CN105085295A - Amphiphilic derivative of tranexamic acid, and use thereof - Google Patents

Abstract

The invention relates to an amphiphilic derivative of tranexamic acid, and a use thereof, and also relates to a liposome prepared from the above amphiphilic compound. The use is the use of the liposome as a drug carrier delivery system. The liposome prepared from the amphiphilic compound can be compounded with gene drug siRNA to form a compound with small particle size and uniform particle size distribution. The amphiphilic compound is electrically neutral in environment with the pH value of 7.4, and the amphiphilic compound contains a cyclohexane structure, so the in vivo stability of the lipid compound is increased, and the cytotoxicity caused by too many positive charges is reduced. The liposome can specifically inhibit gene expression in human non-small cell lung cancer H1299-Pg13 cells in vitro, and can specifically transship fluorescence gene drugs into normal mouse liver cells in vivo.

Description

Amphipathic derivatives of tranexamic acid and uses thereof

Technical field

The invention belongs to gene therapy technology field, be specifically related to amphipathic derivatives of a kind of tranexamic acid and uses thereof.

Background technology

Gene therapy (genetherapy) refers to and external source normal gene is imported target cell, to correct or compensator gene defect and exception, thus reaches a revolutionary new technology of therapeutic purpose.Gene therapy grows up based on modern molecular biology technique, is the novel method of the disease that a kind of healing is caused by gene unconventionality.Nineteen ninety, gene therapy was successfully applied to clinical trial by American Studies person, and this measure has started the research boom of scientific researchers to gene therapy.Afterwards near during the decade, the treatment of gene therapy to various diseases achieves significance progress.

Common genomic medicine has plasmid DNA (plasmidDNA, pDNA), antisense oligonucleotide (antisenseODN), siRNA (siRNA) and little hairpin RNA (shRNA).They are all with poly-rna structure for skeleton, with gene or genetic expression path for action target spot, by regulating genetic expression in target cell, realize therapeutic action.Dissimilar nucleic acid, the effect risen at the level of molecule and gene is also different.

SiRNA (SmallinterferingRNA), is also called siRNA, is the double-stranded RNA of length 20 to 25 Nucleotide.Combined by the mRNA of complementary with it, impel mRNA to degrade, the gene expression inhibition of mediate transcription level, thus entice cell shows the phenotype of specific gene disappearance.The regulatory mechanism of siRNA carries out silence by complementary pairing to the expression of corresponding Target genes, therefore has the specificity of height.So it can be used as medicine, have vast potential for future development, the treatment for a series of illness such as malignant tumour, HIV is significant.

The key of gene therapy is transported to target cell by genomic medicine body, makes it play a role.Foreign gene medicine usually have molecular weight greatly, easily by the feature of nuclease degradation.If it directly imported in body, by nuclease degradation in vivo, therapeutic action can be lost.Although development in recent years has gone out several genes transfer techniques, as: cytology, virus vector, direct injection etc.But research shows, only have and utilize carrier that genomic medicine could be helped to successfully pass complicated blood environment, enrichment in target cell, plays a role.Therefore, efficient, safe genes delivery system is that gene therapy develops bottleneck problem urgently to be resolved hurrily.

Genophore will experience the process of multiple complexity when transporting gene: arrive target cell by blood circulation, cellular uptake, the escape of endosome, motion in born of the same parents, carrier release genetic stew.Obstacle in the cell of its major obstacle mainly the extracellular obstacle of complicated blood environment and lysosomal enzyme degraded.Therefore find good genophore, making target gene arrive target spot and play effectiveness, is genophore investigator problem demanding prompt solution.

At present, diversity, non-immunogenicity and the non-viral carrier systems being easy to production control receive much concern in recent years, and apply to some extent in a lot for the treatment of field.Conventional non-viral carrier systems mainly lipid (cationiclipids) carrier.

The positive charge of cation lipid is combined with electronegative genomic medicine by electrostatic interaction, thus genetic stew is concentrated the particle be packaged into compared with small particle size.The chance that the particle diameter that mixture is less reduces macrophage identification in body, engulfs, removes, bioavailability in the body that improve medicine.Meanwhile, be directed to tumor tissues, mixture comparatively small particle size more easily utilize infiltration and retention effect from vascular endothelial cell gap through entering tumor epithelial cell, be increased in the drug accumulation of tumor tissues.In transfection, because cell surface is slightly with negative electricity, the liposome of positively charged is more easily adsorbed onto cell surface, enters cell by mechanism such as endocytosis, considerably increases the transfection abilities of liposome.

Current cation lipid is as genophore because its structure is simple, easy and simple to handle, biological safety high becomes the non-virus carrier be most widely used at present, but most of preparation process is complicated, not easily carries out amplification and produces.Therefore the present invention attempts utilizing tranexamic acid, with it for architecture basics, using the carrier system of its amphipathic derivatives as genomic medicine.This analog derivative synthesis step is simple, and starting material are simple and easy to get, and meanwhile, energy is delivery of gene medicine better, successfully solves the problems referred to above, reaches good gene therapy effect.

Summary of the invention

The object of the invention is to the shortcoming overcoming the existence of above-mentioned prior art, provide amphipathic derivatives of a kind of tranexamic acid and uses thereof; This derivative be based on tranexamic acid structure based on a series of amphipathic derivative compound.The nitrogenous head base of amphipathic derivative compound of the present invention has different ionized states under different pH, wherein at lower ph, as pH4-5, and its positively charged.Thus its micella be prepared from and liposome, can with electronegative genomic medicine compound, can particle diameter be formed less, the complex nanometer granule that is evenly distributed.Simultaneously, the nitrogenous head base of compound is not charged or electronegative under pH7.4 condition, make mixture not charged or electronegative under pH7.4 environment like this, reduce the chance with electronegative protein adsorption in body inner blood environment, add the body internal stability of lipid complex, reduce the cytotoxicity because too much positive charge causes.Liposome provided by the invention, can by external for luciferase siRNA high-efficiency delivery in Non-small cell lung carcinoma H1299-Pgl3 cell, specific inhibition of gene expression.Carrier system can enter normal mouse liver cell by body internal specific reprinting fluorogene medicine simultaneously.

The object of the invention is to be achieved through the following technical solutions:

First aspect, the present invention relates to a kind of amphipathic derivatives of tranexamic acid, and its structural formula is as shown in formula I:

wherein, R3, R4 are methyl, and R1 is Long carbon chain alkyl or alkyl, and R2 is Long carbon chain alkyl or alkyl.Also can be expressed as: R1 is carbochain or linolic acid chain, R2 is carbochain or linolic acid chain.This structure is as having following characteristics: R3andR4-amino-CH2-cyclohexyl-ester key-R1andR2.

