CN104840975B - A kind of super-molecule assembling body and preparation method of targeted delivery siRNA - Google Patents
A kind of super-molecule assembling body and preparation method of targeted delivery siRNA Download PDFInfo
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Abstract
A kind of super-molecule assembling body of targeted delivery siRNA, to modify hyaluronic acid, adamantane polyamine compounds, Cucurbituril [6] and the quaternary super-molecule assembling body of siRNA synthesis based on beta cyclodextrin, quaternary super-molecule assembling body noncovalent interaction strong between beta cyclodextrin and adamantane and adamantane polyamines chain and Cucurbituril [6], between adamantane polyamines chain and siRNA based on electrostatic interaction, the supramolecular nanoparticles using hydrophilic hyaluronic acid as shell are formed.It is an advantage of the invention that：The super-molecule assembling body, synthesizes and constructs that route is simple, production cost is low and yield is higher, suitable for amplification synthesis and production application；The quaternary super-molecule assembling body is brought among cancer cell with targetting for the endocytosis of medium by the hyaluronic acid receptor of malignant cell surface overexpression, realizes the effective reticence of exogenous gene expression, while toxic side effect substantially reduces.
Description
【Technical field】
The present invention relates to target gene delivery technical field, particularly a kind of oversubscription subgroup of targeted delivery siRNA
Fill body and preparation method.
【Background technology】
Today's society is due to the influence of the factors such as haze, environmental pollution, ionising radiation, food security and heredity, cancer
The incidence of disease rise year by year, serious threat has arrived the health of the mankind.Therefore, operative treatment, chemotherapy and radiotherapy
Main Means as clinical anticancer are able to extensive use.However, these therapies due to therapeutic effect it is bad, it is easy recurrence with
And serious toxic side effect etc. causes cancer can not be captured so far by the mankind.Therefore, chemist, material scholar and biomedicine
Worker develops various drug delivery systems such as liposome, inorganic nano-particle, vesica and carbon nanomaterial etc. to increase
The bio-compatibility and targeting of strong drug delivery system, to therapeutic effect to be reinforced and reduce toxic side effect.However, these
Therapy is all cured the symptoms, not the disease, and wants thoroughly to effect a radical cure cancer it is necessary to contain it from the source of pathogenesis of cancer.Closely
The gene therapy occurred in several years opens a fan gate to capture cancer.Its principle is by nucleic acid (such as matter with biological function
Grain and linear dsdna, or siRNA (siRNA) etc.) transfer or be transported in cancer cell, repair damage gene or
Person suppresses the expression of intracellular aberrant gene, then induces the cancer cell of not apoptosis that apoptosis occurs, and cancer is cured so as to reach
Purpose.But the poor efficiency of gene transfection becomes the restriction wide variety of bottleneck of this method.Due to potential safety issue
And expensive cost, viral vector are not widely developed, therefore design with constructing with bio-compatibility and can
The controlled release playing function and target gene delivery system for possessing high efficiency gene transfection becomes the study hotspot in forward position.This
Outside, noncovalent interaction carrier is due to its excellent slow release effect, stronger binding ability and relatively low toxic side effect etc.
Advantage, it is widely used on the medicine/gene carrier occurred recently.
【The content of the invention】
The purpose of the present invention is for above-mentioned technical Analysis and problem be present, there is provided a kind of targeted delivery siRNA
Super-molecule assembling body and preparation method, the super-molecule assembling body can be specific endogenous and foreign gene in effective reticence cancer cell
Expression, and its toxic side effect is very low, preparation method is simple and yield is higher, suitable for amplification synthesis and production application.
