CN104146964A - Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof - Google Patents
Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a multipurpose polylysine fluorescence self-assembly nano microsphere carrier and a preparation method and application thereof. By nano self-assembly technique, rhodamine marked polylysine fluorescence self-assembly nano microspheres can be prepared, azide group side chains with disulfide bonds can be modified, and by use of a click chemistry method, an alkynyl modified drug is connected with the fluorescent nano microspheres. The polylysine self-assembly nano microspheres linked with the drug can be used for tracing imaging analysis of the modified drug in animal and cell level. At the same time, the polylysine self-assembly nano microspheres can also be used to capture binding proteins of the modified drug, can release target binding proteins and associated proteins by cleavage of the disulfide bonds, and can be used for the study of drug effect targets. The invention provides a simple and fast tool and method for the study of the drug effect targets, and the method integrates drug tracing, target positioning and target protein capture and separation functions, is simple and practical, and has good application prospect in the study of drug action mechanisms and target discovery.
Description
Technical field
The invention belongs to biological medicine technology field, relate to a kind of microsphere supported preparation technology of polylysine nanoassemble who is connected with fluorescent tracing group, and further adopt click chemistry technology covalent coupling by the method for modified medicaments.And utilize the prepared medicine carrying microballoons of the present invention, and to the spike imaging analysis of the whole animal of being undertaken by modified medicaments and cellular level, drug target location, target point protein is caught the application of separation aspect.
Background technology
Epsilon-polylysine (Poly-L-lysine, PL) is the homotype monomer straight chain polymer being connected in sequence by amido link by 25～30 lysine residues.Polylysine is white or pale yellow powder, and hygroscopicity is strong, and slightly bitterness is not affected by pH value.Epsilon-polylysine is a kind of polypeptide with bacteriostasis efficacy, and it has, and antimicrobial spectrum is wide, water solublity is large, Heat stability is good, biodegradable and to advantages such as human and environment nonhazardouss, has become good food preservative freshness retaining agent.Epsilon-polylysine can be decomposed into lysine in human body, and lysine is the aminoacid of needed by human, is also that countries in the world allow the aminoacid of strengthening in food.Therefore epsilon-polylysine is a kind of auxotype antibacterial, safe in other chemical preservatives, and its acute oral toxicity is 5 g/kg.The antimicrobial spectrum of epsilon-polylysine is wide, for the sharp-pointed candida mycoderma in Saccharomyces, method Rhodothece glutinis, product film pichia yeast, Flos Rosae Rugosae shadow yeast; Heat-resisting fatty bacillus cereus in gram positive bacteria, Bacillus coagulans, bacillus subtilis; And aerogenesis arthrobacterium, escherichia coli etc. in gram negative bacteria cause that alimentary toxicosis and corrupt bacterium have strong inhibitory action.Its bacteriostatic activity of the epsilon-polylysine of molecular weight between 3600～4300 is best, and when molecular weight is lower than 1300 time, epsilon-polylysine loses bacteriostatic activity.
In addition, due to epsilon-polylysine positively charged, can there is strong electrostatic attraction with electronegative material, easily by biomembrane and difficult decomposition, can effectively reduce the transport efficacy of medicine resistance raising medicine.Polylysine also has energy Promote cell's growth and adhesion and promotes the effects such as cells play normal function, has therefore been widely used in biomedicine field as slow release and the targeting vector epsilon-polylysine of medicine.As bioabsorbable polymer material skeleton, Chinese patent (application number: the preparation method that 2014100723354) discloses a kind of solubilized thrombin nano-particle, this nano-particle is taking thrombin as core, and shell is the nanoparticle being cross-linked to form by water solublity amylose and polylysine; Chinese patent (application number: 2013107289643,2013106763740) preparation method of epsilon-polylysine hydrogel is disclosed respectively, and the wound tissue being derived by this invention healing material; Chinese patent (application number: the nano-carrier preparation that 2012101329801) a kind of amphipathic three block copolymer is provided, this copolymer forms by comprising polyethyleneglycol derivative, polylysine and poly-leucic linear polymer, in aqueous solution, can self assembly form the nano-carrier with three-decker, and energy payload micromolecule hydrophobic drug, gene and protein or polypeptide.
As pharmaceutical carrier, Chinese patent (application number: the sensitive response type polymer nano-micelle that 2013106705336) a kind of carrier as hydrophobic drug is provided, its composition is the poly-cysteine of n-butylamine-polylysine (folic acid/2,3-dimethyl maleic acid)-b-; Chinese patent (application number: 2004100680618) disclose a kind of short peptide modified polylysine and polylactic copolymer nano particle, this nanoparticle comprises that medicine and the polylysine-polylactic copolymer nano particle carrier by Arg-Gly-Asp sequence small peptide, modification form, and can be used for organic drug, water soluble drug or water-insoluble cancer therapy drug; Chinese patent (application number: 2004100466794) preparation method of the synthetic galactose sodium alginate of a kind of application " plastic phenomenon " and poly-D-lysine nanometre glue is provided, and the hepatic targeting nano-gene carrier system obtaining can provide material for the target gene therapy of primary hepatocarcinoma; In addition; Chinese patent (application number: 2013101802363) also disclose a kind of sensitive disintegratable formula nano vesicle pharmaceutical carrier, wherein sensitive amphipathic nature block polymer forms by reducing the polylysine of the carbon-to-nitrogen double bon bridging hydrophilic polyglycol of responsive disulfide bond and pH sensitivity and the protection of hydrophobicity benzyloxycarbonyl group.This carrier formulation has hydrophobic bimolecular film and hydrophilic inner chamber, hydrophobic bimolecular film load dewatering medicament, hydrophilic inner chamber load hydrophilic drugs, can effectively utilize reducing environment and sour environment in tumor cell to make carrier formulation disintegrate release medicine realize targeting drug release.
