CN103951763B - O-alkyl carbamate chitosan, transgenic compound particles and preparation method thereof - Google Patents
O-alkyl carbamate chitosan, transgenic compound particles and preparation method thereof Download PDFInfo
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Abstract
The preparation method that the invention discloses a kind of O-alkyl carbamate chitosan, and disclose a kind of by its ternary transgenic compound particles as carrier。The present invention reacts preparation O-alkyl carbamate chitosan by the coupling of the phthalimide of amino of chitosan, chitosan and alkylamine and the deprotection etc. of amino of chitosan, it is achieved that to chitosan repetitive C6The selective modification of position hydroxyl, remains chitosan simultaneously and has the free amino group of bioactive functions, and with C8-C20Straight chain alkyl amine be alkyl donor, obtain the alkyl carbamate chitosan that acid-sensitive amino-formate bond is connecting key, for ternary transgenic compound particles prepared by carrier, there is cytotoxicity with it low, the transfection feature that transfection efficiency is high, has broad application prospects in field of gene。
Description
Technical field
The present invention relates to the carrier of a kind of transgenic compound particles and a kind of ternary transgenic compound particles, specifically, relate to a kind of lipid-polymer-DNA ternary transgenic compound particles, and preparation method thereof, belong to preparation method and the technology of the new and effective non-viral gene vector of field of gene。
Background technology
Gene therapy, by being imported to by exogenous gene to repair the deleterious gene causing that the dcc gene of disease or suppression cause disease in target cell core, so that body recovery normal function, reaches the purpose for the treatment of disease。The foundation of molecular biological development and human gene bank has promoted the clinical practice of gene therapy so that it is not only makes the most of the advantage in the treatment field such as heritability congenital disorders and malignant tumor, and is increasingly widely used in the treatment of various universality disease。Gene therapy it is critical only that exploitation safe and efficient gene delivery vector [1. biotechnology circular, 2010,2,38-40, gene therapy import carrier progress]。
Gene delivery vector is divided into virus and non-virus carrier two class。In recent years, non-virus carrier is rapidly developed owing to immunogenicity is low, carry gene ability strong, be prone to the advantages such as batch production。Non-viral gene vector mainly includes liposome and cationic polymer such as chitosan, polymine etc.。Chitosan is a kind of natural cationic alkaline polysaccharide, there is the advantages such as good biological degradability, biocompatibility, low cytotoxicity, its entrained amino cation can form Nano microsphere, one of popular research contents just becoming non-viral gene vector by compound DNA。But chitosan transfection efficiency low [2.J.ControlledRelease, 2001,70,399-421.Chitosan-DNAnanoparticlesasgenecarriers: synthesis, characterizationandtransfectionefficiency]。In order to improve the efficiency gene transfection of chitosan, research worker has carried out substantial amounts of chitin modified modification and has studied [3.Prog.Polym.Sci., 2007.32,726-753.Chemicalmodificationofchitosanasagenecarrierinvi troandinvivo]。Wherein N-alkylated chitosan non-viral gene vector has that hydrophilic and hydrophobic is adjustable, cytotoxicity is low, transfection efficiency advantages of higher [4. Chinese invention patent, patent publication No. CN1386548A, 20021225, the preparation method of transgenic compound particles of alkylated chitosan]。
The alkyl-modified direct modification amino of chitosan of chitosan of bibliographical information, and amino of chitosan is to provide the most important functional group of its bioactive functions, and bibliographical information alkyl modified chitosan is tightr to the compression of DNA, it is unfavorable for that complex enters the release [5.BioconjugateChem.2003 of DNA after cell, 14,782-789, N-AlkylatedChitosanasaPotentialNonviralVectorforGeneTran sfection.]。From the finding of current document, not yet there is any report to be not take up amino of chitosan and chitosan carries out entering the alkyl-modified modification of easy fracture after cell, and it is combined into transgenic compound particles uses。
Summary of the invention
For solving the alkyl-modified direct modification amino of chitosan of chitosan existed in prior art, the problem taking its biological activity important group, the preparation method that a kind of O-alkyl carbamate chitosan is provided, first adopt phthalimide by the amido protecting of chitosan, carry out alkyl-modified again, finally carry out deaminizating protection, prepare a kind of O-alkyl carbamate chitosan gene vector, the present invention also provides for a kind of ternary transgenic compound particles and preparation method thereof, the O-alkyl carbamate chitosan of the present invention is the non-viral gene vector of a kind of high-efficiency low-toxicity。
