CN104844730A - Low molecular heparin-glycyrrhetinic acid polymer and synthetic method and application thereof - Google Patents
Low molecular heparin-glycyrrhetinic acid polymer and synthetic method and application thereof Download PDFInfo
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Abstract
The invention relates to a low molecular heparin-glycyrrhetinic acid polymer and its synthetic method and application. The synthetic method comprises the following steps: firstly synthesizing low molecular heparin-adipodihydrazide; synthesizing glycyrrhetinic acid succinate; preparing an active ester solution of glycyrrhetinic acid succinate; and finally synthesizing the low molecular heparin-glycyrrhetinic acid polymer. According to the invention, heparin is used as a water-soluble skeleton material, and glycyrrhetinic acid is used to carry out lyophobic modification on the water-soluble skeleton through succinic anhydride and adipodihydrazide used as linkers so as to prepare a glycyrrhetinic acid-mediated nano-carrier material with targeting capability. The heparin-glycyrrhetinic acid polymer has good biocompatibility, biodegradability and non-immunogenicity. Prepared hepatocyte-targeted drug-loaded nano-micelles have high entrapment rate and drug loading capacity, have excellent slow-release property, can enhance drug treatment efficacy and reduce non-specific toxic and side effect, and have a good application prospect.
Description
Technical field
The present invention relates to the synthesis of a kind of nanometer polymer self-assembled micelle material (Low molecular heparin-glycyrrhetinic acid polymkeric substance), refer in particular to the synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance and prepare the application method of antitumor drug nano-micelle preparations using this polymkeric substance as biomaterial.The invention belongs to bio-medical material and nano-drug preparation field.
Background technology
In decades, the incidence of malignant tumour and mortality ratio present obvious ascendant trend, become one of frequently-occurring disease of serious threat human life health.Have research prediction to the year two thousand fifty only the malignant tumour new cases of the Asian-Pacific area and death toll just will reach 7,300,000 and 5,500,000, be more than 2 times in 2000.And wherein liver cancer is called as " king in cancer ", its curative ratio is low, and mortality ratio is high.And China is the district occurred frequently of liver cancer in the world, over nearly 20 years, mortality of liver cancer adds 41.7%, and the whole world about has 1,250,000 people to die from liver cancer every year, and wherein nearly half occurs in China.Early hepatocarcinoma symptom is comparatively hidden and is not easily discovered, but once show manifest symptom, its grade malignancy is high, development rapidly, if treatment not in time or treatment plan select improper, mean survival time (MST), is only half a year.At present, for the clinical treatment of liver cancer, in order to make up the no thoroughness of operative therapy and easily bring out the shortcoming of transfer, chemotherapy as auxiliary treatment means still in occupation of very important status.But traditional also also exists stronger cytotoxicity to the internal organs of human normal based on Cytotoxic chemotherapeutic agent while obtaining result for the treatment of by Formulations for systemic administration mode, then causes serious toxic side effect.Such as taxol is a kind of Anti-cancer treatment that extraction and isolation obtains from Chinese yew genus plants.As the efficient cell toxicant kind anti-cancer drugs of one, its clinical preparation PTX injection liquid once long-time first-line drug as mammary cancer, ovarian cancer, prostate cancer and acute leukemia uses.But its solvent polyoxyethylenated castor oil easily produces the toxic side effect such as such as neurotoxicity, cardiac toxic, renal toxicity, anaphylaxis, in addition the whole body distribution of taxol can cause serious cardiac toxic and renal toxicity, and therefore how liver cancer patient after surgery uses chemotherapy means that are gentle, that can tolerate to be the problem needing field of medicaments researchist to need to need badly solution with the potentially dangerous reducing chemotherapy and bring with it.