As the skeleton of fluorescence imaging nano material, Chinese patent (application number: the preparation method that 2012102228846) discloses a kind of functional near-infrared fluorescent nanoparticle.This nanoparticle mean diameter is in about 15nm, using the near infrared fluorescent dye that loaded as the centre of luminescence, taking chitosan, polylysine as basic framework, is rolled into shell is prepared from through sodium alginate self assembly; Chinese patent (application number: 201310470907X) also provides a kind of preparation method of signal scale-up version immune fluorescent probe.The preparation method of this probe comprises: under the existence of condensing agent, p-phenylenediamine (PPD) and trimesic acid obtain many carboxyls macromole through condensation reaction, after many carboxyls macromole is activated, add successively antibody, polylysine to react, then make described probe with fluorescent labeling substance markers.This probe mark has more fluorescent marker, and Stability Analysis of Structures can be used for fluorescence immunoassay and detects, and has the features such as detection sensitivity is high, detection time is short, cost is low.In addition, Chinese patent (application number: 2013101802363) also disclose the poly-polypeptide ICG micelle of a kind of amphipathic three blocks, this micelle has comprised the kernel that is dispersed with indocyanine green and is formed by poly-leucine, and the intermediate layer being formed by polylysine around kernel and be partly interspersed in the shell being formed by Polyethylene Glycol in described intermediate layer.This micelle possesses good spatial stability and outstanding fluorescence property, photo-thermal transfer capability, can realize tumor cell or tissue are carried out to optical imagery and photo-thermal therapy.
In sum, at present both at home and abroad all carry out the exploitation of associated materials taking polylysine or other macromolecular materials of blend as skeleton.But there is not yet with polylysine from as skeleton, on the one hand by the covalently bound fluorescence molecule of amido link (rhodamine), and prepare nano-particle by self-assembling technique, adopt on the other hand click chemistry technology covalent bond releasable medicaments molecule; Can carry out by the inside and outside spike imaging analysis of modified medicaments, target spot location and target point protein are caught separation simultaneously, the report of the polylysine nano material that set above-mentioned functions purposes is integrated.
Summary of the invention
The object of the invention is to utilize nanoassemble technology, prepare polylysine medicament-carrying nano-microsphere by introducing fluorophor and disulfide bond group that can cleaved release, and utilize the biocompatibility of polylysine to realize by modified medicaments in animal tissue and intracellular spike, and target point protein and associated protein thereof catch separation, provide a kind of multipurpose polylysine fluorescence self-assembled nanometer microsphere supported preparation method and application.
The present invention can solve prior art be difficult to realize medicine spike and target spot catch integrated problem.And taking the medicine that contains hydroxyl or carboxyl as example, modify and introduce alkynyl by the amidatioon of acetylenecarboxylic acid esterification or propargylamine, and by with microsphere on the azido group modified click chemistry reaction by medicine be connected to microsphere supported on, use same nano drug-carrying microsphere, realized respectively the spike of medicine internal metabolism, thin inner cellular localization and target point protein and caught the different application such as separation.
The invention provides a kind of multipurpose polylysine fluorescence self-assembled nanometer microsphere supported, this carrier is covalently boundly on framework material to have fluorescent labeling group and drug molecule to make; Described framework material is the homotype monomer-polymer being connected in sequence by amido link by containing 25～30 lysine residues; Lysine residue on described framework material has been modified azido group side chain and the fluorescent marker with disulfide bond simultaneously, can prepare Nano microsphere by self assembly, and can be used for the connection of drug molecule.
Described fluorescent labeling group is the labelled molecule that contains rhodamine.Specifically, by commercially available rhodamine Acibenzolar (NHS-rhodamine), be connected with Nano microsphere carrier with the form of amido link with the amino of polylysine.
The described azido group side chain with disulfide bond, specifically, by commercially available Azide reagent (Sulfo-SADP), its one end is Acibenzolar, is connected with the form of amido link by the amino of polylysine with Nano microsphere carrier.
Described drug molecule, it is alkynyl-modified medicaments derivative, carry out esterification by acetylenecarboxylic acid and the medicine that contains hydroxyl and introduce alkynyl, or carry out amidation process by propargylamine and the medicine that contains carboxylic acid and introduce alkynyl, and further the method by click chemistry by its alkynyl-modified medicaments derivative with contain nitrine side chain Nano microsphere carrier and be connected.
