CN110214145A - CP-iRGD polypeptide, iDPP nanoparticle, load medicine compound and its preparation method and application - Google Patents

CP-iRGD polypeptide, iDPP nanoparticle, load medicine compound and its preparation method and application Download PDF

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CN110214145A
CN110214145A CN201880008394.1A CN201880008394A CN110214145A CN 110214145 A CN110214145 A CN 110214145A CN 201880008394 A CN201880008394 A CN 201880008394A CN 110214145 A CN110214145 A CN 110214145A
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irgd
mpeg
idpp
dotap
nanoparticle
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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 field of medicaments, provide a kind of CP-iRGD polypeptide, iRGD-DOTAP-mPEG-PLA nanoparticle, carry medicine compound and its preparation method and application.The present invention modifies amphiphilic mPEG-PLA diblock copolymer using positively charged amphiphilic species DOTAP and the CP-iRGD polypeptide being modified with cancer target effect, a kind of iRGD-DOTAP-mPEG-PLA nanoparticle of degradability genophore, i.e. iDPP nanoparticle have been prepared using the method for self assembly.Gene plasmid can be imported into tumour cell by the nanoparticle.

Description

CP-iRGD polypeptide, iDPP nanoparticle, load medicine compound and its preparation method and application Technical field
The invention belongs to field of medicaments, and in particular to CP-iRGD polypeptide, iDPP nanoparticle, load medicine compound and its preparation method and application.
Background technique
Gene delivery system has important application in gene functional research and gene therapy.Gene delivery system used at present mainly includes two major classes: viral vectors and non-virus carrier.Viral vectors large-scale production difficulty is big, and the gene content that can be delivered is small, easily causes to be immunoreacted, and has potential bio-safety risk.The features such as non-viral gene vector includes liposome, cation nanometer grain, inorganic nano-particle subcarrier etc., has immunogenicity low, and safety is preferable, is easily mass produced is current international research hotspot.
Methoxy polyethylene glycol-polylactic acid (Methoxy poly (ethylene glycol)-poly (lactide), abbreviation mPEG-PLA) double block polymers are a kind of degradable, good biocompatibility amphiphilic polymers.In the 1990s, the approval for having obtained U.S. FDA containing the drug that PEG or PLA transport vehicle loads is applied to clinic, there is good application prospect in drug, Gene delivery system.Currently, the formulation for paclitaxel of the bis- block polymer nanoparticulate carriers packages of mPEG-PLA has been applied to the clinical treatment of breast cancer in South Korea and Europe, II clinical trial phase is also entered in the U.S..When PEG-PLA nanoparticle is used for Gene delivery system, the simple difficulty for loading gene using mPEG-PLA nanoparticle is big, and transfection efficiency is low.Further mPEG-PLA nanoparticle is physically or chemically modified, it can prepare with the Performances of Novel Nano-Porous grain of rice that gene, transfection efficiency are high, toxicity is low, degradable is easily loaded, have good application prospect in gene functional research, gene therapy research and clinical application.
Summary of the invention
Technical problem solved by the invention is to provide the new modification means of one kind to modify mPEG-PLA diblock copolymer.Inventor modifies iRGD using suitable method, to be prepared yield high, purity is high, dissolubility it is good can target tumor CP-iRGD polypeptide, amphipathic mPEG-PLA diblock copolymer is modified using CP-iRGD polypeptide and DOTAP again, a kind of genophore that novel targeting is degradable is prepared so as to the method using self assembly, that is iRGD-DOTAP-mPEG-PLA cation nanometer grain, referred to as iDPP nanoparticle.IDPP nanoparticle of the present invention has good DNA binding ability, with DNA ining conjunction with after obtained iDPP nano DNA compound in electroneutral, effectively can there is the plasmid of purpose gene to imported into tumour cell load, have the advantages that transfection efficiency height, cytotoxicity are low etc..
Amphipathic mPEG-PLA copolymer employed in the present invention, chemical name are methoxy polyethylene glycol-polylactic acid, abbreviation mPEG-PLA.Methoxy polyethylene glycol-polylactic acid nano particle has amphipathic, good biodegradability and biocompatibility, avoids monocyte phagocytosis, increases drug circulation time in blood and bioavilability, targeted delivery increase drug effect, reduce side effect.
Amphipathic cationic substance DOTAP employed in the present invention, chemical name are (2,3-Dipropyloxypropyl)trimethylammonium chloride, abbreviation DOTAP.
Employed in the present invention to have amphiphilic C18-PEG-iRGD polypeptide, abbreviation CP-iRGD polypeptide is the iRGD by modification.CP-iRGD polypeptide can target tumor tissue, while enhancing the permeability of tumor tissues, tumor tissues can be improved to its intake ability in nanoparticle after the modification of CP-iRGD polypeptide, increase drug in the aggregation of tumor locus, reduce toxic side effect；And modification the resulting CP-iRGD polypeptide of iRGD have it is amphipathic, object can be prepared by way of self assembly.IRGD be existing polypeptide, structural formula such as following formula I:
First technical problem to be solved by this invention is to provide a kind of CP-iRGD polypeptide, structural formula such as following formula II:
Second technical problem to be solved by this invention is to provide the preparation method of above-mentioned CP-iRGD polypeptide.Method includes the following steps:
A, using polyethylene glycol and stearic acid, as raw material, separating-purifying obtains C18-PEG-OH after reaction；
B, it reacts to obtain C18-PEG-Phe-Fmoc with C18-PEG-OH and Fmoc- phenylalanine；
C, C18-PEG-Phe-Fmoc protecting group Fmoc is sloughed, C18-PEG-Phe-NH is obtained 2；
D, by C18-PEG-Phe-NH 2It reacts to obtain C18-PEG-BMPS with 3- maleimidopropionic acid N-hydroxy-succinamide ester；
E, C18-PEG-BMPS is reacted to obtain target compound C18-PEG-iRGD with sulfydryl by maleimide base group with iRGD.
Preferably, in above-mentioned preparation method step C, C18-PEG-Phe-Fmoc protecting group Fmoc is sloughed using 1,8- diazabicyclo [5.4.0], 11 carbon -7- alkene.
Preferably, in above-mentioned preparation method step E, by the PBS buffer solution and dimethyl sulfoxide of pH=7.3,1 ﹕ 1 is mixed the solvent that the reaction uses by volume.
Third problem to be solved by this invention is the method preparation iRGD-DOTAP-mPEG-PLA nanoparticle solution using self assembly, takes raw material, solvent to be prepared according to following proportion relations:
Raw material: the quality proportioning of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide are as follows: 70~99 parts of mPEG-PLA copolymer, 1~30 part of DOTAP, 1~5 part of CP-iRGD；
Solvent: at least one of volatile solvent such as methylene chloride, chloroform, acetone, tetrachloromethane, ethyl alcohol, methanol, ether, pentane, ethyl acetate, hexamethylene；
Rehydration solution: at least one of distilled water, deionized water, pure water, physiological saline, glucose solution；
Preparation method: mPEG-PLA copolymer, DOTAP, CP-iRGD polypeptide are dissolved in solvent respectively and mix, and then evaporate solvent, and concentration needed for adding appropriate aquation solution water to be melted into, acquired solution is iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution.
