CN108531514B - Endogenous hyperbranched polyspermine cationic gene vector and preparation method and application thereof - Google Patents
Endogenous hyperbranched polyspermine cationic gene vector and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an endogenous hyperbranched polyspermine cationic gene vector and a preparation method and application thereof. The preparation method of the gene vector comprises the following steps: (1) dissolving endogenous monomers in an organic solvent, adding a protective agent under the anhydrous and anaerobic conditions, and stirring for reaction at the temperature of 20-30 ℃ to obtain protected endogenous monomers I; (2) dissolving an endogenous monomer I in water, adding a biodegradable linking agent, performing addition reaction at the temperature of 60-80 ℃, and adding diethyl ether to precipitate a product to obtain a polymer II; (3) adding a deprotection agent into the polymer II for reaction, filtering, freezing and drying to obtain the endogenous hyperbranched polyspermine cationic gene vector. The gene vector obtained in the invention has biodegradability and nontoxic metabolism, can aggregate and convey nucleic acid drugs, is compounded with the nucleic acid drugs, can obtain nano composite particles for treating DME, and realizes safe and efficient gene therapy.
Description
Technical Field
The invention belongs to the field of biomedical engineering materials, and particularly relates to an endogenous hyperbranched polyspermine cationic gene vector as well as a preparation method and application thereof.
Background
Among all complications of diabetes, the average prevalence of diabetic retinopathy is as high as 50%. Diabetic retinopathy is complicated after the retina is affected by diabetes, wherein Diabetic Macular Edema (DME) is the leading cause of the visual deterioration of a diabetic patient, and the complete blindness of the diabetic patient can be caused by traction retinal detachment at the later stage of the disease process. Gene therapy becomes a potential therapeutic strategy for DME, especially ribonucleic acid interference technology, and provides a new method for researching gene expression, regulation and control of organisms and a new way for research, prevention and treatment of DME. In the field of gene therapy, RNAi (RNA interference) is a powerful tool for silencing a specific gene, and is widely used in various research fields. In the aspect of DME treatment research, VEGF (vascular endothelial growth factor) and a receptor VEGFR thereof are used as target genes, siRNA or dsRNA synthesized in vitro is introduced, and the result shows that the corresponding siRNA can obviously reduce the expression of VEGF or VEGFR, effectively relieve the induction effect of VEGF on DME formation, inhibit the formation of ocular DME, and treat DME. Compared with the traditional drug treatment mode, the gene therapy has incomparable advantages: (1) the characteristics of the vector can be utilized to directionally transfect target cells by gene delivery technology; (2) products expressed by gene therapy are endogenous proteins, and the safety and the tolerance of an organism are better than those of exogenous drugs; (3) can stably express the anti-angiogenesis target factor in local for a long time, and avoids the possible complications caused by repeated local injection[1-4]。
One of the key technologies for gene therapy is to select a safe and efficient gene vector. The gene vector is divided into two main types of virus vector and non-virus vector. The virus gene carrier replaces the pathogenic gene in the virus with the therapeutic gene, and the gene for controlling the virus to enter the cell, transport in the cell and enter the cell nucleus part is also on the virus carrier, so the virus carrier has the advantages of high target gene expression efficiency, immunogenicity, possibility of activating proto-oncogene to trigger tumor, difficulty in mass preparation and the like, thereby limiting the clinical application of the virus carrier. Non-viral geneThe carrier has the advantages of weak immunogenicity, convenient preparation and less requirement limitation on gene materials. At present, liposome, Polyethyleneimine (PEI), dendritic Polyamidoamine (PAMAM) and the like are mostly used as non-viral gene delivery vectors, and the defects of high toxicity of the vectors, difficult degradation or high toxicity of degradation products and the like generally exist. The preparation of a safe, high-efficiency and degradable and metabolizable gene delivery vector is an important subject to be solved urgently in the field of gene therapy[5-7]。
Hyperbranched polymers are a class of highly branched macromolecules with a three-dimensional spherical or spheroidal structure, which have a number of distinct advantages: no entanglement among molecular chains, good solubility, large internal cavity and a large number of active reaction groups[8]. Based on the structural advantages of hyperbranched polymers, the application of the hyperbranched polymers in the field of gene vectors has outstanding advantages: the hyperbranched polymer cation contains a large amount of amines at all levels on the surface and in the interior, and the three-dimensional structure and good molecular toughness of the hyperbranched polymer cation enable the hyperbranched polymer cation to form a sphere with a compact structure with DNA or siRNA as a gene carrier, so that the transfection efficiency is greatly improved. Research on application of dendritic polylysine modified amphiphilic hyperbranched polyglycidyl ether derivatives in drug and gene co-delivery tumor treatment[9]And redox-responsive hyperbranched polyamidoamine derivatives are used for gene therapy research of tumors and the like, and good therapeutic effects are achieved, but from the current reports, most of the monomers selected by hyperbranched polycation carriers are human exogenous molecules, the safety and cytotoxicity of the hyperbranched polycation carriers need to be further improved, and the safety of degradation products of the hyperbranched polyamidoamine derivatives needs to be studied for a long time.