Concrete preparation method is as follows:
1st, in the mixing organic facies being formed by gasoline and carbon tetrachloride (volume ratio of gasoline and carbon tetrachloride can be 1:3 to 3:1) that contains 1% to 5% Span-80, add NHS-rhodamine to dissolve, and add again the aqueous phase solution of the polylysine that is dissolved with 0.2%-2%.Wherein the ratio of organic facies and water is 1:1 to 5:1, and the consumption of NHS-rhodamine is polylysine 0.5% to 10%.10,000 to 20,000 obtain emulsion after turning high-speed stirred.
2nd, above-mentioned emulsion is transferred in separatory funnel, add respectively petroleum ether and the aqueous solution of above-mentioned volume ratio 1:1 to 5:1, stratification after mix homogeneously, collects water, the centrifugal precipitation that discards, supernatant is the polylysine fluorescent nanometer microsphere of self assembly;
3rd, in the Nano microsphere solution obtaining to the 2nd step, add Azide reagent Sulfo-SADP, stirring at room temperature reaction 0.5 hour to 5 hours, obtains the Nano microsphere that nitrine is modified, and wherein the consumption of Sulfo-SADP is 10% to 100% of polylysine consumption;
4th, further alkynyl-modified drug slow is splashed in the Nano microsphere solution that the 3rd step obtains, under stirring condition, add the catalyst of click chemistry reaction, room temperature reaction 0.5 hour to 5 hours, reactant liquor, through ultrafiltration and concentration, is the fluorescent nanometer microsphere that is connected with medicine.Wherein the consumption of alkynyl-modified medicine is 10% to 100% of polylysine consumption; Described alkynyl-modified medicine, is the propiolate that adopts acetylenecarboxylic acid and the medicine that contains hydroxyl to prepare by the mode of esterification, or the propioloyl amine derivative that adopts propargylamine and the medicine that contains carboxyl to prepare by amidated mode.
In addition, the present invention also provides the application aspect catching at medicine spike and target point protein of above-mentioned polylysine fluorescence self-assembled nanometer microsphere.Comprising by the imaging analysis of the animal of modified medicaments arctigenin and cellular level; And the Nano microsphere that utilizes enoxolone to modify catches the process of enoxolone target point protein, comprise by nanofiltration classification and hold back separation, cracked disulfide bond discharges the concrete steps of target point protein.
One of application of Nano microsphere provided by the invention: the application aspect medicine spike.
First the present invention has prepared the polylysine fluorescence self-assembled nanometer microsphere that is loaded with arctigenin, and by laser particle analyzer, tem study particle diameter and the shape of above-mentioned microsphere; Adopt fluorescence spectrophotometer to investigate its fluorescent characteristic; And further evaluate its distribution situation at body and cellular level by laser confocal microscope and small animal living body imaging technique.
Two of the application of Nano microsphere provided by the invention: target point protein is caught the application of aspect.
Target point protein described in the present invention is caught the application of aspect, utilize ultrafiltration classification to hold back separating trap target point protein, comprise the following steps: incubated cell, lysis, centrifugal, ultrafiltration remove impurity, washing, reduction discharge the steps (accompanying drawing 1) such as albumen, ultrafiltration eluting.Concrete operations are as follows:
In people's lung epithelial BEAS-2B cell, add the polylysine fluorescence self-assembled nanometer microsphere that connects enoxolone, cultivate after 48 hours, through trypsinization, ultrasonication, centrifugal, obtain being combined with the polylysine Nano microsphere suspension of target proteins.The above-mentioned solution that contains Nano microsphere is joined in the super filter tube that molecular cut off is 3 kD, the centrifugal micromolecule that discards, trapped fluid is joined in the super filter tube that molecular cut off is 100 kD, centrifugally discard unconjugated high molecular weight protein, further in trapped fluid, add dithiothreitol, DTT (DTT) solution reduction disulfide bond, again join in the super filter tube that molecular cut off is 100 kD, centrifugal, collect filtrate and obtain target point protein and the associated protein of being combined with enoxolone.
In sum, the invention provides a kind of drugs and distribute and the needed simple and efficient carrier tool and method of action target spot, this polylysine medicine carrying fluorescent nanometer microsphere, collection target spot location, target point protein is caught with separation function in one, can be used for the research of mechanism of drug action.
advantage of the present invention and good effect:
The invention provides a kind of new method of preparing multipurpose polylysine fluorescence nano self assembly medicine carrying microballoons, and introduce rhodamine fluorescent labeling when novelty and can be reduced the azido group of the disulfide bond of cracking, react the alkynyl-modified medicine of connection by click chemistry.Prepared nanoassemble medicine carrying microballoons is spherical in shape, and granularity is uniformly dispersed, and mean diameter is 38 nm, has good colloidal nature, and maximum emission wavelength is 600 nm, can effectively avoid the interference of biological specimen self, by fluoroscopic examination and spike.This Nano microsphere good biocompatibility, is conducive to through cell membrane simultaneously, can successfully indicate and catch target point protein, and can discharge target point protein and related protein thereof by cracked disulfide bond.The method preparation technology is simple and easy to do, prepared medicament-carrying nano-microsphere collection target spot localization, target point protein catch and separation function in one, can be used for by the imaging analysis of the animal of modified medicaments and cellular level, and target point protein isolation identification, aspect the research of mechanism of drug action, there iing good application prospect.
