A kind of radiography material, its preparation method and application
Technical field
This application involves a kind of radiography material, and in particular to a kind of multi-modal target that can be used for ultrasound, magnetic resonance and fluorescence
To type radiography material.
Background technology
Cancer becomes endangers human life and one of major disease of health in world wide, cancer has been in the late three decades
As first cause of the death of the mankind, more than AIDS, tuberculosis, the summation of the lethal case of malaria.Currently for the best treatment of cancer
Means are early discovery, early treatment.By current medical level, 80%~90% early-stage cancer patient can cure.According to report
Road, the cancer detection of world standard, it is possible to find the infantile tumour cell of 15mm or so sizes, and in China, can be with by instrument
It was found that, probably it is more than several centimetres of Advanced cancers cell, therefore the reduction of contrast signal enhancing function of contrast agent seems
It is particularly important that.But the contrast agent listed at present is just for single detection pattern, targeting can force difference, it is difficult to which or neoplastic disease can not be reached
Stove position plays contrasting effects.
To realize the Accurate Diagnosis of tumour early stage, the toxic side effect that multiple medication produces is reduced, multi-modality imaging technology should
Transport and give birth to, it is learnt from other's strong points to offset one's weaknesses by mutually merging a variety of imaging diagnostic modes with reaching, the purpose of mutual supplement with each other's advantages.With multimode
The development of state imaging technique, to improve the clinic and economic benefit of new imaging device, there is an urgent need to develop with multi-modality imaging
The corresponding new multi-modal contrast agent of system, i.e., only need to then can be achieved two or more imaging functions using a kind of contrast agent.It
It is capable of providing faster, accurate, high-res, high-resolution structure and functional imaging.With the hair of visualization treatment technology
Exhibition, have concurrently imaging, targeting, even treatment function multi-modal contrast agent become current biological medical domain study frontier and
Hot spot.Therefore, the targeting ability to improve diagnosing tumor efficiency, reducing toxic side effect, lift radiography material, it is necessary to develop one
The strong multi-modal radiography material of targeting type of kind good water solubility, radiography function.
The content of the invention
To solve the above problems, the purpose of the application is to provide the multi-modal of a kind of ultrasound, magnetic resonance and fluorescence
Targeting type radiography material, with the targeting ability for improving diagnosing tumor efficiency, reducing toxic side effect, lifting radiography material.It is described to make
Shadow material is the bovine serum albumin microspheres for being at least enclosed with hydrophilic magnetic nano-particle and gas A;The bovine serum albumin
White microsphere surface contains the west that the lysine residue in bovine serum albumin(BSA) is formed with the compound crosslink at least containing 2 aldehyde radicals
Buddhist alkali;The bovine serum albumin microspheres surface contains the target ligand with tumor-targeting function;The hydrophilic magnetic nanometer
Particle is the magnetic nano-particle of hydrophilic polymer cladding, the hydrophilic polymer optionally from glucan and its derivative,
One or more in Chitosan-phospholipid complex, cellulose and its derivates, the magnetic nano-particle optionally have magnetic certainly
One or more in the metal oxide nanoparticles of property;The gas A is optionally from air, nitrogen, helium, argon gas, dioxy
Change the one or more in carbon.
Preferably, the particle diameter of the radiography material is 0.5 μm~3 μm.It is further preferred that the particle diameter of the radiography material
Range limit is optionally from 2.5 μm, 2 μm, 1.5 μm or 1.3 μm;Optional 0.8 μm or 1 μm of particle size range lower limit.
The hydrophilic magnetic nano-particle is the magnetic nano-particle of hydrophilic polymer cladding, wherein hydrophilic polymer
The mass ratio of thing and magnetic nano-particle is not less than 1: 5;Preferably, the mass ratio of hydrophilic polymer and magnetic nano-particle is
1: 4~2: 1;It is further preferred that the mass ratio of hydrophilic polymer and magnetic nano-particle is 1: 3~2: 1;Further preferably
The mass ratio of ground, hydrophilic polymer and magnetic nano-particle is 1: 2~1: 1.
