WO2021031839A9 - Targeted polypeptide-modified drug-loaded lipoprotein nano-drug delivery system, and preparation and application thereof - Google Patents

Targeted polypeptide-modified drug-loaded lipoprotein nano-drug delivery system, and preparation and application thereof Download PDF

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WO2021031839A9
WO2021031839A9 PCT/CN2020/106735 CN2020106735W WO2021031839A9 WO 2021031839 A9 WO2021031839 A9 WO 2021031839A9 CN 2020106735 W CN2020106735 W CN 2020106735W WO 2021031839 A9 WO2021031839 A9 WO 2021031839A9
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drug
delivery system
modified
drug delivery
nano
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WO2021031839A1 (en
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江淦
高小玲
陈欢
宋清香
陈红专
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上海交通大学医学院
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    • 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
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the field of biotechnology, in particular to a drug-loaded lipoprotein nano drug delivery system modified by a targeted polypeptide, and its preparation and application.
  • Tumor stem cell subsets cause the recurrence, invasion and metastasis of residual tumors, which cannot be completely removed by surgery and are resistant to radiotherapy and chemotherapy.
  • Traditional methods of targeting cancer stem cells include targeting specific receptors, targeting specific signaling pathways, targeting special microenvironments, and direct immunotherapy, but they all have certain limitations, and there are more targeted drug delivery methods.
  • Challenges such as difficulty in passing through the blood-brain barrier and blood tumor barrier: the biological barrier and complex tumor microenvironment make it difficult for most drugs to enter and effectively distribute inside the tumor; it is difficult to effectively enrich the tumor stem cell subpopulations in recurrent tumors.
  • single-target drugs have limited curative effects on highly heterogeneous cancer stem cells with complex signal regulation pathways, and most of them currently use a single receptor and ligand binding mode for targeted delivery, which is highly heterogeneous
  • the effect of killing cancer stem cells within the tumor is limited.
  • the ideal targeted treatment method should meet various needs. Therefore, the construction of suitable nanocarriers can not only overcome the barrier, but also enable cancer stem cells to actively and efficiently target the uptake of nanocarriers carrying multi-target regulatory drugs, resulting in higher production
  • the concentration of the enrichment and release of the drug can overcome the existing bottleneck and provide a new strategy for the treatment of tumors.
  • Giant pinocytosis plays an important role in non-selective uptake of nutrients such as extracellular solution and protein in glioma cells, pancreatic cancer cells, intestinal cancer cells, macrophages, dendritic cells and other cells. It is regulated by a variety of gene proteins related to actin, microfilament remodeling, wrinkle formation, and vesicle formation, including Ras protein, Cdc42, Rac1, Src, etc. Studies have shown that the giant pinocytosis pathway is an important protein uptake pathway for tumor cells such as pancreatic cancer, glioma, and lung small cell cancer cells.
  • the upstream of these major proteins is regulated by a variety of membrane surface receptors, most of which belong to the family of cytokine receptors, including epidermal growth factor receptors, interleukin receptors, granulocyte colony stimulating factor receptors, etc., so they are in different cells
  • cytokine receptors most of which belong to the family of cytokine receptors, including epidermal growth factor receptors, interleukin receptors, granulocyte colony stimulating factor receptors, etc.
  • High-density lipoprotein is a natural nanoparticle in the human body, mainly composed of phospholipids, apolipoprotein, cholesterol and a small amount of fatty acids.
  • As a natural nanocarrier it has good biocompatibility and excellent stability, and plays an important role in the delivery of imaging reagents and therapeutic drugs.
  • the delivery of drugs in vivo will encounter challenges such as easy degradation, stimulating the secretion of inflammatory cytokines, cytotoxicity, short half-life, difficulty in passing through the blood-brain barrier, off-target effects, etc. Therefore, it is necessary to deliver drugs through nanocarriers for effective targeted drug delivery. Can efficiently load related drugs.
  • gene drugs including (DNA), oligonucleotides (RNA), microRNA, small interfering nucleotides (siRNA) and small hairpin RNA (shRNA) can be loaded, as well as small molecule targeted drugs and chemotherapy Drugs, etc., for the complex pathways of cancer stem cells, microRNAs with multi-target regulation capabilities can be selected for loading, and multiple pathways can be regulated at the same time to inhibit their self-renewal ability.
  • the drug-loaded lipoprotein nanocarrier is modified and further modified by targeting peptides, which is the nano-delivery system of the present invention.
  • the existing published nano-delivery system technologies targeting tumor stem cells mainly focus on the binding of simple receptors and ligands.
  • the existing published technologies include CN201810317543 using exosomal membrane-wrapped nanocarriers to use their CD81 and CD9 membrane proteins to produce cancer stem cells; CN201711273795 and CN201710684008 use the high affinity activity of VAP peptides and GRP78 proteins to produce cancer stem cells.
  • CN201710471867 uses nanoliposomes or micelles modified with a peptide chain targeting CD133 (CX1X2X3X4X5X6X7X8LX9) to target tumor stem cells;
  • CN201710198933 uses A15 (RNA aptamer)-polyethylene glycol-octadecyl alcohol grafting Drug-modified lipid nanocarriers target CD133 to target cancer stem cells;
  • CN201610708677 uses hyaluronic acid-modified ferroferric oxide to carry DAPT drugs to increase the killing effect on cancer stem cells under an external magnetic field; by coating the tumor stem cell antigen Oct4 (15 peptides) cationic liposomes targeting the C-type lectin receptor of dendritic cells induce specific T cells to kill tumor stem cells;
  • CN20141026081 targets H460 lung cancer stem cells through magnetic nanocarriers modified with HCPB-1 polypeptide;
  • the currently published nanomedicines targeting cancer stem cells also have the effect of killing and regulating cancer stem cells by carrying drugs, including CN107375213 that produces certain killing through polyethylene glycol-doxorubicin and irinotecan.
  • the effect of cancer stem cells CN201710097349 inhibits the stemness and differentiation of cancer stem cells by carbon nanomaterial SWCNT; CN201610805898 inhibits cancer stem cells by co-loading microRNA with ferrocenyl retinoic acid/paclitaxel nanocarriers; CN201510500260 is modified by Angiopep-2 and encapsulated salt mold
  • the lipoprotein nano-carriers of vitamin C can simultaneously kill tumor cells, tumor stem cells and tumor angiogenesis at multiple targets.
  • the first objective of the present invention is to use the macropinocytosis mechanism of tumor stem cells to be activated to provide a drug-loaded lipoprotein nano-delivery system for targeted polypeptide modification.
  • the second objective of the present invention is to provide a targeted polypeptide that produces a modified apolipoprotein nano-drug delivery system that activates and enhances the mechanism of giant pinocytosis.
  • the third objective of the present invention is to provide a method for preparing a drug-loaded lipoprotein nano-drug delivery system modified by a targeted polypeptide.
  • the fourth objective of the present invention is to provide an application of a targeted polypeptide-modified drug-laden lipoprotein nano-drug delivery system in the preparation of drugs for the prevention or treatment of tumors or central nervous system diseases.
  • the present invention provides a targeted polypeptide modified apolipoprotein nano-drug delivery system, characterized in that the drug delivery system includes lipid, apolipoprotein, loaded drug and target
  • the targeting polypeptide is formed by covalently connecting the end of the nanocarrier and the peptide chain that activates the macropinocytosis function by a bridge structure, the bridge structure includes Cys-Val, Cys-Phe, Cys-Leu, Cys -Ile, Cys-Gln, Leu-Glu, Gly-Ser-Gly, Ala-Pro-Ala, Cys-Pro-Cys, Gly-Phe-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu , Gly-Phe-Leu-Gly, Gly-Gly-Gly-Gly-Ser, Val-Arg-Gly-Asp-Val, Pro-Ala-Pro-Ala-Pro,
  • the sequence of the targeting polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH, SEQ ID NO. 1), FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR, SEQ ID NO. 2) and FH38 (AC -FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH, SEQ ID NO. 3).
  • the lipid is egg phospholipid, soybean phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, lysophospholipid, sphingosine One or more of ceramide, sphingomyelin, cerebroside, cholesterol, cholesterol ester, glyceride and derivatives thereof.
  • the molar ratio of the targeting polypeptide to lipid is 1:10-1:300, preferably 1:100.
  • the apolipoprotein is one or more of ApoE, ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, and ApoC-III.
  • the mass ratio of the apolipoprotein to the targeting polypeptide is 1:10-1:100, preferably 1:30.
  • the drug delivery system further includes a solid phase core, the solid phase core is composed of drug molecules, insoluble or poorly soluble inorganic salts, the drug molecules are loaded in the solid phase core, the insoluble or poorly soluble inorganic salts
  • the solid phase core is composed of drug molecules, insoluble or poorly soluble inorganic salts
  • the drug molecules are loaded in the solid phase core, the insoluble or poorly soluble inorganic salts
  • biodegradable calcium phosphate precipitation calcium carbonate precipitation, calcium sulfate precipitation, calcium fluoride precipitation, calcium silicate precipitation, calcium alginate precipitation, magnesium sulfate precipitation, magnesium phosphate precipitation, magnesium carbonate precipitation, magnesium fluoride precipitation, silicon
  • magnesium oxide precipitation, barium sulfate precipitation, barium phosphate precipitation, barium carbonate precipitation, barium fluoride precipitation, and barium silicate precipitation are examples of magnesium oxide precipitation, barium sulfate precipitation, barium phosphate precipitation
  • the present invention provides a targeting polypeptide for modifying a drug-loaded lipoprotein nano-drug delivery system, characterized in that the sequence of the targeting polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH) , FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) and FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH).
  • FH27 AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH
  • FH29 AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR
  • FH38 AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH
  • the present invention provides a method for preparing the above-mentioned targeted polypeptide-modified drug-laden lipoprotein nano-drug delivery system, which includes the following steps:
  • c) by adding the targeting polypeptide to the nano drug delivery system solution prepared in b) above, and then adding the apolipoprotein to prepare a drug delivery lipoprotein nano drug delivery system modified by the targeting polypeptide.
  • the present invention provides the application of a targeted polypeptide modified apolipoprotein nano-drug delivery system in the preparation of drugs for the prevention or treatment of tumors or central nervous system diseases.
  • the tumor is glioma and pancreatic cancer.
  • the advantage of the present invention is that the targeted polypeptide modified apolipoprotein nano-drug delivery system is based on the principle of giant pinocytosis.
  • the targeting peptide By linking the targeting peptide, it can target the high expression receptors on the surface of tumor stem cells while activating giant cells.
  • the drinking pathway forms the effect of using metabolic differences to form targeted enrichment.
  • Figure 1 is a transmission electron microscope observation of the morphology of the recombinant lipoprotein containing NC-microRNA drugs without targeting peptides and containing different targeting peptides (FH27, FH29, FH38), scale: 50nm.
  • Figure 2 shows human-derived glioma stem cell-like cells (A) and pancreatic cancer cell line-enriched stem cell-like cells (B) vs. cells without targeting peptides and fluorescently labeled apolipoprotein nanocarriers modified with different targeting peptides Ingestion, *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001 indicates that there is a significant difference.
  • Figure 3 shows the uptake and co-localization of the giant pinocytotic vesicle-specific marker Dextran by human glioma stem cell-like cells stimulated by drug-loaded lipoprotein nanocarriers without targeting peptides and modified with different targeting peptides And the inhibition of uptake after adding EIPA giant pinocytosis inhibitor, *p ⁇ 0.05, **p ⁇ 0.01 indicates that there is a significant difference from the apolipoprotein nanocarrier group without targeting peptide.
  • Figure 4 shows the human-derived glioma stem cell-like cells (A) and pancreatic cancer cell lines enriched stem cell-like cells (B) under different expression levels of the giant pinocytosis activating receptor CXCR4.
  • the cellular uptake of peptide-modified fluorescently labeled lipoprotein nanocarriers, *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001 indicates that there is a significant difference in cellular uptake compared with the CXCR4 non-knockdown group .
  • Figure 5 shows the co-localization of the fluorescently labeled apolipoprotein nanocarriers without targeting peptides and containing different targeting peptide modifications with the giant pinocytosis-activated receptor CXCR4 on the surface of human glioma stem cell-like cells (A). Co-localization of pinocytosis-activated receptor CXCR4 with low expression (B), scale: 100 ⁇ m.
  • Figure 6 shows the injection of human glioma stem cell-like cells (A) and the giant pinocytosis receptor CXCR4 low-expression human glioma stem cell-like cells (B) into the brain of NOD/SCID mice to construct an in situ tumor-bearing cell Mouse model to study the permeability of fluorescently labeled drug-loaded lipoprotein nanocarriers modified by different targeting peptides to the blood-brain barrier of tumor-bearing mice in situ, and the drug-loaded lipoprotein nanocarriers without targeting peptide modification were used as control preparations. Scale bar: 100 ⁇ m.
  • Figure 7 shows the injection of human glioma stem cell-like cells and human glioma stem cell-like cells with low expression of the giant pinocytosis receptor CXCR4 into the brain of NOD/SCID mice to construct an orthotopic tumor-bearing mouse model.
  • Fig. 8 shows that lipoprotein nanocarriers carrying miR-34a microRNAs modified by targeting polypeptides are used to knock down the stemness-related SOX2 protein expression of human glioma stem cell-like cells (A) and inhibit the self-renewal ability of tumor cells (B).
  • Figure 9 shows the determination of the IC50 of temozolomide after the combination of targeted peptide-modified miR-34a microRNA lipoprotein nanocarriers and the chemotherapeutic drug temozolomide.
  • Targeted peptide-modified miR-34a microRNA lipoprotein nanocarrier is used to inhibit the proliferation of NOD/SCID mouse tumor cells bearing human glioma stem cell-like cells in situ (A) and prolong the survival time of mice (B) .
  • Example 1 Preparation and Characterization of a Targeted Polypeptide Modified Apolipoprotein Nano-Drug Delivery System
  • the drug-loaded calcium phosphate solid phase core was prepared by the reverse microemulsion method.
  • the calcium phase was prepared, 300 ⁇ L of 2.5M CaCl 2 solution was incubated with NC-miRNA, pipetting several times, and then dispersed in 20 mL of the oil phase to form a uniformly dispersed water-in-oil reverse microemulsion.
  • the phosphorus phase was prepared by dispersing 300 ⁇ L of 12.5mM Na 2 HPO 4 solution in another 20 mL oil phase, stirring for 10 minutes, and adding 100 ⁇ L of 20 mg/mL 1,2-oleoylphosphatidic acid ( 1,2-dioleoyl phosphatidic acid, DOPA) solution.
  • the two phases are mixed and stirred for 45 minutes.
  • 40 mL of absolute ethanol was added to the above mixed microemulsion to break the emulsion for 10 minutes.
  • the mixed solution is centrifuged at a high speed (12,500 g) for about 20 minutes to remove excess surfactant and cyclohexane.
  • the precipitate obtained by centrifugation was the DOPA-modified drug-loaded calcium phosphate, which was dispersed in 1 mL of chloroform and stored in a glass bottle for subsequent experiments.
  • the thin-film hydration method was used to prepare drug-loaded ordinary liposomes: weigh the lipids (2-10mg) into a 500mL round-bottomed flask, add 2mL ether, evaporate dry to remove the water in the phospholipids, and then add the drug-loaded phosphoric acid prepared above Calcium 750 ⁇ L and 2mL chloroform solution were placed on the rotary evaporator and vacuumed for 1h. Add 4mL 0.01M PBS solution (pH 7.4), shake intermittently for 10 minutes in a 40°C water bath until the film is hydrated and falls off to obtain liposomes. Probe ultrasound further reduces the particle size of liposomes, and obtains common liposomes (CaP-LNC) containing drug-loaded calcium phosphate (CaP-LNC).
  • CaP-LNC common liposomes
  • the solid-phase peptide synthesis method was used to synthesize targeted peptides FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH), FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) and FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH).
  • the bridge structure can also be replaced with Cys-Val, Cys-Phe, Cys-Leu, Cys-Ile, Cys-Gln, Leu-Glu, Gly-Ser-Gly, Ala-Pro-Ala, Cys-Pro-Cys, Gly-Phe-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Gly-Gly-Ser, Val-Arg-Gly-Asp-Val, One or more of Pro-Ala-Pro-Ala-Pro, Pro-Leu-Gly-Leu-Trp-Ala, Arg-Val-Leu-Ala-Glu-Ala, and polyethylene glycol.
  • the specific method insert amino acid into chloromethyl polystyrene resin, and remove amino protecting group under the protection of trifluoroacetic acid. Then it was cut by hydrogen fluoride, ether was precipitated in an ice bath, acetonitrile was dissolved and then revolved, and the acetonitrile water system was used for further purification.
  • the prepared targeting polypeptide was added to ordinary liposomes at a molar ratio of 1:30-1:100 to phospholipids, incubated at 120 rpm and 37°C for 24 hours, and then apolipoproteins such as ApoE or ApoA-I (0.1-10 mg) Add to the above solution (the total lipid mass is 4 mg), mix gently, place on a shaking shaker at 120 rpm, and incubate at 37° C. for 24 hours to obtain a lipoprotein nanocarrier modified by targeting polypeptides and NC-miRNA drugs.
