| Nanomedicine is an important strategy for malignant tumor treatment.Recently,clinical demands for anticancer nanomedicines have emerged widely and rapidly.However,nanomedicines are facing the problem of low efficacy in clinical research.The major reason is that the current nanomedicines are mainly designed based on the EPR effect,which is only suitable for animal models.Tumors in patients lack the EPR effect and are highly heterogeneous,thus it is hard for nanomedicines to extravasate from blood vessels and penetrate into tumors,leading to poor anticancer efficacy and failures in clinical trials.Therefore,it is essential to develop an EPR-independent strategy to enhance the vascular extravasation and tumor penetration.In previous work,a charge-reversal polymer catalyzed by tumor-overexpressingγ-glutamyl transpeptidase(GGT)has achieved active vascular extravasation and tumor penetration,which are the major bottlenecks for nanomedicines.However,this single enzyme-responsive system cannot be applied to all kinds of tumors.The correlations of molecular structure with enzyme-responsive hydrolysis property and in vivo drug delivery efficiency are unknown,and the study has not achieved excellent therapeutic effect on matrix-rich orthotopic pancreatic tumors.This thesis has developed two novel enzyme-responsive charge-reversal drug delivery systems to guide the structural optimization of active tumor penetrating nanomedicines and solve the dilemma in matrix-rich tumor penetration,which includes the following two studies:The first study(Chapter 2&3)developed a novel enzyme-responsive chargereversal system targeting aminopeptidase N(APN),in order to solve the problem of limited applicability of the GGT-responsive charge reversal namomedicine due to the tumor heterogeneity.The correlations between charge-reversal polymer structure with enzymatic responsiveness and biological properties were studied to expand the application scope of enzyme-responsive charge-reversal nanomedicines.The main findings and contributions include:(1)The fabrication of various APN-responsive charge reversal polymers and polymer-drug conjugates with different monomeric unit structure and chain structure and the investigation of the relationship between molecular structure and APNresponsive hydrolysis properties.APN-responsive monomers with different αsubstituent(methyl,ethyl,iso-butyl and benzyl)were synthesized and copolymerized with 7-ethyl-10-hydroxycamptothecin(SN38)monomers to construct homopolymers HP-X,random copolymers RP-X and block copolymers BP-X with different chain structure.The APN-responsive charge reversal rates were found to be tuned by monomeric unit structure(α-substituent of the amide)and polymer chain structure.(2)The elucidation of the correlations of charge reversal property with endocytosis,transcytosis and the overall drug delivery efficiency of RP-X and BP-X.The endocytosis rate,cytotoxicity,transcytosis efficiency and tumor spheroid penetration of RP-X and BP-X were positively correlated with the corresponding charge reversal property.However,the charge reversal rates were not positively correlated with the blood circulation time,tumor accumulation and antitumor efficacy in vivo.The highly APN-responsive conjugate BP-Bn exhibited short blood circulation time and poor tumor accumulation due to its strong hydrophobicity,thus resulting in unsatisfactory antitumor efficacy.On the other hand,the conjugate BP-iBu with a moderate charge reversal rate had prolonged circulation time,high drug accumulation and deep tumor penetration,and achieved an ideal tumor inhibition rate of 97.3%.The second study(Chapter 4&5)constructed a dual enzyme-responsive chargereversal drug delivery system for the matrix-rich microenvironment in pancreatic cancer and achieved efficiently active transcytosis among different types of cells,deep tumor penetration and potent anti pancreatic tumor efficacy.The main findings and contributions include:(1)The design of cell-specific dual enzyme-responsive charge reversal polymers and polymer-drug conjugates for the special microenvironment in pancreatic tumor and the investigation of the effect of hydrophobicity on enzymatic responsiveness.Due to the characteristics of pancreatic tumors,with a small portion of GGT-overexpressing tumor cells scattering in a large amount of fibroblast activation protein(FAP)overexpressing fibroblasts,FAP&GGT dually responsive monomers MFG were synthesized and copolymerized with SN38 monomers to construct homopolymers PFG and polymer-drug conjugates PFG-SN38 with different drug loading content(DLC).As the hydrophobicity increased,FAP&GGT hydrolysis rate of PFG and PFG-SN38 became faster.Only PFG-SN38 with higher DLC had GGT-catalyzed charge reversal property,while FAP catalyzed all the polymers and conjugates for charge reversal.(2)The preliminary revelation of the mechanism of active tumor peneration achieved by PFG-SN38 and the evaluation of anti pancreatic tumor efficacy.The endocytosis rates of the polymers and conjugates became faster as the increase of hydrophobicity,while only PFG-SN38 with higher DLC showed effective endocytosis in both fibroblasts and tumor cells.PFG-SN38 with higher DLC distributed more in Golgi apparatus,and induced efficient exocytosis in the form of extracellular vesicles,thus resulting in mutual transcytosis between fibroblasts and tumor cells,deep penetration in pancreatic tumors,long blood circulation time,and high tumor accumulation.PFG-SN3 8 with higher DLC achieved tumor eradication and inhibited recurrence of cell-establishing subcutaneous pancreatic tumors,patient-derived xenograft(PDX)subcutaneous pancreatic tumors,and orthotopic pancreatic tumors with prolonged survival and excellent biocompatibility.This thesis filled in the blank of the unknown structure-activity relationship of enzyme-responsive charge-reversal nanomedicines,and provided a new strategy for enhancing tumor infiltration and antitumor efficacy in matrix-rich tumors and a new direction for the design of active tumor penetrating nanomedicines. |
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