CN105085292B - Amphipathic derivatives of 3 ((2 (dimethylamino) ethyl group) (methyl) amino) propionic acid and application thereof - Google Patents

Abstract

The present invention relates to a kind of amphipathic derivatives of 3 ((2 (dimethylamino) ethyl group) (methyl) amino) propionic acid and application thereof；The invention further relates to the liposome by described amphiphilic compound preparation；The purposes is purposes of the liposome as pharmaceutical carrier induction system.The liposome that the amphiphilic compound of the present invention is prepared, at acidic can be with more positive charge, and energy preferably complex gene medicine siRNA, formation particle diameter is smaller, the compound of even particle size distribution.It is in simultaneously electroneutral under pH7.4 environment, adds stability inside lipid complex, reduce the cytotoxicity caused by excessive positive charge.Liposome provided by the invention, can In-vitro specificity suppress the intracellular gene expression of Non-small cell lung carcinoma H1299 Pgl3, and can the fluorogene medicine of specificity reprinting in vivo enter normal mouse liver cell.

Description

The amphipathic derivatives of 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid And application thereof

Technical field

The invention belongs to gene therapy technology field, and in particular to a kind of 3- ((2- (dimethylamino) ethyl group) (methyl) Amino) propionic acid amphipathic derivatives and application thereof.

Background technology

Gene therapy (gene therapy) refers to external source normal gene importing target cell, to correct or compensate Yin Jiyin Defect and abnormal caused disease, to reach therapeutic purposes.Between past more than 20 years, gene therapy is in many disease treatments Field, which will be studied, from preclinical has pushed clinic to, and for as caused by gene unconventionality, insoluble disease is such as so far for the world of medicine Tumour etc. has the advantage that can not be substituted.Common genomic medicine has DNA (plasmid DNA, pDNA), antisense oligonucleotides Sour (antisense ODN), siRNA (siRNA) and small hairpin RNA (shRNA)., can be special with RNAi disturbing effects Property silence target gene siRNA be current gene therapy research emphasis.

SiRNA (Small interfering RNA), also known as siRNA, it is the double of 20 to 25 nucleotides of length Chain RNA.It is combined by the complementary mRNA of sequence therewith, promotes mRNA to degrade, the horizontal gene expression inhibition of mediate transcription, from And lure cells show into and go out the phenotype of specific gene missing.SiRNA regulatory mechanism is come to corresponding target position by complementary pairing The expression of gene carries out silence, therefore the specificity with height.So as medicine, with before wide development Scape, the treatment for a series of illnesss such as malignant tumour, HIV are significant.

The key of gene therapy is will to be transported to target cell in genomic medicine body, it is played a role.However, by external source base Internal because introducing, it can be degraded by internal nuclease, before target cell is introduced into, just be degraded to micromolecule nucleotide, So as to lose therapeutic action.Therefore, the key for realizing gene therapy is efficient, safe genes delivery system.

Genophore will undergo multiple complicated processes when gene is transported：Target cell is reached by blood circulation, Cellular uptake, the escape of endosome, intracellular motion, carrier release genetic stew.Its major obstacle is mainly complicated blood environment Extracellular obstacle and lysosomal enzyme degraded intracellular obstacle.Therefore good genophore is found so that target gene reaches Target spot is played effectiveness, and is genophore researcher's urgent problem to be solved.

At present, two major classes are broadly divided into gene delivery carrier system aspects：First, virus carrier system；It is second, non-viral Carrier system.Viral vector is a kind of natural bearer resource, and Organization of viral genome is simple, and transfection efficiency is high, and target cell is special It is different in nature strong, but its guidance quality is poor, carrying capacity is low, the limitation of immunogenicity and potential oncogenicity, makes it be difficult to reach clinic The requirement of application.Therefore the non-viral carrier systems of diversity, non-immunogenicity and easily controllable production receive much concern in recent years, And applied in many therapy fields.Conventional non-viral carrier systems are mainly lipid (cationic lipids) carrier.

The positive charge of cation lipid is combined by electrostatic interaction with electronegative genomic medicine, so as to which genetic stew is dense Contracting is packaged into the particle compared with small particle.Genomic medicine siRNA can be concentrated and is assembled into particle diameter by the cationic-liposome of positively charged Less nano-particle.Cationic-liposome shows other types fat in terms of gene delivery carrier is prepared with cell transfecting The incomparable advantage of plastid.The less particle diameter of compound is reduced by the identification of internal macrophage, phagocytosis, the chance removed, Improve bioavilability inside medicine.Meanwhile be directed to tumor tissues, compound compared with small particle be easier using infiltration and Retention effect, through tumor epithelial cell is entered, increases the drug accumulation in tumor tissues from vascular endothelial cell gap.In transfection side Face, because cell surface slightly carries negative electricity, the liposome of positively charged is easier to be adsorbed onto cell surface, passes through the mechanism such as endocytosis Into cell, the transfection abilities of liposome are considerably increased.

At present cation lipid as genophore because the features such as its is simple in construction, easy to operate, biological safety is high into For the non-virus carrier being most widely used at present, but most of preparation process is complicated, is not easy to be amplified production.Therefore originally Invention is attempted to utilize 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid, using it as architecture basics, its is amphipathic Carrier system of the derivative as genomic medicine.This analog derivative synthesis step is simple, and raw material are simple and easy to get, meanwhile, can be compared with Good delivery of gene medicine, successfully solves above mentioned problem, has reached preferable gene therapy effect.

The content of the invention

A kind of the shortcomings that it is an object of the invention to overcome above-mentioned prior art to exist, there is provided 3- ((2- (dimethylamino) second Alkyl) (methyl) amino) propionic acid amphipathic derivatives and application thereof.The derivative is to be based on 3- ((2- (dimethylamino) ethane Base) (methyl) amino) a series of amphipathic derivative compounds based on propionic acid structure.The amphipathic derivative compound of the present invention Nitrogenous head base there is different ionized states under different pH, wherein at lower ph, such as pH4-5, its positively charged.So as to it The micella and liposome being prepared, can be compound with electronegative genomic medicine, can form that particle diameter is smaller, and what is be evenly distributed answers Compound nano-particle.Meanwhile the nitrogenous head base of compound is not charged or negatively charged under the conditions of pH7.4, so that compound It is not charged or negatively charged under pH7.4 environment, the chance with negatively charged protein adsorption in internal blood environment is reduced, is added Stability inside lipid complex, reduce the cytotoxicity caused by excessive positive charge.Liposome provided by the invention, it can incite somebody to action The external high-efficiency deliveries of luciferase siRNA are intracellular to Non-small cell lung carcinoma H1299-Pgl3, specific inhibition of gene expression. Carrier system can enter normal mouse liver cell by specific fluorogene medicine of reprinting in vivo simultaneously.