  • the targeting peptide and apolipoprotein were added to ordinary liposomes and incubated for 48 hours.
  • Targeting peptide modified drug-loaded lipoprotein nanocarriers were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscope.
  • the particle size and surface potential of the laser particle size analyzer were compared with those of ordinary liposomes containing calcium phosphate and drug-loaded calcium phosphate without incubating the targeting peptide and ApoE.
  • the recombinant lipoprotein containing drug-loaded calcium phosphate is uniformly spherical, uniformly dispersed, and the particle size is about 30nm-50nm.
  • the particle size of the nanocarrier linked to the FH38 targeting peptide is relatively large, and the particle size by laser
  • the surface potential measured by the instrument is about -25mV.
  • Example 2 A variety of tumor stem cells efficiently uptake targeted polypeptide-modified drug-loaded lipoprotein nanocarriers
  • the drug-loaded calcium phosphate was prepared by the reverse microemulsion method, and then the drug-loaded ordinary liposomes were prepared by the membrane hydration method, and phosphatidylcholine, phosphatidic acid (2-10mg) and the fluorescent dye DiI( 20-100 ⁇ g) into a 500ml round-bottomed flask, prepare fluorescently labeled ordinary liposomes as in Example 1, add the targeting peptide to the ordinary liposomes at a molar ratio of 1:100 to the phospholipids, and incubate at 120rpm, 37°C After 24 hours, add ApoE or ApoA-I and other apolipoproteins (0.5-10mg) to the above solution (total lipid weight 4mg), mix gently, and place in a shaking shaker at 120rpm and incubate at 37°C for 24h. A fluorescently labeled drug-loaded lipoprotein nanocarrier modified by a targeted polypeptide is obtained.
  • the uptake efficiency of the lipoprotein nanocarrier containing the FH38 targeting peptide is 3.0 times that of the lipoprotein nanoformulation without the targeting peptide and containing the FH29 target.
  • the uptake efficiency of drug-loaded lipoprotein nanocarriers to peptides is 1.7 times that of lipoprotein nano-preparations without targeting peptides, while the uptake efficiency of drug-loaded lipoprotein nanocarriers containing FH27 targeting peptides is that of lipoproteins without targeting peptides. 1.1 times of nano formulations.
  • the uptake is closely related to apolipoprotein, and simply incubating the targeting peptide without apolipoprotein has no obvious enhancement effect.
  • the uptake efficiency of the drug-laden lipoprotein nanocarrier containing the FH38 targeting peptide is 1.5-1.8 that of the lipoprotein nano-preparation without the targeting peptide. Times, indicating that the targeted peptide-modified fluorescent drug-loaded lipoprotein nanocarriers have the effect of enhancing uptake in a variety of tumor models.
  • the uptake efficiency of co-incubating the targeting peptide is lower than the efficiency of incubating the targeting peptide first and then incubating apolipoprotein.
  • Example 3 Targeting peptide-modified drug-loaded lipoprotein nanocarriers improve the uptake efficiency of tumor stem cells by enhancing macropinocytosis
  • the cell suspension was subjected to flow cytometry to detect the fluorescence intensity of FITC-Dextran. After further treatment with the giant pinocytosis inhibitor EIPA 150 ⁇ M for 1.5 hours, the uptake and co-localization of the preparation and Dextran were observed using a laser confocal microscope.
  • the apolipoprotein nanocarrier containing the FH38 targeting peptide can enhance the uptake of the giant pinocytotic vesicle specific marker Dextran by 1.3 times, indicating This enhancement mechanism is closely related to the giant pinocytosis pathway. It can be observed that the preparation and dextran are highly co-localized, and EIPA can effectively inhibit the uptake of the preparation and dextran.
  • Example 4 Comparison of the uptake of targeted polypeptide-modified apolipoprotein nanocarriers by multiple tumor stem cells with different expression levels of the giant pinocytosis activating receptor (CXCR4).
  • CXCR4 giant pinocytosis activating receptor
  • phosphatidylcholine, phosphatidic acid (2-10mg) and fluorescent dye DiI (20-100 ⁇ g) into a 500ml round-bottomed flask, prepare fluorescent-labeled ordinary liposomes as in Example 1, and combine the targeting peptides with Phospholipid molar ratio 1:100 was added to ordinary liposomes, incubated at 120 rpm, 37°C for 24 hours, and then ApoE or ApoA-I and other apolipoproteins (0.5-10 mg) were added to the above solution (total lipid weight 4 mg) , Mix gently, place on a shaking shaker at 120 rpm, and incubate at 37°C for 24 hours to obtain the targeted polypeptide modified fluorescently labeled drug-laden lipoprotein nanocarrier.
  • Targeted peptide-modified drug-loaded lipoprotein nanocarriers enhance the uptake of cancer stem cells and are related to the expression level of cell giant pinocytosis receptors
  • pancreatic cancer cell line enriched stem cell-like cells at 10 5 cells were plated in 12-well plate, cultured overnight, CXCR4shRNA lentiviral vector system transfected cells (10 ⁇ l / ml), Negative NC shRNA lentiviral vector system was used as a control. After 18 hours of transfection, the medium containing the lentivirus was aspirated and centrifuged to change to fresh medium. After continuing the culture for 3 days, 2 ⁇ g/ml puromycin was added to the culture medium to kill the cells that were not successfully transfected.
  • the human-derived glioma stem cell-like cells, pancreatic cancer cell line-enriched stem cell-like cells and the control group knocked down by giant pinocytosis receptors were inoculated into a 24-well plate with 100 balls, incubated overnight in a 5% carbon dioxide incubator, and then added Carrying fluorescent probe DiI containing targeting peptide-modified nanocarriers and DMEM solution containing no targeting peptide-modified nanocarriers, DMPC concentration is 20 ⁇ g/ml, incubate for 4 hours, wash once with PBS, gently pipette 50 times, After the tumor ball is blown away, it is passed through a cell sieve with a pore size of 100 ⁇ m, and then fixed with paraformaldehyde. The cell suspension was passed through a flow cytometer to detect the fluorescence intensity of the ingested preparation.
  • Example 5 Evaluation of targeted uptake of apolipoprotein nanocarriers modified by targeting polypeptides via macropinocytosis activated receptor (CXCR4)
  • the preparation method was the same as that in Example 4.
  • the fluorescently labeled apolipoprotein nanocarriers containing no targeting polypeptides and containing different targeting polypeptide modifications were prepared, and they were named DiI-CaP-rHDL, FH27-DiI-CaP-rHDL, FH29- DiI-CaP-rHDL and FH38-DiI-CaP-rHDL.
  • Targeted peptide-modified fluorescently labeled drug-loaded lipoprotein nanocarriers are targeted for uptake by tumor stem cell giant pinocytosis receptors.
  • the human-derived glioma stem cell-like cells with and without giant pinocytosis receptor knockdown were seeded in a 24-well plate with 100 spheres and incubated overnight in a 5% carbon dioxide incubator. Then, a fluorescent probe containing DiI containing targeting was added.
  • the fluorescently labeled lipoprotein nanocarriers modified by targeting peptides accumulate in large amounts in human glioma stem cell-like cells and interact with the cells.
  • the surface giant pinocytosis receptor CXCR4 is co-localized; as shown in Figure 5B, when the giant pinocytosis receptor CXCR4 is knocked down, the uptake of the preparation by human glioma stem cell-like cells is reduced, indicating that the fluorescent label modified by the targeted peptide It is recorded that the drug lipoprotein nanocarriers are targeted for uptake through tumor stem cell giant pinocytosis receptors.
  • Example 6 Evaluation of the permeability of the blood-brain barrier by drug-loaded lipoprotein nanocarriers modified by different targeting polypeptides
  • Cy5-labeled non-effect negative control microRNA calcium phosphate drug-loaded recombinant lipoprotein (Cy5-CaP-rHDL).
  • the targeting peptide was added to ordinary liposomes at a molar ratio of 1:100 to the phospholipids, and incubated at 120 rpm and 37°C for 24 hours, and then ApoE or ApoA-I and other apolipoproteins (0.5-10 mg) were added to the above solution ( The total lipid mass is 4 mg), gently mix, place on a shaking shaker at 120 rpm, and incubate at 37° C. for 24 hours to obtain a targeted peptide-modified Cy5 fluorescently labeled apolipoprotein nanocarrier.
  • mice After administration for 4 hours, the mice were anesthetized and fixed with 5% chloral hydrate, the thoracic cavity was cut open, the heart was fully exposed, the scalp needle was inserted into the left ventricle, the right atrial appendage was cut open, and 0.9% normal saline was immediately perfused to the outflow perfusion.
  • the solution is bloodless, and then fixed by perfusion with 4% paraformaldehyde solution until the liver, limbs, and tail become hard, remove the heart, liver, spleen, lung, kidney and brain (tumor) tissues, rinse with saline and place them in a living animal
  • the imager collects images. The results of the experiment are shown in Figure 6A.
  • the drug lipoprotein nanocarrier control group shows that the targeting ability of the vector is related to the expression level of cell giant pinocytosis receptors. The higher the expression level of giant pinocytosis receptors, the stronger the targeting ability of the vector. .
  • Example 7 Establishment of NOD/SCID mouse model of glioma in situ bearing human glioma stem cell-like cells to evaluate the tumor targeting ability of drug-loaded recombinant lipoproteins modified by different targeting polypeptides
  • Cy5 fluorescent-labeled drug-loaded lipoprotein nanocarriers modified with different targeting polypeptides were prepared in the same manner as in Example 6, and the Cy5 fluorescent-labeled drug-loaded lipoprotein nanocarriers (Cy5-CaP-rHDL) that did not contain the targeting polypeptide modification (Cy5-CaP-rHDL) For the control.
  • mice After 4 hours of administration, the mice were anesthetized and fixed, and brain (tumor) tissues were taken out after cardiac perfusion. After trimming the tumor site and washing it with HBSS solution, cut the tumor tissue into a homogenate shape with a razor blade and move it to a petri dish. Add 5ml 0.05% EDTA-pancreatin, 2.5ml HBSS solution, 2.5ml type IV collagenase (2000U/ ml) compound digestive enzyme solution, placed in a 37°C incubator for 15-20min. Then, the digestion was terminated with 10ml of termination digestion solution containing equivalent trypsin inhibitor and DNase, and the digestion was repeated 4 times.
  • the cell suspension was collected, filtered with a 75 ⁇ m filter, and centrifuged at 1000rpm for 20min.
  • the cells were collected in a 1.5ml centrifuge tube, fixed in 4% paraformaldehyde for 20 minutes at room temperature, shaken, centrifuged at 800 rpm, and 100 ⁇ l 0.1% Triton-X was added, and the cells were permeabilized for 20 minutes. PBS was carefully washed once, and 100 ⁇ l 4% BSA was added to block for 40 min.
  • Cy5 labeled and targeted peptide-modified drug-loaded recombinant lipoproteins were injected through the tail vein.
  • Cy5-FH29-CaP-rHDL and Cy5-FH38-CaP-rHDL were injected through the tail vein.
  • Cy5-CaP-rHDL was injected through the tail vein.
  • Cy5-CaP-rHDL was injected through the tail vein.
  • Cy5-FH29-CaP-rHDL and Cy5-FH38-CaP-rHDL Cy5-loaded recombinant lipoprotein
  • mice After administration for 4 hours, the mice were anesthetized and fixed, and the heart was perfused. The whole brains of the tumor-bearing mice were taken, placed in 4% paraformaldehyde and then fixed for 24 hours. After rinsing with PBS, they were placed in 15% and 30% sucrose solutions for dehydration. Shen was then embedded in OCT and frozen at -20°C to make continuous frozen coronal sections with a thickness of 14 ⁇ m. After rinsing with PBS, the brain slices were blocked with 4% BSA at room temperature for 1 hour, incubated with cell stem antibody SOX2 at 4°C overnight, and then with Alexa Incubate with 488-labeled fluorescent secondary antibody.
  • the fluorescence intensity at the tumor site was significantly increased; however, in the mouse glioma tissue slices with the knockdown of the giant pinocytosis receptor, the fluorescence intensity of the targeted peptide-modified drug-loaded recombinant lipoprotein carrier at the tumor site was significantly reduced, indicating that the targeting
  • the polypeptide-modified drug-loaded recombinant lipoprotein carrier has excellent tumor targeting and can be widely distributed in tumor tissues, and this targeting is related to the expression level of the giant pinocytosis receptor.
  • Example 8 Evaluation of in vitro pharmacodynamics of targeted polypeptide modified apolipoprotein nanocarriers
  • the solid phase core was prepared by the reverse microemulsion method, 300-600 ⁇ L of 2.5M CaCl 2 solution was incubated with miR-34a, pipetting several times, and then dispersed in 20mL oil phase to form a uniformly dispersed water-in-oil reverse phase Microemulsion.
  • the phosphorus phase is prepared by dispersing 300-600 ⁇ L of 12.5mM Na 2 HPO 4 solution in another 20mL oil phase. After stirring for 10 minutes, add 100 ⁇ L of 20mg/mL 1,2-oleoyl phospholipid to the phosphorus phase. Acid solution. After the two phases are evenly dispersed, the two phases are mixed and stirred for 45 minutes.
  • the logarithmic growth phase human glioma stem cell-like cells were seeded in a 6-well plate at a density of 5 ⁇ 10 4 per well, and after 12 hours of culture, drug treatment was performed when the cell confluence reached 50%.
  • Different nano preparations were given to different multiple wells, including DMEM control group, empty recombinant lipoprotein (CaP-rHDL), non-interfering NC miRNA and recombinant lipoprotein (NC-CaP-rHDL) modified by different target peptides.
  • miR-34a loaded recombinant lipoproteins (miR-34a-CaP-rHDL, FH27 -miR-34a-CaP-rHDL, FH29-miR-34a-CaP-rHDL and FH38-miR-34a-CaP-rHDL) administration group, administered at a concentration of 100nM miRNA and incubated at 37°C for 12, 24, and 48 hours respectively , Cells were lysed and collected, and the experiment was carried out according to the WB procedure to detect the SOX2 protein expression level of the glioma cells treated with each group of preparations.
  • the SOX2 protein expression level of the cells in the targeted polypeptide-modified drug-loaded lipoprotein nanocarrier treatment group was significantly lower than that of the non-targeted polypeptide-modified treatment group; after 48 hours of incubation, all lipoprotein nanoparticles loaded with miR-34a After carrier treatment, the level of SOX2 protein in human glioma stem cell-like cells decreased by 50-60%, indicating that compared with the unmodified preparation (miR-34a-CaP-rHDL), the targeted peptide-modified drug delivery Lipoprotein nanocarriers can release drugs more efficiently and quickly, and effectively inhibit the growth of tumor stem cells.
  • Example 9 Evaluation of in vitro pharmacodynamics of drug-targeted polypeptide-modified apolipoprotein nanocarriers combined with chemotherapeutic drugs
  • miR-34a microRNA recombinant lipoprotein (miR-34a-CaP-rHDL) was prepared as in Example 8 and incubated with different target polypeptides.
  • the experimental results are shown in Figure 9.
  • the targeted peptide modified apolipoprotein nanocarrier (FH38-MiR34a-CaP-rHDL) combined with the chemotherapeutic drug temozolomide (TMZ) can significantly reduce compared with the TMZ treatment group alone.
  • Example 10 Evaluation of the in vivo pharmacodynamics of targeted polypeptide modified apolipoprotein nanocarrier
  • the lipoprotein nanocarrier carrying miR-34a microRNA was prepared as in Example 8, and incubated with the FH38 targeting polypeptide, and named FH38-miR-34a-CaP-rHDL, and the preparation without targeting polypeptide modification was named miR-34a- CaP-rHDL.
  • mice bearing human-derived glioma stem cell-like cells for glioma in situ were randomly divided into 6 groups, namely normal saline, temozolomide (TMZ), and miR-34a-loaded lipoprotein nanocarrier (miR- 34a-CaP-rHDL), targeting polypeptide-modified lipoprotein nanocarriers carrying miR-34a (FH38-miR-34a-CaP-rHDL), TMZ and miR-34a-CaP-rHDL combined administration group, TMZ and FH38 -miR-34a-CaP-rHDL combined administration group.
  • 6 groups namely normal saline, temozolomide (TMZ), and miR-34a-loaded lipoprotein nanocarrier (miR- 34a-CaP-rHDL), targeting polypeptide-modified lipoprotein nanocarriers carrying miR-34a (FH38-miR-34a-CaP-rHD
  • temozolomide was administered intragastrically (miRNA dosage: 0.36 mg/kg, TMZ dosage: 100 mg/m 2 ). The death time of each group of tumor-bearing mice was recorded, and the survival curve was drawn.
  • mice bearing BxPC3 pancreatic cancer stem cell-like cells were randomly divided into 6 groups, namely, normal saline, gemcitabine (GEM), miR-34a-loaded lipoprotein nanocarrier (miR-34a-CaP-rHDL), targeting Polypeptide modified miR-34a-loaded lipoprotein nanocarrier (FH38-miR-34a-CaP-rHDL), GEM and miR-34a-CaP-rHDL combined administration group, GEM and FH38-miR-34a-CaP-rHDL combined G.
