CN104840975A - Supermolecular assembly for small interfering RNA targeting delivery and preparation method - Google Patents
Supermolecular assembly for small interfering RNA targeting delivery and preparation method Download PDFInfo
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Abstract
The invention relates to a supermolecular assembly for small interfering RNA targeting delivery. The supermolecular assembly is a four-element supermolecular assembly synthesized on the basis of beta-cyclodextrin modified hyaluronic acid, an adamantine polyamine compound, cucurbituril [6] and small interfering RNA. The four-element supermolecular assembly is based on the strong non-covalent interaction between beta-cyclodextrin and adamantine and between adamantine polyamine chain and cucurbituril [6], and the electrostatic interaction between the adamantine polyamine chain and small interfering RNA, and forms supermolecular nanoparticles with hydrophilic hyaluronic acid as the shell. The supermolecular assembly provided by the invention has the advantages of simple synthesis and construction route, low production cost and high yield, and is suitable for amplified synthesis and actual production application. Through the endocytosis of a malignant tumor cell surface overexpressed hyaluronic acid receptor as a medium, the four-element supermolecular assembly can be brought into cancer cells targetedly, thus realizing effective silencing to exogenous gene expression and significantly reducing the toxic and side effect at the same time.
Description
[technical field]
The present invention relates to target gene delivery technical field, particularly a kind of super-molecule assembling body of targeted delivery siRNA and preparation method.
[background technology]
Society is due to the impact of the factors such as haze, environmental pollution, ionizing radiation, food safety and heredity, and the sickness rate of cancer is rising year by year, and the health of the mankind has been arrived in serious threat.For this reason, operative treatment, chemotherapy and radiotherapy are able to extensive use as the Main Means of clinical anticancer.But, these therapies due to not good, the easy recurrence of therapeutic effect and serious toxic and side effects etc. make cancer so far cannot capture by the mankind.Therefore, chemist, material scholar and biomedical worker develop various drug delivery system as bio-compatibility and the targeting in order to strengthen drug delivery system such as liposome, inorganic nano-particle, vesicle and carbon nanomaterial, reduces toxic and side effects to therapeutic effect to be reinforced.But these therapies are all cured the symptoms, not the disease, want thoroughly to effect a radical cure cancer, will be contained it from the source of pathogenesis of cancer.The gene therapy occurred in recent years opens a fan gate for capturing cancer.Its principle be by there is biological function nucleic acid (as plasmid and linear dsdna; or siRNA (siRNA) etc.) shift or be transported in cancerous cell; repair the expression of aberrant gene in the gene of damage or T suppression cell; then induce the cancerous cell generation apoptosis of not apoptosis, thus reach the object of curing cancer.But the poor efficiency of gene transfection becomes the bottleneck of restriction the method extensive use.Due to the cost of potential safety issue and costliness, viral vector is not developed widely, therefore designs and has bio-compatibility and controllable release function and the target gene delivery system possessing high efficiency gene transfection becomes the study hotspot in forward position with constructing.In addition, noncovalent interaction carrier, due to advantages such as its excellent slow release effect, stronger binding ability and lower toxic and side effects, the medicine/gene carrier recently occurred widely is applied.
[summary of the invention]
The object of the invention is for above-mentioned technical Analysis and existing problems, a kind of super-molecule assembling body and preparation method of targeted delivery siRNA are provided, this super-molecule assembling body can the expression of specific endogenous and exogenous gene in effective reticent cancerous cell, and its toxic and side effects is very low, preparation method is simple and productive rate is higher, is suitable for amplifying synthesis and production application.