Administration nano-drug administration system is antitumor drug formulation development and send the study hotspot in field in recent years.It by nano carrier material by chemicals parcel or be scattered in the middle of nano level matrix, effectively improve the solvability of insoluble drug, prolong drug cycling time in blood, change the distribution in vivo of medicine, improve drug bioavailability, and there is certain targeting.These advantages effectively can reduce the non-specific untoward reaction in chemotherapy process, improve therapeutic efficacy, are particularly applicable to the treatment of tumour.Wherein, its hypotoxicity of polymer micelle system addresses and satisfactory stability enjoy Chinese scholars to pay close attention at the solubilising of insoluble drug with the field of sending.It is by amphipathic high molecular polymer in water by the molecule of hydrophobic grouping and the spontaneous a kind of self-assembled structures with typical nucleocapsid structure formed of Intermolecular Forces.This micellar system is made up of hydrophobic inner core and hydrophilic shell closely, and because its kernel can carry some insoluble drugs by bags such as hydrophobic forces, therefore this system is considered to a kind of delivery vector of promising insoluble drug.But these administration nano-drug administration systems do not have the ability of escaping from reticuloendothelial system, are easy in vivo by monokaryon macrophage phagocytic and remove.On the other hand, the passive target that this dependence preparation Nano Particle realizes still exist the picked-up of tumor locus autopolymer insufficient, even cause multidrug resistance thus the phenomenon lessened the curative effect.In order to complete the target administration of tumour nanosystems, utilizing the specific molecular recognition mechanism in body, having become a kind of comparatively ripe target strategy in nano-carrier surface coupling target molecules.
As far back as early 1990s, namely Negishi confirms containing a large amount of glycyrrhetinic acid binding sites in rat hepatocytes membrane component, and presents high degree of specificity.The solid support material modified by target molecules with glycyrrhetinic acid is subsequently reported by domestic and international many scholars, such as, sulfuric ester chitosan polymer modified by the hyaluronic acid polymer that the glycyrrhetinic acid building polymer latex beam system using glycyrrhetinic acid as hydrophobic grouping is modified and glycyrrhetinic acid; By water-soluble PEG long-chain, glycyrrhetinic acid is distributed in PEG – b-poly (γ-benzyl L-glutamate) polymer micelle of the glycyrrhetinic acid modification of polymer surfaces and the chitosan/PEG nanoparticle etc. of glycyrrhetinic acid modification as targeted molecular.Result shows, and such solid support material has significant Liver targeting ability.Therefore the liver-targeted nanometer drug delivery system that group can research and develop the novel glycyrrhetinic acid mediation of a class is mediated using glycyrrhetinic acid as Liver targeting.Chinese patent CN101254308A discloses a kind of glycyrrhetinic acid-Polyethylene Glycol/Chitosan liver target composite drug administration system and preparation method, and illustrates that this targeting drug delivery system has very strong binding ability to liver cancer cell; Chinese patent CN101642573A discloses a kind of chitosan-based hepatic-targeted nano-particle drug delivery system and preparation method thereof, glycyrrhetinic acid is coupled in sulfuric ester chitosan or cm-chitosan as hydrophobic grouping while target molecules, thus builds formation liver-targeted nanometer drug delivery system; Chinese patent CN102336802A discloses glycyrrhetinic acid and modifies lipid, Liver targeting liposome, micella and mixture and method for making, by glycyrrhetinic acid and phosphatide or the cholesterol lipid that obtained glycyrrhetinic acid is modified under condensing agent condition, and with it for the Liver targeting liposome and micellar carrier that glycyrrhetinic acid mediates prepared by basic material.As can be seen here, glycyrrhetinic acid applies to hepatic-targeted delivery system structure as target group and has been subjected to vast focus of attention.
A kind of animal mucopolysaccharide sulfuric ester that heparin mainly extracts from ox lung or pig intestinal mucosa, gains the name because of initial discovery in liver.Heparin is mainly by glucosamine, and the water-soluble long-chain macromole of the structural unit compositions such as L-idose aldehyde glycosides, N-Acetyl-D-glucosamine and D-Glucose aldehydic acid, molecular-weight average is at about 12000Da.Heparin is just used for clinical study as blood anticoagulant as far back as nineteen thirty-nine because of the binding ability of itself and antithrombin uniqueness.But, the clinical application of heparin also easily causes the appearance of such as hemorrhage, thrombocytopenia, thus limits its further application at field of medicaments.Low molecular heparin is the degraded product of unfractionated heparin.Compared with unfractionated heparin, Low molecular heparin is while having similar or more excellent anticoagulation, anti-inflammatory, antiangiogenic and antitumor properties, and its possible untoward reaction significantly reduces.Therefore, Low molecular heparin is more safe and reliable for treating correlative diseases.Low molecular heparin because of molecular weight little, skeleton rigidity reduces and has more excellent water-soluble.In addition, containing modifiable free group active in a large number in heparin skeleton, its new performance can be given by chemically modified.Therefore heparin is except being used as anti-coagulate active components, with water-soluble low-molecular heparin for basic framework framework administration nano-drug administration system has attracted the concern of numerous pharmaceutical preparation research worker.