Preferably, described R1 is the Long carbon chain alkyl or the alkyl that contain 12 ~ 18 carbon atoms, and R2 is the Long carbon chain alkyl or the alkyl that contain 12 ~ 18 carbon atoms.

More preferably, described R1 is the Long carbon chain alkyl for double bond or the 9th, 12 carbon atom positions being double bond on the 9th carbon atom position.

More preferably, described R2 is the Long carbon chain alkyl for double bond or the 9th, 12 carbon atom positions being double bond on the 9th carbon atom position.

The amphipathic derivatives of above-mentioned tranexamic acid comprises following structure:

Preferably, described R1, R2 are identical group.

Preferably, its structural formula is as shown in formula II:

Second aspect, the invention still further relates to a kind of preparation method of amphipathic derivatives of above-mentioned tranexamic acid, described method comprises the steps:

A, under the toluene solution existent condition of catalyst of tetrahydrofuran, red aluminum solutions, at room temperature there is reduction reaction in the straight chain fatty acid of carbon atoms 12-18, obtains intermediate product alcohol 2;

B, under the effect of triethylamine, DMAP, methanesulfonic acid anhydride catalyzer, intermediate product 2 occur at 0 DEG C sulphonateization reaction, obtain intermediate product 3;

C, under dimethyl formamide, lithiumbromide catalyst action, intermediate product 3 is obtained by reacting middle bromide 4 at 45 DEG C;

D, under magnesium, anhydrous diethyl ether, ethyl formate catalyst action, intermediate product 4 generates intermediate products 5 at 40 DEG C of grignard reactions;

E, under sodium hydroxide, tetrahydrofuran (THF) effect, intermediate product 5 65 DEG C issue raw hydrolysis reaction generate intermediate product 6;

F, under catalyzer formic acid, formaldehyde effect, tranexamic acid occur at 80 DEG C dehydrogenation reaction generate intermediate product 10;

G, under DIPEA, DMAP, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt phosphate catalyst effect, intermediate product 10 and intermediate product 6 react at 30 DEG C, obtain described amphipathic derivatives.

The straight chain fatty acid structural formula of above-mentioned carbon atoms 12-18 is: wherein arbitrary integer in m=1-7, and the 6th carbon atom place is singly-bound or double bond, the 9th carbon atom place is singly-bound or double bond.

The third aspect, the invention still further relates to a kind of liposome containing above-mentioned amphipathic derivatives, described liposome comprises amphipathic derivatives, choline, the cholesterol of described tranexamic acid; In described liposome, the weight percent content of choline is 10% ~ 50%, and the weight percent content of cholesterol is 10% ~ 30%.

Preferably, described choline is dipalmitoyl phosphatidylcholine.

Fourth aspect, the invention still further relates to a kind of preparation method of above-mentioned liposome, described method comprises the steps:

A, by the ethanolic soln of the amphipathic derivatives of described tranexamic acid, choline ethanolic soln, the mixing of cholesterol ethanolic soln, form lipid ethanolic soln;

B, described lipid ethanolic soln is joined in the HEPES damping fluid of pH<5.0, stir, obtained liposome turbid liquor;

C, the HEPES damping fluid of described liposome turbid liquor at pH<5.0 to be dialysed, remove ethanol; Obtain described liposome.

In step B, containing 30wt.% ethanol in liposome turbid liquor.In step C, dialysis is the 4h that dialyses at 4 DEG C.

5th aspect, the invention still further relates to a kind of above-mentioned liposome and is preparing the purposes in genomic medicine delivery vehicles, described liposome entrapment DNA, RNA, hyaluronic acid or polypeptide.Described genomic medicine is DNA, RNA, hyaluronic acid or polypeptide.This liposome has the ability helping genomic medicine through obstacle in extracellular obstacle and cell.

6th aspect, the invention still further relates to a kind of lipid/gene composite, and described lipid/gene composite is made up of above-mentioned liposome entrapment gene biological molecule.Its encapsulation rate is more than 30%.This gene biological molecule comprises DNA, RNA, hyaluronic acid or polypeptide.

7th aspect, the invention still further relates to a kind of preparation method of above-mentioned lipid/gene composite, described method comprises the steps:

A, by the ethanolic soln of the amphipathic derivatives of described tranexamic acid, choline ethanolic soln, the mixing of cholesterol ethanolic soln, form lipid ethanolic soln;

B, described lipid ethanolic soln is joined in the HEPES damping fluid of pH<5.0, stir, obtained liposome turbid liquor;

C, in described liposome turbid liquor, add the DEPC aqueous solution of gene biological molecule, hatch 1-2h for 37 DEG C;

D, to dialyse at the HEPES damping fluid of pH<5.0, remove ethanol; Continue in PBS solution dialysis, adjustment system pH, to neutral, obtains described lipid/gene composite.

In step B, containing 30wt.% ethanol in liposome turbid liquor.In step D, in HEPES damping fluid, dialysis is the 4h that dialyses at 4 DEG C; In PBS solution, dialysis is the 12h that dialyses at 4 DEG C; Wherein the pH value of PBS solution is 7.4.In step D, preferred adjustment system pH to 7.4.

Compared with prior art, beneficial effect of the present invention is as follows:

1, the present invention is based on based on tranexamic acid structure, the amphipathic derivative compound of preparation, thus improvement optimize traditional genes delivery system synthesis step complexity, not easily amplifies the problem of production;

2, the present invention is based on based on tranexamic acid structure, the amphipathic derivative compound of preparation, adds the stability of liposome;

3, the liposome that is prepared from of amphipathic derivatives of the present invention, after genomic medicine siRNA compound, can form particle diameter less, the mixture be evenly distributed.Simultaneously not charged or electronegative under pH7.4 environment, add the body internal stability of lipid complex, reduce the cytotoxicity because too much positive charge causes, it can realize safety to siRNA, effectively send as gene drug carriers;

4, the liposome that is prepared from of amphipathic derivatives of the present invention, can enter cell by external effective reprinting genomic medicine, the reticent goal gene of specificity;

5, the liposome that is prepared from of amphipathic derivatives of the present invention, under the acidic conditions of lysosomal pH 4.0, compound ionization positively charged, thus can with the phosphatide negatively charged ion effect in lysosome membrane, formed and adapt to non-double-deck ion pair, then destroy endosome film, realize endosome and escape.Meanwhile, effectively can reprint genomic medicine obstacle in blood circulation and cell in body, enter liver cell.

