CN104892807B - 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 sugar-modified polymer micelle in a kind of surface and its preparation method and application, belong to medicine and pharmacy
Field.
Background technology
Chemotherapy used as being applied to one for the treatment of of cancer most efficient method at this stage.Its effect is often not ideal enough, mainly
Reason is the target administration poor performance of chemotherapy, while the toxic and side effect of normal tissue is easily caused, and in long-term use mistake
Cheng Zhong, chemotherapy easily produces drug resistance.The application of pharmaceutical carrier then can be by realizing that cancer therapy drug targets conveying and then reaches
The purpose for the treatment of cancer.In recent years, many carrier materials such as polymer micelle, nano liposomes, dendritic macromole and have
Machine inorganic hybrid nano particle etc. has been widely deployed and has carried out and has been used as targeting conveying pharmaceutical carrier, to reach treatment
The research of the purpose of cancer.
Amphiphilic polymers can be self-assembly of polymer micelle in water, and its kernel can be used to dredge for hydrophobic cavity
Containing for aqueous pharmaceutical, with contained medicine stability is increased, extends the function of its circulation time in vivo.Meanwhile, form poly-
Compound micella has suitable particle diameter, and (passive target is made can be enriched in tumor locus by strengthening infiltration delay (EPR) effect
With), so as to reduce the adverse reaction of cancer therapy drug normal tissue.But passive target effect is only relied on to realize that carrier is thin to cancer
The Selective depression of intracellular growth has then been short of, therefore in order to further enhance the targeting of nano-carrier, generally its surface is entered
The modification of row targeted molecular, such as utilization are bonded antibody, polypeptide, folic acid being capable of specific recognition cancer cell surfaces overexpression acceptor
Property, carrier can be absorbed by Receptor recognition mode by cancer cell, so as to realize the carrier selectively contained medicine of conveying extremely
The purpose of cancer cell, and then reduce the toxic and side effect of cancer therapy drug.
The content of the invention
Mannose receptor is many agglutinin receptors, can be combined with mannose, N- acetylaminos by way of Receptor recognition
Glucose and fucose are the carbohydrate molecule of end.In recent years, had been reported that proof modifies mannose molecules to be carried in medicine
Body surface face, it is possible to use the acceptor of its identification cancer cell surfaces overexpression is so as to realize targeting conveying medicine to cancer cell.But
Realize that the targeted drug conveying of cancer cell reduces cancer therapy drug to normal cell toxicant simultaneously with carbohydrate molecule modified polymer micella
There is not been reported for the research of side effect.
First purpose of the invention is to provide a kind of sugar-modified polymer, the polymer with the carbohydrate molecule modified be parent
Aqueous fractions, polymer carbon is hydrophobic parts, and micella is self-assembly of in water, while completing the bag of hydrophobic drug
Carry, its composition is as shown in Equation 1：
In equation 1 above：
Z is selected from following one or more identical or different chemical functionals group or function fragment：Mannose, gala
Sugar, glucose, lactose, and other carbohydrate molecules that end is above-mentioned molecule；
B is the connecting key selected from following carbohydrate and polymer：Ester bond (- COO -), amido link (- CONR1-, R1=
H, CH3, or-CH2-), disulfide bond (- S-S -), ehter bond (- O -), carbonnitrogen bond (- C-N (R2)-, R2=H, CH3, or
CH3CH2Deng) and 1,3- triazoles ring ()；
N is 10-300.
The structure comprising polymer, is polymethyl acid glycidyl in one embodiment of the invention in formula 1
Ester.
