CN101461947A - Polypeptide tree-shaped macromolecule as gene vector and use thereof - Google Patents
Polypeptide tree-shaped macromolecule as gene vector and use thereof Download PDFInfo
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- CN101461947A CN101461947A CNA2008101716386A CN200810171638A CN101461947A CN 101461947 A CN101461947 A CN 101461947A CN A2008101716386 A CNA2008101716386 A CN A2008101716386A CN 200810171638 A CN200810171638 A CN 200810171638A CN 101461947 A CN101461947 A CN 101461947A
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Abstract
The invention relates to a polypeptide dendric macromolecule used as a gene vector and application thereof. The polypeptide dendric macromolecule used for preparing a dendric polypeptide macromolecule vector system for loading treating genes consists of treating genes, dendric polypeptide macromolecules or dendric polypeptide macromolecule connected with cation macromolecules. The structural unit of the dendric polypeptide macromolecule is a natural amino acid which can be single amino acid or formed by different amino acids. The dendric polypeptide macromolecule vector system can be salt compounds and ester compounds accepted in the pharmacology. The dendric polypeptide macromolecule vector system for loading treating genes can treat inherited diseases due to gene defect, immune deficiency or tumors due to inactivation of cancer inhibiting genes.
Description
Technical field
The present invention relates to polypeptide tree-shaped macromolecule and application thereof as genophore, purpose is to provide a kind of therapeutic gene that mediates effectively to send the compositions that is delivered in the target cell, be used for carrying out effectively the compositions that DNA send the biocompatible of passing, thereby cause the transfection of non-cell toxicity ground in target cell.Thisly can be used for preparing the tree-shaped polypeptide macromolecule carrier system that loads therapeutic gene, by therapeutic gene, tree-shaped polypeptide macromolecule or have the tree-shaped polypeptide macromolecule that is connected with cation high molecular and form.The construction unit of tree-shaped polypeptide macromolecule is natural aminoacid, can be single aminoacid, also can be made up of different aminoacid.The tree-shaped polypeptide macromolecule carrier system can be acceptable salt compounds of pharmacy and ester type compound.Diseases such as tumor due to the inactivation of heredopathia, immunodeficiency or the antioncogene of the tree-shaped polypeptide macromolecule carrier system of this loading therapeutic gene due to can the therapeutic gene defective.
Background technology
Prevention of disease and treatment, gene therapy is a kind of effective and economic method.Gene therapy is that people's normal gene or medicative gene are imported defective or the performance therapeutical effect of people's somatic target cell to correct gene by certain way, thereby reaches the biomedical technology of treatment disease purpose.Gene therapy be contemporary medical science and biology a new research field.It attempt that dcc gene from the gene level regulating cell is expressed or with normal gene correct, the replace defective gene, reach the diseases such as tumor due to the inactivation of heredopathia, immunodeficiency or antioncogene due to the therapeutic gene defective.
Gene transfer efficiently and expression are the key technologies of gene therapy.Genophore commonly used at present comprises viral vector and non-virus carrier, and the above virus of utilizing is transported genes of interest for carrier in the clinical trial.Though viral vector has higher transfection efficiency, also exist simultaneously and much be difficult to the shortcoming that overcomes, as have immunogenicity, cytotoxicity, lack tissue specificity or the like.Therefore the research to non-virus carrier is subject to people's attention, seeks new efficiently, the non-virus carrier of safety, targeting becomes present research focus.
In recent years, nano-carrier causes more and more the concern with the characteristics of its transfection efficiency height, safety and low toxicity, non-immunogenicity.Especially the cationic polymer nano-gene carrier is that the most promising non-viral gene imports one of carrier system.For example poly--L-relies atmosphere acid (PLL) (people such as M.A.wolfert, " characteristic description of the gene therapy vector that is formed by DNA and synthetic block copolymer self assembly " " human gene therapy " 1996,7,2123-2133; AV Kabanov ﹠amp; VA Kabanov " in genetic stocks being sent the dna complex that is delivered in the cell " with polycation, Bioconj.chem.1995,6:7-20).