Accompanying drawing explanation

By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

Fig. 1 is the structure iron of tranexamic acid amphipathic derivatives MC7;

Fig. 2 is the reaction process schematic diagram of tranexamic acid amphipathic derivatives MC7;

Fig. 3 is the nuclear-magnetism figure of tranexamic acid amphipathic derivatives MC7;

Fig. 4 is the mass spectrum of tranexamic acid amphipathic derivatives MC7;

Fig. 5 is the structure iron of tranexamic acid amphipathic derivatives M1;

Fig. 6 is the structure iron of tranexamic acid amphipathic derivatives M2;

Fig. 7 is that MC7 lipid/luciferase-siRNA mixture gel blocking electrophoresis investigates encapsulation rate schematic diagram;

Fig. 8 is that M1 lipid/luciferase-siRNA mixture gel blocking electrophoresis investigates encapsulation rate schematic diagram;

Fig. 9 is that M2 lipid/luciferase-siRNA mixture gel blocking electrophoresis investigates encapsulation rate schematic diagram;

Figure 10 is MC7 lipid/luciferase-siRNA mixture H1299 cell transfecting gene silencing result schematic diagram;

Figure 11 is MC7 lipid/luciferase-siRNA mixture H1299 cell transfecting BCA albumen result schematic diagram;

Figure 12 is M1 lipid/luciferase-siRNA mixture H1299 cell transfecting gene silencing schematic diagram;

Figure 13 is M1 lipid/luciferase-siRNA mixture H1299 cell transfecting BCA albumen result schematic diagram;

Figure 14 is M2 lipid/luciferase-siRNA mixture H1299 cell transfecting gene silencing schematic diagram;

Figure 15 is M2 lipid/luciferase-siRNA mixture H1299 cell transfecting BCA albumen result schematic diagram;

Figure 16 is the liver cell distribution schematic diagram of MC7 lipid/Cy-5-siRNA mixture in vivo after administration;

Figure 17 is the liver cell distribution schematic diagram of M1 lipid/Cy-5-siRNA mixture in vivo after administration;

Figure 18 is the liver cell distribution schematic diagram of M2 lipid/Cy-5-siRNA mixture in vivo after administration.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.The test method of unreceipted actual conditions in the following example, usually conveniently condition, or by the condition that chapter manufacturer advises.

embodiment 1, tranexamic acid amphipathic derivatives MC7

Fig. 1 is amphiphilic cpds MC7 structure iron; Its preparation method as shown in Figure 2, is specially: select tranexamic acid, linolic acid etc. as raw material, obtains MC7 lipid.Fig. 3 is the nuclear magnetic spectrum of amphiphilic cpds MC7, and Fig. 4 is the mass spectrum of amphiphilic cpds MC7, and carry out mass-spectrogram to it and analyze known, gained MC7 compound purity is greater than 95%, and its molecular weight is 696g/mol, conforms to theoretical value.Synthesis step is as follows:

Reaction raw materials:

Linolic acid, tetrahydrofuran (THF) (THF), red aluminum solutions (vitride), toluene (toluene), anhydrous sodium sulphate, ethyl acetate, pure water, methylene dichloride, triethylamine, DMAP (DMAP), EDCHCl (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride), methanesulfonic acid anhydride, PMA (1-Methoxy-2-propyl acetate), hydrochloric acid, sulfuric acid, sodium-chlor, DMF (dimethyl formamide), lithiumbromide (LiBr), normal hexane, anhydrous diethyl ether, methylene bromide, Mg, ethyl formate, acetone, NaOH, silica gel, DMAP, EDCHCl, DCM (methylene dichloride), DIPEA (N, N-diisopropylethylamine), bromo-propionic acid, Trans-4-(aminomethyl) naphthenic acid, formaldehyde, hydrochloric acid, salt of wormwood, DMF, NaI

Step one:

30g linolic acid (reactant 1) and THF (320ml) are added in reactor.The toluene solution of the red aluminum solutions (60%wt/vol) of 73ml is slowly dropped in reactor, in dropping process, maintains the temperature at about 0 DEG C.After dropwising, room temperature reaction 2 hours.Solution is cooled to 0 DEG C, slowly adds saturated metabisulfite solution.After dropwising, instillation 130ml ethyl acetate in 30min, and vigorous stirring.Reacting liquid filtering, solid with ethyl acetate rinses, and merges organic phase, concentrated.Product is dissolved in 80ml ethyl acetate, washes twice with water, and uses anhydrous sodium sulfate drying.Filter, concentrated organic phase, except desolventizing, obtains product 2 (28.8g).

Step 2:

Add 25g product 2 and 210ml methylene dichloride (DCM) in 500ml reactor, then add 53ml triethylamine and 1.15gDMAP (2.0mol), solution is cooled to-10 DEG C.32.7g methanesulfonic acid anhydride is dissolved in 45mlDCM, slowly drops in reactor, and keeps reacting liquid temperature below 0 DEG C.Dropwise, continue maintenance 0 DEG C reaction 1 hour.After completion of the reaction, 80ml frozen water adds in reaction solution, aqueous phase DCM extracting.Merge organic phase, organic phase dilute hydrochloric acid, water and saturated brine washing, anhydrous sodium sulfate drying.Filter, concentrated organic phase removing organic solvent, obtains product 3 (32.3g).

Step 3:

In glass reactor, 110mlDMF and 30g product 3, is cooled to-10 DEG C.11.5gLiBr is dissolved in 110mlDMF, stirs and slowly drops in reactor, and keeping reacting liquid temperature below 0 DEG C.After dropwising, reaction solution is warming up to 45 DEG C, and stirring is spent the night.After completion of the reaction, add 300ml water, and with the extracting of 240ml normal hexane, aqueous phase continues with the extracting of 2*45ml normal hexane.Merge organic phase, with water and saturated brine washing, sodium sulfate (17g) is dry.Filter, concentrated organic phase removing organic solvent, obtains crude product 27.5g.With 60-120 order silica gel purification (normal hexane is moving phase), obtain sterling 4 about 23g.

Step 4:

2.21gMg and 12ml anhydrous diethyl ether is added in there-necked flask.Argon gas is filled with in reactor.20g product 4 is dissolved in 40ml anhydrous diethyl ether.Under argon shield, this solution of 8ml drops in reactor, and continues to add 0.2ml methylene bromide.Reaction solution is warming up to 40 DEG C in a water bath.After reaction starts, remove thermal source, residue 32ml solution is dropped in reactor, allow mixture keep gentle reflux state.After dropwising, heating makes it keep reflux state to react.After completion of the reaction, reaction solution is cooled with an ice bath to less than 10 DEG C, then slowly adds the diethyl ether solution (2.2ml is dissolved in 32ml ether) of ethyl formate.After dropwising, room temperature reaction spends the night.Then add the sulphuric acid soln of 56ml frozen water and 10%, be separated organic phase, aqueous phase ether extraction.Merge organic phase, use salt water washing, dried over sodium sulfate.Filter, concentrated organic phase removing organic solvent, obtains crude product (alcohol and benzoate mixtures) 16g.Crude product 100mlTHF dissolves, and adds NaOH solution (7.5g is dissolved in 150ml water), is heated to 65 DEG C of reactions 18 hours.After reacting completely, reactant is cooled to room temperature, and with ether extraction, merges organic phase, with the water washing of 40ml salt.Dried over sodium sulfate.Filter, concentrated organic phase.Crude product, with 60-120 order silica gel purification (4% ether/normal hexane), obtains sterling DLM6a (11.6g) (productive rate 40%).

