CN108392471A - Nano medication based on end sulfur-bearing caprylyl star-type polymer - Google Patents

Nano medication based on end sulfur-bearing caprylyl star-type polymer Download PDF

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CN108392471A
CN108392471A CN201810172582.XA CN201810172582A CN108392471A CN 108392471 A CN108392471 A CN 108392471A CN 201810172582 A CN201810172582 A CN 201810172582A CN 108392471 A CN108392471 A CN 108392471A
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star
polymer
caprylyl
bearing
type polymer
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CN108392471B (en
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程茹
王秀秀
钟志远
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Suzhou University
Zhangjiagang Institute of Industrial Technologies Soochow University
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Suzhou University
Zhangjiagang Institute of Industrial Technologies Soochow University
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    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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Abstract

The invention discloses a kind of Nano medications based on end sulfur-bearing caprylyl star-type polymer.The star-type polymer of side chain sulfur-bearing caprylyl is obtained by esterification; LA degree of substitution controllably has excellent biocompatibility; it can be used for controlled drug delivery systems; stablize long circulating in the polymer nano-particle Nano medication support of the sensitive reversible crosslink of reduction of the cancer targeting of preparation; but it is enriched in cancerous tissue height and efficiently enters cell; quickly solution is crosslinked, releases drug in the cell, efficiently specifically kills cancer cell, effectively inhibits the growth of cancer without causing toxic side effect.

Description

Nano medication based on end sulfur-bearing caprylyl star-type polymer
Technical field
The present invention relates to a kind of biocompatible polymeric material and its applications, and in particular to one kind is pungent based on end sulfur-bearing The Nano medication of the star-like biocompatible polymer of acyl group, belongs to medical material field.
Background technology
High molecular material with good biocompatibility and biodegradability has been widely used in biomedicine Field includes organizational project and drug controlled release field, and good application prospect has been shown based on this Nano medication, but By the polymer nanocomposite drug of prior art preparation, that there are body-internal-circulations is unstable, tumour cell intake is low, Intracellular drug is dense The slow problem of low, intracellular drug release rate is spent, causes the drug effect of Nano medication not high, the poison caused there is also drug leakage is secondary Effect；For example BIND-014 does not reach expected in clinical two interim results, due to the stabilization of the Nano medication in vivo Caused by property deficiency.
Cancer is to threaten the primary killers of human health, and morbidity and mortality are in the trend risen year by year.Nanometer medicine It is new uncommon that the appearance of object is that treating cancer is brought, but in the prior art, still lack stable circulation, cancer specific in vivo The small efficient nano drug of targeting, intracellular quick response release drug, toxic side effect, is especially the absence of cyclic process in vivo It can keep stablizing the polymer nanoparticle drug carriers with rapid delivery of pharmaceuticals in the cell.Based on this, exploitation one kind can extend medicine Object circulation time in vivo and can in tumour cell the nano-carrier of fast immediate release drug it is extremely urgent.
Invention content
The object of the present invention is to provide a kind of nanometer medicines based on the star-like biocompatible polymer of end sulfur-bearing caprylyl Object.
In order to achieve the above objectives, the specific technical solution of the present invention is：One kind being based on end sulfur-bearing caprylyl star polymerization The Nano medication of object, the Nano medication based on end sulfur-bearing caprylyl star-type polymer by reversible crosslink there is reduction to respond Property polymer nano-particle load drug obtain；
The reversible crosslink have reduction response polymer nano-particle by end sulfur-bearing caprylyl star-type polymer with Second polymer assembles be prepared altogether；The second polymer is amphipathic nature polyalcohol and/or target direction amphipathic nature polymer；
The chemical structural formula of the star-type polymer of the end sulfur-bearing caprylyl is as follows：
Wherein, x:Y is (1~3): 1.
The star-type polymer of sulfur-bearing caprylyl in end disclosed by the invention is properly termed as sP-LA, and molecular weight is 10000~ 75000。
The preparation of the star-type polymer of above-mentioned end sulfur-bearing caprylyl includes the following steps：
（1）Under nitrogen environment, under the conditions of ice-water bath, by N, N- dicyclohexylcarbodiimide solution is added dropwise in lipoic acid solution； After being added dropwise to complete, sealing room temperature reaction 10~15 hours；Obtain lipoic acid anhydride solution；
（2）Under vacuum environment, using polyhydroxy glucose as initiator, under the catalytic action of stannous octoate, lactide and second Lactide reacts 8 hours at 160 DEG C；Obtain star-type polymer；
（3）Under nitrogen environment, lipoic acid anhydride solution is added to organic containing 4-dimethylaminopyridine and star-type polymer In solvent, under air-proof condition, reacted 1~3 day in 30 DEG C；Obtain the star-type polymer of end sulfur-bearing caprylyl.
Specifically, the preparation method can be as follows：
（1）In a nitrogen environment, lipoic acid is molten in organic solvent, prepares lipoic acid solution；By N, N- dicyclohexyls carbon two is sub- Amine prepares N, N- dicyclohexylcarbodiimide solution in organic solvent；Then under ice-water bath, by N, N- dicyclohexyls carbon two is sub- Amine aqueous solution is added dropwise in lipoic acid solution；After being added dropwise to complete, sealing room temperature reaction 10~15 hours；After reaction, filtering is anti- Liquid is answered, lipoic acid anhydride solution is obtained；
（2）In N2Under environment, polyhydroxy glucose, lactide and glycolide are added in confined reaction bottle, it then will catalysis Agent stannous octoate adds in reaction bulb and is uniformly mixed all materials；Then reaction bulb is vacuumized-replace N2Three times, most Reaction bulb is vacuumized 30 minutes afterwards, seals reaction bulb；Polymerisation is reacted 8 hours in vacuum tank under the conditions of 160 DEG C；Reaction After, product dissolves in methylene chloride, is then precipitated in ice methanol, filters and be dried in vacuo to obtain star polymerization Object；
（3）Under nitrogen environment, lipoic acid anhydride solution is added to organic containing 4-dimethylaminopyridine and star-type polymer In solvent, under air-proof condition, reacted 1~3 day in 30 DEG C；Then reactant is precipitated in ice ether, then through filtering simultaneously vacuum Dry cake obtains the star-type polymer (sP-LA) of end sulfur-bearing caprylyl.
The preparation method of above-mentioned Nano medication, includes the following steps：
（1）Under nitrogen environment, under the conditions of ice-water bath, by N, N- dicyclohexylcarbodiimide solution is added dropwise in lipoic acid solution； After being added dropwise to complete, sealing room temperature reaction 10~15 hours；Obtain lipoic acid anhydride solution；
（2）Under vacuum environment, using polyhydroxy glucose as initiator, under the catalytic action of stannous octoate, lactide and second Lactide reacts 8 hours at 160 DEG C；Obtain star-type polymer；
（3）Under nitrogen environment, lipoic acid anhydride solution is added to organic containing 4-dimethylaminopyridine and star-type polymer In solvent, under air-proof condition, reacted 1~3 day in 30 DEG C；Obtain the star-type polymer of end sulfur-bearing caprylyl；
（4）The star-type polymer of end sulfur-bearing caprylyl is assembled in a solvent with second polymer, small-molecule drug and is received Rice drug.
In the present invention, N, solvent is dichloromethane in N- dicyclohexylcarbodiimide solution；Solvent is in lipoic acid solution Dichloromethane；Organic solvent is dichloromethane in organic solvent containing 4-dimethylaminopyridine and star-type polymer.
In the present invention, polyhydroxy glucose, lactide, glycolide molar ratio be 1:（50~55）∶（60~65）；It prepares Star-type polymer structural formula it is as follows：
In the present invention, N, N- dicyclohexylcarbodiimides, lipoic acid molar ratio be 2: 1；Lipoic acid acid anhydride, 4- dimethylamino pyrroles Pyridine, star-type polymer molar ratio be 7.5: 10: 1.
The star-type polymer of the end sulfur-bearing caprylyl of the present invention can be with polypeptide with different target functions or more Amphipathic nature polyalcohol such as cRGD-PEG-PDLLA, GE11-PEG-PDLLA, TAT-PEG-PDLLA, HA-b- of glycan molecule modification PDLLA is assembled jointly to which preparation has the polypeptides such as different targeted moleculars such as cRGD, GE11, TAT or HA and polysaccharide is repaiied The nano-particle of decorations has targeting, bio-compatible to obtain the polymer nano-particle of different cancer cell special targets Property.The nano-particle that such polymer is formed has good stability, higher efficiency of loading, while can be specifically It is targeted to cancer cell.