Further, the method for above-mentioned self assembly prepares iRGD-DOTAP-mPEG-PLA nanoparticle solution, takes raw material to be prepared according to following proportion relations:
Raw material: the quality proportioning of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide are as follows: 85~95 parts of mPEG-PLA copolymer, 5~15 parts of DOTAP, 1~5 part of CP-iRGD.
In above-mentioned technical proposal, solvent usage is can dissolve raw material.
The present invention also provides the iRGD-DOTAP-mPEG-PLA nanoparticle solution that the method by above-mentioned self assembly is prepared.
The present invention also provides iRGD-DOTAP-mPEG-PLA nanoparticles and preparation method thereof.The preparation method is the following steps are included: be drying to obtain iRGD-DOTAP-mPEG-PLA nanoparticle for iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution.
The average grain diameter of gained iRGD-DOTAP-mPEG-PLA nanoparticle of the invention is 139.16 ± 1.56nm, and average potential is 43.1 ± 6.8mV, has good DNA binding ability.Compared with gold marks transfection Materials PEI 25K, iDPP nanoparticle has higher transfection abilities and lower cytotoxicity；Compared with DOTAP-mPEG-PLA (abbreviation DPP) nanoparticle, iDPP nanoparticle equally has higher transfection efficiency and tumour cell targeting.
Gained iRGD-DOTAP-mPEG-PLA nanoparticle of the invention can be used for containing active constituent, especially gene, chemicals or protein, to obtain iRGD-DOTAP-mPEG-PLA nanoparticle compound.
Preferably, in parts by mass, 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1 part of plasmid.
IRGD-DOTAP-mPEG-PLA nanoparticle category biodegradability cation nanometer grain, is a kind of novel gene import system non-virus carrier.The nanoparticle can be by electrostatic interaction combination DNA, and the iDPP nano-complex in conjunction with DNA is in electroneutral, has long circulating action.By the administration mode of intravenous injection, effectively target gene, chemicals, the targeting of protein isoreactivity ingredient can be imported in tumour cell, itself have many characteristics, such as that cytotoxicity is low, transfection efficiency is high.
Such as, VSVMP plasmid can be contained using iRGD-DOTAP-mPEG-PLA nanoparticle and obtain iDPP/VSVMP compound using the plasmid (VSVMP plasmid) of iRGD-DOTAP-mPEG-PLA nanoparticle delivering expression vesicular stomatitis virus stromatin.Abbreviation iDPP/MP compound.
IDPP/VSVMP compound of the present invention, raw material and auxiliary material including following proportion relations:
Raw material: 1~99 part of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of VSVMP plasmid；
Appropriate osmotic pressure regulator, dosage reach physiological osmotic pressure to prepare gained iDPP/VSVMP compound；
Solvent: at least one of water for injection, distilled water, deionized water, pure water or physiological saline；
The preparation method is as follows:
Above-mentioned raw materials and solvent are successively mixed according to osmotic pressure regulator, solvent, iRGD-DOTAP-mPEG-PLA nanoparticle and VSVMP plasmid up to iDPP/VSVMP compound, acquired solution reaches physiological osmotic pressure.
Further, iDPP/VSVMP compound of the present invention takes raw material to be prepared according to following proportion relations: iRGD-DOTAP-mPEG-PLA nanoparticle is 90~99 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of VSVMP plasmid with VSVMP plasmid quality proportioning.
Further, iDPP/VSVMP compound of the present invention takes raw material to be prepared according to following proportion relations: iRGD-DOTAP-mPEG-PLA nanoparticle is 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1 part of VSVMP plasmid with VSVMP plasmid quality proportioning.
Inventor's discovery mediates VSVMP plasmid to can be applied in vitro and in vivo treatment melanoma using iRGD-DOTAP-mPEG-PLA nanoparticle.
In vitro, inventor detects iDPP/VSVMP compound to the inhibiting effect of B16-F10 melanoma cell growth with mtt assay, and induces cell apoptosis situation with Flow cytometry iDPP/VSVMP compound.Inventor's discovery mediates VSVMP plasmid to import in B16-F10 melanoma cells using iDPP nanoparticle, and iDPP/VSVMP compound passes through the apoptosis-induced growth that can obviously inhibit B16-F10 melanoma cells.
In vivo, inventor establishes melanin subcutaneous implantation tumor model and melanoma Lung metastases model, compares each group gross tumor volume and weight, Lung metastases comparison of tumor tubercle number.Inventor has found that iDPP/VSVMP compound can reduce the generation of the load and Lung metastases of mouse tumor significantly.Experimental data shows that iDPP nanoparticle delivering VSVMP plasmid can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.
So the present invention also provides above-mentioned iDPP/VSVMP compound application in preparations of anti-tumor drugs.Preferably, the tumour is melanoma, oophoroma or lung cancer.
At the same time it can also using the plasmid of iRGD-DOTAP-mPEG-PLA nanoparticle delivering expression IL-12, i.e., contain IL-12 plasmid using iRGD-DOTAP-mPEG-PLA nanoparticle and obtain iDPP/IL-12 compound.
IDPP/IL-12 compound of the present invention, raw material and auxiliary material including following proportion relations:
Raw material: 1~99 part of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of IL-12 plasmid；
Appropriate osmotic pressure regulator, dosage reach physiological osmotic pressure to prepare gained iDPP/IL-12 compound；
Solvent: at least one of water for injection, distilled water, deionized water, pure water or physiological saline；
The preparation method is as follows:
Above-mentioned raw materials and solvent are successively mixed according to osmotic pressure regulator, solvent, iRGD-DOTAP-mPEG-PLA nanoparticle and IL-12 plasmid up to iDPP/IL-12 compound, acquired solution reaches physiological osmotic pressure.
Further, iDPP/IL-12 compound of the present invention takes raw material to be prepared according to following proportion relations: iRGD-DOTAP-mPEG-PLA nanoparticle is 90~99 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of IL-12 plasmid with IL-12 plasmid quality proportioning.
Further, iDPP/IL-12 compound of the present invention takes raw material to be prepared according to following proportion relations: iRGD-DOTAP-mPEG-PLA nanoparticle is 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1 part of IL-12 plasmid with IL-12 plasmid quality proportioning.
Inventor's discovery mediates IL-12 plasmid to can be applied in vitro and in vivo treatment melanoma using iRGD-DOTAP-mPEG-PLA nanoparticle.
In vitro, situation is induced cell apoptosis using Flow cytometry iDPP/IL-12 compound.It was found that mediating IL-12 plasmid B16-F10 melanoma cells using iDPP nanoparticle, iDPP/IL-12 compound passes through apoptosis-induced, hence it is evident that inhibits the growth of B16-F10 melanoma cells.