Brief description of the drawings
Fig. 1 is embodiment 1,2 and 3, the synthetic route chart of the polylysine fluorescent nanometer microsphere that nitrine is modified;
Fig. 2 is embodiment 4, the alkynyl of drug molecule arctigenin modify and with polylysine fluorescent nanometer microsphere adapter path schematic diagram;
Fig. 3 is embodiment 5, and the morphology of polylysine fluorescence nano self assembly medicament-carrying nano-microsphere and fluorescent characteristic are investigated.Wherein, A disperses situation photo in Nano microsphere solution; B is the colloid property schematic diagram of Nano microsphere; C is Nano microsphere particle size distribution figure; D is the overall photo of Nano microsphere TEM transmission electron microscope; E is the enlarged photograph of Nano microsphere TEM transmission electron microscope; F is the fluorescence emission spectrogram of rhodamine solution and polylysine fluorescence nano self-assembly microspheres colloid solution;
Fig. 4 is embodiment 6, the alkynyl of drug molecule enoxolone modify and with polylysine fluorescent nanometer microsphere adapter path schematic diagram;
Fig. 5 is embodiment 7, the cell imaging figure of the polylysine fluorescent nanometer microsphere of arctigenin medicine carrying and mice living imaging spike picture.A organizes the negative control that adds the blank Nano microsphere of the prepared polylysine fluorescence of embodiment 1 for cell, and wherein, A1 is cell DAPI dyeing picture, and A2 is cell fluorescence photo, and A3 is the stack photo of A1 and A2; B organizes the photo that adds the polylysine fluorescence nano medicine carrying microballoons of the prepared aretigenin modification of embodiment 2 for cell, and wherein, B1 is cell DAPI dyeing picture, and B2 is cell fluorescence photo, and B3 is the stack photo of B1 and B2; The mice vivo tracking picture that C group is the prepared polylysine fluorescence nano medicine carrying microballoons of embodiment 2; Wherein, C1 is first 0 minute of administration, and C2 is the living imaging picture of 60 minutes after administration;
Fig. 6 is embodiment 8, and polylysine nano drug-carrying microsphere is caught the operation chart of target point protein;
Fig. 7 is embodiment 8, and the prepared enoxolone medicament-carrying nano-microsphere of embodiment 5 is caught the SDS-PAGE electrophoresis detection figure of target point protein.Wherein, the 1st swimming lane is albumen Marker; The 2nd swimming lane is lysis protein sample; The 3rd swimming lane is the filtered solution through 3 kD super filter tube ultrafiltration; The 4th swimming lane is to hold back albumen in filtrate; After the 5th swimming lane is DTT reduction, through the trapped fluid of 100 kD super filter tube ultrafiltration; After the 6th swimming lane is DTT reduction, the filtered solution after the super filter tube ultrafiltration of 100 kD.
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Detailed description of the invention
the preparation of embodiment 1, polylysine height fluorescence decorated nanometer microsphere and Azide are modified
Get 5 g polylysine hydrochlorates and be dissolved in 500 mL pure water, be splined on 001 × 7 cation exchange resin (2 × 80cm) desalination of activation, loading speed is 1 mL/ minute.After 1500 mL pure water balance to effluent are neutrality, with 5% ammonia eluting, collect eluent, distilling under reduced pressure, concentrate drying, obtains white to faint yellow solid 1.9 g.
Get polylysine 200 mg after above-mentioned desalination in 10 mL pure water, stir and make it dissolve to obtain aqueous phase solution completely; Respectively 13 mL gasoline, 12 mL carbon tetrachloride and 1.25mL Span-80 are mixed, acquisition organic facies stirs simultaneously.In organic facies, add 20 mg NHS-rhodamines (NHS-Rhodamine, Pierce 46406), stirring and evenly mixing, places for subsequent use.Above-mentioned water is poured into rapidly in organic facies, and 12,000 turn stirring 30 seconds, repeat 3 times, obtain uniform emulsion, are transferred in separatory funnel.Add respectively 50 mL petroleum ether, stirring at room temperature 5 minutes, then add 10 mL pure water, and continuing to stir 5 minutes, stratification, collects water, obtains fluorescent nanometer microsphere crude product.Crude product 12,000 is left to the heart 10 minutes, discard precipitation, supernatant is the nanoassemble microsphere that polylysine fluorescenceization is modified.
