CN104013967A - Gadolinium-loaded polymer vesicle and preparation method and application thereof - Google Patents
Gadolinium-loaded polymer vesicle and preparation method and application thereof Download PDFInfo
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- CN104013967A CN104013967A CN201410172409.1A CN201410172409A CN104013967A CN 104013967 A CN104013967 A CN 104013967A CN 201410172409 A CN201410172409 A CN 201410172409A CN 104013967 A CN104013967 A CN 104013967A
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Abstract
The invention provides a gadolinium-loaded polymer vesicle and a preparation method and application thereof. The gadolinium-loaded polymer vesicle comprises a polymer vesicle body and metal gadolinium borne by the polymer vesicle, wherein the polymer vesicle is in a structure which has asymmetric membranes and is formed by self-assembling two polymers together; the two polymers are respectively FA-PGAx-b-Pbetaz and epsilon-PGAy-b-Pbetaz; the metal gadolinium is complexed to epsilon. The preparation method comprises the following steps: respectively synthesizing two polymers, complexing the metal gadolinium to epsilon, and finally generating the gadolinium-loaded polymer vesicle. The gadolinium-loaded polymer vesicle is stable in structure, has high biocompatibility and biodegradability, has high targeting property on cancer cells, can serve as a nuclear magnetic resonance developing agent for performing targeted recognition on the cancer cells, also can be coated with hydrophilic or hydrophobic drugs and can perform controlled drug delivery on a targeted part.
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, about Amphipathilic block polymer, in aqueous solution, carry out self assembly formation vesicle or micelle and as the research of pharmaceutical carrier, enjoy the concern of vast research worker.Amphipathilic block polymer can carry out self assembly and form vesicle duplicature in water, but the less stable of the liposome class vesicle of extensive use at present is easily broken and produces the intermembranous fusion of vesicle.And polymer vesicle can significantly improve the mechanical performance of vesicle, the focus that the biodegradable polymers vesicle being comprised of polycarbonate-based and poltpeptides is studied especially.
In order to improve the targeting of pharmaceutical carrier, carrier surface often connects some targeting groups.Folic acid (Folic acid, FA) essence is a kind of micromolecule vitamin, claims to talk endlessly again phthalein glutamic acid, and it had both participated in the pyrimidine of cell DNA and RNA prerequisite and synthesizing of purine nucleotides, participated in again the synthetic of methionine.The research of folacin receptor shows, the tumor of the many epithelial origins of the mankind, as activity and the quantity of the folacin receptor of the tumor cell surfaces such as ovarian cancer, cervical cancer, breast carcinoma, nasopharyngeal carcinoma, hepatocarcinoma are significantly higher than normal tissue cell, is overexpression.And normal zooblast lacks the synthetic key enzyme of folic acid biological, folic acid specific bond albumen that can only dependent cells surface (is folacin receptor, Folate receptors, FR) initiatively absorb exogenous folic acid and maintain normal vital movement, thereby expression is extremely low.Therefore,, according to the differential expression of normal cell and cancerous cell surface folacin receptor, can prepare the polymer vesicle with folate-targeted group, to improve the identification ability of polymer vesicle to cancerous cell.