Step 5:

There-necked flask adds DLM1.1g and 10mlDCM.Add bromo-propionic acid 477mg successively, EDC.HCL747mg, DMAP38mg, DIPEA0.8ml.Reactor is warming up to 50 DEG C in oil bath, stirring reaction 18-20 hour.After completion of the reaction, 15ml water adds in reaction solution, solution layering, aqueous phase DCM extracting.Merge organic phase, organic phases washed with brine, dried over sodium sulfate.Filter, concentrated organic phase removing organic solvent, obtains thick product 1.5g.With 60-120 order silica gel purification (normal hexane is moving phase), obtain sterling 8 about 800mg (57.8%).

Step 6:

9 (5g), formaldehyde 264mg, formic acid 920mg is added successively in flask.Oil bath is heated to 110 DEG C, and stirring reaction, after 20 hours, adds hydrochloric acid, oil bath 80 DEG C of stirring reactions 4 hours.After completion of the reaction, reaction solution is concentrated, add toluene and again concentrate, until crude product is not at irritant smell.Add ethanol, again concentrated dry, obtain white solid 10 (5g) (70%).Compound 10: ¹hNMR (400MHz, D ₂o) δ 1.05-1.98 (9H), 2.29 (1H), 2.81 (6H), 2.94-2.96 (2H).

Step 7:

There-necked flask adds DLM831mg, adds DCM30ml and makes it dissolve completely, and add 10 (700mg) successively, EDC.HCL451mg, DMAP23mg, DIPEA811mg, reaction solution is warming up to 30 DEG C in a water bath, stirring reaction 36 hours.After completion of the reaction, wash with water, merge organic phase, anhydrous sodium sulfate drying, filtration.Concentrated organic phase removing organic solvent, obtains thick product 1.2g.With 60-120 order silica gel purification (1% ethanol/methylene is moving phase), obtain sterling 11 (380mg) (38%).

Lipid compounds DLin-MC7-DMA:

MS(ES ⁺):C ₄₇H ₈₅NO ₂,calculated696.6580,found696.6655； ¹HNMR(400MHZ,CDCl ₃)δ5.36-5.39(8H)，4.9(1H)，2.77-2.8(4H)，2.21(7H)；2.04-2.09(17H)；1.5(8H)；1.28-1.39(34H)；0.89-0.92(6H)。

Carry out mass-spectrogram, nuclear magnetic spectrum analysis to amphiphilic cpds MPZ, from Fig. 3,4, gained MPZ compound purity is greater than 95%, and its molecular weight is close with theoretical value, is molecular weight 696g/mol.

embodiment 2, tranexamic acid amphipathic derivatives M1

Fig. 5 is the structure iron of tranexamic acid amphipathic derivatives M1; Its preparation is specially: select tranexamic acid, palmitinic acid etc. as raw material, and according to the synthesis of embodiment 1 synthetic method, institute's difference is: in step 1, and reactant 1 is palmitinic acid, and other synthesis steps, composition principle and raw material are identical.Obtain M1 lipid; All through HPLC purifying and Mass Spectrometric Identification, purity is greater than 95%, and molecular weight conforms to theoretical value;

embodiment 2, tranexamic acid amphipathic derivatives M2

Fig. 6 is the structure iron of tranexamic acid amphipathic derivatives M1; Its preparation is specially: select tranexamic acid, oleic acid etc. as raw material, and according to the synthesis of embodiment 1 synthetic method, institute's difference is: in step 1, and reactant 1 is oleic acid, and other synthesis steps, composition principle and raw material are identical.Obtain M1 lipid; All through HPLC purifying and Mass Spectrometric Identification, purity is greater than 95%, and molecular weight conforms to theoretical value;

embodiment 4, M1 liposome

The present embodiment utilizes amphipathic derivative compound M1 and helper lipids, has prepared liposome according to different ratios.

Its preparation method comprises the steps:

1. prepare sample solution

M1 ethanolic soln, dipalmitoyl phosphatidylcholine (DPPC) ethanolic soln, cholesterol (Cholesterol, CHOL) ethanolic soln preparation: take a certain amount of with electronic balance, add dehydrated alcohol to make it to become 10mg/ml, and using as stock solution;

The preparation of 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid (HEPESbuffer): take HEPES with electronic balance, add deionized water, adjust pH with hydrochloric acid soln, make it to become 5mMpH4.0, diethylpyrocarbonate (DEPC) process, after sterilizing, using as stock solution;

2. the preparation of liposome:

1) M1 compound is taken out, cholesterol (CHOL), dipalmitoyl phosphatidylcholine (DPPC) stock solution, equilibrate at room temperature half an hour;

2) press the mol ratio shown in table 1 respectively, measure M1 ethanolic soln, DPPC ethanolic soln, CHOL ethanolic soln mix to 5ml centrifuge tube;

3) 5MmpH4.0HEPESBuffer is got in 5ml centrifuge tube, 30 DEG C of preheatings, 5-10min; Vortex vortex instrument is adjusted to shakeII shelves, under vortex stirs, is slowly added in 5mMpH4.0HEPESbuffer by lipid alcohol mixeding liquid, vortex stirs 1 ~ 2min, and preparation is containing the liposome turbid liquor of 30% ethanol;

4) by step 3) liposome turbid liquor that obtains is placed in dialysis tubing, and the 12h that dialyses at being placed in 5mMpH4.0HEPESbuffer 4 DEG C removes ethanol.

5) laser diffraction particle size analyser (PCS) measures nano particle diameter distribution, eletrokinetic potential (zeta current potential), the instrument used is the ZetaSizer3000H laser particle analyzer of Malvern company of Britain, He-Ne ion laser (λ 0=633nm) is used to be incident light, kinetics light scattering test is carried out at 25 DEG C, reflection angle is 1.33, and angle is 90 °.The mean value that continuous detecting is three times is as the data obtained.

Compliance test result:

As shown in table 1 by the sign of the M1 liposome prepared different lipid composition composition, different lipid composition ratio;

Table 1

embodiment 5, M2 liposome

The present embodiment utilizes amphipathic derivative compound M2 and helper lipids, has prepared liposome according to different ratios.

Its preparation and characterization method is as embodiment 3 method for preparing lipidosome.

Compliance test result:

The sign of M2 liposome prepared by different material, different ratios is as shown in table 2 below:

Table 2

Lipid components	zeta	Particle diameter	PDI
				M2/DPPC/Chol(40/20/40mol)	63.00±0.19	146.3±1.7	0.105±0.002
M2/DPPC/Chol(40/30/40mol)	68.00±0.31	169.2±1.0	0.133±0.011

embodiment 6, MC7 liposome

The present embodiment utilizes amphipathic derivative compound MC7 and helper lipids, has prepared liposome according to different ratios.

