Design Of Supramolecular Nanoassemblies Based On The Regulatory Effects Of NO For Tumor Therapy

The effectivenesses of common tumor treatments are limited by the resistance of tumor cells and tumor microenvironment,while tumor-specific microenvironment can also be used to improve the therapeutic efficacy through rational design.Nitric oxide（NO）,as an endogenous gaseous biological messenger,can affect tumor-related behaviors in many ways.Compared with the direct inhibition of tumors,the multiple regulatory effects of NO on tumor intracellular environment and tumor tissue microenvironment should be paid more attentions.How to amplify the regulatory effects of NO on tumors and to develop tumor combination strategies based on these regulatory effects are key issues for achieving synergy.At the same time,the effective delivery and tumor-specific release of NO are the prerequisites for NO to exert multiple tumor regulatory effects.Therefore,this thesis proposes combination strategies using cyclodextrin-based assembly to design supramolecular nanoplatforms,which can co-deliver NO donors and other therapeutic agents.These strategies not only achieve precise delivery and stimuli-response release of NO,but also systematically explore the mechanism and potential of NO as a“synergist”that synergistically enhances photodynamic efficacy,reverses tumor multidrug resistance and inhibits tumor metastasis,which may well serve as an inspiration for future creative approaches of NO-based gas therapy.This thesis contains the researches in the following aspects:1.Nitric Oxide-based Nanoplatform as an All-Rounder to Enhance Photodynamic Therapeutic EfficacyA synergistic approach was developed by employing the multi-functional NO as an all-rounder to improve the therapeutic performance of photodynamic therapy（PDT）.Supramolecular nanocarriers（α-CD-Ce6-NO NPs）were fabricated by the host-guest interactions.Polyethylene glycol-poly（2-methacryl-oyloxyethyl phosphorylcholine）diblock copolymer（PEG-b-PMPC）acted as the guest molecule.The prodrug（α-CD-Ce6）ofα-cyclodextrin modified with chlorin e6（Ce6）and prodrug（α-CD-NO）ofα-cyclodextrin modified with nitric oxide（NO）donor served as host molecules.The integrated system was expected to kill three birds with one stone:Firstly,NO caused smooth muscle cells relaxation can increase the blood flow and relief tumor hypoxia,thus increase oxygen required for PDT to promote ROS generation.Secondly,Intracellular glutathione（GSH）-released NO can also accelerate the metabolism of GSH.Such GSH-initiated depletion of GSH could be regarded as a new strategy to inhibit the consumption of reactive oxygen species（ROS）by GSH.Last but not least,GSH-released NO and light-triggered ROS can successively react to generate the peroxynitrite anion（ONOO^-）,which greatly boosted the biocidal activity compared with ROS.The research results showed that the designedα-CD-Ce6-NO NPs can deplete GSH level,relief tumor hypoxia,promote ROS generation and generate ONOO^-both in vitro and in vivo.Therefore,laser irradiatedα-CD-Ce6-NO NPs could remarkably inhibit cancer cell proliferation and suppress tumor growth via the excellent synergistic effects of NO and PDT.2.Supramolecular Prodrug Nano-Assemblies for Reactive Nitrogen Species-Potentiated Cisplatin Chemotherapy of Liver CancerA supramolecular prodrug nano-assemblies strategy for reactive nitrogen species（RNS）-potentiated chemotherapy by the combination of CDDP and NO was reported.The prodrug（β-CD-Pt）ofβ-cyclodextrin modified with cisplatin（Pt（IV））and prodrug（β-CD-NO）ofβ-cyclodextrin modified with nitric oxide（NO）donor served as host molecules.Lactose-ended poly（ethylene glycol）-block-polylysine with pendant adamantane（Lac-PEG-b-PLys（Ad））acted as the guest molecule.Lactose which can efficiently target hepatoma cells overexpressing asialoglycoprotein receptor via receptor mediated endocytosis was used as a targeting ligand.Liver cancer-targeted nano-assemblies(T-SPNA_Pt/NO)that co-delivery of NO donor and O₂^·-inducing-Pt（IV）were prepared by supramolecular self-assembly by the host-guest interactions.After targeted delivery of T-SPNA_Pt/NO into liver cancer cells through endocytosis,Pt（II）and NO were expected to be released under intracellular reductive microenvironment.Released Pt（II）would activate NOXs to produce O₂^·-,which will further react with the released NO nearby to generate high toxic ONOO^-with strong oxidizing and nitrating capability.Due to the continuous generation of ONOO^-,the levels of glutathione reductase（GR）and GSH could be effectively suppressed by nitration modification,thus decreasing the formation of detoxified Pt-GSH adducts and increasing the formation of lethal Pt-DNA adducts to enhance DNA damage.Furthermore,ONOO^-is also expected to down-regulate an essential DNA damage repair protein,xeroderma pigmentosum group A（XPA）,to block the repair of damaged DNA.The outstanding efficacy of T-SPNA_Pt/NO was demonstrated on subcutaneous hepatoma xenograft model,orthotopic CDDP-resistant hepatoma model and patient-derived hepatoma xenograft model.This strategy shows the great potential of RNS-mediated cancer therapy and may improve Pt-based therapeutic efficacy in clinic.3.Precise Subcellular Delivery of NO by p H-Activated Mitochondria-targeted Nanoplatform for Drug Resistance Reversal and Metastasis InhibitionAn acid-activated charge-reversal drug nanocarrier（α-CD-DOX-NO-DA NPs）was reported for precise mitochondria-targeted delivery of NO to overcome drug resistance and metastasis in cancer treatment.The PEG conjugated mitochondria-penetrating peptide PEG-（KLAKLAK）₂CGKRK was modified with acid-cleavable dimethylmaleic anhydride（DA）as protecting groups,and then self-assembled withα-cyclodextrin（α-CD）based doxorubicin（DOX）and NO prodrugs via LEGO like host-guest interaction.The obtainedα-CD-DOX-NO-DA nanoparticles with negatively charged acid-cleavable DA could achieve prolonged blood circulation time.Under tumor extracellular p H,the amide bonds could be hydrolyzed to promote the internalization of the nanoparticles with selectively restoring mitochondria-targeting capability of the（KLAKLAK）₂CGKRK sequences.After accumulated in mitochondria,NO could be readily released and then cause mitochondrial dysfunctions to cut off the energy supply to inhibit P-glycoprotein（P-gp）related bioactivities and the formation of Tumor-derived microvesicles（TMV）,resulting in the overcoming of drug resistance and cancer metastasis.The precise subcellular delivery strategy proved crucial to maximize the therapeutic effects of NO on mitochondria and the designedα-CD-DOX-NO-DA NPs can efficiently reverse tumor resistance and inhibit tumor metastasis both in vitro and in vivo.Furthermore,the whole-genetic expression analysis indicted that genes relevant to drug resistance and cancer metastasis could be well regulated by mitochondria targeted precise delivery of NO.Such multifunctional tumor microenvironment-activated mitochondria-targeted NO-generating nanoplatforms are of great significance for basic material designing and may have profound therapeutic implications for more effective cancer therapy in clinic.