CN110003185B - Macrocyclic polyamine amphiphilic compound based on green fluorescent protein chromophore BI and preparation method and application thereof - Google Patents
Macrocyclic polyamine amphiphilic compound based on green fluorescent protein chromophore BI and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a macrocyclic polyamine amphiphilic compound based on a green fluorescent protein chromophore BI, and a preparation method and application thereof. The compound disclosed by the invention is mainly prepared by carbonyl ammonia condensation, 1, 3 dipolar cycloaddition, esterification reaction and Click reaction synthesis. The compound provided by the invention has good two-photon fluorescence property, and has the advantages of emission wavelength, high 3D resolution, low self-luminescence and the like; the invention artificially simulates the color generation mechanism of green fluorescent protein; the compound provided by the invention can form a pH-responsive small-molecule non-viral gene vector with dioleoyl phosphatidylethanolamine (DOPE), thereby promoting the release of DNA, having extremely high transfection efficiency and being capable of targeting cell nucleus.
Description
Technical Field
The invention relates to a non-viral gene vector, in particular to a macrocyclic polyamine amphiphilic compound based on a green fluorescent protein chromophore BI, and a preparation method and application thereof.
Background
Gene therapy is to repair or replace defective genes by introducing normal genes (e.g., DNA, siRNA, mRNA and shRNA) into diseased cells. In recent decades, gene therapy has attracted much attention from researchers as a promising therapeutic approach.
The vectors are divided into two major types, viral gene vectors and non-viral gene vectors. Viral gene vectors can transfect DNA at very high efficiency at low doses, but viral gene vectors also have a risk of carcinogenicity and immunogenicity, have limitations on vector capacity (typically 2-3kb) and are more demanding to manipulate. To address these problems, researchers have developed a variety of non-viral gene vectors as alternatives to viruses. The non-viral gene vector has the advantages of no vector capacity limitation, no infectivity, clear and adjustable chemical structure, easy mass synthesis, simple and convenient use and operation and the like, so the non-viral gene vector has important function in gene therapy. However, conventional non-viral delivery systems still have many problems in clinical treatment, such as accumulation of compounds in vivo, escape of endosomes, single performance, high toxicity and low transfection efficiency. In order to solve these problems, researchers turned to study nature, and viruses, which are the best of nature, could be transfected with very high efficiency by a complex mechanism. They contain different functional components and can perform gradual molecular transformation according to the change of microenvironment, thereby performing high-efficiency transfection. Throughout the transfection process, the gene delivery system is affected by pH changes, oxidation-reduction potential, enzyme activity, and the like. Therefore, designing and synthesizing a gene vector capable of responding to microenvironment stimuli is an important method for improving the transfection efficiency of non-viral gene vectors.
Therefore, the synthesis of a non-viral gene vector which has multiple functions, can stimulate response and has high transfection efficiency has great research value.
Disclosure of Invention
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.
The invention also aims to provide a macrocyclic polyamine amphiphilic compound based on a green fluorescent protein chromophore BI, which takes the BI as a core, the head of the BI unit is connected with a hydrophobic long alkyl chain, and the tail of the BI unit is connected with a hydrophilic macrocyclic polyamine unit to form a hydrophilic and lipophilic amphiphilic compound; the compound can be self-assembled into nano particles with DNA in water; the ternary complex formed by combining the compound with DOPE and DNA can emit green fluorescence, and the light-emitting mechanism of green fluorescent protein is simulated; the compound has two-photon effect after being combined with DOPE and DNA, can be excited by long wave, and has the advantages of high 3D resolution, low self-luminescence and the like; after the compound is self-assembled with DOPE and DNA, the gene is transfected into a cell nucleus, so that the compound has strong fluorescence, and the compound can be used as a non-viral gene vector and can trace the transfection process of the gene. The compound can perform stimulation response to pH, and is more favorable for the release of DNA.
It is also an object of the present invention to provide a process for the preparation of macrocyclic polyamine amphiphiles based on the green fluorescent protein chromophore BI and the use of such compounds.
To achieve these objects and other advantages in accordance with the present invention, there is provided a macrocyclic polyamine-based amphiphilic compound based on the green fluorescent protein chromophore BI, having the following structural formula (I):
in the formula (I), R is a hydrocarbyl structural unit.
Preferably, wherein R is a linear alkyl structural unit.
