CN105343890A - Heparin or heparin salt modified graphene oxide and preparation method and application thereof - Google Patents

Abstract

The present invention discloses heparin or heparin salt modified graphene oxide and a preparation method and application thereof, the modified graphene oxide can be used as a drug, particularly an anti-cancer drug carrier material. By use of pH sensitive principle, adipic dihydrazide is used as a linker molecule, or by use of redox principle, cystamine is used as a linker molecule, heparin is used for chemically modifying graphene oxide, and the heparin has the advantages of increase of water solubility and anti-cancer drug tumor synergistic effect of graphene oxide, and solves the problems of poor water solubility and easy aggregation and the like of the graphene oxide.

Description

Graphene oxide that a kind of heparin or its salt are modified and preparation method thereof and application

Technical field

The present invention relates to pharmaceutical synthesis field, be specifically related to a kind of graphene oxide modified for heparin or its salt and preparation method thereof and application.

Background technology

Graphene oxide (Grapheneoxide, GO are shown in Fig. 1) is the derivant (Carbon, 2011,49:1126-1132) of the Graphene with monoatomic layer structure.Compared with the Graphene (Graphene) on the surface only containing carbon-carbon double bond, containing a large amount of oxy radicals such as hydroxyl, epoxy radicals, carboxyl in graphene oxide self structure, and there is good hydrophilic, (JControlRelease in biomedicine can be widely used in, 2014,173:75-88).Because graphene oxide is monoatomic layer structure, having larger specific surface area (has document to be measured as 736.6m ²/ g, is shown in Langmuir, 2013,29:13443-13448), its two sides is all combined with medicine by covalency, noncovalent interaction, therefore has higher drug load amount.It, by stronger physisorption and fragrant lopps medicine Non-covalent binding, can deliver some insoluble drugs, transport have great importance (Science, 2004,306:666) in the body particularly to most of slightly solubility cancer therapy drug.Graphene oxide is hydroaropic substance, has good biocompatibility.Research finds, a kind of quite safe material at cellular level graphene oxide, there is no obvious cytotoxicity (ToxicolLett, 2011,200:201), therefore graphene oxide is more and more subject to the attention of scientific research personnel as the carrier of targeted drug delivery, and current disclosed patent is more.

As patent CN201210509075 reports the graphene oxide of a kind of glucosan chemical graft haemachrome modification, for biomedical Diagnosis and Treat such as drug conveying.Patent CN201310450626 reports a kind of preparation method of graphene oxide-boron-modifiedphenolic phenolic resin, not only maintain the stuctures and properties of Graphene, and the part oxygen-containing functional group retained can solve the difficult problems such as graphene dispersion, dissolubility and poor in processability well.Patent CN201310185529 reports the preparation method of a kind of graphene oxide and aqueous polyurethane nano composite, can reduce the hydrophilic of graphene oxide lamella, improve its dispersibility in organic solvent and and polymer between intermiscibility.Patent CN201210038641 reports a kind of GO-SAN complex and preparation method thereof.In this complex, acted on by non-covalent π-π between graphene oxide carrier and Sanguinarine and combining, significantly can improve dissolubility and the stability of Sanguinarine, also can make it have targeting, and be expected to be widely used as a kind of anticancer preparation had a high potential.Patent CN201110361574 reports a kind of method of polystyrene graft graphene oxide.Patent CN201110227223 reports the method for polyethyleneglycol modified graphene oxide.

Graphene oxide can change reproducibility graphene oxide (reducedGO, rGO are shown in Fig. 2) into after reducing agent (as hydrazine, sodium borohydride, 1,2,3,-thrihydroxy-benzene, ascorbic acid, thiourea etc.) reduction.Compared with graphene oxide, rGO eliminates more oxy radical, maintains planar structure simultaneously, but there is the feature of irreversible aggrengation, or under the condition lacking macromolecule dispersing agent or surfactant, have the tendency (Carbon, 2012,50:3210-3228) being piled into graphite.RGO not only drug carrying ability improves greatly, and ultrared ability within the scope of Absorbable rod 650-900nm and produce heat (photo-thermal effect) and greatly strengthen (JAmChemSoc, 2009,131:11027-11032).As for doxorubicin, its drug loading improves 10 times (ACSNano, 2013,7:6735-6746), 808nm near infrared light and energy is 0.6W/cm ²illumination under, its photo-thermal effect adds 6 times (JAmChemSoc, 2011,133:6825-6831).Utilize the higher medicine carrying of rGO and stronger photo-thermal effect, greatly enhance antineoplastic targeting and therapeutic effect, have also more report at present.

Bibliographical information, for increasing the transport efficacy at target cancerous cell of cancer therapy drug, reduces other Normocellular toxicity, improve its curative effect, graphene oxide needs through certain chemical modification before pharmaceutical carrier, and its active anticancer of competence exertion is (as NanoLett.2010,10:3318-3323, AdvMater.2012,24:1722-1728, ACSNano.2011,5:7000-7009, Biomaterials.2014,35:4986-4995 etc.).As graphene oxide its water solublity after polyethyleneglycol modified strengthens (JAmChemSoc.2008 further; 130:10876-10877).And for example under certain condition, by graphene oxide and sodium chloroacetate (ClCH ₂cOONa) mix, the hydroxyl in graphene oxide, epoxy radicals, ester group is made to be converted to carboxyl, sulfonated again, generate the graphene oxide derivative containing a large amount of sulfonic group and carboxyl, thus improve the stability (Small of graphene oxide in physiological solution, 2010,6 (4): 37).Carboxyl (COOH) in this derivant can with the amino (NH in folic acid ₂) covalent bond generation folic acid-graphene oxide conjugate.Because folic acid is alternative by folacin receptor identification, and folacin receptor great expression in human breast cancer cell, therefore, the medicine of load on the graphene oxide of folic acid finishing can realize target administration (NanoRes, 2008,1:203-212).By pi-pi accumulation and the hydrophobic interaction of graphene oxide, this conjugate by cancer therapy drug camptothecine (DNA topoisomerase I inhibitor), doxorubicin (DNA Topoisomerase II inhibitors) physical absorption in surface of graphene oxide, and can form the complex containing cancer therapy drug.This complex is transported to the position (as human breast cancer cell) of folacin receptor great expression through blood (pH7.35 ~ 7.45), through receptor-mediated endocytosis, complex containing anticarcinogen is transported in cancerous cell, in the acid destruction of endocytosis body (pH5.0 ~ 6.5) and lysosome (pH4.5 ~ 5.0), discharge cancer therapy drug (Biomaterials, 2013,34:3647-3657).In addition, because growth of tumour cell is fast, generate than acidic metabolites such as the more lactic acid of normal structure in breeding, therefore, the tissue fluid pH value of tumor cell periphery is lower than normal cell, aobvious faintly acid (pH6.5 ~ 7.2, Biomacromolecules2009; 10 (7): 1727-1735; JAmChemSoc2005,127 (51): 17982-17983).And mostly anticarcinogen is alkalescent medicine, as the pKa=8.63 (CurrMedChem.2011 of camptothecine, 18 (9): 1367-1372), the pKa=8.22 (AnalyticalProfilesofDrugSubstancesvol9:260) of doxorubicin, by the process of blood to the outer liquid of tumor cell, and endocytosis body in cancerous cell and lysosomal destructive process, all become weakly acidic process from weakly alkaline environment, drug molecule changes ionic state into by molecular state, its dissolubility raises, namely fat-solublely hydrophilic is become, increase from surface of graphene oxide release, be dissolved in intracellular fluid, be combined with DNA topoisomerase, this utilizes graphene oxide to play the toxic treatment effect of medicine as carrier by the principle of pH sensitivity.