Nuclear magnetic resonance (MRI) technology first Application in 1973 is in human diagnosis, and through the development of four more than ten years, this technology is developed rapidly and extensive use in fields such as biology, medical science.Compare with X-CT, ultrasonic imaging, MRI technology has many outstanding advantages: without ionizing radiation; Can realize the imaging of multinuclear multiparameter; Can implement the scanning of arbitrary orientation aspect; Higher spatial resolution and contrast; Without sclerotin artifact; Can reflection be detected tissue water proton surrounding and obtain relevant Physiology and biochemistry information; Can improve diagnosis of heart, trunk morphology and function etc.So MRI technology has become one of detection means the strongest in current clinical diagnosis.This technology has been widely used in the radiography of head, nervous system, abdominal part and the blood vessel of human body, effective to detecting tissue necrosis, ischemia and various malignant change, is used in particular for the early diagnosis of tumor, can make to treat success rate and have significant improvement.Clinically, for guaranteeing the accuracy of diagnosis, more than 45% MRI checks and need to use contrast agent to improve picture contrast, wherein more than 30% uses containing gadolinium contrast agent.Yet, through repeatedly clinical research discovery, free metal gadolinium ion has very strong toxic and side effects, can cause the serious fibrosis of kidney, the gadolinium contrast agent using at present clinically is all prepared from after chelating agent effect, DTPA-Gd for example, DOTA-Gd, gadodiamide (Gadodiamide), Magnevist Solution (Gadopcntetate dimeglumine), Gadobenate Dimeglumine (Gadobenate dimeglemine), gadoteridol (Gadoteridol), gadoterlc acid meglumine saltlniection (Gadoterate meglumine) etc., but the gadolinium contrast agent that these use clinically still exists sensitivity not high, biocompatibility is poor, be difficult for degraded, the problems such as the low and imaging effect of carrying drug ratio is poor.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, main purpose is to provide a kind of and has no side effect, carrying drug ratio is high, imaging effect good, easily degraded and good biocompatibility carries 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 year gadolinium polymer vesicle, comprises by two kinds of polymer and is jointly self-assembled into the metal gadolinium that has the polymer vesicle of asymmetric membrane structure and be subject to polymer vesicle carrying, two kinds of polymer are respectively FA-PGA
x-b-P β
zand ε-PGA
y-b-P β
z, FA is folic acid group, the degree of polymerization that x and y are PGA and x > y, and ε is the chelating agent for complexing metal gadolinium, P β
zfor hydrophobic segment, FA-PGA
x-b-P β
zin PGA
xform the dizzy layer in outside of polymer vesicle, ε-PGA
y-b-P β
zin PGA
yform the dizzy layer in inside of polymer vesicle, the P β in two kinds of polymer
zform the rete of polymer vesicle.
Above-mentioned hydrophobic segment is a kind of in polycaprolactone, polytrimethylene carbonate and polylactic acid; Or chelating agent is diethyl pentetic acid and Isosorbide-5-Nitrae, 7,10-tetraazacyclododecanand-Isosorbide-5-Nitrae, a kind of in 7,10-tetrabasic carboxylic acid; Or the mol ratio of polymer vesicle and metal gadolinium is 1:(0.1～2).The particle diameter of above-mentioned polymer vesicle is 60～350nm; Or the particle diameter that carries gadolinium polymer vesicle is 80～400nm.
A method of preparing above-mentioned year gadolinium polymer vesicle, comprises the steps:
(1), P β
zthe synthesis step of-NH-Boc:
The ethanolamine of being protected by Boc, β and catalyst be take to mol ratio as 1:(20～100): (0.005～0.05) reaction 12～48 hours, obtains P β after processing
z-NH-Boc;
(2), P β
z-NH
2synthesis step:
By the P β of step (1) gained
z-NH-Boc is dissolved in organic solvent, adds excessive trifluoroacetic acid, reacts 2～24 hours under room temperature, obtains P β after processing
z-NH
2;
(3), PBLG
x-b-P β
zand PBLG
ythe synthesis step of-b-P β z:
By the P β of step (2) gained
z-NH
2the ratio that is 1 ︰ x according to mol ratio with the NCA monomer of glutamic acid is dissolved in dry DMF, reacts 12～48 hours under room temperature, obtains PBLG after processing
x-b-P β
z;
By the P β of step (2) gained
z-NH
2the ratio that is 1 ︰ y according to mol ratio with the NCA monomer of glutamic acid is dissolved in dry DMF, reacts 12～48 hours under room temperature, obtains PBLG after processing
y-b-P β
z;
(4), FA-PBLG
x-b-P β
zand ε-PBLG
y-b-P β
zsynthesis step:
By step (3) gained PBLG
x-b-P β
zbe dissolved in anhydrous DMSO with FA, add dicyclohexylcarbodiimide and N-hydroxy-succinamide, under room temperature, stir 5～48 hours, after processing, obtain FA-PBLG
x-b-P β
z.
By step (3) gained PBLG
y-b-P β
zbe dissolved in anhydrous DMSO with ε, add dicyclohexylcarbodiimide and N-hydroxy-succinamide, under room temperature, stir 5～48 hours, after processing, obtain ε-PBLG
y-b-P β
z.
