CN105131277A - Polymer material containing cholic acid and liposome modified by same - Google Patents
Polymer material containing cholic acid and liposome modified by same Download PDFInfo
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- CN105131277A CN105131277A CN201510468166.0A CN201510468166A CN105131277A CN 105131277 A CN105131277 A CN 105131277A CN 201510468166 A CN201510468166 A CN 201510468166A CN 105131277 A CN105131277 A CN 105131277A
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Abstract
The invention provides a polymer material, which is represented by the formula I and contains cholic acid, and a preparation method thereof. The invention further provides a liposome, which takes phosphatide and cholesterol as the carriers and is modified by the polymer material represented by the formula I. Drugs are coated in the phosphatide bi-molecule layer. The provided liposome has double functions of active targeting and passive targeting; and furthermore, the preparation method is simple and practical and can be applied to massive production. The in-vivo experiment results show that the liposome has a strong targeting performance on livers and thus has a good application prospect.
Description
Technical field
The present invention relates to a kind of liposome containing cholic acid macromolecular material and modification thereof, belong to pharmaceutics field.
Background technology
For chronic diseases such as hepatitis, long term injections medication can bring a lot of misery to patient, oral administration is convenient, there is the plurality of advantages such as better conformability, and after oral administration, often medicine can not be delivered to effectively hepatic disease position, want to reach due curative effect, liver itself has tolerance to medicine in addition, and drug dose hour effect is very micro-; Heavy dose of medication can cause organ failure by grievous injury other organs undoubtedly.Medicine can be delivered to the diseased region of liver by oral hepatic-targeted delivery system effectively, reduces the distribution of its whole body simultaneously, improves the therapeutic index of medicine and reduces untoward reaction, having positive pushing effect to the treatment of hepatic diseases.Therefore, oral hepatic targeting drug preparation is developed current significant.
Drug-loaded liposome is a kind of important way realizing target administration.Liposome can protect drug molecule, by containing the material of specific functional groups in the finishing of liposome, by the receptor-ligand effect of cell surface, nano particle can be targeted to specific cell.
Cholic acid molecules circulates between human body liver sausage, promotes the absorption and digestion of lipid material.Human body cholic acid total amount, at 3 ~ 5g, circulates 6 ~ 10 times every day, and such every day, global cycle amount was 20 ~ 30g, and therefore the bile acid transporter expression amount of surface of hepatocytes is large, strong to cholic acid molecules effect; On the other hand in human body viscera, only have liver organization to express and have bile acid transporter, therefore cholic acid molecules and bile acid transporter have extremely strong tissue specificity.The cholic acid that this interaction force is strong, specificity is good-bile acid transporter system, can be used as the means of liver cell targeting drug delivery.
Chinese scholars with cholic acid be Liver targeting group develop multiple liver-targeted nanometer drug delivery system (Yuan Zhi, looks into auspicious great waves, Du Tian, etc. preparation method [P] .2006 of nano liver-target biodegradating medicine carrier material, CN1743008A; Jiang Guoqiang, Tang Shifu, Yu Yang, etc. a kind of containing the macromolecular material of cholic acid and liver targeted drug delivery nanoparticle [P] .2012, the CN102351967A of modification thereof).This nano material is strong not as liposome bilayers structure-biological consistency, and vivo degradation situation is unclear.Cholate-the PEG that had scholar to synthesize
2000-cholate, and modified liposome, aglucon physical adsorption is on liposome, stable structure can not be formed with liposome, destructurized (the Zhi-PengChen of possibility after oral administration, Jia-BiZhu, Hong-XuanChen, etal.Synthesisofanovelpolymerbilesalts-(polyethyleneglycol) 2000-bilesaltsanditsapplicationtotheliver-selectivetarge tingofliposomalDDB [J] .DrugDevelopmentandIndustrialPharmacy, 2010; 36 (6): 657 – 665).
The macromolecular material that Chinese invention patent CN103784965A and Chinese invention patent CN103735504A all adopts cholic acid to modify prepares nano-emulsion as target tumor target head, utilize this material as the aglucon strengthening cancer target inrichment, and and not mentioned its as liver target aglucon may.In addition, nano-emulsion contains a large amount of cosurfactant ethanol and propylene glycol, and pungency is large, poor storage stability.
The oral Liver targeting material of current synthesis, is prepared into the liposome administration system technology with oral active Liver targeting and there is not yet pertinent literature report.
Summary of the invention
The object of the invention is to overcome prior art above shortcomings, interact based on cholic acid-bile acid transporter, a kind of macromolecular material containing cholic acid molecules and preparation method thereof is provided, and modify drug-loaded liposome with it, obtain medicinal liposome of a kind of chlolic acid derivatives modification and preparation method thereof, obtain the liposome administration system with oral Liver targeting performance.
The object of the invention is to be achieved through the following technical solutions:
A macromolecular material containing cholic acid molecules, its structural formula is such as formula shown in I:
The preparation method of described macromolecular material comprises the steps:
Step a, is dissolved in cholic acid in DMF (DMF), reacts under room temperature;
Step b, by DSPE-amino-polyethyleneglycols (DSPE-PEG
2000-NH
2) be dissolved in organic solvent, then be added in DMF liquid, react under room temperature;
Step c, add water termination reaction, and purifying obtains the macromolecular material containing cholic acid molecules.
Further, described cholic acid and DSPE-PEG
2000-NH
2mass ratio be 1:1 ~ 10; Further, be 1:3 ~ 5.
Further, for forward step a reaction is carried out, catalyzer can be added, described catalyzer is selected from 2-(7-azo benzotriazole)-N, N, any one or a few in N', N'-tetramethyl-urea phosphofluoric acid ester (HATU), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), I-hydroxybenzotriazole (HOBT) or DMAP (DMAP).Further, described catalyzer is EDC.HCL, HOBT and DMAP.
Further, for forward step b reaction is carried out, can add catalyzer, described catalyzer is triethylamine.
Further, organic solvent described in step b is any one in methylene dichloride, trichloromethane, acetone, ethyl acetate.
