CN104892807A - Surface saccharide-modified polymer micelle, and preparation method and application thereof - Google Patents
Surface saccharide-modified polymer micelle, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a surface saccharide-modified polymer micelle, and a preparation method and application thereof, belonging to the fields of drugs and pharmaceutics. The polymer is an amphiphilic macromolecule, and can be self-assembled to form a micelle in water by using the surface modification saccharide as the hydrophilic part and the polymer carbon chain as the hydrophobic part while completing coating on the hydrophobic drug; and meanwhile, the surface modification saccharide molecule has the characteristic of selectively identifying the tumor cell surface receptor and implementing targeted delivery of the carried drug. The surface saccharide-modified polymer micelle has low toxicity, can achieve the goal of targeted drug delivery by identifying the cancer cell surface overexpressed saccharide receptor, lowers the toxic or side effect on normal tissues, and can be used as a delivery carrier for various anticancer drugs.
Description
Technical field
The present invention relates to a kind of surperficial sugar-modified polymer micelle and its preparation method and application, belong to medicine and art of pharmacy.
Background technology
Chemotherapy is applied to one of most effective means of cancer therapy as present stage.Its effect is often not ideal enough, and major cause is the target administration poor performance of chemotherapy, easily cause the toxic side effect of normal tissue, and in life-time service process, chemotherapy easily produces resistance simultaneously.The application of pharmaceutical carrier then can by realizing cancer therapy drug targeted and then reaching the object of Therapeutic cancer.In recent years, many solid support materials such as polymer micelle, nanometer liposome, dendritic macromole and organic-inorganic hybrid nanometer particle etc. have been widely deployed and have carried out used as targeted pharmaceutical carrier, to reach the research of the object of Therapeutic cancer.
Amphiphilic polymers self-assembly can form polymer micelle in water, and its kernel is that the bag that hydrophobic cavity can be used for hydrophobic drug carries, and has and increases contained medicine stability, extend the function of its circulation time in vivo.Meanwhile, form polymer micelle there is suitable particle diameter, be detained (EPR) effect be enriched in tumor locus (passive target effect) by strengthening infiltration, thus reduce the untoward reaction of cancer therapy drug normal tissue.But only rely on passive target to be used for realizing the Selective depression of carrier to growth of cancer cells to be then short of to some extent, therefore in order to strengthen the targeting of nano-carrier further, usually targeted molecular modification is carried out to its surface, can the character of specific recognition cancer cell surfaces overexpression acceptor as utilized key to connect antibody, polypeptide, folic acid etc., carrier can be absorbed by cancer cells by Receptor recognition mode, thus realize carrier selectivity and carry contained medicine to the object of cancer cells, and then reduce the toxic side effect of cancer therapy drug.
Summary of the invention
Mannose receptor is many agglutinin receptors, the carbohydrate molecule that it is end that the mode by Receptor recognition combines with seminose, 2-Acetamido-2-deoxy-D-glucose and Fucose.In recent years, there is report to prove mannose molecules to be modified in pharmaceutical carrier surface, its acceptor identifying cancer cell surfaces overexpression can have been utilized thus realize targeted medicine to cancer cells.But with carbohydrate molecule modified polymer micella realize cancer cells targeted drug conveying simultaneously reduce cancer therapy drug to the research of normal cellulotoxic side effect, there is not been reported.
First object of the present invention is to provide a kind of sugar-modified polymkeric substance, this polymkeric substance is with the carbohydrate molecule modified for hydrophilic parts, and polymer carbon is hydrophobic parts, and in water, self-assembly forms micella, the bag simultaneously completing hydrophobic drug carries, and it forms as shown in Equation 1:
In formula 1 above:
Z is selected from following one or more identical or different chemical functional group or function fragment: seminose, semi-lactosi, glucose, lactose, and end is other carbohydrate molecules of above-mentioned molecule;
B is selected from following carbohydrate and the connecting key of polymkeric substance: ester bond (-COO-), amido linkage (-CONR
1-, R
1=H, CH
3, or-CH
2-), disulfide linkage (-S-S-), ehter bond (-O-), carbonnitrogen bond (-C-N (R
2)-, R
2=H, CH
3, or CH
3cH
2deng) and 1,3-triazole ring (
);
N is 10-300.
