CN104013967B - A kind of gadolinium polymer vesicle and its preparation method and application that carries - Google Patents
A kind of gadolinium polymer vesicle and its preparation method and application that carries Download PDFInfo
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Abstract
The invention provides a kind of gadolinium polymer vesicle and its preparation method and application that carries, within this year, gadolinium polymer vesicle comprises polymer vesicle and is subject to the metal gadolinium of described polymer vesicle carrying, polymer vesicle is self-assembled into the structure with asymmetric membrane jointly by two kinds of polymer, and two kinds of polymer are respectively FA-PGAx-b-PβzAnd ε-PGAy-b-Pβz, the complexing of metal gadolinium is on ε; Its preparation method is: first synthesize respectively two kinds of polymer, then make the complexing of metal gadolinium also finally generate and carry a gadolinium polymer vesicle on ε; This year gadolinium polymer vesicle Stability Analysis of Structures, there is good biocompatibility and biological degradability, cancer cell is had to good targeting, not only can be used as the NMRI agent for target identification cancer cell, can also wrap up hydrophilic or dewatering medicament, targeting moiety is carried out to controllable drug delivery.
Description
Technical field
The invention belongs to polymeric material field, relate to a kind of polymer vesicle and its preparation method and application.
Background technology
In recent years, pharmaceutical carrier has obtained research widely. Wherein, carry out in the aqueous solution about Amphipathilic block polymer certainlyAssembling formation vesica or micella enjoy numerous researchers' concern as the research of pharmaceutical carrier. Amphipathilic block polymer is at waterIn can carry out self assembly and form vesica duplicature, but the less stable of the liposome class vesica of extensive use is at present easily brokenSplit and produce the intermembranous fusion of vesica. And polymer vesicle can significantly improve the mechanical performance of vesica, by polycarbonate-based and polypeptide classThe focus that the biodegradable polymers vesica of polymer composition is studied especially.
In order to improve the targeting of pharmaceutical carrier, carrier surface often connects some target groups. Folic acid (Folicacid, FA) originallyMatter is a kind of little molecule vitamin, claims again the phthalein glutamic acid of talking endlessly, and it had both participated in pyrimidine and the purine core of cell DNA and RNA prerequisiteSynthesizing of thuja acid, participate in again the synthetic of methionine. The research of folacin receptor shows, the tumour of the many epithelial origins of the mankind asActivity and the quantity of the folacin receptor of the tumor cell surfaces such as oophoroma, cervical carcinoma, breast cancer, nasopharyngeal carcinoma, liver cancer are significantly higher thanNormal tissue cell, is overexpression. And normal zooblast lacks the synthetic key enzyme of folic acid biological, can only dependent cellsThe folic acid specific bond albumen (being folacin receptor, Folatereceptors, FR) on surface initiatively absorbs exogenous folic acid and remains normalVital movement, thereby expression is extremely low. Therefore, according to the differential expression of normal cell and cancer cell surface folacin receptor, canTo prepare the polymer vesicle with folate-targeted group, to improve the recognition capability of polymer vesicle to cancer cell.
Magnetic resonance imaging (MRI) technology first Application in 1973 is in human diagnosis, and through the development of four more than ten years, this technologyDeveloped rapidly and extensive use in fields such as biology, medical science. Compared with X-CT, ultrasonic imaging, MRI technology hasMany outstanding advantages: without ionising radiation; Can realize the imaging of multinuclear multi-parameter; Can implement the scanning of arbitrary orientation aspect; HigherSpatial resolution and contrast; Without sclerotin artifact; Can reflection be detected tissue water proton surrounding environment and obtain relevant Physiology and biochemistryInformation; Can improve diagnosis to heart, trunk morphology and function etc. So MRI technology has become in current clinical diagnosisOne of the strongest detection means. This technology has been widely used in the radiography of head, nervous system, belly and the blood vessel of human body,Effective to detecting necrosis, ischaemic and various malignant change, be used in particular for the early diagnosis of tumour, can make to treat successfullyRate has significant improvement. Clinically, for guaranteeing the accuracy of diagnosis, more than 45% MRI checks need to use contrast preparationImprove picture contrast, wherein more than 30% use containing gadolinium contrast preparation. But, through repeatedly clinical research discovery, free goldBelong to gadolinium ion and there is very strong toxic and side effect, can cause the serious fibrillatable of kidney, the gadolinium contrast preparation using at present be clinically all throughCross and be prepared from after complexing agent effect, for example DTPA-Gd, DOTA-Gd, gadodiamide (Gadodiamide), Magnevist Solution(Gadopcntetatedimeglumine), Gadobenate Dimeglumine (Gadobenatedimeglemine), Gadoteridol (Gadoteridol),Gadoterlc acid meglumine saltlniection (Gadoteratemeglumine) etc., but these gadolinium contrast preparation that use clinically still exist sensitivity not high,The problems such as biocompatibility is poor, be difficult for degraded, carrying drug ratio is low and imaging effect is poor.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, main purpose is to provide one to have no side effect, carrying drug ratio is high, imaging effectGood, easily degraded and good biocompatibility carry gadolinium polymer vesicle.
