CN110214145B - CP-iRGD polypeptide, iDPP nanoparticle, drug-loaded compound and preparation method and application thereof - Google Patents

CP-iRGD polypeptide, iDPP nanoparticle, drug-loaded compound and preparation method and application thereof Download PDF

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CN110214145B
CN110214145B CN201880008394.1A CN201880008394A CN110214145B CN 110214145 B CN110214145 B CN 110214145B CN 201880008394 A CN201880008394 A CN 201880008394A CN 110214145 B CN110214145 B CN 110214145B
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苟马玲
魏于全
罗丽
杨玉屏
陈雨文
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Sichuan University
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Abstract

The invention belongs to the field of medicines, and provides CP-iRGD polypeptide, iRGD-DOTAP-mPEG-PLA nanoparticles, a drug-loaded compound, and a preparation method and application thereof. The invention adopts amphiphilic substance DOTAP with positive charge and modified CP-iRGD polypeptide with tumor targeting effect to modify amphiphilic mPEG-PLA diblock copolymer, and prepares iRGD-DOTAP-mPEG-PLA nano-particles, namely iDPP nano-particles, of a degradable gene carrier by using a self-assembly method. The nanoparticle can introduce gene plasmids into tumor cells.

Description

CP-iRGD polypeptide, iDPP nanoparticle, drug-loaded compound and preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to CP-iRGD polypeptide, iDPP nanoparticles, a medicine-carrying compound, and a preparation method and application thereof.
Background
The gene transfer system has important application in gene function research and gene therapy. Currently used gene introduction systems mainly include two main types: viral vectors and non-viral vectors. The virus vector has large scale production difficulty, small deliverable gene capacity, easy immune reaction causing and potential biological safety risk. The non-viral gene vector comprises liposome, cationic nanoparticles, inorganic nanoparticle vector and the like, has the characteristics of low immunogenicity, better safety, easy large-scale production and the like, and is a research hotspot in the world at present.
A Methoxy polyethylene glycol-polylactic acid (Methoxy polyethylene glycol) -poly (lactic acid), abbreviated as mPEG-PLA) diblock polymer is a degradable amphiphilic polymer with good biocompatibility. In the 90 s of the 20 th century, drugs loaded with PEG or PLA delivery vectors have been approved by the U.S. FDA for clinical application, and have good application prospects in drug and gene introduction systems. At present, paclitaxel formulations coated with mPEG-PLA diblock polymer nanoparticle carriers have been used for clinical treatment of breast cancer in korea and europe, and have also entered phase II clinical trials in the us. When the PEG-PLA nanoparticles are used for a gene transfer system, the difficulty of loading genes by simply adopting the mPEG-PLA nanoparticles is high, and the transfection efficiency is low. The mPEG-PLA nanoparticles are further subjected to physical or chemical modification, so that novel nanoparticles which are easy to load genes, high in transfection efficiency, low in toxicity and degradable can be prepared, and the novel nanoparticles have good application prospects in gene function research, gene therapy research and clinical application.
Disclosure of Invention
The technical problem solved by the invention is to provide a novel modification means for modifying mPEG-PLA diblock copolymer. The inventor adopts a proper method to modify iRGD, thereby preparing CP-iRGD polypeptide which has high yield, high purity and good solubility and can target tumors, and then adopts CP-iRGD polypeptide and DOTAP to modify amphiphilic mPEG-PLA diblock copolymer, thereby preparing a novel targeted degradable gene vector, namely iRGD-DOTAP-mPEG-PLA cationic nanoparticles, which are abbreviated as iDPP nanoparticles, by adopting a self-assembly method. The iDPP nanoparticle has good DNA binding capacity, is electrically neutral with an iDPP nano DNA compound obtained after DNA binding, can effectively introduce plasmids loaded with target genes into tumor cells, and has the advantages of high transfection efficiency, low cytotoxicity and the like.
The amphiphilic mPEG-PLA copolymer adopted in the invention is chemically named methoxy polyethylene glycol-polylactic acid, which is called mPEG-PLA for short. The methoxy polyethylene glycol-polylactic acid nano-particles have amphipathy, good biodegradability and biocompatibility, avoid mononuclear phagocytosis, increase the circulation time and bioavailability of the medicine in blood, increase the medicine effect through targeted delivery and reduce side effects.
The amphiphilic cationic substance DOTAP adopted in the invention is chemically named as (2,3-dioleoxypropyl) trimethyl ammonium chloride, which is called DOTAP for short.
The C18-PEG-iRGD polypeptide with amphipathy, CP-iRGD polypeptide for short, adopted in the invention is modified iRGD. The CP-iRGD polypeptide can target tumor tissues and enhance the permeability of the tumor tissues, and the nanoparticles can improve the uptake capacity of the tumor tissues after being modified by the CP-iRGD polypeptide, increase the aggregation of medicines at tumor parts and reduce toxic and side effects; the CP-iRGD polypeptide obtained by modifying the iRGD has amphipathy, and a target object can be prepared in a self-assembly mode. iRGD is the existing polypeptide, and the structural formula is shown as the following formula I:
Figure GPA0000269492980000041
the invention aims to solve the first technical problem of providing a CP-iRGD polypeptide, the structural formula of which is shown as the following formula II:
Figure GPA0000269492980000051
the second technical problem to be solved by the invention is to provide a preparation method of the CP-iRGD polypeptide. The method comprises the following steps:
A. after the reaction of polyethylene glycol and stearic acid as raw materials, separating and purifying to obtain C18-PEG-OH;
B. C18-PEG-OH reacts with Fmoc-phenylalanine to obtain C18-PEG-Phe-Fmoc;
C. removing the Fmoc protecting group of the C18-PEG-Phe-Fmoc protecting group to obtain C18-PEG-Phe-NH 2 ；
D. Reacting C18-PEG-Phe-NH 2 Reacting with 3-maleimide propionic acid N-hydroxysuccinimide ester to obtain C18-PEG-BMPS;
E. C18-PEG-BMPS and iRGD react with sulfydryl through maleimide group to obtain the target compound C18-PEG-iRGD.
Preferably, in step C of the above preparation method, the Fmoc protecting group of C18-PEG-Phe-Fmoc is removed using 1,8-diazabicyclo [5.4.0] undec-7-ene.
Preferably, in step E of the above preparation method, the solvent used in the reaction is a mixture of PBS buffer solution with pH =7.3 and dimethyl sulfoxide at a volume ratio of 1: 1.
