CN108309937A - A kind of preparation method for containing the reduction response type nano material of antitumor drug - Google Patents

A kind of preparation method for containing the reduction response type nano material of antitumor drug Download PDF

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CN108309937A
CN108309937A CN201810328003.6A CN201810328003A CN108309937A CN 108309937 A CN108309937 A CN 108309937A CN 201810328003 A CN201810328003 A CN 201810328003A CN 108309937 A CN108309937 A CN 108309937A
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mpeg
hnts
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喻永杰
胡志国
王小坤
赵沙沙
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Henan Normal University
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
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    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

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Abstract

The technical problem to be solved by the present invention is to provide a kind of preparation methods for containing the reduction response type nano material of antitumor drug, by carrying out using silane coupling agent covalent modification to the surfaces HNTs, containing cystine linkage nanotube of the end with amino is accessed, while HNTs polymer nanocomposites are formed by the electrostatic interaction between nanotube ends amino and block polymer PEO b PAA terminal carboxyl groups.Redox response group cystine linkage is added in design can be used for the carrier of antitumor drug, while reaching and allowing pharmaceutical carrier that long-range can be kept to recycle and quickly release the drug in diseased region in vivo.The cross-linked structure that polymer micelle is stablized can improve the stability of micella, can enhance medicine stability as pharmaceutical carrier, and the micella based on cystine linkage can realize that drug is discharged in tumor locus, improve therapeutic effect.

Description

A kind of preparation method for containing the reduction response type nano material of antitumor drug
Technical field
The invention belongs to the preparing technical fields of nano-medicament carrier, and in particular to a kind of for containing antitumor drug Restore the preparation method of response type nano material.
Background technology
Tumour is one of the major disease for threatening human health in China, is most had currently, chemotherapy is clinical treatment tumour The method of effect, but the selectivity of chemotherapeutics and water solubility are poor, and toxic side effect, while killing tumour cell Also normal histocyte can be killed.In addition, kinds of tumor cells is to the insensitivity of chemotherapeutics and its intrinsic multiple medicine Drug resistance further reduced therapeutic effect.
Nano-medicament carrier because its with targeting, specificity, absorb it is fast, quantitative it is accurate, drugloading rate is high and can control Drug releasing rate extends the features such as drug treating time and raising medicine stability and is concerned by people.Therefore we It can be studied in terms of following two：(1) antitumor drug is carried out to structurally-modified, the antitumor precursor medicine of synthesizing new Object；(2) nanotechnology is combined with pharmaceutical carrier, researchs and develops nano-medicament carrier delivery system.
Halloysite nanotubes (HNTs) have the characteristics that environmental-friendly, good biocompatibility and nature is abundant forms because of it For the important nano material that development is biomedical.The characteristics of using hydroxyl is all contained inside and outside halloysite nanotubes, it is connect Branch modification can improve its structural property, and make halloysite nanotubes functionalization.Up to the present, HNTs- polymer nanocomposites are compound Material is widely used in the fields such as tissue engineering bracket, drug conveying, cancer cell isolation, bone implant and cosmetics.
Reduction response type drug conveying carrier has many advantages, such as the rate of release for reducing multidrug resistance, improving drug.This Disulfide bond is modified halloysite nanotubes and is introduced in invention, makes it have reduction response, is contained end by solwution method Cystine linkage-the HNTs and block polymer MPEG-b-PAA of amino be combined with each other to obtain with reduction response type by electrostatic interaction HNTs-PAA-MPEG nanocomposites, then contain antitumor drug adriamycin as reduced form pharmaceutical carrier.
Invention content
The technical problem to be solved by the present invention is to provide a kind of reduction response type nano materials for containing antitumor drug The preparation method of material uses silane coupling agent covalent modification, access end to contain cystine linkage with amino by being carried out to the surfaces HNTs Nanotube, while being formed by the electrostatic interaction between nanotube ends amino and block polymer PEO-b-PAA terminal carboxyl groups HNTs- polymer nanocomposites.In design, addition redox response group cystine linkage can be used for antitumor The carrier of drug, while reaching and allowing pharmaceutical carrier that long-range can be kept to recycle and quickly release the drug in diseased region in vivo.Polymer The cross-linked structure that micella is stablized can improve the stability of micella, can enhance medicine stability as pharmaceutical carrier, based on double The micella of sulfide linkage can realize that drug is discharged in tumor locus, improve therapeutic effect.
The present invention adopts the following technical scheme that solve above-mentioned technical problem：A kind of reduction for containing antitumor drug The preparation method of response type nano material, it is characterised in that detailed process is：
(1) drawn as Material synthesis macromolecular using the poly glycol monomethyl ether of different molecular weight (MPEG) and dibromo-isobutyl acylbromide Send out agent, then under the conditions of cuprous bromide (CuBr) and pentamethyldiethylenetriamine (PMDETA) are existing by atom transfer oneself Amphiphilic block polymer MPEG-b-PtBA is synthesized by base polymerization, then block polymer MPEG-b-PAA is obtained by acidolysis；
(2) covalent modification is carried out to the surfaces HNTs with silane coupling agent aminopropyl triethoxysilane (APTES), in HNTs It is reacted with succinic anhydride after surface access amino and introduces carboxyl in end, then reacted with cystamine and introduce cystine linkage base on the surfaces HNTs Group；
(3) end amino-containing cystine linkage-HNTs and block polymer MPEG-b-PAA are passed through by electrostatic by solwution method Effect be combined with each other to obtain the reduction response type HNTs-PAA-MPEG nano materials conveyed for drug.
