WO2018137658A1 - CP-iRGD多肽、iDPP纳米粒、载药复合物及其制备方法和应用 - Google Patents

CP-iRGD多肽、iDPP纳米粒、载药复合物及其制备方法和应用 Download PDF

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WO2018137658A1
WO2018137658A1 PCT/CN2018/073997 CN2018073997W WO2018137658A1 WO 2018137658 A1 WO2018137658 A1 WO 2018137658A1 CN 2018073997 W CN2018073997 W CN 2018073997W WO 2018137658 A1 WO2018137658 A1 WO 2018137658A1
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irgd
idpp
mpeg
dotap
pla
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PCT/CN2018/073997
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French (fr)
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苟马玲
魏于全
罗丽
杨玉屏
陈雨文
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四川大学
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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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/54—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 compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a CP-iRGD polypeptide, an iDPP nanoparticle, a drug-loaded composite, a preparation method thereof and an application thereof.
  • Gene introduction systems have important applications in gene function research and gene therapy.
  • Currently used gene introduction systems mainly include two major categories: viral vectors and non-viral vectors.
  • the scale of virus vector production is difficult, and the gene capacity that can be delivered is small, which is easy to cause immune response and has potential biosafety risks.
  • Non-viral gene carriers include liposomes, cationic nanoparticles, inorganic nanoparticle carriers, etc., which have the characteristics of low immunogenicity, good safety and easy mass production, and are currently hot research topics in the world.
  • Methoxy poly(ethylene glycol-poly(lactide), abbreviated as mPEG-PLA) is a biodegradable, biocompatible amphiphilic polymer. .
  • drugs loaded with PEG or PLA delivery carriers have been approved by the US FDA for clinical use, and have good application prospects in drugs and gene delivery systems.
  • the paclitaxel preparation encapsulated by the mPEG-PLA diblock polymer nanoparticle carrier has been applied to the clinical treatment of breast cancer in Korea and Europe, and has also entered the phase II clinical trial in the United States.
  • the technical problem solved by the present invention is to provide a new modification means for modifying the mPEG-PLA diblock copolymer.
  • the inventors modified the iRGD by a suitable method to prepare a tumor-producing CP-iRGD polypeptide with high yield, high purity and good solubility, and then modified the amphiphilic mPEG- using CP-iRGD polypeptide and DOTAP- PLA diblock copolymer, so that a novel target degradable gene carrier can be prepared by self-assembly method, namely iRGD-DOTAP-mPEG-PLA cationic nanoparticles, referred to as iDPP nanoparticles.
  • the iDPP nanoparticle of the invention has good DNA binding ability, and the iDPP nano DNA complex obtained by binding with DNA is electrically neutral, and can effectively introduce the plasmid carrying the target gene into the tumor cell, and has a high transfection rate. Low cytotoxicity.
  • the amphiphilic mPEG-PLA copolymer used in the present invention is chemically named methoxypolyethylene glycol-polylactic acid, abbreviated as mPEG-PLA.
  • the methoxypolyethylene glycol-polylactic acid nanoparticles have amphiphilicity, good biodegradability and biocompatibility, avoid monocyte phagocytosis, increase the cycle time and bioavailability of the drug in the blood, Targeted delivery increases drug efficacy and reduces side effects.
  • the amphiphilic cationic material DOTAP used in the present invention is chemically named (2,3-dioleyloxypropyl)trimethylammonium chloride, abbreviated as DOTAP.
  • the amphiphilic C18-PEG-iRGD polypeptide used in the present invention is a modified iRGD.
  • CP-iRGD polypeptide can target tumor tissue and enhance the permeability of tumor tissue.
  • the modification of CP-iRGD polypeptide can improve the uptake capacity of tumor tissue, increase the accumulation of drugs in tumor sites, and reduce toxic and side effects.
  • the CP-iRGD polypeptide obtained by modifying iRGD is amphiphilic, and the target can be prepared by self-assembly.
  • iRGD is an existing polypeptide with the following structural formula I:
  • a second technical problem to be solved by the present invention is to provide a method for producing the above CP-iRGD polypeptide.
  • the method includes the following steps:
  • step C 1,8-diazabicyclo[5.4.0]undec-7-ene is used to remove the C18-PEG-Phe-Fmoc protecting group Fmoc.
  • the third problem to be solved by the present invention is to prepare an iRGD-DOTAP-mPEG-PLA nanoparticle solution by self-assembly method, and prepare the raw material and solvent according to the following ratio:
  • Raw material mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide mass ratio: 70-99 parts of mPEG-PLA copolymer, 1-30 parts of DOTAP, 1-5 parts of CP-iRGD;
  • Solvent at least one of a volatile solvent such as dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate or cyclohexane;
  • a volatile solvent such as dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate or cyclohexane
  • Hydration solution at least one of double distilled water, deionized water, pure water, physiological saline, and glucose solution;
  • Preparation method mPEG-PLA copolymer, DOTAP, CP-iRGD polypeptide are respectively dissolved in a solvent and mixed, then the solvent is evaporated, and an appropriate amount of the hydration solution is added to hydrate to a desired concentration, and the obtained solution is iRGD-DOTAP-mPEG- PLA nanoparticle aqueous solution.
  • the above self-assembly method prepares the iRGD-DOTAP-mPEG-PLA nanoparticle solution, and prepares the raw materials according to the following ratio:
  • Raw materials mass ratio of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide: 85-95 parts of mPEG-PLA copolymer, 5-15 parts of DOTAP, and 1-5 parts of CP-iRGD.
  • the amount of the solvent is such that the raw material can be dissolved.
  • the present invention also provides an iRGD-DOTAP-mPEG-PLA nanoparticle solution prepared by the above self-assembly method.
  • the invention also provides iRGD-DOTAP-mPEG-PLA nanoparticles and a preparation method thereof.
  • the preparation method comprises the steps of: drying an aqueous solution of iRGD-DOTAP-mPEG-PLA nanoparticles to obtain iRGD-DOTAP-mPEG-PLA nanoparticles.
  • the iRGD-DOTAP-mPEG-PLA nanoparticles obtained by the invention have an average particle diameter of 139.16 ⁇ 1.56 nm and an average potential of 43.1 ⁇ 6.8 mV, and have good DNA binding ability.
  • iDPP nanoparticles Compared with the gold standard transfection material PEI 25K, iDPP nanoparticles have higher transfection ability and lower cytotoxicity; iDPP nanoparticles are also higher than DOTAP-mPEG-PLA (DPP) nanoparticles. Transfection efficiency and tumor cell targeting.
  • the iRGD-DOTAP-mPEG-PLA nanoparticles obtained in the present invention can be used for encapsulating active ingredients, especially genes, chemicals or proteins, to obtain iRGD-DOTAP-mPEG-PLA nanoparticle composites.
  • iRGD-DOTAP-mPEG-PLA nanoparticles and 1 part of plasmid are in parts by mass.
  • iRGD-DOTAP-mPEG-PLA Nanoparticle is a biodegradable cationic nanoparticle, which is a novel gene introduction system non-viral vector.
  • the nanoparticles can bind to DNA by electrostatic interaction, and the iDPP nanocomposite combined with DNA is electrically neutral and has a long circulation effect.
  • the intravenous administration method can effectively introduce active components such as a target gene, a chemical drug, and a protein into tumor cells, and has the characteristics of low cytotoxicity and high transfection rate.
  • a plasmid expressing a vesicular virulence virus matrix protein can be delivered using iRGD-DOTAP-mPEG-PLA nanoparticles, i.e., iVD-DOTAP-mPEG-PLA nanoparticles are used to coat the VSVMP plasmid to obtain an iDPP/VSVMP complex. Referred to as iDPP/MP complex.
  • the iDPP/VSVMP composite of the invention comprises raw materials and auxiliary materials with the following ratio:
  • Raw materials 1 to 99 parts of iRGD-DOTAP-mPEG-PLA nanoparticles, 1 to 10 parts of VSVMP plasmid;
  • the osmotic pressure adjusting agent is suitable in an amount to prepare the obtained iDPP/VSVMP complex to reach physiological osmotic pressure;
  • Solvent at least one of water for injection, double distilled water, deionized water, pure water or physiological saline;
  • the preparation method is as follows:
  • the above raw materials and solvents were sequentially mixed according to an osmotic pressure adjusting agent, a solvent, iRGD-DOTAP-mPEG-PLA nanoparticles and a VSVMP plasmid to obtain an iDPP/VSVMP complex, and the resulting solution reached a physiological osmotic pressure.
  • the iDPP/VSVMP complex of the present invention is prepared according to the following ratio: the mass ratio of iRGD-DOTAP-mPEG-PLA nanoparticles to VSVMP plasmid is 90-99 parts of iRGD-DOTAP-mPEG-PLA nanoparticles. , VSVMP plasmid 1 to 10 parts.
  • iDPP/VSVMP complex of the present invention is prepared according to the following ratio: iRGD-DOTAP-mPEG-PLA nanoparticles and VSVMP plasmid mass ratio is 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticles, VSVMP 1 part of plasmid.
  • the inventors have found that the use of iRGD-DOTAP-mPEG-PLA nanoparticles to mediate VSVMP plasmids can be applied to the treatment of melanoma in vitro and in vivo.
  • the inventors used MTT assay to detect the inhibitory effect of iDPP/VSVMP complex on the growth of B16-F10 melanoma cells, and detected the apoptosis of iDPP/VSVMP complex by flow cytometry.
  • the inventors found that iDPP/VSVMP complex can significantly inhibit the growth of B16-F10 melanoma cells by inducing apoptosis by using iDPP nanoparticles to mediate the introduction of VSVMP plasmid into B16-F10 melanoma cells.
  • the inventors established a subcutaneous melanoma model and a melanoma lung metastasis model, comparing tumor volume and weight in each group, and comparing the number of tumor nodules in lung metastasis.
  • the inventors found that the iDPP/VSVMP complex can significantly reduce the burden of tumors and the production of lung metastases in mice.
