Active-regulated And Hierarchical Targeting Strategy For Polymeric Nanocarrier-directed In Vivo Drug Delivery

Polymeric nanocarrier for anticancer drug delivery to maximize therapeutic efficacy and minimize the adverse side effects can be realized through overcoming a series of physiological obstacles.Particularly,through systemic administration,the drug delivery systemsneed achieve prolonged blood circulation,high tumor accumulation,deep tissue penetration,cellular internalization,and drug release inside cells or organelles.At different stages,nanocarriers should possess special features to overcome the related barriers.Over the past few decades,polymeric nanocarriers have been developed from static and biologically inert entities to more dynamic and smarter systems,which further enhanced the final therapeutic efficacy.However,suboptimal and unpredictable clinical outcomes are still obtained.For overcoming biological barriers and complex tumor microenvironment heterogeneity,more efficient strategies need to be explored.Therefore,this dissertation is mainly focus on using active regulation and hierarchical targeting strategy to achieve polymeric nanocarrier-directed in vivo drug delivery.This dissertation can be further categorized into five main parts as follows:1.Multi-layer nonviral PEG-b-polycation-based gene carriers for efficient systemic delivery.The design of safe and efficient gene delivery vectors is a critical issue for successful gene therapy.Traditional nonviral gene carriers are unstable in the blood circulation because of the attack of DNase enzyme and other biomolecules,which leads to low transfection efficiency in systemic gene delivery.In this project,we continued utilizing hydrophobic effect to stabilize and protect polycation-based gene carriers.We constructed an efficient nonviral systemic gene vector by engineering the coverage of polyplex micelle through incorporation of a hydrophobic intermediate layer between PEG shells and complexed pDNA cores,via facile complexation between pDNA and mixed block copolymers of poly?ethylene glycol?-b-poly{N'-[N-?2-aminoethyl?-2-aminoethyl]aspartamide}?PEG-b-PAsp?DET??and poly?N-isopropylacrylamide?-b-poly{N'-[N-?2-aminoethyl?-2-aminoethyl]aspartamide}?PNIPAM-b-PAsp?DET??at room temperature,followed by heating to body temperature.This polyplex micelles with the temperature-responsive formation of intermediate barrier to complexed pDNA cores in addition to PEG shells showed more compact pDNA condensation,enhanced tolerability against nuclease attack and counter polyanion exchange,resulting in significantly prolonged blood circulation and tumor accumulation.Combined with high gene transfection efficacy in tumor tissue,polyplex micelles loading therapeutic genes achieved potent therapeutic efficacy to tumor.2.PEG-sheddable supramolecular 'Trojan Horse' as smart delivery system.The PEG surface layer of pegylated nanocarrier will hamper their association with cells reducing the efficiency of cellular uptake and endosomal escape,although PEGylated nanocarrier were recognized as promising delivery vectors to realize systemic delivery via prolonged circulation due to reduced interaction with proteins and blood components.To address the PEG dilemma of PEGylated nanocarrier,we developed PEG-sheddable nanocarriers poly?ethylene glycol?-GPLGVRG-b-poly{N'-[N-?2-aminoethyl?-2-aminoethyl]aspartamide}(PEG-GPLGVRG-PAsp?DET??and poly?ethylene glycol?-GPLGVRGDG-b-poly[??-benzyl-L-aspartate?-co-?aspartic acid??PEG-GPLGVRGDG-P?BLA-co-Asp??as smart gene/drug delivery vectors responsive to matrix metalloproteinases?MMPs?highly expressed in tumor tissues.Our results demonstrated that PEG-sheddable nanocarrier exhibits higher cellular uptake,improved endosomal escape,high-efficiency gene transfection or strong cytotoxicity after dePEGylation in the presence of MMP-2.We further constructed an MMP-responsive asymmetric polymersome self-assembled from triblock copolymer,poly?ethylene glycol?-GPLGVRG-b-poly??-carprolactone?-b-poly?3-guanidinopropyl methacrylamide??PEG-GPLGVRG-b-PCL-b-PGPMA?.PGPMA segment is a cell-penetrating peptide?CPP?-mimicking polymer.The asymmetric structure of the vesicle with the majority of PGPMA block distributed in the inner shells?approximately 90%?would be as a Trojan-horse to camouflage the desired functionality.In the presence of MMP-2,the stealth components?PEG?in the outer shell of the polymersomes were cleaved leading to structural rearrangement of the polymersomes and then flipping of the inner shell,thus enabling exposure of inner cell-penetrating components?PGPMA?.Consequently,the proposed system could be utilized as dynamic self-assemblies for drug delivery to promote the specific uptake of the encapsulated drugs by MMP-enriched tumors.Furthermore,we used this polymersome to simultaneously encapsulate hydrophilic MMP inhibitor and hydrophobic anticancer drug within the vesicular interior and hydrophobic membranes,respectively.The feedback-programmed release of MMP inhibitor and enhanced cellular internalization were demonstrated.Intracellular anticancer drug release was observed.The appropriate MMP inhibition and intracellular anticancer drug delivery in tumor tissue resulted in growth and metastasis inhibition of highly metastatic 4T1 breast cancer.3.Mulitistage delivery system for overcoming multiple physiological obstacles.Apart from blood circulation,tumor accumulation,and cellular internalization,polymeric nanocarriers also face another barrier in tumor tissue:tissue penetration,which makes a significant contribution to drug resistance at the tumor tissue level.To massively deliver antitumor drugs throughout the entire tumor tissue via deep tumor tissue penetration and high-efficiency cellular internalization,we prepared an endogenous stimuli-responsive multistage polyplex drug delivery system via electrostatic complexation between anionic block copolymers,poly?ethylene glycol?