Technical scheme of the present invention:
A kind of super-molecule assembling body of targeted delivery siRNA, for the quaternary super-molecule assembling body synthesized based on beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds, Cucurbituril [6] and siRNA, wherein beta-cyclodextrin modified hyaluronan molecule formula is (C
14h
21nO
11)
98(C
58h
95n
3o
44)
17, an average macromolecular chain modifies 17 cyclodextrin units, and diamantane (obsolete) polyamine compounds chemical molecular formula is C
32h
56br
4n
6o
4; This quaternary super-molecule assembling body is with beta-schardinger dextrin-and diamantane (obsolete) and noncovalent interaction strong between diamantane (obsolete) polyamines chain and Cucurbituril [6], and between diamantane (obsolete) polyamines chain and siRNA based on electrostatic interaction, form the supermolecule nano particle being shell with hydrophilic hyaluronic acid, nano particle diameter is 30-40nm.
A preparation method for the super-molecule assembling body of described targeted delivery siRNA, step is as follows:
(1) the hyaluronic preparation of beta-cyclodextrin modified
By molecular weight be 46000 hyaluronate sodium be dissolved in that pH is 7.2, concentration is in the phosphate buffer solution of 1mmol/L, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy thiosuccinimide again, stirring reaction 30 minutes, obtains reactant liquor; It is in the phosphate buffer solution of 7.2 that 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-is dissolved in pH, the amount ratio of 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-and phosphate buffer solution is 0.1mol/L, join in above-mentioned reactant liquor again, stir 24 hours under 25 DEG C of conditions, then loading the scope that retains is dialyse 5 days continuously in the bag filter of 8000-14000, by the lyophilizing of gained solution, obtained beta-cyclodextrin modified hyaluronic acid;
(2) preparation of diamantane (obsolete) polyamine compounds
1) by two azido-methyl oxybenzene compounds (compound 1), diamantane (obsolete) bromomethyl ketone and Anhydrous potassium carbonate mix in acetone, be heated to 56 DEG C of return stirring reaction 10-12h, then filter, filtrate is spin-dried for, add chloroform that volume ratio is 1:1 and water mixed liquid extracts, organic facies is dry with anhydrous magnesium chloride, distilling under reduced pressure removes desolventizing and obtains crude product, then with petroleum ether and ethyl acetate mixtures for developing solvent, the volume ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is 8:1, be separated with 200-300 order silicagel column, the sterling of obtained two azido-methyl phenyl adamantane compounds (compound 2),
2) two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine are dissolved in anhydrous tetrahydro furan, stir 12 hours at ambient temperature, then distilling under reduced pressure is except desolventizing, gained solid is dissolved in ethyl acetate, adding concentration is obtain white precipitate after the hydrochloric acid of 36.5wt%, collecting precipitation also with ethyl acetate washing, obtains the sterling of diamidogen aminomethyl phenyl adamantane compound (compound 3);
3) by diamidogen aminomethyl phenyl adamantane compound (compound 3), Pentafluorophenol polyamine compounds (compound 4) and the triethylamine of benzyl chloroformate protection are dissolved in dichloromethane, stirred at ambient temperature reacts 20 hours, distilling under reduced pressure is except desolventizing, add dichloromethane that volume ratio is 1:1 and water mixed liquid extracts, organic facies anhydrous magnesium sulfate drying, then distilling under reduced pressure removes desolventizing and obtains crude product, with ethyl acetate and petroleum ether mixed liquor for developing solvent, the volumetric ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is followed successively by 1:2 and fades to 4:1 to fade to 6:1 be finally pure ethyl acetate, be separated with 200-300 order silicagel column, the sterling of the diamantane (obsolete) polyamine compounds (compound 5) of obtained benzyl chloroformate protection,
4) the diamantane (obsolete) polyamine compounds (compound 5) that benzyl chloroformate is protected is dissolved in the mixed solution of hydrobromic acid and acetic acid, hydrobromic acid is being 33wt% with the concentration in the mixed solution of acetic acid, stirring at room temperature reacts 10 hours, add excessive ether and produce white precipitate, centrifugal and carry out with ether washing the sterling that can obtain diamantane (obsolete) polyamine compounds;
(3) preparation of the super-molecule assembling body of targeted delivery siRNA
By beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds and Cucurbituril [6] mixing and soluble in water, carry out ultrasonic dissolution, then the aqueous solution that concentration is the siRNA of 20 μm of ol/L is added, 20 times are are repeatedly blown and beaten, the quaternary super-molecule assembling body of obtained targeted delivery siRNA with 200 μ L liquid-transfering guns.