Its preparation and characterization method is as embodiment 3 method for preparing lipidosome.

Compliance test result:

The sign of MC7 liposome prepared by different material, different ratios is as shown in table 3 below:

Table 3

Lipid components	zeta	Particle diameter	PDI
				MC7/DPPC/Chol(40/19/27mol)	65.31±0.22	154.8±3.0	0.131±0.035
MC7/DPPC/Chol(40/16/30mol)	62.24±0.21	156.2±1.0	0.105±0.011

embodiment 7, MC7 liposome/gene composite

The liposome that the present embodiment utilizes MC7 lipid to prepare, has prepared lipid/gene composite according to different ratios N/P and siRNA, and has characterized it.

Its method is as follows:

1. blank liposome preparation: preparation method and prescription ratio are as embodiment 3 method for preparing lipidosome;

2. lipid/gene composite preparation:

1) prepare siRNA (siRNA) solution: siRNA is used DEPC water dissolution, makes it to become 1mg/ml, and using as stock solution;

2) by different siRNA/ lipid total amount (N/P) ratio, get appropriate siRNA solution, the liposome turbid liquor of not dialysing is placed on vortex vortex instrument, slowly adds siRNA solution, hatch 1-2h for 37 DEG C;

3) mixture prepared is placed in 5mMpH4.0HEPESbuffer, dialyse at 4 DEG C 4h, the ethanol in removing system.Then take out, continue in 0.01MpH7.4PBS solution, dialyse at 4 DEG C 12h, and system pH is adjusted to 7.4, close to body fluid pH, is lipid/gene composite.Dialysis end is collected in 1.5ml centrifuge tube, and 4 DEG C save backup;

3. charged situation and the particle uniformity of lipid/gene composite under different pH environment and the encapsulating situation of siRNA is characterized:

1) laser diffraction particle size analyser (PCS) to be determined in 0.01MpH7.4PBS size distribution and the particle zeta current potential of the lipid/gene composite of dialysis removing ethanol, investigates the charged situation of mixture under pH7.4 neutrallty condition and size distribution;

2) laser diffraction particle size analyser (PCS) to be determined in 5mMpH4.0HEPESbuffer size distribution and the particle zeta current potential of the lipid/gene composite of dialysis removing ethanol, investigates the charged situation of mixture under pH4.0 condition and size distribution;

3) with gel-electrophoretic apparatus, utilize the dual function of " molecular sieve " and " electrophoresis ", be separated the siRNA of unstable parcel, from slice result analysis, situation encapsulated to siRNA.

Compliance test result:

(1) sign of the MC7 liposome/gene composite of different material ratio, different N/P ratio preparation is as shown in table 4, as can be seen from result, after liposome genomic medicine siRNA prepared by MC7 amphiphilic cpds, define the less and composite nanoparticle be evenly distributed of particle diameter.Meanwhile, mixture is electronegativity under the condition of nearly blood environment pH7.4, the toxicity that the positive polarity substantially reducing cation lipid surplus causes.And wrapped up lipid/gene composite positively charged under the condition of nearly endosome pH4.0 of siRNA, illustrate that it can merge because of charge reaction and electronegative film after entering intracellular endosome, endosome of escaping out, from enzyme liberating inactivation.Simultaneously; as seen from Figure 7 from gel electrophoresis retardance result; the mixture electrophoretic band prepared under 100:1,50:1,20:1 tri-compound ratios does not almost show; and the mixture of 20:1 group; liposome is by after rupture of membranes; observation has obvious band, therefore illustrates that MC7 liposome has carried out encapsulating completely to siRNA, can protect it not by the destruction of external environment.Therefore, as can be seen from above 2, liposome prepared by MC7 amphiphilic cpds has the ability helping genomic medicine through obstacle in extracellular obstacle and cell.

Table 4

embodiment 8, M1 liposome/gene composite

The liposome that the present embodiment utilizes M1 amphiphilic cpds to prepare, has prepared lipid/gene composite according to different ratios N/P and siRNA, and has characterized it.

Its method is as follows:

Experimental technique such as embodiment 7 mixture characterizes.

Compliance test result:

(1) sign of the M1 liposome/gene composite of different material ratio, different N/P ratio preparation is as shown in table 5, as can be seen from result, after liposome genomic medicine siRNA prepared by M1 amphiphilic cpds, define the less and composite nanoparticle be evenly distributed of particle diameter.Meanwhile, mixture is electronegativity under the condition of nearly blood environment pH7.4, the toxicity that the positive polarity substantially reducing cation lipid surplus causes.And wrapped up lipid/gene composite positively charged under the condition of nearly endosome pH4.0 of siRNA, illustrate that it can merge because of charge reaction and electronegative film after entering intracellular endosome, endosome of escaping out, from enzyme liberating inactivation.Therefore, as can be seen from the above, the liposome that prepared by M1 lipid has the ability helping genomic medicine through obstacle in extracellular obstacle and cell.Simultaneously; result is blocked by the gel electrophoresis of Fig. 8; the mixture electrophoretic band prepared under 100:1,50:1,20:1 tri-compound ratios does not almost show; and the mixture of 20:1 group; liposome is by after rupture of membranes; observation has obvious band, therefore illustrates that M1 liposome has carried out encapsulating completely to siRNA, can protect it not by the destruction of external environment.

Table 5

embodiment 9, M2 liposome/gene composite

The liposome that the present embodiment utilizes M2 amphiphilic cpds to prepare, has prepared lipid/gene composite according to different ratios N/P and siRNA, and has characterized it.

Its method is as follows:

Experimental technique such as embodiment 7 mixture characterizes.

Compliance test result:

(1) sign of the M2 liposome/gene composite of different material ratio, different N/P ratio preparation is as shown in table 6, as can be seen from result, after liposome genomic medicine siRNA prepared by M2 amphiphilic cpds, define the less and composite nanoparticle be evenly distributed of particle diameter.Meanwhile, mixture is electronegativity under the condition of nearly blood environment pH7.4, the toxicity that the positive polarity substantially reducing cation lipid surplus causes.And wrapped up lipid/gene composite positively charged under the condition of nearly endosome pH4.0 of siRNA, illustrate that it can merge because of charge reaction and electronegative film after entering intracellular endosome, endosome of escaping out, from enzyme liberating inactivation.Therefore, as can be seen from the above, the liposome that prepared by M2 amphiphilic cpds has the ability helping genomic medicine through obstacle in extracellular obstacle and cell.Simultaneously; result is blocked by the gel electrophoresis of Fig. 9; the mixture electrophoretic band prepared under 100:1,50:1,20:1 tri-compound ratios does not almost show; and the mixture of 20:1 group; liposome is by after rupture of membranes; observation has obvious band, therefore illustrates that M1 liposome has carried out encapsulating completely to siRNA, can protect it not by the destruction of external environment.