Spermine is a polyamino molecule of a professional coacervate gene in human sperm; in recent years, a linear polyspermine cationic carrier having spermine as a skeleton-constituting unit (polyamino acid ester having spermine as a skeleton[10]Poly (dithioamines)[11]SPE-alt-PEG polyprenamine[12]) All show gene transfection activity several orders higher than PEI and cytotoxicity several orders lower than PEI[13]。
How to obtain the hyperbranched gene vector with good biocompatibility, biodegradability and nontoxic metabolism has become an important subject to be solved urgently in the field of current biomedical engineering materials. So far, the construction of endogenous hyperbranched cationic gene vectors and the application thereof by combining hyperbranched structures with endogenous monomers have not been reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of an endogenous hyperbranched polyspermine cationic gene vector.
The invention also aims to provide the endogenous hyperbranched polyspermine cationic gene vector prepared by the method.
The invention also aims to provide application of the endogenous hyperbranched polyspermine cationic gene vector.
The purpose of the invention is realized by the following technical scheme: a preparation method of an endogenous hyperbranched polyspermine cationic gene vector comprises the following steps:
(1) dissolving an endogenous monomer in an organic solvent, adding a protective agent under the anhydrous and anaerobic conditions, and then stirring for reaction at the temperature of 20-30 ℃ to obtain a protected endogenous monomer I;
(2) dissolving the endogenous monomer I obtained in the step (1) in water, adding a biodegradable linking agent, performing addition reaction at the temperature of 60-80 ℃, and adding ether to precipitate a product after the reaction is finished to obtain a polymer II;
(3) adding a deprotection agent into the polymer II obtained in the step (2), then stirring and reacting at 30-50 ℃, filtering after the reaction is finished, and freeze-drying to obtain the endogenous hyperbranched polyspermine cationic gene vector.
The endogenous monomer in the step (1) is at least one of spermine, spermidine and arginine.
The organic solvent in the step (1) is chloroform; preferably anhydrous chloroform.
The protective agent in the step (1) is at least one of trifluoroacetic acid and 2-acetyl-5, 5-dimethyl-1, 3-cyclohexanedione.
The molar ratio of the endogenous monomer to the protective agent in the step (1) is 1: 2-4.
The stirring reaction conditions in the step (1) are as follows: stirring at 300-700 rpm for 24-36 h; preferably: stirring at 500-600 rpm for 24h at 25 ℃.
The biodegradable connecting agent in the step (2) is at least one of trimethylolpropane triacrylate and trimethylolbutane triacrylate containing ester bonds.
The molar ratio of the endogenous monomer I to the biodegradable connecting agent in the step (2) is 3-5: 1.
The conditions of the addition reaction in the step (2) are as follows: stirring at 400-700 rpm for 5-7 days; preferably: stirring at 500-600 rpm for 6-6.5 days.
The temperature of the addition reaction described in step (2) is preferably 65 ℃.
The deprotection agent in the step (3) is at least one of triethylamine, ammonia water, imidazole, N-methylpyrrolidone and DMF (N, N-dimethylformamide); preferably, the deprotection solution is prepared by mixing imidazole, N-methylpyrrolidone and DMF.