Get above-mentioned 2 mL polylysine fluorescent nanometer microsphere solution, add Azide reagent 10 mg Sulfo-SADP(Pierce 21553) (0.02 mmoL), stirring at room temperature 0.5 hour, must be with the nanoassemble polylysine Nano microsphere of disulfide bond nitrine modification, 4 DEG C save backup.Synthetic route as shown in Figure 1.
the preparation of embodiment 2, polylysine fluorescent nanometer microsphere and Azide are modified
Polylysine 50 mg that get after above-described embodiment 1 desalination are dissolved in 10 mL pure water, obtain aqueous phase solution; Respectively 5mL gasoline, 15mL carbon tetrachloride and 0.5 mL Span-80 are mixed, acquisition organic facies stirs simultaneously.In organic facies, add 0.1 mg NHS-rhodamine (NHS-Rhodamine, Pierce 46406), stirring and dissolving, places for subsequent use.Above-mentioned water is poured into rapidly in organic facies, and 20,000 turn high-speed stirred 15 seconds, repeat 3 times, obtain uniform emulsion, are transferred in separatory funnel.Add respectively 20 mL petroleum ether, stir 5 minutes, then add 10 mL pure water, continue to stir 5 minutes, stratification, collects water, obtains fluorescent nanometer microsphere crude product.Crude product 12,000 is left to the heart 10 minutes, discard precipitation, supernatant is polylysine fluorescence nano self-assembly microspheres.Get above-mentioned polylysine fluorescent nanometer microsphere solution 2 mL, add Azide reagent 50 mg Sulfo-SADP(Pierce 21553) (0. 1 mmoL), stirring at room temperature 5 hours, obtains the nanoassemble polylysine Nano microsphere that Azide is modified, and 4 DEG C for subsequent use.The same Fig. 1 of synthetic route.
the preparation of embodiment 3, polylysine fluorescent nanometer microsphere is modified with height Azide
Polylysine 20 mg that get after above-described embodiment 1 desalination are dissolved in 10 mL pure water, obtain aqueous phase solution; Respectively 15 mL gasoline, 5 mL carbon tetrachloride and 0.2mL Span-80 are mixed, acquisition organic facies stirs simultaneously.In organic facies, add 2 mg NHS-rhodamines (NHS-Rhodamine, Pierce 46406) (0.002 mmoL), stirring and dissolving, places for subsequent use.Above-mentioned water is poured into rapidly in organic facies, and 10,000 turn high-speed stirred 20 seconds, repeat 3 times, obtain uniform emulsion, are transferred in separatory funnel.Add respectively 10 mL petroleum ether, stir 5 minutes, then add 10 mL pure water, continue to stir 5 minutes, stratification, collects water, obtains fluorescent nanometer microsphere crude product.Crude product 12,000 is left to the heart 10 minutes, discard precipitation, supernatant is polylysine fluorescence nano self-assembly microspheres.Get above-mentioned polylysine fluorescent nanometer microsphere solution 4 mL, add Azide reagent 200 mg Sulfo-SADP(Pierce 21553) (0.4 mmoL), stirring at room temperature 5 hours, can obtain the nanoassemble polylysine Nano microsphere that height Azide is modified, and 4 DEG C for subsequent use.The same Fig. 1 of synthetic route.
the alkynyl of embodiment 4, arctigenin modify and with being connected of polylysine fluorescent nanometer microsphere
Taking the medicine aretigenin that contains phenolic hydroxyl group as example, by carrying out the alkynyl modification of medicine with the esterification of acetylenecarboxylic acid, the polylysine fluorescent nanometer microsphere coupling that the Azide of further reacting prepared with embodiment 1 by click chemistry is modified, synthetic route is shown in Fig. 2.Specifically be implemented as follows:
In round-bottomed flask, add respectively 0.372 g arctigenin (1 mmoL), 0.198 g EDCHCl (2 mmoL), and the CH of 5 mL pre-coolings
2cl
2solvent, magnetic agitation 30 minutes, adds 0.116 g N-hydroxy-succinamide (NHS, 2 mmoL) more successively after dissolving, and 0.140 g acetylenecarboxylic acid (2 mmoL) keeps ice bath reaction 4 hours.After question response finishes, in reactant liquor, add 15 mL CH
2cl
2dilution, then uses 1M HCl, 5% NaHCO successively
3solution, and saturated common salt water washing.Collect organic facies, add anhydrous Na
2sO
4dry, to filter, distilling under reduced pressure is concentrated, obtains grease and is arctigenin propiolate crude product.
Crude product is crossed to silicagel column purification, obtain white solid powder, arctigenin propiolate 0.196 g that weighs to obtain, productive rate is about 47%.
Rf?=0.7?(PE?/?EtOAc?1:1);?
