CN102181053B - Hydrophobic-group-modified polyethyleneimine derivative and application thereof - Google Patents
Hydrophobic-group-modified polyethyleneimine derivative and application thereof Download PDFInfo
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- CN102181053B CN102181053B CN 201110046746 CN201110046746A CN102181053B CN 102181053 B CN102181053 B CN 102181053B CN 201110046746 CN201110046746 CN 201110046746 CN 201110046746 A CN201110046746 A CN 201110046746A CN 102181053 B CN102181053 B CN 102181053B
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Abstract
The invention belongs to the field of polymer modification, and particularly relates to an acid-sensitive hydrophobic modified polyethyleneimine and application thereof as a gene vector. The derivative contains an acid-sensitive acetal functional group; and specifically, the derivative is an acetal-molecular trimethoxy benzylacetal-trihydroxy ethylacetal (TMB-THME) modified polyethyleneimine derivative, named polyethyleneimine-(trimethoxy benzylacetal-trihydroxy ethane). The polyethyleneimine-(trimethoxy benzylacetal-trihydroxy ethane) provided by the invention enhances the DNA (deoxyribonucleic acid) compounding capability of PEI (polyetherimide) and the interaction with cells, can convert a hydrophobic group into a hydrophilic group in an endosome, and implements the dissociation of the DNA compound and the intracellular release of the DNA; and the cytotoxicity is low.
Description
Technical field
The invention belongs to the polymer modification field, be specifically related to a kind of polymine of acid-sensitive sense hydrophobically modified and as the application of genophore.
Background technology
Gene therapy has broad application prospects for the treatment of human numerous disease such as cancer, cardiovascular disorder, communicable disease, heredopathia etc.For the gene therapy of success, must therapeutic gene be targeted to lesions position.Virus vector all is at present efficient gene carrier.But virus vector has some intrinsic shortcomings, like immunogenicity, target property is poor, the DNA tonburden is limited, and produce and service routine complicated.In recent years, non-viral gene vector particularly polycation carrier has caused that investigators pay close attention to widely, has many advantages because it is compared with virus vector.For example, the structure of carrier and character are controlled, the DNA tonburden is big, repeatedly duplicate injection, be produced on a large scale etc.In addition, polymer carrier can prolong its body-internal-circulation time through suitably modifying, and target transports therapeutic gene to lesions position.Yet, to compare with virus vector, existing polymer carrier transfection efficiency is lower.Lacking safely and efficiently, gene vehicle system is the maximum bottleneck of present clinical gene therapy.
In in the past several years, obtained huge development as genophore safely and efficiently based on " artificial viral " of polycation.For example Langer etc. has developed a series of urethane as genophore, has both helped the rapid screening of transfection reagent, can help again to understand relation between structure-toxicity-transfection (Angew. Chem. Int. Ed. 2003,42,3153-3158).Utilize similar method, stand-alone developments such as Feijen and Goh a series of hyperbranched urethane and studied its in-vitro transfection (J. Control. Release 2005,109,317-329; Biomacromolecules 2005,6,3166-3173).Kim, Park and Hennink etc. have developed dissimilar degradable polycations as non-viral gene vector (J. Am. Chem. Soc. 2000,122,6524-6525; J. Am. Chem. Soc. 1999,121,5633-5639; Bioconjugate Chem. 2006,17,1077-1084; J Control. Release 2008,126,97-110).Engbersen and Kim etc. have designed the responsive urethane of a series of reduction and have been used for gene transfection (Bioconjugate Chem. 2006,17,1233-1240 in the cell; Bioconjugate Chem. 2007,18,138-145).
Except designing novel polycation, the modification of the traditional polymer carrier especially modification of polymine (PEI) obtains paying close attention to.Because its high electric density and endosome pH surge capability, PEI shows high transfection efficiency in various kinds of cell.The transfection of PEI depends primarily on its molecular structure and molecular weight.Wherein, the linear PEI of 25 kDa branching PEI (25 kDa bPEI) and 22 kDa (22 kDa lPEI) are proved to be present best transfection reagent, and are acknowledged as the golden standard of present non-viral gene vector.Yet PEI has toxicity in various degree, and to compare transfection efficiency lower with virus vector.Recent years, based on the hypotoxicity of lower molecular weight PEI, people design and have synthesized multiple degradable PEI polymer and crosslinked body and function is done the outer-gene transfection.Degradable PEI compares low-molecular-weight PEI and has much higher transfection efficiency; Some in addition be higher than 25 kDa bPEI; Utilizing hydrophobic grouping to modify PEI is a kind of method of effective raising lower molecular weight PEI gene transfection efficient, and the transfection efficiency of 1.8 kDa PEI in various kinds of cell after for example Kim seminar report SUV (cholesterol) is modified compared 1.8 kDa PEI tool large increase (Bioconjugate Chem 2001; 12 (3): 337-345).Klibanov etc. have reported that the transfection efficiency of 2 kDa PEI of laurostearic acid esterification compares 25 kDa PEI and improved 5 times of (Proc Natl Acad Sci U S A 2002; 99 (23): 14640-14645).Uludag etc. have reported that the 2 kDa PEI that fatty lipid is modified have transfection efficiency (the Mol Pharm 2009 suitable with 25 kDa PEI; 6 (6): 1798-1815).Ramezani etc. reported the alkyl oligomeric amine receive in N2A mouse neuroblast, have on the 10 kDa PEI with 25 kDa PEI quite the transfection efficiency of level (Biomaterials 2009; 30 (25): 4187-4194).
