Biodegradable POLY-PROTAC Nanoparticles For Tumor-targeted Protein Degradation To Enhance Antitumor Therapy

Proteolysis Targeting Chimeras（PROTACs）have received considerable attention because of their potential to circumvent drug resistance of small molecule inhibitors and target traditionally"undruggable"proteins through event-driven catalytic degradation.Although PROTACs can inactivate disease-causing proteins directly,the poor pharmacokinetics profile and tumor specificity limit their efficacy.Thus,we proposed a biodegradable and reduced environment-activated polymer PROTAC（POLY-PROTAC）and co-assembled with CRGDK-functionalized ligands to obtain POLY-PROTAC prodrug nanoparticles（RPG7）for tumor-targeted delivery of PROTACs,integration of treatment and anti-tumor combinatory therapy.RPG7 NPs can increase the uptake of tumor cells by recognizing the highly expressed NRP-1receptor on the surface of tumor cells and can release PROTACs under the stimulation of reducing environment.The RPG7 NPs remarkably degraded the protein of interest（POI）and downregulated downstream c-Myc oncogene.Nanoprobes integrating diagnosis and treatment（RPG7@TQTCD）were constructed by non-covalently encapsulating the near-infrared Ⅱ fluorescent probe（TQTCD）,and the CRGDK ligand-modified strategy can improve the accumulation of NPs at tumor sites,as confirmed by fluorescence imaging in vivo.In addition,we constructed chemotherapeutic drug-loaded POLY-PROTAC prodrug NPs（RPG7@DOX）and confirmed by western-blot analysis that doxorubicin（DOX）and ARV-771 can synergistically activate the caspase-3 pathway,which enhances the cell cytotoxicity of POLY-PROTAC prodrug NPs on tumor cells for the combinatory therapy of triple-negative breast cancer（TNBC）.The main research work of this paper is as follows:Chapter one.IntroductionThis chapter mainly introduces the development of PROTACs,the current research status,and their applications in disease treatment;design and application of tumor microenvironment-responsive nanocarriers;application of NIR-Ⅱ fluorescent probes.Finally,the main research and implications of this work are discussed.Chapter two.Synthesis and Physicochemical Characterization of POLY-PROTAC NPsIn this chapter,we firstly synthesized the VHL ligand-based PROTAC molecule（ARV-771）andreduction-sensitive/insensitivederivatives（DT-ARV-771/SA-ARV-771）.Then,the ability of the derivatives to release ARV-771in response to reducing environment was demonstrated in vitro.POLY-PROTAC prodrug（PEG-b-PLGA-DT-ARV-771）was subsequently synthesized using a one-step esterification reaction and CRGDK functionalized ligand（CRGDK-PEG-b-PLGA）was synthesized using an efficient thiol-ene reaction.RPG7 NPs were fabricated using a nanoprecipitation method.They were spherical structures with a hydrodynamic diameter of～70 nm and can effectively release the drug under the reducing environment.In addition,POLY-PROTAC prodrugs can be used as active carriers to prepare NPs（RPG7@TQTCD）for diagnosis and treatment by non-covalent encapsulation of hydrophobic NIR-Ⅱ fluorescent dye（TQTCD）,and to prepare NPs for chemotherapy combinatory therapy（RPG7@DOX）by non-covalent encapsulation of chemotherapeutic drug（DOX）.Physichemical characterization confirmed that non-covalent encapsulation does not affect the morphology of RPG7NPs with the slightly increased hydrodynamic diameter and does not affect the NIR-Ⅱ imaging capability of TQTCD using spectral testing.This strategy provides a new idea for the design of tumor smart delivery PROTAC and multifunctional nano-delivery platforms.Chapter three.Anti-tumor Study of POLY-PROTAC NPs in VitroIn this chapter,we tested the protein degradation ability and cell cytotoxicity of PROTAC molecule（ARV-771）and