CN105617394A - Self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates as well as preparation method and application thereof - Google Patents
Self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates as well as a preparation method and application thereof. (1) Unsaturated fatty acid-anti-tumor drug conjugates can be self-assembled in water to form spherical nano-particles with uniform particle sizes and good stability; and (2) in a preparation process, an amphiphilic polymer material such as DSPE-PEG can be optionally added, so that the particle sizes of the self-assembled nano-particles can be further reduced, and the stability of the self-assembled nano-particles can be further enhanced. The self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates, provided by the invention, is simple in preparation method and easy in industrial production and has the advantages of high drug loading capacity, strong stability, good safety and the like; and growth inhibition tests of tumor cells in vitro prove that the self-assembled nano-system has favorable anti-tumor effects on multiple tumor cells.
Description
Technical field
The present invention relates to nano-drug preparation technical field, self-assembled nanometer system being specifically related to unsaturated fatty acid-antitumor medicine conjugate and its preparation method and application.
Background technology
Chemotherapeutics is that clinical antineoplastic treats one of conventional means, but the poisonous solvent owing to containing in preparation is likely to cause obvious adverse reaction (polyoxyethylene castor oil added in taxol be likely to cause allergic reaction and neurotoxicity etc.), and free drug is distributed non-selectivity at whole body and is likely to cell killing also non-selectivity cause serious toxic and side effects (such as the cardiac toxicity etc. that amycin causes) etc. significantly to constrain its application and development.
Nano-delivery system has the advantages such as slow release, passive target, raising bioavailability, the toxic and side effects that extends circulation time in vivo, improve pharmacokinetics behavior and reduce in disease medication process, thus, develop suitable antitumor drug nano-delivery system in recent decades and have become as study hotspot both domestic and external.
But, the drug loading of the nano-delivery system depending on carrier is generally relatively low, and utilizes the form of carrier bag medicine carrying thing, and the medicine in carrier can be revealed and cause not expected toxic and side effects.
DNAcarrier free self-assembled nanometer system has the advantage (such as passive target ability, circulation time in vivo prolongation and pharmacokinetics behavior improvement etc.) of administration nano-drug administration system, overcome again the low deficiency such as grade of drug loading of conventional carriers nano-delivery system simultaneously, there is good application prospect.
Summary of the invention
The present invention overcomes the deficiencies in the prior art, it is provided that self-assembled nanometer system of a kind of unsaturated fatty acid-antitumor medicine conjugate and its preparation method and application. Applicants have unexpectedly found that, use unsaturated fatty acid and antitumor drug coupling, the conjugate obtained, when not needing conventional nano pharmaceutical carrier, can form nanosystems from group. Further, when adding a small amount of (compared to as pharmaceutical carrier, its consumption is greatly decreased) amphipathy macromolecule material, the character (such as particle diameter etc.) of self-assembled nanometer system makes moderate progress. The purpose of the present invention is that use unsaturated fatty acid is to modify antitumor drug, still to be kept pharmaceutically active but to have the conjugate nanosystems of self-assembly property.
First aspect, the present invention relates to the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate.
The self-assembled nanometer system of a kind of unsaturated fatty acid-antitumor medicine conjugate, described nanosystems includes unsaturated fatty acid-antitumor medicine conjugate, aqueous solution and the amphipathy macromolecule material optionally employed.
Antitumor medicine conjugate, aqueous solution and the amphipathy macromolecule material optionally employed that preferably described nanosystems is modified by unsaturated fatty acid form.
Described unsaturated fatty acid-antitumor medicine conjugate is the compound that unsaturated fatty acid is formed with antitumor drug coupling.
Described unsaturated fatty acid is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29, it is preferable that n is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. Described unsaturated fatty acid is preferably oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA etc. More preferably described unsaturated fatty acid is conjugated linoleic acid. Preferably conjugated linoleic acid is along 9, anti-11-conjugated linoleic acid or anti-10, along 12-conjugated linoleic acid.
Antitumor drug of the present invention includes but not limited to yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament etc.
Described taxanes series antineoplastic medicament includes paclitaxel, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-difference to paclitaxel etc.
Described camptothecin antineoplastic agents includes camptothecine, SN38 (SN38), 9-dimethylamine methyl isophthalic acid 0-hydroxy camptothecin, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, GI147211 and DX-8951f etc.
Described Antitumor Agents Vinblastine Analogues includes vinblastine, vincristine, vindesine, vinorelbine and 20 ', 20 '-two fluoro-3 ', 4 '-dihydro NVB etc.
Described antibiont series antineoplastic medicament includes amycin and epirubicin etc.