  • GEM gemcitabine
  • miR-34a-loaded lipoprotein nanocarrier miR-34a-CaP-rHDL
  • FH38-miR-34a-CaP-rHDL targeting Polypeptide modified miR-34a-loaded lipoprotein nanocarrier
  • GEM and miR-34a-CaP-rHDL combined administration group GEM and FH38-miR-
  • the preparation was injected into the tail vein on the 7, 10, 13, 16 and 19 days, and GEM and miRNA drugs were administered to the tail vein (miRNA administration amount: 0.36 mg/kg, GEM administration amount: 10 mg/ kg). The death time of each group of tumor-bearing mice was recorded, and the survival curve was drawn.
  • the survival time of tumor-bearing mice was investigated. As shown in Figure 10B, temozolomide and miR-34a-loaded lipoprotein nanocarriers modified by targeting peptides were administered in combination (TMZ+FH38-miR-34a-CaP-rHDL) mice The average survival time was 59 days, which was significantly higher than the Saline group (21 days), the temozolomide administration group (24 days), and the miR-34a lipoprotein nanocarrier group without targeting peptide modification (28 days).
  • the above experimental results indicate that the lipoprotein nanocarriers carrying miR-34a modified by targeting peptides have excellent anti-glioma effects and can significantly enhance the effects of existing chemotherapeutics.
  • the average survival time of gemcitabine and miR-34a-loaded lipoprotein nanocarriers modified by targeting peptides was At 96 days, it was significantly higher than the Saline group (29 days), the simple gemcitabine administration group (35 days), the miR-34a lipoprotein nanocarrier group without targeted peptide modification (66 days), and the pure targeted peptide modification group
  • the above experimental results indicate that the targeted peptide modified carrier
  • the miR-34a lipoprotein nanocarrier has excellent anti-pancreatic cancer effect, and can also significantly enhance the effect of existing chemotherapeutic drugs, showing that the drug can be used in multiple tumor models and has good application prospects.

Abstract

Provide are a targeted polypeptide-modified drug-loaded lipoprotein nano-drug delivery system, and preparation and application thereof. The drug delivery system comprises a lipid, an apolipoprotein, a loaded drug, and a targeted polypeptide, and the targeted polypeptide is formed by covalently linking, by a bridge structure, a nanocarrier linking end and a peptide chain which activates a macropinocytosis function. The drug delivery system modified by a polypeptide actively targets and can effectively regulate a variety of tumor stem cells; the use of the lipoprotein nano-drug delivery system in combination with mediation of enhanced macropinocytosis mechanism achieves pinocytosis of more lipoprotein nano-drugs by tumor stem cells, and targeted regulation of tumor stem cells in vivo and in vitro can be implemented safely and effectively with a lower dosage. The nano-drug delivery system can be applied to prevention or treatment of a variety of tumors or central nervous system diseases, and has a simple and safe preparation process, and good research value and clinical application prospects.

Description

一种靶向多肽修饰的载药脂蛋白纳米递药系统及其制备和应用Targeted polypeptide modified drug-loaded lipoprotein nano drug delivery system and preparation and application thereof 技术领域Technical field
本发明涉及生物技术领域，尤其涉及一种靶向多肽修饰的载药脂蛋白纳米递药系统及其制备和应用。The present invention relates to the field of biotechnology, in particular to a drug-loaded lipoprotein nano drug delivery system modified by a targeted polypeptide, and its preparation and application.
背景技术Background technique
肿瘤干细胞亚群造成残余肿瘤的复发、侵袭转移，手术无法完全切除且自身耐受放化疗。传统靶向肿瘤干细胞的方法包括靶向特异性受体、靶向特异信号通路、靶向特殊的微环境以及直接免疫治疗等，但都存在一定局限性，对其进行靶向递药面临较多的挑战，比如难以通过血脑屏障及血瘤屏障：生物屏障及复杂的肿瘤微环境使得绝大多数药物难以进入且有效分布于肿瘤内部；难以对复发肿瘤内的肿瘤干细胞亚群进行有效的富集和靶向；单一靶点的药物对高度异质性的具有复杂信号调控通路的肿瘤干细胞疗效局限，且目前大多数以单一的受体与配体结合模式进行靶向递送，对高度异质性的肿瘤内杀伤肿瘤干细胞的效果有限。理想的靶向治疗方式应需要满足多方面的需求，因此构建合适的纳米载体，不仅能克服屏障同时能够使肿瘤干细胞主动高效的靶向摄取载带多靶点调控药物的纳米载体，产生更高的富集浓度，并将药物释放，能够克服现有的瓶颈，为肿瘤的治疗提供新的策略。Tumor stem cell subsets cause the recurrence, invasion and metastasis of residual tumors, which cannot be completely removed by surgery and are resistant to radiotherapy and chemotherapy. Traditional methods of targeting cancer stem cells include targeting specific receptors, targeting specific signaling pathways, targeting special microenvironments, and direct immunotherapy, but they all have certain limitations, and there are more targeted drug delivery methods. Challenges such as difficulty in passing through the blood-brain barrier and blood tumor barrier: the biological barrier and complex tumor microenvironment make it difficult for most drugs to enter and effectively distribute inside the tumor; it is difficult to effectively enrich the tumor stem cell subpopulations in recurrent tumors. Collection and targeting; single-target drugs have limited curative effects on highly heterogeneous cancer stem cells with complex signal regulation pathways, and most of them currently use a single receptor and ligand binding mode for targeted delivery, which is highly heterogeneous The effect of killing cancer stem cells within the tumor is limited. The ideal targeted treatment method should meet various needs. Therefore, the construction of suitable nanocarriers can not only overcome the barrier, but also enable cancer stem cells to actively and efficiently target the uptake of nanocarriers carrying multi-target regulatory drugs, resulting in higher production The concentration of the enrichment and release of the drug can overcome the existing bottleneck and provide a new strategy for the treatment of tumors.
肿瘤细胞重要的特征之一是能量代谢的改变，经典的Warburg效应提示肿瘤达到能量与物质合成的平衡与正常细胞所经历的途径不同。在多种肿瘤细胞中存在明显的蛋白质及糖类能量代谢的改变，在复发的肿瘤患者中甘氨酸、丙氨酸、谷氨酸盐等代谢水平明显增强。这提示我们，与复发相关的肿瘤干细胞会获取更多的蛋白质以及糖类等营养产生特异性的能量代谢改变。因此通过代谢水平的差异进行靶向是一种良好的策略。而研究发现肿瘤干细胞可以通过巨胞饮这条与正常细胞具有明显差异的代谢通路对周围蛋白质相关的营养及药物进行主动的吞饮。One of the important characteristics of tumor cells is the changes in energy metabolism. The classic Warburg effect suggests that tumors achieve a balance of energy and substance synthesis differently from the pathways experienced by normal cells. There are obvious changes in protein and carbohydrate energy metabolism in a variety of tumor cells, and the metabolic levels of glycine, alanine, and glutamate are significantly enhanced in patients with relapsed tumors. This suggests that cancer stem cells related to recurrence will acquire more protein and carbohydrates and other nutrients to produce specific energy metabolism changes. Therefore, targeting based on differences in metabolic levels is a good strategy. Studies have found that cancer stem cells can actively swallow nutrients and drugs related to surrounding proteins through the metabolic pathway of giant pinocytosis, which is significantly different from normal cells.
巨胞饮在胶质瘤细胞、胰腺癌细胞、肠癌细胞、巨噬细胞、树突状细胞等多种细胞中起到重要的非选择性摄取胞外溶液、蛋白等营养物质的作用。受多种与肌动蛋白、微丝重构、皱褶形成、囊泡形成相关的基因蛋白调控，主要包括Ras蛋白、Cdc42、Rac1、Src等。研究表明巨胞饮通路是肿瘤细胞如胰腺癌、 胶质瘤、肺小细胞癌细胞重要的摄取蛋白质的通路。这些主要蛋白的上游又受多种膜表面受体的调控，大多属于细胞因子受体家族，主要包括表皮生长因子受体、白介素受体、粒细胞集落刺激因子受体等，因此在不同的细胞中激活其相关的表面受体即可通过不同调节通路、不同程度的引起激活巨胞饮的增强现象。Giant pinocytosis plays an important role in non-selective uptake of nutrients such as extracellular solution and protein in glioma cells, pancreatic cancer cells, intestinal cancer cells, macrophages, dendritic cells and other cells. It is regulated by a variety of gene proteins related to actin, microfilament remodeling, wrinkle formation, and vesicle formation, including Ras protein, Cdc42, Rac1, Src, etc. Studies have shown that the giant pinocytosis pathway is an important protein uptake pathway for tumor cells such as pancreatic cancer, glioma, and lung small cell cancer cells. The upstream of these major proteins is regulated by a variety of membrane surface receptors, most of which belong to the family of cytokine receptors, including epidermal growth factor receptors, interleukin receptors, granulocyte colony stimulating factor receptors, etc., so they are in different cells The activation of its related surface receptors in the medium can induce the enhancement of activation of giant pinocytosis to different degrees through different regulatory pathways.
高密度脂蛋白是一种人体内天然的纳米颗粒，主要由磷脂、载脂蛋白、胆固醇和少量脂肪酸组成。作为天然纳米载体，其生物相容性好且稳定性优良，在递送成像试剂和治疗药物中发挥重要作用。然而体内药物的递送会遇到易降解、刺激炎症细胞因子分泌、细胞毒性、半衰期短、难以通过血脑屏障、脱靶效应等挑战，故而需要将药物通过纳米载体进行有效的靶向药物递送，同时可以高效负载相关药物。通过载药可以包载包括(DNA)、寡核苷酸(RNA)、microRNA、小干扰核苷酸(siRNA)以及小发夹RNA(shRNA)在内的基因药物以及小分子靶向药物、化疗药物等，针对肿瘤干细胞的复杂通路，可以选择具有多靶点调控能力的microRNA进行负载，同时调控多条通路从而抑制其自我更新的能力。对该载药的脂蛋白纳米载体进行靶向肽的修饰和进一步改造，即为本发明的纳米递送系统。High-density lipoprotein is a natural nanoparticle in the human body, mainly composed of phospholipids, apolipoprotein, cholesterol and a small amount of fatty acids. As a natural nanocarrier, it has good biocompatibility and excellent stability, and plays an important role in the delivery of imaging reagents and therapeutic drugs. However, the delivery of drugs in vivo will encounter challenges such as easy degradation, stimulating the secretion of inflammatory cytokines, cytotoxicity, short half-life, difficulty in passing through the blood-brain barrier, off-target effects, etc. Therefore, it is necessary to deliver drugs through nanocarriers for effective targeted drug delivery. Can efficiently load related drugs. Through drug loading, gene drugs including (DNA), oligonucleotides (RNA), microRNA, small interfering nucleotides (siRNA) and small hairpin RNA (shRNA) can be loaded, as well as small molecule targeted drugs and chemotherapy Drugs, etc., for the complex pathways of cancer stem cells, microRNAs with multi-target regulation capabilities can be selected for loading, and multiple pathways can be regulated at the same time to inhibit their self-renewal ability. The drug-loaded lipoprotein nanocarrier is modified and further modified by targeting peptides, which is the nano-delivery system of the present invention.
现有已公开的靶向肿瘤干细胞的纳米递送系统技术主要集中于单纯受体与配体的结合。如现有的公开技术包括CN201810317543采用外泌体膜包裹纳米载体利用其CD81、CD9膜蛋白产生肿瘤干细胞的靶向效果；CN201711273795、CN201710684008采用VAP肽与GRP78蛋白的高亲和活性效果产生肿瘤干细胞的靶向效果；CN201710471867利用靶向CD133的肽链(CX1X2X3X4X5X6X7X8LX9)修饰的纳米脂质体或胶束进行肿瘤干细胞的靶向；CN201710198933利用A15(RNA适配体)-聚乙二醇-十八醇嫁接物修饰的脂质纳米载体靶向CD133进行肿瘤干细胞的靶向；CN201610708677通过透明质酸修饰的四氧化三铁携带DAPT药物在外加磁场下增加对肿瘤干细胞的杀伤作用；通过包裹有肿瘤干细胞抗原Oct4(15肽)的靶向树突状细胞C-型凝集素受体的阳离子脂质体诱导特异性T细胞杀伤肿瘤干细胞；CN20141026081通过HCPB-1多肽修饰的磁性纳米载体靶向H460肺癌干细胞；The existing published nano-delivery system technologies targeting tumor stem cells mainly focus on the binding of simple receptors and ligands. For example, the existing published technologies include CN201810317543 using exosomal membrane-wrapped nanocarriers to use their CD81 and CD9 membrane proteins to produce cancer stem cells; CN201711273795 and CN201710684008 use the high affinity activity of VAP peptides and GRP78 proteins to produce cancer stem cells. Targeting effect; CN201710471867 uses nanoliposomes or micelles modified with a peptide chain targeting CD133 (CX1X2X3X4X5X6X7X8LX9) to target tumor stem cells; CN201710198933 uses A15 (RNA aptamer)-polyethylene glycol-octadecyl alcohol grafting Drug-modified lipid nanocarriers target CD133 to target cancer stem cells; CN201610708677 uses hyaluronic acid-modified ferroferric oxide to carry DAPT drugs to increase the killing effect on cancer stem cells under an external magnetic field; by coating the tumor stem cell antigen Oct4 (15 peptides) cationic liposomes targeting the C-type lectin receptor of dendritic cells induce specific T cells to kill tumor stem cells; CN20141026081 targets H460 lung cancer stem cells through magnetic nanocarriers modified with HCPB-1 polypeptide;
除此之外现有已公开的靶向肿瘤干细胞的纳米药物还有通过携带药物起 到杀伤调控肿瘤干细胞的药效，包括CN107375213通过聚乙二醇-阿霉素和伊立替康产生一定的杀伤肿瘤干细胞的效果；CN201710097349通过碳纳米材料SWCNT抑制肿瘤干细胞的干性和分化；CN201610805898通过二茂铁基维甲酸/紫杉醇纳米载体共载microRNA抑制肿瘤干细胞；CN201510500260通过Angiopep-2修饰并包载盐霉素的脂蛋白纳米载体同时对肿瘤细胞、肿瘤干细胞及肿瘤新生血管进行多靶点杀伤。In addition, the currently published nanomedicines targeting cancer stem cells also have the effect of killing and regulating cancer stem cells by carrying drugs, including CN107375213 that produces certain killing through polyethylene glycol-doxorubicin and irinotecan. The effect of cancer stem cells; CN201710097349 inhibits the stemness and differentiation of cancer stem cells by carbon nanomaterial SWCNT; CN201610805898 inhibits cancer stem cells by co-loading microRNA with ferrocenyl retinoic acid/paclitaxel nanocarriers; CN201510500260 is modified by Angiopep-2 and encapsulated salt mold The lipoprotein nano-carriers of vitamin C can simultaneously kill tumor cells, tumor stem cells and tumor angiogenesis at multiple targets.
发明内容Summary of the invention
本发明的第一个目的在于，利用肿瘤干细胞可被激活的巨胞饮机制，提供靶向多肽修饰的载药脂蛋白纳米递药系统。The first objective of the present invention is to use the macropinocytosis mechanism of tumor stem cells to be activated to provide a drug-loaded lipoprotein nano-delivery system for targeted polypeptide modification.
本发明的第二个目的在于，提供一种产生激活增强巨胞饮机制的修饰载药脂蛋白纳米递药系统的靶向多肽。The second objective of the present invention is to provide a targeted polypeptide that produces a modified apolipoprotein nano-drug delivery system that activates and enhances the mechanism of giant pinocytosis.
本发明的第三个目的在于，提供一种靶向多肽修饰的载药脂蛋白纳米递药系统的制备方法。The third objective of the present invention is to provide a method for preparing a drug-loaded lipoprotein nano-drug delivery system modified by a targeted polypeptide.
本发明的第四个目的在于，提供一种靶向多肽修饰的载药脂蛋白纳米递药系统在制备预防或治疗肿瘤或中枢神经系统疾病的药物中的应用。The fourth objective of the present invention is to provide an application of a targeted polypeptide-modified drug-laden lipoprotein nano-drug delivery system in the preparation of drugs for the prevention or treatment of tumors or central nervous system diseases.
