CN102876718A - Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof - Google Patents
Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof Download PDFInfo
- Publication number
- CN102876718A CN102876718A CN2012103564312A CN201210356431A CN102876718A CN 102876718 A CN102876718 A CN 102876718A CN 2012103564312 A CN2012103564312 A CN 2012103564312A CN 201210356431 A CN201210356431 A CN 201210356431A CN 102876718 A CN102876718 A CN 102876718A
- Authority
- CN
- China
- Prior art keywords
- tree
- amide
- polyamide
- gene transfection
- low
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Polyamides (AREA)
Abstract
The invention provides a novel gene transfection vector based on low-algebraic polyamidoamine. The gene transfection vector comprises low-algebraic polyamidoamine and a difunctional cross-linking agent which contains a disulfide bond and of which the reaction points to amino, wherein a primary amine group of the low-algebraic polyamidoamine is reacted and connected with active groups at two ends of the difunctional cross-linking agent. The invention also provides a method for preparing the gene transfection vector and application of the gene transfection vector which is used as a nucleic acid molecule delivery vector in vivo or in vitro. The gene transfection vector is low in synthetic cost and small in cytotoxicity, can deliver gene molecules to cells effectively and safely and can be used as the gene transfection vector which has the advantages of high efficiency, low toxicity, low cost and the like.
Description
Technical field
The present invention relates to polymer chemistry and technical field of biological materials, be specifically related to a kind of based on tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide and its preparation method and application.
Background technology
Gene transfection refers to the foreign gene transfered cell, thereby obtains the process of new inherited character.The gene transfection carrier is the core of Gene transfer techniques, and desirable gene transfection carrier should possess following features: high transfection efficiency, and low cytotoxicity, biological safety is good, and is cheap etc.The gene transfection carrier of current use mainly comprises viral vector and non-viral vector.The high viral vector of transfection efficiency is still adopted in main applied research, but there is problems such as carrying the limited in one's ability and security hidden danger of gene in viral vector.
The tree-like polymer of polyamide-amide (polyamidoamine, PAMAM) in 1985 by Tomalia D.A. reported first, now be widely used in catalysis, photochemistry, electrochemistry, supramolecular chemistry, the energy, Pollution abatement, nano material, the various fields such as biomedicine.Application facet in the gene transportation; the tree-like polymer of polyamide-amide of uncle's amino end group has unique structure and performance; make it form nano-complex in conjunction with gene molecule easily; and transport it in the cell by the endocytosis mode; avoid degrading in endosome by proton sponge effect protection gene molecule subsequently, further therefrom escape and gene molecule is discharged into tenuigenin, nucleus carries out transcribing of gene; the processes such as translation realize the efficient gene conveying.In addition, the monomeric compound that the tree-like high molecular chemical constitution of polyamide-amide is non-immunogenic has reduced the potential safety hazard of virus type and protein gene transfection carrier during as the gene transfection carrier.Therefore, efficient, safe characteristics are so that the tree-like polymer of polyamide-amide becomes the potential substitute of virus, liposome and polyelectrolyte genoid carrier.At present, take the tree-like polymer of polyamide-amide as the basis the gene transfection carrier as
With
Successful commercialization.
But the tree-like polymer of polyamide-amide is high material cost and the incident high cell toxicity of high gene transfection efficiency for the general Problems of gene transfection carrier on basis.The tree-like Polymers Synthesizing Technology of polyamide-amide is complicated, need to expend a large amount of manpower and financial resources, and along with the increase of tree-like polymer algebraically, material cost becomes geometricprogression to increase.Usually only have the tree-like polymer of polyamide-amide of high algebraically (greater than the 5th generation) just to have desirable efficiency gene transfection, and concerning cationic gene transfection carrier, high efficiency gene transfection is accompanied by again high cytotoxicity.In recent years, researchers pass through the tree-like macromolecule surface grafting of the polyamide-amide of high algebraically amino acid, aliphatic chain and targeted molecular etc. carry out chemical modification to it and modify, although improved to a certain extent the tree-like high molecular efficiency gene transfection of polyamide-amide or reduced its cytotoxicity, but tree-like polymer genoid transfection carrier is expensive, and the high cell toxicity problem that the high gene transfection efficiency is followed does not still improve.