  • Experimental data indicate that the delivery of VSVMP plasmid by iDPP nanoparticles can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.
  • the present invention also provides the use of the above iDPP/VSVMP complex in the preparation of an antitumor drug.
  • the tumor is melanoma, ovarian cancer or lung cancer.
  • the plasmid expressing interleukin-12 can also be delivered using iRGD-DOTAP-mPEG-PLA nanoparticles, i.e., the iRGD-DOTAP-mPEG-PLA nanoparticles are used to encapsulate the IL-12 plasmid to obtain the iDPP/IL-12 complex.
  • the iDPP/IL-12 composite of the invention comprises the following raw materials and auxiliary materials of the ratio:
  • Raw materials 1 to 99 parts of iRGD-DOTAP-mPEG-PLA nanoparticles, and 1 to 10 parts of IL-12 plasmid;
  • the osmotic pressure adjusting agent is suitable in an amount to prepare the obtained iDPP/IL-12 complex to reach physiological osmotic pressure;
  • Solvent at least one of water for injection, double distilled water, deionized water, pure water or physiological saline;
  • the preparation method is as follows:
  • the above raw materials and solvent were sequentially mixed according to an osmotic pressure adjusting agent, a solvent, iRGD-DOTAP-mPEG-PLA nanoparticles and an IL-12 plasmid to obtain an iDPP/IL-12 complex, and the resulting solution reached physiological osmotic pressure.
  • iDPP/IL-12 complex of the present invention is prepared according to the following ratio: iRGD-DOTAP-mPEG-PLA nanoparticles and IL-12 plasmid mass ratio is iRGD-DOTAP-mPEG-PLA nanoparticles 90 to 99 parts, 1 to 10 parts of IL-12 plasmid.
  • iDPP/IL-12 complex of the present invention is prepared according to the following ratio: iRGD-DOTAP-mPEG-PLA nanoparticles and IL-12 plasmid mass ratio is iRGD-DOTAP-mPEG-PLA nanoparticles 25 parts, 1 part of IL-12 plasmid.
  • IL-12 plasmids mediated by iRGD-DOTAP-mPEG-PLA nanoparticles can be used to treat melanoma in vitro and in vivo.
  • a melanoma subcutaneous tumor model was established and the tumor volume and weight of each group were compared.
  • the inventors have found that the iDPP/IL-12 complex can significantly reduce the burden of tumors in mice and enhance anti-tumor immunity in mice.
  • Experimental data indicate that the iDPP nanoparticle delivery IL-12 plasmid can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.
  • the invention also provides the use of the above iDPP/IL-12 complex in the preparation of an antitumor drug.
  • the tumor is melanoma, ovarian cancer or lung cancer.
  • the present invention selects a suitable method for modifying iRGD, and obtains a tumor-targeting CP-iRGD polypeptide with high yield and purity.
  • the CP-iRGD polypeptide has good solubility and can be self-assembled.
  • Degradable cationic nanoparticles iRGD-DOTAP-mPEG-PLA nanoparticles were prepared. The nanoparticle can effectively bind DNA, and the nanocomposite of DNA binding is electrically neutral, and the therapeutic gene can be effectively introduced into tumor cells by intravenous injection, which has high transfection rate and low cytotoxicity. It has a good application prospect in gene function research, gene therapy research and clinical application.
  • the iRGD-DOTAP-mPEG-PLA nanoparticles can mediate the efficacy of active components such as iRGD-DOTAP-mPEG-PLA nanoparticles, which are mediated by plasmids carrying the VSVMP gene and plasmids of IL-12 gene, which are effective in vitro and in vivo. Inhibition of the growth of B16-F10 melanoma cells.
  • iDPP nanoparticles are a relatively safe and degradable non-viral gene vector.
  • the prepared iDPP/VSVMP complex and iDPP/IL-12 complex provide a new idea and potential choice for the treatment of melanoma.
  • Figure 1 (A) Molecular structural formula of PEG-PLA; (B) Molecular structural formula of DOTAP; (C) Synthetic route diagram of C18-PEG-iRGD.
  • Figure 2 is a schematic diagram of the synthesis of DIP nanoparticles.
  • Figure 3IDPP nanoparticles particle size and potential distribution map (A) particle size distribution of iDPP nanoparticles; (B) potential distribution of iDPP nanoparticles; (C) scanning transmission electron micrograph of iDPP nanoparticles; (D) iDPP nanoparticle gel retardation analysis.
  • A particle size distribution of iDPP nanoparticles
  • B potential distribution of iDPP nanoparticles
  • C scanning transmission electron micrograph of iDPP nanoparticles
  • D iDPP nanoparticle gel retardation analysis.
  • the mass ratio of iDPP to DNA is 25:1, the iDPP nanoparticles can completely bind to the DNA plasmid.
  • Figure 4IDPP/VSVMP complex particle size and potential distribution map (A) particle size distribution of iDPP/VSVMP complex; (B) potential distribution of iDPP/VSVMP complex; (C) iDPP/VSVMP complex Scanning transmission electron micrograph; (D) iDPP/VSVMP complex gene gradient potential map.
  • Figure 5IDPP nanoparticles cytotoxicity and transfection rate of B16-F10 (A) In B16-F10 cells, the cytotoxicity of iDPP nanoparticles is lower than that of PEI 25K; (B) iDPP nanoparticles, DPP nanoparticles, PEK25K Fluorescence map of B16-F10 cells (nanoparticles: DNA: 25:1, 25:1 and 1:1, respectively); (C) Flow chart statistics of cell transfection.
  • Figure 6 Anti-tumor ability of B16-F10 cells outside the DPP/VSVMP complex: (A) MTT assay of iDPP/VSVMP complex; (B) Flow diagram of iDPP/VSVMP complex proapoptosis, iDPP/VSVMP complex It is to inhibit the growth of tumor cells by inducing apoptosis.
  • Figure 7 iDPP nanoparticle targeting and intratumoral injection of iDPP/VSVMP complex anti-tumor activity: (A) intravenous iDPP/pGL-6 complex, luciferase expression in vivo imaging; (B) iDPP/VSVMP complex Tumor volume curve for treatment; (C) Tumor weight statistics for iDPP/VSVMP complex treatment.
  • Figure 8 Antitumor activity of intravenous iDPP/VSVMP complex: (A) intravenous injection of iDPP/VSVMP complex targeted therapy for B16-F10 subcutaneous tumor map; (B) iDPP/VSVMP complex for treatment of tumor weight statistical map; (C Intravenous iDPP/VSVMP complex inhibits melanoma lung metastasis; (D) weight of lung treated with intravenous iDPP/VSVMP complex.
  • FIG. 10 Antitumor activity of intravenous iDPP/IL-12 complex: (A) intravenous injection of iDPP/IL-12 complex for treatment of B16-F10 subcutaneous tumor map; (B) tumor tissue IFN- ⁇ secretion level; (C) Statistical analysis of tumor tissue NK cell infiltration flow analysis; (D) Statistical analysis of tumor tissue CD8 + T cell infiltration flow analysis.
  • the present invention first provides a CP-iRGD polypeptide having the formula II:
  • the invention also provides a preparation method of the above CP-iRGD polypeptide, comprising the following steps:
  • step C 1,8-diazabicyclo[5.4.0]undec-7-ene is used to remove the C18-PEG-Phe-Fmoc protecting group Fmoc.
  • the preparation method of the above CP-iRGD polypeptide comprises the following steps:
  • Reaction solvent organic solvents such as dichloromethane, chloroform, acetone, etc.;
  • the third step removing C18-PEG-Phe-Fmoc (Compound 2) with 1,8-diazabicyclo[5.4.0]undec-7-ene (abbreviated as DBU) to obtain C18-PEG-Phe- NH 2 (compound 3);
  • the fourth step reacting C18-PEG-Phe-NH 2 (compound 3) with 3-maleimidopropionic acid N-hydroxysuccinimide ester (BMPS for short) to obtain C18-PEG-Phe-BMPS (compound) 4); the purity of the product in this step is >90%, and the yield is 68%;
  • the fifth step reacting C18-PEG-Phe-BMPS (Compound 4) with iRGD through a maleimide group and a thiol group to obtain the target compound C18-PEG-iRGD (Compound 5).
  • the synthetic circuit diagrams of mPEG-PLA, DOTAP molecular structure and CP-iRGD are shown in Figure 1.
  • the iDPP nanoparticles are prepared by self-assembly of mPEG-PLA, DOTAP and CP-iRGD.
  • the structure is shown in Figure 2.
  • mPEG-PLA copolymer 45 mg
  • DOTAP 5 mg
  • CP-iRGD 0.5 mg
  • Solvent double distilled water
  • the preparation method is as follows: the above raw materials and solvent are sequentially mixed together according to glucose, double distilled water, iRGD-DOTAP-mPEG-PLA nanoparticles and VSVMP plasmid, and the final glucose concentration of the composite is controlled to be 5%.
  • iDPP nanoparticles and empty plasmids were mixed with empty plasmid (pVAX).
  • the mass was 0.3 ⁇ g, and the total volume of the mixed solution was adjusted to 5 ul using a DNase-free aqueous solution, and the mixture was incubated at room temperature for 30 minutes.
  • a 1% agarose gel was then prepared and electrophoresed at 100 V for 30 minutes. Finally, the gel was taken out and observed under the irradiation of an ultraviolet lamp. The results are shown in 2.2.
  • VSVMP plasmid treated in the above step (1) was added to the iDPP nanoparticles treated in the step (2), and incubated at room temperature for 30 minutes. The potential was measured, the potential detection was the same as 1.3.1, and the result was found in 2.3.2.
  • Seeds were seeded in a 96-well plate at 5 ⁇ 10 3 cells/well, and 100 ⁇ l of a 293T cell suspension was added to each well, and incubated at 37 ° C for 24 hours in a 5% CO 2 cell incubator.