Dendritic particular structure and character and the utility tree dendritic polymer is made drug release carrier or supporting agent has had report [R.Esfsndando, D.A.Tomalia, DDT.2001,6:427-436; US5338532 and 5527524], the functional character of outer surface of dendritic and the new method that inner morphological characteristic helps to develop medicine sustained release and targeted drug delivery system have especially particularly been proposed.It is to carry out gene transfection as non-virus gene transfection agents that dendrimer is used a most active field.Polyamine class dendrimer such as PAMAM and the commercializations such as (PPI) of polypropylene imines, they and the compound also existing of dna molecular are studied.Under the physiological condition of pH7.4, they have the trend that forms polycation, thereby are used to gene therapy.PolyFect is commercial outer-gene transfection agents, is obtained through the part chemical hydrolysis by the PAMAM dendrimer.
The polypeptide dendritic macromole is meant the dendritic macromole that includes polypeptide in molecular structure.In the polypeptide dendritic macromole, aminoacid or polypeptide are connected in the whole molecule with the covalent bond form as initiated core or cladodification unit.The dendroid polypeptide macromolecule has the universal feature of general dendritic macromole, has the biological nature of polypeptide simultaneously again.The characteristic of dendroid polypeptide macromolecule mainly comprises the following aspects: (1) has the chondritic of albuminoid, can be as the receptor of native ligand; (2) the multivalence structure makes it take place to interact or be connected numerous useful groups with two or more parts simultaneously; (3) good biocompatibility, cytotoxicity is low; (4) good water solubility; (5) the high degree of branching is difficult for by proteasome degradation it; (6), can be used as the transport vehicle of therapeutic gene easily by cell endocytic.Because these characteristics make the polypeptide dendritic macromole at biological, field of medicaments potential widely application be arranged.
Summary of the invention
The present invention relates to polypeptide tree-shaped macromolecule and application thereof as genophore.Thisly can be used for preparing the tree-shaped polypeptide macromolecule carrier system that loads therapeutic gene, by therapeutic gene, tree-shaped polypeptide macromolecule or have the tree-shaped polypeptide macromolecule that is connected with cation high molecular and form.
The construction unit of tree-shaped polypeptide macromolecule is natural aminoacid, can be single aminoacid, also can be made up of different aminoacid.Tree-shaped polypeptide macromolecule is construction unit with the natural amino acid, aminoacid such as glycine, lysine, glutamic acid, proline preferably, and tree-shaped polypeptide macromolecule can have with the seed amino acid cell formation, also can be made up by aminoacid not of the same race.Its synthetic method is identical with the traditional tree dendritic macromolecules, can be divided into the method for dispersing and convergence method two big classes.The method of dispersing is a kind of direct synthetic method, begins progressively to grow from initiated core, constantly outwards disperses, and forms dendritic macromole.Convergence method is a kind of indirect synthetic method, and the synthetic earlier functional group that needs is connected on the tree-shaped core with specific chemical method behind the purification.The preferred synthetic method that adopts the method for dispersing and convergence method to combine at first with the synthetic segmental dendroid polypeptide macromolecule segment of the method for dispersing, then by convergence method and specific core bonding, obtains ball-type, the dumbbell shape dendroid polypeptide macromolecule of different structure.No matter disperse method or convergence method, can use solid phase, liquid phase or solid-liquid associated methods synthetic method to realize.
Being used for preparing the tree-shaped polypeptide macromolecule carrier system carrier system that loads therapeutic gene can form by being connected with the nanoparticle that the cation macromolecular tree-shaped polypeptide macromolecule constructs, and cation macromolecular is preferably from following compounds: chitosan, Polyethylene Glycol (PEG), polymine etc.For example tree-shaped polypeptide macromolecule contains the Polyethylene Glycol unit of covalently bound certain percentage, tree-shaped polypeptide macromolecule can be adjusted by changing reaction condition with the ratio of Polyethylene Glycol unit, (wherein the amino of 5-30% and PEG put together this synthetic carrier in the tree-shaped polypeptide macromolecule, and remaining amino still remains unsubstituted free radical) can form stable and soluble complex with nucleic acid, it can carry out transfection then effectively.Compare with independent use tree-shaped polypeptide macromolecule, this grafted peg moiety makes the better and cytotoxicity of the dissolubility of nucleic acid/carrier complex descend.
