CN102746513B - Polyamino acid segmented copolymer serving as siRAN carrier and preparation method as well as composite particle - Google Patents
Polyamino acid segmented copolymer serving as siRAN carrier and preparation method as well as composite particle Download PDFInfo
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- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 title abstract 5
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- Macromolecular Compounds Obtained By Forming Nitrogen-Containing Linkages In General (AREA)
- Polyamides (AREA)
Abstract
The invention provides a polyamino acid segmented copolymer serving as a siRAN carrier shown in a formula (I). A preparation method for the polyamino acid segmented copolymer comprises the following steps of: enabling beta-benzyl-L-aspartic acid-N-carboxylic acid anhydride to react with cladodification polyethyleneimine in an organic solvent under the protection of inert gas to obtain an intermediate product; enabling the obtained intermediate product to react with polyethyleneimine under the action of a catalytic agent to obtain the polyamino acid segmented copolymer serving as the siRAN carrier. The invention provides a composite particle which comprises the polyamino acid segmented copolymer serving as the siRAN carrier and siRNA; and the siRNA is the Luc siRNA of silent luciferase. The polyamino acid segmented copolymer serving as the siRAN carrier provided by the invention has concentrated electric charge density, strong composite capacity and good biological compatibility; and the toxicity of the polyamino acid segmented copolymer is effectively reduced.
Description
Technical field
The present invention relates to superpolymer field, particularly as polyamino acid block copolymer, preparation method and the composite particles of siRNA carrier.
Background technology
Along with development and human gene bank constantly perfect of biotechnology in modern age, making the mankind be familiar with some disease root from molecular level progressively becomes possibility, for gene therapy provides theoretical basis.In concrete enforcement, how obtaining genophore safely and effectively becomes the bottleneck problem that restricts gene therapy clinical application day by day.Utilizing virus vector mediated gene to shift is most widely used method in gene therapy, comprising carriers such as retrovirus, adenovirus, adeno-associated virus, hsv, vaccinia viruss.But viral vector exists very large potential safety hazard in clinical application, cause gene therapy the first death event and famous France " bubble baby " event in history.With respect to the insecurity of virus gene carrier, the cationic polymers of synthetic is because it is without immunogenicity, and molecular designing is various, and size is controlled and can connect targeting substance and become gradually the study hotspot in this field.
In numerous Poly-cations of having reported, polymine (PEI) PEI class carrier has been proved to be has good effect in plasmid transfection system, and is also playing a role aspect siRNA transfection.The advantage of PEI is that electric density is concentrated, and genetic stew is had to strong compound ability, has inferior quality (m/m) than obtaining best transfection efficiency with genetic stew.But polycation with a large amount of positive charges; in the time that dosage increases, can there is strong effect and cause necrocytosis (Grayson A.C, Doody A.M.et al.Biophysical and structural characterization of polyethyleniminemediated siRNA delivery in vitro Pharm.Res 2006 with cell surface; (23): 1868-1876).
The polyamino acid (ester) of synthetic has the character similar to natural polypeptides, can be degraded by enzyme in vivo, has good biological degradability and biocompatibility, is one of medical material desirable in organizational project and drug delivery system solid support material.The investigators such as recent Kim are successfully grafted to PEI molecule on the poly aspartic acid skeleton of line style by aminolysis reaction, prepare line style poly aspartic acid grafting polyethylene imine copolymer, and confirm that by experiment this cationic copolymer is a kind of high-efficiency low-toxicity degradable outer-gene transfection carrier (Kim H.J., Ishii A., et al.Introduction of stearoyl moieties into a biocompatible cationic polyaspartamide derivative, PAsp (DET), with endosomal escaping function for enhanced siRNA-mediated gene knockdown, Journal of Controlled Release, 2010, (145): 141-148).But this carrier need could be realized best transfection effect at higher mass ratio, line style poly aspartic acid grafting polyethylene imine copolymer electric density is concentrated not as can be seen here, a little less than complex gene physical capacity.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of polyamino acid block copolymer as siRNA carrier and preparation method thereof, and the polyamino acid block copolymer electric density obtaining is concentrated, and the ability of compound siRNA is strong.
