CN104672462A - Multi-tooth bionic ligand for improving biocompatibility and stability of nano particles as well as preparation method of multi-tooth bionic ligand - Google Patents
Multi-tooth bionic ligand for improving biocompatibility and stability of nano particles as well as preparation method of multi-tooth bionic ligand Download PDFInfo
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Abstract
The invention provides multi-tooth bionic ligand for improving biocompatibility and stability of nano particles as well as a preparation method of the multi-tooth bionic ligand. The synthesis bionic ligand is formed by coupling hydroxyl or carboxyl terminated mono-capped alkoxy polyethylene glycol and a carboxylic group of a carboxylic acid compound with a catechol group with an amino group of chitooligosaccharide. Due to the fact that three different kinds of interface action force including coordinate bond of the catechol group and metal, wrapping of the nano particles by micelle formed by a zwitterionic structure and electrostatic attraction between positive charges of the ligand and the nano particles exist between the bionic ligand and nano particles, accordingly, self-assembly modification is performed on the surfaces of the nano particles through triggering of different kinds of interface action force, compact protection films are prepared on the surfaces of the particles, the nano particles can be transferred into a water phase, and the nano particles with good dispersion and stability are obtained. The water-soluble nanometer particles obtained with the method can be applied to the bioimaging field such as MRI (magnetic resonance imaging) contrast agents and the like.
Description
Technical field
The invention belongs to technical field of nanometer material preparation, particularly a kind ofly strengthen multiple tooth bionical part of nanoparticle biocompatibility and stability and preparation method thereof.
Background technology
Magnetic nano-particle, the Strange properties being different from conventional material that physics, chemical property show, illustrates wide application prospect in biological medicine, catalysis, optical filtering absorption, magneticmedium and novel material etc.Particularly, the detection for major diseases such as tumours utilizing magnetic nano-particle to carry out and Therapy study also achieve the achievement of attracting attention, comprising with magnetic nano-particle be the mr molecular image probe of core for diagnosing tumor provides effective research means.
The application of magnetic Nano material has higher requirement to its preparation method and surface property, and at present, comparatively conventional has metal alloy, ferric oxide, ferrite, chromic oxide etc.In order to meet the application requiring of magnetic and stability, preparation shape magnetic nano-particle that is identical, size uniformity is the study hotspot of Materials science always.More specifically, clinically at present coprecipitation method is adopted to prepare with commercial magnetic nano-particle, in coprecipitation method, divalence and ferric ion generate water miscible nanoparticle in the basic conditions, this preparation method is comparatively simple, but the size of particles monodispersity obtained is poor and easily reunite.
In recent years, large quantifier elimination shows, adopt high-temperature decomposition can overcome above-mentioned faced defect, high and the nanoparticle of uniform particle diameter of crystal mass can be obtained, but the hydrophobic particles that this method obtains is coated by oil soluble molecule, can only be dissolved or dispersed in the organic solvent of nonpolar or low-pole, need when biologic applications to exchange or other finishing through water soluble ligand, but the ligand requirements and the iron ion that are applicable to these methods have stronger coordination ability, and take into account water miscible effect, for improving the efficiency of surface ligand free exchange, develop and be a kind ofly adsorbed on nanoparticle surface with stronger effect and can maintain again particle-stabilised ligand molecular better and just become important target.
In existing technological method, first traditional silane coupling agent is hydrolyzed and produces silanol key (Si-OH),-OH the condensation contained with ferric oxide nano particles rich surface again, form Fe-O-Si key, but also may there is crosslinking condensation reaction between silane molecule simultaneously, thus form polymolecular layer in nanoparticle surface, have impact on the water-soluble of nanoparticle and size and magnetic induction density.In recent years, Journal of American Chemical Society, 2005,127,12387 disclose a kind of hydrophobic ligand of wetting ability DMSA (DMSA) ligand exchange nanoparticle surface that uses prepares and can dissolve in water and in PBS and stable Fe
3o
4the method of nanoparticle.Although the nanoparticle hydration radius obtained by this kind of ligand-displacement method is relatively little, still comparatively difficult to the control of displacement efficiency and degree.
Langmuir, 2005,21,3096 adopt phosphonate ester (alkylphosphonate) class tensio-active agents and ethoxylized fatty alcohol or phosphatide compound system, obtain water miscible MFe
2o
4(M=Fe, Mn, Co) nanoparticle.But the hydrophobic layer that the method for this amphiphilic polymers parcel is introduced at particle periphery likely can reduce magnetic nano-particle magnetic property, and then reduce mr angiography reinforced effects, and add the hydration radius of particle, and then affect distribution in vivo behavior and the blood circulation behavior of particle.