Being used for preparing the tree-shaped polypeptide macromolecule carrier system tree-shaped polypeptide macromolecule that loads therapeutic gene, can be the acceptable salt compounds of pharmacy: for alkaline tree-shaped polypeptide macromolecule carrier preferably fluoride, chloride, bromide, iodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalates, succinate, tartrate, malate, mandelate, mesylate or tosilate; For the cation of acid tree-shaped polypeptide macromolecule carrier based on alkali metal and alkaline-earth metal, as sodium salt, lithium salts, potassium salt, calcium salt, magnesium salt, aluminum salt etc., and ammonium, quaternary ammonium and amine cation, comprising but be not limited to: ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethamine etc. and other the representative organic amine that is used to form base addition salts comprise diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine etc.
Be used for preparing the tree-shaped polypeptide macromolecule carrier system tree-shaped polypeptide macromolecule that loads therapeutic gene, it can be pharmacy acceptable esters chemical compound, finger is the ester of hydrolysis in vivo, being included in degrades in the human body discharges the ester of parent compound or its salt, suitable ester group for example comprises: derived from pharmaceutically acceptable aliphatic carboxylic acid or aliphatic family alcohol, especially alkanoic acid (alcohol), alkenoic acid (alcohol), aphthenic acids (alcohol) and alkane dicarboxylic acid's's (glycol) ester, wherein preferred each alkyl or alkenyl partly is less than 6 carbon atoms.
Being used for preparing the tree-shaped polypeptide macromolecule carrier system carrier that loads therapeutic gene and the ratio of therapeutic gene is 0.5～20:1.
Be used for preparing the gene order that the tree-shaped polypeptide macromolecule carrier system therapeutic gene that loads therapeutic gene contains the genetic coding labelling, described genetic marker is selected from luciferase genes, the general enzyme gene of p one galactose, hygromycin Hangzhoupro property gene and neomycin Hangzhoupro property gene and the dead drunk transferase gene of chloromycetin second, is selected from the gene inhibition agent of low density lipoprotein receptor, coagulation factors, tumor, main tissue compatible protein, Hangzhoupro oncogene, P16, p53, the general kinases of breast, IL2, IL4 and TNF α.
Therapeutic gene contains the former DNA sequence in the viral Hangzhoupro of coding described in the tree-shaped polypeptide macromolecule carrier system of preparation loading therapeutic gene, is selected from the RNA of RNA, antisense RNA and ribozyme; Described nucleic acid coding agglutinin, mannose receptor, sialoadhesin or retrovirus trans-activation are because of in (TAT).
Cell contacts under certain conditions with the tree-shaped polypeptide macromolecule carrier system that loads therapeutic gene, and wherein said compositions enters into said cell and discharges the nucleic acid of said compositions under described condition.
The present invention relates to the carrier system complex that forms between the tree-shaped polypeptide macromolecule of electronegative therapeutic gene and positively charged, is electrostatic on the relation nature between wherein said nucleic acid and the described tree-shaped polypeptide macromolecule.The loading therapeutic gene carrier system of said positively charged is by tree-shaped polypeptide macromolecule or have a tree-shaped polypeptide macromolecule that is connected with cation high molecular.On the amino part of tree-shaped polypeptide macromolecule, add cation macromolecular such as Polyethylene Glycol and can effectively prevent the precipitation and the gathering of the complex (or nano-particle) that forms by carrier macromole and nucleic acid, thereby increased the dissolubility of complex. also play fused cell film and the effect that prevents the degraded of nucleic acid-protein enzymolysis as the Polyethylene Glycol that is connected with tree-shaped polypeptide macromolecule, thereby improved transfection efficiency and directive efficiency.
Tree-shaped polypeptide macromolecule carrier system of the present invention can form stable and soluble complex with therapeutic gene, and it is the mammiferous cell of transfection effectively.Described therapeutic gene can be selected in following material: (1) genetic marker, the gene that has resistance such as luciferase genes, the general enzyme gene of p one galactose, chloramphenicol acetyl transferasegene, to antibiotic (such as hygromycin or neomycin); (2) be used for the treatment of the gene of purpose, as be coded in the gene of the low density lipoprotein receptor of (liver) defective under the hypercholesteremic situation, the gene of coding coagulation factors, the suppressor gene of tumor, gene, antioncogene, RNA and the antisense RNA of the main tissue compatible protein of coding and the gene of encoding ribozyme; (3) has the gene of vaccine purpose, as the gene of coding virus antigen.