Find in tumor research, disulfide bond (-S-S-) is stable existence under the temperate condition of extracellular, and in cell, run into reducing substances to be swift in response decomposition by disulfide exchange, these reducing substances are glutathion (glutathione mainly, GSH, MethodsEnzymol, 1995,251:8-28; AnnuRevMicrobiol, 1997,51:179-202).(about 0.5 ~ 10mM) is there is with millimolar concentration high level in GSH in Cell sap and subcellular fraction core, but due in blood plasma rapidly enzymatic degradation dropped to reduced levels (about 2 ~ 20 μm), namely Cytoplasm and endonuclear reduction potential are approximately 100 ~ 1000 times of (CellBiochemFunct of the body fluid such as body fluid or extracellular fluid, 2004,22:343-352; FreeRadicBiolMed, 2001,30:1191-1212).Tumor cell is fast owing to growing, oxygen consumption is larger, the content of GSH is caused to be at least 4 times of (CancerRes of normal structure, 2002,62 (1): 307-312), therefore, oxidoreduction principle is adopted to play therapeutical effect by cancer therapy drug or gene delivery to tumor cell.

Heparin (heparin, see Fig. 3) be the linear polysaccharide compounds be made up of 1 → 4 glycuronic acid connected and glucamine disaccharide found for 1916, nineteen thirty-five starts clinically for anticoagulant, its activity is because it can combine with serpin antithrombase, this inhibitor is caused to become the thrombin (JMedChem of non-activity, 2003,46:2551-64).Common heparin normally extracts from the natural tissues such as pig small intestine or pulmonis Bovis seu Bubali, and mean molecule quantity is about 15kDa, about has 20 disaccharidase structures, chemical constitution and molecular weight height uneven, polydispersity is 1.2 ~ 1.4, and molecular weight ranges is 5 ~ 40kDa.Sodium salt is easily molten in water, common are indissoluble in machine solvent, and at water Middle molecule chain-unfolding curl, the heparin length of 12 dissacharide units is about 5nm, infers that the length of heparin is about 9nm.Each disaccharide unit contains a carboxyl, one or more 1 ° or 2 ° of hydroxyls, 2 ~ 2.5 sulfates, wherein N-sulphuric acid fiduciary point 75 ~ 85%, O-sulphuric acid fiduciary point 15 ~ 25%, the adjacent hydroxyl also containing 15 ~ 25% in each disaccharide unit structure.Each chain contains the reducing end under neutral of a hemiacetal, masks aldehyde radical (CHO).The each chain of heparin is containing the 0.3 free amino (NH that has an appointment ₂).Highly-hydrophilic, even if in dry conditions, still with 2 ~ 10% water.Owing to containing carboxyl (pKa is 3.3) and sulfate (pKa of O-sulfate and N-sulfate is 1.0 ~ 1.5) in structure, make heparin altitudinal belt negative charge (about-75/ chain), make can with somatomedin, protease, the multiple proteins generation electrostatic interactions such as cytokine (NatProdRep, 2002, 19:312-331), cause protein stabilization or add the affinity (JCellPhysiol to cell receptor, 1986, 128:475-484), this stability is for fibroblast growth factor (fibroblastgrowthfactor, and VEGF (vascularendothelialgrowthfactor FGF), VEGF) can be made into the drug delivery system platform (ChemSocRev of tissue regeneration engineering carrier bracket and Drug controlled release, 2013, 42:7335-7372).As anticoagulant, the side effect that heparin is more, as bleeding complications, thrombocytopenia, non-vein administration artifact availability low (JClinPharmacol, 1992,32:584-596).

Low molecular heparin (low-molecularweightheparin, LMWH) chemical composition is definite, and biological half-life extends, untoward reaction less (Science, 2011,334:498-501).Low molecular heparin (CAS:9041-08-1) has multiple medicinal forms, as medicinal low molecular weight heparin sodium, low molecular weight calcium heparin, Ardeparin Sodium (ArdeparinSodium), Bemiparin sodium (BemiparinSodium), Certoparin Sodium (CertoparinSodium), dalteparin sodium (DalteparinSodium), Enoxaparin Sodium (EnoxaparinSodium), nadroparin calcium (NadroparinCalcium), Parnaparin Sodium (ParnaparinSodium), Clivarin sodium (ReviparinSodium), (the Martindale such as tinzaparin sodium (Tinzaparinsodium), 36 editions 1329 pages).Research display, compared with common unassorted heparin, LMWH is by regulating the somatomedin (as FGF and VEGF) relevant to multiple blood vessel, more effective to the growth of Tumor suppression tissue.In addition, LMWH also can disturb neoplasm metastasis, or with P-selectin competitive binding, what Tumor suppression was little sticks (NatRevCancer, 2002,2:521-528).Recent research finds, heparin also can with transcription factor effect, cause apoptotic death (ChemBiol, 2004,11:420-422).Therefore, LMWH is widely used in Tumor Targeting Drug Delivery System, also has the LMWH derivant of some chemical modifications, as LMWH-deoxycholic acid, anticoagulating active reduces, and anti-angiogenesis activity raises (JControlledRelease, 2010,148:317-326).LMWH can be used as antifibrotic agents treatment hepatitis B (Biomaterials, 2011,32:1438-1445).Heparin modified PLGA-pluronic F-127 can be used for the release (Biomaterials controlling somatomedin, 2006,27:2621-2626), the people such as Kipper utilize the compound polyelectrolyte nanoparticle (polyelectrolytecomplexnanoparticles of polyanion chitosan-heparin and polycation Chitosan-Hyaluronic Acid polymer electrolyte confrontation composition, PCN), for the release of controlled release FGF-2, can mode slow releasing 30 days (ActaBiomater of Zero order release, 2012,8:1551-1559).With the nanoparticle that chlorination N-trimethyl chitosan TMC and heparin non-covalent bond form, take VEGF as model drug, prominent releasing acts on minimizing, 14 days zero-order fashion release about 49% (MacromolRapidCommun, 2012,33:2015-2022).The people such as Mi have synthesized heparin modified chitosan/Polyurethane-epoxy resin nanoparticle, due to gamma-glutamic acid and heparin electronegative, chitosan band on schedule, material is responsive to pH, stable under pH6.0 condition, expands gradually but non-disintegrate under pH6.6 condition, but rapid disintegrate under pH7.4 condition, this is because this nanoparticle loses electrostatic interaction (Biomaterials, 2010,31:9320-9332).With heparin and bFGF for model, under the micro-acid environment of ischemic tissue, nanoparticle keeps complete, and slow releasing medicine, in repair tissue (pH7.4), can discharge heparin rapidly, and these characteristics can suppress vascular thrombosis to be formed again.The release of bFGF can significantly make human foreskin fibroblast breed to be increased, and effectively promotes Human umbilical vein endothelial cells segment dislocation.

Although graphene oxide self has more oxy radical (as hydroxyl, epoxy radicals and carboxyl), Fig. 4 is the typical IR collection of illustrative plates of graphene oxide, but because precursor structure has a large amount of carbon-carbon double bonds, therefore, water solublity is not good especially, concentration as usual in graphene oxide is when being greater than 5mg/mL, and long-term placement easily forms g., jelly-like material; When concentration is at 20 more than μ g/mL, graphene oxide starts to be gathered into lamellar structure, and when concentration is 50 μ g/mL, concentration class reaches 15% (Langmuir, 2013,29:13443-13448).In inorganic salt solution (as sodium chloride, sodium phosphate etc.), graphene oxide can become lamellar structure by self-assemble, and produces precipitation, therefore, if as drug carrier material, need carry out modifying to improve its water solublity.For reproducibility graphene oxide, Fig. 5 is the typical IR collection of illustrative plates of reproducibility graphene oxide, and because oxy radical reduces in a large number, therefore, water solublity is poorer, need improve its water solublity with more hydrophilic radical.Research finds, heparin is a kind of good water soluble molecules (1 part of heparin sodium is dissolved in 20 parts of water, Martindale, 36 editions: 1301 pages), and it utilizes self has blood vessel formation against function and play active anticancer.Administration together with cancer therapy drug, has synergistic therapeutic action.At present, graphene oxide combines with heparin or its salt and is applied on drug carrier material and there is not yet relevant report.

Summary of the invention

An object of the present invention is to provide graphene oxide that a kind of heparin or its salt modifies and preparation method thereof and application, heparin or its salt modify after graphene oxide can be used as the carrier material of medicine particularly cancer therapy drug.