为了实现上述第一个目的，本发明提供了一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述递药系统包括脂质、载脂蛋白、载带药物和靶向多肽，所述靶向多肽由桥联结构将链接纳米载体端和激活巨胞饮功能的肽链共价连接形成，所述桥联结构包括Cys-Val、Cys-Phe、Cys-Leu、Cys-Ile、Cys-Gln、Leu-Glu、Gly-Ser-Gly、Ala-Pro-Ala、Cys-Pro-Cys、Gly-Phe-Leu、Gly-Phe-Leu-Gly、Ala-Leu-Ala-Leu、Gly-Phe-Leu-Gly、Gly-Gly-Gly-Gly-Ser、Val-Arg-Gly-Asp-Val、Pro-Ala-Pro-Ala-Pro、Pro-Leu-Gly-Leu-Trp-Ala、Arg-Val-Leu-Ala-Glu-Ala、聚乙二醇中的一种或多种。In order to achieve the above-mentioned first objective, the present invention provides a targeted polypeptide modified apolipoprotein nano-drug delivery system, characterized in that the drug delivery system includes lipid, apolipoprotein, loaded drug and target The targeting polypeptide is formed by covalently connecting the end of the nanocarrier and the peptide chain that activates the macropinocytosis function by a bridge structure, the bridge structure includes Cys-Val, Cys-Phe, Cys-Leu, Cys -Ile, Cys-Gln, Leu-Glu, Gly-Ser-Gly, Ala-Pro-Ala, Cys-Pro-Cys, Gly-Phe-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu , Gly-Phe-Leu-Gly, Gly-Gly-Gly-Gly-Ser, Val-Arg-Gly-Asp-Val, Pro-Ala-Pro-Ala-Pro, Pro-Leu-Gly-Leu-Trp-Ala , Arg-Val-Leu-Ala-Glu-Ala, one or more of polyethylene glycol.
作为一个优选方案，所述靶向多肽的序列为FH27(AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH，SEQ ID NO.1)、FH29(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR，SEQ ID NO.2)以及FH38(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH，SEQ ID NO.3)中的一种。As a preferred solution, the sequence of the targeting polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH, SEQ ID NO. 1), FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR, SEQ ID NO. 2) and FH38 (AC -FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH, SEQ ID NO. 3).
作为一个优选方案，所述脂质为蛋磷脂、豆磷脂、磷脂酰胆碱、磷脂酰乙 醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇、磷脂酸、心磷脂、溶血磷脂、鞘氨醇、神经酰胺、鞘磷脂、脑苷脂、胆固醇、胆固醇酯、甘油酯及其衍生物中的一种或多种。As a preferred solution, the lipid is egg phospholipid, soybean phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, lysophospholipid, sphingosine One or more of ceramide, sphingomyelin, cerebroside, cholesterol, cholesterol ester, glyceride and derivatives thereof.
作为一个优选方案，所述靶向多肽与脂质的摩尔比为1:10-1:300，优选1:100。As a preferred solution, the molar ratio of the targeting polypeptide to lipid is 1:10-1:300, preferably 1:100.
作为一个优选方案，所述载脂蛋白为ApoE，ApoA-I、ApoA-II、ApoA-IV，ApoC-I、ApoC-II、ApoC-III中的一种或多种。As a preferred solution, the apolipoprotein is one or more of ApoE, ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, and ApoC-III.
作为一个优选方案，所述载脂蛋白与靶向多肽的质量比为1:10-1:100，优选1:30。As a preferred solution, the mass ratio of the apolipoprotein to the targeting polypeptide is 1:10-1:100, preferably 1:30.
作为一个优选方案，所述递药系统还包括固相内核，所述固相内核由药物分子、不溶性或难溶性无机盐构成，药物分子负载在固相内核中，所述不溶性或难溶性无机盐为可生物降解的磷酸钙沉淀、碳酸钙沉淀、硫酸钙沉淀、氟化钙沉淀、硅酸钙沉淀、海藻酸钙沉淀、硫酸镁沉淀、磷酸镁沉淀、碳酸镁沉淀、氟化镁沉淀、硅酸镁沉淀、硫酸钡沉淀、磷酸钡沉淀、碳酸钡沉淀、氟化钡沉淀、硅酸钡沉淀的一种或多种。As a preferred solution, the drug delivery system further includes a solid phase core, the solid phase core is composed of drug molecules, insoluble or poorly soluble inorganic salts, the drug molecules are loaded in the solid phase core, the insoluble or poorly soluble inorganic salts For biodegradable calcium phosphate precipitation, calcium carbonate precipitation, calcium sulfate precipitation, calcium fluoride precipitation, calcium silicate precipitation, calcium alginate precipitation, magnesium sulfate precipitation, magnesium phosphate precipitation, magnesium carbonate precipitation, magnesium fluoride precipitation, silicon One or more of magnesium oxide precipitation, barium sulfate precipitation, barium phosphate precipitation, barium carbonate precipitation, barium fluoride precipitation, and barium silicate precipitation.
为了实现上述第二个目的，本发明提供了一种修饰载药脂蛋白纳米递药系统的靶向多肽，其特征在于，所述靶向多肽的序列为FH27(AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH)、FH29(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR)以及FH38(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH)中的一种。In order to achieve the above-mentioned second object, the present invention provides a targeting polypeptide for modifying a drug-loaded lipoprotein nano-drug delivery system, characterized in that the sequence of the targeting polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH) , FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) and FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH).
为了实现上述第三个目的，本发明提供了上述靶向多肽修饰的载药脂蛋白纳米递药系统的制备方法，包括如下步骤：In order to achieve the above-mentioned third objective, the present invention provides a method for preparing the above-mentioned targeted polypeptide-modified drug-laden lipoprotein nano-drug delivery system, which includes the following steps:
a)采用固相多肽合成法合成上述靶向多肽；a) Synthesize the above-mentioned targeted peptides by solid-phase peptide synthesis;
b)采用常规方法制备载药脂质纳米递药系统；b) Prepare the drug-loaded lipid nano-drug delivery system by conventional methods;
c)通过在上述b)制备的纳米递药系统溶液中先加入所述靶向多肽，再加入载脂蛋白，制备得到靶向多肽修饰的载药脂蛋白纳米递药系统。c) by adding the targeting polypeptide to the nano drug delivery system solution prepared in b) above, and then adding the apolipoprotein to prepare a drug delivery lipoprotein nano drug delivery system modified by the targeting polypeptide.
为了实现上述第四个目的，本发明提供了靶向多肽修饰的载药脂蛋白纳米递药系统在制备预防或治疗肿瘤或中枢神经系统疾病的药物中的应用。In order to achieve the fourth objective mentioned above, the present invention provides the application of a targeted polypeptide modified apolipoprotein nano-drug delivery system in the preparation of drugs for the prevention or treatment of tumors or central nervous system diseases.
作为一个优选方案，所述肿瘤为脑胶质瘤和胰腺癌。As a preferred solution, the tumor is glioma and pancreatic cancer.
本发明的优点在于，靶向多肽修饰的载药脂蛋白纳米递药系统基于巨胞饮的原理，通过链接靶向肽，能够靶向在肿瘤干细胞表面高表达受体的同时又能够激活巨胞饮通路，形成利用代谢差异形成靶向富集的效果。利用肿瘤干细胞通过增强的巨胞饮通路主动摄取载药脂蛋白纳米载体，进一步携带药物，进行多通路的抑制，由此克服目前的多重阻碍，大大提高了对肿瘤干细胞的体内外靶向效率和调控能力。The advantage of the present invention is that the targeted polypeptide modified apolipoprotein nano-drug delivery system is based on the principle of giant pinocytosis. By linking the targeting peptide, it can target the high expression receptors on the surface of tumor stem cells while activating giant cells. The drinking pathway forms the effect of using metabolic differences to form targeted enrichment. The use of cancer stem cells to actively uptake the drug-laden lipoprotein nanocarriers through the enhanced giant pinocytosis pathway, to further carry drugs, and to inhibit multiple pathways, thereby overcoming the current multiple obstacles and greatly improving the efficiency of in vivo and in vitro targeting of cancer stem cells. Control ability.
附图说明Description of the drawings
图1为透射电镜观察不含靶向肽以及含不同靶向肽(FH27、FH29、FH38)的载NC-microRNA药物的重组脂蛋白形态，标尺：50nm。Figure 1 is a transmission electron microscope observation of the morphology of the recombinant lipoprotein containing NC-microRNA drugs without targeting peptides and containing different targeting peptides (FH27, FH29, FH38), scale: 50nm.
图2为人源胶质瘤干细胞样细胞(A)以及胰腺癌细胞系富集干细胞样细胞(B)对不含靶向多肽以及含不同靶向肽修饰的荧光标记载药脂蛋白纳米载体的细胞摄取情况，*p<0.05,**p<0.01，****p<0.0001表示存在显著性差异。Figure 2 shows human-derived glioma stem cell-like cells (A) and pancreatic cancer cell line-enriched stem cell-like cells (B) vs. cells without targeting peptides and fluorescently labeled apolipoprotein nanocarriers modified with different targeting peptides Ingestion, *p<0.05, **p<0.01, ****p<0.0001 indicates that there is a significant difference.
图3为在不含靶向多肽以及经不同靶向多肽修饰的载药脂蛋白纳米载体刺激下，人源胶质瘤干细胞样细胞对巨胞饮小泡特异性标记物Dextran的摄取、共定位以及加入EIPA巨胞饮抑制剂后的抑制摄取情况，*p<0.05,**p<0.01表示与不含靶向多肽的载药脂蛋白纳米载体组存在显著性差异。Figure 3 shows the uptake and co-localization of the giant pinocytotic vesicle-specific marker Dextran by human glioma stem cell-like cells stimulated by drug-loaded lipoprotein nanocarriers without targeting peptides and modified with different targeting peptides And the inhibition of uptake after adding EIPA giant pinocytosis inhibitor, *p<0.05, **p<0.01 indicates that there is a significant difference from the apolipoprotein nanocarrier group without targeting peptide.
图4为不同巨胞饮激活受体CXCR4表达水平下的人源胶质瘤干细胞样细胞(A)以及胰腺癌细胞系富集干细胞样细胞(B)对不含靶向多肽以及含不同靶向多肽修饰的荧光标记载药脂蛋白纳米载体的细胞摄取情况，*p<0.05,**p<0.01，****p<0.0001表示与CXCR4未敲低组相比，细胞摄取存在显著性差异。Figure 4 shows the human-derived glioma stem cell-like cells (A) and pancreatic cancer cell lines enriched stem cell-like cells (B) under different expression levels of the giant pinocytosis activating receptor CXCR4. The cellular uptake of peptide-modified fluorescently labeled lipoprotein nanocarriers, *p<0.05, **p<0.01, ****p<0.0001 indicates that there is a significant difference in cellular uptake compared with the CXCR4 non-knockdown group .
图5为不含靶向多肽以及含不同靶向多肽修饰的荧光标记载药脂蛋白纳米载体与人源胶质瘤干细胞样细胞表面巨胞饮激活受体CXCR4的共定位情况(A)，巨胞饮激活受体CXCR4低表达时的共定位情况(B)，标尺：100μm。Figure 5 shows the co-localization of the fluorescently labeled apolipoprotein nanocarriers without targeting peptides and containing different targeting peptide modifications with the giant pinocytosis-activated receptor CXCR4 on the surface of human glioma stem cell-like cells (A). Co-localization of pinocytosis-activated receptor CXCR4 with low expression (B), scale: 100μm.
图6为在NOD/SCID小鼠脑内注射人源胶质瘤干细胞样细胞(A)以及巨胞饮受体CXCR4低表达人源胶质瘤干细胞样细胞(B)，构建原位荷瘤小鼠模型，研究不同靶向多肽修饰的荧光标记载药脂蛋白纳米载体对原位荷瘤鼠血脑屏障的透过性，以不含靶向多肽修饰的载药脂蛋白纳米载体作为对照制剂，标尺：100μm。Figure 6 shows the injection of human glioma stem cell-like cells (A) and the giant pinocytosis receptor CXCR4 low-expression human glioma stem cell-like cells (B) into the brain of NOD/SCID mice to construct an in situ tumor-bearing cell Mouse model to study the permeability of fluorescently labeled drug-loaded lipoprotein nanocarriers modified by different targeting peptides to the blood-brain barrier of tumor-bearing mice in situ, and the drug-loaded lipoprotein nanocarriers without targeting peptide modification were used as control preparations. Scale bar: 100μm.
图7为在NOD/SCID小鼠脑内注射人源胶质瘤干细胞样细胞以及巨胞饮受体CXCR4低表达人源胶质瘤干细胞样细胞，构建原位荷瘤小鼠模型，研究：(A)不同靶向多肽修饰的载药重组脂蛋白载体与对人源胶质瘤干细胞样细胞靶向能力；(B)不同靶向多肽修饰的载药重组脂蛋白载体在胶质瘤组织的分布与巨胞饮受体的表达水平的相关性，以不含靶向多肽修饰的载药脂蛋白纳米载体作为对照制剂，标尺：100μm。Figure 7 shows the injection of human glioma stem cell-like cells and human glioma stem cell-like cells with low expression of the giant pinocytosis receptor CXCR4 into the brain of NOD/SCID mice to construct an orthotopic tumor-bearing mouse model. Research:( A) Drug-loaded recombinant lipoprotein carriers modified by different targeting peptides and their ability to target human glioma stem cell-like cells; (B) Distribution of drug-loaded recombinant lipoprotein carriers modified by different targeting peptides in glioma tissues The correlation with the expression level of the giant pinocytosis receptor, the drug-laden lipoprotein nanocarrier without targeted polypeptide modification was used as the control preparation, and the scale: 100 μm.
图8为靶向多肽修饰的载miR-34a microRNA的脂蛋白纳米载体用于敲低人源胶质瘤干细胞样细胞干性相关SOX2蛋白表达(A)，抑制肿瘤细胞自我更新能力(B)。Fig. 8 shows that lipoprotein nanocarriers carrying miR-34a microRNAs modified by targeting polypeptides are used to knock down the stemness-related SOX2 protein expression of human glioma stem cell-like cells (A) and inhibit the self-renewal ability of tumor cells (B).
图9为靶向多肽修饰的载miR-34a microRNA脂蛋白纳米载体与化疗药物替莫唑胺联合用药后，替莫唑胺IC50的测定。Figure 9 shows the determination of the IC50 of temozolomide after the combination of targeted peptide-modified miR-34a microRNA lipoprotein nanocarriers and the chemotherapeutic drug temozolomide.
图10为靶向多肽修饰的载miR-34a microRNA脂蛋白纳米载体用于抑制原位荷人源胶质瘤干细胞样细胞NOD/SCID鼠肿瘤细胞增殖(A)，延长小鼠生存期(B)。Figure 10: Targeted peptide-modified miR-34a microRNA lipoprotein nanocarrier is used to inhibit the proliferation of NOD/SCID mouse tumor cells bearing human glioma stem cell-like cells in situ (A) and prolong the survival time of mice (B) .
具体实施方式detailed description
以下，结合具体实施方式对本发明的技术进行详细描述。应当知道的是，以下具体实施方式仅用于帮助本领域技术人员理解本发明，而非对本发明的限制。Hereinafter, the technology of the present invention will be described in detail in combination with specific embodiments. It should be understood that the following specific implementations are only used to help those skilled in the art understand the present invention, but not to limit the present invention.
实施例1.靶向多肽修饰的载药脂蛋白纳米递药系统的制备和表征Example 1. Preparation and Characterization of a Targeted Polypeptide Modified Apolipoprotein Nano-Drug Delivery System
(1)制备(1) Preparation
采用反相微乳法制备载药的磷酸钙固相内核。首先制备钙相，将300μL浓度为2.5M的CaCl 2溶液与NC-miRNA共孵育，吹打数次，而后分散于20mL油相中形成分散均一的油包水反相微乳。磷相的制备为将300μL浓度为12.5mM Na 2HPO 4溶液分散于另一份20mL油相中，搅拌10min后，向磷相加入100μL浓度为20mg/mL的1,2-油酰磷脂酸(1,2-dioleoyl phosphatidic acid，DOPA)溶液。待两相分散均匀后，将两相混合搅拌45min。此时，将40mL无水乙醇加入到上述混合微乳中破乳10min。破乳后的混合液通过高速离心(12,500g)约20min除去多余的表面活性剂及环己烷。在同样离心操作进行三次后，离心得到的沉淀即为载DOPA修饰的载药磷酸钙，将其分散于1mL 氯仿中存放于玻璃瓶中用于后续实验。 The drug-loaded calcium phosphate solid phase core was prepared by the reverse microemulsion method. First, the calcium phase was prepared, 300 μL of 2.5M CaCl 2 solution was incubated with NC-miRNA, pipetting several times, and then dispersed in 20 mL of the oil phase to form a uniformly dispersed water-in-oil reverse microemulsion. The phosphorus phase was prepared by dispersing 300 μL of 12.5mM Na 2 HPO 4 solution in another 20 mL oil phase, stirring for 10 minutes, and adding 100 μL of 20 mg/mL 1,2-oleoylphosphatidic acid ( 1,2-dioleoyl phosphatidic acid, DOPA) solution. After the two phases are evenly dispersed, the two phases are mixed and stirred for 45 minutes. At this time, 40 mL of absolute ethanol was added to the above mixed microemulsion to break the emulsion for 10 minutes. After demulsification, the mixed solution is centrifuged at a high speed (12,500 g) for about 20 minutes to remove excess surfactant and cyclohexane. After the same centrifugation operation was performed three times, the precipitate obtained by centrifugation was the DOPA-modified drug-loaded calcium phosphate, which was dispersed in 1 mL of chloroform and stored in a glass bottle for subsequent experiments.