Another object of the present invention is to provide the preparation method of a kind of above-mentioned year gadolinium polymer vesicle.
The 3rd object of the present invention is to provide the purposes of above-mentioned year gadolinium polymer vesicle.
For achieving the above object, solution of the present invention is:
A kind of gadolinium polymer vesicle that carries, comprises by two kinds of polymer and is jointly self-assembled into the polymer vesicle with asymmetric membrane structureAnd the metal gadolinium that carried by polymer vesicle, two kinds of polymer are respectively FA-PGAx-b-PβzAnd ε-PGAy-b-Pβz, FA is leafAcid groups, the degree of polymerization that x and y are PGA and x > y, ε is the complexing agent for complexing metal gadolinium, P βzFor hydrophobic segment,FA-PGAx-b-PβzIn PGAxForm the dizzy layer in outside of polymer vesicle, ε-PGAy-b-PβzIn PGAyForm polymer vesicleThe dizzy layer in inside, the P β in two kinds of polymerzForm the rete of polymer vesicle.
Above-mentioned hydrophobic segment is the one in polycaprolactone, polytrimethylene carbonate and PLA; Or complexing agent is diethylPentaacetic acid and Isosorbide-5-Nitrae, 7,10-tetraazacyclododecanand-Isosorbide-5-Nitrae, the one in 7,10-tetrabasic carboxylic acid; Or polymer vesicleWith the mol ratio of metal gadolinium be 1:(0.1～2). The particle diameter of above-mentioned polymer vesicle is 60～350nm; Or carry gadolinium polymeric bladderThe particle diameter of bubble is 80～400nm.
A method of preparing above-mentioned year gadolinium polymer vesicle, comprises the steps:
(1)、PβzThe synthesis step of-NH-Boc:
By the monoethanolamine of being protected by Boc, β and catalyst taking mol ratio as 1:(20～100): (0.005～0.05) reaction 12～48 hours, after processing, obtain P βz-NH-Boc；
(2)、Pβz-NH2Synthesis step:
By the P β of step (1) gainedz-NH-Boc is dissolved in organic solvent, adds excessive trifluoroacetic acid, under room temperature, react 2～24 hours, after processing, obtain P βz-NH2；
(3)、PBLGx-b-PβzAnd PBLGyThe synthesis step of-b-P β z:
By the P β of step (2) gainedz-NH2The ratio that is 1 ︰ x according to mol ratio with the NCA monomer of glutamic acid is dissolved in anhydrousIn DMF, under room temperature, react 12～48 hours, after processing, obtain PBLGx-b-Pβz；
By the P β of step (2) gainedz-NH2The ratio that is 1 ︰ y according to mol ratio with the NCA monomer of glutamic acid is dissolved in anhydrousIn DMF, under room temperature, react 12～48 hours, after processing, obtain PBLGy-b-Pβz；
(4)、FA-PBLGx-b-PβzAnd ε-PBLGy-b-PβzSynthesis step:
By step (3) gained PBLGx-b-PβzBe dissolved in anhydrous DMSO with FA, add dicyclohexylcarbodiimide and N-HOSu NHS, stirs under room temperature 5～48 hours, obtains FA-PBLG after processingx-b-Pβz。
By step (3) gained PBLGy-b-PβzBe dissolved in anhydrous DMSO with ε, add dicyclohexylcarbodiimide and N-hydroxylBase succinimide, stirs under room temperature 5～48 hours, obtains ε-PBLG after processingy-b-Pβz。
(5)、FA-PGAx-b-PβzAnd ε-PGAy-b-PβzSynthesis step:
By step (4) gained FA-PBLGx-b-PβzBe dissolved in 33% HBr/CH3In COOH solution, react 2～12 hours,After processing, obtain FA-PGAx-b-Pβz；
By step (4) gained ε-PBLGy-b-P β z is dissolved in 33% HBr/CH3In COOH solution, react 2～12 hours,After processing, obtain ε-PGAy-b-Pβz；
(6), carry the preparation process of gadolinium polymer vesicle:
FA-PGA step (5) being obtained according to the concentration of 1～30mg/mlx-b-PβzAnd ε-PGAy-b-PβzBe dissolved in togetherIn DMF, lucifuge stirs and drips with the speed of 5～50d/min the gadolinium ion aqueous solution that doubles DMF volume, and dialysis is removedDMF, obtains carrying gadolinium polymer vesicle solution;
β is the one in caprolactone, trimethyl carbonic ester and lactic acid, the degree of polymerization that z is β.