Summary of the invention
The present invention overcomes the deficiency of the tree-like polymer genoid of prior art transfection material, the present invention has proposed a kind of based on the tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide with innovating, be the gene transfection material that is cross-linked to form by the low tree-like polymer of algebraically polyamide-amide, can be used as efficient, safe, inexpensive gene transfection carrier and use.Preparation method of the present invention utilizes the tree-like polymer of polyamide-amide of low algebraically (4 generations of 1st generation to the) as raw material, synthetic cost is lower, cytotoxicity is less, take its primary functional elements as the gene transfection carrier, obtain to possess simultaneously the gene transfection carrier of high transfection efficiency, low toxicity, advantage of low cost.
The invention provides a kind of suc as formula shown in (I) based on the tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide, comprise the tree-like polymer of polyamide-amide and contain disulfide linkage and the amino bi-functional cross-linking agent of reaction sensing, the active group reaction at the primary amine group of the tree-like macromolecule surface of described polyamide-amide and described bi-functional cross-linking agent two ends links to each other with amido linkage;
Wherein, R is the tree-like polymer of polyamide-amide, is the tree-like polymer that synthesizes as monomer with quadrol and methyl acrylate, and end group is primary amino group; Its structure is suc as formula shown in (II):
In the formula (II), M is the tree-like high molecular centronucleus of described polyamide-amide, and M is ammonia, quadrol, butanediamine, hexanediamine, octamethylenediamine or certain herbaceous plants with big flowers diamines.N is the tree-like high molecular algebraically of described polyamide-amide, and n is the integer of 1-4.M is the functionality of the tree-like polymer centronucleus of described polyamide-amide, and m is the integer of 2-4.
In the formula (I), k is the integer of 1-6; X is the tree-like high molecular mol ratio of bi-functional cross-linking agent and polyamide-amide, and x is 0.25-4.0.
Among the present invention, by the tree-like polymer of polyamide-amide of low algebraically and contain disulfide linkage and reaction point to amino bi-functional cross-linking agent crosslinked consist of a kind of novel polymer-based because of transfection carrier, the tree-like polymer of described polyamide-amide is the tree-like polymer that synthesizes as monomer with quadrol and methyl acrylate, and the tree-like high molecular end group of this polyamide-amide is primary amino group.Described low algebraically is 4 generations of 1st generation to the, and namely n is the integer of 1-4.
Among the present invention, " contain disulfide linkage and amino bi-functional cross-linking agent is pointed in reaction " refer to chain-like structure, two ends be can with the active group of amino reaction, and be a class linking agent of the disulfide linkage structure of isotope of redox-sensitive in the middle of the segment.Described can with the active group of amino reaction, comprise the N-hydroxy-succinamide ester, imidazoles-1-manthanoate, imidoether etc.The disulfide linkage structure of described isotope of redox-sensitive can be sheared by reductive agents such as intracellular gsh.
Among the present invention, the described bi-functional cross-linking agent that contains disulfide linkage and reaction sensing amino is dithio dicarboxylic acid two (N-maloyl imines ester), and wherein, k is the integer of 1-6.Form structure shown in (Y):
The invention allows for a kind of preparation method based on the low tree-like high molecular novel gene transfection carrier of algebraically polyamide-amide, take the tree-like polymer of described polyamide-amide with describedly contain disulfide linkage and reaction is pointed to amino bi-functional cross-linking agent as raw material, in at first that the tree-like polymer of described polyamide-amide is the water-soluble or damping fluid, in this solution, dropwise drip the solution that is dissolved with described bi-functional cross-linking agent afterwards, after being added dropwise to complete with this reaction soln stirring reaction at a certain temperature, with damping fluid and distilled water dialysis postlyophilization, obtain as claimed in claim 1 based on the tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide respectively.
Among the preparation method of the present invention, described pH of cushioning fluid is 6.0 to 12.0.
Among the preparation method of the present invention, the solution that is dissolved with described bi-functional cross-linking agent comprises water, damping fluid, methyl-sulphoxide, DMF, tetrahydrofuran (THF).
Among the preparation method of the present invention, the described reaction times is 2 hours to 96 hours.
Among the preparation method of the present invention, described temperature of reaction is 0 degree centigrade to 50 degrees centigrade.
Among the preparation method of the present invention, it is 1000Da or 3500Da that described dialysis is blocked molecular weight with dialysis tubing.
Among the preparation method of the present invention, the tree-like polymer of described polyamide-amide is 1st generation to the tree-like polymer of polyamide-amide in 4 generations for the tree-like polymer of polyamide-amide of low algebraically.
Among the preparation method of the present invention, the bi-functional cross-linking agent that the tree-like polymer of polyamide-amide of low algebraically is amino with containing disulfide linkage and reaction sensing is by the active group at the tree-like high molecular terminal primary amine groups of polyamide-amide and described bi-functional cross-linking agent two ends, N-hydroxy-succinamide ester for example, imidazoles-1-manthanoate, imidoether, reaction generates crosslinked supramolecular structure and is prepared from.