The graphene oxide that heparin or its salt are modified, prepares one of by the following method:

(1) according to pH sensitivity principle, be connect molecule with adipic dihydrazide, heparin or be connected by amido link between its salt and graphene oxide and obtain the graphene oxide that heparin or its salt modifies;

(2) according to oxidoreduction principle, be connect molecule with cystamine, heparin or be connected by disulfide bond between its salt and graphene oxide and obtain the graphene oxide that heparin or its salt modifies.

In method (1), comprise the following steps: adopt adipic dihydrazide to modify heparin or its salt, obtain the heparin after adipic dihydrazide modification or its salt, then the graphene oxide after the heparin after the adipic dihydrazide obtained modification or its salt and activation is reacted 6 ~ 24h at 25 ~ 30 DEG C, obtain stopping using adipic dihydrazide as the graphene oxide that the heparin or its salt that connect molecule are modified polymer solution.

Wherein, the method that described adipic dihydrazide modifies heparin or its salt is: heparin sodium is soluble in water, add adipic dihydrazide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) and I-hydroxybenzotriazole (HOBt), regulate solution ph to 6.0 ~ 7.0.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted starting material dialysis removing, dry, obtain the heparin (product A) after the modification of product adipic dihydrazide.Described heparin sodium and the mol ratio of water are 1:(10000 ~ 100000); Described heparin sodium and the mol ratio of adipic dihydrazide are 1:(2 ~ 60); Described heparin sodium and the mol ratio of EDCHCl are 1:(2 ~ 60); Described heparin sodium and the mol ratio of HOBt are 1:(2 ~ 60).Adipic dihydrazide in the present invention modifies the method for heparin or its salt, and adipic dihydrazide modifies the good connecting effect of heparin or its salt, improves the productive rate of product in end reaction.But the preparation method of adipic dihydrazide-heparin of the present invention or its salt is not limited only to the method.

The preparation method of the graphene oxide after activation is: get graphene oxide solution, add EDCHCl and N-hydroxy-succinamide sulfonate sodium (sulfur-NHS), regulate solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C, obtains the graphene oxide after activating.Surface of graphene oxide functional group is carried out priming reaction by the method for the active oxidation Graphene in the present invention, makes activation effect better, improves the productive rate of product in end reaction.But the preparation method of active oxidation Graphene (GO) of the present invention is not limited only to the method.

In method (2), comprise the following steps: adopt cystamine to modify heparin or its salt, obtain the heparin after cystamine modification or its salt, then the graphene oxide after the heparin after the cystamine obtained modification or its salt and activation is reacted 6 ~ 24h at 25 ~ 30 DEG C, obtain stopping using cystamine as the graphene oxide that the heparin or its salt that connect molecule are modified polymer solution.

Wherein, the method that described cystamine modifies heparin or its salt is: heparin sodium is soluble in water, adds cystamine (Cys), EDCHCl and HOBt, regulates solution ph to 6.0 ~ 7.0.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted starting material dialysis removing, dry, obtain the heparin (product B) after the modification of product cystamine.Described heparin sodium and the mol ratio of water are 1:(10000 ~ 100000); Described heparin sodium and the mol ratio of cystamine are 1:(2 ~ 60); Described heparin sodium and the mol ratio of EDCHCl are 1:(2 ~ 60); Described heparin sodium and the mol ratio of HOBt are 1:(2 ~ 60).Cystamine in the present invention modifies the method for heparin, and cystamine modifies the good connecting effect of heparin, improves the productive rate of product in end reaction.But the preparation method of cystamine-heparin of the present invention is not limited only to the method.

The preparation method of the graphene oxide after activation is: get graphene oxide solution, add EDCHCl and sulfur-NHS, regulates solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.

Described graphene oxide solution concentration is 0.5 ~ 1.5mg/mL; Described graphene oxide and the weight ratio of product B are 1:(2 ~ 20); Described product B and the weight ratio of EDCHCl are 1:(1 ~ 5); Described product A and the weight ratio of sulfur-NHS are 1:(1 ~ 5).

Another object of the present invention is to provide a kind of anticancer compound, this anticancer compound prepares by the following method: the graphene oxide modified at described heparin or its salt, add cancer therapy drug (Drug) solution, stir, dialysis, the medicine of removing unentrapped, obtains anticancer drug complex.The principle that the graphene oxide that medicine is modified with heparin or its salt is combined is physical absorption, forms complex.

Graphene oxide described in the present invention refers to graphene oxide or the reproducibility graphene oxide after reducing agent reduction.The present invention can adopt the graphene oxide of two kinds of forms, with the reaction effect of heparin or its salt is good and efficiency is high.

In the present invention, preferably, the preparation method of described graphene oxide (GO) is: graphite powder, under the effect of concentrated sulphuric acid, potassium peroxydisulfate and phosphorus pentoxide, obtains the graphite powder (or being called the graphite powder of expansion) of pre-oxidation.Under the graphite powder concentrated sulphuric acid of pre-oxidation and potassium permanganate effect, generate graphene oxide.After adding the water liquid cessation reaction of hydrogen peroxide, obtain the precipitation of graphene oxide, more respectively by dilute hydrochloric acid solution, water washing precipitation, and with purified water dialysis, ultrasonic, obtain the solution of graphene oxide.Adopt the graphene oxide that the method obtains, effect is better, provides the good basis connecting heparin or its molecules of salt.But the preparation method of graphene oxide of the present invention (GO) is not limited only to the method.

The preparation method of described reproducibility graphene oxide (rGO) is: get described GO colloid solution, add vitamin C, ultrasonic under heating condition, add sodium chloride, leave standstill, centrifugal, collect to obtain precipitation, respectively wash 2 times with ethanol and pure water respectively, centrifugal reproducibility graphene oxide, and suspend by purified water, ultrasonic, the solution of the rGO obtained.Adopt the reproducibility graphene oxide that the method obtains, effect is better, provides the good basis connecting heparin or its molecules of salt.But the preparation method of reproducibility graphene oxide of the present invention is not limited only to the method.

Heparin sodium of the present invention refers to one or both the combination wherein of unfractionated heparin sodium, low molecular sodium heparin; The medicinal forms of described low molecular sodium heparin is medicinal low molecular sodium heparin, low molecular heparin calcium, Certoparin Sodium, Bemiparin sodium, dalteparin sodium, Enoxaparin Sodium, nadroparin calcium, Parnaparin Sodium, Clivarin sodium, tinzaparin sodium, one or more combination of Ardeparin Sodium.Adopt the heparin sodium of above form, with the reaction effect of graphene oxide is good and efficiency is high.

According to the principle of pH sensitivity, concrete preparation method is as follows:

A: graphene oxide-heparin stops compound/cancer therapy drug

Graphene oxide (GO) prepare graphite powder under the effect of concentrated sulphuric acid, potassium peroxydisulfate and phosphorus pentoxide, obtain the graphite powder (or being called the graphite powder of expansion) of pre-oxidation.Under the graphite powder concentrated sulphuric acid of pre-oxidation and potassium permanganate effect, generate graphene oxide.After adding the water liquid cessation reaction of hydrogen peroxide, obtain the precipitation of graphene oxide, more respectively by dilute hydrochloric acid solution, water washing precipitation, and with purified water dialysis, ultrasonic, obtain the colloid solution of graphene oxide.

The heparin (ADH-UFH) modified of adipic dihydrazide to prepare heparin sodium soluble in water, add adipic dihydrazide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) and I-hydroxybenzotriazole (HOBt), regulate solution ph to 6.0 ~ 7.0.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted starting material purified water dialysis removing, lyophilization, obtains product A DH-UFH.Described heparin sodium and the mol ratio of water are 1:(10000 ~ 100000); Described heparin sodium and the mol ratio of adipic dihydrazide are 1:(2 ~ 60); Described heparin sodium and the mol ratio of EDCHCl are 1:(2 ~ 60); Described heparin sodium and the mol ratio of HOBt are 1:(2 ~ 60).

(3) graphene oxide solution is got in the preparation of the GO (GO-ADH-UFH) of ADH-UFH modification, add EDCHCl and N-hydroxy-succinamide sulfonate sodium (sulfur-NHS), regulate solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add ADH-UFH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and ADH-UFH are removed in dialysis, obtain GO-ADH-UFH and to stop polymer solution.