Summary of the invention
The purpose of this invention is to provide the polyethylenimine derivates that a kind of hydrophobic grouping is modified.
For achieving the above object; The concrete technical scheme of the present invention is: the polyethylenimine derivates that a kind of hydrophobic grouping is modified; Said verivate contains acid-sensitive sense degradable acetal functional group; Particularly, said verivate is the polyethylenimine derivates that acetal molecule trimethoxy-benzene methylal-trihydroxy-ethane acetal (TMB-THME) is modified, and its general structure is as follows:
When the number-average molecular weight of main chain polymine was 1.5～2 kDa, the grafting number of main chain polymine grafting acetal molecule trimethoxy-benzene methylal-trihydroxy-ethane acetal was 1～5; When the number-average molecular weight of main chain polymine is 9.5～10.5 kDa, and the grafting number of main chain polymine grafting acetal molecule trimethoxy-benzene methylal-trihydroxy-ethane acetal is 5～15.
In the technique scheme, trimethoxy-benzene methylal-trihydroxy-ethane acetal molecule is linked to polymine through the amine ester bond, the polyethylenimine derivates called after polymine that obtains-(trimethoxy-benzene methylal-trihydroxy-ethane) (PEI-
g-(TMB-THME)
n).
The method for preparing above-mentioned polyethylenimine derivates may further comprise the steps:
(1) synthesizing trimethoxy benzene methylal-trihydroxy-ethane (TMB-THME): with 1; 1, in 1-trihydroxy-ethane and the p-methyl benzenesulfonic acid dissolving THF, then with 2; 4; 6-TMB and 4 dust molecular sieves add wherein, and 45～55 ℃ obtain trimethoxy-benzene methylal-trihydroxy-ethane behind reaction 8～16h down, and its reaction process is as follows:
(2) p-nitrophenyl chloroformate ester (
p-NC) activation trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME-PC): trimethoxy-benzene methylal-trihydroxy-ethane is dissolved in the methylene dichloride; Under protection of inert gas; Triethylamine, pyridine, p-nitrophenyl chloroformate ester are added wherein; After reacting 8～16h under the room temperature, obtain activatory trimethoxy-benzene methylal-trihydroxy-ethane, its reaction process is as follows:
(3) preparation polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) (PEI-
g-(TMB-THME)
n): (PEI) is dissolved in the methylene dichloride with polymine; Activatory trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME-PC) is dissolved in the methylene dichloride, under the protection of inert gas, the dichloromethane solution of TMB-THME-PC slowly is added drop-wise in the dichloromethane solution of PEI; Dropwise; React 20～30h under the room temperature, obtain polymine-(trimethoxy-benzene methylal-trihydroxy-ethane), its reaction process is as follows:
In the preparation process, in the step (3), can control the consumption of activatory trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME-PC) and adjust the proportionlity of m and n.
In the technique scheme; Polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) is solvable in water; For example: for 10 kDa polymines, the TMB-THME modification degree is equal water solubles 5,9 and 14 (the per molecule polymine contains the number of TMB-THME); For 1.8 kDa polymines, the TMB-THME modification degree is 1.3,2.1 and 4.2 equal water solubles; And polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) is relatively stable pH 7.4 times, but (like pH 5.0) hydrolysis fast under weak acid environment changes hydrophobic modification into hydrophilic modification.