Table 6

the ionizable cationic-liposome of embodiment 10, MC7/gene composite people lung cancer H1299-pGL3 cell transfecting effect

The liposome that the present embodiment utilizes MC7 amphiphilic cpds to prepare/luciferase-siRNA mixture carries out cell transfecting to Non-small cell lung carcinoma H1299 cell, by its Gene silencing efficacy, observes the transhipment situation of carrier lipid to genomic medicine.

1. the preparation of lipid/gene composite:

MC7/ gene composite preparation method, as embodiment 7, prepares liposome with MC7/DPPC/Chol-(40/40/30mol) mol ratio, wraps up luciferase luciferase-siRNA, prepare lipid/gene composite according to N/P-20/1;

2.H1299 cell harvesting and cultivation:

Non-small cell lung carcinoma H1299-pGL3 clone obtains [DifferentialenhancementofaCutaneouHPVPromoterby △ NP63 α JunandMutantp53. [CellCycle4:5,689-696 by laboratory passage; May2005], adopt the RPMI1640 containing 10% calf serum to cultivate based on 37 DEG C, the cultivation of 5%CO2 incubator, 2 ~ 4 days replaced medium once, cultivate, and the phase cell of taking the logarithm is tested by 1:3 routine passage;

3. mixture and cell incubation:

Cell presses 10 in first 24 hours at transfection experiment ⁵/ Well is inoculated in 24 orifice plates, observes after 18h, about grows to 70-80%.Wash cell once with the substratum not containing serum before transfection, the appropriate Opti-MEM substratum of sample is diluted, adds 24 orifice plates with every hole 400 μ l/well.To put in incubator transfection after 2.5 ~ 4 hours, change into after continuing to cultivate 36-48 hour containing blood serum medium, luciferase detector (Luminometer) detects (RLU) value, the expression of measurement report gene, BCA determining the protein quantity calculates the every porin content of cell plate, use blank PBS group, naked siRNA group in contrast, investigates the ability of carrier rotaring redyeing gene medicine simultaneously;

1) luciferase vitality detects:

The detecting step of the transfection results of Luciferase reporter gene measures according to the operation instructions of LuciferaseAssaySystem, uses luciferase detector (Luminometer) to detect relative light unit;

I. 5XCCLR (cell pyrolysis liquid) is diluted to 1X, and the luciferase substrate (LuciferaseAssaySubstance) getting packing treats that it recovers room temperature and uses;

II. use PBS rinse 3 times after the cell after transfection being removed substratum, blotted by the PBS in hole after rinse, then the 1X cell pyrolysis liquid of 200 μ l is added in 37 DEG C of 30min in every hole;

III. fully after cracking, mixture is moved in centrifuge tube, with the centrifugal 3min of the rotating speed of 13000rpm, with supernatant liquor as detection sample;

IV. each detection sample is got 10 μ l fully mix (blowing and beating 10 times with liquid-transfering gun) in detector tube with the luciferase substrate of 10 μ l after and is put into the luminous value (RLU) that luciferase detector (Luminometer) measures Luciferase.

2) BCA determining the protein quantity method:

The detecting step of every porocyte protein concentration measures according to the operation instructions of BCAProteinAssayKit, uses microplate reader to detect optical density(OD) (OD) value;

I. drawing standard curve;

II. configuration BCA working reagent (WR), in 96 orifice plates, every hole adds the WR of 200 μ l, then as add in hole 10 μ l samples or above-mentioned centrifugal after supernatant detection sample;

III. after adding sample, make it fully react as placing 30min in 37 DEG C of climatic chambers 96 orifice plates, then measure optical density(OD) (OD) value by microplate reader at 562nm place;

IV. the optical density value drawing standard curve of according to standard sample, then obtains the protein content in regression equation calculation detection sample according to standard curve fit.

Compliance test result:

(1) MC7/luciferase-siRNA mixture size distribution and uniformity result as shown in table 7;

(2) MC7/luciferase-siRNA mixture transfection H1299-pGL3 cell, gene silencing result is as shown in Figure 10;

(3) MC7/luciferase-siRNA mixture transfection H1299-pGL3 cell, BCA albumen result is as shown in figure 11;

As can be seen from result, the liposome siRNA prepared through MC7 amphiphilic cpds enters cell, and the reticent goal gene of specificity, compared with PBS blank and naked siRNA group, substantially increase the efficiency of gene silencing.And the display of cell protein BCA experimental result, with blank PBS compared with siRNA group, MC7 liposome siRNA preparation protein B CA level is close, does not cause cytotoxicity.

Table 7

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				MC7 lipid/luciferase-siRNA	154.2±2.0	0.120±0.035	100μg/ml

embodiment 11, M1 liposome/gene composite people lung cancer H1299-pGL3 cell transfecting effect

The liposome that the present embodiment utilizes M1 lipid to prepare/luciferase-siRNA mixture carries out cell transfecting to Non-small cell lung carcinoma H1299 cell, by its Gene silencing efficacy, observes the transhipment situation of carrier lipid to genomic medicine.

1. the preparation of lipid/gene composite:

M1/ gene composite preparation method, as embodiment 8, prepares liposome with M1/DPPC/Chol-(40/20/40mol) mol ratio, wraps up luciferase luciferase-siRNA, prepare lipid/gene composite according to N/P-20/1;

2.H1299 cell harvesting and cultural method are as embodiment 10;

3. mixture and cell incubation and measuring method are as embodiment 10;

Compliance test result:

(1) M1/luciferase-siRNA mixture size distribution and uniformity result as shown in table 8;

(2) M1/luciferase-siRNA mixture transfection H1299-pGL3 cell, gene silencing result is as shown in figure 12;

(3) M1/luciferase-siRNA mixture transfection H1299-pGL3 cell, BCA albumen result is as shown in figure 13;

As can be seen from result, after the liposome siRNA prepared through M1 lipid enters cell, specificity is reticent goal gene, compared with PBS blank and naked siRNA group, substantially increases the efficiency of gene silencing.

And the display of cell protein BCA experimental result, with blank PBS compared with siRNA group, TMEA liposome siRNA preparation protein B CA level is close, does not cause cytotoxicity.

Table 8

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				M1 lipid/luciferase-siRNA	153.6±6.3	0.141±0.054	100.0μg/ml

embodiment 12, M2/ gene composite people lung cancer H1299-pGL3 cell transfecting effect

The liposome that the present embodiment utilizes M2 amphiphilic cpds to prepare/luciferase-siRNA mixture carries out cell transfecting to Non-small cell lung carcinoma H1299 cell, by its Gene silencing efficacy, observes the transhipment situation of carrier lipid to genomic medicine.