The invention provides the polyamino acid block copolymer as siRNA carrier shown in formula (I),
R is selected from the one in structure shown in formula (11) ~ formula (14):
The NH-of described R is connected with the C atom in formula (I);
Ph is
Wherein, x
1, y
1, z
1, x
2, y
2, z
2, x
3, y
3, z
3, m, n, a, b and c are the polymerization degree, x
1, y
1, x
3, y
3, z
3and z
1for nonnegative integer; M, n, x
2, y
2, z
2, a, b and c are positive integer;
9≥a≥1；41≥b+c≥13；41≥m+n≥13；100≥x
1+x
2+x
3≥1；100≥y
1+y
2+y
3≥1；100≥z
1+z
2+z
3≥1。
The preparation method who the invention provides a kind of polyamino acid block copolymer as siRNA carrier, comprises the following steps:
(A), under protection of inert gas, β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride reacts in organic solvent with cladodification polymine, obtains intermediate product;
(B) intermediate product step (A) being obtained reacts under catalyst action with polymine, obtains the polyamino acid block copolymer as siRNA carrier;
The described polyamino acid block copolymer as siRNA carrier is as shown in the formula (I):
R is selected from the one in structure shown in formula (11) ~ formula (14):
The NH-of described R is connected with the C atom in formula (I);
Ph is
Wherein, x
1, y
1, z
1, x
2, y
2, z
2, x
3, y
3, z
3, m, n, a, b and c are the polymerization degree, x
1, y
1, x
3, y
3, z
3and z
1for nonnegative integer; M, n, x
2, y
2, z
2, a, b and c are positive integer;
9≥a≥1；41≥b+c≥13；41≥m+n≥13；100≥x
1+x
2+x
3≥1；100≥y
1+y
2+y
3≥1；100≥z
1+z
2+z
3≥1。
Preferably, in described step (A), described β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride and cladodification polymine mole proportioning are 100～10000:1～10.
Preferably, in described step (A), the mol ratio of described β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride and organic solvent is 5～10:50～1000.
Preferably, in described step (A), described organic solvent is chloroform, methylene dichloride or N ', N '-dimethyl formamide.
Preferably, in described step (B), described catalyzer is 2 hydroxy pyrimidine.
Preferably, in described step (B), the mol ratio of described intermediate product and polymine is 1～10:6～600.
Preferably, in described step (B), the time of described reaction is 10～80 hours.
Preferably, in described step (B), the temperature of described reaction is 15～50 ℃.
The invention provides a kind of composite particles, comprise polyamino acid block copolymer and siRNA as siRNA carrier described in technique scheme, the Luc siRNA that described siRNA is reticent luciferase.
Compared with prior art, polyamino acid block copolymer as siRNA carrier of the present invention as shown in the formula (I), take 3 poly-β-benzyl-L-Aspartic acid chains being connected to cladodification polymine as skeleton, and on poly-β-benzyl-L-Aspartic acid, be also grafted with the side chain of multiple polymines, thereby form branched structure.Because the present invention has multiple branches as the polyamino acid block copolymer of siRNA carrier, and on branch, be also grafted with cationic polymine, therefore electric density is concentrated, and siRNA is had to strong compound ability.Meanwhile, described polyamino acid block copolymer comprises poly-β-benzyl-L-Aspartic acid skeleton, and it has good biocompatibility, effectively reduces the toxicity of polyamino acid block copolymer.
Accompanying drawing explanation
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram as the polyamino acid block copolymer of siRNA carrier prepared by embodiment 9;
Fig. 2 is polyamino acid block copolymer, PEI25k and the transfection reagent Lipofectamine as siRNA carrier of embodiment 9,15,20 preparations
tMthe toxotest result figure of 200 pairs of HeLa cells.
Embodiment
In order further to understand the present invention, below in conjunction with embodiment, the preferred embodiment of the invention is described, but should be appreciated that these are described is for further illustrating the features and advantages of the present invention, rather than limiting to the claimed invention.