1H?NMR?[CDCl
3,?400?MHz]?6.98-6.94?(m,?1H),?6.79-6.75?(m,?2H),?6.68-6.65?(m,?1H),?6.58-6.50?(m,?2H),?3.84(s,?3H),?3.81?(s,?3H),?3.76-3.74?(m,?2H),?2.97-2.95?(m,?2H),?2.83?(s,?1H),?2.66-2.48?(m,?4H)。
The 5 mg arctigenin propiolates (0.01 mmoL) of getting above-mentioned gained are dissolved in 100 μ L dimethyl sulfoxide (DMSO), and it is slowly splashed in the polylysine self-assembled nanometer microspheres solution that 1 mL nitrine prepared by embodiment 1 modifies, finally add catalyst (0.2 mmol/L Tris-triazoleamine, the 1.0 mmol/L CuSO of click chemistry reaction
4with 2.0 mmol/L sodium ascorbates), stirring at room temperature 1 hour.After finishing, reaction joins in the super filter tube that molecular cut off is 3 MilliporekD, 3000 leave the heart 30 minutes, discard filtrate, respectively with 0.2 mmol/L phosphate buffer 2 mL washing 2 times, collect trapped fluid, be the Nano microsphere of the height fluorescenceization modification that is connected with arctigenin.
the morphology of embodiment 5, polylysine fluorescent nanometer microsphere and fluorescent characteristics
The polylysine fluorescence nano medicine carrying microballoons that the prepared arctigenin of above-described embodiment 3 is modified presents orange-yellow in aqueous solution, and be uniformly dispersed (Fig. 3 A).Irradiate by line source, can obviously see and Tyndall phenomenon prove colloid solution, as shown in Fig. 3 B.Through laser particle size analyzer analysis, its mean diameter is 38 nm(Fig. 3 C).Adopt TEM transmission electron microscope to see and look into, the size of this polylysine Nano microsphere is even, is rule spherical (Fig. 3 D), further amplifies observation and shows that its inside is hollow shape, as shown in Fig. 3 E.
Adopt fluorescence spectrophotometer respectively the rhodamine solution to same amount and polylysine fluorescent nanometer microsphere carried out fluorescence spectrum scanning, investigate the fluorescent characteristic of nanoassemble ball.As shown in Fig. 3 F, under the excitation wavelength of 507nm, the maximum emission wavelength of rhodamine solution is 590 nm, and the maximum emission wavelength of polylysine fluorescent nanometer microsphere approaches 600 nm.But the fluorescence intensity of the two is obviously different, and the fluorescence intensity of rhodamine solution is 625 a.u, and the fluorescence intensity of fluorescent nanometer microsphere to be 1149 a.u almost increased by one times.This is because polylysine is assembled into fluorescent dye rhodamine the inside of Nano microsphere more concentratedly, the fluorescence quantum efficiency raising of microsphere inside causes fluorescence intensity to be enhanced, meet the basic demand of near infrared fluorescent probe, can effectively avoid self disturbing of biological specimen, be applicable to the tracer analysis of drug molecule.
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the alkynyl of embodiment 6, enoxolone modify and with being connected of polylysine fluorescent nanometer microsphere
Taking the medicine enoxolone that contains carboxylic acid as example, by carrying out the alkynyl modification of medicine with the amidation process of propargylamine, the polylysine fluorescent nanometer microsphere coupling that the height Azide of further reacting prepared with embodiment 2 by click chemistry is modified, synthetic route is shown in Fig. 4.Specifically be implemented as follows:
In round-bottomed flask, add respectively 0.941 g enoxolone (2 mmoL), 0.460 g EDCHCl (2.4 mmoL), 0.324 g HOBt (2.4 mmoL), and the CH of 5 mL pre-coolings
2cl
2solvent, magnetic agitation 30 minutes, drips triethylamine (TEA) 0.7g (approximately 7 mmoL) after dissolving, and continues to stir 30 minutes, adds 0.165 g propargylamine (3 mmoL), keeps ice bath reaction to spend the night.After question response finishes, in reactant liquor, add 15 ml CH
2cl
2dilution, then uses 1M HCl, 5% NaHCO successively
3solution, and saturated common salt water washing.Collect organic facies, add anhydrous Na
2sO
4dry, to filter, distilling under reduced pressure is concentrated, obtains grease and be the propioloyl amine derivative crude product of enoxolone.
In further adopting, the standby liquid phase of compacting is carried out purification, obtains white solid powder, propioloyl amination enoxolone product 0.482 g that weighs to obtain, and productive rate is about 48%.
1H?NMR?(400?MHz,?CDCl
3)?δ?5.93?(t,?
J?=?5.0?Hz,?1H),?5.70?(s,?1H),?4.13?(ddd,?
J?=?17.5,?5.4,?2.5?Hz,?1H),?4.03?(ddd,?
J?=?17.5,?4.9,?2.5?Hz,?1H),?3.24?(dd,?
J?=?10.5,?5.8?Hz,?1H),?2.80?(dt,?
J?=?13.3,?3.3?Hz,?1H),?2.35?(s,?1H),?2.26?(t,?
J?=?2.5?Hz,?1H),?2.17?(dd,?
J?=?12.2,?5.1?Hz,?1H),?2.11?–?2.00?(m,?2H),?1.96?(d,?
J?=?10.4?Hz,?1H),?1.90?–?1.74?(m,?3H),?1.72?–?1.58?(m,?4H),?1.51?–?1.43?(m,?2H),?1.42?–?1.37?(m,?6H),?1.25?–?1.18?(m,?1H),?1.18?–?1.12?(m,?9H),?1.05?–?1.00?(m,?4H),?0.96?(dd,?