The tree-shaped polypeptide macromolecule carrier system of loading therapeutic gene involved in the present invention is used for cells transfected can be selected from following cell: hematopoietic cell; Hepatocyte; Skeletal Muscle Cell; Skin Cell is as fibroblast, horn cell, dendritic cell or melanocyte; Cells of vascular wall is as endotheliocyte or smooth muscle cell; The epithelial cell of respiratory tract; The cell of central nervous system; Cancerous cell; Immune cell is allly known lymphocyte, macrophage, NK cell etc.
The tree-shaped polypeptide macromolecule carrier system that loads therapeutic gene is used for diseases such as tumor, the especially Xiang Guan tumor disease due to the inactivation of heredopathia, immunodeficiency or antioncogene due to the systematic treating genetic flaw.
Wherein said neoplastic disease is selected from leukemia and (comprises acute leukemia, the acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin, the non-hodgkin's disease, multiple myeloma, idiopathic macroglobulinemia disease, solid tumor, sarcoma and cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma of nipple, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterus carcinoma, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, durosarcoma, melanoma, neuroblastoma and retinal neuronal cell tumor etc.
The specific embodiment
Specify the present invention with embodiment below, disclosure and description the present invention is used for the compositions and the method for gene delivery, be not limited to concrete structure disclosed herein, method step and amino acid monomer, this is because such structure, method step and amino acid monomer can change.Term used herein is only used for describing specific embodiments rather than limits, and this is because scope of the present invention will only be subjected to the restriction of claims and equivalents thereof.
The present invention relates to a kind ofly can form tree-shaped polypeptide macromolecule carrier system of loading therapeutic gene stable, soluble complex and preparation method thereof with nucleic acid, described compositions contains and the covalently bound tree-shaped polypeptide macromolecules in cation macromolecular unit such as Polyethylene Glycol.In dissociated process, this carrier system complex can discharge nucleic acid so that the cell of transfection several types.
The following example should not be construed as the limitation of the present invention that goes up in all senses.
Synthesizing of embodiment 1 lysine base branch polypeptide macromolecule (1)
Lysine is to use maximum cladodification unit when making up polypeptide tree-shaped macromolecule, is nuclear with the .alpha.-aminodiphenylmethane., and the L-lysine of Boc protection is the cladodification unit, and DCC is a coupling agent, and trifluoroacetic acid is a deprotection agent, carries out the reaction of coupling-deprotection repeatedly.In the building-up process each all needs to separate purification with column chromatography for product, and then carries out follow-on synthesizing, so efficient is lower, but the structure of the product of gained is very regular, and theoretical molecular is almost consistent with mass spectrometric measurement gained result.The structure of this dendroid polypeptide macromolecule is very regular, and outer amino quantity can accurately control, the typical characteristic with dendritic macromole as: in the molecular structure pine outer tight, molecular dimension is single, solution viscosity is little etc.The coupling agent that adopts in the reaction is phosphorous condensing agent HBTU and HOBt.Side reaction is many in the reaction of DCC, and impurity is difficult to remove: and side reaction is few in the reaction of HBTU, and impurity is removed easily, therefore the normal phosphorous condensing agents such as BOP or HBTU that use in polypeptide tree-shaped macromolecule synthetic.The adding of HOBt is in order further to reduce side reaction, to reduce racemization simultaneously.Can obtain a generation, secondary by popular response to polybasic lysine base dendroid polypeptide macromolecule (1G-1,2G-1.......7G-1).
