CN107308113B - Macrocyclic polyamines [12] based on tetraphenylethylenes]aneN3Cationic lipid, transgenic vector and preparation method thereof - Google Patents
Macrocyclic polyamines [12] based on tetraphenylethylenes]aneN3Cationic lipid, transgenic vector and preparation method thereof Download PDFInfo
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Abstract
The invention provides a tetraphenylethylene-based macrocyclic polyamine [12]]aneN3And a transgenic vector containing the cationic lipid. The invention also discloses a preparation method of the cationic lipid and the transgenic vector. The cationic lipid provided by the invention not only contains saturated fatty chains, but also introduces tetraphenyl ethylene parent substance, so that the cationic lipid has double heads and double tails, the coagulation concentration of DNA is effectively reduced, and the hydrophobic function of the saturated fatty chains and the DNA form nanoparticles which enter through endocytosisThe gene is beneficial to transmembrane, and a transgenic vector formed by adopting cationic lipid has low cytotoxicity and higher transfection efficiency; the preparation method of the cationic lipid and the transgenic vector is simple, mature and easy to control.
Description
Technical Field
The invention relates to cationic lipid and a transgenic vector, in particular to tetraphenylethylene-based macrocyclic polyamine [12]]aneN3The cationic lipid, the transgenic vector containing the cationic lipid and the preparation method thereof.
Background
The gene therapy is to introduce exogenous normal genes into target cells by a certain method to replace or compensate some defective or abnormal genes so as to achieve the aim of treatment. Gene therapy offers the possibility of treating a number of diseases, such as: cancer, diabetes, cystic fibrosis, AIDS, cardiovascular diseases, etc.
The key of gene therapy is to introduce therapeutic gene into cell efficiently, however, naked DNA generally exists in the form of extended linear helix, has large volume, and DNA molecules are negatively charged anions, and can generate electrostatic repulsion with anions on the outer layer of cell membrane when entering into cell. These disadvantages make it impossible for DNA molecules to pass through cell membranes independently without the aid of other technical means, and therefore the development of effective gene vectors is a prerequisite for the success of gene therapy.
The gene vector includes two types of viral vectors and non-viral vectors. Viral vectors are highly effective transfection reagents, but they have disadvantages such as immunogenicity, carcinogenicity, and the possibility that the size of genes is easily limited by the size of viruses and variation exists, so that safety is concerned. Viral vectors are non-immunogenic, convenient for large-scale production, but have lower transfection efficiency than viral vectors. Therefore, the development of a non-viral gene vector with low toxicity and high efficiency is of great significance.
Disclosure of Invention
As a result of a variety of extensive and intensive studies and experiments, the inventors of the present inventionIt has been found that in macrocyclic polyamines [12]]aneN3The saturated fatty chain is introduced into the cation-like lipid, so that the condensation concentration of DNA can be effectively reduced, the hydrophobic function of the saturated fatty chain and the DNA form nano particles which enter cells through endocytosis, transmembrane spanning is facilitated, and a transgenic vector formed by the cation-like lipid has low cytotoxicity and high transfection efficiency. On the basis of this finding, the present inventors have completed the present invention based on the finding that a cationic lipid can be made double-headed and double-tailed to have a higher transfection efficiency.
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is a further object of the present invention to provide a macrocyclic polyamine [12] having multiple functions]aneN3The transgenic carrier of the cationic lipid not only has low cytotoxicity, but also has higher transfection efficiency at the same time, so as to promote the development of gene therapy.
It is a further object of the present invention to provide macrocyclic polyamines [12] based on tetraphenylethylenes]aneN3The method for producing a cationic lipid and the method for producing a transgenic vector containing the cationic lipid.
To achieve these objects and other advantages in accordance with the present invention, there is provided a macrocyclic polyamine [12] based on tetraphenylethylene]aneN3The cationic lipid of (1), the cationic lipid having the following structural formula (I):
wherein, in the formula (I), n is a positive integer, and R is a hydrocarbyl group.
Preferably, n is 1 or 2, and R is a straight-chain alkyl group.
Preferably, wherein R is
Or
The object of the invention is furthermore achieved by macrocyclic polyamines [12] containing tetraphenylethylene]aneN3By a process for the preparation of cationic lipids of (1), which is carried out by macrocyclic polyamines [12]]aneN3The derivative is reacted with tetraphenylethylene derivative.
Preferably, the specific synthetic route of the preparation method is as follows:
wherein n, R are defined as in claim 1, the specific synthesis steps comprising:
step one, adding sodium azide into an N, N-dimethylformamide solution in which a compound of a formula 1 is dissolved, reacting for 12 hours at 80 ℃, adding a sodium chloride aqueous solution and ethyl acetate for extraction, combining organic phases, drying, concentrating under reduced pressure to remove a solvent, adding paratoluensulfonyl chloride and triethylamine, stirring at room temperature for reacting for 4 hours, and separating by column chromatography to obtain a compound of a formula 3;
step two, dissolving the compound of the formula 3 obtained in the step one and the compound of the formula 4 in acetonitrile, adding potassium carbonate, performing reflux reaction for 36 hours, and performing column chromatography separation to obtain a compound of the formula 5;
reacting the compound shown in the formula 6 and the compound shown in the formula 7 with zinc powder and titanium tetrachloride in anhydrous tetrahydrofuran under the protection of argon, performing reflux reaction for 8-10 h, quenching the reaction by using a 10% potassium carbonate solution, extracting with ethyl acetate, combining organic phases, performing reduced pressure concentration to remove a solvent, and performing column chromatography separation to obtain the compound shown in the formula 8;
step four, dissolving the compound of the formula 8 obtained in the step three and long-chain bromoalkane in acetonitrile, adding potassium carbonate, reacting for 24 hours at 90 ℃, and separating by column chromatography to obtain a compound of a formula 9;
step five, reacting the compound of the formula 5 obtained in the step two with the compound of the formula 9 obtained in the step four to obtain a compound of a formula 10;
and step six, reacting the compound of the formula 10 obtained in the step five with an ethyl acetate solution of hydrochloric acid to obtain a compound of a formula 11.