The invention also discloses the polymer nano-particles that a kind of reversible crosslink has reduction response, are contained by above-mentioned side chain The star-type polymer of sulphur caprylyl is assembled with second polymer and is prepared altogether；The second polymer is amphipathic nature polyalcohol And/or target direction amphipathic nature polymer；Preferably, the second polymer dosage is the star polymerization of above-mentioned end sulfur-bearing caprylyl The 10%~60% of amount of substance；When second polymer is amphipathic nature polyalcohol and target direction amphipathic nature polymer, target direction amphipathic nature is poly- The mass percent for closing object is less than 70%；Amphipathic nature polyalcohol is carried without targeted molecular, target direction amphipathic nature polymer in the present invention Polypeptides and the polysaccharide such as targeted molecular such as cRGD, GE11, TAT or HA；It is preferred that second polymer is amphipathic nature polyalcohol and target To amphipathic nature polyalcohol.Polymer nano-particle i.e. of the invention by above-mentioned side chain sulfur-bearing caprylyl star-type polymer and amphiphilic Property polymer jointly assembling be prepared；Or star-type polymer and grafting targeted molecular by above-mentioned side chain sulfur-bearing caprylyl Assembling is prepared amphipathic nature polyalcohol jointly；Or divided with grafting targeting by the star-type polymer of above-mentioned side chain sulfur-bearing caprylyl Assembling is prepared the amphipathic nature polyalcohol and amphipathic nature polyalcohol of son jointly, for example the star-like of above-mentioned side chain sulfur-bearing caprylyl gathers The amphipathic nature polyalcohol for closing object and grafting targeted molecular is mixed with amphipathic nature polyalcohol according to different proportion, can be prepared with difference The polymer nano-particle of density is targeted to get to the polymer nano-particle with cancer cell active targeting function, Ke Yizeng Load intake of the medicine polymer nano-particle in cancer cell.
There is the star-type polymer of side chain sulfur-bearing caprylyl disclosed by the invention excellent biocompatibility and biology can drop Xie Xing forms polymer nano-particle by being assembled jointly with second polymer, can be in such as two thio Soviet Union of reducing agent of catalytic amount Sugar alcohol（DTT）Or glutathione（GSH）Under the conditions of existing, cross-linked polymer nano particles are prepared or have to cancer cell There are the cross-linked polymer nano particles of active targeting function, 70~180 nanometers of grain size can be as the load of the drug for the treatment of cancer Body；Hydrophobic small molecules anti-lung-cancer medicament adriamycin (DOX), taxol can be loaded in polymer nano-particle（PTX）、 Docetaxel（DTX）Deng, the bioavilability of dewatering medicament in vivo is improved, the circulation time of drug is extended, raising drug In the enriching quantity of tumor locus.Therefore, Nano medication disclosed by the invention loads small molecule medicine by cross-linked polymer nano particles Object obtains.Cross-linked polymer nano particles prepared by the star-type polymer of side chain sulfur-bearing caprylyl disclosed by the invention are in interior karyomorphism At the chemical crosslinking stablized, so as to stablize long circulating in vivo；But endocytosis enters after cancer cell can reproducibility in the cell Under environment, quickly solution crosslinking, quick release go out drug, efficiently kill cancer cell.So the present invention is claimed above-mentioned side chain and contains Star-type polymer, polymer nano-particle, cross-linked polymer nano particles, the Nano medication of sulphur caprylyl are preparing treatment tumour Application in drug such as melanoma, lung cancer and three cloudy breast cancer medicines.
Due to the application of the above technical scheme, compared with prior art, the present invention haing the following advantages：
1. the present invention reacts to obtain the controllable side chain sulfur-bearing caprylyl of degree of substitution using star-type polymer with lipoic acid acid anhydride Star-type polymer assigns star-type polymer new function, enriches the type of star-type polymer.
2. the star-type polymer of side chain sulfur-bearing caprylyl disclosed by the invention has excellent biocompatibility and biology can Degradability can prepare polymer nano-particle and the polymer nano-particle with cancer cell active targeting function, load not Same drug, and disulfide bond crosslinking can be formed, stable cross-linked polymer Nano medication is obtained, to overcome the prior art Middle Nano medication body-internal-circulation is unstable, the easy premature disconnection of drug, the defect for causing toxic side effect.
3. cross-linked polymer Nano medication disclosed by the invention, crosslinking is reversible, i.e. long circulating in support, can be in cancer Cell height is enriched with；But crosslinking but can be quickly solved after entering in cancer cell, drug is released, realization is efficiently specifically killed Cancer cell is without toxic side effect；Overcome the Nano medication being chemically crosslinked in the prior art excessively stablize and in the cell Drug release slowly, cause the defect of drug resistance.
4. biocompatible polymeric nanoparticles disclosed by the invention and the polymerization with cancer cell active targeting function Object nano-particle can form the sensitive disulfide bond crosslinking of reduction in preparation process, and preparation method is easy, existing to overcome The defects of complicated operation and purification process being needed when preparing crosslinking nano drug in technology.
5. the star-type polymer of side chain sulfur-bearing caprylyl disclosed by the invention assembles preparation jointly with amphipathic nature polyalcohol Cross-linked polymer nano particles can be used for the control delivery systme of hydrophobic anticancer drug, to overcome in the prior art without energy Efficiently load and stablize the defect of the hydrophobic anticancer drug of body-internal-circulation；Further, bonding targeted molecular, in cancer Efficient targeted therapy in terms of have wider application value.
Description of the drawings
Fig. 1 is the hydrogen nuclear magnetic spectrogram of the star-type polymer sP-LA of one side chain sulfur-bearing caprylyl of embodiment；
Fig. 2 is amphipathic nature polyalcohol PEG-PDLLA (A), cRGD-PEG-PDLLA (B) in embodiment two, three, four, five, The nuclear magnetic spectrogram of GE11-PEG-PDLLA (C) and TAT-PEG-PDLLA (D)；
Fig. 3 is amphipathic nature polyalcohol HA- in embodiment sixbThe nuclear magnetic spectrogram of-PDLLAPEG-PDLLA；
Fig. 4 is the particle diameter distribution and transmission electron microscope figure of cross-linking nanoparticles sPLy XNPs in embodiment seven（A）, crosslinking Nano-particle stability（B）, reduction response test（C）And external release profile（D）；
Fig. 5 is in embodiment eight using cRGD as the particle diameter distribution of the cross-linking nanoparticles cRGD-XNPs of targeted molecular（A）And transmission Electron microscope picture（B）, restore response test chart（C）And external release profile（D）；
Fig. 6 is in embodiment 11 using HA as the particle diameter distribution of the cross-linking nanoparticles HA-sPLy XNPs of targeted molecular（A）And Transmission electron microscope figure（B）, restore response test chart（C）And external release profile（D）；
Fig. 7 is that difference nano-particle sPLy XNPs are at B16F10 cells (A) in embodiment 17, and cRGD-XNPs is in B16F10 Cell (B), GE11/TAT-XNPs to MDA-MB-231 cells (C) and HA-sPLy XNPs A549 cells (D) intake The figure of amount；
Fig. 8 is that blank nano-particle sPLy XNPs are to B16F10 cells (A) in embodiment 18, and cRGD-XNPs is to B16F10 Cell (B), GE11/TAT-XNPs is to MDA-MB-231 cells (C) and HA-sPLy XNPs to A549 cells (D) toxicity knot Fruit is schemed；
Fig. 9 is medicine-carried nano particles DOX-sPLy XNPs in embodiment 19 to B16F10 cells (A), DOX-cRGD XNPs To B16F10 cells (B), DTX-GE11/TAT XNPs are to MDA-MB-231 cells and DTX-HA-sPLy XNPs to A549 The toxicity data figure of cell；
Figure 10 is medicine-carried nano particles DOX-sPLy XNPs (A), DOX-cRGD in embodiment 20,21,23 The blood circulation result of study figure of XNPs (B), DTX-HA-sPLy XNPs (C) in Mice Body；
Figure 11 is medicine-carried nano particles DOX-cRGD XNPs in embodiment 24 and 26 in lotus B16F10 melanomas (A) and bio distribution figures of the DTX-HA-sPLy XNPs in A549 lung carcinoma subcutaneous tumor (B) Mice Body；
Figure 12 is to target cross-linking nanoparticles DOX-cRGD by the load DOX of targeted molecular of cRGD in embodiment 27 XNPs tumor suppression situation maps in lotus B16F10 melanoma Mice Bodies, wherein A are tumor growth curve, and B changes for mouse weight, C curves for survival；
Figure 13 is to target cross-linking nanoparticles DTX- by the load DTX of targeted molecular of GE11 and TAT in embodiment 28 For GE11/TAT XNPs in the tumor suppression situation map in He Sanyin breast cancer MDA-MB-231 subcutaneous tumors nude mouses, wherein A is tumour Growth curve, B are tumour picture after mouse treatment, and C is changes of weight, D curves for survival；
Figure 14 is to target cross-linking nanoparticles DTX-HA-sPLy by the load DTX of targeted molecular of HA in embodiment 29 Tumor suppression situation maps of the XNPs in lotus A549 lung carcinoma subcutaneous tumor nude mouses, wherein A are tumor growth curve, and B becomes for weight Change, C is tumour inhibiting rate, and D is tumour picture after mouse treatment.