The Z, is mannose, galactolipin, glucose, lactose, 1,2,3,4,6- in one embodiment of the invention
Five-O- acetyl group-the D-MANNOSE,-O- acetyl group -1- propargyls-α of 2,3,4,6- tetra--D-MANNOSE, propargyl-α-D- pyrans
The mannose,-O- acetyl group -1- propargyl-α-D- galactolipins of the 2,3,4,6- tetra-,-O- acetyl group-D- galas of 1,2,3,4,6- five
Sugar, propargyl-α-D- the galactopyranoses ,-O- acetyl-D-glucoses of the 1,2,3,4,6- five ,-O- acetyl group of 2,3,4,6- tetra--
1- propargyls-alpha-D-glucose, propargyl-α-D- glucopyranoses etc..
Described polymer, is that D-MANNOSE modification polymethylacrylic acid shrinks in one embodiment of the invention
Glyceride.
Second object of the present invention is to provide a kind of preparation method of the polymer, it is characterised in that methods described
Comprise the following steps：
(1) the certain poly (glycidyl methacrylate) of the degree of polymerization is prepared；
(2) resulting polymers in (1) are carried out into azido or amino or sulfydryl modification；
(3) resulting polymers in (2) are carried out into surface carbohydrates molecular modification.
The degree of polymerization in the step (1) is 10-300.
In one embodiment of the invention, the carbohydrate is in mannose, galactolipin, glucose and lactose
Plant or various, and the carbohydrate that end is these molecules.
Methods described, in one embodiment of the invention, specifically：
(1) by a certain amount of GMA (GMA), dissolved with dimethyl sulfoxide (DMSO) (DMSO).In argon gas
Under protective condition, cuprous bromide (CuBr) and bipyridyl (Bpy) are successively added.Finally α-bromo-propionic acid first is added with microsyringe
Ester (MBrP) initiator, sealing argon gas protection, with a certain amount of tetrahydrofuran (THF) terminating reaction after reaction.By neutral alumina
Aluminium post removes copper part, after filtrate is concentrated, precipitates in methyl alcohol, dries gained sediment for polymethyl acid glycidyl
Ester (PGMA).
(2) will in (1) be obtained PGMA, a certain amount of Sodium azide, the chloride leach of catalytic amount is in N, N '-dimethyl formamide
(DMF) in, sealing, 50 DEG C of oil bath reactions are filtered to remove solid impurity, after filtrate is concentrated, are precipitated in water, dry sediment
Obtain the PGMA (PGMA-N of azido substitution3)。
(3) by gained PGMA-N in (2)3It is dissolved in DMF, leads to oxygen in argon gas removal system.Separately by carbohydrate molecule and sulphur
Sour copper is dissolved in distilled water, under argon gas protective condition, adds sodium ascorbate.DMF and the aqueous solution will mix above, sealing,
In 60 DEG C of reactions of oil bath, dialysed (Mw 2000) after being filtered to remove insoluble matter, solution obtains sugar-modified in freeze-drying bag filter
Polymer.
The synthesis of polymer used of the invention can use the method for existing any synthetic polymer, such as active free radical polymerization
(including ATRP (ATRP), reversible addition and fragmentation chain transfer radical polymerization (RAFT) etc.), anion gather
Close etc..
Third object of the present invention is to provide a kind of sugar-modified polymer in surface prepared using the polymer
Micella.
The particle diameter of the sugar-modified polymer micelle in surface, in one embodiment of the invention, 10-500nm it
Between.
The average grain diameter of the sugar-modified polymer micelle in surface, in one embodiment of the invention, in 10-
200nm。
In one embodiment of the invention, the Z-shaped hydrophilic segment into the sugar-modified polymer micelle in surface, functions simultaneously as
Active targeting group.
The sugar-modified polymer micelle in surface, in one embodiment of the invention, is prepared as follows
Arrive：Purified by dialysis and gel chromatography, by controlling polymer molecule concentration in aqueous dense higher than critical micell
After degree, ultrasonically treated promotion micelle forma-tion obtains final product polymer micelle solution.It is spherical, uniform point to measure resulting polymers micella
Cloth, between particle size range 10-500nm.
Fourth object of the present invention is to provide the sugar-modified polymer micelle in the surface in terms of as pharmaceutical carrier
Using.