-b-poly[?N'-dimethylmaleoyl-2-aminoethyl?aspartamide]?PEG-b-PAsp?EDA-DM??,and platinum?IV?-conjugated cationic poly?amidoamine??PAMAM-Pt?IV??dendrimer prodrugs.The polyplexes exhibited triggered release of secondary PAMAM-Pt?IV?dendrimer nanocarriers at pH 6.8 due to acid-labile charge-reversal properties of PAsp?EDA-DM?.The released small PAMAM delivery nanocarriers were demonstrated to exhibit significantly enhanced tumor tissue penetration and efficient cellular internalization,followed by release of active cisplatin anticancer drug under intracellular reducing medium.However,the results revealed that the stability of the polyplex micelles was not high enough to perform in vivo anti-tumor experimental evaluation.So we introduced poly??-caprolactone??PCL?hydrophobic segments to form a triblock copolymer,poly?ethylene glycol?-b-poly[?N'-dimethylmaleoyl-2-aminoethyl?aspartamide]-b-poly??-carprolacton e??PEG-b-PAsp?EDA-DM?-b-PCL?.The in vivo investigation revealed that the Pt?IV?-loading micelleplexes via electrostatic complexation between PEG-b-PAsp?EDA-DM?-b-PCL micelle and PAMAM-Pt?IV?with hydrophobic effect significantly suppressed tumor growth via intravenous injection due to combination of long circulation in bloodstream,high tumor accumulation,deep tumor tissue penetration,and efficient cellular internalization in tumor tissue.Actually,the limited penetration depth of photosensitizers-loaded nanocarriers in heterogeneous tumors as well as tumor hypoxia might compromise photodynamic therapy?PDT?efficiency.To address these PDT issues,hydrogen peroxide?H₂O₂?and poly?amidoamine?dendrimer conjugating chlorin e6/cypate?CC-PAMAM?were co-assembled with reactive oxygen species?ROS?responsive triblock copolymer into the polymeric vesicles.The vesicles exhibited the light-triggered thermal effect that is able to decompose thermally unstable H₂O₂ into O2,which distinctly ensured the alleviation of tumor hypoxia at tumor.Meanwhile,the vesicles were rapidly destabilized through singlet oxygen-mediated cleavage of copolymer under light irradiation,and thus allowed the release of photo-active CC-PAMAM,followed by their deep penetration in the poorly permeable BxPC-3 tumor.4.Self-sufficing tumoral hydrogen peroxide-responsive multifunctional nanocarriers.As compared with normal tissues,tumor tissue microenvironment shows distinct features which offer possibility for endogenous stimuli-responsiveness of responsive polymers,such as MMP and pH-responsive block copolymers used in the previous chapters.However,the subtle differences between normal tissues and tumor tissues and complex tumor microenvironment heterogeneity hamper the in vivo applications of stimuli-responsive polymers as drug nanocarriers.We proposed the strategy of specific upregulation of tumor tissue signals to amplify the signal differences between normal tissues and tumor tissue via multifunctional polymeric nanoparticles.Palmitoyl ascorbate?PA?as a prooxidant for hydrogen peroxide?H₂O₂?production in tumor tissue is strategically compiled into a H₂O₂-responsive camptothecin?CPT?polymer prodrug micelle,which endowed the nanocarriers with self-sufficing H₂O₂ stimuli in tumor tissues.H₂O₂ production was demonstrated to specifically sustain in tumors,which not only induced tumor cell apoptosis by elevated oxidation stress but also served as autochthonous H₂O₂ resource to trigger CPT release for chemotherapy.Systemic therapeutic trial revealed potent tumor suppression of the proposed formulation via synergistic oxidation-chemotherapy.5.Therapeutic vesicular nanoreactors with tumor-specific activation and self-destructing ability for amplified oxidation therapy.Enzyme-loaded nanoreactors have attracted tremendous research interest with respect to therapeutic and diagnostic applications in various diseases.Here,we developed glucose oxidase-loaed therapeutic nanoreactors?theraNR?with self-destructing ability for amplified oxidation therapy,which was assembled from H₂O₂ and pH dual-responsive amphiphilic block copolymers poly?ethylene glycol?-b-poly[2-????4-?4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl?benzyl?oxy?carbo nyl?-oxy?ethyl methacrylate-co-2-?piperidin-1-yl?ethyl methacrylate]?PEG-b-PBN?with glucose oxidase.TheraNR could successfully deliver glucose oxidase into tumor site and reserve enzyme activity for a long-time.Upon exposure to acidic and high-glucose tumor environment,an enzyme cascade reaction selectively occurred in tumor site to generate H₂O₂ and release quinone methide?QM?simultaneously.Quinone methide with glutathione?GSH?-depletion ability and produced H₂O₂ act in a synergistic manner to amplify tumor oxidative stress for realizing nanorector-directed amplified oxidation therapy.