CN103224610B - Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof - Google Patents
Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof Download PDFInfo
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- CN103224610B CN103224610B CN201310150710.8A CN201310150710A CN103224610B CN 103224610 B CN103224610 B CN 103224610B CN 201310150710 A CN201310150710 A CN 201310150710A CN 103224610 B CN103224610 B CN 103224610B
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Abstract
The invention discloses a polymer prepared by polymerization with a framework of ethylene glycol diglycidyl ether and a diamines orthoester monomer (4'-methyleneoxy-di-(2-aminoethoxy-1,3-dioxolane) according to a molar ratio of 1:1-2. With different ratios, polymers with different branched degrees can be obtained by polymerization. The polymer has a branched structure, and has the characteristics of acid sensitivity, controllable degradation, low cytotoxicity, and high transfection efficiency. As a high-efficiency gene vector, the material has a wide application prospect in gene treatment. Through the comparison on the properties (such as DNA coating capacity and transfection effect) of polymers with different branched degrees, valuable experiences are provided for future gene vector researches.
Description
Technical field
The invention belongs to biological medicine, polymer medical material tech field, be specifically related to a class with ethylene glycol diglycidylether and a kind of two ends containing the amino new monomer of ortho ester for raw material prepare have branched structure, the synthetic method of the cationic polymers of acid-sensitive and application.
Background technology
Gene therapy is a kind of brand-new disease treatment pattern, is normal gene or tool medicative channel genes human body target cell suitably to be expressed by certain way, to correct or to improve the defect that disease gene produces, reaches the object of disease therapy.Along with deepening continuously of gene therapy research, lacking safe and effective gene delivery system is the Main Bottleneck that restriction gene therapy is implemented.So, develop suitable carrier, especially acid-sensitive, the genophore with sustained release performance in born of the same parents, making safe target cell, the controlled and high expression of goal gene, is the key of gene therapy success.
The main partitivirus carrier of gene therapy vector and non-virus carrier.Virus vector has higher transfection efficiency, but there is the drawbacks such as immunogenicity is high, toxicity is large, targeting specific is poor, seriously limits their application clinically.Non-virus carrier mainly refers to positively charged cationic gene carriers, comprises cationic-liposome and cationic polymers, have low toxicity, low immune response, without advantages such as gene insert size restrictions.
Common cationic polymers, as poly-acetimide (PEI), polylysine, chitosan, tree dress macromole etc., forms compound polyelectrolyte by electrostatic interaction and DNA, can effectively condensation plasmid DNA.But strong electrostatic interaction also limit gene and enters from the release mixture after enchylema, confine expression of genes.Cell components such as tenuigenin, endosome, lysosome, endoplasmic reticulum, golgi body, plastosome, nucleus all maintain unique pH value (pH value from lysosomal 4.5 to mitochondrial 8.0).The pH value gradient of different components and the change of significant physico-chemical property in cell, the gene delivery system with acid-sensitive produces actively impact by the intracellular delivery of gene.Therefore, pH acid-sensitive polymer support, can by changing self macromolecular conformation, the stimulation of structure microenvironment to external world makes response, effectively can solve the drawback of conventional polymer carrier for DNA " packaging easily, disassemble difficulty ", application prospect is good.
At present, poly-acetimide PEI has good DNA stowage capacity, and can promote that DNA escapes to " proton sponge " characteristic tenuigenin from endosome, and transfection efficiency is high, is cationic polymer gene vector studied at present, there are some researches show, the increase of PEI degree of branching can strengthen the ability of its parcel DNA, but its non-degradable, transfection efficiency is higher, and cytotoxicity is also larger.The cationic polymers with branched structure of synthesis in this research, have stronger parcel DNA ability, and cytotoxicity is low, the acid-sensitive performance that it possesses makes again it possess the ability of good released dna, can meet clinical service requirements.Further, by the genophore of more different degree of branching, its character of integrated survey (as DNA wraps up ability, transfection, cytotoxicity, palliating degradation degree) etc., provide possibility for seeking best genophore.
Summary of the invention
The object of the present invention is to provide a kind of synthesis, there is branched structure, acid-sensitive, cytotoxicity is low, transfection efficiency is high cationic polymer gene vector.This cationic polymer gene vector has that preparation method is simple, cost is low, cytotoxicity is low, safe and practical, good biocompatibility, controlled degradation property, efficiency gene transfection advantages of higher.