1. the preparation of lipid/gene composite:

M2 lipid/gene composite preparation method, as embodiment 9, prepares liposome with M2/DPPC/Chol-(40/30/40mol) mol ratio, wraps up luciferase luciferase-siRNA, prepare lipid/gene composite according to N/P-20/1;

2.H1299 cell harvesting and cultural method are as embodiment 10;

3. mixture and cell incubation and measuring method are as embodiment 10;

Compliance test result:

(1) M2 lipid/luciferase-siRNA mixture size distribution and uniformity result as shown in table 9;

(2) M2 lipid/luciferase-siRNA mixture transfection H1299-pGL3 cell, gene silencing result is as shown in figure 14;

(3) M2 lipid/luciferase-siRNA mixture transfection H1299-pGL3 cell, BCA albumen result is as shown in figure 15;

As can be seen from result, after the liposome siRNA prepared through M2 lipid enters cell, specificity is reticent goal gene, compared with PBS blank and naked siRNA group, substantially increases the efficiency of gene silencing.

And the display of cell protein BCA experimental result, with blank PBS compared with siRNA group, TMEA liposome siRNA preparation protein B CA level is close, does not cause cytotoxicity.

Table 9

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				M2 lipid/luciferase-siRNA	164.2±2.0	0.120±0.035	100μg/ml

after embodiment 16, tissue slice, confocal microscopy MC7/Cy-5-siRNA mixture distributes in liver

Lipid prepared by the liposome Cy-5-siRNA that the present embodiment utilizes MC7 amphipathic derivatives to prepare/siRNA mixture, after animals administer, after liver tissue slices, observe MC7 lipid/siRNA mixture distribution under Laser Scanning Confocal Microscope, thus find out the transhipment situation of carrier lipid to genomic medicine.

1.MC7 lipid/Cy5-siRNA mixture preparation: MC7 lipid/Cy5-siRNA mixture preparation method and ratio are as embodiment 7;

2. tissue slice

(1) draw materials: get adult healthy ICR mouse 20g, tail vein injection MC7 lipid/Cy5-siRNA mixture 200 μ L, about 10 μ gCy5-siRNA, after 4h, cervical dislocation, dissects, and takes out liver;

(2) quick-frozen: the liver of taking-up is accomplished fritter, is placed in OCT and embeds, and is placed in rapidly liquid nitrogen quick-frozen, is transferred to freezing microtome immediately after becoming block, prepares section;

(3) cut into slices: freezing microtome thermostat container is adjusted to-25 degree, embedded block is trimmed to rectangle or square along slice direction, and the fritter of organizing after finishing is put into specimen disc, slice thickness is adjusted to 20 μm, serial section, directly with pretreated slide glass bonding die, and put into paraformaldehyde and fix 5min;

(4) rinsing: the section after fixing is transferred to and is equipped with in the staining jar of 1xPBS, rinsing 3min, rinsing 3 times;

(5) close: the section lens wiping paper after rinsing is dried the moisture around tissue, draw a circle around tissue with immunohistochemical methods pen, add the Donkeyserum of 5%, 37 degrees Celsius of closed 30min in wet box;

(6) primary antibodie is hatched: absorb confining liquid, add the primary antibodie of having diluted immediately, in wet box, close 12-16h for 37 degree;

(7) two anti-hatch: sop up primary antibodie, add dilute two anti-, in wet box, hatch about 30min for 37 degree;

(8) DAPI dyeing: sop up two anti-diluents, add DAPI, hatch about 30min for 37 degrees Celsius in wet box;

(9) mounting: with Vector company anti-fluorescent quenching mountant mounting, and by nail varnish closing cap slide surrounding.

3. confocal microscope (ConfocalMicroscopy) signals collecting

(1) DAPI scanning sequence: (Excitation:405nm, Emission:419-460nm);

(2) AlexaFluor488 scanning sequence: (Excitation:500-550nm);

(3) Dylight549 scanning sequence: (Excitation:561nm, Emission:559-610nm);

(4) Cy5-siRNA scanning sequence: (Excitation:633nm, Emission:650-750nm).

Compliance test result:

Table 10

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				MC7 lipid/luciferase-siRNA	176.8±2.0	0.123±0.102	100μg/ml

After MC7 lipid/Cy5-siRNA mixture tissue slice, confocal microscopy mixture distributes in liver, as shown in figure 16; Blue signal is the nucleus of DAPI dyeing, and star green is kupffer cell, and danger signal is MC7 lipid/Cy5-siRNA.As seen from the figure, administration is after 4 hours in vivo, and the Cy5-siRNA danger signal of a large amount of MC7 cationic-liposome parcel is distributed in liver cell.If mixture danger signal is engulfed by scavenger cell kupffer cell green, can in yellow.Therefore as can be seen from Figure 16, minority Cy5-siRNA is by macrophage phagocytic, and more Cy5-siRNA signal has gathered in hepatic parenchymal cells.Illustrate that MC7 lipid carrier system can be delivered to liver by safe and efficient in Cy5-siRNA body, more valuable, be delivered in hepatic parenchymal cells by siRNA more, had very large advantage for treatment hepatic diseases.

after embodiment 17, tissue slice, confocal microscopy M1 lipid/Cy-5-siRNA mixture distributes in liver

Lipid/siRNA mixture that the present embodiment utilizes M1 cation lipid parcel Cy-5-siRNA to prepare, after animals administer, after liver tissue slices, observe TMEA lipid/siRNA mixture distribution under Laser Scanning Confocal Microscope, thus find out the transhipment situation of carrier lipid to genomic medicine.

1.M1 lipid/Cy5-siRNA mixture preparation: M1 lipid/Cy5-siRNA mixture preparation method and ratio are as embodiment 8;

2. tissue section method is as embodiment 16;

3. Laser Scanning Confocal Microscope Confocal (LeicaTSSP8) observation experiment method is as embodiment 16.

Compliance test result:

After M1 lipid/Cy5-siRNA mixture tissue slice, confocal microscopy mixture distributes in liver, as shown in figure 17; Blue signal is the nucleus of DAPI dyeing, and star green is kupffer cell, and danger signal is M1 lipid/Cy5-siRNA.As seen from the figure, administration is after 4 hours in vivo, and the Cy5-siRNA danger signal of a large amount of M1 cationic-liposome parcel is distributed in liver cell.If mixture danger signal is engulfed by scavenger cell kupffer cell green, can in yellow.Therefore as can be seen from Figure 17, minority Cy5-siRNA is by macrophage phagocytic, and more Cy5-siRNA signal has gathered in hepatic parenchymal cells.Illustrate that M1 lipid carrier system can be delivered to liver by safe and efficient in Cy5-siRNA body, more valuable, be delivered in hepatic parenchymal cells by siRNA more, had very large advantage for treatment hepatic diseases.