Preferably, in the step five, the specific steps include: dissolving the compound shown in the formula 5 and the compound shown in the formula 9 in tetrahydrofuran, adding a copper sulfate solution and a vitamin C sodium solution, reacting for 6 hours at room temperature under the protection of argon, and separating by column chromatography to obtain the compound shown in the formula 10.
Preferably, in the sixth step, the specific steps include: dissolving the compound of formula 10 in ethyl acetate, adding 2M ethyl acetate solution of hydrochloric acid, reacting for 4 hours in ice water bath, decompressing and concentrating to remove the solvent, washing the obtained solid with diethyl ether, and drying in vacuum to obtain the compound of formula 11.
To achieve these objects and other advantages in accordance with the present invention, there is also provided a transgenic vector consisting of a macrocyclic polyamine [12] containing tetraphenyl ethylene based]aneN3The cationic lipid, the dioleoylphosphatidylethanolamine and the high-purity sterilization buffer solution, wherein the molar ratio of the cationic lipid to the dioleoylphosphatidylethanolamine is 2:1, 1:1 or 1: 2.
The object of the present invention can be further achieved by a method for preparing a transgenic vector, which specifically comprises the steps of: adding the cationic lipid, dioleoyl phosphatidylethanolamine and anhydrous chloroform into a flask sterilized at high temperature, dissolving, concentrating under reduced pressure to obtain a transgenic carrier membrane, carrying out vacuum drying on the transgenic carrier membrane to remove residual chloroform, mixing the dried transgenic carrier membrane with a trihydroxymethyl aminomethane-hydrochloric acid buffer solution preheated to 70 ℃ in advance to prepare a solution with required concentration, and carrying out ultrasonic treatment to obtain the transgenic carrier, wherein the molar concentration of the trihydroxymethyl aminomethane-hydrochloric acid buffer solution is 10mM, and the pH value is 7.4.
The object of the invention is furthermore achieved by macrocyclic polyamines [12] containing tetraphenylethylene]aneN3The use of the cationic lipid of (4) in the preparation of a transgenic vector.
The invention at least comprises the following beneficial effects:
1. the cationic lipid containing the hydrophobic saturated fatty chain can effectively reduce the condensation concentration of DNA, and form nanoparticles with proper size with the DNA by utilizing the hydrophobic function of the cationic lipid and enter cells through endocytosis;
2. by introducing the parent tetraphenylethylene, the transgenic vector formed by the cationic lipid of the hydrophobic saturated fatty chain has double heads and double tails, has stronger transfection efficiency and lower cytotoxicity, and is somewhat more than or even more than the commercialized Lipofectamine 2000, which contains [12]]aneN3The unit compound is used as a gene vector to be well paved for further research;
3. the preparation method of the cationic lipid and the transgenic vector is simple, mature and easy to control;
4. the transgenic vector has good compatibility with cells and has the potential of being used as a non-viral vector in gene therapy;
5. the transgenic vector can completely wrap DNA and can be used as a good vector for transmembrane and transport of the DNA;
6. particle size of the transgenic vector after condensation with DNA. Form particles with the size of about 100nm with DNA, which has important significance for successfully penetrating cell membranes to realize gene transfection.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the result of agarose gel electrophoresis of a transgenic vector and DNA complex of the present invention;
FIG. 2 is a cytotoxicity diagram of the complex of the transgenic vector and the plasmid DNA in A549, Hela and HepG2 cells;
FIG. 3 is a surface topography of a complex of a transgenic vector of the present invention and plasmid DNA;
FIG. 4 is a graph showing the transfection efficiency of the complex of the transgenic vector of the present invention and DNA in Hela cells.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
< example 1>
A cationic lipid based on tetraphenylethylene comprising macrocyclic polyamine [12] aneN3, the cationic lipid having the formula:
wherein, in the formula (I), n is a positive integer, and R is a hydrocarbyl group;
wherein n is 1 or 2, R is
Or
The cationic lipid is synthesized by the following steps:
the method comprises the following specific steps:
step one, after a reaction of 12 hours at 80 ℃ to a solution of a compound of formula 1, bromoethanol (1.24g, 10mmol), sodium azide (3.25 g, 50mmol), and 10mL of DMF. 30mL of a saturated aqueous NaCl solution was added, extraction was performed with ethyl acetate (25 mL. times.3), the organic phases were combined, washed with a saturated aqueous NaCl solution (50 mL. times.2), the organic phase was dried over anhydrous sodium sulfate powder, filtered, and the solvent was distilled off under reduced pressure to obtain a colorless liquid, and the colorless compound was dissolved in 15mL of dichloromethane in a 50mL single-neck bottle, and triethylamine (2.02g, 20mmol) and p-toluenesulfonyl chloride (2.29g, 12mmol) were added thereto, and after four hours of reaction at room temperature, the mixture was purified by column chromatography (PE: EA. RTM.8: 1) to obtain a compound of formula 3 as a pale yellow liquid in 54% to 63% yield. The reaction using bromopropanol as the starting material is similar. When n is 1 or 2, the compound of formula 3 is defined as 3a and 3b respectively;