采用薄膜水化法制备载药普通脂质体：称取脂质(2-10mg)置于500mL圆底烧瓶中，加入2mL乙醚，挥干除去磷脂中的水分，再加入上述制备的载药磷酸钙750μL及2mL氯仿溶液，置旋转蒸发仪上抽真空1h。加入4mL 0.01M PBS溶液(pH 7.4)，于40℃水浴间歇振摇10min至薄膜水化脱落得到脂质体。探头超声进一步减小脂质体粒径，得到含载药磷酸钙的普通脂质体(CaP-LNC)。The thin-film hydration method was used to prepare drug-loaded ordinary liposomes: weigh the lipids (2-10mg) into a 500mL round-bottomed flask, add 2mL ether, evaporate dry to remove the water in the phospholipids, and then add the drug-loaded phosphoric acid prepared above Calcium 750μL and 2mL chloroform solution were placed on the rotary evaporator and vacuumed for 1h. Add 4mL 0.01M PBS solution (pH 7.4), shake intermittently for 10 minutes in a 40°C water bath until the film is hydrated and falls off to obtain liposomes. Probe ultrasound further reduces the particle size of liposomes, and obtains common liposomes (CaP-LNC) containing drug-loaded calcium phosphate (CaP-LNC).
采用固相多肽合成法，合成靶向多肽FH27(AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH)、FH29(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR)以及FH38(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH)。也可将桥联结构替换为Cys-Val、Cys-Phe、Cys-Leu、Cys-Ile、Cys-Gln、Leu-Glu、Gly-Ser-Gly、Ala-Pro-Ala、Cys-Pro-Cys、Gly-Phe-Leu、Gly-Phe-Leu-Gly、Ala-Leu-Ala-Leu、Gly-Phe-Leu-Gly、Gly-Gly-Gly-Gly-Ser、Val-Arg-Gly-Asp-Val、Pro-Ala-Pro-Ala-Pro、Pro-Leu-Gly-Leu-Trp-Ala、Arg-Val-Leu-Ala-Glu-Ala、聚乙二醇中的一种或多种。The solid-phase peptide synthesis method was used to synthesize targeted peptides FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH), FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) and FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH). The bridge structure can also be replaced with Cys-Val, Cys-Phe, Cys-Leu, Cys-Ile, Cys-Gln, Leu-Glu, Gly-Ser-Gly, Ala-Pro-Ala, Cys-Pro-Cys, Gly-Phe-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Gly-Gly-Ser, Val-Arg-Gly-Asp-Val, One or more of Pro-Ala-Pro-Ala-Pro, Pro-Leu-Gly-Leu-Trp-Ala, Arg-Val-Leu-Ala-Glu-Ala, and polyethylene glycol.
具体方法：在氯甲基聚苯乙烯树脂上接入氨基酸，在三氟乙酸的保护下脱氨基保护基团。而后通过氟化氢进行切割，乙醚冰浴沉淀，乙腈溶解后旋蒸，并采用乙腈水体系进一步纯化。将制得靶向多肽按照与磷脂的摩尔比1：30-1:100加入普通脂质体中，120rpm，37℃孵育24小时，而后将ApoE或ApoA-I等载脂蛋白(0.1-10mg)加入到上述溶液中(脂质总质量4mg)，轻轻混匀，置于震荡摇床于120rpm，37℃孵育24h，得到靶向多肽修饰的载NC-miRNA药物的脂蛋白纳米载体。同时作为一种优选方案的对照，靶向肽与载脂蛋白共同加入普通脂质体中孵育48小时。The specific method: insert amino acid into chloromethyl polystyrene resin, and remove amino protecting group under the protection of trifluoroacetic acid. Then it was cut by hydrogen fluoride, ether was precipitated in an ice bath, acetonitrile was dissolved and then revolved, and the acetonitrile water system was used for further purification. The prepared targeting polypeptide was added to ordinary liposomes at a molar ratio of 1:30-1:100 to phospholipids, incubated at 120 rpm and 37°C for 24 hours, and then apolipoproteins such as ApoE or ApoA-I (0.1-10 mg) Add to the above solution (the total lipid mass is 4 mg), mix gently, place on a shaking shaker at 120 rpm, and incubate at 37° C. for 24 hours to obtain a lipoprotein nanocarrier modified by targeting polypeptides and NC-miRNA drugs. At the same time, as a control for a preferred scheme, the targeting peptide and apolipoprotein were added to ordinary liposomes and incubated for 48 hours.
(2)表征(2) Characterization
靶向多肽修饰的载药脂蛋白纳米载体磷钨酸负染，透射电镜观察形态。激光粒度仪测定其粒径和表面电位，与未孵育靶向肽和ApoE的含磷酸钙载药普通脂质体比较。如图1，在电镜下含载药磷酸钙的重组脂蛋白呈均一的球形，分散均匀，粒径大小在30nm-50nm左右，链接FH38靶向肽的纳米载体粒径 相对较大，通过激光粒度仪测定其表面电位约为-25mV左右。Targeting peptide modified drug-loaded lipoprotein nanocarriers were negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscope. The particle size and surface potential of the laser particle size analyzer were compared with those of ordinary liposomes containing calcium phosphate and drug-loaded calcium phosphate without incubating the targeting peptide and ApoE. As shown in Figure 1, under the electron microscope, the recombinant lipoprotein containing drug-loaded calcium phosphate is uniformly spherical, uniformly dispersed, and the particle size is about 30nm-50nm. The particle size of the nanocarrier linked to the FH38 targeting peptide is relatively large, and the particle size by laser The surface potential measured by the instrument is about -25mV.
实施例2.多种肿瘤干细胞高效摄取靶向多肽修饰的载药脂蛋白纳米载体Example 2. A variety of tumor stem cells efficiently uptake targeted polypeptide-modified drug-loaded lipoprotein nanocarriers
(1)制备(1) Preparation
同实施例1采用反相微乳法制备载药磷酸钙，后续采用薄膜水化法制备载药的普通脂质体，称取磷脂酰胆碱、磷脂酸(2-10mg)和荧光染料DiI(20-100μg)放入500ml圆底烧瓶中，同实施例1制备荧光标记的普通脂质体，将靶向多肽按照与磷脂的摩尔比1:100加入普通脂质体中，120rpm，37℃孵育24小时，而后将ApoE或ApoA-I等载脂蛋白(0.5-10mg)加入到上述溶液中(脂质总质量4mg)，轻轻混匀，置于震荡摇床于120rpm，37℃孵育24h，得到靶向多肽修饰的荧光标记载药脂蛋白纳米载体。As in Example 1, the drug-loaded calcium phosphate was prepared by the reverse microemulsion method, and then the drug-loaded ordinary liposomes were prepared by the membrane hydration method, and phosphatidylcholine, phosphatidic acid (2-10mg) and the fluorescent dye DiI( 20-100μg) into a 500ml round-bottomed flask, prepare fluorescently labeled ordinary liposomes as in Example 1, add the targeting peptide to the ordinary liposomes at a molar ratio of 1:100 to the phospholipids, and incubate at 120rpm, 37°C After 24 hours, add ApoE or ApoA-I and other apolipoproteins (0.5-10mg) to the above solution (total lipid weight 4mg), mix gently, and place in a shaking shaker at 120rpm and incubate at 37°C for 24h. A fluorescently labeled drug-loaded lipoprotein nanocarrier modified by a targeted polypeptide is obtained.
(2)将人源胶质瘤干细胞样细胞、胰腺癌细胞系富集干细胞样细胞以100个球接种于24孔板，5％二氧化碳培养箱培养过夜后，加入载荧光探针DiI的含靶向肽修饰的纳米载体和不含靶向肽修饰的纳米载体的DMEM溶液，DMPC浓度为20μg/ml，孵育4小时，PBS清洗一次后，轻柔吹打50次，将肿瘤球吹散后，过100μm孔径的细胞筛，而后采用多聚甲醛固定。将细胞悬液经过流式细胞仪检测其摄取制剂的荧光强度。(2) Human-derived glioma stem cell-like cells and pancreatic cancer cell line-enriched stem cell-like cells were seeded in a 24-well plate with 100 balls, incubated overnight in a 5% carbon dioxide incubator, and then a target containing fluorescent probe DiI was added. To the peptide-modified nanocarriers and the DMEM solution without targeting peptide-modified nanocarriers, the DMPC concentration is 20μg/ml, incubate for 4 hours, wash once with PBS, gently pipette 50 times, blow the tumor ball away, pass 100μm Cell sieve with pore size, and then fixed with paraformaldehyde. The cell suspension was passed through a flow cytometer to detect the fluorescence intensity of the ingested preparation.
结果如图2A所示，在人源胶质瘤干细胞样细胞中，含FH38靶向肽的载药脂蛋白纳米载体的摄取效率是不含靶向肽脂蛋白纳米制剂的3.0倍，含FH29靶向肽的载药脂蛋白纳米载体的摄取效率是不含靶向肽脂蛋白纳米制剂的1.7倍，而含FH27靶向肽的载药脂蛋白纳米载体的摄取效率是不含靶向肽脂蛋白纳米制剂的1.1倍。且该摄取与载脂蛋白密切相关，无载脂蛋白单纯孵育靶向肽无明显增强效果。同样在人胰腺癌细胞系富集干细胞样细胞模型中，如图2B所示，含FH38靶向肽的载药脂蛋白纳米载体的摄取效率是不含靶向肽脂蛋白纳米制剂的1.5-1.8倍，表明靶向多肽修饰的载荧光的载药脂蛋白纳米载体在多种肿瘤模型中均具有增强摄取的效果。作为一种优选方案的对比，共同孵育靶向肽的摄取效率要低于先孵育靶向肽后孵育载脂蛋白的效率。The results are shown in Figure 2A. In human glioma stem cell-like cells, the uptake efficiency of the lipoprotein nanocarrier containing the FH38 targeting peptide is 3.0 times that of the lipoprotein nanoformulation without the targeting peptide and containing the FH29 target. The uptake efficiency of drug-loaded lipoprotein nanocarriers to peptides is 1.7 times that of lipoprotein nano-preparations without targeting peptides, while the uptake efficiency of drug-loaded lipoprotein nanocarriers containing FH27 targeting peptides is that of lipoproteins without targeting peptides. 1.1 times of nano formulations. And the uptake is closely related to apolipoprotein, and simply incubating the targeting peptide without apolipoprotein has no obvious enhancement effect. Also in the human pancreatic cancer cell line enriched stem cell-like cell model, as shown in Figure 2B, the uptake efficiency of the drug-laden lipoprotein nanocarrier containing the FH38 targeting peptide is 1.5-1.8 that of the lipoprotein nano-preparation without the targeting peptide. Times, indicating that the targeted peptide-modified fluorescent drug-loaded lipoprotein nanocarriers have the effect of enhancing uptake in a variety of tumor models. As a comparison of a preferred scheme, the uptake efficiency of co-incubating the targeting peptide is lower than the efficiency of incubating the targeting peptide first and then incubating apolipoprotein.
实施例3.靶向肽修饰的载药脂蛋白纳米载体通过增强巨胞饮提高肿瘤干细胞的摄取效率Example 3. Targeting peptide-modified drug-loaded lipoprotein nanocarriers improve the uptake efficiency of tumor stem cells by enhancing macropinocytosis
(1)制备(1) Preparation
采用薄膜水化法制备载药的普通脂质体，称取磷脂酰胆碱、磷脂酸(2-10mg)放入500ml圆底烧瓶中，同实施例1制备普通脂质体，将靶向肽按照与磷脂的摩尔比1:100加入普通脂质体中，120rpm，37℃孵育24小时，而后将ApoE或ApoA-I等载脂蛋白(0.5-10mg)加入到上述溶液中(脂质总质量4mg)，轻轻混匀，置于震荡摇床于120rpm，37℃孵育24h，得到靶向多肽修饰的载药脂蛋白纳米载体。Prepare drug-loaded common liposomes by membrane hydration method, weigh phosphatidylcholine and phosphatidic acid (2-10mg) into a 500ml round-bottomed flask, prepare common liposomes as in Example 1, and combine the targeting peptide Add ordinary liposomes according to the molar ratio of 1:100 to phospholipids, incubate at 120rpm and 37°C for 24 hours, and then add ApoE or ApoA-I and other apolipoproteins (0.5-10mg) to the above solution (total lipid mass 4mg), mix gently, place on a shaking shaker at 120rpm, and incubate at 37°C for 24h to obtain the targeted polypeptide modified drug-loaded lipoprotein nanocarrier.
(2)将肿瘤干细胞以100个球接种于24孔板，5％二氧化碳培养箱培养过夜后，加入含靶向肽修饰的纳米载体和不含靶向肽修饰的纳米载体的DMEM溶液，DMPC浓度为20μg/ml，孵育2.5小时，而后加入巨胞饮通路标记物FITC-Dextran(1mg/ml)，再共孵育1.5小时，弃去培养液后PBS清洗一次，轻柔吹打50次，将肿瘤球吹散后，过100μm孔径的细胞筛，而后采用多聚甲醛固定。将细胞悬液经过流式细胞仪检测其FITC-Dextran的荧光强度。进一步通过巨胞饮抑制剂EIPA 150μM处理1.5小时后采用激光共聚焦显微镜观察制剂与Dextran的摄取和共定位情况。(2) 100 spheres of tumor stem cells were inoculated on a 24-well plate and cultured in a 5% carbon dioxide incubator overnight. Then, add the targeted peptide-modified nanocarrier and the DMEM solution without the targeted peptide-modified nanocarrier, and the concentration of DMPC Incubate for 2.5 hours at 20μg/ml, then add FITC-Dextran (1mg/ml), a marker of the giant pinocytosis pathway, and incubate for a total of 1.5 hours. Discard the culture medium and wash with PBS once, gently pipette 50 times to blow the tumor ball After dispersing, pass through a cell sieve with a pore size of 100 μm, and then fix with paraformaldehyde. The cell suspension was subjected to flow cytometry to detect the fluorescence intensity of FITC-Dextran. After further treatment with the giant pinocytosis inhibitor EIPA 150 μM for 1.5 hours, the uptake and co-localization of the preparation and Dextran were observed using a laser confocal microscope.
结果如图3所示，在人源胶质瘤干细胞样细胞中，含FH38靶向肽的载药脂蛋白纳米载体的能够增强巨胞饮小泡特异性标记物Dextran的摄取量1.3倍，表明该增强机制与巨胞饮通路密切相关。可观察到制剂与dextran高度共定位，且EIPA能够有效抑制制剂和dextran的摄取。The results are shown in Fig. 3, in human glioma stem cell-like cells, the apolipoprotein nanocarrier containing the FH38 targeting peptide can enhance the uptake of the giant pinocytotic vesicle specific marker Dextran by 1.3 times, indicating This enhancement mechanism is closely related to the giant pinocytosis pathway. It can be observed that the preparation and dextran are highly co-localized, and EIPA can effectively inhibit the uptake of the preparation and dextran.
实施例4.不同巨胞饮激活受体(CXCR4)表达水平的多肿瘤干细胞对靶向多肽修饰的载药脂蛋白纳米载体摄取情况对比。Example 4. Comparison of the uptake of targeted polypeptide-modified apolipoprotein nanocarriers by multiple tumor stem cells with different expression levels of the giant pinocytosis activating receptor (CXCR4).
(1)制备(1) Preparation
称取磷脂酰胆碱、磷脂酸(2-10mg)和荧光染料DiI(20-100μg)放入500ml圆底烧瓶中，同实施例1制备荧光标记的普通脂质体，将靶向肽按照与磷脂的摩尔比1:100加入普通脂质体中，120rpm，37℃孵育24小时，而后将ApoE或ApoA-I等载脂蛋白(0.5-10mg)加入到上述溶液中(脂质总质量4mg)，轻轻混匀，置于震荡摇床于120rpm，37℃孵育24h，得到靶向多肽修饰荧光标记载药脂蛋白纳米载体。Weigh phosphatidylcholine, phosphatidic acid (2-10mg) and fluorescent dye DiI (20-100μg) into a 500ml round-bottomed flask, prepare fluorescent-labeled ordinary liposomes as in Example 1, and combine the targeting peptides with Phospholipid molar ratio 1:100 was added to ordinary liposomes, incubated at 120 rpm, 37°C for 24 hours, and then ApoE or ApoA-I and other apolipoproteins (0.5-10 mg) were added to the above solution (total lipid weight 4 mg) , Mix gently, place on a shaking shaker at 120 rpm, and incubate at 37°C for 24 hours to obtain the targeted polypeptide modified fluorescently labeled drug-laden lipoprotein nanocarrier.
