Remarkably,MTX-Anis-RCCPs have long elimination half-life of 5.24 h?18 times higher than free MTX?and accumulate in H460 tumors by 5.3%ID/g,4.5 or 9 fold of nontargeting counterparts?MTX-RCCPs?and commercial MTX injection Trexall.Moreover,they can significantly inhibit tumor growth in H460 bearing mice,yet without causing side effects thus greatly prolong the survival rates compared with MTX-RCCPs and Trexall.The present MTX-Anis-RCCPs nanomedicines extend the applications of MTX in cancer therapy.In chapter 3,PDMA has been replaced with polyacrylic acid?PAA?in the triblock copolymers to finally afford negatively charged polymersome inner shell for loading positively charged granzyme B?GrB??GrB-Anis-BCPs?for targeted treatment of H460 bearing mice.Nearly quantitative protein loading has been obtained for GrB-Anis-BCPs and loading contents can reach 36.5 wt.%,and the IC₅₀ was as low as 7.8nM.Polymersomes can penetrate deep into the tumor tissue and delivery GrB into H460cells,inducing cell apoptosis,tumor shrinkage and prolonged survival rates while without systemic toxicity.GrB-Anis-BCPs nanomedicines present as a new strategy for protein delivery in cancer therapy.Biodegradability is one of key factors for the clinical applications of nanomedicines to prevent the cumulative toxicity.Then we design reversibly crosslinked chimaeric polymersomes based on PEG-P?TMC-DTC?-PEI as versatile nanoplatform for loading hydrophilic PEM,MTX or GrB for NSCLC targeted therapy.Here PTMC is an FDA approved biodegradable polymer,and DTC is our proprietary monomer.The dithiolane rings of P?TMC-DTC?in side chains form disulfide crosslinking of polymersomes.Positively charged PEI as polymersome inner shell can encapsulate drugs and help escape from endosomes.In chapter 4,cyclic peptide CC₉?CSNIDARAC?coupled,PEM loaded PEG-P?TMC-DTC?-PEI polymersomes?PEM-CC₉-BCCPs?have been prepared.They are ca.60 nm with high PEM loading content of 14.2 wt%.Notably,PEM-CC₉-BCCPs have long elimination half-life of 5.21 h?vs.0.24 h of free PEM?,enhanced tumor inhibition and prolonged survival rates?58 d vs.36 d or 31 d of nontargeting or free PEM?.Selective cell penetrating peptide?SCPP?can significantly increase cellular uptake,without nonspecific selectivity of normal CPPs.In chapter 5,SCPP?RLWMRWYSPRTRAYGC?to lung tumor is applied to construct MTX loaded PEG-P?TMC-DTC?-PEI polymersomes?MTX-SCPP-PS?for treatment of A549 bearing mice.From both in vitro cell experiments and in vivo animal study,MTX-SCPP-PS demonstrates excellent penetration,cytotoxicity,tumor growth inhibition without toxicity to normal tissues.Chapter 6 further reports that SCPP-PS based on PEG-P?TMC-DTC?-PEI delivers GrB?GrB-SCPP-PS?into orthotopic A549 tumor mice.Polymersomes with high protein loading content of 17.2 wt%are obtained.In A549-Luc bearing mice,GrB-SCPP-PS significantly improves the stability,reduces side-effects,enhances antitumor efficacy and suppresses the tumor metastasis.These results point to that lung tumor specific SCPP is highly attractive in building tumor targeting therapy.The two sets of disulfide crosslinked chimaeric polymersome nanomedicine platform technology in this thesis are robust,low toxicity and versatile in targeting moieties and drugs,demonstrating potential in clinical trails.Chapter 7 summarizes the whole thesis and gives future perspectives.