Comprising the structure of polymkeric substance in formula 1, in one embodiment of the invention, is poly (glycidyl methacrylate).
Described Z, in one embodiment of the invention, for seminose, semi-lactosi, glucose, lactose, 1, 2, 3, 4, 6-five-O-ethanoyl-D-MANNOSE, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-α-D-MANNOSE, propargyl-α-D-mannopyranose, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-α-D-semi-lactosi, 1, 2, 3, 4, 6-five-O-ethanoyl-D-semi-lactosi, propargyl-α-D-galactopyranose, 1, 2, 3, 4, 6-five-O-acetyl-D-glucose, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-alpha-D-glucose, propargyl-α-D-Glucopyranose etc.
Described polymkeric substance, in one embodiment of the invention, for D-MANNOSE modifies poly (glycidyl methacrylate).
Second object of the present invention is to provide a kind of preparation method of described polymkeric substance, it is characterized in that, described method comprises the steps:
(1) poly (glycidyl methacrylate) that the polymerization degree is certain is prepared;
(2) resulting polymers in (1) is carried out azido-or amino or sulfydryl modification;
(3) resulting polymers in (2) is carried out surface carbohydrates molecular modification.
The polymerization degree in described step (1) is 10-300.
In one embodiment of the invention, described carbohydrate is one or more in seminose, semi-lactosi, glucose and lactose, and end is the carbohydrate of these molecules.
Described method, in one embodiment of the invention, specifically:
(1) by a certain amount of glycidyl methacrylate (GMA), dissolve by dimethyl sulfoxide (DMSO) (DMSO).Under argon shield condition, successively add cuprous bromide (CuBr) and dipyridyl (Bpy).Finally add α-methyl bromide c (MBrP) initiator with microsyringe, sealing argon shield, by a certain amount of tetrahydrofuran (THF) (THF) termination reaction after reaction.By neutral alumina column removing copper part, after filtrate is concentrated, precipitate in methyl alcohol, dry gained throw out is poly (glycidyl methacrylate) (PGMA).
(2) by PGMA obtained in (1), a certain amount of sodium azide, the chloride leach of catalytic amount is in N, in N '-dimethyl methane amide (DMF), sealing, 50 DEG C of oil bath reactions, solids removed by filtration impurity, after filtrate is concentrated, precipitate in water, dry sediment obtains the PGMA (PGMA-N that azido-replaces
3).
(3) by gained PGMA-N in (2)
3be dissolved in DMF, oxygen in logical argon gas removal system.Separately carbohydrate molecule and copper sulfate are dissolved in distilled water, under argon shield condition, add sodium ascorbate.To DMF and aqueous solution above, sealing, in oil bath 60 DEG C reaction, cross after filtering insolubles and dialyse (Mw 2000), in lyophilize dialysis tubing, solution obtains sugar-modified polymkeric substance.
The method of the available existing any synthetic polymer of synthesis of the present invention's polymkeric substance used, as active free radical polymerization (comprising atom transfer radical polymerization (ATRP), reversible addition and fragmentation chain transfer radical polymerization (RAFT) etc.), anionoid polymerization etc.
3rd object of the present invention is to provide the sugar-modified polymer micelle in a kind of surface utilizing described polymkeric substance to prepare.
The particle diameter of the sugar-modified polymer micelle in described surface, in one embodiment of the invention, between 10-500nm.
The median size of the sugar-modified polymer micelle in described surface, in one embodiment of the invention, at 10-200nm.
In one embodiment of the invention, the hydrophilic segment of the surperficial sugar-modified polymer micelle of Z-shaped one-tenth, serves as active targeting group simultaneously.
The sugar-modified polymer micelle in described surface, in one embodiment of the invention, prepare as follows: by dialysis and gel chromatography purifying, by controlling polymer molecule concentration in aqueous higher than after micelle-forming concentration, supersound process promotes micelle formation, obtains polymer micelle solution.Record resulting polymers micella be spherical, be uniformly distributed, between particle size range 10-500nm.