Further, described purification process can adopt this area ordinary method, comprises extraction, gel column chromatography, dialysis etc.
The invention provides the liposome that a kind of cholic acid macromolecular material is modified, its raw material mainly comprises carrier, medicine, decorative material, it is characterized in that, described liposome vectors mainly comprises phosphatide and cholesterol, decorative material is formula I, by drug encapsulation in phospholipid bilayer.
Further, the concentration of described phosphatide is 1 ~ 20mg/mL; Further, be 5 ~ 10mg/mL; Further, be 8 ~ 10mg/mL.
Further, the mass ratio of described phosphatide and cholesterol is 3 ~ 10:1; Be further 5 ~ 10:1; Be further 8 ~ 10:1; Be further 10:1.
Further, the mass ratio of described medicine and phosphatide is 1:5 ~ 100; Further, the mass ratio of medicine and phosphatide is 1:5 ~ 20; Be further 1:8 ~ 15; Be further 1:8 ~ 10.
Further, the mass ratio of described formula I and phosphatide is 1:10 ~ 50; Further, the mass ratio of formula I and phosphatide is 1:15 ~ 30; Further, the mass ratio of formula I and phosphatide is 1:20.
Described medicine is selected from the fat-soluble medicines such as silibinin, curcumine, Quercetin, baicalin, cucurbitacin, podophyllotoxin, Oleanolic Acid, Cantharidin, hydroxycamptothecine.
Above-mentioned liposome adopts alcohol injection preparation, specifically comprises the steps:
Step a, prepares aqueous phase;
Step b, is dissolved in dehydrated alcohol by medicine, phosphatide, cholesterol, formula I, forms oil phase;
Step c, drops to oil phase in aqueous phase, forms the uniform dispersion emulsion of water oil;
Steps d, the dispersion emulsion obtained by step c carries out ultrasonic disperse after removing ethanol, namely obtains liposome.
Further, aqueous phase described in step a is 1: 1 ~ 5 with oil phase volume ratio described in step b;
Further, the concentration of phosphatide described in step b is 1 ~ 20mg/mL; Further, be 5 ~ 10mg/mL; Further, be 8 ~ 10mg/mL.
Further, the mass ratio of phosphatide described in step b and cholesterol is 3 ~ 10:1; Be further 5 ~ 10:1; Be further 8 ~ 10:1; Be further 10:1.
Further, the mass ratio of described medicine and phosphatide is 1:5 ~ 100; Further, the mass ratio of medicine and phosphatide is 1:5 ~ 20; Be further 1:8 ~ 15; Be further 1:8 ~ 10.
Further, the mass ratio of described formula I and phosphatide is 1:10 ~ 50; Further, the mass ratio of formula I and phosphatide is 1:15 ~ 30; Further, the mass ratio of formula I and phosphatide is 1:20.
Further, the particle diameter of described liposome administration system is 50 ~ 400nm.
Further, after formation liposome, add lyophilized vaccine, described lyophilized vaccine is selected from glucose-N.F,USP MANNITOL or sucrose; Further be preferably sucrose.
Advantage of the present invention and beneficial effect:
1, the present invention has prepared the composite drug administration system of the cholic acid modification having active targeting and passive target double effects concurrently.
2, present invention achieves cholic acid molecules C
24position carboxyl scion grafting to main polymer chain does not affect the identification of bile acid transporter to wherein cholic acid molecules structure.The principle of building-up process and simple to operate.Material of the present invention has amphipathic, is convenient to be modified in liposomal phospholipids bilayer.Material has good biocompatibility, can be targeted to the specific function of liver in conjunction with it, is a kind of decorative material of desirable liver-targeted nanometer drug-loading system.
The modification of the hydrophilic polyglycol 3, in material of the present invention effectively can avoid the non-specific adsorption with cell and albumen.
4, the preparation method of this liposome administration system is simple and practical, is easy to amplify produce, and the liposome that experiment in vivo display cholic acid is modified has stronger target ability to liver and has a good application prospect.
Accompanying drawing explanation
Fig. 1 is cholic acid macromolecular material qualitative identification thin-layer chromatogram.
Figure A developer is phospho-molybdic acid, and 1 is cholic acid, and 2,3,4 is synthetic product;
Figure B developer is iodine vapor, and 1 is cholic acid, and 2 is DSPE-PEG
2000-NH
2, 3 is synthetic product;
Figure C developer is triketohydrindene hydrate, and 1 is cholic acid, and 2 is DSPE-PEG
2000-NH
2, 3,4 is synthetic product.
Fig. 2 is liposome transmission electron microscope picture.
Fig. 3 is the different preparations of silibinin release in vitro results under pH2 condition.
Fig. 4 is the different preparations of silibinin release in vitro results under pH7.4 condition.
Embodiment
The synthesis of embodiment 1 cholic acid macromolecular material
Take 25mg cholic acid and be dissolved in 4mLN, in dinethylformamide (DMF), add 14mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), 10mg1-hydroxybenzotriazole (HOBT) and 0.37mg4-Dimethylamino pyridine (DMAP), under room temperature, react 1h.Take 90mgDSPE-PEG
2000-NH
2be dissolved in 4mL methylene dichloride (DCM), DCM liquid be added at a slow speed in DMF liquid, under room temperature, react 48h.By the termination reaction that adds water in reaction system.
The synthesis of embodiment 2 cholic acid macromolecular material
Take 8.7mg cholic acid and be dissolved in 3mLN, in dinethylformamide (DMF), add 12.2436mg2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester (HATU), take 40mgDSPE-PEG
2000-NH
2with triethylamine 6.5mg, be dissolved in 3mL methylene dichloride (DCM), DCM liquid be added at a slow speed in DMF liquid, under room temperature, react 30min.By the termination reaction that adds water in reaction system.