The technical purpose of the present invention is achieved through the following technical solutions:
The preparation method of a kind of O-alkyl carbamate chitosan, comprises the following steps:
1. the phthalimide of amino of chitosan: chitosan is generated phthalimide with phthalic anhydride；
2. the coupling of chitosan and alkylamine: phthalimide step 1. obtained is distributed in dry N-methylpyrrolidone, add coupling agent, the mol ratio of coupling agent and chitosan repetitive is 0.1-2:1, described coupling agent one in carbonyl dimidazoles, phosgene and dimethyl carbonate, in 30-80 DEG C of stirring reaction 1-5h under normal pressure, nitrogen atmosphere, add C8-C20Straight chain alkyl amine, be 0.1-2:1 with the mol ratio of chitosan repetitive, in 30-80 DEG C of stirring reaction 1h-5h under normal pressure, nitrogen atmosphere, obtain reactant liquor；
3. the deprotection of amino in reactant: add dehydrated alcohol, sucking filtration in step reactant liquor 2., collect precipitation; add the mixed liquor of hydrazine hydrate solution and methanol, backflow, slough phthalyl group; collect product, be purified by surname extraction, obtain finished product then through vacuum drying。
Preparation method of the present invention, step chitosan preferable weight-average molecular weight 1. is 2-10 ten thousand, and deacetylation is 65%-95%。
Preparation method of the present invention, described C8-C20Straight chain alkyl amine refer to C8-C20Linear paraffin in be positioned at any one H at two ends compound replaced by amido, it is preferable that from lauryl amine, C8Straight chain alkyl amine, C10Straight chain alkyl amine, C14Straight chain alkyl amine, C16Straight chain alkyl amine and C18One in straight chain alkyl amine。
Preparation method of the present invention, step 1. in realize the protection to amino by the amino of chitosan with the reaction of phthalic anhydride generation phthalimideization。Solvent used in reaction is DMF and water mixed liquid, and in solvent, the mass fraction of water is 1%-10%。Before reaction, first chitosan is dispersed in DMF/aqueous solvent, swelling 24h-72h。The mol ratio of phthalic anhydride and chitosan repetitive is 1-6:1, reacts 3-10h in 80 DEG C-150 DEG C under normal pressure, nitrogen atmosphere, and wherein reaction temperature is more preferably 90 DEG C-140 DEG C；Reactant liquor is poured in frozen water, sucking filtration after terminating by reaction, collects precipitation。
Step 2. in selected coupling agent be carbonylation agent, be very easily hydrolyzed, so each step of O-alkyl carbamateization reaction must carry out in water-less environment。
Step be 3. by step 2. in product in amino slough the process of phthalimide, the addition of dehydrated alcohol is 1-10 times of the reactant liquor volume that 2. step obtains, in the mixed liquor of hydrazine hydrate solution and methanol, hydrazine hydrate solution and methanol volume ratio are 1-3:1's, described hydrazine hydrate solution is the aqueous solution of concentration 20%-80%, being more preferably 40～80%, reaction condition is 30-80 DEG C of backflow 2-20h。It is dehydrated alcohol that described surname extraction is purified the solvent of use, and extraction time is 12-36h。
Adopt the O-alkyl carbamate chitosan prepared by method made above。
Another technical purpose of the present invention is in that to provide the liposome-O-alkyl carbamate chitosan-DNA ternary transgenic compound particles prepared by described O-alkyl carbamate chitosan, described ternary transgenic compound particles includes plasmid DNA, O-alkyl carbamate chitosan and liposome, and the mass ratio of three is 1:1-10:1-10。
Ternary transgenic compound particles of the present invention, described plasmid DNA one in PGL3 plasmid and PGFP-N2 plasmid。Described liposome one in transfection reagent DOTAP, Invitrogen company of Roche company transfection reagent lipofectin and lipofectamineTM。
Another technical purpose of the present invention is in that the preparation method providing above-mentioned ternary transgenic compound particles, comprise the following steps: O-alkyl carbamate chitosan is dissolved in the phosphate buffer that pH is 3-8, add plasmid DNA aqueous solution, room temperature stands self assembly and hatches, add liposome aqueous solution mixing, room temperature stands self assembly and hatches, and the mass ratio that plasmid DNA, O-alkyl carbamate chitosan and liposome add is 1:1-10:1-10。
The preparation method of ternary transgenic compound particles of the present invention, it is preferable that described O-alkyl carbamate chitosan mass concentration in phosphate buffer is 0.1-2mg·mL-1, the mass concentration of described plasmid DNA aqueous solution is 0.1-1mg mL-1, the mass concentration of described liposome aqueous solution is 0.3-3mg mL-1；The time that twice self assembly is hatched is both preferably 30min。