In vivo, melanin subcutaneous implantation tumor model is established, each group gross tumor volume and weight are compared.Inventor has found that iDPP/IL-12 compound can reduce the load of mouse tumor significantly, enhances mouse antineoplastic immune.Experimental data shows that iDPP nanoparticle delivering IL-12 plasmid can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.
The present invention also provides above-mentioned iDPP/IL-12 compound application in preparations of anti-tumor drugs.Preferably, the tumour is melanoma, oophoroma or lung cancer.
The present invention has selected a kind of suitable method to modify iRGD, obtained yield, purity it is higher can target tumor CP-iRGD polypeptide, the CP-iRGD polypeptide dissolubility is good, prepares degradable cationic nanoparticle iRGD-DOTAP-mPEG-PLA nanoparticle so as to the method by self assembly.The nanoparticle can effectively combine DNA, nano-complex in conjunction with DNA is in electroneutral, pass through the administration mode of intravenous injection, effectively therapeutic gene can be imported into tumour cell, have the characteristics that transfection efficiency is high, cytotoxicity is low, has good application prospect in gene functional research, gene therapy research and clinical application.IRGD-DOTAP-mPEG-PLA nanoparticle can mediate gene isoreactivity ingredient to play curative effect, as iRGD-DOTAP-mPEG-PLA nanoparticle mediates load to have the plasmid of VSVMP gene and the plasmid of IL-12 gene that can effectively inhibit the growths of B16-F10 melanoma cells in vitro and in vivo.IDPP nanoparticle is a kind of comparatively safe degradability non-viral gene vector, prepares gained iDPP/VSVMP compound and iDPP/IL-12 compound as treatment melanoma and provides the new thinking of one kind and potentially selection.
Detailed description of the invention
The molecular structural formula of Fig. 1 (A) PEG-PLA；(B) molecular structural formula of DOTAP；(C) synthetic route chart of C18-PEG-iRGD.
The synthesis schematic diagram of Fig. 2 iDPP nanoparticle.
The partial size and potential image of Fig. 3 iDPP nanoparticle: the grain size distribution of (A) iDPP nanoparticle；(B) potential image of iDPP nanoparticle；(C) the scanning transmission electron microscope photo of iDPP nanoparticle；(D) iDPP nanoparticle gel retardation assay.When the mass ratio of iDPP and DNA is 25 ﹕ 1, iDPP nanoparticle can be completely combined DNA plasmid.
The partial size and potential image of Fig. 4 iDPP/VSVMP compound: the grain size distribution of (A) iDPP/VSVMP compound；(B) potential image of iDPP/VSVMP compound；(C) the scanning transmission electron microscope photo of iDPP/VSVMP compound；(D) iDPP/VSVMP Complex Gene gradient potential diagram.
Fig. 5 iDPP nanoparticle detects B16-F10 cytotoxicity and transfection efficiency: (A) in B16-F10 cell, the cytotoxicity of iDPP nanoparticle is lower than PEI 25K；(B) iDPP nanoparticle, DPP nanoparticle, PEK25K transfection B16-F10 cell fluorescence figure (nanoparticle: DNA is respectively 25 ﹕ 1,25 ﹕ 1 of ﹕ 1 and 1)；(C) the streaming figure statistics of cell transfecting.
The outer anti-tumor capacity to B16-F10 cell of Fig. 6 iDPP/VSVMP composite body: (A) iDPP/VSVMP compound MTT detection figure；(B) iDPP/VSVMP compound promotees Apoptosis streaming figure, and iDPP/VSVMP compound is the growth for inhibiting tumour cell by inducing cell apoptosis.
The anti-tumor activity of Fig. 7 iDPP nanoparticle targeting and intratumor injection iDPP/VSVMP compound: (A) is injected intravenously iDPP/pGL-6 compound, luciferase expression situation living imaging figure；(B) the gross tumor volume curve graph of iDPP/VSVMP complex therapies；(C) the tumor weight statistical chart of iDPP/VSVMP complex therapies.
The anti-tumor activity of Fig. 8 intravenous injection iDPP/VSVMP compound: (A) is injected intravenously iDPP/VSVMP compound targeted therapy B16-F10 subcutaneous tumor figure；(B) iDPP/VSVMP complex therapies tumor weight statistical chart；(C) intravenous injection iDPP/VSVMP compound inhibits melanoma Lung metastases figure；(D) it is injected intravenously the weight of the lung of iDPP/VSVMP complex therapies.
The outer anti-tumor capacity to B16-F10 cell of the external iDPP/IL-12 composite body of Fig. 9: (A) iDPP/IL-12 compound Apoptosis streaming figure；(B) apoptosis statistical chart.
The anti-tumor activity of Figure 10 intravenous injection iDPP/IL-12 compound: (A) is injected intravenously iDPP/IL-12 complex therapies B16-F10 subcutaneous tumor figure；(B) tumor tissues IFN-γ secretion is horizontal；(C) tumor tissues NK cellular infiltration flow cytometer showed statistical chart；(D) tumor tissues CD8 +T cell infiltrates flow cytometer showed statistical chart.
Specific embodiment
Present invention firstly provides a kind of CP-iRGD polypeptide, structural formula such as following formula II:
The present invention also provides the preparation methods of above-mentioned CP-iRGD polypeptide, comprising the following steps:
A, using polyethylene glycol and stearic acid, as raw material, separating-purifying obtains C18-PEG-OH after reaction；
B, it reacts to obtain C18-PEG-Phe-Fmoc with C18-PEG-OH and Fmoc- phenylalanine；
C, C18-PEG-Phe-Fmoc protecting group Fmoc is sloughed, C18-PEG-Phe-NH is obtained 2；
D, by C18-PEG-Phe-NH 2It reacts to obtain C18-PEG-BMPS with 3- maleimidopropionic acid N-hydroxy-succinamide ester；
E, C18-PEG-BMPS is reacted to obtain target compound C18-PEG-iRGD with sulfydryl by maleimide base group with iRGD.
Preferably, in above-mentioned preparation method step C, C18-PEG-Phe-Fmoc protecting group Fmoc is sloughed using 1,8- diazabicyclo [5.4.0], 11 carbon -7- alkene.
Preferably, in above-mentioned preparation method step E, by the PBS buffer solution and dimethyl sulfoxide of pH=7.3,1 ﹕ 1 is mixed the solvent that the reaction uses by volume.
Specifically, the preparation method of above-mentioned CP-iRGD polypeptide, comprising the following steps:
Step 1: being that CH is prepared in raw material with polyethylene glycol (PEG) and stearic acid (C17COOH) 3(CH 2) 16COO-PEG-OH (compound 1)；With polyethylene glycol (PEG, Mw=1000,2000, each molecular weight such as 4000,8000) and straight-chain carboxylic acid (CH 3(CH 2) 16COOH CH) is prepared for raw material 3(CH 2) 16CO-PEG-OH (compound 1)；This step products therefrom purity >=90%, yield 70%；
Reaction dissolvent: methylene chloride, chloroform, acetone and other organic solvent all may be used；
Reaction condition: room temperature, stirring, 12h；
Wherein, polyethylene glycol (PEG) molecular weight can be Mw=1000, each different molecular weights such as 2000,4000,8000.