The structural formula of reduction response type nano material of the present invention for for containing antitumor drug is：
Preparation method of the present invention for containing the reduction response type nano material of antitumor drug, it is characterised in that The specific steps are：
(1) dry MPEG is dissolved in anhydrous tetrahydro furan, triethylamine is added, in ice-water bath by the synthesis of MPEG-Br And 2- bromine isobutyl acylbromides are added dropwise under stirring condition, 2- bromine isobutyl acylbromides in 0 DEG C react 0.5h after dripping, then anti-at room temperature 12h is answered, then is warming up to 20 DEG C and 35 DEG C successively and reacts 12h respectively, is filtered after the reaction was complete and obtains light yellow filtrate, filtrate rotation Steaming obtains light yellow residue, is dissolved with dry methylene chloride, and is precipitated in ether, gained crude product again through dissolving twice, Product is obtained in 35 DEG C of vacuum drying after filtering, precipitation, which is MPEG-Br；
(2) synthesis of MPEG-b-PtBA is separately added into MPEG-Br, ligand pentamethyldiethylenetriamine in ampere bottle (PMDETA), tert-butyl acrylate (PtBA) and acetone, reaction system twice, are added by freezing, pumping, thaw cycles CuBr, then by reaction system again by freezing, pumping, thaw cycles three times, closed be placed in 60 DEG C of oil baths is reacted, then It takes out ampere bottle and is put into liquid nitrogen to terminate reaction, after crude product is dissolved in tetrahydrofuran, filled out in neutral alumina column It is filtered to remove colored bivalent cupric ion in material and obtains light yellow filtrate, removing unreacted is vacuumized in 60 DEG C after filtrate concentration Tert-butyl acrylate, then the dope of gained is dissolved with n,N-Dimethylformamide, be fitted into 2000Da bag filters thoroughly Analysis 3 days, will wherein water be appeared with acetone, remaining liquid be transferred in round-bottomed flask revolving remove solvent obtain it is faint yellow sticky Product, the Product Labeling are MPEG-b-PtBA；
(3) MPEG-b-PtBA is dissolved in anhydrous methylene chloride by acidolysis, and trifluoro second is added under 0 DEG C of stirring condition Acid continues to be stirred to react at room temperature then at 0 DEG C of reaction 3h postposition, and revolving removes solvent after the reaction was complete, then after being dissolved with DMF It is fitted into 3500Da bag filters and dialyses, MPEG-b-PAA is then obtained by vacuum freeze drying；
(4)HNTs-NH2Synthesis, weigh the anhydrous HNTs of 1.000g in 100mL round-bottomed flasks, be added 12mL it is anhydrous Toluene is vigorously stirred 2h, adds 4mL 3- aminopropyl triethoxysilanes (APTES), and ultrasonic disperse 30min waits for that dispersion is equal It is even to be placed in 120 DEG C of oil bath pans back flow reaction for 24 hours, dry water-less environment is kept with calcium chloride tube in reflux course, Solid is centrifuged with 8000rpm after reaction, is used in combination toluene to wash repeatedly to remove unreacted three second of 3- aminopropyls Oxysilane, obtained solid product are placed in vacuum drying chamber in 40 DEG C of dry 48h, and products therefrom is labeled as HNTs-NH2；
(5) synthesis of HNTs-COOH weighs the HNTs-NH of 0.907g2In 100mL round-bottomed flasks, 30mL dryings are added N,N-Dimethylformamide, ultrasonic disperse 30min, the succinic anhydride for weighing 3.008g are added in dispersion liquid, room temperature reaction For 24 hours, solid is centrifuged after reaction with 8000rpm, ethyl alcohol is used in combination to be washed repeatedly to remove unreacted succinic anhydride, Obtained solid product is placed in vacuum drying chamber in 40 DEG C of dry 48h, and products therefrom is labeled as HNTs-COOH；
(6)HNTs-S-S-NH2Synthesis, weigh the HNTs-COOH of 0.869g in 100mL round-bottomed flasks, be added 40mL Brand-new MES buffer solutions, ultrasonic disperse 30min, the carbodiimides (EDCI) for weighing 0.891g is added in dispersion liquid, to be mixed The n-hydroxysuccinimide (NHS) of 0.276g is added after dissolving, cystamine and the mixing of MES is added dropwise in stirring and dissolving 15min Liquid, the mixed liquor are to weigh the 2-aminoethyl disulfide dihydrochloride of 0.901g to be dissolved in the MES buffer solutions of 5mL obtained, react at room temperature 8h, Solid is centrifuged with 8000rpm after reaction, ethyl alcohol is used in combination to wash repeatedly, obtained solid product is placed in vacuum drying chamber In in 40 DEG C of dry 48h, products therefrom is labeled as HNTs-S-S-NH2；
(7) synthesis of HNTs-PAA-MPEG weighs the HNTs-S-S-NH of 0.082g2In 10mL centrifuge tubes, add 2mL's The MPEG-b-PAA of 0.078g is dissolved in 2mL ethyl alcohol by ethyl alcohol, ultrasonic disperse 10min, is then transferred into above-mentioned centrifuge tube In, 48h is stirred at room temperature, solid is centrifuged with 8000rpm after stirring, ethyl alcohol is used in combination to wash repeatedly, obtained solid production Object is placed in vacuum drying chamber and obtains the reduction response type nano material HNTs-PAA- conveyed for drug in 40 DEG C of dry 48h MPEG。
