CN109554396B - Imidazole derivative and gene delivery system thereof - Google Patents
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Abstract
The invention belongs to the technical field of pharmacy and biological materials, relates to an imidazole derivative and a gene delivery system thereof, and particularly relates to 2-aminoimidazole with high transfection efficiency and good biocompatibility and a gene delivery system thereof. Experiments prove that the 2-aminoimidazole modified cationic material serving as a novel non-viral vector has high transfection efficiency and good biocompatibility in various cell lines, and the transfection efficiency reaches the levels of PEI25K and lipofectamine 2000; the non-viral vector entraps the therapeutic gene pORF-hTRAIL, and the killing effect on tumor cells is obviously improved.
Description
Technical Field
The invention belongs to the technical field of pharmacy and biological materials, relates to an imidazole derivative and a gene delivery system thereof, and particularly relates to 2-aminoimidazole with high transfection efficiency and good biocompatibility and a gene delivery system thereof.
Background
Gene therapy is to introduce a target gene into a specific tissue cell of a patient through a vector for expression so as to correct or improve defects generated by a pathogenic gene, so that the gene level treatment of some chronic diseases becomes possible. The current gene-carrying vectors are mainly divided into two categories: viral vectors and non-viral vectors. The virus vector makes full use of the infectivity and the parasitism of virus evolution, has high transfection efficiency, and adopts the virus vector in over 70 percent of the current clinical research of gene therapy. However, viral vectors have the disadvantages of complex preparation, small gene capacity, high immunogenicity, potential tumorigenicity, etc., and the wide application thereof is limited. Compared with viral vectors, non-viral vectors have the advantages of controllable quality, high carrying capacity, low immunogenicity and the like, and are favored. However, non-viral vectors have the fatal disadvantage of low transfection efficiency, which limits their clinical application. Therefore, the research for improving the gene transfection efficiency of the non-viral vector is very important.
The ideal non-viral vector needs to overcome the following physiological barriers during gene delivery: (1) The blood barrier protects DNA to effectively avoid phagocytosis of reticuloendothelial system and degradation of nuclease in blood; (2) The cellular barrier carries DNA across the negatively charged amphiphilic cell membrane into the cell; (3) Intracellular transport barriers assist DNA in achieving endosome/lysosome escape, crossing the nuclear membrane, and DNA transfection, with each physiological barrier directly affecting the transfection efficiency of DNA.
Functional molecule modification is one of strategies for overcoming the physiological barriers, and hydrophobic molecules, targeting molecules, membrane penetrating peptides, imidazolyl, guanidino, fusion proteins, nuclear localization signal peptides and the like are modified on the existing non-viral vectors so as to enhance the capacities of DNA compression, DNA carrying in cells, endosome/lysosome escape, nuclear membrane crossing and the like. Among the functional molecules, both the imidazolyl group and the guanidinyl group are derived from natural amino acids, and the functions of both are emphasized in the gene delivery system. The imidazolyl comes from histidine, has strong buffering capacity in the pH range of the endosome, and is favorable for assisting DNA in realizing endosome escape according to a proton sponge effect. The guanidino group is derived from arginine and has the ability to compress DNA, cross cell and nuclear membranes, due to its ability to form bidentate hydrogen bonds with phosphate groups on DNA and biological membranes.
The invention aims to provide a functional molecule which simultaneously compresses DNA, carries the DNA to enter cells, escapes from endosomes/lysosomes and spans nuclear membranes, and is modified on a cationic material to construct a novel non-viral vector so as to achieve the purpose of improving the gene transfection efficiency.
Disclosure of Invention
The invention aims to provide a functional molecule which has the capabilities of compressing DNA, carrying the DNA into cells, escaping from endosomes/lysosomes and crossing nuclear membranes, in particular to an imidazole derivative with high transfection efficiency and good biocompatibility and a gene delivery system thereof.