In above-mentioned steps (1), z is 30～60; Preferably, in the time that β is caprolactone, z is 30 or 60; When β is threeMethyl carbonic, z is 30; In the time that β is lactic acid, z is 50. Catalyst in step (1) is stannous octoate; Or step(2) organic solvent in is carrene.
In above-mentioned steps (3), x is that 50～100, y is 10～50; Preferably, in the time that β is caprolactone, x be 70 or100, y is 20 or 30; When β is trimethyl carbonic ester, x is that 80, y is 25; In the time that β is lactic acid, x is that 75, y is15。
Above-mentioned year gadolinium polymer vesicle applied in the carrier of the medicine for the preparation for the treatment of cancer or pharmaceutical composition.
Within above-mentioned year, gadolinium polymer vesicle is applied in the NMRI agent for the preparation of target identification cancer cell.
Owing to adopting such scheme, the invention has the beneficial effects as follows:
The first because wherein on a kind of polymer with folate-targeted group, when multiple polymer form while carrying gadolinium polymer vesicle,It is upper that folate-targeted group is positioned at the outside dizzy layer of vesica, and that folate-targeted group has cancer cell surface folacin receptor is higher affinePower, can be enriched in so carry gadolinium polymer vesicle the tumor locus that is rich in cancer cell; Be filled with and control when carrying in gadolinium polymer vesicleWhile treating the medicine of cancer, it has good cancer cell targeting as pharmaceutical carrier, can carry out target administration to cancer cell,Carry out after specific binding at the folacin receptor on folate-targeted group and cancer cell surface, can greatly improve medicine and enter cancer cellSpeed, thereby improved inhibition or the killing effect to cancer cell.
The second, because in the time that gadolinium polymer vesicle is carried in multiple polymer formation, the end of this year inner dizzy layer of gadolinium polymer vesicle hasComplexing agent, this complexing agent and the complexing of metal gadolinium phase, so this year gadolinium polymer vesicle clinically can be simultaneously as nuclear magnetic resonanceContrast preparation use, and higher than the current sensitivity of DTPA-Gd using clinically.
The 3rd, in the time that a year gadolinium polymer vesicle wraps up hydrophilic anticarcinogen DOXHCl, carry the dizzy layer of a gadolinium polymer vesicle hydrophilic segment sideChain carboxyl and DOXHCl have electrostatic interaction, make its carrying drug ratio can reach 40%～60%, and the polymer vesicle pair of current techniquesThe carrying drug ratio of DOXHCl only has 20%～30%, and therefore carrying drug ratio is approximately doubled.
The 4th, the bi-block copolymer that the polymer that carries the use of gadolinium polymer vesicle is degradable polypeptide and polycarbonate-based composition,Under protease or lipase effect, easily there is biodegradation, simultaneously because the bag of vesica carries effect, greatly reduce metal gadoliniumTo the toxic and side effect of human body, improve the security of medication and development.
The 5th, carry gadolinium polymer vesicle and there are two kinds of polymer, the segment ratio difference of these two kinds of polymer, therefore, polymerSelf assembling process make to carry gadolinium polymer vesicle and become the structure of asymmetric membrane, convenient to dizzy layer inside and outside this year gadolinium polymer vesicleCarry out functionalized design.
Brief description of the drawings
Fig. 1 is the DLS result figure that carries gadolinium mixed with polymers vesica of the embodiment of the present invention one gained.
Fig. 2 is the TEM result figure that carries gadolinium mixed with polymers vesica of the embodiment of the present invention one gained.
Fig. 3 is the relaxation rate r that carries gadolinium mixed with polymers vesica of the embodiment of the present invention one gained1Test result figure.
Detailed description of the invention
Below in conjunction with accompanying drawing illustrated embodiment, the present invention is further illustrated.
Embodiment mono-
The present embodiment provides a kind of gadolinium polymer vesicle that carries, by polymer vesicle and be subject to the metal gadolinium of this polymer vesicle carryingComposition.