The third problem to be solved by the invention is that the iRGD-DOTAP-mPEG-PLA nanoparticle solution is prepared by a self-assembly method, and the raw materials and the solvent are taken according to the following proportioning relation to prepare the solution:
raw materials: the mass ratio of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide is as follows: 70-99 parts of mPEG-PLA copolymer, 1-30 parts of DOTAP and 1-5 parts of CP-iRGD;
solvent: at least one volatile solvent selected from dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate, and cyclohexane;
hydration solution: at least one of double distilled water, deionized water, pure water, normal saline and glucose solution;
the preparation method comprises the following steps: respectively dissolving mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide in a solvent, uniformly mixing, evaporating the solvent, adding a proper amount of a water liquefaction solution, and hydrating to obtain a required concentration to obtain an iRGD-DOTAP-mPEG-PLA nanoparticle water solution.
Further, the iRGD-DOTAP-mPEG-PLA nanoparticle solution prepared by the self-assembly method is prepared by taking the following raw materials according to the following proportioning relation:
raw materials: the mass ratio of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide is as follows: 85-95 parts of mPEG-PLA copolymer, 5-15 parts of DOTAP and 1-5 parts of CP-iRGD.
In the technical scheme, the solvent is used in an amount capable of dissolving the raw materials.
The invention also provides the iRGD-DOTAP-mPEG-PLA nanoparticle solution prepared by the self-assembly method.
The invention also provides the iRGD-DOTAP-mPEG-PLA nanoparticle and a preparation method thereof. The preparation method comprises the following steps: and drying the iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution to obtain the iRGD-DOTAP-mPEG-PLA nanoparticle.
The average particle size of the iRGD-DOTAP-mPEG-PLA nanoparticle is 139.16 +/-1.56 nm, the average potential is 43.1 +/-6.8 mV, and the iRGD-DOTAP-mPEG-PLA nanoparticle has good DNA binding capacity. Compared with gold-labeled transfection material PEI 25K, the iDPP nanoparticle has higher transfection capability and lower cytotoxicity; compared with DOTAP-mPEG-PLA (DPP for short), the iDPP nanoparticle has higher transfection efficiency and tumor cell targeting property.
The iRGD-DOTAP-mPEG-PLA nanoparticle can be used for encapsulating active ingredients, particularly genes, chemical drugs or proteins, so as to obtain an iRGD-DOTAP-mPEG-PLA nanoparticle compound.
Preferably, the plasmid comprises 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticles and 1 part of plasmid in parts by mass.
The iRGD-DOTAP-mPEG-PLA nanoparticle belongs to a biodegradable cationic nanoparticle, and is a novel non-viral vector of a gene transfer system. The nanoparticles can be combined with DNA through electrostatic interaction, and the iDPP nano compound combined with the DNA is neutral in electricity and has a long-circulating effect. The active ingredients such as target genes, chemical drugs, proteins and the like can be effectively targeted and introduced into tumor cells by an intravenous injection administration mode, and the tumor cells have the characteristics of low cytotoxicity, high transfection efficiency and the like.
For example, an iRGD-DOTAP-mPEG-PLA nanoparticle can be used to deliver a plasmid expressing a vesicular stomatitis virus matrix protein (VSVMP plasmid), i.e., an iRGD-DOTAP-mPEG-PLA nanoparticle is used to entrap the VSVMP plasmid to obtain an iDPP/VSVMP complex. Abbreviated as iDPP/MP complexes.
The iDPP/VSPMP compound comprises the following raw materials and auxiliary materials in proportion:
raw materials: 1-99 parts of iRGD-DOTAP-mPEG-PLA nano-particles and 1-10 parts of VSVMP plasmid;
proper amount of osmotic pressure regulator, and the dosage of the osmotic pressure regulator is that the iDPP/VSPMP compound prepared reaches physiological osmotic pressure;
solvent: at least one of water for injection, double distilled water, deionized water, pure water or physiological saline;
the preparation method comprises the following steps:
mixing the raw materials and the solvent in sequence according to an osmotic pressure regulator, the solvent, the iRGD-DOTAP-mPEG-PLA nano-particles and the VSVMP plasmid to obtain the iDPP/VSVMP compound, wherein the obtained solution reaches physiological osmotic pressure.
Further, the iDPP/VSVMP compound is prepared by taking the following raw materials according to the following proportion: the mass ratio of the iRGD-DOTAP-mPEG-PLA nano-particles to the VSVMP plasmid is 90-99 parts of the iRGD-DOTAP-mPEG-PLA nano-particles and 1-10 parts of the VSVMP plasmid.
Further, the iDPP/VSPMP compound is prepared by taking the raw materials according to the following proportioning relation: the mass ratio of the iRGD-DOTAP-mPEG-PLA nano-particles to the VSVMP plasmid is 25 parts of the iRGD-DOTAP-mPEG-PLA nano-particles and 1 part of the VSVMP plasmid.
The inventor finds that the iRGD-DOTAP-mPEG-PLA nanoparticle mediated VSVMP plasmid can be applied to in vitro and in vivo treatment of melanoma.
In vitro, the inventor uses MTT method to detect the inhibition effect of iDPP/VSVMP compound on the growth of B16-F10 melanoma cells, and uses flow cytometry to detect the condition that iDPP/VSVMP compound induces apoptosis. The inventor finds that by using the iDPP nanoparticle to mediate VSVMP plasmid to be introduced into B16-F10 melanoma cells, the iDPP/VSVMP compound can obviously inhibit the growth of the B16-F10 melanoma cells by inducing apoptosis.
In vivo, the inventors established a melanoma subcutaneous implantation tumor model and a melanoma lung metastasis model, compared the tumor volumes and weights of the groups, and compared the tumor node numbers of lung metastasis. The inventors found that the iDPP/VSVMP complex can significantly reduce tumor burden and lung metastasis production in mice. Experimental data show that the iDPP nanoparticle delivered VSVMP plasmid can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.
Therefore, the invention also provides the application of the iDPP/VSPMP compound in preparing antitumor drugs. Preferably, the tumor is melanoma, ovarian cancer or lung cancer.
Meanwhile, the iRGD-DOTAP-mPEG-PLA nanoparticle can be used for delivering the plasmid for expressing interleukin-12, namely the iRGD-DOTAP-mPEG-PLA nanoparticle is used for encapsulating the IL-12 plasmid to obtain the iDPP/IL-12 compound.
The iDPP/IL-12 compound comprises the following raw materials and auxiliary materials in proportion relationship:
raw materials: 1-99 parts of iRGD-DOTAP-mPEG-PLA nanoparticles and 1-10 parts of IL-12 plasmid;
the proper amount of osmotic pressure regulator is used for preparing the iDPP/IL-12 compound 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 comprises the following steps:
mixing the raw materials and the solvent in sequence according to an osmotic pressure regulator, the solvent, the iRGD-DOTAP-mPEG-PLA nano-particles and the IL-12 plasmid to obtain the iDPP/IL-12 compound, wherein the obtained solution reaches physiological osmotic pressure.
Further, the iDPP/IL-12 compound is prepared by taking the following raw materials according to the proportion relation: the mass ratio of the iRGD-DOTAP-mPEG-PLA nano-particles to the IL-12 plasmid is 90-99 parts of the iRGD-DOTAP-mPEG-PLA nano-particles and 1-10 parts of the IL-12 plasmid.