(2)靶向多肽修饰的载药的脂蛋白纳米载体增强肿瘤干细胞的摄取与细胞巨胞饮受体表达水平相关(2) Targeted peptide-modified drug-loaded lipoprotein nanocarriers enhance the uptake of cancer stem cells and are related to the expression level of cell giant pinocytosis receptors
将人源胶质瘤干细胞样细胞、胰腺癌细胞系富集干细胞样细胞以10 5个细胞密度接种于12孔板，培养过夜后，加入CXCR4shRNA慢病毒载体系统转染细胞(10μl/ml)，阴性NC shRNA慢病毒载体系统作为对照。转染18h后，吸除含慢病毒的培养基，离心换为新鲜培养基。继续培养3天后，向培养基中加入2μg/ml的嘌呤霉素杀死未转染成功的细胞。 After the human glioma stem-like cells, pancreatic cancer cell line enriched stem cell-like cells at 10 5 cells were plated in 12-well plate, cultured overnight, CXCR4shRNA lentiviral vector system transfected cells (10μl / ml), Negative NC shRNA lentiviral vector system was used as a control. After 18 hours of transfection, the medium containing the lentivirus was aspirated and centrifuged to change to fresh medium. After continuing the culture for 3 days, 2μg/ml puromycin was added to the culture medium to kill the cells that were not successfully transfected.
将巨胞饮受体敲低的人源胶质瘤干细胞样细胞、胰腺癌细胞系富集干细胞样细胞和对照组以100个球接种于24孔板，5％二氧化碳培养箱培养过夜后，加入载荧光探针DiI的含靶向肽修饰的纳米载体和不含靶向肽修饰的纳米载体的DMEM溶液，DMPC浓度为20μg/ml，孵育4小时，PBS清洗一次后，轻柔吹打50次，将肿瘤球吹散后，过100μm孔径的细胞筛，而后采用多聚甲醛固定。将细胞悬液经过流式细胞仪检测其摄取制剂的荧光强度。The human-derived glioma stem cell-like cells, pancreatic cancer cell line-enriched stem cell-like cells and the control group knocked down by giant pinocytosis receptors were inoculated into a 24-well plate with 100 balls, incubated overnight in a 5% carbon dioxide incubator, and then added Carrying fluorescent probe DiI containing targeting peptide-modified nanocarriers and DMEM solution containing no targeting peptide-modified nanocarriers, DMPC concentration is 20μg/ml, incubate for 4 hours, wash once with PBS, gently pipette 50 times, After the tumor ball is blown away, it is passed through a cell sieve with a pore size of 100 μm, and then fixed with paraformaldehyde. The cell suspension was passed through a flow cytometer to detect the fluorescence intensity of the ingested preparation.
结果如图4所示，当CXCR4表达水平降低时，人源胶质瘤干细胞样细胞以及胰腺癌细胞系富集干细胞样细胞对含靶向肽修饰的纳米载体的摄取显著减少，表明了巨胞饮激活受体(CXCR4)高表达的肿瘤干细胞更容易摄取靶向多肽修饰的载药脂蛋白纳米载体。The results are shown in Figure 4, when the expression level of CXCR4 decreases, the uptake of human-derived glioma stem cell-like cells and pancreatic cancer cell lines-enriched stem cell-like cells to the targeted peptide-modified nanocarriers is significantly reduced, indicating that giant cells Cancer stem cells with high expression of CXCR4 are more likely to take up targeted peptide-modified apolipoprotein nanocarriers.
实施例5.靶向多肽修饰的载药脂蛋白纳米载体通过巨胞饮激活受体(CXCR4)靶向摄取的评价Example 5. Evaluation of targeted uptake of apolipoprotein nanocarriers modified by targeting polypeptides via macropinocytosis activated receptor (CXCR4)
(1)制备(1) Preparation
制备方法同实施例4，制得不含靶向多肽以及含不同靶向多肽修饰的荧光标记载药脂蛋白纳米载体，分别命名为DiI-CaP-rHDL,FH27-DiI-CaP-rHDL,FH29-DiI-CaP-rHDL和FH38-DiI-CaP-rHDL。The preparation method was the same as that in Example 4. The fluorescently labeled apolipoprotein nanocarriers containing no targeting polypeptides and containing different targeting polypeptide modifications were prepared, and they were named DiI-CaP-rHDL, FH27-DiI-CaP-rHDL, FH29- DiI-CaP-rHDL and FH38-DiI-CaP-rHDL.
(2)靶向多肽修饰的荧光标记载药脂蛋白纳米载体通过肿瘤干细胞巨胞饮受体靶向摄取。(2) Targeted peptide-modified fluorescently labeled drug-loaded lipoprotein nanocarriers are targeted for uptake by tumor stem cell giant pinocytosis receptors.
将巨胞饮受体敲低与不敲低的人源胶质瘤干细胞样细胞以100个球接种于24孔板，5％二氧化碳培养箱培养过夜后，加入载荧光探针DiI的含靶向肽修饰的纳米载体和不含靶向肽修饰的纳米载体的DMEM溶液，DMPC浓度为20μg/ml，孵育3.5小时。PBS洗一遍后，4％多聚甲醛室温固定15min，0.1％Triton-X通透20min，PBS洗一遍，加入4％BSA封闭40min。与细胞干性相关抗体SOX2在4℃孵育过夜，再与Alexa
Figure PCTCN2020106735-appb-000001
488标记的荧光二抗孵育， PBS洗涤后用100ng/mL DAPI染色10min，加入抗荧光猝灭封片剂滴入玻璃底小皿中，荧光共聚焦显微镜观察。 The human-derived glioma stem cell-like cells with and without giant pinocytosis receptor knockdown were seeded in a 24-well plate with 100 spheres and incubated overnight in a 5% carbon dioxide incubator. Then, a fluorescent probe containing DiI containing targeting was added The peptide-modified nanocarrier and the DMEM solution without the targeting peptide-modified nanocarrier, the DMPC concentration is 20 μg/ml, are incubated for 3.5 hours. After washing with PBS, 4% paraformaldehyde was fixed at room temperature for 15 minutes, 0.1% Triton-X was permeated for 20 minutes, PBS was washed again, and 4% BSA was added to block for 40 minutes. Incubate with cell stemness-related antibody SOX2 overnight at 4°C, and then with Alexa
Figure PCTCN2020106735-appb-000001
Incubate with 488-labeled fluorescent secondary antibody, wash with PBS and stain with 100ng/mL DAPI for 10 minutes, add anti-fluorescence quenching mounting tablets and drop them into a glass-bottomed small dish, observe with a fluorescent confocal microscope.
结果如图5A所示，相比不含靶向多肽的制剂DiI-CaP-rHDL，靶向多肽修饰的荧光标记载药脂蛋白纳米载体在人源胶质瘤干细胞样细胞内部大量积聚且与细胞表面巨胞饮受体CXCR4存在共定位；如图5B所示，当巨胞饮受体CXCR4敲低后，人源胶质瘤干细胞样细胞对制剂的摄取减少，表明靶向多肽修饰的荧光标记载药脂蛋白纳米载体通过肿瘤干细胞巨胞饮受体靶向摄取。The results are shown in Figure 5A. Compared with the preparation DiI-CaP-rHDL without targeting peptides, the fluorescently labeled lipoprotein nanocarriers modified by targeting peptides accumulate in large amounts in human glioma stem cell-like cells and interact with the cells. The surface giant pinocytosis receptor CXCR4 is co-localized; as shown in Figure 5B, when the giant pinocytosis receptor CXCR4 is knocked down, the uptake of the preparation by human glioma stem cell-like cells is reduced, indicating that the fluorescent label modified by the targeted peptide It is recorded that the drug lipoprotein nanocarriers are targeted for uptake through tumor stem cell giant pinocytosis receptors.
实施例6.不同靶向多肽修饰的载药脂蛋白纳米载体对于血脑屏障透过性的评价Example 6. Evaluation of the permeability of the blood-brain barrier by drug-loaded lipoprotein nanocarriers modified by different targeting polypeptides
(1)制备(1) Preparation
同实施例1采用反相微乳法制备载Cy5标记的无作用阴性对照microRNA磷酸钙载药的重组脂蛋白(Cy5-CaP-rHDL)。将靶向肽按照与磷脂的摩尔比1:100加入普通脂质体中，120rpm，37℃孵育24小时，而后将ApoE或ApoA-I等载脂蛋白(0.5-10mg)加入到上述溶液中(脂质总质量4mg)，轻轻混匀，置于震荡摇床于120rpm，37℃孵育24h，得到靶向多肽修饰的载Cy5荧光标记的载药脂蛋白纳米载体。As in Example 1, a reverse microemulsion method was used to prepare Cy5-labeled non-effect negative control microRNA calcium phosphate drug-loaded recombinant lipoprotein (Cy5-CaP-rHDL). The targeting peptide was added to ordinary liposomes at a molar ratio of 1:100 to the phospholipids, and incubated at 120 rpm and 37°C for 24 hours, and then ApoE or ApoA-I and other apolipoproteins (0.5-10 mg) were added to the above solution ( The total lipid mass is 4 mg), gently mix, place on a shaking shaker at 120 rpm, and incubate at 37° C. for 24 hours to obtain a targeted peptide-modified Cy5 fluorescently labeled apolipoprotein nanocarrier.
(2)采用NOD/SCID小鼠脑定位注射巨胞饮受体未敲低以及敲低后的人源胶质瘤干细胞样细胞，构建原位荷胶质瘤小鼠模型，评价靶向多肽修饰的载药脂蛋白纳米载体对于荷瘤小鼠血脑屏障的透过性以及与细胞巨胞饮受体表达水平的相关性(2) Using NOD/SCID mouse brain-localized injection of human-derived glioma stem cell-like cells without knockdown and knockdown of the giant pinocytosis receptor, construct an in situ glioma-bearing mouse model and evaluate targeted peptide modification The permeability of the drug-loaded lipoprotein nanocarrier to the blood-brain barrier of tumor-bearing mice and its correlation with the expression level of cell giant pinocytosis receptors
用截留分子量3kD的超滤离心管浓缩Cy5标记、靶向多肽修饰的载药重组脂蛋白和不经靶向多肽修饰的载药重组脂蛋白溶液，按miRNA 0.36mg/kg的剂量给药。荷瘤小鼠造模7天后，尾静脉给予载Cy5标记的靶向多肽修饰及无靶向多肽修饰的载药重组脂蛋白制剂溶液。给药4h后，用5％水合氯醛麻醉并固定小鼠，剪开胸腔，充分暴露心脏，将头皮针头刺入左心室，剪开右心耳，立即用0.9％的生理盐水灌流至流出的灌流液无血色，然后以4％多聚甲醛溶液灌流固定，直至肝脏、四肢、尾变硬，取出心、肝、脾、肺、肾和脑(肿瘤)组织，生理盐水冲洗后置于小动物活体成像仪采集图像。实验结果如图6A所示，在巨胞饮受体CXCR4未敲低的荷瘤小鼠中，含FH38靶向肽的载药脂 蛋白纳米载体给药组(FH38-Cy5-CaP-rHDL)小鼠脑部荧光强度高于其他组，表明相对于不含靶向多肽修饰的载体，含靶向多肽修饰的载药脂蛋白纳米载体可以高效透过血脑屏障分布于肿瘤部位。实验结果如图6B所示，在巨胞饮受体CXCR4敲低的荷瘤小鼠中，FH38-Cy5-CaP-rHDL处理组的小鼠脑部荧光强度显著低于不含靶向多肽的载药脂蛋白纳米载体对照组(Cy5-CaP-rHDL)，表明载体的这种靶向能力与细胞巨胞饮受体表达水平相关，巨胞饮受体表达水平越高，载体靶向能力越强。Concentrate the Cy5-labeled, targeted polypeptide-modified drug-loaded recombinant lipoprotein and the drug-loaded recombinant lipoprotein solution not modified by the targeted polypeptide with an ultrafiltration centrifuge tube with a molecular weight cutoff of 3kD, and administer the miRNA at a dose of 0.36 mg/kg. Seven days after the tumor-bearing mice were modeled, a Cy5-labeled targeted polypeptide modification and a drug-loaded recombinant lipoprotein preparation solution without targeted polypeptide modification were administered to the tail vein. After administration for 4 hours, the mice were anesthetized and fixed with 5% chloral hydrate, the thoracic cavity was cut open, the heart was fully exposed, the scalp needle was inserted into the left ventricle, the right atrial appendage was cut open, and 0.9% normal saline was immediately perfused to the outflow perfusion. The solution is bloodless, and then fixed by perfusion with 4% paraformaldehyde solution until the liver, limbs, and tail become hard, remove the heart, liver, spleen, lung, kidney and brain (tumor) tissues, rinse with saline and place them in a living animal The imager collects images. The results of the experiment are shown in Figure 6A. In tumor-bearing mice that did not knock down the giant pinocytosis receptor CXCR4, the drug-loaded lipoprotein nanocarrier group containing FH38 targeting peptide (FH38-Cy5-CaP-rHDL) was small. The fluorescence intensity of mouse brain was higher than that of other groups, indicating that compared with the carrier without targeting polypeptide modification, the drug-loaded lipoprotein nanocarrier containing targeting polypeptide modification can efficiently pass through the blood-brain barrier and distribute to the tumor site. The experimental results are shown in Figure 6B. Among tumor-bearing mice with knockdown of the giant pinocytosis receptor CXCR4, the fluorescence intensity of the brains of the mice in the FH38-Cy5-CaP-rHDL treatment group was significantly lower than that of the mice without the targeted peptide. The drug lipoprotein nanocarrier control group (Cy5-CaP-rHDL) shows that the targeting ability of the vector is related to the expression level of cell giant pinocytosis receptors. The higher the expression level of giant pinocytosis receptors, the stronger the targeting ability of the vector. .
实施例7.建立荷人源胶质瘤干细胞样细胞原位胶质瘤NOD/SCID小鼠模型，评价不同靶向多肽修饰的载药重组脂蛋白的肿瘤靶向能力Example 7. Establishment of NOD/SCID mouse model of glioma in situ bearing human glioma stem cell-like cells to evaluate the tumor targeting ability of drug-loaded recombinant lipoproteins modified by different targeting polypeptides
(1)制备(1) Preparation
同实施例6制备得到不同靶向多肽修饰的载Cy5荧光标记的载药脂蛋白纳米载体，以不含靶向多肽修饰的载Cy5荧光标记的载药脂蛋白纳米载体(Cy5-CaP-rHDL)为对照。The Cy5 fluorescent-labeled drug-loaded lipoprotein nanocarriers modified with different targeting polypeptides were prepared in the same manner as in Example 6, and the Cy5 fluorescent-labeled drug-loaded lipoprotein nanocarriers (Cy5-CaP-rHDL) that did not contain the targeting polypeptide modification (Cy5-CaP-rHDL) For the control.
(2)采用NOD/SCID小鼠脑定位注射人源胶质瘤干细胞样细胞，构建原位荷胶质瘤小鼠模型，评价靶向多肽修饰的载药脂蛋白纳米载体的体内肿瘤靶向能力。(2) Using NOD/SCID mouse brain to inject human-derived glioma stem cell-like cells to construct an in situ glioma-bearing mouse model to evaluate the in vivo tumor targeting ability of targeted peptide-modified apolipoprotein nanocarriers .
通过流式细胞术，评价靶向多肽修饰的载药重组脂蛋白在原位脑肿瘤小鼠肿瘤组织中的摄取情况。通过尾静脉给予荷瘤鼠浓缩后的载Cy5标记、不同靶向多肽修饰的载药重组脂蛋白(Cy5-FH27-CaP-rHDL、Cy5-FH29-CaP-rHDL以及Cy5-FH38-CaP-rHDL)和不经靶向多肽修饰的载药重组脂蛋白(Cy5-CaP-rHDL)溶液。给药4h后，麻醉固定小鼠，心脏灌流后取出脑(肿瘤)组织。修剪肿瘤部位并用HBSS液冲洗后，用刀片将肿瘤组织切成匀浆状，移至培养皿中，加入含5ml 0.05％EDTA-胰酶、2.5ml HBSS液、2.5ml四型胶原酶(2000U/ml)的复合消化酶液中，置于37℃培养箱中消化15-20min。然后用含等效胰酶抑制剂及DNA酶的终止消化液10ml终止消化，反复剥离消化4次，收集细胞混悬液，用75μm滤网过滤，1000rpm离心20min。收集细胞于1.5ml离心管中，4％多聚甲醛室温固定20min，震摇，800rpm离心后加入100μl 0.1％Triton-X，通透20min。PBS小心洗一次，加入100μl 4％BSA封闭40min。与细胞干性相关抗体SOX2在4℃孵育过夜，再与Alexa
Figure PCTCN2020106735-appb-000002
488标记的荧光二抗孵育，PBS洗一次后将细胞悬液经过流式细胞仪检测SOX2阳性的人源胶质瘤干细胞样细胞中Cy5标记的载药重组脂蛋白的荧光强度。实验结果如图7A所示，SOX2阳性人源胶质瘤干细胞样细胞中，对Cy5-FH38-CaP-rHDL的摄取量是未经靶向多肽修饰Cy5-CaP-rHDL摄取量的10.6倍，表明靶向多肽修饰的载药重组脂蛋白载体具有良好的人源胶质瘤干细胞样细胞靶向性。 Through flow cytometry, the uptake of drug-loaded recombinant lipoprotein modified by targeted peptides in tumor tissues of mice with brain tumors in situ was evaluated. Concentrated Cy5-labeled and drug-loaded recombinant lipoproteins modified with different targeting peptides (Cy5-FH27-CaP-rHDL, Cy5-FH29-CaP-rHDL and Cy5-FH38-CaP-rHDL) were administered through the tail vein to tumor-bearing mice And a solution of drug-loaded recombinant lipoprotein (Cy5-CaP-rHDL) not modified by the targeting polypeptide. After 4 hours of administration, the mice were anesthetized and fixed, and brain (tumor) tissues were taken out after cardiac perfusion. After trimming the tumor site and washing it with HBSS solution, cut the tumor tissue into a homogenate shape with a razor blade and move it to a petri dish. Add 5ml 0.05% EDTA-pancreatin, 2.5ml HBSS solution, 2.5ml type IV collagenase (2000U/ ml) compound digestive enzyme solution, placed in a 37°C incubator for 15-20min. Then, the digestion was terminated with 10ml of termination digestion solution containing equivalent trypsin inhibitor and DNase, and the digestion was repeated 4 times. The cell suspension was collected, filtered with a 75μm filter, and centrifuged at 1000rpm for 20min. The cells were collected in a 1.5ml centrifuge tube, fixed in 4% paraformaldehyde for 20 minutes at room temperature, shaken, centrifuged at 800 rpm, and 100 μl 0.1% Triton-X was added, and the cells were permeabilized for 20 minutes. PBS was carefully washed once, and 100 μl 4% BSA was added to block for 40 min. Incubate with cell stemness-related antibody SOX2 overnight at 4°C, and then with Alexa
Figure PCTCN2020106735-appb-000002
The 488-labeled fluorescent secondary antibody was incubated, washed once with PBS, and the cell suspension was subjected to flow cytometry to detect the fluorescence intensity of Cy5-labeled drug-loaded recombinant lipoprotein in SOX2-positive human glioma stem cell-like cells. The experimental results are shown in Figure 7A. In SOX2-positive human glioma stem cell-like cells, the uptake of Cy5-FH38-CaP-rHDL is 10.6 times that of Cy5-CaP-rHDL that has not been modified by the targeted peptide, indicating The drug-loaded recombinant lipoprotein carrier modified by targeting polypeptide has good targeting of human glioma stem cell-like cells.