Preferably, wherein R is
The object of the present invention can be further achieved by a process for the preparation of a macrocyclic polyamine amphiphile based on the green fluorescent protein chromophore BI, comprising the steps of:
1) preparing a BI derivative;
2) preparing a macrocyclic polyamine derivative;
3) the BI derivative reacts with the macrocyclic polyamine derivative to prepare the macrocyclic polyamine amphipathic compound (I) based on the green fluorescent protein chromophore BI.
Preferably, wherein the preparation of the BI derivative in step 1) specifically comprises: step one, preparing a compound shown in a formula (II) through a nucleophilic substitution reaction; secondly, carrying out esterification reaction on the compound shown in the formula (II) and the diazanazobenzoic acid to prepare a compound shown in the formula (III);
preferably, the first step specifically comprises: dissolving 1.0 equivalent of the compound shown in the formula (IV), 2.0 equivalents of bromoalkane and 4.0 equivalents of potassium carbonate in 20mL of acetone, heating and refluxing for reaction for 48 hours, filtering and collecting filtrate, and performing column chromatography purification to obtain a compound shown in the formula (II);
preferably, the second step specifically comprises: equivalent amounts of the compound of formula (II) and 3, 5-bis (azidomethyl) benzoic acid, catalytic amount of DMAP were added to 30mL of DCM, and then 1.5 equivalents of DCC in DCM solution was slowly added dropwise, stirred at room temperature for reaction for 2 days, and purified by column chromatography to give the compound of formula (III).
The aim of the invention can be further realized by the application of the macrocyclic polyamine amphipathic compound based on the green fluorescent protein chromophore BI in a non-viral gene vector.
The object of the present invention can be further achieved by the use of macrocyclic polyamine amphiphiles based on the green fluorescent protein chromophore BI as DNA tracers.
The invention at least comprises the following beneficial effects:
1. the compound of the invention takes a BI unit as a center, the head part of the compound is modified with a hydrophobic long alkyl chain, and the tail part of the compound is connected with a hydrophilic macrocyclic polyamine [12 ]]aneN3Forming an amphiphilic compound that can effectively condense DNA to form nanoparticles therewith;
2. the ternary compound formed by self-assembling the compound, DOPE and DNA has two-photon effect, and has the characteristics of long-wave excitation and low self-luminescence. The light-emitting mechanism of the green fluorescent protein is simulated artificially;
3. the compounds of the invention can respond to pH stimuli and release condensed DNA at pH 5;
4. the compound of the invention can be used as a non-viral gene vector, wherein the transfection efficiency of the compound BI-B given in the example figures is far more than that of a commercial transfection reagent Lipofectamine2000, and the transfection efficiency of the compound BI-B in partial cells is more than 10 times that of Lipofectamine 2000;
5. the compound of the invention can carry out single photon and two-photon imaging tracing on the gene transfection process respectively, is convenient for researching the transfection mechanism, and lays a foundation for the research and development of novel transfection reagents.
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 graph of agarose gel retardation experiments for compounds of the invention and for cationic liposomes prepared from compounds of the invention;
wherein, FIG. 1A is the agarose gel retardation experiment chart of compound BI-A on pUC18 DNA; FIG. 1B is a graph of agarose gel retardation experiments on pUC18 DNA with the compound BI-A/DOPE; FIG. 1C is a graph of an agarose gel block experiment of pUC18 DNA with compound BI-B; FIG. 1D is a graph of an agarose gel retardation experiment of pUC18 DNA with compound BI-B/DOPE; FIG. 1E is a graph of agarose gel retardation experiments for pUC18 DNA with compound BI-C; FIG. 1F is a drawing of an agarose gel retardation experiment of pUC18 DNA with the compound BI-C/DOPE;
FIG. 2 is a graph showing the result of ctDNA fluorescence titration of cationic liposomes prepared from the compounds of the present invention;
wherein, FIG. 2A is a graph of ctDNA fluorescence titration result of cationic liposome BI-A/DOPE; FIG. 2B is a graph of ctDNA fluorescence titration results for cationic liposomes BI-B/DOPE; FIG. 2C is a graph of ctDNA fluorescence titration results for cationic liposomes BI-C/DOPE; FIG. 2D is a graph showing the result of ctDNA fluorescence titration of cationic liposome BI-D/DOPE;
FIG. 3 is a diagram showing agarose gel electrophoresis experiments of cationic liposomes prepared from the compounds of the present invention on pUC18 DNA release;
FIG. 4 is a graph showing the results of luciferase expression in Hela cells of the compound BI-B of the present invention and cationic liposomes formed from it and DOPE;
FIG. 5 is a graph showing the results of expressing luciferase from the compounds BI-A/DOPE to BI-D/DOPE in different cells according to the present invention;
wherein, fig. 5A is the luciferase expression result of the liposome in HeK293T cells; FIG. 5B shows the results of luciferase expression of the liposome in Hela cells; FIG. 5C shows the luciferase expression of the liposome in HepG2 cells; fig. 5D shows the results of luciferase expression of the liposome in a549 cells;
FIG. 6 is a red fluorescent protein transfection expression diagram of compounds BI-A/DOPE-BI-D/DOPE of the present invention for pERFP-N1 gene;
FIG. 7 is a diagram of single-photon confocal imaging of cellular uptake of complexes of cationic liposomes BI-B/DOPE and Cy5-DNA of the present invention at various time periods;
FIG. 8 is a two-photon confocal map of cellular uptake of cationic liposome BI-B/DOPE of the present invention in complexes with Cy5-DNA at different time periods.