Specific implementation mode
With reference to embodiment and attached drawing, the invention will be further described：
Embodiment one synthesizes the star-type polymer of side chain sulfur-bearing caprylyl
Synthesize star-type polymer and linear polymer
Star-type polymer can be used as initiator by polyhydroxy glucose, under the catalytic action of stannous octoate, cause third and hand over The ring-opening polymerization of ester and glycolide synthesizes.In N2Under environment, by 0.18 g（1 mmol）Polyhydroxy glucose, 7.5 g（52 mmol）Lactide and 7.5 g（65 mmol）Glycolide is added in confined reaction bottle, then by 4.73 mg octoate catalysts Stannous adds in reaction bulb and is uniformly mixed all materials.Then reaction bulb is vacuumized-replace N2It three times, finally will be anti- It answers bottle to vacuumize 30 minutes, seals reaction bulb.Polymerisation is reacted 8 hours in vacuum tank under the conditions of 160 DEG C.Crude product dissolves In methylene chloride, it is then precipitated in ice methanol, filter and is dried in vacuo to obtain star-type polymer.1H NMR (600 MHz, CDCl3): δ 5.22 (-OOCCH 2O-), 4.83 (-OOCCH(CH3)O-), 2.45 (, -OCCH 2-), 1.6 (-CH(CH 3)O-)。
Linear polymer is using 1,4-butanediol as initiator, under the catalytic action of stannous octoate, causes lactide and second The ring-opening polymerization of lactide synthesizes.In N2Under environment, by 0.9 g（1 mmol）1,4-butanediol, 7.5 g（52 mmol）Third Lactide and 7.5 g（65 mmol）Glycolide is added in confined reaction bottle, then adds 4.73 mg octoate catalyst stannous instead It answers in bottle and is uniformly mixed all materials.Then reaction bulb is vacuumized-replace N2Three times, finally reaction bulb is taken out true It is 30 minutes empty, seal reaction bulb.Polymerisation is reacted 5 hours in vacuum tank under the conditions of 200 DEG C.Crude product is dissolved in dichloromethane It in alkane, is then precipitated in ice methanol, filter and is dried in vacuo to obtain linear polymer.
Synthesize lipoic acid acid anhydride LAA：In a nitrogen environment, in a nitrogen environment, 30 mg（0.15 mmol）Lipoic acid（LA） It is dissolved in 1 mL dichloromethane, stirring in two neck bottles is added and extremely dissolves, 60 mg（0.3 mmol）N, N- dicyclohexylcarbodiimide （DCC）In 0.5 mL dichloromethane, under ice-water bath, it is added dropwise in LA solution.Continue to lead to 5 minutes nitrogen, by two necks Bottle is sealed, and at room temperature, reacts 12 hours；After reaction, it filters out the precipitation that reaction generates and obtains lipoic acid anhydride solution, it will Lipoic acid acid anhydride（LAA）Solution is concentrated into 0.5 ml.
Synthesize the star-type polymer (sP-LA) of side chain sulfur-bearing caprylyl：Under nitrogen environment, by lipoic acid acid anhydride（LAA）30mg （0.075 mmol）Solution is added to 150 mg（0.01 mmol）Star-type polymer and 12 mg（0.1 mmol）2 mL of DMAP In dichloromethane solution, continue to lead to 5 minutes nitrogen, sealed flask is placed in 30 DEG C of oil baths and reacts 48 h；Then by product in ice It is precipitated in ether, filter and is dried in vacuo to obtain the star-type polymer sP-LA of side chain sulfur-bearing caprylyl, yield 86.7%.1H NMR (600 MHz, CDCl3): δ 5.22 (-OOCCH 2O-), 4.83 (-OOCCH(CH3)O-), 3.57 (- CH2CHCH2CH2S2), 3.17 (-CH2CHCH2CH 2S2), 2.45 (-OCCH 2-), 1.6 (-CH(CH 3)O-).Nuclear-magnetism calculates The grafting rate of LA is 90%, sees Fig. 1.
Two synthesizing amphipathic polymer P EG-PDLLA of embodiment
Amphipathic nature polyalcohol PEG-PDLLA can cause D by macromole evocating agent PEG, prepared by L- lactide ring-opening polymerisations. N2Under environment, to 2.5 mL PEG (M n=5.0 kg/mol, 0.5 g, 0.1 mmol) and D, L- lactide (0.4 g, 2.8 mmol) anhydrous toluene solution in, rapidly join the toluene storing solution of 0.5 mL (0.2 mol/L) stannous octoate. After reacting 48 h in 110 DEG C of constant temperature oil baths, glacial acetic acid is added and terminates reaction.Product is precipitated in ice ether then, is filtered simultaneously Vacuum drying obtains PEG-PDLLA, and yield is：88.9 %.1H NMR (600 MHz, CDCl3): δ 5.16 (-CH(CH3) O- ), 3.65 (-CH 2CH 2O-), 3.38 (CH 3O-), 1.56 (-CH(CH 3) O-), see Fig. 2 (A).M n (1H NMR) =8.9 kg/mol,M n(GPC)=15.9 kg/mol,M w/M n (GPC) = 1.3。
Three synthesizing amphipathic target polymer cRGD-PEG-PDLLA of embodiment
Target polymer cRGD-PEG-PDLLA is obtained by two-step reaction.Synthesis maleimide amino-functionalization is amphipathic first Then polymer MAL-PEG-PDLLA further synthesizes cRGD by the polypeptide cRGD-SH Michael's additions of MAL and sulfhydrylation Peptide modified amphipathic nature polyalcohol cRGD-PEG-PDLLA.The MAL-PEG-PDLLA of maleimide amino-functionalization passes through MAL- PEG causes D, prepared by L- lactide ring-opening polymerisations.In N2Under environment, to 2.5 mL MAL-PEG (M n=5.0 kg/mol, 0.5 G, 0.1 mmol) and the anhydrous toluene solution of D, L- lactide (0.4 g, 2.8 mmol) in, rapidly join 0.5 mL The toluene storing solution of (0.2 mol/L) stannous octoate.After reacting 48 h in 110 DEG C of constant temperature oil baths, glacial acetic acid is added and terminates instead It answers.Product is precipitated in ice ether then, filter and is dried in vacuo to obtain MAL-PEG-PDLLA.Then by MAL-PEG- PDLLA and cRGD-SH is dissolved in DMF, reacts 24 h at room temperature.Product is dialysed in DMF 48 h, then again Dialyse 24 h in deionized water, is finally freeze-dried product.Yield：85.4%.1H NMR (600 MHz, DMSO-d 6): δ 7.0-7.4 cRGD, 5.16 (-CH(CH3)O- ), 3.65 (-CH 2CH 2O-), 1.56 (-CH(CH 3) O-), see Fig. 2 （B）.M n (1H NMR) = 8.8 kg/mol。 M n (GPC) = 13.9 kg/mol。M w/M n(GPC) = 1.3.CRGD's connects It is 94% that branch rate tests to obtain by BCA.