step two, a 100mL single-necked flask was charged with the compound of formula 4 (0.371g, 1mmol), the compound of formula 3a or formula 3b (1.1mmol), potassium carbonate (0.690g, 5mmol), and 20mL acetonitrile, and refluxed for 36 hours. After the reaction, the reaction mixture was filtered, and the filtrate was subjected to distillation under reduced pressure to remove the solvent and purified by column chromatography (PE: EA ═ 5:1) to obtain the compound of formula 5 as a pale yellow oil in a yield of 43% to 51%. When n is 1 or 2, the compound of formula 5 is defined as 5a, 5b, respectively;
step three, a 250mL two-necked flask was charged with the compound of formula 7 (2.00g, 6.89mmol), the compound of formula 6 (1.77g, 8.27mmol), zinc powder (2.68g, 41.3mmol), argon was introduced three times, and then 50mL of freshly distilled tetrahydrofuran was added via syringe. Slowly injecting titanium tetrachloride (2.4 mL, 20.7mmol) in an ice-water bath, removing the ice-water bath, continuously stirring for 30 minutes at room temperature, performing reflux reaction for 8-10 hours, cooling the reaction system to room temperature the next day, performing reduced pressure distillation to remove most of the solvent, and quenching the reaction with 10% potassium carbonate aqueous solution until no bubbles are generated. Ethyl acetate was extracted several times, the organic phases were combined, dried over anhydrous sodium sulfate powder, filtered, the solvent was distilled off under reduced pressure, and column chromatography purification (PE: EA ═ 5:1) gave 0.500g (1.06mmol) of the compound of formula 8 as a pale yellow solid, in yield: 15 percent;
step four, reacting the compound in the formula 8 with different long-chain alkyl bromides to obtain a compound in a formula 9, wherein R is a saturated fatty chain with different lengths, and the reaction is as follows: adding the compound of formula 8 (0.100g, 0.21 mmol), bromoalkane (0.63mmol), potassium carbonate (0.146g, 1.06mmol) and 10mL of DMF into a 50mL single-neck flask, reacting at 90 deg.C for 24 hr, distilling under reduced pressure to remove most of the solvent after the reaction is finished, adding 10mL of dichloromethane, filtering, washing with a small amount of dichloromethane, distilling under reduced pressure to remove the solvent from the filtrate, and purifying by column chromatography(PE: EA ═ 30: 1) to give the compound of formula 9 as a yellow oil in 47% to 54% yield. Wherein when R is
Compounds of formula 9 are defined as 9a, 9b, respectively;
step five, reacting the compound shown in the formula 5a and the formula 5b with the compound shown in the formula 9a and the formula 9b to obtain the compound shown in the formula 10, wherein the reaction process is as follows: in a 25mL two-necked flask, the compound of formula 5 (0.25mmol), the compound of formula 9 (0.1mmol), 0.5mmol/mL aqueous copper sulfate solution, 0.1mL each of aqueous sodium ascorbate solution, and 2mL of tetrahydrofuran were added under argon, and the reaction was stirred at room temperature for 6 hours. After the reaction was completed, preparative TLC separation (dichloromethane: methanol ═ 20:1) gave the compound of formula 10 as a yellow oil in 58% to 69% yield. Wherein n is 1 and R is
When, compounds of formula 10 are defined as 10a, 10b, respectively; wherein n is 2 and R is
When, the compounds of formula 10 are defined as 10c, 10d, respectively;
reacting the compound of formula 10 obtained in the sixth step and the fifth step with ethyl acetate solution of hydrochloric acid to obtain cationic lipid of formula 11, wherein n is 1 or 2, R is different saturated aliphatic chains, and the synthesis method is similar to that of the compound of formula 10: in a 10mL single-neck flask, the compound of formula 10 (0.05mmol) and 2mL of ethyl acetate were added and dissolved, and 2mL of 2M HCl-EA solution was slowly added dropwise in an ice-water bath, and after stirring and reacting for 4 hours, a large amount of solid precipitated. Distilling under reduced pressure to remove solvent, adding a small amount of diethyl ether for solidification, filtering, washing with a small amount of diethyl ether, and vacuum drying the filter residue for 12 hours to obtain light yellow or white solid type 11 cationic lipid with a yield of 55-98%.
Structural formula of compound of formula 5a, nuclear magnetism: (1H-NMR，13C NMR), infrared and mass spectra (HR-MS or ESI-MS) were characterized as follows:
nuclear magnetism1H NMR(400MHz,CDCl3)δ3.34(dt,J＝13.6,6.7Hz,8H),3.28(t,J＝6.1 Hz,2H),2.62(t,J＝6.1Hz,2H),2.48(t,J＝6.4Hz,4H),1.89–1.82(m,2H), 1.81–1.74(m,4H),1.45(s,18H).