(5), FA-PGA
x-b-P β
zand ε-PGA
y-b-P β
zsynthesis step:
By step (4) gained FA-PBLG
x-b-P β
zbe dissolved in 33% HBr/CH
3in COOH solution, react 2～12 hours, after processing, obtain FA-PGA
x-b-P β
z;
By step (4) gained ε-PBLG
y-b-P β z is dissolved in 33% HBr/CH
3in COOH solution, react 2～12 hours, after processing, obtain ε-PGA
y-b-P β
z;
(6), carry the preparation process of gadolinium polymer vesicle:
FA-PGA step (5) being obtained according to the concentration of 1～30mg/ml
x-b-P β
zand ε-PGA
y-b-P β
zbe dissolved in together in DMF, lucifuge stirs and doubles with the speed dropping of 5～50d/min the gadolinium ion aqueous solution of DMF volume, and DMF is removed in dialysis, obtains carrying gadolinium polymer vesicle solution;
β is a kind of 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, when β is caprolactone, z is 30 or 60; When β is trimethyl carbonic ester, z is 30; When β is lactic acid, z is 50.Catalyst in step (1) is stannous octoate; Or the organic solvent in step (2) is dichloromethane.
In above-mentioned steps (3), x is that 50～100, y is 10～50; Preferably, when β is 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 is lactic acid, x is that 75, y is 15.
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 targeting identification cancerous cell.
Owing to adopting such scheme, the invention has the beneficial effects as follows:
First, because wherein on a kind of polymer with folate-targeted group, when a plurality of polymer formation carry gadolinium polymer vesicle, folate-targeted group is positioned on the outside dizzy layer of vesicle, and folate-targeted group has higher affinity to cancerous cell surface folacin receptor, so carry gadolinium polymer vesicle, can be enriched in the tumor locus that is rich in cancerous cell; While being filled with the medicine for the treatment of cancer in carrying gadolinium polymer vesicle, it has good cancerous cell targeting as pharmaceutical carrier, can carry out target administration to cancerous cell, folacin receptor on folate-targeted group and cancerous cell surface carries out after specific binding, can greatly improve the speed that medicine enters cancerous cell, thereby improve the inhibition of cancerous cell or killing effect.
Second, because when a plurality of polymer formation carry gadolinium polymer vesicle, the end of this year inner dizzy layer of gadolinium polymer vesicle has chelating agent, this chelating agent and the complexation of metal gadolinium phase, so this year gadolinium polymer vesicle clinically can be simultaneously used as the contrast agent of nuclear magnetic resonance, NMR, and higher than the sensitivity of the current DTPA-Gd using clinically.
The 3rd, when a year gadolinium polymer vesicle wraps up hydrophilic anticarcinogen DOXHCl, carry gadolinium polymer vesicle dizzy layer hydrophilic segment side chain carboxyl group and DOXHCl and have electrostatic interaction, make its carrying drug ratio can reach 40%～60%, and the polymer vesicle of current techniques only has 20%～30% to the carrying drug ratio of DOXHCl, so 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, bag due to vesicle carries effect simultaneously, greatly reduce the toxic and side effects of metal gadolinium to human body, improved the safety of medication and development.
The 5th, carry gadolinium polymer vesicle and have two kinds of polymer, the segment ratio of these two kinds of polymer is different, therefore, the self assembling process of polymer makes to carry the structure that gadolinium polymer vesicle becomes asymmetric membrane, conveniently dizzy layer inside and outside this year gadolinium polymer vesicle is carried out to functionalized design.
Accompanying drawing explanation
Fig. 1 is the DLS result figure that carries gadolinium polymer mixed vesicle of the embodiment of the present invention one gained.
Fig. 2 is the TEM result figure that carries gadolinium polymer mixed vesicle of the embodiment of the present invention one gained.
Fig. 3 is the relaxation rate r that carries gadolinium polymer mixed vesicle of the embodiment of the present invention one gained
1test result figure.
The specific embodiment
Below in conjunction with accompanying drawing illustrated embodiment, the present invention is further illustrated.
Embodiment mono-
The present embodiment provides a kind of year gadolinium polymer vesicle, by polymer vesicle and formed by the metal gadolinium of this polymer vesicle carrying.
