Background technology
Cancer has become the main disease of harm humans health, and one of important means for the treatment of cancer is Drug therapy, yet many cancer therapy drugs exist defects such as being insoluble in water, poor stability.If camptothecine, paclitaxel, amycin, 5-fluorouracil etc. are all because poor solubility is difficult to be utilized well by organism, solve the crux that its water solublity problem is this class pharmaceutical preparation clinical practice.In addition, its effect of oncotherapy and diagnostic medicine is nonselective mostly, and some normal structure organs often have more distribution, and under therapeutic dose, normal tissue organ toxic and side effects is large.Therefore how to solve solubilising slightly solubility cancer therapy drug and increase medicine stability, improve extremely urgent to the selectivity of cancerous cell.Therefore, finding a kind of reliable target medicine carrier is the key that solves above two problems.
What macromolecule micelle pharmaceutical carrier generally adopted is all linear polymeric micelle.The research work that Bae seminar carries out is the most noticeable, and it points out in delivered research paper, with hydrazone key, Dox key is linked on main polymer chain, and is utilized in cancerous cell microenvironment pH to be faintly acid to make hydrazone bond fission, and the target spot of having realized Dox discharges.And, one piece of paper (Bae Y, Jang W-D, Nishiyama N, Fukushima S, Kataoka K. that Bae delivered on Molecular BioSystems magazine in 2005mol BioSyst2005; 1 (3): 242-250.), first simultaneously by Fol and Dox respectively key link PEG and PASP(poly-aspartate) on, thereby the targeted delivery and the target spot that have medicine concurrently discharge multifunctionality, and fluidic cell experiment (FCM) shows that cell is not more significantly increased containing the polymer micelle of Fol the intake of polymer micelle.Gong Shaoqin seminar has also been engaged in similar research.It is with polycaprolactone random copolymer is kernel, and PEG is shell, has prepared polymer nanocomposite microcapsule.With hydrazone key, connect Dox, and utilize Fol to modify PEG end group, prepared pH responsive type target drug-carrying micelle, cellular uptake amount and cytotoxicity have been tested, proof Dox under acid condition discharges (Yang X, Grailer JJ, Pilla S fast due to the fracture of hydrazone key, Steeber DA, Gong S.bioconjugate Chem2010; 21 (3): 496-504.).
Although linear polymer micelle, as the carrier of poorly water soluble drugs, has demonstrated huge advantage and potentiality in passing medicine process, but still there is micelle poor stability, solubilizing effect is not obvious, drug release rate is low problem.The drug loading of polymer micelle can only reach 5% left and right conventionally at present, obtain higher drug loading very difficult.Research discovery, polymer connects medicine monomer by chemical bond, can reach higher drug loading.And linear block copolymers load capacity is often on the low side, if but cross multikey to connect its micellar structure of drug molecule unstable again.And star-type polymer can overcome this deficiency.
The Yan De of Shanghai Communications University high mountain (Liu J, Pang Y, Huang W, Huang X, Meng L, Zhu X, Zhou Y, Yan D.biomacromolecules2011; 12 (5): 1567-1577.) synthesized dendroid poly phosphate-polylactic-acid block copolymer and be applied to pharmaceutical carrier research, after parcel Dox, HeLa cell has been had to obvious inhibitory action.Another representative research is that Chinese University of Science and Technology's Liu's generation is brave, utilizes functionalizationcyclodextrin has been prepared the polymaleic anhydride star-type polymer of modifying with Fol and Dox, and key has connected the gadolinium element to magnetic resonance effect sensitivity simultaneously, in mouse experiment, finds, the gathering of polymer micelle at liver, kidney position has obvious reinforcement (Liu T, Li X, Qian Y, Hu X, Liu S.biomaterials2012; 33 (8): 2521-2531.).