The application, is that medicine is wrapped in into the sugar-modified polymer micelle in surface in one embodiment of the invention
Inside, so improve medicine water solubility, while using micellar surface sugar identification cell surface saccharide acceptor act on, completion to institute
Carry the targeting transport of medicine.Packaging medicine method is：Medicine and polymer molecule are dissolved in DMSO, addition water promotes medicine
Contain, dialysis removes DMSO, lyophilized to obtain final product drug-carrying polymer micelle.
The medicine, is cancer therapy drug, antiviral drugs, treatment diabetes in one embodiment of the invention
The medicine of medicine or treatment cardiovascular and cerebrovascular disease.
The medicine, is adriamycin or camptothecine specifically in one embodiment of the invention.
Beneficial effects of the present invention：
(1) the surface carbohydrates modified polymer micella that polymer of the invention is prepared is spherical, is uniformly distributed；
(2) classical micellar medicine-carried system, because its biocompatibility is poor, and easily causes general distribution, to normal group
Weave into larger toxic and side effect.And polymer micelle prepared by the present invention has relatively low cytotoxicity, under experimental conditions, gather
Compound micella and human breast carcinoma MDA-MB-231 cells are incubated 72 hours jointly, and cell survival rate is higher than 90%；
(3) surface of the invention is bonded the polymer micelle of carbohydrate, can specific recognition cancer-cell surface receptors, pass through
Receptor recognition selective action enters cancer cell, and by the release of contained cancer therapy drug, medicine suppresses cancer cell life after entering nucleus
It is long；Surface enters less in breast cancer cell without the polymer micelle that carbohydrate molecule is modified, and surface has what carbohydrate molecule was modified
Polymer micelle can rapidly enter breast cancer cell.
Brief description of the drawings
Fig. 1：The proton nmr spectra of PGMA-Mannose；
Fig. 2：The gel permeation chromatography figure of PGMA-Mannose；
Fig. 3：PGMA-Mannose blank micellas transmission electron microscope photo (A) and grain size distribution (B)；
Fig. 4：Human breast carcinoma MDA-MB-231 cells and people's kidney epithelium HEK293 cells are in various concentrations polymer micelle
Relative survival rate after being cultivated 72 hours under PGMA-Mannose existence conditions；
Fig. 5：A small amount of drug-carrying polymer micelle is thin by people's kidney epithelium after confocal microscope photo proves culture in 1 hour
Born of the same parents HEK293 is absorbed；A：The nucleus of DAPI dyeing；B：DOX fluorescence imagings；C：The merging figure of first two imaging；
Fig. 6：A large amount of drug-carrying polymer micelles are thin by human breast carcinoma after confocal microscope photo proves culture in 1 hour
Born of the same parents MDA-MB-231 is absorbed；A：The nucleus of DAPI dyeing；B：DOX fluorescence imagings；C：The merging figure of first two imaging.
Specific embodiment
Abbreviation：
PGMA, poly (glycidyl methacrylate)；GMA, GMA；MBrP, α-bromo-propionic acid first
Ester；CuBr, cuprous bromide；Bpy, bipyridyl；THF, tetrahydrofuran；PGMA-N3, the polymethylacrylic acid shrink of azido substitution
Glyceride；PGMA-Mannose, D-MANNOSE modification poly (glycidyl methacrylate)；TMSOTf, TFMS front three
Base silicone grease；PGMA-Galactose, the sugar-modified poly (glycidyl methacrylate) of gala；PGMA-Glucose, glucose is repaiied
Decorations poly (glycidyl methacrylate).