By imitating chemical structure and the performance with specific function organism, for people's design and synthesis novel material provides thinking and approach.Ocean bivalves almost can by themselves on the surface of any material, found by research, a kind of material 3 extracted from the bivalves of ocean sticking what play a major role in process, catechol group (catechol) in 4-dopa (DOPA), it can form very strong hydrogen bond with other materials surface, is therefore widely used in design and synthesis new function material.
Summary of the invention
The object of the invention is that providing a kind of strengthens multiple tooth bionical part of nanoparticle biocompatibility and stability and preparation method thereof, the anchoring group (anchor group) when described multiple tooth bionical part is provided in nanoparticle surface modified by bionics principle.Utilize bionical ligand modified water soluble nanometer particles highly stable in aqueous, and low to the toxicity of live body, can be applied in matrix material, electronic material, biological diagnosis and treatment.
The multiple tooth bionical part of enhancing nanoparticle biocompatibility of the present invention and stability, it is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl by end, the hydroxy-acid group of carboxylic acid cpd with catechol group is coupled to form with the amino of Chitosan poly oligosaccharide respectively; End is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is 0-60% to the substitution value of the amino of Chitosan poly oligosaccharide, and the carboxylic acid cpd with catechol group is 0-60% to the substitution value of the amino of Chitosan poly oligosaccharide and is not 0.
Described end is the number-average molecular weight of the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is 300-100000.
Described end is the methoxy poly (ethylene glycol) that the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is selected from that end is hydroxyl or carboxyl.
The carboxylic acid cpd with catechol group is selected from 3,4-dihydroxyphenyl propionic acid, 3-(3,4-dihydroxy phenyl)-ALANINE.
The method of described coupling is that carbodlimide method, mixed anhydride method or azide are legal; Preferred carbodlimide method.
Described nanoparticle is selected from metal, metal chalcogenide compound, magneticsubstance, semiconductor material.
The particle diameter of described nanoparticle is 1-1000nm, preferred 2-100nm.
Described nanoparticle is hydrophobic nano particle, namely at nanoparticle surface parcel hydrophobic surface stablizer.
Described hydrophobic surface stablizer is oleic acid, oleyl amine.
Described metal is selected from Pt, Pd, Ag, Cu, Au, Ru, Rh and Os.
The metallic element of described metal chalcogenide compound is selected from one or more in Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zr, Mo, Ru, Rh, Ag, W, Re, Ta, Zn.
Described magneticsubstance is selected from CoCu, CoPt, FePt, CoSm, NiFe, CoAu, CoAg, CoPtAu, CoPtAg and NiFeCo.
Described semiconductor material is selected from: by one or more semiconductor materials formed in O, S, Se of one or more in Zn, Cd, Hg of II race and VI race; By one or more semiconductor materials formed in P, As, Sb of one or more in B, Al, Ga, In of III-th family and V race; By one or more semiconductor materials formed in Si, Ge, Pb, Sn of IV race; By one or more semiconductor materials formed in O, S, Se of one or both in Si, Ge of IV race and VI race; By one or more semiconductor materials formed in O, S, Se of one or more in P, As, Sb, Bi of V race and VI race.
The preparation method of the multiple tooth bionical part of enhancing nanoparticle biocompatibility of the present invention and stability, its concrete steps are: end is that to join concentration be in the Chitosan poly oligosaccharide aqueous solution of 5-40 grams per liter for the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and N-hydroxy-succinamide by (1), add 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide again, then room temperature reaction 8-24 hour; Wherein end is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide, the mol ratio of N-hydroxy-succinamide is (1:1:1)-(1:10:10), and end is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and the mol ratio of Chitosan poly oligosaccharide is 1:1-1:10; (2) reacted rear dialysis, lyophilize obtains amphipathic ethylene glycol graft shell poly oligosaccharide multipolymer; (3) by concentration be the methanol solution of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide of 5-40 grams per liter add concentration be 5-40 grams per liter have in the dimethyl formamide solution of the carboxylic acid cpd of catechol group, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide is (1:1)-(5:1) with the mol ratio of the carboxylic acid cpd with catechol group, reacts 0.5-6 hour under room temperature; (4) polyoxyethylene glycol graft shell poly oligosaccharide multipolymer 0.1-2.0 gram step (2) obtained adds in the reaction soln of 5-20mL step (3); ambient temperature under nitrogen protective reaction is after 12-36 hour; ether is separated out; dialysis, lyophilize is enhanced the multiple tooth bionical part of nanoparticle biocompatibility and stability.