Step 2: C18-PEG-OH (compound 1) and Fmoc- phenylalanine react to obtain C18-PEG-Phe-Fmoc (compound 2)；This step products purity > 90%, yield 90%；
Step 3: 11 carbon -7- alkene (abbreviation DBU) of 8- diazabicyclo [5.4.0] sloughs C18-PEG-Phe-Fmoc (compound 2) with 1, C18-PEG-Phe-NH is obtained 2(compound 3)；
Step 4: by C18-PEG-Phe-NH 2(compound 3) reacts to obtain C18-PEG-Phe-BMPS (compound 4) with 3- maleimidopropionic acid N-hydroxy-succinamide ester (abbreviation BMPS)；This step products purity > 90%, yield 68%；
Step 5: C18-PEG-Phe-BMPS (compound 4) is reacted to obtain target compound C18-PEG-iRGD (compound 5) with sulfydryl by maleimide base group with iRGD.By the PBS buffer solution and dimethyl sulfoxide of pH=7.3,1 ﹕ 1 is mixed the solvent that the reaction uses by volume.
The specific embodiment of form by the following examples is described in further detail above content of the invention again, illustrates but does not limit the present invention.
The synthetic line figure of mPEG-PLA, DOTAP molecular structural formula and CP-iRGD are shown in that Fig. 1, iDPP nanoparticle are prepared by the method for mPEG-PLA, DOTAP and CP-iRGD self assembly, and structural schematic diagram is shown in Fig. 2.
1, experimental method
1.1 preparation synthesis C18-PEG-iRGD compound
1.1.1 synthesis C18-PEG-OH (hereinafter referred to as compound 1) is prepared
Taking polyethylene glycol (1mmol), stearic acid (C 17COOH) (1mmol) is dissolved in methylene chloride, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDCHCl is added, 3mmol) and N- hydroxysuccinimide (NHS, 0.3mmol), it is stirred overnight at room temperature after reaction, successively washed 3 times with 1N HCl solution, aqueous solution and saturated common salt, organic phase is dry with anhydrous sodium sulfate, filter, dissolved solids are removed under reduced pressure after collection filtrate isolates and purifies (Er Lv Jia Wan ﹕ methanol=20 ﹕ 1) with column chromatography chromatogram, obtains compound 1；
1.1.2 synthesis C18-PEG-Phe-Fmoc (abbreviation compound 2) is prepared
Compound 1 (500mg) and Fmoc-L- phenylalanine (142mg) is taken to be dissolved in methylene chloride, EDCHCl (140mg) and DMAP (9mg) is added, ambient temperature overnight, sample collection and the same 1.1.1 of processing, obtain compound 2.
1.1.3 synthesis C18-PEG-Phe-NH2 (abbreviation compound 3) is prepared:
Compound 2 (500mg) is dissolved in methylene chloride, is slowly added to DBU (100uL), stirring at normal temperature reacts 3h.Except column chromatography chromatogram isolates and purifies (Er Lv Jia Wan ﹕ methanol=25 ﹕ 1), sample collection and the same 1.1.1 of processing obtain compound 3.
1.1.4 synthesis C18-PEG-Phe-BMPS (abbreviation compound 4) is prepared:
It takes compound 3 (200mg) and BMPS (49mg) to be dissolved in methylene chloride, 0.2% triethylamine is added, stirring at normal temperature reacts 12h, sample collection and the same 1.1.1 of processing, obtains compound 4.
1.1.5 synthesising target compound C18-PEG-iRGD (abbreviation compound 5) is prepared:
C18-PEG-Phe-BMPS (300mg) is dissolved in acetone; iRGD (150mg) is dissolved in water; iRGD is dissolved in the mixed solvent solution of PBS buffer solution (PH=7.3) and dimethyl sulfoxide; again with C18-PEG-Phe-BMPS solution therewith; 0.2% triethylamine is added dropwise; room temperature, vacuum, nitrogen protection, reaction is overnight.Dialysis (Mw=2000) 2 days, freeze-drying, 4 DEG C are sealed, spare.
1.2iDPP nanoparticle and iDPP/VSVMP compound preparation method
1.2.1iDPP the preparation of nanoparticle
MPEG-PLA copolymer (45mg), DOTAP (5mg) and CP-iRGD (1mg) are dissolved in respectively in 4mL dichloromethane solution, then is transferred in flask and mixes；Mixed solution is placed in 60 DEG C of water-baths, is rotated 30 minutes under vacuum condition.Add appropriate distilled water aquation at required concentration, 60 DEG C of water-baths are gently vibrated, and until being completely dissolved, acquired solution is iDPP nanoparticle aqueous solution, are placed in 4 DEG C of refrigerators and are saved backup.Gained iDPP nanoparticle aqueous solution is drying to obtain iDPP nanoparticle.
1.2.2iDPP/VSVMP compound preparation method
Raw material: 5 μ g of VSVMP plasmid；125 μ g of iRGD-DOTAP-mPEG-PLA nanoparticle；50% glucose；
Solvent: distilled water；
The preparation method is as follows: above-mentioned raw materials and solvent are mixed according to glucose, distilled water, iRGD-DOTAP-mPEG-PLA nanoparticle and VSVMP plasmid sequencing, the control final concentration of glucose of compound is 5%.
Partial size, current potential and the Senile Mouse of 1.3iDPP nanoparticle
1.3.1iDPP the partial size of nanoparticle, current potential
1.2.1 the partial size and current potential size for the iDPP nanoparticle being prepared are detected using Zetasizer Nano ZS Malvern ParticleSizer (Malvern Instruments, Worcestershire, Britain).As a result the average value of 3 measurements is taken.As a result see 2.1.1.
1.3.2iDPP the Senile Mouse of nanoparticle
1.2.1 the morphology for the iDPP nanoparticle being prepared is observed by scanning transmission electron microscope (scanning transmission electron microsope, STEM).As a result see 2.1.1.
The research of 1.4iDPP nanoparticle and DNA binding ability
The DNA binding ability of iDPP nanoparticle is detected with gel retardation assay experiment.First different quality ratio (1 ﹕ 1,5 ﹕ 1,10 ﹕ 1,15 ﹕ 1,20 ﹕, 1,25 ﹕ 1) iDPP nanoparticle and empty plasmid (pVAX) mixing, the quality of empty plasmid (pVAX) be 0.3 μ g, it uses the aqueous solution without DNA enzymatic to adjust mixed overall solution volume as 5ul, is stored at room temperature incubation 30 minutes.Then 1% Ago-Gel, electrophoresis 30 minutes under 100V voltage are prepared.Gel is finally taken out to be observed under the irradiation of ultraviolet lamp and take figure.As a result see 2.2.
Partial size, current potential and the Senile Mouse of 1.5iDPP/VSVMP compound
1.5.1iDPP/VSVMP the partial size of compound, current potential
1.2.2 the same 1.3.1 of measurement method of the iDPP/VSVMP compound partial size, current potential that are prepared, is as a result shown in 2.3.1.