The present invention passes through the protection to amino of chitosan and deprotection, it is achieved that to chitosan repetitive C6Remain chitosan while the selective modification of position hydroxyl and there is the free amino group of bioactive functions。Adopt the one in carbonyl dimidazoles, phosgene or dimethyl carbonate as coupling agent, with C6-C20Straight chain alkyl amine be alkyl donor, obtain the alkyl carbamate chitosan that acid-sensitive amino-formate bond is connecting key。The present invention is also with it for carrier, lipid-polymer-DNA ternary transgenic compound particles is prepared with liposome and plasmid DNA self assembly compound in aqueous by O-alkyl carbamate chitosan, described ternary transgenic compound particles cytotoxicity is low, transfection efficiency is high, has the great potential of clinical practice。
The invention have the advantages that
1. the present invention passes through the protection to amino of chitosan and deprotection, it is achieved that to chitosan repetitive C6The selective modification of position hydroxyl, remains chitosan simultaneously and has the free amino group of bioactive functions, is conducive to this non-viral gene vector to the protection of DNA and delivery。
2. the one in carbonyl dimidazoles, phosgene, dimethyl carbonate coupling reagent is adopted, with C6-C20Straight chain alkyl amine be alkyl donor, obtain the alkyl carbamate chitosan that acid-sensitive amino-formate bond is connecting key, make this carrier delivery DNA enter after cell and be expected to quick released dna。
3. transgenic compound particles is obtained with liposome and plasmid DNA self assembly compound in aqueous by O alkyl carbamate chitosan, the advantage that this ternary transgenic compound has both O-alkyl carbamate chitosan and liposomal delivery gene simultaneously, there is cytotoxicity low, the transfection feature that transfection efficiency is high, has broad application prospects in field of gene。
Accompanying drawing explanation
Fig. 1. the transfection efficiency figure of the comparative example product of embodiment 1～6 and correspondence thereof；
Fig. 2. the cell survival rate figure of the comparative example product of embodiment 1～6 and correspondence thereof。
Detailed description of the invention
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way。
Embodiment 1
1. the phthalimide of amino of chitosan: chitosan (Mw=4 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 36h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 130 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 3 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 4h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 24h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 1:6:1。
Embodiment 2
Except step 4. in by O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add mass ratio change 2:6:1 into, other are with embodiment 1。
Embodiment 3
Except step 4. in by O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add mass ratio change 4:6:1 into, other are with embodiment 1。
Embodiment 4
Except step 4. in by O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add mass ratio change 6:6:1 into, other are with embodiment 1。
Embodiment 5
Except step 4. in by O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add mass ratio change 8:6:1 into, other are with embodiment 1。
Embodiment 6
Except step 4. in by O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add mass ratio change 10:6:1 into, other are with embodiment 1。
Embodiment 7
1. the phthalimide of amino of chitosan: chitosan (Mw=5 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 36h。Add the phthalic anhydride that amount of substance is chitosan repetitive 5 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 2h, add the lauryl amine that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 2h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 3 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 24h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, wherein the mass ratio of O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid is 10:6:1。
Embodiment 8
1. the phthalimide of polysaccharide amino: chitosan (Mw=10 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 150 DEG C of reaction temperatures, stirring reaction 7h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 80%; at 80 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 8:6:1。
Embodiment 9
1. the phthalimide of amino of chitosan: chitosan (Mw=2 ten thousand) is dispersed in DMF solvent (mass fraction of water is 5%), swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 5 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 10h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 3:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 12h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 8:6:1。
Embodiment 10