Further, the iDPP/IL-12 compound is prepared by taking the following raw materials according to the proportion relation: the mass ratio of the iRGD-DOTAP-mPEG-PLA nano-particles to the IL-12 plasmid is 25 parts of the iRGD-DOTAP-mPEG-PLA nano-particles and 1 part of the IL-12 plasmid.
The inventor finds that the iRGD-DOTAP-mPEG-PLA nanoparticle mediated IL-12 plasmid can be applied to in vitro and in vivo treatment of melanoma.
In vitro, flow cytometry was used to detect apoptosis induced by iDPP/IL-12 complex. The finding shows that the iDPP nanoparticle mediated IL-12 plasmid is used for treating B16-F10 melanoma cells, and the iDPP/IL-12 compound obviously inhibits the growth of the B16-F10 melanoma cells by inducing apoptosis.
In vivo, a model of melanoma was established subcutaneously and tumor volumes and weights were compared for each group. The inventors found that the iDPP/IL-12 complex can significantly reduce the tumor burden of mice and enhance the anti-tumor immunity of the mice. Experimental data show that the iDPP nanoparticle can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo by delivering the IL-12 plasmid.
The invention also provides application of the iDPP/IL-12 compound in preparation of antitumor drugs. Preferably, the tumor is melanoma, ovarian cancer or lung cancer.
According to the invention, an appropriate method is selected to modify iRGD, so that the CP-iRGD polypeptide which has high yield and purity and can target tumors is obtained, and the CP-iRGD polypeptide has good solubility, so that the degradable cationic nanoparticle iRGD-DOTAP-mPEG-PLA nanoparticle can be prepared by a self-assembly method. The nanoparticle can be effectively combined with DNA, the nano compound combined with the DNA is neutral in electricity, the therapeutic gene can be effectively introduced into tumor cells in an intravenous injection administration mode, and the nanoparticle has the characteristics of high transfection rate, low cytotoxicity and the like, and has good application prospects in gene function research, gene therapy research and clinical application. The iRGD-DOTAP-mPEG-PLA nanoparticles can mediate active ingredients such as genes to exert curative effects, for example, the iRGD-DOTAP-mPEG-PLA nanoparticles mediate plasmids loaded with VSPMP genes and plasmids loaded with IL-12 genes to effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo. The iDPP nanoparticle is a relatively safe degradable non-viral gene vector, and the prepared iDPP/VSPMP compound and the iDPP/IL-12 compound provide a new idea and potential choice for treating melanoma.
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FIG. 1 (A) molecular structural formula of PEG-PLA; (B) molecular structural formula of DOTAP; (C) synthetic roadmap for C18-PEG-iRGD.
FIG. 2 schematic synthesis of iDPP nanoparticles.
Fig. 3 particle size and potential distribution diagram of the iDPP nanoparticle: (A) a particle size distribution diagram of the iDPP nanoparticles; (B) the potential distribution diagram of the iDPP nanoparticles; (C) scanning transmission electron microscope photos of iDPP nanoparticles; (D) iDPP nanoparticle gel blocking assay. When the mass ratio of the iDPP to the DNA is 25: 1, the iDPP nanoparticle can be completely combined with the DNA plasmid.
FIG. 4 particle size and potential profiles of iDPP/VSPMP complexes: (A) particle size distribution profile of the iDPP/VSPMP complex; (B) the potential profile of the iDPP/VSPMP complex; (C) scanning transmission electron micrographs of the iDPP/VSPMP complex; (D) iDPP/VSPMP complex gene gradient potential map.
FIG. 5iDPP nanoparticle is used for detecting B16-F10 cytotoxicity and transfection rate: (A) In B16-F10 cells, the cytotoxicity of iDPP nanoparticles is lower than that of PEI 25K; (B) iDPP nanoparticle, DPP nanoparticle, PEK25K transfection B16-F10 cell fluorescence map (nanoparticle: DNA is 25: 1, 25: 1 and 1: 1 respectively); (C) flow chart statistics of cell transfection.
FIG. 6iDPP/VSVMP complex antitumor ability against B16-F10 cells in vitro: (A) an iDPP/VSVMP complex MTT detection profile; (B) iDPP/VSVMP complex promotes apoptosis flow pattern, and iDPP/VSVMP complex inhibits tumor cell growth by inducing apoptosis.
FIG. 7iDPP nanoparticle targeting and antitumor activity of intratumoral injection iDPP/VSPMP complexes: (A) Intravenous injection of iDPP/pGL-6 complex, luciferase expression and in vivo imaging; (B) Tumor volume profile for iDPP/VSVMP complex treatment; (C) Statistical plots of tumor weights for iDPP/VSPMP complex treatment.
FIG. 8 antitumor Activity of the intravenous iDPP/VSPMP Complex: (A) Intravenous iDPP/VSPMP complex targeted therapy B16-F10 subcutaneous tumor maps; (B) iDPP/VSVMP complex treatment tumor weight statistics; (C) Intravenous injection of the iDPP/VSPMP complex inhibits melanoma lung metastasis; (D) Weight of lungs treated with intravenous iDPP/VSPMP complex.
FIG. 9 in vitro antitumor Capacity of iDPP/IL-12 complexes against B16-F10 cells in vitro: (A) iDPP/IL-12 complex apoptotic flow map; (B) apoptosis statistical map.
FIG. 10 antitumor Activity of intravenous iDPP/IL-12 Complex: (A) Intravenous iDPP/IL-12 complex for treatment of B16-F10 subcutaneous tumors; (B) tumor tissue IFN- γ secretion levels; (C) tumor tissue NK cell infiltration flow analysis statistical chart; (D) tumor tissue CD8+ T cell infiltration flow analysis statistical chart.
Detailed Description
The invention firstly provides a CP-iRGD polypeptide, the structural formula is as the following formula II:
Figure GPA0000269492980000101
the invention also provides a preparation method of the CP-iRGD polypeptide, which comprises the following steps:
A. after the reaction of polyethylene glycol and stearic acid as raw materials, separating and purifying to obtain C18-PEG-OH;
B. C18-PEG-OH reacts with Fmoc-phenylalanine to obtain C18-PEG-Phe-Fmoc;
C. removing the Fmoc protecting group of the C18-PEG-Phe-Fmoc protecting group to obtain C18-PEG-Phe-NH 2 ；
D. Reacting C18-PEG-Phe-NH 2 Reacting with 3-maleimide propionic acid N-hydroxysuccinimide ester to obtain C18-PEG-BMPS;
E. C18-PEG-BMPS and iRGD react with sulfydryl through a maleimide group to obtain a target compound C18-PEG-iRGD.
Preferably, in step C of the above preparation method, the C18-PEG-Phe-Fmoc protecting group Fmoc is removed using 1,8-diazabicyclo [5.4.0] undec-7-ene.