The purpose of the present invention is achieved through the following technical solutions：

In a first aspect, the present invention relates to a kind of the amphipathic of 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid Derivative, shown in its structural formula such as formula (I)：

(I), wherein, n=1,2,3 or 4, R1 are Long carbon chain alkyl or alkyl, R2 is Long carbon chain alkyl or alkyl.It can also be expressed as：R1 is carbochain or linoleic acid chain, and R2 is carbochain or linoleic acid chain.Its Structure such as has the characteristics that：Dimethylaminoethane base-(methyl) amino-(CH2) n- ester bond-R1and R2.

Preferably, the R1 is Long carbon chain alkyl or alkyl containing 12~18 carbon atoms, and R2 is containing 12~18 carbon The Long carbon chain alkyl or alkyl of atom.

It is highly preferred that the R1 is to be to be on the 9th carbon atom position on double bond or the nine, the 12 carbon atom positions The Long carbon chain alkyl of double bond.

It is highly preferred that the R2 is to be to be on the 9th carbon atom position on double bond or the nine, the 12 carbon atom positions The Long carbon chain alkyl of double bond.

Above-mentioned amphipathic derivatives include each material containing following structure：

Preferably, described R1, R2 are identical group.

Preferably, shown in its structural formula such as formula (II)：

Second aspect, the present invention relates to a kind of preparation method of above-mentioned amphipathic derivatives, methods described includes as follows Step：

A, catalyst of tetrahydrofuran, red aluminum solution toluene solution existing under the conditions of, carbon atoms 16-18 straight chain Reduction reaction occurs at room temperature for aliphatic acid, obtains intermediate product alcohol 2；

B, in the presence of triethylamine, DMAP, Loprazolam acid anhydride catalyst, intermediate product alcohol is at 20 DEG C Generation sulfonic acid esterification obtains intermediate product 3；

C, under dimethylformamide, lithium bromide catalyst action, intermediate product 3 reacts at 45 DEG C obtains middle bromination Thing 4；

D, under magnesium, absolute ether, Ethyl formate catalyst action, grignard reaction generation occurs at 40 DEG C for intermediate product 4 Intermediate product 5；

E, under sodium hydroxide, tetrahydrofuran effect, intermediate product 5 issues raw hydrolysis generation intermediate product at 65 DEG C 6；

F, it is sub- in DIPEA, DMAP, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two Under amine hydrochlorate catalyst action, esterification occurs at 50 DEG C and produces intermediate product 8 for intermediate product 6；

G, dimethylformamide, sodium iodide, potassium carbonate catalyst action under, intermediate product 8 at 80 DEG C with N, N, Ammoxidation occurs for N '-ethylenediamine, produces the amphipathic derivatives.

Above-mentioned carbon atoms 12-18 straight chain fatty acid structural formula is：Wherein m= Any integer in 1-7, and be singly-bound or double bond at the 6th carbon atom, it is singly-bound or double bond at the 9th carbon atom.

The third aspect, the present invention relates to a kind of liposome containing above-mentioned amphipathic derivatives, the liposome includes institute State amphipathic derivatives, choline, cholesterol；The weight percent content of choline is 20%~50% in the liposome, and courage is solid The weight percent content of alcohol is 10%~30%.

Preferably, the choline is DPPC.

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

A, the ethanol solution of the amphipathic derivatives, choline ethanol solution, cholesterol ethanol solution are mixed, forms fat Matter ethanol solution；

B, the lipid ethanol solution is added to pH<In 5.0 HEPES buffer solution, stirring, liposome is made and is suspended Liquid；

C, by the liposome turbid liquor in pH<5.0 HEPES buffer solution dialysis, removes ethanol；Produce the lipid Body.

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

5th aspect, the present invention relates to a kind of purposes of above-mentioned liposome in genomic medicine delivery vehicles are prepared, institute State liposome entrapment DNA, RNA, hyaluronic acid or polypeptide.The genomic medicine is DNA, RNA, hyaluronic acid or polypeptide.The fat Plastid has the ability for helping genomic medicine through extracellular obstacle and intracellular obstacle.

6th aspect, the present invention relates to a kind of lipid/gene composite, the lipid/gene composite is by above-mentioned Liposome entrapment gene biological molecule and form.Its envelop rate is more than 30%.The gene biological molecule include DNA, RNA, Hyaluronic acid or polypeptide.

7th aspect, the present invention relates to a kind of preparation method of above-mentioned lipid/gene composite, methods described is included such as Lower step：

A, the ethanol solution of the amphipathic derivatives, choline ethanol solution, cholesterol ethanol solution are mixed, forms fat Matter ethanol solution；

B, the lipid ethanol solution is added to pH<In 5.0 HEPES buffer solution, stirring, liposome is made and is suspended Liquid；

C, the DEPC aqueous solution of gene biological molecule, 37 DEG C of incubation 1-2h are added in the liposome turbid liquor；

D, in pH<5.0 HEPES buffer solution dialysis, removes ethanol；Continue to dialyse in PBS solution, system pH is into for adjustment Property, produce the lipid/gene composite.

In step B, ethanol containing 30wt.% in liposome turbid liquor.In step D, dialysis is 4 in HEPES buffer solution Dialyse 4h at DEG C；Dialysis is the 12h that dialysed at 4 DEG C in PBS solution；Wherein the pH value of PBS solution is 7.4.It is excellent in step D It is pH to 7.4 to recruit overall.

Compared with prior art, beneficial effects of the present invention are as follows：

(1) present invention is based on based on 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid structure, preparation Amphipathic derivative compound, traditional genes delivery system synthesis step complexity is optimized so as to improve, is not easy asking for amplification production Topic；

(2) liposome that amphipathic derivatives of the invention are prepared, can increase at acidic with more positive charge To genomic medicine siRNA delivered payload capability, after compound the compound that particle diameter is smaller, is evenly distributed can be formed.Simultaneously in pH7.4 It is not charged or negatively charged under environment, stability inside lipid complex is added, reduces the cell caused by excessive positive charge Toxicity, it can be used as gene drug carriers to realize siRNA and safely, effectively deliver；

(3) liposome that amphipathic derivatives of the invention are prepared, genomic medicine can be effectively reprinted in vitro and is entered carefully Born of the same parents, specific silence target gene；

(4) liposome that amphipathic derivatives of the invention are prepared, under the acid condition of lysosomal pH 4.0, change Compound ionizes positively charged, so as to be acted on the phosphatide anion in lysosome membrane, forms the ion for adapting to non-double-decker It is right, endosome film is then destroyed, realizes that endosome is escaped.Meanwhile can effectively reprint in vivo genomic medicine by blood circulation and Intracellular obstacle, into liver cell.