In the technique scheme; Said polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) can be used to prepare the DNA nano-complex that meets the transfection requirement; Because the introducing of hydrophobic molecule; Modified PE I is when forming mixture with the DNA effect, and static and hydrophobic effect simultaneously form mixture more closely, so the PEI-after the modification
g-(TMB-THME)
nCan load DNA and form the mixture that particle diameter is less, surface potential is higher.For example: modification 10 kDa PEI gained PEI-
g-(TMB-THME)
nMore than or equal to 5/1 o'clock, can compress DNA at N/P ratio effectively, form the mixture of particle diameter 100 ~ 170 nm, surface potential+25 ~+43 mV, be 10/1 o'clock at N/P ratio, PEI-
g-(TMB-THME)
nThe mixture particle diameter that loads DNA formation is 110 ~ 160 nm, and surface potential is+25 ~+28 mV, and the mixture particle diameter that 10 kDa PEI and DNA form is 175 nm, and surface potential is+18 mV.Equally, gained PEI-behind the TMB-THME modification 1.8 kDa PEI
g-(TMB-THME)
nAlso less with DNA formation particle diameter; The mixture that surface potential is higher for example, is 20/1 o'clock at N/P ratio; The mixture particle diameter that modification 1.8 kDa PEI form is 120 ~ 190 nm; Surface potential is+22 ~+26 mV, and 1.8 kDa PEI to form particle diameter be 450 nm, surface potential is+the DNA mixture of 22 mV.In addition, TMB-THME modified PE I does not have influence on the pH surge capability of PEI.
In the technique scheme, the hydrolysis situation of acetal bonds under different pH can record through ultraviolet in said polymine-(the trimethoxy-benzene methylal-trihydroxy-ethane).With PEI (10 kDa)-
g-(TMB-THME)
9Be example, the transformation period that the result is presented under pH 4.0,5.0 and 6.0 is respectively 1.3h, 2.8h and 11h, and the hydrolysis in 7.4 times 24h of pH seldom (<12%).Simultaneously, through DLS and the gel release behavior of DNA under pH 5.0 and 7.4 in the mixture that postponed experimental study.With PEI-
g-(TMB-THME)
9The DNA mixture that forms is an example, and the result is illustrated in 3 h, increases to more than 800 nm at 5.0 times mixture particle diameters of pH, and has only small variation at 7.4 times particle diameters of pH.Simultaneously, surface potential is reduced to about-25 mV in 5.0 times 6 h of pH, and constant basically at pH 7.4 lower surface current potentials.Gel postpones experimental result and shows that further modification 10 kDa PEI/DNA mixtures have advantages of higher stability 7.4 times at pH, but then discharges soon at 5.0 times DNA of pH.
In the technique scheme, the mixture that adopts polymine according to the invention-(trimethoxy-benzene methylal-trihydroxy-ethane) and DNA to form can be further used for gene transfection in the cell.With the pGL3 expressing luciferase is that the outer-gene transfection experiment of reporter gene shows: gained PEI-of the present invention
g-(TMB-THME)
nHeLa and 293T cell are being had under the condition with serum-free N/P ratio 10/1 and 20/1 time with the mixture of DNA and all to have high transfection efficiency.For example, PEI-
g-(TMB-THME)
14/ DNA mixture is compared with 10 kDa bPEI/DNA mixtures under its optimum N/P ratio; Transfection efficiency under the condition that has with serum-free has improved 235 and 175 times respectively, compares the transfection efficiency of 25 kDa bPEI under its optimum N/P ratio and has improved 16 and 7 times respectively.And the toxic result who measures polymkeric substance through the MTT method shows: modification 10 kDa PEI gained PEI-
g-(TMB-THME)
n(0.6 to 2.4 μ mol/L) is nontoxic under the required concentration of transfection.
Because gained polymine of the present invention-(trimethoxy-benzene methylal-trihydroxy-ethane) has above-mentioned character; Therefore; Polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) can effectively compress DNA for 7.4 times at pH and form the positively charged nano-complex in surface (< 250 nm), and this DNA mixture is relatively stable pH 7.4 times, but (like pH 5.0) then can be owing to the quick hydrolysis of acetal causes complex dissociation under weak acid environment; Released dna; Therefore, can efficiently deliver DNA in cell, produce high transfection efficiency; And polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) has lower cytotoxicity.
Therefore; The present invention requires to protect the application of above-mentioned polymine-(trimethoxy-benzene methylal-trihydroxy-ethane) as dna vector simultaneously; In the concrete application process; N/P ratio between said dna vector and DNA relation is: main chain is that the N/P ratio of the dna vector of the PEI of 9.5～10.5 kDa when loading DNA is 10/1～20/1, and main chain is that the N/P ratio of the dna vector of the PEI of 1.5～2 kDa when loading DNA is 20/1 ~ 40/1.
Because the application of technique scheme, the present invention compared with prior art has the following advantages:
(1) in polymine according to the invention-(the trimethoxy-benzene methylal-trihydroxy-ethane) owing to introduce hydrophobic modification, therefore, can strengthen PEI the DNA compound ability and with the interaction of cell.