Based on the structure-activity relationship of imidazolyl and guanidino, the invention designs a functional molecule-2-aminoimidazole for gene delivery, and the functional molecule is connected with a cationic material through a chemical bond to obtain a novel non-viral vector, which is named as R-AM and has the following structure:
structural formula of R-AM
Wherein AM represents 2-aminoimidazole; r represents a cationic material which can be polyethyleneimine, polyamide-amine, chitosan, polycation amino acid, polypropylene imine, polyurethane, polycarbonate, polyphosphate, protamine, spermine, cationic liposome and the like; x represents a connecting group which can be a carbon-nitrogen bond, an amido bond, a carbonate bond, a phosphate bond, urea, thiourea, amidine and the like. n represents a modification ratio.
In the present invention, R-AM is capable of forming a gene complex with a reporter gene or a therapeutic gene, wherein the gene may be DNA, siRNA, mRNA, shRNA, antisense nucleotide, or the like. The buffering capacity of R-AM in the pH range of lysosomes is beneficial to carrying genes to realize lysosome escape. R-AM has good gene compression, cellular uptake and nuclear membrane spanning ability, high gene transfection efficiency and good biocompatibility.
Specifically, the invention is realized by the following technical scheme:
obtaining 2-N-tert-butyloxycarbonyl- (4 formyl) -1H imidazole according to a synthetic route shown in the figure, and obtaining G2-AM after removing a protecting group through the reaction of the amino of 2 generation (G2) of polyamide-amine and the aldehyde group of the compound; as a control, the same reaction links imidazole-4-carbaldehyde and G2 to give G2-M. Acid-base titration experiments, micro-fluorescence methods, flow cytometry and other experiments show that G2-AM has good gene compression, cell uptake, endosome/lysosome escape and nuclear membrane crossing capabilities. Cell qualitative and quantitative transfection experiments show that G2-AM has high transfection efficiency on cells such as HEK 293T, U87, heLa and the like, and has very significant difference compared with G2 and G2-M; cell pharmacodynamic experiments show that the G2-AM entrapped therapeutic gene can improve the killing effect on tumor cells.
The synthetic routes of the G2-AM and the G2-M are as follows:
drawings
FIG. 1: nuclear magnetic resonance hydrogen spectrum. Wherein, FIG. 1a is a nuclear magnetic resonance hydrogen spectrum of N-tert-butyloxycarbonyl-2-amino-4 (dimethoxymethyl) -imidazole, and FIG. 1b is a nuclear magnetic resonance hydrogen spectrum of N-tert-butyloxycarbonyl- ((2-N-tert-butyloxycarbonyl) -4 (dimethoxymethyl)) -imidazole; 1c is the nuclear magnetic resonance hydrogen spectrum of 2-N-tert-butyloxycarbonyl- (4-formyl) -1H imidazole, 1d is the nuclear magnetic resonance hydrogen spectrum of G2-AM, and 1f is the nuclear magnetic resonance hydrogen spectrum of G2-M.
FIG. 2 is a schematic diagram: characterization of the Gene Complex. Wherein, the figure a is the vector/pGL 3 Particle size and potential of (d); FIG. b is a photograph of agarose gel electrophoresis of vector/pGL 3; FIG. c is vector/pGL 3 Transmission electron microscope pictures.
FIG. 3: buffer capacity curve.
FIG. 4: cytotoxicity of G2-AM on HEK 293T cells, U87 cells and HeLa cells.
FIG. 5 is a schematic view of: G2-AM/pGL 3 Cellular uptake in HEK 293T cells. Wherein, the picture a is a micro fluorescence photo, and the picture b is the data measured by the flow cytometer.
FIG. 6: G2-AM/pEGFP-N2 in vitro qualitative transfection. FIG. a is a microscopic fluorescence photograph of HEK 293T cells; panel b is flow cytometry data for three cells.
FIG. 7 is a schematic view of: G2-AM/pGL 3 Quantitative transfection of HEK 293T cells in vitro.
FIG. 8: apoptosis data of G2-AM/pORF-hTRAIL on HeLa cells.
Detailed Description
The following examples will help to further understand the present invention, but do not limit the content of the present invention.