The object of the present invention is to provide above-mentioned have branched structure, acid-sensitive, the preparation method of cytotoxicity is low, transfection efficiency is high cationic polymer gene vector.This preparation method's technique is simple, is easy to control, with low cost.
The object of the present invention is to provide the application as genophore in gene therapy of above-mentioned cationic polymers.
The object of the present invention is to provide a kind of have branched structure, acid-sensitive, cytotoxicity is low, transfection efficiency is high cationic polymer gene vector, with ethylene glycol diglycidylether and a kind of diamine ortho ester monomer (4 '-dimethylene oxygen-two-(2-amino ethoxy-1,3-dioxolane)
for skeleton, the ratio of 1: 1-2 is polymerized in molar ratio.
The preparation method of cationic polymer gene vector provided by the invention comprises the following steps: get ethylene glycol diglycidylether and be placed in two-neck bottle, add appropriate DMF, after stirring and dissolving, add diamin type ortho ester monomer (ethylene glycol diglycidylether: diamin type ortho ester monomer mole ratio=1-2: 1) wherein to stir, under anhydrous and oxygen-free room temperature condition, react 48h; Reaction product being loaded cutoff is in the dialysis tubing of 3000-4000, and dialyse distilled water (wherein adding 0.01% triethylamine) 24-48h; The lyophilize of accurate measuring 1mL dialysis product, obtains the series polymer that degree of branching is different.
Cationic polymers provided by the invention is formed by ethylene glycol diglycidylether and diamine ortho ester monomer-polymer, when both ratios are different, along with the increase of ethylene glycol diglycidylether mol ratio, polymkeric substance is progressively cross-linked by linear structure (branching), final formation 3 D stereo reticulated structure.Contained amino with positive charge in polymkeric substance, can DNA be compressed, form nano level mixture, make it can by delivery of nucleic acids in cell.Comprise polyamines and the responsive ortho ester structure of pH in this polymkeric substance, there is micro-acid response and controlled feature of degrading, make this polymkeric substance the genomic medicine of parcel can be made to be easy to discharge from endosome and lysosome as genophore, be conducive to improving transfection efficiency; Its degradability also makes this polymkeric substance have good biocompatibility, and cytotoxicity reduces greatly compared with the PEI that can not degrade.Polymkeric substance of the present invention had both remained the high transfection efficiency suitable with PEI, greatly reduced cytotoxicity again, and possessed acid-sensitive and controlled feature of degrading.
The invention provides the polymkeric substance of different degree of branching, by comparing its character, reach a conclusion: degree of branching is higher larger to DNA parcel ability, but cytotoxicity is corresponding increase also, transfection efficiency also can reduce, and therefore selects the polymkeric substance of suitable degree of branching to obtain the highest transfection efficiency.
Accompanying drawing explanation
Fig. 1 is that ethylene glycol diglycidylether and diamine ortho ester monomer synthesize the schematic diagram of the polymkeric substance 1,2,3 obtained according to the ratio that mol ratio is 1: 1,1.5: 1 and 2: 1.
Fig. 2 is that mtt assay measures polymkeric substance 1,2,3 cytotoxicity schematic diagram; Wherein, Fig. 2-2 is the partial enlarged drawing of 2-1;
Fig. 3 is the gel electrophoresis figure of the mixture that polymkeric substance 1,2,3 and DNA are formed with different mass ratio;
Fig. 4-1 is the particle size analysis figure of the mixture that polymkeric substance 1,2,3 and DNA are formed with different mass ratio;
Fig. 4-2 is the potentiometric analysis figure of the mixture that polymkeric substance 1,2,3 and DNA are formed with different mass ratio;
Fig. 5 is the gene transfection situation map of the mixture that polymkeric substance 1,2,3 and DNA are formed with different mass ratio;
Embodiment
Below by embodiment, the present invention is further described.The present invention is not limited to following embodiment, in the scope illustrated by the claims in the present invention, can carry out various change and equivalent replacement to the present invention.