The synthesis of embodiment 3 cholic acid macromolecular material
Take 25mg cholic acid and be dissolved in 4mLN, in dinethylformamide (DMF), add 14mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), 8.4mgN-N-Hydroxysuccinimide (NHS), under room temperature, react 4h.Take 90mgDSPE-PEG
2000-NH
2be dissolved in 4mL methylene dichloride (DCM), DCM liquid be added at a slow speed in DMF liquid, under room temperature, react 48h.By the termination reaction that adds water in reaction system.
The synthesis of embodiment 4 cholic acid macromolecular material
Take 45mg cholic acid and be dissolved in 4mLN, in dinethylformamide (DMF), add 14mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), 10mg1-hydroxybenzotriazole (HOBT) and 0.37mg4-Dimethylamino pyridine (DMAP), under room temperature, react 1h.Take 90mgDSPE-PEG
2000-NH
2be dissolved in 4mL trichloromethane, DCM liquid be added at a slow speed in DMF liquid, under room temperature, react 48h.By the termination reaction that adds water in reaction system.
The purifying of embodiment 5 cholic acid macromolecular material
The product of Example 1 ~ 3 preparation, extracts with DCM respectively; Extraction liquid pours 5%NaHCO into
3unnecessary cholic acid is removed in solution (100mL × 3 time); Then DCM layer water (100mL × 2 time), 0.1mol/L hydrochloric acid (100mL × 2 time), water (100mL × 2 time), saturated aqueous common salt (100mL × 2 time) wash successively, anhydrous sodium sulfate drying, filtration, decompression and solvent recovery.Dissolve with methanol, upper gel column sephdexLH-20.Lyophilize obtains white solid.
The purified rear ultimate yield of embodiment 1 ~ 3 is respectively 70%, 60%, 30%.
The purifying of embodiment 6 cholic acid macromolecular material
The product of Example 1 ~ 3 preparation, extracts with DCM respectively; Extraction liquid pours 5%NaHCO into
3unnecessary cholic acid is removed in solution (100mL × 3 time).Then DCM layer water (100mL × 2 time), 0.1mol/L hydrochloric acid (100mL × 2 time), water (100mL × 2 time), saturated aqueous common salt (100mL × 2 time) wash successively, anhydrous sodium sulfate drying, filtration, decompression and solvent recovery.Gained solids DMF dissolution with solvents, molecular weight cut-off is 1000 dialysis tubing dialysis 48h.Lyophilize obtains white solid.
The preparation of embodiment 7 silibinin liposome
Silibinin 4mg, soybean phospholipid 40mg, cholesterol 4mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 10mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 5 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 147nm, and drug loading is 8.33%.
The preparation of embodiment 8 silibinin liposome
Silibinin 3mg, soybean phospholipid 40mg, cholesterol 8mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 5mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 1 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 170.4nm, and drug loading is 5.88%.
The preparation of embodiment 9 silibinin liposome
Silibinin 3.5mg, soybean phospholipid 40mg, cholesterol 10mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 8mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 163.0nm, and drug loading is 6.54%.
Prepared by embodiment 10 curcumin liposome
Bisdemethoxycurcumin .5mg, soybean phospholipid 40mg, cholesterol 6.7mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 113.9nm, PDI=0.260, and drug loading is 6.7%.
Embodiment 11 Cantharidin liposomal preparation
Cantharidin 3.5mg, soybean phospholipid 40mg, cholesterol 6.7mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 61.68nm, PDI=0.239, and drug loading is 6.7%.
Embodiment 12 Quercetin liposomal preparation
Quercetin 4mg, soybean phospholipid 40mg, cholesterol 8mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 133.2nm, PDI=0.289, and drug loading is 7.4%.
Prepared by embodiment 13 oleanolic acid liposome
Oleanolic Acid 2mg, soybean phospholipid 40mg, cholesterol 10mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 135.6nm, PDI=0.213, and drug loading is 3.7%.
Prepared by embodiment 1410-Hydroxycamptothecin liposome
10-hydroxycamptothecine 0.5mg, soybean phospholipid 40mg, cholesterol 10mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 150.4nm, PDI=0.272, and drug loading is 1%.
Embodiment 15 trans-resveratrol liposomal preparation
Trans-resveratrol 4mg, soybean phospholipid 40mg, cholesterol 6.7mg, cholic acid macromolecular material 2mg are dissolved in dehydrated alcohol, phospholipid concentration is 20mg/ml, form oil phase, at 50 DEG C, slowly dropped in the aqueous phase of tween 80 by oil phase under magnetic agitation 800r/min, aqueous phase is 1: 2 with oil phase volume ratio; Drip off rear continuation and stir removal ethanol; After ethanol volatilizes completely, carry out ultrasonic disperse by ultrasonic apparatus, namely obtain liposome.Particle diameter is 114.6nm, PDI=0.253, and drug loading is 7.59%.
Embodiment 16
Liposome prepared by Example 7, according to sucrose: the ratio of phosphatide=3:1 adds sucrose, lyophilize.After redissolving, particle diameter is 125.4nm, PDI=0.269.
Embodiment 17
Liposome prepared by Example 8, according to glucose-N.F,USP MANNITOL (3:1): the ratio of phosphatide=5:1 adds glucose-N.F,USP MANNITOL, lyophilize.After redissolving, particle diameter is 153.7nm, PDI=0.344.
Embodiment 18
Prepare in liposome process in embodiment 7, add lyophilized vaccine sucrose in aqueous phase, the ratio of described lyophilized vaccine sucrose and phosphatide is 3:1.After lyophilize redissolution, particle diameter is 108.5nm, PDI=0.2.
The qualification of embodiment 19 cholic acid macromolecular material
Product embodiment 1 prepared is identified as follows by gained white solid after embodiment 5 purifying:
1 thin layer detects
Product is dissolved in methylene dichloride, developping agent: methylene dichloride: methyl alcohol: water: Glacial acetic acid (3:1:0.5:0.05), developer: 1. 5% phospho-molybdic acid (cholic acid and product colour developing can be made); 2. iodine steam (can make cholic acid, DSPE-PEG
2000-NH
2all develop the color with product); 3. triketohydrindene hydrate (can make DSPE-PEG
2000-NH
2colour developing).