Preferably, the mass percentage of the hydrophilic magnetic nano-particle in the bovine serum albumin microspheres for 10~
95%.It is further preferred that the mass percentage model of the hydrophilic magnetic nano-particle in the bovine serum albumin microspheres
Limit is placed optionally from 90%, 85%, 80%, 75%, 70%, 65%;Lower limit is optionally from 15%, 20%, 25%, 30%, 35%,
40%th, 45%.
The lysine residue in bovine serum albumin(BSA) contained by the bovine serum albumin microspheres surface is with least containing 2
The Schiff 's base that the compound of a aldehyde radical is formed, by any one aldehyde radical in the compound containing 2 aldehyde radicals and lysine residue
Nitrogen-atoms in amino, connects to be formed by-N=C- double bonds.Preferably, the west contained by the bovine serum albumin microspheres surface
Buddhist alkali and the molar ratio of lysine residue in bovine serum albumin(BSA) are not less than 1: 9;It is further preferred that the bovine serum albumin
The molar ratio of lysine residue is 3: 7~7: 3 in Schiff 's base and bovine serum albumin(BSA) contained by white microsphere surface;It is further excellent
Selection of land, the molar ratio of lysine residue in the Schiff 's base and bovine serum albumin(BSA) contained by the bovine serum albumin microspheres surface
For 2: 3~3: 2.
Preferably, matter of the target ligand on the bovine serum albumin microspheres surface in the bovine serum albumin microspheres
It is 1~20% to measure accounting.It is further preferred that the target ligand on the bovine serum albumin microspheres surface is pure in the ox blood
Quality accounting range limit in protein microsphere is optionally from 20%, 18%, 15%;Lower limit is optionally from 1%, 3% or 5%.
Preferably, the hydrophilic polymer is optionally from the one or more in dextran, dextran derivative.
In the application, the magnetic nano-particle optionally contains ferromagnetic material certainly, it is preferable that optionally from the oxidation of iron
Thing nano-particle, the oxide nano-particles of cobalt, nickel oxide nano-particles in one or more.
In the application, the magnetic nano-particle has the particle diameter of nanoscale.According to this area public office general knowledge, nanometer ruler
Spend for 1nm to 100nm.Those skilled in the art, can select to close according to actually detected needs in the range of 1nm to 100nm
The magnetic nano-particle of suitable scale.Preferably, the particle size range of the hydrophilic magnetic nano-particle is 5~25nm.
Preferably, the magnetic nano-particle is Fe3O4And/or γ-Fe2O3Nano-particle.
Preferably, the compound at least containing 2 aldehyde radicals is 2~20 changes containing 2 aldehyde radicals optionally from carbon atom
Compound.Carbon atom is 2~20 compounds containing 2 aldehyde radicals, and it is 2~20 organic matters containing 2 aldehyde radicals to refer to carbon number, institute
Two aldehyde functions can only be contained by stating organic matter, other functional groups can also be included, such as aliphatic radical, carboxyl, halogenic substituent
Etc..The structural formula of the organic matter can be can also contain side chain for straight chain.
Preferably, the upper limit of the compound institute carbon atom quantity at least containing 2 aldehyde radicals is optionally from 18,15,10;Institute
The lower limit of the compound institute carbon atom quantity at least containing 2 aldehyde radicals is stated optionally from 3,5,8.
Preferably, the compound at least containing 2 aldehyde radicals is glutaraldehyde.
In the application, the target ligand with tumor-targeting function is referred to for clearly carcinogenic site
(protein molecular of inside tumor cells, or a genetic fragment) combines the group having an effect.
Preferably, the target ligand with tumor-targeting function is the folic acid with bovine serum albumin(BSA) coupling.