The preparation method of the multiple tooth bionical part of above-mentioned enhancing nanoparticle biocompatibility and stability, its concrete steps are: (1) by concentration be the methanol solution of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide of 5-40 grams per liter add concentration be 5-40 grams per liter have in the dimethyl formamide solution of the carboxylic acid cpd of catechol group, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide is (1:1)-(5:1) with the mol ratio of the carboxylic acid cpd with catechol group, reacts 0.5-6 hour under room temperature; (2) Chitosan poly oligosaccharide 0.1-0.8 gram is added in the reaction soln of 5-20mL step (1); ambient temperature under nitrogen protective reaction is after 12-36 hour; ether is separated out; dialysis, it be the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is the enhancing nanoparticle biocompatibility of 0 and the multiple tooth bionical part of stability to the substitution value of the amino of Chitosan poly oligosaccharide that lyophilize obtains end.
A kind of preparation method of water soluble nanometer particles is: be scattered in organic solvent by nanoparticle and the multiple tooth bionical part strengthening nanoparticle biocompatibility and stability, after stirred at ambient temperature 0.5-4 hour, removing organic solvent, then water filtration is added, remove unnecessary multiple tooth bionical part, namely obtain water soluble nanometer particles.
Water soluble nanometer particles of the present invention is preparing the application in magnetic resonance imaging contrast.
The bionical part of design and synthesis of the present invention comprises the catechol group playing main adhesion function in Chitosan poly oligosaccharide molecule of the skeleton, polyoxyethylene glycol and ocean bivalves surface chemical structure.Wherein, (1) polyoxyethylene glycol (Polyethyleneglycol, PEG) be a kind of amphipathic nature polyalcohol, both water-soluble, be dissolved in again most organic solvent, and its good biocompatibility, there is the features such as nontoxic, immunogenicity is low, can help to avoid reticuloendothelial system phagocytic, excrete by kidney, can not accumulate in vivo; (2) the degraded product Chitosan poly oligosaccharide (Chitooligosaccharide of chitosan, COS) be the oligose be formed by connecting by β-1-4 glycosidic link by 2-20 glucosamine, good water solubility, and due to its molecular weight very little, hemato encephalic barrier (Blood-brain barrier, BBB) can be passed through smoothly; (3) catechol group on the Chitosan poly oligosaccharide molecule of the skeleton in bionical part has affinity to nanoparticle, can play grappling effect.Due to the interface interaction power that bionical part is different from there being three kinds between nanoparticle: comprise catechol group and be combined with the coordination of metal, catechol group can carry out bonding with hydrophobic nano-particles surface, both sexes structure forms micella Encapsulation nanoparticle, part with positive charge and nanoparticle between electrostatic attraction, therefore, undertaken self-assembled modified by the initiation different reactive force at interface and the surface of nanoparticle, thus the protective membrane of one deck densification is prepared at particle surface, can nano particle be transferred in aqueous phase, obtain the nanoparticle of good dispersion and stability.For these reasons, the water soluble nanometer particles that the present invention obtains can be used for multiple fields, comprises the biomedical applications such as organizational engineering application, heat cure and drug delivery such as bio-imaging application, the cell therapies such as nuclear magnetic resonance (MRI) contrast medium.
Accompanying drawing explanation
Fig. 1 is the chemical structure of bionical part in the present invention and the stabilization schematic diagram to water-insoluble nanoparticle;
Fig. 2 is the building-up reactions figure of the multiple tooth bionical part strengthening nanoparticle biocompatibility and stability;
Fig. 3 is the HNMR spectrogram of polyoxyethylene glycol graft shell poly oligosaccharide multipolymer prepared by embodiment 1;
Fig. 4 is the HNMR spectrogram of the enhancing nanoparticle biocompatibility of embodiment 1 preparation and the multiple tooth bionical part of stability;
Fig. 5 is X-ray diffraction (XRD) figure of ferriferous oxide (12nm) nanoparticle of the bionical part parcel of preparation in embodiment 2;
Fig. 6 is the solvability (B) of the solvability (A) of the iron oxide nanoparticle wrapped up by organic surface stabilizer in organic solvent in embodiment 2, the ferriferous oxide wrapped up by bionical part in aqueous;
Fig. 7 is transmission electron microscope figure (a) of the iron oxide nanoparticle (12nm) wrapped up by organic surface stabilizer in embodiment 2, and transmission electron microscope figure (b) of the ferriferous oxide (12nm) wrapped up by bionical part;
The stability of Fig. 8 bionical ligand modified water-soluble iron oxide nanoparticle prepared by embodiment 2 under different pH value and ionic strength;
The fourier transform infrared spectrometry (FTIR) of the iron oxide nanoparticle of bionical part parcel prepared by Fig. 9 embodiment 2;
C1s, O1s, N1s and Fe2p region amplifying X-ray photoelectron spectrum (XPS) of the iron oxide nanoparticle of bionical part parcel prepared by Figure 10 embodiment 2;
Utilize bionical ligand modified water-soluble iron oxide nanoparticle as contrast medium in Figure 11 embodiment 2 and MRI photo in the Mice Body utilizing nuclear magnetic resonance (MRI) equipment to obtain.