Embodiment 1：The synthesis of-O- the acetyl group of 1,2,3,4,6- five-D-MANNOSE
The pyridine solution of 80mL, after the D-MANNOSE of 5.4g, and 70mL acetic anhydride, room temperature are added in 250mL flasks
Stirring reaction, period is detected with thin-layered chromatography (TLC) and reacted, after as a result showing reaction completely.Solution in flask is transferred to
1000mL separatory funnels, add 100mL dichloromethane solutions to be extracted, with water (3 × 100mL), saturated sodium bicarbonate (3 ×
100mL), and after 1M salt acid elutions, dichloromethane layer is taken.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulfate is done
It is dry, be filtered to remove anhydrous sodium sulfate, and rotary evaporation removes solvent, obtained after vacuum drying 1,2,3,4,6- six-O- acetyl group-
D-MANNOSE.
Embodiment 2：The synthesis of-O- acetyl group -1- propargyls-the α of 2,3,4,6- tetra--D-MANNOSE
During 4.8g compounds 2 and a certain amount of pre-activate molecular sieve added into 250mL flasks, in vacuumizing 3h on vavuum pump
Afterwards, flask is sealed, to anhydrous methylene chloride 60mL is added in flask, is placed on ice bath in whole system, after stirring 15min.
Under argon gas protective condition, to addition 2.8mL TFMS trimethyls silicone grease (TMSOTf) in flask.After adding reagent, remove
Remove ice bath, sealing argon gas protection reaction 2d.TLC is detected after completion of the reaction, added in system, 50mL saturations K2CO3Terminate anti-
Should.Mixture in flask is transferred in 500mL separatory funnels, with saturation K2CO3(50mL × 3) wash, saturation NaCl (50mL
× 3), and after anhydrous sodium sulfate drying, after being filtered to remove solid anhydrous sodium sulfate, rotary evaporation concentration gained liquid, through silicagel column
Isolate and purify, obtain white powder for 2,3,4,6- six-O- acetyl group -1- propargyls-α-D-MANNOSE.
Embodiment 3：The synthesis of propargyl-α-D- mannopyranoses
To in the flask of 100mL, 1g compounds 3, the methyl alcohol of 20mL, and catalytic amount sodium methoxide are added.Reaction 30min
Afterwards, it is complete through TLC detection reactions.Add hydrogen type cation exchange resin in and solution acidic, be filtered to remove hard resin, rotate
Evaporation removes residual solvent, after vacuum drying 12h, obtains white powder as final product propargyl-α-D- mannopyranoses.
Embodiment 4：The synthesis of-O- acetyl group-D- the galactolipins of 1,2,3,4,6- five
The pyridine solution of addition 80mL in 250mL flasks, the D- galactolipins of 5.4g, and after 70mL acetic anhydride, room temperature
Stirring reaction, period is detected with thin-layered chromatography (TLC) and reacted, after as a result showing reaction completely.Solution in flask is transferred to
1000mL separatory funnels, add 100mL dichloromethane solutions to be extracted, with water (3 × 100mL), saturated sodium bicarbonate (3 ×
100mL), and after 1M salt acid elutions, dichloromethane layer is taken.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulfate is done
It is dry, be filtered to remove anhydrous sodium sulfate, and rotary evaporation removes solvent, obtained after vacuum drying 1,2,3,4,6- six-O- acetyl group-
D- galactolipins.
Embodiment 5：The synthesis of-O- acetyl group -1- propargyl-α-D- the galactolipins of 2,3,4,6- tetra-
During 4.8g compounds 2 and a certain amount of pre-activate molecular sieve added into 250mL flasks, in vacuumizing 3h on vavuum pump
Afterwards, flask is sealed, to anhydrous methylene chloride 60mL is added in flask, is placed on ice bath in whole system, after stirring 15min.
Under argon gas protective condition, to addition 2.8mL TFMS trimethyls silicone grease (TMSOTf) in flask.After adding reagent, remove
Remove ice bath, sealing argon gas protection reaction 2d.TLC is detected after completion of the reaction, added in system, 50mL saturations K2CO3Terminate anti-
Should.Mixture in flask is transferred in 500mL separatory funnels, with saturation K2CO3(50mL × 3) wash, saturation NaCl (50mL
× 3), and after anhydrous sodium sulfate drying, after being filtered to remove solid anhydrous sodium sulfate, rotary evaporation concentration gained liquid, through silicagel column
Isolate and purify, obtain white powder for 2,3,4,6- six-O- acetyl group -1- propargyl-α-D- galactolipins.