Preparation method of the present invention for containing the reduction response type nano material of antitumor drug, feature exist In：
MPEG1.9KThe specific building-up process of-b-PtBA is：
(1)MPEG1.9K5.006g is dried MPEG by the synthesis of-Br1.9kIt is dissolved in the anhydrous tetrahydro furan of 40mL, then adds Enter 1.20mL triethylamines, 1.02mL 2- bromine isobutyl acylbromides are added dropwise under ice-water bath and stirring condition, 2- bromine isobutyl acylbromides drip 0.5h is reacted in 0 DEG C afterwards, 12h is then reacted at room temperature, then be warming up to 20 DEG C and 35 DEG C successively and react 12h respectively, has reacted Light yellow filtrate is obtained at rear suction filtration, which rotates to obtain light yellow residue, is dissolved with dry methylene chloride, and in ether Middle precipitation, gained crude product obtain product after dissolving, filtering twice, precipitate in 35 DEG C of vacuum drying again, which is MPEG1.9K-Br；
(2)MPEG1.9KThe synthesis of-b-PtBA is separately added into 0.998g MPEG in 50mL amperes of bottles1.9k-Br、0.12mL Ligand pentamethyldiethylenetriamine (PMDETA), 3.81mL tert-butyl acrylates (PtBA) and 3.00mL acetone, reaction system are logical It crosses freezing, pumping, thaw cycles twice, 0.088g CuBr is added, then follow reaction system by freezing, pumping, defrosting again Ring three times, react by closed be placed in 60 DEG C of oil baths, then takes out ampere bottle and is put into terminate reaction in liquid nitrogen, thick to produce After object is dissolved in tetrahydrofuran, it is filtered to remove colored bivalent cupric ion in neutral alumina column packing and obtains light yellow filter Liquid vacuumizes in 60 DEG C after filtrate concentration and removes unreacted tert-butyl acrylate, then by the dope N, N- bis- of gained Methylformamide dissolves, and is fitted into the bag filter of 2000Da and dialyses 3 days, will wherein water be appeared with acetone, remaining liquid transfer Solvent is removed to revolving in 50mL round-bottomed flasks and obtains faint yellow viscous product, which is MPEG1.9K-b-PtBA；
MPEG5KThe specific building-up process of-b-PtBA is：
(1)MPEG5K11.065g is dried MPEG by the synthesis of-Br5kIt is dissolved in the anhydrous tetrahydro furan of 40mL, then adds Enter 1.52mL triethylamines, 1.24mL 2- bromine isobutyl acylbromides are added dropwise under ice-water bath and stirring condition, 2- bromine isobutyl acylbromides drip 0.5h is reacted in 0 DEG C afterwards, 12h is then reacted at room temperature, then be warming up to 20 DEG C and 35 DEG C successively and react 12h respectively, has reacted Light yellow filtrate is obtained at rear suction filtration, which rotates to obtain light yellow residue, is dissolved with dry methylene chloride, and in ether Middle precipitation, gained crude product obtain product after dissolving, filtering twice, precipitate in 35 DEG C of vacuum drying again, which is MPEG5K-Br；
(2)MPEG5KThe synthesis of-b-PtBA is separately added into 1.502g MPEG in 50mL amperes of bottles5k- Br, 0.08mL match Body pentamethyldiethylenetriamine (PMDETA), 3.6mL tert-butyl acrylates (PtBA) and 3.00mL acetone, reaction system pass through cold Jelly, pumping, thaw cycles twice, are added 0.047g CuBr, reaction system are then passed through freezing, pumping, thaw cycles three again Secondary, closed be placed in 60 DEG C of oil baths is reacted, and is then taken out ampere bottle and is put into terminate reaction in liquid nitrogen, crude product is molten Solution is filtered to remove colored bivalent cupric ion in neutral alumina column packing and obtains light yellow filtrate after tetrahydrofuran, It is vacuumized in 60 DEG C after filtrate concentration and removes unreacted tert-butyl acrylate, then by dope N, the N- dimethyl of gained Formyl amine solvent is fitted into the bag filter of 2000Da and dialyses 3 days, will wherein water be appeared with acetone, remaining liquid is transferred to Revolving removes solvent and obtains faint yellow viscous product in 50mL round-bottomed flasks, which is MPEG5K-b-PtBA。
Preparation method of the present invention for containing the reduction response type nano material of antitumor drug is specific to prepare Process is：Claim the adriamycin hydrochlorides (DOXHCl) of 7.5mg in 50mL round-bottomed flasks, addition 15mL absolute ethyl alcohols dissolve, The reduction response type nano material HNTs-PAA-MPEG that 30mg is weighed for drug conveying is added in round-bottomed flask, ultrasound point Dissipate 1h, be subsequently vacuumed out and stir 30min, stopping, which vacuumizes, to be continued to stir 30min, repeats this process 2 times, after solid It is centrifugally separating to obtain reduced form pharmaceutical carrier with 8000rpm, which makees at reducing agent homoglutathion (GSH) It is significantly improved with the rate of release of lower antitumor drug adriamycin hydrochlorides.
