CN102746513A - 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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- 239000011246 composite particle Substances 0.000 title claims abstract description 19
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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 the superpolymer field, particularly as polyamino acid segmented copolymer, preparation method and the composite particles of siRNA carrier.
Background technology
Along with modern age development of biology and human gene bank constantly perfect, making human progressively becomes possibility from some disease root of molecular level understanding, for gene therapy provides theoretical basis.In practical implementation, how obtaining safely and effectively, genophore becomes the bottleneck problem that restricts the gene therapy clinical application day by day.Utilizing the virus vector mediated gene to shift is most widely used method in the gene therapy, comprising carriers such as retrovirus, adenovirus, adeno-associated virus, hsv, vaccinia viruss.But viral vector exists very big potential safety hazard in clinical application, has caused the first in history death incident of gene therapy and famous France " bubble baby " incident.With respect to the insecurity of virus gene carrier, the cationic polymers of synthetic is owing to its no immunogenicity, and molecular designing is various, and also can connect targeting substance becomes this hot research fields to controllable size gradually.
In numerous polycation carriers of having reported, polymine (PEI) PEI class carrier has been proved to be has good effect in the plasmid transfection system, and is also playing a role aspect the siRNA transfection.The advantage of PEI is that electric density is concentrated, and genetic stew is had strong compound ability, has inferior quality (m/m) than obtaining best transfection efficiency with genetic stew.But a large amount of positive charge that polycation has; When dosage increases, can the intensive effect be arranged 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 of synthetic (ester) has the character similar with natural polypeptides, can be degraded by enzyme in vivo, has good biological degradability and biocompatibility, is one of ideal medical material in organizational project and the drug delivery system solid support material.Investigators such as recent Kim successfully are grafted to the PEI molecule on the SAP 73 skeleton of line style through aminolysis reaction; Prepared line style SAP 73 grafting polyethylene imine copolymer; And through experiment confirm this cationic copolymer be a kind of high-efficiency low-toxicity and 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).Yet this carrier need this shows that line style SAP 73 grafting polyethylene imine copolymer electric density is concentrated inadequately, a little less than the complex gene physical capacity in higher quality than realizing best transfection effect.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of polyamino acid segmented copolymer that is used as the siRNA carrier and preparation method thereof, and the polyamino acid segmented copolymer electric density that obtains is concentrated, and the ability of compound siRNA is strong.
The invention provides the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier,
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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
1Be 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 invention provides a kind of preparation method who is used as the polyamino acid segmented copolymer of siRNA carrier, may further comprise the steps:
(A) under protection of inert gas, β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and cladodification polymine react in organic solvent, obtain intermediate product;
(B) intermediate product and the polymine that step (A) are obtained react under catalyst action, obtain the polyamino acid segmented copolymer as the siRNA carrier;
Said polyamino acid segmented copolymer as the siRNA carrier is suc as formula shown in (I):
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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
1Be 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 the said step (A), said β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and cladodification polymine mole proportioning are 100～10000:1～10.
Preferably, in the said step (A), the mol ratio of said β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and organic solvent is 5～10:50～1000.
Preferably, in the said step (A), said organic solvent is chloroform, methylene dichloride or N ', N '-N.
Preferably, in the said step (B), said catalyzer is a 2 hydroxy pyrimidine.
Preferably, in the said step (B), the mol ratio of said intermediate product and polymine is 1～10:6～600.
Preferably, in the said step (B), the time of said reaction is 10～80 hours.
Preferably, in the said step (B), the temperature of said reaction is 15～50 ℃.
The invention provides a kind of composite particles, comprise technique scheme described polyamino acid segmented copolymer and siRNA as the siRNA carrier, said siRNA is the Luc siRNA of reticent luciferase.
Compared with prior art; The polyamino acid segmented copolymer that is used as the siRNA carrier of the present invention is suc as formula shown in (I); To gather β-benzyl-L-aspartic acid chain be skeleton to be connected 3 of cladodification polymine; And gathering the side chain that also is grafted with a plurality of polymines on β-benzyl-L-aspartic acid, dividing dendritic structure thereby form.Because the present invention has a plurality of branches as the polyamino acid segmented copolymer of siRNA carrier, and on branch, also be grafted with and have cationic polymine, so electric density concentrates, siRNA is had strong compound ability.Simultaneously, comprise in the said polyamino acid segmented copolymer and gather β-benzyl-L-aspartic acid skeleton that it has excellent biological compatibility, effectively reduces the toxicity of polyamino acid segmented copolymer.