1. the phthalimide of polysaccharide amino: chitosan (Mw=10 ten thousand) is dispersed in DMF/water (mass fraction of water is 10%) solvent, swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 5 times, under normal pressure, nitrogen atmosphere and 150 DEG C of reaction temperatures, stirring reaction 3h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 80 DEG C of 3h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 6:6:1。
Embodiment 11
1. the phthalimide of polysaccharide amino: chitosan (Mw=3 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 24h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 6h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 2 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 4h, adds the C that amount of substance is chitosan repetitive 1 times14Straight chain alkyl amine, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 2h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 24h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 8:6:1。
Embodiment 12
1. the phthalimide of polysaccharide amino: chitosan (Mw=5 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the carbonyl dimidazoles that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, adds the C that amount of substance is chitosan repetitive 0.5 times16Straight chain alkyl amine, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 60%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 8:6:1。
Embodiment 13
Except step 2. in C18Straight chain alkyl amine replaces C16Outside straight chain alkyl amine, other are with embodiment 12。
Embodiment 14
Except step 2. in C20Alkanamine replaces C16Outside straight chain alkyl amine, other are with embodiment 12。
Embodiment 15
Except step 2. in C10Straight chain alkyl amine replaces C16Straight chain alkyl amine, carbonyl dimidazoles and C10The addition of straight chain alkyl amine becomes outside 1 times of chitosan repetitive, and other are with embodiment 12。
Embodiment 16
Except step 2. in C8Straight chain alkyl amine replaces C16Straight chain alkyl amine, carbonyl dimidazoles and C8The addition of straight chain alkyl amine becomes outside 1 times of chitosan repetitive, and other are with embodiment 12。
Embodiment 17
Except step 2. in dimethyl carbonate replace carbonyl dimidazoles, other are with embodiment 12。
Embodiment 18
1. the phthalimide of polysaccharide amino: chitosan (Mw=4 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 36h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 130 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the dimethyl carbonate that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 3 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 4h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 24h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 8:6:1。
Embodiment 19
1. the phthalimide of polysaccharide amino: chitosan (Mw=5 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 36h。Add the phthalic anhydride that amount of substance is chitosan repetitive 5 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the dimethyl carbonate that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 2h, add the lauryl amine that amount of substance is chitosan repetitive 0.5 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 2h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 3 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 10:6:1。
Embodiment 20
1. the phthalimide of polysaccharide amino: chitosan (Mw=10 ten thousand) is dispersed in DMF/water (mass fraction of water is 10%) solvent, swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 5 times, under normal pressure, nitrogen atmosphere and 150 DEG C of reaction temperatures, stirring reaction 3h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the dimethyl carbonate that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, add the lauryl amine that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 50%; at 80 DEG C of 3h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 10:6:1。
Embodiment 21
Except step 2. in dimethyl carbonate replace carbonyl dimidazoles, with C18Straight chain alkyl amine replaces C16Outside straight chain alkyl amine, other are with embodiment 12。
Embodiment 22
Except step 2. in dimethyl carbonate replace carbonyl dimidazoles, with C10Straight chain alkyl amine replaces C16Straight chain alkyl amine, dimethyl carbonate and C10The addition of straight chain alkyl amine becomes outside 1 times of chitosan repetitive, and other are with embodiment 12。
Embodiment 23
1. the phthalimide of polysaccharide amino: chitosan (Mw=5 ten thousand) is dispersed in DMF/water (mass fraction of water is 5%) solvent, swelling 72h。Add the phthalic anhydride that amount of substance is chitosan repetitive 3 times, under normal pressure, nitrogen atmosphere and 110 DEG C of reaction temperatures, stirring reaction 5h, make amino of chitosan phthalimide。After reaction terminates, reactant liquor is poured in frozen water, sucking filtration, collects precipitation。