Polymer vesicle is formed by two kinds of common self assemblies of polymer, has asymmetric membrane structure.These two kinds of polymer are respectively FA-PGA
x-b-P β
zand ε-PGA
y-b-P β
z.Wherein, FA is folic acid group, as the targeting group of cancerous cell surface folacin receptor, works.PGA represents polyglutamic acid (Polyglutamic Acid, PGA), and it works as hydrophilic segment; X and y are the degree of polymerization and the x > y of PGA, and x can be 10～50 for 50～100, y.ε is the chelating agent for complexing metal gadolinium, can be diethyl pentetic acid (Diethylene Triamine Pentacetate Acid, DTPA) and 1,4,7,10-tetraazacyclododecanand-1, any one in 4,7,10-tetrabasic carboxylic acid (DOTA).P β
zfor hydrophobic segment, z represents the degree of polymerization of this hydrophobic segment, and z can be 30～60.When a plurality of polymer formation polymer vesicle, polymer FA-PGA
x-b-P β
zin PGA
xform the dizzy layer in outside of polymer vesicle, polymer ε-PGA
y-b-P β
zin PGA
yform the dizzy layer in inside of polymer vesicle, the P β in two kinds of polymer
zform the rete of polymer vesicle.
The complexation of metal gadolinium is on ε.In carrying 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 β
z(be PCL
z) degree of polymerization z be preferably 30.At polymer FA-PGA
x-b-P β
zin, PGA
xdegree of polymerization x be preferably 70; At polymer ε-PGA
y-b-P β
zin, PGA
ydegree of polymerization y be preferably 20.Chelating agent ε is preferably DTPA.Now, the structural formula of two kinds of polymer is respectively: FA-PGA
70-b-PCL
30and DTPA-PGA
20-b-PCL
30.
The preparation method of carrying gadolinium polymer vesicle in the present embodiment comprises the steps:
(1) PCL
30the synthesis step of-NH-Boc;
The ratio that is 1 ︰ 30 ︰ 0.005 according to mol ratio by the sub-stannum of the ethanolamine of the dried tert-butyl group (Boc) protection, caprolactone monomer (CL) and octoate catalyst joins in round-bottomed flask; at 110 ℃, react 48 hours; then add methylene chloride to dissolve; in methanol, precipitate; sucking filtration; vacuum drying, obtains white powder polymer PC L
30-NH-Boc.
(2) macromole evocating agent PCL
30-NH
2synthesis step;
By step (1) resulting polymers PCL
30-NH-Boc is dissolved in the middle of organic solvent dichloromethane, adds excessive trifluoroacetic acid (TFA), and under room temperature, stirring reaction is 12 hours, concentrated by rotary evaporation, precipitates in methanol, sucking filtration, vacuum drying, repetitive operation three times, obtains white powder macromole evocating agent PCL
30-NH
2.
(3) two kinds of bi-block copolymer PBLG
70-b-PCL
30and PBLG
20-b-PCL
30synthesis step;
By step (2) gained macromole evocating agent PCL
30-NH
2the ratio that is 1 ︰ 70 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, in ether, precipitates, and sucking filtration, vacuum drying, obtains white polymer PBLG
70-b-PCL
30.B represents block, i.e. segment.
By step (2) gained macromole evocating agent PCL
30-NH
2the ratio that is 1 ︰ 20 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, in ether, precipitates, and sucking filtration, vacuum drying, obtains white polymer PBLG
20-b-PCL
30.
(4) FA-PBLG
70-b-PCL
30and DTPA-PBLG
20-b-PCL
30synthesis step;
By step (3) resulting polymers PBLG
70-b-PCL
30with folic acid (FA) is dissolved in anhydrous DMSO, add a certain amount of dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS), under room temperature, stir 24 hours, concentrated by rotary evaporation, precipitates in ether, sucking filtration, vacuum drying, obtains yellow polymer FA-PBLG
70-b-PCL
30.
By step (3) resulting polymers PBLG
20-b-PCL
30be dissolved in anhydrous DMSO with DTPA, add a certain amount of dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS), under room temperature, stir 24 hours, revolve steaming, precipitation, sucking filtration, vacuum drying, obtains white polymer DTPA-PBLG
20-b-PCL
30.
(5) amphipathic nature polyalcohol FA-PGA
70-b-PCL
30and DTPA-PGA
20-b-PCL
30synthesis step;
By step (4) gained FA-PBLG
70-b-PCL
30be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder FA-PGA
70-b-PCL
30.
By step (4) gained DTPA-PBLG
20-b-PCL
30be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder DTPA-PGA
20-b-PCL
30.