The tree-like Polymers Synthesizing Technology of prior art is complicated, need to expend a large amount of manpower and materials, and along with its synthetic cost of increase of tree-like polymer algebraically is the geometricprogression increase.Compare with high algebraically like product on prior art and the market, formula of the present invention (I) is to be prepared from as elementary cell by the tree-like polymer that hangs down algebraically based on the low tree-like high molecular novel gene transfection carrier of algebraically polyamide-amide, can effectively and safely gene molecule be transported in the cell, with synthetic with low cost, the preparation method is easy, transfection efficiency is high, low toxin.
The invention allows for a kind of based on the application as the delivery vehicles of nucleic acid molecule in external or body of the low tree-like high molecular gene transfection carrier of algebraically polyamide-amide.Described nucleic acid comprise DNA, siRNA, shRNA or modified after nucleic acid etc.
The invention allows for a kind of mixture, it comprises and contains describedly based on the tree-like high molecular gene transfection carrier of low algebraically polyamide-amide and nucleic acid, and polymer-based carries described nucleic acid because of transfection carrier by described based on low algebraically polyamide-amide is tree-like.
As reporter gene, utilize transfection carrier of the present invention to carry plasmid with green fluorescence protein gene and luciferase gene, experiment shows that the present invention has the following advantages: the present invention keeps preferably biocompatibility when keeping high efficiency gene transfection.Find by the gene transfection experiment, be that the efficient of transfection luciferase and green fluorescent protein under 40: 1 the condition and transfection efficiency that bPEI 25kD N/P ratio under optimal conditions is at 10: 1 o'clock are suitable at N/P ratio, be higher than the tree-like high molecular transfection efficiency of the 5th PAMAM far away; Find that by MTT cytotoxicity test experience the cytotoxicity of rotaring redyeing gene carrier of the present invention is less, far below with bPEI 25kD under the concentration and the tree-like polymer of the 5th PAMAM.
Description of drawings
Fig. 1 is synthetic route and the genophore structure iron of preparation gene transfection carrier G2DSP1-x among the embodiment 1.
Fig. 2 is the nuclear magnetic resonance map of the gene transfection carrier G2DSP1-x of gained among the embodiment 1.
Fig. 3 is the agarose gel electrophoresis figure of gene transfection carrier G2DSP1-1 and the mixture that DNA forms of gained among the embodiment 1.
Fig. 4 is the agarose gel electrophoresis figure of gene transfection carrier G2DSP1-1 and the mixture antagonism DNA enzyme liberating that DNA forms of gained among the embodiment 1.
Fig. 5 is the gene transfection carrier G2DSP1-1 of gained among the embodiment 1 and other transfection carrier dyes luciferase gene at the HEK293 transit cell transfection efficiency comparison diagram.
Fig. 6 is that the gene transfection carrier G2DSP1-1 of gained among the embodiment 1 and other transfection carrier are at the transfection efficiency comparison diagram of HEK293 transit cell green colouring fluorescence protein gene.
Fig. 7 is that the gene transfection carrier G2DSP1-1 of gained among the embodiment 1 is having or not reducing agent dithiothreitol (DTT) to have Transfection of Enhanced Green Fluorescent efficient comparison diagram in the situation.
Fig. 8 be the gene transfection carrier G2DSP1-1 of gained among the embodiment 1 and other transfection carrier to (a) HeLa, (b) the toxicity comparison diagram of HEK293 cell.
Fig. 9 is the gene transfection carrier G2DSP1-1 of gained among the embodiment 1 and other transfection carrier toxicity comparison diagram to the HeLa cell after forming mixture with DNA.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope that do not deviate from inventive concept, variation and advantage that those skilled in the art can expect all are included in the present invention, and take appending claims as protection domain.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content of mentioning specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
Embodiment 1: preparation is based on the tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide
Take based on low algebraically polyamide-amide tree-like polymer-based because of transfection carrier G2DSP1-x as example, as shown in Figure 1, wherein, G2 is the tree-like polymer of s-generation polyamide-amide, centronucleus is quadrol, end group is primary amine group, n is that 3, m is 2; DSP is 3, and 3 '-dithiodipropionic acid two (N-maloyl imines ester), 1-x are the cross liner DS P of the corresponding x molar equivalent of 1 molar equivalent G2, x=0.25-4.0, and its structural formula is:
In the formula (I), R is the tree-like polymer G2 of s-generation polyamide-amide; Its structure is suc as formula shown in (II):
In the formula (II), M is the centronucleus of the tree-like polymer G2 of s-generation polyamide-amide: quadrol.
