WO2024055804A1 - 靶向降解致病蛋白的纳米复合体及其制备方法和应用 - Google Patents

靶向降解致病蛋白的纳米复合体及其制备方法和应用 Download PDF

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WO2024055804A1
WO2024055804A1 PCT/CN2023/113588 CN2023113588W WO2024055804A1 WO 2024055804 A1 WO2024055804 A1 WO 2024055804A1 CN 2023113588 W CN2023113588 W CN 2023113588W WO 2024055804 A1 WO2024055804 A1 WO 2024055804A1
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protein
nrs
mutant
tumor
cells
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张云娇
黄晓婉
曹紫洋
杨显珠
温龙平
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华南理工大学
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present application belongs to the technical field of nano-biopharmaceuticals, and in particular relates to a nano-complex targeting the degradation of pathogenic proteins and its preparation method and application.
  • Abnormal protein homeostasis and accumulation of pathogenic proteins are the causes of various diseases such as tumors and Huntington's disease. Therefore, inducing the degradation of pathogenic proteins and reducing the levels of pathogenic proteins is an effective way to treat related diseases.
  • proteasome-targeting chimeras PROTACs
  • LYTACs lysosome-targeting protein chimeras
  • PROTACs cannot effectively degrade certain large molecular weight proteins or protein aggregates, while LYTACs can only be used to degrade secreted proteins and cell membrane proteins, but cannot degrade cytoplasmic proteins.
  • the embodiments of this application disclose a nanocomplex targeted at degrading pathogenic proteins and its preparation method and application, aiming to solve the problem that PROTACs cannot effectively degrade large molecular weight pathogenic proteins or protein aggregates, and LYTACs cannot degrade cytoplasmic proteins. question.
  • the first aspect provides a nanocomplex for targeted degradation of pathogenic proteins, which contains a nanocarrier and a pathogenic protein-targeted binding peptide grafted on the nanocarrier.
  • the nanocarrier is a nanoassembly of maleimide-polyethylene glycol-polylactic acid and cationic lipids.
  • a pathogenic protein-targeted binding peptide is grafted onto a nano-assembly carrier composed of cationic lipids and maleimide-polyethylene glycol-polylactic acid, so that the nano-assembly carrier can be targeted to bind to the pathogenic protein.
  • the peptides have a synergistic effect based on their respective functions. Specifically, maleimide-polyethylene glycol-polylactic acid can not only be combined with cationic lipids to assemble to form a nano-assembly carrier, but also can target pathogenic proteins.
  • the peptide is efficiently clicked through "thiol-maleimide” and covalently grafted onto the nanoassembly carrier to obtain a stable nanocomplex.
  • cationic lipids can induce significant autophagy effects in cells and promote the formation and accumulation of autophagosomes
  • the results show that the intervention of maleimide-polyethylene glycol-polylactic acid can better promote cationic lipids. related functions.
  • the process of nanocomplexes inducing the degradation of pathogenic proteins in tumor cells is an autophagy-dependent and ubiquitination-dependent process, and the degradation of pathogenic proteins by nanocomplexes can inhibit mutant proteins in tumor cells.
  • Gained functions such as inhibiting the proliferation and migration of tumor cells, enhancing tumor cell death, enhancing the sensitivity of tumor cells to anti-tumor active ingredients, etc.
  • the nanocomplex not only has good binding properties with pathogenic proteins, but also can effectively reduce the toxicity to normal cells and can be used to induce the degradation of different types of pathogenic proteins with broad spectrum.
  • the preparation method of nanocarriers in the embodiments of the present application includes:
  • the organic solvent After dissolving the cationic lipid and maleimide-polyethylene glycol-polylactic acid in an organic solvent, the organic solvent is dropped into the buffer and stirred to obtain a nanocarrier solution.
  • the nanocarrier solution can be subjected to purification treatment including rotary evaporation to remove the solvent, and centrifugation to obtain the supernatant.
  • the centrifugation conditions are: centrifugation at a speed of 2000-4000rpm/min for 3-10 minutes, specifically centrifugation at a speed of 3000rpm/min for 5 minutes.
  • the organic solvent is preferably tetrahydrofuran, which is beneficial to the dissolution and mixing of maleimide-polyethylene glycol-polylactic acid and cationic lipids;
  • the cationic lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3-dioleoyloxypropyl-1-trimethylamine bromide (DOTMA) .
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DOTMA 2,3-dioleoyloxypropyl-1-trimethylamine bromide
  • 1,2-dioleoyl-3-trimethylammonium-propane is preferred to obtain the best autophagy effect.
  • the "polyethylene glycol/polylactic acid" block in maleimide-polyethylene glycol-polylactic acid has a molecular weight of 1000-3000.
  • it can be 1000, 2000, 3000, etc.
  • the mass ratio of cationic lipids to maleimide-polyethylene glycol-polylactic acid is (5-40):100. Among them, this mass ratio is conducive to improving the level of induced cell autophagy by nanocarriers.
  • the disease-causing protein is selected from one of mutant p53 protein and mutant huntingtin protein.
  • the mutant p53 protein contains at least one of S241F, R175H, R248W, and R280K mutation sites; the mutant huntingtin protein includes Htt-(Q74).
  • the source of p53 protein can be ES-2, MDA-MB-231, MIAPaCa-2 and SK-BR-3 cells; the source of huntingtin protein can be PC-12/GFP-Htt(Q74) and Neuro2A/GFP-Htt(Q74) and other cells.
  • the nanocomplexes of the embodiments of the present application when used to degrade mutant p53 protein, they can also effectively inhibit the acquired function (GOF) of the mutant protein in tumor cells, such as inhibiting the proliferation and migration ability of tumor cells, and enhancing tumor Cell death and enhanced sensitivity of tumor cells to active ingredients of drugs, etc.
  • GAF acquired function
  • the mutant p53 protein targeting binding peptide has the amino acid sequence of SEQ ID NO.1; the mutant huntingtin protein targeting binding peptide has the amino acid sequence of SEQ ID NO.2, and the two targeting binding peptides can efficiently Targeted binding to the target protein, and the "thiol" in the molecular structure allows it to click efficiently with maleimide-polyethylene glycol-polylactic acid through "thiol-maleimide", making the targeted binding peptide efficient and stably grafted onto nanocarriers.
  • the second aspect provides a method for preparing the nanocomposite, including:
  • the nanocomplex is obtained by subjecting the pathogenic protein targeting binding peptide to a click reaction to a solution containing a nanocarrier, and subjecting the reaction product to purification treatment including dialysis, ultrafiltration and concentration.
  • dialysis methods include:
  • the molar ratio of the pathogenic protein-targeting binding peptide to the nanocarrier is (1-3):1. Among them, this molar ratio is conducive to efficient and sufficient loading of the peptide on the surface of the nanocarrier, improving the capture effect of the target protein.
  • the third aspect provides the use of the nanocomposite for preparing anti-tumor nanomedicines and Huntington's disease inhibitory drugs.
  • the nanocomplex has the ability to bring pathogenic protein aggregates to autophagosomes for degradation through the autophagy pathway. Therefore, after the nanocomplex is used to prepare anti-tumor nanomedicines, it can effectively inhibit the proliferation and migration of tumor cells, enhance tumor cell death, and enhance the sensitivity of tumor cells to active ingredients such as cisplatin (CDDP), showing that Produce good synergistic inhibition effect.
  • CDDP cisplatin
  • the fourth aspect provides an anti-tumor nanomedicine, which contains a nanocomplex and an anti-tumor active ingredient loaded in the nanocomplex.
  • the anti-tumor active ingredient includes cisplatin, Pt(IV) prodrug.
  • Pt(IV) prodrugs can be prepared according to the literature Stimuli-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy.
  • loading anti-tumor active ingredients on nanocomposites can not only enhance the sensitivity of tumor cells to anti-tumor nanomedicines, but also anti-tumor nanomedicines can show good synergistic anti-tumor effects with patient-derived xenograft (PDX) ovarian cancer. tumor effects.
  • PDX patient-derived xenograft
  • the fifth aspect provides a preparation method of anti-tumor nanomedicine, including:
  • a solution containing maleimide-polyethylene glycol-polylactic acid, cationic lipids and anti-tumor active ingredients is self-assembled in a buffer and dialyzed to obtain a solution containing drug-loaded nanoparticles;
  • a click reaction occurs between a solution containing drug-loaded nanoparticles and a pathogenic protein-targeting binding peptide, and the reaction product is sequentially dialyzed, ultrafiltrated, and concentrated to obtain an anti-tumor nanomedicine.
  • the first dialysis can effectively remove unreacted anti-tumor active ingredients; the second dialysis can effectively remove unreacted peptides.
  • the advantages or beneficial effects of the embodiments of the present application at least include:
  • the nanocomposite in the embodiment of the present application grafts the pathogenic protein-targeting binding peptide onto a nanoassembly carrier assembled from cationic lipids and maleimide-polyethylene glycol-polylactic acid.
  • a nanoassembly carrier assembled from cationic lipids and maleimide-polyethylene glycol-polylactic acid.
  • the complex can simulate key receptor proteins in the selective autophagy pathway, so that pathogenic proteins are brought into autophagosomes and degraded through the autophagy pathway, which can effectively solve the problem of PROTACs' inability to degrade large molecular weight protein aggregates and LYTACs' inability to degrade Cytoplasmic proteins;
  • the nanocomplex can form good binding properties with a variety of pathogenic proteins and effectively reduce the toxicity to normal cells. It can be used to induce the degradation of different types of pathogenic proteins and has a broad spectrum.
