CN101704949A - Polyethyleneimine modified with acrylamide monomers, preparation method and application in gene delivery - Google Patents
Polyethyleneimine modified with acrylamide monomers, preparation method and application in gene delivery Download PDFInfo
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- CN101704949A CN101704949A CN200910217849A CN200910217849A CN101704949A CN 101704949 A CN101704949 A CN 101704949A CN 200910217849 A CN200910217849 A CN 200910217849A CN 200910217849 A CN200910217849 A CN 200910217849A CN 101704949 A CN101704949 A CN 101704949A
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Abstract
The invention relates to polyethyleneimine modified with acrylamide monomers, a preparation method and application in gene delivery. In the invention, polyethyleneimine is modified with the acrylamide monomers or N-isopropyl acrylamide monomers, thereby the cytotoxicity is reduced, and the transfection efficiency is improved. When the polyethyleneimine modified with the acrylamide monomers or the N-isopropyl acrylamide monomers is used as a carrier material of non-viral genes, the highest transfection efficiency is more than 9 times that of commercial PE125K, and the cytotoxicity at the concentration of 20 gammas/ml is only one third of that of the PEK125, thereby the polyethyleneimine modified with the acrylamide monomers or the N-isopropyl acrylamide monomers is a non-viral gene carrier with low toxicity and high efficiency, and has wider application prospect.
Description
Technical field
The present invention relates to polymine, method for making and the application in gene transmits of modified with acrylamide monomers.
Background technology
The gene transmission has caused scientific research scholar's very big interest for many years as a kind of emerging medical procedure that may fundamentally treat disease, and a development that key factor is a gene vector material of restriction gene transmission development.Gene vector material generally is divided into virus type and non-virus type, viral vector comprises adenovirus, adeno-associated virus, retrovirus etc., their transfection efficiency is higher, but loading capacity is limited, the cost height, and the most key be its potential safety hazard, may cause and canceration, genovariation etc. greatly limit its development.Non-viral gene vector arises at the historic moment, it is various that they have structure, and physicochemical property determine that stowage space is big, advantages such as easily a large amount of preparations, but transfection efficiency lower [referring to Wong SY, Pelet JM, Putnam D.Polymer systems for gene delivery-past, present, and future.PROGRESS INPOLYMER SCIENCE, 2007,32 (8-9): 799-837].The most famous in the non-viral gene vector material is that hyperbranched weight-average molecular weight is the polymine of 25K, be called for short PEI25K, has relative higher transfection efficiency, the researcher with its owing to " proton sponge effect " [referring to Boussif O, Zanta M.A, Behr J.P, et al.A versatile vector for gene and oligonucleotide transfer into cells inculture and in vivo-polyethylenimine.PROCEEDINGS OF THE NATIONALACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995,92 (16): 7297-7301].Yet, its nondegradable characteristic and very high cytotoxicity have greatly limited its application prospect [referring to Lungwitz U, Breunig M, Blunk T, et al.Polyethylenimine-basednon-viral gene delivery systems.EUROPEAN JOURNAL OF PHARMACEUTICSAND BIOPHARMACEUTICS, 2005,60 (2): 247-266].
Summary of the invention
In order to solve the problem that prior art exists, the invention provides polymine, method for making and the application in gene transmits of modified with acrylamide monomers.
The present invention has not only reduced cytotoxicity, and has greatly improved transfection efficiency by using the modified with acrylamide monomers polymine, has a good application prospect.
The polymine of modified with acrylamide monomers, its structural formula is as follows:
In the formula, the R group is-H or sec.-propyl; Described acrylamide monomers is acrylamide or N-N-isopropylacrylamide.
The preferably polyethylene imines is that hyperbranched weight-average molecular weight is the polymine of 25k.
The polymine preparation method's of modified with acrylamide monomers step and condition are as follows:
(1) is 0.05～0.3g/ml according to polymine concentration in chloroform, polymine is dissolved in the chloroform, be re-introduced in the reactor;
(2) then, be 88: 1～400: 1 according to acrylamide monomers with polymine amount of substance ratio, take by weighing acrylamide monomers and add in the chloroformic solution of above-mentioned polymine, 50 ℃ were reacted 4 days;
(3) after reaction finishes, reaction solution is spin-dried for, the crude product that obtains is dissolved in the deionized water, in molecular weight cut-off be in 3,500 the dialysis tubing to deionized water dialysis 3 days, the G4 sand core funnel filters, freeze-drying obtains the polymine of target product modified with acrylamide monomers.
The application of the polymine of modified with acrylamide monomers in gene transmits, its in in-vitro transfection or body in the transfection as gene delivery vector.