In implementation of the present invention, M can also be ammonia, quadrol, butanediamine, hexanediamine, octamethylenediamine or certain herbaceous plants with big flowers diamines.
The preparation synthetic method: the tree-like polymer of 2nd generation polyamide-amide of getting 100 milligrams is dissolved in 3 milliliters of PBS damping fluids (pH 7.4), dropwise drip in this solution be dissolved with 3.1 milligrams to 49.7 milligrams 3,1.5 milliliters of the dimethyl sulfoxide solutions of 3 '-dithiodipropionic acid two (N-maloyl imines ester), after being added dropwise to complete this reaction soln was continued stirring reactions 12 hours under 25 degrees centigrade, then repeatedly dialyse with PBS damping fluid and distilled water, last lyophilize, can obtain outward appearance is the gene transfection carrier G2DSP1-x of white powder, wherein, x is the mole number of cross liner DS P corresponding to 1 molar equivalent G2.
The gene transfection carrier G2DSP1-x for preparing by above-mentioned steps is carried out nuclear magnetic resonance spectroscopy, as shown in Figure 2.Fig. 2 is respectively from top to bottom x and equals 0.25,0.5, the one dimension nucleus magnetic hydrogen spectrum of 1,2,4 o'clock gained samples.As seen from Figure 2, the tree-like polymer of G2 polyamide-amide and 3, crosslinking reaction has occured in 3 '-dithiodipropionic acid two (N-maloyl imines ester), along with linking agent 3, the concentration of 3 '-dithiodipropionic acid two (N-maloyl imines ester) increases, the corresponding enhancing of the proton signal of the linking agent that detects on the nuclear magnetic resonance spectrum.
In subsequent embodiment, mainly take gene transfection carrier G2DSP1-1 as example Benq because of the effect in the transfection experiment.
Embodiment 2: the stability of gene transfection carrier G2DSP1-1 and DNA mixture
Gene transfection agent G2DSP1-1 prepared among the embodiment 1 and green fluorescent protein plasmid DNA (pEGFP-1) are at room temperature formed mixture, detect the ability of the compound DNA of gene transfection agent G2DSP1-1 by agarose gel electrophoresis.
Concrete grammar is: gene transfection agent G2DSP1-1 (was respectively 0: 1 by different N/P ratios from green fluorescent protein plasmid DNA (pEGFP-1), 0.5: 1,1: 1,2: Isosorbide-5-Nitrae: 1 and 8: 1) mix under the room temperature, hatched 30 minutes, then with the dilution of DNA sample-loading buffer, at last sample is depressed at 90 voltaisms and carried out the agarose gel electrophoresis experiment, experimental period is 50 minutes, and the concentration of sepharose is 1%.Adopt the tree-like polymer G2 of 2nd generation polyamide-amide and the tree-like polymer G5 of the 5th PAMAM in contrast in the experiment.
Do not assessed the stability of G2DSP1-1/DNA mixture by the ability of DNA enzyme liberating by detecting GSDSP1-1 protection plasmid DNA; concrete grammar is: gene transfection agent G2DSP1-1 and 0.8 microgram green fluorescent protein plasmid DNA (pEGFP-1) were at room temperature hatched 30 minutes by N/P ratio at 8: 1; then add 3 unit of activity/microlitre DNA enzyme 1 microlitre, hatched 2 hours for 37 degrees centigrade.Further analyze the stability of G2DSP1-1/DNA mixture by the polyanion competition law: the heparin sodium aqua of 40 mg/ml is joined in the G2DSP1-1/DNA complex solution incubated at room 10 minutes; In another parallel sample, after G2DSP1-1/DNA mixture and DNA enzyme hatched, with enzyme deactivation, and add heparin sodium.At last sample is depressed at 90 voltaisms and carried out the agarose gel electrophoresis experiment, experimental period is 50 minutes, and the concentration of sepharose is 1%.
Experimental result: the agarose gel electrophoresis figure of the mixture that gene transfection carrier G2DSP1-1 as shown in Figure 3 and DNA form, wherein, N/P is N/P ratio.Fig. 3 shows that gene transfection agent G2DSP1-1 forms stable mixture when mixing with the N/P ratio more than 2: 1 with DNA.