Embodiment 1 has the preparation method of the acid-sensitive cationic polymer gene vector of branched structure
Get ethylene glycol diglycidylether 0.283g and be placed in two-neck bottle, add 1mL DMF, after stirring and dissolving, (i.e. ethylene glycol diglycidylether: diamin type acid-sensitive monomer mole ratio=1: 1) stir, reacts 48h under anhydrous and oxygen-free room temperature condition to add 0.5g diamin type acid-sensitive monomer wherein; Reaction product being loaded cutoff is in the dialysis tubing of 3000-4000, and dialyse distilled water (wherein adding 0.01% triethylamine) 24-48h; The lyophilize of accurate measuring 1mL dialysis product, takes the quality after freeze-drying, namely obtains the concentration of polymkeric substance in 1mL dialysis solution.Residual content is freezen protective in liquid form.This polymerisate is polymkeric substance 1.
Get ethylene glycol diglycidylether 0.424g and be placed in two-neck bottle, add 1mL DMF, after stirring and dissolving, (i.e. ethylene glycol diglycidylether: diamin type acid-sensitive monomer mole ratio=1.5: 1) stir, reacts 48h under anhydrous and oxygen-free room temperature condition to add 0.5g diamin type acid-sensitive monomer wherein; Reaction product being loaded cutoff is in the dialysis tubing of 3000-4000, and dialyse distilled water (wherein adding 0.01% triethylamine) 24-48h; The lyophilize of accurate measuring 1mL dialysis product, takes the quality after freeze-drying, namely obtains the concentration of polymkeric substance in 1mL dialysis solution.Residual content is freezen protective in liquid form.This polymerisate is polymkeric substance 2.
Get ethylene glycol diglycidylether 0.565g and be placed in two-neck bottle, add 1mL DMF, after stirring and dissolving, (i.e. ethylene glycol diglycidylether: diamin type acid-sensitive monomer mole ratio=2: 1) stir, reacts 48h under anhydrous and oxygen-free room temperature condition to add 0.5g diamin type acid-sensitive monomer wherein; Reaction product being loaded cutoff is in the dialysis tubing of 3000-4000, and dialyse distilled water (wherein adding 0.01% triethylamine) 24-48h; The lyophilize of accurate measuring 1mL dialysis product, takes the quality after freeze-drying, namely obtains the concentration of polymkeric substance in 1mL dialysis solution.Residual content is freezen protective in liquid form.This polymerisate is polymkeric substance 3.
As shown in Figure 1, by diamin type acid-sensitive monomer from ethylene glycol diglycidylether under the condition of different mol ratios, ring opening polyaddition, formed microtexture be linearly, cladodification or reticular pattern superpolymer.When both mol ratios are 1: 1, polymkeric substance mainly exists in linear form; Along with the increase of monomer 2, start between linear polymer to be cross-linked (cladodification), thus formed netted; When monomer 1: the mol ratio of monomer 2 is 1: 2, and polymkeric substance will form 3 D stereo reticulated structure (Fig. 1).
Embodiment 2 measures the cytotoxicity of polymkeric substance with mtt assay
NIH/3T3 cell to be inoculated in 96 orifice plates (1 × 10
4individual/hole), every hole adds containing 10% foetal calf serum DMEM substratum 200 μ L37 DEG C, 5%CO
224h is cultivated under condition.Abandon substratum, add 180 μ L fresh DMEM medium to each hole and with 20 μ L with the cationic polymer solution of 20mM HEPES solution allocation series concentration, make polymkeric substance final concentration be: 1mg/mL, 5mg/mL, 10mg/mL, 50mg/mL, 100mg/mL, 500mg/mL, 1000mg/mL, 5000mg/mL.37 DEG C, 5%CO
224h is cultivated again under condition.Every hole adds MTT solution (5mg/mL) 20 μ L, and 37 DEG C are continued to cultivate 4h.Stop cultivating, discard culture supernatant in hole, add 100 μ L dimethyl sulfoxide (DMSO) (DMSO), shake 10min gently.Measure each hole in the light absorption value of 570nm, calculate cytoactive (see Fig. 2).
As figure learns, the raising along with degree of branching of polymkeric substance 1,2,3, cytotoxicity increases.But polymkeric substance 1,2,3 is much less than the cytotoxicity of PEI.PEI is when concentration is 25mg/mL, and cell survival rate is 50%.And polymkeric substance 1,2,3 is respectively when 3500mg/mL, 1000mg/mL and 275mg/mL, cell survival rate is 50%.