Brief description of the drawings

The detailed description made by reading with reference to the following drawings to non-limiting example, further feature of the invention, Objects and advantages will become more apparent upon：

Fig. 1 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives TMEA structure chart；

Fig. 2 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives TMEA reaction process Schematic diagram；

Fig. 3 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives TMEA mass spectrogram；

Fig. 4 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives TMEA nuclear-magnetism figure；

Fig. 5 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T1 structure chart；

Fig. 6 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T2 structure chart；

Fig. 7 is that TMEA lipids/luciferase-siRNA compound gel blockings electrophoresis investigates envelop rate schematic diagram；

Fig. 8 is that T1 lipids/luciferase-siRNA compound gel blockings electrophoresis investigates envelop rate schematic diagram；

Fig. 9 is that T2 lipids/luciferase-siRNA compound gel blockings electrophoresis investigates envelop rate schematic diagram；

Figure 10 is TMEA lipids/luciferase-siRNA compound H1299 cell transfecting gene silencing result schematic diagrams；

Figure 11 is TMEA lipids/luciferase-siRNA compound H1299 cell transfecting BCA albumen result schematic diagrams；

Figure 12 is T1 lipids/luciferase-siRNA compound H1299 cell transfecting gene silencing schematic diagrames；

Figure 13 is T1 lipids/luciferase-siRNA compound H1299 cell transfecting BCA albumen result schematic diagrams；

Figure 14 is T2 lipids/luciferase-siRNA compound H1299 cell transfecting gene silencing schematic diagrames；

Figure 15 is T2 lipids/luciferase-siRNA compound H1299 cell transfecting BCA albumen result schematic diagrams；

Figure 16 is the liver cell distribution schematic diagram after TMEA lipids/Cy-5-siRNA compounds are administered in vivo；

Figure 17 is the liver cell distribution schematic diagram after T1 lipids/Cy-5-siRNA compounds are administered in vivo；

Figure 18 is the liver cell distribution schematic diagram after T2 lipids/Cy-5-siRNA compounds are administered in vivo.

Embodiment

With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection domain.The test method of unreceipted actual conditions in the following example, generally according to normal condition, or manufacture by chapter Condition proposed by manufacturer.

Embodiment 1,3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives TMEA

Fig. 1 is amphiphilic compound TMEA structure charts；It is prepared as shown in Fig. 2 being specially：From N, N, N '-ethylenediamine, Linoleic acid etc. is used as raw material, 2-in-1 into obtaining TMEA lipids according to reacting flow chart.Fig. 3 is carried out for amphiphilic compound TMAEA Nuclear magnetic resonance map result, Fig. 4 are its mass-spectrogram result.

Reaction raw materials：

Linoleic acid, tetrahydrofuran (THF), red aluminum solution (vitride), toluene (toluene), anhydrous sodium sulfate, acetic acid Ethyl ester, pure water, dichloromethane, triethylamine, DMAP (DMAP), EDCHCl (1- (3- dimethylaminopropyls)- 3- ethyl-carbodiimide hydrochlorides), Loprazolam acid anhydride, PMA (propylene glycol methyl ether acetate), hydrochloric acid, sulfuric acid, sodium chloride, DMF (dimethylformamide), lithium bromide (LiBr), n-hexane, absolute ether, methylene bromide, Mg, Ethyl formate, acetone, NaOH, silicon Glue, DMAP, EDCHCl, DCM (dichloromethane), DIPEA (DIPEA), bromo-propionic acid, Trans-4- (ammonia first Base) naphthenic acid, formaldehyde, hydrochloric acid, (N, N, N ')-trimethyl ethylenediamine, potassium carbonate, DMF, NaI

Step 1：

30g linoleic acid (reactant 1) and THF (320ml) are added in reactor.By 73ml red aluminum solution (60%wt/ Vol toluene solution) is slowly added dropwise into reactor, and 0 DEG C or so is maintained the temperature at during dropwise addition.After being added dropwise, room temperature Reaction 2 hours.Solution is cooled to 0 DEG C, is slowly added to saturation metabisulfite solution.After being added dropwise, 130ml second is instilled in 30min Acetoacetic ester, and be stirred vigorously.Reacting liquid filtering, solid ethyl acetate rinse, merge organic phase, concentration.Product is dissolved in In 80ml ethyl acetate, it is washed with water twice, and uses anhydrous sodium sulfate drying.Filtering, concentration organic phase remove solvent, obtain product 2(28.8g)。

Step 2：

25g products 2 and 210ml dichloromethane (DCM) are added in 500ml reactors, then add 53ml triethylamines and 1.15g DMAP (2.0mol), solution is cooled to -10 DEG C.32.7g Loprazolam acid anhydrides are dissolved in 45ml DCM, are slowly added dropwise to anti- Answer in device, and keep reacting liquid temperature below 0 DEG C.It is added dropwise, continues to be kept for 0 DEG C react 1 hour.After completion of the reaction, 80ml frozen water is added in reaction solution, and aqueous phase is extracted with DCM.Merge organic phase, organic phase watery hydrochloric acid, water and saturated salt washing Wash, anhydrous sodium sulfate drying.Filtering, concentration organic phase remove organic solvent, obtain product 3 (32.3g).

Step 3：

110ml DMF and 30g product 3, is cooled to -10 DEG C in glass reactor.11.5g LiBr are dissolved in 110ml DMF, Stir and be slowly added dropwise into reactor, and keep reacting liquid temperature below 0 DEG C.After being added dropwise, reaction solution is warming up to 45 DEG C, it is stirred overnight.After completion of the reaction, 300ml water is added, and is extracted with 240ml n-hexanes, aqueous phase continues to use 2*45ml n-hexanes Extracting.Merge organic phase, washed with water and saturated brine, sodium sulphate (17g) is dried.Filtering, concentration organic phase remove organic molten Agent, obtain crude product 27.5g.With 60-120 mesh silica gel purification (n-hexane is mobile phase), the about 23g of sterling 4 is obtained.