The agarose gel electrophoresis figure of the mixture antagonism DNA enzyme liberating that gene transfection carrier G2DSP1-1 as shown in Figure 4 and DNA form.Swimming lane 1 is green fluorescent protein plasmid DNA among the figure; Swimming lane 2 is the mixture that G2DSP1-1 and DNA form with N/P ratio at 8: 1; Swimming lane 3 is for processing plasmid DNA 2 hours with the DNA enzyme; Swimming lane 4 is for processing G2DSP1-1 and DNA with the mixture of N/P ratio formation in 8: 12 hours with the DNA enzyme; Swimming lane 5 is to process G2DSP1-1/DNA mixture, the DNA of release with heparin; Swimming lane 6 is for processing the G2DSP1-1/DNA mixture 2 hours with the DNA enzyme, and then heating makes the DNA enzyme deactivation, processes at last the DNA of release with heparin.
Fig. 4 shows that G2DSP1-1 can effectively protect DNA to avoid the degraded of DNA enzyme, and DNA discharges from the G2DSP1-1/DNA mixture in the presence of heparin sodium.In the situation that the DNA enzyme exists, exposed plasmid DNA can be degraded, and DNA and G2DSP1-1 compound under 8: 1 condition of N/P ratio has prevented the degraded of DNA effectively.Adding heparin can discharge the DNA in the G2DSP1-1/DNA mixture, in addition, G2DSP1-1/DNA mixture and DNA enzyme is hatched 2 hours, processes with heparin, still can extract greatly DNA.This explanation G2DSP1-1 can protect plasmid DNA effectively, makes it not to be degraded.
Embodiment 3: the efficiency gene transfection of gene transfection carrier G2DSP1-1
Gene transfection agent G2DSP1-1 prepared among the embodiment 1 and luciferase plasmids or green fluorescent protein plasmid are at room temperature formed mixture; then in HEK293 cell and HeLa cell, carry out transfection, assess the efficiency gene transfection of carrier by the expression amount that detects luciferase or green fluorescent protein.Concrete grammar is: HEK293 or HeLa cell cultures were hatched in 24 orifice plates 48 hours, with 1.6 microgram luciferases or green fluorescent protein plasmid from join mixing in the substratum after G2DSP1-1 mixes with different N/P ratios; Hatch with cell and to add substratum 500 microlitres that contain 10% serum after 6 hours.24 hours aftertreatment cells are used trysinization, and the detection method of luciferase expression amount is with reference to going out the operation instruction that manufacturer (Promega company) provides.The tree-like polymer of 2nd generation polyamide-amide is as negative control, and the tree-like polymer of the 5th PAMAM and bPEI 25kD are as positive control.In the transfection experiment of green fluorescent protein plasmid, adopt the efficient of flow cytometric analysis transfection green fluorescent protein.Concrete grammar is: Transfection of Enhanced Green Fluorescent is after 24 hours, and is with cell dissociation, and resuspended with 300 microlitre PBS damping fluids with pancreatin, immediately detects the cell per-cent of successful transfection green fluorescent protein with flow cytometer.
Experimental result: (1) gene transfection carrier G2DSP1-1 and other transfection carrier as shown in Figure 5 dyes the transfection efficiency comparison diagram of luciferase gene at the HEK293 transit cell, and wherein, N/P is N/P ratio.Fig. 5 shows that the transfection efficiency of G2DSP1-1 is suitable with the efficient of the transfection reagent bPEI 25kD of the marketization, and far above the s-generation and the tree-like high molecular efficient of the 5th PAMAM.Be carrier with G2DSP1-1, luciferase gene is reporter gene, N/P ratio is that the transfection efficiency of 40: 1 o'clock transfected HEK 293s and efficient that bPEI 25kD N/P ratio under optimal conditions is at 10: 1 o'clock are suitable, and far above 2nd generation and the tree-like high molecular transfection efficiency of the 5th PAMAM.
(2) same, gene transfection carrier G2DSP1-1 as shown in Figure 6 and other transfection carrier are at the transfection efficiency comparison diagram of HEK293 transit cell green colouring fluorescence protein gene, and wherein, N/P is N/P ratio.Fig. 6 shows that the cell per-cent of G2DSP1-1 success transfection green fluorescent protein is higher than bPEI 25kD, and the s-generation and the tree-like polymer of the 5th PAMAM.Be carrier with G2DSP1-1, green fluorescence protein gene is reporter gene, N/P ratio be the efficient (34.80%) of 40: 1 o'clock transfected HEK 293s to be higher than bPEI 25kD N/P ratio under optimal conditions be 10: 1 o'clock transfection efficiency (29.17%), far above 2nd generation and the tree-like polymer of the 5th PAMAM transfection efficiency (being respectively 0.18% and 10.58%) under optimal conditions separately.