  • Figure 1 is a schematic structural diagram of NRs
  • Figure 2 is a fluorescence image of the autophagy effect of DOTAP on MDA-MB-231-EGFP-LC3 cells;
  • Figure 3 is a western blot diagram of the degradation of mutant p53 protein by nanocomplexes containing neutral lipids, anionic lipids and cationic lipids;
  • Figure 4 shows the particle size distribution diagram and Zeta potential diagram of mNRs, dNRs and NRs
  • Figure 5 shows the particle size distribution diagram of mNRs, dNRs and NRs in PBS solution containing 10% FBS;
  • Figure 6 shows the transmission electron microscope images of mNRs, dNRs and NRs
  • Figure 7 shows the circular dichroism spectrum of mutant p53 protein after treatment with PBS, MBP, dNRs and NRs;
  • Figure 8 shows the SPR patterns of mutant p53 protein after different treatments
  • Figure 9 shows the STORM electron microscope image of NRs
  • Figure 10 is a western blot diagram of NRs containing different concentrations of DOTAP degrading mutant p53 protein in ES-2 cells;
  • Figure 11 shows the cell viability of NRs containing different concentrations of DOTAP in HEK 293T cells
  • Figure 12 is an immunofluorescence image of MBP, mNRs, dNRs and NRs degrading mutant p53 protein in ES-2 cells;
  • Figure 13 shows the changes in p53 mRNA in ES-2 cells after NRs treatment
  • Figure 14 is a western blot diagram of NRs degrading mutant p53 protein in ES-2, MDA-MB-231, MIAPaCa-2 and SK-BR-3 cells;
  • Figure 15 is a western blot diagram to detect the specific degradation pathway of NRs degrading mutant p53 protein in ES-2 cells;
  • Figure 16 is a western blot diagram for detecting the ubiquitination dependence of NRs degradation in the process of degrading mutant p53 protein in ES-2 cells;
  • Figure 17 is a bioelectron microscope image of NRs triggering autophagy in ES-2 cells
  • Figure 18 is a fluorescence image of NRs triggering complete autophagy in ES-2 cells
  • Figure 19 is a western blot diagram showing that NRs triggers the degradation of mutant p53 protein in ES-2 cells and requires the participation of ubiquitinase;
  • Figure 20 is a western blot diagram showing that NRs triggers an increase in ubiquitination levels in ES-2 cells
  • Figure 21 shows the cell viability of HUVEC, H1299, HEK 293T, A549, HCT 116, MIAPaCa-2, SK-BR-3, MDA-MB-231 and ES-2 after NRs treatment;
  • Figure 22 is a picture of the sphere formation experiment of MBP, mNRs, dNRs and NRs in ES-2 cells;
  • Figure 23 shows the Transwell results of MBP, mNRs, dNRs and NRs in ES-2 cells
  • Figure 24 is a Hoechst/PI double-staining image of MBP, mNRs, dNRs and NRs in ES-2 cells;
  • Figure 25 is the biodistribution map of DiD-NRs in ES-2 tumor-bearing mice
  • Figure 26 is a diagram of the in vivo treatment experiment of nanoparticles in ES-2 tumor-bearing mice
  • Figure 27 is a western blot diagram of p53 in tumors of mice in each group of ES-2 tumor-bearing mice;
  • Figure 28 is an immunofluorescence image of p53 and LC3 in tumor sections of mice in each group of ES-2 tumor-bearing mice;
  • Figure 29 is a diagram of the in vivo treatment experiment of nanoparticles in PDX model mice.
  • Figure 30 is a western blot diagram of p53 in tumors of mice in each group of PDX model mice;
  • Figure 31 shows immunohistochemistry and TUNEL staining of p53 in tumor sections of mice in each group of PDX model mice;
  • Figure 32 is a western blot diagram of NRs-Htt causing autophagy effects and degrading GFP-Htt (Q74) protein in PC-12/GFP-Htt (Q74) and Neuro 2A/GFP-Htt (Q74) cells;
  • Figure 33 is an immunofluorescence image of NRs-Htt degrading GFP-Htt (Q74) protein in PC-12/GFP-Htt (Q74) and Neuro 2A/GFP-Htt (Q74) cells.
  • This embodiment provides a method for preparing nanocomplex NRs targeting mutant proteins, which specifically includes:
  • NPs@DOTAP nanocarrier dNRs solution
  • FIG. 1 is a schematic structural diagram of NRs.
  • the nanocomplex consists of self-assembled carrier particles of Mal-PEG 2000 -PLA 2000 and cationic lipids, and pathogenic protein-binding peptides modified on the surface of the self-assembled carrier particles.
  • nanocomposites NPs@GTP, NPs@PL, NPs@DPPG, NPs@DOPG and NPs@DOTMA after replacing DOTAP with neutral lipids (GTP, PL), anionic lipids (DPPG, DOPG) and cationic lipids (DOTMA), nanocomposites NPs@GTP, NPs@PL, NPs@DPPG, NPs@DOPG and NPs@DOTMA.
  • the addition amount of DOTAP is adjusted, and nanocomposite mNRs (NRs@0% DOTAP), NRs@5% DOTAP, NRs@10% DOTAP, NRs@20% D-OTAP and NRs@40%DOTAP.
  • This embodiment provides a method for preparing anti-tumor nanomedicine NRs/Pt(IV), which specifically includes:
  • the chemical reagents, biological raw materials, etc. used in the test are commercially available, and the reagent concentration, overnight culture, etc. refer to the conventional requirements, such as overnight culture (37°C, 5% CO 2 ).
  • MDA-MB-231 cells were transfected with EGFP-LC3 plasmid to obtain MDA-MB-231-EGFP-LC3 cells, and then seeded into a 24-well cell culture plate with circular slides (density approximately 3 ⁇ 10 4 /well) medium, cultured overnight, and added PBS and DOTAP (2 ⁇ g/mL) respectively. After 6 h of culture, fluorescence was detected, and the results are shown in Figure 2. Among them, Figure 2 is a fluorescence image of the autophagy effect of DOTAP on MDA-MB-231-EGFP-LC3 cells.
  • LC3 green fluorescent protein in cells showed obvious point-like aggregation, proving that DOTAP can induce significant autophagy effects in cells.
  • MDA-MB-231-EGFP-LC3 cells were seeded in a 24-well cell culture plate (density approximately 3 ⁇ 10 4 /well), cultured overnight, and added with NPs@GTP, NPs@PL, NPs@DPPG, and NPs@ respectively.
  • DOPG, NPs@DOTMA and NPs@DOTAP(dNRs) after culturing for 6 hours, the LC3 protein level in the cells was detected by western blot.
  • Figure 3 is a western blot diagram of the degradation of mutant p53 protein by nanocomplexes containing neutral lipids, anionic lipids and cationic lipids.
  • Figure 4a shows the particle size distribution diagram of mNRs, dNRs and NRs
  • Figure 4b shows the Zeta potential diagram of mNRs, dNRs and NRs.
  • mNRs, dNRs and NRs were dispersed in PBS solution containing 10% fetal bovine serum (FBS) at a concentration of 1.0 mg/mL, and incubated at 37°C for 0, 1, 2, 4, 8, and 12 respectively. and 24h, the particle size was measured with a nanoparticle size analyzer, and the results are shown in Figure 5.
  • Figure 5 shows the particle size distribution diagram of mNRs, dNRs and NRs in PBS solution containing 10% FBS.
  • Figure 6 shows the transmission electron microscope images of mNRs, dNRs and NRs.
  • the spectrum of the mutant p53 protein showed two negative peaks at 208nm and 222nm, which correspond to the secondary structure of the mutant p53 protein; while incubated with NRs
  • the spectrum of the mutant p53 has a minimum peak at 231 nm, indicating that the secondary structure of the mutant p53 protein has undergone a significant transformation, proving that NRs can bind to the mutant p53 protein, causing changes in its secondary structure.
  • SPR Surface plasmon resonance
  • each CM5 sensor chip consists of 8 identical experimental channels, each channel is divided into two flow cells (flow-cell 1 and flow-cell 2).
  • flow-cell 1 was always left blank as a reference, while flow-cell 2 (Fc2) was used to study the interaction of NRs with mutant p53 protein.
  • mutant p53 protein (p53S241F) and NRs
  • 5.21 ⁇ M mutant p53 protein solution was injected onto the NRs-modified sensor surface.
  • the injection time was 3 min with a flow rate of 30 ⁇ L/min, followed by a 5 min dissociation step.
  • mutant p53 proteins with concentrations of 2500nM, 500nM, 100nM, 20nM and 4nM were injected into the channel, and the binding confidence was determined in each cycle.
  • BIAcore 8K Evaluation software was used to calculate the binding kinetic constants between NRs and mutant p53 protein using a 1:1 binding model. The results are shown in Figure 8. Among them, Figure 8 shows the SPR patterns of mutant p53 protein after different treatments.
  • ES-2 cells were seeded in a 24-well cell culture plate (density approximately 5 ⁇ 10 4 /well) and cultured overnight. PBS and NRs containing different concentrations of DOTAP (0%, 5%, 10%, 20% and 40%) were added respectively. After incubation for 12 hours, western blot detection was performed. The results are shown in Figure 10. Among them, Figure 10 shows the western blot diagram of NRs containing different concentrations of DOTAP degrading mutant p53 protein in ES-2 cells.
  • HEK 293T cells were seeded in a 96-well cell culture plate (density was approximately 1 ⁇ 10 4 /well) and cultured overnight. PBS and NRs with different concentrations of DOTAP (0%, 5%, 10%, 20% and 40%) were added, and after incubation for 24 hours, MTT detection was performed. The results are shown in Figure 11. Among them, Figure 11 shows the cell viability of NRs containing different concentrations of DOTAP in HEK 293T cells.
  • ES-2 cells were seeded in a 24-well cell culture plate (density approximately 3 ⁇ 10 4 /well) with circular slides placed in advance, and cultured overnight.
  • PBS, free MBP peptide (250 ⁇ g/mL), and mNRs, dNRs, and NRs all at a concentration of 500 ⁇ g/mL were added respectively.
  • immunofluorescence detection was performed. The results are shown in Figure 12. Among them, Figure 12 shows the immunofluorescence image of MBP, mNRs, dNRs and NRs degrading mutant p53 protein in ES-2 cells.
  • ES-2 cells were seeded in a 6-well cell culture plate (density approximately 3 ⁇ 10 5 /well) and cultured overnight. PBS and NRs (500 ⁇ g/mL) were added respectively, and after 12 h of culture, RNA was extracted using TRIzol RNA extraction reagent (Invitrogen), and the p53 mRNA level was detected using RT-PCR. The results are shown in Figure 13. Among them, Figure 13 shows the changes in p53 mRNA in ES-2 cells after NRs treatment.
  • ES-2, MDA-MB-231, MIAPaCa-2 and SK-BR-3 cells were seeded in a 24-well cell culture plate (density approximately 5 ⁇ 10 4 /well) and cultured overnight. PBS and 250 ⁇ g/mL and 500 ⁇ g/mL NRs were added respectively. After 12 h of culture, the level of mutant p53 protein was detected by western blot. The results are shown in Figure 14. Among them, Figure 14 shows the western blot diagram of NRs degrading mutant p53 protein in ES-2, MDA-MB-231, MIAPaCa-2 and SK-BR-3 cells.
  • ES-2 cells were seeded in a 24-well cell culture plate (density approximately 5 ⁇ 10 4 /well) and cultured overnight.
  • PBS 500 ⁇ g/mL NRs
  • autophagy inhibitor Wortmannin abbreviated as Wort, 1 ⁇ M
  • 500 ⁇ g/mL NRs+Wort autophagy inhibitor
  • proteasome inhibitor MG-132 10 ⁇ M
  • 500 ⁇ g/mL NRs+ 500 ⁇ g/mL NRs+
  • Figure 15 is a western blot diagram to detect the specific degradation pathway of NRs degrading mutant p53 protein in ES-2 cells.