Figure 12 is the transmission electron microscope figure of the water soluble oxidized Fe nanometer particles (12nm) that polyoxyethylene glycol graft shell poly oligosaccharide multipolymer (PEG-g-COS) of embodiment 5 preparation is modified.
Embodiment
By following exemplary illustration, but the embodiment of indefiniteness, better can understand the present invention.
[embodiment 1]
1, the synthesis of polyoxyethylene glycol graft shell poly oligosaccharide multipolymer: by 1.9 grams of methoxy poly (ethylene glycol) carboxylic acid (0.9mmol, mPEG-COOH, Mn=2000) and 0.5 gram of N-hydroxy-succinamide (NHS, 4.5mmol) join in the 30 ml water solution containing 0.5 gram of Chitosan poly oligosaccharide, then, by 0.86 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC, 4.5mmol) join in three batches in above-mentioned solution, room temperature reaction 24 hours; In retaining relative molecular mass about 12, carry out dynamic dialysis 2 days in the dialysis tubing of 000 to distilled water, freeze-drying obtains white fluffy solid polyoxyethylene glycol-g-Chitosan poly oligosaccharide (mPEG-COS),
1hNMR as shown in Figure 3.
2, the methanol solution that 5 milliliters contain 0.13 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (0.68mmol) is joined 2 milliliters containing 0.06 gram 3,4-dihydroxyphenyl propionic acid (HCA, in anhydrous dimethyl formamide solution 0.34mmol), room temperature reaction 1 hour; Then 0.54 gram of polyoxyethylene glycol graft shell poly oligosaccharide multipolymer is joined in above-mentioned solution, react 24 hours under nitrogen protection; After completing reaction, reactant is separated out in ether, in retaining relative molecular mass about 12, carry out dynamic dialysis 2 days to distilled water (pH=5.5) in the dialysis tubing of 000, freeze-drying is enhanced the multiple tooth bionical part (BCOS1) of nanoparticle biocompatibility and stability
1hNMR as shown in Figure 4.
The enhancing nanoparticle biocompatibility of above-mentioned preparation and the multiple tooth bionical part of stability, it is coupled to form with the amino of Chitosan poly oligosaccharide respectively by the hydroxy-acid group of methoxy poly (ethylene glycol) carboxylic acid, 3,4-dihydroxyphenyl propionic acid; Methoxy poly (ethylene glycol) carboxylic acid is 25%, 3,4-dihydroxyphenyl propionic acid to the substitution value of the amino of Chitosan poly oligosaccharide is 28% to the substitution value of the amino of Chitosan poly oligosaccharide.
Utilize the multiple tooth bionical part of enhancing nanoparticle biocompatibility obtained above and stability to prepare water-soluble magnetic ferric oxide nano particles: magnetic ferric oxide nano particles and 50 milligrams of multiple tooth bionical parts obtained above of the 12 nanometer oleic acid modifieds synthesized 10 milligrams are in organic solvent dispersed in 10 milliliters of chloroforms, at room temperature stir 1 hour; After removing chloroform, 5 ml distilled waters are added in resistates, the pin type sample filter of the soln using 200nm obtained is filtered, in order to remove unreacted bionical part, by filtering the solution use centrifugal filter obtained, (retaining relative molecular mass is 10,0000) repeat filter 23-5 times, obtain water-soluble magnetic iron oxide nanoparticle.Understand according to the XRD result in Fig. 5, use the crystal face parameter corresponding to diffraction peak and the spinel type magnetite Fe of the water-soluble magnetic iron oxide nanoparticle of the bionical ligands stabilize prepared by method of the present invention
30
4standard diagram (JCPDS 19-0629) consistent, show the crystallinity not larger change of bionical ligand modified front and back nanoparticle.