The dosage of the imidazole is preferably calculated according to 5ml of deprotection solution per gram (g) of imidazole.
The volume ratio of the N-methylpyrrolidone to the DMF is preferably 4: 1.
The amount of deprotecting agent described in step (3) is preferably calculated as 0.6mg of polymer II per milliliter (ml) of deprotecting agent.
The stirring reaction conditions in the step (3) are as follows: stirring at 300-700 rpm for 4-6 h; preferably: stirring at 500-600 rpm for 4-5 h.
The temperature of the stirring reaction in the step (3) is preferably 40 ℃.
Filtering in the step (3) by adopting an ultrafiltration tube with the molecular weight cutoff of 30000-60000, wherein the centrifugation speed is 4000-6000 rpm; preferably, an ultrafiltration tube with the molecular weight cutoff of 40000-60000 is adopted for filtration, and the centrifugation speed is 4000-5000 rpm;
an endogenous hyperbranched polyspermine cationic gene vector prepared by any one of the methods.
The endogenous hyperbranched polyspermine cationic gene vector is applied to the preparation of nano composite particles for treating DME (diabetic macular edema).
The nano composite particle for treating DME is obtained by uniformly mixing the endogenous hyperbranched polyspermine cationic gene vector and a nucleic acid drug and then incubating for 20-30 minutes at room temperature.
The nucleic acid medicament is at least one of DNA vaccine, DNA, siRNA and microRNA.
The mass ratio of the endogenous hyperbranched polyspermine cationic gene vector to the nucleic acid drug is 1: 1-30.
The particle size of the nano composite particles (endogenous hyperbranched cation and nucleic acid drug composite) for treating DME is 100-300 nanometers.
Compared with the prior art, the invention has the following advantages and effects:
(1) the endogenous hyperbranched cationic gene vector with high product yield and easily controlled structure is prepared by using an endogenous monomer as a basic structural unit, protecting the endogenous monomer with a protective agent, carrying out Michael addition reaction on the protected endogenous monomer and a biodegradable connecting agent, deprotecting the protected endogenous monomer, removing impurities by ultrafiltration, and freeze-drying.
(2) The invention relates to a nano composite particle for treating DME (dimethyl ether) prepared by synthesizing an endogenous hyperbranched gene vector through Michael addition under the electrostatic action with a nucleic acid drug, belonging to the first synthesis of an endogenous hyperbranched cationic gene vector.
(3) The gene vector can agglomerate and convey nucleic acid medicine, has particle size of about 100 nm after compounding in relatively high mass ratio, and can release therapeutic gene via proton sponge effect to reach the treating effect.
(4) The invention combines endogenous monomers with hyperbranched structures, has biodegradability and nontoxic metabolism (toxicity is not shown in cytotoxicity experiments), constructs endogenous hyperbranched gene vectors, and is expected to realize safe and efficient gene therapy.
(5) The gene vector of the present invention can complete non-toxic metabolism in vivo, and may be used in gene therapy of various diseases via intravenous injection.
(6) The material disclosed by the invention is simple in component, easy to obtain raw materials and good in biocompatibility, provides support for the application of the material in preparation of biomedical engineering materials, and is expected to be widely applied to the field of biomedical engineering materials.
Drawings
FIG. 1 is a scheme of the synthesis of hyperbranched polyprenamines.
FIG. 2 is a nuclear magnetic hydrogen spectrum of hyperbranched polyprenamine in example 2.
FIG. 3 is a graph of the particle size of the hyperbranched polyprenamine complex with pDNA in example 4.
FIG. 4 is a potential diagram of the complex of hyperbranched polypiperazine and pDNA in example 4.
FIG. 5 is an agarose gel electrophoresis of the hyperbranched polyprenamine complex with pDNA of example 6.