Example IV synthesizing amphipathic target polymer GE11-PEG-PDLLA
The synthesis of target polymer GE11-PEG-PDLLA peptide modified GE11 and the cRGD modifications in embodiment three The building-up process method of cRGD-PEG-PDLLA is similar.MAL-PEG-PDLLA and GE11-SH are dissolved in DMF, in room temperature 24 h of lower reaction.Product is dialysed in DMF 48 h, and then dialyse 24 h in deionized water again, is finally freezed product dry It is dry.Yield：89.6%.1H NMR (600 MHz, DMSO-d 6): δ 6.5-7.1 GE11, 5.16 (-CH(CH3)O- ), 3.65 (-CH 2CH 2O-), 1.56 (-CH(CH 3) O-), see Fig. 2（C）.M n (1H NMR) = 8.8 kg/mol。 M n (GPC) = 13.9 kg/mol。M w/M n(GPC) = 1.3.It is 96% that the grafting rate of cRGD is tested to obtain by BCA.
Five synthesizing amphipathic target polymer TAT-PEG-PDLLA of embodiment
The cRGD- of the synthesis of target polymer TAT-PEG-PDLLA peptide modified TAT and the cRGD modifications in embodiment three The building-up process method of PEG-PDLLA is similar.MAL-PEG-PDLLA and TAT-SH are dissolved in DMF, reacted at room temperature 24 h.Product is dialysed in DMF 48 h, and then dialyse 24 h in deionized water again, is finally freeze-dried product.Production Rate：84.3%.1H NMR (600 MHz, DMSO-d 6): δ 7.0-7.4 TAT, 5.16 (-CH(CH3)O- ), 3.65 (- CH 2CH 2O-), 1.56 (-CH(CH 3) O-), see Fig. 2（D）.M n (1H NMR) = 8.8 kg/mol。M n (GPC) = 13.9 kg/mol。 M w/M n(GPC) = 1.3.It is 98% that the grafting rate of TAT is tested to obtain by BCA.
The embodiment six directions is at target polymer HA-b-PDLLA
HA-bPDLLA (the N that-PDLLA passes through the HA and nitrine sealing end of alkynyl3- PDLLA) click chemistry be obtained by the reaction.It is first First, aldimine condensation reaction generation schiff bases occurs for the amino on the aldehyde radical and propargylamine on hyaluronic acid, then by cyano boron hydrogen Change sodium reduction and product Alkyn-HA is made for imines；The specific steps are：Under nitrogen protection, oligomerization hyaluronic acid（HA, 1360 Mg, 0.17 mmol）, propargylamine（37.4 mg, 0.68 mmol）, sodium cyanoborohydride（42.8 mg, 0.68 mmol）Addition is gone Ionized water（15 mL）In, it is stirred to react two days for 60 DEG C under confined conditions, then adjusting the temperature to 40 DEG C, the reaction was continued two days. Reaction solution dialysis, be lyophilized white solid is intermediate product Alkyn-HA.Yield：89%.1H NMR (600 MHz, D2O)： 1.94 (s, -OCH3), 2.91 (t, -CH2CH2N-), 4.38-4.48 (m, HA)。
Under glove box nitrogen protection, D, L- lactides are placed in closed reactor, and 1.2 mL dichloromethane, which are added, makes it fill Divide dissolving.Then sequentially add azidoethyl alcohol（4.35 mg, 0.05 mmol）With tri- azabicyclics of 1,5,7- [4.4.0] decyl- 5- alkene（TBD, 6.95 mg, 0.05 mmol）As initiator and catalyst.It is sufficiently stirred system, gloves are transferred out of after closed Case.4 h are reacted at 30 DEG C, then plus two drop glacial acetic acids terminate reaction.In 15 ~ 20 times of ice for being in excess in reaction solution volume of volume Freeze precipitated product in anhydrous ether.It is dried in vacuo after filtering, finally obtains sticky faint yellow solid, as intermediate product N3- PDLLA）.Yield：91%.1H NMR (600 MHz, CDCl3): 5.16 (-CH(CH3)O- ), 1.56 (-CH(CH 3) O-).Molecular weight is calculated in nuclear-magnetism：5.9 kDa.GPC surveys molecular weight：11.0 kDa, molecular weight distribution：1.2.
Pass through the click chemistry of alkynyl and nitrine（Click chemistry）Prepare amphipathic nature polyalcohol.Take above-mentioned experiment The derivatives of hyaluronic acids Alkyn-HA for being modified with alkynyl of synthesis（500mg, 0.06mmol）And N3-PDLLA (416 mg, 76 μm of ol) it is dissolved in DMSO respectively, two neck flasks are transferred to after it is fully dissolved.Holding DMSO total volumes are 15 mL.With Lead to nitrogen afterwards, excludes the oxygen in solution.By Salzburg vitriol（1.5 mg, 0.006 mmol）Sodium ascorbate（2.4 mg , 0.012 mmol）It is dissolved in respectively in 10 μ L deionized waters, reaction system is sequentially added after to be dissolved.Reaction temperature, which is arranged, is 50 DEG C, nitrogen protection in reaction process, 24 h of reaction time.Yellow transparent liquid is obtained after reaction.Use molecular cut off Unreacted N is removed for the 15000 first dialysis in DMSO of bag filter3- PDLLA, then successively edta salt solution and go from It dialyses 2 days in sub- water.Concentrated frozen is dried after dialysis, and it is final product HA- to finally obtain white solidb-PDLLA 。 Nuclear-magnetism is surveyed in sampling after drying.Yield：86%.1H NMR (600 MHz, D2O/DMSO-d 6 = 1/9) δ (ppm): 1.79, 3.03-3.67, and 4.41-4.49 (HA);1.56 and 5.16 (PDLLA), are shown in Fig. 3.
Embodiment seven prepares cross-linked polymer nano particles and non-crosslinked nano-particle
10 mg sP-LA and 10 mg PEG-PDLLA are dissolved in respectively in 1mL DMSO, the solution of 10 mg/mL is made into, takes 100 mL sP-LA solution and 30 mLPEG-PDLLA are uniformly mixed, and are added drop-wise to 870mL phosphate buffer solutions（PB, 10 mM, pH 7.4）In, it is stirred at room temperature 0.5 h, under condition of nitrogen gas, the DTT solution of 10 mM, sealed vial, at 37 DEG C, 100 is added 10 h are incubated in rpm constant-temperature tables.Then the solution is placed in bag filter（MWCO3500）It is interior, by it in largely dialysis medium （PB, 10 mM, pH 7.4）Middle dialysis 24 hours, changes five water, obtains cross-linked polymer nano particles sPLy XNPs.It obtains The size of cross-linked polymer nano particles is by dynamic light scattering particle size analyzer（DLS）The nano-particle of the formation of survey is 73 Nm, particle diameter distribution is very narrow, sees Fig. 4 A, by Fig. 4 A it is found that it is solid ball shape structure, cross-linking nanoparticles that TEM, which measures nano-particle, Constant grain size and particle diameter distribution are still maintained in the presence of dilution for many times and fetal calf serum（Fig. 4 B）, but in simulation tumour cell Fast-swelling under reducing environment, solution crosslinking（Fig. 4 C）.It follows that the solution that obtained cross-linking nanoparticles have reduction sensitive is handed over The property of connection is suitable for pharmaceutical carrier.
Non-crosslinked nano-particle preparation is similar with cross-linking nanoparticles, but DTT need not be added to be crosslinked.Non-crosslinked nanoparticle Son two groups of controls of setting, respectively assemble system jointly by the star-type polymer of embodiment one and linear polymer with PEG-PDLLA It is standby.Specially：Take 100 mL star-type polymers or linear polymer solution (10 mg/mL in DMSO) and 30 mL PEG- PDLLA solution (10 mg/mL in DMSO) is uniformly mixed, and is added drop-wise to 870mL phosphate buffer solutions（PB, 10 mM, pH 7.4）In, 0.5 h is stirred at room temperature, the solution is then placed in bag filter（MWCO3500）It is interior, by it in largely dialysis medium （PB, 10 mM, pH 7.4）Middle dialysis 24 hours, changes five water, obtains non-cross-linked polymer nano-particle sPLy NPs, lPLy NPs.The size of obtained polymer nano-particle is by dynamic light scattering particle size analyzer（DLS）The nano-particle of the formation of survey is 85 nm, particle diameter distribution are very narrow.
Embodiment eight prepares the targeting cross-linking nanoparticles and non-crosslinked nano-particle that cRGD is targeted molecular
CRGD is the preparation of the targeting cross-linking nanoparticles of targeted molecular：Certain mass ratio is pressed in 30 mL DMSO by PEG- PDLLA and cRGD-PEG-PDLLA dissolvings, using PEG-PDLLA and cRGD-PEG-PDLLA as second polymer, with reference to embodiment Seven method prepares targeting cross-linking nanoparticles, is solid ball shape structure.PEG-PDLLA and cRGD-PEG-PDLLA is not by year-on-year Example mixing can prepare cross-linking nanoparticles of the surface with different quality than targeted molecular, when cRGD-PEG-PDLLA is poly- second When content in conjunction object is 50 wt.%, DLS measures targeting crosslinking vesicle size as 85 nm or so, and particle diameter distribution is relatively narrow.With CRGD is that the targeting cross-linking nanoparticles of targeted molecular are referred to as cRGD XNPs.
