CN101638484A - Polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine copolymer, preparation method thereof and application thereof - Google Patents
Polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine copolymer, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention relates to polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine copolymer, a preparation method thereof and application thereof. The preparation method comprises the step of directly condensing carboxyl in the polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine segmented copolymer with amino in polyethyleneimine to form the graft copolymer. The copolymer is a polycation gene carrier, integrates the advantages of polyethylene glycol, Makrolan and polyethyleneimine, and has high transfection efficiency, wherein the highest transfection efficiency to the medium luciferase plasmid of african green monkey kidney cell is 14 times of that of American Invitrogen biological company transfection reagent Lipofetamine<TM>2000, can effectively antagonize the inhibiting effect of blood serum to the transfection, and has less cell toxicity, wherein consistency thereof is not higher than 200 microgramme/ml and cell survival rate is more than 80%.
Description
Technical field
The present invention relates to poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer and method for making and application.
Background technology
Gene therapy is hoped by scientist becomes the important medical procedure that does not solve difficult and complicated illness, yet how to endure therapeutic gene fragment efficient transfer to the fullest extent puzzlement to the focus cell always.Be widely used in clinical viral vector at present and exist great potential safety hazard, caused the first in history death incident of gene therapy and famous France " bubble baby " incident.With respect to the insecurity of virus gene carrier, the cationic polymers of synthetic is owing to its no immunogenicity, and molecular designing is various, and controllable size also can connect the research focus that targeting substance becomes this field gradually.
In numerous polycation carriers of having reported, polymine (PEI) can realize that owing to it has unique " proton sponge effect " gene transmission efficiently receives much attention [referring to Boussif O, ZantaM.A, Behr J.P, et al.A versatile vector for gene and oligonucleotide transfer intocells in culture and in vivo-polyethylenimine.PNAS, 1995; 92:7297-7301].The transfection performance of PEI depends on molecular weight strongly, and the PEI (PEI25k) that generally uses molecular weight 25000 is as transfection carrier.The advantage of PEI is that electric density concentrates, and genetic stew is had strong compound ability, at low N/P ratio (N/P) than obtaining best transfection efficiency.But use homopolymer PEI as genophore merely, often be subject to its non-degradable and high cytotoxicity.
Polyoxyethylene glycol (PEG) is because the inhibition non-specific adsorption ability and the good biocompatibility of its excellence, the modification that often is applied to the positively charged ion genophore in existing report is [referring to Petersen H, Fechner P.M, Fischer D, Kissel T.Synthesis, characterization, and biocompatibility ofpolyethylenimine-graft-poly (ethylene glycol) block copolymers.Macromolecules, 2002; 35:6867-6874].Various studies show that after PEG modifies, the nano-particle complex of PEI and genetic stew can be avoided self cohesion, reduces granular size, and effectively antagonism serum reduces erythrocytic gathering to the inhibition of transfection.PEGization can shield the positive charge on composite particles surface, increases its stability in hypersaline environment in vivo, reduces cytotoxicity.
Polycarbonate is a kind of macromolecular material that has good biocompatibility and can realize biological vivo degradation, the polycarbonate that some of them have the functionalization group is applied to drug release carrier (reference Hu X.L by wide coverage, Chen X.S, Liu S, Shi Q, Jing X.B.Novel aliphaticpoly (ester-carbonate) with pendant allyl ester groups and its folic acidfunctionalization.Journal of Polymer Science:Part A:Polymer Chemistry, 2007; 1852-1861].)
Summary of the invention
In order to solve the problem that prior art exists, the object of the present invention is to provide poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer and method for making and application.Institute's synthetic poly glycol monomethyl ether-poly-the 2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is a degradable cationic polymer, does not see bibliographical information.Poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer had the advantage of polyoxyethylene glycol, polycarbonate and polymine concurrently, overcome the shortcoming of PEI homopolymer high toxicity, non-degradable.Poly glycol monomethyl ether-poly-the 2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is to have excellent biological compatibility, and antagonism serum transmits carrier to the high efficiency gene of the inhibition of transfection effectively.