In described step (1), the amount ratio of hyaluronate sodium and phosphate buffer solution is 0.073mmol/L, and the mol ratio of hyaluronate sodium, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxy thiosuccinimide and 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-is 0.0044:1.75:1.75:1.
1 of described step (2)) in the amount ratio of two azido-methyl oxybenzene compounds (compound 1) and acetone be 0.026mol/L, two azido-methyl oxybenzene compounds (compound 1) are 1:0.74:3 with the mol ratio of diamantane (obsolete) bromomethyl ketone and Anhydrous potassium carbonate.
2 of described step (2)) in the amount ratio of two azido-methyl phenyl adamantane compounds (compound 2) and anhydrous tetrahydro furan be 0.097mol/L, two azido-methyl phenyl adamantane compounds (compound 2) are 1:3 with the mol ratio of triphenylphosphine, and anhydrous tetrahydro furan and concentration are the volume ratio of the hydrochloric acid of 36.5wt% is 40:1.
3 of described step (2)) in the amount ratio of diamidogen aminomethyl phenyl adamantane compound (compound 3) and dichloromethane be 0.015mol/L, the mol ratio of the Pentafluorophenol polyamine compounds (compound 4) that diamidogen aminomethyl phenyl adamantane compound (compound 3) and benzyl chloroformate are protected and triethylamine is 1:2.5:4.
4 of described step (2)) in the amount ratio of mixed solution of diamantane (obsolete) polyamine compounds (compound 5) and hydrobromic acid-acetic acid of benzyl chloroformate protection be 0.04mol/L.
In described step (3), the amount ratio of beta-cyclodextrin modified hyaluronic acid and water is 2.0 × 10
-4mol/L, the mol ratio of beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds and Cucurbituril [6] is 1:17:34, and the volume ratio of beta-cyclodextrin modified hyaluronic acid/diamantane (obsolete) polyamine compounds/Cucurbituril [6] mixed solution and siRNA aqueous solution is 1:1.
Advantage of the present invention is: the quaternary super-molecule assembling body of this targeted delivery siRNA, Cucurbituril [6] makes the nitrogen-atoms pKa value of polyamines chain change with the bonding of polyamines chain, the electropositivity of polyamines chain is strengthened greatly, bonding can be carried out with electronegative siRNA consumingly, avoid siRNA due to hydrolysis and with RNA catabolic enzyme effect and decompose inactivation; In addition, the hyaluronic acid shell of this quaternary super-molecule assembling body can carry out specificity bonding action with the hyaluronic acid receptor of cancer cell surfaces overexpression, then quaternary super-molecule assembling body is entered in the middle of cancerous cell specifically by the endocytosis of cell, make quaternary assemble physical ability by the hydrolysis of the hyaluronidase of overexpression in cancerous cell again access effectively degraded thus siRNA is discharged, substantially increase the efficiency gene transfection of cell, make endogenous in cell and ectogenic specific gene obtain effective silence; This assembly targeting is good, and cellulotoxic side effect is very low, and transfection efficiency is higher, and preparation method more for convenience, the gene therapy of following cancer has good application prospect.
[accompanying drawing explanation]
Fig. 1 is the synthetic route chart of diamantane (obsolete) polyamine compounds.
Fig. 2 be this quaternary super-molecule assembling body construct route schematic diagram.
Fig. 3 is the transmission electron microscope figure of this quaternary super-molecule assembling body.
Fig. 4 is the electrophoretogram of this quaternary super-molecule assembling body and siRNA bonding.
