Mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer and preparation method thereof with and the preparation method of polypeptide drug-loaded micelle solution
Technical field
The present invention relates to a kind of amphipathic nature block polymer, particularly a kind of mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.The preparation method who also relates to this mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.The preparation method who also relates to this mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Background technology
Contain hydrophilic segment and hydrophobic segment in the amphipathic nature block polymer molecule, similar to the small molecules tensio-active agent, this type of polymer molecule independently exists when lower concentration; After concentration increases to certain value, microphase-separated occurs in the aqueous solution under the promotions such as hydrophobic interaction, electrostatic interaction, hydrogen bond and Van der Waals force, self-assembly forms the copolymer micelle with spherical nucleocapsid structure, and its hydrophobic segment consists of the kernel of micella, and hydrophilic segment consists of shell.Amphipathic nature block polymer is because of advantages such as its good biocompatibility, biodegradable in vivo, and the carrier as the medicine parcel demonstrates good performance.In the aqueous solution, hydrophobic oil phase kernel can provide several hydrophobic microenvironments for poorly water soluble drugs, thereby medicine is played solublization.
The Chinese invention patent of document 1 " patent publication No. is CN 101829046A ", a kind of Amphipathilic block polymer micelle nano medicament carrying system and preparation method are disclosed, hydrophilic segment for the preparation of the amphipathic nature block polymer of micella comprises: polyoxyethylene glycol (PEG), polyoxyethylene (PEO) etc., wherein, security is widely used PEG in good biocompatibility and the body owing to it has.Hydrophobic segment comprises: polyester (such as poly(lactic acid) (PLA), poly(lactic acid)/oxyacetic acid (PLGA), polycaprolactone (PCL) etc.), the synthetic phospholipid such as phosphatidylethanolamine and phosphatidylcholine, oligomeric hydrophobic amino acid (such as polyhistidyl, polyphenylalanine etc.).Wherein, oligomeric hydrophobic amino acid has vast potential for future development as hydrophobic segment, because it can be degraded in vivo, degraded product is amino acid, can not produce and accumulate and toxic side effect.
Different from the combination of poorly water soluble drugs according to copolymer micelle, the medicine-carrying method of copolymer micelle mainly is divided three classes: physical method, chemical bonding and electrostatic interaction method.Wherein, physics mode is the major way that micella wraps up medicine.The kernel of micella is the combining site of poorly water soluble drugs, and the character of hydrophobic segment directly affects physico-chemical property and the drug loading of micella.According to the similar compatibility principle, when copolymer micelle hydrophobic segment during with the group similar to drug molecule, the physico-chemical property of hydrophobic segment and medicine approaches, consistency is better, micella hydrophobic cores this moment planar water insoluble drug to greatest extent, and can obtain the lower stabilising system of energy.Analyze and find, many typical poorly water soluble drugs, for example, nimodipine, taxol, Ibuprofen BP/EP, tamoxifen etc. all contain phenyl ring in their molecular structure.
The Chinese invention patent of document 2 " publication number is CN101284896A " discloses a kind of chitosan oligosaccharide-poly L-beta-indole alanine graft copolymer and homogeneous phase synthetic method thereof.L-Trp and triphosgene are reacted to get L-Trp ring inner-acid anhydride.Then the ethyl acetate solution with L-Trp ring inner-acid anhydride is added drop-wise in the oligochitosan aqueous solution, react to get the mixture of product and L-Trp and oligochitosan, filter residue is used tetrahydrofuran (THF), distilled water wash successively, remove remaining L-Trp and oligochitosan, vacuum-drying gets the canescence chitosan oligosaccharide-poly L-beta-indole alanine graft copolymer.Document 2 described synthetic employing solution methods need to be carried out subsequent reactions with the L-Trp activation again with triphosgene when synthetic, the preparation process contaminate environment, and reactions steps is many, condition is harsh.
Summary of the invention
In order to overcome existing preparation method when preparation gathers the tryptophane multipolymer, main reaction activates L-Trp first before occuring, reactivation process need to be used this highly toxic substance of Triphosgene, easily cause the deficiency of environmental pollution, the invention provides a kind of mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer and preparation method thereof, with the poly-tryptophane that contains phenyl ring and nitrogen heterocyclic as hydrophobic segment, utilize the stannous octoate catalyzed polymerization, L-Trp is connected with the amino poly glycol monomethyl ether of end by amido linkage, has obtained mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.Preparation process does not have environmental pollution.
The present invention also provides the preparation method of this mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.As solid support material, prepare mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution by emulsion process with mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
The technical solution adopted for the present invention to solve the technical problems is: a kind of mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer, and its structural formula is:
The preparation method of a kind of above-mentioned mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer is characterized in that adopting following step:
(a) be that the activation of 2000 or 5000 end methoxy poly (ethylene glycol) is the amino poly glycol monomethyl ether of end with molecular weight.
(b) the amino poly glycol monomethyl ether of the end that step (a) is obtained and L-Trp mixed in 1: 2 in mass ratio~1: 9, added sealed tube.
