Design,Fabrication And In Vitro Anticancer Properties Of HCPT-based Tumor Microenvironment-sensitive Polyprodrugs

Millions of people in the world are currently battling cancer.Cytotoxic chemotherapy compared to the other treatments?e.g.surgery,radiation therapy?remains the preferred front line strategy used against most types of cancer.Hydroxycamptothecin?HCPT?is a pentacyclic alkaloid originally isolated from Camptothecinacuminate Decene,a deciduous tree native to South China that has gained great attention for its significant antitumor activities in the 1960s and 1970s.HCPT has significant antitumor activitiesover 30-fold greater than camptothecin,no cross-resistance with other commonly used anticancer drugs,a broad anti-tumor spectrum and less toxic side effects.Unfortunately,conventional chemotherapeutic agents always exhibit inherent limitations such as nonspecific distribution,rapid blood clearance and poor solubility in physiological environments and the treatment-related side effects resulting from exposure of normal tissues to the drug.Besides,the lactone ring structure is sensitive to light and acid,and the intact hydroxyl-lactone must be present for the expression of the in vivo antitumor activity.Approaches for addressing these issues are often related to the modulation of polymeric prodrug-based self-assemblies for drug delivery,which protect the conjugated drugs from rapid blood/renal clearance and achieve the preferential accumulation of drugs within solid tumors via the so-called enhanced permeability and retention?EPR?effect.For this purpose,the active free drugs are generally conjugatedtothepolymerchainviavarioustypesoftumor microenvironme nt-cleavable linkers.Therefore the for mulatio n of polymeric prodrugs provides a powerful tool to improve the pharmaceutical properties of the active pharmacologic agent via a proper chemical modification.This strategy could maintain the advantages and overcome the disadvantages of physical encapsulation approach,including the improvement of water solubility of hydrophobic drugs,great enhancement of the stability of drug molecules in the blood circulation,significant reduction of premature drug release and non-specific release behavior toward minimized side effects,rapid degradation of polymeric prodrugs for enhanced therapeutic efficiency.In other words,polymeric prodrugs not only reduce the toxic side effects of free drugs on normal tissues,but also achieve efficient accumulation and enhanced therapeutic effects of drug molecules in the tumor tissues.However,a survey of literature indicated that polymer prodrugs developed so far generally suffered from complicated and laborious preparation and purification steps,low drug conjugation efficiency with unsatisfactorily therapeutic efficencey,and insufficient stability for drug leakage.To address these issues,we developed several strategies to fabricate HCPT-based polymeric prodrugs in this thesis.A series of tumor microenvironment-sensitive polymeric prodrugs were designed and evaluated.Their self-assembly behaviors and in vitro antitumor properties were investigated in detail.The details are listed as the following five parts,1.Polymeric prodrugs with precisely controlled drug loading content and rapid intracellular destabilization generally require complicated chemistry that hinder large scale manufacture.In Chapter 1,we reported a facile construction of reduction-sensitive amphiphilic polyprodrugs with an anti-cancer drug,10-hydroxycamptothecin?HCPT?and a hydrophilic poly?ethylene oxide??PEG?moiety as the alternating building blocks of the multi-block copolymer using Cu?I?-catalyzed azide-alkyne cycloaddition?CuAAc?click coupling between azide-SS-HCPT-SS-azide and alkyne-PEG-alkyne.Adoption of PEGs with two different molecular weights?MWs?of 400 and 1450 Da?PEG400 and PEG1450?afforded two polyprodrugs with different DLCs.Both formulations can self-assemble into spherical micelles with hydrodynamic diameter smaller than 200 nm,and exhibit glutathione?GSH?-triggered degradation for promoted drug release.A further comparison study revealed that the PEG1450-based polyprodrug is a better formulation than the analogue constructed from PEG400 in terms of micelle stability,DLC,in vitro drug release behaviors,cellular uptake,and cytotoxicity.This work thus provided a facile yet efficient strategy toward polymeric prodrugs with precisely controlled DLC and reduction-triggered degradation for enhanced anticancer drug delivery.2.In Chapter 2,we adopted a simpler and more efficient strategy,compared to the click polymerization used in the first charpter,to synthesize a polymeric prodrug capable of responding to the acidic pH of the tumor microenvironment.The amphiphilic alternating polymeric prodrug was prepared by one-step acetal reaction of the HCPT prodrug and PEG,where the hydroxyl groups of PEG were polymerized with the oxyethylene-modified HCPT prodrug by condensation polymerization.Since the acetal bonds can be hydrolyzed under the mildly acidic conditions,the destabilization of resulting polymeric prodrugs could be realized at the localized tumortissues.PolymericprodrugsP?HCPT/Acetal/PEG400?and P?HCPT/Acetal/PEG 1450?were synthesized using PEG?PEG400 and PEG1450?with molecular weights of 400 and 1450.P?HCPT/Acetal/PEG 1450?with a drug loading content of 4.5%,can self-assemble into spherical micelles with a diameter of