Polymer vesicle is formed by two kinds of common self assemblies of polymer, has asymmetric membrane structure. These two kinds of polymer are respectivelyFA-PGAx-b-PβzAnd ε-PGAy-b-Pβz. Wherein, FA is folic acid group, as the target group of cancer cell surface folacin receptorAnd work. PGA represents polyglutamic acid (PolyglutamicAcid, PGA), and it works as hydrophilic segment; X and yFor the degree of polymerization and the x > y of PGA, x can be that 50～100, y can be 10～50. ε is the complexing for complexing metal gadoliniumAgent, can be diethyl pentetic acid (DiethyleneTriaminePentacetateAcid, DTPA) and Isosorbide-5-Nitrae, 7,10-Tetraazacyclododecanand-Isosorbide-5-Nitrae, any one in 7,10-tetrabasic carboxylic acid (DOTA). P βzFor hydrophobic segment, z represents that this is hydrophobicThe degree of polymerization of segment, z can be 30～60. In the time that multiple polymer form polymer vesicle, polymer FA-PGAx-b-PβzInPGAxForm the dizzy layer in outside of polymer vesicle, polymer ε-PGAy-b-PβzIn PGAyThe inside that forms polymer vesicle is dizzyLayer, the P β in two kinds of polymerzForm the rete of polymer vesicle.
The complexing of metal gadolinium is on ε. In year gadolinium polymer vesicle, the mol ratio of polymer vesicle and metal gadolinium is 1:(0.1～2),In the present embodiment, be 1:0.5. The particle diameter of polymer vesicle is 60～350nm, and the particle diameter that carries gadolinium polymer vesicle is 80～400nm.
In the present embodiment, β can be caprolactone (Caprolactone, CL), P βz(be PCLz) degree of polymerization z be preferably 30.At polymer FA-PGAx-b-PβzIn, PGAxDegree of polymerization x be preferably 70; At polymer ε-PGAy-b-PβzIn, PGAyPoly-Right y is preferably 20. Complexing agent ε is preferably DTPA. Now, the structural formula of two kinds of polymer is respectively: FA-PGA70-b-PCL30And DTPA-PGA20-b-PCL30。
The preparation method of carrying gadolinium polymer vesicle in the present embodiment comprises the steps:
(1)PCL30The synthesis step of-NH-Boc;
By the monoethanolamine of the dried tert-butyl group (Boc) protection, caprolactone monomer (CL) and octoate catalyst Asia tin according to moleJoin in round-bottomed flask than the ratio that is 1 ︰ 30 ︰ 0.005, at 110 DEG C, react 48 hours, then add solvent dichloromethaneAlkane dissolves, in methyl alcohol, precipitates, and suction filtration, vacuum drying, obtains white powder polymer PC L30-NH-Boc。
(2) macromole evocating agent PCL30-NH2Synthesis step;
By step (1) resulting polymers PCL30-NH-Boc is dissolved in the middle of organic solvent dichloromethane, adds excessive trifluoroacetic acid(TFA), stirring reaction 12 hours under room temperature, concentrated by rotary evaporation, precipitates in methyl alcohol, suction filtration, vacuum drying, repeats threeInferior, obtain white powder macromole evocating agent PCL30-NH2。
(3) two kinds of bi-block copolymer PBLG70-b-PCL30And PBLG20-b-PCL30Synthesis step;
By step (2) gained macromole evocating agent PCL30-NH2With the NCA monomer of glutamic acid be 1 ︰ 70 according to mol ratio respectivelyRatio be dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, in ether, precipitate, suction filtration, vacuum is dryDry, obtain white polymer PBLG70-b-PCL30. B represents block, i.e. segment.
By step (2) gained macromole evocating agent PCL30-NH2With the NCA monomer of glutamic acid be 1 ︰ 20 according to mol ratio respectivelyRatio be dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, in ether, precipitate, suction filtration, vacuum is dryDry, obtain white polymer PBLG20-b-PCL30。
(4)FA-PBLG70-b-PCL30And DTPA-PBLG20-b-PCL30Synthesis step;
By step (3) resulting polymers PBLG70-b-PCL30With folic acid (FA) is dissolved in anhydrous DMSO, add a certain amount ofDicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS), under room temperature, stir 24 hours, concentrated by rotary evaporation,In ether, precipitate, suction filtration, vacuum drying, obtains yellow polymer FA-PBLG70-b-PCL30。
By step (3) resulting polymers PBLG20-b-PCL30Be dissolved in anhydrous DMSO with DTPA, add a certain amount of twoCarbodicyclo hexylimide (DCC) and N-hydroxy-succinamide (NHS), stir under room temperature 24 hours, revolves steaming, and precipitation, takes outFilter, vacuum drying, obtains white polymer DTPA-PBLG20-b-PCL30。
(5) amphipathic nature polyalcohol FA-PGA70-b-PCL30And DTPA-PGA20-b-PCL30Synthesis step;
By step (4) gained FA-PBLG70-b-PCL30Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction 5 hours,In ether, precipitate, suction filtration, vacuum drying, obtains polymer powder FA-PGA70-b-PCL30。
By step (4) gained DTPA-PBLG20-b-PCL30Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction 5 is littleTime, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder DTPA-PGA20-b-PCL30。
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml70-b-PCL30And DTPA-PGA20-b-PCL30OneRise and be dissolved in DMF, under the condition of lucifuge vigorous stirring, drip with the speed of 10d/min the gadolinium that doubles DMF volumeThe aqueous solution of ion, organic solvent DMF is removed in lucifuge dialysis, obtains light blue solution and is year gadolinium polymer vesicle solution.