The present invention has the advantages that compared with prior art：
1, there is reduction response type HNTs-PAA-MPEG nanocomposites by solwution method synthesis, effectively increases it Dispersibility in blood；
2, can reach allow HNTs-PAA-MPEG nano-medicament carriers keep in vivo long-range recycle and it is fast in diseased region Quick-release medicine；
3, compared with covalent modification, agent approach assembly operation is simple and fast, and can by arbitrarily adjust block ratio come It influences to carry medicine Release Performance；
4, medicine stability can be enhanced as pharmaceutical carrier, after reducing agent glutathione (GSH) cuts off cystine linkage, Drug release rate dramatically increases, and can improve therapeutic effect.
Description of the drawings
Fig. 1 is scanning electron microscope, A-HNTs, B-HNTs-PAA4-MPEG1.9K；
Fig. 2 is thermogravimetric curve, a-HNTs, b-HNTs-NH2, c-HNTs-COOH, d-HNTs-S-S-NH2, e-HNTs- PAA4-MPEG1.9K, f-HNTs-PAA11-MPEG1.9K, g-HNTs-PAA11-MPEG5K；
Fig. 3 is Zeta potential figure；
Fig. 4 is the release profiles (37 DEG C, PBS=7.4) of adriamycin, a-HNTs-PAA11-MPEG5K；b-HNTs-PAA4- MPEG1.9K；c-HNTs-PAA11-MPEG1.9K, the release profiles (37 DEG C, 10mM GSH-PBS=7.4) of adriamycin, d-HNTs- PAA11-MPEG5K, e-HNTs-PAA4-MPEG1.9K, f-HNTs-PAA11-MPEG1.9K。
Specific implementation mode
The above of the present invention is described in further details by the following examples, but this should not be interpreted as to this The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on the above of the present invention belong to this hair Bright range.
Embodiment 1
(1)MPEG1.9KThe synthesis of-Br：
MPEG1.9KThe synthesis of-Br：
5.006g is dried into MPEG1.9k(2.632mmol hydroxyl) is dissolved in the anhydrous tetrahydro furan of 40mL, is added 1.02mL 2- bromine isobutyl acylbromides are added dropwise in ice-water bath and under being vigorously stirred dropwise for the triethylamine for entering 1.20mL (8.421mmol) (7.895mmol)([OH]:[Br]:[triethylamine]=1:3:3.2).After acylbromide adds, 0.5h is reacted at 0 DEG C followed by room temperature Lower reaction 12h is warming up to 20 DEG C, 35 DEG C of difference the reaction was continued 12h, is filtered after the completion of reaction and obtain light yellow filtrate successively, should Filtrate rotates to obtain light yellow residue, is dissolved with a small amount of dry methylene chloride, and precipitated in ether, gained crude product is again Product is obtained in 35 DEG C of vacuum drying after dissolving, filtering twice, precipitate, which is MPEG1.9K-Br。
MPEG5KThe synthesis of-Br：
Synthesize MPEG5KDrug and reagent dosage needed for-Br is as follows：Dry MPEG5k 11.065g(2.213mmol Hydroxyl), anhydrous tetrahydro furan 40mL, triethylamine 1.52mL (10.411mmol), 2- bromine isobutyl acylbromides 1.24mL (10.030mmol)([OH]:[Br]:[triethylamine]=1:5:5.2).Experimental implementation is according to MPEG1.9KThe experimental implementation of-Br into Row.