  • the cell density was 2 ⁇ 10 5 /well, and B16-F10 cells (purchased from ATCC) were seeded in a 6-well plate, and 2 ml of a cell suspension was added to each well.
  • the medium of the 6-well plate treated in the step (1) was changed to 800 ul of serum-free and antibiotic-free 1640 medium, followed by the iDPP/pGFP complex obtained in the step (4), the DPP/pGFP complex, and the PEI.
  • the 25K/pGFP complex was separately added to a 6-well plate and incubated for 4 to 8 hours at 37 ° C in a 5% CO 2 cell incubator.
  • the medium in the 6-well plate was replaced with 2 ml of 1640 medium containing antibiotics, and placed in an incubator for further 40 hours.
  • the anti-tumor activity of the iDPP/VSVMP complex on B16-F10 cells was tested in vitro by two methods: MTT assay and flow cytometry.
  • VSVMP plasmid and empty plasmid were separately diluted in 25 ⁇ l of serum-free antibiotic-free 1640 medium, and gently mixed;
  • the transfection method is detailed in 1.8(4)-(6), and the groups NS, iDPP, iDPP/pVAX, DPP/MP, iDPP/MP are obtained respectively; wherein, for the NS group, steps (2) and ( 3) Preparing 25 ⁇ l of serum-free antibiotic-free 1640 medium; for the iDPP group, step (2) preparing 25 ⁇ l of serum-free antibiotic-free 1640 medium;
  • VSVMP plasmid and empty plasmid were diluted in 50 ⁇ l of serum-free antibiotic-free 1640 medium, and gently mixed;
  • the transfection method is detailed in 1.8(4)-(6), and the groups NS, iDPP, iDPP/pVAX, DPP/MP, iDPP/MP are obtained respectively; wherein, for the NS group, steps (2) and ( 3) Preparing 25 ⁇ l of serum-free antibiotic-free 1640 medium; for the iDPP group, step (2) preparing 25 ⁇ l of serum-free antibiotic-free 1640 medium;
  • each mouse was injected with the iDPP/pGL-6 complex prepared by the following method via the tail vein.
  • 8 ⁇ g of luciferase-expressing plasmid pGL-6 and 200 ⁇ g of iDPP nanoparticles were diluted in 50 ⁇ l of 5% dextrose water, respectively, and incubated at 1.8 (4) to obtain iDPP/pGL-6 complex.
  • mice were sacrificed by cervical dislocation, and the tumor, heart, liver, spleen, lung and kidney were collected, and the tumor weight was weighed. See 2.7.2 for results.
  • mice were treated with reference to 1.11.1 (5). See 2.7.3 for the results.
  • mice were treated with reference to 1.11.1 (5), and the number of tumor lung metastasis nodules was recorded. The results are shown in 2.7.4.
  • the anti-tumor activity of iDPP/IL-12 complex on B16-F10 cells was detected by flow cytometry in vitro.
  • the detection method refers to 1.9.2, and is grouped into NS, iDPP, iDPP/pVAX, iDPP/pIL12. The results are shown in 2.8.1.
  • the dosage form is: each mouse is administered in a volume of 100 ⁇ l per dose; 100 ⁇ l of physiological saline in the NS group, 100 ⁇ l of a solution containing 125 ⁇ g of DPP nanoparticles in the iDPP group, 100 ⁇ l of a solution containing 5 ⁇ g of pVAX empty plasmid and 125 ⁇ g of DPP nanoparticles; iDPP/pIL12 is a solution containing 5 ⁇ g of pIL12 plasmid and 125 ⁇ g of DPP nanoparticles. 100 ⁇ l;
  • mice were treated with reference to 1.11.1 (5). See 2.8.2 for results.
  • the tumor tissues collected in 1.11.1 (5) were analyzed for the level of IFN- ⁇ secretion by elisa analysis.
  • the experimental data were expressed as mean ⁇ standard error (mean ⁇ SD), and the data was analyzed by SPSS 17.0 statistical software. The mean comparison and t test were used to compare the mean. P ⁇ 0.05 was statistically different, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the average particle size of the iDPP nanoparticles was 139 ⁇ 1.5 nm, and the average potential was +43 ⁇ 3.9 mV.
  • the particle size distribution and potential distribution of the nanoparticles (Fig. 3A, 3B).
  • the iDPP Under scanning TEM, the iDPP has a diameter of about 50 m (Fig. 3C).
  • iDPP/VSVMP is a relatively uniform spherical particle with a diameter of approximately 54 m (Fig. 4C).
  • PEI 25K cells were more toxic with IC 50 ⁇ 10 ⁇ g/mL.
  • the iDPP nanocarriers were very toxic to cells with IC50 > 200 ⁇ g/mL (Fig. 5A).
  • the iDPP nanoparticles can deliver the pGFP plasmid into the cell.
  • the transfection rate of iDPP nanoparticles was significantly higher than that of DPP (88.37% ⁇ 2.24%VS 42.87% ⁇ 5.68%). It is indicated that iDPP nanoparticles are a novel non-viral gene carrier with low degradability, low cytotoxicity and high transfection rate.
  • the iDPP/VSVMP complex significantly inhibited the growth of B16-F10 tumor cells.
  • the results of flow cytometry showed that the number of apoptotic cells induced by iDPP/VSVMP complex was significantly higher than that of the other four groups (Fig. 9B). Therefore, the iDPP nanoparticles can efficiently transfect the VSVMP gene plasmid into B16-F10 cells, and inhibit tumor cell growth by inducing apoptosis and the like.
  • plasmid pGL-6 which iDPP complex-expressing luciferase, can be expressed at the tumor site 72 hours after injection into the tail vein of the mouse, indicating that the iDPP complex has good tumor targeting.
  • mice In the mouse B16-F10 subcutaneous tumor implanted tumor model, treatment was performed by tail vein injection in mice, and tumor volume was recorded. We took the mouse subcutaneous tumor and photographed it (Fig. 8A), and weighed the tumor (Fig. 8B). The tumor weight of the iDPP/VSVMP group was significantly lower than that of the other four groups. The intravenous injection of iDPP/VSVMP complex could target B16. -F10 subcutaneous tumor, inhibiting tumor growth.
  • the weight ratio of mPEG-PLA to DOTAP is: 99:1, 90:10, 85:15, 80:20, 70:30, 60:40, the weight ratio of CP-iRGD to mPEG-PLA, DOTAP
  • the iRGD-DOTAP-mPEG-PLA nanoparticles were prepared at 1:100, 5:100, 10:100, 10:100, 20:100.
  • iRGD-DOTAP-mPEG-PLA had good transfection in the range of 70-99 parts of mPEG-PLA copolymer, 1-30 parts of DOTAP and 1-10 parts of CP-iRGD polypeptide. effectiveness. On this basis, the ratio was further reduced to 85-95 parts of mPEG-PLA copolymer, 5-15 parts of DOTAP, and 1-5 parts of CP-iRGD. Then, cell transfection experiments were carried out, and it was found that there was a higher turn in this range. Dyeing efficiency.
  • the content of the gene is set to 1% to 50%, and the gel retardation analysis and cell transfection experiments show that the iDPP nanoparticles can effectively bind the genes in this ratio range and have better results. Transfection efficiency.
  • the solvent for dissolving the CP-iRGD polypeptide was investigated to be volatile such as dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate or cyclohexane.
  • the solvent was found to completely dissolve the CP-iRGD polypeptide.
  • the hydration solution for hydrating the mixture of mPEG-PLA, DOTAP and CP-iRGD polypeptides may be double distilled water, deionized water, pure water, physiological saline, etc., and finally the iDPP nanoparticle solution can be obtained. However, it is preferred to use double distilled water as the hydration solution.
  • the total molecular weight of mPEG-PLA ranges from 4000 Da to 8000 Da.
  • the present invention provides a novel modification means for modifying the mPEG-PLA diblock copolymer.
  • the inventors used the positively charged amphiphilic material DOTAP and the CP-iRGD modified parent with tumor targeting.
  • the mPEG-PLA diblock copolymer was prepared by self-assembly method, which is a novel target degradable gene carrier, namely iRGD-DOTAP-mPEG-PLA cationic nanoparticles.
  • the nanoparticle has good DNA binding ability, and the iDPP complex combined with DNA is electrically neutral, and the gene plasmid can be effectively introduced into the tumor cell by intravenous injection, and the transfection rate is high.
  • the advantage of low cytotoxicity is a novel modification means for modifying the mPEG-PLA diblock copolymer.
  • the iDPP/VSVMP complex prepared from iRGD-DOTAP-mPEG-PLA nanoparticles can significantly inhibit tumor growth and lung metastasis of tumors.
  • the experimental data show that the iDPP nanoparticles can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo. It effectively inhibits the growth of melanoma cells in vitro and in vivo.
  • iDPP nanoparticles are a relatively safe, degradable non-viral gene vector.