The DLS result of carrying gadolinium polymer vesicle solution of the present embodiment gained as shown in Figure 1. This year gadolinium polymer vesicle solutionTME result as shown in Figure 2. The relaxation rate r of this year gadolinium polymer vesicle solution1Test result as shown in Figure 3.
Year gadolinium polymer vesicle solution in the present embodiment can be used for for the preparation of the NMRI agent of target identification cancer cell,Carrier or the pharmaceutical composition of medicine that can also be used for for the preparation for the treatment of cancer.
The gadolinium polymer vesicle that carries in the present embodiment is easily degraded under the effect of protease or lipase, therefore bio-compatibleProperty is good, can not produce toxicological effect to people's body.
Embodiment bis-
In embodiment mono-, two kinds of polymer that carry in gadolinium polymer vesicle are FA-PGA70-b-PCL30And DTPA-PGA20-b-PCL30。In fact the Amphipathilic block polymer, carrying in gadolinium polymer vesicle can also be FA-PGA100-b-PCL60WithDTPA-PGA30-b-PCL60. Now, FA-PGA100-b-PCL60The degree of polymerization x of middle GA is that the degree of polymerization z of 100, CL is 60,DTPA-PGA30-b-PCL60The degree of polymerization x of middle GA is that the degree of polymerization z of 30, CL is 60.
The preparation method of carrying gadolinium polymer vesicle in the present embodiment comprises the steps:
(1)PCL60The synthesis step of-NH-Boc;
Be 1 by sub-to monoethanolamine, caprolactone monomer (CL) and the octoate catalyst of dried tert-butyl group protection tin according to mol ratioThe ratio of ︰ 60 ︰ 0.005 joins in round-bottomed flask, reacts 48 hours at 110 DEG C, adds methylene chloride to dissolve,In methyl alcohol, precipitate, suction filtration, vacuum drying, obtains white powder polymer PC L60-NH-Boc。
(2) macromole evocating agent PCL60-NH2Synthesis step;
By step (1) resulting polymers PCL60-NH-Boc is dissolved in the middle of carrene, adds excessive trifluoroacetic acid, under room temperatureStirring reaction 8 hours, concentrated by rotary evaporation, precipitates in methyl alcohol, suction filtration, vacuum drying, repeats three times, obtains white powderShape macromole evocating agent PCL60-NH2。
(3) two kinds of bi-block copolymer PBLG100-b-PCL60And PBLG30-b-PCL60Synthesis step;
By step (2) gained macromole evocating agent PCL60-NH2With the NCA monomer of glutamic acid be 1 ︰ 100 according to mol ratio respectivelyRatio be dissolved in dry DMF, room temperature lower seal stirring reaction 48 hours, precipitates in ether, suction filtration, vacuum drying,Obtain white polymer PBLG100-b-PCL60。
By step (2) gained macromole evocating agent PCL60-NH2With the NCA monomer of glutamic acid be 1 ︰ 30 according to mol ratio respectivelyRatio be dissolved in dry DMF, room temperature lower seal stirring reaction 48 hours, precipitates in ether, suction filtration, vacuum drying,Obtain white polymer PBLG30-b-PCL60。
(4)FA-PBLG100-b-PCL60And DTPA-PBLG30-b-PCL60Synthesis step;
By step (3) resulting polymers PBLG100-b-PCL60Be dissolved in anhydrous DMSO with folic acid, add a certain amount of DCCAnd NHS, under room temperature, stirring 24 hours, concentrated by rotary evaporation, precipitates in ether, suction filtration, vacuum drying, obtains yellow polymerFA-PBLG100-b-PCL60。
By step (3) resulting polymers PBLG30-b-PCL60Be dissolved in anhydrous DMSO with DTPA, add a certain amount of twoCarbodicyclo hexylimide (DCC) and N-hydroxy-succinamide (NHS), stir under room temperature 24 hours, revolves steaming, and precipitation, takes outFilter, vacuum drying, obtains white polymer DTPA-PBLG30-b-PCL60。
(5) amphipathic nature polyalcohol FA-PGA100-b-PCL60And DTPA-PGA30-b-PCL60Synthesis step;
By step (4) resulting polymers FA-PBLG100-b-PCL60Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction 5Hour, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder FA-PGA100-b-PCL60。
By step (4) resulting polymers DTPA-PBLG30-b-PCL60Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction5 hours, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder DTPA-PGA30-b-PCL60。
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml100-b-PCL60And DTPA-PGA30-b-PCL60Be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip and to double DMF volume with the speed of 10d/minThe aqueous solution of gadolinium ion, organic solvent is removed in lucifuge dialysis, obtains light blue solution and is year gadolinium polymer vesicle solution.