Description of drawings
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram of the polyamino acid segmented copolymer that is used as the siRNA carrier of embodiment 9 preparations;
Fig. 2 is polyamino acid segmented copolymer, PEI25k and the transfection reagent Lipofectamine as the siRNA carrier of embodiment 9,15,20 preparations
TMThe toxotest of 200 pairs of HeLa cells is figure as a result.
Embodiment
In order further to understand the present invention, below in conjunction with embodiment the preferred embodiment of the invention is described, describe just to further specifying feature and advantage of the present invention but should be appreciated that these, rather than to the restriction of claim of the present invention.
The embodiment of the invention discloses the polyamino acid segmented copolymer shown in a kind of formula (I) as the siRNA carrier,
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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
1Be 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 said polyamino acid segmented copolymer that is used as the siRNA carrier is suc as formula shown in (I); To gather β-benzyl-L-aspartic acid chain be skeleton to be connected 3 of cladodification polymine; And gathering the side chain that also is grafted with a plurality of polymines on β-benzyl-L-aspartic acid, dividing dendritic structure thereby form.Because polyamino acid segmented copolymer of the present invention has a plurality of branches, and on branch, also be grafted with and have cationic polymine, so electric density concentrates, siRNA is had strong compound ability.
The invention also discloses a kind of preparation method who is used as the polyamino acid segmented copolymer of siRNA carrier, may further comprise the steps:
(A) under protection of inert gas, β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and cladodification polymine react in organic solvent, obtain intermediate product;
(B) intermediate product and the polymine that step (A) are obtained react under catalyst action, obtain the polyamino acid segmented copolymer as the siRNA carrier;
Said polyamino acid segmented copolymer as the siRNA carrier is suc as formula shown in (I):
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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
1Be 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 an amino acid monomer with β-benzyl-L-aspartic acid-N-carboxylic acid anhydride at first, and the cladodification polymine is that initiator reacts in organic solvent.Said being reflected under the protection of inert gas carried out, and the present invention does not have particular restriction to rare gas element, can be nitrogen or argon gas.The present invention does not have particular restriction to the source of said β-benzyl-L-aspartic acid-N-carboxylic acid anhydride, is bought by market to get final product.The present invention does not have particular restriction to the molecular weight of cladodification polymine, is preferably 400 ~ 3000, and more preferably 600 ~ 1800.Said organic solvent is preferably chloroform, methylene dichloride or N ', N '-N.The mol ratio of said 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; 300 ~ 800:1 ~ 10 more preferably; 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; 11 ~ 14h more preferably, temperature of reaction is preferably 0 ~ 50 ℃, more preferably 10 ~ 40 ℃.
After reaction finishes, preferably obtain intermediate product through sedimentation, filtration, drying.Polycondensation is taken place under catalyst action for intermediate product and polymine, and said polymine can be the polymine of cladodification, also can be the polymine of line style.Because the polymine of cladodification has a plurality of amidos, said amido all can react with intermediate product.So after the polymine reaction of said intermediate product and cladodification, being used as in the polyamino acid segmented copolymer of siRNA carrier shown in the formula that obtains (I), R has formula (11) ~ formula (13) structure; Behind said intermediate product and linear polyethylene imine reaction, being used as in the polyamino acid segmented copolymer of siRNA carrier shown in the formula that obtains (I), R has formula (14) structure.In said reaction process, the polymerization degree is x
2Repeating unit can be x with the polymerization degree
3Repeating unit take place to transform each other, the polymerization degree is y
2Repeating unit can be y with the polymerization degree
3Repeating unit take place to transform each other, the polymerization degree is z
2Repeating unit can be z with the polymerization degree
3Repeating unit take place to transform each other.