Result: see Fig. 1.Wherein, figure A is phospho-molybdic acid colour developing result, and phospho-molybdic acid can make cholic acid and the colour developing of formula I, as seen from the figure, has formula I to produce in synthetic product;
Figure B is iodine vapor colour developing result, and iodine vapor can make raw material cholic acid, DSPE-PEG
2000-NH
2all develop the color with formula I, as seen from the figure, not containing raw material cholic acid and DSPE-PEG in synthetic product
2000-NH
2;
Figure C is triketohydrindene hydrate colour developing result, and triketohydrindene hydrate can make raw material DSPE-PEG
2000-NH
2free-NH
2colour developing, as seen from the figure, synthetic product does not develop the color, and free-NH is described
2with cholic acid C
24-the COOH of position there occurs reaction, defines amido linkage, and figure C shows the cholic acid macromolecular material finally synthesized shown in formula I, and cholic acid molecules passes through C
24position carboxyl scion grafting is on main polymer chain.
2
1h-NMR characterizes
Take a certain amount of synthetic product, with deuterated trichloromethane for solvent, carry out
1h-NMR (600MHz) analyzes.
From
1find out in H-NMR figure, δ (ppm) 4.457-4.437 (t, 2H) is DSPE-PEG
2000-NH
2on methylene radical, δ (ppm) 0.715 (s) is CH on cholic acid
3, illustrate all have cholic acid and DSPE-PEG
2000-NH
2characteristic peak, cholic acid peak area is methylene radical 1.10 times, illustrates that synthetic product purity is probably: 2 × 1.10 ÷ 3 × 100%=73%.
3 infrared measurements
IR (v, cm-1): 3434.98vs (v ,-OH); 3479.34vs (v ,-OH); 3507.31vs (v, amido-NH-C=O); 2918.10vs, 2872.77vs, 2850.59vs (v, Alkyl); (1738.71s v, O=C-O); 1651.92s, 1344.29s (v, carbonyl O=C-NH); 1459.05,1467.73s (δ, Alkyl); 111.89s (v, C-O); 952.77,842.83 (r, Alkyl)
Without-COOH charateristic avsorption band (s, 2500-3500cm
-1), there is amido linkage characteristic peak ,-COOH and-NH are described
2define amido linkage.
4MALDITOF mass spectral characteristi
Solid nitrogen laser emission wavelength is 355nm, and whole experiment matrix used is the saturated solution of a-cyano group-4-hydroxycinnamic acid in tetrahydrofuran (THF), DSPE-PEG
2000-NH
2to be dissolved in chloroform the concentration being made into 10mg/mL for subsequent use with Product samples, and reflector mode is positive ion mode, and acceleration voltage is 20KV.
Starting material and product MALDI-TOF mass spectroscopy, the composition being applicable to polydisperse polymer, non uniform is analyzed, and the molecular-weight average due to starting material MALDI is 2986, and the molecular-weight average of synthetics MALDI is 3348, so raw-material synthesis is successful.
Comprehensive above qualification result, show that building-up reactions finally generates formula I, and cholic acid molecules passes through C
24-the COOH of position and DSPE-PEG
2000-NH
2-NH in molecule
2to form amido linkage scion grafting on main polymer chain.
The safety evaluation of embodiment 20 cholic acid macromolecular material
The product that Example 1 synthesizes carries out by embodiment 5 the cholic acid macromolecular material (DSPE-PEG-cholic acid) that purifying obtains and tests as follows.
To take the logarithm cell in vegetative period, blow and beat into single cell suspension gently after 0.25% tryptic digestion, adjustment cell concn is 5 × 10
4individual/mL, is inoculated in 96 orifice plates, every hole 200 μ L, inoculates 2 pieces of culture plates altogether, is placed in 37 DEG C, 5%CO
2, cultivate in saturated humidity incubator, after cell attachment, discard original fluid, add different substances, grouping situation is as follows: blank control wells, not inoculating cell, only add nutrient solution, the MTT solution containing DMSO; Solvent control hole: inoculating cell, adds nutrient solution, the MTT solution containing DMSO; Test group, inoculating cell, adding containing DSPE-PEG-Bile acid concentrations is 5,10,25,40,55,70,85,100 μ g/mL nutrient solution 200 μ L, often group establishes 6 multiple holes, after cultivating 24h, discard substratum, add 200uL substratum, 20 μ LMTT (5mg/mL) cultivate 4h, sucking-off supernatant liquor, every hole adds 150 μ LDMSO, and the purple crystal produced in cell is fully dissolved, survey OD value by microplate reader at wavelength 570nm place, calculate cell survival rate.
Experimental result shows, material when concentration is lower than 85 μ g/mL, to colon cancer cell Caco-2 and human liver cancer cell HepG-224 hour equal free of toxic effects.
Table 1 synthetic materials safety evaluation result (n=6)
Embodiment 21 liposomal particle size measures and transmission electron microscope observing form
The present embodiment liposome used is liposome prepared by embodiment 7
1 particle size determination
Get liposome liquid, by the size distribution under Malvern Nano-ZS particle size analyzer determination 25 DEG C of conditions, parallel 3 times, each 12 circulations.Median size is 147nm, PDI=0.27.
2 transmission electron microscopes
The liposome liquid of preparation 1mg/mL silibinin concentration, dilutes 1000 times, draws 6 μ L and drip on 300 object copper mesh, naturally dry in air, and rear use 2% phospho-wolframic acid dyeing 10min, observes particle shape under transmission electron microscope.See Fig. 2.
As can be seen from Electronic Speculum, said preparation has bilayer structure, illustrates that the preparation formed is liposome.
Study on the stability in embodiment 22 preparation simulated gastric fluid, simulated intestinal fluid, rat plasma
The liposome that the present embodiment " modified liposome " used is prepared for embodiment 7; Lack target material (cholic acid macromolecular material) in " conventional liposome " prescription, all the other are identical with embodiment 7 method for preparing lipidosome.