Table 11

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				M1 lipid/luciferase-siRNA	163.6±6.3	0.141±0.054	100μg/ml

after embodiment 18, tissue slice, confocal microscopy M2 lipid/Cy-5-siRNA mixture distributes in liver

Lipid prepared by the liposome Cy-5-siRNA that the present embodiment utilizes M2 amphipathic derivatives compound to prepare/siRNA mixture, after animals administer, after liver tissue slices, observe M2 lipid/siRNA mixture distribution under Laser Scanning Confocal Microscope, thus find out the transhipment situation of carrier lipid to genomic medicine.

1.M2 lipid/Cy5-siRNA mixture preparation: M2 lipid/Cy5-siRNA mixture preparation method and ratio are as embodiment 8;

2. tissue section method is as embodiment 16;

3. Laser Scanning Confocal Microscope Confocal (LeicaTSSP8) observation experiment method is as embodiment 16.

Compliance test result:

After M2 lipid/Cy5-siRNA mixture tissue slice, confocal microscopy mixture distributes in liver, as shown in figure 18; Blue signal is the nucleus of DAPI dyeing, and star green is kupffer cell, and danger signal is M2 lipid/Cy5-siRNA.As seen from the figure, administration is after 4 hours in vivo, and the Cy5-siRNA danger signal of a large amount of M2 cationic-liposome parcel is distributed in liver cell.If mixture danger signal is engulfed by scavenger cell kupffer cell green, can in yellow.Therefore as can be seen from Figure 18, minority Cy5-siRNA is by macrophage phagocytic, and more Cy5-siRNA signal has gathered in hepatic parenchymal cells.Illustrate that M2 lipid carrier system can be delivered to liver by safe and efficient in Cy5-siRNA body, more valuable, be delivered in hepatic parenchymal cells by siRNA more, had very large advantage for treatment hepatic diseases.

Table 12

Prescription	Mixture particle diameter	Mixture PDI	SiRNA concentration
				M2 lipid/luciferase-siRNA	169.6±6.3	0.141±0.054	100μg/ml

Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims (13)

1. an amphipathic derivatives for tranexamic acid, is characterized in that, its structural formula is as shown in formula I:

wherein, R3, R4 are methyl, and R1 is Long carbon chain alkyl or alkyl, and R2 is Long carbon chain alkyl or alkyl.

2. amphipathic derivatives as claimed in claim 1, is characterized in that, described R1 is the Long carbon chain alkyl or the alkyl that contain 12 ~ 18 carbon atoms, and R2 is the Long carbon chain alkyl or the alkyl that contain 12 ~ 18 carbon atoms.

3. amphipathic derivatives as claimed in claim 2, is characterized in that, described R1 is the Long carbon chain alkyl for double bond or the 9th, 12 carbon atom positions being double bond on the 9th carbon atom position.

4. amphipathic derivatives as claimed in claim 2 or claim 3, is characterized in that, described R2 is the 9th carbon atom position is the Long carbon chain alkyl for double bond in double bond or the 9th, 12 carbon atom positions.

5. amphipathic derivatives as claimed in claim 1, it is characterized in that, described R1, R2 are identical group.

6. amphipathic derivatives as claimed in claim 5, it is characterized in that, its structural formula is as shown in formula II:

7. a preparation method for the amphipathic derivatives according to any one of claim 1 ~ 6, is characterized in that, described method comprises the steps:

A, under the toluene solution existent condition of catalyst of tetrahydrofuran, red aluminum solutions, at room temperature there is reduction reaction in the straight chain fatty acid of carbon atoms 12-18, obtains intermediate product alcohol 2;

B, under the effect of triethylamine, DMAP, methanesulfonic acid anhydride catalyzer, intermediate product alcohol 2 occur at 0 DEG C sulphonateization reaction, obtain intermediate product 3;

C, under dimethyl formamide, lithiumbromide catalyst action, intermediate product 3 is obtained by reacting middle bromide 4 at 45 DEG C;

D, under magnesium, anhydrous diethyl ether, ethyl formate catalyst action, intermediate product 4 generates intermediate products 5 at 40 DEG C of grignard reactions;

E, under sodium hydroxide, tetrahydrofuran (THF) effect, intermediate product 5 65 DEG C issue raw hydrolysis reaction generate intermediate product 6;

F, under catalyzer formic acid, formaldehyde effect, tranexamic acid occur at 80 DEG C dehydrogenation reaction generate intermediate product 10;

G, under DIPEA, DMAP, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt phosphate catalyst effect, intermediate product 10 and intermediate product 6 react at 30 DEG C, obtain described amphipathic derivatives.

8. the liposome containing the amphipathic derivatives according to any one of claim 1 ~ 6, it is characterized in that, described liposome comprises described amphipathic derivatives, choline, cholesterol; In described liposome, the weight percent content of choline is 10% ~ 50%, and the weight percent content of cholesterol is 10% ~ 30%.

9. liposome as claimed in claim 8, it is characterized in that, described choline is dipalmitoyl phosphatidylcholine.

10. a preparation method for liposome as claimed in claim 8, is characterized in that, described method comprises the steps:

A, by the ethanolic soln of the amphipathic derivatives of described tranexamic acid, choline ethanolic soln, the mixing of cholesterol ethanolic soln, form lipid ethanolic soln;

B, described lipid ethanolic soln is joined in the HEPES damping fluid of pH<5.0, stir, obtained liposome turbid liquor;

C, the HEPES damping fluid of described liposome turbid liquor at pH<5.0 to be dialysed, remove ethanol; Obtain described liposome.

11. 1 kinds of liposomes as claimed in claim 8, preparing the purposes in genomic medicine delivery vehicles, is characterized in that, described liposome entrapment DNA, RNA, hyaluronic acid or polypeptide.

12. 1 kinds of lipid/gene composites, is characterized in that, described lipid/gene composite is made up of liposome entrapment gene biological molecule as claimed in claim 8.

The preparation method of 13. 1 kinds of lipid/gene composites as claimed in claim 12, is characterized in that, described method comprises the steps:

A, by the ethanolic soln of the amphipathic derivatives of described tranexamic acid, choline ethanolic soln, the mixing of cholesterol ethanolic soln, form lipid ethanolic soln;

B, described lipid ethanolic soln is joined in the HEPES damping fluid of pH<5.0, stir, obtained liposome turbid liquor;

C, in described liposome turbid liquor, add the DEPC aqueous solution of gene biological molecule, hatch 1-2h for 37 DEG C;

D, to dialyse at the HEPES damping fluid of pH<5.0, remove ethanol; Continue in PBS solution dialysis, adjustment system pH, to neutral, obtains described lipid/gene composite.