Example 1
Synthesis of compound 2-N-tert-butoxycarbonyl- (4-formyl) -1H-imidazole
Pyruvic acid dimethylacetal (20g, 0.1699 mol) was dissolved in 400mL of CCl 4 In (1), the reaction solution is cooled to 0 ℃ and Br 2 (32.45g, 0.202mol) was slowly added dropwise to the above system, reacted at room temperature under nitrogen for 12 hours, cooled to 0 ℃ and 100mL of NaHCO was added 3 The saturated solution was dropped into the system. The organic phase was separated via a funnel and washed 3 times with NaCl solution (3X 100 ml), dried over anhydrous sodium sulfate and evaporated to remove the organic solvent to give intermediate 3, 3-dimethoxy-2-oxo bromopropane, which was used in the next reaction without further purification.
Boc-guanidine (10g, 63mmol) and intermediate 3, 3-dimethoxy-2-oxobromopropane (18.5g, 76mmol) were dissolved in 200ml of THF, and tetraisopropyl titanate (5.96g, 21mmol) was added to the reaction solution, followed by stirring at room temperature. After 48 hours the organic solvent was filtered and evaporated to give crude N-tert-butoxycarbonyl-2-amino-4 (dimethoxymethyl) -imidazole. The crude product was purified by column chromatography (eluent: dichloromethane/methanol/triethylamine = 3/1/0.5) to give 13.68g of white solid in 84.5% yield.
Intermediate N-tert-Butoxycarbonyl-2-amino-4 (dimethoxymethyl) -imidazole (5.0g, 19.45mmol) was added to N 2 Dissolved in 50ml of THF under protection, diisopropylethylenediamine (3.01g, 23.34mmol) and di-tert-butyl dicarbonate (5.08g, 23.34mmol) were added to the reaction system, the mixture was stirred at room temperature for 12 hours, the organic solvent was evaporated under reduced pressure, and CH was used 2 Cl 2 Redissolved, washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: dichloromethane/ethyl acetate = 5/1) to give 6.42g of N-tert-butoxycarbonyl- ((2-N-tert-butoxycarbonyl) -4 (dimethoxymethyl)) -imidazole as a colored solid in 92.1% yield.
The compound N-t-butyloxycarbonyl- ((2-N-t-butyloxycarbonyl) -4 (dimethoxymethyl)) -imidazole (2.0g, 5.6 mmol) was dissolved in 20ml of CH 2 Cl 2 In (1), the reaction system is cooled to 0 ℃ and pyridine hydrochloride is addedThe reaction solution was added, stirred overnight at room temperature and checked by TLC for progress. After completion of the reaction, 10ml of water were poured into the solution, washed 3 times with saturated sodium chloride solution (3X 10 ml) and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: dichloromethane/methanol = 10/1) to give 0.99g of 2-N-tert-butoxycarbonyl- (4-formyl) -1H-imidazole as a white solid in 84.0% yield.
Example 2
Synthesis of G2-AM and G2-M
G2 and 2-N-tert-butoxycarbonyl- (4-formyl) -1H-imidazole were dissolved in methanol, stirred at 45 ℃ for 24 hours under nitrogen protection, added with sodium borohydride in an amount equivalent to the molar amount of 2-N-tert-butoxycarbonyl- (4-formyl) -1H-imidazole, and stirred at room temperature overnight. The organic solvent was evaporated under reduced pressure to give a yellow solid, which was then dissolved in CH containing 20% trifluoroacetic acid 2 Cl 2 And stirring the solution at room temperature for 8 hours, dialyzing (molecular weight cut-off is 500-1000 Da), wherein the dialyzate is distilled water containing a small amount of ammonia water, freeze-drying the dialyzate after 48 hours to obtain G2-AM, and determining the modification ratio to be 11 by nuclear magnetic resonance hydrogen spectrum.