Embodiment bis-gained carry distinguishing without remarkable in purposes of gadolinium polymer vesicle and embodiment mono-, and just the degree of polymerization is different,The particle diameter that carries gadolinium polymer vesicle obtaining can be variant, can different in the situation that, select to be suitable for.
Embodiment tri-
Compared with embodiment mono-, in the polymer of year gadolinium polymer vesicle, P βzIt can also be polytrimethylene carbonate (PTMC).In the time that multiple polymer form polymer vesicle, the PTMC of these polymer forms the rete of polymer vesicle.
The preparation method of carrying gadolinium polymer vesicle in the present embodiment comprises the steps:
(1)PTMC30The synthesis step of-NH-Boc;
Be 1 ︰ 30 ︰ by sub-to monoethanolamine, TMC monomer and the octoate catalyst of dried tert-butyl group protection tin according to mol ratio0.005 ratio joins in round-bottomed flask, reacts 40 hours at 110 DEG C, adds methylene chloride to dissolve, in methyl alcohol, precipitate,Suction filtration, vacuum drying, obtains white powder polymer P TMC30-NH-Boc。
(2) macromole evocating agent PTMC30-NH2Synthesis step;
By step (1) resulting polymers PTMC30-NH-Boc is dissolved in carrene, adds excessive trifluoroacetic acid, under room temperatureStirring reaction 8 hours, concentrated by rotary evaporation, precipitates in methyl alcohol, suction filtration, vacuum drying, repeats three times, obtains white powderShape macromole evocating agent PTMC30-NH2。
(3) two kinds of bi-block copolymer PBLG80-b-PTMC30And PBLG25-b-PTMC30Synthesis step;
By step (2) gained macromole evocating agent PTMC30-NH2With the NCA monomer of glutamic acid be 1 ︰ according to mol ratio respectively80 ratio is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, suction filtration, and vacuum drying,Obtain white polymer PBLG80-b-PTMC30。
By step (2) gained macromole evocating agent PTMC30-NH2With the NCA monomer of glutamic acid be 1 ︰ according to mol ratio respectively25 ratio is dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, precipitates suction filtration, vacuum in etherDry, obtain white polymer PBLG25-b-PTMC30。
(4)FA-PBLG80-b-PTMC30And DTPA-PBLG25-b-PTMC30Synthesis step;
By step (3) resulting polymers PBLG80-b-PTMC30Be dissolved in anhydrous DMSO with folic acid, add a certain amount of DCCAnd NHS, under room temperature, stirring 24 hours, concentrated by rotary evaporation, precipitates in ether, suction filtration, vacuum drying, obtains yellow polymerFA-PBLG80-b-PTMC30。
By step (3) resulting polymers PBLG25-b-PTMC30Be dissolved in anhydrous DMSO with DTPA, add a certain amount of twoCarbodicyclo hexylimide and N-hydroxy-succinamide, stir under room temperature 24 hours, revolves steaming, precipitation, and suction filtration, vacuum drying,Obtain white polymer DTPA-PBLG25-b-PTMC30。
(5) amphipathic nature polyalcohol FA-PGA80-b-PTMC30And DTPA-PGA25-b-PTMC30Synthesis step;
By step (4) resulting polymers FA-PBLG80-b-PTMC30Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction5 hours, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder FA-PGA80-b-PTMC30。
By step (4) resulting polymers DTPA-PBLG25-b-PTMC30Be dissolved in 33%HBr/CH3In COOH solution, stir anti-Answer 5 hours, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder DTPA-PGA25-b-PTMC30。
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml80-b-PTMC30And DTPA-PGA25-b-PTMC30Be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip and to double DMF volume with the speed of 10d/minThe aqueous solution of gadolinium ion, DMF is removed in lucifuge dialysis, obtains light blue solution and is year gadolinium polymer vesicle solution.
Embodiment tetra-
Compared with embodiment mono-, in the polymer of year gadolinium polymer vesicle, P βzIt can also be PLA (PLA). When multiple poly-When compound forms polymer vesicle, the PLA of these polymer forms the rete of polymer vesicle.