CRGD is the preparation of the non-crosslinked nano-particle of targeting of targeted molecular：Certain mass ratio is pressed in 30 mL DMSO will PEG-PDLLA and cRGD-PEG-PDLLA dissolving, using PEG-PDLLA and cRGD-PEG-PDLLA as second polymer, respectively with The star-type polymer of embodiment one is assembled with linear polymer, and prepared by the method for reference implementation example seven targets non-crosslinked nanoparticle Son.PEG-PDLLA and cRGD-PEG-PDLLA can prepare surface with different quality than targeted molecular by different proportion mixing Cross-linking nanoparticles, when contents of the cRGD-PEG-PDLLA in second polymer is 50 wt.%, DLS measures targeting crosslinking Vesicle size is 90 nm or so, and particle diameter distribution is relatively narrow.It is referred to as by the non-crosslinked nano-particle of the targeting of targeted molecular of cRGD cRGD NPs。
Embodiment nine prepares the targeting cross-linking nanoparticles that GE11 polypeptides are targeted molecular
It is assembled by second polymer and sP-LA of PEG-PDLLA and GE11-PEG-PDLLA, as the method for embodiment seven prepares target To cross-linking nanoparticles.When contents of the GE11-PEG-PDLLA in second polymer is 20 wt.%, DLS measures targeting crosslinking Vesicle size is 87 nm or so, and particle diameter distribution is relatively narrow.
Embodiment ten prepares the bis- targeting cross-linked polymer nano particles of GE11/TAT
It is assembled by second polymer and sP-LA of PEG-PDLLA, GE11-PEG-PDLLA and TAT-PEG-PDLLA, such as embodiment Seven method prepares cross-linking nanoparticles.In second polymer, the content of GE11-PEG-PDLLA is 20 wt.%, TAT-PEG- The content of PDLLA is 20 wt.%, and it is 104 nm or so that DLS, which measures targeting crosslinking vesicle size, and particle diameter distribution is relatively narrow.
Embodiment 11 prepares the targeting cross-linking nanoparticles and non-crosslinked nano-particle that HA is targeted molecular
With reference to the method for embodiment seven, PEG-PDLLA is replaced with into HA-b- PDLLA and sP-LA is assembled altogether, obtains cross-linked polymeric The nano-particle of object nano-particle HA-sPLy XNPs, the formation that DLS is surveyed are 90 nm, and particle diameter distribution is very narrow, sees Fig. 6 A；By scheming 6B it is found that TEM measure nano-particle be solid ball shape structure, cross-linking nanoparticles in the presence of dilution for many times and fetal calf serum still The grain size and particle diameter distribution so remained unchanged（Fig. 6 C）, but the quick release in the case where simulating tumour cell reducing environment, solution crosslinking（Figure 6D）.It follows that the crosslinked property of solution that obtained cross-linking nanoparticles have reduction sensitive, is suitable for pharmaceutical carrier.
With reference to the method for embodiment seven, PEG-PDLLA is replaced with into HA-b- PDLLA is star-like poly- with embodiment one respectively Object and linear polymer self assembly are closed, non-cross-linked polymer nano-particle HA-sPLy NPs, HA-lPLy NPs are obtained, formation Nano-particle is respectively 105 nm, 110 nm, and particle diameter distribution is very narrow.
12 crosslinking nano drug of embodiment and non-crosslinked Nano medication load adriamycin and release in vitro
Targeting cross-linking nanoparticles are prepared with solvent displacement, prepared by load medicine cross-linking nanoparticles, the preparation side of airborne nanoparticles Method is similar.Specifically, taking 100 mL sP-LA solution and 30 mL PEG-PDLLA, the DMSO solution of 15 mL DOX is added (10 mg/mL) is uniformly mixed, and mixed liquor is added drop-wise to 850mL phosphate buffer solutions（PB, 10 mM, pH 7.4）In, room temperature Stir 0.5 h.Under condition of nitrogen gas, the DTT solution of 10 mM, sealed vial, in 37 DEG C, 100 rpm constant-temperature tables is added It is incubated 10 h.Then the solution is placed in bag filter（MWCO3500）It is interior, by it in largely dialysis medium（PB, 10 mM, pH 7.4）Middle dialysis 24 hours, changes five water, obtains carrying medicine（DOX）Cross-linking nanoparticles, size are analyzed by dynamic light scattering particle size Instrument（DLS）It is 85 nm to survey, and particle diameter distribution is very narrow.In addition, also it is prepared for noncrosslinking nano-particle as a control group, preparation side Method is identical as cross-linking nanoparticles, but DTT need not be added to be crosslinked.Obtained Nano medication is named as DOX-sPLy XNPs, DOX- SPLy NPs, DOX-lPLy NPs indicate that the drug carried is DOX, and XNPs indicates that cross-linking nanoparticles, NPs indicate non-crosslinked and receive Rice corpuscles, other names and so on.
The load different proportion medicine obtained using the DOX DMSO solution of various concentration（10%-30wt.%）Cross-linked polymer The grain size of nano-particle is in 130-150 nm, and particle diameter distribution is in 0.15-0.19；Fluorescence Spectrometer measures Inventive polymers nanometer Particle is 85%-98% to the package efficiency of DOX.
Load medicine cross-linking nanoparticles (DOX-sPLy XNPs) are to the extracorporeal releasing experiment of DOX in 37 DEG C of constant-temperature tables Concussion（200 rpm）It carries out, every group respectively there are three Duplicate Samples.First group, 10 mM are being added in the cross-linking nanoparticles for carrying DOX GSH is simulated in intracellular reducing environment PB (10 mM, pH 7.4)；Second group, the cross-linking nanoparticles of DOX are carried in PB (10 MM, pH 7.4) in；A concentration of 100 mg/L for carrying medicine cross-linking nanoparticles, takes 0.5 mL to be put into bag filter（MWCO: 12, 000）In, corresponding dialysis 25 mL of solvent is added in each test tube and takes out outside 5.0 mL bag filters in predetermined time interval Portion's medium is used as test, while 5.0 mL respective medias are added into test tube.Drug concentration in solution is measured using luminoscope. Attached drawing 4D is the relationship of DOX cumulative release amounts and time, it can be seen from the figure that being added in simulation tumour cell after GSH, is released The sample for being signifi-cantly more rapidly than and not adding GSH is put, illustrates to carry medicine cross-linking nanoparticles in the presence of the GSH of 10 mM, can be released effectively Drug.
The load non-crosslinked nano-particle of medicine is to the extracorporeal releasing experiment and cross-linking nanoparticles of DOX under similarity condition setting It carries out.Attached drawing 4D is the relationship of DOX cumulative release amounts and time, it can be seen from the figure that GSH in simulation tumour cell is added Afterwards, DOX-sPLy NPs, DOX-lPLy NPs do not have apparent humidification to the burst size of DOX, and in normal physiological conditions Under burst size it is similar, slow sustained release behavior is presented.
Release in vitro of the embodiment 13 cRGD targetings crosslinking/non-crosslinked nano-particle to DOX
The method of reference implementation example eight and embodiment 12 prepares cRGD targeting crosslinkings and carries medicine（DOX）Nano-particle (DOX-cRGD XNPs), cRGD targets non-crosslinked medicine-carried nano particles (DOX-cRGD NPs), carries out DOX's according to the method for embodiment 12 Extracorporeal releasing experiment.
Attached drawing 5D is that the relationship of DOX cumulative release amounts and time adds it can be seen from the figure that for DOX-cRGD XNPs Enter and simulate in tumour cell after GSH, release is signifi-cantly more rapidly than the sample for not adding GSH, illustrates to carry medicine cross-linking nanoparticles 10 In the presence of the GSH of mM, drug can be released effectively.The introducing of cRGD does not have an impact release behavior of the nano-particle to DOX.
Attached drawing 5D is the relationship of DOX cumulative release amounts and time, it can be seen from the figure that being added in simulation tumour cell After GSH, DOX-cRGD NPs do not have apparent humidification to the burst size of DOX, with the burst size under normal physiological conditions It is similar, slow sustained release behavior is presented, and there is no changed its release behavior by the introducing of cRGD.