(2) polymine according to the invention-(trimethoxy-benzene methylal-trihydroxy-ethane) is under low pH environment; Acid-sensitive sensitive group degraded; Hydrophobic modification changes hydrophilic modification into; Helping dissociating of DNA mixture discharges with the born of the same parents of DNA are interior; The polymine that 10 kDa bPEI obtain behind acid-sensitive sense hydrophobic modification among the present invention-(trimethoxy-benzene methylal-trihydroxy-ethane) improved 175 times to the transfection efficiency of HeLa cell, compares with transfection efficiency under the optimum N/P ratio of the bPEI of 25 kDa and improved 16 times.
(3) polymine according to the invention-(trimethoxy-benzene methylal-trihydroxy-ethane) strengthened PEI the DNA compound ability and with the interaction of cell; Can change hydrophobic grouping into hydrophilic radical in the endosome; Realize that dissociating of DNA mixture discharges with the born of the same parents of DNA are interior, and cytotoxicity is low.
Description of drawings
Fig. 1. modified polyethyleneimine is as gene release vehicle synoptic diagram among the embodiment;
Fig. 2. trimethoxy-benzene methylal among the embodiment one and two-trihydroxy-ethane (TMB-THME) (A) with activation after trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME-NC) proton magnetic chart spectrum (B);
Fig. 3. PEI among the embodiment five (10 kDa)-
g-(TMB-THME)
9Proton magnetic chart spectrum;
Fig. 4. the surge capability synoptic diagram of modification 10 kDa PEI among the embodiment five;
Fig. 5. the surge capability synoptic diagram of modification 1.8 kDa PEI among the embodiment five;
Fig. 6. PEI among the embodiment five (10 kDa)-
g-(TMB-THME)
nSize distribution of/DNA mixture (A) and surface potential (B) synoptic diagram;
Fig. 7. PEI among the embodiment five (1.8 kDa)-
g-(TMB-THME)
nSize distribution of/DNA mixture (A) and surface potential (B) synoptic diagram;
Fig. 8. PEI among the embodiment five (10 kDa)-
g-(TMB-THME)
nThe gel electrophoresis experimental result of/DNA mixture;
Fig. 9. PEI among the embodiment six (10 kDa)-
g-(TMB-THME)
9The hydrolysis trend of acetal bonds under different pH in the/DNA mixture;
Figure 10. PEI among the embodiment seven (10 kDa)-
g-(TMB-THME)
9The hydrolysis of/DNA mixture acetal bonds under different pH causes that DNA discharges the particle diameter (A) cause and the variation of surface potential (B) contrasts;
Figure 11. PEI among the embodiment seven (10 kDa)-
g-(TMB-THME)
9The acetal bonds hydrolysis under different pH of/DNA mixture causes the gel electrophoresis experimental result that DNA discharges;
Figure 12. the modification 10 toxicity results of kDa PEI under the different N volumetric molar concentration among the embodiment eight;
Figure 13. PEI among the embodiment nine (10 kDa)-
g-(TMB-THME)
n/ DNA mixture is in the HeLa cell under the serum-free condition (A) and the transfection results of (B) under the serum condition is arranged;
Figure 14. PEI among the embodiment nine (10 kDa)-
g-(TMB-THME)
n/ DNA mixture is in the 293T cell under the serum-free condition (A) and the transfection results of (B) under the serum condition is arranged.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one: trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME) synthetic
1,1,1-trihydroxy-ethane (THME; 6.667 g, 55.5 mmol) and p-methyl benzenesulfonic acid (0.5278 g, 2.775 mmol) at first under 50 ℃, be dissolved in the 100 mL THFs; Then 2,4,6-TMB (TMB; 3.63 g, 18.5 mmol) and the adding of 7.5 g, 4 dust molecular sieves is wherein.After 50 ℃ reaction is spent the night down, add 7 mL triethylamines, and dilute with 75 mL methylene dichloride.Filter out molecular sieve and use washed with dichloromethane; Dried white solid is revolved in decompression; Solid extracts three times with 0.1 M pH, 8.0 PB buffered soln with methylene dichloride dissolving back; Use anhydrous magnesium sulfate drying to spend the night after collecting organic phase, filtered out sal epsom in second day and the dried white solid that obtains is revolved in decompression, white solid in vacuum drying oven dry 2 days.Productive rate: 54.6 %.
The nuclear-magnetism of TMB-THME characterizes sees accompanying drawing 2 A:
1H NMR (400 MHz, CDCl
3): d 6.16 (s, 2H), d 6.06 (s, 1H), d 4.23 (s, 2H), d 3.98 (s, 2H), d 3.88 (s, 6H), d 3.86 (s, 3H), d 3.62 (s, 2H), d 0.76 (s, 3H).