Synthesizing of embodiment 2 lysine base tree-shaped polypeptide macromolecule-polyethylene coupling compounds (1-PEG)
In the nitrogen protection ice-water bath, the Polyethylene Glycol 0.042M of various molecular weight ranges is dissolved among the anhydrous DMF of 0.5mL.The IBCF (0.036M) that will be dissolved in 0.5mL NMF dropwise adds in the above-mentioned solution.This mixture was stirred in ice-water bath 30 minutes, dropwise join then and contain 25mg lysine base dendroid polypeptide macromolecule 1HBr) and the anhydrous dimethyl sulfoxide of 1mL (DMSO) of 10 μ L TEA in, restir 3 hours at room temperature then. precipitated product in the absolute ether of 100mL, and with resolution of precipitate in the 10mL distilled water.With its NaHCO at 0.01M
3Dialysis (MWCO of bag filter is 25,000) in the solution, and then in the hydrochloric acid solution of 0.1M, dialyse, in distilled water, dialyse again afterwards.The product of last lyophilization dialysis gained.By
1H-NMR calculates mol ratio amino among the nG-1-PEG.
NG-1-PEG's
1H-NMR analyzes:
In water, carry out 1-PEG's that the step by embodiment 1 and 2 makes
1In the H-NMR collection of illustrative plates, approximately the peak at 3.5ppm place shows and exists PEG in the said composition.By with PEG (CH
2CH
2-, s, 3.4-3.7ppm) peak and 1 side chain (CH
2CH
2CH
2-, m, 1.1-1.8ppm) peak is associated, from NMR spectrum, calculate the content of PEG. in four kinds of illustrative carrier compositions that confirmed to make by this method by the present invention PEG content such as down: be respectively 7 (5G-1-PEG), 11 (4G-1-PEG), 20 (2G-1-PEG) and 30 (7G-1-PEG) mole %.
Synthesizing of embodiment 3 glutamic acid base tree-shaped polypeptide macromolecules (2)
With L-ethyl glutamate and (Z)-L-glutamic acid as the cladodification unit, HBTU and HOBt utilize liquid phase method to synthesize segmental dendroid polypeptide macromolecule segment as coupling agent, by convergence method and specific core bonding, obtain ball-type, the dumbbell shape dendroid polypeptide macromolecule of different structure then.Can obtain a generation, secondary by popular response to polybasic glutamic acid base dendroid polypeptide macromolecule (1G-2,2G-2.......7G-2).
Synthesizing of embodiment 4 glutamic acid base tree-shaped polypeptide macromolecule-polyethylene coupling compounds (2-PEG)
Building-up process is with reaction embodiment 2.Obtain 1G-2-PEG, 7G-2-PEG......7G-2-PEG.By
1H-NMR calculates mol ratio amino among the nG-2-PEG.
Synthesizing of embodiment 5 proline base tree-shaped polypeptide macromolecules (3)
Proline makes it have unique space chemistry character owing to have a circulus and imines in the molecule.The proline oligomer has two kinds of different conformations.In organic solvent, polyproline is right hand helix chain structure (PPI); In aqueous solution, polyproline is left-handed coiled strand structure (PPII).Based on this specific character, make up polypeptide tree-shaped macromolecule with the oligomerization proline, be expected to develop a kind of functional pharmaceutical carrier.Medicine can be wrapped in organic solvent in the coiled strand that is in PPI structure picture, and when in the human body environment, the conformation of oligomerization proline will change to PPII from PPI, thereby medicine obtains discharging.The synthetic available strategy of proline polypeptide tree-shaped macromolecule is to adopt the convergence solid-phase synthesis.Can obtain a generation, secondary by popular response to polybasic proline base dendroid polypeptide macromolecule (1G-3,2G-3.......7G-3).
Synthesizing of embodiment 6 glutamic acid base tree-shaped polypeptide macromolecule-polyethylene coupling compounds (3-PEG)
Building-up process is with reaction embodiment 2.Obtain 1G-3-PEG, 7G-3-PEG......7G-3-PEG.By
1H-NMR calculates mol ratio amino among the nG-3-PEG.
Synthesizing of embodiment 7 tree-shaped polypeptide macromolecule carrier-green fluorescence protein gene (pEGFP) complexs
Get the tree-shaped polypeptide macromolecule nano-particle aqueous suspensions of 20 μ L concentration, 1 μ g/ μ l, add 6 μ gpEGFP plasmid DNA, mix homogeneously, room temperature was placed 30 minutes.Can obtain nG-1-pEGFP thus, nG-2-pEGFP, nG-3-pEGFP and nG-1-PEG-pEGFP, nG-2-PEG-pEGFP, the genophore complex of nG-3-PEG-pEGFP.
