| Cancers are serious threats to human's health.Surgical resection,radiotherapy and chemotherapy are the main regimens for clinical cancer treatment.Clinically,chemotherapy has direct and potent anticancer efficacy,whereas suffers from several drawbacks,including poor tumor selectivity,low drug utilization,and severe toxicity to normal tissues and organs at the same time of killing tumor cells.Anticancer proteins such as granzyme B(Gr B),cytochrome C(CC),and saporin(SAP))that act intracellularly have attracted wide attention due to their high efficiency,high specificity,and low toxicity.However,the clinical translation of anticancer proteins is greatly limited owing to their poor plasma stability and low cell selectivity and uptake,as well as inefficient intracellular trafficking.To resolve this crucial issue,a variety of nanocarriers such as liposomes,polymersomes,and nanogels have been extensively developed for the targeted protein treatment of cancers.In chapter 1,we presented a brief introduction on the current situation of nanocarrier systems for protein loading and controlled release with particular emphasis on the application of coiled coil motif in nanosystems.In chapter 2,based on the targeting of hyaluronic acid(HA)to CD44 as well as the overexpression of CD44 on the surface of MCF-7 breast cancer cells,we designed and constructed p H-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels(HAc NGs)for encapsulation of saporin(a ribosome inactivating protein toxin)(SAP loaded HAc NGs was denoted as HA-c NGs-SAP)and its targeted intracellular protein delivery to MCF-7 cells.HA was conjugated with E3(Sequence: GY(EIAALEK)3GC)and K3(GY-(KIAALKE)3GC),respectively,to give conjugates of HA-E3 and HA-K3.By mixing equivalent of HA-E3 and HA-K3,HA-c NGs nanogels were prepared via nanoprecipitation in acetone with a mean size of 176 nm.The formation of coiled-coil structures between E3 and K3 peptides at p H 7.4 while fast uncoiling at p H 5.0 was proved by circular dichroism(CD).The model protein cytochrome C(CC)was then efficiently loaded into HA-c NGs(HA-c NGs-CC)and released in a p H-dependent profile.The cumulative release of CC at p H 7.4,6.0,and 5.0 in 24 h was 18.4%,76.8% and 91.4%,respectively.Both confocal microscopy and flow cytometry indicated efficient internalization of HA-c NGs-CC by MCF-7 cancer cells.Furthermore,the coiled coil structure of HA-c NGs collapsed rapidly in acidic endosomes,and the uncoiled E3 and K3 further fused with endosomal membrane,which assists the endosomal escape of CC.Subsequently,SAP was loaded into HA-c NGs for targeted delivery to MCF-7 cells,demonstrating potent in vitro anticancer efficacy with an IC50 of 12.2 n M(in terms of SAP).The in vitro antitumor activity of HA-c NGs-SAP against MCF-7 cells was further confirmed by Annexin V-FITC/PI staining assays.In addition,the preliminary animal study showed the targeted accumulation of HA-c NGs-CC in subcutaneous MCF-7 tumor,displaying promising results.Based on the results of Chapter 3,we further investigated the in vitro anticancer activity of HA-c NGs-SAP against CD44-overexpressing triple-negative MDA-MB-231 breast cancer cells in Chapter 3.Cell study indicated that HA-c NGs-SAP had potent antiproliferation efficacy against MDA-MB-231 cells with an IC50 of 14.3 n M,comparable to that for MCF-7 cells.In addition,scratch assay showed the migration inhibition of HAc NGs-SAP for MDA-MB-231 cells.Preliminary animal study also indicated the targeted accumulation of HA-c NGs-CC in subcutaneous MDA-MB-231 breast cancer.Chapter 4 is a summary of the whole study,followed by a brief perspective of this thesis. |
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