Release in vitro of 14 cross-linking nanoparticles of embodiment to docetaxel (DTX)
The method of reference implementation example 12 prepares crosslinking medicine-carried nano particles (DTX-sPLy XNPs), according to embodiment 12 Method carry out DTX extracorporeal releasing experiment, use high performance liquid chromatograph measure solution in DTX drug concentrations.It is accumulated from DTX As can be seen that being added in simulation tumour cell after GSH in burst size and the relationship of time, release, which is signifi-cantly more rapidly than, does not add GSH Sample, illustrate carry medicine cross-linking nanoparticles in the presence of the GSH of 10 mM, drug can be released effectively.
15 polypeptide of embodiment targets release of the cross-linking nanoparticles to docetaxel (DTX)
The method of reference implementation example nine, embodiment ten and embodiment 12 prepares GE11 targeting crosslinking medicine-carried nano particles (DTX- GE11 XNPs), GE11/TAT targeting crosslinking medicine-carried nano particles (DTX-GE11/TAT XNPs), according to embodiment 12 Method carries out the extracorporeal releasing experiment of DTX, and DTX drug concentrations in solution are measured using high performance liquid chromatograph.It is released from DTX accumulations It is high-volume simulated in tumour cell after GSH with the relationship of time as can be seen that being added, release, which is signifi-cantly more rapidly than, does not add GSH's Sample illustrates to carry medicine cross-linking nanoparticles in the presence of the GSH of 10 mM, can be released effectively drug.The introducing of GE11 and TAT Do not have an impact release behavior of the nano-particle to DOX.
Embodiment 16 HA targetings crosslinking/release of the non-crosslinked nano-particle to docetaxel (DTX)
The method of reference implementation example nine, embodiment ten and embodiment 12 prepares HA targeting crosslinking medicine-carried nano particles (DTX- HA-sPLy XNPs), HA target non-crosslinked medicine-carried nano particles (DTX-HA-sPLy NPs, DTX-HA-lPLy NPs), according to The method of embodiment 12 carries out the extracorporeal releasing experiment of DTX, and it is dense to measure DTX drugs in solution using high performance liquid chromatograph Degree.
Attached drawing 6D is the relationship of DTX cumulative release amounts and time, it can be seen from the figure that being added in simulation tumour cell After GSH, release is signifi-cantly more rapidly than the sample for not adding GSH, illustrates to carry medicine cross-linking nanoparticles in the presence of the GSH of 10 mM, Drug can be released effectively.
Attached drawing 6D is the relationship of DTX cumulative release amounts and time, it can be seen from the figure that under normal physiological conditions, DTX-HA-sPLy NPs, DTX-HA-lPLy NPs are significantly more than cross-linking nanoparticles in similarity condition to the burst size of DTX Under burst size, illustrate that the colloidal stability of non-crosslinked nano-particle is poor.
Influence of the 17 Nano medication surface polypeptide density of embodiment to cell endocytic nano-particle
With 5 × 106A/hole supports cell kind per hole 1mL to cell adherent 70% or so in 6 orifice plates after 24 hours.Then, It is separately added into the nano-particle sample for targeting density containing different polypeptides in each hole of experimental group, each group cell disappears after being incubated 4 h Change centrifugation, is put into dedicated stream measuring pipe after being disperseed again with 500 μ L PBS, is measured by flow cytometer；It is wherein real It tests group and is set as Lipo-DOX（It is existing）、DOX-cRGD XNPs、DOX-sPLy XNPs、DOX-cRGD NPs、DOX-sPLy NPs sets sample blank control wells.
It is measured using cRGD as the endocytosis behavior of the targeted nano-particle of targeted molecular, Fig. 7 in B16F10 cells（A）It is aobvious Show, cross-linking nanoparticles show stronger fluorescence signal due to quickly responding release；The crosslinking nano grain of cRGD modifications exists There is highest fluorescence intensity in cell, see Fig. 7（B）；
The nano-particle marked as the targeting of targeted molecular, Cy5 using GE11 and TAT is measured in MDA-MB-231 breast cancer cells Endocytosis amount, experimental group be Cy5 label, with different TAT ratios double targeting cross-linking nanoparticles（GE11/(10、15、 20、25、30)%TAT XNPs）, Cy5 label the cross-linking nanoparticles with GE11 targeted moleculars（GE11 XNPs）, Cy5 mark The cross-linking nanoparticles (XNPs) of note, Fig. 7（C）Show that the endocytosis amount of the targeting cross-linking nanoparticles with GE11 targeted moleculars is bright Aobvious to be higher than cross-linking nanoparticles, in addition, increasing with TAT amounts, intensity of cellular fluorescence enhances therewith；Next cell is real It is GE11/20%TAT XNPs (later referred to as GE11/TAT XNPs), GE11 to test the experimental group selected with zoopery XNPs、XNPs。
It is measured in A549 cells using HA as targeted molecular, Cy5 labels cross-linking nanoparticles Cy5-HA-sPLy The specific binding Fig. 7 of XNPs to A549 cells（D）.
The cytotoxicity of 17 mtt assay blank testing polymer of embodiment
Mtt assay uses mouse melanin tumor cell（B16F10）, Non-small cell lung carcinoma cell (A549), three cloudy breast cancer cells (MDA-MB-231) cytotoxicity of blank testing nano-particle.With 4 × 103A/hole, by cell kind in 96 orifice plates, per hole 100 μ L are supported after 24 hours to cell adherent 70% or so.Then, it is separately added into containing various concentration in each hole of experimental group（0.1- 1 mg/mL) nano-particle sample, separately set cell blank control wells and culture medium blank well（Multiple 4 holes）.After culture 24 hours, MTT is added per hole（5.0 mg/mL）The crystallization that 150 μ L DMSO dissolvings generate is added in 10 μ L per hole after continuing culture 4 hours, Absorbance value is surveyed at 490 nm with microplate reader（A）, returned to zero with culture medium blank well, calculate cell survival rate.
Attached drawing 8（A）It is sPLy XNPs, sPLy NPs, lPLy NPs to the cytotoxicity result of B16F10, it can be seen that when When the concentration of cross-linked polymer nano particles is from 0.1 increasing to 0.5 mg/mL, the survival rate of B16F10 remains above 90%, illustrates side chain The star-type polymer of sulfur-bearing caprylyl has good biocompatibility；Fig. 8（B）After showing that cRGD XNPs, cRGD NPs are incubated B16F10 cell survival rates be above 95%；Fig. 8（C）Display removes the nano-particle that surface is TAT entirely, other group of nanoparticle Son（GE11/TAT XNPs、GE11 XNPs、XNPs）The MDA-MB-231 cell survival rates of incubation are above 90%；Attached drawing 8（D） HA-sPLy XNPs and HA-sPLy NPs, HA-lPLy NPs are shown after A549 cell incubations, cell survival rate is all higher than 90%。
The above blank nano-particle illustrates that these carriers all have good bio-compatible to the equal very little of the toxicity of cell Property.
18 mtt assay of embodiment surveys toxicity of the drug-carrying polymer nano-particle to cell
DOX-sPLy XNPs, DOX-sPLy NPs, DOX-lPLy NP prepared by testing example 12（DOX is a concentration of 0.001 to 20 μ g/mL）To the cytotoxicity of B16F10.The culture of cell and embodiment 14 are identical, and co-incubation is after 4 hours, Sample replaced with fresh medium is sucked out to continue after being incubated 44 h, MTT then is added, handles and measures absorbance with embodiment ten Seven.
Attached drawing 9 (A) has highest cell inhibitory effect the results show that carrying medicine cross-linking nanoparticles DOX-sPLy XNPs Effect, to the half lethal concentration of cell（IC50）For 1.8 mg/mL, respectively than non-cross-linked control group DOX-sPLy NPs, DOX- The IC of lPLy NPs50 It has been worth low 2.4 and 4.2 times, has illustrated that drug is transmitted into the cell by the cross-linking nanoparticles well, And effectively discharge, finally kill cancer cell.