The embodiment of the invention discloses the polyamino acid block copolymer as siRNA carrier shown in a kind of formula (I),
R is selected from the one in structure shown in formula (11) ~ formula (14):
The NH-of described R is connected with the C atom in formula (I);
Ph is
Wherein, x
1, y
1, z
1, x
2, y
2, z
2, x
3, y
3, z
3, m, n, a, b and c are the polymerization degree, x
1, y
1, x
3, y
3, z
3and z
1for nonnegative integer; M, n, x
2, y
2, z
2, a, b and c are positive integer;
9≥a≥1；41≥b+c≥13；41≥m+n≥13；100≥x
1+x
2+x
3≥1；100≥y
1+y
2+y
3≥1；100≥z
1+z
2+z
3≥1。
The described polyamino acid block copolymer as siRNA carrier as shown in the formula (I), take 3 poly-β-benzyl-L-Aspartic acid chains being connected to cladodification polymine as skeleton, and on poly-β-benzyl-L-Aspartic acid, be also grafted with the side chain of multiple polymines, thereby form branched structure.Because polyamino acid block copolymer of the present invention has multiple branches, and on branch, be also grafted with cationic polymine, therefore electric density is concentrated, and siRNA is had to strong compound ability.
The preparation method who the invention also discloses a kind of polyamino acid block copolymer as siRNA carrier, comprises the following steps:
(A), under protection of inert gas, β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride reacts in organic solvent with cladodification polymine, obtains intermediate product;
(B) intermediate product step (A) being obtained reacts under catalyst action with polymine, obtains the polyamino acid block copolymer as siRNA carrier;
The described polyamino acid block copolymer as siRNA carrier is as shown in the formula (I):
R is selected from the one in structure shown in formula (11) ~ formula (14):
The NH-of described R is connected with the C atom in formula (I);
Ph is
Wherein, x
1, y
1, z
1, x
2, y
2, z
2, x
3, y
3, z
3, m, n, a, b and c are the polymerization degree, x
1, y
1, x
3, y
3, z
3and z
1for nonnegative integer; M, n, x
2, y
2, z
2, a, b and c are positive integer;
9≥a≥1；41≥b+c≥13；41≥m+n≥13；100≥x
1+x
2+x
3≥1；100≥y
1+y
2+y
3≥1；100≥z
1+z
2+z
3≥1。
The present invention is first take β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride as amino acid monomer, and cladodification polymine is that initiator reacts in organic solvent.Described reaction is carried out under protection of inert gas, and the present invention is not particularly limited rare gas element, can be nitrogen or argon gas.The present invention is not particularly limited the source of described β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride, is bought by market.The present invention is not particularly limited the molecular weight of cladodification polymine, is preferably 400 ~ 3000, and more preferably 600 ~ 1800.Described organic solvent is preferably chloroform, methylene dichloride or N ', N '-dimethyl formamide.The mol ratio of described β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride and organic solvent is preferably 5～10:50～1000, more preferably 6 ~ 9:150～800.β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride is that the mol ratio of amino acid monomer and cladodification polymine is preferably 100～10000:1～10, more preferably 300 ~ 800:1 ~ 10, β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride is the reaction times of amino acid monomer and cladodification polymine to be preferably 10 ~ 15h, more preferably 11 ~ 14h, temperature of reaction is preferably 0 ~ 50 ℃, more preferably 10 ~ 40 ℃.
After reaction finishes, preferably through sedimentation, filtration, the dry intermediate product that obtains.Intermediate product and polymine, under catalyst action, polycondensation is occurred to, and described polymine can be the polymine of cladodification, can be also the polymine of line style.Because the polymine of cladodification has multiple amidos, described amido all can react with intermediate product.Therefore after described intermediate product reacts with the polymine of cladodification, shown in the formula (I) obtaining as in the polyamino acid block copolymer of siRNA carrier, R has formula (11) ~ formula (13) structure; When after described intermediate product and linear polyethylene imine reaction, being used as in the polyamino acid block copolymer of siRNA carrier shown in the formula (I) obtaining, R has formula (14) structure.In described reaction process, the polymerization degree is x
2repeating unit can be x with the polymerization degree
3repeating unit occur mutually to transform, the polymerization degree is y
2repeating unit can be y with the polymerization degree
3repeating unit occur mutually to transform, the polymerization degree is z
2repeating unit can be z with the polymerization degree
3repeating unit occur mutually to transform.