In recent years, to Low molecular heparin skeleton carry out hydrophobically modified and in an aqueous medium build polymer micelle attracted the research interest of numerous experts in medicine scholar.Hydrophobic small molecules to be introduced in heparin skeleton by chemical means thus build amphipathic nature polyalcohol and lure that it self-assembly polymerization occurs in an aqueous medium and becomes the nucleocapsid structure micella with hydrophobic inner core into.But these nano-micelle systems do not have the ability of escaping from reticuloendothelial system under normal circumstances, are easy in vivo by monokaryon macrophage phagocytic and remove.On the other hand, the passive target that this dependence preparation Nano Particle realizes still exist the picked-up of tumor locus autopolymer insufficient, even cause multidrug resistance thus the phenomenon lessened the curative effect.In order to complete the target administration of tumour nanosystems, utilize the specific molecular recognition mechanism in body, in nano-carrier building process, coupling target molecules has become a kind of comparatively ripe target strategy.
In sum, using Low molecular heparin as water-soluble backbone material construction polymer micelle, Low molecular heparin molecule is introduced glycyrrhetinic acid and carries out hydrophobically modified, make it possess performance that in aqueous solution self-assembly assembles the hepatic targeting drug utilizing the specific binding capacity of itself and liver cancer parenchyma to realize nano-micelle are sent.This method of design is at increase drug solubility, change medicine distribution in vivo, prolong drug in vivo while the residence time, and plays the Targeting Performance of glycyrrhetinic acid decorative material, and its method of design has very large research and development prospect in tumor diagnosis and therapy.This design at present there is not yet relevant report.In addition, use the same line antitumor drug of cancer therapy drug sensitizer to carry out drug combination, reverse the multidrug resistance effect that single long-term prescription causes, strengthen the result for the treatment of of antitumor drug.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of Low molecular heparin-glycyrrhetinic acid polymkeric substance, and provide its synthetic method, and it is as the application of biological degradable material for the preparation of antitumor drug liver-targeted nanometer preparation.
The present invention is achieved by the following technical solutions:
The synthetic method of Low molecular heparin-glycyrrhetinic acid polymkeric substance, comprises step as follows:
(1) synthesis of Low molecular heparin-adipic dihydrazide: Low molecular heparin is dissolved in distilled water, add adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 1-hydroxy benzo triazole wherein successively, reaction system pH=6-7 is regulated with sodium hydroxide, room temperature reaction 20-30 hour, obtains Low molecular heparin-adipic dihydrazide;
(2) synthesis of glycyrrhetinic acid succinate: glycyrrhetinic acid is dissolved in anhydrous tetrahydro furan, add Succinic anhydried and DMAP, lucifuge stirred at ambient temperature is after 20-30 hour, revolve and steam removing organic solvent, then add acetic acid ethyl dissolution, washing, purifying obtain glycyrrhetinic acid succinate;
(3) preparation of glycyrrhetinic acid succinate active ester solution: glycyrrhetinic acid succinate is dissolved in N, in N-dimethylformamide, then 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide is added, after stirring at room temperature 3-5 hour, add organic bases, obtain glycyrrhetinic acid succinate active ester solution;
(4) synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance: the Low molecular heparin-adipic dihydrazide of gained in step (1) is dissolved in distilled water, stirring makes its fully swelling, dissolving, then DMF dilution is added, for subsequent use; Gained glycyrrhetinic acid succinate active ester solution in step (3) is instilled in above-mentioned Low molecular heparin-adipic dihydrazide solution under vigorous stirring, stirring at room temperature reaction 40-50 hour, obtain reactant solution, namely reaction soln purifying, lyophilize obtain Low molecular heparin-glycyrrhetinic acid polymkeric substance.
In above-mentioned synthetic method, the mass ratio of Low molecular heparin described in step (1), distilled water, adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 1-hydroxy benzo triazole is 0.5-0.6:100:6-7:0.9-1:0.6-0.7.The molecular weight of Low molecular heparin is 3kD ~ 6kD.The adipic dihydrazide substitution value of Low molecular heparin-adipic dihydrazide is 43%.
In step (2), the mol ratio of glycyrrhetinic acid, Succinic anhydried, DMAP is 1:4:0.1; The consumption of anhydrous tetrahydro furan is that every gram of glycyrrhetinic acid adds 25 milliliters; The volume ratio of anhydrous tetrahydro furan and ethyl acetate is 1:2.