为了从脑切片水平评价靶向多肽修饰的载药重组脂蛋白在原位脑肿瘤小鼠脑内的分布情况，通过尾静脉分别注射载Cy5标记、靶向多肽修饰的载药重组脂蛋白(Cy5-FH27-CaP-rHDL、Cy5-FH29-CaP-rHDL以及Cy5-FH38-CaP-rHDL)和不经靶向多肽修饰的载药重组脂蛋白(Cy5-CaP-rHDL)溶液至荷瘤鼠体内。给药4h后，麻醉固定小鼠，心脏灌流，取荷瘤小鼠完整的大脑，置4％多聚甲醛中后固定24h，PBS漂洗后依次置于15％和30％蔗糖溶液中脱水至下沉，然后用O.C.T.包埋并–20℃冷冻，作连续冰冻冠状切片，片厚为14μm。PBS漂洗后,脑切片用4％BSA常温封闭1h，与细胞干性抗体SOX2在4℃孵育一夜后，再与Alexa
Figure PCTCN2020106735-appb-000003
488标记的荧光二抗孵育。PBS漂洗后用100ng/mL DAPI染色10min，PBS漂洗，拭去水渍，加入抗荧光猝灭剂封片，激光共聚焦显微镜观察。实验结果如图7B所示，相比于不经靶向多肽修饰的对照组(Cy5-CaP-rHDL)，靶向多肽修饰后的载体在胶质瘤部位有更多的蓄积，且肿瘤组织深处的荧光强度明显增强；然而在巨胞饮受体敲低的小鼠脑胶质瘤组织切片中，靶向多肽修饰的载药重组脂蛋白载体在肿瘤部位的荧光强度显著减弱，表明靶向多肽修饰的载药重组脂蛋白载体具有优良的肿瘤靶向性，可在肿瘤组织内部广泛分布，且这种靶向性与巨胞饮受体的表达水平相关。 In order to evaluate the distribution of targeted peptide-modified drug-loaded recombinant lipoproteins in the brain of mice with in situ brain tumors from the brain slice level, Cy5 labeled and targeted peptide-modified drug-loaded recombinant lipoproteins (Cy5) were injected through the tail vein. -FH27-CaP-rHDL, Cy5-FH29-CaP-rHDL and Cy5-FH38-CaP-rHDL) and a drug-loaded recombinant lipoprotein (Cy5-CaP-rHDL) that is not modified by a targeting polypeptide into the tumor-bearing mice. After administration for 4 hours, the mice were anesthetized and fixed, and the heart was perfused. The whole brains of the tumor-bearing mice were taken, placed in 4% paraformaldehyde and then fixed for 24 hours. After rinsing with PBS, they were placed in 15% and 30% sucrose solutions for dehydration. Shen was then embedded in OCT and frozen at -20°C to make continuous frozen coronal sections with a thickness of 14μm. After rinsing with PBS, the brain slices were blocked with 4% BSA at room temperature for 1 hour, incubated with cell stem antibody SOX2 at 4°C overnight, and then with Alexa
Figure PCTCN2020106735-appb-000003
Incubate with 488-labeled fluorescent secondary antibody. After rinsing with PBS, staining with 100ng/mL DAPI for 10 minutes, rinsing with PBS, wiping off water stains, adding anti-fluorescence quencher to mount, and observing by laser confocal microscope. The experimental results are shown in Figure 7B. Compared with the control group (Cy5-CaP-rHDL) that is not modified by the targeted peptide, the carrier modified by the targeted peptide has more accumulation in the glioma site, and the tumor tissue is deeper. The fluorescence intensity at the tumor site was significantly increased; however, in the mouse glioma tissue slices with the knockdown of the giant pinocytosis receptor, the fluorescence intensity of the targeted peptide-modified drug-loaded recombinant lipoprotein carrier at the tumor site was significantly reduced, indicating that the targeting The polypeptide-modified drug-loaded recombinant lipoprotein carrier has excellent tumor targeting and can be widely distributed in tumor tissues, and this targeting is related to the expression level of the giant pinocytosis receptor.
实施例8.靶向多肽修饰的载药脂蛋白纳米载体的体外药效评价Example 8. Evaluation of in vitro pharmacodynamics of targeted polypeptide modified apolipoprotein nanocarriers
(1)制备(1) Preparation
通过反向微乳法制备固相内核，将300-600μL浓度为2.5M的CaCl 2溶液与miR-34a共孵育，吹打数次，而后分散于20mL油相中形成分散均一的油包水反相微乳。磷相的制备为将300-600μL浓度为12.5mM Na 2HPO 4溶液分散于另一份20mL油相中，搅拌10min后，向磷相加入100μL浓度为20mg/mL 的1,2-油酰磷脂酸溶液。待两相分散均匀后，将两相混合搅拌45min。此时，将40mL无水乙醇加入到上述混合微乳中破乳10min。破乳后的混合液通过高速离心(12,500g)约20min除去多余的表面活性剂及环己烷。进一步通过薄膜水化法将固相内核与磷脂制备载miR-34a microRNA或者无干扰作用的NC microRNA的重组脂蛋白(miR-34a-CaP-rHDL、NC-CaP-rHDL)，并分别与靶向多肽进行孵育，以无靶向多肽修饰的重组脂蛋白纳米载体为对照。 The solid phase core was prepared by the reverse microemulsion method, 300-600μL of 2.5M CaCl 2 solution was incubated with miR-34a, pipetting several times, and then dispersed in 20mL oil phase to form a uniformly dispersed water-in-oil reverse phase Microemulsion. The phosphorus phase is prepared by dispersing 300-600μL of 12.5mM Na 2 HPO 4 solution in another 20mL oil phase. After stirring for 10 minutes, add 100μL of 20mg/mL 1,2-oleoyl phospholipid to the phosphorus phase. Acid solution. After the two phases are evenly dispersed, the two phases are mixed and stirred for 45 minutes. At this time, 40 mL of absolute ethanol was added to the above mixed microemulsion to break the emulsion for 10 minutes. After demulsification, the mixed solution is centrifuged at a high speed (12,500 g) for about 20 minutes to remove excess surfactant and cyclohexane. The solid-phase core and phospholipids were further used to prepare miR-34a microRNA or non-interfering NC microRNA-containing recombinant lipoproteins (miR-34a-CaP-rHDL, NC-CaP-rHDL) by the thin-film hydration method. The polypeptides were incubated, and the recombinant lipoprotein nanocarrier without targeted polypeptide modification was used as a control.
(2)靶向多肽修饰的载药脂蛋白纳米载体对人源胶质瘤干细胞样细胞SOX2蛋白表达的敲低作用及抑制自我更新能力的评价(2) Evaluation of the knockdown effect of targeted polypeptide-modified drug-loaded lipoprotein nanocarriers on SOX2 protein expression in human glioma stem cell-like cells and the ability to inhibit self-renewal
将对数生长期人源胶质瘤干细胞样细胞按每孔5×10 4的密度接种于6孔板中，培养12小时后，待细胞汇合度达到50％时进行药物处理。不同的复孔分别给予不同的纳米制剂，包括DMEM对照组、空载重组脂蛋白(CaP-rHDL)、载无干扰作用的NC miRNA且不同靶向多肽修饰的重组脂蛋白(NC-CaP-rHDL、FH27-NC-CaP-rHDL、FH29-NC-CaP-rHDL及FH38-NC-CaP-rHDL)、载miR-34a且不同靶向多肽修饰的重组脂蛋白(miR-34a-CaP-rHDL、FH27-miR-34a-CaP-rHDL、FH29-miR-34a-CaP-rHDL及FH38-miR-34a-CaP-rHDL)给药组，按100nM miRNA浓度给药，37℃分别孵育12,24,48h后，细胞裂解收样，按照WB操作步骤进行实验，检测各组制剂处理后的胶质瘤细胞的SOX2蛋白表达水平。实验结果如图8A所示，孵育12h后，与无靶向多肽修饰的制剂处理组相比，经FH38靶向肽与FH27靶向肽修饰的制剂处理的细胞，其SOX2的蛋白表达水平减低了30-40％，表明miR-34a被有效释放出来抑制了肿瘤干性细胞的增值。孵育24h后，靶向多肽修饰的载药脂蛋白纳米载体处理组细胞的SOX2蛋白表达水平均显著低于无靶向多肽修饰的处理组；孵育48h后，所有载有miR-34a的脂蛋白纳米载体处理后的人源胶质瘤干细胞样细胞中SOX2蛋白水平均下降了50-60％，表明与无修饰的制剂(miR-34a-CaP-rHDL)相比，靶向多肽修饰后的载药脂蛋白纳米载体可以更高效快速地释放药物，有效抑制肿瘤干性细胞的增值。 The logarithmic growth phase human glioma stem cell-like cells were seeded in a 6-well plate at a density of 5×10 4 per well, and after 12 hours of culture, drug treatment was performed when the cell confluence reached 50%. Different nano preparations were given to different multiple wells, including DMEM control group, empty recombinant lipoprotein (CaP-rHDL), non-interfering NC miRNA and recombinant lipoprotein (NC-CaP-rHDL) modified by different target peptides. , FH27-NC-CaP-rHDL, FH29-NC-CaP-rHDL and FH38-NC-CaP-rHDL), miR-34a loaded recombinant lipoproteins (miR-34a-CaP-rHDL, FH27 -miR-34a-CaP-rHDL, FH29-miR-34a-CaP-rHDL and FH38-miR-34a-CaP-rHDL) administration group, administered at a concentration of 100nM miRNA and incubated at 37°C for 12, 24, and 48 hours respectively , Cells were lysed and collected, and the experiment was carried out according to the WB procedure to detect the SOX2 protein expression level of the glioma cells treated with each group of preparations. The experimental results are shown in Figure 8A. After 12 hours of incubation, compared with the preparation treatment group without targeting peptide modification, cells treated with FH38 targeting peptide and FH27 targeting peptide modified preparations have reduced SOX2 protein expression levels. 30-40%, indicating that miR-34a was effectively released and inhibited the proliferation of tumor stem cells. After 24 hours of incubation, the SOX2 protein expression level of the cells in the targeted polypeptide-modified drug-loaded lipoprotein nanocarrier treatment group was significantly lower than that of the non-targeted polypeptide-modified treatment group; after 48 hours of incubation, all lipoprotein nanoparticles loaded with miR-34a After carrier treatment, the level of SOX2 protein in human glioma stem cell-like cells decreased by 50-60%, indicating that compared with the unmodified preparation (miR-34a-CaP-rHDL), the targeted peptide-modified drug delivery Lipoprotein nanocarriers can release drugs more efficiently and quickly, and effectively inhibit the growth of tumor stem cells.
将对数生长期的人源胶质瘤干细胞样细胞以及胰腺癌细胞系富集干细胞样细胞按每孔5×10 4的密度接种于6孔板中，培养12小时后，待细胞汇合度达到50％时进行药物处理。不同的复孔分别给予不同的纳米制剂，包括DMEM 对照组和不同靶向多肽修饰的载miR-34a重组脂蛋白(miR-34a-CaP-rHDL)给药组。分别按5,50,100nM miRNA浓度给药，37℃孵育48h。更换成完全培养液后继续培养，72h后弃培养液，用PBS洗涤1遍，加入适量4％多聚甲醛固定细胞，然后用0.5％结晶紫溶液染色3min,水洗至背景清晰后拍照观察并统计细胞克隆数。实验结果如图8B所示，与无靶向多肽修饰的miR-34a-CaP-rHDL处理组相比，FH38靶向肽修饰的miR-34a-CaP-rHDL处理组形成的细胞克隆数更少且形态更小，表明靶向多肽修饰后的载药脂蛋白纳米载体可以有效递送至肿瘤干性细胞，从而影响肿瘤干性细胞的自我更新能力。 Human-derived glioma stem cell-like cells and pancreatic cancer cell line-enriched stem cell-like cells in the logarithmic growth phase were seeded in a 6-well plate at a density of 5×10 4 per well. After 12 hours of culture, the cell confluence reached Drug treatment was performed at 50%. Different nano preparations were given to different multiple holes, including DMEM control group and miR-34a recombinant lipoprotein (miR-34a-CaP-rHDL) administration group modified with different targeting peptides. They were administered at concentrations of 5, 50, and 100 nM miRNA, respectively, and incubated at 37°C for 48 hours. Change to complete culture medium and continue culturing. After 72 hours, discard the culture medium, wash once with PBS, add an appropriate amount of 4% paraformaldehyde to fix the cells, then stain with 0.5% crystal violet solution for 3 minutes, wash with water until the background is clear, take pictures, observe and count The number of cell clones. The experimental results are shown in Figure 8B. Compared with the miR-34a-CaP-rHDL treatment group without targeting peptide modification, the FH38 targeting peptide modified miR-34a-CaP-rHDL treatment group formed fewer cell clones and The smaller shape indicates that the drug-loaded lipoprotein nanocarrier modified by the targeted polypeptide can be effectively delivered to cancer stem cells, thereby affecting the self-renewal ability of cancer stem cells.
实施例9.靶向多肽修饰的载药脂蛋白纳米载体与化疗药物联合用药的体外药效评价Example 9. Evaluation of in vitro pharmacodynamics of drug-targeted polypeptide-modified apolipoprotein nanocarriers combined with chemotherapeutic drugs
(1)制备(1) Preparation
同实施例8制备载miR-34a microRNA重组脂蛋白(miR-34a-CaP-rHDL)，并与不同靶向多肽进行孵育。The miR-34a microRNA recombinant lipoprotein (miR-34a-CaP-rHDL) was prepared as in Example 8 and incubated with different target polypeptides.
(2)靶向多肽修饰的载药脂蛋白纳米载体与化疗药物替莫唑胺联合用药，抑制人源胶质瘤干细胞样细胞增殖能力的评价。通过CCK-8考察不同浓度的(5μM,20μM,50μM,100μM和200μM)替莫唑胺联合靶向多肽修饰后的载MiR34a(50nM)药物的脂蛋白纳米载体对其测定抑制细胞活力的效果。通过在37℃药物处理24小时后，加入10μl CCK-8，450nm波长酶标仪读值。(2) Evaluation of the ability to inhibit the proliferation of human glioma stem cell-like cells by combining drug-loaded lipoprotein nanocarriers modified by targeting peptides and the chemotherapeutic drug temozolomide. CCK-8 was used to investigate the effect of different concentrations of temozolomide (5μM, 20μM, 50μM, 100μM and 200μM) in combination with lipoprotein nanocarriers containing MiR34a (50nM) drug modified by targeting peptides. After 24 hours of drug treatment at 37℃, add 10μl CCK-8, 450nm wavelength microplate reader to read the value.