1.5.2iDPP/VSVMP the Senile Mouse of compound
1.2.2 as a result the iDPP/VSVMP compound morphologic detection being prepared, the same 1.3.2 of operating method are shown in 2.3.1.
After 1.6iDPP nanoparticle is in conjunction with VSVMP, potential change situation
Control the iDPP nanoparticle and VSVMP plasmid of following each group different quality ratio (5 ﹕, 1,10 ﹕, 1,15 ﹕, 1,20 ﹕, 1,25 ﹕, 1,30 ﹕, 1,35 ﹕ 1).
(1) VSVMP plasmid is diluted in respectively in 400 μ l deionized waters, is mixed gently.
(2) it takes iDPP nanoparticle to be diluted in 400 μ l deionized waters respectively, mixes gently.
(3) by above-mentioned steps (1) treated VSVMP plasmid is added to step (2) treated iDPP nanoparticle, it is stored at room temperature incubation 30 minutes.Current potential is measured, as a result the same 1.3.1 of potentiometric detection is shown in 2.3.2.
The cytotoxicity of 1.7iDPP nanoparticle detects
IDPP nanoparticle detects the toxic effect of 293T cell (being purchased from ATCC) by cell viability analysis
(1) with 5 × 10 3A cells/well is inoculated in 96 orifice plates, and every hole is loaded 100 μ l 293T cell suspending liquids, is put into 37 DEG C, 5%CO 2It is incubated for 24 hours in cell constant temperature incubator.
(2) the iDPP nanoparticle solution and PEI 25K solution of a series of concentration (0 μ g/ml, 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 400 μ g/ml) are prepared, every hole is separately added into the solution of the 100 above-mentioned various concentrations of μ l in 96 orifice plates, and each concentration sets 6 multiple holes.At 37 DEG C after the completion of dosing, 5%CO 2Continue to be incubated for 48 hours in cell constant temperature incubator.
(3) it after the completion of being incubated for, is detected using mtt assay, as a result sees 2.4.
1.8iDPP nanoparticle transfects B16-F10 cell
(1) cell density is 2 × 10 5B16-F10 cell (being purchased from ATCC) is inoculated in 6 orifice plates by/hole, and every hole adds cell suspending liquid 2ml.
(2) plasmid (pGFP) of 2 μ g expressing green fluorescent proteins is diluted in 1640 culture mediums of 50 μ l serum-free antibiotic-frees, is mixed gently.
(3) it takes the iDPP nanoparticle prepared, 50 μ g of DPP nanoparticle to be diluted in 1640 culture mediums of 50 μ l serum-free antibiotic-frees respectively, mixes gently.It takes 2 μ gPEI 25K solution (1 μ g/ μ l) to be diluted in 1640 culture mediums of 50 μ l serum-free antibiotic-frees, mixes gently.
(4) by step (2) treated pGFP is added separately to iDPP nanoparticle solution that step (3) obtains, DPP nanoparticle solution, PEI 25K solution, it is stored at room temperature incubation 30 minutes.
(5) culture medium of step (1) treated 6 orifice plates is changed into 1640 culture medium 800ul of serum-free antibiotic-free, then iDPP/pGFP compound, DPP/pGFP compound, PEI 25K/pGFP compound that step (4) obtains are separately added into 6 orifice plates, 37 DEG C are put into, 5%CO 2It is incubated for 4~8 hours in cell constant temperature incubator.
(6) it changes the culture medium in 6 orifice plates into 1640 culture medium 2ml that serum has antibiotic again, is put into incubator and continues culture 40 hours.
(7) egfp expression situation in Transfected cells is observed under inverted fluorescence microscope.Cell is collected, row Flow cytometry determines transfection efficiency, as a result sees 2.5.
1.9 detection iDPP/VSVMP compounds are in vitro to the anti-tumor activity of B16-F10 cell
In vitro mainly with two methods detection iDPP/VSVMP compound to the anti-tumor activity of B16-F10 cell: mtt assay and flow cytometry.
1.9.1 inhibiting effect B16-F10 cell grown with mtt assay detection iDPP/VSVMP compound
(1) 96 orifice plate method of B16-F10 cell inoculation is detailed in 1.7 (1)；
(2) it takes 1 μ g VSVMP plasmid and empty plasmid (pVAX) to be diluted in 1640 culture mediums of 25 μ l serum-free antibiotic-frees respectively respectively, mixes gently；
(3) DPP nanoparticle, iDPP nanoparticle take 25 μ g to be diluted in 1640 culture mediums of 25 μ l serum-free antibiotic-frees respectively；
(4) transfection method is detailed in 1.8 (4)-(6), respectively obtains group NS, iDPP, iDPP/pVAX, DPP/MP, an iDPP/MP；Wherein, for NS group, 1640 culture mediums of 25 μ l serum-free antibiotic-frees of step (2) and (3) preparation；For iDPP group, step (2) prepares 1640 culture mediums of 25 μ l serum-free antibiotic-frees；
(5) it is detected using mtt assay, detection method is detailed in 1.7 (3), as a result sees 2.6.
1.9.2 with the apoptosis situation of Flow cytometry iDPP/VSVMP compound induction B16-F10 cell
(1) B16-F10 cell inoculation 6 orifice plates method is detailed in 1.8 (1)；
(2) it takes 2 μ g VSVMP plasmids and empty plasmid (pVAX) to be diluted in 1640 culture mediums of 50 μ l serum-free antibiotic-frees respectively, mixes gently；
(3) DPP nanoparticle, iDPP nanoparticle take 50 μ g to be diluted in 1640 culture mediums of 50 μ l serum-free antibiotic-frees respectively；
(4) transfection method is detailed in 1.8 (4)-(6), respectively obtains group NS, iDPP, iDPP/pVAX, DPP/MP, an iDPP/MP；Wherein, for NS group, 1640 culture mediums of 25 μ l serum-free antibiotic-frees of step (2) and (3) preparation；For iDPP group, step (2) prepares 1640 culture mediums of 25 μ l serum-free antibiotic-frees；
(5) the apoptosis situation that Flow cytometry B16-F10 cell is carried out referring to the bis- transfection reagent box specifications of Biolegend company annexin V-FITC/PI, is as a result shown in 2.6.
The tumor-targeting of 1.10 detection iDPP nanoparticles, iDPP/VSVMP compound
(1) foundation of B16-F10 melanin subcutaneous implantation tumor model: the female C57BL/6 mouse 5 (Sichuan University's Experimental Animal Center) of 6-8 week old, SPF grades of raisings, every mouse inoculation cell concentration are 2 × 10 5100 μ l of cell suspending liquid is subcutaneously injected in a cell.
(2) it is inoculated with after two weeks, the iDPP/pGL-6 compound that every mouse is prepared through tail vein injection following methods.The plasmid pGL-6 of 8 μ g expressing luciferases and 200 μ g iDPP nanoparticles are diluted in 50 μ l, 5% glucose water respectively, obtain iDPP/pGL-6 compound after being incubated for by 1.8 (4).