The colloid solution concentration of described graphene oxide is 0.5 ~ 1.5mg/mL; Described graphene oxide and the weight ratio of product A DH-UFH are 1:(2 ~ 20); The weight ratio of described product A DH-UFH and EDCHCl is 1:(1 ~ 5); The weight ratio of described ADH-UFH and sulfur-NHS is 1:(1 ~ 5).

(4) the GO-ADH-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (GO-ADH-UFH/Drug).

B: reproducibility graphene oxide-heparin stops compound/cancer therapy drug

Graphene oxide preparation with in above-mentioned A (1).

Adipic dihydrazide modify heparin preparation with in above-mentioned A (2).

(3) the colloid solution of GO is got in the preparation of reproducibility graphene oxide (rGO), add vitamin C, ultrasonic under heating condition, add sodium chloride, leave standstill, centrifugal, collect to obtain precipitation, respectively wash 2 times with ethanol and pure water respectively, centrifugal reproducibility graphene oxide, and suspend by purified water, ultrasonic, the colloid solution of the rGO obtained.

(4) the colloid solution of rGO is got in the preparation of the rGO (rGO-ADH-UFH) of ADH-UFH modification, adds EDCHCl and sulfur-NHS, regulates solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add ADH-UFH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and ADH-UFH are removed in dialysis, obtain GO-ADH-UFH and to stop polymer solution.

The colloid solution concentration of described rGO is 0.5 ~ 1.5mg/mL; The weight ratio of described rGO and ADH-UFH is 1:(2 ~ 20); The weight ratio of described ADH-UFH and EDCHCl is 1:(1 ~ 5); The weight ratio of described ADH-UFH and sulfur-NHS is 1:(1 ~ 5).

(5) the rGO-ADH-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (rGO-ADH-UFH/Drug).

C: graphene oxide-Low molecular heparin stops compound/cancer therapy drug

Graphene oxide (GO) preparation with in above-mentioned A (1).

The Low molecular heparin (ADH-LMWH) modified of adipic dihydrazide to prepare low molecular sodium heparin soluble in water, add adipic dihydrazide (ADH), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) and I-hydroxybenzotriazole (HOBt), regulate value to 6.0 ~ 7.0 of solution.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product A DH-LMWH.

Described low molecular sodium heparin and the mol ratio of water are 1:(10000 ~ 100000); Described heparin sodium and the mol ratio of adipic dihydrazide are 1:(2 ~ 60); Described heparin sodium and the mol ratio of EDCHCl are 1:(2 ~ 60); Described heparin sodium and the mol ratio of HOBt are 1:(2 ~ 60).

(3) graphene oxide solution is got in the preparation of the GO (GO-ADH-LMWH) of ADH-LMWH modification, add EDCHCl and N-hydroxy-succinamide sulfonate sodium (sulfur-NHS), regulate solution value to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add ADH-LMWH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and ADH-LMWH are removed in dialysis, obtain GO-ADH-LMWH and to stop polymer solution.

Described graphene oxide solution concentration is 0.5 ~ 1.5mg/mL; Described graphene oxide and the weight ratio of ADH-LMWH are 1:(2 ~ 20); The weight ratio of described ADH-LMWH and EDCHCl is 1:(1 ~ 5); The weight ratio of described ADH-LMWH and sulfur-NHS is 1:(1 ~ 5).

(4) the GO-ADH-LMWH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (GO-ADH-LMWH/Drug).

D: reproducibility graphene oxide-Low molecular heparin stops compound/cancer therapy drug

Graphene oxide preparation with in above-mentioned A (1).

Adipic dihydrazide modify Low molecular heparin preparation with in above-mentioned C (2).

Reproducibility graphene oxide (rGO) preparation with in above-mentioned B (3).

(4) the colloid solution of rGO is got in the preparation of the rGO (rGO-ADH-LMWH) of ADH-LMWH modification, adds EDCHCl and sulfur-NHS, regulates solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add ADH-LMWH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and ADH-LMWH are removed in dialysis, obtain GO-ADH-LMWH and to stop polymer solution.

Described reproducibility graphene oxide colloid solution concentration is 0.5 ~ 1.5mg/mL; The weight ratio of described reproducibility graphene oxide and ADH-LMWH is 1:(2 ~ 20); The weight ratio of described ADH-LMWH and EDCHCl is 1:(1 ~ 5); The weight ratio of described ADH-LMWH and sulfur-NHS is 1:(1 ~ 5).

(5) the rGO-ADH-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (rGO-ADH-UFH/Drug).

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide, described graphene oxide refers to graphene oxide or the reproducibility graphene oxide after reducing agent reduction;

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide, described heparin sodium refers to unfractionated heparin sodium or low molecular sodium heparin;

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide is using adipic dihydrazide as connecting molecule and the graphene oxide of unfractionated heparin modification.

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide is using adipic dihydrazide as connecting molecule and the reproducibility graphene oxide of unfractionated heparin modification.

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide is using adipic dihydrazide as connecting molecule and the graphene oxide of Low molecular heparin modification.

Utilize pH sensitivity principle, a kind of heparin modified graphene oxide is using adipic dihydrazide as connecting molecule and the reproducibility graphene oxide of Low molecular heparin modification.

According to oxidoreduction principle, concrete preparation method is as follows:

E: graphene oxide-heparin stops compound/cancer therapy drug

Graphene oxide (GO) preparation with in above-mentioned A (1).

The heparin (Cys-UFH) modified of cystamine to prepare heparin sodium soluble in water, add cystamine (Cys), EDCHCl and HOBt, regulate pH value to 6.0 ~ 7.0 of solution.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product C ys-UFH.

Described heparin sodium and the mol ratio of water are 1:(10000 ~ 100000); Described heparin sodium and the mol ratio of cystamine are 1:(2 ~ 60); Described heparin sodium and the mol ratio of EDCHCl are 1:(2 ~ 60); Described heparin sodium and the mol ratio of HOBt are 1:(2 ~ 60)

(3) graphene oxide solution is got in the preparation of the GO (GO-ss-UFH) of Cys-UFH modification, adds EDCHCl and sulfur-NHS, regulates solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add Cys-UFH, stirring is spent the night, and unreacted EDCHCl, sulfur-NHS and Cys-UFH are removed in dialysis, obtains GO-ss-UFH and to stop polymer solution.

Described graphene oxide colloid solution concentration is 0.5 ~ 1.5mg/mL; The weight ratio of described graphene oxide and Cys-UFH is 1:(2 ~ 20); The weight ratio of described Cys-UFH and EDCHCl is 1:(1 ~ 5); The weight ratio of described Cys-UFH-LMWH and sulfur-NHS is 1:(1 ~ 5).

(4) the GO-ss-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (GO-ss-UFH/Drug).

F: reproducibility graphene oxide-heparin stops compound/cancer therapy drug

Graphene oxide preparation with in above-mentioned A (1).

Cystamine modify heparin (Cys-UFH) preparation with in above-mentioned E (2).

(3) GO colloid solution is got in the preparation of reproducibility graphene oxide (rGO), add vitamin C, ultrasonic under heating condition, add sodium chloride, leave standstill, centrifugal, collect to obtain precipitation, respectively wash 2 times with ethanol and pure water respectively, centrifugal reproducibility graphene oxide, and suspend by purified water, ultrasonic, the colloid solution of the rGO obtained.

(4) rGO solution is got in the preparation of the rGO (rGO-ss-UFH) of Cys-UFH modification, adds cystamine (Cys), EDCHCl and HOBt, regulates pH value to 6.0 ~ 7.0 of solution, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

Described rGO colloid solution concentration is 0.5 ~ 1.5mg/mL; Described rGO and the weight ratio of product B are 1:(2 ~ 20); The weight ratio of described Cys-UFH and EDCHCl is 1:(1 ~ 5); The weight ratio of described product C ys-UFH and sulfur-NHS is 1:(1 ~ 5).

(5) the rGO-ss-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (rGO-ss-UFH/Drug).

G: graphene oxide-Low molecular heparin stops compound/cancer therapy drug

Graphene oxide (GO) preparation with in above-mentioned A (1).