The mol ratio of described intermediate product and polymine is preferably 1～10:6～600, more preferably 1～10:20～300.Described reaction is preferably carried out in organic solvent, and described organic solvent is preferably chloroform, methylene dichloride or N ', N '-dimethyl formamide, and the quality of described polymine and the volume ratio of organic solvent are preferably 5～10:50～1000.Described catalyzer is preferably 2 hydroxy pyrimidine.The time of described reaction is preferably 10～80 hours, and more preferably 30 ~ 60 hours, the temperature of described reaction was preferably 15～50 ℃, more preferably 30 ~ 40 ℃.After described reaction finishes, the dialysis band dialysis that is preferably 1000～10000 through molecular weight cut-off at least 24 hours, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier.
The invention also discloses a kind of composite particles, comprise polyamino acid block copolymer and siRNA as siRNA carrier described in technique scheme, the Luc siRNA that described siRNA is reticent luciferase.
The sequence of described Luc siRNA is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ', and the present invention is not particularly limited the synthetic method of Luc siRNA, synthetic according to method well known to those skilled in the art, also can buy acquisition.Describedly be preferably 1 ~ 40:1, more preferably 2 ~ 15:1 as the polyamino acid block copolymer of siRNA carrier and the mass ratio of siRNA.
The present invention is not particularly limited the preparation method of described composite particles, is preferably in sterilized water, described polyamino acid block copolymer as siRNA carrier and siRNA is carried out to static compound, obtains composite particles.
Describedly be preferably 1 ~ 40:1, more preferably 2 ~ 15:1 as the polyamino acid block copolymer of siRNA carrier and the mass ratio of siRNA.The compound time of described static is preferably 10 ~ 30 minutes.
Described composite particles can be for external transfection, and transfection efficiency is high.After its transfection, gene silencing efficiency is high, and cytotoxicity is little.
In order further to understand the present invention, below in conjunction with embodiment, polyamino acid block copolymer, preparation method and the composite particles as siRNA carrier provided by the invention described, protection scope of the present invention is not limited by the following examples.
Embodiment 1
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 600 by 19mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 21000.
Embodiment 2
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 600 by 3.8mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 100800.
Embodiment 3
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 1200 by 11.8mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 33600.
Embodiment 4
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 1200 by 2.4mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 162000.
Embodiment 5
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 1800 by 7.6mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 55500.
Embodiment 6
Under anhydrous and oxygen-free condition, 4.7 grams of β-benzyl-L-Aspartic acid-N-carboxylic acid anhydride are joined in dry reaction ampulla, be filled with nitrogen protection, add the anhydrous N ' of 300mL, N '-dimethyl formamide, stirs and makes its dissolving.
The polymine of the cladodification that is 1800 by 1.5mL weight-average molecular weight is dissolved in anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in reaction ampulla, temperature control was 30 ℃ of stirring reactions 72 hours.After having reacted, sedimentation in the ether of 3000mL, filters and dry cake, obtains poly-β-benzyl-L-aspartate, and its molecular weight is 250100.
Embodiment 7
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 103 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 1, molecular weight is 30480, and benzyl ester decreasing ratio is 100%.
Embodiment 8
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 423 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 2; Wherein x
1+ x
2=20; y
1+ y
2=20; z
1+ z
2=20; Molecular weight is 43260, and benzyl ester decreasing ratio is 100%.
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 600 linear polyethylene imines 40mmol, 2 hydroxy pyrimidine 40mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 3; Wherein x
1+ x
2=20; y
1+ y
2=20; z
1+ z
2=20; Molecular weight is 56060, and benzyl ester decreasing ratio is 100%.