Fig. 5 is the cytotoxicity experiment result of this quaternary super-molecule assembling body.
Fig. 6 is that this quaternary super-molecule assembling body is to cell exogenous green fluorescence protein gene silencing efficiency figure.
[detailed description of the invention]
Below by example, the present invention is described further:
Embodiment:
A kind of super-molecule assembling body of targeted delivery siRNA, for the quaternary super-molecule assembling body synthesized based on beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds, Cucurbituril [6] and siRNA, wherein beta-cyclodextrin modified hyaluronan molecule formula is (C
14h
21nO
11)
98(C
58h
95n
3o
44)
17, an average macromolecular chain modifies 17 cyclodextrin units, and diamantane (obsolete) polyamine compounds chemical molecular formula is C
32h
56br
4n
6o
4; This quaternary super-molecule assembling body is with beta-schardinger dextrin-and diamantane (obsolete) and noncovalent interaction strong between diamantane (obsolete) polyamines chain and Cucurbituril [6], and between diamantane (obsolete) polyamines chain and siRNA based on electrostatic interaction, form the supermolecule nano particle being shell with hydrophilic hyaluronic acid, nano particle diameter is 30-40nm.
A preparation method for the super-molecule assembling body of described targeted delivery siRNA, step is as follows:
(1) the hyaluronic preparation of beta-cyclodextrin modified
The hyaluronate sodium being 46000 by 100mg (0.0022mmol) molecular weight is dissolved in that 30mL pH is 7.2, concentration is in the phosphate buffer solution of 1mmol/L, add 167.7mg (0.875mmol) 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 190mg (0.875mmol) N-hydroxy thiosuccinimide again, stirring reaction 30 minutes, obtains reactant liquor; It is in the phosphate buffer solution of 7.2 that 588.5mg (0.5mmol) 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-is dissolved in 5mL pH, join in above-mentioned reactant liquor again, stir 24 hours under 25 DEG C of conditions, then loading the scope that retains is dialyse 5 days continuously in the bag filter of 8000-14000, by the lyophilizing of gained solution, obtained HACD (HACD).
The hyaluronic nuclear-magnetism of beta-cyclodextrin modified prepared by detection display the present invention characterizes as follows:
1hNMR (400MHz, D
2o, TMS, ppm): δ 1.97 (s, 3H, H of methyl group of HA), 2.84-3.93 (m, 16.24H, H of HA and C-3, C-5, C-6, C-2, C-4of β-CD), 4.45-4.50 (m, 2H, H of HA), 5.04-5.10 (m, 1.06H, H of C-1of β-CD).By beta-cyclodextrin modified hyaluronan molecule formula can be obtained to nuclear magnetic spectrogram integral and calculating for (C
14h
21nO
11)
98(C
58h
95n
3o
44)
17, an average macromolecular chain modifies 17 beta-schardinger dextrin-unit.
(2) preparation of diamantane (obsolete) polyamine compounds
1) by 265mg (1.3mmol) two azido-methyl oxybenzene compound (compound 1), 247mg (0.962mmol) diamantane (obsolete) bromomethyl ketone and 540mg (3.9mmol) Anhydrous potassium carbonate mix in 50mL acetone, be heated to 56 DEG C of return stirring reaction 12h, then filter, filtrate is spin-dried for, add 50mL chloroform and 50mL water extracts, organic facies is dry with anhydrous magnesium chloride, distilling under reduced pressure removes desolventizing and obtains crude product, then with petroleum ether and ethyl acetate mixtures for developing solvent, the volume ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is 8:1, carrying out with 200-300 order silicagel column the sterling that separation can obtain two azido-methyl phenyl adamantane compounds (compound 2) is colorless oil,
The nuclear-magnetism of two azido-methyl phenyl adamantane compounds (compound 2) prepared by detection display the present invention characterizes as follows:
1h NMR (400MHz, CDCl
3, ppm) and δ 6.87 (s, 1H), 6.79 (s, 2H), 4.89 (s, 2H), 4.33 (s, 4H), 2.10 (m, 3H), 1.94 (m, 6H), 1.78 (m, 6H).