Technical scheme：
A kind of super-molecule assembling body of targeted delivery siRNA, for based on beta-cyclodextrin modified hyaluronic acid, adamantane
The quaternary super-molecule assembling body of polyamine compounds, Cucurbituril [6] and siRNA synthesis, wherein beta-cyclodextrin modified are transparent
Matter acid molecule formula is (C14H21NO11)98(C58H95N3O44)17, 17 cyclodextrin units, Buddha's warrior attendant are averagely modified on a macromolecular chain
Alkane polyamine compounds chemical molecular formula is C32H56Br4N6O4；The quaternary super-molecule assembling body with beta-schardinger dextrin and adamantane and
Strong noncovalent interaction between adamantane polyamines chain and Cucurbituril [6], and adamantane polyamines chain and electrostatic phase between siRNA
Based on interaction, the supramolecular nanoparticles using hydrophilic hyaluronic acid as shell, nano particle diameter 30- are formed
40nm。
A kind of preparation method of the super-molecule assembling body of the targeted delivery siRNA, step are as follows：
(1) preparation of beta-cyclodextrin modified hyaluronic acid
The Sodium Hyaluronate that molecular weight is 46000 is dissolved in the phosphate buffer solution that pH is 7.2, concentration is 1mmol/L,
Add 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride and N- hydroxy thiosuccinimides, stirring reaction
30 minutes, obtain reaction solution；6- is deoxygenated into -6- ethylenediamines-beta-schardinger dextrin to be dissolved in the phosphate buffer solution that pH is 7.2,6- is gone
The amount ratio of oxygen -6- ethylenediamines-beta-schardinger dextrin and phosphate buffer solution is 0.1mol/L, is then added in above-mentioned reaction solution,
Stirred 24 hours under the conditions of 25 DEG C, continuous dialysis 5 days in the bag filter that retention scope is 8000-14000 are then charged into, by gained
Solution freezes, and beta-cyclodextrin modified hyaluronic acid is made；
(2) preparation of adamantane polyamine compounds
1) by two azido-methyl oxybenzene compounds (compound 1), adamantane bromomethyl ketone and Anhydrous potassium carbonate in acetone
Mixing, 56 DEG C of return stirring reaction 10-12h are heated to, then filters, filtrate is spin-dried for, it is 1 to add volume ratio:1 three chloromethanes
Alkane and water mixed liquid are extracted, and organic phase is dried with anhydrous magnesium chloride, are evaporated under reduced pressure removing solvent and are obtained crude product, then with stone
Oily ether and ethyl acetate mixtures are solvent, and the volume ratio of the mixed liquor petrochina ether and ethyl acetate is 8:1, use 200-
300 mesh silicagel columns are separated, and the sterling of two azido-methyl phenyl adamantane compounds (compound 2) is made；
2) two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine are dissolved in anhydrous tetrahydro furan,
Stir at ambient temperature 12 hours, be then evaporated under reduced pressure and remove solvent, gained solid is dissolved in ethyl acetate, add concentration
To obtain white precipitate after 36.5wt% hydrochloric acid, collect and precipitate and washed with ethyl acetate, obtain diamines aminomethyl phenyl Buddha's warrior attendant
The sterling of hydride compounds (compound 3)；
3) Pentafluorophenol protected diamines aminomethyl phenyl adamantane compound (compound 3), benzyl chloroformate is polyaminated
Compound (compound 4) and triethylamine are dissolved in dichloromethane, at room temperature stirring reaction 20 hours, are evaporated under reduced pressure and are removed solvent, add
Enter volume ratio for 1:1 dichloromethane and water mixed liquid is extracted, and organic phase is dried with anhydrous magnesium sulfate, is then evaporated under reduced pressure
Remove solvent and obtain crude product, using ethyl acetate and petroleum ether mixed liquor as solvent, the mixed liquor petrochina ether and ethyl acetate
Volumetric ratio be followed successively by 1:2 fade to 4:1 fades to 6:1 is finally pure ethyl acetate, is separated, made with 200-300 mesh silicagel columns
Obtain the sterling of the adamantane polyamine compounds (compound 5) of benzyl chloroformate protection；
4) mixing that the adamantane polyamine compounds (compound 5) that benzyl chloroformate is protected are dissolved in hydrobromic acid and acetic acid is molten
In liquid, concentration of the hydrobromic acid in the mixed solution with acetic acid is 33wt%, and reaction 10 hours is stirred at room temperature, adds excessive second
Ether produces white precipitate, centrifuges and is carried out washing the sterling that can be prepared by adamantane polyamine compounds with ether；
(3) preparation of the super-molecule assembling body of targeted delivery siRNA
By the mixing of beta-cyclodextrin modified hyaluronic acid, adamantane polyamine compounds and Cucurbituril [6] and soluble in water, progress
Ultrasonic dissolution, the aqueous solution for the siRNA that concentration is 20 μm of ol/L is then added, is blown and beaten repeatedly 20 times with 200 μ L liquid-transfering guns,
The quaternary super-molecule assembling body of targeted delivery siRNA is made.
The amount ratio of Sodium Hyaluronate and phosphate buffer solution is 0.073mmol/L in the step (1), hyaluronic acid
Sodium, 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, N- hydroxy thiosuccinimides and 6- deoxidation -6- second
The mol ratio of diamines-beta-schardinger dextrin is 0.0044:1.75:1.75:1.