The DNA-nano-particle complex that obtains is added in the 200 μ l serum-free mediums, and mixing was placed 60 minutes.Culture medium dropwise adds to cultivate to be had in human breast cancer cell's the culture dish, and co-cultivation 4-6 hour, and then add the complete medium co-cultivation.Cultivate after 20～40 hours the fluorescence of observation of cell under fluorescence microscope, carry out genetically modified detection.
Method of the same race can prepare tree-shaped polypeptide macromolecule carrier-red color fluorescence protein gene complex
Embodiment 8 tree-shaped polypeptide macromolecules carrier-green fluorescence protein gene in-vitro transfection experiment
With insect cell line Sfg is test cell system.Cell culture processes: the cell of the trophophase of taking the logarithm, cell degree of converging reaches at 90% o'clock, adds a certain amount of fresh Grace ' s insect cell culture medium that contains 10% hyclone.The cell dispersion agglomerate divides to install in the culture bottle, is positioned in 27 ℃ of incubators and cultivates.
The complex solution for preparing the tree-shaped polypeptide macromolecule carrier-plasmid DNA of a series of different quality ratios with reference to the method for embodiment 6 respectively.It is identical in quality to guarantee to be coated in every kind of proportioning plasmid DNA.
Transfection method: preceding 24 hours of transfection, with 1 * 10
6The density of individual cells/ml is inoculated in 24 orifice plates, makes cell degree of converging reach 75%.Every hole adds 1 milliliter of Grace ' s insect cell culture medium that contains serum.Transfection same day, former culture medium is gone in suction, and once with the cell culture medium washed cell, then with the above-mentioned carrier of 100 microlitres/plasmid dna complex compound and 400 microlitre serum-free medium mixings, and add equably in every hole, cultivated 5-8 hour for 27 ℃, inhale and removed to add after the former culture medium 1 milliliter of Grace ' s insect cell culture medium culturing that contains serum 6-9 days, observe with laser confocal microscope then and take pictures.The transfection efficiency result shows, the genophore of various molecular weight, and the efficient that green fluorescence protein gene is expressed all is higher than the red fluorescent protein gene.Contrast finds that what transfection efficiency was the highest is nG-2 and nG-3-PEG sample, surpasses 50%.By optimizing the molecular weight of these carriers, its transfection efficiency is expected to further raising
Synthesizing of embodiment 9 tree-shaped polypeptide macromolecules carrier-pSV1-β-gal gene vector system complex
The tree-shaped polypeptide macromolecule carrier of varying number (0.1-10 μ g) adds the matter of 2 μ g to and draws among the DNA, and at room temperature cultivates 30 minutes.The gel electrophoresis sample buffer is joined in each sample, adopt electrophoresis system under 100V, to come sample separation in 90 minutes then by electrophoresis on 1% (w/v) agarose gel.Use TBE (pH8.0) buffer is as electrophoretic buffer for 45mMTris-borate, 1mM EDTA. with Australia's second ingot (1 μ g/mL) gel-colored 30 minutes are also thrown light on to show the position of DNA with the ultra-violet light-emitting device.Employing is gone into the label of DNA (Promega) as the DNA size by EcoRI digestion.Can obtain nG-1-pSV1-β-gal thus, nG-2-pSV1-β-gal, nG-3-pSV1-β-gal and nG-1-PEG-pSV1-β-gal, nG-2-PEG-pSV1-β-gal, the genophore complex of nG-3-PEG-pSV1-β-gal.