Cytotoxicities of same method and condition test DOX-cRGD XNPs and the DOX-cRGD NPs to B16F10.Knot Fruit shows that targeting cross-linking nanoparticles have higher cytotoxicity, IC to B16F10 cells50For 0.92 mg/mL, difference Lower than DOX-XNPs, DOX-cRGD NPs and DOX NPs 1.95 times, 3.48 times and 4.70 times, are shown in Fig. 9（B）；Illustrate the targeting Cross-linking nanoparticles have good targeting, can effectively be transmitted to drug into the cell, and effectively discharge, finally It is thin to kill cancer.
The same method and condition test bis- polypeptides of GE11 and TAT are the cross-linking nanoparticles pair of the load DTX of targeted molecular The killing ability of MDA-MB-231 cells.It is most strong that experimental result shows that DTX-GE11/TAT XNPs have MDA-MB-231 Inhibition cell Proliferation ability, IC50For 0.05 mg/mL, DTX-GE11 XNPs, DTX-XNPs, free DTX are low respectively 4.6 times, 9.8 times and 16.2 times, see Fig. 9（C）；Illustrate that this pair targeting cross-linking nanoparticles have to MDA-MB-231 cells Drug can efficiently be transmitted into the cell, and quickly discharge by good targeting, and final kill cancer is thin.
Same method and condition test DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLy NPs To the anti tumor activity in vitro of A549 cells；MTT results show that DTX-HA-sPLy XNPs there is most strong anti-cell to increase A549 Grow effect, IC50For 0.18 mg/mL, respectively than DTX-HA-sPLy NPs, DTX-HA-lPLy NPs are low with free DTX 2.1 times, 6.6 times and 4.6 times, see Fig. 9（D）；Illustrate that the targeting cross-linking nanoparticles have and work is targeted well to A549 cells With efficiently drug capable of being transmitted into the cell, and quickly discharges, final to kill cancer thin.
Embodiment 19 carries the blood circulation of powder
All zoopery operations meet University Of Suzhou's animal experimental center regulation.Experiment select weight be 18 ~ 20 grams or so, 4 The Balb/C nude mices of ~ 6 week old.DOX-sPLy XNPs, DOX-sPLy NPs, DOX-lPLy NPs make all in accordance with embodiment 12 It is standby.Each group load medicine cross-linking nanoparticles are passed through in Tail Vein injection Mouse body（DOX doses are 10 mg/kg）, 0.083, 0.25,0.5,1,2,4,8,12 and 24 hour fixed point takes blood, and blood sample is centrifuged（3000 rpm, 6 min）, 20 μ L of serum are taken, Again plus 100 μ L, 1% Qula is logical and the anhydrous DMSO extractions of 500 μ L（DTT containing 20 mM）；It is surveyed by Fluorescence Spectrometer every The amount of a time point DOX.Figure 10（A）Middle abscissa is the time, and ordinate is the concentration of DOX.As seen from the figure, DOX-sPLy There is XNPs longer circulation time, about 4.94 h to be higher than non-crosslinked nano-particle (DOX-sPLy NPs:4.43 h, DOX-lPLy NPs:3.46 h), thus carry medicine cross-linked polymer vesica in Mice Body relative to non-crosslinked nano-particle more Stablize, there are longer cycle times.
The blood circulation of 20 DOX-cRGD XNPs, DOX-cRGD NPs of embodiment
Animal is the same as embodiment 17.The preparation method such as embodiment eight of DOX-cRGD XNPs, DOX-cRGD NPs, test method With embodiment 19.Figure 10（B）Abscissa is the time, and ordinate is the concentration of DOX.As seen from the figure, DOX-cRGD XNPs have Longer circulation time, about 5.16 h are higher than non-crosslinked nano-particle DOX-cRGD NPs, so carrying medicine cross-linked polymer capsule Bubble is more stable relative to non-crosslinked nano-particle in Mice Body, has longer cycle times, and the introducing of cRGD polypeptides will not King-sized influence is generated on the circulation time in vivo of nano-particle.
Embodiment 21 carries the blood circulation of DTX particles
Animal is the same as embodiment 17.The preparation method of DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-sPLy XNPs Such as embodiment nine, test method surveys the amount of each time point DTX by high performance liquid chromatography with embodiment 19.In Figure 10 (C) Abscissa is the time, and ordinate is the concentration of DTX.As seen from the figure, DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX- There is XNPs similar circulation time, about 5 h to be all considerably longer than free DTX（0.34 h）.So carrying medicine cross-linked polymer Nano-particle has longer cycle times in Mice Body, and the introducing especially introducing of TAT of polypeptide is not to nano-particle Cycle when have a huge impact.
The blood circulation of 22 HA target drug-carrying particles of embodiment
Animal is the same as embodiment 17.The preparation of DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs Method such as embodiment eight, test method survey the amount of each time point DTX by high performance liquid chromatography with embodiment 19.Figure 10 (D) abscissa is the time in, and ordinate is the concentration of DTX.As seen from the figure, when DTX-HA-sPLy XNPs have longer cycle Between, about 4.18 h are higher than non-crosslinked nano-particle DTX-HA-sPLy NPs (3.5), DTX-HA-lPLGA NPs (2.97) With free DTX (0.23)；So carry medicine cross-linked polymer nano particles in Mice Body relative to non-crosslinked nano-particle more Stablize, there are longer cycle times.
23 DOX particles of embodiment are distributed in the vivo biodistribution of lotus B16F10 melanotic tumor mouse
Animal is being subcutaneously injected 1 × 10 with embodiment 175A B16F10 human lung carcinoma cells, after about 7 days, tumor size is 100~200 mm3When start to test.DOX-cRGD XNPs, DOX-cRGD NPs are prepared according to embodiment eight, tail vein injection is small In mouse body（DOX：10 mg/kg）, mouse is put to death after 8 hours, tumour and the heart, liver, spleen, lung and nephridial tissue are taken out, cleaning claims It the Qula of 500 μ L 1% is added after weight crosses refiner all and grind, add the anhydrous DMSO extractions of 900 μ L（Wherein contain 20 The DTT of mM）.Centrifugation（20000 revs/min, 20 minutes）Afterwards, supernatant liquor is taken, each tissue the inside is measured by fluorescence spectrum The amount of DOX.Abscissa is histoorgan in Figure 11 (A), and ordinate is that the DOX in every gram of tumour or tissue accounts for total DOX injection volumes （ID%/g）.It is DOX-cRGD NPs that it is 10.96ID%/g that DOX-cRGD XNPs, which inject 8 hours DOX amounts in tumor accumulation, 2.1 times, it is more to illustrate that load medicine DOX-cRGD XNPs are accumulated by active targeting in tumor locus, there is preferable tomour specific Property targeting.
24 DTX particles of embodiment are distributed in the vivo biodistribution of lotus MDA-MB-231 breast cancer mouses
The inoculation of tumour and tail vein administration are the same as embodiment 18 in biodistribution experiments.It is prepared first according to embodiment nine DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-sPLy XNPs.In Tail Vein injection Mouse body（DTX：10 mg/ kg）, using the method for embodiment 23, the amount of each tissue the inside DTX is measured by high performance liquid chromatography.In Figure 11 (B) Abscissa is histoorgan, and ordinate is that the DTX in every gram of tumour or tissue accounts for total DOX injection volumes（ID%/g）.DTX-GE11/ It is 9.72 ID%/g that TAT XNPs, which inject 8 hours DTX amounts in tumor accumulation, is DTX-GE11 XNPs, DTX-sPLy respectively 1.88,3.12,8.75 times of XNPs and free DTX, illustrate DTX-GE11/TAT XNPs by active targeting in tumor locus Accumulate it is more, have preferable fluorescent dye with tumour-specific targeting effect.
Embodiment 25 HA targetings carry DTX particles and are distributed in the vivo biodistribution of lotus A549 lung cancer in mice
The inoculation of tumour and tail vein administration are the same as embodiment 18 in biodistribution experiments.It is prepared first according to embodiment nine DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs, using the method for embodiment 24.Figure 11 (C) abscissa is histoorgan in, and ordinate is that the DTX in every gram of tumour or tissue accounts for total DOX injection volumes（ID%/g）.DTX- It is DTX-HA-sPLy NPs, DTX- respectively that it is 9.48ID%/g that HA-sPLy XNPs, which inject 8 hours DTX amounts in tumor accumulation, 1.5,2.0,3.9 times of HA-lPLGA NPs and free DTX, illustrate DTX-HA-sPLy XNPs by active targeting in tumour Position accumulation is more, has preferable fluorescent dye with tumour-specific targeting effect.