As the usage of gene delivery vector, its step and condition are as follows in in-vitro transfection for the polymine of modified with acrylamide monomers:
(1) cultivation of cell
Cell placed 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
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 and the serum-free of gene transfection or contain volume fraction be the DMEM substratum of 10% foetal calf serum to final volume 200 μ l, continue to cultivate 48 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 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 that adds the cell sample hole of polymers soln, A
ControlBe the absorption that does not add the cell sample hole of polymers soln, every group of experiment triplicate.
The polymine of modified with acrylamide monomers in vivo in the transfection as the usage of gene delivery vector, its 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, treat that diameter of tumor grows to 0.4cm after, be divided into two groups at random, every group 6, 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: when the polymine of modified with acrylamide monomers provided by the invention is used as the non-viral gene vector material, its high transfection efficiency of PEN175 is more than 9 times of commercial PEI25K, and cytotoxicity only is about 1/3 of PEI25K when concentration 20 μ g/ml, be a kind of low toxicity non-viral gene vector efficiently, be with a wide range of applications.
Description of drawings
Fig. 1 is the transfection efficiency in vitro column diagram of the polymine of using the modification of N-N-isopropylacrylamide of embodiment 1.
Fig. 2 is the cytotoxicity curve of the target product of embodiment 1 and 2 preparations.■ is PEI25k, ▲ be PEA100,
Be PEA200, ● be PEA400, △ is PEN88,
Be PEN175, zero is PEN350.
Embodiment
Embodiment 1: use the preparation of the polymine of N-N-isopropylacrylamide (being called for short NIPA) modification
(1) takes by weighing the polymine that the hyperbranched weight-average molecular weight of 1.5g is 25K (being called for short PEI25k) respectively, be dissolved in respectively in the chloroform, be injected in the reactor according to table 1;
(2) then, the amount of substance ratio according to the N-N-isopropylacrylamide of table 1 and PEI25k is 88: 1,175: 1 and 350: 1 respectively, takes by weighing the N-N-isopropylacrylamide and adds in the chloroformic solution of above-mentioned polymine, 50 ℃ of reactions 4 days;
(3) after reaction finishes, reaction solution is spin-dried for, the crude product that obtains is dissolved in the deionized water, in molecular weight cut-off is 3, to deionized water dialysis 3 days, the G4 sand core funnel filtered, freeze-drying in 500 the dialysis tubing, obtain polymine PEN88, PEN175 and PEN350 that the N-N-isopropylacrylamide is modified respectively, see table 1 for details.
Table 1: use the preparation of the polymine of N-N-isopropylacrylamide modification
| The product numbering | ?NIPA∶PEI25k(mol) | PEI25k concentration (g/ml) | NIPA number in the product |
| ??PEN88 | ?88∶1 | ??0.3 | ??79.8 |
| ??PEN175 | ?175∶1 | ??0.15 | ??128.6 |
| ??PEN350 | ?350∶1 | ??0.05 | ??131.1 |
Embodiment 2: use the preparation of the polymine of acrylamide (being called for short AA) modification
(1) takes by weighing the polymine that the hyperbranched weight-average molecular weight of 15g is 25K (being called for short PEI25k) respectively, be dissolved in respectively in the chloroform, be injected in the reactor according to table 2;
(2) then, the amount of substance ratio according to the acrylamide of table 2 and PEI25k is 100: 1,200: 1 and 400: 1 respectively, takes by weighing acrylamide and adds in the chloroformic solution of above-mentioned polymine, 50 ℃ of reactions 4 days;
(3) after reaction finishes, reaction solution is spin-dried for, the crude product that obtains is dissolved in the deionized water, in molecular weight cut-off is 3, to deionized water dialysis 3 days, the G4 sand core funnel filtered, freeze-drying in 500 the dialysis tubing, obtain polymine PEA88, PEA175 and PEA350 that acrylamide is modified respectively, see table 2 for details.
Table 2: the preparation of the polymine of modifying with acrylamide
| The product numbering | ?AA∶PEI25k(mol) | PEI25k concentration (g/ml) | AA number in the product |
| ??PEA100 | ?100∶1 | ??0.3 | ??129.0 |
| ??PEA200 | ?200∶1 | ??0.15 | ??190.4 |
| ??PEA400 | ?400∶1 | ??0.05 | ??366.0 |
Embodiment 3: target product mediation luciferase plasmids is to the in-vitro transfection of human cervical carcinoma cell (HeLa cell)
1, the cultivation of HeLa cell
Get the HeLa 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
In preceding 24 hours of the transfection, the HeLa 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 volume fraction is 5%CO
2, continue to be cultured to degree of converging in 37 ℃ of incubators and arrive 80～90%.During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, change 200 μ l/ holes and contain the fresh DMEM nutrient solution of volume fraction 10% foetal calf serum, select for use luciferase plasmids (pGL3) to add all types of target product that embodiment 1-2 obtains respectively again and the composite particles of pGL3 and PEI25k/pGL3 carries out the transfection contrast, continue to cultivate 48 hours;
3, the mensuration of transfection efficiency in vitro
Take out culture plate, inhale and abandon nutrient solution, phosphate buffered saline buffer (PBS) washing 2 times, add the cell pyrolysis liquid cracking, add the luciferase substrate then, use luxmeter to measure transfection efficiency, be expressed as every milligram of proteic luminescence unit number (RLU/mg Protein), test result is seen Fig. 1.