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Abstract

本发明属于医药领域，提供了一种CP-iRGD多肽、iRGD-DOTAP-mPEG-PLA纳米粒、载药复合物及其制备方法和应用。本发明采用带正电荷的两亲性物质DOTAP和具有肿瘤靶向作用的被修饰过的CP-iRGD多肽来修饰两亲性的mPEG-PLA双嵌段共聚物，利用自组装的方法制备出了一种可降解性基因载体的iRGD-DOTAP-mPEG-PLA纳米粒，即iDPP纳米粒。该纳米粒可将基因质粒导入到肿瘤细胞中。

Description

CP-iRGD多肽、iDPP纳米粒、载药复合物及其制备方法和应用 技术领域
本发明属于医药领域，具体涉及CP-iRGD多肽、iDPP纳米粒、载药复合物及其制备方法和应用。
背景技术
基因导入系统在基因功能研究和基因治疗中有重要应用。目前使用的基因导入系统主要包括两大类：病毒载体和非病毒载体。病毒载体规模生产难度大，可递送的基因容量小，易引起免疫反应，有潜在的生物安全风险。非病毒基因载体包括脂质体、阳离子纳米粒、无机纳米粒子载体等，具有免疫原性低，安全性较好，易大规模生产等特点，是当前国际上的研究热点。
甲氧基聚乙二醇-聚乳酸(Methoxy poly(ethylene glycol)-poly(lactide)，简称mPEG-PLA)双嵌段聚合物是一种可降解、生物相容性好的两亲性聚合物。在20世纪90年代，含有PEG或PLA传输载体装载的药物已获得了美国FDA的批准应用于临床，在药物、基因导入系统中有很好的应用前景。目前，mPEG-PLA双嵌段聚合物纳米粒载体包裹的紫杉醇制剂已在韩国和欧洲应用于乳腺癌的临床治疗，在美国也已进入II期临床试验。在将PEG-PLA纳米粒用于基因导入系统时，单纯采用mPEG-PLA纳米粒装载基因的难度大，且转染效率低。进一步对mPEG-PLA纳米粒进行物理或化学修饰，可制备出具有易装载基因、转染效率高、毒性低、可降解的新型纳米粒，在基因功能研究、基因治疗研究及临床应用中有很好的应用前景。
发明内容
本发明所解决的技术问题是提供一种新的修饰手段用以修饰mPEG-PLA双嵌段共聚物。发明人采用合适的方法对iRGD进行修饰，从而制备得到了产率高、纯度高、溶解性好的可靶向肿瘤的CP-iRGD多肽，再采用CP-iRGD多肽和DOTAP修饰两亲性mPEG-PLA双嵌段共聚物，从而可采用自组装的方法制备出一种新型的靶向可降解的基因载体，即iRGD-DOTAP-mPEG-PLA阳离子纳米粒，简称为iDPP纳米粒。本发明iDPP纳米粒具有良好的DNA结合能力，与DNA结合后得到的iDPP纳米DNA复合物呈电中性，可有效地将负载有目的基因的质粒导入到肿瘤细胞中，具有转染率高、细胞毒性低等优点。
本发明中所采用的两亲性mPEG-PLA共聚物，化学命名为甲氧基聚乙二醇-聚乳酸， 简称mPEG-PLA。甲氧基聚乙二醇-聚乳酸纳米颗粒具有两亲性、良好的生物可降解性和生物相容性、避免了单核细胞吞噬、增加了药物在血液中的循环时间和生物利用度，靶向传递增加了药效、减小了副作用。
本发明中所采用的两亲性阳离子物质DOTAP，化学命名为(2,3-二油氧基丙基)三甲基氯化铵，简称DOTAP。
本发明中所采用的具有两亲性的C18-PEG-iRGD多肽，简称CP-iRGD多肽，是经过修饰的iRGD。CP-iRGD多肽能够靶向肿瘤组织，同时增强肿瘤组织的渗透性，纳米粒经过CP-iRGD多肽的修饰后可提高肿瘤组织对其的摄取能力，增加药物在肿瘤部位的聚集，减少毒副作用；且修饰iRGD所得的CP-iRGD多肽具有两亲性，可通过自组装的方式制备目标物。iRGD为现有多肽，结构式如下式Ⅰ：
Figure PCTCN2018073997-appb-000001
本发明所要解决的第一个技术问题是提供一种CP-iRGD多肽，结构式如下式Ⅱ：
Figure PCTCN2018073997-appb-000002
本发明所要解决的第二个技术问题是提供上述CP-iRGD多肽的制备方法。该方法包括以下步骤：
A、以聚乙二醇和硬酯酸为原料反应结束后分离提纯得到C18-PEG-OH；
B、以C18-PEG-OH和Fmoc-苯丙氨酸反应得到C18-PEG-Phe-Fmoc；
C、脱去C18-PEG-Phe-Fmoc保护基Fmoc，得到C18-PEG-Phe-NH 2；
D、将C18-PEG-Phe-NH 2与3-马来酰亚胺丙酸N-羟基琥珀酰亚胺酯反应得到C18-PEG-BMPS；
E、将C18-PEG-BMPS与iRGD通过马来酰亚胺基团与巯基反应得到目标化合物C18-PEG-iRGD。
优选的，上述制备方法步骤C中，采用1,8-二氮杂双环[5.4.0]十一碳-7-烯脱去C18-PEG-Phe-Fmoc保护基Fmoc。
优选的，上述制备方法步骤E中，所述反应采用的溶剂由pH＝7.3的PBS缓冲液和二甲基亚砜按体积比1﹕1混合而成。
本发明所要解决的第三个问题是采用自组装的方法制备iRGD-DOTAP-mPEG-PLA 纳米粒溶液，按照下述配比关系取原料、溶剂进行制备：
原料：mPEG-PLA共聚物、DOTAP与CP-iRGD多肽的质量配比为：mPEG-PLA共聚物70～99份、DOTAP 1～30份、CP-iRGD 1～5份；
溶剂：二氯甲烷、三氯甲烷、丙酮、四氯甲烷、乙醇、甲醇、乙醚、戊烷、乙酸乙酯、环己烷等易挥发的溶剂中的至少一种；
水化溶液：双蒸水、去离子水、纯水、生理盐水、葡萄糖溶液中的至少一种；
制备方法：mPEG-PLA共聚物、DOTAP、CP-iRGD多肽分别溶于溶剂中并混匀，然后将溶剂蒸发，加适量水化溶液水化成所需浓度，所得溶液即为iRGD-DOTAP-mPEG-PLA纳米粒水溶液。
进一步的，上述自组装的方法制备iRGD-DOTAP-mPEG-PLA纳米粒溶液，按照下述配比关系取原料进行制备：
原料：mPEG-PLA共聚物、DOTAP与CP-iRGD多肽的质量配比为：mPEG-PLA共聚物85～95份、DOTAP 5～15份、CP-iRGD 1～5份。
上述技术方案中，溶剂用量以能够溶解原料即可。
本发明还提供了由上述自组装的方法制备得到的iRGD-DOTAP-mPEG-PLA纳米粒溶液。
本发明还提供了iRGD-DOTAP-mPEG-PLA纳米粒及其制备方法。该制备方法包括以下步骤：将iRGD-DOTAP-mPEG-PLA纳米粒水溶液干燥即得iRGD-DOTAP-mPEG-PLA纳米粒。
本发明所得iRGD-DOTAP-mPEG-PLA纳米粒的平均粒径为139.16±1.56nm，平均电位为43.1±6.8mV，具备良好的DNA结合能力。与金标转染材料PEI 25K相比，iDPP纳米粒具有更高的转染能力和较低的细胞毒性；与DOTAP-mPEG-PLA(简称DPP)纳米粒相比，iDPP纳米粒同样具有更高的转染效率和肿瘤细胞靶向性。
本发明所得iRGD-DOTAP-mPEG-PLA纳米粒可用于包载活性成分，尤其是基因、化学药物或蛋白质，从而得到iRGD-DOTAP-mPEG-PLA纳米粒复合物。
优选的，按质量份计，iRGD-DOTAP-mPEG-PLA纳米粒25份，质粒1份。
iRGD-DOTAP-mPEG-PLA纳米粒属生物可降解性阳离子纳米粒，是一种新型基因导入系统非病毒载体。该纳米粒能通过静电作用结合DNA，与DNA结合的iDPP纳米复合物呈电中性，具有长循环作用。通过静脉注射的给药方式，可有效地将目的基因、 化学药物、蛋白质等活性成分靶向导入肿瘤细胞中，其本身具有细胞毒性低、转染率高等特征。
如，可以采用iRGD-DOTAP-mPEG-PLA纳米粒递送表达水泡口炎病毒基质蛋白的质粒(VSVMP质粒)，即采用iRGD-DOTAP-mPEG-PLA纳米粒包载VSVMP质粒得到iDPP/VSVMP复合物。简称iDPP/MP复合物。
本发明iDPP/VSVMP复合物，包括下述配比关系的原料及辅料：
原料：iRGD-DOTAP-mPEG-PLA纳米粒1～99份，VSVMP质粒1～10份；
渗透压调节剂适量，用量以配制所得iDPP/VSVMP复合物达到生理渗透压即可；
溶剂：注射用水、双蒸水、去离子水、纯水或生理盐水中的至少一种；
制备方法如下：
将上述原料及溶剂按照渗透压调节剂、溶剂、iRGD-DOTAP-mPEG-PLA纳米粒和VSVMP质粒依次混合即得iDPP/VSVMP复合物，所得溶液达到生理渗透压。
进一步的，本发明iDPP/VSVMP复合物按照下述配比关系取原料进行制备：iRGD-DOTAP-mPEG-PLA纳米粒与VSVMP质粒质量配比为iRGD-DOTAP-mPEG-PLA纳米粒90～99份，VSVMP质粒1～10份。
进一步的，本发明iDPP/VSVMP复合物按照下述配比关系取原料进行制备：iRGD-DOTAP-mPEG-PLA纳米粒与VSVMP质粒质量配比为iRGD-DOTAP-mPEG-PLA纳米粒25份，VSVMP质粒1份。
发明人发现利用iRGD-DOTAP-mPEG-PLA纳米粒介导VSVMP质粒可应用于体外和体内治疗黑色素瘤。