The application further object is to provide a kind of method for preparing the radiography material, it is characterised in that at least contains
Following steps:
A) to being dispersed with the system of magnetic nano-particle, add hydrophilic polymer, after stirring evenly, through Magneto separate,
After washing, hydrophilic magnetic nano-particle is obtained;
B) by the obtained hydrophilic magnetic nano-particles of step a), it is added to containing bovine serum albumin(BSA) and at least contains
In the liquid-phase system of the compound of 2 aldehyde radicals, after mixing, gas A is contained in the gas contacted with liquid-phase system surface
Under conditions of, air-liquid two-phase interface is ultrasonically treated, obtains being enclosed with hydrophilic magnetic nano-particle and gas A and table
Contain the bovine serum albumin microspheres of Schiff 's base in face;
C) by the compound of the target ligand containing tumor-targeting function with step b) is obtained is enclosed with hydrophily magnetic
The bovine serum albumin microspheres that Schiff 's base is contained on property nano-particle and gas A and surface are uniformly mixed, by isolated described
Radiography material.
In the application, in the preparation process of step a) hydrophilic magnetic nano-particles, the addition of hydrophilic polymer should be able to
Enough abundant coated magnetic nano-particles, within this range, those skilled in the art can be required according to specific, and selection is suitable
The ratio of hydrophilic polymer and magnetic nano-particle.Preferably, the step a) hydrophilic polymers and magnetic nano-particle
Mass ratio be not less than 1: 5.Further preferred range is that the mass ratio of the hydrophilic polymer and magnetic nano-particle is
1: 2~1: 1.
Preferably, the step a) hydrophilic polymers are dextran and/or the derivative of dextran.
Preferably, step a) the hydrophilic magnetic nano-particles are the magnetic nano-particle of cladding dextran.
Preferably, the preparation of the step a) systems for being dispersed with magnetic nano-particle at least contains following steps:Filling
In the container of full gas A, under stirring condition, ammonium hydroxide is added dropwise to after precipitating completely to the solution containing metal ion, lasting stirring
For a period of time, up to the system for being dispersed with magnetic nano-particle;The metal ion is optionally from divalent cobalt ion, trivalent cobalt
At least one of ion, ferrous ion, ferric ion or bivalent nickel ion;Described lasting stirring a period of time, be
Persistently stirred at 30~80 DEG C 0.5~24 it is small when.Preferably, persistently stirred at 30~60 DEG C 0.5~12 it is small when;It is further excellent
Selection of land, persistently stirred at 40~60 DEG C 0.5~2 it is small when.
Preferably, the liquid-phase system of the step b) compounds containing bovine serum albumin(BSA) and at least containing 2 aldehyde radicals,
It is shape after 6.5~7.5 phosphate buffered saline solutions that compound for bovine serum albumin(BSA) and at least containing 2 aldehyde radicals, which is dissolved in pH,
Into.
Preferably, step b) the hydrophilic magnetic nano-particles and the mass ratio of bovine serum albumin(BSA) in liquid-phase system are
0.1~19.It is further preferred that the mass ratio of the hydrophilic magnetic nano-particle and bovine serum albumin(BSA) in liquid-phase system
Range limit is optionally from 18,15,12 or 10;Lower limit is optionally from 0.2,0.5,1,2,5,6 or 8.
Preferably, the liquid-phase system of the step b) compounds containing bovine serum albumin(BSA) and at least containing 2 aldehyde radicals
In, the mass concentration of bovine serum albumin(BSA) is 1~8%;The volumetric concentration of compound at least containing 2 aldehyde radicals is 3~6%.
According to general knowledge known in this field, air-liquid two-phase interface is ultrasonically treated described in step b), is referred in air-liquid
Placement frequency is higher than 20000 hertz of ultrasonic wave at two-phase interface.Preferably, used ultrasonic frequency range for 20K~
25K hertz.
Preferably, the compound of the target ligand containing tumor-targeting function described in step c) is folic acid active ester.It is described
Folic acid activity fat is prepared by the reaction of folic acid, n-hydroxysuccinimide and dicyclohexylcarbodiimide.Folic acid active ester with
Bovine serum albumin(BSA) is connected to the microsphere surface of bovine serum albumin(BSA) by removing the coupling reaction of amide groups.