(3) working solution (15mg/ml) of normal saline D- fluorescein sodium salt, 0.22 μm of membrane filtration degerming are used.
(4) after step (2) are administered 72 hours, the working solution for the D- fluorescein sodium salt prepared in 200 μ l steps (3) of intravenous injection, after twenty minutes, the imaging of mouse living imaging instrument.As a result see 2.7.1.
Anti-tumor capacity in 1.11 detection iDPP/VSVMP composite bodies
1.11.1 the research of intratumor injection iDPP/VSVMP compound and its antitumor mechanism
(1) foundation of B16-F10 melanin subcutaneous implantation tumor model: every mouse inoculation cell concentration is 2 × 10 5100 μ l of cell suspending liquid is subcutaneously injected in a cell；
(2) the 6th day after inoculation, C57 mouse random grouping: is randomly divided into 5 groups, every group 5；
(3) intratumor injection is administered: the 6th day after inoculation, by five groups of grouping administrations of NS, iDPP, iDPP/pVAX, DPP/MP, iDPP/MP, each administered volume of every mouse was 100 μ l；Wherein, NS group is 100 μ l physiological saline, and iDPP group is that solution 100 the μ l, iDPP/pVAX containing 125 μ g iDPP nanoparticles are the 100 μ l of solution containing 5 μ g pVAX empty plasmids, 125 μ giDPP nanoparticles；DPP/MP is the 100 μ l of solution containing 5 μ gVSVMP plasmids, 125 μ gDPP nanoparticles；IDPP/MP is the 100 μ l of solution containing 5 μ gVSVMP plasmids, 125 μ giDPP nanoparticles；
(4) it is administered once within every 2 days, with the length and width of vernier caliper measurement tumour, and records, treat 4 times altogether；
(5) 1 week after the completion for the treatment of, mouse is put to death with dislocation of cervical vertebra method, collects tumour and conscience spleen and lung kidney, weighs knurl weight.As a result see 2.7.2.
1.11.2 it is injected intravenously the research of iDPP/VSVMP compound and its antitumous effect
(1) foundation of B16-F10 melanin subcutaneous implantation tumor model: method refers to 1.11.1 (1)；
(2) the 6th day after inoculation, C57 mouse random grouping: is randomly divided into 5 groups, every group 5；
(3) intravenously administrable: the 6th day after inoculation, being administered by 1.11.1 (3) and be grouped, mouse tail vein injection；
(4) it is administered once within every 2 days, and with the length and width of vernier caliper measurement tumour, and records, treat 7 times altogether；
(5) 1 week after the completion for the treatment of, mouse is handled referring to 1.11.1 (5).As a result see 2.7.3.
1.11.3 it is injected intravenously iDPP/VSVMP compound and its inhibits the research of melanoma Lung metastases
(1) foundation of B16-F10 melanoma Lung metastases model: every mouse inoculation cell concentration is 1 × 10 5A cell, 100 μ l of mouse tail vein injection cell suspending liquid；
(2) the 6th day after inoculation, C57 mouse random grouping: is randomly divided into 5 groups, every group 5；
(3) intravenously administrable: the 6th day after inoculation, method referred to 1.11.2 (3)；
(4) it is administered once within every 2 days, treats 7 times altogether；
(5) 1 week after the completion for the treatment of, mouse is handled referring to 1.11.1 (5), and record neoplasm lung metastasis tubercle number.As a result see 2.7.4.
1.12 detection iDPP/IL-12 compounds are in vitro to the anti-tumor activity of B16-F10 cell
In vitro mainly using fluidic cell apoptosis detection iDPP/IL-12 compound to the anti-tumor activity of B16-F10 cell.Detection method refers to 1.9.2, is grouped into NS, iDPP, iDPP/pVAX, iDPP/pIL12.As a result see 2.8.1.
Anti-tumor capacity in 1.13 detection iDPP/IL-12 composite bodies
1.13.1 it is injected intravenously the research of iDPP/IL-12 compound antitumor mechanism
(1) foundation of B16-F10 melanin subcutaneous implantation tumor model: the same 1.10.1 of method (1)；
(2) the 6th day after inoculation, C57 mouse random grouping: is randomly divided into 5 groups, every group 5；
(3) intravenously administrable: the 6th day after inoculation, by NS, iDPP, iDPP/pVAX, tetra- groups of grouping administrations of iDPP/pIL12, administration mode are as follows: each administered volume of every mouse is 100 μ l；Wherein, NS group is 100 μ l physiological saline, and iDPP group is that solution 100 the μ l, iDPP/pVAX containing 125 μ giDPP nanoparticles are the 100 μ l of solution containing 5 μ g pVAX empty plasmids, 125 μ giDPP nanoparticles；IDPP/pIL12 is the 100 μ l of solution containing 5 μ g pIL12 plasmids, 125 μ giDPP nanoparticles；
(4) it is administered once within every 2 days, with the length and width of vernier caliper measurement tumour, and records, treat 5 times altogether.
(5) 1 week after the completion for the treatment of, mouse is handled referring to 1.11.1 (5).As a result see 2.8.2.
1.13.2 intravenous injection iDPP/IL-12 compound immune detection analysis
(1) tumor tissues for collecting 1.11.1 (5) are horizontal using elisa analysis detection IFN-γ secretion.
(2) flow cytometry is used, to tumor tissues CD8+T cell, NK cellular infiltration situation analysis.As a result see 2.8.2.
1.14 statistical analysis
Experimental data indicates (mean ± SD) with average ± standard error, with SPSS17.0 statistical software carries out data analysis, it is main with average, t is examined and is carried out mean comparative analysis.The statistics of P < 0.05 is variant, p < 0.001 * p < 0.05, * * p < 0.01, * * *.
2 results
The feature of 2.1iDPP nanoparticle
2.1.1iDPP the partial size of nanoparticle, current potential and morphology
The average grain diameter of iDPP nanoparticle is 139 ± 1.5nm, and average potential is+43 ± 3.9mV.The particle diameter distribution and Potential distribution (Fig. 3 A, 3B) of nanoparticle.Under scanning transmission electron microscope, iDPP diameter about 50m (Fig. 3 C).
The binding ability of 2.2iDPP nanoparticle and DNA
We have detected the DNA binding ability of iDPP nanoparticle with gel retardation assay experiment.When the mass ratio of iDPP and DNA is 25 ﹕ 1, iDPP nanoparticle can be completely combined DNA (Fig. 3 D).
The feature of 2.3iDPP/VSVMP compound
2.3.1iDPP/VSVMP the partial size of compound, current potential and morphology
The average grain diameter of iDPP nanoparticle is 142 ± 1.0nm, and average potential is 0.26 ± 0.2mV.The particle diameter distribution and Potential distribution (Fig. 4 A, 3B) of nanoparticle.Under scanning transmission electron microscope, iDPP/VSVMP is the more uniform spherical particles of size, diameter about 54m (Fig. 4 C).