Embodiment 3 mixture is formed
With green fluorescent protein plasmid pEG-N1 for research object investigate DNA and cationic polymers in conjunction with situation.The pEG-N1 of DNA purity > 95% is dissolved in 20mM HEPES solution, is configured to the DNA liquid storage of 0.1 μ g/ μ L; With 20mM HEPES solution, polymkeric substance 1,2,3 is diluted to suitable concn gradient.Get 50 μ L DNA liquid storages (namely DNA amount is 5 μ g), add 50 μ L mixture gradient concentration solution wherein, make final volume be 100 μ L, and make DNA: polymkeric substance (mass ratio) is respectively 8: 1,4: 1,2: 1,1: 1,1: 2,1: 4,1: 8,1: 16.By mixture vortex oscillation 30s, room temperature is placed 20min and be get final product.
The sepharose retardation experiment of embodiment 4 mixture and DNase I are to the enzymolysis analysis of mixture
By DNA: polymkeric substance (mass ratio) 8: 1,4: 1,2: 1,1: 1,1: 2,1: 4,1: 8,1: 16 configures mixture, often in group, DNA amount is 5 μ g, and mixture final volume is 100 μ L.Vortex oscillation 30s mixes mixture, standing at room temperature 20min.Get 10 μ L mixture agarose gel electrophoresis and detect mixture retarding degree (see (A) in Fig. 3).
Utilize DNase I enzyme to investigate mixture to the parcel situation of DNA, reaction system is as follows:
Mixture 89 μ L
DNase I buffer 10μL
DNase I 1μL (0.1U/μL)
Above system 37 DEG C insulation 10min, gets 10 μ L agarose gel electrophoresis and detects DNA degradation degree (see (B) in Fig. 3).
Along with the increase of polymer quality ratio, stable mixture can be formed with DNA gradually.Polymkeric substance with positive charge in various degree, can shield DNA phosphate group with positive charge, can not present in the electric field by the movement of negative pole to positive pole.Namely the motion of mixture in gel is blocked.In stable mixture, the mass ratio of polymkeric substance/DNA is enough large, can wrap up DNA completely, thus blocking dna degrading enzyme DNase I is to the Degradation of DNA, makes it can not be degraded to the smear of < 200bp.And not formed in the gradient of stable compound, DNA is all degraded to the smear of < 200bp.
As shown in the figure, during polymer1:DNA ratio (W/W) > 4: 1, stable mixture can be formed.
During Polymer2:DNA ratio (W/W) > 2: 1, stable mixture can be formed.
During Polymer3:DNA ratio (W/W) > 4: 1, stable mixture can be formed.
The mensuration of embodiment 5 mixture particle diameter and the mensuration of Zeta potential
By DNA: polymkeric substance (mass ratio) 8: 1,4: 1,2: 1,1: 1,1: 2,1: 4,1: 8,1: 16 configures mixture, often in group, DNA amount is 5 μ g, and mixture final volume is 100 μ L.Vortex oscillation 30s mixes mixture, standing at room temperature 20min.Malvern particle diameter and potentiometric analyzer is utilized to measure particle diameter (see Fig. 4-1) and the current potential (see Fig. 4-2) of each mixture.
As figure learns, when the mass ratio of polymkeric substance 1,2,3 and DNA be 4: 1 and higher time, polymkeric substance all can compress DNA and form stable, the diameter mixture at about 150-250nm.And the increase of the mass ratio along with polymkeric substance, mixture particle diameter diminishes, and when mass ratio reaches 16: 1, particle diameter can reach 150nm.In the mixture that polymkeric substance 1,2,3 is formed, the particle diameter of polymkeric substance 2 is minimum, and illustrate that polymkeric substance 1,2,3 all can form stable mixture with DNA, wherein the compression to DNA of polymkeric substance 2 is the most effective.