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml
70-b-PCL
30and DTPA-PGA
20-b-PCL
30be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip the aqueous solution of the gadolinium ion that doubles DMF volume with the speed of 10d/min, 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.The TME result of this year gadolinium polymer vesicle solution as shown in Figure 2.The relaxation rate r of this year gadolinium polymer vesicle solution
1test 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 targeting identification cancerous cell, can also being used for for the preparation of carrier or the pharmaceutical composition for the treatment of the medicine of cancer.
The gadolinium polymer vesicle that carries in the present embodiment is easily degraded under the effect of protease or lipase, so good biocompatibility, to people's body, can not produce toxicological effect.
Embodiment bis-
In embodiment mono-, two kinds of polymer that carry in gadolinium polymer vesicle are FA-PGA
70-b-PCL
30and DTPA-PGA
20-b-PCL
30.In fact, the Amphipathilic block polymer carrying in gadolinium polymer vesicle can also be FA-PGA
100-b-PCL
60and DTPA-PGA
30-b-PCL
60.Now, FA-PGA
100-b-PCL
60the degree of polymerization x of middle GA is that the degree of polymerization z of 100, CL is 60, DTPA-PGA
30-b-PCL
60the 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) PCL
60the synthesis step of-NH-Boc;
The ratio that is 1 ︰ 60 ︰ 0.005 according to mol ratio by the sub-stannum of ethanolamine, caprolactone monomer (CL) and the octoate catalyst of dried tert-butyl group protection joins in round-bottomed flask; at 110 ℃, react 48 hours; add methylene chloride to dissolve; in methanol, precipitate; sucking filtration; vacuum drying, obtains white powder polymer PC L
60-NH-Boc.
(2) macromole evocating agent PCL
60-NH
2synthesis step;
By step (1) resulting polymers PCL
60-NH-Boc is dissolved in the middle of dichloromethane, adds excessive trifluoroacetic acid, and under room temperature, stirring reaction is 8 hours, and concentrated by rotary evaporation, precipitates in methanol, sucking filtration, and vacuum drying, repetitive operation three times, obtains white powder macromole evocating agent PCL
60-NH
2.
(3) two kinds of bi-block copolymer PBLG
100-b-PCL
60and PBLG
30-b-PCL
60synthesis step;
By step (2) gained macromole evocating agent PCL
60-NH
2the ratio that is 1 ︰ 100 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains white polymer PBLG
100-b-PCL
60.
By step (2) gained macromole evocating agent PCL
60-NH
2the ratio that is 1 ︰ 30 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains white polymer PBLG
30-b-PCL
60.
(4) FA-PBLG
100-b-PCL
60and DTPA-PBLG
30-b-PCL
60synthesis step;
By step (3) resulting polymers PBLG
100-b-PCL
60be dissolved in anhydrous DMSO with folic acid, add a certain amount of DCC and NHS, stir 24 hours under room temperature, concentrated by rotary evaporation, precipitates in ether, sucking filtration, and vacuum drying, obtains yellow polymer FA-PBLG
100-b-PCL
60.
By step (3) resulting polymers PBLG
30-b-PCL
60be dissolved in anhydrous DMSO with DTPA, add a certain amount of dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS), under room temperature, stir 24 hours, revolve steaming, precipitation, sucking filtration, vacuum drying, obtains white polymer DTPA-PBLG
30-b-PCL
60.
(5) amphipathic nature polyalcohol FA-PGA
100-b-PCL
60and DTPA-PGA
30-b-PCL
60synthesis step;
By step (4) resulting polymers FA-PBLG
100-b-PCL
60be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder FA-PGA
100-b-PCL
60.
By step (4) resulting polymers DTPA-PBLG
30-b-PCL
60be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder DTPA-PGA
30-b-PCL
60.
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml
100-b-PCL
60and DTPA-PGA
30-b-PCL
60be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip the aqueous solution of the gadolinium ion that doubles DMF volume with the speed of 10d/min, 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, and 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-
Compare with embodiment mono-, in carrying the polymer of gadolinium polymer vesicle, P β
zcan also be polytrimethylene carbonate (PTMC).When a plurality of polymer formation 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) PTMC
30the synthesis step of-NH-Boc;
The ratio that is 1 ︰ 30 ︰ 0.005 according to mol ratio by the sub-stannum of ethanolamine, TMC monomer and the octoate catalyst of dried tert-butyl group protection joins in round-bottomed flask; at 110 ℃, react 40 hours; add methylene chloride to dissolve; in methanol, precipitate; sucking filtration; vacuum drying, obtains white powder polymer P TMC
30-NH-Boc.