在体外，发明人用MTT法检测iDPP/VSVMP复合物对B16-F10黑色素瘤细胞生长的抑制作用，并用流式细胞术检测iDPP/VSVMP复合物诱导细胞凋亡情况。发明人发现利用iDPP纳米粒介导VSVMP质粒导入B16-F10黑色素瘤细胞中，iDPP/VSVMP复合物通过诱导凋亡可以明显抑制B16-F10黑色素瘤细胞的生长。
在体内，发明人建立黑色素皮下种植瘤模型和黑色素瘤肺转移模型，比较各组肿瘤体积和重量，肺转移比较肿瘤结节数。发明人发现iDPP/VSVMP复合物可以显著地减少小鼠肿瘤的负荷和肺转移的产生。实验数据表明，iDPP纳米粒递送VSVMP质粒可以在体外和体内有效抑制B16-F10黑色素瘤细胞的生长。
所以，本发明还提供了上述iDPP/VSVMP复合物在制备抗肿瘤药物中的应用。优选 的，所述肿瘤为黑色素瘤、卵巢癌或肺癌。
同时，还可以采用iRGD-DOTAP-mPEG-PLA纳米粒递送表达白介素-12的质粒，即采用iRGD-DOTAP-mPEG-PLA纳米粒包载IL-12质粒得到iDPP/IL-12复合物。
本发明iDPP/IL-12复合物，包括下述配比关系的原料及辅料：
原料：iRGD-DOTAP-mPEG-PLA纳米粒1～99份，IL-12质粒1～10份；
渗透压调节剂适量，用量以配制所得iDPP/IL-12复合物达到生理渗透压即可；
溶剂：注射用水、双蒸水、去离子水、纯水或生理盐水中的至少一种；
制备方法如下：
将上述原料及溶剂按照渗透压调节剂、溶剂、iRGD-DOTAP-mPEG-PLA纳米粒和IL-12质粒依次混合即得iDPP/IL-12复合物，所得溶液达到生理渗透压。
进一步的，本发明iDPP/IL-12复合物按照下述配比关系取原料进行制备：iRGD-DOTAP-mPEG-PLA纳米粒与IL-12质粒质量配比为iRGD-DOTAP-mPEG-PLA纳米粒90～99份，IL-12质粒1～10份。
进一步的，本发明iDPP/IL-12复合物按照下述配比关系取原料进行制备：iRGD-DOTAP-mPEG-PLA纳米粒与IL-12质粒质量配比为iRGD-DOTAP-mPEG-PLA纳米粒25份，IL-12质粒1份。
发明人发现利用iRGD-DOTAP-mPEG-PLA纳米粒介导IL-12质粒可应用于体外和体内治疗黑色素瘤。
在体外，采用流式细胞术检测iDPP/IL-12复合物诱导细胞凋亡情况。发现利用iDPP纳米粒介导IL-12质粒治疗B16-F10黑色素瘤细胞，iDPP/IL-12复合物通过诱导凋亡，明显抑制B16-F10黑色素瘤细胞的生长。
在体内，建立黑色素皮下种植瘤模型，比较各组肿瘤体积和重量。发明人发现iDPP/IL-12复合物可以显著地减少小鼠肿瘤的负荷，增强小鼠抗肿瘤免疫。实验数据表明，iDPP纳米粒递送IL-12质粒可以在体外和体内有效抑制B16-F10黑色素瘤细胞的生长。
本发明还提供了上述iDPP/IL-12复合物在制备抗肿瘤药物中的应用。优选的，所述肿瘤为黑色素瘤、卵巢癌或肺癌。
本发明选择了一种合适的方法对iRGD进行修饰，得到了产率、纯度均较高的可靶向肿瘤的CP-iRGD多肽，该CP-iRGD多肽溶解性好，从而可以通过自组装的方法制备可 降解阳离子纳米粒iRGD-DOTAP-mPEG-PLA纳米粒。该纳米粒可有效结合DNA，结合DNA的纳米复合物呈电中性，通过静脉注射的给药方式，可有效地将治疗基因导入到肿瘤细胞中，具有转染率高、细胞毒性低等特点，在基因功能研究、基因治疗研究及临床应用中有很好的应用前景。iRGD-DOTAP-mPEG-PLA纳米粒可介导基因等活性成分发挥疗效，如iRGD-DOTAP-mPEG-PLA纳米粒介导负载有VSVMP基因的质粒和IL-12基因的质粒可以在体外和体内有效抑制B16-F10黑色素瘤细胞的生长。iDPP纳米粒是一种相对安全的可降解性非病毒基因载体，制备所得iDPP/VSVMP复合物以及iDPP/IL-12复合物为治疗黑色素瘤提供了一种新的思路和潜在的选择。
附图说明
图1(A)PEG-PLA的分子结构式；(B)DOTAP的分子结构式；(C)C18-PEG-iRGD的合成路线图。
图2iDPP纳米粒的合成示意图。
图3iDPP纳米粒的粒径及电位分布图：(A)iDPP纳米粒的粒径分布图；(B)iDPP纳米粒的电位分布图；(C)iDPP纳米粒的扫描透射电镜照片；(D)iDPP纳米粒凝胶阻滞分析。当iDPP与DNA的质量之比为25﹕1时，iDPP纳米粒可以完全结合DNA质粒。
图4iDPP/VSVMP复合物的粒径及电位分布图：(A)iDPP/VSVMP复合物的粒径分布图；(B)iDPP/VSVMP复合物的电位分布图；(C)iDPP/VSVMP复合物的扫描透射电镜照片；(D)iDPP/VSVMP复合物基因梯度电位图。
图5iDPP纳米粒对B16-F10细胞毒性以及转染率检测：(A)在B16-F10细胞中，iDPP纳米粒的细胞毒性低于PEI 25K；(B)iDPP纳米粒、DPP纳米粒、PEK25K转染B16-F10细胞荧光图(纳米粒：DNA分别为25﹕1、25﹕1和1﹕1)；(C)细胞转染的流式图统计。
图6iDPP/VSVMP复合物体外对B16-F10细胞的抗肿瘤能力：(A)iDPP/VSVMP复合物MTT检测图；(B)iDPP/VSVMP复合物促细胞凋亡流式图，iDPP/VSVMP复合物是通过诱导细胞凋亡来抑制肿瘤细胞的生长。
图7iDPP纳米粒靶向性以及瘤内注射iDPP/VSVMP复合物的抗肿瘤活性：(A)静脉注射iDPP/pGL-6复合物，荧光素酶表达情况活体成像图；(B)iDPP/VSVMP复合物治疗的肿瘤体积曲线图；(C)iDPP/VSVMP复合物治疗的肿瘤重量统计图。
图8静脉注射iDPP/VSVMP复合物的抗肿瘤活性：(A)静脉注射iDPP/VSVMP复合物靶向治疗B16-F10皮下肿瘤图；(B)iDPP/VSVMP复合物治疗肿瘤重量统计图；(C)静脉注射iDPP/VSVMP复合物抑制黑色素瘤肺转移图；(D)静脉注射iDPP/VSVMP复合物治疗的肺的重量。
图9体外iDPP/IL-12复合物体外对B16-F10细胞的抗肿瘤能力：(A)iDPP/IL-12复合物细胞凋亡流式图；(B)凋亡统计图。
图10静脉注射iDPP/IL-12复合物的抗肿瘤活性：(A)静脉注射iDPP/IL-12复合物治疗B16-F10皮下肿瘤图；(B)肿瘤组织IFN-γ分泌水平；(C)肿瘤组织NK细胞浸润流式分析统计图；(D)肿瘤组织CD8 +T细胞浸润流式分析统计图。
具体实施方式
本发明首先提供了一种CP-iRGD多肽，结构式如下式Ⅱ：
Figure PCTCN2018073997-appb-000003
本发明还提供了上述CP-iRGD多肽的制备方法，包括以下步骤：
A、以聚乙二醇和硬酯酸为原料反应结束后分离提纯得到C18-PEG-OH；
B、以C18-PEG-OH和Fmoc-苯丙氨酸反应得到C18-PEG-Phe-Fmoc；
C、脱去C18-PEG-Phe-Fmoc保护基Fmoc，得到C18-PEG-Phe-NH 2；
D、将C18-PEG-Phe-NH 2与3-马来酰亚胺丙酸N-羟基琥珀酰亚胺酯反应得到 C18-PEG-BMPS；
E、将C18-PEG-BMPS与iRGD通过马来酰亚胺基团与巯基反应得到目标化合物C18-PEG-iRGD。
优选的，上述制备方法步骤C中，采用1,8-二氮杂双环[5.4.0]十一碳-7-烯脱去C18-PEG-Phe-Fmoc保护基Fmoc。
优选的，上述制备方法步骤E中，所述反应采用的溶剂由pH＝7.3的PBS缓冲液和二甲基亚砜按体积比1﹕1混合而成。
具体的，上述CP-iRGD多肽的制备方法，包括以下步骤：
第一步：以聚乙二醇(PEG)和硬酯酸(C17COOH)为原料制备得到CH 3(CH 2) 16COO-PEG-OH(化合物1)；以聚乙二醇(PEG，Mw＝1000，2000，4000，8000等各个分子量)和直链羧酸(CH 3(CH 2) 16COOH)为原料制备得到CH 3(CH 2) 16CO-PEG-OH(化合物1)；本步骤所得产物纯度≥90％，产率70％；
反应溶剂：二氯甲烷、氯仿、丙酮等有机溶剂皆可；
反应条件：室温，搅拌，12h；
其中，聚乙二醇(PEG)分子量可以是Mw＝1000，2000，4000，8000等各个不同分子量。
第二步：C18-PEG-OH(化合物1)和Fmoc-苯丙氨酸反应得到C18-PEG-Phe-Fmoc(化合物2)；本步骤产物纯度＞90％，产率90％；
第三步：用1,8-二氮杂双环[5.4.0]十一碳-7-烯(简称DBU)脱去C18-PEG-Phe-Fmoc(化合物2)，得到C18-PEG-Phe-NH 2(化合物3)；
第四步：将C18-PEG-Phe-NH 2(化合物3)与3-马来酰亚胺丙酸N-羟基琥珀酰亚胺酯(简称BMPS)反应得到C18-PEG-Phe-BMPS(化合物4)；本步骤产物纯度>90％，产率68％；