The preparation method of carrying gadolinium polymer vesicle in the present embodiment comprises the steps:
(1)PLA50The synthesis step of-NH-Boc;
Be 1 ︰ by sub-to monoethanolamine, lactic acid monomer (LA) and the octoate catalyst of dried tert-butyl group protection tin according to mol ratioThe ratio of 50 ︰ 0.005 joins in round-bottomed flask, reacts 48 hours at 125 DEG C, adds methylene chloride to dissolve, in methyl alcoholPrecipitation, suction filtration, vacuum drying, obtains white powder polymer P LA50-NH-Boc。
(2) macromole evocating agent PLA50-NH2Synthesis step;
By step (1) resulting polymers PLA50-NH-Boc is dissolved in the middle of carrene, adds excessive trifluoroacetic acid (TFA),Stirring reaction 8 hours under room temperature, concentrated by rotary evaporation, precipitates in methyl alcohol, suction filtration, vacuum drying, repeats three times, obtains whiteToner powder macromole evocating agent PLA50-NH2。
(3) two kinds of bi-block copolymer PBLG75-b-PLA50And PBLG15-b-PLA50Synthesis step;
By step (2) gained macromole evocating agent PLA50-NH2With the NCA monomer of glutamic acid be 1 ︰ 75 according to mol ratio respectivelyRatio be dissolved in dry DMF, room temperature lower seal stirring reaction 48 hours, precipitates in ether, suction filtration, vacuum drying,Obtain white polymer PBLG75-b-PLA50。
By step (2) gained macromole evocating agent PLA50-NH2With the NCA monomer of glutamic acid be 1 ︰ 15 according to mol ratio respectivelyRatio be dissolved in dry DMF, room temperature lower seal stirring reaction 48 hours, precipitates in ether, suction filtration, vacuum drying,Obtain white polymer PBLG15-b-PLA50。
(4)FA-PBLG75-b-PLA50And DTPA-PBLG15-b-PLA50Synthesis step;
By step (3) resulting polymers PBLG75-b-PLA50With folic acid (FA) is dissolved in anhydrous DMSO, add a certain amount ofDCC and NHS, under room temperature lucifuge stir 24 hours, concentrated by rotary evaporation, precipitates in ether, suction filtration, vacuum drying, obtainsYellow polymer FA-PBLG75-b-PLA50。
By step (3) resulting polymers PBLG15-b-PLA50Be dissolved in anhydrous DMSO with DTPA, add a certain amount of DCCAnd NHS, under room temperature, stir 24 hours, revolve steaming, precipitation, suction filtration, vacuum drying, obtains white polymer DTPA-PBLG15-b-PLA50。
(5) amphipathic nature polyalcohol FA-PGA75-b-PLA50And DTPA-PGA15-b-PLA50Synthesis step;
By step (4) resulting polymers FA-PBLG75-b-PLA50Be dissolved in 33%HBr/CH3In COOH solution, stirring reaction 5Hour, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder FA-PGA75-b-PLA50。
By step (4) resulting polymers DTPA-PBLG15-b-PLA50Be dissolved in respectively 33%HBr/CH3In COOH solution, stirMix reaction 5 hours, in ether, precipitate, suction filtration, vacuum drying, obtains polymer powder DTPA-PGA15-b-PLA50。
(6) carry the preparation process of gadolinium polymer vesicle;
According to the concentration of the 3mg/ml polymer FA-PGA that just step (5) obtains75-b-PLA50And DTPA-PGA15-b-PLA50Be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip and to double DMF volume with the speed of 10d/minContaining the aqueous solution of a certain amount of gadolinium, DMF is removed in lucifuge dialysis, obtains light blue solution and is year gadolinium polymer vesicle solution.
The above-mentioned description to embodiment is can understand and use the present invention for ease of those skilled in the art. Be familiar withThe personnel of art technology obviously can easily make various amendments to these embodiment, and should General Principle described hereinUse in other embodiment and needn't pass through performing creative labour. Therefore, the invention is not restricted to above-described embodiment, art technologyPersonnel are according to announcement of the present invention, and not departing from improvement and the amendment that category of the present invention makes all should be in protection scope of the present inventionWithin.
Claims (11)
1. carry a gadolinium polymer vesicle, it is characterized in that: comprise by two kinds of polymer and be jointly self-assembled into and there is asymmetric membrane structurePolymer vesicle and be subject to the metal gadolinium of described polymer vesicle carrying, described two kinds of polymer be respectively FA ?PGAx‐b‐PβzWithε‐PGAy‐b‐Pβz, FA is folic acid group, the degree of polymerization that x and y are PGA and x > y, and ε is the network for metal gadolinium described in complexingMixture, P βzFor hydrophobic segment, FA ?PGAx‐b‐PβzIn PGAxForm described polymer vesicle outside dizzy layer ， ε ?PGAy‐b‐PβzIn PGAyForm the dizzy layer in inside of described polymer vesicle, the P β in described two kinds of polymerization single polymerization monomerszForm described polymer vesicleRete.