J?=?12.5,?4.7?Hz,?1H),?0.87?–?0.78?(m,?6H),?0.71?(d,?
J?=?11.7?Hz,?1H)；
13C?NMR?(101?MHz,?CDCl3)?δppm?200.18,?175.51,?169.09,?128.54,?79.78,?78.78,?71.67,?61.83,?54.95,?48.04,?45.37,?43.54,?43.19,?41.77,?39.16,?37.35,?37.08,?32.76,?31.90,?31.44,?29.31,?28.40,?28.11,?27.28,?26.43,?23.35,?18.67,?17.48,?16.37,?15.58。
Get enoxolone propioloyl amine derivative 100 mg(approximately 0.2 mmoL of above-mentioned gained) be dissolved in 500 μ L DMSO, and it is slowly splashed in the polylysine self-assembled nanometer microspheres solution that 4 mL nitrine prepared by embodiment 2 modify, finally add catalyst (2 mmol/L Tris-triazoleamine, the 15 mmol/L CuSO of click chemistry reaction
4with 30 mmol/L sodium ascorbates), stirring at room temperature 2 hours.After finishing, reaction joins in the Millipore super filter tube that molecular cut off is 3 kD, 3000 leave the heart 30 minutes, discard filtrate, respectively with 0.2 mmol/L phosphate buffer 5 mL washing 3 times, collect trapped fluid, be the fluorescent nanometer microsphere that is connected with enoxolone medicine, can be used for catching and separating of target point protein.
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embodiment 7, polylysine fluorescent nanometer microsphere are applied to cell and living imaging
Get respectively above-described embodiment 1 and 2 prepared polylysine fluorescence nano self-assembly microspheres 30 μ L, join in the cultured cell of people's lung epithelial BEAS-2B, adopt Laser Scanning Confocal Microscope TCS SP5 to observe, use 543 nm exciting lights, 570 nm utilizing emitted lights detect the distribution situation of intracellular medicament-carrying nano-microsphere.Result as shown in Figure 5A, hatch after 3h by the Nano microsphere that does not connect medicine, and cell, with after DAPI dyeing, is washed away to free fluorescent dye repeatedly, through observing the blue-fluorescence that can only see nucleus DAPI, almost can't see the red fluorescence of rhodamine.And coupling has the Nano microsphere of arctigenin, equally after 3 hours hatch, carry out DAPI dyeing, and repeatedly wash away free fluorescent dye, Microscopic observation finds that nucleus is blue, and in endochylema, have very strong red fluorescence, but in cell membrane and nucleus, all there is no the obvious red fluorescence (Fig. 5 B) of rhodamine.Result shows, the medicament-carrying nano-microsphere that this patent is invented has good biocompatibility, can effectively enter in target cell, for further catching with enrichment of target point protein provides feasibility.
For the spike in Mice Body of medicament-carrying nano-microsphere that further prepared by observation station changes, get kunming mice the prepared fluorescent nanometer microsphere of tail vein injection 100 μ L embodiment 2, and adopt the distribution situation of small animal living body imaging system observation Nano microsphere in Mice Body.Result as shown in Figure 5 C, compared with the administration matched group of first 0 minute, administration had obvious rhodamine fluorescence to assemble in the bottom, abdominal cavity of mice after 60 minutes, showed that medicine carrying polylysine fluorescent nanometer microsphere is through after the circulation of mice body fluid, by metabolism and transferred in the bladder of mice.
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embodiment 8, enoxolone target point protein catch the electrophoretic analysis with SDS-PAGE
People's lung epithelial BEAS-2B cell of cultivation is laid in six orifice plates, treats that degrees of fusion reaches more than 80%, density reaches 10
5after, add respectively the polylysine fluorescent nanometer microsphere 100 μ L of the prepared connection enoxolone of embodiment 5.Adopt DMEM complete medium to continue to cultivate 48 hours; Through trypsinization 30 seconds, collecting cell and by cell suspension in PBS, supersound process in ice bath, smudge cells.4 DEG C, 10,000 leave the heart 10 minutes, discard cell debris and broken cell, get supernatant and obtain mixed solution (Fig. 6, the step 1) of the polylysine Nano microsphere that includes target point protein.
Above-mentioned mixed solution is joined in the Millipore super filter tube that molecular cut off is 3 kD, and 3000 leave the heart, discard micromolecule through normal saline suspendible recentrifuge, collect respectively filtrate 1 and trapped fluid (Fig. 6, step 2); Trapped fluid is joined in the Millipore super filter tube that molecular cut off is 100 kD, and 3,000 leave the heart, discard the unconjugated high molecular weight protein of Nano microsphere through normal saline suspendible recentrifuge, collect respectively filtrate 2 and trapped fluid (Fig. 6, step 3); To the DTT solution 100 μ L that add 100 mM in trapped fluid, leave standstill and after 15 minutes, again join in the Millipore super filter tube that molecular cut off is 100 kD, 3,000 leave the heart, collect filtrate 3 and trapped fluid 4(Fig. 6, step 4); Concrete operation step as shown in Figure 6.