(2) synthesis of amphiphilic block copolymer MPEG-b-PtBA：
MPEG1.9KThe synthesis of-b-PtBA：
0.998g MPEG are separately added into 50mL amperes of bottles1.9k- Br (0.526mmol), 0.12mL ligands pentamethyl two Ethyl triamine (PMDETA) (0.575mmol, l eq.), tert-butyl acrylate (3.81mL, 0.026mmol), acetone (3.00mL), by freezing, pumping, thaw cycles twice, 0.088g CuBr (0.614mmol) are added in reaction system, then anti- Answer system again by freezing, pumping, thaw cycles three times after, closed be placed in 60 DEG C of oil baths is reacted.Then, by it is certain when Between be spaced respectively taking-up ampere bottle, and put into liquid nitrogen with terminate react.After crude product is dissolved in a small amount of tetrahydrofuran, It is filtered to remove the impurity such as colored bivalent cupric ion in neutral alumina column packing and obtains lurid filtrate.In 60 after concentration It DEG C vacuumizes and to remove unreacted tert-butyl acrylate, then the dope of gained is dissolved with a small amount of DMF, is packed into the saturating of 2000Da It dialyses 3 days in analysis bag, will wherein water be appeared with acetone, remaining liquid is transferred to revolving removing solvent in 50mL round-bottomed flasks and obtains To faint yellow viscous product, which is MPEG1.9K-b-PtBA。
MPEG5KThe synthesis of-b-PtBA：
MPEG5k- Br 1.502g (0.301mmol), pentamethyldiethylenetriamine (PMDETA) 0.08mL (0.384mmol, l Eq.), tert-butyl acrylate 3.60mL (0.0248mmol), acetone (3.00mL), CuBr 0.047g (0.330mmol).Experiment Operation is according to above-mentioned MPEG1.9KThe synthetic operation of-b-PtBA carries out.
(3) acidolysis：
It takes a certain amount of polymer MPEG-b-PtBA to be dissolved in anhydrous methylene chloride, adds under 0 DEG C of intense agitation Enter trifluoroacetic acid (molar ratio：Trifluoroacetic acid:TBA=5:1), 3h postpositions are reacted in 0 DEG C to continue to be stirred to react at room temperature, react Revolving removes solvent after complete, is dialysed with bag filter (3500Da) is packed into after appropriate DMF dissolvings, is obtained by vacuum freeze drying To MPEG-b-PAA.
(4)HNTs-NH2Synthesis：
The anhydrous HNTs of 1.000g is weighed in 100mL round-bottomed flasks, 12mL dry toluenes are added, are vigorously stirred 2h.So 4mL 3- aminopropyl triethoxysilanes (APTES) are added with disposable syringe afterwards, ultrasonic disperse 30min waits being uniformly dispersed After be put into 120 DEG C of oil bath pans, back flow reaction for 24 hours, dry water-less environment is kept in reflux course with calcium chloride tube.Instead Solid is centrifuged with 8000rpm after answering, is used in combination toluene to wash repeatedly to remove unreacted APTES.Obtained solid Product is placed in vacuum drying chamber in 40 DEG C of dry 48h, and products therefrom is labeled as HNTs-NH2。
(5) synthesis of HNTs-COOH：
Weigh the HNTs-NH of 0.907g2In 100mL round-bottomed flask, 30mL is added and dries DMF, ultrasonic disperse 30min, The succinic anhydride for weighing 3.008g is added in dispersion liquid, and room temperature reaction is for 24 hours.Solid is centrifuged with 8000rpm after reaction Separation, is used in combination ethyl alcohol to wash repeatedly to remove unreacted succinic anhydride.Obtained solid product is placed in vacuum drying chamber in 40 DEG C dry 48h, products therefrom are labeled as HNTs-COOH.
(6)HNTs-S-S-NH2Synthesis：
The HNTs-COOH of 0.869g is weighed in 100mL round-bottomed flasks, 40mL brand-new MES buffer solutions, ultrasonic disperse is added 30min, the EDCI for weighing 0.891g are added in dispersion liquid, and the NHS of 0.276g, stirring and dissolving are added after dissolving to be mixed Cystamine/MES mixed liquors are added dropwise in 15min (2-aminoethyl disulfide dihydrochloride for weighing 0.901g is dissolved in the MES buffer solutions of 5mL). React at room temperature 8h.Solid is centrifuged with 8000rpm after reaction, ethyl alcohol is used in combination to wash repeatedly.Obtained solid product is set In vacuum drying chamber HNTs-S-S-NH is labeled as in 40 DEG C of dry 48h, products therefrom2。
(7) synthesis of HNTs-PAA-MPEG：
Weigh the HNTs-S-S-NH of 0.082g2In 10mL centrifuge tubes, add the ethyl alcohol of 2mL, ultrasonic disperse 10min.It will The MPEG of 0.078g1.9K-b-PAA4It is dissolved in 2mL ethyl alcohol, is then transferred into above-mentioned centrifuge tube.The total 5mL of reaction solution, room temperature Stir 48h.Solid is centrifuged with 8000rpm after stirring, ethyl alcohol is used in combination to wash repeatedly.Obtained solid product is placed in very In empty drying box HNTs-PAA is labeled as in 40 DEG C of dry 48h, products therefrom4-MPEG1.9K。
HNTs-PAA11-MPEG1.9KSynthesis：The HNTs-S-S-NH of 0.101g is used in experiment2, the MPEG of 0.105g1.9K- b-PAA11.It tests operating process with reference to HNTs-PAA4-MPEG1.9KPreparation process.
HNTs-PAA11-MPEG5KSynthesis：The HNTs-S-S-NH of 0.104g is used in experiment2, the MPEG of 0.107g5K-b- PAA11.It tests operating process with reference to HNTs-PAA4-MPEG1.9KPreparation process.