The structural formula of poly glycol monomethyl ether provided by the invention-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is as follows:
Wherein, PEI is a polymine, and structure is line style or branching, and its structural formula is as follows:
Wherein polymerization degree m, n, a, b and c are equal to or greater than 1.
The preparation process and the condition of poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer are as follows:
(1) the mole proportioning according to poly glycol monomethyl ether (mPEG) and 2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate (MBC) is 1: 10～1: 100, both are dosed in the exsiccant reactor, with the proportioning of the volume ml of the total mass g of mPEG and MBC and toluene is 1: 5～50 to add dry toluenes, 130 ℃ of azeotropic water removings at least 2 hours, after azeotropic water removing is finished, temperature control steams part toluene for 130～150 ℃, proportioning with the volume ml of the total mass g of mPEG and MBC and remaining toluene is 1: 2～20, as the distillation end point; Is that 0.05～0.5mmol/ml is dissolved in the dry toluene with the catalyzer zinc ethyl with concentration, add zinc ethyl/toluene solution with zinc ethyl and the monomeric mole of MBC proportioning 1: 100～300,100～130 ℃ of stirring reactions of temperature control at least 12 hours, sedimentation in normal hexane after reaction is finished, filter, dry cake obtains poly glycol monomethyl ether-poly-2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate ester block copolymer (mPEG-b-PMBC);
Proportioning 1: 5～50 according to mPEG-b-PMBC quality g and tetrahydrofuran (THF) volume ml will be dosed in the reactor behind both mixed dissolutions together; Be to measure methyl alcohol at 2～5: 1 according to tetrahydrofuran (THF) and methyl alcohol volume proportion again, take by weighing palladium hydroxide/carbon with catalyzer palladium hydroxide/carbon and mPEG-b-PMBC quality proportioning 1: 2～10 and evenly be suspended in the methyl alcohol, be dosed in the reactor together then; Charge into nitrogen, hydrogenation is to pressure 0.5～2MPa, 40～60 ℃ of temperature controls, stirring reaction 24～72 hours after reaction is finished, removes by filter palladium hydroxide/carbon, filtrate is sedimentation in ether, filter, dry cake obtains poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer (mPEG-b-PMCC);
(2) proportioning according to mPEG-b-PMCC quality g and methyl-sulphoxide volume ml is 1: 10～100, both are dosed in the reactor, stirring and dissolving, mole proportioning with carboxyl among the mPEG-b-PMCC and polymine is 1: 1～4, polymine is added in the reactor, mole proportioning according to the carboxyl among the mPEG-b-PMCC and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC.HCl) is 1: 1～2 again, also be added to EDC.HCl in the reactor, 20～30 ℃ of temperature controls, stirring reaction at least 2 hours, after reaction is finished, with the reaction solution molecular weight cut-off of packing into is to deionized water dialysis at least 24 hours in 1000～5000 the dialysis tubing, with the liquid freezing drying after the dialysis, obtain poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer (mPEG-b-PMCC-g-PEI is called for short polyethylene glycol-carbonic ether grafting polyethylene imine copolymer).