Preferably, it is in step c), the compound of the target ligand containing tumor-targeting function and step b) is obtained
It is enclosed with hydrophilic magnetic nano-particle and gas A and bovine serum albumin microspheres that Schiff 's base is contained on surface is uniformly mixed, keeps away
After light reaction for a period of time, by the isolated radiography material.
Preferably, when the time of the lucifuge reaction is 4~48 small;When further preferred range is 8~36 small;Into one
Walk preferable scope for 12~24 it is small when.
Preferably, the step c) separation methods are dialysis separation and/or column chromatography for separation.
As one preferred embodiment of the application, the preparation process of the radiography material is:
(1) Fe of dextran parcel is prepared3O4Nano-particle:Nitrogen (N is passed through to 250mL conical flasks2) 10~
30min is spare, weighs divalent iron salt (FeCl2·6H2O) 0.25~0.35g, in molar ratio (divalent iron salt: trivalent iron salt=1: 1
~1: 2) weigh appropriate trivalent iron salt, be dissolved in after mixing in 30~50mL water, be placed in N2It is in the conical flask of protection and quick
Stir (200~1000rpm), 30~80mL ammonium hydroxide is added dropwise into mixed solution with 0.2~2mL/s speed, 30~60 DEG C of temperature
10~60min of lower quick stirring;Appropriate dextran is weighed after the completion of stirring in mass ratio to be added dropwise in mixed solution, 30~60
30~120min is quickly stirred at a temperature of DEG C;Gained mixed solution is through Magneto separate, 8000~15000rpm centrifugations, spend from
Sub- water is resuspended after cleaning 1~3 time, and 0.004~0.009g of citric acid, 0~4 DEG C of preservation is added dropwise.
(2) ultrasonic cavitation method prepares the Fe for being enclosed with dextran3O4Western Buddhist is contained on magnetic nano-particle and nitrogen and surface
The bovine serum albumin microspheres of alkali:The bovine serum albumin(BSA) (BSA) for weighing mass fraction (1~8%) is dissolved in the PBS of 1~5mL
Mixed in buffer solution, while add appropriate glutaraldehyde solution and mix, stand 20~60sec;Dextrorotation obtained by a dropping step (1)
Sugared acid anhydride wraps up Fe3O40.5~2mL of nano-particle is into mixed solution and mixes;Ultrasonic probe is placed in liquid phase and gas phase (N2) boundary
Face top layer, takes out ultrasonic probe after ultrasonic, collect the Fe for being enclosed with dextran3O4Magnetic nano-particle and nitrogen and surface contains
There are the microvesicle of the bovine serum albumin microspheres of Schiff 's base and 0~4 DEG C of preservation.
(3) bovine serum albumin microspheres surface obtained by step (2) is folacin coupled:0.2~0.4g of folic acid is weighed, is dissolved in 5
In the anhydrous dimethyl sulfoxide (DMSO) of~20mL, the stirring of 20~60 μ L lucifuges of triethylamine is added dropwise, N- hydroxyls are added dropwise after being completely dissolved
Succinimide (NHS) 0.05~0.15g and dicyclohexylcarbodiimide (DCC) 0.1~0.2g into mixed solution and continue
Overnight, next day filters rear gained folic acid active ester for lucifuge reaction;It is added dropwise what 0.5~2mL folic acid active ester was collected into step (2)
In 1~4mL of microvesicle, room temperature lucifuge reacts 30~120min, and through dialysis, gained is the radiography material after post separation.
A diameter of 4~8mm of ultrasonic probe in the step (2).
Ultrasonic time is 10~180sec in the step (2), and effective ultrasonic time is 20~50sec.
Step (3) folic acid: NHS: DCC molar ratio is 1: 1: 1~1: 1.5: 1.5.