Embodiment two: p-nitrophenyl chloroformate ester (
p-NC) activation trimethoxy-benzene methylal-trihydroxy-ethane (TMB-THME-PC)
White product TMB-THME (2.31 g among the embodiment one; 7.8 mmol) be dissolved in the 70 mL methylene dichloride; Leading under the nitrogen protection triethylamine (2.36 g, 23.4 mmol), pyridine (0.60 g, 7.8 mmol), p-nitrophenyl chloroformate ester (1.565 g; 9.13 mmol) add wherein, reaction is spent the night under the room temperature.Reaction finishes, and reaction solution is added to (deposition triethylamine hydrochloride) in the 300 mL anhydrous diethyl ethers, crosses filtering filtrating and be deposited in the ice normal hexane, crosses and filters little brown solid, vacuum-drying 2 days.Productive rate: 60 %.
The nuclear-magnetism of TMB-THME-PC characterizes sees accompanying drawing 2 B:
1H NMR (400 MHz, CDCl
3): d 8.30 (d, 2H), d 7.42 (d, 2H), d 6.10 (s, 2H), d 6.00 (s; 1H), and d 4.77 (s, 2H), d 4.05 (d, 2H), d 3.83 (s, 6H); D 3.79 (s, 3H), d 3.68 (d, 2H), d 0.90 (s, 3H).
Embodiment three: modification 10 kDa PEI (PEI (10 kDa)-
g-(TMB-THME)
n) synthetic
10 kDa PEI (0.3 g, 6.97 mmol) are dissolved in the 6 mL methylene dichloride, and TMB-THME-PC (0.497 g, 1.1 mmol) is dissolved in CH
2Cl
2In, under the nitrogen protection, dropwise TMB-THME-PC is added drop-wise in the dichloromethane solution of 10 kDa PEI, dropwise, reaction is 24 hours under the room temperature.After reaction finished, reaction solution at first precipitated three times in the ice ether, uses secondary water dissolution product afterwards again, removed p-NP with the dialysis of MWCO 3500 dialysis tubings, and lyophilize obtained little yellow solid in 2 days.Obtain PEI (10 kDa)-
g-(TMB-THME)
9, productive rate 73 %.
Wherein PEI (10 kDa)-
g-(TMB-THME)
9Nuclear-magnetism characterize and to see accompanying drawing 3 as follows:
1H NMR (400 MHz, CDCl
3): d 6.07 (s, 2H), d 5.93 (s, 1H), d 4.50 (s, 2H), d 3.97 (s, 2H), d 3.81 (s, 6H), d 3.75 (s, 3H), d 3.57 (s, 2H), d 0.74 (s, 3H), d 3.00-2.50 (PEI).Through regulating the number 9,12,17 that feeds intake of hydrophobic molecule, obtain containing different hydrophobic molecule numbers and be respectively 5,9,14 modification 10 kDa PEI.The TMB-THME modification degree is equal water solubles 5,9 and 14 (the per molecule polymine contains the number of TMB-THME).Concrete outcome sees the following form 1:
The essential property of the modification 10kDa PEI of table 1. hydrophobic modification characterizes
| | Modification | 10 kDa PEI | The theoretical grafting number of the TMB-THME of design | The actual grafting number of the TMB-THME that nuclear-magnetism calculates | Surge capability (%) |
| 1 | PEI- g-(TMB-THME) 5 | 9 | 5 | 13.9 | |
| 2 | PEI- g-(TMB-THME) 9 | 12 | 9 | 13.7 | |
| 3 | PEI- g-(TMB-THME) 14 | 17 | 14 | 13.0 | |
| 4 | 10 kDa PEI | - | - | 13.5 |
Embodiment four: modification 1.8 kDa PEI (PEI (1.8 kDa)-
g-(TMB-THME)
n) synthetic
1.8 kDa PEI (0.184 g; 4.279 mmol) be dissolved in the 6 mL methylene dichloride, TMB-THME-PC (0.385 g, 0.8558 mmol) is dissolved in the methylene dichloride; Under the nitrogen protection; Dropwise TMB-THME-PC is added drop-wise in the dichloromethane solution of 1.8 kDa PEI, dropwises, reaction is 24 hours under the room temperature.After reaction finished, reaction solution at first vacuum rotary steam was used the secondary water dissolution afterwards to doing, and removed p-NP with the dialysis of MWCO 1000 dialysis tubings then, and dialysis finishes to remove by filter earlier the TMB-THME part of separating out, and the filtrating lyophilize obtained little yellow solid in 2 days.Obtain PEI (1.8 kDa)-
g-(TMB-THME)
4.2, productive rate 46 %.