Embodiment 4: the degradable behavior of gene transfection carrier G2DSP1-1
The whether degradable behavior of the essential and exploration G2DSP1-1 of high efficiency gene transfection institute among the embodiment 2 of disulfide linkage among the research embodiment 1 among the gene transfection carrier G2DSP1-1 of preparation.Concrete grammar is: the DTT of G2DSP1-1 and 5 times of molar equivalents hatch after and green fluorescence protein gene compound, wherein, N/P ratio is 40: 1 in the mixture, by the fluorescence microscope transfection efficiency, result gene transfection carrier G2DSP1-1 is as shown in Figure 7 having or not reducing agent dithiothreitol (DTT) to have Transfection of Enhanced Green Fluorescent efficient comparison diagram in the situation after the transfection.。The result shows: after G2DSP1-1 is hatched through DTT, no matter be to HEK293 cell or HeLa cell, section shows extremely low Transfection of Enhanced Green Fluorescent efficient, illustrate that the disulfide linkage among the G2DSP1-1 can be reduced, illustrate that also disulfide linkage plays vital effect to the efficiency gene transfection of G2DSP1-1 simultaneously.Fig. 7 shows that G2DSP1-1 is degraded under reductive agent DTT existence condition, thereby efficiency gene transfection reduces greatly.
Embodiment 5: the cytotoxicity of gene transfection carrier G2DSP1-1 and G2DSP1-1 and DNA form behind the mixture impact on cytoactive
Among the research embodiment 1 gene transfection carrier G2DSP1-1 with and the mixture that forms with DNA to the activity influence of cell.Concrete experimental technique is: hatch HEK293 or HeLa cell in 96 orifice plates, cell density is 10
4Cells/well is cultivated and after 12 hours substratum is shifted out, and adds the fresh culture (containing 10% serum) that 100 microlitres contain a certain amount of G2DSP1-1 or G2DSP1-1/DNA mixture, cultivates 48 hours.Detect cytoactive by mtt assay.
Experimental result: gene transfection carrier G2DSP1-1 as shown in Figure 8 and other transfection carrier be to (a) HeLa, (b) the toxicity comparison diagram of HEK293 cell.Fig. 8 shows that G2DSP1-1 has lower cytotoxicity with respect to bPEI 25kD and the tree-like polymer of the 5th PAMAM, and toxicity is only suitable with the tree-like polymer of s-generation polyamide-amide.G2DSP1-1 still can make HeLa cell (shown in Fig. 8 a) or HEK293 cell (shown in Fig. 8 b) keep about 90% cytoactive under the concentration conditions of 400 ug/ml.Simultaneously, by relatively seeing, under the concentration conditions of 200 ug/ml and 400 ug/ml, transfection carrier G2DSP1-1 of the present invention in the HeLa cell biocompatibility in addition be better than the tree-like polymer G2 of 2nd generation polyamide-amide.And the tree-like polymer G5 of the 5th PAMAM and bPEI 25kD show higher cytotoxicity in HeLa cell and HEK293 cell under equal concentration conditions.
Gene transfection carrier G2DSP1-1 as shown in Figure 9 and other transfection carrier after forming mixture with DNA to the toxicity comparison diagram of HeLa cell, Fig. 9 show G2DSP1-1 and DNA form mixture after cytotoxicity suitable with independent G2DSP1-1, the cytotoxicity of mixture relative other mixture in the HeLa cell is lower.Do not produce extra cytotoxicity after G2DSP1-1 and DNA are compound, illustrate that gene transfection carrier of the present invention has higher biological safety.
Just for technical conceive of the present invention and characteristics are described, its purpose is to allow the one of ordinary skilled in the art can understand content of the present invention and implements according to this above-described embodiment, can not limit protection scope of the present invention with this.Variation or the modification of every equivalence that the essence of content has been done according to the present invention all should be encompassed in protection scope of the present invention.
Claims (11)
- One kind suc as formula shown in (I) based on the tree-like high molecular gene transfection carrier of low algebraically polyamide-amide, it is characterized in that, comprise the low tree-like polymer of algebraically polyamide-amide and contain disulfide linkage and the amino bi-functional cross-linking agent of reaction sensing, the tree-like high molecular primary amine group of described low algebraically polyamide-amide links to each other with the active group reaction at described bi-functional cross-linking agent two ends;
Wherein, R is the tree-like polymer of polyamide-amide, and its structure is suc as formula shown in (II):
In the formula (II), M is ammonia, quadrol, butanediamine, hexanediamine, octamethylenediamine or certain herbaceous plants with big flowers diamines; N is the integer of 1-4;M is the integer of 2-4;K is the integer of 1-6;X is 0.25-4.0. - 2. as claimed in claim 1ly it is characterized in that based on the tree-like high molecular gene transfection carrier of low algebraically polyamide-amide, describedly contain disulfide linkage and reaction to point to amino bi-functional cross-linking agent be dithio dicarboxylic acid two (N-maloyl imines ester).