4th object of the present invention is to provide the sugar-modified polymer micelle in described surface as the application in pharmaceutical carrier.
Described application, in one embodiment of the invention, be that pharmaceutical pack to be rolled in surperficial sugar-modified polymer micelle inner, and then improve water-soluble, the identification cell surface saccharide acceptor effect that simultaneously utilizes micellar surface sugar of medicine, complete and the target of contained medicine is transported.Packaging medicine method is: be dissolved in DMSO by medicine and polymer molecule, adds water and promotes drug encapsulation, and dialysis removing DMSO, namely freeze-drying obtains drug-carrying polymer micelle.
Described medicine in one embodiment of the invention, is the medicine of cancer therapy drug, antiviral, treatment diabetes or the medicine for the treatment of cardiovascular and cerebrovascular diseases.
Described medicine in one embodiment of the invention, particularly, is Zorubicin or camptothecine.
Beneficial effect of the present invention:
(1) the surface carbohydrates modified polymer micella that polymkeric substance of the present invention prepares is spherical, is uniformly distributed;
(2) classical micellar medicine-carried system, because its biocompatibility is poor, and easily cause general to distribute, normal tissue causes larger toxic side effect.And polymer micelle prepared by the present invention has lower cytotoxicity, under experimental conditions, polymer micelle and human breast carcinoma MDA-MB-231 cell hatch 72 hours jointly, and cell survival rate is higher than 90%;
(3) surperficial key of the present invention connects the polymer micelle of carbohydrate, can specific recognition cancer-cell surface receptors, enters cancer cells by Receptor recognition selective action, and by institute's loaded with anti-cancer medicine release, medicine enters anticancer growth after nucleus; The polymer micelle that surface is modified without carbohydrate molecule enters in breast cancer cell less, and the polymer micelle that surface has carbohydrate molecule to modify can enter breast cancer cell fast.
Accompanying drawing explanation
The proton nmr spectra of Fig. 1: PGMA-Mannose;
The gel permeation chromatography figure of Fig. 2: PGMA-Mannose;
Fig. 3: PGMA-Mannose blank micella transmission electron microscope photo (A) and grain size distribution (B);
Fig. 4: human breast carcinoma MDA-MB-231 cell and people's kidney epithelium HEK293 cell cultivate the relative survival rate after 72 hours under different concns polymer micelle PGMA-Mannose existence condition;
Fig. 5: confocal microscope photo proves that cultivating rear a small amount of drug-carrying polymer micelle for 1 hour is absorbed by people's renal epithelial cell HEK293; The nucleus of A:DAPI dyeing; B:DOX fluorescence imaging; C: the merging figure of first two imaging;
Fig. 6: confocal microscope photo proves that cultivating rear a large amount of drug-carrying polymer micelle for 1 hour is absorbed by human breast cancer cell MDA-MB-231; The nucleus of A:DAPI dyeing; B:DOX fluorescence imaging; C: the merging figure of first two imaging.
Embodiment
Abbreviation:
PGMA, poly (glycidyl methacrylate); GMA, glycidyl methacrylate; MBrP, α-methyl bromide c; CuBr, cuprous bromide; Bpy, dipyridyl; THF, tetrahydrofuran (THF); PGMA-N
3, the poly (glycidyl methacrylate) that azido-replaces; PGMA-Mannose, D-MANNOSE modifies poly (glycidyl methacrylate); TMSOTf, trifluoromethanesulfonic acid trimethylammonium silicone grease; PGMA-Galactose, poly (glycidyl methacrylate) modified by semi-lactosi; PGMA-Glucose, glucose modified poly (glycidyl methacrylate).