The Low molecular heparin (Cys-LMWH) modified of cystamine to prepare low molecular sodium heparin soluble in water, add adipic dihydrazide (ADH), EDCHCl and HOBt, regulate pH value to 6.0 ~ 7.0 of solution.Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis dialysis removing, lyophilization, obtains product.

Described low molecular sodium heparin and the mol ratio of water are 1:(10000 ~ 100000); The mol ratio of described low molecular sodium heparin and cystamine is 1:(2 ~ 60); Described low molecular sodium heparin and the mol ratio of EDCHCl are 1:(2 ~ 60); Described low molecular sodium heparin and the mol ratio of HOBt are 1:(2 ~ 60).

(3) graphene oxide solution is got in the preparation of the GO (GO-ss-LMWH) of Cys-LMWH modification, adds EDCHCl and sulfur-NHS, regulates solution value to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C.Add Cys-LMWH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and Cys-LMWH are removed in dialysis, obtain GO-ss-LMWH and to stop polymer solution.

Described graphene oxide solution concentration is 0.5 ~ 1.5mg/mL; Described graphene oxide and the weight ratio of Cys-UFH are 1:(2 ~ 20); The weight ratio of described Cys-UFH and EDCHCl is 1:(1 ~ 5); The weight ratio of described Cys-UFH and sulfur-NHS is 1:(1 ~ 5).

(4) the GO-ss-LMWH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (GO-ss-LMWH/Drug).

H: reproducibility graphene oxide-heparin stops compound/cancer therapy drug

Graphene oxide preparation with in above-mentioned A (1).

Cystamine modify heparin preparation with in above-mentioned G (2).

Reproducibility graphene oxide (rGO) preparation with in above-mentioned B (3).

(4) the colloid solution of rGO is got in the preparation of the rGO (rGO-ss-LMWH) of Cys-LMWH modification, adds EDCHCl and sulfur-NHS, regulates solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3h at 25 ~ 30 DEG C.Add Cys-LMWH, stirring reaction 6 ~ 24 hours at 25 ~ 30 DEG C, unreacted EDCHCl, sulfur-NHS and Cys-LMWH are removed in dialysis, obtain GO-ss-LMWH and to stop polymer solution.

The colloid solution concentration of described rGO is 0.5 ~ 1.5mg/mL; The weight ratio of described rGO and Cys-LMWH is 1:(2 ~ 20); The weight ratio of described Cys-LMWH and EDCHCl is 1:(1 ~ 5); The weight ratio of described Cys-LMWH and sulfur-NHS is 1:(1 ~ 5).

(5) the rGO-ADH-UFH that is prepared in containing anticancer drug complex stops polymer solution, adds cancer therapy drug (Drug) solution, stirs, dialysis, and the medicine of removing unentrapped, obtains anticancer drug complex (rGO-ss-UFH/Drug).

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide, described graphene oxide refers to graphene oxide or the reproducibility graphene oxide after reducing agent reduction.

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide, described heparin sodium refers to unfractionated heparin sodium or low molecular sodium heparin.

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide is using cystamine as connecting molecule and the graphene oxide of unfractionated heparin modification.

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide is using cystamine as connecting molecule and the reproducibility graphene oxide of unfractionated heparin modification.

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide is using cystamine as connecting molecule and the graphene oxide of Low molecular heparin modification.

Utilize oxidoreduction principle, a kind of heparin modified graphene oxide is using cystamine as connecting molecule and the reproducibility graphene oxide of Low molecular heparin modification.

According to quality standard WS ₁-(X-149)-2005Z, low molecular sodium heparin is with heparin sodium through nitrous acid cleavage, the refining and sodium salt of the CSSO3 that obtains (new drug become a full member standard the 66th 193-196 page).

According to quality standard WS ₁-(X-147)-2005Z, low molecular heparin calcium is with heparin sodium through nitrous acid cleavage, the refining and calcium salt of the CSSO3 that obtains (new drug become a full member standard the 66th 176-179 page).The weight average molecular weight of low molecular sodium heparin and low molecular heparin calcium is all less than 8000, and the component that molecular weight is less than 8000 is all no less than 60% of total amount.

Certoparin Sodium is produced by AspenPharma company, and trade name has: obtain according to nitrous depolymerisation method, its mean molecule quantity is 6000, and its component molecules amount of 70% is not more than 10000 (Martindale, 36 editions 1242 pages).

Bemiparin is produced by Rovi drugmaker, and trade name has: with ), mean molecule quantity is 3000-4200, and its characteristic molecular amount is 3600, and the molecular weight of about 85% is less than 6000, and disaccharide unit is containing 2.0 sulphuric acid unit (Drugs.2003,63 (21): 2357-2377 of having an appointment; Martindale, 36 editions 1223 pages).

Dalteparin sodium is by Pfizer's production and selling, and trade name has: ), obtain according to nitrous depolymerisation method, its mean molecule quantity is 5600-6400, its characteristic molecular amount is 6000, the component being less than 3000 molecular weight is not more than 13%, be greater than the component of 8000 molecular weight within the scope of 15-25%, disaccharide unit contains 2.0-2.5 sulphuric acid unit (European Pharmacopoeia 7.0 editions 1788-1789 pages; Martindale, 36 editions 1255 pages).

Enoxaparin Sodium is by matching Norfin, Inc's production and selling, and trade name has: with ), gather method obtain according to carrying out alkaline hydrolysis after Benzylation, its mean molecule quantity is 3800-5000, and its characteristic molecular amount is 4500 (European Pharmacopoeia 7.0 editions 1920-1921 pages).

Nadroparin calcium is by matching Norfin, Inc and GSK company production and selling, and trade name has: by NobleMolecules company production and selling, trade name: adopt nitrous depolymerisation method to obtain, its mean molecule quantity is 3600-5000, and its characteristic molecular amount is 4300, and the component being less than 2000 molecular weight is not more than 15% (European Pharmacopoeia 7.0 editions 2543-2545 pages).

Parnaparin Sodium is by Italian AlfaWassermann drugmaker production and selling, and trade name has: pharmaceutical industries company of Hibiscus rosa-sinensis of Japan production and selling, trade name: aginomoto drugmaker of Japan trade name: obtain according to peroxidative depolymerization method, its mean molecule quantity is 4000-6000, and its characteristic molecular amount is 5000, and the component being less than 3000 molecular weight is not more than 30% (European Pharmacopoeia 7.0 editions the 2672nd page).

Clivarin sodium is by German KnollAG company production and selling, and trade name has: nitrous depolymerisation method is adopted to obtain, its mean molecule quantity is 3900 (ExpertOpinPharmacother.2002,3 (2): 173-182) also report is had, its mean molecule quantity is 3150-5150, containing 2.1 sulfate groups (Martindale, 36 editions: 1388 pages) in its disaccharide unit.

Tinzaparin sodium by Aktiebolaget Leo (SE) Box 941, S-251 09 Helsingborg, Sweden of Denmark production and selling, trade name: by Novo company production and selling trade name: use heparinase to carry out β-elimination depolymerization to obtain, its mean molecule quantity is 5500-7500, and its characteristic molecular amount is 6500, and the component being less than 2000 molecular weight is not more than 10% (European Pharmacopoeia 7.0 editions 3098-3099 pages).

Ardeparin Sodium is produced by Wyeth Pharmaceuticals, trade name: Normiflo, obtain according to peroxidative depolymerization, its component molecules amount of 98% is at 2000-15000, its mean molecule quantity is 5500-6500, containing 2.7 sulfate group (Martindale in its disaccharide unit, 36 editions 1216 pages), but not reason due to safety or effectiveness but within 2000, remove city (JThrombThrombolysis.2001,11 (3): 247-259 from American market due to the reason of regulation; CarbohydrPolym.2013,97 (2): 684-689).