The 1 of the step (2)) in the amount ratios of two azido-methyl oxybenzene compounds (compound 1) and acetone be
0.026mol/L, the mol ratio of two azido-methyl oxybenzene compounds (compound 1) and adamantane bromomethyl ketone and Anhydrous potassium carbonate
It is 1:0.74:3.
The 2 of the step (2)) in two azido-methyl phenyl adamantane compounds (compound 2) and anhydrous tetrahydro furan
Amount ratio is 0.097mol/L, and the mol ratio of two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine is 1:
3, the volume ratio for the hydrochloric acid that anhydrous tetrahydro furan is 36.5wt% with concentration is 40:1.
The 3 of the step (2)) in diamines aminomethyl phenyl adamantane compound (compound 3) and dichloromethane amount ratio
For 0.015mol/L, diamines aminomethyl phenyl adamantane compound (compound 3) and the Pentafluorophenol polyamines of benzyl chloroformate protection
Compound (compound 4) and the mol ratio of triethylamine are 1:2.5:4.
The 4 of the step (2)) in benzyl chloroformate adamantane polyamine compounds (compound 5) and the hydrobromic acid-second protected
The amount ratio of the mixed solution of acid is 0.04mol/L.
The amount ratio of beta-cyclodextrin modified hyaluronic acid and water is 2.0 × 10 in the step (3)-4Mol/L, beta-schardinger dextrin
The mol ratio for modifying hyaluronic acid, adamantane polyamine compounds and Cucurbituril [6] is 1:17:34, beta-cyclodextrin modified hyalomitome
The volume ratio of acid/adamantane polyamine compounds/Cucurbituril [6] mixed solution and the siRNA aqueous solution is 1:1.
It is an advantage of the invention that：The quaternary super-molecule assembling body of the targeted delivery siRNA, Cucurbituril [6] and polyamines
The bonding of chain causes the nitrogen-atoms pKa value of polyamines chain to change so that the electropositivity of polyamines chain greatly enhances, can be consumingly
It is bonded with electronegative siRNA, avoids siRNA from decomposing inactivation due to hydrolysis and with RNA decomposition enzyme effects；
In addition, the hyaluronic acid shell of the quaternary super-molecule assembling body can enter with the hyaluronic acid receptor of cancer cell surfaces overexpression
The specific bonding action of row, then quaternary super-molecule assembling body cancer cell is specifically entered by the endocytosis of cell and worked as
In, then by cancer cell the hyaluronidase of overexpression hydrolysis quaternary assemble physical efficiency access effective degraded
So that siRNA is released, the efficiency gene transfection of cell is substantially increased so that intracellular endogenous and exogenous
Specific gene obtains effective reticence；The assembly targeting is good, and cellulotoxic side effect is very low, and transfection efficiency is higher, preparation method
It is more convenient, there is preferable application prospect in the gene therapy of following cancer.
【Brief description of the drawings】
Fig. 1 is the synthetic route chart of adamantane polyamine compounds.
Fig. 2 constructs route schematic diagram for the quaternary super-molecule assembling body.
Fig. 3 is the transmission electron microscope figure of the quaternary super-molecule assembling body.
Fig. 4 is the electrophoretogram that the quaternary super-molecule assembling body is bonded with siRNA.
Fig. 5 is the cytotoxicity experiment result of the quaternary super-molecule assembling body.
Fig. 6 is the quaternary super-molecule assembling body to the exogenous green fluorescence protein gene silencing efficiency figure of cell.