Step 4：

2.21gMg and 12ml absolute ethers are added in three-necked flask.Argon gas is filled with reactor.20g products 4 are dissolved in In 40ml absolute ethers.Under argon gas protection, the 8ml solution is added dropwise in reactor, and continuously adds 0.2ml methylene bromides.Instead Liquid is answered to be warming up to 40 DEG C in a water bath.After reaction starts, thermal source is removed, remaining 32ml solution is added dropwise in reactor, allowed mixed Compound keeps gentle reflux state.After being added dropwise, heating makes it maintain the reflux for state response.After completion of the reaction, reaction solution is used Ice bath is cooled to less than 10 DEG C, is then slowly added into the diethyl ether solution of Ethyl formate (2.2ml is dissolved in 32ml ether).Drip Bi Hou, room temperature reaction is overnight.Then 56ml frozen water and 10% sulfuric acid solution are added, separates organic phase, aqueous phase ether extraction. Merge organic phase, with salt water washing, sodium sulphate is dried.Filtering, concentration organic phase remove organic solvent, obtain crude product (alcohol and first Acid ester mixtures) 16g.Crude product is dissolved with 100mlTHF, is added NaOH solution (7.5g is dissolved in 150ml water), is heated to 65 DEG C Reaction 18 hours.After reaction completely, reactant is cooled to room temperature, and with ether extraction, merges organic phase, with the washing of 40ml salt Wash.Sodium sulphate is dried.Filtering, concentrate organic phase.Crude product obtains sterling with 60-120 mesh silica gel purification (4% ether/n-hexane) DLM6a (11.6g) (yield 40%).

Step 5：

Three-necked flask adds DLM1.1g and 10ml DCM.Bromo-propionic acid 477mg, EDC.HCL747mg are sequentially added, DMAP38mg, DIPEA0.8ml.Reactor is warming up to 50 DEG C in oil bath, stirring reaction 18-20 hours.After completion of the reaction, 15ml water is added in reaction solution, and solution layering, aqueous phase is extracted with DCM.Merge organic phase, organic phase salt water washing, sodium sulphate Dry.Filtering, concentration organic phase remove organic solvent, obtain crude product 1.5g.With 60-120 mesh silica gel purification, (n-hexane is stream Dynamic phase), obtain the about 800mg of sterling 8 (57.8%).

Step 6：

Three-necked flask adds 8 (500) mg, and adding DMF30ml is completely dissolved it, sequentially adds N, N, N '-ethylenediamine (420mg), NaI112mg, K₂CO₃311mg, reactor are warming up to 80 DEG C in oil bath, stirring reaction 36 hours.Reaction finishes Afterwards, water 20ml is added, dichloromethane extraction, merges organic phase, anhydrous sodium sulfate drying, filtering.It is organic to concentrate organic phase removing Solvent, obtain crude product 800mg.With 60-120 mesh silica gel purification (1% ethanol/methylene is mobile phase), sterling 13 is obtained (160mg) (31%).

Mass-spectrogram, nuclear magnetic spectrum analysis are carried out to amphiphilic compound MPZ, from Fig. 3,4, gained MPZ compounds Purity is more than 95%, and its molecular weight is close with theoretical value, is molecular weight 685g/mol.

Lipid compounds DLin-MC2-TMEA12：

MS(ES⁺):C₄₅H₈₄N₂O₂,calculated685.6533,found685.6627；

¹HNMR(400MHz,CDCl₃) δ 5.36-5.39 (8H), 4.9 (1H), 2.72-2.79 (4H), 2.48-2.51 (9H)； 2.29(4H)；2.04-2.07(8H)；1.51-1.52(8H)；1.28-1.37(36H)；0.89-0.92(6H).

Embodiment 2,3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T1

Fig. 5 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T1 structure chart；Its Preparation is specially：From N, N, N '-ethylenediamine, palmitic acid etc. be used as raw material, synthesized according to the synthetic method of embodiment 1, institute is different Part is：In step 1, reactant 1 is palmitic acid, and other synthesis steps, composition principle and raw material are identical.Obtain T1 fat Matter；It is more than 95% by HPLC purifying and Mass Spectrometric Identification, purity, molecular weight is consistent with theoretical value.

Embodiment 3,3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T2

Fig. 6 is 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid amphipathic derivatives T2 structure charts；It is made It is standby to be specially：From N, N, N '-ethylenediamine, oleic acid etc. is used as raw material, synthesized according to the synthetic method of embodiment 1, institute's difference It is：In step 1, reactant 1 is oleic acid, and other synthesis steps, composition principle and raw material are identical.Obtain T2 lipids. By HPLC purifying and Mass Spectrometric Identification, purity is more than 95%, and molecular weight is consistent with theoretical value.

Embodiment 4, T1 liposomes

The present embodiment utilizes amphipathic derivative compound T1 and helper lipids, and liposome is prepared for according to different proportion.

Its preparation method comprises the following steps：

1. prepare sample solution

T1 ethanol solutions, DPPC (DPPC) ethanol solution, cholesterol (Cholesterol, CHOL) Ethanol solution is prepared：Weighed with electronic balance a certain amount of, add absolute ethyl alcohol and make 10mg/ml, and using as storeing Liquid；

The preparation of 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) buffer solution (HEPES buffer)：Weighed with electronic balance HEPES, deionized water is added, pH is adjusted with hydrochloric acid solution, made 5mM pH4.0, pyrocarbonic acid diethyl ester (DEPC) processing, go out After bacterium, stock solution is used as using it；

2. the preparation of liposome：

1) T1 compounds are taken out, cholesterol (CHOL), DPPC (DPPC) storing solution, are balanced at room temperature Half an hour；

2) certain mol proportion is pressed respectively, is measured T1 ethanol solutions, DPPC ethanol solutions, CHOL ethanol solutions to 5ml and is centrifuged Pipe mixes；

3) 5Mm pH4.0HEPES Buffer are taken in 5ml centrifuge tubes, 30 DEG C of preheatings, 5-10min；Vortex is vortexed Instrument is adjusted to shake II shelves, and under the stirring that is vortexed, lipid alcohol mixeding liquid is slowly added into 5mM pH4.0HEPES buffer In, be vortexed 1~2min of stirring, prepares the liposome turbid liquor containing 30% ethanol；

4) liposome turbid liquor for obtaining step 3) is initially positioned in 5mM pH4.0HEPES buffer and dialysed at 4 DEG C 12h goes out ethanol.

5) distribution of laser diffraction particle size analyzer (PCS) measure nano particle diameter, eletrokinetic potential (zeta current potentials), are used Instrument be Malvern company of Britain ZetaSizer3000H laser particle analyzers, use He-Ne ion lasers (λ 0=633nm) For incident light, dynamics light scattering test is 90 ° in 25 DEG C of progress, angle of reflection 1.33, angle.Continuous detection being averaged three times Value is as obtained data.

Compliance test result：

The sign of T1 liposomes by being prepared to different lipid compositions composition, different lipid composition ratios is as shown in table 1；

Table 1

Lipid components	Zeta	Particle diameter	PDI
				T1/DPPC/Chol(40/40/30-mol/mol)	79.23±0.21	153.9±1.2	0.125±0.006
T1/DPPC/Chol(40/20/40-mol/mol)	78.27±0.32	162.8±1.0	0.103±0.017

Embodiment 5, T2 liposomes

The present embodiment utilizes amphipathic derivative compound T2 and helper lipids, and liposome is prepared for according to different proportion.