The stability of 1 silibinin under pH2 and 7.4 conditions
Preparation 3%SDS solution, pH is regulated to be 2 and 7.4 respectively, get appropriate silibinin bulk drug in above-mentioned solution, with 0.45 μm of water system membrane filtration, filtrate is placed in 37 DEG C of water-baths, respectively at 0,1,2,3,4,5,6,12, measure content under 24h condition, calculate RSD value, under pH2 condition, 24h stability RSD is 1.45%, pH7.4 condition lower 24 hours stability RSD is 3.35%.
Result shows, silibinin has good stability for 24 hours under pH2 and 7.4 conditions.
2 liposomes are study on the stability in simulated gastric fluid, simulated intestinal fluid, rat plasma
Simulated gastric fluid: get dilute hydrochloric acid 16.4mL, add water about 800mL and stomach en-10g, and after shaking up, the title that adds water is interpreted into 1000ml and get final product.Wherein, dilute hydrochloric acid is 1mol/L hydrochloric acid.
Simulated intestinal fluid: get potassium primary phosphate 6.8g and to add water 500mL.Sodium hydroxide solution with 0.4% regulates pH to 6.8; Separately get pancreatin 10g to add water and make dissolving in right amount, after two liquid mixing, add water and be settled to 1000mL.
Liposome turbid liquor is joined in equal-volume simulated gastric fluid and cultivate 2h under 37 DEG C of conditions, and then join in simulated intestinal fluid and cultivate 6h under 37 DEG C of conditions.2h, 4h, 8h, 12h sampling and measuring particle diameter in 0h, 1h, 2h, 4h, 6h and rat plasma in 0h, 0.5h, 1h, 1.5h, 2h and simulated intestinal fluid in simulated gastric fluid respectively, and measure encapsulation rate.
The results are shown in Table 2-4.Result shows, two kinds of liposomes under simulated gastric fluid, simulated intestinal fluid and rat plasma condition, its particle diameter and encapsulation rate there are no significant change.
Stability result (n=2) in table 2 simulated gastric fluid
Stability result (n=2) in table 3 simulated intestinal fluid
Stability result (n=2) in table 4 rat plasma
Note: in table 2-4, "-" is for measure
3 liposome release in vitro are investigated
3.1 silibinins are equilibrium solubility in 3%SDS solution
Get excessive silibinin in the 10mL3%SDS aqueous solution, ultrasonic 30min, sampling, carry out assay, sample introduction 10 μ L, under 37 DEG C of water-soluble conditions, reciprocating frequence 100rpm, samples after 24h, 0.45 μm of filtering with microporous membrane, sample introduction 10 μ L.
Experimental result: after ultrasonic 30min, silibinin concentration is 84.00ug/mL, after balance dissolving 24h, concentration is 107.48 μ g/mL.
Therefore release in vitro investigation will be carried out containing 1mg silibinin liposome in 100mL3%SDS release liquid, meet sink conditions.
3.2 release in vitro are investigated
Absorption 1ml silibinin bulk drug and modified liposome are placed in the dialysis tubing processed respectively, sealing, are placed on 100ml containing in the PBS solution of 3% sodium lauryl sulphate, pH is regulated to be 2 and 7.4 respectively, dialyse under 37 DEG C of 100rpm magnetic agitation, get 1mL at every turn, supply 1mL.Sampling spot 0.5,1,2,4,6,8,12,24, measures cumulative release percentage.
The results are shown in Table 5-6, Fig. 3-4.Result shows: silibinin bulk drug release in vitro is very fast, 4h discharges completely substantially, and modified liposome slow releasing under pH=2 and 7.4 conditions, said preparation is described, and stability is better in the gastrointestinal tract, is more conducive to medicine with complete formutatibh form target in liver.
The different preparations of table 5 silibinin release in vitro result (n=3) under pH2 condition
The different preparations of table 6 silibinin release in vitro result (n=3) under pH7.4 condition
The research of the distribution in vivo experiment of the silibinin drug-loaded liposome that embodiment 23 cholic acid macromolecular material is modified
The foundation of silibinin analytical procedure in 1 mice plasma sample
1.1 chromatographic condition
Chromatographic column: C
18chromatographic column (Kromasil, 250 × 4.6mm, 5 μm); Moving phase: methyl alcohol-1% acetic acid water (48:52); Determined wavelength: 287nm; Flow velocity: 1.0mLmin
-1; Column temperature: 30 DEG C.
The Extraction and isolation of 1.2 mice plasma samples
Draw mice plasma 100 μ L, add 100 μ L1mol/L Sodium phosphate dibasic 1mL, vortex 1min, adds 1mL extracted with diethyl ether, eddy mixer vibration 3min, 10000rmin
-1high speed centrifugation 10min, Aspirate supernatant, coextraction twice, merges supernatant liquor, N
2dry up, 100uL moving phase is redissolved, 40 μ L sample introductions.
1.3 chromatographic behavior
Get blank mice plasma, add the mice plasma of silibinin reference substance and oral silibinin after mice plasma, by processing under " 1.2 " item, sample introduction analysis, result shows, in mice plasma, impurity does not disturb silibinin sample tests.
The preparation of 1.4 mice plasma sample standard curves
Precision takes 4.26mg silibinin reference substance in 25mL measuring bottle, and dissolve with methanol is also diluted to scale, and obtaining concentration is 170.4 μ g/mL storing solutions.Get above-mentioned storing solution, being diluted to concentration respectively with methyl alcohol is 0.6656,1.33125,2.6625,5.325,10.65,21.3,42.6,85.2 μ g/mL solution, respectively getting 10 μ L adds in blank 1.5mL centrifuge tube, add mouse blank plasma 100 μ L, vortex mixed 30s, process by under " 1.2 " item, sample introduction analysis.Press with silibinin concentration C (μ g/mL) for X-coordinate, with silibinin peak area A for ordinate zou, carry out linear regression, regression equation is Y=120362 – 2960.8, r=0.9991.Show that in mice plasma, silibinin concentration is good in 0.066 ~ 8.52 μ g/mL linear relationship, peak area and plasma drug level are good linear relationship.