Nuclear magnetism13C NMR(101MHz,CDCl3)δ156.43,79.12 50.11,50.02,49.11,43.23, 28.13,27.12,26.34,25.20.
Infrared IR (KBr, cm)-1):2927,2361,2326,2097,1690,1474,1414,1364,1250,1172,769.
Mass spectrum HR-MS (high-resolution mass spectrometry) M/z 441.3191([ M + H)]+).
Structural formula of compound of formula 9, nuclear magnetism: (1H-NMR，13C NMR), infrared and mass spectra (HR MS) were characterized as follows:
9a compound, nuclear magnetism1H NMR(400MHz,CDCl3) δ 6.91(dd, J ═ 15.5,8.3Hz, 8H),6.69(d, J ═ 8.3Hz,4H),6.61(d, J ═ 8.3Hz,4H),4.61(d, J ═ 2.3Hz,4H), 3.86(t, J ═ 6.6Hz,4H),2.50(d, J ═ 2.4Hz,2H),1.80-1.56(m,4H), 1.47-1.38 (m,4H), nuclear magnetic resonance (nmr13C NMR(101MHz,CDCl3)δ156.61,155.00,138.27,136.90,135.59, 131.65,113.11,112.70,75.84,74.48,66.94,54.90,30.94,28.62,28.26,25.21, 21.79,13.23.
Infrared IR (KBr, cm)-1):3291,3036,2926,2855,2122,1889,1605,1573,1507,1470,1458,1375,1289,1242,1219,1174,1031,1031,974,924,831,809,669,637.
Mass spectrum HR MS M/z 697.4257([ M + H)]+).
9b compound, nuclear magnetism1H NMR(400MHz,CDCl3)δ6.91(dd,J＝15.7,8.3Hz, 8H),6.69(d,J＝8.3Hz,4H),6.61(d,J＝8.6Hz,4H),4.61(d,J＝2.2Hz,4H), 4.61(d,J＝2.2Hz,4H),3.86(t,J＝6.6Hz,4H),2.50(d,J＝2.4Hz,2H), 1.77-1.68(m,4H),1.46-1.38(m,4H),1.30-1.20(m,32H),0.87(t,J＝6.6Hz6H) nuclear magnetism13C NMR(101MHz,CDCl3)157.58,155.97,139.24,137.87,136.56, 132.62,114.08,113.67,78.78,75.47,67.89,55.84,32.03,29.88–29.35(m), 26.19,22.80,14.24.
Infrared IR (KBr, cm)-1):3306,3291,3037,2924,2853,2326,2050,2050,1888,1605,1573,1507,1466,1373,1288,1241,1220,1174,1111,1031,974,924,831,771,724,669,632,603.
Mass spectrum HR MS M/z 809.5448([ M + H)]+).
Structural formula of compound of formula 10a-10d, nuclear magnetism: (1H-NMR), infrared and mass spectra (HR MS) were characterized as follows:
10a Compound: nuclear magnetism1H NMR(400MHz,CDCl3) δ 7.61(s,2H),6.92(dd, J ═ 11.6,8.7Hz,8H),6.70(d, J ═ 8.7Hz,4H),6.62(d, J ═ 8.7Hz,4H),5.11(s,4H), 4.40(t, J ═ 6.1Hz,4H),3.88(t, J ═ 6.6Hz,4H),3.24(dt, J ═ 12.1,6.4Hz,16H), 2.93(t, J ═ 6.2Hz,4H),2.49(t, J ═ 6.1Hz,8H), 1.79-1.64 (m,12H),1.55(s, 4H),1.48(d, J ═ 31.5Hz,36H), 1.41-1.88 (t, 17.88H), 6.9, 17, 8H, 6, 8H), infrared (br), 6, 4H),1.48 (t, 17, 6, 4H), and 18H) respectively-1):3743,2925,2854,2362,2326,2101,1690,1506,1464,1363, 1293,1243,1175,1018,916,817,767,664.
Mass spectrum HR MS M/z 790.0296([ M + 2H)]2+).
10b Compound: nuclear magnetism1H NMR(400MHz,CDCl3)δ7.60(s,2H),6.92(dd,J＝ 11.3,8.7Hz,8H),6.70(d,J＝8.6Hz,4H),6.62(d,J＝8.6Hz,4H),5.11(s,4H), 4.40(s,4H),3.88(t,J＝6.5Hz,4H),3.25(dd,J＝16.6,9.8Hz,16H),2.93(s,4H), 2.49(s,8H),1.72(dd,J＝19.3,11.4Hz,12H),1.51(s,4H),1.53–1.36(m,36H), 1.26(s,36H),0.88(t,J＝6.8Hz,6H).
Infrared IR (KBr, cm)-1):3738,2965,2926,2854,2361,2328,2099,1690,1605,1507,1458,1414,1364,1242,1173,983,832,770.
Mass spectrum HR MS M/z 845.6003([ M + 2H)]2+).
10c Compound: nuclear magnetism1H NMR(400MHz,CDCl3)δ7.59(s,2H),6.92(dd,J＝ 14.9,8.6Hz,8H),6.72(d,J＝8.8Hz,4H),6.62(d,J＝8.7Hz,4H),5.12(s,4H), 4.37(t,J＝7.0Hz,4H),3.88(t,J＝6.6Hz,4H),3.42–3.23(m,16H),2.44(dd,J ＝14.4,8.2Hz,12H),2.09–2.02(m,4H),1.89–1.81(m,4H),1.73(d,J＝6.3Hz, 8H),1.52(s,4H),1.45(s,36H),1.29(d,J＝10.7Hz,20H),0.88(t,J＝6.8Hz, 6H).