【Embodiment】
Below by example, the present invention is described further：
Embodiment：
A kind of super-molecule assembling body of targeted delivery siRNA, for based on beta-cyclodextrin modified hyaluronic acid, adamantane
The quaternary super-molecule assembling body of polyamine compounds, Cucurbituril [6] and siRNA synthesis, wherein beta-cyclodextrin modified are transparent
Matter acid molecule formula is (C14H21NO11)98(C58H95N3O44)17, 17 cyclodextrin units, Buddha's warrior attendant are averagely modified on a macromolecular chain
Alkane polyamine compounds chemical molecular formula is C32H56Br4N6O4；The quaternary super-molecule assembling body with beta-schardinger dextrin and adamantane and
Strong noncovalent interaction between adamantane polyamines chain and Cucurbituril [6], and adamantane polyamines chain and electrostatic phase between siRNA
Based on interaction, the supramolecular nanoparticles using hydrophilic hyaluronic acid as shell, nano particle diameter 30- are formed
40nm。
A kind of preparation method of the super-molecule assembling body of the targeted delivery siRNA, step are as follows：
(1) preparation of beta-cyclodextrin modified hyaluronic acid
By the Sodium Hyaluronate that 100mg (0.0022mmol) molecular weight is 46000 be dissolved in 30mL pH be 7.2, concentration be
In 1mmol/L phosphate buffer solution, 167.7mg (0.875mmol) 1- ethyls-(3- dimethylaminopropyls) carbon two is added
Inferior amine salt hydrochlorate and 190mg (0.875mmol) N- hydroxy thiosuccinimides, stirring reaction 30 minutes, obtain reaction solution；Will
588.5mg (0.5mmol) 6- deoxidations -6- ethylenediamines-beta-schardinger dextrin is dissolved in the phosphate buffer solution that 5mL pH are 7.2, then is added
Enter into above-mentioned reaction solution, stirred 24 hours under the conditions of 25 DEG C, be then charged into the bag filter that retention scope is 8000-14000
In continuous dialysis 5 days, resulting solution is freezed, HACD (HACD) is made.
The nuclear-magnetism of beta-cyclodextrin modified hyaluronic acid prepared by the detection display present invention is characterized as below：1H NMR(400MHz,
D2O,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-1ofβ-CD).It is by the way that beta-cyclodextrin modified hyaluronan molecule formula can be obtained to nuclear magnetic spectrogram integral and calculating
(C14H21NO11)98(C58H95N3O44)17, 17 beta-schardinger dextrin units are averagely modified on a macromolecular chain.
(2) preparation of adamantane polyamine compounds
1) by the azido-methyl oxybenzene compounds (compound 1) of 265mg (1.3mmol) two, 247mg (0.962mmol) Buddha's warrior attendant
Alkane bromomethyl ketone and 540mg (3.9mmol) Anhydrous potassium carbonates mix in 50mL acetone, are heated to 56 DEG C of return stirring reactions
12h, then filter, filtrate is spin-dried for, add 50mL chloroforms and 50mL water is extracted, organic phase is done with anhydrous magnesium chloride
It is dry, it is evaporated under reduced pressure removing solvent and obtains crude product, then using petroleum ether and ethyl acetate mixtures as solvent, stone in the mixed liquor
The volume ratio of oily ether and ethyl acetate is 8:1, carry out separation with 200-300 mesh silicagel column and can be prepared by two azido-methyl phenyl gold
The sterling of firm hydride compounds (compound 2) is colorless oil；
The nuclear-magnetism of two azido-methyl phenyl adamantane compounds (compound 2) prepared by the detection display present invention characterizes such as
Under：1H NMR(400MHz,CDCl3,ppm)δ6.87(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) by the azido-methyl phenyl adamantane compounds (compound 2) of 738mg (1.94mmol) two and 1563mg
(5.96mmol) triphenylphosphine is dissolved in 20mL anhydrous tetrahydro furans, is stirred 12 hours, is then evaporated under reduced pressure at ambient temperature
Solvent is removed, gained solid is dissolved in ethyl acetate, 0.5mL is added, white sink is obtained after the hydrochloric acid that concentration is 36.5wt%
Form sediment, collect and precipitate and washed with ethyl acetate, obtain the sterling of diamines aminomethyl phenyl adamantane compound (compound 3)；
The nuclear-magnetism of diamines aminomethyl phenyl adamantane compound (compound 3) prepared by the detection display present invention is characterized as below：1H NMR(400MHz,D2O,ppm)δ7.00(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) diamines aminomethyl phenyl adamantane compound (compound 3), 400mg (0.69mmol) chlorine
The Pentafluorophenol polyamine compounds (compound 4) and 0.2mL triethylamines of benzyl formate protection are dissolved in 20mL dichloromethane, room temperature