2) 738mg (1.94mmol) two azido-methyl phenyl adamantane compound (compound 2) and 1563mg (5.96mmol) triphenylphosphine are dissolved in 20mL anhydrous tetrahydro furan, stir 12 hours at ambient temperature, then distilling under reduced pressure is except desolventizing, gained solid is dissolved in ethyl acetate, add 0.5mL, concentration is obtain white precipitate after the hydrochloric acid of 36.5wt%, collecting precipitation also with ethyl acetate washing, obtains the sterling of diamidogen aminomethyl phenyl adamantane compound (compound 3);
The nuclear-magnetism of diamidogen aminomethyl phenyl adamantane compound (compound 3) prepared by detection display the present invention characterizes as follows:
1h NMR (400MHz, D
2o, ppm) δ 7.00 (s, 1H), 6.92 (s, 2H), 5.09 (s, 2H), 4.05 (s, 4H), 1.94 (m, 3H), 1.80 (m, 6H), 1.61 (m, 6H).
3) by 120mg (0.3mmol) diamidogen aminomethyl phenyl adamantane compound (compound 3), Pentafluorophenol polyamine compounds (compound 4) and the 0.2mL triethylamine of the protection of 400mg (0.69mmol) benzyl chloroformate are dissolved in 20mL dichloromethane, stirred at ambient temperature reacts 20 hours, distilling under reduced pressure is except desolventizing, add 50mL dichloromethane and 50mL water extracts, organic facies anhydrous magnesium sulfate drying, then distilling under reduced pressure removes desolventizing and obtains crude product, with ethyl acetate and petroleum ether mixed liquor for developing solvent, the volume ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is followed successively by 1:2 and fades to 4:1 to fade to 6:1 be finally pure ethyl acetate, be separated with 200-300 order silicagel column, the sterling of the diamantane (obsolete) polyamine compounds (compound 5) of obtained benzyl chloroformate protection is white solid,
The nuclear-magnetism of the diamantane (obsolete) polyamine compounds (compound 5) of benzyl chloroformate protection prepared by detection display the present invention characterizes as follows:
1h NMR (400MHz, CDCl
3, ppm) and δ 7.32 (m, 20H), 6.62 (m, 3H), 5.11 (s, 4H), 5.05 (s, 4H), 4.80 (s, 2H), 4.31 (m, 4H), 3.90 (s, 4H) .3.33 (m, 4H), 3.10 (m, 4H), 2.05 (m, 3H), 1.88 (m, 6H), 1.72 (m, 6H), 1.52-1.42 (m, 8H).
4) the diamantane (obsolete) polyamine compounds (compound 5) that 211.5mg (0.2mmol) benzyl chloroformate is protected is dissolved in the mixed solution of 5mL hydrobromic acid and acetic acid, hydrobromic acid is being 33wt% with the concentration in the mixed solution of acetic acid, stirring at room temperature reacts 10 hours, add excessive ether and produce white precipitate, centrifugal and to carry out washing the sterling that can obtain diamantane (obsolete) polyamine compounds with ether be white solid;
The nuclear-magnetism of diamantane (obsolete) polyamine compounds prepared by detection display the present invention characterizes as follows:
1h NMR (400MHz, CDCl
3, ppm) and δ 6.66 (s, 1H), 6.51 (s, 2H), 4.94 (s, 2H), 4.15 (s, 4H), 3.69 (s, 4H), 2.88 (m, 4H), 2.78 (m, 4H) .1.82 (m, 3H), 1.68 (m, 6H), 1.54 (m, 14H).