Infrared IR (KBr, cm)-1):3901,3742,3673,3649,3624,3591,3568,2922,2852,2361,2335,2328,2104,1699,1653,1560,1540,1513,1506,1363,1347,1297,1245,1176,1017,916,815,750,665
Mass spectrum HR MS M/z 804.0451([ M + 2H)]2+).
10d Compound: nuclear magnetism1H NMR(400MHz,CDCl3)δ7.59(s,2H),6.92(dd,J＝ 15.6,8.5Hz,8H),6.72(d,J＝8.8Hz,4H),6.62(d,J＝8.4Hz,4H),5.12(s,4H), 4.37(s,4H),3.88(t,J＝6.5Hz,4H),3.34(d,J＝5.3Hz,16H),2.69(s,4H),2.43 (s,8H),2.05(s,4H),1.85(s,4H),1.73(d,J＝7.0Hz,8H),1.51(s,4H),1.52– 1.36(m,36H),1.28(d,J＝15.9Hz,36H),0.88(t,J＝6.8Hz,6H).
Infrared IR (KBr, cm)-1):2923,2853,2360,2327,2104,1690,1681,1506,1457,1414,1364,1286,1243,1174,1016,916,817,766,751,665,554.
Mass spectrum HR MS M/z 860.1077([ M + 2H)]2+).
Structural formula of compound of formula 11a-11d, nuclear magnetism: (1H-NMR，13C NMR), infrared and mass spectra (HR MS or ESI-MS) were characterized as follows:
11a Compound: nuclear magnetism1H NMR(400MHz,D2O) δ 7.93(s,2H),6.84(s,4H),6.74 (s,4H),6.64(s,4H),6.36(s,4H),4.93(s,4H),4.46(s,4H),3.53(s,4H),3.17(d, J ═ 59.6Hz,16H),2.88(s,4H),2.65(s,8H),2.15(s,4H),1.80(s,8H),1.47(s, 4H),1.09(s,20H),0.69(s,6H). nuclear magnetic resonance13C NMR(101MHz,D2O)δ181.96,156.12, 155.32,142.70,137.87,136.96,136.35,135.33,131.34,126.45,123.97,120.89, 112.61,66.22,60.22,50.73,48.44,45.65,40.95,40.34,30.68,28.19,24.93,21.49, 19.71,17.61,12.87.
Infrared IR (KBr, cm)-1):3399,2953,2928,2854,2354,1604,1507,1456,1384,1242,1173,1110,1009,830,745,596,502,472.
Mass Spectrometry ESI-MS:589.47([ M +2H ]]2+).
11b Compound: nuclear magnetism1H NMR(400MHz,D2O)δ7.73(s,2H),6.70(s,8H),6.50 (s,4H),6.28(s,4H),4.28(s,4H),3.45(s,4H),3.23(s,16H),2.95(d,J＝32.3Hz, 12H),2.13(s,4H),1.92(s,12H),1.38(s,4H),1.03(s,36H),0.61(s,6H).
Nuclear magnetism13C NMR(101MHz,DMSO)156.90,156.07,142.68,138.32,137,32, 136.62,136.02,131.95,124.65,113.86,113.65,46.27,67.21,40.15,31.27,29.00, 28.98,28.95,28.73,28.68,25.49,22.07,17.10,14.22,13.93.
Infrared IR (KBr, cm)-1):3419,2926,2853,2736,2634,2369,2323,2193,1604,1461,1287,1241,1173,1109,1048,1010,829,806,503,457.
Mass Spectrometry ESI-MS:645.54([ M +2H ]]2+).
11c Compound: nuclear magnetism1H NMR(400MHz,D2O)δ7.81(s,2H),6.80(s,8H),6.60 (s,4H),6.37(s,4H),4.38(s,4H),3.54(s,4H),3.32(d,J＝19.2Hz,16H),3.01(d, J＝37.1Hz,12H),2.24(s,4H),2.01(s,12H),1.46(s,4H),1.09(s,20H),0.68(s, 6H).
Nuclear magnetism13C NMR(101MHz,DMSO)157.49,156.90,143.22,138.85,138.21, 137.17,136.56,132.50,125.23,114.40,114.20,67.76,61.50,51.89,47.26,46.82, 41.66,31.76,29.28,29.19,26.07,24.97,21.39,17.63,14.50.
Infrared IR (KBr, cm)-1):3402,2956,2927,2854,2727,2631,2361,2340,1604,1507,1460,1384,1286,1242,1172,1109,1031,829,803,744,650,596,566,502,480, 449.
Mass spectrum HR-MS:603.4391([ M +2H ]]2+).
11d Compound: nuclear magnetism1H NMR(400MHz,D2O)δ7.73(s,2H),6.70(s,8H),6.50 (s,4H),6.28(s,4H),4.28(s,4H),3.45(s,4H),3.23(s,16H),2.95(d,J＝32.3Hz, 12H),2.39–1.64(m,20H),1.38(s,4H),1.03(s,36H),0.61(s,6H).