Described ucleosides antitumor drug includes gemcitabine, 5-fluorouracil and cytosine arabinoside etc.
Described platinum series antineoplastic medicament includes cisplatin, carboplatin, nedaplatin and oxaliplatin etc.
The preferred paclitaxel of antitumor drug of the present invention, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-poor to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin.
It is highly preferred that antitumor drug of the present invention is paclitaxel, SN38, gemcitabine, vinblastine, adriamycin and Platinol cisplatin.
The optional pure water of described aqueous solution, 5% glucose solution, normal saline, phosphate buffer etc., it is preferable that pure water.
Described amphipathy macromolecule material includes but not limited to DSPE-PEG, PLGA-PEG, polyoxyethylene castor oil, Tweens, lecithin etc., it is preferable that DSPE-PEG.
Optional 40:1��the 1:80 of mass ratio of amphipathy macromolecule material and unsaturated fatty acid-antitumor medicine conjugate, it is preferable that 20:1��1:40, more preferably 7:1��1:20, it is most preferred that 1:1��1:20.
Second aspect, the invention provides unsaturated fatty acid-antitumor medicine conjugate that can be used for self-assembled nanometer system and preparation method thereof.
Described unsaturated fatty acid-antitumor medicine conjugate is the compound that unsaturated fatty acid is formed with antitumor drug coupling.
Described antitumor drug includes but not limited to yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament etc.
Described taxanes series antineoplastic medicament includes paclitaxel, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-difference to paclitaxel etc.
Described camptothecin antineoplastic agents includes camptothecine, SN38 (SN38), 9-dimethylamine methyl isophthalic acid 0-hydroxy camptothecin, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, GI147211 and DX-8951f etc.
Described Antitumor Agents Vinblastine Analogues includes vinblastine, vincristine, vindesine, vinorelbine and 20 ', 20 '-two fluoro-3 ', 4 '-dihydro NVB etc.
Described antibiont series antineoplastic medicament includes amycin and epirubicin etc.
Described ucleosides antitumor drug includes gemcitabine, 5-fluorouracil and cytosine arabinoside etc.
Described platinum series antineoplastic medicament includes cisplatin, carboplatin, nedaplatin and oxaliplatin etc.
The preferred paclitaxel of antitumor drug of the present invention, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-poor to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin.
It is highly preferred that antitumor drug of the present invention is paclitaxel, SN38, gemcitabine, vinblastine, adriamycin and Platinol cisplatin.
Unsaturated fatty acid of the present invention, is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29, it is preferable that n is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. Described unsaturated fatty acid is preferably oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA etc.
The preparation method of described conjugate comprises the steps:
Described method includes:
(1), by antitumor drug it is dissolved in a certain amount of suitable solvent, adds catalyst and dehydrant, under stirring condition, add unsaturated fatty acid, continue stirring reaction;
(2), it is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration;
(3), gained filtrate ambient temperature under nitrogen dries up or reduced vacuum dries, and obtains the antitumor medicine conjugate that unsaturated fatty acid is modified.
Described antitumor drug and unsaturated fatty acids acid range as defined above, and described unsaturated fatty acid modify antitumor medicine conjugate do not include conjugated linoleic acid-paclitaxel and conjugated linoleic acid-gemcitabine.
Solvent described in step (1) is selected from the one in dichloromethane, chloroform, DMF, dimethyl sulfoxide, dioxane or oxolane or its mixed solution, it is preferable that dichloromethane;
Catalyst described in step (1) is dimethylamino naphthyridine;
Dehydrant described in step (1) is selected from the one in dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide or DIC or its mixture, it is preferable that dicyclohexylcarbodiimide;
The temperature of the reaction in step (1) is room temperature��80 DEG C, different according to different unsaturated fatty acid Optimal Temperature, it is preferable that room temperature; Reaction condition is under indoor conditions or nitrogen protection, it is preferable that nitrogen protection condition; Response time is 4-72 hour; Preferably 12��24 hours;
In step (2), thin-layer developing solvent for use is volume ratio is the methylene chloride/methanol solution of 1:1��40:1; Preferred 1:1��20:1;
Wherein antitumor drug is 1:0.1��1:10 with the molar ratio of unsaturated fatty acid; Preferred 1:1��1:4;
The mol ratio of described catalyst and antitumor drug is 1:0.1��1:10; Preferred 1:1��4:1;
Described dehydrant and antitumor drug are 1:0.1��1:10 with mol ratio; Preferred 1:1��4:1.