The stability assessment of water-soluble magnetic ferric oxide nano particles in the aqueous solution:
A. the solubleness analysis of water-soluble magnetic ferric oxide nano particles
The magnetic ferric oxide nano particles of the oleic acid modified synthesized in organic solvent is dissolved in normal hexane, adds water subsequently, the water-soluble magnetic ferric oxide nano particles of preparation is dissolved in water simultaneously, adds normal hexane subsequently.Then the change of the solubleness caused by the surface of nanoparticle replaces is analyzed.
Can confirm that hydrophobic nanoparticle surface oleic acid molecular is by the displacement of bionical part parcel from Fig. 6, thus hydrophobic nano particle is converted into water soluble nanometer particles.Therefore, can confirm without precipitation or agglomeration with visual inspection, known, water-soluble magnetic ferric oxide nano particles good dispersion in aqueous phase.
B. the analysis of transmission electron microscope (TEM) is used
By hydrophobic oxide nanoparticle dispersion in organic solvent (as normal hexane etc.), being dripped to by this solution scribbles on the TEM grid of carbon film, drying about 20 minutes, observes with electron microscope (Hitachi H7650TEM, acceleration voltage 100kV).Being dripped to by the aqueous solution comprising water-soluble magnetic ferric oxide nano particles scribbles on the TEM grid of carbon film, drying about 30 minutes, observes with electron microscope (Hitachi H7650TEM, acceleration voltage 100kV).As shown in Fig. 7 a to 7b, use by method of the present invention prepare bionical ligand modified before and modify after, magnetic ferric oxide nano particles has substantially identical form and size.
C. the hydrodynamic diameter analysis of dynamic light scattering technique (DLS) is used
Centrifugal 5 minutes of the aqueous solution 5000g first will comprising water-soluble magnetic ferric oxide nano particles, gets upper strata sample solution and utilizes the pin type sample filter of 200nm to filter.Sample after filtration is directly loaded onto in sample chamber.As shown in Figure 8, the bionical ligand modified water soluble oxidized Fe nanometer particles using method of the present invention to prepare under different pH value and ionic strength disperses and has been stabilized in aqueous phase.More specifically, can find out, compared with the water soluble oxidized Fe nanometer particles modified with polyoxyethylene glycol graft shell poly oligosaccharide multipolymer (PEG-g-COS) not containing catechol group, the bionical ligand modified water-soluble oxide nanoparticle utilizing method of the present invention to prepare is all more stable in harsh aqueous phase.
Utilize the surface composition analysis of bionical ligand modified water-soluble magnetic iron oxide nanoparticle:
In order to analyze above-mentioned nanoparticle surface composition, fourier transform infrared spectrometry (FTIR) and x-ray photoelectron power spectrum (XPS) is adopted to measure the nano grain surface state of preparation, as shown in Figure 9 and Figure 10, can confirm, bionical part is well attached to the ferric oxide nano particles surface of preparation.
Figure 11 illustrates and utilizes bionical ligand modified water soluble oxidized Fe nanometer particles as contrast medium and MRI photo in the Mice Body utilizing nuclear magnetic resonance (MRI) equipment to obtain.Specifically, the contrast medium that prepared by method of the present invention to the photo display that (Figure 11 A), 2 hours afterwards (Figure 11 B) and 24 hours afterwards (Figure 11 C) before injected in mice obtain not only has excellent colloidal stability and blood compatibility, and there is longer circulation time in vivo, show the superperformance as MRI contrast agent.
[embodiment 2]
1, the synthesis of polyoxyethylene glycol graft shell poly oligosaccharide multipolymer: by 1.9 grams of methoxy poly (ethylene glycol) carboxylic acid (0.9mmol, mPEG-COOH, Mn=2000) and 0.5 gram of N-hydroxy-succinamide (NHS, 4.5mmol) join in the 15 ml water solution containing 0.25 gram of Chitosan poly oligosaccharide, then, by 0.86 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC, 4.5mmol) join in three batches in above-mentioned solution, room temperature reaction 24 hours; In retaining relative molecular mass about 12, carry out dynamic dialysis 2 days in the dialysis tubing of 000 to distilled water, freeze-drying obtains white fluffy solid polyoxyethylene glycol-g-Chitosan poly oligosaccharide (mPEG-COS).