But because polymer architecture in star-type polymer is comparatively complicated, prepared pharmaceutical carrier occurs that biocompatibility is poor, drug encapsulation ability declines, the problems such as dendrimer surface drug macromolecule water-solubility reduction.In addition, dendrimer complex structure, synthetic cost is higher.These problems have all limited the application of star-type polymer nano medicament carrying system.Therefore, no matter be property polymer or common star-type polymer pharmaceutical carrier, can not possess: target-oriented drug simultaneously, high drug loading ability, micellar structure is stable, the pH sensitivity that target spot discharges, high these several the clinical practices to polymer drug carrier system of biocompatibility play the performance of most important effect.
Summary of the invention
The preparation method that the object of the invention is to set up a kind of tumor cell microenvironment responsive nano microcapsule, this pharmaceutical carrier micelle has the following advantages: belong to nanoparticle; Can realize medicine discharges pH sensitivity in cancerous cell targeted delivery and cancerous cell; Drug loading is large; Good stability; Its target function can effectively reduce medicine normal tissue organ toxic and side effects.
This micelle is a kind of star block copolymer with hydrophilic and hydrophobicity block; Amycin key can be connected with block polymer on; Macromolecular material has two hydrophilic segments, a hydrophobic segment: its hydrophilic section of macromolecular material is polypyrrole alkane ketone, by folic acid, modifies.Hydrophobic section is for poly-threonine, by hydrazone keyed jointing branch amycin; High molecular micellar structure has nucleocapsid double-decker, and skin is hydrophilic polypyrrole alkane ketone, and internal layer is drug molecule integument.
Described block polymer is the polymer in following structural formula:
The technology of preparing scheme of pharmaceutical carrier micelle is as follows:
1) prepare nitrine micromolecule initiator
2) atom transfer radical polymerization of polypyrrole alkane ketone
3) the poly-O-tert-butyl group-L-threonine of both arms alkynes end group is synthetic
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4) Dox molecule is connected with the hydrazone key of the poly-O-tert-butyl group-L-threonine
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5) star block copolymer is synthetic
6) prepare capsule of nano
By dialysis, prepare polymer nanocomposite microcapsule.Concrete grammar is: synthetic star-type polymer is dissolved in respectively after THF, moves in bag filter, with 1000 mL pure water dialysis, refresh the water periodically.Dialysis solution is with after membrane filtration, and lyophilization obtains copolymer carrier micelle.
By above technical scheme, tool of the present invention has the following advantages: 1) this tumor cell microenvironment responsive nano microcapsule volume is little, has nanoscale structures, can pass through various barriers in human body;
2) this tumor cell microenvironment responsive nano microcapsule critical micelle concentration is little, Stability Analysis of Structures;
3) this tumor cell microenvironment responsive nano microcapsule medicine load capacity is large, and good biocompatibility;
4) this tumor cell microenvironment responsive nano microcapsule has targeting release pharmic function, can effectively reduce medicine normal tissue organ toxic and side effects.
The specific embodiment
For making enforcement object of the present invention, technical scheme and advantage more clear, below in conjunction with the drawings and specific embodiments of the present invention, the present invention is described in detail:
As shown in Figure 1, be the chemical constitution schematic diagram of this tumor cell microenvironment responsive nano microcapsule.This macromolecule is AB22. type block macromolecular, have two hydrophilic section, and 3. a hydrophobic section forms.Key on each segment has connected different molecular structures:
2. be to prepare by Transfer Radical Polymerization;
Utilize the resulting five-membered ring of 1,3-Dipolar Cycloaddition and poly-threonine 3. to carry out coupling;
Utilize hydrazone key by segment 3. with amycin molecule 4. key link up.
As shown in Figure 2, a kind of this tumor cell microenvironment responsive nano microcapsule is by this AB2assorted arm star block macromolecular assembling forms.Its structure is respectively by 1. active end group; 2. polypyrrole alkane ketone molecule; 3. gather threonine; 4. the composition such as amycin molecule.Macromolecule by hydrophobic interaction, can self assembly be spherical micellar structure in aqueous solution, and high molecular micellar structure has nucleocapsid double-decker:
Skin be hydrophilic polypyrrole alkane ketone 2.;
Internal layer, for gathering threonine 3., is drug molecule integument, and internal layer can make drug molecule be wrapped in spheroid inside, thereby realizes medicine delivery function.