In step (3), the mol ratio of glycyrrhetinic acid succinate, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, organic bases is 1:2:2:2.Described organic bases is triethylamine, N, N, N', N'-Tetramethyl Ethylene Diamine or DIPEA.The consumption of DMF is that every gram of glycyrrhetinic acid succinate adds 70-300 milliliter.
In step (4), the mass ratio of Low molecular heparin-adipic dihydrazide and glycyrrhetinic acid succinate active ester is 1:0.2-0.6.The volume ratio of distilled water and DMF is 1:3, and every gram of Low molecular heparin-adipic dihydrazide adds 100ml distilled water.The substitution value scope of glycyrrhetinic acid is 3%-10% (substitution value according to required for the physico-chemical property adjustment of required self-assembly system).
Low molecular heparin-glycyrrhetinic acid polymkeric substance that aforesaid method is obtained.
Described Low molecular heparin-glycyrrhetinic acid polymkeric substance is as the application of solid support material in receptor-mediated liver target drug-carrying nanoassemble micell formulations preparation of insoluble anti-tumor medicament.During embody rule, preparation method is: by Low molecular heparin-glycyrrhetinic acid polymkeric substance, in ultrasonic disperse deionized water; By antitumor drug, or antitumor drug and antitumor drug sensitizer are dissolved in organic solvent, slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, room temperature continues violent stirring, uses probe type ultrasonic process, is transferred in dialysis tubing by solution and dialyses to water after ultrasonic, then by gained solution centrifugal, to remove non-encapsulated medicine, supernatant liquor then crosses 0.8 μm of filter membrane, obtains the nano-micelle preparations of antitumor drug.
Preferred preparation method is as follows: take 50mg Low molecular heparin-glycyrrhetinic acid polymkeric substance, ultrasonic disperse is for subsequent use in 5mL deionized water; Another by 5 ~ 40mg antitumor drug, or antitumor drug and antitumor drug sensitizer are dissolved in 1mL organic solvent, slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, after room temperature continues violent stirring 4h, process three times under 120W power condition with probe type ultrasonic, each 2min, temperature 4 DEG C ~ 8 DEG C, pulse is opened 2s and is stopped 4s, solution is transferred in dialysis tubing after ultrasonic and water is dialysed 24 hours, then by the centrifugal 20min of gained solution 4000r/min, to remove non-encapsulated medicine.Supernatant liquor then crosses 0.8 μm of filter membrane, obtains the nano-micelle preparations of antitumor drug, and 4 DEG C of preservations or freeze-drying obtain the lyophilized powder of medicament-carried nano micelle.
Described insoluble anti-tumor medicament is taxol, Zorubicin, Docetaxel etc.; Described antitumor drug sensitizer is curcumine, Quercetin etc.
Described organic solvent is tetrahydrofuran (THF), DMF or dimethyl sulfoxide (DMSO).
Low molecular heparin of the present invention-glycyrrhetinic acid polymkeric substance, adopt water miscible Low molecular heparin as basic framework, carry out hydrophobicity targeting modification to it, the amphipathic nature polyalcohol obtained can carry out self assembling process in an aqueous medium and form targeted nano micella, has the following advantages:
1, solid support material Low molecular heparin-glycyrrhetinic acid polymkeric substance that prepared by the present invention has excellent biocompatibility, degradability and non-immunogenicity, and preparation technology is simple, and mild condition is a kind of excellent liver-targeted nanometer pharmaceutical carrier.
2, the medicament-carried nano micelle form rounding prepared of the present invention, present spherical uniformly, particle diameter is less, has higher encapsulation rate and drug loading, and good stability.
3, the nano-micelle prepared of the present invention is in self-assembly building process, as this acid of Radix Glycyrrhizae of hydrophobic grouping while kernel stablize in formation, gives the Liver targeting characteristic that solid support material is potential, and mentality of designing is reasonable, it is easy to operate.
4, the medicament-carried nano micelle that prepared by the present invention overcomes the defect of insoluble drug poorly water-soluble, substantially improves the solubleness of insoluble anti-tumor medicament, for its targeted therapy provides a desirable novel carriers.
The antitumor drug of insoluble and sensitizer bag thereof are loaded in micell formulations by the medicament-carried nano micelle preparation that 5, prepared by the present invention simultaneously, effectively can control the generation of multidrug resistance, reduce dosage, provide medication effect.