Dissolving G2 and imidazole-4-formaldehyde in methanol, stirring for 24 hours at 45 ℃ under the protection of nitrogen, adding sodium borohydride with the same molar quantity as imidazole-4-formaldehyde, stirring overnight at room temperature, dialyzing (molecular weight cut-off is 500-1000 Da), wherein the dialyzate is distilled water, freeze-drying the dialyzate after 48 hours to obtain G2-M, determining the modification ratio to be 11 through nuclear magnetic resonance hydrogen spectrum, and using the G2-M as a control of subsequent experiments.
Example 3
Preparation and characterization of G2-AM/pDNA
Mixing pGL 3 Isovolumetrically mixing the solution and the carrier material solution, immediately whirling for 30s when the final concentration of pGL3 is 40 mu g/mL, standing at room temperature for 30min to obtain a freshly prepared compound solution, wherein the proportion of the carrier to pGL3 in the compound is represented by mass ratio, and the particle size and the Zeta potential of each sample are respectively measured by a Malvern NanoZS particle size analyzer, and the result is shown in figure 2 a; dropping 10 μ l of the complex on a copper net, dispersing, sucking off the liquid with filter paper, staining with phosphotungstic acid, oven drying, observing with a transmission electron microscope and taking picturesAs shown in figure 2 c. The results show that G2-AM can compress pGL3 to form nanoparticles with the particle size of less than 100nm when the mass ratio is 12, and the potential is about 18 mv.
The agarose gel electrophoresis retardation experiment examines the compression capacity of the vector material on pGL3, and the specific experimental scheme is as follows: 0.3g Agarose was weighed into a small Erlenmeyer flask, 30ml of 1 XTAE buffer was added, and heating was carried out in a microwave oven (medium high fire) for 2-3 times in a discontinuous manner until the Agarose was completely dissolved. Cooling to 50 deg.C at room temperature, adding 3 μ L gel red, mixing, pouring gel, and inserting into comb. Standing at room temperature for 40min or a little longer, and pulling out the comb after the gel is solidified. Pouring 1 XTAE into the electrophoresis tank in advance, putting the gel, and allowing the liquid to submerge the gel surface. Subsequently, the samples (prepared as above) were added to the sample wells, respectively, and subjected to electrophoresis [ electrophoresis conditions: voltage 120V,1 XTAE, 40min, 10. Mu.l per well ]. The DNA bands were observed under UV and photographed. As a result, as shown in FIG. 2c, when the mass ratio of G2-AM to pGL3 was 4, G2-AM completely compressed the pDNA.
Example 4
Buffered titration experiments on materials
The acid-base titration experiment examines the buffer capacity of the material, and respectively examines the buffer capacity of G2-AM, G2-M and G2, and PEI25K is used as a control group. The three materials are all prepared into 30mL of aqueous solution with the concentration of 0.2mg/mL, and the pH values are respectively adjusted to be about 10 by using 0.1M NaOH solution. Titration with 0.1M HCl at room temperature with vigorous stirring was carried out using 20. Mu.L of HCl each time. The pH of the solution was measured with a pH meter. According to the experimental result, the buffer index mapping is calculated, and the result is shown in figure 3, the G2-AM buffer capacity is obviously better than that of G2 and G2-M in the pH value range of 4.2-5.8, and lysosome escape is realized after the G2-AM carrying gene enters the cell.
Example 5
Cytotoxicity assay of materials
The cells were plated in 96-well plates, and 200. Mu.l of 10% serum-containing DMEM medium containing 3X 10 cells per well 3 cells/well, 37 ℃,5% CO 2 Culturing overnight under the conditions, adding the materials into 96-well plate, setting each well with 10 μ l, setting each sample with 3 multiple wells, placing into cell incubator, incubating for 4h, changing DMEM culture solution containing 10% serum, culturing for 48h, and preparing 5mThe MTT solution of g/mL and a filter membrane of 0.22 μm are filtered, added into a 96-well plate, 20 μ l of each well is cultured for 4h, the cell culture solution is discarded, 200 μ l of DMSO is added into each well, the mixture is placed at room temperature for 30min and then shaken to completely dissolve purple crystals, and an enzyme-linked immunosorbent assay (ELIASA) is used for measuring the absorbance at the wavelength of 490nm, and the result is shown in figure 4: among the three cells, G2-AM shows lower toxicity, and the cell survival rate of the three concentrations is over 90 percent; as a positive control group, PEI25K showed higher toxicity, and the survival rate of the three cells was below 30% at high concentration.