According to this area public office general knowledge, the dextran is a kind of is made of many glucose molecules and the Portugal of side chain gathers
Sugar, chemical formula can be abbreviated as H (C6H10O5)xOH。
The Schiff 's base is also referred to as schiff bases, contains N=C double bonds.In the application, the bad ammonia on bovine serum albumin microspheres surface
Sour residue forms Schiff 's base with the compound crosslink at least containing 2 aldehyde radicals, by the aldehyde in the compound at least containing 2 aldehyde radicals
Nitrogen-atoms in base carbon atom, with the amino of the lysine residue on bovine serum albumin microspheres surface, is connected by-N=C- double bonds
Connect to be formed.The target ligand with tumor-targeting function, the i.e. folic acid with bovine serum albumin(BSA) coupling, pass through folic acid activity
Ester occurs coupling reaction with the amino on bovine serum albumin(BSA) and obtains.
In the application, MRI is writing a Chinese character in simplified form for Magnetic resonance imaging;BSA is writing a Chinese character in simplified form for bovine serum albumin(BSA);PBS buffer is
Phosphate buffered saline solution is write a Chinese character in simplified form;GA is writing a Chinese character in simplified form for glutaraldehyde;FA is writing a Chinese character in simplified form for folic acid.
Herein described technical solution has the beneficial effect that:
(1) radiography material provided, has that particle diameter distribution is uniform, size is controllable, good water solubility, good biocompatibility etc.
Advantage;
(2) radiography material provided, is used equally for magnetic resonance imaging contrast, ultrasonic imaging contrast agent, fluoroscopic visualization
Agent and the separation of targeted drug and cell etc.;
(3) radiography material provided, has the function of medical MRI, ultrasound and fluoroscopic visualization, with medically clinical practice
MRI, ultrasound and fluorescent contrast agent are compared, and radiography performance is significantly improved, early detection and diagnosis available for tumour.
(4) method for preparing radiography material provided, using gentle aqueous phase system, method is simple, is easily enlarged metaplasia
Production.
It is to be understood that in the range of herein disclosed technical solution, above-mentioned each technical characteristic of the application and below (such as
Embodiment) in specifically describe each technical characteristic between can be combined with each other, so as to form new or preferable technical solution.
As space is limited, not repeated them here.
Unless otherwise defined, all professional and scientific terms used in text and meaning known to one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the application method.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
Brief description of the drawings
Fig. 1 be sample 1# structure diagram and embodiment 1 in main preparation process schematic diagram;(a) it is structure diagram,
(b) it is preparation process schematic diagram.
Fig. 2 is the grain size distribution of sample 1#.
Fig. 3 is bovine serum albumin(BSA)-glutaraldehyde fluorescence spectra;(a) be excitation wavelength be 468nm bovine serum albumin(BSA)s,
The fluorescence spectra of glutaraldehyde, bovine serum albumin(BSA)-glutaraldehyde;(b) it is that excitation wavelength is 536nm bovine serum albumin(BSA)s, penta 2
The fluorescence spectra of aldehyde, bovine serum albumin(BSA)-glutaraldehyde;(c) it is that excitation wavelength is 630nm bovine serum albumin(BSA)s, glutaraldehyde, ox
The fluorescence spectra of seralbumin-glutaraldehyde.
Fig. 4 is the cytotoxicity analysis figure of sample 1#.
Fig. 5 is the ultrasonic imaging figure of sample 1#;(a) it is comparison diagram;(b) it is to be enclosed with the Fe of dextran3O4Magnetic Nano
The microcapsular ultrasound image of the bovine serum albumin microspheres of Schiff 's base is contained on particle and nitrogen and surface.
Fig. 6 is the fluorescence imaging figure of sample 1#;(a) it is the image under fluorescence microscope common views;(b) shown for fluorescence
Green fluorescence image of the micro mirror in the case where excitation wavelength is 536nm;(c) it is fluorescence microscope in the case where excitation wavelength is 630nm
Red fluorescence image;(d) it is b figures and the merging figure of c figures.
Fig. 7 is the magnetic resonance imaging figure of sample 1#.
Embodiment
The features described above that the application mentions, or the feature that embodiment is mentioned can be in any combination.Disclosed in this case specification
All features can be used in combination with any composition form, each feature disclosed in specification, can by it is any provide it is identical,
The alternative characteristics substitution of impartial or similar purpose.Therefore except there is special instruction, revealed feature is only impartial or similar spy
The general example of sign.