The third aspect, the preparation method that the present invention relates to the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate, preparation technology is simple, and preparation method comprises the steps:
Unsaturated fatty acid-antitumor medicine conjugate is dissolved in a certain amount of suitable organic solvent and forms solution, pipette in the aqueous solution that above-mentioned dropwise instills certain volume, and be stirred continuously, remove organic solvent with deionized water dialysis, obtain self-assembled nanometer grain dispersion liquid (1).
Unsaturated fatty acid of the present invention, is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29, it is preferable that n is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. Described unsaturated fatty acid is preferably oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA etc. More preferably described unsaturated fatty acid is conjugated linoleic acid. Preferably conjugated linoleic acid is along 9, anti-11-conjugated linoleic acid or anti-10, along 12-conjugated linoleic acid.
Antitumor drug of the present invention includes but not limited to yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament etc.
Described taxanes series antineoplastic medicament includes paclitaxel, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-difference to paclitaxel etc.
Described camptothecin antineoplastic agents includes camptothecine, SN38 (SN38), 9-dimethylamine methyl isophthalic acid 0-hydroxy camptothecin, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, GI147211 and DX-8951f etc.
Described Antitumor Agents Vinblastine Analogues includes vinblastine, vincristine, vindesine, vinorelbine and 20 ', 20 '-two fluoro-3 ', 4 '-dihydro NVB etc.
Described antibiont series antineoplastic medicament includes amycin and epirubicin etc.
Described ucleosides antitumor drug includes gemcitabine, 5-fluorouracil and cytosine arabinoside etc.
Described platinum series antineoplastic medicament includes cisplatin, carboplatin, nedaplatin and oxaliplatin etc.
The preferred paclitaxel of antitumor drug of the present invention, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-poor to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin.
It is highly preferred that antitumor drug of the present invention is paclitaxel, SN38, gemcitabine, vinblastine, adriamycin and Platinol cisplatin.
The water-soluble solvents such as the optional dimethyl sulfoxide of described organic solvent, acetone, ethanol, oxolane, DMF, methanol, acetonitrile, it is preferable that dimethyl sulfoxide, acetone and ethanol, it is most preferred that dimethyl sulfoxide.
The optional pure water of described aqueous solution, 5% glucose solution, normal saline, phosphate buffer etc., it is preferable that pure water.
The solution of medicine and organic solvent formation and the optional 1:1��1:100 of volume ratio of aqueous solution, it is preferable that 1:1��1:50, more preferably 1:1��1:20, it is most preferred that 1:3��1:10.
Optional 50��1500 revs/min of speed of agitator during preparation, it is preferable that 300��500 revs/min.
Optional 24��72 hours of dialysis time, it is preferable that 36��54 hours, it is preferable that 36-48 hour.
Fourth aspect, the present invention relates to the further optimization of unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer system, and preparation method comprises the steps:
Unsaturated fatty acid-antitumor medicine conjugate and amphipathy macromolecule material mixing are dissolved in a certain amount of suitable organic solvent, pipette above-mentioned dropwise and instill in a certain amount of aqueous solution, and be stirred continuously, organic solvent is removed with deionized water dialysis, obtain self-assembled nanometer grain dispersion liquid (2), optionally can centrifugal concentrating further by gained nanoparticle dispersion liquid (2).
Described unsaturated fatty acid is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29, it is preferable that n is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. Described unsaturated fatty acid is preferably oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA etc. More preferably described unsaturated fatty acid is conjugated linoleic acid. Preferably conjugated linoleic acid is along 9, anti-11-conjugated linoleic acid or anti-10, along 12-conjugated linoleic acid.
Antitumor drug of the present invention includes but not limited to yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament etc.
Described taxanes series antineoplastic medicament includes paclitaxel, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-difference to paclitaxel etc.
Described camptothecin antineoplastic agents includes camptothecine, SN38 (SN38), 9-dimethylamine methyl isophthalic acid 0-hydroxy camptothecin, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, GI147211 and DX-8951f etc.
Described Antitumor Agents Vinblastine Analogues includes vinblastine, vincristine, vindesine, vinorelbine and 20 ', 20 '-two fluoro-3 ', 4 '-dihydro NVB etc.
Described antibiont series antineoplastic medicament includes amycin and epirubicin etc.
Described ucleosides antitumor drug includes gemcitabine, 5-fluorouracil and cytosine arabinoside etc.
Described platinum series antineoplastic medicament includes cisplatin, carboplatin, nedaplatin and oxaliplatin etc.
The preferred paclitaxel of antitumor drug of the present invention, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-poor to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin.
It is highly preferred that antitumor drug of the present invention is paclitaxel, SN38, gemcitabine, vinblastine, adriamycin and Platinol cisplatin.