(3) preparation of the super-molecule assembling body of targeted delivery siRNA
1.20mg beta-cyclodextrin modified hyaluronic acid, 0.29mg diamantane (obsolete) polyamine compounds and 0.64mg Cucurbituril [6] are mixed and is dissolved in 80 μ L water, carry out ultrasonic dissolution, then the aqueous solution that 80 μ L concentration are 20 μMs of siRNAs is added, 20 times are are repeatedly blown and beaten, the quaternary super-molecule assembling body of obtained targeted delivery siRNA with 200 μ L liquid-transfering guns.
Fig. 1 is the synthetic route chart of diamantane (obsolete) polyamine compounds.
Fig. 2 be this quaternary super-molecule assembling body construct route schematic diagram.
Fig. 3 is the transmission electron microscope figure of this quaternary super-molecule assembling body, characterized by transmission electron microscope and can draw the supermolecule nano particle that this quaternary super-molecule assembling body is formed based on electrostatic interaction between strong noncovalent interaction between cyclodextrin-diamantane (obsolete), positive charge polyamines-Cucurbituril [6] and positive charge polyamines-siRNA, nano particle diameter size is 30-40nm.
Fig. 4 is the electrophoretogram of this quaternary super-molecule assembling body and siRNA bonding.Compare simple siRNA (the 1st row) and siRNA+beta-cyclodextrin modified hyaluronic acid+diamantane (obsolete) polyamine compounds (the 2nd row) complex, adding of Cucurbituril [6] effectively can strengthen the binding ability (3-5 row) of super-molecule assembling body to siRNA, beta-cyclodextrin modified hyaluronic acid under last stoichiometric proportion+diamantane (obsolete) polyamine compounds+Cucurbituril [6] (beta-cyclodextrin modified hyaluronic acid, the mol ratio of diamantane (obsolete) polyamine compounds and Cucurbituril [6] is 1:17:34) to the binding ability of siRNA the strongest (the 6th row).
Embody rule effect of the present invention is as follows:
PC-3 cell (human prostate cancer cells) is layered in 96 orifice plates of the RPMI-1640 culture medium containing 10% hyclone and cultivates 24 hours, add 160 μ L respectively, 80 μ L, 40 μ L, diamantane (obsolete) polyamine compounds (a) of 20 μ L, diamantane (obsolete) polyamine compounds+Cucurbituril [6] (b), diamantane (obsolete) polyamine compounds+beta-cyclodextrin modified hyaluronic acid (c), and Cucurbituril [6]+diamantane (obsolete) polyamine compounds+beta-cyclodextrin modified hyaluronic acid (d), continuous culture 24 hours, the cells survival rate under each experiment condition is measured with mtt assay.Result as shown in Figure 5, in 24 hours window, diamantane (obsolete) polyamine compounds under each concentration has shown certain cytotoxicity because it has positively charged polyamines chain, and after adding beta-cyclodextrin modified hyaluronic acid, due to hyaluronic targeting, make that beta-cyclodextrin modified hyaluronic acid-diamantane (obsolete) polyamine compounds assembly is entered in cell by endocytosis in a large number, the cytotoxicity of diamantane (obsolete) polyamine compounds is enhanced.And adding of Cucurbituril [6] makes it be bonded on positive charge polyamines chain, greatly can reduce the cytotoxicity of assembly, cells survival rate is generally more than 90%.As can be seen here, the quaternary super-molecule assembling body that we construct has shown very low cytotoxicity under each concentration, and safety significantly promotes, and is conducive to the evaluation of gene delivery effect below.On the other hand, as shown in Figure 6, PC-3 cell is layered in 6 orifice plates of the RPMI-1640 culture medium containing 10% hyclone and cultivates 24 hours, cellular expression is made to go out green fluorescent protein with Lipofactamine 2000 and EGFP-pDNA process cell in advance, then the quaternary super-molecule assembling body (g-i) of Lipofacyamine 2000+EGFP-siRNA (d-f) and load EGFP-siRNA is added respectively, act on and change fresh culture into cell after 6 hours, cultivate 24 hours again, with the silencing efficiency of fluorescence microscope green fluorescence protein gene.As shown in fig. 6a-6c, PC-3 cell effective expression after proceeding to EGFP-pDNA has gone out green fluorescent protein, has then shown bright green fluorescence; Commercial reagents Lipofacyamine 2000 bonding EGFP-siRNA has shown the good silence efficiency to green fluorescent protein, and the transfection of EGFP-siRNA can effectively enter in PC-3 cell by the quaternary super-molecule assembling body of the load EGFP-siRNA that we prepare, fluorecyte quantity and cell fluorescence intensity are reduced greatly, show Gene silencing efficacy to green fluorescent protein more better than commercial reagents, there is good gene delivery application prospect.