As seen from Figure 1, unmodified nanotube surface is smooth, and the nanotube surface of cement-based powder material cladding becomes thick It is rough, thus one can consider that polymer success coating decoration nanotube.
As seen from Figure 2, each step modification of halloysite nanotubes successfully completes, and final polymer wrapped HNTs-PAA11-MPEG1.9K covering amounts highest in nanotube.
As seen from Figure 3, polymer may indicate that by the variation for connecting Zeta electric potential value after different groups on nanotube Successfully it is connected on nanotube.
(8) medicine and vitro drug release are carried：
Carry medicine：In in 50mL round-bottomed flasks, addition 15mL absolute ethyl alcohols dissolve the accurate DOXHCl for weighing 7.5mg.Claim Take 30mg HNTs-PAA4-MPEG1.9KIt is added in round-bottomed flask, ultrasonic disperse 1h, is subsequently vacuumed out and stirs 30min, stop Vacuumize and continue to stir 30min, repeat this process 2 times, after solid centrifuged with 8000rpm.Filtrate is collected, filter is surveyed UV absorption of the liquid at 481nm.
HNTs-PAA11-MPEG1.9KAnd HNTs-PAA11-MPEG5KThe experiment of load medicine with reference to HNTs-PAA4-MPEG1.9KCarry medicine Experiment carries out.
Vitro drug release is tested：A, the drug release in PBS buffer solution (pH=7.4)；B, in the GSH- of 10mmol Drug release in PBS buffer solution (pH=7.4).
Utilize DOXHCl of the carrier micelle of the above-mentioned preparation of determined by ultraviolet spectrophotometry in different time release liquid Concentration draws its In-vitro release curves, as shown in Figure 4.
By load medicine and the drug release test to nanotube, obtain in a concentration of 0.5mg/mL of DOXHCl, nanometer The drugloading rate and encapsulation rate of pipe be respectively：HNTs-PAA4-MPEG1.9KLC (%)=10.07%, EE (%)=48.41%； HNTs-PAA11-MPEG1.9KLC (%)=10.02%, EE (%)=48.08%；HNTs-PAA11-MPEG5KLC (%)= 9.92%, EE (%)=47.58%；The nanotube of different polymer overmolds differs not the drugloading rate and encapsulation rate of adriamycin Greatly, this may be to be entered by vacuum process filling due to containing drug, be physical adsorption process.
By Fig. 4, it can be seen that, the nanotube drug release rate of coated polymer is slower, and only 10% or so；And when molten In liquid when homoglutathion containing reducing agent (GSH), carries medicine rate and significantly improve, total volume is between 30%-50%.This may It is since halloysite nanotubes surface has the adsorption and de-adsorption process to drug molecule, when surface coats last layer polymer Afterwards so that the rate of release of drug is more slow；Simultaneously inside and outside tube chamber liquid since that there are exchange rates is opposite for polymeric layer It is relatively low, it causes to be loaded into intraluminal drug release rate and is declined.And in the presence of reducing agent GSH, polymer film is fast Speed cut-out, drug release rate improve.
Embodiment above describes the basic principles and main features and advantage of the present invention, and the technical staff of the industry should Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe the originals of the present invention Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within In the scope of protection of the invention.

Claims (5)

1. a kind of preparation method for containing the reduction response type nano material of antitumor drug, it is characterised in that detailed process For：
（1）Using the poly glycol monomethyl ether of different molecular weight and dibromo-isobutyl acylbromide as Material synthesis macromole evocating agent, then It is embedding by atom transfer radical polymerization method synthesis amphiphilic under the conditions of existing for cuprous bromide and pentamethyldiethylenetriamine Section polymer MPEG-b-PtBA, then block polymer MPEG-b-PAA is obtained by acidolysis；
（2）Covalent modification is carried out to the surfaces HNTs with silane coupling agent aminopropyl triethoxysilane, ammonia is accessed on the surfaces HNTs It is reacted with succinic anhydride after base and introduces carboxyl in end, then reacted with cystamine and introduce cystine linkage group on the surfaces HNTs；
（3）End amino-containing cystine linkage-HNTs and block polymer MPEG-b-PAA are passed through into electrostatic interaction by solwution method It is combined with each other to obtain the reduction response type HNTs-PAA-MPEG nano materials conveyed for drug.