FIG. 6 is a graph of toxicity of hyperbranched polypiperazine on retinal endothelial cells in example 8.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1: preparation of hyperbranched polyprenamine cations
(1) 2mg of spermine was weighed out and dissolved in 5ml of anhydrous chloroform, and two primary amine groups thereof were protected with 2-acetyl-5, 5-dimethyl-1, 3-cyclohexanedione (Dde-OH) (5.4mg) under anhydrous and oxygen-free conditions. The reaction was carried out at a rotation speed of 600rpm and a temperature of 25 ℃ for 24 hours. Removing impurities after the reaction is finished to obtain a protected spermine monomer; wherein the molar ratio of spermine to Dde-OH is 1: 3.
(2) Weighing (5mg) protected spermine monomer, dissolving in 10m L water, adding trimethylolpropane triacrylate (0.96mg), stirring and reacting at the rotation speed of 600rpm and the temperature of 65 ℃ for 6 days, adding anhydrous ether to precipitate a polymer after the reaction is finished, adding 10ml deprotection solution (2.01g of imidazole, 8m L of N-methylpyrrolidone and 2m L of DMF) to the precipitated polymer, stirring and reacting for 4 hours (40 ℃ and 600rpm), ultrafiltering and washing the product for four times after the reaction, removing unreacted spermine monomer and trimethylolpropane triacrylate through an ultrafiltration tube with the molecular weight cutoff of 40000 at the rotation speed of 4000rpm, and freeze-drying to obtain the hyperbranched polyspermine, wherein the molar ratio of the protected spermine monomer to the trimethylolpropane triacrylate is 3: 1.
Example 2: hydrogen spectrum nuclear magnetic characterization of hyperbranched polyspermine cations
The hyperbranched polyspermine cation obtained in example 1 was dissolved in deuterated water for hydrogen spectrum nuclear magnetic characterization. Chemical shifts of hydrogen atoms in hyperbranched polypiperazine cations have been labeled as in fig. 2, and the results in fig. 2 demonstrate that the reaction of this step successfully synthesizes hyperbranched polypiperazine cations.
Example 3: preparation of hyperbranched polyspermine cation and pDNA nano-composite
(1) Weighing the hyperbranched polyspermine cation prepared in the embodiment 1, and dissolving the hyperbranched polyspermine cation in water to obtain a hyperbranched polyspermine cation solution;
(2) preparing pDNA (DNA, Jima pharmaceutical technology, Suzhou) into a 1mg/ml stock solution;
(3) preparing a hyperbranched polypiperazine cation and pDNA nano compound: diluting the hyperbranched polyspermine cation solution by 1mg/ml according to a set series of mass ratios (1, 5, 10, 15, 20 and 30), adding 1, 5, 10, 15, 20 and 30ml of the polyspermine cation solution into each test tube, quickly adding 1ml of pDNA stock solution, and incubating for 30 minutes at 25 ℃ to obtain a series of nano-composites with different surface charge properties.
Example 4: testing of particle size and Zeta potential of hyperbranched polyspermine cation and pDNA nano-composite
Nanoparticle composite solutions of different mass ratios were prepared according to the preparation method of hyperbranched polypiperazine cations and pDNA nanocomposites of example 3, with the required mass ratios of hyperbranched polypiperazine cations to pDNA of 5, 7, 10, 15, 20, 30, respectively. The sample size was 1 ml. At room temperature, the nano laser particle analyzer is preheated for 20 minutes, the lml nano particle solution is sucked and added into a trace sample pool, then the trace sample pool is placed into a test slot of the particle analyzer, the test temperature is set to be 25 ℃, and the medium is ultrapure water. For the nanoparticle size distribution test, each sample was tested 3 times with a run time of 2 minutes, and the average of the particle size and its polydispersity were recorded for each sample. For the Zeta potential testing of the nanoparticles, 3 times per sample were tested, 12 times per automatic run, and the average value of Zeta potential and its mobility were recorded for each sample.
As a result: the particle size distribution of the hyperbranched polyspermine and pDNA complex is shown in figure 3, the Zeta potential test result is shown in figure 4, and it can be seen from the figure that the particle size of the hyperbranched polyspermine cation and pDNA nano complex with the mass ratio of more than 15 is stabilized at 100-300 nanometers, and the Zeta potential distribution of the nano particles is 20-30 mV.