Wherein PEI (1.8 kDa)-
g-(TMB-THME)
4.2Nuclear-magnetism characterize as follows:
1H NMR (400 MHz, CDCl
3): d 6.07 (s, 2H), d 5.93 (s, 1H), d 4.50 (s, 2H), d 3.97 (s, 2H), d 3.81 (s, 6H), d 3.75 (s, 3H), d 3.57 (s, 2H), d 0.74 (s, 3H), d 3.00-2.50 (PEI).The number ratio that feeds intake through regulating hydrophobic molecule is 1.6,4.2,8.4, obtains containing different hydrophobic molecule numbers and is respectively 1.3,2.1,4.2 modification 1.8 kDa PEI.The TMB-THME modification degree is 1.3,2.1 and 4.2 equal water solubles.Concrete outcome sees the following form 2:
The essential property of the modification 1.8 kDa PEI of table 2. hydrophobic modification characterizes
| Sequence number | Modification 1.8 kDa PEI | The theoretical grafting number of the TMB-THME of design | The actual grafting number of the TMB-THME that nuclear-magnetism calculates | Surge capability (%) |
| 1 | PEI- g-(TMB-THME) 1.3 | 1.6 | 1.3 | 13.0 |
| 2 | PEI- g-(TMB-THME) 2.1 | 4.2 | 2.1 | 12.7 |
| 3 | PEI- g-(TMB-THME) 4.2 | 8.4 | 4.2 | 9.9 |
| 4 | 1.8 kDa PEI | - | - | 13.2 |
Embodiment five: the preparation of mixture and sign
1H NMR is with deuterochloroform (CDCl with Varian Inova 400 type resonance wave spectrometers
3) for solvent records, confirm the substitution value of verivate through nuclear-magnetism.PEI-g-(TMB-THME)
nThe surge capability of verivate is to record in pH 10.0 ~ 2.0 scopes through acid base titration.In brief, PEI-g-(TMB-THME)
nVerivate (0.1 mmol N) is dissolved in earlier among 5 mL, the 150 mM NaCl, uses the NaOH of 1 M to regulate pH to 10.0 then, uses the quick titration sample of 0.1 M HCl afterwards and measures pH.As a comparison, 10 kDa, 1.8 kDa, 25 kDa PEI titration after the same method.Surge capability is defined as between the pH 7.4 to 5.1 amido by protonated ratio, and calculates according to following formula: Buffer capacity (%)=100! ⊿ V
HCl* 0.1 M)/N
Mol
， ⊿ V here
HClBe with the pH value of solution from 7.4 be adjusted to 5.1 needed HCl volume (0.1 M), N
MolIt is the ratio of the amido that all can be protonated in the polymkeric substance.
With reference to Fig. 1, with the concentration of 1 mg/mL with PEI (10 kDa)-
g-(TMB-THME)
nBe dissolved in the HEPES buffered soln (20 mM, pH 7.4).Calculate required amount by N/P ratio 10/1,20/1,40/1, with 600 μ L PEI (10 kDa)-
g-(TMB-THME)
nHEPES solution be added in the DNA (37.5 μ g/mL) of 150 μ L, eddy current shook 5 seconds, at room temperature cultivated then.Measure the particle diameter and the surface potential of mixture behind 30 min.
Press preceding method with N/P ratio 1/1,2/1,3/1,5/1,8/1 preparation PEI (10 kDa)-
g-(TMB-THME)
n/ DNA mixture.In the mixture of preparation, add pyridine of a certain amount of bromination second and tetrabromophenol sulfonphthalein solution, in TAE buffered soln, ran glue 40 minutes with 110 V on the agarose of 0.8 % afterwards, experiment finishes the observation experiment result.
By identical method 20/1,40/1,60/1 time preparation of N/P ratio PEI (1.8 kDa)-
g-(TMB-THME)
n/ DNA mixture is also measured particle diameter and surface potential.
The result of this embodiment is referring to Fig. 4～8, and wherein, Fig. 4 ~ 5 show that TMB-THME modified PE I does not have influence on the pH surge capability of PEI.Fig. 6 shows modification 10 kDa PEI gained PEI-
g-(TMB-THME)
nMore than or equal to 5/1 o'clock, can compress DNA at N/P ratio effectively, form the mixture of particle diameter 100 ~ 170 nm, surface potential+25 ~+43 mV, be 10/1 o'clock at N/P ratio, PEI-
g-(TMB-THME)
nThe mixture particle diameter that loads DNA formation is 110 ~ 160 nm, and surface potential is+25 ~+28 mV, and the mixture particle diameter that 10 kDa PEI and DNA form is 175 nm, and surface potential is+18 mV.Fig. 7 shows gained PEI-behind the TMB-THME modification 1.8 kDa PEI
g-(TMB-THME)
nAlso less with DNA formation particle diameter; The mixture that surface potential is higher for example, is 20/1 o'clock at N/P ratio; The mixture particle diameter that modification 1.8 kDa PEI form is 120 ~ 190 nm; Surface potential is+22 ~+26 mV, and 1.8 kDa PEI to form particle diameter be 450 nm, surface potential is+the DNA mixture of 22 mV.Fig. 8 show behind all modifying and decoratings 10 kDa PEI the N/P ratio be 3/1 time all can be effectively compound with DNA.