Embodiment 6：The synthesis of propargyl-α-D- galactopyranoses
To in the flask of 100mL, 1g compounds 3, the methyl alcohol of 20mL, and catalytic amount sodium methoxide are added.Reaction 30min
Afterwards, it is complete through TLC detection reactions.Add hydrogen type cation exchange resin in and solution acidic, be filtered to remove hard resin, rotate
Evaporation removes residual solvent, after vacuum drying 12h, obtains white powder as final product propargyl-α-D- galactopyranoses.
Embodiment 7：The synthesis of-O- the acetyl-D-glucoses of 1,2,3,4,6- five
The pyridine solution of 80mL, after the D-Glucose of 5.4g, and 70mL acetic anhydride, room temperature are added in 250mL flasks
Stirring reaction, period is detected with thin-layered chromatography (TLC) and reacted, after as a result showing reaction completely.Solution in flask is transferred to
1000mL separatory funnels, add 100mL dichloromethane solutions to be extracted, with water (3 × 100mL), saturated sodium bicarbonate (3 ×
100mL), and after 1M salt acid elutions, dichloromethane layer is taken.With saturated aqueous common salt (2 × 100mL), anhydrous sodium sulfate is done
It is dry, be filtered to remove anhydrous sodium sulfate, and rotary evaporation removes solvent, obtained after vacuum drying 1,2,3,4,6- six-O- acetyl group-
D-Glucose.
Embodiment 8：The synthesis of-O- acetyl group -1- the propargyls of 2,3,4,6- tetra--alpha-D-glucose
During 4.8g compounds 2 and a certain amount of pre-activate molecular sieve added into 250mL flasks, in vacuumizing 3h on vavuum pump
Afterwards, flask is sealed, to anhydrous methylene chloride 60mL is added in flask, is placed on ice bath in whole system, after stirring 15min.
Under argon gas protective condition, to addition 2.8mL TFMS trimethyls silicone grease (TMSOTf) in flask.After adding reagent, remove
Remove ice bath, sealing argon gas protection reaction 2d.TLC is detected after completion of the reaction, added in system, 50mL saturations K2CO3Terminate anti-
Should.Mixture in flask is transferred in 500mL separatory funnels, with saturation K2CO3(50mL × 3) wash, saturation NaCl (50mL
× 3), and after anhydrous sodium sulfate drying, after being filtered to remove solid anhydrous sodium sulfate, rotary evaporation concentration gained liquid, through silicagel column
Isolate and purify, obtain white powder for 2,3,4,6- six-O- acetyl group -1- propargyls-alpha-D-glucose.
Embodiment 9：The synthesis of propargyl-α-D- glucopyranoses
To in the flask of 100mL, 1g compounds 3, the methyl alcohol of 20mL, and catalytic amount sodium methoxide are added.Reaction 30min
Afterwards, it is complete through TLC detection reactions.Add hydrogen type cation exchange resin in and solution acidic, be filtered to remove hard resin, rotate
Evaporation removes residual solvent, after vacuum drying 12h, obtains white powder as final product propargyl-α-D- glucopyranoses.