In the present invention, the mean molecule quantity of unfractionated heparin sodium (UFH) calculates with 1.5 ten thousand, and the mean molecule quantity of Low molecular heparin calculates with 0.5 ten thousand, feeds intake with mol ratio.Graphene oxide or reproducibility graphene oxide due to laminated structure not of uniform size, the molecular weight self do not determined or average molecular weight range, separately because ingredient proportion is different, the heparin of the adipic dihydrazide obtained or cystamine chemical modification and the graphene oxide of final chemical modification or reproducibility graphene oxide substitution value all different, therefore in the present invention, all feed intake with weight ratio containing adipic dihydrazide or the heparin of cystamine chemical modification and the material of graphene oxide or reproducibility graphene oxide.

The invention has the beneficial effects as follows:

(1) contain a large amount of oxy radicals such as hydroxyl, epoxy radicals, carboxyl in graphene oxide self structure, and there is good hydrophilic, can be widely used in biomedicine.Because graphene oxide is monoatomic layer structure, having larger specific surface area (has document to be measured as 736.6m ²/ g, is shown in Langmuir, 2013,29:13443-13448), its two sides is all combined with medicine by covalency, noncovalent interaction, therefore has higher drug load amount.It, by stronger physisorption and fragrant lopps medicine Non-covalent binding, can deliver some insoluble drugs, transports and have great importance in the body particularly to most of slightly solubility cancer therapy drug.Heparin is a kind of good water soluble molecules, and it utilizes self has blood vessel formation against function and play active anticancer.Administration together with cancer therapy drug, has synergistic therapeutic action.The present invention utilizes graphene oxide and heparin in the advantage of drug world, according to research, is connected by heparin with the carrying out of graphene oxide, obtain heparin modified after graphene oxide.

(2) the present invention is according to the connectivity problem of two kinds of principle research heparin and graphene oxide, and the first scheme is according to the principle of pH sensitivity.Namely utilize in tumor cell and extracellular fluid has lower pH, carboxyl (COOH) and the active group (O=C-NH-NH in adipic dihydrazide (ADH) structure of graphene oxide or reproducibility graphene oxide ₂) form biamide structure (O=C-NH-NH-C=O), be prepared into the compound that stops (conjugates) of pH sensitivity.In the blood (pH7.35 ~ 7.45) of meta-alkalescence, this biamide structure can not be hydrolyzed, heparin is positioned at the both sides of graphene oxide, the water solublity of graphene oxide can be increased, utilize its pi-pi accumulation and hydrophobic interaction simultaneously, antitumor drug in physical absorption, forms complex (complexs); When this complex is transported to target cell (as cancerous cell) place, utilize strengthen penetrating and retention effect (edit see Lu Bin. novel pharmaceutical formulation and new technique, the second edition, 63rd page), enter in target cell, in the slant acidity microenvironment of target cell, complex discharges cancer therapy drug, simultaneously, due to biamide structure (O=C-NH-NH-C=O) fracture stopped in compound, define heparin and graphene oxide, the former has blood vessel formation against function, plays synergistic therapeutic action to anticancer.

First scheme is according to oxidoreduction principle.Namely utilize reproducibility in tumor cell comparatively strong, the glutathion (GSH) containing higher concentration, the compound containing (-S-S-) can be reduced into the compound containing sulfydryl (SH).Cystamine (NH ₂cH ₂cH ₂sH) dimer (NH formed ₂cH ₂cH ₂s-SCH ₂cH ₂nH ₂) amino (NH of one end ₂) be combined into amido link (O=C-NH) with the carboxyl of graphene oxide, the amino of the other end and the carboxyl of heparin are combined into amido link, what formed stops compound after cancer therapy drug in physical bond, arrives cancerous cell through blood, after in the born of the same parents that transduce, under the effect of GSH, disulfide bond is degraded, and dissociate heparin, simultaneously because pH in born of the same parents is lower, the medicine discharged increases, and heparin adds the therapeutical effect of cancer therapy drug.

(3) the present invention heparin modified after graphene oxide can be used as the carrier material of medicine particularly cancer therapy drug.Utilize pH sensitivity principle or oxidoreduction principle, and adipic dihydrazide or cystamine are for connecting molecule, with heparin, chemical modification is carried out to graphene oxide, there is the water solublity, the synergistic advantage of cancer therapy drug antitumor that increase graphene oxide, solve the problems such as graphene oxide water solublity is poor, easy gathering.Utilize heparin to be combined with cytokine profiles specifically, inhibiting angiogenesis simultaneously, play synergistic antitumor effect.

Accompanying drawing explanation

The structure of Fig. 1 graphene oxide, containing a large amount of carbon-carbon double bonds in this structure, simultaneously containing hydroxyl (OH), carboxyl (COOH) and epoxy bond (C-O-C).

The structure of Fig. 2 reproducibility graphene oxide, this structure is compared with graphene oxide, and its oxy radical greatly reduces, and has a large amount of hydroxy-acid groups at structural edge place.

The structure of Fig. 3 heparin, the linear polysaccharide compounds that this structure is made up of 1 → 4 glycuronic acid connected and glucamine disaccharide, each disaccharide unit contains a carboxyl, one or more hydroxyl, 2 ~ 2.5 sulfates, and each chain is containing 0.3 free amino of having an appointment.

The typical IR collection of illustrative plates of Fig. 4 graphene oxide, in this collection of illustrative plates, 3885cm ^-1wider peak is attributed to the stretching vibration peak of hydroxyl (O-H), due to containing water, defines hydrogen bond with water, defines associative structure, therefore is wider peak; 2975cm ^-1peak, 2928cm ^-1peak and 2892cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1648cm ^-1peak is attributed to the stretching vibration peak of carboxyl (O=C-OH), 1420cm ^-1peak and 1383cm ^-1peak is attributed to the stretching vibration peak of carbon hydroxyl (C-OH), 1090cm ^-1peak and 1050cm ^-1peak is attributed to the stretching vibration peak of the carbon oxygen (C-O-C) of epoxide group.

The typical IR collection of illustrative plates of Fig. 5 reproducibility graphene oxide, in this collection of illustrative plates, 3885cm ^-1wider peak, peak is attributed to the stretching vibration peak of hydroxyl (O-H), 2912cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1631cm ^-1peak is attributed to the stretching vibration peak of carboxyl (O=C-OH), 1574cm ^-1peak is attributed to carbon-carbon double bond (C=C) stretching vibration peak of aromatic rings, the stretching vibration peak of carbon hydroxyl (C-OH), 1431cm ^-1peak is attributed to the stretching vibration peak of carbon hydroxyl (C-OH), 1060cm ^-1peak and 1030cm ^-1peak is attributed to the stretching vibration peak of the carbon oxygen (C-O-C) of epoxide group.

The typical IR collection of illustrative plates of the heparin that Fig. 6 adipic dihydrazide is modified, in this collection of illustrative plates, 3423cm ^-1peak and 3310cm ^-1wider peak, these two, peak is attributed to the stretching vibration peak of hydroxyl (O-H) and amino (N-H), due to containing water, defines hydrogen bond with water, defines associative structure, therefore be wider peak; 2972cm ^-1peak and 2934cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1655cm ^-1peak is attributed to the asymmetrical stretching vibration peak of carboxyl (O=C-OH), 1701cm ^-1peak is attributed to amide I peaks, 1483cm ^-1peak is attributed to amide II peak, 1466cm ^-1peak is attributed to the symmetrical stretching vibration peak of carboxyl (O=C-OH), 1228cm ^-1peak is attributed to the asymmetrical stretching vibration peak of thiocarbonyl (S=O), 1032cm ^-1peak and 1006cm ^-1the symmetrical stretching vibration peak of peak thiocarbonyl (S=O).

The typical IR collection of illustrative plates of the Low molecular heparin that Fig. 7 adipic dihydrazide is modified, in this collection of illustrative plates, 3445cm ^-1peak is attributed to the stretching vibration peak of hydroxyl (O-H) and amino (N-H), due to containing water, defines hydrogen bond with water, defines associative structure, therefore be wider peak; 2933cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1660cm ^-1peak is attributed to the asymmetrical stretching vibration peak of carboxyl (O=C-OH), 1701cm ^-1peak is attributed to amide I peaks, 1548cm ^-1the stretching vibration of amino (N-H) and bending vibration (amide II peak) on secondary amide, 1385cm ^-1peak is attributed to the symmetrical stretching vibration peak of carboxyl (O=C-OH), 1228cm ^-1peak is attributed to the asymmetrical stretching vibration peak of thiocarbonyl (S=O), 1031cm ^-1peak and 985cm ^-1the symmetrical stretching vibration peak of peak thiocarbonyl (S=O).