2. the preparation method according to claim 1 for containing the reduction response type nano material of antitumor drug, Be characterized in that the specific steps are：
（1）Dry MPEG is dissolved in anhydrous tetrahydro furan, adds triethylamine, in ice-water bath and stir by the synthesis of MPEG-Br Under the conditions of mixing be added dropwise 2- bromine isobutyl acylbromides, 2- bromine isobutyl acylbromides drip after in 0 DEG C react 0.5h, then react at room temperature 12h, then be warming up to 20 DEG C and 35 DEG C successively and react 12h respectively, it is filtered after the reaction was complete and obtains light yellow filtrate, filtrate revolving Obtain light yellow residue, dissolved with dry methylene chloride, and precipitated in ether, gained crude product again through dissolving twice, mistake Product is obtained in 35 DEG C of vacuum drying after filter, precipitation, which is MPEG-Br；
（2）The synthesis of MPEG-b-PtBA is separately added into MPEG-Br, ligand pentamethyldiethylenetriamine, propylene in ampere bottle Tert-butyl acrylate and acetone, reaction system twice, are added CuBr, then lead to reaction system again by freezing, pumping, thaw cycles Cross freezing, pumping, thaw cycles three times, closed be placed in 60 DEG C of oil baths is reacted, and is then taken out ampere bottle and is put into liquid To terminate reaction in nitrogen, after crude product is dissolved in tetrahydrofuran, colored two are filtered to remove in neutral alumina column packing Valence copper ion obtains light yellow filtrate, is vacuumized in 60 DEG C after filtrate concentration and removes unreacted tert-butyl acrylate, then will The dope of gained is dissolved with n,N-Dimethylformamide, is fitted into 2000Da bag filters and is dialysed 3 days, with acetone will wherein water it is saturating Go out, remaining liquid is transferred to revolving removing solvent in round-bottomed flask and obtains faint yellow viscous product, which is MPEG- b-PtBA；
（3）MPEG-b-PtBA is dissolved in anhydrous methylene chloride by acidolysis, and trifluoroacetic acid is added under 0 DEG C of stirring condition, then 3h postpositions are reacted in 0 DEG C to continue to be stirred to react at room temperature, revolving removes solvent after the reaction was complete, then is packed into after being dissolved with DMF It dialyses in 3500Da bag filters, MPEG-b-PAA is then obtained by vacuum freeze drying；
（4）HNTs-NH2Synthesis, weigh the anhydrous HNTs of 1.000g in 100mL round-bottomed flasks, be added 12mL dry toluenes, It is vigorously stirred 2h, adds 4mL 3- aminopropyl triethoxysilanes, ultrasonic disperse 30min, waiting being uniformly dispersed is placed on 120 DEG C Back flow reaction for 24 hours, keeps dry water-less environment with calcium chloride tube in reflux course, will consolidate after reaction in oil bath pan Body is centrifuged with 8000rpm, is used in combination toluene to wash repeatedly to remove unreacted 3- aminopropyl triethoxysilanes, gained is solid Body product is placed in vacuum drying chamber in 40 DEG C of dry 48h, and products therefrom is labeled as HNTs-NH2；
（5）The synthesis of HNTs-COOH weighs the HNTs-NH of 0.907g2In 100mL round-bottomed flasks, 30mL is added and dries N, N- Dimethylformamide, ultrasonic disperse 30min, the succinic anhydride for weighing 3.008g are added in dispersion liquid, are reacted at room temperature for 24 hours, instead Solid is centrifuged with 8000rpm after answering, is used in combination ethyl alcohol to be washed repeatedly to remove unreacted succinic anhydride, gained is solid Body product is placed in vacuum drying chamber in 40 DEG C of dry 48h, and products therefrom is labeled as HNTs-COOH；
（6）HNTs-S-S-NH2Synthesis, weigh the HNTs-COOH of 0.869g in 100mL round-bottomed flasks, be added 40mL brand-news MES buffer solutions, ultrasonic disperse 30min, the carbodiimides for weighing 0.891g are added in dispersion liquid, are added after dissolving to be mixed Cystamine and the mixed liquor of MES is added dropwise in the n-hydroxysuccinimide of 0.276g, stirring and dissolving 15min, which is to claim It takes the 2-aminoethyl disulfide dihydrochloride of 0.901g to be dissolved in the MES buffer solutions of 5mL obtained, reacts at room temperature 8h, will consolidate after reaction Body is centrifuged with 8000rpm, and ethyl alcohol is used in combination to wash repeatedly, and obtained solid product is placed in vacuum drying chamber in 40 DEG C of dryings 48h, products therefrom are labeled as HNTs-S-S-NH2；
（7）The synthesis of HNTs-PAA-MPEG weighs the HNTs-S-S-NH of 0.082g2In 10mL centrifuge tubes, add the ethyl alcohol of 2mL, The MPEG-b-PAA of 0.078g is dissolved in 2mL ethyl alcohol by ultrasonic disperse 10min, is then transferred into above-mentioned centrifuge tube, room temperature 48h is stirred, solid is centrifuged with 8000rpm after stirring, ethyl alcohol is used in combination to wash repeatedly, obtained solid product is placed in very In empty drying box the reduction response type nano material HNTs-PAA-MPEG conveyed for drug is obtained in 40 DEG C of dry 48h.