Preferably, in step E of the above preparation method, the solvent used in the reaction is a mixture of PBS buffer solution with pH =7.3 and dimethyl sulfoxide in a volume ratio of 1: 1.
Specifically, the preparation method of the CP-iRGD polypeptide comprises the following steps:
the first step is as follows: preparing CH by using polyethylene glycol (PEG) and stearic acid (C17 COOH) as raw materials 3 (CH 2 ) 16 COO-PEG-OH (Compound 1); polyethylene glycol (PEG, mw =1000, 2000, 4000, 8000, etc. each molecular weight) and straight chain carboxylic acid (CH) 3 (CH 2 ) 16 COOH) as raw material to obtain CH 3 (CH 2 ) 16 CO-PEG-OH (Compound 1); the purity of the product obtained in the step is more than or equal to 90 percent, and the yield is 70 percent;
reaction solvent: organic solvents such as dichloromethane, chloroform, and acetone;
reaction conditions are as follows: stirring at room temperature for 12h;
the molecular weight of polyethylene glycol (PEG) may be Mw =1000, 2000, 4000, 8000, or the like.
The second step is that: C18-PEG-OH (compound 1) and Fmoc-phenylalanine react to obtain C18-PEG-Phe-Fmoc (compound 2); the purity of the product in the step is more than 90 percent, and the yield is 90 percent;
the third step: with 1,8-diazabicyclo [5.4.0]]Undec-7-ene (DBU for short) is subjected to C18-PEG-Phe-Fmoc (compound 2) removal to obtain C18-PEG-Phe-NH 2 (Compound 3);
the fourth step: reacting C18-PEG-Phe-NH 2 (compound 3) reacts with 3-maleimide propionic acid N-hydroxysuccinimide ester (abbreviated as BMPS) to obtain C18-PEG-Phe-BMPS (compound 4); the purity of the product in the step is more than 90 percent, and the yield is 68 percent;
the fifth step: C18-PEG-Phe-BMPS (compound 4) and iRGD react with sulfydryl through maleimide group to obtain a target compound C18-PEG-iRGD (compound 5). The solvent adopted by the reaction is formed by mixing PBS buffer solution with pH =7.3 and dimethyl sulfoxide according to the volume ratio of 1: 1.
The present invention will be described in further detail below with reference to specific embodiments of examples, but the present invention is not limited thereto.
The synthesis circuit diagram of the mPEG-PLA, the DOTAP molecular structural formula and the CP-iRGD is shown in figure 1, the iDPP nano-particles are prepared by the mPEG-PLA, the DOTAP and the CP-iRGD through a self-assembly method, and the structural schematic diagram is shown in figure 2.
1. Experimental method
1.1 preparation of synthetic C18-PEG-iRGD Compounds
1.1.1 preparation Synthesis of C18-PEG-OH (hereinafter referred to as Compound 1)
Dissolving polyethylene glycol (1 mmol) and stearic acid (C17 COOH) (1 mmol) in dichloromethane, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl,3 mmol) and N-hydroxysuccinimide (NHS, 0.3 mmol), stirring at room temperature overnight for reaction, washing with 1N HCl solution, aqueous solution and saturated salt solution for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, collecting the filtrate, removing the solvent solid under reduced pressure, and separating and purifying by column chromatography (dichloromethane: methanol = 20: 1) to obtain compound 1;
1.1.2 preparation of C18-PEG-Phe-Fmoc (Compound 2 for short)
Compound 1 (500 mg) and Fmoc-L-phenylalanine (142 mg) were dissolved in methylene chloride, EDC. HCl (140 mg) and DMAP (9 mg) were added thereto overnight at room temperature, and the same samples as 1.1.1 were collected and treated to give Compound 2.
1.1.3 preparation of synthetic C18-PEG-Phe-NH2 (Compound 3 for short):
compound 2 (500 mg) was dissolved in methylene chloride, DBU (100 uL) was slowly added thereto, and the reaction was stirred at ordinary temperature for 3 hours. The same sample was collected and treated as 1.1.1 except for column chromatography separation and purification (dichloromethane: methanol = 25: 1) to obtain compound 3.
1.1.4 preparation of synthetic C18-PEG-Phe-BMPS (Compound 4 for short):
compound 3 (200 mg) and BMPS (49 mg) were dissolved in methylene chloride, 0.2% triethylamine was added, the reaction was stirred at room temperature for 12 hours, and the same sample collection and treatment as 1.1.1 gave compound 4.
1.1.5 preparation and synthesis of a target compound C18-PEG-iRGD (compound 5 for short):
dissolving C18-PEG-Phe-BMPS (300 mg) in acetone, dissolving iRGD (150 mg) in water, dissolving iRGD in a mixed solvent solution of PBS buffer (PH = 7.3) and dimethyl sulfoxide, then mixing and dissolving with the C18-PEG-Phe-BMPS solution, dropwise adding 0.2% triethylamine, and reacting at room temperature under vacuum and nitrogen protection overnight. Dialyzed (Mw = 2000) for 2 days, lyophilized, and stored sealed at 4 ℃ until use.
1.2 iDPP nano particle and iDPP/VSPMP compound preparation method
1.2.1 Preparation of iDPP nanoparticles
Respectively dissolving mPEG-PLA copolymer (45 mg), DOTAP (5 mg) and CP-iRGD (1 mg) in 4mL dichloromethane solution, transferring into a flask, and mixing uniformly; and (3) placing the mixed solution in a water bath kettle at 60 ℃, and performing rotary evaporation for 30 minutes under a vacuum condition. Adding a proper amount of double distilled water to hydrate to a required concentration, slightly oscillating in a water bath at 60 ℃ until the double distilled water is completely dissolved to obtain a solution, namely the iDPP nanoparticle aqueous solution, and storing the solution in a refrigerator at 4 ℃ for later use. And drying the obtained iDPP nanoparticle aqueous solution to obtain the iDPP nanoparticles.
1.2.2 iDPP/VSPMP compound preparation method
Raw materials: VSVMP plasmid 5. Mu.g; 125 mu g of iRGD-DOTAP-mPEG-PLA nano-particles; 50% glucose;
solvent: double distilled water;
the preparation method comprises the following steps: mixing the raw materials and the solvent according to the sequence of glucose, double distilled water, iRGD-DOTAP-mPEG-PLA nanoparticles and VSVMP plasmid, and controlling the final glucose concentration of the compound to be 5%.
1.3 Particle size, potential and morphology study of iDPP (ion-doped dipeptidyl peptidase) nanoparticles
1.3.1 Particle size and potential of iDPP (ion-doped dipeptidyl peptidase) nanoparticles
1.2.1 particle size and potential size of the iDPP nanoparticles prepared were measured using a Zetasizer Nano ZS Malvern particle sizer (Malverm Instruments, worcestershire, UK). Results were averaged over 3 measurements. The results are shown in 2.1.1.