Embodiment 1:1,2,3,4,6-five synthesis of-O-ethanoyl-D-MANNOSE
In 250mL flask, add the pyridine solution of 80mL, the D-MANNOSE of 5.4g, and after 70mL acetic anhydride, stirring at room temperature is reacted, period with tlc (TLC) detection reaction, after result display reacts completely.Solution in flask is transferred to 1000mL separating funnel, adds 100mL dichloromethane solution and extract, with water (3 × 100mL), saturated sodium bicarbonate (3 × 100mL), and after 1M salt acid elution, get dichloromethane layer.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulphate carries out drying, crosses and filters anhydrous sodium sulphate, and rotary evaporation is except desolventizing, obtains 1,2,3,4,6-six-O-ethanoyl-D-MANNOSE after vacuum-drying.
Embodiment 2:2,3,4,6-tetra-synthesis of-O-ethanoyl-1-propargyl-α-D-MANNOSE
4.8g compound 2 and a certain amount of pre-activate molecular sieve are added in 250mL flask, vacuumize 3h on vacuum pump after, flask is sealed, in flask, adds anhydrous methylene chloride 60mL, be placed on ice bath in whole system, after stirring 15min.Under argon shield condition, in flask, add 2.8mL trifluoromethanesulfonic acid trimethylammonium silicone grease (TMSOTf).After adding reagent, remove ice bath, sealing argon shield reaction 2d.After TLC detection reaction, add in system, the saturated K of 50mL
2cO
3termination reaction.Mixture in flask is transferred in 500mL separating funnel, with saturated K
2cO
3(50mL × 3) wash; saturated NaCl (50mL × 3); after anhydrous sodium sulfate drying; after solids removed by filtration anhydrous sodium sulphate, rotary evaporation concentrates gained liquid, through silicagel column separation and purification; obtaining white powder is 2; 3,4,6-six-O-ethanoyl-1-propargyl-α-D-MANNOSE.
Embodiment 3: the synthesis of propargyl-α-D-mannopyranose
To in the flask of 100mL, add the methyl alcohol of 1g compound 3,20mL, and catalytic amount sodium methylate.After reaction 30min, complete through TLC detection reaction.Add hydrogen type cation exchange resin neutralization solution acid, solids removed by filtration resin, rotary evaporation removing residual solvent, after vacuum-drying 12h, obtains white powder and is final product propargyl-α-D-mannopyranose.
Embodiment 4:1,2,3,4,6-five synthesis of-O-ethanoyl-D-semi-lactosi
In 250mL flask, add the pyridine solution of 80mL, the D-semi-lactosi of 5.4g, and after 70mL acetic anhydride, stirring at room temperature is reacted, period with tlc (TLC) detection reaction, after result display reacts completely.Solution in flask is transferred to 1000mL separating funnel, adds 100mL dichloromethane solution and extract, with water (3 × 100mL), saturated sodium bicarbonate (3 × 100mL), and after 1M salt acid elution, get dichloromethane layer.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulphate carries out drying, crosses and filters anhydrous sodium sulphate, and rotary evaporation is except desolventizing, obtains 1,2,3,4,6-six-O-ethanoyl-D-semi-lactosi after vacuum-drying.
Embodiment 5:2,3,4,6-tetra-synthesis of-O-ethanoyl-1-propargyl-α-D-semi-lactosi
4.8g compound 2 and a certain amount of pre-activate molecular sieve are added in 250mL flask, vacuumize 3h on vacuum pump after, flask is sealed, in flask, adds anhydrous methylene chloride 60mL, be placed on ice bath in whole system, after stirring 15min.Under argon shield condition, in flask, add 2.8mL trifluoromethanesulfonic acid trimethylammonium silicone grease (TMSOTf).After adding reagent, remove ice bath, sealing argon shield reaction 2d.After TLC detection reaction, add in system, the saturated K of 50mL
2cO
3termination reaction.Mixture in flask is transferred in 500mL separating funnel, with saturated K
2cO
3(50mL × 3) wash; saturated NaCl (50mL × 3); after anhydrous sodium sulfate drying; after solids removed by filtration anhydrous sodium sulphate, rotary evaporation concentrates gained liquid, through silicagel column separation and purification; obtaining white powder is 2; 3,4,6-six-O-ethanoyl-1-propargyl-α-D-semi-lactosi.