3. the preparation method according to claim 1 for containing the reduction response type nano material of antitumor drug, It is characterized in that：
MPEG1.9KThe specific building-up process of-b-PtBA is：
（1）MPEG1.9K5.006g is dried MPEG by the synthesis of-Br1.9kIt is dissolved in the anhydrous tetrahydro furan of 40mL, adds 1.02mL 2- bromine isobutyl acylbromides are added dropwise, after 2- bromine isobutyl acylbromides drip in 1.20mL triethylamines under ice-water bath and stirring condition 0.5h is reacted in 0 DEG C, then reacts 12h at room temperature, then is warming up to 20 DEG C and 35 DEG C successively and reacts 12h respectively, reaction is completed It filters afterwards and obtains light yellow filtrate, which rotates to obtain light yellow residue, is dissolved with dry methylene chloride, and in ether Precipitation, gained crude product obtain product after dissolving, filtering twice, precipitate in 35 DEG C of vacuum drying again, which is MPEG1.9K-Br；
（2）MPEG1.9KThe synthesis of-b-PtBA is separately added into 0.998g MPEG in 50mL amperes of bottles1.9k- Br, 0.12mL ligands Pentamethyldiethylenetriamine, 3.81mL tert-butyl acrylates and 3.00mL acetone, reaction system are followed by freezing, pumping, defrosting Ring twice, be added 0.088g CuBr, then by reaction system again by freezing, pumping, thaw cycles three times, it is closed to be placed on It reacts, then take out ampere bottle and is put into terminate reaction in liquid nitrogen in 60 DEG C of oil baths, crude product is dissolved in tetrahydrofuran Afterwards, colored bivalent cupric ion is filtered to remove in neutral alumina column packing and obtains light yellow filtrate, after filtrate concentration in 60 DEG C vacuumize the unreacted tert-butyl acrylate of removing, then dissolve the dope of gained with n,N-Dimethylformamide, It is fitted into the bag filter of 2000Da and dialyses 3 days, will wherein water be appeared with acetone, remaining liquid is transferred in 50mL round-bottomed flasks Revolving removes solvent and obtains faint yellow viscous product, which is MPEG1.9K-b-PtBA；
MPEG5KThe specific building-up process of-b-PtBA is：
（1）MPEG5K11.065g is dried MPEG by the synthesis of-Br5kIt is dissolved in the anhydrous tetrahydro furan of 40mL, adds 1.24mL 2- bromine isobutyl acylbromides are added dropwise, after 2- bromine isobutyl acylbromides drip in 1.52mL triethylamines under ice-water bath and stirring condition 0.5h is reacted in 0 DEG C, then reacts 12h at room temperature, then is warming up to 20 DEG C and 35 DEG C successively and reacts 12h respectively, reaction is completed It filters afterwards and obtains light yellow filtrate, which rotates to obtain light yellow residue, is dissolved with dry methylene chloride, and in ether Precipitation, gained crude product obtain product after dissolving, filtering twice, precipitate in 35 DEG C of vacuum drying again, which is MPEG5K-Br；
（2）MPEG5KThe synthesis of-b-PtBA is separately added into 1.502g MPEG in 50mL amperes of bottles5k- Br, 0.08mL ligands five Methyl diethyl triamine, 3.6mL tert-butyl acrylates and 3.00mL acetone, reaction system pass through freezing, pumping, thaw cycles two It is secondary, be added 0.047g CuBr, then by reaction system again by freezing, pumping, thaw cycles three times, it is closed to be placed on 60 DEG C It reacts, then take out ampere bottle and is put into terminate reaction in liquid nitrogen in oil bath, after crude product is dissolved in tetrahydrofuran, It is filtered to remove colored bivalent cupric ion in neutral alumina column packing and obtains light yellow filtrate, in 60 DEG C of pumpings after filtrate concentration Unreacted tert-butyl acrylate is removed in vacuum, then dissolves the dope of gained with n,N-Dimethylformamide, is packed into It dialyses 3 days, will wherein water be appeared with acetone, remaining liquid is transferred in 50mL round-bottomed flasks and rotates in the bag filter of 2000Da It removes solvent and obtains faint yellow viscous product, which is MPEG5K-b-PtBA。
4. the reduction response type nano material described in claim 1 for drug conveying contains the reduced form medicine of antitumor drug Object carrier, reduced form pharmaceutical carrier rate of release of antitumor drug under the effect of reducing agent homoglutathion significantly improve.
5. the reduction response type nano material for drug conveying described in claim 4 contains the reduced form medicine of antitumor drug The preparation method of object carrier, it is characterised in that specifically preparation process is：Claim the adriamycin of 7.5mg.Hydrochloride is burnt in 50mL round bottoms In bottle, the dissolving of 15mL absolute ethyl alcohols is added, weighs reduction response type nano material HNTs-PAA-s of the 30mg for drug conveying MPEG is added in round-bottomed flask, ultrasonic disperse 1h, is subsequently vacuumed out and stirs 30min, and stopping, which vacuumizes, to continue to stir 30min repeats this process 2 times, after solid reduced form pharmaceutical carrier is centrifugally separating to obtain with 8000rpm, the reduced form Pharmaceutical carrier antitumor drug adriamycin under the effect of reducing agent homoglutathion.The rate of release of hydrochloride significantly improves.
CN201810328003.6A 2018-04-12 2018-04-12 A kind of preparation method for containing the reduction response type nano material of antitumor drug Pending CN108309937A (en)

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