(c) take by weighing the inferior tin of 5 ‰ octoate catalyst that quality is the L-Trp quality, be configured to 0.02% dichloromethane solution, drip in the sealed tube, methylene dichloride is removed in decompression, treats the methylene dichloride volatilization fully, with the sealed tube sealing by fusing.
(d) sealed tube that sealing by fusing is good is put into vacuum drying oven, and at vacuum environment, 225 ℃~230 ℃ lower reaction 5~8h, the question response product is cooled to room temperature, obtains yellow to brown semi-solid mixture.
(e) yellow that step (d) is obtained leaves standstill 8~15min to brown semi-solid mixture dissolve with ethanol, treats that upper solution becomes clarification, takes out the desolventizing of supernatant liquid rotary evaporation, obtains the yellow solid powder.
(f) the yellow solid powder that step (e) is obtained dissolves with methylene dichloride, drips precipitation in ether, filters, and discards brown filter residue, and the desolventizing of filtrate rotary evaporation obtains yellow semi-solid material.
(g) the yellow semi-solid material acetone solution that step (f) is obtained, the rotary evaporation desolventizing obtains the yellow solid powder, i.e. mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
The preparation method of a kind of above-mentioned mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution is characterized in that adopting following step:
(h) took by weighing in mass ratio mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer and medicine in 4: 1~10: 1, be that the ratio of 1mg: 1ml is measured methylene dichloride in mono methoxy polyethylene glycol-poly-L-Trp and methylene dichloride, mono methoxy polyethylene glycol-poly-L-Trp and medicine dissolution in methylene dichloride, are obtained solution.
(i) measure ultrapure water, the volume ratio of ultrapure water and methylene dichloride is 5: 1~8: 1, the solution that under the rapid stirring step (h) is prepared is added drop-wise in the ultrapure water, 1/6s of rate of addition, get the O/W emulsion, dropwise and continue to stir 4~7 hours, until the organic solvent volatilization fully, obtain mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Described medicine is poorly water soluble drugs, namely nimodipine, taxol, Ibuprofen BP/EP or tamoxifen any.
The invention has the beneficial effects as follows: since the present invention with the poly-tryptophane that contains phenyl ring and nitrogen heterocyclic as hydrophobic segment, utilize the stannous octoate catalyzed polymerization, L-Trp is connected with the amino poly glycol monomethyl ether of end by amido linkage, has obtained mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.Reactivation process does not need to use Triphosgene, so preparation process does not have environmental pollution.
Below in conjunction with the drawings and specific embodiments the present invention is elaborated.
Description of drawings
Fig. 1 is the FT-IR spectrogram of the mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer of the inventive method preparation.
Fig. 2 is the mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer of the inventive method preparation
1The H-NMR spectrogram.
Fig. 3 is the TEM photo of the mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution of the inventive method preparation.
Embodiment
Embodiment 1: with molecular weight be the activation of 5000 end methoxy poly (ethylene glycol) for the amino poly glycol monomethyl ether of end, take by weighing 10mg, mix the adding sealed tube with the 25mg L-Trp.Take by weighing the 1.25mg stannous octoate, be configured to 0.02% dichloromethane solution, be added drop-wise in the sealed tube, methylene dichloride is removed in decompression, treats the methylene dichloride volatilization fully, with the sealed tube sealing by fusing, puts into vacuum drying oven.At vacuum environment, 225 ℃ of lower reaction 8h, the question response product is cooled to room temperature, use the hot ethanol lysate, leave standstill 15min, treat that upper solution becomes clarification, take out the desolventizing of supernatant liquid rotary evaporation, the yellow solid powder that obtains dissolves with methylene dichloride, remove byproduct of reaction after the precipitation in the ether, the desolventizing of filtrate rotary evaporation, the semi-solid material obtains the yellow solid powder with again rotary evaporation desolventizing of acetone solution, i.e. mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
Embodiment 2: with molecular weight be the activation of 2000 end methoxy poly (ethylene glycol) for the amino poly glycol monomethyl ether of end, take by weighing 10mg, mix the adding sealed tube with the 35mg L-Trp.Take by weighing the 1.75mg stannous octoate, be configured to 0.02% dichloromethane solution, be added drop-wise in the sealed tube, methylene dichloride is removed in decompression, treats the methylene dichloride volatilization fully, with the sealed tube sealing by fusing, puts into vacuum drying oven.At vacuum environment, 227 ℃ of lower reaction 7h, the question response product is cooled to room temperature, use the hot ethanol lysate, leave standstill 13min, treat that upper solution becomes clarification, take out the desolventizing of supernatant liquid rotary evaporation, the yellow solid powder that obtains dissolves with methylene dichloride, remove byproduct of reaction after the precipitation in the ether, the desolventizing of filtrate rotary evaporation, the semi-solid material obtains the yellow solid powder with again rotary evaporation desolventizing of acetone solution, i.e. mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