Embodiment 6: the synthesis of propargyl-α-D-galactopyranose
To in the flask of 100mL, add the methyl alcohol of 1g compound 3,20mL, and catalytic amount sodium methylate.After reaction 30min, complete through TLC detection reaction.Add hydrogen type cation exchange resin neutralization solution acid, solids removed by filtration resin, rotary evaporation removing residual solvent, after vacuum-drying 12h, obtains white powder and is final product propargyl-α-D-galactopyranose.
Embodiment 7:1,2,3,4,6-five synthesis of-O-acetyl-D-glucose
In 250mL flask, add the pyridine solution of 80mL, the D-Glucose of 5.4g, and after 70mL acetic anhydride, stirring at room temperature is reacted, period with tlc (TLC) detection reaction, after result display reacts completely.Solution in flask is transferred to 1000mL separating funnel, adds 100mL dichloromethane solution and extract, with water (3 × 100mL), saturated sodium bicarbonate (3 × 100mL), and after 1M salt acid elution, get dichloromethane layer.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulphate carries out drying, crosses and filters anhydrous sodium sulphate, and rotary evaporation is except desolventizing, obtains 1,2,3,4,6-six-O-acetyl-D-glucose after vacuum-drying.
Embodiment 8:2,3,4,6-tetra-synthesis of-O-ethanoyl-1-propargyl-alpha-D-glucose
4.8g compound 2 and a certain amount of pre-activate molecular sieve are added in 250mL flask, vacuumize 3h on vacuum pump after, flask is sealed, in flask, adds anhydrous methylene chloride 60mL, be placed on ice bath in whole system, after stirring 15min.Under argon shield condition, in flask, add 2.8mL trifluoromethanesulfonic acid trimethylammonium silicone grease (TMSOTf).After adding reagent, remove ice bath, sealing argon shield reaction 2d.After TLC detection reaction, add in system, the saturated K of 50mL
2cO
3termination reaction.Mixture in flask is transferred in 500mL separating funnel, with saturated K
2cO
3(50mL × 3) wash; saturated NaCl (50mL × 3); after anhydrous sodium sulfate drying; after solids removed by filtration anhydrous sodium sulphate, rotary evaporation concentrates gained liquid, through silicagel column separation and purification; obtaining white powder is 2; 3,4,6-six-O-ethanoyl-1-propargyl-alpha-D-glucose.
Embodiment 9: the synthesis of propargyl-α-D-Glucopyranose
To in the flask of 100mL, add the methyl alcohol of 1g compound 3,20mL, and catalytic amount sodium methylate.After reaction 30min, complete through TLC detection reaction.Add hydrogen type cation exchange resin neutralization solution acid, solids removed by filtration resin, rotary evaporation removing residual solvent, after vacuum-drying 12h, obtains white powder and is final product propargyl-α-D-Glucopyranose.
The synthesis of embodiment 10:PGMA
In 50mL flask, add the GMA monomer (in advance by Flash silica post removing stopper) of the 1.5g accurately taken, then use 2.5mL dimethyl sulfoxide (DMSO) (DMSO) to dissolve.Logical argon gas 15min in flask, oxygen in removal system.Then, under argon shield condition, the CuBr catalyzer of 64.4mg and the Bpy part of 189.7mg is successively added.Finally in anaerobic system, add 81 μ L initiator MBrP with microsyringe, under sealing argon shield condition, 40 DEG C of reaction 2h.After having reacted, open sealing system, in flask, add 10mL THF termination reaction.When solution from brown become blackish green time, mixture solution is passed through neutral alumina pillar to remove copper part, obtain colourless liquid, after gained filtrate is concentrated, precipitate in large volume methyl alcohol, under 35 DEG C of conditions, dry gained throw out can obtain white solid poly (glycidyl methacrylate) (PGMA).By the ratio of control GMA monomer and initiator MBrP, the PGMA of obtained different polymerization degree respectively.
Embodiment 11:PGMA-N
3synthesis
By the PGMA solid of 100.0mg, the ammonium chloride powder of 56.0mg, and the sodiumazide of 68.5mg, add in 50mL flask, then continue to add 7mLN in flask, dinethylformamide (DMF) liquid, by flask encapsulation process, 50 DEG C of oil bath reactions, after 12h, cross the solid impurity filtering reaction system, after concentrated gained filtrate, precipitate in cardinal principle ponding, vacuum-drying gained throw out can obtain the PGMA (PGMA-N that azido-replaces
3).