Described amphipathy macromolecule material includes but not limited to DSPE-PEG, PLGA-PEG, polyoxyethylene castor oil, Tweens, lecithin etc., it is preferable that DSPE-PEG.
Optional 40:1��the 1:80 of mass ratio of amphipathy macromolecule material and unsaturated fatty acid-antitumor medicine conjugate, it is preferable that 20:1��1:40, more preferably 7:1��1:40, it is most preferred that 1:1��1:20.
The water-soluble solvents such as the optional dimethyl sulfoxide of described organic solvent, acetone, ethanol, oxolane, DMF, methanol and acetonitrile, it is preferable that dimethyl sulfoxide, acetone and ethanol, it is most preferred that dimethyl sulfoxide.
The optional pure water of described aqueous solution, 5% glucose solution, normal saline, phosphate buffer etc., preferred pure water.
The solution of medicine and organic solvent formation and the optional 1:1��1:100 of volume ratio of aqueous solution, it is preferable that 1:1��1:50, more preferably 1:1��1:20, it is most preferred that 1:3��1:10.
Optional 50��1500 revs/min of speed of agitator during preparation, it is preferable that 100��1000 revs/min, more preferably 300��500 revs/min.
Optional 12��72 hours of dialysis time, it is preferable that 36��54 hours, more preferably 36-48 hour.
Optional 500��15000 revs/min of centrifugal force, it is preferable that 1000��10000 revs/min, more preferably 2000��4000 revs/min.
5th aspect, the invention still further relates to unsaturated fatty acid-antitumor medicine conjugate and the application in preparing antitumor drug of the self-assembled nanometer system thereof, described tumor includes ovarian cancer, breast carcinoma, pulmonary carcinoma, colorectal cancer, melanoma, incidence cancer, lymphoma, cerebroma, leukemia, nasopharyngeal carcinoma, lymphoma, uterus carcinoma. Preferred breast carcinoma, melanoma and cerebral glioma.
In the present invention, by tumor cell extracorporeal growth inhibition test is shown: tumor cell is had certain inhibitory action by unsaturated fatty acid of the present invention-antitumor medicine conjugate self-assembled nanometer system in vitro.
Sulforhodamine B (SRB) colorimetry that assay method is bibliographical information adopted in this research, concrete operations are as follows:
(1) tumor cell is inoculated in 96 orifice plates with suitable cell density, hatches 24h standby.
(2) nanoparticle dispersion liquid is after 0.2 micron of sterilised membrane filter filtration sterilization, is diluted to the nanoparticle diluent of a series of concentration by culture medium.
(3) after discarding former culture medium, it is separately added into nanoparticle diluent, to add complete medium for comparison, after hatching 48 hours, discard nanoparticle diluent, cell 1 hour (4 DEG C) fixed by 10% trichloroacetic acid, use distilled water cyclic washing, air dries naturally, 0.4%SRB dye liquor dyes 20 minutes in room temperature, and 0.1% acetic acid cyclic washing adds 10 micromoles per liter Tris-base solution after naturally drying in air, after putting shaking table concussion 30 minutes, measure each group of light absorption value at 540nm place by microplate reader.
(4) survival rate is calculated: survival rate=(dosing group cell average absorbance value/cellular control unit average absorbance value) �� 100%.
Result shows that kinds of tumor cells is all had certain Developing restraint effect by the unsaturated fatty acid-paclitaxel conjugate self-assembled nanometer grain of each concentration in vitro.
Advantages of the present invention:
(1) the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate provided by the invention, compared to traditional sequestered anti-tumor medicinal preparation, there is passive target ability, the bioavailability of chemotherapeutics can be improved, extend its circulation time in vivo, improve the advantages such as its pharmacokinetics behavior and the toxic and side effects that reduces in chemotherapy process.
(2) the self-assembled nanometer system of unsaturated fatty acid provided by the invention-tumour medicine conjugate comes from the self assembly of conjugate itself, compared with conventional carriers dependent form nanosystems, has the advantages such as drug loading height, stability is strong, safety is good.
(3) the invention provides the further prioritization scheme of the self-assembled nanometer system of unsaturated fatty acid-tumour medicine conjugate, by introducing a small amount of amphipathy macromolecule material (such as DSPE-PEG etc.) in unsaturated fatty acid-tumour medicine conjugate self assembling process, can make that the nanoparticle particle diameter being self-assembly of is less, stability is better.
(4) the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate of the present invention not only has good antitumous effect, also has preparation method simple, it is easy to the advantages such as industrialized production.