Example 5: transmission electron microscope observation of hyperbranched polyspermine cation and pDNA nano composite
Nanoparticles were prepared in a mass ratio of 30 according to the preparation method of the hyperbranched polypiperazine cation and pDNA nanocomposite of example 3. Firstly, 10 mul of nanoparticle solution is absorbed and slowly dripped on a 400-mesh copper net, and the appearance of a sample is observed by using a transmission electron microscope after being dried (the particle size of the nanoparticles is conveniently and visually displayed). The experimental results were consistent with the particle size results of the dynamic light scattering measurements.
Example 6: agarose gel electrophoresis of hyperbranched polyspermine cation and pDNA nano-composite with different mass ratios
Weighing 1g of agarose, adding the agarose into 100ml of 1 × TAE buffer solution, heating in a microwave oven to completely dissolve agarose particles, cooling to 50-60 ℃, adding Ethidium Bromide (EB) solution, slightly shaking, uniformly mixing, pouring into a gel tank when the gel is cooled to about 50 ℃, putting a comb into the gel tank, after the gel is formed, carefully pulling out the comb, taking out the gel, putting the gel into an electrophoresis tank containing the electrophoresis buffer solution, enabling the liquid level of the electrophoresis buffer solution to be 2-3 mm higher than that of the gel, waiting for sample loading, preparing hyperbranched polyspermine cation and pDNA nano-composite with different mass ratios of 1, 3, 5, 10, 20, 30 and 50, respectively taking 5 mu l of sample to be tested by using a liquid transfer gun, uniformly mixing with the sample loading buffer solution, carefully adding the sample loading buffer solution into a sample loading hole of the gel, opening an electrophoresis apparatus, carrying out electrophoresis for 30min at a voltage of 100V, taking out the gel, and placing the gel on an ultraviolet transilluminator to observe electrophoresis results.
And (4) analyzing results: FIG. 5 shows an electrophoresis chart that when the mass ratio of the hyperbranched polyprenamine cation to the pDNA is 5, the migration of pDNA molecules in an electric field is partially blocked, with the gradual increase of the mass ratio of the hyperbranched polyprenamine cation to the pDNA, when the mass ratio of the hyperbranched polyprenamine cation to the pDNA reaches 20-30, the hyperbranched polyprenamine cation completely blocks the migration of the pDNA, even under the condition of larger mass ratio, the hyperbranched polyprenamine cation can more fully wrap the pDNA, and the movement of the pDNA cannot be completely observed in the electrophoresis chart.
Example 7: degradation and metabolic processes of hyperbranched polyprenamides cations
2mg of hyperbranched polyspermine cation were dissolved in 10ml of PBS buffer (pH 7.4), and samples were taken every 2 days for 20 consecutive days. Samples were taken and stored in a refrigerator at-20 ℃, after the samples were collected, the molecular weight of each sample was measured by Gel Permeation Chromatography (GPC), and the degradability was investigated by plotting the degradation curve of the polymer with time as abscissa and the weight-average molecular weight as ordinate. The degradation capability of the hyperbranched polyprenamine cation is tested.
The experimental results are as follows: the molecular weight of the hyperbranched polypiperazines decreases significantly with increasing time. On day 10, its molecular weight was only the first 40%. Only the first 15% of its molecular weight was found at day 20. The experimental result shows that the hyperbranched polyspermine has good biodegradability.
Example 8: cytotoxicity assay for hyperbranched polypiperazine cations
The cytotoxicity of the hyperbranched polypspermine cation is determined by a CCK-8 method. After sterilization by filtration, the hyperbranched polyspermine cation prepared in example 1 and PEI (polyetherimide) with a comparative sample molecular weight of 25000 were diluted with DMEM medium according to a concentration gradient (0.1, 1, 5, 10, 50, 100. mu.g/ml) and co-cultured in human retinal endothelial cells (human retinal endothelial cells, Shanghai and Nippon Biotech Co., Ltd.) with a fusion degree of 80%, and the volume of the hyperbranched polyspermine cation solution added to each well was 100. mu.l, each concentration was 3 duplicate wells. After 24 hours, the cytotoxicity of the material was measured by the CCK-8 method.