Lower stirring reaction 20 hours, it is evaporated under reduced pressure and removes solvent, adds 50mL dichloromethane and 50mL water is extracted, organic phase nothing
Water magnesium sulfate is dried, and is then evaporated under reduced pressure removing solvent and is obtained crude product, should using ethyl acetate and petroleum ether mixed liquor as solvent
The volume ratio of mixed liquor petrochina ether and ethyl acetate is followed successively by 1:2 fade to 4:1 fades to 6:1 is finally pure ethyl acetate, is used
200-300 mesh silicagel columns are separated, and the sterling that the adamantane polyamine compounds (compound 5) of benzyl chloroformate protection are made is
White solid；
The nuclear-magnetism table of the adamantane polyamine compounds (compound 5) of benzyl chloroformate protection prepared by the detection display present invention
Sign is as follows：1H NMR(400MHz,CDCl3,ppm)δ7.32(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 adamantane polyamine compounds (compound 5) that 211.5mg (0.2mmol) benzyl chloroformate is protected are dissolved in 5mL
In the mixed solution of hydrobromic acid and acetic acid, concentration of the hydrobromic acid in the mixed solution with acetic acid is 33wt%, is stirred at room temperature anti-
Answer 10 hours, add excessive ether and produce white precipitate, centrifuge and carry out washing with ether to can be prepared by adamantane polyaminated
The sterling of compound is white solid；
The nuclear-magnetism of adamantane polyamine compounds prepared by the detection display present invention is characterized as below：1H NMR(400MHz,
CDCl3,ppm)δ6.66(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
By 1.20mg beta-cyclodextrin modifieds hyaluronic acid, 0.29mg adamantane polyamine compounds and 0.64mg Cucurbiturils [6]
Mix and be dissolved in 80 μ L water, carry out ultrasonic dissolution, the aqueous solution of the 80 μ L concentration for 20 μM of siRNAs is then added, with 200
μ L liquid-transfering guns are blown and beaten 20 times repeatedly, and the quaternary super-molecule assembling body of targeted delivery siRNA is made.
Fig. 1 is the synthetic route chart of adamantane polyamine compounds.
Fig. 2 constructs route schematic diagram for the quaternary super-molecule assembling body.
Fig. 3 is the transmission electron microscope figure of the quaternary super-molecule assembling body, and being characterized by transmission electron microscope can be with
Draw the quaternary super-molecule assembling body between cyclodextrin-adamantane, positive charge polyamines-Cucurbituril [6] it is strong it is non-covalent mutually
The supramolecular nanoparticles formed between effect and positive charge polyamines-siRNA based on electrostatic interaction, nano-particle
Particle size is 30-40nm.
Fig. 4 is the electrophoretogram that the quaternary super-molecule assembling body is bonded with siRNA.Compared to simple siRNA (
1 row) and siRNA+beta-cyclodextrin modified hyaluronic acid+adamantane polyamine compounds (the 2nd row) compound, Cucurbituril [6]
Addition can effectively strengthen binding ability (3-5 row) of the super-molecule assembling body to siRNA, under last stoichiometric proportion
Beta-cyclodextrin modified hyaluronic acid+adamantane polyamine compounds+(beta-cyclodextrin modified hyaluronic acid, adamantane are more for Cucurbituril [6]
The mol ratio of amines and Cucurbituril [6] is 1:17:34) it is most strong (the 6th row) to the binding ability of siRNA.
The concrete application effect of the present invention is as follows：
PC-3 cells (human prostate cancer cells) are layered on to 96 of the RPMI-1640 culture mediums containing 10% hyclone
Cultivated 24 hours in orifice plate, be separately added into 160 μ L, 80 μ L, 40 μ L, 20 μ L adamantane polyamine compounds (a), adamantane polyamines
Compound+Cucurbituril [6] (b), adamantane polyamine compounds+beta-cyclodextrin modified hyaluronic acid (c), and Cucurbituril [6]+gold
Firm alkane polyamine compounds+beta-cyclodextrin modified hyaluronic acid (d), continuous culture 24 hours, each experiment condition is measured with mtt assay
Under cells survival rate.As a result as shown in figure 5, in the range of 24 hours, adamantane polyamine compounds under each concentration due to
It has positively charged polyamines chain and has shown certain cytotoxicity, and after adding beta-cyclodextrin modified hyaluronic acid,
Due to the targeting of hyaluronic acid so that a large amount of quilt of beta-cyclodextrin modified hyaluronic acid-adamantane polyamine compounds assembly
Endocytosis enters into the cell so that the cytotoxicity of adamantane polyamine compounds is strengthened.And the addition of Cucurbituril [6] makes its bonding