Nuclear magnetism13C NMR(101MHz,DMSO)δ156.95,156.36,142.68,138.31,137,67, 136.62,136.02,131.95,124.65,113.86,113.65,46.27,67.21,60.97,40.15,31.27, 29.00,28.98,28.95,28.73,28.68,25.49,22.07,17.10,13.93.
Infrared IR (KBr, cm)-1):3446,3421,3413,2926,2853,2735,2634,2538,2496,2370,1604,1466,1459,1288,1242,1174,1111,1049,1010,831,595.
Mass spectrum HR-MS:659.5017([ M +2H ]]2+).
< example 2>
Preparation of transgenic vectors containing the cationic lipid Compounds of formulas 11a-11d in example 1
The starting materials included the cationic lipid compounds of formulae 11a-11d prepared in example 1, dioleoylphosphatidylethanolamine DOPE (dioleylphosphatidylethanolamine), and anhydrous chloroform. Dissolving the synthesized cationic lipid compound of formula 1 (0.005mmol) and DOPE in a molar ratio of 2:1 or 1:2 in 2.5mL of anhydrous chloroform in a high-temperature sterilized flask, thoroughly mixing and dissolving, spin-drying the solvent at room temperature under reduced pressure to obtain a transgenic carrier membrane, drying the mixture in a vacuum drying oven (drying time 12 hours, temperature 25 ℃) to remove residual chloroform, mixing the dried transgenic carrier membrane with a Tris-HCl buffer (10mM, pH 7.4) preheated to 70 ℃ in advance, and adding a buffer solution in an amount to make the final concentration of the lipid 1.0 mM. Finally, the mixture is subjected to ultrasonic treatment at 60 ℃ for 20 minutes and stored in a refrigerator at 4 ℃ for later use.
< example 3>
Example 2 the transgenic vector prepared was tested for the following:
(1) agarose gel electrophoresis experiment of transgenic vector and pUC 18 plasmid compound
Preparation of transgenic vector and pUC 18 plasmid Complex
The transgenic vectors prepared in 11a-11d were added to PE tubes at room temperature, respectively, wherein the concentration of the transgenic vectors was 0,5,10,15,20,25, 30. mu.M, and the concentration of pUC 18 plasmid was 9. mu.g/mL, and a corresponding volume of ultrapure water was added to maintain the final volume of the reaction solution at 20. mu.L, followed by centrifugation and uniform mixing. After incubation in a 37 ℃ thermostatic water bath for 1 hour, 2. mu.L of 10 × Loading Buffer was added to terminate the reaction.
Preparation of agarose gel
280mg of agarose was weighed, 40mL of 1 XTAE buffer solution was added, and the agarose beads were completely dissolved by microwave heating to obtain a colorless transparent liquid. When the temperature is reduced to about 60 ℃,2 mu L of Goldview II nucleic acid color developing agent is added, and the mixture is uniformly mixed and poured into a glue making groove with a comb while the mixture is hot. After cooling for 40 minutes until the gel is completely solidified, carefully pulling out the comb, placing the gel preparation tank with the gel into the electrophoresis tank, and adding 1 XTAE electrophoresis buffer to slightly submerge the gel surface.
Agarose gel electrophoresis experiment of the complexes
The transgenic vector and pUC 18 plasmid complex 9. mu.L prepared above were added to the wells on the gel. Covering an electrophoresis cover, stopping electrophoresis under the voltage of 80V for 40 minutes, taking out the gel, and exposing and sampling in a gel electrophoresis phase forming system.
As shown in FIG. 1, FIGS. 1(A), 1(B), 1(C) and 1(D) are schematic diagrams showing the results of agarose gel electrophoresis experiments of transgenic vectors prepared from 11a-11D and plasmid DNA, respectively, and we observed the action of the transgenic vectors prepared from cationic transgenic vectors 11a-11D and pUC 18 plasmid by using the agarose gel electrophoresis experiments. As can be seen from FIG. 1, most of the pUC 18 plasmid was retained in the loading well at a concentration of 10. mu.M of the transgenic vector, and all of the pUC 18 plasmid was retained in the loading well at a concentration of 15. mu.M, indicating that the pUC 18 plasmid was completely encapsulated by the transgenic vector.
(2) Transgenic vector cytotoxicity assay
Trypsinization of logarithmic growth phase A549, Hela, HepG2 cells approximately 7000 cells per well were seeded into 96-well plates containing 5% CO at 37 deg.C2The culture box is cultured for 24 hours, so that the cells grow to 70-80% of fusion. Discarding original culture medium in the holes, washing with PBS buffer solution for 1-2 times, adding 100 μ L of the prepared transgenic vectors with different concentrations into each hole, and setting three parallel holes for each concentration; using transgenes not containingDMEM media with vehicle was used as a blank, DMEM without cells only as a blank. After 4 hours all the medium was aspirated, 200. mu.L of DMEM containing 10% FBS was added to each well, after 24 hours incubation in an incubator, 10. mu.L of MTT solution was added to each well, after 4 hours all the addition was aspirated, the resulting violet-blue crystals were dissolved in 150. mu.L of DMSO, and the absorbance value at 490nm was measured in each well by a microplate reader after 10 minutes shaking on a shaker. The cell survival rate (%) - [ A490 test-blank was calculated as follows]/[ A490 control-blank]×100。
As shown in FIG. 2, FIGS. 2(A), 2(B) and 2(C) are the cytotoxicity diagrams of the complex of the transgenic vector of the present invention and plasmid DNA in A549 cells, Hela cells and HepG2 cells, respectively, wherein 1, 2, 3 and 4 in the abscissa correspond to the transgenic vectors prepared from the compounds 11a-11d, respectively, and the cytotoxicity of the four transgenic vectors was determined in A549 cells, Hela cells and HepG2 cells, and it was found that the cytotoxicity was low. The overall presentation rule is: with increasing concentration, the number of apoptotic cells increased, since the transgene vector nitrogen atom can increase cytotoxicity. Therefore, it is important to select a proper concentration for achieving low cytotoxicity and high transfection efficiency.