The results of the cytotoxicity test of the hyperbranched polypiperazine are shown in fig. 6. When the hyperbranched polyspermine reaches 100 mu g/ml, the cell still maintains the survival rate of more than 80 percent; compared with PEI, the PEI shows obvious cytotoxicity, and the cell survival rate has obvious concentration dependence. The following conclusions can be drawn: the hyperbranched polyspermine has no toxicity to human retinal endothelial cells within the working concentration range and has good biocompatibility.
Example 9: cell transfection of hyperbranched polyprenamine cation and pDNA nanocomposite
Selecting human retinal endothelial cells growing in logarithmic phase as receptor cells, and adjusting cell concentration to 10 per well5The cells were seeded in 24-well cell culture plates at 37 ℃ with 5% CO2Cultured under conditions to a cell confluence of 70%. Then, the Opti-MEM serum-free cell culture medium is replaced for incubation for 2 hours, and serum-free culture solution of the hyperbranched polypiperazine cation and pDNA compound is added into a culture dish at the same time, so that the content of DNA in each hole is ensured to be 1 mu g; the mass ratio of the hyperbranched polyspermine cation to the pDNA nano is 5, 10, 20, 30 and 50 respectively. PEI-25k/DNA complex was used as a positive control group, and naked pDNA was used as a negative control group. The 24-well plate was then placed in an incubator at 37 ℃ with 5% CO2After culturing for 4 hours, the culture medium was replaced, and a serum-containing culture medium was added thereto to continue the culture for 24 hours. The expression of green fluorescent protein was qualitatively studied under a fluorescence microscope. Then sucking out the culture solution, adding pancreatin digestive cells and adding a culture medium to stop pancreatin action, centrifuging the cell sample, then resuspending the cell sample in PBS, and quantitatively analyzing the cell transfection number, namely the gene transfection efficiency of the sample by adopting a flow cytometer.
The experimental results are as follows: the transfection efficiency tended to increase with increasing mass ratio. And at the mass ratio of 30, the transfection efficiency is 5% higher than that of a positive control (40%). However, at a mass ratio of 50, the transfection efficiency was slightly decreased. It is believed that transfection efficiency is reduced because the vector is too tightly packed around the pDNA to be released.
Example 10: effect of hyperbranched polyspermine cationic gene vector on VEGF-siRNA delivery to VEGF protein in human retinal endothelial cells
VEGF-siRNA (Suzhou Jima gene) was selected as the nucleic acid drug. Co-culturing human retinal endothelial cells and the hyperbranched polyspermine cation and VEGF-siRNA nano-composite with the mass ratio of 30 for 24 hours. Thereafter, the cells were washed three times with PBS, lysed with SDS lysis buffer on ice for 30 minutes to sufficiently lyse the cells, and then centrifuged at 13000rpm at 4 ℃ for 15 minutes, and the supernatant was collected. Protein concentration was determined using the Bio-Rad protein assay. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred to PVDF membranes at 300mA, and the membranes were blocked with 5% skim milk for 1 hour. The membranes were then incubated with the VEGF antibody overnight at 4 ℃. The membrane was then incubated with secondary antibody for 1 hour at 25 ℃ and washed three times with TBST solution. Thereafter, the samples were examined using a chemiluminescence visualizer, GAPDH protein as a control. Experimental results show that the hyperbranched polyspermine cationic gene vector loaded with VEGF-siRNA can well reduce the expression of VEGF protein. The result proves that the bionic nano erythrocyte gene carrier can safely and efficiently transport the therapeutic gene to the focus.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Reference to the literature
[1]P.Chanphai and H.A.Tajmir-Riahi,Journal of Photochemistry andPhotobiology B-Biology,2016,155,13-19.
[2]D.Ge,K.Higashi,D.Ito,K.Nagano,R.Ishikawa,Y.Terui,K.Higashi,K.Moribe,R.J.Linhardt and T.Toida,Chemical&Pharmaceutical Bulletin,2016,64,390-398.
[3]N.Kasyanenko and B.Dribinsky,International Journal of BiologicalMacromolecules,2016,86,216-223.
[4]Z.J.Wang,H.J.Zou,Z.R.Wang,J.M.Wu,Z.S.Xia and M.Feng,InternationalJournal of Pharmaceutics,2016,505,24-34.