Embodiment 12:PGMA-NH
2synthesis
By PGMA solid, palladium carbon dust, and methanol solution, add in flask, in displacement flask, gas is hydrogen, add hydrogen balloon, after reaction 12h, cross the solid impurity filtering reaction system, after concentrated gained filtrate, precipitate in cardinal principle ponding, vacuum-drying gained throw out can obtain the amino PGMA (PGMA-NH replaced
2).
By similar approach, the compound that connecting key b is sulfydryl (-SH) and carboxyl (-COOH) can be prepared.
The synthesis of embodiment 13:PGMA-Mannose
By the PGMA-N of 60.0mg
3solid is placed in the flask of 50mL, adds 15mL DMF solution, be stirred to dissolution of solid in flask, and in flask, logical argon gas 15min is with oxygen in removal system.Separately propargyl-α-D-mannopyranose the powder of 125.2mg and the cupric sulfate pentahydrate crystal of 90.5mg are added in 5mL distilled water; concussion stirring makes it thoroughly dissolve; logical argon gas 15min in system; to remove oxygen in flask; then, under argon shield condition, in system, the sodium ascorbate powder of 248.1mg is added.The above-mentioned DMF solution system eliminating oxygen is mixed with water solution system, sealing, under the condition of argon shield, 60 DEG C of reaction 24h.After having reacted, after mixture in system was passed through to filter insolubles, concentrated gained filtrate is after dialyzate dialysis (MW 2000) 2d with distilled water, cross the solid filtering dialysis and produce, lyophilize gained filtrate can obtain the amphiphilic compound PGMA-Mannose of target.Proton nmr spectra and gel permeation chromatography demonstrate chemical structure and the relative molecular weight (as Fig. 1 and Fig. 2) of obtained PGMA-Mannose.
The polymer micelle made with PGMA-Mannose is spherical, be uniformly distributed (as Fig. 3, A, B), and there is lower cytotoxicity, under experimental conditions, polymer micelle and human breast carcinoma MDA-MB-231 cell hatch 72 hours jointly, and cell survival rate is higher than 92% (as shown in Figure 4).The glycan molecule adopting embodiment 6 or 9 to synthesize, also has similar effect.
The synthesis of embodiment 14:PGMA-CONH-Mannose
By the PGMA-NH of 60.0mg
2solid is placed in the flask of 50mL, adds 15mL DMF solution, is stirred to dissolution of solid, add carboxyl modified seminose in solution in flask, sealing, under the condition of argon shield, and 60 DEG C of reaction 24h.After having reacted, after mixture in system was passed through to filter insolubles, concentrated gained filtrate, be after dialyzate dialysis (MW 2000) 2d with distilled water, cross the solid filtering dialysis and produce, lyophilize gained filtrate can obtain the amphiphilic compound PGMA-CONH-Mannose of target.
Sugar-modified polymer micelle with prepared by PGMA-CONH-Mannose: uniform particle diameter; Cytotoxicity is low, and jointly hatch 72 hours with human breast carcinoma MDA-MB-231 cell, cell survival rate is higher than 92%; Compared with the polymer micelle modified without carbohydrate molecule with other surfaces, breast cancer cell can be entered fast.The glycan molecule adopting embodiment 6 or 9 to synthesize, also has similar effect.
Embodiment 15:PGMA-Mannose micella bag carries Zorubicin
First take 10mg PGMA-Mannose sample, add 1mL DMSO, stirring 2h to dissolving completely, adding the Doxorubicin solution that 100 μ L are pre-configured wherein.After stirring 5min, under the condition of vigorous stirring, slowly drip 1mL distilled water wherein, dialysis tubing dialysis removing DMSO solvent (Mw=1000,24h).Filter liquid in dialysis tubing, gained liquid freezing is dry, be the PGMA-Mannose micella sample that bag carries Zorubicin.