With reference to embodiment, the application is expanded on further.It is to be understood that these embodiments be merely to illustrate the application without
For limiting scope of the present application.The experimental method of actual conditions is not specified in the following example, usually according to normal condition or
According to the condition proposed by manufacturer.
In the case of not doing specified otherwise, raw material used in this application, is bought by commercial sources, not specially treated
Directly use.
Specified otherwise is not done, and the instrument and test condition used in embodiment is as follows:
In sample preparation procedure, ultrasonic wave is added at air-liquid two-phase interface using Biosafer900-92 types Ultrasound Instrument,
Ultrasonic wave range is 20K~25K hertz.
Particle diameter distribution carries out material particle size test on zetasizer Nano ZS type dynamic light scattering particle size analyzers.
Spectrofluorimetry carries out sample spectra analysis in Hitachi F-4600 types Fluorescence Spectrometer.
Cytotoxicity analysis carry out cytotoxicity analysis using mtt assay on UV754PC type ultraviolet-uisible spectrophotometers.
Ultrasonic imaging analysis steps progress sample imaging research on auspicious diasonograph in DP-10 types.
Fluorescence imaging analysis carries out sample imaging research on LWD300-38LFT type inverted fluorescence microscopes.
Magnetic resonance imaging analysis carries out sample imaging research on Micro MR type Low-field magnetic resonance imaging instrument.
The preparation of 1 sample 1# of embodiment
(1) Fe of dextran cladding is prepared3O4Nano-particle:It is spare that nitrogen 30min is passed through to 250mL conical flasks, is claimed
Take divalent iron salt (FeCl2·4H2O) 0.2982g, weighs trivalent iron salt (FeCl3·6H2O) 0.5406g, is dissolved in after mixing
In 30mL water, N is placed in2(800rpm) in the conical flask of protection and quickly is stirred, 10mL ammonium hydroxide is added dropwise to mixed with 1mL/s speed
Close in solution, 30min is quickly stirred at a temperature of 50 DEG C;Weighed in mass ratio after the completion of stirring 0.15g dextrans be added dropwise to it is mixed
Close in solution, 60min is quickly stirred at a temperature of 50 DEG C;Gained mixed solution is through Magneto separate, 10000rpm centrifugations, spend from
After sub- water cleans 1~3 time, add 30ml ultra-pure waters and be resuspended, citric acid 0.006g is added dropwise, obtain wrapping up Fe containing dextran3O4
The liquid of nano-particle, in 4 DEG C of preservations.
(2) weigh the bovine serum albumin(BSA) 0.05g that mass fraction is 5% be dissolved in 1mL by disodium hydrogen phosphate dodecahydrate and
Two hypophosphite monohydrate sodium dihydrogens configuration pH is to be mixed in 7.2 PBS buffer, while it is 25% penta 2 to add 120 μ L mass concentrations
Aldehyde solution simultaneously mixes, and stands 60sec;Contain dextran parcel Fe obtained by a dropping step (1)3O4The liquid 1mL of nano-particle
Into mixed solution and mix;Ultrasonic probe is placed in liquid phase and gas phase nitrogen interface, ultrasound is taken out after ultrasonic 60sec and visits
Head, obtains the Fe with parcel dextran cladding3O4The bovine serum albumin(BSA) of Schiff 's base is contained on nano-particle and nitrogen and surface
The system of microballoon.
(3) folic acid 0.25g is weighed, is dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, 40 μ L lucifuges of triethylamine are added dropwise and stir
Mix, n-hydroxysuccinimide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g is added dropwise after being completely dissolved to mixing
In solution and continue lucifuge reaction overnight, gained folic acid active ester after next day filters;0.005g folic acid active ester is added dropwise to step
(2) in gained system 2mL, room temperature lucifuge reaction 60min, after dialysis, post separation, gained is to wrap up dextran parcel
Fe3O4The bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nano-particle and nitrogen, surface, is denoted as sample
Product 1#.The structure diagram of gained sample 1# is shown in Fig. 1 (a), and the schematic diagram of step (2) and (3) is shown in Fig. 1 (b).