  • the autophagy inhibitor Wortmannin can effectively inhibit the degradation of mutant p53 protein triggered by NRs, but the proteasome inhibitor MG-132 cannot. This indicates that the degradation of mutant p53 protein triggered by NRs is mediated through the autophagy pathway.
  • HEK293T cells were seeded in a 6-well cell culture plate (density of about 3 ⁇ 10 5 /well) and cultured overnight.
  • the control shRNA (shNC) and shATG5 for knocking out ATG5 were co-transfected with the lentiviral vector system using lipofectamine 3000. into HEK 293T cells. 48 h after transfection, the viral supernatant was collected and used to infect ES-2 cells. 24 hours later, the virus-infected ES-2 cells were seeded in 24-well plates, treated with PBS or 500 ⁇ g/mL NRs for 12 hours, and western blot detection was performed. The results are shown in Figure 16. Among them, Figure 16 is a western blot diagram for detecting the autophagy dependence in the degradation of mutant p53 protein by NRs.
  • ES-2 cells were seeded in a 10 mm cell culture dish (density approximately 5 ⁇ 10 6 /dish) and cultured overnight. PBS and NRs with a concentration of 500 ⁇ g/mL were added. After 12 h of culture, the cell pellets were collected and fixed overnight at 4°C using electron microscopy fixative. The cells were then embedded and cut into ultrathin sections, stained with uranyl acetate and lead citrate, and bioelectron microscopy was used to observe the autophagosomes in the cytoplasm. The results are shown in Figure 17. Among them, Figure 17 is a bioelectron microscope image of NRs triggering autophagy in ES-2 cells.
  • MDA-MB-231 cells were transfected with EGFP-mCherry-LC3 plasmid to obtain MDA-MB-231-EGFP-mCherry-LC3 cells, which were seeded in a 24-well cell culture plate (density) with circular slides placed in advance. About 3 ⁇ 10 4 /well), culture it overnight, add DiD-mNRs and DiD-NRs at a concentration of 500 ⁇ g/mL respectively (wherein, refer to the method of mNRs and NRs in Example 1, and incorporate DiD fluorescent dye during preparation, DiD-labeled DiD-mNRs and DiD-NRs were obtained. After culturing for 12 hours, fluorescence detection was performed. The results are shown in Figure 18. Among them, Figure 18 shows the fluorescence image of NRs triggering complete autophagy in ES-2 cells.
  • ES-2 cells were seeded in a 24-well cell culture plate (density of about 5 ⁇ 10 4 cells/well) and cultured overnight.
  • PBS, NRs with a concentration of 500 ⁇ g/mL, PYR-41 (5 ⁇ M) and 500 ⁇ g/mL were added respectively.
  • NRs+PYR-41 5 ⁇ M).
  • FIG. 19 is a western blot diagram for detecting the ubiquitination dependence of NRs in degrading mutant p53 protein in ES-2 cells.
  • the ubiquitinase inhibitor PYR-41 can effectively inhibit the degradation of mutant p53 protein caused by NRs, indicating that the degradation of mutant p53 protein by NRs depends on the ubiquitination modification of mutant p53 protein.
  • HEK 293T cells were seeded in a 6-well cell culture plate (density approximately 5 ⁇ 10 5 cells/well) and cultured overnight. Add PBS or NRs at a concentration of 500 ⁇ g/mL, and after culturing for 12 h, use IP lysis buffer to lyse the cells, and incubate cell extracts containing equal amounts of protein with protein A/G-agarose and p53 antibodies (2 ⁇ g each) overnight. After washing with PBST buffer, the precipitate was separated on SDS-PAGE, and western blot analysis was performed using Ub antibody and K63-Ub antibody. The results are shown in Figure 20. Among them, Figure 20 is a western blot diagram showing that NRs triggers an increase in ubiquitination levels in ES-2 cells.
  • NRs degrades mutant p53 and inhibits the gain of function (GOF) of mutant p53
  • Normal cells (HUVEC and HEK 293T), tumor cells not expressing p53 (H1299), tumor cells expressing wild-type p53 protein (A549 and HCT 116), and tumor cells expressing mutant p53 protein (MIAPaCa-2, SK-BR -3. MDA-MB-231 and ES-2) were seeded in a 96-well cell culture plate (density approximately 1 ⁇ 10 4 /well) and cultured overnight. Add PBS and 500 ⁇ g/mL NRs, incubate for 24 hours, and perform MTT detection. The results are shown in Figure 21.
  • Figure 21 shows the cell viability of HUVEC, H1299, HEK 293T, A549, HCT 116, MIAPaCa-2, SK-BR-3, MDA-MB-231 and ES-2 after NRs treatment.
  • NRs has little effect on the viability of normal cells and tumor cells that do not express p53 and express wild-type p53 protein, but specifically reduce the viability of tumor cells that express mutant p53 protein, among which in ES-2 cells The reducing effect on cell viability is the most obvious.
  • ES-2 cells were seeded into a 24-well low-adhesion cell culture plate containing serum-free medium (density 1 ⁇ 10 3 /well), and 2% B27 and basic fibroblast growth factor (20ng/mL) were added. and epidermal growth factor (20ng/mL).
  • PBS, free MBP peptide (250 ⁇ g/mL), mNRs (500 ⁇ g/mL), dNRs (500 ⁇ g/mL) and NRs (500 ⁇ g/mL) were added respectively. After 12 h of culture, they were removed and replaced with fresh medium. After 7 days of culture, an inverted phase contrast microscope was used to record the diameters of the spheroids in different groups, and the results are shown in Figure 22. Among them, Figure 22 is a picture of the sphere formation experiment of MBP, mNRs, dNRs and NRs in ES-2 cells.
  • the diameter of the cell spheroids after NRs treatment was significantly smaller, indicating that NRs can effectively inhibit the proliferation of ES-2 cells.
  • ES-2 cells were seeded into the upper chamber in serum-free medium (density approximately 5 ⁇ 10 3 /well), and medium containing 10% FBS was added to the lower chamber.
  • PBS, free MBP peptide (250 ⁇ g/mL), mNRs (500 ⁇ g/mL), dNRs (500 ⁇ g/mL) and NRs (500 ⁇ g/mL) were added to the upper chamber.
  • cells that migrated to the lower surface of the membrane were treated with methanol. Fixed and stained using 0.1% crystal violet. After washing three times with PBS, the cells were subjected to microscopic imaging, and the results are shown in Figure 23. Among them, Figure 23 shows the Transwell results of MBP, mNRs, dNRs and NRs in ES-2 cells.
  • ES-2 cells were seeded in a 24-well cell culture plate (density approximately 5 ⁇ 10 3 /well) and cultured overnight.
  • PBS, free MBP peptide (250 ⁇ g/mL), and mNRs, dNRs, and NRs with a concentration of 500 ⁇ g/mL were added respectively.
  • Hoechst (10 ⁇ g/mL) dye was used to stain the cell nuclei, and propidium iodide (10 ⁇ g/mL) was used. ) to stain the dead cells and then detect them using a fluorescence microscope.
  • Figure 24 shows the Hoechst/PI double staining diagram of MBP peptide, mNRs, dNRs and NRs in ES-2 cells.
  • ES-2 cells were subcutaneously injected into the right side of BALB/c nude mice to construct an ES-2 tumor-bearing nude mouse model.
  • BALB/c nude mice carrying ES-2 xenografts were injected intravenously (iv) with 3 mg of DiD fluorescent dye-labeled NRs/Pt (DiD-NRs/Pt , refer to the preparation method of NRs/Pt(IV) in Example 2, incorporate DiD fluorescent dye when preparing nanoparticles to obtain DiD-labeled DiD-NRs/Pt(IV)), and then use the Xenogen IVIS Lumina system in 2 and 4 , 8, 12, 24 and 48h imaging.
  • DiD-NRs/Pt DiD fluorescent dye-labeled NRs/Pt
  • Figure 25 shows the biodistribution map of DiD-NRs in ES-2 tumor-bearing mice.
  • mice were randomly divided into the following 7 groups, with 6 mice in each group, namely: PBS group, dNRs group, CDDP (cisplatin) group, dNRs/Pt (IV) group, and NRs group. , NRs+CDDP group and NRs/Pt(IV) group, in which the equivalent injection doses of platinum, dNRs and NRs are 1.0mg/kg, 25mg/kg and 25mg/kg respectively.
  • Figures 26, 27 and 28 are shown in Figures 26, 27 and 28.
  • Figure 26 is a picture of the in vivo treatment experiment of nanoparticles in ES-2 tumor-bearing mice;
  • Figure 27 is a western blot picture of p53 in the tumors of each group of ES-2 tumor-bearing mice;
  • Figure 28 is a picture of ES-2 tumor-bearing mice. Immunofluorescence images of p53 and LC3 in tumor sections of mice in each group.
  • mice in each group did not change significantly, proving that the components of each experimental group did not cause obvious systemic toxicity to the mice, and also reflected that NRs has good biocompatibility.
  • the mouse tumor growth curve in Figure 26b and the in vitro tumor picture in Figure 26c show that compared with the PBS, dNRs, CDDP and dNRs/Pt(IV) groups, the NRs group and NRs+CDDP group can effectively inhibit tumor growth. growth, the NRs/Pt(IV) group further inhibited the growth of tumor volume.
  • the tumor weights of mice in each group in Figure 26d show that compared with other treatment groups, the combined treatment group of NRs and chemotherapy drug CDDP effectively inhibited tumor growth, and NRs/Pt(IV) was further reduced. tumor weight.
  • Figures 27 and 28 show that, consistent with the results obtained in cell lines, autophagy levels were significantly enhanced in mouse tumor tissues treated with NRs (including NRs, NRs+CDDP and NRs/Pt groups), and mutant p53 Protein levels were significantly reduced.
  • patient-derived p53 mutant (P72R+/+, C141Y+/+ and L350P+/-) ovarian cancer samples were transplanted subcutaneously into NOD/SCID mice to construct a PDX ovarian cancer model.
  • the tumor volume reached approximately 100 mm
  • the mice were randomly divided into 4 groups, with 6 mice in each group.
  • the mice were injected with PBS, CDDP, NRs or NRs/Pt through the tail vein, in which the doses of platinum and NRs were 10 mg/ kg and 25mg/kg.
  • Administration was given once every two days, and the body weight and tumor volume of the mice were recorded at the same time.
  • Figure 29 is a picture of the in vivo treatment experiment of nanoparticles in PDX model mice
  • Figure 30 is a western blot picture of p53 in the tumors of each group of mice in the PDX model mice
  • Figure 31 is a picture of the tumors of each group of mice in the PDX model mice Immunohistochemistry and TUNEL staining of p53 in sections.