Meanwhile, bag carries the PGMA-Mannose micella of Zorubicin, specificity can select cancer-cell surface receptors, cancer cells is entered by Receptor recognition selective action, in cell, the effect such as lysosome, discharges into nucleus by institute's loaded with anti-cancer medicine, reaches the object of anticancer growth.Demonstrate in the incubation time of 1 hour by laser confocal microscope test, the polymer micelle that surface is modified without carbohydrate molecule enters less in breast cancer cell (as shown in Figure 5), and the polymer micelle that surface has carbohydrate molecule to modify can enter breast cancer cell (as shown in Figure 6) fast.
Embodiment 16:PGMA-Galactose micella bag carries Zorubicin
First take 10mg PGMA-Galactose sample, add 1mL DMSO, stirring 2h to dissolving completely, adding the Doxorubicin solution that 100 μ L are pre-configured wherein.After stirring 5min, under the condition of vigorous stirring, slowly drip 1mL distilled water wherein, dialysis tubing dialysis removing DMSO solvent (Mw=1000,24h).Filter liquid in dialysis tubing, gained liquid freezing is dry, be the PGMA-Galactose micella sample that bag carries Zorubicin.
Embodiment 17:PGMA-Glucose micella bag carries Zorubicin
First take 10mg PGMA-Glucose sample, add 1mL DMSO, stirring 2h to dissolving completely, adding the Doxorubicin solution that 100 μ L are pre-configured wherein.After stirring 5min, under the condition of vigorous stirring, slowly drip 1mL distilled water wherein, dialysis tubing dialysis removing DMSO solvent (Mw=1000,24h).Filter liquid in dialysis tubing, gained liquid freezing is dry, be the PGMA-Glucose micella sample that bag carries Zorubicin.
Embodiment 18:PGMA-Mannose micella bag carries camptothecine
Replace the Zorubicin in case study on implementation 15 by camptothecine, other operation is with embodiment 15.Prepare the PGMA-Mannose micella sample that bag carries camptothecine.
Embodiment 19: the cell endocytic of carrier micelle
The two kinds of cells choosing logarithmic phase growth are inoculated in Tissue Culture Dish respectively, put incubator and hatch 24h and make it adherent.The nutrient solution added containing carrier micelle (40 μ g/mL) continues to cultivate the regular hour.Discard nutrient solution, clean 2 times with the phosphate buffered saline buffer (PBS) of pH 7.4.Cell 4.0% formaldehyde is at room temperature fixed 15min.After PBS cleans 2 times, with 4 ', 6-diamidino-2-phenylindone (DAPI, 1 μ g/mL) transfect cell core 15min.By the distribution of confocal laser scanning microscope carrier micelle at cell interior, excitation wavelength is 405/561nm, and emission wavelength is 417-477/570-1000nm.
Embodiment 20: blank micella toxicity assessment
Mtt assay is adopted to investigate the cytotoxicity of carrier micelle.The cell suspension of MDA-MB-231 or HEK293 is planted in 96 orifice plates, 10000, every hole cell.Cultivate 24h in incubator after, clean 2 times with the PBS of pH 7.4, add the substratum containing carrier micelle or former medicine Zorubicin, the mass concentration making Zorubicin contained in system is 2 μ g/mL.After cultivating the regular hour, clean 2 times with the PBS of pH 7.4, every hole adds 100 μ L MTT solution (1mg/mL) and continues to cultivate 4h.Discard nutrient solution in hole, every hole adds 100 μ L DMSO, vibration 10min, and application microplate reader measures the light absorption value (OD) in each hole at 490nm place, calculate cell survival rate.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a sugar-modified polymkeric substance, is characterized in that, described sugar-modified polymkeric substance has the structure shown in formula 1:
Wherein, n is 10-300;
Z is the chemical functional group that obtains of following any one or multiple identical or different molecular combinations or function fragment: seminose, semi-lactosi, glucose, lactose or the carbohydrate containing above molecule;
B is the one in the connecting key of following any carbohydrate and polymkeric substance: ester bond, amido linkage, disulfide linkage, ehter bond, carbonnitrogen bond or 1,3-triazole ring
2. polymkeric substance according to claim 1, it is characterized in that, Z in described formula 1 be following any one: seminose, semi-lactosi, glucose, lactose, 1, 2, 3, 4, 6-five-O-ethanoyl-D-MANNOSE, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-α-D-MANNOSE, propargyl-α-D-mannopyranose, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-α-D-semi-lactosi, 1, 2, 3, 4, 6-five-O-ethanoyl-D-semi-lactosi, propargyl-α-D-galactopyranose, 1, 2, 3, 4, 6-five-O-acetyl-D-glucose, 2, 3, 4, 6-tetra--O-ethanoyl-1-propargyl-alpha-D-glucose, propargyl-α-D-Glucopyranose.