The typical IR collection of illustrative plates of Fig. 8 heparin-adipic dihydrazide-reproducibility graphene oxide, in this collection of illustrative plates, 3441cm ^-1peak is attributed to the stretching vibration peak of hydroxyl (O-H) and amino (N-H); 2921cm ^-1peak and 2848cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1641cm ^-1peak is attributed to the asymmetrical stretching vibration peak of carboxyl (O=C-OH), 1549cm ^-1the stretching vibration of amino (N-H) and bending vibration (amide II peak) on secondary amide, 1385cm ^-1peak is attributed to the symmetrical stretching vibration peak of carboxyl (O=C-OH), 1212cm ^-1peak is attributed to the asymmetrical stretching vibration peak of thiocarbonyl (S=O), 1026cm ^-1peak and 1000cm ^-1the symmetrical stretching vibration peak of peak thiocarbonyl (S=O).

The typical IR collection of illustrative plates of Fig. 9 Low molecular heparin-adipic dihydrazide-reproducibility graphene oxide, in this collection of illustrative plates, 3425cm ^-1peak is attributed to the stretching vibration peak of hydroxyl (O-H) and amino (N-H); 2925cm ^-1peak is attributed to the stretching vibration peak of hydrocarbon (C-H), 1648cm ^-1peak is attributed to the asymmetrical stretching vibration peak of carboxyl (O=C-OH), 1552cm ^-1peak is attributed to stretching vibration and the bending vibration (amide II peak) of amino (N-H) on secondary amide, 1447cm ^-1peak is attributed to the symmetrical stretching vibration peak of carboxyl (O=C-OH), 1216cm ^-1peak is attributed to the asymmetrical stretching vibration peak of thiocarbonyl (S=O), 1031cm ^-1peak and 1000cm ^-1the symmetrical stretching vibration peak of peak thiocarbonyl (S=O).

Detailed description of the invention

A kind of heparin modified graphene oxide and preparation method thereof, is described in further detail the present invention by following embodiment, but the present invention is not confined to following embodiment.

Embodiment 1

Graphene oxide-heparin stops compound

(1) graphite powder 0.40kg is got in the preparation of graphene oxide (GO), slowly add concentrated sulphuric acid 2.40 liters, potassium peroxydisulfate 0.60kg and phosphorus pentoxide 0.60kg, is heated to 80 DEG C, stir 10 ~ 24h, obtain navy blue mixed liquor, be cooled to room temperature gradually, add purified water 10 liters, filter, collect residue, 60 DEG C of dried overnight, obtain the graphite powder of pre-oxidation.Get the graphite powder 0.20kg of pre-oxidation, slowly join 15 liters (0 DEG C) in the concentrated sulphuric acid of ice bath, under condition of ice bath, stir and slowly add potassium permanganate 2.5kg.Add rear continuation and stir half an hour, reactant liquor is heated up gradually be heated to 40 DEG C afterwards, stir 8h.Slowly the mixed liquor adding purified water 75 liters and hydrogen peroxide (30wt%) 3 liters stops above-mentioned reaction.Hold over night, the crystallization of question response liquid goes out precipitation, abandoning supernatant, uses dilute hydrochloric acid solution (1:10, v/v) and water washing to precipitate respectively twice to three time, filter, must precipitate, again use aqueous suspension, and dialyse with water, under the filtrate ice bath obtained, ultrasonic 2 ~ 3h (solution temperature < 15 DEG C), obtains the brown GO colloid solution of normal distribution.

Adipic dihydrazide modify heparin (ADH-UFH) prepare in water-soluble 0.4 liter of heparin sodium 5g, add adipic dihydrazide (ADH) 2.2g, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) 3.2g and I-hydroxybenzotriazole (HOBt) 2.2g, regulates solution ph to 6.8.Stirring reaction 24h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product, and Fig. 6 is the typical IR collection of illustrative plates of the heparin that adipic dihydrazide is modified.

(3) 1mg/mL graphene oxide solution 1 liter is got in the preparation of the GO (GO-ADH-UFH) of ADH-UFH modification, add EDCHCl28.7g and N-hydroxy-succinamide sulfonate sodium (sulfur-NHS) 32.5g, regulate solution ph 6.4, stirring reaction 2h at 25 ~ 30 DEG C, with activating carboxy acid's group.Add ADH-UFH10g, stirring reaction 12h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ADH-UFH and to stop polymer solution.

Embodiment 2

Reproducibility graphene oxide-heparin stops compound

(1) above-described embodiment 1 (1) middle 1 liter, GO colloid solution is got in the preparation of reproducibility graphene oxide (rGO), makes 0.1g/L aqueous solution, adds Catergen g, ultrasonic 30min at 60 DEG C, adds sodium chloride 5g afterwards, leaves standstill 2h, centrifugal 1000min under 4000rpm, collecting precipitation, respectively washs 2 times with ethanol and pure water, centrifugal 1000min under 4000rpm respectively, reproducibility graphene oxide, and suspend by purified water, ultrasonic 1h, the colloid solution of the rGO obtained.

(2) 1mg/mLrGO solution 1 liter is got in the preparation of the rGO (rGO-ADH-UFH) of ADH-UFH modification, adds EDCHCl28.7g and sulfur-NHS32.5g, and stirring reaction 1.5h at adjustment solution ph 6.4,25 ~ 30 DEG C, with activating carboxy acid's group.Add above-described embodiment 1 (2) in ADH-UFH10g, stirring reaction 20h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains rGO-ADH-UFH and to stop polymer solution.Fig. 8 is the typical IR collection of illustrative plates of heparin-adipic dihydrazide-reproducibility graphene oxide.

Embodiment 3

Graphene oxide-Low molecular heparin stops compound

(1) the preparation of the Low molecular heparin (ADH-LMWH) of adipic dihydrazide modification is got in water-soluble 1 liter of low molecular sodium heparin 10g, adds ADH2g, EDCHCl8g and HOBt6g, regulates solution ph to 6.6.Stirring reaction 36h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.Fig. 7 is the typical IR collection of illustrative plates of the Low molecular heparin that adipic dihydrazide is modified.

(2) above-described embodiment 1 (1) middle 1mg/mL graphene oxide solution 1 liter is got in the preparation of the GO (GO-ADH-LMWH) of ADH-UFH modification, add EDCHCl26g and sulfur-NHS30g, stirring reaction 2h at adjustment solution ph 6.2,25 ~ 30 DEG C, with activating carboxy acid's group.Add ADH-LMWH10g, stirring reaction 18h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ADH-LMWH and to stop polymer solution.

Embodiment 4

Graphene oxide-Low molecular heparin stops compound

(1) the preparation of the Enoxaparin Sodium (ADH-Eno) of adipic dihydrazide modification is got in water-soluble 1 liter of Enoxaparin Sodium (Eno) 12g, adds ADH4g, EDCHCl3g and HOBt3g, regulates solution ph to 6.6.Stirring reaction 40h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 1 (1) middle 1mg/mL graphene oxide solution 1.2 liters is got in the preparation of the GO (GO-ADH-Eno) of ADH-UFH modification, add EDCHCl26g and sulfur-NHS30g, stirring reaction 2h at adjustment solution ph 6.2,25 ~ 30 DEG C, with activating carboxy acid's group.Add ADH-Eno12g, stirring reaction 18h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ADH-Eno and to stop polymer solution.

Embodiment 5

Reproducibility graphene oxide-Low molecular heparin stops compound

(1) adipic dihydrazide the preparing in water-soluble 1 liter of low molecular sodium heparin 10g of Low molecular heparin (ADH-LMWH) of modifying, add ADH3g, EDCHCl3g and HOBt3g, regulate solution ph to 6.2.Stirring reaction 36h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 2 (1) middle 1mg/mLrGO solution 1 liter is got in the preparation of the rGO (rGO-ADH-LMWH) of ADH-LMWH modification, adds EDCHCl30g and sulfur-NHS35g, stirring reaction 2h at adjustment solution ph 6.5,25 ~ 30 DEG C.Add middle ADH-LMWH10g, stirring reaction 12h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains rGO-ADH-UFH and to stop polymer solution.Fig. 9 is the typical IR collection of illustrative plates of Low molecular heparin-adipic dihydrazide-reproducibility graphene oxide.