  • the body weight of the mice in the CDDP group was slightly lower than that of other mice, but there was no significant difference in the body weight of the mice among the four treatment groups. This preliminarily shows that the various treatments have no obvious effect on the mice. toxic side effect.
  • the tumor volume of mice in the PBS-treated group reached more than 1000 mm2 at the end of treatment, while the tumor volume growth of mice in the NRs-treated group and CDDP-treated group slowed down significantly, and NRs/ The average tumor volume of mice in the Pt-treated group was only about 300 mm 2 , proving that NRs/Pt further enhanced the tumor inhibitory effect of NRs nanoparticles.
  • the tumor weight plot of mice in each group in Figure 29c shows that compared with the PBS group, the tumor weight of the NRs alone treatment group was significantly reduced, and the tumor weight of the NRs/Pt group was further reduced.
  • the NRs treatment group and the NRs/Pt treatment group effectively reduced the mutant p53 protein level in the tumor tissue of PDX model mice, while enhancing the autophagy level in the tumor tissue; finally, the tumor tissue sections in Figure 31
  • the results of immunohistochemistry and TUNEL staining showed that the level of mutant p53 protein in tumor tissues of the NRs-treated group and the NRs/Pt(IV)-treated group was significantly reduced, while the level of cell apoptosis was significantly enhanced.
  • PC-12 and Neuro 2A cells were seeded in a 24-well cell culture plate (density approximately 5 ⁇ 10 4 /well) and cultured overnight. Lipofectamine 3000 transfection reagent was used to transfect GFP-Htt(Q74) plasmid into PC-12 and Neuro 2A cells respectively, to obtain PC-12/GFP-Htt(Q74) stably expressing GFP-Htt(Q74) mutant huntingtin protein. and Neuro 2A/GFP-Htt(Q74) cells.
  • Figure 32 is a western blot diagram of NRs-Htt causing autophagy effects and degrading GFP-Htt (Q74) protein in PC-12/GFP-Htt(Q74) and Neuro 2A/GFP-Htt(Q74) cells;
  • NRs-Htt can effectively enhance the level of autophagy-related LC3II protein, trigger the autophagy effect, and reduce the levels of autophagy in PC-12/GFP-Htt (Q74) and Neuro 2A/GFP-Htt (Q74) cells. Mutant huntingtin protein levels.
  • Figure 32 is an immunofluorescence image of NRs-Htt degrading GFP-Htt(Q74) protein in PC-12/GFP-Htt(Q74) and Neuro 2A/GFP-Htt(Q74) cells.
  • NRs-Htt can effectively reduce the level of mutant huntingtin protein in PC-12/GFP-Htt (Q74) and Neuro 2A/GFP-Htt (Q74) cells.

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Abstract

提供一种用于靶向降解致病蛋白的纳米复合体及其制备方法和应用，属于纳米生物药物技术领域。提供用于靶向降解致病蛋白的纳米复合体，其含有纳米载体以及接枝于纳米载体上的蛋白靶向结合肽，其中，纳米载体为马来酰亚胺-聚乙二醇-聚乳酸与阳离子脂质的纳米组装体；在另一方面提供纳米复合体用于制备包括抗肿瘤纳米药物和亨廷顿舞蹈症抑制药物中的应用。该纳米复合体可以模拟选择性自噬途径中的关键受体蛋白，从而能够将待降解致病蛋白带到自噬体中通过自噬途径降解，有效解决PROTACs不能降解大分子量蛋白聚集体，以及LYTACs不能降解细胞质蛋白的问题。

Description

靶向降解致病蛋白的纳米复合体及其制备方法和应用 技术领域
本申请属于纳米生物药物技术领域，尤其涉及一种靶向降解致病蛋白的纳米复合体及其制备方法和应用。
背景技术
蛋白质稳态异常和致病蛋白积累是引发肿瘤、亨廷顿舞蹈症等多种疾病的诱因。因此，诱导致病蛋白降解，降低致病蛋白水平是治疗相关疾病的有效办法。
目前，相关技术中可以使用蛋白酶体靶向嵌合体(PROTACs)或溶酶体靶向蛋白嵌合体(LYTACs)促进靶蛋白通过蛋白酶体或溶酶体途径降解。
但是，受限于蛋白酶体的狭窄空腔，使得PROTACs不能有效降解某些大分子量蛋白或蛋白聚集体，LYTACs则只能用于分泌蛋白和细胞膜蛋白的降解，无法降解细胞质蛋白。
发明内容
本申请实施例公开一种靶向降解致病蛋白的纳米复合体及其制备方法和应用，旨在解决PROTACs不能有效降解大分子量的致病蛋白或蛋白聚集体，以及LYTACs不能降解细胞质蛋白的技术问题。
为实现上述目的，第一方面提供一种靶向降解致病蛋白的纳米复合体，其含有纳米载体以及接枝于纳米载体上的致病蛋白靶向结合肽。其中，纳米载体为马来酰亚胺-聚乙二醇-聚乳酸与阳离子脂质的纳米组装体。
本申请实施例将致病蛋白靶向结合肽接枝于阳离子脂质与马来酰亚胺-聚乙二醇-聚乳酸的纳米组装载体上，从而使纳米组装载体与致病蛋白靶向结合肽各自发挥作用的基础上产生相互协同作用，具体的如马来酰亚胺-聚乙二醇-聚乳酸既可结合阳离子脂质共同组装形成纳米组装载体，又可以使致病蛋白靶向结合肽通过“巯基-马来酰亚胺”高效点击并以共价键接枝于纳米组装载体上，从而获得体系稳定的纳米复合体。特别地，基于阳离子脂质在细胞内可以诱导显著的细胞自噬效应，并能促进自噬体的形成和积累的特点，我们深入研究马来酰亚胺-聚乙二醇-聚乳酸与阳离子脂质的纳米组装载体被摄取到细胞内是否可以提高自噬体的数量并被自噬体有效吞噬，结果显示马来酰亚胺-聚乙二醇-聚乳酸的介入更能促进阳离子脂质的相关功能。因此，我们将致病蛋白靶向结合肽接枝于纳米组装载体表面形成纳米复合体，以期通过特异性结合致病蛋白并介导其通过自噬途径降解，结果显示纳米复合体可以有效将致病蛋白带入自噬体中高效降解，可有效解决PROTACs不能降解大分子量蛋白聚集体以及LYTACs不能降解细胞质蛋白的问题。同时，进一步的研究表明纳米复合体诱导肿瘤细胞中致病蛋白降解的过程是一种自噬依赖和泛素化依赖的过程，并且纳米复合体对致病蛋白的降解能够抑制肿瘤细胞中突变蛋白的获得性功能(GOF)，例如抑制肿瘤细胞的增殖和迁移能力、增强肿瘤细胞死亡、增强肿瘤细胞对抗肿瘤活性成分的敏感性等。此外，该纳米复合体不仅与致病蛋白具有良好的结合性，而且可以有效降低对正常细胞毒性，可用于诱导不同类型的致病蛋白降解，具有广谱性。
在一些实施方案中，本申请实施例纳米载体的制备方法包括：
使阳离子脂质和马来酰亚胺-聚乙二醇-聚乳酸溶于有机溶剂后，将有机溶剂滴入到缓冲液中搅拌，即可获得纳米载体溶液。其中，可以将纳米载体溶液实施包括旋蒸除溶剂，以及离心后取上清液的纯化处置。离心条件为：在2000-4000rpm/min的转速下离心处置3-10min，具体为在3000rpm/min的转速下离心处置5min。
需要说明的是，有机溶剂优选为四氢呋喃,从而有利于马来酰亚胺-聚乙二醇-聚乳酸和阳离子脂质的溶解混合；缓冲液优选为pH＝7.4的0.01M的PBS缓冲液，从而可以模拟正常生理环境以制成等渗的纳米载体溶液。
在一些实施方案中，阳离子脂质包括1,2-二油酰-3-三甲基铵-丙烷(DOTAP)、2,3-二油酰氧丙基-1-溴化三甲胺(DOTMA)。其中，优选为1,2-二油酰-3-三甲基铵-丙烷，以获得最佳的细胞自噬效应。
在一些实施方案中，马来酰亚胺-聚乙二醇-聚乳酸中的嵌段“聚乙二醇/聚乳酸”的分子量为1000-3000。比如，可以为1000、2000和3000等。
在一些实施方案中，阳离子脂质与马来酰亚胺-聚乙二醇-聚乳酸的质量比为(5-40)：100。其中，该质量比有利于提高纳米载体的诱导细胞自噬水平。
在一些实施方案中，致病蛋白选自突变p53蛋白和突变亨廷顿蛋白中的一种。其中，突变p53蛋白含有S241F、R175H、R248W、R280K突变位点中的至少一个；突变亨廷顿蛋白包括Htt-(Q74)。
需要说明的是，p53蛋白的来源可以为ES-2、MDA-MB-231、MIAPaCa-2和SK-BR-3等细胞；亨廷顿蛋白的来源可以为PC-12/GFP-Htt(Q74)和Neuro2A/GFP-Htt(Q74)等细胞。