It is prepared and the characterizing method such as method for preparing lipidosome of embodiment 3.

Compliance test result：

T2 liposomes prepared by different material, different proportion are characterized as below shown in table 2：

Table 2

Lipid components	zeta	Particle diameter	PDI
				T2/DPPC/Chol(40/20/40mol)	79.00±0.19	166.6±1.7	0.135±0.002
T2/DPPC/Chol(40/30/40mol)	78.00±0.31	161.7±1.0	0.143±0.011

Embodiment 6, TMEA liposomes

The present embodiment utilizes amphipathic derivative compound TMEA and helper lipids, and liposome is prepared for according to different proportion.

It is prepared and the characterizing method such as method for preparing lipidosome of embodiment 3.

Compliance test result：

TMEA liposomes prepared by different material, different proportion are characterized as below shown in table 3：

Table 3

Lipid components	zeta	Particle diameter	PDI
				TMEA/DPPC/Chol(40/10/40mol)	80.00±0.79	112.6±1.7	0.255±0.002
TMEA/DPPC/Chol(40/20/40mol)	81.00±0.31	126.7±1.0	0.103±0.011

Embodiment 7, TMEA liposomes/gene composite

The present embodiment utilizes liposome prepared by TMEA lipids, and lipid/base is prepared for according to different proportion N/P and siRNA Characterized because of compound, and to it.

Its method is as follows：

1. prepared by blank liposome：Preparation method and the prescription ratio such as method for preparing lipidosome of embodiment 3；

2. prepared by lipid/gene composite：

1) siRNA (siRNA) solution is prepared：SiRNA DEPC water is dissolved, makes 1mg/ml, and with it As stock solution；

2) by different siRNA/ lipids total amounts (N/P) ratio, appropriate siRNA solution is taken, the liposome turbid liquor that will do not dialysed It is placed in vortex vortex instruments, is slowly added to siRNA solution, 37 DEG C of incubation 1-2h；

3) compound prepared is placed in 5mM pH4.0HEPES buffer, dialyse 4h at 4 DEG C, in removing system Ethanol.Then take out, continue in 0.01M pH7.4PBS solution, dialysed 12h at 4 DEG C, and system pH is adjusted into 7.4, is approached Body fluid pH, as lipidic gene compound.Dialysis end is collected in 1.5ml centrifuge tubes, and 4 DEG C save backup；

3. powered situation and particle uniformity of the lipid/gene composite under different pH environment and the bag to siRNA Envelope situation characterizes：

1) dialysis in 0.01M pH7.4PBS of laser diffraction particle size analyzer (PCS) measure removes lipid/base of ethanol Because of the particle diameter distribution and particle zeta current potentials of compound, powered situation and particle diameter of the compound under pH7.4 neutrallty conditions are investigated Distribution；

2) laser diffraction particle size analyzer (PCS) measure dialysis in 5mM pH4.0HEPES buffer removes ethanol The particle diameter distribution and particle zeta current potentials of lipid/gene composite, investigate powered situation and grain of the compound under the conditions of pH4.0 Footpath is distributed；

3) gel-electrophoretic apparatus is used, using the double action for utilizing " molecular sieve " and " electrophoresis ", the unstable parcel of separation SiRNA, situation is encapsulated to siRNA from slice result analysis.

Compliance test result：

(1) different material ratio, different N/P are more as shown in table 4 than the sign of TMEA liposomes/gene composite of preparation, It can be seen from the results that after liposome genomic medicine siRNA prepared by TMEA amphiphilic compounds, it is smaller to form particle diameter And the composite nanoparticle being evenly distributed.Meanwhile compound is elecrtonegativity under conditions of nearly blood environment pH7.4, is subtracted significantly Toxicity caused by the superfluous electropositive of small cation lipid.And siRNA lipid/gene composite has been wrapped up in nearly endosome Positively charged under conditions of pH4.0, illustrate its enter after intracellular endosome can because charge reaction merges with electronegative film, Endosome is escaped out, degrades and inactivates from enzyme.Meanwhile as seen from Figure 7 from the point of view of result is blocked in gel electrophoresis, 100：1、 50:1、20:1 three compound compound electrophoretic bands than lower preparation almost do not show, and 20:1 group of compound, liposome After rupture of membranes, observation has obvious band, therefore illustrates that TMEA liposomes have carried out complete encapsulating to siRNA, can protect it from The destruction of external environment.Therefore, 2 points of liposomes that can be seen that the preparation of TMEA amphiphilic compounds have help base more than Because medicine passes through the ability of extracellular obstacle and intracellular obstacle.

Table 4

Embodiment 8, T1 liposomes/gene composite

The present embodiment utilizes liposome prepared by T1 amphiphilic compounds, and fat is prepared for according to different proportion N/P and siRNA Matter/gene composite, and it is characterized.

Its method is as follows：

The experimental method such as compound of embodiment 7 characterizes.

Compliance test result：

(1) different material ratio, different N/P are more as shown in table 5 than the sign of T1 liposomes/gene composite of preparation, by As a result as can be seen that T1 amphiphilic compounds prepare liposome genomic medicine siRNA after, form particle diameter it is smaller and point The uniform composite nanoparticle of cloth.Meanwhile compound is elecrtonegativity under conditions of nearly blood environment pH7.4, is substantially reduced Toxicity caused by the superfluous electropositive of cation lipid.And siRNA lipid/gene composite has been wrapped up in nearly endosome Positively charged under conditions of pH4.0, illustrate its enter after intracellular endosome can because charge reaction merges with electronegative film, Endosome is escaped out, degrades and inactivates from enzyme.From the point of view of Fig. 8 gel electrophoresis retardance result, 100：1、50:1、20:1 three The compound compound electrophoretic band than lower preparation almost do not show, and 20:1 group of compound, liposome are observed by after rupture of membranes There is obvious band, therefore illustrate that MPZ liposomes have carried out complete encapsulating to siRNA, the destruction of external environment can be protected it from. Therefore, from the above, it can be seen that liposome prepared by T1 lipids, which has, helps genomic medicine through extracellular obstacle and intracellular The ability of obstacle.

Table 5

Embodiment 9, T2 liposomes/gene composite

The present embodiment utilizes liposome prepared by T2 amphiphilic compounds, and fat is prepared for according to different proportion N/P and siRNA Matter/gene composite, and it is characterized.

Its method is as follows：

The experimental method such as compound of embodiment 7 characterizes.