2.3.2iDPP/VSVMP compound gradient potential change
Prepare the iDPP/VSVMP compound of different proportion, potential change.- 19.7 ± 0.62mV (Fig. 4 D) is changed by+43 ± 3.9mV.
The cytotoxicity of 2.4iDPP nanoparticle detects
In 293T cell, PEI 25K cell is more toxic, IC 50<10μg/mL.And iDPP nano-carrier is very low to the toxicity of cell, the μ of IC50 > 200 g/mL (Fig. 5 A).
2.5iDPP nanoparticle transfects B16-F10 cell
As shown in Fig. 5 B-C, pGFP plasmid can be transmitted in cell by iDPP nanoparticle.The transfection efficiency of iDPP nanoparticle is apparently higher than the transfection efficiency (88.37% ± 2.24%VS 42.87% ± 5.68%) of DPP.Show that iDPP nanoparticle is a kind of novel targeted non-viral gene vector, there is degradability, the feature that cytotoxicity is low and transfection efficiency is high.
2.6iDPP/VSVMP compound is in vitro to the anti-tumor capacity of B16-F10 cell
As shown in Figure 6A, iDPP/VSVMP compound can obviously inhibit the growth of B16-F10 tumour cell.Flow cytometry is the result shows that display, the apoptosis-induced cell number of iDPP/VSVMP compound are apparently higher than other four groups (Fig. 9 B).Therefore, VSVMP gene plasmid can be effectively transfected into B16-F10 cell by iDPP nanoparticle, inhibit growth of tumour cell by the mechanism such as apoptosis-induced.
The tumor-targeting and Anticancer effect in vivo of 2.7iDPP compound
2.7.1iDPP the tumor-targeting of compound
The plasmid pGL-6 of the compound expressing luciferase of iDPP can be expressed for 72 hours after mouse tail vein injection in tumor locus known to Fig. 7 A, show that iDPP compound has good tumor-targeting.
2.7.2iDPP/VSVMP compound intratumor injection inhibits the growth of subcutaneous transplantation tumor
In mouse B16-F10 subcutaneous tumors plantation tumor model, treated through intratumor injection, and record gross tumor volume (Fig. 7 B), iDPP/VSVMP group gross tumor volume increasess slowly, and shows that iDPP/VSVMP compound can significantly inhibit tumour growth.We remove mouse subcutaneous tumors, and carry out tumour weighing (Fig. 7 C).
2.7.3iDPP/VSVMP compound tail vein injection inhibits the growth of subcutaneous transplantation tumor
In mouse B16-F10 subcutaneous tumors plantation tumor model, treated through mouse tail vein injection, and record gross tumor volume.Mouse subcutaneous tumors are removed and take pictures (Fig. 8 A) by we, and carry out tumour weighing (Fig. 8 B), iDPP/VSVMP group tumor weight be significantly lower than other four groups, intravenous injection iDPP/VSVMP compound can targeted therapy B16-F10 subcutaneous tumors, inhibit tumour growth.
2.7.4iDPP/VSVMP compound tail vein injection inhibits melanotic tumor Lung metastases
In mouse B16-F10 Pulmonary metastases model, treated through mouse tail vein injection.Mouse lung is removed and is taken pictures (Fig. 8 C) by we, and iDPP/VSVMP group neoplasm lung metastasis tubercle number is considerably less than other four groups, and tail vein injection iDPP/VSVMP compound can significantly inhibit melanoma Lung metastases, and is weighed (Fig. 8 D).
2.8iDPP/IL-12 compound is in vitro to the anti-tumor capacity of B16-F10 cell
2.8.1iDPP/IL-12 compound is in vitro to the anti-tumor capacity of B16-F10 cell
By FCM analysis result (Fig. 9 A-B), the apoptosis-induced cell number of iDPP/IL-12 compound is apparently higher than other four groups.Anti tumor activity in vitro is the results show that IL-12 gene plasmid effectively can be effectively transfected into B16-F10 cell by iDPP nanoparticle, by apoptosis-induced to inhibit the proliferation of tumour cell.
2.8.2iDPP/IL-12 compound tail vein injection inhibits the growth of subcutaneous transplantation tumor
In mouse B16-F10 subcutaneous tumors plantation tumor model, treated through mouse tail vein injection, and record gross tumor volume.Mouse subcutaneous tumors are removed and take pictures (Figure 10 A) by we, and tumor tissues IFN-γ secretion situation (Figure 10 B) is tested and analyzed using elisa, and use flow cytometry tumor tissues NK cell (Figure 10 C) and CD8+T cell (Figure 10 D) Infiltrating.Intravenous injection iDPP/IL-12 compound can target B16-F10 subcutaneous tumors plantation tumor, by inducing cell apoptosis, increase mouse antineoplastic immune, inhibit tumour growth.
3. screening test early period:
3.1 inventor modifies water-soluble iRGD in the experimentation of early period
3.1. the weight ratio of mPEG-PLA and DOTAP are set to: 99 ﹕, 1,90 ﹕, 10,85 ﹕, 15,80 ﹕ 20, the weight ratio of 70 ﹕ 30,60 ﹕ 40, CP-iRGD and mPEG-PLA, DOTAP are set to 1 ﹕, 100,5 ﹕ 100,10 ﹕, 100,10 ﹕, 100,20 ﹕ 100 prepares iRGD-DOTAP-mPEG-PLA nanoparticle.
Cell transfection assays are carried out, find there is preferable transfection efficiency in the proportional region of 1~10 part of 1~30 part of 70~99 parts of raw material mPEG-PLA copolymer, DOTAP, the CP-iRGD polypeptide of iRGD-DOTAP-mPEG-PLA.It will further be scaled to 85~95 parts of mPEG-PLA copolymer, 5~15 parts of DOTAP, CP-iRGD1~5 part proportional region on this basis, then carry out cell transfection assays discovery has higher transfection efficiency within this range.
3.2. in the preparation of gene formulations, the content of gene is set to 1%~50%, carries out gel retardation assay and cell transfection assays discovery, iDPP nanoparticle effectively in conjunction with gene and there can be preferable transfection efficiency in this proportional region.
3.3. on the basis of early period, gene content is further contracted to 1%~10%, carries out gel retardation assay and cell transfection assays discovery again, gene efficiently in conjunction with iDPP nanoparticle and there can be better transfection efficiency in this proportional region.
When 3.4. preparing nanoparticle, the solvent for having investigated dissolution CP-iRGD polypeptide is the volatile solvents such as methylene chloride, chloroform, acetone, tetrachloromethane, ethyl alcohol, methanol, ether, pentane, ethyl acetate, hexamethylene, and discovery can be such that CP-iRGD polypeptide is completely dissolved.
It 3.5. can be distilled water, deionized water for the rehydration solution of aquation mPEG-PLA, DOTAP and CP-iRGD mixtures of polypeptides, pure water, iDPP nanoparticle solution finally can be obtained in physiological saline etc..It is rehydration solution it is preferable to employ distilled water.