The transfection of embodiment 9 tree-shaped polypeptide macromolecules carrier-pSV1-β-gal
Adopt Hep G2 cell line to estimate the in-vitro transfection rate of tree-shaped polypeptide macromolecule carrier-pSV1-β-gal.Under 37 ℃, at 5% CO
2In the incubator, in the MEM of 1mM Sodium Pyruvate and 10%FBs culture medium, keep cell, use not contain the cell culture medium of FBs as mixed culture medium.In-vitro transfection carries out as follows usually.With the HepGZ cell with 20 * 10
4The density of individual cell/mL is seeded in the flat microdetermination flat board in 96 holes, and cultivates 24 hours before adding plasmid DNA/carrier complex or single adding carrier.Prepare tree-shaped polypeptide macromolecule carrier-pSV1-β-gal complex by the pSV1-β-gal of mixing 1 μ g and the carrier of varying number in the cell culture medium that does not contain FBS at 100 μ L. and at room temperature cultivated 30 minutes.Add FBs and chloroquine with the ultimate density of 10% (v/v) and 100 μ M respectively.Replace the culture medium in each hole in the 96 hole flat boards with the transfection mixture of 100 μ L.Then under 37 ℃ at 5%CO
2Cultured cell is 4 hours in the incubator.After 4 hours, remove transfection mixture and in each hole, add the fresh culture that 100 μ L contain 10%FBS.Cultured cell 48 hours again under 37 ℃.Measure transfection efficiency by measuring by the activity of the general enzyme of beta galactose in pSV1-β-gal introducing cell.
Embodiment 10 tree-shaped polypeptide macromolecules carrier-pSV1-β-gal cytotoxicity
Tree-shaped polypeptide macromolecule carrier-pSV1-β-gal transfection Hep G2 cell line, the different genophore of concentration (being 30 μ g/mL in the cell culture medium) test with each hole 3 μ g in 96 orifice plates. genophore was cultivated 4 hours with cell, measured the survival of cell then.The result demonstrates the cytotoxicity at the synthetic vectors of Hep G2 cell.Under the situation that has 30 μ g/mL genophores to exist, cultivated Hep G2 cell 4 hours.For tree-shaped polypeptide macromolecule carrier-pSV1-β-gal, in Hep G2 cell, do not observe cytotoxicity, tree-shaped polypeptide macromolecule then presents medium cytotoxicity to height.The difference of PEG substitute proportion is not brought significant variation aspect cell survival.This can obtain differing the further support of microscopic observation, promptly tree-shaped polypeptide macromolecule carrier-pSV1-β-gal and Hep G2 cell is not together cultivated and is brought any remarkable influence for the viability of cell.The tree-shaped polypeptide macromolecule carrier is cultivated the form that changed cell in 4 hours with cell, but tree-shaped polypeptide macromolecule carrier-pSV1-β-gal does not change the form of cell significantly under identical concentration and incubation time.
Claims (9)
1. one kind can be used for preparing the tree-shaped polypeptide macromolecule carrier system that loads therapeutic gene, it is characterized in that by therapeutic gene, tree-shaped polypeptide macromolecule or have the tree-shaped polypeptide macromolecule that is connected with cation high molecular forming.
2. press the tree-shaped polypeptide macromolecule carrier system of the described loading therapeutic gene of claim 1, it is characterized in that described carrier system can be made up of the tree-shaped polypeptide macromolecule nanoparticle, its construction unit is natural aminoacid, can be single aminoacid, also can be made up of different aminoacid, wherein aminoacid is preferably from following compounds: lysine, glutamic acid, proline and hydroxyproline etc.; Described tree-shaped polypeptide macromolecule is that 1-7 is for chemical compound.
3. press the tree-shaped polypeptide macromolecule carrier system of the described loading therapeutic gene of claim 1, it is characterized in that described carrier system can form by being connected with the nanoparticle that the cation macromolecular tree-shaped polypeptide macromolecule constructs, cation macromolecular is preferably from following compounds: chitosan, Polyethylene Glycol, polymine etc.