1.5 the rate of recovery
The method rate of recovery: get silibinin reference substance solution 10 μ L and put in 1.5mL centrifuge tube, adds blank mice plasma 100 μ L, make silibinin concentration in blood plasma be respectively 0.26625,2.13,8.52ug/mL, add 1mL extracted with diethyl ether.Process by under " 1.2 " item, sample introduction analysis.Substitute into typical curve and calculate the blood plasma content of dispersion recorded, compare with theoretical value, the method for calculation rate of recovery, the results are shown in Table 7.The method rate of recovery meets Pharmacokinetic experiments requirement.
Table 7 plasma sample method determination of recovery rates result (n=3)
Recovery of extraction: get silibinin reference substance solution 10 μ L and put in 1.5mL centrifuge tube, adds blank mice plasma 100 μ L, make silibinin concentration in blood plasma be respectively 0.26625,2.13,8.52ug/mL.Process by under " 1.2 " item, sample introduction analysis.Separately substitute with distilled water the preparation that blank plasma carries out aforementioned sample, sample introduction analysis.The plasma sample peak area of each for gained concentration and distilled water sample peak area are compared, calculates recovery of extraction, the results are shown in Table 8.Recovery of extraction meets Pharmacokinetic experiments requirement.
Table 8 plasma sample recovery of extraction measurement result (n=3)
1.6 precision
Getting silibinin reference substance solution 10 μ L puts in 1.5mL centrifuge tube, air blow drying, adds blank mice plasma 100 μ L, make silibinin concentration in blood plasma be respectively 0.26625,2.13,8.52ug/mL.Each concentration prepares 5 increment product, processes by under " 1.2 " item.By the obtained sample mix of similar concentration level.Each concentration is in early, middle and late and second day, the 3rd day difference sample introduction, and gained peak area substitutes into typical curve calculating drug level and also compares, and calculates withinday precision and day to day precision, the results are shown in Table 9-10.Precision meets Pharmacokinetic experiments requirement.
Table 9 withinday precision (n=3)
Table 10 day to day precision (n=3)
1.7 matrix effect
Get blank plasma, after plasma sample disposal methods, add and mix reference substance solution (n=3) with each of Quality Control sample equivalent, dry up redissolution; Separately get the mixing reference substance solution of above-mentioned basic, normal, high concentration, dry up redissolution.By two kinds of sample feedings, calculate peak area ratio.Result is as shown in table 11, shows that the matrix effect of basic, normal, high concentration samples all meets Pharmacokinetic experiments requirement.
Table 11 plasma sample matrix effect investigates result (n=3)
1.8 stability
1.8.1 plasma containing drug stability: get silibinin reference substance solution 10 μ L and put in 1.5mL centrifuge tube, air blow drying, adds blank mice plasma 100 μ L, make silibinin concentration in blood plasma be respectively 0.26625,2.13,8.52ug/mL, prepare each 5 parts of plasma containing drug, mixing, room temperature is placed.Draw 100 μ L plasma containing drugs respectively in 0,12,24h, process by under " 1.2 " item, sample introduction analysis.Gained peak area substitutes into typical curve calculating concentration, compares the change in concentration of different time, evaluates the stability of plasma containing drug.The results are shown in Table 12.Plasma containing drug stability meets Pharmacokinetic experiments requirement.
Table 12 is containing silibinin plasma sample stability (n=3)
1.8.2 freeze-thaw stability: get silibinin reference substance solution 10 μ L and put in 1.5mL centrifuge tube, air blow drying, add blank mice plasma 100 μ L, make silibinin concentration in blood plasma be respectively 0.26625,2.13,8.52ug/mL, prepare each 3 parts of plasma containing drug, multigelation 3 aftertreatments in-20 DEG C of refrigerators, investigate multigelation stability.The results are shown in Table 13.Stability meets Pharmacokinetic experiments requirement.
Table 13 plasma sample freeze-thaw stability investigates result (n=3)
1.9 lowest detectable limit
Under this experiment chromatographic condition, be minimumly quantitatively limited to 66ng/mL (S/N >=3), reach the requirement of silibinin In vivo analysis.
The foundation of 2 mouse liver sample analysis methods
2.1 chromatographic condition
Chromatographic condition is with " 1.1 ".
The Extraction and isolation of 2.2 mouse liver samples
Mouse is isolated mouse liver rapidly, carefully blots the remained blood on liver, weigh after taking off cervical vertebra execution.Physiological saline is added, homogenate in the ratio of 1.5mL/g.Get homogenate 1mL, add 1mL1mol/L disodium phosphate soln, vortex mixing 1min, adds 2mL extracted with diethyl ether, eddy mixer vibration 3min, 12000rmin-1 high speed centrifugation 10min, Aspirate supernatant, extracting twice, combining extraction liquid, N
2dry up, add 0.5mL acetonitrile precipitation albumen, vortex 1min, 12000rmin-1 high speed centrifugation 10min, Aspirate supernatant, N
2dry up, 100 vibration of μ L moving phase solution 3min, 12000rmin-1 high speed centrifugation 10min, get supernatant liquor 40 μ L sample introduction.
2.3 chromatographic behavior
Get blank mouse liver tissue homogenate, add the mouse liver tissue homogenate of silibinin reference substance and oral administration after mouse liver tissue homogenate, process by under " 2.4.2 " item, sample introduction is analyzed, the relatively color atlas of three, result shows that in mouse liver, impurity does not disturb silibinin sample tests.
The preparation of 2.4 mouse liver sample standard curves
Get blank mouse liver, add physiological saline homogenate by the amount of 1.5mL/g, obtain blank hepatic homogenate liquid.In 4mL centrifuge tube, add concentration is respectively 0.665625,1.33125,2.6625,5.325,10.65,21.3,42.6,85.2 μ g/mL silibinin reference substance 10 μ L, respectively add the blank hepatic homogenate liquid of 1mL, vortex mixed 30s, 13.3125,26.625,53.25,106.5,213,426,852,1704ng/g, process by under " 2.2 " item, sample introduction analysis.Pressing silibinin content C (ng/g) in liver is X-coordinate, with silibinin peak area A for ordinate zou, carries out linear regression.Obtaining regression equation is A=595.44C-4234.4, r=0.9989.Show that in mouse liver, silibinin concentration is in 13.31 ~ 1704ng/g, peak area and liver drug concentration are good linear relationship.