第五步：将C18-PEG-Phe-BMPS(化合物4)与iRGD通过马来酰亚胺基团与巯基反应得到目标化合物C18-PEG-iRGD(化合物5)。所述反应采用的溶剂由pH＝7.3的PBS缓冲液和二甲基亚砜按体积比1﹕1混合而成。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明，说明但不限制本发明。
mPEG-PLA、DOTAP分子结构式和CP-iRGD的合成线路图见图1，iDPP纳米粒通过mPEG-PLA、DOTAP和CP-iRGD自组装的方法制备而成，结构示意图见图2。
1、实验方法
1.1制备合成C18-PEG-iRGD化合物
1.1.1制备合成C18-PEG-OH(以下简称化合物1)
取聚乙二醇(1mmol)、硬酯酸(C 17COOH)(1mmol)溶于二氯甲烷，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl，3mmol)和N-羟基丁二酰亚胺(NHS,0.3mmol)，室温搅拌过夜反应后，依次用1N HCl溶液、水溶液及饱和食盐水洗3次，有机相用无水硫酸钠干燥、过滤、收集滤液后减压除去溶剂固体用柱层析色谱分离纯化(二氯甲烷﹕甲醇＝20﹕1)，得到化合物1；
1.1.2制备合成C18-PEG-Phe-Fmoc(简称化合物2)
取化合物1(500mg)和Fmoc-L-苯丙氨酸(142mg)溶于二氯甲烷，加入EDC·HCl(140mg)和DMAP(9mg)，室温过夜，样品收集和处理同1.1.1，得到化合物2。
1.1.3制备合成C18-PEG-Phe-NH2(简称化合物3)：
将化合物2(500mg)溶于二氯甲烷，缓慢加入DBU(100uL)，常温搅拌反应3h。除柱层析色谱分离纯化(二氯甲烷﹕甲醇＝25﹕1)，样品收集和处理同1.1.1，得到化合物3。
1.1.4制备合成C18-PEG-Phe-BMPS(简称化合物4)：
取化合物3(200mg)和BMPS(49mg)溶解于二氯甲烷，加入0.2％的三乙胺，常温搅拌反应12h，样品收集和处理同1.1.1，得到化合物4。
1.1.5制备合成目标化合物C18-PEG-iRGD(简称化合物5)：
将C18-PEG-Phe-BMPS(300mg)溶于丙酮，将iRGD(150mg)溶于水，将iRGD溶于PBS缓冲液(PH＝7.3)和二甲基亚砜的混合溶剂溶液，再与C18-PEG-Phe-BMPS溶液混溶，滴加0.2％三乙胺，室温，真空、氮气保护，反应过夜。透析(Mw＝2000)2天，冻干，4℃密封保存，备用。
1.2iDPP纳米粒及iDPP/VSVMP复合物制备方法
1.2.1iDPP纳米粒的制备
将mPEG-PLA共聚物(45mg)、DOTAP(5mg)和CP-iRGD(1mg)分别溶于4mL二氯甲烷溶液中，再转入烧瓶中混匀；将混合溶液置于60℃水浴锅中，真空条件下旋蒸30分钟。加适量双蒸水水化成所需浓度，60℃水浴轻轻振荡，直到完全溶解，所得溶液即为iDPP纳米粒水溶液，置于4℃冰箱保存备用。将所得iDPP纳米粒水溶液干燥即得 iDPP纳米粒。
1.2.2iDPP/VSVMP复合物制备方法
原料：VSVMP质粒5μg；iRGD-DOTAP-mPEG-PLA纳米粒125μg；50％葡萄糖；
溶剂：双蒸水；
制备方法如下：将上述原料及溶剂按照葡萄糖、双蒸水、iRGD-DOTAP-mPEG-PLA纳米粒和VSVMP质粒先后顺序混合在一起，控制复合物最终葡萄糖浓度为5％。
1.3iDPP纳米粒的粒径、电位和形态学研究
1.3.1iDPP纳米粒的粒径、电位
1.2.1制备得到的iDPP纳米粒的粒径和电位大小使用Zetasizer Nano ZS马尔文粒度仪(Malvern Instruments，Worcestershire，英国)检测。结果取3次测量的平均值。结果见2.1.1。
1.3.2iDPP纳米粒的形态学研究
1.2.1制备得到的iDPP纳米粒的形态学通过扫描透射电子显微镜(scanning transmission electron microsope，STEM)来观察。结果见2.1.1。
1.4iDPP纳米粒与DNA结合能力的研究
用凝胶阻滞分析实验来检测iDPP纳米粒的DNA结合能力。首先把不同质量比例(1﹕1，5﹕1，10﹕1，15﹕1，20﹕1，25﹕1)的iDPP纳米粒和空载质粒(pVAX)混合，空载质粒(pVAX)的质量为0.3μg，采用不含DNA酶的水溶液调节混合后的溶液总体积为5ul，室温静置孵育30分钟。然后配制1％的琼脂糖凝胶，在100V电压下电泳30分钟。最后取出凝胶在紫外灯的照射下进行观察并取图。结果见2.2。
1.5iDPP/VSVMP复合物的粒径、电位和形态学研究
1.5.1iDPP/VSVMP复合物的粒径、电位
1.2.2制备得到的iDPP/VSVMP复合物粒径、电位的测量方法同1.3.1，结果见2.3.1。
1.5.2iDPP/VSVMP复合物的形态学研究
1.2.2制备得到的iDPP/VSVMP复合物形态学检测，操作方法同1.3.2，结果见2.3.1。
1.6iDPP纳米粒与VSVMP结合后，电位变化情况
控制以下各组不同质量比例(5﹕1，10﹕1，15﹕1，20﹕1，25﹕1，30﹕1，35﹕1)的iDPP纳米粒与VSVMP质粒。
(1)将VSVMP质粒分别稀释于400μl去离子水中，轻轻混匀。
(2)取iDPP纳米粒分别稀释于400μl去离子水中，轻轻混匀。
(3)将上述步骤(1)处理后的VSVMP质粒加入到步骤(2)处理后的iDPP纳米粒中，室温静置孵育30分钟。测定电位，电位检测同1.3.1，结果见2.3.2。
1.7iDPP纳米粒的细胞毒性检测
iDPP纳米粒对293T细胞(购于ATCC)的毒性作用通过细胞活力分析来检测
(1)以5×10 3个细胞/孔接种于96孔板，每孔加样100μl 293T细胞悬浮液，放入37℃，5％CO 2细胞恒温培养箱中孵育24小时。
(2)配制一系列浓度(0μg/ml、6.25μg/ml、12.5μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml)的iDPP纳米粒溶液和PEI 25K溶液，96孔板中每孔分别加入100μl上述不同浓度的溶液，每个浓度设6个复孔。加药完成后在37℃，5％CO 2细胞恒温培养箱中继续孵育48小时。
(3)孵育完成后，采用MTT法检测，结果见2.4。
1.8iDPP纳米粒转染B16-F10细胞
(1)细胞密度为2×10 5/孔，将B16-F10细胞(购于ATCC)接种于6孔板中，每孔加细胞悬浮液2ml。
(2)将2μg表达绿色荧光蛋白的质粒(pGFP)稀释于50μl无血清无抗生素的1640培养基中，轻轻混匀。
(3)取制备好的iDPP纳米粒、DPP纳米粒50μg分别稀释于50μl无血清无抗生素的1640培养基中，轻轻混匀。取2μgPEI 25K溶液(1μg/μl)稀释于50μl无血清无抗生素的1640培养基中，轻轻混匀。
(4)将步骤(2)处理后的pGFP分别加入到步骤(3)得到的iDPP纳米粒溶液、DPP纳米粒溶液、PEI 25K溶液中，室温静置孵育30分钟。
(5)将步骤(1)处理后的6孔板的培养基换成无血清无抗生素的1640培养基800ul，接着将步骤(4)得到的iDPP/pGFP复合物、DPP/pGFP复合物、PEI 25K/pGFP复合物分别加入6孔板中，放入37℃，5％CO 2细胞恒温培养箱中孵育4～8小时。
(6)将6孔板中的培养基再换成有血清有抗生素的1640培养基2ml，放入孵箱继续培养40小时。
(7)倒置荧光显微镜下观察转染后细胞内绿色荧光蛋白表达情况。收集细胞，行流式细胞术检测，确定转染率，结果见2.5。
1.9检测iDPP/VSVMP复合物在体外对B16-F10细胞的抗肿瘤活性
在体外主要用两种方法检测iDPP/VSVMP复合物对B16-F10细胞的抗肿瘤活性：MTT法和流式细胞术。
1.9.1用MTT法检测iDPP/VSVMP复合物对B16-F10细胞生长的抑制作用
(1)B16-F10细胞接种96孔板方法详见1.7(1)；
(2)分别取1μg VSVMP质粒和空载质粒(pVAX)分别稀释于25μl无血清无抗生素的1640培养基中，轻轻混匀；
(3)DPP纳米粒、iDPP纳米粒取25μg分别稀释于25μl无血清无抗生素的1640培养基中；
(4)转染方法详见1.8(4)-(6)，分别得到组NS、iDPP、iDPP/pVAX、DPP/MP、iDPP/MP；其中，对于NS组而言，步骤(2)和(3)配制25μl无血清无抗生素的1640培养基；对于iDPP组而言，步骤(2)配制25μl无血清无抗生素的1640培养基；
(5)采用MTT法检测，检测方法详见1.7(3)，结果见2.6。
1.9.2用流式细胞术检测iDPP/VSVMP复合物诱导B16-F10细胞的凋亡情况
(1)B16-F10细胞接种6孔板方法详见1.8(1)；
(2)取2μg VSVMP质粒和空载质粒(pVAX)分别稀释于50μl无血清无抗生素的1640培养基中，轻轻混匀；
(3)DPP纳米粒、iDPP纳米粒取50μg分别稀释于50μl无血清无抗生素的1640培养基中；