Embodiment 6 in-vitro transfection is tested
50000 l cell NIH/3T3 are inoculated in the 12 every holes of orifice plate, and 37 DEG C of 5%CO2 cultivate 24h.Plasmid DNA is diluted to suitable concn, makes in the DNA solution of 25 μ L containing 2 μ gDNA.By polymer dilution to finite concentration, by DNA: the polymkeric substance of polymkeric substance (mass ratio) 1: 4,1: 8,1: 16,1: 32 different amount for gradient adds, makes the polymer volume finally added be 25 μ L; 25 μ L polymkeric substance are joined inside 25 μ LDNA, blows and beats 10 times, to ensure to mix; Leave standstill 30min.Obtain transfection composite.Get the cell having cultivated 24h, absorb cells and supernatant, with PBS rinse twice, change 1mL plasma-free DMEM medium; Mixture 50 μ L is added, and weak vibrations, makes mixture be uniformly dispersed; Cultivate 4 hours; Absorb supernatant; With PBS rinse twice, then change full culture medium culturing 24h.Use the transfection in each hole of fluorescence microscope.PEI, the better transfection gradient of polymkeric substance 1,2,3 is have chosen: time PEI/DNA=16: 1 (w/w) in Fig. 5, time polymkeric substance 1/DNA=192: 1 (w/w), time polymkeric substance 2/DNA=128: 1 (w/w), transfection figure time polymkeric substance 3/DNA=64: 1 (w/w).Have figure known, the toxicity of PEI is comparatively large, and 16: 1 time, transfection efficiency is the highest, and when PEI concentration being improved, toxicity is excessive again, and transfection efficiency declines.The toxicity of polymkeric substance 1,2,3 is much smaller compared with PEI, and the mass ratio listing polymkeric substance 1,2,3 and DNA in figure reaches transfection when 192: 1,28: 1,64: 1, well a lot of compared with the transfection of PEI the best.Illustrate that in the present invention, polymkeric substance has good application prospect in field of gene.In addition, the transfection efficiency of polymkeric substance 2 is best.Illustrate that structure and the degree of branching of polymkeric substance 2 are most suitable, both effectively can wrap up DNA, again can effective released dna, and cytotoxicity is also lower, is a polymkeric substance having the prospect of application.
Claims (7)
1. one kind has the acid-sensitive cationic polymer gene vector of branched structure, it is characterized in that this cationic polymer gene vector is with ethylene glycol diglycidylether and a kind of diamine ortho ester monomer 4,4 '-dimethylene oxygen-two-(2-amino ethoxy-1,3-dioxolane) be polymerized, the structure of this diamine ortho ester monomer is
2. cationic polymer gene vector as claimed in claim 1, it is characterized in that it is formed by ethylene glycol diglycidylether and a kind of diamine ortho ester monomer polymerization, comprise the structure of polyamines and the responsive ortho ester of pH, there is micro-acid response and controlled feature of degrading.
3. there is the preparation method of the acid-sensitive cationic polymer gene vector of branched structure as claimed in claim 1, it is characterized in that comprising the following steps: get ethylene glycol diglycidylether and be dissolved in DMF, press diamine ortho ester monomer wherein: the mol ratio 1: 1-2 of ethylene glycol diglycidylether drops into diamine ortho ester monomer, stir, under anhydrous and oxygen-free room temperature condition, react 48h; Reaction product being loaded cutoff is in the dialysis tubing of 3000-4000, and dialyse 24-48h in the distilled water adding 0.01% triethylamine; After product lyophilize and get final product.
4. there is the preparation method of the acid-sensitive cationic polymer gene vector of branched structure as claimed in claim 3, it is characterized in that forming with ethylene glycol diglycidylether and a kind of diamine ortho ester monomer polymerization, and ethylene glycol diglycidylether is different with the ratio of diamine ortho ester monomer when being polymerized, the structure of the cationic polymers obtained is just different with degree of branching, when diamine ortho ester monomer and ethylene glycol diglycidylether mol ratio are 1: 1, polymkeric substance mainly exists in linear form; Along with the mol ratio of ethylene glycol diglycidylether increases, between linear polymer, start crosslinked, branched, thus formed netted; When the mol ratio of diamine ortho ester monomer and ethylene glycol diglycidylether is 1: 2, polymkeric substance will form 3 D stereo reticulated structure.
5. the acid-sensitive cationic polymer gene vector with branched structure according to claim 1, is characterized by the purposes for non-viral gene vector; This carrier is nano level, and can form nano-complex with genomic medicine, genomic medicine comprises DNA or RNA.
6. according to the preparation method of the nanocomposite described in claim 5, it is characterized by: by cationic polymers according to claim 1 and genomic medicine DNA/RNA deionized water dissolving, mixing, vortex oscillation 30s, the static 30min of room temperature, cationic polymers and DNA/RNA are self-assembled into nano-complex by electrostatical binding.
7. nano-complex as claimed in claim 6, it is characterized in that for genomic medicine being effectively delivered to cell and effectively discharging genomic medicine, because it has branched structure in various degree, be convenient to find different genes medicine at the trim point transmitted and in release two, thus meet the requirement of different genes medicine.
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