2.5 the rate of recovery
The method rate of recovery: get silibinin reference substance solution and put in 4mL centrifuge tube and dry up, add blank mouse liver tissue homogenate 1mL, obtain liver drug concentration be 53.25,426,1704ng/g hepatic homogenate liquid.Process by under " 2.2 " item, sample introduction analysis.Substitute into typical curve and calculate the liver medicine content recorded, compare with theoretical value, the method for calculation rate of recovery, the results are shown in Table 14.The method rate of recovery meets the requirement of liver medicine dynamic experiment.
Table 14 method rate of recovery result (n=3)
Recovery of extraction: get silibinin reference substance solution 10 μ L and put in 1.5mL centrifuge tube, add blank mouse liver tissue homogenate 1mL, obtain liver drug concentration be 53.25,426,1704ng/g.Process by under " 1.2 " item, sample introduction analysis.Separately substitute with distilled water the preparation that blank liver carries out aforementioned sample, sample introduction analysis.The liver specimens peak area of each for gained concentration and distilled water sample peak area are compared, calculates recovery of extraction, the results are shown in Table 15.Recovery of extraction meets Pharmacokinetic experiments requirement.
Table 15 recovery of extraction result (n=3)
2.6 precision
Get silibinin reference substance solution 10 μ L to put in 1.5mL centrifuge tube, add blank mouse liver tissue homogenate 1mL, obtain liver drug concentration be 53.25,426,1704ng/g.Each concentration prepares 5 increment product, processes by under " 2.2 " item.By the obtained sample mix of similar concentration level.Each concentration is in early, middle and late sample introduction respectively, and gained peak area substitutes into typical curve and calculates drug level and compare, and calculates withinday precision and day to day precision, the results are shown in Table 16-17.Precision meets Pharmacokinetic experiments requirement.
Table 16 withinday precision (n=3)
Table 17 day to day precision (n=3)
2.7 matrix effect
Get blank hepatic homogenate liquid, after liver specimens disposal methods, add each reference substance solution (n=3) with Quality Control sample equivalent, dry up redissolution; Separately get the reference substance solution of above-mentioned basic, normal, high concentration, dry up redissolution.By two kinds of sample feedings, calculate peak area ratio.The results are shown in Table 18.It is as shown in the table, shows that the matrix effect of basic, normal, high concentration samples all meets the requirements.
Table 18 matrix effect experimental result (n=3)
2.8 stability
2.8.1 pastille hepatic homogenate liquid stability
Getting silibinin reference substance solution 10 μ L puts in 1.5mL centrifuge tube, add blank mouse liver tissue homogenate 1mL, make silibinin concentration in hepatic homogenate liquid be respectively 53.25,426,1704ng/g, prepare each 5 parts of pastille hepatic homogenate liquid, mixing, room temperature is placed.Draw 1mL pastille hepatic homogenate liquid respectively in 0,12,24h, process by under " 2.2 " item, sample introduction analysis.Gained peak area substitutes into typical curve calculating concentration, compares the change in concentration of different time, evaluates the stability of pastille hepatic homogenate liquid.The results are shown in Table 19.Pastille hepatic homogenate liquid stability meets Pharmacokinetic experiments requirement.
Table 19 investigates (n=3) containing silibinin hepatic homogenate liquid chamber temperature shelf-stability
2.8.2 freeze-thaw stability
Getting silibinin reference substance solution 10 μ L puts in 1.5mL centrifuge tube, add blank mouse liver tissue homogenate, make silibinin concentration in hepatic homogenate liquid be respectively 53.25,426,1704ng/g, prepare each 3 parts of pastille hepatic homogenate liquid, multigelation 3 aftertreatments in-20 DEG C of refrigerators, investigate multigelation stability.The results are shown in Table 20.
Table 20 investigates (n=3) containing silibinin hepatic homogenate liquid freeze-thaw stability
2.9 lowest detectable limit
Under this experiment chromatographic condition, be minimumly quantitatively limited to 13.31ng/g (S/N >=3), reach the requirement of silibinin In vivo analysis.
3 administrations
With male weight range the ICR mouse of 18-22g for animal model, mouse is divided into 3 groups, often organizes 18.The preparation being given suitable 100mg/kg dosage silibinin respectively by gavage (is respectively silibinin bulk drug group, silibinin conventional liposome group, silibinin modified liposome group), the time period set, often organize taking-up 3 mouse, after eye socket gets blood, de-neck is put to death, take out liver, extract the silibinin in blood plasma and liver, and measured by HPLC-UV, calculate the content of silibinin in blood plasma and liver, and calculate target index, selectivity index, target efficiency and relative target efficiency.Calculation formula is as follows:
After DTI (target index)=give targeting preparation T moment I organ the non-targeted preparation of medication amount/give after the medication amount of T moment I organ
Medication amount/T moment the blood of DSI (selectivity index)=T moment target organ or the medication amount of non-target organ
Below the Drug-time curve of DTE (target efficiency)=target organ/Drug-time curve of blood or non-target organ
After RTE (relative target efficiency)=give targeting preparation, after the area under the drug-time curve of target organ/give non-targeted preparation, the area under the drug-time curve of target organ
Note: the liposome that silibinin modified liposome adopts embodiment 7 to prepare; Lack cholic acid macromolecular material in the prescription of silibinin conventional liposome, all the other are identical with embodiment 7 method for preparing lipidosome.
Experimental result is in table 21.
Table 21 silibinin and preparation oral Liver targeting parameter thereof
Experimental result shows: compare with conventional liposome with silibinin solution, and the Targeting Effect of modified liposome drug delivery system significantly improves, and illustrates that this drug delivery system not only has passive target effect, has active targeting in liver effect simultaneously.