The polyamino acid block copolymer as siRNA carrier shown in the formula obtaining (I) is carried out to nuclear magnetic resonance spectroscopy, result is referring to Fig. 1, Fig. 1 is the nuclear magnetic resonance map as the polyamino acid block copolymer of siRNA carrier prepared by embodiment 9, as can be seen here, embodiment 9 has prepared the polyamino acid block copolymer as siRNA carrier shown in formula (I).
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 600 linear polyethylene imines 10mmol, 2 hydroxy pyrimidine 10mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 4; Wherein x
1+ x
2=20; y
1+ y
2=20; z
1+ z
2=20; Molecular weight is 43150, and benzyl ester decreasing ratio is 87%.
Embodiment 11
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 1200 linear polyethylene imines 10mmol, 2 hydroxy pyrimidine 10mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 5; Wherein x
1+ x
2=20; y
1+ y
2=20; z
1+ z
2=20; Molecular weight is 57100, and benzyl ester decreasing ratio is 85%.
Embodiment 12
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 1, molecular weight is 1800 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 6, wherein x
1+ x
2=20; y
1+ y
2=20; z
1+ z
2=20; Molecular weight is 74300, and benzyl ester decreasing ratio is 100%.
Embodiment 13
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 103 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 7, wherein x
1+ x
2=19; y
1+ y
2=19; z
1+ z
2=19; Molecular weight is 44560, and benzyl ester decreasing ratio is 88%.
Embodiment 14
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 423 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 8, wherein x
1+ x
2=19; y
1+ y
2=19; z
1+ z
2=19; Molecular weight is 63500, and benzyl ester decreasing ratio is 93%.
Embodiment 15
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 600 linear polyethylene imines 40mmol, 2 hydroxy pyrimidine 40mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 9, wherein x
1+ x
2=19; y
1+ y
2=19; z
1+ z
2=19; Molecular weight is 76030, and benzyl ester decreasing ratio is 100%.
Embodiment 16
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 600 linear polyethylene imines 10mmol, 2 hydroxy pyrimidine 10mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 10, molecular weight is 63120, and benzyl ester decreasing ratio is 82%.
Embodiment 17
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 1200 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 11, wherein x
1+ x
2=19; y
1+ y
2=19; z
1+ z
2=19; Molecular weight is 80870, and benzyl ester decreasing ratio is 93%.
Embodiment 18
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 3, molecular weight is 1800 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 12, wherein x
1+ x
2=19; y
1+ y
2=19; z
1+ z
2=19; Molecular weight is 86260, and benzyl ester decreasing ratio is 100%.
Embodiment 19
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 103 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 13, wherein x
1+ x
2=22; y
1+ y
2=22; z
1+ z
2=22; Molecular weight is 60970, and benzyl ester decreasing ratio is 95%.
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 42 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and is in 3500 different dialysis tubings deionized water dialysis 7 days, the liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 14, wherein x
1+ x
2=22; y
1+ y
2=22; z
1+ z
2=22; Molecular weight is 70870, and benzyl ester decreasing ratio is 93%.
Embodiment 21
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 600 linear polyethylene imines 40mmol, 2 hydroxy pyrimidine 40mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 15; Wherein x
1+ x
2=22; y
1+ y
2=22; z
1+ z
2=22; Molecular weight is 86070, and benzyl ester decreasing ratio is 100%.
Embodiment 22
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 600 linear polyethylene imines 10mmol, 2 hydroxy pyrimidine 10mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 16, molecular weight is 73120, and benzyl ester decreasing ratio is 82%.
Embodiment 23
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 1200 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 17, molecular weight is 92220, and benzyl ester decreasing ratio is 100%.