The mol ratio of said intermediate product and polymine is preferably 1～10:6～600, more preferably 1～10:20～300.Said reaction is preferably carried out in organic solvent, and said organic solvent is preferably chloroform, methylene dichloride or N ', N '-N, and the quality of said polymine is preferably 5～10:50～1000 with the volume of organic solvent ratio.Said catalyzer is preferably 2 hydroxy pyrimidine.The time of said reaction is preferably 10～80 hours, and more preferably 30 ~ 60 hours, the temperature of said reaction was preferably 15～50 ℃, more preferably 30 ~ 40 ℃.After said reaction finishes, preferably be 1000～10000 dialysis band dialysis at least 24 hours through molecular weight cut-off, the liquid freezing after the dialysis is dry, obtain polyamino acid segmented copolymer as the siRNA carrier.
The invention also discloses a kind of composite particles, comprise technique scheme described polyamino acid segmented copolymer and siRNA as the siRNA carrier, said siRNA is the Luc siRNA of reticent luciferase.
The sequence of said Luc siRNA is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ', the present invention does not have particular restriction to the compound method of Luc siRNA, gets final product according to method well known to those skilled in the art is synthetic, can buy acquisition yet.Said polyamino acid segmented copolymer and the mass ratio of siRNA as the siRNA carrier is preferably 1 ~ 40:1, more preferably 2 ~ 15:1.
The present invention does not have particular restriction to the preparation method of said composite particles, is preferably in sterilized water, and it is compound that said polyamino acid segmented copolymer and siRNA as the siRNA carrier carried out static, obtains composite particles.
Said polyamino acid segmented copolymer and the mass ratio of siRNA as the siRNA carrier is preferably 1 ~ 40:1, more preferably 2 ~ 15:1.The said static compound time is preferably 10 ~ 30 minutes.
Said composite particles can be used for external transfection, and transfection efficiency is high.After its transfection, gene silencing efficient is high, and cytotoxicity is little.
In order further to understand the present invention, below in conjunction with embodiment polyamino acid segmented copolymer, preparation method and the composite particles as the siRNA carrier provided by the invention described, protection scope of the present invention is not limited by the following examples.
Embodiment 1
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 19mL weight-average molecular weight is 600 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 21000.
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 3.8mL weight-average molecular weight is 600 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 100800.
Embodiment 3
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 11.8mL weight-average molecular weight is 1200 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 33600.
Embodiment 4
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 2.4mL weight-average molecular weight is 1200 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 162000.
Embodiment 5
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 7.6mL weight-average molecular weight is 1800 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 55500.
Embodiment 6
Under the anhydrous and oxygen-free condition, 4.7 gram β-benzyl-L-aspartic acid-N-carboxylic acid anhydride are joined in the dry reaction peace bottle, charge into nitrogen protection, add the anhydrous N ' of 300mL, N '-N stirs and makes its dissolving.
The polymine that with the 1.5mL weight-average molecular weight is 1800 cladodification is dissolved in the anhydrous chloroform, obtains polymine/chloroformic solution that concentration is 0.01mmol/mL.Get 19mL polymine/chloroformic solution and join in the reaction peace bottle, temperature control was 30 ℃ of stirring reactions 72 hours.After reaction was accomplished, sedimentation in the ether of 3000mL was filtered and dry cake, obtains poly-, and its molecular weight is 250100.
Embodiment 7
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 103 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 1; Molecular weight is 30480, and benzyl ester decreasing ratio is 100%.
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 423 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 2; X wherein
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%.
Embodiment 9
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 600 linear polyethylene imines 40mmol, and 2 hydroxy pyrimidine 40mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 3; X wherein
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 segmented copolymer as the siRNA carrier to shown in the formula (I) that obtains carries out nuclear magnetic resonance spectroscopy; The result is referring to Fig. 1; Fig. 1 is the nuclear magnetic resonance map of the polyamino acid segmented copolymer that is used as the siRNA carrier of embodiment 9 preparations; This shows that embodiment 9 has prepared the polyamino acid segmented copolymer as the siRNA carrier shown in the formula (I).