Below introduce poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer in in-vitro transfection as gene delivery vector.The step and the condition of its usage are as follows:
(1) cultivation of cell
Cell places and contains the nutrient solution that volume fraction is 10% foetal calf serum, contains cultured continuously in the incubator that volume fraction is 5% carbonic acid gas at 37 ℃;
(2) in-vitro transfection
The transfection of I serum-free
In preceding 24 hours of the transfection, the cell in vegetative period of taking the logarithm is improved the dilution of Iger (DMEM) substratum with Da Erbaike after the trysinization, by every hole 1 * 10
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%, during transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after phosphate buffered saline buffer (PBS) washed twice, the composite particles of adding genome transfection and the DMEM substratum of serum-free are to final volume 200 μ/hole, continue to cultivate after 4 hours, the nutrient solution in the Tissue Culture Plate is abandoned in suction, change 200 μ l/ holes and contain the fresh DMEM nutrient solution that volume fraction is 10% foetal calf serum, continue to cultivate 20 hours;
II contains the serum transfection
In preceding 24 hours of the transfection, the cell in vegetative period of taking the logarithm is improved the dilution of Iger (DMEM) substratum with Da Erbaike after the trysinization, by every hole 1 * 10
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%, during transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after phosphate buffered saline buffer (PBS) washed twice, add the composite particles of genome transfection and contain volume fraction be the DMEM substratum of 10% foetal calf serum to final volume 200 μ l/ holes, continue to cultivate 24 hours;
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution,, add the cell pyrolysis liquid cracking, add the luciferase substrate then, measure transfection efficiency with luxmeter with PBS washing 2 times;
(4) cytotoxicity test
Adopt the cytotoxicity of tetrazolium bromide (MTT) colorimetry comparative evaluation gene vector material;
Test in preceding 24 hours, the cell in vegetative period of taking the logarithm with the dilution of DMEM substratum, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and arrives 80～90%, with the material of different concns and cell co-cultivation after 24 hours, every hole adds 20 μ l respectively and contains the PBS solution that massfraction is 0.5%MTT, mixture was 37 ℃ of continuation effects 4 hours, add 200 μ l dmso solution MTT Jia Za crystallizations 10 minutes, test the absorption value in every hole then with microplate reader, the test wavelength is selected 492nm for use, and cell survival rate is pressed formula and calculated:
Cell survival rate (%)=(A
Sample/ A
Control) * 100
A
SampleBe the absorption value in the cell sample hole after the transfection, A
ControlBe not with the absorption value in the cell sample hole of complex solution effect, every group of experiment triplicate.
Poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer in vivo in the transfection as gene delivery vector.The step and the condition of its usage are as follows:
Get the crust match nude mice in 5 weeks, at the epithelial cell strain of veutro subcutaneous vaccination human cervical carcinoma, treat that diameter of tumor grows to 0.4cm after, be divided into two groups at random, every group 9, contain the composite particles solution 100 μ l of the genome transfection of 5 μ g luciferase plasmids respectively at intratumor injection; Duplicate injection in second day was once injected back 48 hours, with transfection effect in the true living imaging instrument observation of the smart promise body; Before carrying out the living imaging observation, with mouse anesthesia, anaesthetize after 5 minutes, in the mouse body, inject 200 μ l luciferase substrates, after 10 minutes, with transfection effect in the living imaging systematic observation body.
Beneficial effect: prepared poly glycol monomethyl ether-poly-the 2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is a kind of polycation gene carrier, integrated polyoxyethylene glycol, polycarbonate and polymine advantage separately, the transfection efficiency height, it under equal conditions is up to the commercial transfection reagent Lipofectamine of American I nvitrogen biotech firm to the transfection efficiency of the cell-mediated luciferase plasmids of African green monkey kidney cell
TM2000 14 times, and antagonism serum is to the restraining effect of transfection effectively, cytotoxicity is little, and its concentration is not in being higher than 200 mcg/ml scopes, and cell survival rate is more than 80%.
Prepared poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate the grafting polyethylene imine copolymer of the present invention has low toxicity and biodegradable, because its selected polyethylene glycol-carbonic ether grafting skeleton has excellent biological compatibility, under physiological condition, can degrade voluntarily, collapse and metabolism, and then absorbed by organism or excrete, human body is had no side effect.
Institute of the present invention synthetic poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer has very high use value, has wide practical use in the gene transmission.
Description of drawings
Fig. 1 is the cytotoxicity test result figure of contrast different carriers material under series concentration.Test cell is selected African green monkey kidney cell (Cos-7 cell) for use, test concentrations is 0～200 mcg/ml, and test material is respectively representative PPP-11, PPP-14 and PPP-17 and the PEI25k and the commercial transfection reagent Lipofectamine of poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer
TM2000, test result is represented with the cell survival rate (%) after handling.