Figure 3 shows that AB2the AFM scanogram of the assorted arm star block copolymer globular micelle structure that self assembly forms in aqueous solution.Proof macromolecule forms globular micelle structure homogeneous, and good dispersion is not reunited in aqueous solution, is conducive to realize the delivery of drug molecule and release function.
Provide embodiment below so that the present invention is specifically described; but it is worthy of note that following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that the person skilled in the art in this field makes the present invention according to the invention described above content and adjustment still belong to protection scope of the present invention.
Embodiment 1:
1. for nitrine micromolecule initiator
Step 1: 2-chloroethoxy ethanol 5mL is dissolved in 25mL butanone, adds 4.5g NaN in solution3, 2.5gBu4nI, 10mg bicyclohexane also-hexaoxacyclooctadecane-6-6, by mixture be heated to boiling, stir 24 hours.Mixture is filtered, and precipitation is carried out cleaning down with acetone.Resulting mixed solution is the crude product of product, after mixed solution is concentrated, 90oc distills and obtains pure substance.Resulting 2-nitrine ethoxy ethanol1h NMR (CDCl3):δ3.70 (t, 2 H, Ch2oH), 3.65 (t,2h, HOCH2ch2o), 3.56 (t, 2H, N3cH2ch2o), 3.37 (t, 2H, Ch2n3), and 2.56 (s, 1H, OH).
Step 2: by a kind of 2-nitrine ethoxy ethanol 2g making of step, alpha-chloro acyl chlorides 5.09g is dissolved in 30mL dichloromethane, and reaction system is transferred in ice territory, slowly adds 6.8g dicyclohexylcarbodiimide in reaction vessel, and stirs.0.4g dimethylamino naphthyridine is dissolved in dichloromethane and was splashed in reaction vessel in 10 minutes.Whole reaction system is 0ounder C condition, react 1 hour, then at room temperature react 24 hours.The cyclohexyl urea being precipitated out in course of reaction is filtered, and wash with dichloromethane.With sodium bicarbonate solution (5%), extract, then with anhydrous magnesium sulfate, be dried.After reduced vacuum is dry, with silica gel chromatographic column, carry out separation, finally obtain product 2-chloro nitrine Ethoxyethane.1H?NMR?(CDCl3):δ4.30?(t,?2H,?CH2OCO),?3.73?(t,?2H,?COOCH2CH2O),?3.67?(t,?2H,?N3CH2CH2O),?3.35?(t,?2H,?CH2N3),?and?1.92?(s,?6H,?(CH3)2C)。
2. the atom transfer radical polymerization of polypyrrole alkane ketone
2-nitrine ethyoxyl bromo acid 12mg by obtaining in step 2, is dissolved in 25mL DMSO and in the ratio of 2:1:1, adds methanol, bipyridyl and cuprous bromide simultaneously with 5.4g vinyl pyrrolidone.By bleeding-ventilate for three times-thaw cycles carries out tube sealing to polymerization pipe.After polymerization 24 hours, with methanol, polymer is precipitated out, filters, dry stand-by.The prepared polypyrrole alkane of 2g ketone is dissolved in 20mL dichloromethane, adds 2.8mL ethylenediamine, the terminal groups of polypyrrole alkane ketone is modified, obtain the polymer of end group band primary amine.The polypyrrole alkane ketone that 2g end group is modified is warming up to 50oc reacts 6h.After having reacted, reactant liquor is packed into bag filter (MWCO=3500), after dialysing 2 days, remove free folic acid in deionized water, change water every day 3 times.Dialysis solution ether sedimentation, vacuum drying obtains product.