2. the coupling of chitosan and alkylamine: step precipitation 1. is distributed in dry N-methylpyrrolidone, add the dimethyl carbonate that amount of substance is chitosan repetitive 1 times, under normal pressure, nitrogen atmosphere and 50 DEG C of reaction temperatures, stirring reaction 3h, adds the C that amount of substance is chitosan repetitive 1 times8Straight chain alkyl amine, under normal pressure, nitrogen atmosphere and 40 DEG C of reaction temperatures, stirring reaction 3h, obtain reactant liquor。
3. the deprotection of amino in reactant: add the dehydrated alcohol of 2 times of volumes, sucking filtration in step reactant liquor 2., collect precipitation。Precipitation is transferred in the mixed solution that hydrazine hydrate solution is 1:1 with methanol volume ratio; wherein the concentration of hydrazine hydrate solution is 60%; at 70 DEG C of 2h that reflux; slough phthalyl group; obtain the O-thick product of alkyl carbamate chitosan, carry out surname extraction 36h, purification with dehydrated alcohol; vacuum drying, obtains O-alkyl carbamate chitosan finished product。
4. the preparation of ternary transgenic compound particles: the O-alkyl carbamate chitosan of above-mentioned preparation is dissolved in the phosphate buffer that pH is 5.5, obtain the O-alkyl carbamate chitosan aqueous solution that mass concentration is 1mg/mL, the PGFP-N2 plasmid solution of above solution, 0.5mg/mL is mixed, incubated at room self assembly 30min, add the liposome DOTAP aqueous solution of 1mg/mL, incubated at room self assembly 30min, the mass ratio that wherein O-alkyl carbamate chitosan, liposome DOTAP and PGFP-N2 plasmid add is 10:6:1。
Embodiment 24
Except step 2. in phosgene replace carbonyl dimidazoles, other are with embodiment 23。
Comparative example 1
A () is with unmodified chitosan for carrier, prepare chitosan transgenic compound particles: chitosan is dissolved in the phosphate buffer that pH is 5.5, obtain the chitosan aqueous solution that mass concentration is 1mg/mL, add PGFP-N2 plasmid DNA (mass concentration is 0.5mg/mL), incubated at room self assembly 30min, obtain chitosan transgenic compound particles, the mass ratio of chitosan and PGFP-N2 plasmid DNA respectively 1:1,2:1,4:1,6:1,8:1 and 10:1, namely distinguish corresponding embodiment 1～6, obtain transgenic compound particles as a comparison。
Comparative example 2
B () prepares liposome transgenic compound particles with liposome for carrier: by the liposome aqueous solution of 1mg/mL, add PGFP-N2 plasmid DNA (mass concentration is 0.5mg/mL), incubated at room self assembly 30min, obtaining liposome transgenic compound particles, it is 6:1 that liposome and PGFP-N2 plasmid DNA add mass ratio。
Comparative example 3
C () prepares liposome-chitosan-DNA ternary transgenic compound particles with unmodified chitosan liposome for carrier: chitosan is dissolved in the phosphate buffer that pH is 5.5, obtain the chitosan aqueous solution that mass concentration is 1mg/mL, add PGFP-N2 plasmid DNA (mass concentration is 0.5mg/mL), incubated at room self assembly 30min, add 1mg/mL liposome aqueous solution, incubated at room self assembly 30min, obtain liposome-chitosan-DNA ternary transgenic compound particles, chitosan, the mass ratio of liposome and PGFP-N2 plasmid DNA respectively 1:6:1, 2:6:1, 4:6:1, 6:6:1, 8:6:1 and 10:6:1, namely corresponding embodiment 1～6 is distinguished, obtain transgenic compound particles as a comparison。
Ternary transgenic compound particles prepared by the present invention and the performance measurement of the transgenic compound particles of comparative example:
(1) mensuration of the cell transfecting efficiency of transgenic compound particles
Transgenic compound particles transfection efficiency of cells in vitro adopts PGL3 albumen to be reporter gene, is evaluated in Hep-2 cell。With 5 × 104Hep-2 cell is inoculated in 24 orifice plates by the density of cells/well, at 37 DEG C, and 5%CO280% is reached to cell fusion degree after hatching 18～24h under condition。2h before transfection, sucks complete medium, washes twice with PBS, adds after the transgenic compound particles of 400 μ L serum-free mediums and preparation hatches 5h, changes serum-free medium into complete medium。After 48h, the multi-functional microplate reader of BioTekSynergy2 detects the intensity of photon。The concentration of total protein is detected with BCA, thus by result unified standard chemical conversion RLU/mg albumen (the relative number of photons corresponding to every milligram of albumen)。
(2) the Cytotoxic mensuration of transgenic compound particles。