Embodiment
Embodiment 1: the preparation of poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer
Respectively according to the listed charging capacity of table 1, take by weighing number-average molecular weight and be 5000 poly glycol monomethyl ether (mPEG
5000) and 2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate (MBC), both are dosed in the different exsiccant reactors, the 130 ℃ of azeotropic water removings of dry toluene 5 hours that add 300ml respectively, after azeotropic water removing is finished, temperature control steams part toluene for 130 ℃, is that 50ml is as distilling end point separately with the volume of remaining toluene; The catalyzer zinc ethyl is configured in the dry toluene with volumetric molar concentration 0.2mmol/ml dissolving, add zinc ethyl/toluene solution to corresponding reactor according to the zinc ethyl charging capacity in the table 1 respectively, and abide by polymerization temperature in the table 1 and polymerization time and carry out separately stirring reaction respectively, sedimentation in normal hexane respectively after reaction is finished, filter, dry cake obtains poly glycol monomethyl ether-poly-2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate ester block copolymer (mPEG-b-PMBC);
Intermediate product mPEG-b-PMBC separately is dissolved in respectively in the tetrahydrofuran (THF) of 100ml, and be dosed in the different reactors, take by weighing the hydroxide palladium/carbon catalyst of corresponding charging capacity shown in the table 1 respectively, and evenly be suspended in the 30ml methyl alcohol separately, be dosed in the corresponding reactor, charge into nitrogen, charge into hydrogen according to corresponding hydrogen pressure in the table 1 respectively again, and carry out separately stirring reaction respectively according to hydrogenation temperature shown in the table 1 and hydrogenation time, after reaction is finished, remove by filter palladium hydroxide/carbon, filtrate sedimentation in ether is respectively filtered, dry cake, obtain poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer (mPEG-b-PMCC), use nmr analysis product number-average molecular weight (Mn), the results are shown in Table 1.
Embodiment 2: the preparation of poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer
Kind according to mPEG-b-PMCC listed in the table 2 takes by weighing corresponding charging capacity respectively, be dosed into together in the different reactors with the methyl-sulphoxide of the respective reaction volume described in the table 2, stirring and dissolving, again according to the polymine shown in the table 2 (PEI) kind and the molar weight that feeds intake, take by weighing PEI respectively and add in the corresponding reactor, again according to the molar weight that feeds intake shown in the table 2, taking by weighing 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC.HCl) respectively also is added in the corresponding reactor, 25 ℃ of temperature controls, respectively according to the reaction times in the table 2, carry out stirring reaction separately, after reaction is finished, it is to deionized water dialysis 72 hours in 3500 the dialysis tubing that reaction solution is respectively charged into molecular weight cut-off, liquid freezing drying after will dialysing respectively at last, obtain poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer (mPEG-b-PMCC-g-PEI), be called for short PPP, use nmr analysis product number-average molecular weight (Mn), the results are shown in Table 2.
Embodiment 3: poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer mediation luciferase plasmids is to the in-vitro transfection of African green monkey kidney cell (Cos-7 cell)
1, the cultivation of Cos-7 cell
Get the Cos-7 cell and place and contain the nutrient solution that volume fraction is 10% foetal calf serum, contain cultured continuously in the incubator that volume fraction is 5% carbonic acid gas at 37 ℃.
2, in-vitro transfection
(1) serum-free transfection
In preceding 24 hours of the transfection, the Cos-7 cell in vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, and placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%.During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after phosphate buffered saline buffer (PBS) washed twice, change 200 μ l/ holes and do not contain the fresh DMEM nutrient solution of foetal calf serum, selecting luciferase plasmids (pGL3) again for use is reporter gene, adds PPP-11/pGL3, PPP-14/pGL3, PPP-17/pGL3, PEI25k/pGL3 and commercialization transfection reagent Lipofectamine respectively
TMThe composite particles of 2000/pGL3 carries out the transfection contrast, cultivates after 4 hours, inhales the nutrient solution of abandoning in the Tissue Culture Plate, changes 200 μ l/ holes and contains the fresh DMEM nutrient solution that volume fraction is 10% foetal calf serum, continues to cultivate 20 hours.