3. the poly-O-tert-butyl group-L-threonine of both arms alkynes end group is synthetic
Step 1: by 16.8g 3,5-methyl dihydroxy benzoate and 26.2g propargyl bromide are dissolved in 300mL acetone, add 15.1g sodium carbonate in solution, 0.1g bicyclohexane also-hexaoxacyclooctadecane-6-6.Reaction solution is heated to reflux, reacts 24 hours.After reaction finishes, by sedimentation and filtration, filtrate is carried out concentrating under reduced pressure, carries out recrystallization in methanol, obtains product 3,5-diacetylene p-methoxybenzoic acid methyl ester.1H?NMR?in?CDCl3,?δ(ppm):?2.55?(2H,?CCH),?3.91(3H,?CH3O),?4.73?(4H,?CH2CCH),?6.82?(1H,?aromatic),?7.29?(2H,?aromatic)。
Step 2: by 6.25g3,5-diacetylene p-methoxybenzoic acid methyl ester is dissolved in 30mL methanol, is dissolved in 73.9g ethylenediamine in 120mL ethylenediamine, and 0ounder C condition, be slowly added dropwise in reaction vessel, dropping process needs about 1 hour, and under room temperature, reacts 96 hours after being added dropwise to complete again.Be less than 40oin the water-bath of C, revolve to steam and remove unnecessary solvent, utilizing the mixed solution of benzene/methanol (9/1 v/v) to carry out recrystallization to mixed solution, in vacuum drying oven, be dried 24 hours, obtain product N-amine ethyl 3,5-diethyl alkynyloxy group aniline.1H?NMR?in?CDCl3,?δ(ppm):?1.61?(2H,
CH2NH2),?2.55?(2H,?CCH),?2.95?(2H,?CH2NH2),?3.49?(2H,?CONHCH2),?4.73?(4H,?CH2CCH),?6.68?(1H,?CONH),?6.76?(1H,?aromatic),?7.05?(2H,?aromatic)。
Step 3: by resulting product N-amine ethyl 3 in 0.16g step 2,5-diethyl alkynyloxy group aniline is as initiator, utilize the ring-opening polymerisation mode polymer poly O-tert-butyl group-L-threonine carboxylic acid anhydrides 5.19g, monomer and initiator are added in polymerization pipe, and add dry DMF 15mL, under room temperature, react 24 hours.Polymer precipitates with absolute ether, dry 24 hours of vacuum drying oven.Obtain the poly-O-tert-butyl group-L-threonine of product both arms alkynes end group.
4. amycin molecule is connected with the hydrazone key of the poly-O-tert-butyl group-L-threonine
The poly-O-tert-butyl group-L-threonine of the both arms alkynes end group obtaining in 5.8mL hydrazine and 2.43g above-mentioned steps is dissolved in the dry DMF of 25mL, reaction system is heated to 40oc, reacts 1 hour.Thick product is precipitated with absolute ether, and by sedimentation and filtration, dry 24 hours of vacuum drying oven, obtains the poly-O-tert-butyl group-L-threonine of amido both arms alkynes end group.This product 5g and 0.34g amycin monomer are dissolved in DMSO, reaction system is heated to 30oc, reacts 48 hours, and the reaction mixture obtaining precipitates with absolute methanol, and by sedimentation and filtration, dry 24 hours of vacuum drying oven.Make the poly-O-tert-butyl group-L-threonine of amycin molecule grafting.
5. AB2synthesizing of assorted arm star block copolymer
The polypyrrole alkane ketone that 3.8g end group is modified and the poly-O-tert-butyl group-L-threonine of 7.8g amycin molecule grafting are dissolved in 20mL dry DMF, and add 6.93mg PMDETA.Reaction system is carried out to bleed-ventilation-thaw cycles three times, add rapidly cuprous bromide 5.47mg.Reaction is 35ounder C condition, react 24 hours.React resulting mixed solution and cross silicagel column to remove unnecessary mantoquita and other impurity.After solution is concentrated, with absolute methanol, precipitate, finally obtain AB2assorted arm star block copolymer.
6. the preparation of polymer nanocomposite microcapsule
By dialysis, prepare polymer nanocomposite microcapsule.Concrete grammar is: one or both star polymers are dissolved in respectively after THF, move in bag filter, with 1000 mL pure water dialysis, refresh the water periodically.Dialysis solution is with after membrane filtration, and lyophilization obtains copolymer carrier micelle.
Finally it should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art should understand, can a minute technical scheme for invention be modified or be replaced on an equal basis, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.