- 3. preparation method based on the low tree-like high molecular gene transfection carrier of algebraically polyamide-amide, it is characterized in that, take the tree-like polymer of described polyamide-amide with describedly contain disulfide linkage and reaction is pointed to amino bi-functional cross-linking agent as raw material, at first with the water-soluble or damping fluid of the tree-like polymer of described polyamide-amide, in this solution, dropwise drip the solution that is dissolved with described bi-functional cross-linking agent afterwards, after being added dropwise to complete with this reaction soln stirring reaction at a certain temperature, with damping fluid and distilled water dialysis postlyophilization, obtain as claimed in claim 1 based on the tree-like high molecular novel gene transfection carrier of low algebraically polyamide-amide respectively.
- 4. preparation method as claimed in claim 3 is characterized in that, described pH of cushioning fluid is 6.0-12.0.
- 5. preparation method as claimed in claim 3 is characterized in that, the solution of described dissolving bi-functional cross-linking agent comprises water, damping fluid, methyl-sulphoxide, DMF, or tetrahydrofuran (THF).
- 6. preparation method as claimed in claim 3 is characterized in that, the described reaction times is 2 hours to 96 hours.
- 7. preparation method as claimed in claim 3 is characterized in that, described temperature of reaction is 0 degree centigrade to 50 degrees centigrade.
- 8. preparation method as claimed in claim 3 is characterized in that, it is 1000Da or 3500Da that described dialysis is blocked molecular weight with dialysis tubing.
- 9. as claimed in claim 1 based on the application as the delivery vehicles of nucleic acid molecule in external or body of the low tree-like high molecular gene transfection carrier of algebraically polyamide-amide.
- 10. application as claimed in claim 9 is characterized in that, described nucleic acid is the nucleic acid of DNA, siRNA, shRNA or modification.
- 11. mixture, it is characterized in that, comprise and contain as claimed in claim 1ly based on the tree-like high molecular gene transfection carrier of low algebraically polyamide-amide and nucleic acid, polymer-based carry described nucleic acid because of transfection carrier by described based on low algebraically polyamide-amide is tree-like.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103564312A CN102876718A (en) | 2012-09-21 | 2012-09-21 | Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103564312A CN102876718A (en) | 2012-09-21 | 2012-09-21 | Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102876718A true CN102876718A (en) | 2013-01-16 |
Family
ID=47478233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012103564312A Pending CN102876718A (en) | 2012-09-21 | 2012-09-21 | Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102876718A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290046A (en) * | 2013-05-27 | 2013-09-11 | 华东师范大学 | Fluoride modification based dendrimer gene transfer vector as well as preparation method and application thereof |
| CN103289101A (en) * | 2013-05-31 | 2013-09-11 | 华东师范大学 | Gene transfer vector and preparation method as well as application thereof |
| CN104449638A (en) * | 2014-10-29 | 2015-03-25 | 中国科学院化学研究所 | Viscosity reducer for crude oil and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102585044A (en) * | 2012-03-09 | 2012-07-18 | 山东轻工业学院 | Preparation method for dendritic polyfunctional group RAFT (Reversible Addition-Fragmentation Chain Transfer) reagent |
| EP2552458A1 (en) * | 2010-03-31 | 2013-02-06 | Wayne State University | Injectable dendrimer hydrogel nanoparticles |
-
2012
- 2012-09-21 CN CN2012103564312A patent/CN102876718A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2552458A1 (en) * | 2010-03-31 | 2013-02-06 | Wayne State University | Injectable dendrimer hydrogel nanoparticles |
| CN102585044A (en) * | 2012-03-09 | 2012-07-18 | 山东轻工业学院 | Preparation method for dendritic polyfunctional group RAFT (Reversible Addition-Fragmentation Chain Transfer) reagent |
Non-Patent Citations (3)
| Title |
|---|
| HONGMEI LIU等: "Disulfide Cross-Linked Low Generation Dendrimers with High Gene Transfection Efficacy, Low Cytotoxicity, and Low Cost", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》, vol. 134, 10 October 2012 (2012-10-10) * |