2, the methanol solution that 5 milliliters contain 0.13 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (0.68mmol) is joined 2 milliliters containing 0.06 gram 3, in the anhydrous dimethyl formamide solution of 4-dihydroxyphenyl propionic acid (0.34mmol), room temperature reaction 1 hour; Then 1.0 grams of above-mentioned polyoxyethylene glycol graft shell poly oligosaccharide multipolymers are joined in above-mentioned solution, react 24 hours under nitrogen protection; After completing reaction, reactant is separated out in ether, in retaining relative molecular mass about 12, carry out dynamic dialysis 2 days to distilled water (pH=5.5) in the dialysis tubing of 000, freeze-drying is enhanced the multiple tooth bionical part (BCOS2) of nanoparticle biocompatibility and stability.
The enhancing nanoparticle biocompatibility of above-mentioned preparation and the multiple tooth bionical part of stability, it is coupled to form with the amino of Chitosan poly oligosaccharide respectively by the hydroxy-acid group of methoxy poly (ethylene glycol) carboxylic acid, 3,4-dihydroxyphenyl propionic acid; Methoxy poly (ethylene glycol) carboxylic acid is 50%, 3,4-dihydroxyphenyl propionic acid to the substitution value of the amino of Chitosan poly oligosaccharide is 28% to the substitution value of the amino of Chitosan poly oligosaccharide.
Multiple tooth bionical part obtained above is utilized to adopt the identical reaction conditions of embodiment 1 to prepare water-soluble magnetic ferric oxide nano particles.
[embodiment 3]
1, the synthesis of polyoxyethylene glycol graft shell poly oligosaccharide multipolymer: by 1.9 grams of methoxy poly (ethylene glycol) carboxylic acid (0.9mmol, mPEG-COOH, Mn=2000) and 0.5 gram of N-hydroxy-succinamide (NHS, 4.5mmol) join in the 30 ml water solution containing 0.5 gram of Chitosan poly oligosaccharide, then, by 0.86 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC, 4.5mmol) join in three batches in above-mentioned solution, room temperature reaction 24 hours; In retaining relative molecular mass about 12, carry out dynamic dialysis 2 days in the dialysis tubing of 000 to distilled water, freeze-drying obtains white fluffy solid polyoxyethylene glycol-g-Chitosan poly oligosaccharide (mPEG-COS),
1hNMR as shown in Figure 3.
2, the methanol solution that 5 milliliters contain 0.13 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (0.68mmol) is joined 2 milliliters containing 0.06 gram 3, in the anhydrous dimethyl formamide solution of 4-dihydroxyphenyl propionic acid (0.34mmol), room temperature reaction 1 hour; Then 0.4 gram of above-mentioned polyoxyethylene glycol graft shell poly oligosaccharide multipolymer is joined in above-mentioned solution, react 24 hours under nitrogen protection; After completing reaction, reactant is separated out in ether, in retaining relative molecular mass about 5, carry out dynamic dialysis 2 days to distilled water (pH=5.5) in the dialysis tubing of 000, freeze-drying is enhanced the multiple tooth bionical part (BCOS3) of nanoparticle biocompatibility and stability.
The enhancing nanoparticle biocompatibility of above-mentioned preparation and the multiple tooth bionical part of stability, it is coupled to form with the amino of Chitosan poly oligosaccharide respectively by the hydroxy-acid group of methoxy poly (ethylene glycol) carboxylic acid, 3,4-dihydroxyphenyl propionic acid; Methoxy poly (ethylene glycol) carboxylic acid is 25%, 3,4-dihydroxyphenyl propionic acid to the substitution value of the amino of Chitosan poly oligosaccharide is 56% to the substitution value of the amino of Chitosan poly oligosaccharide.
Multiple tooth bionical part obtained above is utilized to adopt the identical reaction conditions of embodiment 1 to prepare water-soluble magnetic ferric oxide nano particles.
[embodiment 4]
The methanol solution that 5 milliliters contain 0.13 gram of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (0.68mmol) is joined 2 milliliters containing 0.06 gram 3, in the anhydrous dimethyl formamide solution of 4-dihydroxyphenyl propionic acid (0.34mmol), room temperature reaction 1 hour; Then 0.1 gram of Chitosan poly oligosaccharide is joined in above-mentioned solution, react 24 hours under nitrogen protection; After completing reaction, reactant is separated out in ether, in retaining relative molecular mass about 3, carry out dynamic dialysis 2 days to distilled water (pH=5.5) in the dialysis tubing of 000, freeze-drying is enhanced the multiple tooth bionical part (BCOS4) of nanoparticle biocompatibility and stability.