Accompanying drawing explanation
Fig. 1: the proton nmr spectra of solid support material Low molecular heparin-glycyrrhetinic acid polymkeric substance.
Fig. 2: the grain size distribution carrying the Low molecular heparin-glycyrrhetinic acid nano-micelle of taxol.
Fig. 3: the electromicroscopic photograph carrying the Low molecular heparin-glycyrrhetinic acid nano-micelle of taxol.
Fig. 4: the Zeta potential figure carrying the Low molecular heparin-glycyrrhetinic acid nano-micelle of taxol.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.Following examples are not used for limiting the scope of the invention.
Embodiment 1: the synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance
(1) synthesis of Low molecular heparin-adipic dihydrazide: take 0.5g Low molecular heparin and be dissolved in 100mL distilled water, stirring makes its fully swelling, dissolving, then in this solution, 6.53g adipic dihydrazide is added successively, 0.96g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 0.68g 1-hydroxy benzo triazole, pH value of reaction system is regulated to be 6.8 with sodium hydroxide, room temperature reaction 24 hours, with distill water dialysis three days, lyophilize obtained intermediate product Low molecular heparin-adipic dihydrazide.
(2) synthesis of glycyrrhetinic acid succinate: take 200mg glycyrrhetinic acid ultrasonic dissolution in 5mL anhydrous tetrahydro furan, add the Succinic anhydried of 4 times of molar weights and the DMAP of 0.1 times of molar weight, lucifuge stirred at ambient temperature, after 24 hours, revolves and steams removing organic solvent.Then add 10mL acetic acid ethyl dissolution, successively with 1% hydrochloric acid soln and ultrapure water wash three times respectively, boil off organic solvent after anhydrous sodium sulfate drying spends the night.By silica gel column chromatography method, (eluent is ethanol to product: ethyl acetate (11:3; V/v) purifying obtains glycyrrhetinic acid succinate.
(3) take 20mg glycyrrhetinic acid succinate and be dissolved in 3mL N, in N-dimethylformamide, then 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride being equivalent to glycyrrhetinic acid succinate 2 times of molar weights and the N-hydroxy-succinamide being equivalent to glycyrrhetinic acid succinate 2 times of molar weights is added, in stirring at room temperature after 4 hours, add the N being equivalent to glycyrrhetinic acid succinate 2 times of molar weights, N, N', N'-Tetramethyl Ethylene Diamine, obtains glycyrrhetinic acid succinate active ester solution.
(4) synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance: the Low molecular heparin-adipic dihydrazide taking gained in 100mg step (1) is dissolved in 10mL distilled water, stirring makes its fully swelling, dissolving, then the N of 3 times of volumes is added, N-dimethylformamide is diluted, for subsequent use.Instilled under vigorous stirring in above-mentioned Low molecular heparin-adipic dihydrazide solution by gained glycyrrhetinic acid succinate active ester solution in step (3), stirring at room temperature reacts 48 hours, obtains reactant solution.Reaction soln dialyse three days in the mixing solutions (volume ratio 4:1) of methyl alcohol and water, to remove free glycyrrhetinic acid succinate wherein and by product, then within three days, removes organic solvent to distill water dialysis.Namely lyophilize obtains Low molecular heparin-glycyrrhetinic acid polymkeric substance.
1the substitution value that H NMR quantitative analysis obtains glycyrrhetinic acid is 6.8%.
Low molecular heparin-the adipic dihydrazide (HEP-ADH) of Low molecular heparin (HEP) and gained and Low molecular heparin-glycyrrhetinic acid polymkeric substance (HEP-GA)
1h NMR structure spectrogram is shown in Fig. 1, relative to HEP, in HEP-ADH nucleus magnetic hydrogen spectrum, be positioned at 1.0-1.6ppm, the new characteristic peak of 2.1-2.5ppm belongs to the proton peak of adipic dihydrazide scion grafting chain, this demonstrates adipic dihydrazide and has successfully been coupled on HEP skeleton.And for HEP-GA, chemical shift is the diagnostic protons peak on glycyrrhetinic acid framework ring proton in the new proton peak that 0.5-1.4ppm, 5.52ppm occur.The above results proves that glycyrrhetinic acid has successfully been coupled on Low molecular heparin by adipic dihydrazide.