Accompanying drawing explanation
The Electronic Speculum figure of Fig. 1 conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain
Fig. 2: the Electronic Speculum figure of conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain after introducing DSPE-PEG
Detailed description of the invention
Embodiment 1: the synthesis of oleic acid-paclitaxel conjugate
Under nitrogen protection; by paclitaxel (85mg; 0.1mmol) it is dissolved in appropriate anhydrous methylene chloride; add dimethylamino naphthyridine (14.4mg; 0.1mmol) with dicyclohexylcarbodiimide (24.6mg, 0.2mmol), under stirring condition, add oleic acid (28mg; 0.1mmol), continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains oleic acid-paclitaxel conjugate (productivity 63%).
Embodiment 2: the synthesis of alpha-linolenic acid-paclitaxel conjugate
Under nitrogen protection; by paclitaxel (85mg; 0.1mmol) it is dissolved in appropriate anhydrous methylene chloride; add dimethylamino naphthyridine (14.4mg; 0.1mmol) with dicyclohexylcarbodiimide (24.6mg, 0.2mmol), under stirring condition, add oleic acid (28mg; 0.1mmol), continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separate through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains alpha-linolenic acid-paclitaxel conjugate (productivity 68%).
The synthesis of embodiment 3:DHA-SN38 conjugate
Under nitrogen protection; by SN38 (40mg; 0.1mmol) it is dissolved in appropriate anhydrous N; in dinethylformamide, add dimethylamino naphthyridine (14.4mg, 0.1mmol) and dicyclohexylcarbodiimide (24.6mg; 0.2mmol); add conjugated linoleic acid (33mg, 0.1mmol) under stirring condition, continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains DHA-SN38 conjugate (productivity 63%).
The synthesis of embodiment 4:EPA-amycin conjugate
Under nitrogen protection; by amycin (55mg; 0.1mmol) it is dissolved in appropriate anhydrous methylene chloride, adds O-BTA-tetramethylurea hexafluorophosphate (HBTU, 115mg; 0.15mmol) and N; N-diisopropylethylamine (100mg, 0.77mmol), adds EPA (30mg; 0.1mmol), continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains EPA-amycin conjugate (productivity 61%).
Embodiment 5: the synthesis of conjugated linoleic acid-vinblastine conjugate
Under nitrogen protection; by vinblastine (91mg; 0.1mmol) it is dissolved in appropriate anhydrous methylene chloride; add dimethylamino naphthyridine (14.4mg; 0.1mmol) with dicyclohexylcarbodiimide (24.6mg, 0.2mmol), under stirring condition, add conjugated linoleic acid (28mg; 0.1mmol), continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separate through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains conjugated linoleic acid-vinblastine conjugate (productivity 65%).
Embodiment 6: the synthesis of alpha-linolenic acid-gemcitabine conjugate
Under nitrogen protection; alpha-linolenic acid (278mg; 1mmol) it is dissolved in appropriate anhydrous tetrahydro furan; triethylamine (125mg is added under stirring condition; 1.2mmol); under condition of ice bath; it is added dropwise over ethyl chloroformate (113mg; anhydrous tetrahydrofuran solution 1mmol), stirring reaction 30 minutes, then it is added dropwise over gemcitabine hydrochloride (309mg; 1mmol) with triethylamine (125mg; anhydrous DMF mixed solution 1.2mmol), stirring reaction 48 hours under room temperature condition. Reactant liquor reduced vacuum dries, separate through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains alpha-linolenic acid-gemcitabine conjugate (productivity 60%).
Embodiment 7: the synthesis of conjugated linoleic acid-cisplatin conjugate
Hydrogen peroxide is added dropwise over cisplatin (1g, 3.33mmol), heats and keep reacting fully for 5 hours carrying out to 75 DEG C, in room temperature, lucifuge is placed overnight, product collected by filtration, and pure water is for several times, namely reduced vacuum obtains c, c, t-[Pt (NH after drying3)2Cl2(OH)2] sterling. By c, c, t-[Pt (NH3)2Cl2(OH)2] (34mg, 0.1mmol) it is dissolved in appropriate dimethyl sulfoxide, add dimethylamino naphthyridine (14.4mg, 0.1mmol) with dicyclohexylcarbodiimide (24.6mg, 0.2mmol), add conjugated linoleic acid (29mg, 0.1mmol) under stirring condition, continue reaction 24 hour is stirred at room temperature. After reactant liquor is lyophilised, utilizing acetone recrystallization, centrifugal collection product, reduced vacuum is drying to obtain conjugated linoleic acid-cisplatin conjugate (productivity 61%).