The Cytotoxic mensuration of transgenic compound particles adopts mtt assay to be evaluated。With 1 × 104Hep-2 cell is inoculated in 96 orifice plates by the density of cells/well, at 37 DEG C, and 5%CO2The 20 μ L transgenic compound particles prepared it is separately added into after hatching 24h under condition。After cultivating 24h, every hole adds MTT solution (5mg/mL) 20 μ L, and 37 DEG C are continued to hatch 4h, terminates cultivating。In careful absorption hole after culture supernatant, every hole adds 150 μ LDMSO, continues to hatch 30min at 37 DEG C。Select 570nm wavelength, SUNRISE microplate reader measures each hole light absorption value, automatic mixing 600s before detection。The expression of results of cytoactive is: cell survival rate (%)=A570 (sample)/A570 (control) × 100, the wherein light absorption value in the hole that A570 (sample) adds for compound particles, A570 (control) is the light absorption value in the hole containing only culture medium。
Undertaken carrying out transfection efficiency and Cytotoxic evaluation with O-alkyl carbamate chitosan transgenic compound particles (d) of the present invention product accordingly by comparative example three kinds of compound particles a, b, c, carry out Comparative result。Transfection efficiency result is as shown in Figure 1, in figure, vertical coordinate is the relative number of photons corresponding to every milligram of albumen, abscissa is each product a, b, c in the product d of the present invention and the comparative example 1～3 of correspondence thereof, abscissa such as, marks four products of embodiment 1, d is the product of embodiment 1, and a represents the product of 1:1 corresponding with embodiment 1 in comparative example 1, and b represents the product of comparative example 2, c represents the product of 1:6:1 corresponding with embodiment 1 in comparative example 3, the like。Cytotoxicity is as in figure 2 it is shown, its vertical coordinate represents cell survival rate, abscissa and same meaning in Fig. 1。
Results of comparison shows, compared with chitosan complexes microgranule, liposome complex microgranule, liposome-chitosan-DNA ternary transgenic compound particles, the O-alkyl carbamate chitosan transgenic compound particles of the present invention has the highest transfection efficiency and relatively low cytotoxicity, has huge application potential in field of gene。
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within protection scope of the present invention。
Claims (8)
1. a preparation method for O-alkyl carbamate chitosan, comprises the following steps:
1. the phthalimide of amino of chitosan: chitosan is generated phthalimide with phthalic anhydride；
2. the coupling of chitosan and alkylamine: phthalimide step 1. obtained is distributed in dry N-methylpyrrolidone, add coupling agent, the mol ratio of coupling agent and chitosan repetitive is 0.1-2:1, described coupling agent one in carbonyl dimidazoles, phosgene and dimethyl carbonate, in 30-80 DEG C of stirring reaction 1-5h under normal pressure, nitrogen atmosphere, add C8-C20Straight chain alkyl amine, be 0.1-2:1 with the mol ratio of chitosan repetitive, in 30-80 DEG C of stirring reaction 1h-5h under normal pressure, nitrogen atmosphere, obtain reactant liquor；
3. the deprotection of amino in reactant: add dehydrated alcohol, sucking filtration in step reactant liquor 2., collect precipitation; add the mixed liquor of hydrazine hydrate solution and methanol, backflow, slough phthalyl group; collect product, be purified by surname extraction, obtain finished product then through vacuum drying；
The weight average molecular weight of described chitosan is 2-10 ten thousand, and deacetylation is 65%-95%。
2. the preparation method of O-alkyl carbamate chitosan according to claim 1, it is characterised in that described straight chain alkyl amine is selected from lauryl amine, C8Straight chain alkyl amine, C10Straight chain alkyl amine, C14Straight chain alkyl amine, C16Straight chain alkyl amine and C18One in straight chain alkyl amine。
3. the O-alkyl carbamate chitosan that prepared by the preparation method described in claim 1～2 any one claim。
4. liposome-O-alkyl carbamate chitosan-DNA ternary the transgenic compound particles that prepared by the O-alkyl carbamate chitosan described in claim 3, it is characterized in that described ternary transgenic compound particles includes plasmid DNA, O-alkyl carbamate chitosan and liposome, the mass ratio of three is 1:1-10:1-10。
5. the preparation method of the ternary transgenic compound particles described in claim 4, comprise the following steps: O-alkyl carbamate chitosan is dissolved in the phosphate buffer that pH is 3-8, add plasmid DNA aqueous solution, room temperature stands self assembly and hatches, add liposome aqueous solution mixing, room temperature stands self assembly and hatches, and the mass ratio that plasmid DNA, O-alkyl carbamate chitosan and liposome add is 1:1-10:1-10。
6. preparation method according to claim 5, it is characterised in that described O-alkyl carbamate chitosan mass concentration in phosphate buffer is 0.1-2mg mL-1。
7. preparation method according to claim 5, it is characterised in that the mass concentration of described plasmid DNA aqueous solution is 0.1-1mg mL-1。
8. preparation method according to claim 5, it is characterised in that the mass concentration of described liposome aqueous solution is 0.3-3mg mL-1。
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