On positive charge polyamines chain, the cytotoxicity of assembly can be substantially reduced, cells survival rate is generally more than 90%.Thus may be used
See, the quaternary super-molecule assembling body that we construct has shown very low cytotoxicity under each concentration, and security significantly carries
Rise, be advantageous to the evaluation of gene delivery effect below.On the other hand, as shown in fig. 6, PC-3 cells are layered on containing 10% tire ox
Cultivated 24 hours in 6 orifice plates of the RPMI-1640 culture mediums of serum, use Lipofactamine 2000 and EGFP-pDNA in advance
Processing cell causes cell to give expression to green fluorescent protein, is then respectively adding the small interference of Lipofacyamine 2000+EGFP-
RNA (d-f) and the quaternary super-molecule assembling body (g-i) for loading EGFP- siRNAs, effect change into fresh to cell after 6 hours
Culture medium, it is further cultured for 24 hours, with the silencing efficiency of fluorescence microscope green fluorescence protein gene.As shown in fig. 6a-6c,
PC-3 cells effective expression after EGFP-pDNA is transferred to has gone out green fluorescent protein, has then shown bright green fluorescence；
Commercial reagents Lipofacyamine 2000 be bonded EGFP- siRNAs shown it is good to green fluorescent protein
Silence efficiency, and the quaternary super-molecule assembling body for the load EGFP- siRNAs that we prepare can be effectively small dry by EGFP-
It is intracellular into PC-3 to disturb RNA transfection so that fluorecyte quantity and cell fluorescence intensity substantially reduce, and show and compare commodity
Change reagent preferably to the Gene silencing efficacy of green fluorescent protein, there is good gene delivery application prospect.
Claims (8)
- A kind of 1. super-molecule assembling body of targeted delivery siRNA, it is characterised in that：For based on beta-cyclodextrin modified hyalomitome Acid, adamantane polyamine compounds, Cucurbituril [6] and the quaternary super-molecule assembling body of siRNA synthesis, wherein beta-schardinger dextrin It is (C to modify hyaluronan molecule formula14H21NO11)98(C58H95N3O44)17, 17 cyclodextrin are averagely modified on a macromolecular chain Unit, adamantane polyamine compounds chemical molecular formula are C32H56Br4N6O4；The quaternary super-molecule assembling body is with beta-schardinger dextrin and gold Noncovalent interaction just strong between alkane and adamantane polyamines chain and Cucurbituril [6], and adamantane polyamines chain and siRNA Between based on electrostatic interaction, form the supramolecular nanoparticles using hydrophilic hyaluronic acid as shell, nano particle diameter For 30-40nm.
- A kind of 2. preparation method of the super-molecule assembling body of targeted delivery siRNA as claimed in claim 1, it is characterised in that Step is as follows：(1) preparation of beta-cyclodextrin modified hyaluronic acidThe Sodium Hyaluronate that molecular weight is 46000 is dissolved in the phosphate buffer solution that pH is 7.2, concentration is 1mmol/L, then added Enter 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride and N- hydroxy thiosuccinimides, stirring reaction 30 are divided Clock, obtain reaction solution；6- is deoxygenated into -6- ethylenediamines-beta-schardinger dextrin to be dissolved in the phosphate buffer solution that pH is 7.2,6- deoxidations -6- The amount ratio of ethylenediamine-beta-schardinger dextrin and phosphate buffer solution is 0.1mol/L, is then added in above-mentioned reaction solution, in 25 DEG C of bars Stirred 24 hours under part, be then charged into continuous dialysis 5 days in the bag filter that retention scope is 8000-14000, resulting solution is frozen It is dry, beta-cyclodextrin modified hyaluronic acid is made；(2) preparation of adamantane polyamine compounds1) two azido-methyl oxybenzene compounds (compound 1), adamantane bromomethyl ketone and Anhydrous potassium carbonate are mixed in acetone, 56 DEG C of return stirring reaction 10-12h are heated to, then filters, filtrate is spin-dried for, it is 1 to add volume ratio:1 chloroform and Water mixed liquid is extracted, and organic phase is dried with anhydrous magnesium chloride, is evaporated under reduced pressure removing solvent and is obtained crude product, then with petroleum ether It is solvent with ethyl acetate mixtures, the volume ratio of the mixed liquor petrochina ether and ethyl acetate is 8:1, with 200-300 mesh Silicagel column is separated, and the sterling of two azido-methyl phenyl adamantane compounds (compound 2) is made；2) two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine are dissolved in anhydrous tetrahydro furan, in room Stirred under