The enhancing nanoparticle biocompatibility of above-mentioned preparation and the multiple tooth bionical part of stability, it is by 3, the hydroxy-acid group of 4-dihydroxyphenyl propionic acid and the amino of Chitosan poly oligosaccharide are coupled to form, and 3,4-dihydroxyphenyl propionic acid is 48% to the substitution value of the amino of Chitosan poly oligosaccharide.
Multiple tooth bionical part obtained above is utilized to adopt the identical reaction conditions of embodiment 1 to prepare water-soluble magnetic ferric oxide nano particles.
Following table 1 shows structure determination result and the feature of the multiple tooth bionical part strengthening nanoparticle biocompatibility and stability.
[table 1] strengthens the structural analysis of the multiple tooth bionical part of nanoparticle biocompatibility and stability
The preparation of the water soluble oxidized Fe nanometer particles that [embodiment 5] utilizes polyoxyethylene glycol graft shell poly oligosaccharide multipolymer to modify
50 milligrams of polyoxyethylene glycol graft shell poly oligosaccharides multipolymer (PEG-g-COS) prepared by the magnetic ferric oxide nano particles of the 12 nanometer oleic acid modifieds synthesized 10 milligrams in organic solvent and embodiment 1 are dispersed in 10 milliliters of chloroforms, at room temperature stir 1 hour; After removing chloroform, 5 ml distilled waters are added in resistates, the pin type sample filter of the soln using 200nm obtained is filtered, in order to remove unreacted bionical part, by filtering the solution use centrifugal filter obtained, (retaining relative molecular mass is 10,0000) repeat filter 23-5 times, obtain the water-soluble magnetic iron oxide nanoparticle that polyoxyethylene glycol graft shell poly oligosaccharide multipolymer is modified.The aqueous solution of the water soluble oxidized Fe nanometer particles modified by the polyoxyethylene glycol graft shell poly oligosaccharide multipolymer of preparation drips to and scribbles on the TEM grid of carbon film, drying about 30 minutes, observe with electron microscope (Hitachi H7650TEM, acceleration voltage 100kV).As shown in figure 12, the water soluble oxidized Fe nanometer particles modified by polyoxyethylene glycol graft shell poly oligosaccharide multipolymer has reunion, more specifically, can find out compared with ferric oxide (12nm) nanoparticle (Fig. 7 b) that wraps up with bionical part (BCOS1), utilize the bionical ligand modified nanoparticle prepared according to the present invention there is no obvious agglomeration, and with the oil soluble particle before modifying, there is substantially identical form and size.
[embodiment 6]
The enhancing nanoparticle biocompatibility adopting embodiment 1 to prepare and the multiple tooth bionical part of stability prepare water-soluble mangaic acid Fe nanometer particles, be specially, the mangaic acid Fe nanometer particles modified by the oleic acid synthesized in organic solvent/oleyl amine adopts the reaction conditions identical with embodiment 1 to prepare water-soluble mangaic acid Fe nanometer particles.
[embodiment 7]
The enhancing nanoparticle biocompatibility adopting embodiment 1 to prepare and the multiple tooth bionical part of stability prepare water-soluble gold nanoparticles, be specially, adopt the reaction conditions identical with embodiment 1 to prepare water-soluble gold nanoparticles the golden nanometer particle synthesized in organic solvent.
[embodiment 8]
The enhancing nanoparticle biocompatibility adopting embodiment 1 to prepare and the multiple tooth bionical part of stability prepare water-soluble nuclear-shell (Au@Fe
3o
4) nanoparticle, be specially, by nucleocapsid (the Au@Fe that the oleic acid/oleyl amine synthesized in organic solvent is modified
3o
4) nanoparticle adopt the reaction conditions identical with embodiment 1 prepare water-soluble nuclear-shell (Au@Fe
3o
4) nanoparticle.
Claims (10)
1. one kind strengthens the multiple tooth bionical part of nanoparticle biocompatibility and stability, it is characterized in that, it is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl by end, the hydroxy-acid group of carboxylic acid cpd with catechol group is coupled to form with the amino of Chitosan poly oligosaccharide respectively; End is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is 0-60% to the substitution value of the amino of Chitosan poly oligosaccharide, and the carboxylic acid cpd with catechol group is 0-60% to the substitution value of the amino of Chitosan poly oligosaccharide and is not 0.
2. the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 1 and stability, is characterized in that, described end is the number-average molecular weight of the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is 300-100000.
3. the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 1 and stability, it is characterized in that, described end is the methoxy poly (ethylene glycol) that the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is selected from that end is hydroxyl or carboxyl; The described carboxylic acid cpd with catechol group is selected from 3,4-dihydroxyphenyl propionic acid, 3-(3,4-dihydroxy phenyl)-ALANINE; The method of described coupling is that carbodlimide method, mixed anhydride method or azide are legal; Preferred carbodlimide method.