Embodiment 6

Graphene oxide-heparin stops compound

(1) cystamine the preparing in water-soluble 0.2 liter of heparin sodium 3g of heparin (Cys-UFH) of modifying, add cystamine 2g, EDCHCl2g and HOBt2g, regulate solution ph to 6.5.Stirring reaction 24h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) 1mg/mL graphene oxide solution 1 liter is got in the preparation of the GO (GO-ss-UFH) of Cys-UFH modification, adds EDCHCl28.7g and sulfur-NHS32.5g, and stirring reaction 2h at adjustment solution ph 6.4,25 ~ 30 DEG C, with activating carboxy acid's group.Add Cys-UFH10g, stirring reaction 23h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ss-UFH and to stop polymer solution.

Embodiment 7

Reproducibility graphene oxide-heparin stops compound

(1) cystamine the preparing in water-soluble 0.3 liter of heparin sodium 3g of heparin (Cys-UFH) of modifying, add cystamine 2.5g, EDCHCl3g and HOBt3g, regulate solution ph to 6.5.Stirring reaction 30h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 2 (1) middle 1mg/mLrGO solution 1 liter is got in the preparation of the rGO (rGO-ss-UFH) of Cys-UFH modification, add EDCHCl30g and sulfur-NHS40g, stirring reaction 2h at adjustment solution ph 6.4,25 ~ 30 DEG C, with activating carboxy acid's group.Add Cys-UFH10g, stirring reaction 22h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains rGO-ss-UFH and to stop polymer solution.

Embodiment 8

Graphene oxide-Low molecular heparin stops compound

(1) the preparation of the Low molecular heparin (Cys-LMWH) of cystamine modification is got in water-soluble 0.6 liter of low molecular sodium heparin 3g, adds cystamine 1.5g, EDCHCl1.5g and HOBt1.5g, regulates solution ph to 6.5.Stirring reaction 40h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 1 (1) middle 1mg/mL graphene oxide solution 1 liter is got in the preparation of the GO (GO-ss-LMWH) of Cys-LMWH modification, add EDCHCl26g and sulfur-NHS30g, stirring reaction 2h at adjustment solution ph 6.2,25 ~ 30 DEG C, with activating carboxy acid's group.Add Cys-LMWH7g, stirring reaction 20h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ss-LMWH and to stop polymer solution.

Embodiment 9

Graphene oxide-Low molecular heparin stops compound

(1) the preparation of the Parnaparin Sodium (Cys-Par) of cystamine modification is got in water-soluble 0.2 liter of Parnaparin Sodium (Par) 3g, adds cystamine 2g, EDCHCl2g and HOBt2g, regulates solution ph to 6.5.Stirring reaction 45h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 1 (1) middle 1mg/mL graphene oxide solution 1 liter is got in the preparation of the GO (GO-ss-Par) of Cys-Par modification, add EDCHCl26g and sulfur-NHS30g, stirring reaction 2h at adjustment solution ph 6.2,25 ~ 30 DEG C, with activating carboxy acid's group.Add Cys-Par8g, stirring reaction 22h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains GO-ss-Par and to stop polymer solution.

Embodiment 10

Reproducibility graphene oxide-Low molecular heparin stops compound

(1) the preparation of the Low molecular heparin (Cys-LMWH) of cystamine modification is got in water-soluble 0.25 liter of low molecular sodium heparin 3g, adds cystamine 2.5g, EDCHCl1g and HOBt1g, regulates solution ph to 6.6.Stirring reaction 36h at 25 ~ 30 DEG C.Unreacted reactant use water dialysis removing, lyophilization, obtains product.

(2) above-described embodiment 2 (1) middle 1mg/mLrGO solution 1 liter is got in the preparation of the rGO (rGO-ss-LMWH) of Cys-LMWH modification, adds EDCHCl30g and sulfur-NHS35g, stirring reaction 2h at adjustment solution ph 6.5,25 ~ 30 DEG C.Add middle Cys-LMWH10g, stirring reaction 22h at 25 ~ 30 DEG C, unreacted starting material material is removed in dialysis, obtains rGO-ss-UFH and to stop polymer solution.

By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.

Claims (10)

1. a graphene oxide for heparin or the modification of its salt, be is characterized in that: be prepared by following a kind of wherein method:

(1) be connect molecule with adipic dihydrazide, be connected by amido link between heparin and graphene oxide and obtain heparin modified graphene oxide;

(2) be connect molecule with cystamine, be connected by disulfide bond between heparin and graphene oxide and obtain heparin modified graphene oxide.

2. the graphene oxide of heparin as claimed in claim 1 or the modification of its salt, it is characterized in that: in method (1), adipic dihydrazide is adopted to modify heparin, obtain the heparin after adipic dihydrazide modification, then the heparin after being modified by the adipic dihydrazide obtained and the graphene oxide after activation react 6 ~ 24h at 25 ~ 30 DEG C, obtain stopping using adipic dihydrazide as the heparin modified graphene oxide connecting molecule polymer solution.

3. the graphene oxide of heparin as claimed in claim 2 or the modification of its salt, it is characterized in that: the method that described adipic dihydrazide modifies heparin is: heparin sodium is soluble in water, add adipic dihydrazide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and I-hydroxybenzotriazole, regulate solution ph to 6.0 ~ 7.0; Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C; Unreacted starting material dialysis removing, dry, obtain the heparin after the modification of product adipic dihydrazide.

4. the graphene oxide of heparin as claimed in claim 1 or the modification of its salt, it is characterized in that: in method (2), cystamine is adopted to modify heparin, obtain the heparin after cystamine modification, then the heparin after being modified by the cystamine obtained and the graphene oxide after activation react 6 ~ 24h at 25 ~ 30 DEG C, obtain stopping using cystamine as the heparin modified graphene oxide connecting molecule polymer solution.

5. the graphene oxide of heparin as claimed in claim 4 or the modification of its salt, it is characterized in that: the method that described cystamine modifies heparin is: heparin sodium is soluble in water, add cystamine, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and I-hydroxybenzotriazole, regulate solution ph to 6.0 ~ 7.0; Stirring reaction 12 ~ 48h at 25 ~ 30 DEG C; Unreacted starting material dialysis removing, dry, obtain the heparin after the modification of product cystamine.

6. the graphene oxide that the heparin as described in claim 2 or 4 or its salt are modified, it is characterized in that: get graphene oxide solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide sulfonate sodium, regulate solution ph to 6.0 ~ 7.0, stirring reaction 1 ~ 3 hour at 25 ~ 30 DEG C, obtains the graphene oxide after activating.

7. the graphene oxide that the heparin as described in claim 3 or 5 or its salt are modified, is characterized in that: described heparin sodium refers to one or both the combination wherein of unfractionated heparin sodium, low molecular sodium heparin; The medicinal forms of described low molecular sodium heparin is medicinal low molecular sodium heparin, low molecular heparin calcium, Certoparin Sodium, Bemiparin sodium, dalteparin sodium, Enoxaparin Sodium, nadroparin calcium, Parnaparin Sodium, Clivarin sodium, tinzaparin sodium, one or more combination of Ardeparin Sodium.

8. the graphene oxide that the heparin according to any one of Claims 1 to 5 or its salt are modified, is characterized in that: described graphene oxide is graphene oxide or the reproducibility graphene oxide after reducing agent reduction.

9. the graphene oxide that the heparin according to any one of claim 1 ~ 8 or its salt are modified is preparing the application of pharmaceutical carrier.

10. containing an anticancer drug complex, it is characterized in that, prepare by the following method: the graphene oxide that the heparin according to any one of claim 1 ~ 8 or its salt are modified mixes with cancer therapy drug, stir, dialysis, the medicine of removing unentrapped, obtains anticancer drug complex.