Collect respectively above-mentioned filtrate and trapped fluid, and adopt 10%SDS-PAGE electrophoresis to analyze capture effect.Result is as shown in Figure 7: the 1st swimming lane is albumen Marker, and the 2nd swimming lane is cell pyrolysis liquid sample; Through the super filter tube ultrafiltration of 3 kD, in filtrate, almost there is no obvious protein band, as shown in the 3rd swimming lane; And trapped fluid is gone up again to the super filter tube of 100 kD, and a large amount of protein bands in filtrate, can be detected, be shown to be the cell soluble protein (the 4th swimming lane) of not being combined with Nano microsphere; After trapped fluid is added to DTT reduction, through the super filter tube of 100 kD, the filtrate obtaining is target point protein and the associated protein (the 6th swimming lane) of being combined with enoxolone; And remaining trapped fluid comprises not by almost can't detect soluble protein (the 5th swimming lane) in the Nano microsphere of filter membrane.
The present embodiment shows, polylysine fluorescence nano self assembly medicine carrying ball disclosed by the invention not only can carry out the localization of target point protein, can also carry out the enrichment of catching of target point protein, is having good application prospect aspect the research of mechanism of drug action.
Claims (9)
1. a multipurpose polylysine fluorescence self-assembled nanometer is microsphere supported, and being covalently boundly on framework material has fluorescent labeling group and drug molecule to make; Described framework material is the homotype monomer-polymer being connected in sequence by amido link by containing 25～30 lysine residues; Lysine residue on described framework material has been modified azido group side chain and the fluorescent marker with disulfide bond simultaneously, can prepare Nano microsphere by self assembly, and can be used for the connection of drug molecule.
2. polylysine fluorescence self-assembled nanometer according to claim 1 is microsphere supported, it is characterized in that described fluorescent tracing labelling groups is the labelled molecule that contains rhodamine.
3. polylysine fluorescence self-assembled nanometer according to claim 2 is microsphere supported, it is characterized in that the described fluorescent tag molecule that contains rhodamine is the Acibenzolar by rhodamine, is connected with the form of amido link with the amino of polylysine.
4. polylysine fluorescence self-assembled nanometer according to claim 1 is microsphere supported, it is characterized in that the described azido group side chain with disulfide bond, its one end is Acibenzolar, is connected with the form of amido link by the amino of polylysine with Nano microsphere carrier.
5. polylysine fluorescence self-assembled nanometer according to claim 1 is microsphere supported, it is characterized in that described drug molecule, be alkynyl-modified medicaments derivative, and can it be connected with the azido group side chain of Nano microsphere carrier by the method for click chemistry.
6. the microsphere supported preparation method of polylysine fluorescence self-assembled nanometer described in claim 1, is characterized in that being prepared as follows of the method:
1st, in the mixing organic facies being formed by gasoline and carbon tetrachloride that contains 1% to 5% Span-80, add NHS-rhodamine, wherein the volume ratio of gasoline and carbon tetrachloride can be 1:3 to 3:1, after dissolving, add again the aqueous solution that contains 0.2%-2% polylysine, 10,000 to 20,000 obtains emulsion after turning high-speed stirred; Wherein the ratio of organic facies and water is 1:1 to 5:1, and the consumption of NHS-rhodamine is polylysine 0.5% to 10%;
2nd, above-mentioned emulsion is transferred in separatory funnel, add respectively petroleum ether and the aqueous solution of above-mentioned volume ratio 1:1 to 5:1, stratification after mix homogeneously, collects water, the centrifugal precipitation that discards, supernatant is the polylysine fluorescent nanometer microsphere of self assembly;
3rd, in the Nano microsphere solution obtaining to the 2nd step, add Azide reagent Sulfo-SADP, stirring at room temperature reaction 0.5 hour to 5 hours, obtains the Nano microsphere that nitrine is modified;
Wherein the consumption of Sulfo-SADP is 10% to 100% of polylysine consumption;
4th, further alkynyl-modified drug slow is splashed in the Nano microsphere solution that the 3rd step obtains, under stirring condition, add the catalyst of click chemistry reaction, room temperature reaction 0.5 hour to 5 hours, reactant liquor, through ultrafiltration and concentration, is the fluorescent nanometer microsphere that is connected with medicine; Wherein the consumption of alkynyl-modified medicine is 10% to 100% of polylysine consumption.
7. method according to claim 6, it is characterized in that the alkynyl-modified medicine described in the 4th, the propiolate that adopts acetylenecarboxylic acid and the medicine that contains hydroxyl to prepare by the mode of esterification, or the propioloyl amine derivative that adopts propargylamine and the medicine that contains carboxyl to prepare by amidated mode.
8. the microsphere supported application of polylysine fluorescence self-assembled nanometer claimed in claim 1, for the spike imaging analysis at animal tissue and cellular level by modified medicaments, and target spot Position Research.
9. the microsphere supported application of polylysine fluorescence self-assembled nanometer claimed in claim 1, for catching target point protein at tissue and cellular level, can discharge target point protein and related protein thereof by cracked disulfide bond.
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