4. the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 1 and stability, is characterized in that, described nanoparticle is selected from metal, metal chalcogenide compound, magneticsubstance, semiconductor material; The particle diameter of described nanoparticle is 1-1000nm, preferred 2-100nm.
5. the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 4 and stability, is characterized in that, described nanoparticle is hydrophobic nano particle, namely at nanoparticle surface parcel hydrophobic surface stablizer; Described hydrophobic surface stablizer is oleic acid, oleyl amine.
6. the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 4 and stability, is characterized in that, described metal is selected from Pt, Pd, Ag, Cu, Au, Ru, Rh and Os;
The metallic element of described metal chalcogenide compound is selected from one or more in Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zr, Mo, Ru, Rh, Ag, W, Re, Ta, Zn;
Described magneticsubstance is selected from CoCu, CoPt, FePt, CoSm, NiFe, CoAu, CoAg, CoPtAu, CoPtAg and NiFeCo;
Described semiconductor material is selected from: by one or more semiconductor materials formed in O, S, Se of one or more in Zn, Cd, Hg of II race and VI race; By one or more semiconductor materials formed in P, As, Sb of one or more in B, Al, Ga, In of III-th family and V race; By one or more semiconductor materials formed in Si, Ge, Pb, Sn of IV race; By one or more semiconductor materials formed in O, S, Se of one or both in Si, Ge of IV race and VI race; By one or more semiconductor materials formed in O, S, Se of one or more in P, As, Sb, Bi of V race and VI race.
7. the preparation method of the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 1 and stability, it is characterized in that, its concrete steps are: end is that to join concentration be in the Chitosan poly oligosaccharide aqueous solution of 5-40 grams per liter for the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and N-hydroxy-succinamide by (1), add 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide again, then room temperature reaction 8-24 hour; Wherein end is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide, the mol ratio of N-hydroxy-succinamide is (1:1:1)-(1:10:10), and end is the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl and the mol ratio of Chitosan poly oligosaccharide is 1:1-1:10; (2) reacted rear dialysis, lyophilize obtains amphipathic ethylene glycol graft shell poly oligosaccharide multipolymer; (3) by concentration be the methanol solution of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide of 5-40 grams per liter add concentration be 5-40 grams per liter have in the dimethyl formamide solution of the carboxylic acid cpd of catechol group, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide is (1:1)-(5:1) with the mol ratio of the carboxylic acid cpd with catechol group, reacts 0.5-6 hour under room temperature; (4) polyoxyethylene glycol graft shell poly oligosaccharide multipolymer 0.1-2.0 gram step (2) obtained adds in the reaction soln of 5-20mL step (3); ambient temperature under nitrogen protective reaction is after 12-36 hour; ether is separated out; dialysis, lyophilize is enhanced the multiple tooth bionical part of nanoparticle biocompatibility and stability.
8. the preparation method of the multiple tooth bionical part of enhancing nanoparticle biocompatibility according to claim 1 and stability, it is characterized in that, its concrete steps are: (1) by concentration be the methanol solution of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide of 5-40 grams per liter add concentration be 5-40 grams per liter have in the dimethyl formamide solution of the carboxylic acid cpd of catechol group, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide is (1:1)-(5:1) with the mol ratio of the carboxylic acid cpd with catechol group, 0.5-6 hour is reacted under room temperature, (2) Chitosan poly oligosaccharide 0.1-0.8 gram is added in the reaction soln of 5-20mL step (1), ambient temperature under nitrogen protective reaction is after 12-36 hour, ether is separated out, dialysis, it be the alkoxyl group polyoxyethylene glycol of single end-blocking of hydroxyl or carboxyl is the enhancing nanoparticle biocompatibility of 0 and the multiple tooth bionical part of stability to the substitution value of the amino of Chitosan poly oligosaccharide that lyophilize obtains end.
9. the preparation method of a water soluble nanometer particles, it is characterized in that, the method concrete operations are: be scattered in organic solvent by nanoparticle and the multiple tooth bionical part strengthening nanoparticle biocompatibility and stability, after stirred at ambient temperature 0.5-4 hour, removing organic solvent, then add water filtration, remove unnecessary multiple tooth bionical part, namely obtain water soluble nanometer particles.
10. the water soluble nanometer particles that method according to claim 9 prepares is preparing the application in magnetic resonance imaging contrast.
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