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 600 linear polyethylene imines 10mmol, and 2 hydroxy pyrimidine 10mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 4; X wherein
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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 1200 linear polyethylene imines 10mmol, and 2 hydroxy pyrimidine 10mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 5; X wherein
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%.
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 1 preparation; Molecular weight is 1800 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 103 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 423 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 600 linear polyethylene imines 40mmol, and 2 hydroxy pyrimidine 40mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 600 linear polyethylene imines 10mmol, and 2 hydroxy pyrimidine 10mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 10; Molecular weight is 63120, and benzyl ester decreasing ratio is 82%.
Embodiment 17
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 1200 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 3 preparations; Molecular weight is 1800 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 103 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 42 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Respectively with reaction solution pack into molecular weight cut-off be in 3500 the different dialysis tubings to deionized water dialysis 7 days, the liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier; 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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 600 linear polyethylene imines 40mmol, and 2 hydroxy pyrimidine 40mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 15; X wherein
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 by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 600 linear polyethylene imines 10mmol, and 2 hydroxy pyrimidine 10mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 16; Molecular weight is 73120, and benzyl ester decreasing ratio is 82%.
Embodiment 23
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 1200 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, be designated as PLAA-g-PEI 17; Molecular weight is 92220, and benzyl ester decreasing ratio is 100%.
Embodiment 24
Take by weighing poly-2.1 grams (containing 10mmol carbobenzoxy group) of embodiment 5 preparations; Molecular weight is 1800 linear polyethylene imines 20mmol, and 2 hydroxy pyrimidine 20mmol stirs and makes its dissolving at the anhydrous dimethyl sulphoxide of 200mL; 38 ℃ of temperature controls, stirring reaction 48 hours; After reaction is accomplished; Be to deionized water dialysis 7 days in 3500 the different dialysis tubings with the reaction solution molecular weight cut-off of packing into respectively; Liquid freezing after the dialysis is dry, obtain the polyamino acid segmented copolymer shown in the formula (I) as the siRNA carrier, 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, its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.
The PLAA-g-PEI3, PLAA-g-PEI 9, the PLAA-g-PEI 14 that get 20mg embodiment 9, embodiment 15, embodiment 20 preparations respectively are put in sterilized water; With its 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 with 1mgsiRNA static.
Choosing siRNA is the Luc siRNA of reticent luciferase, its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.Control is Rev siRNA, its sequence is 5 '-AGCUUCAUGAGUCGCAUUCdTdT-3 '.
Huh 7 cells place and contain the nutrient solution that volume(tric)fraction is 10% foetal calf serum, contain cultured continuously in the incubator that volume(tric)fraction is 5% carbonic acid gas at 37 ℃.
In preceding 24 hours of the transfection; The Huh7 cell in vegetative period of taking the logarithm; Dilute with DMEM after the trysinization; Density by every hole 1 * 104 cell is inoculated in 96 well culture plates, and placing 37 ℃, to contain volume(tric)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 previous day is abandoned in suction; After the 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, inhale and remove nutrient solution,, add the cell pyrolysis liquid cracking, add the luciferase substrate then, measure transfection efficiency with luxmeter with phosphate buffered saline buffer washing 2 times.
Gene silencing efficient and the transfection mass ratio of table 1 different carriers material delivery siRNA
| Sequence number | Solid support material | Gene silencing efficient (%) | 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 the table 1, transfection m/m is the mass ratio of carrier and siRNA.
Embodiment 31
Get the HeLa cell and place and contain the nutrient solution that volume(tric)fraction is 10% foetal calf serum, contain cultured continuously in the incubator that volume(tric)fraction is 5% carbonic acid gas at 37 ℃.