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.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
A macrocyclic polyamine amphiphile based on the green fluorescent protein chromophore BI, the compound having the following structural formula (I):
in the formula (I), R is a hydrocarbyl structural unit.
Wherein, when R is a linear alkyl structural unit, and R is
Then, the synthesized macrocyclic polyamine amphiphilic compound based on a green fluorescent protein chromophore BI is marked as BI-A; r is
The synthesized macrocyclic polyamine amphiphilic compound based on the green fluorescent protein chromophore BI is marked as BI-B; r is
The synthesized macrocyclic polyamine amphiphilic compound based on the green fluorescent protein chromophore BI is marked as BI-C; r is
The synthesized macrocyclic polyamine amphiphilic compound based on the green fluorescent protein chromophore BI is recorded as BI-D. The specific synthetic route is as follows:
in the formula, (i) bromoalkane RBr, potassium carbonate and acetone are refluxed for 48 hours; (ii)3, 5-bis (azidomethyl) benzoic acid, DCC, DMAP, DCM for 48 hours; (iii) propargyl [12 ]]aneN3Cuprous bromide and trichloromethane at room temperature for 24 hours; (iv) hydrochloric acid in ethyl acetate at room temperature for 2 hours.
The specific synthesis steps are as follows:
(1) 1.0 equivalent of compound 1 and 2.0 equivalents of bromoalkane and 4.0 equivalents of potassium carbonate were dissolved in 20mL of acetone and heated under reflux for 48 hours. Filtering, collecting filtrate, and purifying by column chromatography, wherein an eluent is as follows: PE and EA are 1:1, and 2-5 of a pure compound is obtained, wherein the yield is 61-84%;
compound 2:1H NMR(600MHz,CDCl3):δ8.09(d,J＝8.8Hz,2H)),7.07(s, 1H),6.93(d,J＝8.9Hz,2H),4.01(t,J＝6.5Hz,2H),3.84(d,J＝4.9Hz,2H), 3.77(t,J＝5.1Hz,2H),2.40(s,3H),1.81-1.76(m,2H),1.60(s,1H),1.52-1.48 (m,2H),0.98(t,J＝7.4Hz,3H).13CNMR(151MHz,CDCl3):δ171.52, 161.29,161.15,136.39,134.28,128.17,126.86,114.90,67.90,61.23,43.73, 31.27,19.29,15.92,13.90.HRMS(ES+)calcd.for C17H22N2O3(M+H)+:303.1703,found:303.1705.
compound 3:1H NMR(600MHz,CDCl3):δ8.08(d,J＝8.5Hz,2H),7.07(s, 1H),6.92(d,J＝8.4Hz,2H),3.99(t,J＝6.6Hz,2H),3.84(t,J＝4.8Hz,2H), 3.77(t,J＝4.8Hz,2H),2.41(s,3H),1.82–1.74(m,2H),1.57(s,1H),1.47– 1.41(m,2H),1.33–1.25(m,8H),0.88(t,J＝6.6Hz,3H).13C NMR(101MHz, CDCl3):171.41,161.46,161.14,136.33,134.28,128.16,126.83,114.89,68.22, 61.00,43.71,31.88,29.42,29.30,29.23,26.08,22.73,15.89,14.18.HRMS(ES+) calcd.for C21H30N2O3(M+H)+:359.2329,found:359.2332.