Embodiment 8: the synthesis of conjugated linoleic acid-paclitaxel conjugate
Under nitrogen protection; by paclitaxel (85mg; 0.1mmol) it is dissolved in appropriate anhydrous methylene chloride; add dimethylamino naphthyridine (14.4mg; 0.1mmol) with dicyclohexylcarbodiimide (24.6mg, 0.2mmol), under stirring condition, add conjugated linoleic acid (28mg; 0.1mmol), continue reaction 12 hour is stirred at room temperature. It is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration, filtrate dries up or reduced vacuum dries through ambient temperature under nitrogen, obtains oleic acid-paclitaxel conjugate (productivity 70%).
Embodiment 9: the preparation of conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain
Conjugated linoleic acid-paclitaxel conjugate is dissolved in appropriate dimethyl sulfoxide the 5mg/ml conjugated linoleic acid-paclitaxel conjugate solution obtained, pipette 1ml and dropwise instill in 3ml pure water, and be stirred continuously, obtain conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain dispersion liquid (CLA-PTXNP), then continue in bag filter, utilize deionized water dialysis can remove dimethyl sulfoxide in 24-48 hour, to obtain final product.
Embodiment 10: introduce the preparation of the conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain after DSPE-PEG
Conjugated linoleic acid-paclitaxel conjugate and DSPE-PEG are mixed and is dissolved in appropriate dimethyl sulfoxide and obtains the 5mg/ml conjugated linoleic acid-paclitaxel conjugate solution containing 10%DSPE-PEG, pipette 1ml and dropwise instill in 3ml pure water, and be stirred continuously, obtain conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain dispersion liquid (CLA-PTXPEGNP), then continue in bag filter, utilize deionized water dialysis can remove dimethyl sulfoxide in 24-48 hour, to obtain final product.
Embodiment 11: the preparation of other unsaturated fatty acids-antitumor medicine conjugate self-assembled nanometer grain
The method of reference example 9-10, unsaturated fatty acid-the antitumor medicine conjugate prepared by embodiment 1-7 is dissolved in appropriate dimethyl sulfoxide respectively and obtains 5mg/ml unsaturated fatty acid-antitumor medicine conjugate solution, pipette 1ml and dropwise instill in 3ml pure water, and be stirred continuously, obtain the unsaturated fatty acid with opalescence-antitumor medicine conjugate self-assembled nanometer grain dispersion liquid, then continue in bag filter, utilize deionized water to dialyse 24-48 hour and remove dimethyl sulfoxide, to obtain final product.
Embodiment 12: introduce the preparation of the unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer grain after DSPE-PEG
Unsaturated fatty acid-the antitumor medicine conjugate (or and DSPE-PEG mixing) prepared by embodiment 1-7 is dissolved in appropriate dimethyl sulfoxide and obtains (or containing 10%DSPE-PEG's) 5mg/ml unsaturated fatty acid-antitumor medicine conjugate solution, pipette 1ml and dropwise instill in 3ml pure water, and be stirred continuously, obtain (or containing DSPE-PEG's) unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer grain dispersion liquid, then continue in bag filter, utilize deionized water dialysis can remove dimethyl sulfoxide in 24-48 hour hour, to obtain final product.
Test example 1: the structural characterization of unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer grain
(1) utilize Malvern ParticleSizer to be prepared particle size distribution and the zeta potential (such as chart 1) of nanoparticle by embodiment 9-11 by Dynamic Light Scattering Determination, measure sample volume: 1ml; Measure temperature 25 DEG C; Disperse medium: water.
As an example, when antitumor drug is paclitaxel, the particle size distribution and the zeta potential that introduce the self-assembled nanometer grain of unsaturated fatty acid-paclitaxel conjugate before and after DSPE-PEG are as follows:
Table 1 introduces particle size distribution and the zeta potential of unsaturated fatty acid before and after DSPE-PEG-paclitaxel conjugate self-assembled nanometer grain
(2) JEM-1400Plus transmission electron microscope observation is utilized to prepare the form of nanoparticle (such as Fig. 1 and Fig. 2) by embodiment 8-11.
Result shows, the nanoparticle that unsaturated fatty acid-antitumor medicine conjugate is self-assembly of is uniform ball shaped nano grain, and uniform particle diameter, and surface potential is nagative potential.
The nanoparticle that after introducing DSPE-PEG, unsaturated fatty acid-antitumor conjugate is self-assembly of is still for uniform ball shaped nano grain, the nanoparticle particle diameter that particle diameter pure unsaturated fatty acid-antitumor medicine conjugate is self-assembly of is less, surface potential is still in nagative potential, it was shown that the introducing of DSPE-PEG is conducive to the self assembly behavior of unsaturated fatty acid-antitumor medicine conjugate.