The silibinin drug-loaded liposome Bel7402 HepG-2 that embodiment 24 cholic acid macromolecular material is modified absorbs experiment
1 tumor cell of liver is to the absorption of liposome
1.1 methodology
1.1.1 the preparation of lysate
Get PMSF-SDN (1:100) and be made into cell pyrolysis liquid, for subsequent use.
1.1.2 the preparation of reference substance solution
Accurate title silibinin 85.2 μ g/mL reference substance solution.The reference substance solution that concentration is 0.0852,0.852,3.408 μ g/mL is diluted to before use with blanc cell suspension after cracking.
1.1.3 sample determination
Get lysis sample, the centrifugal 10min of 10000r/min, get the analysis of supernatant liquor 40 μ L sample introduction.
1.1.4 extraction recovery
By the method under " 1.1.3 " item, be each 3 parts of the quality-control sample of 0.0852,0.852,3.408 μ g/mL containing silibinin with the preparation of blanc cell cracking suspension; Meanwhile, with the sample of lysate preparation with concentration, sample introduction 40 μ L, measures in accordance with the law and records peak area respectively.Compare the average peak area of silibinin in 3 parts of lysis samples and lysate sample, calculate extraction recovery.
Table 22 extraction recovery
1.1.5 precision
By the method under " 1.1.3 " item, be each 1 part of the quality-control sample of 0.0852,0.852,3.408 μ g/mL containing silibinin with the preparation of blanc cell cracking suspension, process in accordance with the law and measure, every day is early, middle and late respectively once, once a day, and METHOD FOR CONTINUOUS DETERMINATION 3d.Calculate RSD, to record in a few days, day to day precision.
Table 23 withinday precision
Table 24 day to day precision
1.1.6 stability
Be each 1 part of the quality-control sample of 0.0852,0.852,3.408 μ g/mL with blanc cell cracking suspension preparation silibinin, room temperature place 0,1,2,4,6,8,12h, calculate RSD value.
Table 25 study on the stability
1.1.7 cellular uptake kinetics
By HepG2 cell cultures in six orifice plates, every hole 10
6individual cell, in 37 DEG C, 5%CO
224h is cultivated in incubator.Change and do not continue to cultivate more than 30min containing the RPMI1640 substratum of serum, add silibinin solution again, conventional liposome, modified liposome, makes its final concentration be 50 μ g/mL, continues in incubator and cultivate, after 2h, remove substratum, 4 DEG C of cold PBS solution rinse twice, termination test.Add 200 μ L cell pyrolysis liquids, place 30min, pipette samples is in 1.5mL plastic centrifuge tube, and the centrifugal 10min of 10000r/min, gets supernatant liquor 100 μ L HPLC method and measure medicament contg, remain 100 μ L BCA kit measurement protein contents.In the sample cell of every hole, the concentration of medicine is divided by every hole total protein concentration standardized data, represents with μ g/ μ gprotein.
Table 26 absorbs experimental result (n=3)
Experimental result shows: compare with conventional liposome with silibinin solution, the hepatocyte picked-up of modified liposome drug delivery system improves 7.4 and 3.2 times respectively, illustrate that this drug delivery system not only has passive target effect, there is active targeting in the effect of liver cancer cell simultaneously.
Claims (10)
1. the macromolecular material containing cholic acid molecules, its structural formula is such as formula shown in I:
2. the preparation method of macromolecular material as claimed in claim 1, it is characterized in that, the method comprises the steps:
Step a, is dissolved in cholic acid in DMF, reacts under room temperature;
Step b, by DSPE-PEG
2000-NH
2be dissolved in organic solvent, then be added in DMF, react under room temperature;
Step c, add water termination reaction, purifying, obtains the macromolecular material containing cholic acid molecules.
3. preparation method as claimed in claim 2, is characterized in that, described cholic acid and DSPE-PEG
2000-NH
2mass ratio be 1:1 ~ 10.
4. preparation method as claimed in claim 2, it is characterized in that, catalyzer is added in step a, described catalyzer is selected from 2-(7-azo benzotriazole)-N, N, any one or a few in N', N'-tetramethyl-urea phosphofluoric acid ester (HATU), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), I-hydroxybenzotriazole (HOBT) or DMAP (DMAP).
5. preparation method as claimed in claim 4, it is characterized in that, described catalyzer is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, I-hydroxybenzotriazole and DMAP.
6. a liposome for cholic acid macromolecular material modification, comprise carrier, medicine, decorative material, it is characterized in that, described liposome vectors mainly comprises phosphatide and cholesterol, and decorative material is formula I, by drug encapsulation in phospholipid bilayer.
7. liposome as claimed in claim 6, it is characterized in that, the concentration of phosphatide is 1 ~ 20mg/mL, and the mass ratio of phosphatide and cholesterol is 3 ~ 10:1, and the mass ratio of medicine and phosphatide is 1:5 ~ 100, and the mass ratio of formula I and phosphatide is 1:10 ~ 50.
8. liposome as claimed in claim 6, is characterized in that, described medicine be selected from silibinin, curcumine, Quercetin, baicalin, cucurbitacin, podophyllotoxin, Oleanolic Acid, Cantharidin, hydroxycamptothecine any one.
9. the preparation method of liposome as claimed in claim 6, is characterized in that comprising the steps
Step a, prepares aqueous phase;
Step b, is dissolved in dehydrated alcohol by medicine, phosphatide, cholesterol, formula I, forms oil phase;
Step c, drops to oil phase in aqueous phase, forms the uniform dispersion emulsion of water oil;
Steps d, the dispersion emulsion obtained by step c carries out ultrasonic disperse after removing ethanol, namely obtains liposome.
10. preparation method as claimed in claim 8, it is characterized in that, the concentration of phosphatide is 1 ~ 20mg/mL, and the mass ratio of phosphatide and cholesterol is 3 ~ 10:1, and the mass ratio of medicine and phosphatide is 1:5 ~ 100, and the mass ratio of formula I and phosphatide is 1:10 ~ 50.
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Application publication date: 20151209 |