Therapeutic effect of the 26 DOX particles of embodiment in the mouse of lotus B16F10 melanomas
The inoculation of tumour and tail vein administration are with embodiment 18, and about after two weeks, tumor size is 30 ~ 50 mm3Shi Kai Begin to test.By DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs, Lipo-DOX and PBS respectively 0,2, 4,6 and 8 days by Tail Vein injection Mouse body（DOX doses are 10 mg/kg）, a height is arranged for DOX-cRGD XNPs Dosage administration group（DOX doses are 20 mg/kg）.At 0 ~ 12 day, the weight of mouse, vernier caliper measurement tumour were weighed every three days Volume, gross tumor volume computational methods are：V=（L×W×W）/ 2,（Wherein L, W, H are respectively the length of tumour, width and thickness）. The existence of continuous observation mouse was by 45 days.By in Figure 12 it is found that DOX-cRGD XNPs（DOX:20 mg/kg）Treatment group was at 12 days When, tumour is significantly suppressed, and effect is suitable with Lipo-DOX groups, and mouse weight does not change significantly, and is worth note Meaning is that Lipo-DOX group mouse weights have dropped about 15%.And DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs group tumours have certain growth, but its growth rate and increment are all significantly lower than PBS groups, and do not cause The changes of weight of mouse illustrates that drug-carrying polymer nano-particle does not have toxic side effect to mouse.DOX-cRGD XNPs（DOX:20 mg/kg）The median survival interval of mouse is 43 days, DOX-cRGD XNPs, DOX-sPLy XNPs, DOX-cRGD NPs and Lipo- The median survival interval of DOX and PBS group mouse is respectively 30,26,25,21 and 14 days.Moreover, all treatment groups remove Lipo-DOX groups Outside mouse main organs are not caused significantly to injure, the liver of Lipo-DOX group mouse, spleen and kidney have obviously Damage.
Therapeutic effect of the 27 DTX particles of embodiment in lotus MDA-MB-231 breast cancer mouses
The inoculation of tumour and tail vein administration are the same as embodiment 19.DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX- XNPs and free DTX and PBS is respectively at 0,3,6 and 9 day by Tail Vein injection Mouse body（DTX doses are 5 mg/kg）. At 0 ~ 18 day, the weight of mouse is weighed every three days, as embodiment 26 measured gross tumor volume and observation mouse by 60 days.By scheming In 13 it is found that when DTX-GE11/TAT XNPs, DTX-GE11 XNPs, DTX-XNPs and free DTX treatment groups 18 days, tumour Inhibited in various degree, and PBS group tumours have apparent growth.It is worth noting that DTX-GE11/TAT XNPs, DTX- GE11 XNPs, DTX-XNPs energy significant effective inhibit the growth of tumour, and significant change does not occur for mouse weight.For Free DTX groups, although tumour growth has obtained certain inhibition, mouse weight declines obviously, illustrates free DTX to small Mouse brings serious system toxicity.
Therapeutic effect of the embodiment 28 HA targetings load DTX particles in lotus A549 lung cancer in mice
The inoculation of A549 subcutaneous tumors and tail vein administration are the same as embodiment 19.By DTX-HA-sPLy XNPs, DTX-HA- SPLy NPs, DTX-HA-lPLGA NPs and free DTX and PBS passed through Tail Vein injection Mouse at 0,4,8 and 12 day respectively In vivo（DTX doses are 5 mg/kg）.As embodiment 20 weighs weight, amount gross tumor volume and the observation of mouse in 0 ~ 16 day Mouse was by 60 days.As shown in Figure 14, DTX-HA-sPLy XNPs, DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs and from When being treated 16 days by DTX, tumour obtains to be inhibited to a certain extent, and PBS group tumours have apparent growth.It is worth noting that, removing Except free DTX groups, mouse weight is without significant change, and due to the toxic side effect that DTX is strong, mouse weight declines seriously, About 15% was had dropped at 16 days, these results suggest that medicine-carried nano particles have preferable biocompatibility, to mouse without poison Side effect.In addition to this, DTX-HA-sPLy XNPs treatment groups are relative to DTX-HA-sPLy NPs, DTX-HA-lPLGA NPs Treatment group has significant difference, shows the effect of most excellent inhibition tumour growth.

Claims (10)

1. a kind of Nano medication based on end sulfur-bearing caprylyl star-type polymer, which is characterized in that described to be based on end sulfur-bearing The Nano medication of caprylyl star-type polymer by reversible crosslink there is the polymer nano-particle loading drug of reduction response to obtain It arrives；
The reversible crosslink have reduction response polymer nano-particle by end sulfur-bearing caprylyl star-type polymer with Second polymer assembles be prepared altogether；The second polymer is amphipathic nature polyalcohol and/or target direction amphipathic nature polymer；
The chemical structural formula of the star-type polymer of the end sulfur-bearing caprylyl is as follows：
Wherein, x:Y is (1~3): 1.
2. the Nano medication according to claim 1 based on end sulfur-bearing caprylyl star-type polymer, which is characterized in that described The preparation method of Nano medication based on end sulfur-bearing caprylyl star-type polymer includes the following steps：
（1）Under nitrogen environment, under the conditions of ice-water bath, by N, N- dicyclohexylcarbodiimide solution is added dropwise in lipoic acid solution； After being added dropwise to complete, sealing room temperature reaction 10~15 hours；Obtain lipoic acid anhydride solution；
（2）Under vacuum environment, using polyhydroxy glucose as initiator, under the catalytic action of stannous octoate, lactide and second Lactide reacts 8 hours at 160 DEG C；Obtain star-type polymer；
（3）Under nitrogen environment, lipoic acid anhydride solution is added to organic containing 4-dimethylaminopyridine and star-type polymer In solvent, under air-proof condition, reacted 1~3 day in 30 DEG C；Obtain the star-type polymer of end sulfur-bearing caprylyl；
（4）The star-type polymer of end sulfur-bearing caprylyl is assembled in a solvent with second polymer, small-molecule drug and is received Rice drug.
3. the Nano medication according to claim 2 based on end sulfur-bearing caprylyl star-type polymer, which is characterized in that step （1）After reaction, filtering reacting liquid obtains lipoic acid anhydride solution；Solvent is two in N, N- dicyclohexylcarbodiimide solution Chloromethanes；Solvent is dichloromethane in lipoic acid solution；N, N- dicyclohexylcarbodiimide, lipoic acid molar ratio be 2: 1.
4. the Nano medication according to claim 2 based on end sulfur-bearing caprylyl star-type polymer, which is characterized in that step （2）Product dissolving in methylene chloride, is then precipitated in ice methanol, is filtered, vacuum drying obtains star-like gather after the completion of reaction Close object；Polyhydroxy glucose, lactide, glycolide molar ratio be 1:（50~55）∶（60~65）.
5. the Nano medication according to claim 2 based on end sulfur-bearing caprylyl star-type polymer, which is characterized in that step （3）Reactant is precipitated in ice ether after reaction, then end sulfur-bearing caprylyl is obtained through filtering simultaneously Vacuum dry filter cake Star-type polymer；Organic solvent is dichloromethane in organic solvent containing 4-dimethylaminopyridine and star-type polymer； Lipoic acid acid anhydride, 4-dimethylaminopyridine, star-type polymer molar ratio be 7.5: 10: 1.
6. the Nano medication according to claim 1 based on end sulfur-bearing caprylyl star-type polymer, it is characterised in that：It is described Amphipathic nature polyalcohol is PEG-PDLLA；The target direction amphipathic nature polymer be cRGD-PEG-PDLLA, GE11-PEG-PDLLA, TAT-PEG-PDLLA、HA-bOne or more of-PDLLA.
7. the Nano medication based on end sulfur-bearing caprylyl star-type polymer described in claim 1, it is characterised in that：Described second Polymer volume is the 10%~60% of the star polymerization amount of substance of end sulfur-bearing caprylyl.
8. the Nano medication according to claim 1 based on end sulfur-bearing caprylyl star-type polymer, it is characterised in that：It is described Drug includes adriamycin, taxol, Docetaxel.
9. the Nano medication according to claim 1 based on end sulfur-bearing caprylyl star-type polymer, it is characterised in that：It is described The molecular weight of the star-type polymer of end sulfur-bearing caprylyl is 10000~75000.
10. the Nano medication based on end sulfur-bearing caprylyl star-type polymer described in claim 1, it is characterised in that：When second When polymer is amphipathic nature polyalcohol and target direction amphipathic nature polymer, the mass percent of target direction amphipathic nature polymer is less than 70%.
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