Embodiment 2: the synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance
(1) synthesis of Low molecular heparin-adipic dihydrazide: take 0.5g Low molecular heparin and be dissolved in 100mL distilled water, stirring makes its fully swelling, dissolving, then in this solution, 6.53g adipic dihydrazide is added successively, 0.96g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 0.68g 1-hydroxy benzo triazole, pH value of reaction system is regulated to be 6.8 with sodium hydroxide, room temperature reaction 24 hours, with distill water dialysis three days, lyophilize obtained intermediate product Low molecular heparin-adipic dihydrazide.
(2) synthesis of glycyrrhetinic acid succinate: take 200mg glycyrrhetinic acid ultrasonic dissolution in 5mL anhydrous tetrahydro furan, add the Succinic anhydried of 4 times of molar weights and the DMAP of 0.1 times of molar weight, lucifuge stirred at ambient temperature, after 24 hours, revolves and steams removing organic solvent.Then add 10mL acetic acid ethyl dissolution, successively with 1% hydrochloric acid soln and ultrapure water wash three times respectively, boil off organic solvent after anhydrous sodium sulfate drying spends the night.By silica gel column chromatography method, (eluent is ethanol to product: ethyl acetate (11:3; V/v) purifying obtains glycyrrhetinic acid succinate.
(3) take 30mg glycyrrhetinic acid succinate and be dissolved in 3mL N, in N-dimethylformamide, then 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride being equivalent to glycyrrhetinic acid succinate 2 times of molar weights and the N-hydroxy-succinamide being equivalent to glycyrrhetinic acid succinate 2 times of molar weights is added, in stirring at room temperature after 4 hours, add the N being equivalent to glycyrrhetinic acid succinate 2 times of molar weights, N-diisopropylethylamine, obtains glycyrrhetinic acid succinate active ester solution.
(4) synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance: the Low molecular heparin-adipic dihydrazide taking gained in 100mg step (1) is dissolved in 10mL distilled water, stirring makes its fully swelling, dissolving, then the N of 3 times of volumes is added, N-dimethylformamide is diluted, for subsequent use.Instilled under vigorous stirring in above-mentioned Low molecular heparin-adipic dihydrazide solution by gained glycyrrhetinic acid succinate active ester solution in step (3), stirring at room temperature reacts 48 hours, obtains reactant solution.Reaction soln dialyse three days in the mixing solutions (volume ratio 4:1) of methyl alcohol and water, to remove free glycyrrhetinic acid succinate wherein and by product, then within three days, removes organic solvent to distill water dialysis.Namely lyophilize obtains Low molecular heparin-glycyrrhetinic acid polymkeric substance.
1the substitution value that H NMR quantitative analysis obtains glycyrrhetinic acid is 8.5%.
Embodiment 3: the Low molecular heparin-glycyrrhetinic acid polymer nano micelle preparation of carrying taxol
By 50mg Low molecular heparin-glycyrrhetinic acid (prepared by embodiment 1), ultrasonic disperse is in 5mL deionized water, for subsequent use; Separately taking 20mg taxol is dissolved in 1mL tetrahydrofuran (THF), slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, after room temperature continues violent stirring 4h, process three times under 120W power condition with probe type ultrasonic, each 2min, temperature remains on 4 DEG C ~ 8 DEG C, pulse is opened 2s and is stopped 4s, solution is transferred in dialysis tubing after ultrasonic and water is dialysed 24 hours, then by the centrifugal 20min of gained solution 4000r/min, to remove non-encapsulated medicine.Supernatant liquor then crosses 0.8 μm of filter membrane, must carry the nano-micelle preparations of taxol, and freeze-drying obtains the lyophilized powder carrying paclitaxel nano micelle.It is 20.6% that high performance liquid chromatography detected result shows its drug loading, and encapsulation rate is 72.0%.
After the nano micellar solution carrying taxol of preparation is diluted suitable multiple, with the particle diameter of Malvern Zetasizer Nano-ZS type laser current potential particle size analyzer determination micella and Zeta potential, measured micella average particle size is 90.2nm, polyphyly dispersion coefficient PI is 0.23, to be-25.2mV, Fig. 2 be Zeta potential value carries the grain size distribution of the Low molecular heparin-glycyrrhetinic acid nano-micelle of taxol.With H-7000 type transmission electron microscope observation micellar surface form, as shown in Figure 3, prepared carrier micelle form rounding, size is homogeneous, is adhered less.Fig. 4 is the Zeta potential figure of this medicament-carried nano micelle.