Compliance test result：

(1) different material ratio, different N/P are more as shown in table 6 than the sign of T2 liposomes/gene composite of preparation, by As a result as can be seen that T2 amphiphilic compounds prepare liposome genomic medicine siRNA after, form particle diameter it is smaller and point The uniform composite nanoparticle of cloth.Meanwhile compound is elecrtonegativity under conditions of nearly blood environment pH7.4, is substantially reduced Toxicity caused by the superfluous electropositive of cation lipid.And siRNA lipid/gene composite has been wrapped up in nearly endosome Positively charged under conditions of pH4.0, illustrate its enter after intracellular endosome can because charge reaction merges with electronegative film, Endosome is escaped out, degrades and inactivates from enzyme.From the point of view of Fig. 9 gel electrophoresis retardance result, 100：1、50:1、20:1 three The compound compound electrophoretic band than lower preparation almost do not show, and 20:1 group of compound, liposome are observed by after rupture of membranes There is obvious band, therefore illustrate that MPZ liposomes have carried out complete encapsulating to siRNA, the destruction of external environment can be protected it from. Therefore, from the above, it can be seen that liposome prepared by T1 lipids, which has, helps genomic medicine through extracellular obstacle and intracellular The ability of obstacle.Therefore, genomic medicine is helped to pass through from the above, it can be seen that liposome prepared by T2 amphiphilic compounds has The ability of extracellular obstacle and intracellular obstacle.

Table 6

The ionizable cationic-liposome of embodiment 10, TMEA/gene composite human lung cancer H1299-pGL3 cell transfectings effect Fruit

Liposome/luciferase-siRNA compounds that the present embodiment is prepared using TMEA amphiphilic compounds are non-to people ED-SCLC H1299 cells carry out cell transfecting, and by its Gene silencing efficacy, genomic medicine is turned to observe carrier lipid Fortune situation.

1. the preparation of lipid/gene composite：

TMEA/ gene composites preparation method such as embodiment 7, with TMEA/DPPC/Chol- (40/40/30mol) mol ratio Liposome is prepared, luciferase luciferase-siRNA is wrapped up according to N/P-20/1, prepares lipid/gene composite；

2.H1299 cell is collected and culture：

Non-small cell lung carcinoma H1299-pGL3 cell lines are obtained by laboratory passage, using containing 10% calf serum RPMI1640 cultures are based on 37 DEG C, 5%CO2 incubator cultures, change culture medium once within 2~4 days, and 1:3 routine passage cultures, take Logarithmic phase cell is tested；

3. compound and cell incubation：

Cell presses 10 in 24 hours before transfection experiment⁵/ Well is inoculated in 24 orifice plates, is observed after 18h, about grows to 70-80%. Cell is washed with the culture medium without serum once, sample is diluted with appropriate Opti-MEM culture mediums, with every μ of hole 400 before transfection L/well adds 24 orifice plates.Put after being transfected 2.5~4 hours in incubator, change serum-containing media into and continue to cultivate 36-48 hours Afterwards, luciferase detector (Luminometer) detection (RLU) value, the expression of measurement report gene, BCA determining the protein quantity Cell plates are calculated per PFP content, while carrier rotaring redyeing gene medicine is investigated as control with blank PBS groups, naked siRNA groups Ability；

1) luciferase vitality detects：

The detecting step of the transfection results of Luciferase reporter genes according to Luciferase Assay System behaviour Measure is explained, relative light unit is detected using luciferase detector (Luminometer)；

5X CCLR (cell pyrolysis liquid) are diluted to 1X by I, take luciferase substrate (the Luciferase Assay of packing Substance) treat that it recovers room temperature and used；

II uses PBS rinses 3 times after the cell after transfection is removed into culture medium, blots the PBS in hole after rinse, then 200 μ l 1X cell pyrolysis liquids are added per hole in 37 DEG C of 30min；

III moves into mixture in centrifuge tube after fully cracking, and centrifuges 3min with 13000rpm rotating speed, is made with supernatant To detect sample；

IV, which each detects sample and takes 10 μ l and 10 μ l luciferase substrate to be sufficiently mixed in detection pipe, (uses liquid-transfering gun Piping and druming 10 times) after be put into the luminous value (RLU) of measure Luciferase in luciferase detector (Luminometer).

2) BCA determining the protein quantity method：

Detecting step per hole cellular protein concentration determines according to BCA Protein Assay Kit operating instruction, makes Optical density (OD) value value is detected with ELIASA；

I draws standard curve；

II configuration BCA working reagents (WR), 200 μ l WR is added per hole in 96 orifice plates, then as adding 10 μ l in hole Supernatant detection sample after sample or above-mentioned centrifugation；

After III adds sample, it is set fully to react as placement 30min in 37 DEG C of climatic chambers 96 orifice plates, then use ELIASA measures optical density (OD) value at 562nm；

The OD value of IV according to standard samples draws standard curve, then obtains recurrence side according to standard curve fit Journey calculates the protein content in detection sample.

Compliance test result：

(1) TMEA/luciferase-siRNA compounds particle diameter distribution and uniformity result are as shown in table 7；

(2) TMEA/luciferase-siRNA compounds transfection H1299-pGL3 cells, gene silencing result such as Figure 10 institutes Show；

(3) TMEA/luciferase-siRNA compounds transfection H1299-pGL3 cells, BCA albumen result such as Figure 11 institutes Show；

The liposome siRNA prepared it can be seen from result through TMEA amphiphilic compounds enters cell, and Specific silence target gene, compared with PBS blank controls and naked siRNA groups, substantially increases the efficiency of gene silencing. And cell protein BCA experimental results are shown, with blank control PBS compared with siRNA groups, TMEA liposome siRNA preparations Protein B CA levels are close, do not cause cytotoxicity.

Table 7

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				TMEA/luciferase-siRNA	163.8±2.0	0.123±0.102	100μg/ml

Embodiment 11, T1 liposomes/gene composite human lung cancer H1299-pGL3 cell transfecting effects

Liposome/luciferase-siRNA compounds that the present embodiment is prepared using T1 lipids are to Non-small cell lung carcinoma H1299 cells carry out cell transfecting, by its Gene silencing efficacy, to observe transhipment situation of the carrier lipid to genomic medicine.

1. the preparation of lipid/gene composite：

T1/ gene composites preparation method such as embodiment 8, prepared with T1/DPPC/Chol- (40/20/40mol) mol ratio Liposome, luciferase luciferase-siRNA is wrapped up according to N/P-20/1, prepares lipid/gene composite；

2.H1299 cell is collected and cultural method such as embodiment 10；

3. compound and cell incubation and assay method such as embodiment 10；

Compliance test result：

(1) T1/luciferase-siRNA compounds particle diameter distribution and uniformity result are as shown in table 8；

(2) T1/luciferase-siRNA compounds transfection H1299-pGL3 cells, gene silencing result such as Figure 12 institutes Show；

(3) T1/luciferase-siRNA compounds transfection H1299-pGL3 cells, BCA albumen results are as shown in figure 13；

After the liposome siRNA prepared it can be seen from result through T1 lipids enters cell, specific silence Target gene, compared with PBS blank controls and naked siRNA groups, substantially increase the efficiency of gene silencing.