(2) macromole evocating agent PTMC
30-NH
2synthesis step;
By step (1) resulting polymers PTMC
30-NH-Boc is dissolved in dichloromethane, adds excessive trifluoroacetic acid, and under room temperature, stirring reaction is 8 hours, and concentrated by rotary evaporation, precipitates in methanol, sucking filtration, and vacuum drying, repetitive operation three times, obtains white powder macromole evocating agent PTMC
30-NH
2.
(3) two kinds of bi-block copolymer PBLG
80-b-PTMC
30and PBLG
25-b-PTMC
30synthesis step;
By step (2) gained macromole evocating agent PTMC
30-NH
2the ratio that is 1 ︰ 80 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains white polymer PBLG
80-b-PTMC
30.
By step (2) gained macromole evocating agent PTMC
30-NH
2the ratio that is 1 ︰ 25 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, in room temperature lower seal stirring reaction 48 hours, in ether, precipitates, and sucking filtration, vacuum drying, obtains white polymer PBLG
25-b-PTMC
30.
(4) FA-PBLG
80-b-PTMC
30and DTPA-PBLG
25-b-PTMC
30synthesis step;
By step (3) resulting polymers PBLG
80-b-PTMC
30be dissolved in anhydrous DMSO with folic acid, add a certain amount of DCC and NHS, stir 24 hours under room temperature, concentrated by rotary evaporation, precipitates in ether, sucking filtration, and vacuum drying, obtains yellow polymer FA-PBLG
80-b-PTMC
30.
By step (3) resulting polymers PBLG
25-b-PTMC
30be dissolved in anhydrous DMSO with DTPA, add a certain amount of dicyclohexylcarbodiimide and N-hydroxy-succinamide, under room temperature, stir 24 hours, revolve steaming, precipitation, sucking filtration, vacuum drying, obtains white polymer DTPA-PBLG
25-b-PTMC
30.
(5) amphipathic nature polyalcohol FA-PGA
80-b-PTMC
30and DTPA-PGA
25-b-PTMC
30synthesis step;
By step (4) resulting polymers FA-PBLG
80-b-PTMC
30be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder FA-PGA
80-b-PTMC
30.
By step (4) resulting polymers DTPA-PBLG
25-b-PTMC
30be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder DTPA-PGA
25-b-PTMC
30.
(6) carry the preparation process of gadolinium polymer vesicle;
Polymer FA-PGA step (5) being obtained according to the concentration of 3mg/ml
80-b-PTMC
30and DTPA-PGA
25-b-PTMC
30be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, drip the aqueous solution of the gadolinium ion that doubles DMF volume with the speed of 10d/min, DMF is removed in lucifuge dialysis, obtains light blue solution and is year gadolinium polymer vesicle solution.
Embodiment tetra-
Compare with embodiment mono-, in carrying the polymer of gadolinium polymer vesicle, P β
zcan also be polylactic acid (PLA).When a plurality of polymer formation 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) PLA
50the synthesis step of-NH-Boc;
The ratio that is 1 ︰ 50 ︰ 0.005 according to mol ratio by the sub-stannum of ethanolamine, lactic acid monomer (LA) and the octoate catalyst of dried tert-butyl group protection joins in round-bottomed flask; at 125 ℃, react 48 hours; add methylene chloride to dissolve; in methanol, precipitate; sucking filtration; vacuum drying, obtains white powder polymer P LA
50-NH-Boc.
(2) macromole evocating agent PLA
50-NH
2synthesis step;
By step (1) resulting polymers PLA
50-NH-Boc is dissolved in the middle of dichloromethane, adds excessive trifluoroacetic acid (TFA), and under room temperature, stirring reaction is 8 hours, and concentrated by rotary evaporation, precipitates in methanol, sucking filtration, and vacuum drying, repetitive operation three times, obtains white powder macromole evocating agent PLA
50-NH
2.
(3) two kinds of bi-block copolymer PBLG
75-b-PLA
50and PBLG
15-b-PLA
50synthesis step;
By step (2) gained macromole evocating agent PLA
50-NH
2the ratio that is 1 ︰ 75 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains white polymer PBLG
75-b-PLA
50.