实验结果如图9所示，靶向多肽修饰的载药脂蛋白纳米载体(FH38-MiR34a-CaP-rHDL)与化疗药物替莫唑胺(TMZ)联合使用后，与单独TMZ处理组相比，可以显著降低替莫唑胺的使用量(TMZ IC50＝15μM)，表明在低剂量联合用药时可以有效促进肿瘤细胞凋亡并减少耐药等不良反应。The experimental results are shown in Figure 9. The targeted peptide modified apolipoprotein nanocarrier (FH38-MiR34a-CaP-rHDL) combined with the chemotherapeutic drug temozolomide (TMZ) can significantly reduce compared with the TMZ treatment group alone. The dosage of temozolomide (TMZ IC50=15μM) indicates that the low-dose combination can effectively promote tumor cell apoptosis and reduce adverse reactions such as drug resistance.
实施例10.靶向多肽修饰的载药脂蛋白纳米载体的体内药效评价Example 10. Evaluation of the in vivo pharmacodynamics of targeted polypeptide modified apolipoprotein nanocarrier
(1)制备(1) Preparation
同实施例8制备载miR-34a microRNA的脂蛋白纳米载体，并与FH38靶向多肽进行孵育，命名为FH38-miR-34a-CaP-rHDL，无靶向多肽修饰的制剂命名为miR-34a-CaP-rHDL。The lipoprotein nanocarrier carrying miR-34a microRNA was prepared as in Example 8, and incubated with the FH38 targeting polypeptide, and named FH38-miR-34a-CaP-rHDL, and the preparation without targeting polypeptide modification was named miR-34a- CaP-rHDL.
(2)靶向多肽修饰的载miR-34a的脂蛋白纳米载体与化疗药物替莫唑胺联合治疗，能够在体内促进人源胶质瘤干细胞样细胞凋亡并延长原位荷脑胶质 瘤小鼠生存期。(2) The combined treatment of targeting polypeptide-modified miR-34a-loaded lipoprotein nanocarriers and the chemotherapeutic drug temozolomide can promote the apoptosis of human glioma stem cell-like cells in vivo and prolong the survival of mice bearing glioma in situ period.
将54只荷人源胶质瘤干细胞样细胞原位胶质瘤的NOD/SCID鼠随机分为6组，分别为生理盐水、替莫唑胺(TMZ)、载miR-34a的脂蛋白纳米载体(miR-34a-CaP-rHDL)、靶向多肽修饰的载miR-34a的脂蛋白纳米载体(FH38-miR-34a-CaP-rHDL)、TMZ与miR-34a-CaP-rHDL联合给药组、TMZ与FH38-miR-34a-CaP-rHDL联合给药组。于人源胶质瘤干细胞样细胞接种后第7、10、13、16和19天尾静脉注射制剂，灌胃给予替莫唑胺(miRNA给药量：0.36mg/kg，TMZ给药量：100mg/m 2)。记录各组荷瘤小鼠死亡时间，绘制生存曲线。 54 NOD/SCID mice bearing human-derived glioma stem cell-like cells for glioma in situ were randomly divided into 6 groups, namely normal saline, temozolomide (TMZ), and miR-34a-loaded lipoprotein nanocarrier (miR- 34a-CaP-rHDL), targeting polypeptide-modified lipoprotein nanocarriers carrying miR-34a (FH38-miR-34a-CaP-rHDL), TMZ and miR-34a-CaP-rHDL combined administration group, TMZ and FH38 -miR-34a-CaP-rHDL combined administration group. On the 7, 10, 13, 16 and 19 days after inoculation of human glioma stem cell-like cells, the preparation was injected into the tail vein, and temozolomide was administered intragastrically (miRNA dosage: 0.36 mg/kg, TMZ dosage: 100 mg/m 2 ). The death time of each group of tumor-bearing mice was recorded, and the survival curve was drawn.
将上述六组小鼠每组随机取3只小鼠，在造模后的第6、13、20天分别对荷瘤鼠脑部进行MRI成像，观察肿瘤增长情况。实验结果如图10A所示，相比生理盐水和单纯TMZ给药组，其他四组荷瘤鼠脑部肿瘤体积增长缓慢，表明载miR-34a的脂蛋白纳米载体有效抑制了肿瘤细胞的增殖。Randomly select 3 mice from each of the above six groups of mice, and perform MRI imaging on the brains of the tumor-bearing mice on the 6, 13, and 20 days after modeling to observe the tumor growth. The experimental results are shown in Figure 10A. Compared with the normal saline and TMZ administration group, the brain tumors of the other four groups of tumor-bearing mice increased slowly, indicating that the lipoprotein nanocarriers carrying miR-34a effectively inhibited the proliferation of tumor cells.
将42只荷BxPC3胰腺癌干细胞样细胞的裸鼠随机分为6组，分别为生理盐水、吉西他滨(GEM)、载miR-34a的脂蛋白纳米载体(miR-34a-CaP-rHDL)、靶向多肽修饰的载miR-34a的脂蛋白纳米载体(FH38-miR-34a-CaP-rHDL)、GEM与miR-34a-CaP-rHDL联合给药组、GEM与FH38-miR-34a-CaP-rHDL联合给药组。于BxPC3胰腺癌干细胞样细胞接种后第7、10、13、16和19天尾静脉注射制剂，尾静脉给予GEM和miRNA药物(miRNA给药量：0.36mg/kg，GEM给药量：10mg/kg)。记录各组荷瘤小鼠死亡时间，绘制生存曲线。42 nude mice bearing BxPC3 pancreatic cancer stem cell-like cells were randomly divided into 6 groups, namely, normal saline, gemcitabine (GEM), miR-34a-loaded lipoprotein nanocarrier (miR-34a-CaP-rHDL), targeting Polypeptide modified miR-34a-loaded lipoprotein nanocarrier (FH38-miR-34a-CaP-rHDL), GEM and miR-34a-CaP-rHDL combined administration group, GEM and FH38-miR-34a-CaP-rHDL combined G. After inoculation with BxPC3 pancreatic cancer stem cell-like cells, the preparation was injected into the tail vein on the 7, 10, 13, 16 and 19 days, and GEM and miRNA drugs were administered to the tail vein (miRNA administration amount: 0.36 mg/kg, GEM administration amount: 10 mg/ kg). The death time of each group of tumor-bearing mice was recorded, and the survival curve was drawn.
荷瘤鼠的生存时间考察结果显示如图10B所示，替莫唑胺与靶向多肽修饰的载miR-34a的脂蛋白纳米载体联合给药组(TMZ+FH38-miR-34a-CaP-rHDL)小鼠的平均生存时间为59天，显著高于Saline组(21天)、单纯替莫唑胺给药组(24天)、单纯无靶向多肽修饰的载miR-34a的脂蛋白纳米载体组(28天)、单纯靶向多肽修饰的载miR-34a脂蛋白纳米载体组(33天)以及替莫唑胺与无靶向多肽修饰的载miR-34a脂蛋白纳米载体联合给药组(30天)，而且在接种肿瘤后的100天，TMZ与FH38-miR-34a-CaP-rHDL联合给药组仍有2只小鼠未死亡(22％存活率)。以上实验结果提示，靶向多肽修饰的载miR-34a的脂 蛋白纳米载体具有优良的抗脑胶质瘤效果，并可显著增强现有化疗药物的效果。The survival time of tumor-bearing mice was investigated. As shown in Figure 10B, temozolomide and miR-34a-loaded lipoprotein nanocarriers modified by targeting peptides were administered in combination (TMZ+FH38-miR-34a-CaP-rHDL) mice The average survival time was 59 days, which was significantly higher than the Saline group (21 days), the temozolomide administration group (24 days), and the miR-34a lipoprotein nanocarrier group without targeting peptide modification (28 days). The miR-34a lipoprotein nanocarrier group modified by pure targeting peptide (33 days) and the co-administration group of temozolomide and miR-34a lipoprotein nanocarrier without targeting peptide modification (30 days), and after tumor inoculation On the 100th day, there were still 2 mice in the combined administration group of TMZ and FH38-miR-34a-CaP-rHDL (22% survival rate). The above experimental results indicate that the lipoprotein nanocarriers carrying miR-34a modified by targeting peptides have excellent anti-glioma effects and can significantly enhance the effects of existing chemotherapeutics.
在荷胰腺癌干细胞样细胞模型中，吉西他滨与靶向多肽修饰的载miR-34a的脂蛋白纳米载体联合给药组(TMZ+FH38-miR-34a-CaP-rHDL)小鼠的平均生存时间为96天，显著高于Saline组(29天)、单纯吉西他滨给药组(35天)、单纯无靶向多肽修饰的载miR-34a脂蛋白纳米载体组(66天)、单纯靶向多肽修饰的载miR-34a脂蛋白纳米载体组(68天)以及替莫唑胺与无靶向多肽修饰的载miR-34a脂蛋白纳米载体联合给药组(69天)，以上实验结果提示，靶向多肽修饰的载miR-34a脂蛋白纳米载体具有优良的抗胰腺癌的效果，并同样可显著增强现有化疗药物的效果，显示该药物可以用于多肿瘤模型，具有较好的应用前景。In the pancreatic cancer stem cell-like cell model, the average survival time of gemcitabine and miR-34a-loaded lipoprotein nanocarriers modified by targeting peptides (TMZ+FH38-miR-34a-CaP-rHDL) mice was At 96 days, it was significantly higher than the Saline group (29 days), the simple gemcitabine administration group (35 days), the miR-34a lipoprotein nanocarrier group without targeted peptide modification (66 days), and the pure targeted peptide modification group The miR-34a lipoprotein nanocarrier group (68 days) and the co-administration group of temozolomide and miR-34a lipoprotein nanocarriers without targeting peptide modification (69 days). The above experimental results indicate that the targeted peptide modified carrier The miR-34a lipoprotein nanocarrier has excellent anti-pancreatic cancer effect, and can also significantly enhance the effect of existing chemotherapeutic drugs, showing that the drug can be used in multiple tumor models and has good application prospects.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered This is the protection scope of the present invention.

Claims (12)

  1. 一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述递药系统包括脂质、载脂蛋白、载带药物和靶向多肽，所述靶向多肽由桥联结构将链接纳米载体端和激活巨胞饮功能的肽链共价连接形成，所述桥联结构包括Cys-Val、Cys-Phe、Cys-Leu、Cys-Ile、Cys-Gln、Leu-Glu、Gly-Ser-Gly、Ala-Pro-Ala、Cys-Pro-Cys、Gly-Phe-Leu、Gly-Phe-Leu-Gly、Ala-Leu-Ala-Leu、Gly-Phe-Leu-Gly、Gly-Gly-Gly-Gly-Ser、Val-Arg-Gly-Asp-Val、Pro-Ala-Pro-Ala-Pro、Pro-Leu-Gly-Leu-Trp-Ala、Arg-Val-Leu-Ala-Glu-Ala、聚乙二醇中的一种或多种。A targeted polypeptide-modified drug-loaded lipoprotein nano-drug delivery system is characterized in that the drug delivery system includes lipids, apolipoproteins, loaded drugs, and targeted polypeptides, and the targeted polypeptides consist of a bridge structure. Covalently linking the end of the linking nanocarrier and the peptide chain that activates the macropinocytosis function, the bridge structure includes Cys-Val, Cys-Phe, Cys-Leu, Cys-Ile, Cys-Gln, Leu-Glu, Gly -Ser-Gly, Ala-Pro-Ala, Cys-Pro-Cys, Gly-Phe-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly -Gly-Gly-Ser, Val-Arg-Gly-Asp-Val, Pro-Ala-Pro-Ala-Pro, Pro-Leu-Gly-Leu-Trp-Ala, Arg-Val-Leu-Ala-Glu-Ala , One or more of polyethylene glycol.
  2. 根据权利要求1所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述靶向多肽的序列为FH27(AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH)、FH29(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR)以及FH38(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH)中的一种。A targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 1, wherein the sequence of the targeted polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH), FH29 (AC- FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) and one of FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH).
  3. 根据权利要求1所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述脂质为蛋磷脂、豆磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇、磷脂酸、心磷脂、溶血磷脂、鞘氨醇、神经酰胺、鞘磷脂、脑苷脂、胆固醇、胆固醇酯、甘油酯及其衍生物中的一种或多种。The drug-loaded lipoprotein nano-delivery system for targeted polypeptide modification according to claim 1, wherein the lipid is egg phospholipid, soybean phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine One or more of, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, lysophospholipid, sphingosine, ceramide, sphingomyelin, cerebroside, cholesterol, cholesterol ester, glyceride and its derivatives Kind.
  4. 根据权利要求3所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述靶向多肽与脂质的摩尔比为1:10-1:300。The targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 3, wherein the molar ratio of the targeted polypeptide to lipid is 1:10-1:300.
  5. 根据权利要求1所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述载脂蛋白为ApoE，ApoA-I、ApoA-II、ApoA-IV，ApoC-I、ApoC-II、ApoC-III中的一种或多种。A targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 1, wherein the apolipoprotein is ApoE, ApoA-I, ApoA-II, ApoA-IV, ApoC-I , One or more of ApoC-II, ApoC-III.
  6. 根据权利要求5所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述载脂蛋白与靶向多肽的质量比为1:10-1:100。The targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 5, wherein the mass ratio of the apolipoprotein to the targeted polypeptide is 1:10-1:100.
  7. 根据权利要求1所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统，其特征在于，所述递药系统还包括固相内核，所述固相内核由药物分子、不 溶性或难溶性无机盐构成，药物分子负载在固相内核中，所述不溶性或难溶性无机盐为可生物降解的磷酸钙沉淀、碳酸钙沉淀、硫酸钙沉淀、氟化钙沉淀、硅酸钙沉淀、海藻酸钙沉淀、硫酸镁沉淀、磷酸镁沉淀、碳酸镁沉淀、氟化镁沉淀、硅酸镁沉淀、硫酸钡沉淀、磷酸钡沉淀、碳酸钡沉淀、氟化钡沉淀、硅酸钡沉淀的一种或多种。The targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 1, wherein the drug delivery system further comprises a solid-phase core, and the solid-phase core is composed of drug molecules, insoluble or insoluble The insoluble or poorly soluble inorganic salt is composed of soluble inorganic salt, and the drug molecule is loaded in the solid core. The insoluble or poorly soluble inorganic salt is biodegradable calcium phosphate precipitation, calcium carbonate precipitation, calcium sulfate precipitation, calcium fluoride precipitation, calcium silicate precipitation, seaweed One of calcium acid precipitation, magnesium sulfate precipitation, magnesium phosphate precipitation, magnesium carbonate precipitation, magnesium fluoride precipitation, magnesium silicate precipitation, barium sulfate precipitation, barium phosphate precipitation, barium carbonate precipitation, barium fluoride precipitation, and barium silicate precipitation Or multiple.
  8. 一种修饰载药脂蛋白纳米递药系统的靶向多肽，其特征在于，所述靶向多肽的序列为FH27(AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH)、FH29(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR)以及FH38(AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH)中的一种。A targeted polypeptide modified a drug-loaded lipoprotein nano-drug delivery system, characterized in that the sequence of the targeted polypeptide is FH27 (AC-FAEKFKEAVKDYFAKFWD-GSG-RFFESH), FH29 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYR) One of FH38 (AC-FAEKFKEAVKDYFAKFWD-GSG-KPVSLSYRAPARFFESH).
  9. 权利要求8所述的一种修饰载药脂蛋白纳米递药系统的靶向多肽在制备载药脂蛋白纳米递药系统中的应用。The application of the targeting polypeptide of a modified apolipoprotein nano-drug delivery system according to claim 8 in the preparation of a drug-laoprotein nano-drug delivery system.
  10. 权利要求1所述一种靶向多肽修饰的载药脂蛋白纳米递药系统的制备方法，包括如下步骤：The preparation method of a targeted polypeptide-modified drug-loaded lipoprotein nano-drug delivery system according to claim 1, comprising the following steps:
    a)采用固相多肽合成法合成权利要求1或2所述靶向多肽；a) Synthesize the targeting polypeptide of claim 1 or 2 by solid-phase polypeptide synthesis;
    b)采用常规方法制备载药脂质纳米递药系统；b) Prepare the drug-loaded lipid nano-drug delivery system by conventional methods;
    c)通过在上述b)制备的纳米递药系统溶液中先加入所述靶向多肽，再加入载脂蛋白，制备得到靶向多肽修饰的载药脂蛋白纳米递药系统。c) by adding the targeting polypeptide to the nano drug delivery system solution prepared in b) above, and then adding the apolipoprotein to prepare a drug delivery lipoprotein nano drug delivery system modified by the targeting polypeptide.
  11. 权利要求1所述的一种靶向多肽修饰的载药脂蛋白纳米递药系统在制备预防或治疗肿瘤或中枢神经系统疾病的药物中的应用。The use of a targeted polypeptide modified apolipoprotein nano-drug delivery system according to claim 1 in the preparation of drugs for the prevention or treatment of tumors or central nervous system diseases.
  12. 根据权利要求11所述的种靶向多肽修饰的载药脂蛋白纳米递药系统在制备预防或治疗肿瘤或中枢神经系统疾病的药物中的应用，其特征在于，所述肿瘤为脑胶质瘤和胰腺癌。The use of a targeted polypeptide-modified drug-loaded lipoprotein nano-delivery system according to claim 11 in the preparation of drugs for preventing or treating tumors or central nervous system diseases, characterized in that the tumor is glioma And pancreatic cancer.
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