The preparation of 2 sample 2# of embodiment
(1) γ-Fe of dextran parcel are prepared2O3Nano-particle:Nitrogen (N is passed through to 250mL conical flasks2) 10~
30min is spare, weighs divalent iron salt (FeCl2·6H2O) 0.5964g, weighs 1.0812g trivalent iron salts (FeCl3·6H2O), mix
It is dissolved in after even in 60mL water, is placed in N2In the conical flask of protection and (1000rpm) quickly is stirred, is dripped with 1.2mL/s speed
60mL ammonium hydroxide (20%) is added to be heated to reflux 90min at a temperature of 95 DEG C into mixed solution;0.32g dextrorotation is weighed after the completion of stirring
Sugared acid anhydride is added dropwise in mixed solution, and 30min is quickly stirred at a temperature of 50 DEG C;Gained mixed solution through Magneto separate, 12000rpm from
The heart precipitates, and 60ml ultra-pure waters are added after cleaning 1~3 time with deionized water and are resuspended, citric acid 0.012g is added dropwise, obtains containing dextrorotation
Sugared acid anhydride parcel γ-Fe2O3The liquid of nano-particle, in 4 DEG C of preservations.
(2) weigh bovine serum albumin(BSA) (BSA) 0.05g that mass fraction is 5% and be dissolved in 1mL by 12 hypophosphite monohydrate hydrogen two
Sodium and two hypophosphite monohydrate sodium dihydrogens configuration pH are to be mixed in 6.8 PBS buffer, while it is 25% to add 120 μ L mass concentrations
Glutaraldehyde solution simultaneously mixes, and stands 60sec;Contain dextran parcel γ-Fe obtained by a dropping step (1)2O3Nano-particle
Liquid 1mL is into mixed solution and mixes;Ultrasonic probe is placed in liquid phase and gas phase (N2) interface top layer, take after ultrasonic 60sec
Go out ultrasonic probe, obtain the γ-Fe with parcel dextran cladding2O3Nano-particle and nitrogen and Schiff 's base is contained on surface
The system of bovine serum albumin microspheres.
(3) folic acid 0.25g is weighed, is dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, 40 μ L lucifuges of triethylamine are added dropwise and stir
Mix, n-hydroxysuccinimide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g is added dropwise after being completely dissolved to mixing
In solution and continue lucifuge reaction overnight, gained folic acid active ester after next day filters;0.005g folic acid active ester is added dropwise to step
(2) in gained system 2mL, room temperature lucifuge reaction 60min, through dialysing, after post separation, gained is to wrap up dextran parcel
γ-Fe2O3The bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nano-particle and nitrogen, surface, is denoted as
Sample 2#.
The preparation of 3 sample 3# of embodiment
(1) γ-Fe of dextran parcel are prepared2O3Nano-particle:Nitrogen (N is passed through to 250mL conical flasks2) 10~
30min is spare, weighs divalent iron salt (FeCl2·6H2O) 0.2982g, weighs 0.5406g trivalent iron salts (FeCl3·6H2O), mix
It is dissolved in after even in 30mL water, is placed in N2In the conical flask of protection and (900rpm) quickly is stirred, is added dropwise with 1mL/s speed
30mL ammonium hydroxide (20%) is heated to reflux 90min into mixed solution at a temperature of 95 DEG C;After 0.5g mannitol is weighed after the completion of stirring
Quick stirring 20min, weighs 0.32g dextrans and is added dropwise in mixed solution, quickly stirred at a temperature of 50 DEG C after the completion of stirring
30min;Through Magneto separate, 12000rpm centrifugations, after cleaning 1~3 time with deionized water, add 30ml and surpass gained mixed solution
Pure water is resuspended, and citric acid 0.01g is added dropwise, and obtains containing dextran parcel γ-Fe2O3The liquid of nano-particle, in 4 DEG C of preservations.