(2) contain the serum transfection
In preceding 24 hours of the transfection, the Cos-7 cell in vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, and placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%.During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after phosphate buffered saline buffer (PBS) washed twice, change 200 μ l/ holes and contain the fresh DMEM nutrient solution that volume fraction is 10% foetal calf serum, selecting luciferase plasmids (pGL3) again for use is reporter gene, adds PPP-11/pGL3, PPP-14/pGL3, PPP-17/pGL3, PEI25k/pGL3 and commercialization transfection reagent Lipofectamine respectively
TMThe composite particles of 2000/pGL3 carries out the transfection contrast, continues to cultivate 24 hours.
3, the mensuration of transfection efficiency in vitro
Take out culture plate, inhale and abandon nutrient solution, PBS washing 2 times adds the cell pyrolysis liquid cracking, adds the luciferase substrate then, uses luxmeter to measure transfection efficiency, is expressed as every milligram of proteic luminescence unit number (RLU/mg Protein).
The transfection efficiency in vitro of table 3 mediation luciferase plasmids
| Sequence number | Solid support material | Whether contain foetal calf serum during transfection | Best transfection efficiency, RLU/mg Protein |
| ??1 | ??PEI25k | Not | ??9.023×10 9 |
| ??2 | ??PEI25k | Be | ??8.331×10 8 |
| ??3 | ??Lipofectamine TM?2000 | Not | ??1.091×10 10 |
| ??4 | ??Lipofectamine TM?2000 | Be | ??1.204×10 9 |
| ??5 | ??PPP-11 | Not | ??1.874×10 10 |
| ??6 | ??PPP-11 | Be | ??8.344×10 9 |
| ??7 | ??PPP-14 | Not | ??2.514×10 10 |
| ??8 | ??PPP-14 | Be | ??1.295×10 10 |
| ??9 | ??PPP-17 | Not | ??2.966×10 10 |
| ??10 | ??PPP-17 | Be | ??1.732×10 10 |
Poly glycol monomethyl ether among the present invention-poly-the 2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is a kind of material of gene delivery vector very efficiently, and has the restraining effect of antagonism serum to transfection.Table 3 has provided PPP-11, PPP-14, PPP-17, PEI25k and Lipofectamine in the in-vitro transfection experiment respectively
TM2000 best transfection efficiency has higher transfection efficiency by the poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer that contrasts PPP-11, PPP-14 and PPP-17 representative among the present invention as can be seen; And the contrast of serum and serum-free experimental result is arranged during by transfection, as can be seen the poly glycol monomethyl ether of PPP-11, PPP-14 and PPP-17 representative-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer effectively antagonism serum to the restraining effect of transfection, with respect to the serum-free transfection, PPP-11, PPP-14 and PPP-17 transfection efficiency (RLU/mg Protein) absolute value drops to 45%, 52%, 58% when the serum transfection is arranged, and PEI25k and Lipofectamine
TM2000 but drop to 9%, 11% respectively.
Embodiment 10: the cytotoxicity test of poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer
1, the cultivation of Cos-7 cell
Get the Cos-7 cell and place and contain the nutrient solution that volume fraction is 10% foetal calf serum, contain cultured continuously in the incubator that volume fraction is 5% carbonic acid gas at 37 ℃.
2, toxotest
Adopt method comparative evaluation different concns PPP-11, PPP-14, PPP-17, PEI25k and the commercial transfection reagent Lipofectamine of MTT
TM2000 material cytotoxicity.Test in preceding 24 hours, the Cos-7 cell in the vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, and placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%.After 24 hours, every hole adds 20 μ l respectively and contains the PBS solution that massfraction is 0.5%MTT with the material of different concns and cell co-cultivation.Mixture adds 200 μ l dmso solution MTT Jia Za crystallizations 10 minutes 37 ℃ of continuation effects 4 hours.Test the absorption value in every hole then with microplate reader, the test wavelength is selected 492nm for use.Cell survival rate is pressed formula and is calculated:
Cell survival rate (%)=(A
Sample/ A
Control) * 100
A
SampleBe the absorption value in the cell sample hole after the transfection, A
ControlBe not with the absorption value in the cell sample hole of complex solution effect, every group of experiment triplicate, test result is seen Fig. 1.