需要说明的是，本申请实施例纳米复合体用于突变p53蛋白的降解时，还可以有效抑制肿瘤细胞中突变蛋白的获得性功能(GOF)，比如抑制肿瘤细胞的增殖和迁移能力、增强肿瘤细胞死亡以及增强肿瘤细胞对药物活性成分的敏感性等。
在一些实施方案中，突变p53蛋白靶向结合肽具备SEQ ID NO.1的氨基酸序列；突变亨廷顿蛋白靶向结合肽具备SEQ ID NO.2的氨基酸序列，该两个靶向结合肽可以高效地靶向结合目标蛋白，并且分子结构中的“巯基”可使其与 马来酰亚胺-聚乙二醇-聚乳酸通过“巯基-马来酰亚胺”高效点击，使得靶向结合肽高效且稳定的接枝于纳米载体上。
第二方面提供一种所述纳米复合体的制备方法，包括：
使致病蛋白靶向结合肽与含有纳米载体的溶液发生点击反应，并使反应产物经包括透析、超滤和浓缩的纯化处置，即得所述纳米复合体。其中，透析方法包括：
将反应产物转移至透析袋(MWCO＝14000Da)中后，置于PBS溶液(pH＝7.4，浓度0.01M)中进行透析。
在一些实施方案中，致病蛋白靶向结合肽与纳米载体的摩尔比为(1-3)：1。其中，该摩尔比有利于促使肽高效且充足的装载于纳米载体表面，提高针对靶标蛋白的捕获效果。
第三方面提供所述纳米复合体用于制备包括抗肿瘤纳米药物和亨廷顿舞蹈症抑制药物中的应用。
其中，基于该纳米复合体具备能够使致病蛋白聚集体被带到自噬体中通过自噬途径降解的性能。因此，该纳米复合体用于制备包括抗肿瘤纳米药物后，能够有效抑制肿瘤细胞的增殖和迁移能力，增强肿瘤细胞死亡，以及增强肿瘤细胞对活性成分如顺铂(CDDP)的敏感性，显示出良好的协同抑制效果。
第四方面提供一种抗肿瘤纳米药物，该抗肿瘤纳米药物含有纳米复合体以及负载于纳米复合体中的抗肿瘤活性成分。
在一些实施方案中，抗肿瘤活性成分包括顺铂、Pt(IV)前药。其中，Pt(IV)前药可根据文献Stimuli-responsive clustered nanoparticles for improved tumor penetration andtherapeutic efficacy制备。
其中，将抗肿瘤活性成分负载于纳米复合体上，不仅能够增强肿瘤细胞对抗肿瘤纳米药物的敏感性，而且抗肿瘤纳米药物可与患者来源的异种移植(PDX)卵巢癌显示出良好的协同抗肿瘤效果。
第五方面提供一种抗肿瘤纳米药物的制备方法，包括：
使含有马来酰亚胺-聚乙二醇-聚乳酸、阳离子脂质和抗肿瘤活性成分的溶液在缓冲液中进行自组装，透析，得到含有载药纳米颗粒的溶液；
使含有载药纳米颗粒的溶液与致病蛋白靶向结合肽发生点击反应，并对反应产物依次进行透析、超滤和浓缩，得到抗肿瘤纳米药物。
其中，第一次透析可以有效除去未反应的抗肿瘤活性成分；第二次透析可以有效除去未反应的肽。
与现有技术相比，本申请实施例的优点或有益效果至少包括：
本申请实施例纳米复合体通过使致病蛋白靶向结合肽接枝于由阳离子脂质和马来酰亚胺-聚乙二醇-聚乳酸共同组装的纳米组装载体上，一方面能够使得纳米复合体可以模拟选择性自噬途径中的关键受体蛋白，从而使得致病蛋白被带到自噬体中并通过自噬途径降解，可有效解决PROTACs不能降解大分子量蛋白聚集体以及LYTACs不能降解细胞质蛋白的问题；另一方面能够使纳米复合体与多种致病蛋白形成良好的结合性，并有效降低对正常细胞毒性，可用于诱导不同类型的致病蛋白降解，具有广谱性。
附图说明
图1为NRs的结构示意图；
图2为DOTAP诱导MDA-MB-231-EGFP-LC3细胞自噬效应的荧光图；
图3为含中性脂质、阴离子脂质和阳离子脂质的纳米复合体降解突变p53蛋白的western blot图；
图4为mNRs、dNRs和NRs的粒径分布图和Zeta电势图；
图5为mNRs、dNRs和NRs在含10％FBS的PBS溶液中的粒径分布图；
图6为mNRs、dNRs和NRs的透射电镜图；
图7为PBS、MBP、dNRs和NRs处理后突变p53蛋白的圆二色光谱图；
图8为不同处理后突变p53蛋白的SPR图谱；
图9为NRs的STORM电镜图；
图10为含不同浓度DOTAP的NRs在ES-2细胞中降解突变p53蛋白的western blot图；
图11为含不同浓度DOTAP的NRs在HEK 293T细胞中的细胞活力；
图12为MBP、mNRs、dNRs和NRs在ES-2细胞中降解突变p53蛋白的免疫荧光图；
图13为NRs处理后ES-2细胞中p53的mRNA变化图；
图14为NRs在ES-2、MDA-MB-231、MIAPaCa-2和SK-BR-3细胞中降解突变p53蛋白的western blot图；
图15为检测NRs在ES-2细胞中降解突变p53蛋白的具体降解通路的western blot图；
图16为检测NRs降解降解ES-2细胞中突变p53蛋白过程中的泛素化依赖性的western blot图；
图17为NRs引发ES-2细胞发生自噬效应的生物电镜图；
图18为NRs引发ES-2细胞发生完整性自噬的荧光图；
图19为NRs引发ES-2细胞中突变p53蛋白降解需要泛素化酶参与的western blot图；
图20为NRs引发ES-2细胞中泛素化水平升高的western blot图；
图21为NRs处理后HUVEC、H1299、HEK 293T、A549、HCT 116、MIAPaCa-2、SK-BR-3、MDA-MB-231和ES-2的细胞活力；
图22为MBP、mNRs、dNRs和NRs在ES-2细胞中的成球实验图；
图23为MBP、mNRs、dNRs和NRs在ES-2细胞中的Transwell结果图；
图24为MBP、mNRs、dNRs和NRs在ES-2细胞中的Hoechst/PI双染图；
图25为DiD-NRs在ES-2荷瘤小鼠的生物分布图；
图26为纳米颗粒在ES-2荷瘤小鼠的体内治疗实验图；
图27为ES-2荷瘤小鼠中各组小鼠肿瘤中p53的western blot图；
图28为ES-2荷瘤小鼠中各组小鼠肿瘤切片中p53和LC3的免疫荧光图；
图29为纳米颗粒在PDX模型小鼠的体内治疗实验图；
图30为PDX模型小鼠中各组小鼠肿瘤中p53的western blot图；
图31为PDX模型小鼠中各组小鼠肿瘤切片中p53的免疫组化以及TUNEL染色图；
图32为NRs-Htt在PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中引起细胞自噬效应并降解GFP-Htt(Q74)蛋白的western blot图；
图33为NRs-Htt在PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中降解GFP-Htt(Q74)蛋白的免疫荧光图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本申请的一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
实施例1
本实施例提供一种靶向突变蛋白的纳米复合体NRs的制备方法，具体包括：
S101-纳米载体dNRs的制备：
将10mg的Mal-PEG2000-PLA2000、2.0mg的阳离子脂质DOTAP溶于1.2mL的四氢呋喃并转移至5mL的EP管中，涡旋混合均匀，得到混合溶液；
搅拌下，将混合溶液逐滴滴入10mL的PBS(pH＝7.4，0.01M，下同)中，室温下继续搅拌2h，得到纳米颗粒溶液；
利用旋转蒸发仪除去纳米颗粒溶液中的四氢呋喃，并在3000rpm/min的转速下离心5min之后，取上清，获得纳米载体dNRs溶液(NPs@DOTAP)；
S102-纳米复合体NRs的制备：
将5.0mg的SEQ ID NO.1所示的MBP肽加入到dNRs溶液中，并在室温下搅拌反应8h，得到含杂产物NRs(MBP-NPs@DOTAP)；
将含杂产物NRs转移至透析袋(MWCO＝14000Da)，并置于2L的PBS溶液中透析过夜后，将NRs溶液经AmiconYM-30离心过滤装置(MWCO＝5000Da)超滤和浓缩，获得图1所示的纯化纳米复合体NRs(MBP-NPs@DOTAP)。其中，图1为NRs的结构示意图。
根据图1可知，纳米复合体由Mal-PEG2000-PLA2000与阳离子脂质的自组装载体颗粒和修饰在自组装载体颗粒表面的致病蛋白结合肽组成。
其中，本实施例将DOTAP分别替换为中性脂质(GTP、PL)、阴离子脂质(DPPG、DOPG)和阳离子脂质(DOTMA)后，制备出纳米复合体NPs@GTP、NPs@PL、NPs@DPPG、NPs@DOPG和NPs@DOTMA。
本实施例调整DOTAP的加入量，并参考实施例1的方法制备出纳米复合体mNRs(NRs@0％DOTAP)、NRs@5％DOTAP、NRs@10％DOTAP、NRs@20％D-OTAP和NRs@40％DOTAP。
以及，本实施例1将5.0mg的SEQ ID NO.2所示的HBP肽加入到dNRs溶液中，制得纳米复合体NRs-Htt(HBP-NPs@DOTAP)。
实施例2
本实施例提供一种抗肿瘤纳米药物NRs/Pt(IV)的制备方法，具体包括：
S201：将10mg的Mal-PEG2000-PLA2000、2.0mg的阳离子脂质DOTAP溶于1.2mL的四氢呋喃，并加入1mg的Pt(IV)前药(DMSO，10mg/mL)后，转移至5mL的EP管中，涡旋混合均匀，得到载药混合溶液；
S202：搅拌下，将载药混合溶液逐滴滴入10mL的PBS中，室温下继续搅拌2h，得到载药纳米颗粒溶液；
S203：利用旋转蒸发仪除去载药颗粒溶液中的四氢呋喃并转移至透析袋(MWCO＝14000Da)透析8h，得到纳米颗粒dNRs/Pt(IV)；
S204：将MBP肽(5.0mg，0.0025mM)加入到dNRs/Pt(IV)溶液中，并在室温下搅拌反应8h，得到含杂NRs/Pt(IV)；
S205：将NRs/Pt(IV)转移至透析袋(MWCO＝14000Da)后，置于2.0L的PBS溶液中透析过夜，并将载药纳米颗粒经AmiconYM-30离心过滤装置超滤和浓缩，获得纯化NRs/Pt(IV)。
各种纳米复合体的测试。其中，测试中所用的化学试剂、生物原料等市购获得，并且试剂浓度、过夜培养等参照常规要求，例如过夜培养(37℃，5％CO2)。
1.阳离子脂质诱导细胞自噬的能力测试
使MDA-MB-231细胞转染EGFP-LC3质粒获得MDA-MB-231-EGFP-LC3细胞后，接种于放有圆形爬片的24孔细胞培养板(密度约3×104/孔)中，过夜培养，并分别加入PBS和DOTAP(2μg/mL)，培养6h后，荧光检测，结果为图2所示。其中，图2为DOTAP诱导MDA-MB-231-EGFP-LC3细胞自噬效应的荧光图。
根据图2可知，DOTAP处理后，细胞中的LC3绿色荧光蛋白出现了明显的点状聚集，证明DOTAP能够诱导细胞发生显著的自噬效应。
2.含中性脂质、阴离子脂质和阳离子脂质的纳米复合体分别诱导细胞自噬能力对比
将MDA-MB-231-EGFP-LC3细胞接种于24孔细胞培养板(密度约3×104/孔)中，过夜培养，并分别加入NPs@GTP、NPs@PL、NPs@DPPG、NPs@DOPG、NPs@DOTMA和NPs@DOTAP(dNRs)，培养6h后，western blot检测细胞中的LC3蛋白水平，结果为图3所示。其中，图3为含中性脂质、阴离子脂质和阳离子脂质的纳米复合体降解突变p53蛋白的western blot图。