the conditions of temperature 12 hours, be then evaporated under reduced pressure and remove solvent, gained solid is dissolved in ethyl acetate, adding concentration is White precipitate is obtained after 36.5wt% hydrochloric acid, collects and precipitates and washed with ethyl acetate, obtain diamines aminomethyl phenyl adamantane The sterling of compound (compound 3)；3) the Pentafluorophenol polyamine compounds for protecting diamines aminomethyl phenyl adamantane compound (compound 3), benzyl chloroformate (compound 4) and triethylamine are dissolved in dichloromethane, at room temperature stirring reaction 20 hours, are evaporated under reduced pressure and are removed solvent, add body Product is than being 1:1 dichloromethane and water mixed liquid is extracted, and organic phase is dried with anhydrous magnesium sulfate, is then evaporated under reduced pressure and is removed Solvent obtains crude product, using ethyl acetate and petroleum ether mixed liquor as solvent, the appearance of the mixed liquor petrochina ether and ethyl acetate Product ratio is followed successively by 1:2 fade to 4:1 fades to 6:1 is finally pure ethyl acetate, is separated with 200-300 mesh silicagel columns, chlorine is made The sterling of the adamantane polyamine compounds (compound 5) of benzyl formate protection；4) the adamantane polyamine compounds (compound 5) that benzyl chloroformate is protected are dissolved in the mixed solution of hydrobromic acid and acetic acid In, concentration of the hydrobromic acid in the mixed solution with acetic acid is 33wt%, and reaction 10 hours is stirred at room temperature, adds excessive ether White precipitate is produced, centrifuges and is carried out washing the sterling that can be prepared by adamantane polyamine compounds with ether；(3) preparation of the super-molecule assembling body of targeted delivery siRNABy the mixing of beta-cyclodextrin modified hyaluronic acid, adamantane polyamine compounds and Cucurbituril [6] and soluble in water, progress ultrasound Dissolving, the aqueous solution for the siRNA that concentration is 20 μm of ol/L is then added, is blown and beaten repeatedly 20 times with 200 μ L liquid-transfering guns, is made The quaternary super-molecule assembling body of targeted delivery siRNA.
- 3. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterised in that： The amount ratio of Sodium Hyaluronate and phosphate buffer solution is 0.073mmol/L in the step (1), Sodium Hyaluronate, 1- ethyls- (3- dimethylaminopropyls) carbodiimide hydrochloride, N- hydroxy thiosuccinimides and 6- deoxidation -6- ethylenediamines-β-ring paste The mol ratio of essence 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 characterised in that： The 1 of the step (2)) in the amount ratio of two azido-methyl oxybenzene compounds (compound 1) and acetone be 0.026mol/L, two fold The mol ratio of N-methyl oxybenzene compound (compound 1) and adamantane bromomethyl ketone and Anhydrous potassium carbonate is 1:0.74:3.
- 5. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterised in that： The 2 of the step (2)) in the amount ratios of two azido-methyl phenyl adamantane compounds (compound 2) and anhydrous tetrahydro furan be The mol ratio of 0.097mol/L, two azido-methyl phenyl adamantane compounds (compound 2) and triphenylphosphine is 1:3, anhydrous four The volume ratio for the hydrochloric acid that hydrogen furans is 36.5wt% with concentration is 40:1.
- 6. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterised in that： The 3 of the step (2)) in the amount ratio of diamines aminomethyl phenyl adamantane compound (compound 3) and dichloromethane be 0.015mol/L, diamines aminomethyl phenyl adamantane compound (compound 3) and the Pentafluorophenol that benzyl chloroformate is protected are polyaminated The mol ratio of compound (compound 4) 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, it is characterised in that： The 4 of the step (2)) in the mixing of adamantane polyamine compounds (compound 5) and hydrobromic acid-acetic acid protected of benzyl chloroformate The amount ratio of solution is 0.04mol/L.
- 8. the preparation method of the super-molecule assembling body of targeted delivery siRNA according to claim 2, it is characterised in that： The amount ratio of beta-cyclodextrin modified hyaluronic acid and water is 2.0 × 10 in the step (3)-4Mol/L, beta-cyclodextrin modified are transparent The mol ratio of matter acid, adamantane polyamine compounds and Cucurbituril [6] is 1:17:34, beta-cyclodextrin modified hyaluronic acid/adamantane The volume ratio of polyamine compounds/Cucurbituril [6] mixed solution and the siRNA aqueous solution is 1:1.
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