Claims (8)
1. the super-molecule assembling body of a targeted delivery siRNA, it is characterized in that: be the quaternary super-molecule assembling body synthesized based on beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds, Cucurbituril [6] and siRNA, wherein beta-cyclodextrin modified hyaluronan molecule formula is (C
14h
21nO
11)
98(C
58h
95n
3o
44)
17, an average macromolecular chain modifies 17 cyclodextrin units, and diamantane (obsolete) polyamine compounds chemical molecular formula is C
32h
56br
4n
6o
4; This quaternary super-molecule assembling body is with beta-schardinger dextrin-and diamantane (obsolete) and noncovalent interaction strong between diamantane (obsolete) polyamines chain and Cucurbituril [6], and between diamantane (obsolete) polyamines chain and siRNA based on electrostatic interaction, form the supermolecule nano particle being shell with hydrophilic hyaluronic acid, nano particle diameter is 30-40nm.
2. a preparation method for the super-molecule assembling body of targeted delivery siRNA as claimed in claim 1, is characterized in that step is as follows:
(1) the hyaluronic preparation of beta-cyclodextrin modified
By molecular weight be 46000 hyaluronate sodium be dissolved in that pH is 7.2, concentration is in the phosphate buffer solution of 1mmol/L, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy thiosuccinimide again, stirring reaction 30 minutes, obtains reactant liquor; It is in the phosphate buffer solution of 7.2 that 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-is dissolved in pH, the amount ratio of 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-and phosphate buffer solution is 0.1mol/L, join in above-mentioned reactant liquor again, stir 24 hours under 25 DEG C of conditions, then loading the scope that retains is dialyse 5 days continuously in the bag filter of 8000-14000, by the lyophilizing of gained solution, obtained beta-cyclodextrin modified hyaluronic acid;
(2) preparation of diamantane (obsolete) polyamine compounds
1) by two azido-methyl oxybenzene compounds (compound 1), diamantane (obsolete) bromomethyl ketone and Anhydrous potassium carbonate mix in acetone, be heated to 56 DEG C of return stirring reaction 10-12h, then filter, filtrate is spin-dried for, add chloroform that volume ratio is 1:1 and water mixed liquid extracts, organic facies is dry with anhydrous magnesium chloride, distilling under reduced pressure removes desolventizing and obtains crude product, then with petroleum ether and ethyl acetate mixtures for developing solvent, the volume ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is 8:1, be separated with 200-300 order silicagel column, the sterling of obtained two azido-methyl phenyl adamantane compounds (compound 2),
2) two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine are dissolved in anhydrous tetrahydro furan, stir 12 hours at ambient temperature, then distilling under reduced pressure is except desolventizing, gained solid is dissolved in ethyl acetate, adding concentration is obtain white precipitate after the hydrochloric acid of 36.5wt%, collecting precipitation also with ethyl acetate washing, obtains the sterling of diamidogen aminomethyl phenyl adamantane compound (compound 3);
3) by diamidogen aminomethyl phenyl adamantane compound (compound 3), Pentafluorophenol polyamine compounds (compound 4) and the triethylamine of benzyl chloroformate protection are dissolved in dichloromethane, stirred at ambient temperature reacts 20 hours, distilling under reduced pressure is except desolventizing, add dichloromethane that volume ratio is 1:1 and water mixed liquid extracts, organic facies anhydrous magnesium sulfate drying, then distilling under reduced pressure removes desolventizing and obtains crude product, with ethyl acetate and petroleum ether mixed liquor for developing solvent, the volumetric ratio of this mixed liquor PetroChina Company Limited. ether and ethyl acetate is followed successively by 1:2 and fades to 4:1 to fade to 6:1 be finally pure ethyl acetate, be separated with 200-300 order silicagel column, the sterling of the diamantane (obsolete) polyamine compounds (compound 5) of obtained benzyl chloroformate protection,