Embodiment six: through the hydrolysis of acetal bonds under different pH in the ultraviolet determination mixture
Through the hydrolysis of ultraviolet at the absorption measurement acetal at 290 nm places.Will the PEI (10 kDa) of N/P ratio 10/1 time preparation-
g-(TMB-THME)
9/ DNA mixture is divided into four parts of equivalent; With the hac buffer of 4.0 M pH 4.0,5.0,6.0 will be wherein three parts be transferred to pH 4.0,5.0,6.0 respectively, another part adds the phosphate buffer solution of isopyknic 4.0 M pH 7.4 to keep the identical salt concn of mixture.At the different time point, get 80 μ L wherein respectively and add 3.5 mL phosphate buffer solutions (0.1 M, pH 7.4) and measure the absorption of ultraviolet at 290 nm places.Final all samples add 2 concentrated hydrochloric acids to guarantee that all acetal bonds complete hydrolysis are as the 100 % benchmark that calculate percent hydrolysis.
The gained result is referring to Fig. 9, and the transformation period that the result is presented under pH 4.0,5.0 and 6.0 is respectively 1.3h, 2.8h and 11h, and the hydrolysis in 7.4 times 24h of pH seldom (< 12%).
Embodiment seven: the DNA release behavior that the acetal bonds hydrolysis causes under the acidic conditions
Will the PEI (10 kDa) of N/P ratio 10/1 time preparation-
g-(TMB-THME)
9/ DNA mixture is divided into two parts of equivalent, with the hac buffer of 4.0 M pH 5.0 a copy of it is regulated pH to 5.0, and another part adds the phosphate buffer solution of isopyknic 4.0 M pH 7.4 to keep identical salt concn.At the different time point; The sample that pH cultivates for 5.0 times adds the phosphate buffer solution of 4.0 M pH 7.4 pH is recalled to 7.4; 4.0 M pH, 7.4 phosphate buffer solutions that the sample of 7.4 times cultivations of pH simultaneously adds equivalent carry out the measurement of particle diameter and surface potential afterwards to keep identical salt concn.The result sees Figure 10.The result is illustrated in 3 h, increases to more than 800 nm at 5.0 times mixture particle diameters of pH, and has only small variation at 7.4 times particle diameters of pH.Simultaneously, surface potential is reduced to about-25 mV in 5.0 times 6 h of pH, and constant basically at pH 7.4 lower surface current potentials.
The PEI (10 kDa) of N/P ratio 10/1 time preparation-
g-(TMB-THME)
9/ DNA mixture is regulated pH and is returned 7.4 by same adjusting pH value and after cultivating 4.5 h, adds a certain amount of DEXTRAVEN SODIUM SULFATE (charge ratio is 40/1) simultaneously, carries out gel with method same among the embodiment five after half a hour and postpones experiment.The result sees Figure 11, its mesopore 1:DNA; Hole 2:10 kDa PEI; Hole 3:PEI-
g-(TMB-THME)
5; Hole 4:PEI-
g-(TMB-THME)
9; Hole 5:PEI-
g-(TMB-THME)
14. hole 2-5 adds a certain amount of DEXTRAVEN SODIUM SULFATE (DSS, the charge ratio of DSS and DNA are 40/1).Gel postpones experimental result and shows that further modification 10 kDa PEI/DNA mixtures have advantages of higher stability 7.4 times at pH, but then discharges soon at 5.0 times DNA of pH.
Embodiment eight: the MTT method studied PEI (10 kDa)-
g-(TMB-THME)
nToxicity
In the 293T cell, studied the toxicity of modification 10 kDa PEI through the MTT method.At first each hole adds 6000 cells and adds the substratum that 100 mL contain 10% serum in 96 orifice plates, is cultured to that cell density reaches 70～80 % in each hole.The PEI (10 kDa) of every hole adding different concentration-
g-(TMB-THME)
nSolution is cultivated 48 h subsequently again.Change substratum with the fresh substratum of 200 μ L.Every hole adds 25 μ L MTT solution (among the 4 mg/mL PBS) and cultivates 4 h down at 37 ℃.The sucking-off substratum, the MTT-first a ceremonial jade-ladle, used in libation that viable cell produces is dissolved among the 150 mL DMSO, uses ELIASA to measure the absorption of each hole at 490 nm places then.The cell relative survival rate through with the hole of having only substratum in the absorption at 490 nm places compare and obtain.Every group of data have 5 parallel appearance.