Embodiment 10：The synthesis of PGMA
(removed beforehand through Flash silica post and inhibited to the GMA monomers of the accurate 1.5g for weighing are added in 50mL flasks
Agent), then dissolved with 2.5mL dimethyl sulfoxide (DMSO)s (DMSO).To logical argon gas 15min, oxygen in removal system in flask.Then exist
Under argon gas protective condition, the CuBr catalyst of 64.4mg and the Bpy parts of 189.7mg are successively added.Finally use microsyringe
To 81 μ L initiator MBrP are added in anaerobic system, under sealing argon gas protective condition, 40 DEG C are reacted 2h.After the completion of reaction, open
Sealing system, to addition 10mL THF terminating reactions in flask.When solution becomes blackish green from brown, mixture solution is led to
Neutral alumina pillar is crossed to remove copper part, colourless liquid is obtained, after gained filtrate is concentrated, precipitated in large volume methyl alcohol,
Gained sediment is dried under the conditions of 35 DEG C and can obtain white solid poly (glycidyl methacrylate) (PGMA).By control
The ratio of GMA monomers and initiator MBrP, is obtained the PGMA of different polymerization degree respectively.
Embodiment 11：PGMA-N3Synthesis
By the PGMA solids of 100.0mg, the sodium azide of the ammonium chloride powder of 56.0mg, and 68.5mg adds to 50mL
In flask, then continue to adding 7mLN, dinethylformamide (DMF) liquid, by flask encapsulation process, 50 DEG C of oil in flask
Bath reaction, after 12h, is filtered to remove the solid impurity of reaction system, after concentration gained filtrate, is precipitated in substantially ponding, vacuum
Dry the PGMA (PGMA-N that gained sediment can obtain azido substitution3)。
Embodiment 12：PGMA-NH2Synthesis
By PGMA solids, palladium carbon powder, and methanol solution are added in flask, and gas is hydrogen, addition in displacement flask
Hydrogen balloon, after reaction 12h, is filtered to remove the solid impurity of reaction system, after concentration gained filtrate, is precipitated in substantially ponding,
Vacuum drying gained sediment is the PGMA (PGMA-NH that can obtain amino substitution2)。
By similar approach, it is sulfydryl (- SH) and the compound of carboxyl (- COOH) that can prepare connecting key b.
Embodiment 13：The synthesis of PGMA-Mannose
By the PGMA-N of 60.0mg3Solid is placed in the flask of 50mL, and to 15mL DMF solutions are added in flask, stirring is extremely
Solid dissolving, to logical argon gas 15min in flask with oxygen in removal system.Another propargyl-α-D- pyrans the sweet dews by 125.2mg
The cupric sulfate pentahydrate crystal of Icing Sugar end and 90.5mg is added in 5mL distilled water, and concussion stirring makes its thorough dissolving, leads to in system
Argon gas 15min, to remove oxygen in flask, then under argon gas protective condition, to the ascorbic acid that 248.1mg is added in system
Sodium powder end.The above-mentioned DMF solution system for eliminating oxygen is mixed with water solution system, is sealed, under conditions of argon gas protection, 60 DEG C
Reaction 24h.After the completion of reaction, after mixture in system is removed by filtration into insoluble matter, concentration gained filtrate is with distilled water
After dialyzate dialysis (MW 2000) 2d, the solid that dialysis is produced is filtered to remove, freeze-drying gained filtrate is that can obtain target pair
Parent's property compound PGMA-Mannose.Proton nmr spectra and gel permeation chromatography demonstrate the change of obtained PGMA-Mannose
Learn structure and relative molecular weight (such as Fig. 1 and Fig. 2).
The polymer micelle being made of PGMA-Mannose is spherical, is uniformly distributed (such as Fig. 3, A, B), and with relatively low
Cytotoxicity, under experimental conditions, polymer micelle and human breast carcinoma MDA-MB-231 cells are incubated 72 hours jointly, cell
Survival rate is higher than 92% (as shown in Figure 4).The glycan molecule synthesized using embodiment 6 or 9, it may have similar effect.