By our cell survival rate after various solid support materials are handled under the different concns as can be seen of Fig. 1.With respect to PEI25k and Lipofectamine
TM2000, the poly glycol monomethyl ether of PPP-11, PPP-14 and PPP-17 representative among the present invention-poly-the 2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is the very little transfection reagent of a kind of pair cell toxicity.
Embodiment 11: transfection experiment in poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer mediation body
Get the crust match nude mice in 5 weeks, at veutro subcutaneous vaccination human cervical carcinoma epithelial cell strain, treat that diameter of tumor grows to 0.4cm after, be divided into two groups at random, every group 9, contain PPP-14/pGL3, PPP-17/pGL3 and the Lipofectamine of 5 μ g luciferase plasmids (pGL3) respectively at intratumor injection
TM2000/pGL3 composite particles solution 100 μ l.Duplicate injection in second day was once injected back 48 hours, with transfection effect in the true living imaging instrument observation of the smart promise body.Before carrying out the living imaging observation, with mouse anesthesia.Anaesthetize after 5 minutes, in the mouse body, inject 200 μ l luciferase substrates.After 10 minutes, with transfection effect in the living imaging systematic observation body.The result shows that the poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer of PPP-14 and PPP-17 representative compares Lipofectamine
TM2000 have higher transfection efficiency.
Claims (6)
1, poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is characterized in that its structural formula is as follows:
PEI is a polymine, and structure is line style or branching, and its structural formula is as follows:
Wherein polymerization degree m, n, a, b and c are equal to or greater than 1.
2, the preparation method of poly glycol monomethyl ether as claimed in claim 1-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is characterized in that step and condition are as follows:
(1) the mole proportioning according to poly glycol monomethyl ether and 2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate is 1: 10～1: 100, both are dosed in the exsiccant reactor, proportioning with poly glycol monomethyl ether and the 2-methyl-total mass g of 2-carbobenzoxy-(Cbz) propylene carbonate and the volume ml of toluene is 1: 5～50 adding dry toluenes, 130 ℃ of azeotropic water removings at least 2 hours, after azeotropic water removing is finished, temperature control steams part toluene for 130～150 ℃, proportioning with poly glycol monomethyl ether and the 2-methyl-total mass g of 2-carbobenzoxy-(Cbz) propylene carbonate and the volume ml of remaining toluene is 1: 2～20, as the distillation end point; Is that 0.05～0.5mmol/ml is dissolved in the dry toluene with the catalyzer zinc ethyl with concentration, add zinc ethyl/toluene solution with zinc ethyl and the monomeric mole of 2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate proportioning 1: 100～300,100～130 ℃ of stirring reactions of temperature control at least 12 hours, sedimentation in normal hexane after reaction is finished, filter, dry cake obtains poly glycol monomethyl ether-poly-2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate ester block copolymer;
Proportioning 1: 5～50 according to poly glycol monomethyl ether-poly-2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate ester block copolymer quality g and tetrahydrofuran (THF) volume ml will be dosed in the reactor behind both mixed dissolutions together; Be 2～5 according to tetrahydrofuran (THF) and methyl alcohol volume proportion again: 1 measures methyl alcohol, take by weighing palladium hydroxide/carbon and evenly be suspended in the methyl alcohol with catalyzer palladium hydroxide/carbon and poly glycol monomethyl ether-poly-2-methyl-2-carbobenzoxy-(Cbz) propylene carbonate quality proportioning 1: 2～10, be dosed in the reactor together then; Charge into nitrogen, hydrogenation is to pressure 0.5～2MPa, 40～60 ℃ of temperature controls, stirring reaction 24～72 hours, after reaction is finished, remove by filter palladium hydroxide/carbon, filtrate is sedimentation in ether, filter, dry cake obtains poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer;
(2) proportioning according to poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer quality g and methyl-sulphoxide volume ml is 1: 10～100, both are dosed in the reactor, stirring and dissolving, with the carboxyl in poly glycol monomethyl ether-poly-2-methyl-2-carboxyl carbon acid propylene ester block copolymer and the mole proportioning of polymine is 1: 1～4, polymine is added in the reactor, be 1: 1～2 according to the carboxyl in poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate and the mole proportioning of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride again, with 1-(3-dimethylamino-propyl)-the 3-ethyl-carbodiimide hydrochloride also is added in the reactor, 20～30 ℃ of temperature controls, stirring reaction at least 2 hours, after reaction is finished, with the reaction solution molecular weight cut-off of packing into is to deionized water dialysis at least 24 hours in 1000～5000 the dialysis tubing, with the liquid freezing drying after the dialysis, obtain poly glycol monomethyl ether-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer.