(4)转染方法详见1.8(4)-(6)，分别得到组NS、iDPP、iDPP/pVAX、DPP/MP、iDPP/MP；其中，对于NS组而言，步骤(2)和(3)配制25μl无血清无抗生素的1640培养基；对于iDPP组而言，步骤(2)配制25μl无血清无抗生素的1640培养基；
(5)参照Biolegend公司annexin V-FITC/PI双染试剂盒说明书进行流式细胞术检测B16-F10细胞的凋亡情况，结果见2.6。
1.10检测iDPP纳米粒、iDPP/VSVMP复合物的肿瘤靶向性
(1)B16-F10黑色素皮下种植瘤模型的建立：6-8周龄的雌性C57BL/6小鼠5只(四川大学实验动物中心)，SPF级饲养，每只小鼠接种细胞量为2×10 5个细胞，皮下注射细胞悬浮液100μl。
(2)接种两周后，每只老鼠经尾静脉注射以下方法制备得到的iDPP/pGL-6复合物。 将8μg表达荧光素酶的质粒pGL-6和200μg iDPP纳米粒分别稀释于50μl 5％葡萄糖水，按1.8(4)孵育后得到iDPP/pGL-6复合物。
(3)用生理盐水配制D-荧光素钠盐的工作液(15mg/ml)，0.22μm滤膜过滤除菌。
(4)经步骤(2)给药72小时后，静脉注射200μl步骤(3)中配制的D-荧光素钠盐的工作液，20分钟后，小鼠活体成像仪成像。结果见2.7.1。
1.11检测iDPP/VSVMP复合物体内抗肿瘤能力
1.11.1瘤内注射iDPP/VSVMP复合物及其抗肿瘤机制的研究
(1)B16-F10黑色素皮下种植瘤模型的建立：每只小鼠接种细胞量为2×10 5个细胞，皮下注射细胞悬浮液100μl；
(2)随机分组：接种后的第6天，将C57小鼠随机分为5组，每组5只；
(3)瘤内注射给药：接种后的第6天，按NS、iDPP、iDPP/pVAX、DPP/MP、iDPP/MP五组分组给药，每只老鼠每次给药体积为100μl；其中，NS组为100μl生理盐水，iDPP组为含125μg iDPP纳米粒的溶液100μl，iDPP/pVAX为含5μg pVAX空载质粒、125μgiDPP纳米粒的溶液100μl；DPP/MP为含5μgVSVMP质粒、125μgDPP纳米粒的溶液100μl；iDPP/MP为含5μgVSVMP质粒、125μgiDPP纳米粒的溶液100μl；
(4)每2天给药一次，用游标卡尺测量肿瘤的长和宽，并记录，共治疗4次；
(5)治疗完成后1周，用颈椎脱位法将小鼠处死，收集肿瘤和心肝脾肺肾，称量瘤重。结果见2.7.2。
1.11.2静脉注射iDPP/VSVMP复合物及其抗肿瘤效果的研究
(1)B16-F10黑色素皮下种植瘤模型的建立：方法参考1.11.1(1)；
(2)随机分组：接种后的第6天，将C57小鼠随机分为5组，每组5只；
(3)静脉给药：接种后的第6天，按1.11.1(3)给药分组，小鼠尾静脉注射；
(4)每2天给药一次，并用游标卡尺测量肿瘤的长和宽，并记录，共治疗7次；
(5)治疗完成后1周，参照1.11.1(5)处理小鼠。结果见2.7.3。
1.11.3静脉注射iDPP/VSVMP复合物及其抑制黑色素瘤肺转移的研究
(1)B16-F10黑色素瘤肺转移模型的建立：每只小鼠接种细胞量为1×10 5个细胞，小鼠尾静脉注射细胞悬浮液100μl；
(2)随机分组：接种后的第6天，将C57小鼠随机分为5组，每组5只；
(3)静脉给药：接种后的第6天，方法参考1.11.2(3)；
(4)每2天给药一次，共治疗7次；
(5)治疗完成后1周，参照1.11.1(5)处理小鼠，并记录肿瘤肺转移结节数。结果见2.7.4。
1.12检测iDPP/IL-12复合物在体外对B16-F10细胞的抗肿瘤活性
在体外主要采用流式细胞凋亡检测iDPP/IL-12复合物对B16-F10细胞的抗肿瘤活性。检测方法参考1.9.2，分组为NS，iDPP，iDPP/pVAX，iDPP/pIL12。结果见2.8.1。
1.13检测iDPP/IL-12复合物体内抗肿瘤能力
1.13.1静脉注射iDPP/IL-12复合物抗肿瘤机制的研究
(1)B16-F10黑色素皮下种植瘤模型的建立：方法同1.10.1(1)；
(2)随机分组：接种后的第6天，将C57小鼠随机分为5组，每组5只；
(3)静脉给药：接种后的第6天，按NS，iDPP，iDPP/pVAX，iDPP/pIL12四组分组给药，给药方式为：每只老鼠每次给药体积为100μl；其中，NS组为100μl生理盐水，iDPP组为含125μgiDPP纳米粒的溶液100μl，iDPP/pVAX为含5μg pVAX空载质粒、125μgiDPP纳米粒的溶液100μl；iDPP/pIL12为含5μg pIL12质粒、125μgiDPP纳米粒的溶液100μl；
(4)每2天给药一次，用游标卡尺测量肿瘤的长和宽，并记录，共治疗5次。
(5)治疗完成后1周，参照1.11.1(5)处理小鼠。结果见2.8.2。
1.13.2静脉注射iDPP/IL-12复合物免疫检测分析
(1)将1.11.1(5)收集的肿瘤组织采用elisa分析检测IFN-γ分泌水平。
(2)采用流式细胞术，对肿瘤组织CD8+T细胞，NK细胞浸润情况分析。结果见2.8.2。
1.14统计分析
实验数据以平均数±标准误来表示(mean±SD)，用SPSS17.0统计软件进行数据分析，主要运用求均值、t检验进行均数比较分析。P<0.05统计学有差异,*p<0.05,**p<0.01,***p<0.001。
2结果
2.1iDPP纳米粒的特征
2.1.1iDPP纳米粒的粒径、电位和形态学
iDPP纳米粒的平均粒径为139±1.5nm，平均电位为+43±3.9mV。纳米粒的粒径分 布和电位分布(图3A、3B)。在扫描透射电镜下，iDPP直径大小约50m(图3C)。
2.2iDPP纳米粒与DNA的结合能力
我们用凝胶阻滞分析实验检测了iDPP纳米粒的DNA结合能力。当iDPP与DNA的质量之比为25﹕1时，iDPP纳米粒可以完全结合DNA(图3D)。
2.3iDPP/VSVMP复合物的特征
2.3.1iDPP/VSVMP复合物的粒径、电位和形态学
iDPP纳米粒的平均粒径为142±1.0nm，平均电位为0.26±0.2mV。纳米粒的粒径分布和电位分布(图4A、3B)。在扫描透射电镜下，iDPP/VSVMP是大小较为均一的球型颗粒，直径大小约54m(图4C)。
2.3.2iDPP/VSVMP复合物梯度电位变化
制备好不同比例的iDPP/VSVMP复合物，电位变化。由+43±3.9mV转变为-19.7±0.62mV(图4D)。
2.4iDPP纳米粒的细胞毒性检测
在293T细胞中，PEI 25K细胞的毒性较大，IC 50<10μg/mL。而iDPP纳米载体对细胞的毒性很低，IC50>200μg/mL(图5A)。
2.5iDPP纳米粒转染B16-F10细胞
如图5B-C所示，iDPP纳米粒可将pGFP质粒传递到细胞中。iDPP纳米粒的转染率明显高于DPP的转染率(88.37％±2.24％VS 42.87％±5.68％)。表明，iDPP纳米粒是一种新型靶向非病毒基因载体，具有可降解性、细胞毒性低且转染率高的特征。
2.6iDPP/VSVMP复合物在体外对B16-F10细胞的抗肿瘤能力
如图6A所示，iDPP/VSVMP复合物可明显抑制B16-F10肿瘤细胞的生长。流式细胞术检测结果表明显示，iDPP/VSVMP复合物诱导凋亡细胞数明显高于其他四组(图9B)。因此，iDPP纳米粒可将VSVMP基因质粒有效转染到B16-F10细胞中，通过诱导凋亡等机制抑制肿瘤细胞生长。
2.7iDPP复合物的肿瘤靶向性以及体内抗肿瘤作用
2.7.1iDPP复合物的肿瘤靶向性
由图7A可知iDPP复合表达荧光素酶的质粒pGL-6经小鼠尾静脉注射后，72小时可在肿瘤部位表达，表明iDPP复合物具有很好的肿瘤靶向性。
2.7.2iDPP/VSVMP复合物瘤内注射抑制皮下移植瘤的生长
在小鼠B16-F10皮下瘤种植瘤模型中，经瘤内注射进行治疗，并记录肿瘤体积(图7B)，iDPP/VSVMP组肿瘤体积增长缓慢，表明iDPP/VSVMP复合物能显著抑制肿瘤生长。我们将小鼠皮下瘤取下，并进行肿瘤称重(图7C)。
2.7.3iDPP/VSVMP复合物尾静脉注射抑制皮下移植瘤的生长
在小鼠B16-F10皮下瘤种植瘤模型中，经小鼠尾静脉注射进行治疗，并记录肿瘤体积。我们将小鼠皮下瘤取下并拍照(图8A)，并进行肿瘤称重(图8B)，iDPP/VSVMP组肿瘤重量明显低于其他四组，静脉注射iDPP/VSVMP复合物可靶向治疗B16-F10皮下瘤，抑制肿瘤生长。
2.7.4iDPP/VSVMP复合物尾静脉注射抑制黑色瘤肺转移
在小鼠B16-F10肺转移肿瘤模型中，经小鼠尾静脉注射进行治疗。我们将小鼠肺取下并拍照(图8C)，iDPP/VSVMP组肿瘤肺转移结节数明显少于其他四组，尾静脉注射iDPP/VSVMP复合物可显著抑制黑色素瘤肺转移，并进行称重(图8D)。
2.8iDPP/IL-12复合物在体外对B16-F10细胞的抗肿瘤能力
2.8.1iDPP/IL-12复合物在体外对B16-F10细胞的抗肿瘤能力