3.6. in mPEG-PLA copolymer needed for preparing iDPP nanoparticle, the total molecular weight range of mPEG-PLA is 4000Da~8000Da.
To sum up, the present invention provides a kind of new modification means to modify mPEG-PLA diblock copolymer, inventor modifies amphipathic mPEG-PLA diblock copolymer using positively charged amphiphilic species DOTAP and the CP-iRGD with cancer target effect, a kind of novel targeting degradability genophore, i.e. iRGD-DOTAP-mPEG-PLA cation nanometer grain have been prepared using the method for self assembly.The nanoparticle has good DNA binding ability, and effectively gene plasmid targeting can be imported into tumour cell by the administration mode of intravenous injection at electroneutral in conjunction with the iDPP compound after DNA, have the advantages that transfection efficiency is high, cytotoxicity is low.The iDPP/VSVMP compound prepared by iRGD-DOTAP-mPEG-PLA nanoparticle can inhibit the Lung metastases of tumour growth and tumour significantly.Experimental data shows that iDPP nanoparticle transmitting VSVMP gene can effectively inhibit the growth of B16-F10 melanoma cells in vitro and in vivo.Effectively inhibit the growth of melanoma cells in vitro and in vivo.IDPP nanoparticle is a kind of comparatively safe degradability non-viral gene vector.The iDPP/IL-12 compound prepared by iRGD-DOTAP-mPEG-PLA nanoparticle equally can effectively inhibit the growth of melanoma in vivo and in vitro.Gained iDPP/VSVMP compound and iDPP/IL-12 compound is prepared to provide the new thinking of one kind for targeted therapy melanoma and potentially select.

Claims (17)

  1. CP-iRGD polypeptide, it is characterised in that: structural formula such as following formula II:
  2. The preparation method of CP-iRGD polypeptide described in claim 1, it is characterised in that: the following steps are included:
    A, using polyethylene glycol and stearic acid, as raw material, separating-purifying obtains C18-PEG-OH after reaction；
    B, it reacts to obtain C18-PEG-Phe-Fmoc with C18-PEG-OH and Fmoc- phenylalanine；
    C, C18-PEG-Phe-Fmoc protecting group Fmoc is sloughed, C18-PEG-Phe-NH2 is obtained；
    D, C18-PEG-Phe-NH2 is reacted to obtain C18-PEG-BMPS with 3- maleimidopropionic acid N-hydroxy-succinamide ester；
    E, C18-PEG-BMPS is reacted to obtain target compound C18-PEG-iRGD with sulfydryl by maleimide base group with iRGD.
  3. The preparation method of iRGD-DOTAP-mPEG-PLA nanoparticle solution, it is characterised in that: take raw material, solvent to be prepared according to following proportion relations:
    Raw material: the quality proportioning of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide described in claim 1 are as follows: 70~99 parts of mPEG-PLA copolymer, 1~30 part of DOTAP, 1~5 part of CP-iRGD；
    Solvent: at least one of methylene chloride, chloroform, acetone, tetrachloromethane, ethyl alcohol, methanol, ether, pentane, ethyl acetate, hexamethylene；
    Rehydration solution: at least one of distilled water, deionized water, pure water, physiological saline, glucose solution；
    Preparation method: mPEG-PLA copolymer, DOTAP, CP-iRGD polypeptide are dissolved in solvent respectively and mix, and then evaporate solvent, add rehydration solution aquation at required concentration, and acquired solution is iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution.
  4. The preparation method of iRGD-DOTAP-mPEG-PLA nanoparticle solution according to claim 3, it is characterised in that: the quality proportioning of mPEG-PLA copolymer, DOTAP and CP-iRGD polypeptide are as follows: 85~95 parts of mPEG-PLA copolymer, 5~15 parts of DOTAP, 1~5 part of CP-iRGD.
  5. The iRGD-DOTAP-mPEG-PLA nanoparticle solution that the preparation method as described in claim 3 or 4 is prepared.
  6. IRGD-DOTAP-mPEG-PLA nanoparticle, it is characterised in that: the iRGD-DOTAP-mPEG-PLA nanoparticle aqueous solution as described in claim 5 is dried to obtain.
  7. IRGD-DOTAP-mPEG-PLA nanoparticle compound, it is characterised in that: obtained by iRGD-DOTAP-mPEG-PLA nanoparticle loaded gene, chemicals or protein as claimed in claim 6.
  8. IDPP/VSVMP compound, it is characterised in that: obtained by the plasmid of iRGD-DOTAP-mPEG-PLA nanometers of particle loading as claimed in claim 6 expression vesicular stomatitis virus stromatin.
  9. The preparation method of iDPP/VSVMP compound according to any one of claims 8, it is characterised in that: raw material and auxiliary material including following proportion relations:
    Raw material mass mixture ratio are as follows: 1~99 part of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of VSVMP plasmid；
    Appropriate osmotic pressure regulator；
    Solvent: at least one of water for injection, distilled water, deionized water, pure water or physiological saline；
    The preparation method is as follows:
    Above-mentioned raw materials and solvent are successively mixed according to osmotic pressure regulator, solvent, iRGD-DOTAP-mPEG-PLA nanoparticle and VSVMP plasmid up to iDPP/VSVMP complex solution, acquired solution reaches physiological osmotic pressure.
  10. The preparation method of iDPP/VSVMP compound according to claim 9, it is characterised in that: raw material mass mixture ratio is 90~99 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of VSVMP plasmid；It is preferred that 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1 part of VSVMP plasmid.
  11. IDPP/VSVMP compound application in preparation of anti-tumor drugs according to any one of claims 8.
  12. Application according to claim 11, it is characterised in that: the tumour is melanoma, oophoroma or lung cancer.
  13. IDPP/IL-12 compound, it is characterised in that: obtained by the plasmid of iRGD-DOTAP-mPEG-PLA nanometers of particle loading as claimed in claim 6 expression IL-12.
  14. The preparation method of iDPP/IL-12 compound described in claim 13, it is characterised in that: raw material and auxiliary material including following proportion relations:
    Raw material mass mixture ratio are as follows: 1~99 part of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of IL-12 plasmid；
    Appropriate osmotic pressure regulator；
    Solvent: at least one of water for injection, distilled water, deionized water, pure water or physiological saline；
    The preparation method is as follows:
    Above-mentioned raw materials and solvent are successively mixed according to osmotic pressure regulator, solvent, iRGD-DOTAP-mPEG-PLA nanoparticle and IL-12 plasmid up to iDPP/IL-12 complex solution, acquired solution reaches physiological osmotic pressure.
  15. The preparation method of iDPP/IL-12 compound according to claim 14, it is characterised in that: raw material mass mixture ratio is 90~99 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1~10 part of IL-12 plasmid；It is preferred that 25 parts of iRGD-DOTAP-mPEG-PLA nanoparticle, 1 part of IL-12 plasmid.
  16. Application of the iDPP/IL-12 compound described in claim 13 in preparation treatment anti-tumor drug.
  17. Application according to claim 16, it is characterised in that: the tumour is melanoma, oophoroma or lung cancer.
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