Embodiment 24
Take 2.1 grams of poly-β-benzyl-L-aspartates (containing 10mmol carbobenzoxy group) prepared by embodiment 5, molecular weight is 1800 linear polyethylene imines 20mmol, 2 hydroxy pyrimidine 20mmol is at the anhydrous dimethyl sulphoxide of 200mL, stirring makes its dissolving, 38 ℃ of temperature controls, stirring reaction 48 hours; After having reacted, respectively reaction solution is packed into molecular weight cut-off and be in 3500 different dialysis tubings deionized water dialysis 7 days, liquid freezing after dialysis is dry, obtain the polyamino acid block copolymer as siRNA carrier shown in formula (I), be designated as PLAA-g-PEI 18, molecular weight is 98260, and benzyl ester decreasing ratio is 100%.‘
Embodiment 25 ~ 29
Choosing siRNA is the Luc siRNA of reticent luciferase, and its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.
Getting respectively PLAA-g-PEI3, PLAA-g-PEI 9, PLAA-g-PEI 14 prepared by 20mg embodiment 9, embodiment 15, embodiment 20 is put in sterilized water, by its with 1mgsiRNA static compound 20 minutes respectively, obtain PLAA-g-PEI 3/siRNA composite particles, PLAA-g-PEI 9/siRNA composite particles and PLAA-g-PEI 14/siRNA composite particles.
Choosing siRNA is the Luc siRNA of reticent luciferase, and its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.Contrast is Rev siRNA, and its sequence is 5 '-AGCUUCAUGAGUCGCAUUCdTdT-3 '.
It is the nutrient solution of 10% foetal calf serum that Huh 7 cells are placed in containing volume fraction, cultured continuously in 37 ℃ contain the incubator that volume fraction is 5% carbonic acid gas.
In first 24 hours of transfection, the Huh7 cell in vegetative period of taking the logarithm, after trysinization, dilute with DMEM, be inoculated in 96 well culture plates by the density of every hole 1 × 104 cell, being placed in 37 ℃ is that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80 ~ 90% containing volume fraction.When transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, with after phosphate buffered saline buffer washed twice, the composite particles of PLAA-g-PEI3 and siRNA composite particles, PLAA-g-PEI9/siRNA composite particles, PLAA-g-PEI14/siRNA composite particles, PEI25k/siRNA composite particles and Lipofectamine TM2000/siRNA carries out reticent effect comparison, continues to cultivate 24 hours.
Take out culture plate, suck nutrient solution, with phosphate buffered saline buffer washing 2 times, add cell pyrolysis liquid cracking, then add luciferase substrate, measure transfection efficiency with luxmeter.
Gene silencing efficiency and the transfection mass ratio of table 1 different carriers material delivery siRNA
| Sequence number | Solid support material | Gene silencing efficiency (%) | Transfection m/m |
| 1 | Rev siRNA | 5.3 | |
| 2 | PEI25k | 38.6 | 1 |
| 3 | Lipofectamine TM2000 | 53.5 | 2.5 |
| 4 | PLAA-g-PEI3 | 78.8 | 5 |
| 5 | PLAA-g-PEI9 | 70.6 | 5 |
| 6 | PLAA-g-PEI14 | 68.4 | 5 |
In table 1, transfection m/m is the mass ratio of carrier and siRNA.
Embodiment 31
Getting that HeLa cell is placed in containing volume fraction is the nutrient solution of 10% foetal calf serum, is cultured continuously in the incubator of 5% carbonic acid gas at 37 ℃ containing volume fraction.
Polyoxyethylene glycol (PEI25k) and transfection reagent Lipofectamine that method comparative evaluation different concns PLAA-g-PEI3, PLAA-g-PEI9, PLAA-g-PEI14, the molecular weight that adopts MTT is 25000
tMthe toxicity of 2000 pairs of cells.Test in first 24 hours, the HeLa cell in the vegetative period of taking the logarithm, dilutes with DMEM after trysinization, by every hole 1 × 10
4the density of cell is inoculated in 96 well culture plates, and being placed in 37 ℃ is that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80 ~ 90% containing volume fraction.By the material of different concns and cell co-culture, after 24 hours, every hole adds respectively the phosphoric acid buffer that 20 μ L are 0.5%MTT containing massfraction.Mixture, 37 ℃ of continuation effects 4 hours, adds 200 μ L dmso solution MTT Jia Za crystallization 10 minutes.Then test the absorption in every hole by microplate reader, test wavelength is selected 492nm.Cell survival rate is pressed formula and is calculated:
Cell survival rate (%)=(A
sample/ A
control) × 100
A
samplethe absorption in the cell sample hole after transfection, A
controlbe not with the absorption in the cell sample hole of complex solution effect, every group of experiment in triplicate, test result is shown in Fig. 2, and Fig. 2 is polyamino acid block copolymer, PEI25k and the transfection reagent Lipofectamine as siRNA carrier of embodiment 9,15,20 preparations
tMthe toxotest result figure of 200 pairs of HeLa cells.