compound 4:1H NMR(600MHz,CDCl3):δ8.09(d,J＝8.8Hz,2H),7.08(s, 1H),6.93(d,J＝8.9Hz,2H),4.00(t,J＝6.6Hz,2H),3.85(t,J＝5.0Hz,2H), 3.78(t,J＝5.0Hz,2H),2.42(s,3H),1.83–1.75(m,2),1.58(s,1H),1.49–1.42 (m,2H),1.34–1.22(m,16H),0.88(t,J＝7.0Hz,3H).13C NMR(151MHz, CDCl3):δ171.51,161.29,161.16,136.37,134.28,128.19,126.85,114.91,68.23, 61.22,43.72,31.99,29.73,29.71,29.67,29.64,29.46,29.42,29.23,26.08,22.76, 15.91,14.19.HRMS(ES+)calcd.for C25H38N2O3(M+H)+:415.2955,found: 415.2951.
compound 5:1H NMR(400MHz,CDCl3):δ8.06(d,J＝8.9Hz,2H),7.03(s, 1H),6.92(d,J＝8.9Hz,2H),3.99(t,J＝6.6Hz,1H),3.80(s,2H),3.73(d,J＝ 4.9Hz,2H),2.36(s,3H),1.81–1.75(m,2H),1.50–1.41(m,2H),1.26(s,24H), 0.88(t,J＝6.9Hz,3H).13C NMR(101MHz,CDCl3):δ171.39,161.47,161.15, 136.31,134.28,128.18,126.83,114.88,68.23,60.96,43.71,32.00,29.78,29.77, 29.76,29.74,29.73,29.72,29.68,29.66,29.47,29.44,29.24,26.08,22.77,15.88, 14.19.HRMS(ES+)calcd.for C29H46N2O3(M+H)+:471.3581,found:471.3578.
(2) 0.25mmol of compound 2-5, 3, 5-bis (azidomethyl) benzoic acid and a catalytic amount of DMAP are added into 30mL of DCM, then 1.5 equivalents of DCM solution of DCC are slowly dropped into the mixture, and the mixture is stirred and reacted for 2 days at room temperature. Purifying by column chromatography, wherein the eluent is: PE and EA are 1:1, and the pure compound 6-9 is obtained, wherein the yield is 52-63%;
compound 6:1H NMR(400MHz,CDCl3):δ8.08(d,J＝8.8Hz,2H),7.91(s, 2H),7.48(s,1H),7.09(s,1H),6.91(d,J＝8.88Hz,2H),4.52(t,J＝5.5Hz,2H), 4.43(s,4H),4.03–3.98(m,4H),2.39(s,3H),1.75(s,2H),1.31(s,2H),0.96(d, J＝8.2Hz,3H).13C NMR(151MHz,CDCl3):δ170.71,165.51,161.18,160.18, 137.09,134.27,132.20,131.89,129.04,128.22,126.84,126.18,114.92,67.90, 62.81,54.13,39.72,35.00,25.54,15.73,13.89.HRMS(ES+)calcd.for C26H28N8O4(M+H)+:517.2306,found:517.2301.
compound 7:1H NMR(400MHz,CDCl3):δ8.11(s,2H),7.92(s,2H),7.49(s, 1H),7.12(s,1H),6.93(d,J＝5.9Hz,2H),4.54(s,2H),4.42(d,J＝15.9Hz, 4H),4.03(s,4H),2.44(s,3H),1.79(s,2H),1.45(s,2H),1.27(d,J＝12.1Hz, 8H),0.89(s,3H).13C NMR(151MHz,CDCl3):δ170.70,165.51,161.18, 160.20,137.09,134.27,132.21,130.77,129.03,128.20,126.83,126.19,114.92, 68.23,62.81,54.12,39.71,31.87,29.40,29.29,29.22,26.07,22.72,15.73,14.16. HRMS(ES+)calcd.for C30H36N8O4(M+H)+:573.2932,found:573.2930.
compound 8:1H NMR(400MHz,CDCl3):δ8.09(s,2H),7.91(s,2H),7.48(s, 1H),7.09(s,1H),6.92(s,2H),4.52(t,J＝5.52Hz,2H),4.43(s,4H),4.02– 3.97(m,4H),2.39(s,3H),1.89(s,2H),1.74-1,71(m,2H),1.27(d,J＝13.5Hz, 16H),0.87(t,J＝6.86Hz,3H).13CNMR(151MHz,CDCl3):δ170.71,165.51, 161.19,160.17,137.10,134.27,132.20,130.78,129.04,128.23,126.83,126.18, 114.93,68.24,62.81,54.13,39.72,35.00,31.99,29.72,29.70,29.63,29.45, 29.41,29.22,26.07,22.76,15.74,14.18.HRMS(ES+)calcd.for C34H44N8O4(M+H)+:629.3558,found:629.3562.