4. the tree-shaped polypeptide macromolecule carrier system of claim 1 arbitrary claim to the claim 4 can be acceptable salt compounds of pharmacy and ester type compound: salt compounds is for alkaline tree-shaped polypeptide macromolecule carrier preferably fluoride, chloride, bromide, iodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalates, succinate, tartrate, malate, mandelate, mesylate or tosilate; For the cation of acid tree-shaped polypeptide macromolecule carrier based on alkali metal and alkaline-earth metal, as sodium salt, lithium salts, potassium salt, calcium salt, magnesium salt, aluminum salt etc., and ammonium, quaternary ammonium and amine cation, comprising but be not limited to: ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethamine etc. and other the representative organic amine that is used to form base addition salts comprise diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine; Ester type compound, finger is the ester of hydrolysis in vivo, being included in degrades in the human body discharges the ester of parent compound or its salt, suitable ester group for example comprises: derived from pharmaceutically acceptable aliphatic carboxylic acid or aliphatic family alcohol, especially alkanoic acid (alcohol), alkenoic acid (alcohol), aphthenic acids (alcohol) and alkane dicarboxylic acid's's (glycol) ester, wherein preferred each alkyl or alkenyl partly is less than 6 carbon atoms.
5. the tree-shaped polypeptide macromolecule carrier system of claim 1 arbitrary claim to the claim 5, the ratio that it is characterized in that described carrier and therapeutic gene is 0.5～20: 1.
6. the tree-shaped polypeptide macromolecule carrier system of claim 1 arbitrary claim to the claim 5, it is characterized in that described carrier and therapeutic gene contain the gene order of genetic coding labelling, described genetic marker is selected from luciferase genes, the general enzyme gene of p one galactose, hygromycin Hangzhoupro property gene and neomycin Hangzhoupro property gene and the dead drunk transferase gene of chloromycetin second, is selected from the gene inhibition agent of low density lipoprotein receptor, coagulation factors, tumor, main tissue compatible protein, Hangzhoupro oncogene, P16, p53, the general kinases of breast, IL2, IL4 and TNF α.
7. the tree-shaped polypeptide macromolecule carrier system of claim 1 arbitrary claim to the claim 5 is characterized in that described therapeutic gene contains the former DNA sequence in the viral Hangzhoupro of coding, is selected from the RNA of RNA, antisense RNA and ribozyme; Described nucleic acid coding agglutinin, mannose receptor, sialoadhesin or retrovirus trans-activation are because of in (TAT).
8. the method for a transfectional cell, described method comprise make said cell with 1 to the claim 5 the tree-shaped polypeptide macromolecule carrier system of arbitrary claim contact under certain conditions, wherein said compositions enters into said cell and discharges the nucleic acid of said compositions under described condition.
9. the tree-shaped polypeptide macromolecule carrier system of claim 1 arbitrary claim to the claim 5, it is characterized in that the diseases such as tumor due to the inactivation of heredopathia, immunodeficiency or antioncogene due to the described carrier system therapeutic gene defective, especially Xiang Guan tumor disease, for example leukemia, colon cancer, carcinoma of prostate, hepatocarcinoma, cancer of pancreas etc.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113368236A (en) * | 2021-04-28 | 2021-09-10 | 温州医科大学附属口腔医院 | Amphiphilic dendritic polypeptide for photodynamic/NO synergistic anti-biofilm infection and wound healing promotion, and preparation method and application thereof |
| CN113999279A (en) * | 2020-11-04 | 2022-02-01 | 中国药科大学 | Dumbbell type amphiphilic peptide dendrimer, synthesis and application of dendrimer as drug delivery system |
| CN114478301A (en) * | 2022-03-11 | 2022-05-13 | 苏州锦博莱生物医药科技有限公司 | Gene delivery vector, and preparation method and application thereof |
-
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- 2008-10-23 CN CNA2008101716386A patent/CN101461947A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999279A (en) * | 2020-11-04 | 2022-02-01 | 中国药科大学 | Dumbbell type amphiphilic peptide dendrimer, synthesis and application of dendrimer as drug delivery system |
| CN113368236A (en) * | 2021-04-28 | 2021-09-10 | 温州医科大学附属口腔医院 | Amphiphilic dendritic polypeptide for photodynamic/NO synergistic anti-biofilm infection and wound healing promotion, and preparation method and application thereof |
| CN114478301A (en) * | 2022-03-11 | 2022-05-13 | 苏州锦博莱生物医药科技有限公司 | Gene delivery vector, and preparation method and application thereof |
| CN114478301B (en) * | 2022-03-11 | 2023-10-20 | 苏州锦博莱生物医药科技有限公司 | Gene delivery vector, preparation method and application thereof |
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