通过流式细胞检测结果(图9A-B)，iDPP/IL-12复合物诱导凋亡细胞数明显高于其他四组。体外抗肿瘤活性结果显示，iDPP纳米粒可有效地将IL-12基因质粒有效转染到B16-F10细胞中，通过诱导凋亡从而抑制肿瘤细胞的增殖。
2.8.2iDPP/IL-12复合物尾静脉注射抑制皮下移植瘤的生长
在小鼠B16-F10皮下瘤种植瘤模型中，经小鼠尾静脉注射进行治疗，并记录肿瘤体积。我们将小鼠皮下瘤取下并拍照(图10A)，并采用elisa检测分析肿瘤组织IFN-γ分泌情况(图10B)，以及采用流式细胞术分析肿瘤组织NK细胞(图10C)和CD8+T细胞(图10D)浸润情况。静脉注射iDPP/IL-12复合物可靶向B16-F10皮下瘤种植瘤，通过诱导细胞凋亡，增加小鼠抗肿瘤免疫，抑制肿瘤生长。
3.前期筛选试验：
3.1在前期的实验过程中，发明人对水溶性iRGD进行修饰
3.1.将mPEG-PLA与DOTAP的重量比定为：99﹕1，90﹕10，85﹕15，80﹕20，70﹕30，60﹕40，CP-iRGD与mPEG-PLA、DOTAP的重量比定为1﹕100，5﹕100，10﹕100，10﹕100，20﹕100制备iRGD-DOTAP-mPEG-PLA纳米粒。
进行细胞转染实验，发现iRGD-DOTAP-mPEG-PLA的原料mPEG-PLA共聚物70～ 99份、DOTAP 1～30份、CP-iRGD多肽1～10份的比例范围内有较好的转染效率。在此基础上进一步将比例缩小至mPEG-PLA共聚物85～95份、DOTAP 5～15份、CP-iRGD1～5份比例范围，然后进行细胞转染实验发现在此范围内有更高的转染效率。
3.2.在基因制剂的制备中，将基因的含量定为1％～50％，进行凝胶阻滞分析和细胞转染实验发现，在这个比例范围内iDPP纳米粒能够有效结合基因并有较好的转染效率。
3.3.在前期的基础上，进一步将基因含量缩小至1％～10％，再次进行凝胶阻滞分析和细胞转染实验发现，在这个比例范围内基因能够高效的结合iDPP纳米粒并有更好的转染效率。
3.4.制备纳米粒时，考察了溶解CP-iRGD多肽的溶剂为二氯甲烷、三氯甲烷、丙酮、四氯甲烷、乙醇、甲醇、乙醚、戊烷、乙酸乙酯、环己烷等易挥发的溶剂，发现均可使CP-iRGD多肽完全溶解。
3.5.用于水化mPEG-PLA、DOTAP和CP-iRGD多肽混合物的水化溶液可以为双蒸水、去离子水，纯水，生理盐水等，最终均可得到iDPP纳米粒溶液。但优选采用双蒸水为水化溶液。
3.6.制备iDPP纳米粒所需的mPEG-PLA共聚物中，mPEG-PLA的总分子量范围为4000Da～8000Da。
综上，本发明提供了一种新的修饰手段用以修饰mPEG-PLA双嵌段共聚物，发明人采用带正电荷的两亲性物质DOTAP和具有肿瘤靶向作用的CP-iRGD修饰两亲性mPEG-PLA双嵌段共聚物，利用自组装的方法制备出了一种新型的靶向可降解性基因载体，即iRGD-DOTAP-mPEG-PLA阳离子纳米粒。该纳米粒具有良好的DNA结合能力，结合DNA后的iDPP复合物成电中性，通过静脉注射的给药方式，可有效地将基因质粒靶向导入到肿瘤细胞中，具有转染率高、细胞毒性低的优点。由iRGD-DOTAP-mPEG-PLA纳米粒制备的iDPP/VSVMP复合物可以显著地抑制肿瘤生长和肿瘤的肺转移。实验数据表明，iDPP纳米粒传递VSVMP基因可以在体外和体内有效抑制B16-F10黑色素瘤细胞的生长。在体外和体内有效抑制黑色素瘤细胞的生长。iDPP纳米粒是一种相对安全的可降解性非病毒基因载体。由iRGD-DOTAP-mPEG-PLA纳米粒制备的iDPP/IL-12复合物同样可以在体内和体外有效抑制黑色素瘤的生长。制备所得iDPP/VSVMP复合物和iDPP/IL-12复合物为靶向治疗黑色素瘤提供了一种新的思路和潜在的选择。

Claims (17)

  1. CP-iRGD多肽，其特征在于：结构式如下式Ⅱ：
    Figure PCTCN2018073997-appb-100001
  2. 权利要求1所述的CP-iRGD多肽的制备方法，其特征在于：包括以下步骤：
    A、以聚乙二醇和硬酯酸为原料反应结束后分离提纯得到C18-PEG-OH；
    B、以C18-PEG-OH和Fmoc-苯丙氨酸反应得到C18-PEG-Phe-Fmoc；
    C、脱去C18-PEG-Phe-Fmoc保护基Fmoc，得到C18-PEG-Phe-NH2；
    D、将C18-PEG-Phe-NH2与3-马来酰亚胺丙酸N-羟基琥珀酰亚胺酯反应得到C18-PEG-BMPS；
    E、将C18-PEG-BMPS与iRGD通过马来酰亚胺基团与巯基反应得到目标化合物C18-PEG-iRGD。
  3. iRGD-DOTAP-mPEG-PLA纳米粒溶液的制备方法，其特征在于：按照下述配比关系取原料、溶剂进行制备：
    原料：mPEG-PLA共聚物、DOTAP与权利要求1所述的CP-iRGD多肽的质量配比为：mPEG-PLA共聚物70～99份、DOTAP 1～30份、CP-iRGD 1～5份；
    溶剂：二氯甲烷、三氯甲烷、丙酮、四氯甲烷、乙醇、甲醇、乙醚、戊烷、乙酸乙酯、环己烷中的至少一种；
    水化溶液：双蒸水、去离子水、纯水、生理盐水、葡萄糖溶液中的至少一种；
    制备方法：mPEG-PLA共聚物、DOTAP、CP-iRGD多肽分别溶于溶剂中并混匀，然后将溶剂蒸发，加水化溶液水化成所需浓度，所得溶液即为iRGD-DOTAP-mPEG-PLA纳米粒水溶液。
  4. 根据权利要求3所述的iRGD-DOTAP-mPEG-PLA纳米粒溶液的制备方法，其特征在于：mPEG-PLA共聚物、DOTAP与CP-iRGD多肽的质量配比为：mPEG-PLA共聚物85～95份、DOTAP 5～15份、CP-iRGD 1～5份。
  5. 由权利要求3或4所述的制备方法制备得到的iRGD-DOTAP-mPEG-PLA纳米粒溶液。
  6. iRGD-DOTAP-mPEG-PLA纳米粒，其特征在于：由权利要求5所述的iRGD-DOTAP-mPEG-PLA纳米粒水溶液干燥得到。
  7. iRGD-DOTAP-mPEG-PLA纳米粒复合物，其特征在于：由权利要求6所述的iRGD-DOTAP-mPEG-PLA纳米粒负载基因、化学药物或蛋白质得到。
  8. iDPP/VSVMP复合物，其特征在于：由权利要求6所述的iRGD-DOTAP-mPEG-PLA纳米粒负载表达水泡口炎病毒基质蛋白的质粒得到。
  9. 权利要求8所述的iDPP/VSVMP复合物的制备方法，其特征在于：包括下述配比关系的原料及辅料：
    原料质量配比为：iRGD-DOTAP-mPEG-PLA纳米粒1～99份，VSVMP质粒1～10份；
    渗透压调节剂适量；
    溶剂：注射用水、双蒸水、去离子水、纯水或生理盐水中的至少一种；
    制备方法如下：
    将上述原料及溶剂按照渗透压调节剂、溶剂、iRGD-DOTAP-mPEG-PLA纳米粒和VSVMP质粒依次混合即得iDPP/VSVMP复合物溶液，所得溶液达到生理渗透压。
  10. 根据权利要求9所述的iDPP/VSVMP复合物的制备方法，其特征在于： 原料质量配比为iRGD-DOTAP-mPEG-PLA纳米粒90～99份，VSVMP质粒1～10份；优选iRGD-DOTAP-mPEG-PLA纳米粒25份，VSVMP质粒1份。
  11. 权利要求8所述的iDPP/VSVMP复合物在制备抗肿瘤药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于：所述的肿瘤为黑色素瘤、卵巢癌或肺癌。
  13. iDPP/IL-12复合物，其特征在于：由权利要求6所述的iRGD-DOTAP-mPEG-PLA纳米粒负载表达白介素-12的质粒得到。
  14. 权利要求13所述的iDPP/IL-12复合物的制备方法，其特征在于：包括下述配比关系的原料及辅料：
    原料质量配比为：iRGD-DOTAP-mPEG-PLA纳米粒1～99份，IL-12质粒1～10份；
    渗透压调节剂适量；
    溶剂：注射用水、双蒸水、去离子水、纯水或生理盐水中的至少一种；
    制备方法如下：
    将上述原料及溶剂按照渗透压调节剂、溶剂、iRGD-DOTAP-mPEG-PLA纳米粒和IL-12质粒依次混合即得iDPP/IL-12复合物溶液，所得溶液达到生理渗透压。
  15. 根据权利要求14所述的iDPP/IL-12复合物的制备方法，其特征在于：原料质量配比为iRGD-DOTAP-mPEG-PLA纳米粒90～99份，IL-12质粒1～10份；优选iRGD-DOTAP-mPEG-PLA纳米粒25份，IL-12质粒1份。
  16. 权利要求13所述的iDPP/IL-12复合物在制备治疗抗肿瘤药物中的应用。
  17. 根据权利要求16所述的应用，其特征在于：所述的肿瘤为黑色素瘤、卵巢癌或肺癌。
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