compound 9:1H NMR(400MHz,CDCl3):δ8.09(d,J＝8.8Hz,2H),7.96– 7.91(m,2H),7.49(s,1H),7.09(s,1H),6.92(d,J＝8.9Hz,2H),4.53(t,J＝5.5 Hz,2H),4.43(s,4H),4.06–3.96(m,4H),2.40(s,3H),1.81–1.76(m,2H), 1.44(m,2H),1.25(s,24H),0.86(d,J＝7.0Hz,3H).13C NMR(101MHz, CDCl3):δ170.71,165.52,161.18,160.20,137.09,134.27,132.23,130.75, 129.04,128.24,126.81,126.21,114.91,68.23,62.82,54.11,39.72,34.05,32.01, 29.78,29.77,29.74,29.67,29.64,29.46,29.44,29.23,26.08,25.70,25.03,22.77, 15.75,14.21.HRMS(ES+)calcd.for C38H52N8O4(M+H)+:685.4184,found:685.4188.
(3) under argon atmosphere, 1 equivalent of compound 6-9, 2.5 equivalents of propargyl [12 ] were added]N3After reacting with a catalytic amount of CuBr, and 5mL of chloroform at room temperature for 24 hours, spin-drying to give a crude product, and column chromatography (eluent: DCM: MeOH ═ 10:1) to give Boc (tert-butyloxycarbonyl) protected products of the corresponding compounds BI-a to BI-D, yields: 51-60 percent. Dissolving the compound in 4mL of ethyl acetate solution of saturated hydrogen chloride, stirring at room temperature for 2 hours, and filtering to obtain corresponding compounds BI-A to BI-D, wherein the yield is as follows: 72 to 90 percent.
Wherein, propargyl [12 ]]aneN3Reference to Bioorganic&Medicinal Chemistry20(2012)801–808。
The compound BI-A:1H NMR(600MHz,DMSO):δ8.54(s,2H),8.34(s,1H),8.16 (d,J＝8.6Hz,1H),7.87(s,2H),7.53(s,1H),7.12–6.92(m,1H),5.77(s,4H), 4.47(s,2H),4.01(q,J＝7.0Hz,4H),3.56(s,4H),3.52–3.35(m,12H),3.26(s, 4H),3.07(s,4H),2.19(s,8H),2.05(s,4H),1.97(s,3H),1.70(s,4H),1.59(s, 2H),1.39(s,2H),1.22(s,4H),0.83(t,J＝6.8Hz,3H).13C NMR(151MHz, DMSO):δ172.46,166.00,165.39,161.23,157.30,137.75,134.66,131.08, 130.86,129.14,128.80,126.69,126.46,115.47,115.44,68.12,63.49,56.57,53.09, 48.09,47.15,41.75,33.81,31.19,25.87,24.95,21.72,19.26,14.29.HRMS(ES+) calcd.for C50H74N14O4(M+H)+:935.6090,found:935.6082.
the compound BI-B:1H NMR(400MHz,DMSO):δ8.42(s,2H),8.16(d,J＝8.7 Hz,2H),7.80(s,2H),7.55–7.44(m,1H),7.37(s,1H),7.05–6.92(m,2H),5.69 (s,4H),4.42(s,4H),4.09–3.92(m,4H),3.81(d,J＝32.5Hz,4H),3.38(d,J＝ 7.1Hz,6H),3.13(d,J＝39.3Hz,12H),2.67(s,3H),2.43–2.33(t,J＝14.6Hz, 8H),2.18(s,4H),2.07(s,4H),1.91(s,4H),1.72(s,2H),1.41(s,2H),1.26(s, 8H),0.86(t,J＝6.5Hz,3H).13C NMR(151MHz,DMSO):δ172.47,166.01, 165.41,163.31,161.17,137.77,134.61,132.72,130.86,129.02,128.64,126.59, 126.52,115.46,115.39,68.30,65.43,63.37,60.28,52.73,46.98,41.48,32.86, 31.74,29.24,29.15,29.10,26.00,22.59,21.62,18.02,15.69,14.47.HRMS(ES+) calcd.for C54H82N14O4(M+H)+:991.6716,found:991.6730.