[5]M.S.Shim and Y.J.Kwon,Biomaterials,2011,32,4009-4020.
[6]X.B.Xiong,H.Uludag and A.Lavasanifar,Biomaterials,2009,30,242-253.
[7]X.B.Xiong,H.Uludag and A.Lavasanifar,Biomaterials,2010,31,5886-5893.
[8]Q.Q.Zheng,H.Li,D.Ma and W.Xue,Journal of Applied Polymer Science,2016,133.
[9]M.Y.Li,X.L.Zeng,C.Y.Wang,J.K.Xu,D.Ma and W.Xue,Journal ofMaterials Chemistry B,2016,4,547-556.
[10]H.L.Jiang,S.H.Hong,Y.K.Kim,M.A.Islam,H.J.Kim,Y.J.Choi,J.W.Nah,K.H.Lee,K.W.Han,C.Chae,C.S.Cho and M.H.Cho,International Journal ofPharmaceutics,2011,420,256-265.
[11]M.Ou,R.Z.Xu,S.H.Kim,D.A.Bull and S.W.Kim,Biomaterials,2009,30,5804-5814.
[12]Y.K.Kim,C.S.Cho,M.H.Cho and H.L.Jiang,Journal of BiomedicalMaterials Research Part A,2014,102,2230-2237.
[13]D.Jere,J.E.Kim,R.Arote,H.L.Jiang,Y.K.Kim,Y.J.Choi,C.H.Yun,M.H.Choand C.S.Cho,Biomaterials,2009,30,1635-1647.
Claims (10)
1. A preparation method of an endogenous hyperbranched polyspermine cationic gene vector is characterized by comprising the following steps:
(1) dissolving an endogenous monomer in an organic solvent, adding a protective agent under the anhydrous and anaerobic conditions, and then stirring for reaction at the temperature of 20-30 ℃ to obtain a protected endogenous monomer I;
(2) dissolving the endogenous monomer I obtained in the step (1) in water, adding a biodegradable linking agent, performing addition reaction at the temperature of 60-80 ℃, and adding ether to precipitate a product after the reaction is finished to obtain a polymer II;
(3) adding a deprotection agent into the polymer II obtained in the step (2), stirring and reacting at 30-50 ℃, filtering after the reaction is finished, and freeze-drying to obtain an endogenous hyperbranched polyspermine cationic gene vector;
the endogenous monomer in the step (1) is spermine;
the protective agent in the step (1) is 2-acetyl-5, 5-dimethyl-1, 3-cyclohexanedione;
the biodegradable connecting agent in the step (2) is trimethylolpropane triacrylate.
2. The method for preparing the endogenous hyperbranched polypiperazine cationic gene vector according to claim 1, characterized in that:
the deprotection agent in the step (3) is at least one of triethylamine, ammonia water, imidazole, N-methylpyrrolidone and N, N-dimethylformamide.
3. The method for preparing the endogenous hyperbranched polypiperazine cationic gene vector according to claim 1, characterized in that:
the molar ratio of the endogenous monomer to the protective agent in the step (1) is 1: 2-4;
the molar ratio of the endogenous monomer I to the biodegradable connecting agent in the step (2) is 3-5: 1.
4. The method for preparing the endogenous hyperbranched polypiperazine cationic gene vector according to claim 1, characterized in that:
the organic solvent in the step (1) is chloroform;
the stirring reaction conditions in the step (1) are as follows: stirring at 300-700 rpm for 24-36 h;
the conditions of the addition reaction in the step (2) are as follows: stirring at 400-700 rpm for 5-7 d;
the stirring reaction conditions in the step (3) are as follows: stirring at 300-700 rpm for 4-6 h.
5. An endogenous hyperbranched polyspermine cationic gene vector, which is characterized in that: prepared by the method of any one of claims 1 to 4.
6. Use of the endogenous hyperbranched polypiperazine cationic gene vector according to claim 5 for preparing nanocomposite particles for treating diabetic macular edema.