4) the diamantane (obsolete) polyamine compounds (compound 5) that benzyl chloroformate is protected is dissolved in the mixed solution of hydrobromic acid and acetic acid, hydrobromic acid is being 33wt% with the concentration in the mixed solution of acetic acid, stirring at room temperature reacts 10 hours, add excessive ether and produce white precipitate, centrifugal and carry out with ether washing the sterling that can obtain diamantane (obsolete) polyamine compounds;
(3) preparation of the super-molecule assembling body of targeted delivery siRNA
By beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds and Cucurbituril [6] mixing and soluble in water, carry out ultrasonic dissolution, then the aqueous solution that concentration is the siRNA of 20 μm of ol/L is added, 20 times are are repeatedly blown and beaten, the quaternary super-molecule assembling body of obtained targeted delivery siRNA with 200 μ L liquid-transfering guns.
3. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterized in that: in described step (1), the amount ratio of hyaluronate sodium and phosphate buffer solution is 0.073mmol/L, and the mol ratio of hyaluronate sodium, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxy thiosuccinimide and 6-deoxidation-6-ethylenediamine-beta-schardinger dextrin-is 0.0044:1.75:1.75:1.
4. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterized in that: 1 of described step (2)) in the amount ratio of two azido-methyl oxybenzene compounds (compound 1) and acetone be 0.026mol/L, two azido-methyl oxybenzene compounds (compound 1) are 1:0.74:3 with the mol ratio of diamantane (obsolete) bromomethyl ketone and Anhydrous potassium carbonate.
5. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterized in that: 2 of described step (2)) in the amount ratio of two azido-methyl phenyl adamantane compounds (compound 2) and anhydrous tetrahydro furan be 0.097mol/L, two azido-methyl phenyl adamantane compounds (compound 2) are 1:3 with the mol ratio of triphenylphosphine, and anhydrous tetrahydro furan and concentration are the volume ratio of the hydrochloric acid of 36.5wt% is 40:1.
6. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2; it is characterized in that: 3 of described step (2)) in the amount ratio of diamidogen aminomethyl phenyl adamantane compound (compound 3) and dichloromethane be 0.015mol/L, the mol ratio of the Pentafluorophenol polyamine compounds (compound 4) that diamidogen aminomethyl phenyl adamantane compound (compound 3) and benzyl chloroformate are protected and triethylamine is 1:2.5:4.
7. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, is characterized in that: 4 of described step (2)) in the diamantane (obsolete) polyamine compounds (compound 5) of benzyl chloroformate protection be 0.04mol/L with the amount ratio of the mixed solution of hydrobromic acid-acetic acid.
8. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, is characterized in that: in described step (3), the amount ratio of beta-cyclodextrin modified hyaluronic acid and water is 2.0 × 10
-4mol/L, the mol ratio of beta-cyclodextrin modified hyaluronic acid, diamantane (obsolete) polyamine compounds and Cucurbituril [6] is 1:17:34, and the volume ratio of beta-cyclodextrin modified hyaluronic acid/diamantane (obsolete) polyamine compounds/Cucurbituril [6] mixed solution and siRNA aqueous solution is 1:1.
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