the compound BI-C:1H NMR(600MHz,DMSO):δ8.50(s,2H),8.11(d,J＝8.0 Hz,2H),7.82(s,2H),7.52(s,1H),6.97(s,3H),5.73(s,4H),4.38(s,4H),4.03– 3.96(m,4H),3.51(s,3H),3.37(s,12H),3.22(s,6H),3.04(s,6H),2.15(s,8H), 2.01(s,4H),1.85(s,3H),1.66(d,J＝6.4Hz,6H),1.56(d,J＝12.2Hz,4H),1.44 (d,J＝12.0Hz,2H),1.39–1.36(m,2H),1.21–1.15(m,4H),1.03–0.99(m, 4H),0.88(t,J＝7.1Hz,3H).1H NMR(101MHz,DMSO):δ172.47,170.85, 166.00,165.39,156.92,137.74,136.99,134.75,132.86,132.78,130.84,129.04, 128.70,115.45,115.43,68.91,68.37,60.33,56.53,52.91,48.14,41.64,33.75, 31.80,29.54,29.52,29.49,29.27,29.22,25.98,25.84,24.91,22.61,21.71,18.03, 14.66,14.49.HRMS(ES+)calcd.for C58H90N14O4(M+H)+:1047.7342,found:1047.7337.
compound BI-D:1H NMR(600MHz,DMSO):δ8.50(s,2H),8.26(s,1H),8.09 (s,1H),7.85(s,2H),7.78(s,1H),7.32(s,1H),6.93(s,2H),5.73(s,4H),4.37(s, 4H),3.97(d,J＝6.7Hz,2H),3.81(s,6H),3.51(s,12H),3.36(s,6H),3.04(s,6H), 2.15(s,8H),2.01(s,4H),1.85(s,3H),1.66(s,4H),1.56(s,4H),1.45(s,2H), 1.35(s,2H),1.17(s,16H),0.78(s,3H).1H NMR(101MHz,DMSO):δ172.48, 170.85,167.59,165.97,161.13,137.94,134.71,133.17,131.17,129.35,128.82, 126.87,126.54,115.70,115.30,68.42,60.47,53.58,53.37,48.02,47.34,42.03, 33.85,31.80,30.86,29.52,29.51,29.30,29.29,29.26,29.20,29.17,29.15,25.90, 25.00,22.63,21.87,21.60,18.29,14.83,14.61.HRMS(ES+)calcd.for C62H98N14O4(M+H)+:1103.7968,found:1103.7983.
< example 2>
Respectively preparing solutions of compounds BI-A-BI-D with different concentrations, placing the BI-A-BI-D and pUC18 plasmid DNA (9 mu g/mL) with different concentrations in a water bath at 37 ℃, incubating for 1h, and then performing a DNA agarose gel retardation experiment to obtain a gel retardation result of the compounds with different concentrations on the pUC18 DNA.
FIGS. 1A to 1H are agarose gel retardation experiments of pUC18 DNA with the compounds BI-A to BI-D and BI-A/DOPE to BI-D/DOPE (compound to DOPE ratio 1: 3), respectively, according to the present invention; the values marked in FIGS. 1A to 1H are test concentrations (. mu.M); FIGS. 1A-1H show that compounds BI-A-BI-D and BI-A/DOPE-BI-D/DOPE (compound to DOPE ratio 1: 3) can completely block DNA migration in agarose at lower concentrations.
From example 2, it can be concluded that the BI unit-based amphiphilic compound prepared by the present invention can effectively aggregate DNA to form nanoparticles, and can be used as a non-viral gene vector.
< example 3>
To solutions of cationic liposomes BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE (compound to DOPE ratio 1: 3) was added ct DNA, and fluorescence intensity was measured and plotted to obtain FIGS. 2A to 2D.
FIGS. 2A-2D are the results of fluorescence titration of ctDNA on complexes BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE (compound to DOPE ratio 1: 3); wherein the X-axis represents the concentration of DNA and the Y-axis represents the fluorescence intensity. As can be seen from example 3, the present invention artificially simulates the process of green fluorescent protein color generation.
< example 4>
Preparing a certain concentration of BI-A/DOPE-BI-D/DOPE (the ratio of a compound to DOPE is 1: 3), forming a compound with pUC18 plasmid DNA, incubating at 37 ℃ for 2 hours, and performing a DNA agarose release experiment under the condition that the pH is 5.2 to obtain the condition that BI-A/DOPE-BI-D/DOPE (the ratio of a compound to DOPE is 1: 3) responds to acid stimulation.