Embodiment 14：The synthesis of PGMA-CONH-Mannose
By the PGMA-NH of 60.0mg2Solid is placed in the flask of 50mL, to 15mL DMF solutions are added in flask, is stirred
To solid dissolving, to carboxyl modified mannose is added in solution, seal, under conditions of argon gas protection, 60 DEG C of reaction 24h.Reaction
After the completion of, after mixture in system is removed by filtration into insoluble matter, concentration gained filtrate is dialysed by dialyzate of distilled water
After (MW 2000) 2d, the solid that dialysis is produced is filtered to remove, freeze-drying gained filtrate is that can obtain the amphiphilic compound of target
PGMA-CONH-Mannose。
The sugar-modified polymer micelle prepared with PGMA-CONH-Mannose：Uniform particle diameter；Cytotoxicity is low, with human milk
Gland cancer MDA-MB-231 cells are incubated 72 hours jointly, and cell survival rate is higher than 92%；Modified without carbohydrate molecule with other surfaces
Polymer micelle compare, breast cancer cell can be rapidly entered.The glycan molecule synthesized using embodiment 6 or 9, it may have similar
Effect.
Embodiment 15：PGMA-Mannose micellas contain adriamycin
10mg PGMA-Mannose samples are weighed first, 1mL DMSO are added, and 2h is to being completely dissolved for stirring, adds thereto
Enter the Doxorubicin solution that 100 μ L are pre-configured with.After stirring 5min, under conditions of being stirred vigorously, 1mL is slowly added dropwise thereto double
Water is steamed, bag filter dialysis removes DMSO solvents (Mw=1000,24h).Liquid in filtering bag filter, gained liquid freezing is done
It is dry, as contain the PGMA-Mannose micella samples of adriamycin.
Meanwhile, contain the PGMA-Mannose micellas of adriamycin, can specific selection cancer-cell surface receptors, by receiving
Body recognition reaction selectively enters cancer cell, is acted on by intracellular lysosome etc., and contained cancer therapy drug is discharged into cell
Core, reaches the purpose for suppressing growth of cancer cells.The incubation time demonstrated at 1 hour is tested by laser confocal microscope
Interior, surface enters in breast cancer cell less (as shown in Figure 5) without the polymer micelle that carbohydrate molecule is modified, and there is carbohydrate on surface
The polymer micelle of molecular modification can rapidly enter breast cancer cell (as shown in Figure 6).
Embodiment 16：PGMA-Galactose micellas contain adriamycin
10mg PGMA-Galactose samples are weighed first, 1mL DMSO are added, and 2h is to being completely dissolved for stirring, thereto
The Doxorubicin solution for adding 100 μ L to be pre-configured with.After stirring 5min, under conditions of being stirred vigorously, 1mL is slowly added dropwise thereto
Distilled water, bag filter dialysis removes DMSO solvents (Mw=1000,24h).Liquid in filtering bag filter, gained liquid freezing is done
It is dry, as contain the PGMA-Galactose micella samples of adriamycin.
Embodiment 17：PGMA-Glucose micellas contain adriamycin
10mg PGMA-Glucose samples are weighed first, 1mL DMSO are added, and 2h is to being completely dissolved for stirring, adds thereto
Enter the Doxorubicin solution that 100 μ L are pre-configured with.After stirring 5min, under conditions of being stirred vigorously, 1mL is slowly added dropwise thereto double
Water is steamed, bag filter dialysis removes DMSO solvents (Mw=1000,24h).Liquid in filtering bag filter, gained liquid freezing is done
It is dry, as contain the PGMA-Glucose micella samples of adriamycin.
Embodiment 18：PGMA-Mannose micellas contain camptothecine
Replace the adriamycin in case study on implementation 15 with camptothecine, other operations are with embodiment 15.Prepare and contain camplotheca acuminata
The PGMA-Mannose micella samples of alkali.
Embodiment 19：The cell endocytic of carrier micelle
Two kinds of cells for choosing logarithmic phase growth are inoculated in Tissue Culture Dish respectively, and putting incubator incubation 24h pastes it
Wall.The nutrient solution containing carrier micelle (40 μ g/mL) is added to continue to cultivate the regular hour.Nutrient solution is discarded, with pH's 7.4
Phosphate buffer (PBS) is cleaned 2 times.Cell is fixed into 15min at room temperature with 4.0% formaldehyde.After PBS 2 times, use
4 ', 6- diamidino -2-phenylindone (DAPI, 1 μ g/mL) dye nucleus 15min.Medicine glue is carried with confocal laser scanning microscope
The distribution in beam portion in the cell, excitation wavelength is 405/561nm, and launch wavelength is 417-477/570-1000nm.