By step (2) gained macromole evocating agent PLA
50-NH
2the ratio that is 1 ︰ 15 according to mol ratio respectively with the NCA monomer of glutamic acid is dissolved in dry DMF, and room temperature lower seal stirring reaction 48 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains white polymer PBLG
15-b-PLA
50.
(4) FA-PBLG
75-b-PLA
50and DTPA-PBLG
15-b-PLA
50synthesis step;
By step (3) resulting polymers PBLG
75-b-PLA
50with folic acid (FA) is dissolved in anhydrous DMSO, add a certain amount of DCC and NHS, under room temperature, lucifuge stirs 24 hours, and concentrated by rotary evaporation, precipitates in ether, sucking filtration, vacuum drying, obtains yellow polymer FA-PBLG
75-b-PLA
50.
By step (3) resulting polymers PBLG
15-b-PLA
50be dissolved in anhydrous DMSO with DTPA, add a certain amount of DCC and NHS, under room temperature, stir 24 hours, revolve steaming, precipitation, sucking filtration, vacuum drying, obtains white polymer DTPA-PBLG
15-b-PLA
50.
(5) amphipathic nature polyalcohol FA-PGA
75-b-PLA
50and DTPA-PGA
15-b-PLA
50synthesis step;
By step (4) resulting polymers FA-PBLG
75-b-PLA
50be dissolved in 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder FA-PGA
75-b-PLA
50.
By step (4) resulting polymers DTPA-PBLG
15-b-PLA
50be dissolved in respectively 33%HBr/CH
3in COOH solution, stirring reaction 5 hours, precipitates in ether, sucking filtration, and vacuum drying, obtains polymer powder DTPA-PGA
15-b-PLA
50.
(6) carry the preparation process of gadolinium polymer vesicle;
According to the concentration of the 3mg/ml polymer FA-PGA that just step (5) obtains
75-b-PLA
50and DTPA-PGA
15-b-PLA
50be dissolved in together in DMF, under the condition of lucifuge vigorous stirring, double the aqueous solution containing a certain amount of gadolinium of DMF volume with the speed dropping of 10d/min, 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.Person skilled in the art obviously can easily make various modifications to these embodiment, and General Principle described herein is applied in other embodiment and needn't passes through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art are according to announcement of the present invention, and not departing from the improvement that category of the present invention makes and revise all should be within protection scope of the present invention.
Claims (9)
1. carry a gadolinium polymer vesicle, it is characterized in that: comprise by two kinds of polymer and be jointly self-assembled into the metal gadolinium that has the polymer vesicle of asymmetric membrane structure and be subject to described polymer vesicle carrying, described two kinds of polymer are respectively FA-PGA
x-b-P β
zand ε-PGA
y-b-P β
z, FA is folic acid group, the degree of polymerization that x and y are PGA and x > y, and ε is the chelating agent for metal gadolinium described in complexation, P β
zfor hydrophobic segment, FA-PGA
x-b-P β
zin PGA
xform the dizzy layer in outside of described polymer vesicle, ε-PGA
y-b-P β
zin PGA
ythe dizzy layer in inside that forms described polymer vesicle, the P β z in described two kinds of polymerization single polymerization monomers forms the rete of described polymer vesicle.