(3) Particle size experiment of transgenic vector and pUC 18 plasmid composition
Preparation of transgenic vector and pUC 18 plasmid Complex
At room temperature, transgenic vectors with a concentration of 20. mu.M and a concentration of pUC 18 plasmid of 9. mu.g/mL were added to PE tubes, respectively, and ultrapure water was added in a corresponding volume to maintain the final volume of the reaction solution at 500. mu.L, followed by centrifugation and uniform mixing. Putting the mixture into a constant-temperature water bath at 37 ℃ and preserving the heat for 1 hour. (in the scanning electron microscope experiment, the compound of the transgenic vector and the DNA is directly dripped on a silicon chip without dilution, and the compound is naturally dried at room temperature).
Particle size and morphology testing
The cuvette was repeatedly rinsed with the reaction solution, and finally 500. mu.L of the reaction solution was transferred to the cuvette for measurement. The test temperature was set at 25.00 ℃ and 10 sets of replicates were collected for each measurement and averaged.
As shown in FIG. 3, FIGS. 3(A), 3(B), 3(C), and 3(D) are surface topography diagrams of the complex of the transgenic vector prepared from 11a-11D and plasmid DNA, respectively, and we studied the particle size of the condensed transgenic vector and DNA by using the dynamic laser light scattering technique. The formed condensed reagent can form particles with the size of about 100nm with DNA, which has important significance for successfully penetrating cell membranes to realize gene transfection.
(4) In vitro transfection experiment of transgenic vector and pGL-3 complex
Measurement of fluorescence intensity
① plating, collecting cells with good growth state, sucking the culture solution in the plate, washing with PBS twice, adding 0.25% trypsin for digestion for about 3min, transferring to a centrifuge tube, centrifuging (1000rpm, 5min), pouring out the supernatant, preparing the cells into about 20000 single cell suspension with DEME culture solution containing 10% FBS (fetal bovine serum albumin), adding into 24-well plate with each well being 0.4mL, dispersing uniformly at 37 deg.C, and adding 5% CO2The culture box is used for culturing for 24 hours.
② preparing solution, adding pGL-3DNA and 1mM compound aqueous solution into a 1.5mL centrifuge tube, and culturing with DMEM to prepare solutions with different concentrations, wherein the total volume is 650 μ L, the DNA concentration is 9 μ g/mL, and the prepared solution is placed at room temperature for balancing for 30min to ensure that the compound and the DNA fully act.
③ transfection, precipitating the original culture solution in 24-well plate, washing each well with DMEM culture solution once, adding the prepared solution, each well is 0.2mL, each sample is divided into three groups, placing at 37 deg.C, and containing 5% CO2Cultured in an incubator. After 4 hours, the added solution was aspirated, and after washing with DMEM once per well, 0.4mL of DEME culture medium containing 10% FBS was added and incubation was continued for 24 hours.
④ lysis, 24 hours later, take out 24-hole plate, suck out the culture medium, each hole add 120 u L1 x CellLysis Buffer cell lysis solution, shake 20min at normal temperature, scrape all cells, blow and break up, and transfer to 1.5mL centrifuge tube, low temperature centrifugation (12000rmp, 30s), take the supernatant, fully lysis sample for use.
⑤ bioluminescence assay, 20. mu.L of the well lysed sample was put in a white 96-well plate, 50. mu.L of luciferase assay Reagent (fluorescein substrate) was added thereto, and after stirring the mixture well, the RLU (relative light unit) value was measured on a microplate reader.
Protein content determination
① working solution preparation, the copper reagent and the BCA diluent in the kit are prepared into the BCA working solution according to the ratio of 1: 50.
② dilution of the standard substance 10. mu.L of the BSA protein standard substance was diluted to 100. mu.L with PBS diluent to give a final concentration of 0.5mg/mL, and the diluted standard substance was added to a clear 96-well plate in the order of 0,2, 4, 6,8, 12, 16, and 20. mu.L, and was again diluted to 20. mu.L with PBS.
③ sample treatment well lysed samples were added to clear 96-well plates at 5. mu.L per well, after which 15. mu.L PBS dilution was added per well and finally 100. mu.L BCA working solution was added per well and incubated for 15min at 37 ℃.
④, measuring and calculating absorbance at 562nm by using an enzyme-labeling instrument, and drawing the numerical value of the standard substance into a working curve according to which the protein content of the sample can be calculated, wherein the ratio of the fluorescence intensity to the protein content is the transfection efficiency, and Lipofectamine 2000 is used as a reference in the experiment.