3, the application of poly glycol monomethyl ether as claimed in claim 1-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is characterized in that, its in in-vitro transfection as gene delivery vector.
4, poly glycol monomethyl ether as claimed in claim 3-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer as the usage of gene delivery vector, is characterized in that step and condition are as follows in in-vitro transfection:
(1) cultivation of cell
Cell places and contains the nutrient solution that volume fraction is 10% foetal calf serum, contains cultured continuously in the incubator that volume fraction is 5% carbonic acid gas at 37 ℃;
(2) in-vitro transfection
The transfection of I serum-free
In preceding 24 hours of the transfection, the cell in vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%, during transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the phosphate buffered saline buffer washed twice, the composite particles of adding genome transfection and the DMEM of serum-free are to final volume 200 μ l/ holes, continue to cultivate after 4 hours, the nutrient solution in the Tissue Culture Plate is abandoned in suction, change 200 μ l/ holes and contain the fresh DMEM that volume fraction is 10% foetal calf serum, continue to cultivate 20 hours;
II contains the serum transfection
In preceding 24 hours of the transfection, the cell in vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80～90%, during transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the phosphate buffered saline buffer washed twice, add the composite particles of genome transfection and contain volume fraction be the DMEM of 10% foetal calf serum to final volume 200 μ l/ holes, continue to cultivate 24 hours;
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution,, add the cell pyrolysis liquid cracking, add the luciferase substrate then, measure transfection efficiency with luxmeter with phosphate buffered saline buffer washing 2 times;
(4) cytotoxicity test
Adopt the cytotoxicity of tetrazolium bromide colorimetry comparative evaluation gene vector material;
Test in preceding 24 hours, the cell in vegetative period of taking the logarithm with the DMEM dilution, is pressed every hole 1 * 10 after the trysinization
4The density of cell is inoculated in 96 well culture plates, placing 37 ℃, to contain volume fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and arrives 80～90%, with the material of different concns and cell co-cultivation after 24 hours, every hole adds 20 μ l respectively and contains the phosphate buffered saline buffer that massfraction is 0.5% tetrazolium bromide, mixture was 37 ℃ of continuation effects 4 hours, add 200 μ l dmso solution tetrazolium bromide Jia Za crystallizations 10 minutes, test the absorption value in every hole then with microplate reader, the test wavelength is selected 492nm for use, and cell survival rate is pressed formula and calculated:
Cell survival rate (%)=(A
Sample/ A
Control) * 100
A
SampleBe the absorption value in the cell sample hole after the transfection, A
ControlBe not with the absorption value in the cell sample hole of complex solution effect, every group of experiment triplicate.
5, the application of poly glycol monomethyl ether as claimed in claim 1-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer is characterized in that, its in vivo in the transfection as gene delivery vector.
6, poly glycol monomethyl ether as claimed in claim 5-poly-2-methyl-2-carboxyl propylene carbonate grafting polyethylene imine copolymer in vivo in the transfection as the usage of gene delivery vector, it is characterized in that step and condition are as follows:
Get the crust match nude mice in 5 weeks, at the epithelial cell strain of veutro subcutaneous vaccination human cervical carcinoma, after treating that diameter of tumor grows to 0.4cm, be divided into two groups at random, every group 9, the composite particles solution 100 μ l that contain the genome transfection of 5 μ g luciferase plasmids respectively at intratumor injection, duplicate injection in second day was once injected back 48 hours, with transfection effect in the true living imaging instrument observation of the smart promise body, before carrying out the living imaging observation, with mouse anesthesia, anaesthetize after 5 minutes, in the mouse body, inject 200 μ l luciferase substrates, after 10 minutes, with transfection effect in the living imaging systematic observation body.
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