reduction-sensitive/insensitive derivatives（DT-ARV-771/SA-ARV-771）in vitro.Both DT-ARV-771 and ARV-771 efficiently degraded BRD4 protein and downregulated downstream c-Myc oncogene in vitro.Moreover,it can kill tumor cells in a dose-dependent manner.Subsequently,the effect of the CRDGK ligand-modified strategy on the ability of cellular uptake and tumor penetration was tested in vitro.CRGDK ligand-modified can increase the uptake of RPG7 NPs by tumor cells up to 7-times that of PG7 NPs by recognizing NRP-1receptors and can significantly enhance the selectivity for tumor cells which are highly expressed NRP-1 receptors.POLY-PROTAC prodrug NPs released ARV-771molecules under the stimulation of glutathione（GSH）to degrade BRD4 protein and downregulate downstream c-Myc oncogene,which leaded to the cytotoxicity of tumor cells.RPG7 NPs can reduce the IC₅₀ value of the ARV-771 molecule to 0.25μM and increase the protein degradation of BRD4 by～2-fold.Moreover,protein degradation ability of ARV-771 molecule and POLY-PROTAC prodrug NPs was confirmed by co-incubation with protease inhibitor（MG132）and VHL ligand,suggesting their protein degradation ability was based on proteasome pathway and VHL.Chapter four.Bio-distribution and Anti-tumor Therapy of POLY-PROTAC NPs in vivoThe work in this chapter firstly tested the bio-biodistribution of NIR-Ⅱ therapeutically integrated POLY-PROTAC prodrug NPs in vivo and compared their tumor penetration behavior in vivo.In addition,the drug distribution of ARV-771 at the tumor site was also explored by HPLC in vivo.Then,the anti-tumor properties and protein degradation of ARV-771 and RPG7 NPs were subsequently tested in MDA-MB-231 tumor-bearing mice.RPG7 NPs were administrated via i.v.injection.The PEG shell prolonged their circulation time,and RPG7 NPs can actively target MDA-MB-231 tumor sites using CRGDK ligands,which promoted their accumulation and tumor penetration at tumor sites by recognizing NRP-1 receptors.We verified that the CRGDK ligand-modified increased the tumor accumulation of PG7 NPs by 2.9-fold in vivo.RPG7 NPs can release ARV-771 molecules via GSH stimulation,then degraded BRD4 protein and downregulated downstream c-Myc oncogene to inhibit tumor growth.We found that RPG7 NPs can significantly enhance the ability of BRD4 degradation and c-Myc oncogene inhibition by using immunofluorescence sections and western-blot analysis in vivo.In addition,RPG7NPs can significantly inhibit tumor growth and did not have toxic effects on normal tissues.This strategy offerss a novel approach to antitumor treatment.Chapter five.Anti-tumor Chemotherapy Combination Therapy of POLY-PROTAC NPs in vivoThe work in this chapter constructed chemotherapeutic drug-loaded RPG7@DOX NPs and tested the cellular cytotoxicity and the mechanism of RPG7@DOX NPs for anti-tumor combinatory therapy in vitro.RPG7@DOX NPs exhibited stronger cellular cytotoxicity than ARV-771 and DOX molecules in vitro,and the DOX combinatory therapy reduced the IC₅₀ value of RPG7 NPs to 0.036μM.Western-blot assay also confirmed that DOX and ARV-771 could synergistically activate the caspase-3 pathway and thus enhance the cellular cytotoxicity of RPG7@DOX NPs.Encouraged by the results of in vitro experiments.we then tested the efficacy of RPG7@DOX NPs in chemotherapy combinatory antitumor therapy in MDA-MB-231 tumor-bearing mice.The results showed that this strategy significantly improved the efficacy of RPG7 NPs and more cellular apoptosis was seen in tumor sites from H&E and TUNEL staining section.This combinatory chemotherapy therapy can significantly inhibit tumor growth and improve half survival of MDA-MB-231 tumor-bearing mice,which provide a practical and effective idea for the treatment of TNBC.