The result is referring to Figure 12, and the result shows: modification 10 kDa PEI gained PEI-
g-(TMB-THME)
n(0.6 to 2.4 μ mol/L) is nontoxic under the required concentration of transfection.
Embodiment nine: PEI (10 kDa)-
g-(TMB-THME)
nThe transfection experiment of/DNA mixture in different cells
With plasmid pGL3 is that reporter gene carries out the transfection in HeLa and 293T cell, and transfection process is referring to Fig. 1.Transfection be used in the PEI (10 kDa) that N/P ratio 10/1 and 20/1 time forms-
g-(TMB-THME)
n/ DNA mixture carries out.Cell is at first planted in 24 orifice plates (cell density is 60000 cells/well), under 37 ℃, keeps the CO of 5 %
2Cultivate in the substratum of concentration 10 % serum to cell density and reach 70 %.Transfection experiment is: cell is earlier with the washing of PBS buffered soln, and every subsequently hole adds the complex solution (DNA that contains 1 mg) of 100 mL and substratum that 400 mL contain 10 % serum or serum-free at 37 ℃ of following cultivation 4 h.Remove mixture afterwards, and add 500 mL and contain 10 % fresh serum substratum, cell was cultivated 2 days again.(Promega USA) goes up the quantitatively determined luciferase at the TD-20/20 photometer to use business-like luciferase detection kit (Pu is grand biological).Transfection efficiency is represented with the proteic relative intensity of fluorescence of every mg.The mixture that forms its optimum N/P ratio 10/1 time with 25 kDa bPEI is standard as a reference.Parallel three groups of all experimental datas.
The result sees Figure 13,14, is that the outer-gene transfection experiment of reporter gene shows: gained PEI-of the present invention with the pGL3 expressing luciferase
g-(TMB-THME)
nHeLa and 293T cell are being had under the condition with serum-free N/P ratio 10/1 and 20/1 time with the mixture of DNA and all to have high transfection efficiency.For example, PEI-
g-(TMB-THME)
14/ DNA mixture is compared with 10 kDa bPEI/DNA mixtures under its optimum N/P ratio; Transfection efficiency under the condition that has with serum-free has improved 235 and 175 times respectively, compares the transfection efficiency of 25 kDa bPEI under its optimum N/P ratio and has improved 16 and 7 times respectively.
Claims (3)
1. the polymine modified of a hydrophobic grouping; It is characterized in that: said polymine is the polymine that acetal molecule trimethoxy-benzene methylal-trimethylolethane acetal is modified; Said polymine contains acid-sensitive sense degradable acetal functional group, and its general structure is as follows:
In the formula, the number-average molecular weight of main chain polymine is 1.5～2 kDa, and the grafting number of main chain polymine grafting acetal molecule trimethoxy-benzene methylal-trimethylolethane acetal is 1～5.
2. the polymine modified of a hydrophobic grouping; It is characterized in that: said polymine is the polymine that acetal molecule trimethoxy-benzene methylal-trimethylolethane acetal is modified; Said polymine contains acid-sensitive sense degradable acetal functional group, and its general structure is as follows:
；
In the formula, the number-average molecular weight of main chain polymine is 9.5～10.5 kDa, and the grafting number of main chain polymine grafting acetal molecule trimethoxy-benzene methylal-trimethylolethane acetal is 5～15.
3. the polymine of claim 1 or 2 said hydrophobic groupings modifications is as the application of dna vector.
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| US8703197B2 (en) * | 2012-09-13 | 2014-04-22 | International Business Machines Corporation | Branched polyamines for delivery of biologically active materials |
| CN104231265A (en) * | 2013-06-19 | 2014-12-24 | 中国医学科学院药物研究所 | Aliphatic group-grafted low molecular weight polyethyleneimine as well as preparation method and application of polyethyleneimine |
| CN103965470B (en) * | 2014-04-30 | 2016-03-23 | 四川大学 | Can the hydrophobically modified polymine whipping agent and its preparation method and application of release of carbon dioxide |
| CN105732981A (en) * | 2014-12-10 | 2016-07-06 | 深圳先进技术研究院 | Modified polyethyleneimine, a gene vector composition, and a preparing method and applications of the gene vector composition |
| WO2018001256A1 (en) * | 2016-06-30 | 2018-01-04 | 苏州大学 | Reversible cross-linked polymer vesicle having asymmetrical film structure, anti-tumour medicine, and preparation method therefor |
| CN107880306B (en) * | 2017-09-30 | 2019-12-10 | 四川大学 | Hydrophobic modified polyethyleneimine foaming agent |
| CN109734921B (en) * | 2017-10-27 | 2021-09-14 | 中国医学科学院药物研究所 | Polyethyleneimine-b-polylactic acid block copolymer, and preparation method and application thereof |
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