1.3.2 Morphological study of iDPP nanoparticles
1.2.1 morphology of the iDPP nanoparticles prepared was observed by Scanning Transmission Electron Microscope (STEM). The results are shown in 2.1.1.
1.4 Research on binding capacity of iDPP (Interleukin-derived DPP) nanoparticles and DNA (deoxyribonucleic acid)
The DNA binding capacity of the iDPP nanoparticles is detected by a gel retardation analysis experiment. Firstly, iDPP nano-particles with different mass ratios (1: 1,5: 1, 10: 1, 15: 1, 20: 1, 25: 1) and no-load plasmid (pVAX) are mixed, the mass of the no-load plasmid (pVAX) is 0.3 mu g, the total volume of the mixed solution is adjusted to be 5ul by adopting an aqueous solution without DNase, and the mixture is kept stand and incubated for 30 minutes at room temperature. Then, a 1% agarose gel was prepared and electrophoresed at 100V for 30 minutes. And finally, taking out the gel, observing under the irradiation of an ultraviolet lamp, and taking a picture. The results are shown in 2.2.
1.5 Particle size, potential and morphology study of iDPP/VSPMP complexes
1.5.1 Particle size and potential of iDPP/VSPMP Complex
1.2.2 the particle size and potential of the iDPP/VSVMP complex prepared were measured by the same method as 1.3.1, and the results are shown in 2.3.1.
1.5.2 Morphological study of iDPP/VSVMP Complex
1.2.2 morphological examination of the iDPP/VSVMP complex prepared by the same method as 1.3.2, and the result is shown in 2.3.1.
1.6 After the iDPP nano-particle is combined with VSPMP, the potential change condition
The iDPP nanoparticles and VSVMP plasmids were controlled in different mass ratios (5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1) for each of the following groups.
(1) VSVMP plasmids were diluted individually in 400. Mu.l deionized water and mixed gently.
(2) And (3) respectively diluting the iDPP nanoparticles into 400 mu l of deionized water, and gently mixing the diluted solution.
(3) And (3) adding the VSVMP plasmid treated in the step (1) into the iDPP nanoparticle treated in the step (2), and standing and incubating for 30 minutes at room temperature. The potential was measured and determined as 1.3.1, and the result was found to be 2.3.2.
1.7 Cytotoxicity detection of iDPP nanoparticles
Toxic effects of iDPP nanoparticles on 293T cells (purchased from ATCC) were examined by cell viability assay
(1) At 5X 10 3 The cells/well were plated in 96-well plates, 100. Mu.l of 293T cell suspension was added to each well, the cells were placed at 37 ℃ and the cell concentration was 5% 2 The cells were incubated in a constant temperature incubator for 24 hours.
(2) A series of concentrations (0. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml, 400. Mu.g/ml) of the iDPP nanoparticle solution and the PEI 25K solution were prepared, and 100. Mu.l of the above solutions with different concentrations were added to each well of a 96-well plate, and 6 duplicate wells were set for each concentration. After the addition, 5% of C0 at 37 ℃ 2 Incubation was continued in the cell incubator for 48 hours.
(3) After the incubation is finished, the MTT method is adopted for detection, and the result is shown in 2.4.
1.8 iDPP nano particle transfection B16-F10 cell
(1) Cell density of 2X 10 5 B16-F10 cells (purchased from ATCC) were seeded in 6-well plates, adding 2ml of cell suspension per well.
(2) Mu.g of a plasmid expressing green fluorescent protein (pGFP) was diluted in 50. Mu.l of serum-free, antibiotic-free 1640 medium and gently mixed.
(3) And (3) respectively diluting the prepared iDPP nanoparticles and 50 mu g of DPP nanoparticles into 50 mu l of serum-free and antibiotic-free 1640 culture medium, and gently mixing uniformly. Mu.g of PEI 25K solution (1. Mu.g/. Mu.l) was diluted in 50. Mu.l of serum-free, antibiotic-free 1640 medium and gently mixed.
(4) And (3) respectively adding the pGFP treated in the step (2) into the iDPP nanoparticle solution, the DPP nanoparticle solution and the PEI 25K solution obtained in the step (3), and standing and incubating for 30 minutes at room temperature.
(5) The medium of the 6-well plate treated in step (1) was changed to 800ul of a serum-free and antibiotic-free 1640 medium, and then the iDPP/pGFP complex, the DPP/pGFP complex, and the PEI 25K/pGFP complex obtained in step (4) were added to the 6-well plate, respectively, and the mixture was charged at 37 ℃ and 5% CO 2 Incubating for 4-8 hours in a constant temperature incubator.
(6) The culture medium in the 6-well plate is replaced by 2ml of 1640 culture medium containing serum and antibiotics, and the obtained product is placed into an incubator to be continuously cultured for 40 hours.
(7) And (3) observing the expression condition of the green fluorescent protein in the transfected cells under an inverted fluorescence microscope. The cells were collected and examined by flow cytometry to determine the transfection efficiency, as shown in 2.5.
1.9 detection of the antitumor Activity of the iDPP/VSVMP Complex in vitro against B16-F10 cells
The antitumor activity of the iDPP/VSPMP complex on B16-F10 cells was tested in vitro mainly by two methods: MTT method and flow cytometry.
1.9.1 MTT method for detecting inhibition effect of iDPP/VSVMP complex on growth of B16-F10 cells
(1) The details of the method for inoculating the B16-F10 cells into the 96-well plate are shown in 1.7 (1);
(2) Respectively diluting 1 mu g of VSVMP plasmid and unloaded plasmid (pVAX) in 25 mu l of 1640 culture medium without serum or antibiotics, and gently mixing;
(3) Respectively diluting 25 mu g of DPP nano-particles and iDPP nano-particles into 25 mu l of 1640 culture medium without serum or antibiotics;
(4) The transfection methods are detailed in 1.8 (4) - (6), and groups NS, iDPP/pVAX, DPP/MP, iDPP/MP are obtained respectively; wherein, for the NS group, steps (2) and (3) prepare 25 μ l of 1640 medium without serum and antibiotics; for the iDPP group, step (2) prepared 25 μ l of 1640 medium without serum and antibiotics;
(5) The MTT method is adopted for detection, the details of the detection method are shown in 1.7 (3), and the result is shown in 2.6.