Result is shown in the nanosystems that unsaturated fatty acid-antitumor medicine conjugate is self-assembly of and introduces minimal amount of DSPE-PEG (such as embodiment 10, the content of DSPE-PEG is less than 10%) particle diameter can be reduced, improve the purpose of stability, this is significantly different with traditional nanosystems (content >=90% of usual carrier material) relying on carrier material, thus unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer system has higher drug loading.
Test example 2: the extracorporeal suppression tumor cell growth effects of conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain is evaluated
(1) by human breast carcinoma MDA-MB-231 cell (1 �� 104Cells/well), Mus source melanoma B16-F1 0 cell (3 �� 103Cells/well) and human glioma U87-MG cell (8 �� 103Cells/well) it is inoculated in respectively in 96 orifice plates, hatch 24h standby.
(2) nanoparticle dispersion liquid after degerming, will be diluted to the nanoparticle diluent of 0.25��32 ��M through 0.2 micron of sterilised membrane filter by culture medium.
(3) after discarding former culture medium, it is separately added into nanoparticle diluent, to add complete medium for comparison, after hatching 48 hours, discard nanoparticle diluent, cell 1 hour (4 DEG C) fixed by 10% trichloroacetic acid, use distilled water cyclic washing, air dries naturally, 0.4%SRB dye liquor dyes 20 minutes in room temperature, and 0.1% acetic acid cyclic washing adds 10 micromoles per liter Tris-base solution after naturally drying in air, after putting shaking table concussion 30 minutes, measure each group of light absorption value at 540nm place by microplate reader.
(4) survival rate is calculated by following equation:
Survival rate=(dosing group cell average absorbance value/cellular control unit average absorbance value) �� 100%
(5) GraphPadPrism6 is utilized to calculate IC50, conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain vitro inhibition difference growth of tumour cell situation:
Table 2 conjugated linoleic acids-paclitaxel conjugate self-assembled nanometer grain vitro inhibition difference growth of tumour cell situation (n=3)
Wherein FreeCLA-PTX represents that conjugated linoleic acid-paclitaxel conjugate adopts the compound method of taxol to prepare the solution (40mg is dissolved in 2.5ml dehydrated alcohol and 2.635g polyoxyethylene castor oil, faces the front 5% glucose injection dilution agent to desired concn of administration) obtained;
CLA-PTX-NPs represents by conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain that embodiment 9 prepares;
CLA-PTXPEGNPs represents by the conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain introducing DSPE-PEG that embodiment 10 prepares.
Result display introduces the conjugated linoleic acid-paclitaxel conjugate self-assembled nanometer grain before and after DSPE-PEG has depression effect to kinds of tumor cells growth in vitro, has good anti-tumor activity.
Above with general explanation, detailed description of the invention and test, the present invention is described in detail, modifications or improvements without departing from theon the basis of the spirit of the present invention, belongs to the scope of protection of present invention.
Claims (10)
1. a self-assembled nanometer system for unsaturated fatty acid-antitumor medicine conjugate, described nanosystems includes unsaturated fatty acid-antitumor medicine conjugate, aqueous solution and the amphipathy macromolecule material optionally contained; Described unsaturated fatty acid-antitumor medicine conjugate is the compound that unsaturated fatty acid is formed with antitumor drug coupling; Described antitumor drug is selected from yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament; Described unsaturated fatty acid is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29; Described amphipathy macromolecule material includes one or more in DSPE-PEG, PLGA-PEG, polyoxyethylene castor oil, Tweens and lecithin.
2. the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate according to claim 1, it is characterised in that:
Described antitumor drug preferably is selected from paclitaxel, Docetaxel, Cephalomannine, 10-go acetyl baccatin III and 7-difference to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin; And/or
Described unsaturated fatty acid is selected from oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA; Preferred described unsaturated fatty acid is conjugated linoleic acid; More preferably conjugated linoleic acid is along 9, anti-11-conjugated linoleic acid or anti-10, along 12-conjugated linoleic acid; And/or
Described aqueous solution is selected from pure water, 5% glucose solution, normal saline, phosphate buffer; And/or
When in nanosystems containing amphipathy macromolecule material, the optional 40:1��1:80 of mass ratio of amphipathy macromolecule material and unsaturated fatty acid-antitumor medicine conjugate, it is preferable that 10:1��1:40, more preferably 1:1��1:20; Described amphipathy macromolecule material is DSPE-PEG.