As shown in FIG. 4, FIGS. 4(A), 4(B), 4(C), and 4(D) are graphs of transfection efficiencies of complexes of the transgenic vectors prepared from 11a to 11D and DNA in Hela cells, respectively, and it can be found through cell transfection experiments that: the transgenic vectors prepared from 11a-11d can carry plasmids into cells, and finally express luciferase. The addition of DOPE during the formation of the transgenic vector allows the compound to condense the DNA into particles of appropriate size, while increasing a certain transfection efficiency. Among them, the complex of compound 15c and DOPE showed the best transfection efficiency, which already exceeded the commercial lipofectamine 2000 with better transfection efficiency. The experimentally synthesized cationic transgenic vector has cell compatibility and potential as a non-viral vector in gene therapy.
In conclusion, the introduction of the aliphatic chain in the present invention can effectively reduce the DNAThe concentration is condensed, and the hydrophobic function of the nano particles is utilized to form nano particles with DNA, and the nano particles enter cells through endocytosis. The four compounds of 11a-11d which are synthesized are designed to be capable of efficiently condensing DNA, MTT experiments of the formed transgenic vector on HepG2, Hela and A549 show that the cytotoxicity is very low, the compound formed by the synthesized transgenic vector and DOPE shows good transfection effect on Hela cells after gene transfection experiments, and some compounds even exceed commercial Lipofectamine 2000, which contains [12]aneN3The unit compound is used as a gene vector to be well paved for further research, and the preparation method is simple, mature and easy to control.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (7)
1. Macrocyclic polyamine [12] based on tetraphenylethylene]aneN3The cationic lipid of (1), the cationic lipid having the following structural formula (I):
wherein, in the formula (I), n is 2, R is
Or
2. A process for preparing the cationic lipid of claim 1 by reacting a macrocyclic polyamine [12]]aneN3The derivative is prepared by reacting with a tetraphenylethylene derivative, and the specific synthetic route is as follows:
wherein n, R are defined as in claim 1, the specific synthesis steps comprising:
step one, adding sodium azide into an N, N-dimethylformamide solution in which a compound of a formula 1 is dissolved, reacting for 12 hours at 80 ℃, adding a sodium chloride aqueous solution and ethyl acetate for extraction, combining organic phases, drying, concentrating under reduced pressure to remove a solvent, adding paratoluensulfonyl chloride and triethylamine, stirring at room temperature for reacting for 4 hours, and separating by column chromatography to obtain a compound of a formula 3;
step two, dissolving the compound of the formula 3 obtained in the step one and the compound of the formula 4 in acetonitrile, adding potassium carbonate, performing reflux reaction for 36 hours, and performing column chromatography separation to obtain a compound of the formula 5;
reacting the compound shown in the formula 6 and the compound shown in the formula 7 with zinc powder and titanium tetrachloride in anhydrous tetrahydrofuran under the protection of argon, performing reflux reaction for 8-10 h, quenching the reaction by using a 10% potassium carbonate solution, extracting with ethyl acetate, combining organic phases, performing reduced pressure concentration to remove a solvent, and performing column chromatography separation to obtain the compound shown in the formula 8;
step four, dissolving the compound of the formula 8 obtained in the step three and long-chain bromoalkane in acetonitrile, adding potassium carbonate, reacting for 24 hours at 90 ℃, and separating by column chromatography to obtain a compound of a formula 9;
step five, reacting the compound of the formula 5 obtained in the step two with the compound of the formula 9 obtained in the step four to obtain a compound of a formula 10;
and step six, reacting the compound of the formula 10 obtained in the step five with an ethyl acetate solution of hydrochloric acid to obtain a compound of a formula 11.
3. The method as claimed in claim 2, wherein in the step five, the specific steps include: dissolving the compound shown in the formula 5 and the compound shown in the formula 9 in tetrahydrofuran, adding a copper sulfate solution and a vitamin C sodium solution, reacting for 6 hours at room temperature under the protection of argon, and separating by column chromatography to obtain the compound shown in the formula 10.
4. The method as claimed in claim 2, wherein in the sixth step, the specific steps include: dissolving the compound of formula 10 in ethyl acetate, adding 2M ethyl acetate solution of hydrochloric acid, reacting for 4 hours in ice water bath, decompressing and concentrating to remove the solvent, washing the obtained solid with diethyl ether, and drying in vacuum to obtain the compound of formula 11.
5. A transgenic vector consisting of the cationic lipid of claim 1, dioleoylphosphatidylethanolamine, and a highly pure sterilization buffer, wherein the molar ratio of the cationic lipid to the dioleoylphosphatidylethanolamine is 2:1, 1:1, or 1: 2.
6. A method of making the transgenic vector of claim 5, the method comprising in particular the steps of: adding the cationic lipid, dioleoyl phosphatidylethanolamine and anhydrous chloroform into a flask sterilized at high temperature, dissolving, concentrating under reduced pressure to obtain a transgenic carrier membrane, carrying out vacuum drying on the transgenic carrier membrane to remove residual chloroform, mixing the dried transgenic carrier membrane with a trihydroxymethyl aminomethane-hydrochloric acid buffer solution preheated to 70 ℃ in advance to prepare a solution with required concentration, and carrying out ultrasonic treatment to obtain the transgenic carrier, wherein the molar concentration of the trihydroxymethyl aminomethane-hydrochloric acid buffer solution is 10mM, and the pH value is 7.4.
7. Use of the cationic lipid of claim 1 for the preparation of a transgenic vector.
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