Embodiment 4: combine the Low molecular heparin-glycyrrhetinic acid polymer nano micelle preparation of carrying taxol, curcumine
By 50mg Low molecular heparin-glycyrrhetinic acid polymkeric substance (prepared by embodiment 1), ultrasonic disperse is in 5mL deionized water, for subsequent use; Separately take 20mg taxol and 20mg curcumine is dissolved in 1mL tetrahydrofuran (THF), slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, after room temperature continues violent stirring 4h, process three times under 120W power condition with probe type ultrasonic, each 2min, temperature remains on 4 DEG C ~ 8 DEG C, pulse is opened 2s and is stopped 4s, solution is transferred in dialysis tubing after ultrasonic and water is dialysed 24 hours, then by the centrifugal 20min of gained solution 4000r/min, to remove non-encapsulated medicine.Supernatant liquor then crosses 0.8 μm of filter membrane, obtains the nano-micelle preparations carrying taxol and curcumine altogether, and freeze-drying obtains the lyophilized powder carrying paclitaxel nano micelle.Drug loading and the encapsulation rate of high performance liquid chromatography detected result display taxol are respectively 17.2% and 63.5%, and the drug loading of curcumine and encapsulation rate are respectively 14.9% and 55.0%.
Embodiment 5: combine the Low molecular heparin-glycyrrhetinic acid polymer nano micelle preparation of carrying Docetaxel, Quercetin
By 50mg Low molecular heparin-glycyrrhetinic acid polymkeric substance (prepared by embodiment 2), ultrasonic disperse is in 5mL deionized water, for subsequent use; Separately take 20mg Docetaxel and 20mg Quercetin is dissolved in 1mL tetrahydrofuran (THF), slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, after room temperature continues violent stirring 4h, process three times under 120W power condition with probe type ultrasonic, each 2min, temperature remains on 4 DEG C ~ 8 DEG C, pulse is opened 2s and is stopped 4s, solution is transferred in dialysis tubing after ultrasonic and water is dialysed 24 hours, then by the centrifugal 20min of gained solution 4000r/min, to remove non-encapsulated medicine.Supernatant liquor then crosses 0.8 μm of filter membrane, obtains the nano-micelle preparations carrying taxol and curcumine altogether, and freeze-drying obtains the lyophilized powder carrying paclitaxel nano micelle.Drug loading and the encapsulation rate of high performance liquid chromatography detected result display Docetaxel are respectively 18.9% and 66.1%, and the drug loading of Quercetin and encapsulation rate are respectively 14.4% and 53.1%.
Claims (10)
1. the synthetic method of Low molecular heparin-glycyrrhetinic acid polymkeric substance, is characterized in that, comprise step as follows:
(1) synthesis of Low molecular heparin-adipic dihydrazide: Low molecular heparin is dissolved in distilled water, add adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 1-hydroxy benzo triazole wherein successively, reaction system pH=6-7 is regulated with sodium hydroxide, room temperature reaction 20-30 hour, obtains Low molecular heparin-adipic dihydrazide;
(2) synthesis of glycyrrhetinic acid succinate: glycyrrhetinic acid is dissolved in anhydrous tetrahydro furan, add Succinic anhydried and DMAP, lucifuge stirred at ambient temperature is after 20-30 hour, revolve and steam removing organic solvent, then add, acetic acid ethyl dissolution, washing, purifying obtain glycyrrhetinic acid succinate;
(3) preparation of glycyrrhetinic acid succinate active ester solution: glycyrrhetinic acid succinate is dissolved in N, in N-dimethylformamide, then 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide is added, after stirring at room temperature 3-5 hour, add organic bases, obtain glycyrrhetinic acid succinate active ester solution;
(4) synthesis of Low molecular heparin-glycyrrhetinic acid polymkeric substance: the Low molecular heparin-adipic dihydrazide of gained in step (1) is dissolved in distilled water, stirring makes its fully swelling, dissolving, then DMF dilution is added, for subsequent use; Gained glycyrrhetinic acid succinate active ester solution in step (3) is instilled in above-mentioned Low molecular heparin-adipic dihydrazide solution under vigorous stirring, stirring at room temperature reaction 40-50 hour, obtain reactant solution, namely reaction soln purifying, lyophilize obtain Low molecular heparin-glycyrrhetinic acid polymkeric substance.
2. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, it is characterized in that, the mass ratio of Low molecular heparin described in step (1), distilled water, adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 1-hydroxy benzo triazole is 0.5-0.6:100:6-7:0.9-1:0.6-0.7.
3. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, is characterized in that, described in step (1), the molecular weight of Low molecular heparin is 3kD ~ 6kD.
4. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, is characterized in that, in step (2), the mol ratio of glycyrrhetinic acid, Succinic anhydried, DMAP is 1:4:0.1; The consumption of anhydrous tetrahydro furan is that every gram of glycyrrhetinic acid adds 25 milliliters; The volume ratio of anhydrous tetrahydro furan and ethyl acetate is 1:2.
5. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, it is characterized in that, in step (3), the mol ratio of glycyrrhetinic acid succinate, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, organic bases is 1:2:2:2.
6. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, is characterized in that, described organic bases is triethylamine, N, N, N', N'-Tetramethyl Ethylene Diamine or DIPEA.
7. the synthetic method of Low molecular heparin according to claim 1-glycyrrhetinic acid polymkeric substance, it is characterized in that, in step (4), the mass ratio of Low molecular heparin-adipic dihydrazide and glycyrrhetinic acid succinate active ester is 1:0.2-0.6, distilled water and N, the volume ratio of N-dimethylformamide is 1:3, and every gram of Low molecular heparin-adipic dihydrazide adds 100ml distilled water.
8. Low molecular heparin-glycyrrhetinic acid polymkeric substance that the method described in any one of claim 1-7 is obtained.
9. Low molecular heparin according to claim 8-glycyrrhetinic acid polymkeric substance is as the application of solid support material in receptor-mediated liver target drug-carrying nanoassemble micell formulations preparation of insoluble anti-tumor medicament.
10. application according to claim 9, is characterized in that, method is: by Low molecular heparin-glycyrrhetinic acid polymkeric substance, in ultrasonic disperse deionized water; By antitumor drug, or antitumor drug and antitumor drug sensitizer are dissolved in organic solvent, slowly drop in above-mentioned Low molecular heparin-glycyrrhetinic acid aqueous solutions of polymers in violent stirring situation, room temperature continues violent stirring, uses probe type ultrasonic process, is transferred in dialysis tubing by solution and dialyses to water after ultrasonic, then by gained solution centrifugal, to remove non-encapsulated medicine, supernatant liquor then crosses 0.8 μm of filter membrane, obtains the nano-micelle preparations of antitumor drug.
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| CN107362348A (en) * | 2017-06-26 | 2017-11-21 | 安徽省黄淮兽药有限公司 | A kind of fish insect killing agent and preparation method thereof |
| CN112138001A (en) * | 2020-09-25 | 2020-12-29 | 山东大学 | Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application |
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| CN101254308A (en) * | 2008-04-08 | 2008-09-03 | 南开大学 | Biogastrone acid-polyethyleneglycol /chitosan liver target composite drug administration system and preparation thereof |
| CN102836435A (en) * | 2012-09-20 | 2012-12-26 | 南开大学 | PH sensitive type liver-targeted compound nano drug delivery system based on sodium alginate and preparation method |
| CN102863556A (en) * | 2012-09-27 | 2013-01-09 | 复旦大学附属上海市第五人民医院 | Lactose acidized glycyrrhetinic chitosan material and preparation method and application thereof |
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| US5474765A (en) * | 1992-03-23 | 1995-12-12 | Ut Sw Medical Ctr At Dallas | Preparation and use of steroid-polyanionic polymer-based conjugates targeted to vascular endothelial cells |
| CN101254308A (en) * | 2008-04-08 | 2008-09-03 | 南开大学 | Biogastrone acid-polyethyleneglycol /chitosan liver target composite drug administration system and preparation thereof |
| CN102836435A (en) * | 2012-09-20 | 2012-12-26 | 南开大学 | PH sensitive type liver-targeted compound nano drug delivery system based on sodium alginate and preparation method |
| CN102863556A (en) * | 2012-09-27 | 2013-01-09 | 复旦大学附属上海市第五人民医院 | Lactose acidized glycyrrhetinic chitosan material and preparation method and application thereof |
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| CN107362348B (en) * | 2017-06-26 | 2021-01-12 | 安徽省凯强生物科技有限公司 | Fishing pesticide and preparation method thereof |
| CN112138001A (en) * | 2020-09-25 | 2020-12-29 | 山东大学 | Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application |
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