Embodiment 3: with molecular weight be the activation of 5000 end methoxy poly (ethylene glycol) for the amino poly glycol monomethyl ether of end, take by weighing 10mg, mix the adding sealed tube with the 90mg L-Trp.Take by weighing the 4.5mg stannous octoate, be configured to 0.02% dichloromethane solution, be added drop-wise in the sealed tube, methylene dichloride is removed in decompression, treats the methylene dichloride volatilization fully, with the sealed tube sealing by fusing, puts into vacuum drying oven.At vacuum environment, 228 ℃ of lower reaction 6h, the question response product is cooled to room temperature, use the hot ethanol lysate, leave standstill 10min, treat that upper solution becomes clarification, take out the desolventizing of supernatant liquid rotary evaporation, the yellow solid powder that obtains dissolves with methylene dichloride, remove byproduct of reaction after the precipitation in the ether, the desolventizing of filtrate rotary evaporation, the semi-solid material obtains the yellow solid powder with again rotary evaporation desolventizing of acetone solution, i.e. mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
Embodiment 4: with molecular weight be the activation of 2000 end methoxy poly (ethylene glycol) for the amino poly glycol monomethyl ether of end, take by weighing 10mg, mix the adding sealed tube with the 35mg L-Trp.Take by weighing the 1.75mg stannous octoate, be configured to 0.02% dichloromethane solution, be added drop-wise in the sealed tube, methylene dichloride is removed in decompression, treats the methylene dichloride volatilization fully, with the sealed tube sealing by fusing, puts into vacuum drying oven.At vacuum environment, 230 ℃ of lower reaction 5h, the question response product is cooled to room temperature, use the hot ethanol lysate, leave standstill 8min, treat that upper solution becomes clarification, take out the desolventizing of supernatant liquid rotary evaporation, the yellow solid powder that obtains dissolves with methylene dichloride, remove byproduct of reaction after the precipitation in the ether, the desolventizing of filtrate rotary evaporation, the semi-solid material obtains the yellow solid powder with again rotary evaporation desolventizing of acetone solution, i.e. mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer.
Embodiment 5: take by weighing mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer 8mg, taxol 2mg, dissolve with the 8ml methylene dichloride, this solution is added drop-wise in the 40ml ultrapure water, 1/6s of rate of addition, dropwise and continue to stir 7h, until the organic solvent volatilization fully, obtain mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Embodiment 6: take by weighing mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer 7mg, Ibuprofen BP/EP 1.6mg, dissolve with the 7ml methylene dichloride, this solution is added drop-wise in the 43ml ultrapure water, 1/6s of rate of addition, dropwise and continue to stir 6h, until the organic solvent volatilization fully, obtain mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Embodiment 7: take by weighing mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer 7mg, tamoxifen 1.6mg, dissolve with the 7ml methylene dichloride, this solution is added drop-wise in the 43ml ultrapure water, 1/6s of rate of addition, dropwise and continue to stir 5h, until the organic solvent volatilization fully, obtain mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Embodiment 8: take by weighing mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer 6mg, nimodipine 0.8mg, dissolve with the 6ml methylene dichloride, this solution is added drop-wise in the 45ml ultrapure water, 1/6s of rate of addition, dropwise and continue to stir 4h, until the organic solvent volatilization fully, obtain mono methoxy polyethylene glycol-poly-L-Trp amphipathic nature block polymer polypeptide drug-loaded micelle solution.
Adopt the KBr pressed disc method, measure with Tenson 27 FT-IR infrared spectrometers, the result as shown in Figure 1.The infrared spectra of the amino poly glycol monomethyl ether of curve 1 expression end, the infrared spectra of curve 2 expression mono methoxy polyethylene glycol-poly-L-Trps.3253cm
-1Be N-H stretching vibration, 2913cm
-1Be CH
2Stretching vibration, 1669cm
-1For acid amides absorbs I band, 1457cm
-1For acid amides absorbs II band, 1233cm
-1For acid amides absorbs the III band.Hold amino poly glycol monomethyl ether to compare with mono methoxy polyethylene glycol-poly-L-Trp, terminal amino group is at 1648cm
-1And 1561cm
-1The biabsorption peak disappear 3452cm
-1Absorption peak move to lower wave number.
Measure with AVANCF300MHZ superconduction fourier numeralization nuclear magnetic resonance spectrometer
1The H-NMR spectrogram, the result as shown in Figure 2.1.2ppm be-CH
3H, 1.8ppm is-CH
2On the NH-H of NH-, 3.7ppm be mono methoxy polyethylene glycol-CH
2H, 5.8~7.2ppm is the H on the tryptophane indole ring, 8.2ppm is the H on the amido linkage.
With the mono methoxy polyethylene glycol of JEM-2000EX transmission electron microscope (TEM) observation post preparation-poly-L-Trp amphipathic nature block polymer carrier micelle pattern.The polypeptide drug-loaded micelle solution of preparation is stained with on copper mesh, treat online drop to do and when not dried, drip 2% Salkowski's solution and carry out negative staining, tweezer plays copper mesh after for some time, draw unnecessary dye liquor with filter paper, copper mesh is seasoning at room temperature, then observes at transmission electron microscope, and the acceleration voltage of transmission electron microscope is 100KV.As shown in Figure 3, observe transmission electron microscope as can be known, mono methoxy polyethylene glycol-poly-L-Trp block polymer forms spherical micelle, particle diameter 60~100nm.