In Fig. 2, curve A is transfection reagent Lipofectamine
tMthe toxotest result of 2000 pairs of cells; Curve B is the toxotest result of PEI25k to cell; Curve C is the toxotest result of PLAA-g-PEI3 to cell; Curve D is the toxotest result of PLAA-g-PEI9 to cell; Curve E is the toxotest result of PLAA-g-PEI14 to cell.As shown in Figure 2, the polyamino acid block copolymer as siRNA carrier that prepared by the embodiment of the present invention is significantly less than PEI25k and Lipofectamine for the toxicity of HeLa cell
tM2000.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
To the above-mentioned explanation of the disclosed embodiments, make professional and technical personnel in the field can realize or use the present invention.To be apparent for those skilled in the art to the multiple modification of these embodiment, General Principle as defined herein can, in the situation that not departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention will can not be restricted to these embodiment shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (1)
1. a composite particles, comprises polyamino acid block copolymer and the siRNA as siRNA carrier shown in formula (I), the Luc siRNA that described siRNA is reticent luciferase;
The polyamino acid block copolymer as siRNA carrier shown in formula (I),
R is selected from the one in structure shown in formula (11)～formula (14):
The NH-of described R is connected with the C atom in formula (I);
Ph is
Wherein, x
1, y
1, z
1, x
2, y
2, z
2, x
3, y
3, z
3, m, n, a, b and c are the polymerization degree, x
1, y
1, x
3, y
3, z
3and z
1for nonnegative integer; M, n, x
2, y
2, z
2, a, b and c are positive integer;
9≥a≥1；41≥b+c≥13；41≥m+n≥13；100≥x
1+x
2+x
3≥1；100≥y
1+y
2+y
3≥1；100≥z
1+z
2+z
3≥1。
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| CN101575416A (en) * | 2009-06-15 | 2009-11-11 | 中国科学院长春应用化学研究所 | Multi-arm polyamino acid (ester) grafted polyethyleneimine copolymer, preparation method and application in gene delivery |
| CN101704949A (en) * | 2009-11-13 | 2010-05-12 | 中国科学院长春应用化学研究所 | Polyethyleneimine modified with acrylamide monomers, preparation method and application in gene delivery |
| WO2010131777A1 (en) * | 2009-05-14 | 2010-11-18 | 国立大学法人東京大学 | Fine particles of crystalline polyol and method of preparing same |
| JP2011026219A (en) * | 2009-07-22 | 2011-02-10 | Univ Of Tokyo | Polyion complex including phd2 expression inhibiting substance |
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| WO2010131777A1 (en) * | 2009-05-14 | 2010-11-18 | 国立大学法人東京大学 | Fine particles of crystalline polyol and method of preparing same |
| CN101575416A (en) * | 2009-06-15 | 2009-11-11 | 中国科学院长春应用化学研究所 | Multi-arm polyamino acid (ester) grafted polyethyleneimine copolymer, preparation method and application in gene delivery |
| JP2011026219A (en) * | 2009-07-22 | 2011-02-10 | Univ Of Tokyo | Polyion complex including phd2 expression inhibiting substance |
| CN101704949A (en) * | 2009-11-13 | 2010-05-12 | 中国科学院长春应用化学研究所 | Polyethyleneimine modified with acrylamide monomers, preparation method and application in gene delivery |
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