approximately 208 nm in water.The in vitro drug release profiles showed that P?HCPT/Acetal/PEG 1450?micelles had a relatively faster release behavior under acidic conditions?pH 5.0?.The in vitro cytotoxicity study revealed a low IC₅₀ value for P?HCPT/Acetal/PEG 1450?formulation.The efficient cellular uptake of this micelle construct was confirmed by fluorescence imaging and flow cytometry analysis.Taken together,this study provided a simple and powerful means toward pH-responsive polymeric prodrugs.3.Synergistic treatments can overcome the limitations of single drug therapy,such as low effective drug concentration,severe side effects,and insufficient therapeutic effects.In other words,synergistic treatments can effectively improve the cytotoxicity of a single drug in vivo and in vitro.For this purpose,we designed in Chapter 3 a polymeric prodrug formulation based on a hydrophilic drug molecule FUDR and a hydrophobic drug molecule HCPT.Firstly,we synthesized a double-head RAFT chain transfer reagent?CTA?with alkynyl termini.Next,the alkynyl-terminated RAFT CTA was converted to a macroRAFT CTA by click polymerization with the azide-modified HCPT prodrug.Finally,the resulting macroCTA was used to mediate the RAFT polymerization of FUDR monomer to produce the target polymeric prodrug P?HCPT/PFUDRM?.Under the reducing environments in cancer cells,both HCPT and 5-FU free drugs were cleaved from the the parent polymeric prodrug formulation,thereby improving the anti-tumor effect.The DLCs of P?HCPT/PFUDRM?polymeric prodrug was 20%?HCPT?and 27.5%?FUDR?for each drug.P?HCPT/PFUDRM?polymeric prodrug can self-assemble into spherical micelles with a size of 164 nm in water and showed proper anti-tumor effects in vitro.Fluorescence imaging and flow cytometry results indicated that the polymeric prodrug micelles could be endocytosed by HeLa cells.In summary,this chapter developed a new method for the construction of a dual-drug polyprodrug self-delivery system with high DLC by RAFT polymerization.4.The preparation of polymeric prodrug generally requires tedious post-polymerization modifications,leading to batch-to-batch variations and scale-up complexity.To address the relatively low and uncontrolled post-polymerization conjugation efficiency of drug molecules,we designed and synthesized in Chapter 4an amphiphilic prodrug monomer and fabricated polyprodrug by a direct polymerization of this monomer.We furtherperformed a detailed comparison study regarding the self-assembly and anticancer properties of the amphiphilic prodrug monomer and the brush-like polyprodrugs.Amphiphilic prodrug monomers with different DLCs,HCPT-PEG 350 and HCPT-PEG 750,were synthesized using PEG?PEG350 and PEG750?with molecular weights of 350 and 750.The ratio of the hydrophilic and hydrophobic segments of HCPT-PEG 350 and HCPT-PEG 750 were set at 1:1 and 1:2,respectively.The effect of this ratio on the structure and properties of polymeric prodrugs was further investigated.The brush-like polymeric prodrugs PHCPT-PEG 750 were subsequently prepared by RAFT polymerization of HCPT-PEG 750 prodrug monomer.The prodrug monomer and resulting polyprodrug shared an identical DLC of 15.7%.PHCPT-PEG 750 and HCPT-PEG 750self-assembled into spherical micelles with a diameter of 341 nm and 220 nm in water,respectively.The in vitro drug release study showed that HCPT-PEG 750 micelles had a faster release behavior relative to PHCPT-PEG 750 micelles,and in vitro cytotoxicity study indicated a lower IC₅₀ value for the HCPT-PEG 750 micelles.Fluorescence imaging results indicated that both polymeric prodrug micelles can be endocytosed by HeLa cells.By flow cytometric quantitative analysis,the HCPT-PEG750 micelles showed higher cellular uptake efficiency.In summary,this chapter confirmed that the micelles self-assembled by HCPT-PEG 750 prodrug monomer had smaller hydrodynamic size,faster in vitro release behavior and greater antitumor effects than the PHCPT-PEG 750 formulations.5.Incorporation of various dynamic stimuli-responsive bonds to the nanocarriers has been repeatedly highlighted to provide an elegant solution to the tradeoff between extracellular stability and intracellular high therapeutic efficiency,however,most of the developed systems still suffered from drug leakage-associated side effects due to insufficient stability and unsatisfactorily therape utic efficiency attributed to low drug loading capacity.To further address these critical issues,we reported in Chapter 5 a coordination-driven formation of biocleavable crosslinked polyprodrug vesicles?CPV?basedonthereversiblecoordinationinteractionsbetweenelectron acceptor-containing polyprodrug and electron donor-containing crosslinker,1,6-hexanediamine.The resulting CPV exhibited a high drug loading content of34.8%,and simultaneously enhanced extracellular micelle stability and promoted intracellular redox-triggered decrosslinking toward accelerated drug release.More importantly,a comparison study further revealed that the CPV outperformed the noncrosslinked analogues in terms of greater stability,faster redox-triggered decrosslinking and drug release,amore compact structure with a smaller size toward higher cellular uptake,and greater in vitro cytotoxicity.This work thus developed a robust reversible crosslinking strategy to address high stability vs.sufficient therapeutic efficency dilemma of polyprodrug-based nanocarriers.