| MICHAEL A. GOSSELIN等: "Efficient Gene Transfer Using Reversibly Cross-Linked Low Molecular Weight Polyethylenimine", 《BIOCONJUGATE CHEM.》, vol. 12, 13 October 2001 (2001-10-13), pages 989 - 994, XP001092541, DOI: doi:10.1021/bc0100455 * |
| YA-NAN XUE等: "Improving Gene Delivery Efficiency of Bioreducible Poly(amidoamine)s via Grafting with Dendritic Poly(amidoamine)s", 《MACROMOLECULAR BIOSCIENCE》, vol. 10, no. 4, 17 December 2009 (2009-12-17), pages 404 - 414 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290046A (en) * | 2013-05-27 | 2013-09-11 | 华东师范大学 | Fluoride modification based dendrimer gene transfer vector as well as preparation method and application thereof |
| CN104017828A (en) * | 2013-05-27 | 2014-09-03 | 华东师范大学 | Fluorine-containing aliphatic chain-modified cationic polymer and application of fluorine-containing aliphatic chain-modified cationic polymer as gene carrier |
| CN103289101A (en) * | 2013-05-31 | 2013-09-11 | 华东师范大学 | Gene transfer vector and preparation method as well as application thereof |
| CN103289101B (en) * | 2013-05-31 | 2015-06-24 | 华东师范大学 | Gene transfer vector and preparation method as well as application thereof |
| CN104449638A (en) * | 2014-10-29 | 2015-03-25 | 中国科学院化学研究所 | Viscosity reducer for crude oil and preparation method thereof |
| CN104449638B (en) * | 2014-10-29 | 2017-04-12 | 中国科学院化学研究所 | Viscosity reducer for crude oil and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Park et al. | pH‐responsive polymers as gene carriers | |
| CN103290046A (en) | Fluoride modification based dendrimer gene transfer vector as well as preparation method and application thereof | |
| Zhang et al. | Synthesis and biological applications of imidazolium‐based polymerized ionic liquid as a gene delivery vector | |
| US11020487B2 (en) | Compositions for introducing nucelic acid into cells | |
| Van der Aa et al. | Optimization of poly (amido amine) s as vectors for siRNA delivery | |
| Xu et al. | Comparison of ethanolamine/ethylenediamine-functionalized poly (glycidyl methacrylate) for efficient gene delivery | |
| Pezzoli et al. | Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior | |
| Scholz et al. | Correlation of length of linear oligo (ethanamino) amides with gene transfer and cytotoxicity | |
| Duan et al. | Amphiphilic graft copolymer based on poly (styrene-co-maleic anhydride) with low molecular weight polyethylenimine for efficient gene delivery | |
| CN105254900B (en) | The high molecular material and preparation method of the modification of bromine substituted aromatic and application | |
| Dong et al. | A Serum‐Tolerant Hydroxyl‐Modified Polyethylenimine as Versatile Carriers of pDNA/siRNA | |
| Tripathi et al. | Polyethylenimine-polyacrylic acid nanocomposites: type of bonding does influence the gene transfer efficacy and cytotoxicity | |
| Duan et al. | Synthetic polyspermine imidazole-4, 5-amide as an efficient and cytotoxicity-free gene delivery system | |
| Fang et al. | Low molecular weight PEIs modified by hydrazone-based crosslinker and betaine as improved gene carriers | |
| CN102876718A (en) | Gene transfection vector based on low-algebraic polyamidoamine and preparation method and application thereof | |
| CN103710383A (en) | Non-virus transgenic vector, preparation method and applications thereof | |
| JPWO2005078084A1 (en) | Polyion complex carrying double-stranded oligonucleic acid, process for producing the same and pharmaceutical composition containing the same | |
| CN104725478B (en) | Polypeptide compound, the assembly of polypeptide compound and siRNA and its application | |
| CN102250348A (en) | Polyethyleneimine derivative and application thereof as gene delivery carrier | |
| CN103881108A (en) | Dendrimer gene carrier modified by fluorine-containing aromatic ring compound as well as preparation method and application thereof | |
| CN103289101B (en) | Gene transfer vector and preparation method as well as application thereof | |
| CN105418939B (en) | The high molecular material of guanidine radicals substituted aromatic modification, preparation method and application | |
| CN101429260B (en) | Polymethacrylate, preparation method and application thereof | |
| CN103804694A (en) | Polymer gene transfection vector based on nucleic acid base derivative and preparation method and application thereof | |
| CN107216445A (en) | A kind of nano-complex and preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130116 |