Embodiment 4: the cytotoxicity test of target product
1, the cultivation of HeLa cell
Get the HeLa 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
All types of target product that the method comparative evaluation embodiment 1-2 of employing MTT obtains and the material cytotoxicity of PEI25k.Test in preceding 24 hours, the HeLa 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, 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%. 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. and mixture was 37 ℃ of continuation effects 4 hours, add 200 μ l dmso solution MTT first Za crystallizations 10 minutes. test the absorption in every hole then with microplate reader, the test wavelength is selected for use the 492nm. cell survival rate to press formula and is calculated:
Cell survival rate (%)=(A
Sample/ A
Control) * 100
A
SampleBe the absorption in the cell sample hole after the transfection, A
ControlBe not with the absorption in the cell sample hole of complex solution effect, every group of experiment triplicate, test result is seen Fig. 2.
Transfection experiment in the body of embodiment 5:PEN175 mediation
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 6, contain the PEN175/pGL3 and the PEI25K/pGL3 composite particles solution 100 μ l of 5 μ g luciferase plasmids (pGL3) 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.
Claims (5)
1. the polymine of modified with acrylamide monomers is characterized in that, its structural formula is as follows:
In the formula, the R group is-H or sec.-propyl; Described acrylamide monomers is acrylamide or N-N-isopropylacrylamide.
2. the polymine of modified with acrylamide monomers as claimed in claim 1 is characterized in that, described polymine is that hyperbranched weight-average molecular weight is the polymine of 25k.
3. the preparation method's of the polymine of modified with acrylamide monomers as claimed in claim 1 step and condition are as follows:
(1) is 0.05～0.3g/ml according to polymine concentration in chloroform, polymine is dissolved in the chloroform, be re-introduced in the reactor;
(2) then, be 88: 1～400: 1 according to acrylamide monomers with polymine amount of substance ratio, take by weighing acrylamide monomers and add in the chloroformic solution of above-mentioned polymine, 50 ℃ were reacted 4 days;
(3) after reaction finishes, reaction solution is spin-dried for, the crude product that obtains is dissolved in the deionized water, in molecular weight cut-off be in 3,500 the dialysis tubing to deionized water dialysis 3 days, the G4 sand core funnel filters, freeze-drying obtains the polymine of target product modified with acrylamide monomers.
4. the application of the polymine of the described modified with acrylamide monomers of claim 1 in gene transmits is characterized in that, its in in-vitro transfection as gene delivery vector.
5. the application of the polymine of the described modified with acrylamide monomers of claim 1 in gene transmits is characterized in that, its in vivo in the transfection as gene delivery vector.
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| CN102276829A (en) * | 2011-05-24 | 2011-12-14 | 浙江大学 | Non-viral gene vector material as well as preparation method and application thereof |
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| CN101880397B (en) * | 2010-07-02 | 2012-06-27 | 中国科学院长春应用化学研究所 | Cross linking polymer and preparation method thereof |
| CN101880397A (en) * | 2010-07-02 | 2010-11-10 | 中国科学院长春应用化学研究所 | Cross linking polymer and preparation method thereof |
| CN101974154A (en) * | 2010-11-10 | 2011-02-16 | 中国科学院长春应用化学研究所 | Modified polyethyleneimine and preparation method thereof |
| CN101974154B (en) * | 2010-11-10 | 2012-07-25 | 中国科学院长春应用化学研究所 | Modified polyethyleneimine and preparation method thereof |
| CN102276829A (en) * | 2011-05-24 | 2011-12-14 | 浙江大学 | Non-viral gene vector material as well as preparation method and application thereof |
| CN102276829B (en) * | 2011-05-24 | 2012-11-21 | 浙江大学 | Non-viral gene vector material as well as preparation method and application thereof |
| CN102337296A (en) * | 2011-07-05 | 2012-02-01 | 天津大学 | Glucan-agmatine polycation transgenic vector, and preparation method and application thereof |
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| CN102746513B (en) * | 2012-07-24 | 2014-05-21 | 中国科学院长春应用化学研究所 | Polyamino acid segmented copolymer serving as siRAN carrier and preparation method as well as composite particle |
| CN108354855A (en) * | 2017-01-26 | 2018-08-03 | 上海氪励铵勤科技发展有限公司 | Composition and preparation method thereof and application method |
| CN114957656A (en) * | 2022-07-01 | 2022-08-30 | 济南大学 | Preparation method of multi-stimulus-responsive fluorescent polyethyleneimine with LCST and UCST |
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