7. A nanocomposite particle for use in the treatment of diabetic macular edema, comprising: the nucleic acid drug is obtained by uniformly mixing the endogenous hyperbranched polypiperazine cationic gene vector disclosed in claim 5 and a nucleic acid drug and then incubating for 20-30 minutes at room temperature.
8. The nanocomposite particles for treating diabetic macular edema according to claim 7, wherein:
the nucleic acid drug is at least one of DNA, siRNA and microRNA.
9. The nanocomposite particles for treating diabetic macular edema according to claim 8, wherein:
the DNA is DNA vaccine.
10. The nanocomposite particles for treating diabetic macular edema according to claim 7, wherein:
the mass ratio of the endogenous hyperbranched polyspermine cationic gene vector to the nucleic acid drug is 1: 1-30.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008075806A1 (en) * | 2006-12-19 | 2008-06-26 | Hak Soo Han | Photo-curable coating composition comprising hyperbranched structure prepolymer, method for preparing the same and product prepared by the same |
| WO2009133071A2 (en) * | 2008-04-30 | 2009-11-05 | Consorzio Per Il Centro Di Biomedicina Molecolare Scrl | Polyelectrolyte with positive net charge for use as medicament and diagnostic for cancer |
| CN102174579A (en) * | 2011-01-19 | 2011-09-07 | 武汉大学 | Reducible and biodegradable comb type high polymer gene vector and preparation method of same |
| CN104226128A (en) * | 2014-09-12 | 2014-12-24 | 浙江大学 | Method for forming amphoteric ion structure on surface of separation membrane and separation membrane |
| CN104906075A (en) * | 2015-06-19 | 2015-09-16 | 中国药科大学 | Simultaneous-delivery nanometer carrier and preparation method thereof |
| CN105461935A (en) * | 2015-12-16 | 2016-04-06 | 浙江大学 | Hyperbranched polymer containing imidazole, preparation method and method thereof for stabilizing nano-silver |
| CN106893054A (en) * | 2016-03-29 | 2017-06-27 | 江苏省肿瘤医院 | A kind of cationic polymer gene vector and its preparation method and application |
-
2018
- 2018-04-10 CN CN201810313557.9A patent/CN108531514B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008075806A1 (en) * | 2006-12-19 | 2008-06-26 | Hak Soo Han | Photo-curable coating composition comprising hyperbranched structure prepolymer, method for preparing the same and product prepared by the same |
| WO2009133071A2 (en) * | 2008-04-30 | 2009-11-05 | Consorzio Per Il Centro Di Biomedicina Molecolare Scrl | Polyelectrolyte with positive net charge for use as medicament and diagnostic for cancer |
| CN102174579A (en) * | 2011-01-19 | 2011-09-07 | 武汉大学 | Reducible and biodegradable comb type high polymer gene vector and preparation method of same |
| CN104226128A (en) * | 2014-09-12 | 2014-12-24 | 浙江大学 | Method for forming amphoteric ion structure on surface of separation membrane and separation membrane |
| CN104906075A (en) * | 2015-06-19 | 2015-09-16 | 中国药科大学 | Simultaneous-delivery nanometer carrier and preparation method thereof |
| CN105461935A (en) * | 2015-12-16 | 2016-04-06 | 浙江大学 | Hyperbranched polymer containing imidazole, preparation method and method thereof for stabilizing nano-silver |
| CN106893054A (en) * | 2016-03-29 | 2017-06-27 | 江苏省肿瘤医院 | A kind of cationic polymer gene vector and its preparation method and application |
Non-Patent Citations (4)
| Title |
|---|
| A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy;Rong-Lin Xie et al.;《International Journal of Pharmaceutics》;20141111;第478卷;第19-30页 * |
| pH敏感基因载体的研究现状;沈银 等;《化学学报》;20131231;第71卷;第323-333页 * |
| Spermine-alt-poly(ethylene glycol) polyspermine as a safe and efficient aerosol gene carrier for lung cancer therapy;You-Kyoung Kim et al.;《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A》;20130813;第102A卷(第7期);第2230-2237页 * |
| 含酯键的可降解聚合物基因载体研究进展;李硕 等;《高分子通报》;20140630(第6期);第46-61页 * |
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