2. according to claim 1 year gadolinium polymer vesicle, is characterized in that: described hydrophobic segment is a kind of in polycaprolactone, polytrimethylene carbonate and polylactic acid; Or described chelating agent is diethyl pentetic acid and Isosorbide-5-Nitrae, 7,10-tetraazacyclododecanand-Isosorbide-5-Nitrae, a kind of in 7,10-tetrabasic carboxylic acid; 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 that 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: comprise the steps:
(1), P β
zthe synthesis step of-NH-Boc:
The ethanolamine of being protected by Boc, β and catalyst be take to mol ratio as 1:(20～100): (0.005～0.05) reaction 12～48 hours, obtains P β after processing
z-NH-Boc;
(2), P β
z-NH
2synthesis step:
By the P β of step (1) gained
z-NH-Boc is dissolved in organic solvent, adds excessive trifluoroacetic acid, reacts 2～24 hours under room temperature, obtains P β after processing
z-NH
2;
(3), PBLG
x-b-P β
zand PBLG
ythe synthesis step of-b-P β z:
By the P β of step (2) gained
z-NH
2the ratio that is 1 ︰ x according to mol ratio with the NCA monomer of glutamic acid is dissolved in dry DMF, reacts 12～48 hours under room temperature, obtains PBLG after processing
x-b-P β
z;
By the P β of step (2) gained
z-NH
2the ratio that is 1 ︰ y according to mol ratio with the NCA monomer of glutamic acid is dissolved in dry DMF, reacts 12～48 hours under room temperature, obtains PBLG after processing
y-b-P β
z;
(4), FA-PBLG
x-b-P β
zand ε-PBLG
ythe synthesis step of-b-P β z:
By step (3) gained PBLG
x-b-P β
zbe dissolved in anhydrous DMSO with FA, add dicyclohexylcarbodiimide and N-hydroxy-succinamide, under room temperature, stir 5～48 hours, after processing, obtain FA-PBLG
x-b-P β
z;
By step (3) gained PBLG
y-b-P β
zbe dissolved in anhydrous DMSO with ε, add dicyclohexylcarbodiimide and N-hydroxy-succinamide, under room temperature, stir 5～48 hours, after processing, obtain ε-PBLG
y-b-P β
z;
(5), FA-PGA
x-b-P β
zand ε-PGA
y-b-P β
zsynthesis step
By step (4) gained FA-PBLG
x-b-P β
zbe dissolved in 33% HBr/CH
3in COOH solution, react 2～12 hours, after processing, obtain FA-PGA
x-b-P β
z;
By step (4) gained ε-PBLG
y-b-P β z is dissolved in 33% HBr/CH
3in COOH solution, react 2～12 hours, after processing, obtain ε-PGA
y-b-P β
z;
(6), carry the preparation process of gadolinium polymer vesicle:
FA-PGA step (5) being obtained according to the concentration of 1～30mg/ml
x-b-P β
zand ε-PGA
y-b-P β
zbe dissolved in together in DMF, lucifuge stirs and doubles with the speed dropping of 5～50d/min the gadolinium ion aqueous solution of DMF volume, and DMF is removed in dialysis, obtains carrying gadolinium polymer vesicle solution;
β is a kind of 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 is 30～60;
Preferably, when β is caprolactone, z is 30 or 60; When β is trimethyl carbonic ester, z is 30; When β is lactic acid, z is 50.
6. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: the catalyst in step (1) is stannous octoate; Or the organic solvent in step (2) is dichloromethane.
7. the method for gadolinium polymer vesicle is carried in preparation according to claim 4, it is characterized in that: in step (3), x is that 50～100, y is 10～50; Preferably, when β is 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 is lactic acid, x is that 75, y is 15.
8. year gadolinium polymer vesicle as described in any one in claims 1 to 3 is in the carrier of the medicine for the preparation for the treatment of cancer or the application in pharmaceutical composition.
9. the application of year gadolinium polymer vesicle as described in any one in claims 1 to 3 in the NMRI agent for the preparation of targeting identification cancerous cell.
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| CN111333835A (en) * | 2020-03-06 | 2020-06-26 | 同济大学 | Bone-targeting polymer, bone-targeting polymer vesicle and preparation method and application thereof |
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| WO2008152490A2 (en) * | 2007-06-14 | 2008-12-18 | Universität Basel | Loaded target-specific vesicles, method of producing the vesicles and their use in medical imaging |
| CN102573814A (en) * | 2009-06-26 | 2012-07-11 | 上海交通大学 | Polymer vesicles of asymmetric membrane |
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| WO2008152490A2 (en) * | 2007-06-14 | 2008-12-18 | Universität Basel | Loaded target-specific vesicles, method of producing the vesicles and their use in medical imaging |
| CN102573814A (en) * | 2009-06-26 | 2012-07-11 | 上海交通大学 | Polymer vesicles of asymmetric membrane |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111333835A (en) * | 2020-03-06 | 2020-06-26 | 同济大学 | Bone-targeting polymer, bone-targeting polymer vesicle and preparation method and application thereof |
| CN111333835B (en) * | 2020-03-06 | 2021-11-05 | 同济大学 | Bone-targeting polymer, bone-targeting polymer vesicle and preparation method and application thereof |
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