3. polymkeric substance according to claim 1, is characterized in that, the b in described formula 1 is ester bond, amido linkage, disulfide linkage or 1,3-triazole ring.
4. a preparation method for the arbitrary described polymkeric substance of claim 1-3, it is characterized in that, described method comprises the steps:
(1) poly (glycidyl methacrylate) that the polymerization degree is certain is prepared;
(2) resulting polymers in (1) is carried out azido-or amino or sulfydryl modification;
(3) resulting polymers in (2) is carried out surface carbohydrates molecular modification.
5. the sugar-modified polymer micelle in surface utilizing the arbitrary described polymkeric substance of claim 1-3 to prepare.
6. the sugar-modified polymer micelle in surface according to claim 5, is characterized in that, the particle diameter of described micella is between 10-500nm.
7. the sugar-modified polymer micelle in surface according to claim 5, it is characterized in that, described polymer micelle prepares by the following method: by dialysis and gel chromatography purifying, by controlling polymer molecule concentration in aqueous higher than after micelle-forming concentration, supersound process promotes micelle formation, obtains polymer micelle solution.
8. surperficial sugar-modified polymer micelle described in claim 5 is as the application in pharmaceutical carrier.
9. application according to claim 8, is characterized in that, described medicine is cancer therapy drug, antiviral, the medicine for the treatment of diabetes or the medicine for the treatment of cardiovascular and cerebrovascular diseases.
10. application according to claim 8, is characterized in that, described medicine is Zorubicin or camptothecine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108403704A (en) * | 2018-05-31 | 2018-08-17 | 江南大学 | Heparin disaccharides is grafted the purposes of sulphation polymethyl acyl ethanol amine |
| CN108498536A (en) * | 2018-04-28 | 2018-09-07 | 江南大学 | Sulfated heparin disaccharides is grafted the purposes of polymethyl acyl ethanol amine |
| CN108641018A (en) * | 2018-04-28 | 2018-10-12 | 江南大学 | A kind of sulfated heparin disaccharides grafting polymethyl acyl ethanol amine and preparation method thereof |
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| FABIAN SURIANO ET AL.: "Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery", 《BIOMATERIALS》 * |
| M. W. JONES ET AL.: "Glycopolymers with secondary binding motifs mimic glycan branching and display bacterial lectin selectivity in addition to affinity", 《CHEMICAL SCIENCE》 * |
| QIANG ZHANG ET AL.: "Terminal functional glycopolymers via a combination of catalytic chain transfer polymerisation (CCTP) followed by three consecutive click reactions", 《POLYMER CHEMISTRY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498536A (en) * | 2018-04-28 | 2018-09-07 | 江南大学 | Sulfated heparin disaccharides is grafted the purposes of polymethyl acyl ethanol amine |
| CN108641018A (en) * | 2018-04-28 | 2018-10-12 | 江南大学 | A kind of sulfated heparin disaccharides grafting polymethyl acyl ethanol amine and preparation method thereof |
| WO2019205256A1 (en) * | 2018-04-28 | 2019-10-31 | 江南大学 | Sulfated heparin disaccharide-grafted polymethylacryloyl ethanolamine and preparation method thereof |
| CN108403704A (en) * | 2018-05-31 | 2018-08-17 | 江南大学 | Heparin disaccharides is grafted the purposes of sulphation polymethyl acyl ethanol amine |
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