1.9.2 flow cytometry detection of iDPP/VSVMP Complex for inducing apoptosis of B16-F10 cells
(1) The details of the method for inoculating the B16-F10 cells into the 6-well plate are shown in 1.8 (1);
(2) Respectively diluting 2 μ g VSVMP plasmid and unloaded plasmid (pVAX) in 50 μ l serum-free and antibiotic-free 1640 culture medium, and mixing gently;
(3) Respectively diluting 50 mu g of DPP nanoparticles and iDPP nanoparticles in 50 mu l of 1640 culture medium without serum or antibiotics;
(4) The transfection methods are detailed in 1.8 (4) - (6), and groups NS, iDPP/pVAX, DPP/MP and iDPP/MP are obtained respectively; wherein, for the NS group, steps (2) and (3) prepare 25 μ l of 1640 medium without serum and antibiotics; for the iDPP group, step (2) prepared 25 μ l of 1640 medium without serum and antibiotics;
(5) The apoptosis of B16-F10 cells was detected by flow cytometry according to the instructions of Biolegend V-FITC/PI double staining kit, and the results are shown in 2.6.
1.10 detection of tumor targeting of iDPP nanoparticle and iDPP/VSVMP Complex
(1) B16-F10 melanoma subcutaneous implantation tumor model establishment: female C57BL/6 mice (Sichuan university laboratory animal center) of 6-8 weeks old were bred in SPF class, and each mouse was inoculated with 2X 10 cells 5 Cells, 100. Mu.l of cell suspension was injected subcutaneously.
(2) Two weeks after inoculation, each mouse was injected tail vein with the iDPP/pGL-6 complex prepared as follows. Mu.g of plasmid pGL-6 expressing luciferase and 200. Mu.g of iDPP nanoparticles are respectively diluted in 50. Mu.l of 5% glucose water, and the iDPP/pGL-6 complex is obtained after incubation according to 1.8 (4).
(3) Working solution (15 mg/ml) of D-fluorescein sodium salt is prepared by normal saline, and is filtered and sterilized by a 0.22 mu m filter membrane.
(4) After 72 hours of administration in the step (2), 200. Mu.l of the working solution of D-fluorescein sodium salt prepared in the step (3) was intravenously injected, and 20 minutes later, the images were taken by a mouse in vivo imager. The results are shown in 2.7.1.
1.11 testing of the antitumor Activity of iDPP/VSVMP Complex in vivo
1.11.1 intratumoral injection of iDPP/VSVMP Complex and study of antitumor mechanism thereof
(1) B16-F10 melanoma subcutaneous implantation tumor model establishment: each mouse was inoculated with 2X 105 cells and injected subcutaneously with 100. Mu.l of cell suspension;
(2) And (3) random grouping: on day 6 post inoculation, C57 mice were randomly divided into 5 groups of 5 mice each;
(3) Intratumoral injection administration: on day 6 after inoculation, NS, iDPP/pVAX, DPP/MP, iDPP/MP are administered in five groups, and the administration volume of each mouse is 100 μ l; wherein, NS group is 100 mul normal saline, iDPP group is 100 mul solution containing 125 mul iDPP nano-particles, iDPP/pVAX is 100 mul solution containing 5 mul pVAX no-load plasmid and 125 mul giDPP nano-particles; DPP/MP is solution 100 mu 1 containing 5 mu g VSVMP plasmid and 125 mu g DPP nano-particle; the iDPP/MP is 100 mul of solution containing 5 mu g of VSVMP plasmid and 125 mu g of DPP nanoparticles;
(4) The medicine is administrated once every 2 days, the length and the width of the tumor are measured by a vernier caliper and recorded for 4 times;
(5) 1 week after completion of treatment, mice were sacrificed by cervical dislocation, tumors and heart, liver, spleen, lung and kidney were collected, and tumor weights were weighed. The results are shown in 2.7.2.
1.11.2 intravenous iDPP/VSVMP complex and research of antitumor effect thereof
(1) B16-F10 melanoma subcutaneous implantation tumor model establishment: method reference 1.11.1 (1);
(2) And (3) random grouping: on day 6 post inoculation, C57 mice were randomly divided into 5 groups of 5 mice each;
(3) Intravenous administration: on the 6 th day after inoculation, the mice were divided into groups according to the 1.11.1 (3) administration and injected intravenously at the tail of the mouse;
(4) The drug is administrated once every 2 days, the length and the width of the tumor are measured by a vernier caliper and recorded for 7 times;
(5) 1 week after completion of treatment, mice were treated with reference to 1.11.1 (5). The result is 2.7.3.
1.11.3 intravenous injection iDPP/VSPMP compound and research on inhibition of melanoma lung metastasis
(1) B16-F10 melanoma lung metastasis model establishment: the cell amount of each mouse was 1X 10 5 For each cell, 100. Mu.l of cell suspension was injected intravenously from the tail of the mouse;
(2) And (3) random grouping: on day 6 post inoculation, C57 mice were randomly divided into 5 groups of 5 mice each;
(3) Intravenous administration: day 6 post inoculation, method reference 1.11.2 (3);
(4) The medicine is administrated once every 2 days, and the treatment is carried out 7 times;
(5) 1 week after completion of treatment, mice were treated with reference to 1.11.1 (5), and tumor lung metastatic nodal counts were recorded. The result is 2.7.4.
1.12 detection of the antitumor Activity of the iDPP/IL-12 Complex on B16-F10 cells in vitro
The anti-tumor activity of the iDPP/IL-12 complex on B16-F10 cells is mainly detected in vitro by adopting flow apoptosis. The detection method refers to 1.9.2, which is grouped as NS, iDPP, iDPP/pVAX, iDPP/pIL12. The results are shown in 2.8.1.
1.13 testing of the antitumor Activity of iDPP/IL-12 Complex in vivo
1.13.1 intravenous injection iDPP/IL-12 complex anti-tumor mechanism research
(1) B16-F10 melanoma subcutaneous implantation tumor model establishment: the method is 1.10.1 (1);
(2) Random grouping: on day 6 post inoculation, C57 mice were randomly divided into 5 groups of 5 mice each;
(3) Intravenous administration: on day 6 post-inoculation, NS, iDPP/pVAX, iDPP/pl 12 were given in groups of four, by: the volume of each mouse for each administration is 100 mul; wherein the NS group is 100 mul of normal saline, the iDPP group is 100 mul of solution containing 125 mul of DPP nano-particles, the iDPP/pVAX group is 100 mul of solution containing 5 mu g of pVAX no-load plasmid and 125 mul of DPP nano-particles; iDPP/pIL12 is 100 mul of solution containing 5 mu g of pIL12 plasmid and 125 mu g of DPP nano-particles;
(4) Once every 2 days, tumor length and width were measured with a vernier caliper and recorded for a total of 5 treatments.
(5) 1 week after completion of treatment, mice were treated with reference to 1.11.1 (5). The results are shown in 2.8.2.
1.13.2 intravenous iDPP/IL-12 Complex immunodetection assay
(1) The tumor tissues collected at 1.11.1 (5) were assayed for IFN-. Gamma.secretion levels using an elisa assay.
(2) And analyzing the infiltration condition of the CD8+ T cells and NK cells of the tumor tissue by adopting flow cytometry. The results are shown in 2.8.2.