FIG. 3 is a graph of the response of a compound of the present invention to acidity; as can be seen from the figure, BI-A/DOPE-BI-D/DOPE (compound to DOPE ratio 1: 3) can release DNA within 2 hours, which shows that the invention can stimulate response under acidic condition.
< example 5>
Incubating complexes formed by compounds BI-B with different concentrations and DOPE with different proportions and PGL-3 DNA at 37 ℃ for 30 minutes, adding the complexes into cultured Hela cells, acting for 5 hours, sucking out the compounds, rinsing with DMEM, and adding complete culture medium for incubation for 40 hours. Finally, a cell lysate was added, the cells were lysed, and the luminescence intensity and protein content were measured, and the transfection efficiency of the compound was expressed as the luminescence intensity per mg of protein (RLU/mg protein) and as the percentage of the commercial transfection reagent lipofectamine2000 (% of Lipo2000) as a standard sample.
FIG. 4 shows the transfection of complexes of compound BI-B of the invention with DOPE in different ratios as non-viral gene vectors under different concentration conditions, referred to the commercial transfection reagent Lipofectamine 2000; in FIG. 4, the X-axis shows the complex formed by the compound and DOPE in various ratios, wherein the last bar represents Lipofectamine 2000; the Y axis represents luciferase expression level;
in the first set of histograms, the luciferase expression levels of compound BI-A at concentrations of 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M and 35. mu.M are shown in the order from left to right; in the second set of histograms, fluorescence expression at concentrations of 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M and 35. mu.M is shown in order from left to right at a ratio of compound to DOPE of 1: 1; in the third group of histogram, the luciferase expression levels at concentrations of 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M and 35. mu.M at a ratio of compound to DOPE of 1: 2 are shown in order from left to right; in the fourth group of histograms, luciferase expression levels at concentrations of 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M and 35. mu.M at a ratio of compound to DOPE of 1: 3 are shown in order from left to right; in the fifth set of histogram, the luciferase expression levels at concentrations of 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M and 35. mu.M at a ratio of compound to DOPE of 1: 4 are shown in order from left to right; the last one represents the luciferase expression level of Lipo2000 at 10. mu.g/mL;
the results show that in Hela cells, when the ratio of the compounds BI-B/DOPE to DOPE is 1: 3, the optimal transfection efficiency is 10.0 times of that of Lipofectamine 2000.
< example 6>
Reacting compounds BI-A/DOPE-BI-D/DOPE (compound to DOPE ratio is 1: 3) with pGL-3 plasmid DNA at different concentrations at 37 deg.C for 30min, adding into HEK293T, Hela, HepG2 and A549 cells, and culturing for 5 h; then replacing the culture solution containing the compound with a fresh complete culture solution for culturing for 40 h; after removal of the medium, 20. mu.L of cell lysate was added and the relative luminescence intensity and protein content were determined separately, and finally the transfection efficiencies of complexes BI-A/DOPE, BI-B/DOPE, BI-C/DOPE and BI-D/DOPE were expressed as relative luminescence intensity per mg of protein (RLU/mg protein) and percentage of commercial transfection reagent lipofectamine2000 (% of Lipo 2000).
FIG. 5 shows the transfection conditions of compounds BI-A/DOPE-BI-D/DOPE complex (compound to DOPE ratio 1: 3, the same applies below) of the present invention as non-viral gene vectors at different concentrations, with reference to commercial transfection reagent Lipofectamine 2000; in FIG. 5, the X-axis represents the different complexes, wherein the last bar represents Lipofectamine 2000; the Y axis represents luciferase expression level;
FIG. 5A shows the results of luciferase expression of complexes BI-A/DOPE-BI-D/DOPE in HEK 293T;
FIG. 5B shows the results of luciferase expression of complexes BI-A/DOPE-BI-D/DOPE in Hela;
FIG. 5C shows the results of luciferase expression of complexes BI-A/DOPE-BI-D/DOPE in HepG 2;
FIG. 5D shows the results of luciferase expression of complexes BI-A/DOPE through BI-D/DOPE in A549;
(1) the results show that in HEK239T cells, the optimal transfection efficiencies of complexes BI-A/DOPE to BI-D/DOPE are 2.5 times, 11.1 times, 4.0 times and 1.4 times of those of Lipofectamine2000, respectively.