And cell protein BCA experimental results are shown, with blank control PBS compared with siRNA groups, TMEA liposomes SiRNA preparation protein B CA levels are close, do not cause cytotoxicity.

Table 8

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				T1 lipids/luciferase-siRNA	136.6±6.3	0.141±0.054	100.0μg/ml

Embodiment 12, T2/ gene composite human lung cancer H1299-pGL3 cell transfecting effects

Liposome/luciferase-siRNA compounds that the present embodiment is prepared using T2 amphiphilic compounds are non-to people small Cell lung cancer H1299 cells carry out cell transfecting, by its Gene silencing efficacy, to observe transhipment of the carrier lipid to genomic medicine Situation.

1. the preparation of lipid/gene composite：

T2 lipids/gene composite preparation method such as embodiment 9, with T2/DPPC/Chol- (40/30/40mol) mol ratio Liposome is prepared, luciferase luciferase-siRNA is wrapped up according to N/P-20/1, prepares lipid/gene composite；

2.H1299 cells are collected and cultural method such as embodiment 10；

3. compound and cell incubation and assay method such as embodiment 10；

Compliance test result：

(1) T2 lipids/luciferase-siRNA compounds particle diameter distribution and uniformity result are as shown in table 9；

(2) T2 lipids/luciferase-siRNA compounds transfection H1299-pGL3 cells, gene silencing result such as Figure 14 It is shown；

(3) T2 lipids/luciferase-siRNA compounds transfection H1299-pGL3 cells, BCA albumen result such as Figure 15 It is shown；

After the liposome siRNA prepared it can be seen from result through T2 lipids enters cell, specific silence Target gene, compared with PBS blank controls and naked siRNA groups, substantially increase the efficiency of gene silencing.

And cell protein BCA experimental results are shown, with blank control PBS compared with siRNA groups, TMEA liposomes SiRNA preparation protein B CA levels are close, do not cause cytotoxicity.

Table 9

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				T2 lipids/luciferase-siRNA	129.2±2.0	0.120±0.035	100μg/ml

Confocal microscopy TMEA/Cy-5-siRNA compounds are distributed in liver after embodiment 16, histotomy

Lipid prepared by the liposome Cy-5-siRNA that the present embodiment is prepared using TMEA amphipathic derivatives/ SiRNA compounds, after animal administration, after liver tissue slices, TMEA lipids/siRNA compounds are observed under Laser Scanning Confocal Microscope Distribution, so as to find out transhipment situation of the carrier lipid to genomic medicine.

It is prepared by 1.TMEA lipids/Cy5-siRNA compounds：TMEA lipids/Cy5-siRNA compounds preparation method and ratio Such as embodiment 7；

2. histotomy

(1) draw materials：The μ L of adult healthy ICR mouse 20g, tail vein injection TMEA lipid/Cy5-siRNA compounds 200 are taken, After about 10 μ g Cy5-siRNA, 4h, cervical dislocation, dissection, liver is taken out；

(2) it is quick-frozen：The liver of taking-up is accomplished into fritter, is placed in OCT and embeds, be immediately placed in it is quick-frozen in liquid nitrogen, after blocking Freezing microtome is immediately transferred into, prepares section；

(3) cut into slices：By freezing microtome insulating box be adjusted to -25 degree, by embedded block along slice direction repair rectangularity or Square, and the tissue fritter after finishing is put into specimen disc, slice thickness is adjusted to 20 μm, serial section, directly with pre- place Slide bonding die after reason, and be put into paraformaldehyde and fix 5min；

(4) rinse：Section after fixation is transferred in the staining jar equipped with 1xPBS, rinses 3min, is rinsed 3 times；

(5) close:Section after rinsing is dried into moisture around tissue with lens wiping paper, with SABC pen in tissue week Enclose and draw a circle, add 5% Donkey serum, in 37 degrees Celsius of closing 30min in wet box；

(6) primary antibody is incubated：Confining liquid is absorbed, adds the primary antibody diluted immediately, in 37 degree of closing 12-16h in wet box；

(7) secondary antibody is incubated：Primary antibody is sopped up, adds the secondary antibody diluted, in 37 degree of incubation about 30min in wet box；

(8) DAPI is dyed：Secondary antibody dilution is sopped up, adds DAPI, in 37 degrees Celsius of incubation about 30min in wet box；

(9) mounting：Cover glass surrounding is closed with the anti-fluorescent quenching mountant mounting of Vector companies, and with nail polish.

Confocal microscope 3. (Confocal Microscopy) signal acquisition

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

(2) Alexa Fluor488 scanning sequences：(Excitation：500-550nm)；

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

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

Compliance test result：

Table 10

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				TMEA lipids/luciferase-siRNA	113.6±6.3	0.141±0.054	100μg/ml

Confocal microscopy compound is distributed in liver after TMEA lipids/Cy5-siRNA compound histotomies, such as Shown in Figure 16；Blue signal is the nucleus of DAPI dyeing, and star green is kupffer cells, danger signal TMEA Lipid/Cy5-siRNA.As seen from Figure 15, after being administered 4 hours in vivo, a large amount of TMEA cationic-liposomes parcels Cy5-siRNA danger signals are distributed in liver cell.If compound danger signal is by macrophage kupffer cells green Signal swallows, and can be in yellow.Therefore as can be seen from Figure 16, a small number of Cy5-siRNA are swallowed by macrophage, more Cy5- SiRNA signals have been gathered in hepatic parenchymal cells.Illustrate that TMEA lipid carrier systems can be passed safe and efficient in Cy5-siRNA bodies Liver is sent to, it is more valuable, siRNA has more been delivered in hepatic parenchymal cells, had for treatment liver diseases very big Advantage.

Confocal microscopy T1 lipids/Cy-5-siRNA compounds are distributed in liver after embodiment 17, histotomy

Lipid/siRNA compounds that the present embodiment is prepared using T1 cation lipids parcel Cy-5-siRNA, animal administration Afterwards, TMEA lipids/siRNA compounds distribution is observed after liver tissue slices, under Laser Scanning Confocal Microscope, so as to find out carrier lipid Transhipment situation to genomic medicine.