2. according to claim 1 year gadolinium polymer vesicle, is characterized in that: described hydrophobic segment is polycaprolactone, poly-threeOne in methyl carbonic and PLA; Or described complexing agent is diethyl pentetic acid and Isosorbide-5-Nitrae, 7,10 ?four nitrogenAssorted cyclododecane ?Isosorbide-5-Nitrae, the one in 7,10 ?tetrabasic carboxylic acids; Or the mol ratio of described polymer vesicle and described metal gadolinium is 1:(0.1～2)。
3. according to claim 1 year gadolinium polymer vesicle, is characterized in that: the particle diameter of described polymer vesicle is 60～350nm; Or the particle diameter of described year gadolinium polymer vesicle is 80～400nm; Or x is that 50～100, y is 10～50,Z is 30～60.
4. prepare the method for carrying gadolinium polymer vesicle as described in any one in claims 1 to 3, it is characterized in that: bagDraw together following steps:
(1)、Pβz?NH ?the synthesis step of Boc:
By the monoethanolamine of being protected by Boc, β and catalyst taking mol ratio as 1:(20～100): (0.005～0.05) reaction 12～48 hours, after processing, obtain P βz‐NH‐Boc；
(2)、Pβz‐NH2Synthesis step:
By the P β of step (1) gainedz?NH ?Boc be dissolved in organic solvent, add excessive trifluoroacetic acid, under room temperature, react 2～24 hours, after processing, obtain P βz‐NH2；
(3)、PBLGx‐b‐PβzAnd PBLGy‐b‐PβzSynthesis step:
By the P β of step (2) gainedz‐NH2The ratio that is 1 ︰ x according to mol ratio with the NCA monomer of glutamic acid is dissolved in anhydrousIn DMF, under room temperature, react 12～48 hours, after processing, obtain PBLGx‐b‐Pβz；
By the P β of step (2) gainedz‐NH2The ratio that is 1 ︰ y according to mol ratio with the NCA monomer of glutamic acid is dissolved in anhydrousIn DMF, under room temperature, react 12～48 hours, after processing, obtain PBLGy‐b‐Pβz；
(4)、FA‐PBLGx‐b‐PβzHe ε ?PBLGy‐b‐PβzSynthesis step:
By step (3) gained PBLGx‐b‐PβzBe dissolved in anhydrous DMSO with FA, add dicyclohexylcarbodiimide and N ?HOSu NHS, stirs under room temperature 5～48 hours, after processing, obtain FA ?PBLGx‐b‐Pβz；
By step (3) gained PBLGy‐b‐PβzBe dissolved in anhydrous DMSO with ε, add dicyclohexylcarbodiimide and N ?hydroxylBase succinimide, stirs under room temperature 5～48 hours, after processing, obtain ε ?PBLGy-b-Pβz；
(5)、FA‐PGAx-b-PβzHe ε ?PGAy-b-PβzSynthesis step
By step (4) gained FA ?PBLGx‐b‐PβzBe dissolved in 33% HBr/CH3In COOH solution, react 2～12 hours,After processing, obtain FA ?PGAx‐b‐Pβz；
By step (4) gained ε ?PBLGy‐b‐PβzBe dissolved in 33% HBr/CH3In COOH solution, react 2～12 hours,After processing, obtain ε ?PGAy‐b‐Pβz；
(6), carry the preparation process of gadolinium polymer vesicle:
FA step (5) being obtained according to the concentration of 1～30mg/ml ?PGAx‐b‐PβzHe ε ?PGAy‐b‐PβzBe dissolved in togetherIn DMF, lucifuge stirs and drips with the speed of 5～50d/min the gadolinium ion aqueous solution that doubles DMF volume, and dialysis is removedDMF, obtains carrying gadolinium polymer vesicle solution;
β is the one in caprolactone, trimethyl carbonic ester and lactic acid, the degree of polymerization that z is β.
5. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: in step (1), z is30～60。
6. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: in step (1), work as βDuring for caprolactone, z is 30 or 60; When β is trimethyl carbonic ester, z is 30; In the time that β is lactic acid, z is 50.
7. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: the catalyst in step (1)For stannous octoate; Or the organic solvent in step (2) is carrene.
8. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: in step (3), x is50～100, y is 10～50.
9. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: in step (3), work as βDuring for caprolactone, x be 70 or 100, y be 20 or 30; When β is trimethyl carbonic ester, x is that 80, y is 25; When β isWhen lactic acid, x is that 75, y is 15.
10. year gadolinium polymer vesicle as described in any one in claims 1 to 3 is at the carrier of the medicine for the preparation for the treatment of cancerOr application in pharmaceutical composition.
11. carry gadolinium polymer vesicle at the nuclear-magnetism for the preparation of target identification cancer cell as described in any one in claims 1 to 3Application in resonance developer.
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