根据图3可知，含中性脂质(GTP、PL)和阴离子脂质(DPPG、DOPG)的纳米复合体处理后的细胞中LC3-II型蛋白没有进一步积累，而含阳离子脂质(DOTMA、DOTAP)的纳米复合体处理后的LC3-II型蛋白明显增多，但含DOTAP的纳米复合体增多最为明显，证明含阳离子脂质的纳米复合体可以诱导细胞发生明显的自噬效应，并且DOTAP诱导的细胞自噬效应最佳。
3.mNRs、dNRs和NRs粒径和表面电势的测定
将mNRs、dNRs和NRs分别以1.0mg/mL的浓度分散于超纯水中后，纳米粒度仪测定粒径和表面电势，结果为图4所示。其中，图4a为mNRs、dNRs和NRs的粒径分布图；图4b为mNRs、dNRs和NRs的Zeta电势图。
根据图4a可知，dNRs、mNRs和NRs的粒径均在100nm左右；根据图4b可知，mNRs呈负电性，含DOTAP的dNRs电势呈较强的正电性，其电势约为+15mV左右，其是因为组分中含有正电性阳离子脂质DOTAP。此外，NRs也呈较强的表面正电性。
4.mNRs、dNRs和NRs稳定性表征
将mNRs、dNRs和NRs分别以1.0mg/mL的浓度分散在含有10％胎牛血清(FBS)的PBS溶液中，并在37℃的温度下分别孵育0、1、2、4、8、12和24h时，纳米粒度仪测定其粒径，结果为图5所示。其中，图5为mNRs、dNRs和NRs在含10％FBS的PBS溶液中的粒径分布图。
根据图5可知，mNRs、dNRs和NRs均在24h内粒径均无明显的变化，表明mNRs、dNRs和NRs在含10％FBS的PBS溶液中具有良好的颗粒稳定性。
5.mNRs、dNRs和NRs形貌表征
分别取10μL的mNRs、dNRs或1.0mg/mL的NRs溶液滴在400目的铜网中，并置于洁净干燥处过夜晾干，然后使用透射电子显微镜观察形貌，结果为图6所示。其中，图6为mNRs、dNRs和NRs的透射电镜图。
根据图6可知，mNRs、dNRs和NRs在电镜下均呈完整的球形形貌，且其尺寸都在100nm左右。
6.圆二色光谱扫描检测NRs和突变蛋白的结合
取1.25mM的突变p53S241F蛋白(Mutp53)溶液，加入等体积的PBS以及MBP肽(1.25mM)、dNRs(5mg/mL)或NRs(5mg/mL)混合后，室温下搅拌结合2h，然后分别将各组混合溶液转移至石英比色皿中，用Chirascan圆二色光谱仪检测突变p53蛋白的圆二色谱(光谱条件：温度为65℃；光谱检测范围为195-260nm)，结果为图7所示。其中，图7为PBS、MBP、dNRs和NRs处理后突变p53蛋白的圆二色光谱图。
根据图7可知，单独Mutp53组以及dNRs与Mutp53共孵育实验组中，突变p53蛋白的光谱在208nm和222nm处均出现两个负峰，其对应突变p53蛋白质的二级结构；而与NRs一起孵育后的突变p53的光谱在231nm处出现最小峰值，表明突变p53蛋白的二级结构发生了明显的转变，证明NRs可以与突变p53蛋白结合，导致其二级结构发生变化。
7.表面等离子共振法(SPR)检测NRs和突变蛋白的结合
使用GE BIAcore 8K仪器在25℃恒温下操作，每个CM5传感器芯片由8个相同的实验通道组成，每个通道分为两个流动池(flow-cell 1和flow-cell 2)。以flow-cell 1(Fc1)始终保持空白作为参考，而flow-cell 2(Fc2)则用于研究NRs与突变型p53蛋白的相互作用。
首先，使用PBS-T缓冲液(20mM的Na-phosphate，150mM的NaCl，0.05％的Tween 20，pH＝7.4)平衡体系；其次，使用EDC(0.2M)和NHS(0.05M)的混合溶液冲洗实验通道6min活化传感器上的羧基；再次，向Fc2池中注射NRs(10mM的乙酸缓冲液，pH＝4.0)7min，通过NRs表面多肽对的氨基与羧基反应在传感器芯片上固定NRs；最后，在Fc1和Fc2池中分别注入1M乙醇胺-HCl溶液，阻断剩余的羧基活性酯。
为观察突变p53蛋白(p53S241F)与NRs间的相互作用，将5.21μM突变p53蛋白溶液注射到NRs修饰的传感器表面。注射时间为3min，流速为30μL/min，然后是5min的解离步骤。为了计算结合常数(Kd值)，在相同的条件下，分别将浓度为2500nM、500nM、100nM、20nM和4nM的突变p53蛋白注入通道，每个周期内都测定结合信合。最后用BIAcore 8K Evaluation软件以1：1结合模型计算NRs与突变p53蛋白间的结合动力学常数。结果为图8所示。其中，图8为不同处理后突变p53蛋白的SPR图谱。
根据图8可知，突变p53S241F蛋白和NRs之间的结合等温线符合标准的1:1结合模型，并且NRs在各种蛋白质浓度下均可有效结合突变p53。进一步，通过单循环动力学测定突变p53蛋白和NRs之间相互作用的Kd值为1.40×10-9。相比之下，无MBP肽修饰的dNRs和突变p53之间的结合效率远低于NRs和突变p53之间的结合效率，表明NRs可以通过特定的高亲和力相互作用有效地结合突变p53。
8.随机光学重建显微镜(STORM)检测NRs纳米颗粒和突变蛋白的结合
为直观地观察NRs表面MBP肽的修饰情况，以及突变p53蛋白(p53S241F)与NRs的结合情况。参照实施例1的制备过程，在制备NRs的过程中掺入罗丹明B(RhoB)标记的PEG-b-PLA(RhoB-PEG-b-PLA)，得到RhoB(红色荧光)标记的纳米颗粒；加入Aleax fluorTM 488-NHS标记的MBP通过点击反应制得RhoB标记内核、Aleax fluorTM 488标记表面MBP的双荧光NRs；将Aleax fluorTM 647-NHS标记的突变p53蛋白加入双荧光NRs中，室温搅拌混合4h后转移至透析袋(MWCO＝14000Da)透析除去未反应的MBP肽；将荧光标记的NRs和突变p53蛋白的混合溶液加入玻底培养皿中，静置10min，使用随机光学重建显微镜采集488nm、530nm和647nm通道的荧光图像，结果为图9所示。其中，图9为NRs的STORM电镜图。
根据图9可知，红色荧光RhoB标记的NPs周围观察到明显的绿色荧光，表明MBP肽被成功修饰在NRs表面。而将Aleax fluorTM 647标记的突变p53蛋白与NRs溶液混合后，突变p53蛋白的蓝色荧光与MBP肽的绿色荧光发生具有较明显的共定位现象，分散在NRs的周围，表明NRs可以通过MBP肽和突变p53蛋白之间的高亲和力相互作用而有效结合突变p53蛋白。
9.NRs诱导突变p53蛋白降解实验
9.1含不同浓度DOTAP的NRs清除突变p53蛋白能力评估
将ES-2细胞接种在24孔细胞培养板(密度约5×104/孔)中，过夜培养。分别加入PBS和含不同浓度DOTAP(0％、5％、10％、20％和40％)的NRs，培养12h后，进行western blot检测，结果为图10所示。其中，图10为含不同浓度DOTAP的NRs在ES-2细胞中降解突变p53蛋白的western blot图。
根据图10可知，随着DOTAP浓度的升高，NRs在ES-2细胞中清除突变p53蛋白的效果逐渐增强。
9.2含不同浓度DOTAP的NRs对正常细胞的毒性评估
将HEK 293T细胞接种在96孔细胞培养板(密度约为1×104/孔)中，过夜培养。加入PBS和不同浓度DOTAP(0％、5％、10％、20％和40％)的NRs，培养24h后，进行MTT检测，结果为图11所示。其中，图11为含不同浓度DOTAP的NRs在HEK 293T细胞中的细胞活力。
根据图11可知，与其他组相比，含40％DOTAP的NRs在HEK 293T细胞中细胞存活率明显降低，说明含40％DOTAP的NRs对正常细胞的毒性较大，所以我们均选用毒性较低、清除突变p53蛋白效果较佳的含20％DOTAP的NRs进行后续实验。
9.3游离MBP肽、mNRs、dNRs和NRs清除突变p53蛋白能力评估
将ES-2细胞接种在预前放有圆形爬片的24孔细胞培养板(密度约3×104/孔)中，过夜培养。分别加入PBS、游离的MBP肽(250μg/mL)以及浓度均为500μg/mL的mNRs、dNRs和NRs，培养6h后，进行免疫荧光检测，结果为图12所示。其中，图12为MBP、mNRs、dNRs和NRs在ES-2细胞中降解突变p53蛋白的免疫荧光图。
根据图12可知，相比于其他组，NRs处理组中标记突变p53蛋白的红色荧光明显降低，证明NRs可以有效降低ES-2细胞中的突变p53蛋白水平。
9.4测试NRs是否会影响突变p53的mRNA水平
将ES-2细胞接种在6孔细胞培养板(密度约3×105/孔)中，过夜培养。分别加入PBS和NRs(500μg/mL)，培养12h后，使用TRIzol RNA提取试剂(Invitrogen)提取RNA，并使用RT-PCR法检测p53的mRNA水平，结果为图13所示。其中，图13为NRs处理后ES-2细胞中p53的mRNA变化图。
根据图13可知，和PBS组相比，NRs处理不影响突变p53的mRNA水平，证明NRs通过降解途径清除ES-2细胞中的突变p53蛋白。
9.5测试NRs降解不同类型突变p53的广谱性
将ES-2、MDA-MB-231、MIAPaCa-2和SK-BR-3细胞接种于24孔细胞培养板(密度约5×104/孔)中，过夜培养。分别加入PBS和250μg/mL、500μg/mL的NRs，培养12h后，western blot检测突变p53蛋白的水平，结果为图14所示。其中，图14为NRs在ES-2、MDA-MB-231、MIAPaCa-2和SK-BR-3细胞中降解突变p53蛋白的western blot图。
根据图14可知，随着NRs浓度的增加，四种含不同p53突变位点的肿瘤细胞中突变p53蛋白均逐渐减少，证明NRs可降解多种不同类型的突变p53，具有一定的广谱性。
10.NRs通过自噬依赖和泛素化依赖的方式诱导突变p53蛋白降解
10.1测试NRs降解细胞中突变p53蛋白的途径
将ES-2细胞接种于24孔细胞培养板(密度约5×104/孔)中，过夜培养。分别加入PBS、浓度为500μg/mL的NRs、自噬抑制剂Wortmannin(简写为Wort，1μM)、500μg/mL的NRs+Wort、蛋白酶体抑制剂MG-132(10μM)、500μg/mL的NRs+MG-132(10μM)，培养12h后，进行western blot检测，结果为图15所示。其中，图15为检测NRs在ES-2细胞中降解突变p53蛋白的具体降解通路的western blot图。
根据图15可知，自噬抑制剂Wortmannin能有效抑制NRs引发的突变p53蛋白降解，而蛋白酶体抑制剂MG-132则不能。说明NRs引发的突变p53蛋白的降解是通过自噬途径介导的。
10.2测试NRs降解突变p53的自噬依赖性
将HEK293T细胞接种在6孔细胞培养板(密度约3×105/孔)中，过夜培养，使用lipofectamine 3000将对照shRNA(shNC)以及用于敲除ATG5的shATG5与慢病毒载体体系共转染到HEK 293T细胞中。转染48h后，收集病毒上清液感染ES-2细胞。24h后，将病毒感染后的ES-2细胞接种于24孔板中，分别用PBS或500μg/mL的NRs处理12h，并进行western blot检测，结果如图16所示。其中，图16为检测NRs降解突变p53蛋白过程中的自噬依赖性的western blot图。