1.14 statistical analysis
The experimental data are expressed as mean ± SD, and the data analysis is performed by SPSS17.0 statistical software, and mean comparison analysis is mainly performed by means of averaging and t-test. Statistical differences of P < 0.05, P < 0.01, P < 0.001.
2 results
2.1 Characteristics of iDPP nanoparticle
2.1.1 Particle size, potential and morphology of iDPP nanoparticles
The average particle diameter of the iDPP nano-particle is 139 +/-1.5 nm, and the average potential is +43 +/-3.9 mV. Particle size distribution and potential distribution of nanoparticles (fig. 3A, 3B). Under scanning transmission electron microscopy, the iDPP diameter is about 50m in size (FIG. 3C).
2.2 Binding capacity of iDPP (Interleukin-derived DPP) nanoparticles and DNA (deoxyribonucleic acid)
We used gel retardation analysis experiments to test the DNA binding ability of iDPP nanoparticles. When the mass ratio of iDPP to DNA is 25: 1, the iDPP nanoparticle can completely bind to DNA (FIG. 3D).
2.3 Characterization of the iDPP/VSVMP Complex
2.3.1 Particle size, potential and morphology of iDPP/VSPMP complexes
The average particle diameter of the iDPP nano-particles is 142 +/-1.0 nm, and the average potential is 0.26 +/-0.2 mV. Particle size distribution and potential distribution of nanoparticles (fig. 4A, 3B). Under a scanning transmission electron microscope, iDPP/VSPMP are spherical particles with uniform size, and the diameter size is about 54m (FIG. 4C).
2.3.2 iDPP/VS VMP complex gradient potential change
Different ratios of iDPP/VSPMP complexes were prepared and the potential varied. The transition from + 43. + -. 3.9mV to-19.7. + -. 0.62mV (FIG. 4D).
2.4 Cytotoxicity detection of iDPP nanoparticles
Among 293T cells, PEI 25K cells were more toxic and IC 50 < 10. Mu.g/mL. Whereas iDPP nanocarriers have very low toxicity to cells with IC50 > 200. Mu.g/mL (FIG. 5A).
2.5 iDPP nano particle transfection B16-F10 cell
As shown in fig. 5B-C, the iDPP nanoparticles can deliver pGFP plasmids into cells. The transfection efficiency of iDPP nanoparticles is significantly higher than that of DPP (88.37% + -2.24% vs 42.87% + -5.68%). The iDPP nanoparticle is a novel targeted non-viral gene vector and has the characteristics of degradability, low cytotoxicity and high transfection efficiency.
2.6 Antitumor capability of iDPP/VSVMP complex on B16-F10 cells in vitro
As shown in FIG. 6A, the iDPP/VSVMP complex significantly inhibited the growth of B16-F10 tumor cells. Flow cytometry detection results showed that the iDPP/VSPMP complex induced significantly higher numbers of apoptotic cells than the other four groups (FIG. 9B). Therefore, the iDPP nanoparticle can effectively transfect the VSVMP gene plasmid into B16-F10 cells, and inhibit the growth of tumor cells through mechanisms such as apoptosis induction and the like.
2.7 Tumor targeting and in vivo anti-tumor effect of iDPP (dipeptidyl peptidase) compound
2.7.1 Tumor targeting of iDPP complexes
From FIG. 7A, it can be seen that plasmid pGL-6 of iDPP complex expressed luciferase can be expressed at tumor site 72 hours after injection via tail vein of mouse, indicating that the iDPP complex has good tumor targeting property.
2.7.2 iDPP/VSVMP complex tumor injection inhibiting growth of subcutaneous transplantation tumor
In the mouse B16-F10 subcutaneous tumor implantation tumor model, treatment was performed by intratumoral injection, and tumor volume was recorded (FIG. 7B), and the iDPP/VSVMP group had slow tumor volume growth, indicating that the iDPP/VSVMP complex significantly inhibited tumor growth. We removed the subcutaneous tumor from the mice and weighed the tumor (fig. 7C).
2.7.3 iDPP/VSVMP compound tail vein injection for inhibiting growth of subcutaneous transplantation tumor
In the mouse B16-F10 subcutaneous tumor implantation tumor model, treatment was performed by mouse tail vein injection, and tumor volumes were recorded. We removed the subcutaneous tumors from mice and photographed (FIG. 8A), and weighed the tumors (FIG. 8B), the iDPP/VSVMP group had significantly lower tumor weight than the other four groups, and intravenous injection of the iDPP/VSVMP complex resulted in targeted treatment of the subcutaneous B16-F10 tumors, inhibiting tumor growth.
2.7.4 iDPP/VSVMP compound tail vein injection for inhibiting melanoma lung metastasis
Treatment was performed via tail vein injection in mice in a B16-F10 lung metastatic tumor model. We removed the mouse lung and photographed (fig. 8C), the tumor lung metastasis nodules were significantly less in iDPP/VSVMP group than in the other four groups, and the lung metastasis of melanoma was significantly inhibited by tail vein injection of iDPP/VSVMP complex and weighed (fig. 8D).
2.8 Antitumor capacity of iDPP/IL-12 complex on B16-F10 cells in vitro
2.8.1 Antitumor capacity of iDPP/IL-12 complex on B16-F10 cells in vitro
The iDPP/IL-12 complex induced a significantly higher number of apoptotic cells than the other four groups as determined by flow cytometry (FIGS. 9A-B). In vitro antitumor activity results show that the iDPP nanoparticles can effectively transfect IL-12 gene plasmids into B16-F10 cells, and inhibit the proliferation of tumor cells by inducing apoptosis.
2.8.2 iDPP/IL-12 complex tail vein injection for inhibiting growth of subcutaneous transplantation tumor
In the mouse B16-F10 subcutaneous tumor implantation tumor model, treatment was performed by mouse tail vein injection, and tumor volumes were recorded. We removed subcutaneous tumors from mice and photographed (fig. 10A), and analyzed the tumor tissue for IFN- γ secretion using the elisa assay (fig. 10B), and tumor tissue for NK cells (fig. 10C) and CD8+ T cell infiltration using flow cytometry (fig. 10D). The intravenous injection of the iDPP/IL-12 compound can target B16-F10 subcutaneous tumor implantation tumor, increase the anti-tumor immunity of mice by inducing apoptosis, and inhibit the tumor growth.
3. Early stage screening test:
3.1 in the course of earlier experiments, the inventors modified water-soluble iRGD
3.1. The weight ratio of mPEG-PLA to DOTAP is defined as: 99: 1, 90: 10, 85: 15, 80: 20, 70: 30, 60: 40, and the weight ratio of CP-iRGD to mPEG-PLA and DOTAP is 1: 100,5: 100, 10: 100, 20: 100.