3. the antitumor medicine conjugate that a unsaturated fatty acid is modified, described antitumor medicine conjugate has the character that can be self-assembled into nanosystems, and described unsaturated fatty acid-antitumor medicine conjugate is the compound that unsaturated fatty acid is formed with antitumor drug coupling; Described antitumor drug is selected from yew alkanes, camptothecin, vinca, antibiotics, ucleosides and platinum series antineoplastic medicament; Described unsaturated fatty acid is structured with: CnH2n-1COOH or CnH2n-3COOH or CnH2n-5COOH or CnH2n-7COOH or CnH2n-9COOH or CnH2n-11COOH, wherein n is the integer of 1-29, and described unsaturated fatty acid-antitumor medicine conjugate is not conjugated linoleic acid-paclitaxel and conjugated linoleic acid-gemcitabine.
4. conjugate according to claim 3, described antitumor drug goes acetyl baccatin III and 7-poor to paclitaxel, gemcitabine, 5-fluorouracil and cytosine arabinoside, SN38, vinblastine, amycin, cisplatin selected from paclitaxel, Docetaxel, Cephalomannine, 10-; Described unsaturated fatty acid is selected from oleic acid, conjugated linoleic acid, alpha-linolenic acid, EPA, DHA.
5. the preparation method of the antitumor medicine conjugate that the unsaturated fatty acid described in any one of claim 3 to 4 is modified, described method includes:
(1), by antitumor drug it is dissolved in a certain amount of suitable solvent, adds catalyst and dehydrant, under stirring condition, add unsaturated fatty acid, continue stirring reaction;
(2), it is filtered to remove precipitation, filtrate decompression vacuum drying, separates through thin layer chromatography after dissolving with a small amount of solvent, after products therefrom band Extraction solvent fully dissolves, sucking filtration;
(3), gained filtrate ambient temperature under nitrogen dries up or reduced vacuum dries, and obtains the antitumor medicine conjugate that unsaturated fatty acid is modified.
6. preparation method according to claim 5, wherein,
Solvent described in step (1) is selected from the one in dichloromethane, chloroform, DMF, dimethyl sulfoxide, dioxane or oxolane or its mixed solution, it is preferable that dichloromethane; And/or
Catalyst described in step (1) is dimethylamino naphthyridine; And/or
Dehydrant described in step (1) is selected from the one in dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide or DIC or its mixture, it is preferable that dicyclohexylcarbodiimide; And/or
The temperature of the reaction in step (1) is room temperature��80 DEG C, different according to different unsaturated fatty acid Optimal Temperature, it is preferable that room temperature; Reaction condition is under indoor conditions or nitrogen protection, it is preferable that nitrogen protection condition; Response time is 4-72 hour;
In step (2), thin-layer developing solvent for use is volume ratio is the methylene chloride/methanol solution of 1:1��40:1; And/or
Wherein antitumor drug is 1:0.1��1:10 with the molar ratio of unsaturated fatty acid; The mol ratio of described catalyst and antitumor drug is 1:0.1��1:10; Described dehydrant and antitumor drug are 1:0.1��1:10 with mol ratio.
7. the preparation method of the self-assembled nanometer system of unsaturated fatty acid-antitumor medicine conjugate according to any one of claim 1-2, comprises the steps:
(1) by unsaturated fatty acid-antitumor medicine conjugate and optionally with amphipathy macromolecule material mixing, be dissolved in a certain amount of suitable organic solvent;
(2) pipette in the aqueous solution that above-mentioned dropwise instills certain volume, and be stirred continuously under 50��1500rpm;
(3) within 12��72 hours, remove organic solvent with deionized water dialysis, obtain self-assembled nanometer grain dispersion liquid;
(4) when in nanosystems containing amphipathy macromolecule material, by the self-assembled nanometer further centrifugal concentrating of grain dispersion liquid obtained above.
8. preparation method according to claim 7, the organic solvent described in step (1) is selected from water-soluble solvents such as dimethyl sulfoxide, acetone, ethanol, oxolane, DMF, methanol and acetonitriles, it is preferable that dimethyl sulfoxide; Solution and the volume ratio of aqueous solution that conjugate and organic solvent are formed are 1:1��1:100, it is preferable that 1:3 to 1:20.
9. conjugate or claim 7 or 8 described in unsaturated fatty acid according to any one of claim 1-2 is modified antitumor medicine conjugate self-assembled nanometer system or claim 3 or 4 prepare unsaturated fatty acid-antitumor medicine conjugate self-assembled nanometer system application in preparing antitumor drug.
10. application according to claim 9, described tumor is selected from ovarian cancer, breast carcinoma, pulmonary carcinoma, colorectal cancer, melanoma, incidence cancer, lymphoma, cerebroma, leukemia, nasopharyngeal carcinoma, lymphoma, uterus carcinoma.
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