Method comparative evaluation different concns PLAA-g-PEI3, PLAA-g-PEI9, PLAA-g-PEI14, the molecular weight that adopts MTT is 25000 polyoxyethylene glycol (PEI25k) and transfection reagent Lipofectamine
TMThe toxicity of 2000 pair cells.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, and placing 37 ℃, to contain volume(tric)fraction be that the incubator of 5% carbonic acid gas continues to be cultured to degree of converging and reaches 80 ~ 90%.After 24 hours, every hole adds 20 μ L respectively and contains the phosphoric acid buffer that massfraction is 0.5%MTT with the material of different concns and cell co-cultivation.Mixture adds 200 μ L dmso solution MTT Jia Za crystallizations 10 minutes 37 ℃ of continuation effects 4 hours.Test the absorption in every hole then with ELIASA, the test wavelength is selected 492nm for use.Cell survival rate is pressed formula and is calculated:
Cell survival rate (%)=(A
Sample/ A
Control) * 100
A
SampleBe the absorption 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, and Fig. 2 is polyamino acid segmented copolymer, PEI25k and the transfection reagent Lipofectamine as the siRNA carrier of embodiment 9,15,20 preparations
TMThe toxotest of 200 pairs of HeLa cells is figure as a result.
Among Fig. 2, curve A is transfection reagent Lipofectamine
TMThe toxotest result of 2000 pair cells; Curve B is the toxotest result of PEI25k pair cell; Curve C is the toxotest result of PLAA-g-PEI3 pair cell; Curve D is the toxotest result of PLAA-g-PEI9 pair cell; Curve E is the toxotest result of PLAA-g-PEI14 pair cell.Can know that by Fig. 2 the polyamino acid segmented copolymer that is used as the siRNA carrier of embodiment of the invention preparation is significantly less than PEI25k and Lipofectamine for the toxicity of HeLa cell
TM2000.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle 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 claim of the present invention.
To the above-mentioned explanation of the disclosed embodiments, make this area professional and technical personnel can realize or use the present invention.Multiple modification to these embodiment will be conspicuous concerning those skilled in the art, and defined General Principle can realize under the situation that does not break away from the spirit or scope of the present invention in other embodiments among this paper.Therefore, the present invention will can not be restricted to these embodiment shown in this paper, but will meet and principle disclosed herein and features of novelty the wideest corresponding to scope.
Claims (10)
- Shown in the formula (I) as the polyamino acid segmented copolymer of siRNA carrier,
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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 1Be 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。 - 2. preparation method as the polyamino acid segmented copolymer of siRNA carrier may further comprise the steps:(A) under protection of inert gas, β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and cladodification polymine react in organic solvent, obtain intermediate product;(B) intermediate product and the polymine that step (A) are obtained react under catalyst action, obtain the polyamino acid segmented copolymer as the siRNA carrier;Said polyamino acid segmented copolymer as the siRNA carrier is suc as formula shown in (I):
R is selected from shown in formula (11) ~ formula (14) a kind of in the structure:
The NH-of said R links to each other with C atom in the 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 1Be 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。 - 3. preparation method according to claim 2 is characterized in that, in the said step (A), said β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and cladodification polymine mole proportioning are 100～10000:1～10.
- 4. preparation method according to claim 2 is characterized in that, in the said step (A), the mol ratio of said β-benzyl-L-aspartic acid-N-carboxylic acid anhydride and organic solvent is 5～10:50～1000.
- 5. preparation method according to claim 4 is characterized in that, in the said step (A), said organic solvent is chloroform, methylene dichloride or N ', N '-N.
- 6. preparation method according to claim 2 is characterized in that, in the said step (B), said catalyzer is a 2 hydroxy pyrimidine.
- 7. preparation method according to claim 2 is characterized in that, in the said step (B), the mol ratio of said intermediate product and polymine is 1～10:6～600.
- 8. preparation method according to claim 2 is characterized in that, in the said step (B), the time of said reaction is 10～80 hours.
- 9. preparation method according to claim 2 is characterized in that, in the said step (B), the temperature of said reaction is 15～50 ℃.
- 10. a composite particles comprises claim 1 described polyamino acid segmented copolymer and siRNA as the siRNA carrier, and said siRNA is the LucsiRNA of reticent luciferase.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104072766A (en) * | 2014-07-09 | 2014-10-01 | 中国科学院长春应用化学研究所 | Carrier for loading medicine and gene, medicine-gene carrier system and preparation method of system |
| CN104072766B (en) * | 2014-07-09 | 2016-08-24 | 中国科学院长春应用化学研究所 | A kind of for carrying medicament with the carrier of gene, medicine-gene vector system and preparation method thereof |
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