根据图16可知，与对照shNC组相比，慢病毒感染后，ES-2细胞中ATG5蛋白表达水平显著降低，且敲除自噬相关蛋白ATG5后明显抑制了NRs引发的突变p53蛋白的降解，证明NRs降解突变p53蛋白是自噬依赖性的。
10.3测试NRs诱导细胞自噬的能力
将ES-2细胞接种于10mm细胞培养皿(密度约5×106/皿)中，过夜培养，分别加入PBS和浓度为500μg/mL的NRs。培养12h后，收集细胞沉淀，使用电镜固定液在4℃固定过夜。然后将细胞包埋并切成超薄切片，醋酸双氧铀和柠檬酸铅染色，并使用生物电镜观察细胞质中的自噬体，结果为图17所示。其中，图17为NRs引发ES-2细胞发生自噬效应的生物电镜图。
根据图17可知，NRs处理后的ES-2细胞中，自噬体的数量明显增加，证明NRs能够有效诱导细胞发生自噬效应。
10.4测试NRs诱导细胞自噬的完整性
将MDA-MB-231细胞转染EGFP-mCherry-LC3质粒，获得MDA-MB-231-EGFP-mCherry-LC3细胞，将其接种在预前放有圆形爬片的24孔细胞培养板(密度约3×104/孔)中，过夜培养，分别加入浓度为500μg/mL的DiD-mNRs和DiD-NRs(其中，参照实施例1中mNRs和NRs的方法，制备时掺入DiD荧光染料，得到DiD标记的DiD-mNRs和DiD-NRs)，培养12h后，进行荧光检测，结果为图18所示。其中，图18为NRs引发ES-2细胞发生完整性自噬的荧光图。
根据图18可知，mNRs处理的细胞中，表示LC3蛋白的mCherry(红色荧光)和EGFP(绿色荧光)出现明显的共定位现象；而NRs处理的细胞中，EGFP荧光由于在自噬溶酶体的酸性条件下容易淬灭，所以绿色荧光明显减少，而红色荧光则比较稳定，荧光强度不变，证明NRs诱导的细胞自噬是一个完整的过程。
10.5测试NRs降解突变p53的泛素化依赖性
将ES-2细胞接种在24孔细胞培养板(密度约5×104个/孔)中，过夜培养，分别加入PBS、浓度为500μg/mL的NRs、PYR-41(5μM)以及500μg/mL的NRs+PYR-41(5μM)。培养12h后，进行western blot检测，结果为图19所示。其中，图19为检测NRs降解ES-2细胞中突变p53蛋白过程中的泛素化依赖性的western blot图。
根据图19可知，泛素化酶抑制剂PYR-41能够有效抑制NRs引发的突变p53蛋白降解，说明NRs降解突变p53蛋白依赖于突变p53蛋白的泛素化修饰。
10.6测试NRs降解突变p53对蛋白泛素化的影响
将HEK 293T细胞接种在6孔细胞培养板(密度约5×105个/孔)中，过夜培养。加入PBS或浓度为500μg/mL的NRs，培养12h后，使用IP裂解液裂解细胞，并将含有等量蛋白质的细胞提取物与蛋白质A/G-琼脂糖和p53抗体(2μg每样)一起孵育过夜。用PBST缓冲液洗涤后，沉淀物在SDS-PAGE上分离，并用Ub抗体、K63-Ub抗体进行western blot分析，结果为图20所示。其中，图20为NRs引发ES-2细胞中泛素化水平升高的western blot图。
根据图20可知，与对照组相比，NRs处理后细胞中突变p53蛋白的总体泛素化和K63泛素化水平明显升高。
11.NRs降解突变p53抑制突变p53的获得性功能(GOF)
11.1测试NRs对多种细胞活力的影响
将正常细胞(HUVEC和HEK 293T)、不表达p53的肿瘤细胞(H1299)、表达野生型p53蛋白的肿瘤细胞(A549和HCT 116)以及表达突变p53蛋白的肿瘤细胞(MIAPaCa-2、SK-BR-3、MDA-MB-231和ES-2)分别接种在96孔细胞培养板(密度约1×104/孔)中，过夜培养。加入PBS和500μg/mL的NRs，培养24h后，进行MTT检测，结果为图21所示。其中，图21为NRs处理后的HUVEC、H1299、HEK 293T、A549、HCT 116、MIAPaCa-2、SK-BR-3、MDA-MB-231和ES-2的细胞活力。
根据图21可知，NRs对正常细胞以及不表达p53和表达野生型p53蛋白的肿瘤细胞的活力几乎没有影响，但特异性地降低表达突变p53蛋白的肿瘤细胞的活力，其中在ES-2细胞中对细胞活力的降低效应最为明显。
11.2测试游离的MBP、mNRs、dNRs和NRs对ES-2细胞增殖能力评估
将ES-2细胞接种到含有无血清培养基的24孔低黏附细胞培养板(密度1×103/孔)中，并添加2％的B27、碱性成纤维细胞生长因子(20ng/mL)和表皮生长因子(20ng/mL)。分别加入PBS、游离的MBP肽(250μg/mL)、mNRs(500μg/mL)、dNRs(500μg/mL)和NRs(500μg/mL)，培养12h后，移除并更换为新鲜的培养基。培养7天后，使用倒置相差显微镜记录不同组中球体的直径，结果为图22所示。其中，图22为MBP、mNRs、dNRs和NRs在ES-2细胞中的成球实验图。
根据图22可知，与其他处理组相比，NRs处理后的细胞球体的直径明显较小，说明NRs可以有效抑制ES-2细胞的增殖能力。
11.3测试游离的MBP肽、mNRs、dNRs和NRs对ES-2细胞迁徙能力评估
将ES-2细胞接种到无血清培养基的上室(密度约5×103/孔)中，并将含有10％FBS的培养基添加到下室。上室中加入PBS、游离MBP肽(250μg/mL)、mNRs(500μg/mL)、dNRs(500μg/mL)和NRs(500μg/mL)，孵育12h后，将迁移到膜下表面的细胞用甲醇固定，并使用0.1％结晶紫染色。待使用PBS洗涤3次后，对细胞进行显微成像，结果为图23所示。其中，图23为MBP、mNRs、dNRs和NRs在ES-2细胞中的Transwell结果图。
根据图23可知，与其他处理组相比，NRs处理后迁移到膜下表面的细胞明显较少，说明NRs可以有效抑制ES-2细胞的迁移能力。
11.4测试游离的MBP、mNRs、dNRs和NRs对ES-2细胞死亡能力评估
将ES-2细胞接种在24孔细胞培养板(密度约5×103/孔)中，过夜培养。分别加入PBS、游离的MBP肽(250μg/mL)以及浓度均为500μg/mL的mNRs、dNRs和NRs，培养24h后，使用Hoechst(10μg/mL)染料染细胞核，碘化丙啶(10μg/mL)对死亡的细胞进行染色，然后使用荧光显微镜检测，结果为图24所示。其中，图24为MBP肽、mNRs、dNRs和NRs在ES-2细胞中的Hoechst/PI双染图。
根据图24可知，与其他处理组相比，NRs处理后的细胞中红色的荧光增强，说明NRs可以有效引发ES-2细胞死亡。
12.NRs降解突变p53在动物水平上的治疗效果
12.1体内生物分布实验
将1×107的ES-2细胞皮下注射到BALB/c裸鼠的右侧，构建ES-2荷瘤裸鼠模型。当小鼠的肿瘤体积达到约100mm3左右时，将携带ES-2异种移植物的BALB/c裸鼠通过静脉内(i.v.)注射3mg的DiD荧光染料标记的NRs/Pt(DiD-NRs/Pt，参照实施例2中NRs/Pt(IV)的制备方法，制备纳米颗粒时掺入DiD荧光染料，得到DiD标记的DiD-NRs/Pt(IV))，然后通过Xenogen IVIS Lumina系统在2、4、8、12、24和48h成像。并在注射48h后牺牲小鼠，收集主要器官和肿瘤组织进行拍照和荧光统计分析，结果为图25所示。其中，图25为DiD-NRs在ES-2荷瘤小鼠的生物分布图。
根据图25可知，小鼠肿瘤部位中纳米颗粒的荧光强度随时间的推移先逐渐升高后降低，在8h处荧光强度最高。主要器官和肿瘤的荧光图片和统计分析显示，NRs/Pt在肿瘤部位的平均荧光强度最强，说明NRs/Pt能有效在肿瘤部位富集。
12.2体内抗肿瘤治疗实验
为探究纳米颗粒在体内的抑瘤效果，我们在BALB/c裸鼠皮下构建了人源ES-2的卵巢癌模型。当肿瘤体积达到约100mm3时，将小鼠随机分为以下7组，每组6只，分别是：PBS组、dNRs组、CDDP(顺铂)组、dNRs/Pt(IV)组、NRs组、NRs+CDDP组和NRs/Pt(IV)组，其中铂、dNRs和NRs的等效注射剂量分别为1.0mg/kg、25mg/kg和25mg/kg。每隔一天给药一次，同时记录小鼠的肿瘤体积和体重，在治疗的第13天，牺牲小鼠并切除肿瘤组织，称重，并检测肿瘤组织中突变p53蛋白和自噬相关蛋白LC3的水平变化。结果为图26、27和28所示。其中图26为纳米颗粒在ES-2荷瘤小鼠的体内治疗实验图；图27为ES-2荷瘤小鼠中各组小鼠肿瘤中p53的western blot图；图28为ES-2荷瘤小鼠中各组小鼠肿瘤切片中p53和LC3的免疫荧光图。
根据图26a所示，在整个治疗过程中，各组小鼠体重未出现明显变化，证明各实验组的组分未对小鼠造成明显的全身毒性，也反映了NRs具有良好的生物相容性；而图26b的小鼠肿瘤生长曲线图和图26c的离体肿瘤图片显示，与PBS、dNRs、CDDP和dNRs/Pt(IV)组相比，NRs组和NRs+CDDP组能有效抑制肿瘤的生长，NRs/Pt(IV)组则进一步抑制了肿瘤体积的增长。此外，与肿瘤体积数据相一致，图26d中各组小鼠的肿瘤重量显示，与其他处理组相比，NRs和化疗药物CDDP联合治疗组有效抑制了肿瘤生长，NRs/Pt(IV)进一步降低了肿瘤的重量。图27和图28显示，与在细胞系中获得的结果一致，用NRs(包括NRs、NRs+CDDP和NRs/Pt组)处理后的小鼠肿瘤组织中细胞自噬水平显著增强，且突变p53蛋白水平显著降低。
同时，为初步了解NRs的潜在临床效应，将患者来源的p53突变型(P72R+/+、C141Y+/+和L350P+/-)卵巢癌样本移植于NOD/SCID小鼠皮下，构建PDX卵巢癌模型。当肿瘤体积达到约100mm3时，随机将小鼠分为4组，每组6只，通过尾静脉向小鼠注射PBS、CDDP、NRs或NRs/Pt，其中铂和NRs的剂量分别为10mg/kg和25mg/kg。每两天给药一次，同时记录小鼠的体重和肿瘤体积，在治疗的第13天，牺牲小鼠切除肿瘤组织并称重。并检测肿瘤组织中突变p53蛋白和自噬相关蛋白LC3的水平变化。结果为图29、30和31所示。其中图29为纳米颗粒在PDX模型小鼠的体内治疗实验图；图30为PDX模型小鼠中各组小鼠肿瘤中p53的western blot图；图31为PDX模型小鼠中各组小鼠肿瘤切片中p53的免疫组化以及TUNEL染色图。
根据图29a所示，CDDP组的小鼠体重较其他小鼠有轻微的降低，但四个处理组之间的小鼠体重并没有显著性差异，初步说明各种处理对小鼠并没有明显的毒副作用。图29b的小鼠肿瘤生长曲线中，我们发现，PBS处理组的小鼠在治疗结束时肿瘤体积已高达1000mm2以上，而NRs处理组和CDDP处理组的小鼠肿瘤体积增长明显减缓，NRs/Pt处理组的小鼠平均肿瘤体积只有300mm2左右，证明NRs/Pt进一步增强了NRs纳米颗粒的肿瘤抑制作用。一致地，图29c中各组小鼠的肿瘤重量图显示，与PBS组相比，单独NRs处理组的肿瘤重量明显降低，且NRs/Pt组的肿瘤重量进一步降低。
