Peptide-modified Chimaeric Polymersomes For Glioblastoma-targeted Delivery Of Protein Drugs

Glioblastoma(GBM)is the most aggressive malignancy in central nervous system.GBM patients have poor prognosis with a median survival time of 14 months.Current first-line treatment of GBM is maximal surgical resection combining with radiotherapy and/or temozolomide(TMZ)chemotherapy.However,high aggression makes complete GBM removal impossible by surgery and relapse is inevitable.radiotherapy is limited to be applied in the central nervous system diseases,considering the severe adverse effect.The majority of chemotherapeutics,even antibody-drug conjugates,fail to penetrate blood-brain barrier(BBB).With the development of nanotechnology,many chemotherapeutics-loaded nanoliposomes were applied in clinical trials of GBM patients,but the results were not satisfying.These results can be attributed to two main reasons that non-targeted nanoliposomes possess poor stability in blood circulation and low drug accumulation through slight EPR effect of cerebral capillary in GBM zone.To improve the chemotherapy effect,various drug delivery systems with BBB crossing and GBM cells targeting ability were designed.Drug delivery systems improve the accumulation of chemotherapeutics and reduce the side effect,but a large number of nanoparticles that accumulate in liver,kidney and spleen after continuous injection release chemotherapeutics gradually after degradation,which brings about severe toxicity toward normal organs.In order to overcome these hinderances,we try to fabricate polymersomes with several advantages:(1)high serum stability and tumor micro-environment responsibility;(2)improved BBB penetration,tumor tissue penetration and cellular endocytosis;(3)enhanced antitumor efficiency and low adverse effect.In the first chapter,we analyzed the biological characterization of GBM,current first line treatment and challenges in clinic,strategies and challenges for delivery of nanomedicine across BBB and carriers for protein delivery.The design of protein drug chaperoned by reduction-sensitive and reversibly crosslinked polymersomes for GBM therapy was proposed.In the second chapter.both BBB and human glioblastoma cells(U-87 MG)dual targeting Angiopep-2(ANG)was modified on saporin(SAP),antitumor protein therapeutics,loaded reduction-sensitive and reversibly crosslinked polymersomes(ANG-CP-SAP).ANG-CP has a high SAP loading of 8.8 wt%and a small size of 76 nm.ANG-CP-SAP was robust in circulation,but de-crosslinked fast and released SAP in cytoplasm to induce apoptosis(half maximal inhibitory concentration,IC50=30.2 nM).ANG-CP-SAP can effectively penetrate BBB,improve drug accumulation in GBM and prolong survival time of mice bearing GBM(43 d)without side effect.In the third chapter,in order to further improve the anti-GBM efficacy,we utilize multi-receptors targeting strategy for drug delivery.ApoE peptide,which targets to low density lipoprotein receptor family(LDLRs,including LDLR,LRP1 and LRP2),was modified on SAP loaded crosslinked polymersomes for effective GBM therapy.ApoE-CP present higher BBB permeability(2.2-fold)and GBM targeting capability(4.26%ID/g vs.1.71%ID/g)than ANG-CP.ApoE-CP-SAP strongly inhibited the proliferation of GBM cells and prolonged the survival time of GBM bearing mice(52 d).In the fourth chapter,to overcome poor tumor tissue penetration of nanomedicine in GBM therapy,iNGR which possess selective penetrating ability based on "C end rule",was introduced to fabricate dual-functionalized ApoE/iNGR-CP-SAP.ApoE/iNGR-CP was biocompatible and simple to prepare than non-specific cell and tissue penetrating peptide directed nanomedicine which often bring about adverse effect.Results showed that ApoE/iNGR-CP-SAP caused enhanced BBB penetration,deep tumor tissue penetration and high endocytosis.The BBB model transport ratio of ApoE/iNGR-CP is significantly higher than ApoE-CP(1.9 fold)and iNGR-CP(3.8 fold).ApoE/iNGR-CP-SAP induce more tumor cell apoptosis in deep tumor parenchyma and prolong the median survival time of GBM bearing mice to 63 d.In the fifth chapter,to explore the possibility of clinical application of GBM targeted protein-loaded polymersomes,spermine(SP),which is human endogenous,was introduced to polymersomes for humanized recombinant granzyme B(GrB)loading to construct a more biocompatible protein nanomedicine(ApoE-BCP-GrB).There was no significant difference between the blood of mice intravenously injected with ApoE-BCP-GrB and the control in routine blood test and blood biochemical analysis of BALB/c mice.The protein loading efficiency of ApoE-BCP was up to 77.3%;ApoE-BCP-GrB presents more toxic to U-87 MG(IC50=4.5 nM);Survival time of GBM bearing mice treated with ApoE-BCP-GrB was up to 54 d which was longer than that of mice administrated with TMZ that is the first-line agent of GBM patients.In the sixth chapter,ApoE-BCP-SAP was applied to the treatment of BM-TNBC bearing mice to improve BBB penetration and MDA-MB-231 endocytosis by LRP1 and LDLR mediating.Higher uptake of ApoE-BCP-SAP by MDA-MB-231 was observed in flow cytometry assay,which led to low IC50 of 26 nM.In anti-BM-TNBC experiment,ApoE-BCP-SAP caused obvious tumor growth inhibition and long survival time of BM-TNBC bearing mice(44 d).In the last chapter,we made a summary of the whole study and give a future perspective of the research.