Example 6
Cell uptake assay
Laser confocal experiments: the HEK 293T cells were trypsinized to a cell concentration of about 4X 10 4 Per mL, add 0.5mL cell suspension per well in the confocal dish, shake well and place in the incubator at 37 ℃ and 5% 2 Incubated overnight under conditions. Gene complexes were prepared with a material/pGL 3 mass ratio of 12, and pGL3 was labeled with TOTO-3 dye according to the same preparation method and concentration as those of example 3. Removing the culture solution from the confocal dish, replacing each well with 0.5mL of cell culture solution without FBS, gently shaking 50 μ L of gene complex in each well, mixing uniformly, placing in an incubator for culturing for 4h, removing the culture solution, fixing cells with paraformaldehyde, staining cell nuclei with DAPI staining solution for 5min, and taking pictures with a laser confocal microscope, wherein the results are shown in figure 5a. pGL3 encapsulated by G2-AM has obvious fluorescence and obvious co-localization with cell nucleus, which shows that the AM modification obviously improves the capability of G2-AM/pGL3 entering cells and crossing nuclear membranes.
Flow-type quantitative experiment: cell culture and resuspension were performed as above, and 1X 10 counts were obtained 5 Adding 1mL of cell suspension into each hole of a 12-hole plate per mL, and culturing overnight to allow cells to adhere to the wall; the administration mode is the same as the above, cells are digested by pancreatin after 4 hours, PBS is resuspended, the detection is carried out by a flow cytometer, the result is shown in a figure 5b, the AM modification obviously improves the cell uptake efficiency of G2-AM/pGL3, and the cell uptake efficiency is slightly higher than that of PEI 25K.
Example 7
Cell transfection assay for Green fluorescent protein plasmids
Cells were plated at 2X 10 4 One well was inoculated into 48-well plates containing 0.5mL of DMEM medium (containing 10% serum) per well and incubated overnight toThe confluence degree of the cells reaches 70-80%, a freshly prepared carrier material/pEGFP-N2 compound (the mass ratio is 12) is added after the equal volume of serum-free culture solution is replaced in each hole, 50 mu l of the culture solution containing 2 mu g of pEGFP-N2 plasmid is cultured in each hole for 4 hours at 37 ℃, the culture solution is replaced by fresh culture solution containing 10% of serum, the culture solution is continuously cultured for 48 hours at 37 ℃, and the culture solution is placed under a fluorescence microscope for observation; discarding culture solution, digesting with pancreatin, centrifuging, suspending the cells in PBS, and detecting the transfection efficiency by a flow cytometer. The result is shown in FIG. 6, and a fluorescent photograph 6a shows that the transfection efficiency of G2-AM/pEGFP-N2 is remarkably improved by AM modification, and the transfection efficiency is equivalent to that of PEI25K and higher than that of lipofectamine; flow results 6b show that AM modification significantly improved the transfection efficiency of G2-AM/pEGFP-N2 in all three cells.
Example 8
Cell quantitative transfection assay of luciferase plasmid
Cells were plated at 2X 10 4 Inoculating each well to 48-well plate containing 0.5mL DMEM culture solution (containing 10% serum), culturing overnight until cell confluence reaches 70-80%, replacing equal volume of serum-free culture solution for each well, and adding freshly prepared carrier material/pGL 3 The complex (12 mass ratio) contained 2. Mu.g of pGL in 50. Mu.l per well 3 Culturing at 37 ℃ for 4h, then replacing with fresh culture solution containing 10% serum, continuously culturing at 37 ℃ for 48h, then discarding the culture solution, adding 100 mu l of cell lysate into each hole, standing for 1-2min, blowing a pipette to assist cell lysis, then transferring the cell lysate into a 1.5mL centrifuge tube, centrifuging at 15000 Xg and 4 ℃ for 2min, and taking the supernatant and transferring the supernatant into an EP tube to be measured.