It is prepared by 1.T1 lipids/Cy5-siRNA compounds：T1 lipids/Cy5-siRNA compounds preparation method and ratio are strictly according to the facts Apply example 8；

2. tissue section method such as embodiment 16；

3. Laser Scanning Confocal Microscope Confocal (Leica TS SP8) observation experiment methods such as embodiment 16.

Compliance test result：

Confocal microscopy compound is distributed in liver after T1 lipids/Cy5-siRNA compound histotomies, such as schemes Shown in 17；Blue signal is the nucleus of DAPI dyeing, and star green be kupffer cells, danger signal for T1 lipids/ Cy5-siRNA.As seen from the figure, after being administered 4 hours in vivo, the Cy5-siRNA of a large amount of T1 cationic-liposomes parcels is red Chrominance signal is distributed in liver cell.If compound danger signal is swallowed by macrophage kupffer cells green, meeting In yellow.Therefore as can be seen from Figure 17, a small number of Cy5-siRNA are swallowed by macrophage, more Cy5-siRNA signals aggregations Arrive in hepatic parenchymal cells.Illustrate that T1 lipid carrier systems can be delivered to liver by safe and efficient in Cy5-siRNA bodies, it is more valuable , siRNA has more been delivered in hepatic parenchymal cells, has had very big advantage for treatment liver diseases.

Table 11

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				T1 lipids/luciferase-siRNA	135.6±6.3	0.101±0.054	100μg/ml

Confocal microscopy T2 lipids/Cy-5-siRNA compounds are distributed in liver after embodiment 18, histotomy

Lipid prepared by the liposome Cy-5-siRNA that the present embodiment is prepared using T2 amphipathic derivatives compound/ SiRNA compounds, after animal administration, T2 lipids/siRNA compounds point are observed after liver tissue slices, under Laser Scanning Confocal Microscope Cloth, so as to find out transhipment situation of the carrier lipid to genomic medicine.

It is prepared by 1.T2 lipids/Cy5-siRNA compounds：T2 lipids/Cy5-siRNA compounds preparation method and ratio are strictly according to the facts Apply example 8；

2. tissue section method such as embodiment 16；

3. Laser Scanning Confocal Microscope Confocal (Leica TS SP8) observation experiment methods such as embodiment 16.

Compliance test result：

Confocal microscopy compound is distributed in liver after T2 lipids/Cy5-siRNA compound histotomies, such as schemes Shown in 18；Blue signal is the nucleus of DAPI dyeing, and star green be kupffer cells, danger signal for T2 lipids/ Cy5-siRNA.As seen from the figure, after being administered 4 hours in vivo, the Cy5-siRNA of a large amount of T2 cationic-liposomes parcels is red Chrominance signal is distributed in liver cell.If compound danger signal is swallowed by macrophage kupffer cells green, meeting In yellow.Therefore as can be seen from Figure 18, a small number of Cy5-siRNA are swallowed by macrophage, more Cy5-siRNA signals aggregations Arrive in hepatic parenchymal cells.Illustrate that T2 lipid carrier systems can be delivered to liver by safe and efficient in Cy5-siRNA bodies, it is more valuable , siRNA has more been delivered in hepatic parenchymal cells, has had very big advantage for treatment liver diseases.

Table 12

Prescription	Compound particle diameter	Compound PDI	SiRNA concentration
				T2 lipids/luciferase-siRNA	138.6±6.3	0.038±0.054	100μg/ml

The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring the substantive content of the present invention.

Claims (7)

1. A kind of 1. amphipathic derivatives of 3- ((2- (dimethylamino) ethyl group) (methyl) amino) propionic acid, it is characterised in that its Structural formula is as follows：
2. 2. a kind of preparation method of amphipathic derivatives as claimed in claim 1, it is characterised in that methods described includes as follows Step：

   A, catalyst of tetrahydrofuran, red aluminum solution toluene solution existing under the conditions of, carbon atoms 12-18 straight chain fatty Reduction reaction occurs at room temperature for acid, obtains intermediate product alcohol 2；

   B, in the presence of triethylamine, DMAP, Loprazolam acid anhydride catalyst, intermediate product alcohol 2 issues at 20 DEG C Raw sulfonic acid esterification obtains intermediate product 3；

   C, under dimethylformamide, lithium bromide catalyst action, intermediate product 3 reacts at 45 DEG C obtains middle bromide 4；

   D, under magnesium, absolute ether, Ethyl formate catalyst action, intermediate product 4 occurs among grignard reaction generation at 40 DEG C Product 5；

   E, under sodium hydroxide, tetrahydrofuran effect, intermediate product 5 issues raw hydrolysis generation intermediate product 6 at 65 DEG C；

   F, in DIPEA, DMAP, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt Under phosphate catalyst effect, esterification occurs at 50 DEG C and produces intermediate product 8 for intermediate product 6；

   G, dimethylformamide, sodium iodide, potassium carbonate catalyst action under, intermediate product 8 at 80 DEG C with N, N, N '-second Ammoxidation occurs for diamines, produces the amphipathic derivatives；

   The straight chain fatty acid of the carbon atoms 12-18 is palmitic acid or oleic acid.
3. 3. a kind of liposome of the amphipathic derivatives containing described in claim 1, it is characterised in that the liposome includes institute State amphipathic derivatives, choline, cholesterol；The weight percent content of choline is 20%~50% in the liposome, and courage is solid The weight percent content of alcohol is 10%~30%；The choline is DPPC.
4. 4. a kind of preparation method of liposome as claimed in claim 3, it is characterised in that methods described comprises the following steps：

   A, the ethanol solution of the amphipathic derivatives, choline ethanol solution, cholesterol ethanol solution are mixed, forms lipid second Alcoholic solution；

   B, the lipid ethanol solution is added in pH ＜ 5.0 HEPES buffer solution, stirred, liposome turbid liquor is made；

   C, HEPES buffer solution of the liposome turbid liquor in pH ＜ 5.0 is dialysed, removes ethanol；Produce the liposome.
5. A kind of 5. purposes of the liposome in genomic medicine delivery vehicles are prepared as claimed in claim 3, it is characterised in that institute State liposome entrapment DNA, RNA, hyaluronic acid or polypeptide.
6. 6. a kind of lipid/gene composite, it is characterised in that the lipid/gene composite is by as claimed in claim 3 The lipid that liposome Cy-5-siRNA is prepared/siRNA compounds.
7. 7. a kind of preparation method of lipid/gene composite as claimed in claim 6, it is characterised in that methods described includes Following steps：

   A, the ethanol solution of the amphipathic derivatives, choline ethanol solution, cholesterol ethanol solution are mixed, forms lipid second Alcoholic solution；

   B, the lipid ethanol solution is added in pH ＜ 5.0 HEPES buffer solution, stirred, liposome turbid liquor is made；

   C, the DEPC aqueous solution of gene biological molecule, 37 DEG C of incubation 1-2h are added in the liposome turbid liquor；The gene Biomolecule is Cy-5-siRNA；

   D, dialysed in pH ＜ 5.0 HEPES buffer solution, remove ethanol；Continue to dialyse in PBS solution, adjustment system pH to neutrality, Produce the lipid/gene composite.