根据图30所示，NRs处理组和NRs/Pt处理组有效降低了PDX模型小鼠肿瘤组织的突变p53蛋白水平，同时增强了肿瘤组织中的自噬水平；最后，图31中肿瘤组织切片的免疫组化和TUNEL染色的结果显示，NRs处理组和NRs/Pt(IV)处理组的肿瘤组织中突变p53蛋白的水平显著降低，而细胞凋亡水平显著增强。
13.NRs-Htt诱导突变亨廷顿蛋白降解实验
将PC-12和Neuro 2A细胞接种在24孔细胞培养板(密度约5×104/孔)中，过夜培养。使用Lipofectamine 3000转染试剂将GFP-Htt(Q74)质粒分别转染到PC-12和Neuro 2A细胞中，得到稳定表达GFP-Htt(Q74)突变亨廷顿蛋白的PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞。
将上述两种细胞分别接种于24孔细胞培养板(密度约3×104/孔)中，过夜培养。分别加入PBS和NRs-Htt(50或100μg/mL)，培养36h，进行western blot检测突变亨廷顿蛋白和自噬相关LC3蛋白的水平，结果为图32所示。其中，图32为NRs-Htt在PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中引起细胞自噬效应并降解GFP-Htt(Q74)蛋白的western blot图；
根据图32可知，NRs-Htt可以有效增强自噬相关LC3II蛋白的水平，引发细胞自噬效应，并降低PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中的突变亨廷顿蛋白水平。
将上述两种细胞分别接种于预前放有圆形爬片的24孔细胞培养板(密度约3×104/孔)中，过夜培养。分别加入PBS和NRs-Htt(100μg/mL)，培养36h后，进行免疫荧光检测，结果为图33所示。其中，图32为NRs-Htt在PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中降解GFP-Htt(Q74)蛋白的免疫荧光图。
根据图33可知，NRs-Htt可以有效降低PC-12/GFP-Htt(Q74)和Neuro 2A/GFP-Htt(Q74)细胞中的突变亨廷顿蛋白水平。
以上实施例仅用以说明本申请的技术方案，而非对本申请限制；尽管参照前述实施例对本申请进行了详细的说明，本领域普通技术人员应当理解：其依然可以对前述实施例记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请技术方案的范围。

Claims (10)

  1. 一种靶向降解致病蛋白的纳米复合体，其特征在于，含有纳米载体以及接枝于所述纳米载体上的致病蛋白靶向结合肽；
    所述纳米载体为马来酰亚胺-聚乙二醇-聚乳酸与阳离子脂质的纳米组装体。
  2. 根据权利要求1所述的纳米复合体，其特征在于，所述阳离子脂质包括1,2-二油酰-3-三甲基铵-丙烷、2,3-二油酰氧丙基-1-溴化三甲胺。
  3. 根据权利要求1所述的纳米复合体，其特征在于，所述阳离子脂质与所述马来酰亚胺-聚乙二醇-聚乳酸的质量比为(5-40)：100。
  4. 根据权利要求1所述的纳米复合体，其特征在于，所述致病蛋白包括突变p53蛋白和突变亨廷顿蛋白；
    所述突变p53蛋白含有S241F、R175H、R248W、R280K突变位点中的至少一个；所述突变亨廷顿蛋白包括Htt-(Q74)突变类型。
  5. 根据权利要求4所述的纳米复合体，其特征在于，突变p53蛋白靶向结合肽具备SEQ ID NO.1的氨基酸序列；
    突变亨廷顿蛋白靶向结合肽具备SEQ ID NO.2的氨基酸序列。
  6. 一种根据权利要求1-5任一所述纳米复合体的制备方法，其特征在于，所述方法包括：
    使致病蛋白靶向结合肽与含有纳米载体的溶液发生点击反应，并对反应产物依次进行透析、超滤和浓缩，得到靶向降解致病蛋白的纳米复合体。
  7. 根据权利要求6所述的制备方法，其特征在于，所述致病蛋白靶向结合肽与所述纳米载体的摩尔比为(1-3)：1。
  8. 权利要求1-5任一所述纳米复合体用于制备包括抗肿瘤纳米药物和亨廷顿舞蹈症抑制药物中的应用。
  9. 一种抗肿瘤纳米药物，其特征在于，含有权利要求1-5任一项所述的纳米复合体，以及负载于所述纳米复合体中的抗肿瘤活性成分；
    其中，所述抗肿瘤活性成分包括顺铂、Pt(IV)前药。
  10. 根据权利要求9所述抗肿瘤纳米药物的制备方法，其特征在于，所述方法包括：
    使含有马来酰亚胺-聚乙二醇-聚乳酸、阳离子脂质和抗肿瘤活性成分的溶液在缓冲液中进行自组装，得到含有载药纳米颗粒的溶液；
    使含有载药纳米颗粒的溶液与蛋白靶向结合肽发生点击反应，并对反应产物依次进行透析、超滤和浓缩，得到抗肿瘤纳米药物。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020009752A1 (en) * 2000-03-16 2002-01-24 Burke James R. Compounds that selectively bind to expanded polyglutamine repeat domains and methods of use thereof
US20120258920A1 (en) * 2008-03-13 2012-10-11 Universita' Degli Studi Di Trieste Peptides and aptamers thereof as specific modulators of mutant p53 function
CN102727907A (zh) * 2011-04-13 2012-10-17 苏州瑞博生物技术有限公司 一种小干扰rna药物的给药系统和制剂

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020009752A1 (en) * 2000-03-16 2002-01-24 Burke James R. Compounds that selectively bind to expanded polyglutamine repeat domains and methods of use thereof
US20120258920A1 (en) * 2008-03-13 2012-10-11 Universita' Degli Studi Di Trieste Peptides and aptamers thereof as specific modulators of mutant p53 function
CN102727907A (zh) * 2011-04-13 2012-10-17 苏州瑞博生物技术有限公司 一种小干扰rna药物的给药系统和制剂

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GUIDA ELISA ET AL: "Peptide aptamers targeting mutant p53 induce apoptosis in tumor cells.", CANCER RESEARCH, vol. 68, no. 16, 15 August 2008 (2008-08-15), US , pages 6550 - 6558, XP002501679, ISSN: 1538-7445, DOI: 10.1158/0008-5472.CAN-08-0137 *
HUANG XIAOWAN, CAO ZIYANG, QIAN JIEYING, DING TAO, WU YANXIA, ZHANG HAO, ZHONG SUQIN, WANG XIAOLI, REN XIAOGUANG, ZHANG WANG, XU Y: "Nanoreceptors promote mutant p53 protein degradation by mimicking selective autophagy receptors", NATURE NANOTECHNOLOGY, London , pages 1 - 20, XP093150239, ISSN: 1748-3387, DOI: 10.1038/s41565-023-01562-5 *
MAN NA, CHEN YONG, ZHENG FANG, ZHOU WEI, WEN LONG-PING: "Induction of genuine autophagy by cationic lipids in mammalian cells", AUTOPHAGY, vol. 6, no. 4, 16 May 2010 (2010-05-16), US , pages 449 - 454, XP093150413, ISSN: 1554-8627, DOI: 10.4161/auto.6.4.11612 *
VINAY KUMAR BELWAL; DEBIKA DATTA; NITIN CHAUDHARY: "The β‐turn‐supporting motif in the polyglutamine binding peptide QBP1 is essential for inhibiting huntingtin aggregation", FEBS LETTERS, vol. 594, no. 17, 11 July 2020 (2020-07-11), NL , pages 2894 - 2903, XP071256991, ISSN: 0014-5793, DOI: 10.1002/1873-3468.13873 *
WANG CHANGLI; MANNA; WEN LONGPING: "Effects and Biological Functions of Nanomaterials Inducing Autophagy", JOURNAL OF SOUTHEAST UNIVERSITY (MEDICAL SCIENCE EDITION), vol. 30, no. 1, 28 February 2011 (2011-02-28), pages 200 - 208, XP009554058, ISSN: 1671-6264 *
ZOU WEI-WEI, ZHANG NA: "Preparation of Cationic Gene Loaded PLA-PEG Nanoparticles by Nanoprecipitation Method", CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS, vol. 30, no. 1, 31 December 2009 (2009-12-31), CN , pages 1 - 5, XP009554057, ISSN: 1005-1678 *

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