Cell transfection experiments are carried out, and the raw materials of iRGD-DOTAP-mPEG-PLA, namely 70-99 parts of mPEG-PLA copolymer, 1-30 parts of DOTAP and 1-10 parts of CP-iRGD polypeptide have better transfection efficiency within the proportion range. On the basis, the proportion is further reduced to the proportion range of 85-95 parts of mPEG-PLA copolymer, 5-15 parts of DOTAP and 1-5 parts of CP-iRGD, and then cell transfection experiments are carried out to find that higher transfection efficiency exists in the range.
3.2. In the preparation of the gene preparation, the content of the gene is determined to be 1% -50%, and gel retardation analysis and cell transfection experiments are carried out to find that the iDPP nanoparticles can be effectively combined with the gene in the proportion range and have better transfection efficiency.
3.3. On the basis of the previous stage, the gene content is further reduced to 1% -10%, and gel retardation analysis and cell transfection experiments are carried out again to find that the gene can be efficiently combined with the iDPP nanoparticles in the proportion range and has better transfection efficiency.
3.4. When the nanoparticles are prepared, the solvents for dissolving the CP-iRGD polypeptide are considered to be dichloromethane, trichloromethane, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate, cyclohexane and other volatile solvents, and the CP-iRGD polypeptide can be completely dissolved.
3.5. The hydration solution for hydrating the mixture of mPEG-PLA, DOTAP and CP-iRGD polypeptide can be double distilled water, deionized water, pure water, normal saline and the like, and the iDPP nanoparticle solution can be finally obtained. But preferably double distilled water is used as the hydration solution.
3.6. In the mPEG-PLA copolymer required for preparing the iDPP nanoparticles, the total molecular weight of mPEG-PLA is 4000 Da-8000 Da.
In conclusion, the invention provides a new modification means for modifying mPEG-PLA diblock copolymer, and the inventor adopts an amphiphilic substance DOTAP with positive charge and CP-iRGD with a tumor targeting effect to modify the amphiphilic mPEG-PLA diblock copolymer, and utilizes a self-assembly method to prepare a novel targeted degradable gene carrier, namely iRGD-DOTAP-mPEG-PLA cation nanoparticles. The nanoparticle has good DNA binding capacity, the iDPP compound bound with DNA becomes neutral in electricity, gene plasmids can be effectively guided into tumor cells in a targeted mode through an intravenous injection administration mode, and the nanoparticle has the advantages of high transfection efficiency and low cytotoxicity. The iDPP/VSPMP compound prepared from the iRGD-DOTAP-mPEG-PLA nano-particles can obviously inhibit tumor growth and lung metastasis of tumors. Experimental data show that the iDPP nanoparticle can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo by transferring the VSPMP gene. Effectively inhibit the growth of melanoma cells in vitro and in vivo. The iDPP nanoparticle is a relatively safe degradable non-viral gene vector. The iDPP/IL-l2 compound prepared from the iRGD-DOTAP-mPEG-PLA nanoparticle can also effectively inhibit the growth of melanoma in vivo and in vitro. The iDPP/VSVMP complex and the iDPP/IL-l2 complex prepared by the method provide a new idea and potential choice for targeted therapy of melanoma.

Claims (7)

  1. An iDPP/IL-12 complex, characterized in that: the interleukin-12 gene vector is obtained by loading plasmids expressing interleukin-12 on iRGD-DOTAP-mPEG-PLA nanoparticles; wherein the iRGD-DOTAP-mPEG-PLA nanoparticle is obtained by drying an iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution;
    the iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution is prepared by taking raw materials and a solvent according to the following proportion:
    raw materials: the mass ratio of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide is as follows: 70-99 parts of mPEG-PLA copolymer, 1-30 parts of DOTAP and 1-5 parts of CP-iRGD;
    solvent: at least one of dichloromethane, trichloromethane, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate and cyclohexane;
    hydration solution: at least one of double distilled water, deionized water, pure water, normal saline and glucose solution;
    the preparation method comprises the following steps: respectively dissolving mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide in a solvent, uniformly mixing, evaporating the solvent, adding a water solution, and hydrating to obtain a required concentration to obtain an iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution;
    wherein, the structural formula of the CP-iRGD polypeptide is shown as the following formula II:
    Figure FDA0004094473890000011
  2. 2. the iDPP/IL-12 complex according to claim 1, wherein: the preparation method of the CP-iRGD polypeptide comprises the following steps:
    A. after the reaction of polyethylene glycol and stearic acid as raw materials, separating and purifying to obtain C18-PEG-OH;
    B. C18-PEG-OH reacts with Fmoc-phenylalanine to obtain C18-PEG-Phe-Fmoc;
    C. removing the Fmoc protecting group of C18-PEG-Phe-Fmoc to obtain C18-PEG-Phe-NH 2 ；
    D. Mixing C18-PEG-Phe-NH 2 Reacting with 3-maleimide propionic acid N-hydroxysuccinimide ester to obtain C18-PEG-BMPS;
    E. C18-PEG-BMPS and iRGD react with sulfydryl through maleimide group to obtain the target compound C18-PEG-iRGD.
  3. 3. The iDPP/IL-12 complex according to claim 1, characterized in that: in the preparation method of the iRGD-DOTAP-mPEG-PLA nanoparticle solution, the mass ratio of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide is as follows: 85-95 parts of mPEG-PLA copolymer, 5-15 parts of DOTAP and 1-5 parts of CP-iRGD.
  4. 4. A process for preparing the iDPP/IL-12 complex according to any one of claims 1 to 3, characterized in that: comprises the following raw materials and auxiliary materials in proportion relationship:
    the raw materials comprise the following components in percentage by mass: 1-99 parts of iRGD-DOTAP-mPEG-PLA nano-particles and 1-10 parts of IL-12 plasmid;
    proper amount of osmotic pressure regulator;
    solvent: at least one of water for injection, double distilled water, deionized water, pure water or physiological saline;
    the preparation method comprises the following steps:
    mixing the raw materials and the solvent in sequence according to an osmotic pressure regulator, the solvent, the iRGD-DOTAP-mPEG-PLA nano-particles and the IL-12 plasmid to obtain the iDPP/IL-12 compound solution, wherein the obtained solution reaches physiological osmotic pressure.
  5. 5. The process for preparing an iDPP/IL-12 complex according to claim 4, wherein: the raw materials comprise, by mass, 90-99 parts of iRGD-DOTAP-mPEG-PLA nanoparticles and 1-10 parts of IL-12 plasmid.
  6. 6. The process for preparing an iDPP/IL-12 complex according to claim 5, wherein: 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticles and 1 part of IL-12 plasmid.
  7. 7. Use of the iDPP/IL-12 complex of any one of claims 1 to 3 for the preparation of a medicament for the treatment of an antineoplastic drug; the tumor is melanoma, ovarian cancer or lung cancer.
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