According to the Promega kit operating instructions, luciferase substrate is prepared into working solution, then 40 mul of supernatant is placed in a test tube, 90 mul of luciferase substrate is added, the test tube is placed in an ultra-weak luminescence analyzer to measure the luminescence intensity, the collection time is 10s, the time interval is 1s, then the total protein content of cells is measured by a MicroBCA method, the transfection result is expressed by the fluorescence intensity emitted by the total protein per unit mass (RLU/mg protein), and the experimental result is shown in figure 7: the transfection efficiency of G2-AM/pGL3 was 100 times that of G2/pGL3 and 1000 times that of G2-M/pGL3, respectively, which was comparable to that of PEI 25K.
Example 9
Apoptosis assay
In vitro apoptosis experimental studies were performed using pORF-hTRAIL as the therapeutic gene to evaluate the ability of AM-modified vector materials to induce apoptosis in tumor cells after gene loading. HeLa cells were cultured at 8X 10 4 The density of each hole is inoculated on a 12-hole plate, each hole contains 1mL of DMEM culture solution (10% serum), the cell confluency is 70% -80% after being cultured overnight, each hole is changed into the DMEM culture solution without serum with the same volume, then a freshly prepared carrier material/pORF-hTRAIL gene compound (the mass ratio is 12) is added, 50 mu L of each hole contains 2 mu g of pORF-hTRAIL, the DMEM culture solution is changed into a fresh culture solution after being cultured for 4h at 37 ℃, the culture solution is continuously cultured for 48h, the culture solution is discarded, the cells are washed for 2 times by PBS, trypsinized and centrifuged, the collected cells are resuspended in 300 mu L of 1 Xbuffer solution, then 3 mu Lannexin V-FITC is added, 3 mu L of PI is added after being uniformly mixed, and the mixture is uniformly mixed. Incubating for 15min at room temperature in dark place, and finally quantitatively determining the apoptosis percentage by using a flow cytometer. The results are shown in FIG. 8, the early and late apoptosis rate and the death rate of HeLa cells in the G2/pORF-hTRAIL group are 8.47%, the G2-M/pORF-hTRAIL group is 7.27%, and the G2-AM/pORF-hTRAIL group is improved to 20.88%, which shows that the killing capacity of the G2-AM/pORF-hTRAIL on the HeLa cells is obviously improved by AM modification.
Claims (6)
1. A non-viral vector with high transfection efficiency and good biocompatibility, characterized in that the non-viral vector is obtained by connecting 2-aminoimidazole and a cationic material through a chemical bond, is named R-AM, and has the structure shown in the following figure: AM represents 2-aminoimidazole, R represents a cationic material, X represents a linking group, n represents a modification ratio,
structural formula of R-AM
R in the R-AM is a cationic material selected from polyethyleneimine, polyamide-amine, chitosan, polycationic amino acid, polypropylene imine, polyurethane, protamine or cationic lipid.
2. The non-viral vector according to claim 1, wherein X in R-AM is a carbon nitrogen bond, an amide bond, a carbonate bond, a phosphate bond, urea, thiourea or amidine.
3. The nonviral vector of claim 1, wherein the R-AM comprises a reporter gene and a therapeutic gene to form a gene delivery system.
4. The non-viral vector according to claim 3, wherein the reporter gene and the therapeutic gene are DNA, siRNA, mRNA, shRNA or antisense nucleotides.
5. The nonviral vector of claim 1, wherein said R-AM has good ability to compact genes, cellular uptake, endosome/lysosome escape, and nuclear membrane spanning.
6. The non-viral vector according to claim 3, wherein the gene delivery system comprising the entrapped therapeutic gene is used for the treatment of cancer, genetic diseases, infectious diseases, cardiovascular diseases and autoimmune diseases.
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