(2) The results show that in Hela cells, the optimal transfection efficiencies of complexes BI-A/DOPE to BI-D/DOPE are 2.3 times, 10.0 times, 4.1 times and 2.6 times of those of Lipofectamine2000, respectively.
(3) The results show that in HepG2 cells, the optimal transfection efficiencies of complexes BI-A/DOPE to BI-D/DOPE were 0.56 times, 4.1 times, 1.2 times and 0.82 times, respectively, that of Lipofectamine 2000.
(4) The results show that in A549 cells, the optimal transfection efficiencies of complexes BI-A/DOPE to BI-D/DOPE are 0.33 times, 5.2 times, 1.1 times and 0.27 times of those of Lipofectamine2000, respectively.
< example 7>
Respectively taking the compounds with the concentrations of 30 mu M, 20 mu M, 15 mu M and 10 mu M to incubate with pERFP for 30 minutes, adding the compounds into Hela cells to culture, then sucking out the culture medium after administration, adding a complete culture medium containing FBS to incubate for 24 hours; finally, sucking out the culture medium, washing the culture medium for 3-5 times by PBS, and taking a picture by a laser confocal scanning microscope; a control group was prepared using lipofectamine2000, a commercial transfection reagent.
As can be seen from example 7, the efficiency of BI-B/DOPE transfection was the highest.
< example 8>
And (2) incubating the cationic liposome BI-B/DOPE and Cy5-DNA for 0.5h, adding the incubated solution into Hela cells for culturing for different times, sucking out the culture medium, washing the culture medium for 3-5 times by PBS (phosphate buffer solution), photographing by a laser confocal scanning microscope, performing biological imaging, and observing the DNA transfection process.
FIG. 7 is a graph of cell uptake obtained by adding the complex BI-B/DOPE-condensed Cy5-DNA to Hela cells and then obtaining the cell uptake profile at various time periods. From example 8, it can be concluded that BI-B/DOPE coagulated DNA into the nucleus after 0.5h, the complex entering the nucleus increased with increasing time.
< example 9>
Experiments were performed according to the above method, and the gene transfection procedure was followed by taking a picture with a two-photon confocal microscope.
FIG. 8 is a graph of cell uptake at various time periods obtained by adding the complex BI-B/DOPE coacervated Cy5-DNA to Hela cells. From example 9 it follows that the complex can be put into two-photon imaging in vivo.
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. A macrocyclic polyamine amphiphile based on the green fluorescent protein chromophore BI, the compound having the following structural formula (I):
in the formula (I), R is
2. A method for preparing the green fluorescent protein chromophore BI based macrocyclic polyamine amphiphile of claim 1, comprising the steps of:
1) preparing a BI derivative;
2) preparing a macrocyclic polyamine derivative;
3) the BI derivative reacts with the macrocyclic polyamine derivative to prepare the macrocyclic polyamine amphipathic compound (I) based on the green fluorescent protein chromophore BI.
3. The method of claim 2, wherein the step 1) of preparing the BI derivative comprises: step one, preparing a compound shown in a formula (II) through a nucleophilic substitution reaction; secondly, carrying out esterification reaction on the compound shown in the formula (II) and the diazanazobenzoic acid to prepare a compound shown in the formula (III);
4. the method of claim 3, wherein the first step specifically comprises: dissolving 1.0 equivalent of the compound shown in the formula (IV), 2.0 equivalents of bromoalkane and 4.0 equivalents of potassium carbonate in 20mL of acetone, heating and refluxing for reaction for 48 hours, filtering and collecting filtrate, and performing column chromatography purification to obtain a compound shown in the formula (II);
5. the method according to claim 3, wherein the second step specifically comprises: equivalent amounts of the compound of formula (II) and 3, 5-bis (azidomethyl) benzoic acid, catalytic amount of DMAP were added to 30mL of DCM, and then 1.5 equivalents of DCC in DCM solution was slowly added dropwise, stirred at room temperature for reaction for 2 days, and purified by column chromatography to give the compound of formula (III).
6. Use of the macrocyclic polyamine amphiphile compound based on the green fluorescent protein chromophore BI as defined in claim 1 for the preparation of non-viral gene vectors.
7. Use of the macrocyclic polyamine amphiphile compound based on the green fluorescent protein chromophore BI as defined in claim 1 for the preparation of DNA tracers.
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