Embodiment 20：Blank micella toxicity assessment
The cytotoxicity of carrier micelle is investigated using mtt assay.By the cell suspension of MDA-MB-231 or HEK293 plant in
In 96 orifice plates, per 10000, hole cell.After 24h is cultivated in the incubator, with the PBS 2 times of pH 7.4, add containing carrying medicine
The culture medium of micella or active compound adriamycin, the mass concentration for making contained adriamycin in system is 2 μ g/mL.When culture is certain
Between after, with the PBS 2 times of pH 7.4, add 100 μ L MTT solution (1mg/mL) to continue to cultivate 4h per hole.Discard in hole and train
Nutrient solution, 100 μ L DMSO are added per hole, vibrate 10min, measure the light absorption value (OD) in each hole at 490nm using ELIASA, are counted
Calculate cell survival rate.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
Claims (6)
1. the sugar-modified polymer micelle in a kind of surface, it is characterised in that the following sugar-modified polymer of application is prepared from, the sugar
Modified polymer has the structure shown in formula 1：
Wherein, n is 10-300；B is the one kind in the connecting key of following any carbohydrate and polymer：Ester bond, amido link, two sulphur
Key, ehter bond, carbonnitrogen bond or 1,3- triazole ringsZ in the formula 1 for it is following any one：Mannose, gala
Sugar, glucose, the lactose,-O- acetyl group of 1,2,3,4,6- five-the D-MANNOSE,-O- acetyl group -1- propargyls-α of 2,3,4,6- tetra- -
D-MANNOSE, propargyl-α-D- the mannopyranoses,-O- acetyl group -1- propargyl-α-D- galactolipins of 2,3,4,6- tetra-, 1,2,3,
- O- acetyl group-D- the galactolipins of 4,6- five, propargyl-α-D- the galactopyranoses ,-O- acetyl group-D- grapes of 1,2,3,4,6- five
The sugar,-O- acetyl group -1- propargyls of 2,3,4,6- tetra--alpha-D-glucose, propargyl-α-D- glucopyranoses.
2. the sugar-modified polymer micelle in surface according to claim 1, it is characterised in that the particle diameter of the micella is in 10-
Between 500nm.
3. the sugar-modified polymer micelle in surface according to claim 1, it is characterised in that the polymer micelle be by with
Lower section method is prepared：Purified by dialysis and gel chromatography, by controlling polymer molecule concentration in aqueous high
In after critical micelle concentration, ultrasonically treated promotion micelle forma-tion obtains final product polymer micelle solution.
4. the sugar-modified polymer micelle in surface described in claim 1 is as the application in terms of pharmaceutical carrier.
5. application according to claim 4, it is characterised in that the medicine is cancer therapy drug, antiviral drugs, treatment sugar
Urinate the medicine of disease or the medicine for the treatment of cardiovascular and cerebrovascular disease.
6. application according to claim 4, it is characterised in that the medicine is adriamycin or camptothecine.
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| Glycopolymers with secondary binding motifs mimic glycan branching and display bacterial lectin selectivity in addition to affinity;M. W. Jones et al.;《Chemical Science》;20140206;第5卷(第4期);1611-1616,supplementary information 1,7,9,38 * |
| Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery;Fabian Suriano et al.;《Biomaterials》;20100113;第31卷(第9期);2637-2645 * |
| Terminal functional glycopolymers via a combination of catalytic chain transfer polymerisation (CCTP) followed by three consecutive click reactions;Qiang Zhang et al.;《Polymer Chemistry》;20120203;第3卷;1016-1023 * |
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