(2) weigh bovine serum albumin(BSA) (BSA) 0.05g that mass fraction is 5% and be dissolved in 1mL by 12 hypophosphite monohydrate hydrogen two
Sodium and two hypophosphite monohydrate sodium dihydrogens configuration pH are to be mixed in 7.0 PBS buffer, while it is 20% to add 120 μ L mass concentrations
Glutaraldehyde solution simultaneously mixes, and stands 60sec;Contain dextran parcel γ-Fe obtained by a dropping step (1)2O3Nano-particle
Liquid 1mL is into mixed solution and mixes;Ultrasonic probe is placed in liquid phase and gas phase (N2) interface top layer, take after ultrasonic 60sec
Go out ultrasonic probe, obtain the γ-Fe with parcel dextran cladding2O3Nano-particle and nitrogen and Schiff 's base is contained on surface
The system of bovine serum albumin microspheres.
(3) folic acid 0.25g is weighed, is dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, 40 μ L lucifuges of triethylamine are added dropwise and stir
Mix, n-hydroxysuccinimide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g is added dropwise after being completely dissolved to mixing
In solution and continue lucifuge reaction overnight, gained folic acid active ester after next day filters;0.004g folic acid active ester is added dropwise to step
(2) in gained system 2mL, room temperature lucifuge reaction 60min, through dialysing, after post separation, gained is to wrap up dextran parcel
γ-Fe2O3The bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nano-particle and nitrogen, surface, is denoted as
Sample 3#.
Embodiment 4
Raw material proportioning, preparation process, condition in the same manner as in Example 1, only change step nitrogen into argon gas, and gained sample is denoted as
Sample 4#.
Embodiment 5
Raw material proportioning, preparation process, condition in the same manner as in Example 2, only by step nitrogen change into carbon dioxide other, gained
Sample is denoted as sample 5#.
Embodiment 6
Raw material proportioning, preparation process, condition, in the same manner as in Example 1, only by the FeCl of 0.5964g2·6H2O and 1.0812g
FeCl3·6H2O changes the CoCl of 2g into2·6H2O, changes nitrogen into helium, gained sample is denoted as sample 6#.
Embodiment 7
Raw material proportioning, preparation process, condition, in the same manner as in Example 1, only by the FeCl of 0.5964g2·6H2O and 1.0812g
FeCl3·6H2O changes the NiCl of 2g into2·6H2O, changes nitrogen into helium, gained sample is denoted as sample 7#.
Embodiment 8
Raw material proportioning, preparation process, condition, in the same manner as in Example 1, the shell that dextran is only changed into equal quality gathers
Sugar, changes nitrogen into air, gained sample is denoted as sample 8#.
Embodiment 9
Dextran, is only changed into the fiber of equal quality by raw material proportioning, preparation process, condition, in the same manner as in Example 1
Glutaraldehyde, is changed into the glyoxal of equivalent molar number by element, and gained sample is denoted as sample 9#.
Embodiment 10
Raw material proportioning, preparation process, condition, in the same manner as in Example 1,1, the 8- that glutaraldehyde is only changed into equivalent molar number is pungent
Dialdehyde, gained sample are denoted as sample 10#.
Comparative example 1
Bovine serum albumin(BSA) (BSA) 0.05g for taking mass percentage to be 5% is dissolved in 1mL by 12 hypophosphite monohydrate hydrogen two
Sodium and two hypophosphite monohydrate sodium dihydrogens configuration pH are to be mixed in 7.2 PBS buffer, while add 100 μ L glutaraldehyde solutions and mix
It is even, stand 60sec;Ultrasonic probe (diameter 6mm, power 490W) is placed in liquid phase and gas phase (N2) interface top layer, ultrasonic 60sec
After take out ultrasonic probe, obtain the system with parcel nitrogen and the surface bovine serum albumin microspheres that contain Schiff 's base.Collect
The microvesicle on system surface and 4 DEG C of preservations, are denoted as sample 11#.
Comparative example 2
Folacin coupled BSA-GA composite materials:Folic acid 0.25g is weighed, is dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL,
The stirring of 40 μ L lucifuges of triethylamine is added dropwise, n-hydroxysuccinimide (NHS) 0.1g and dicyclohexyl carbon two are added dropwise after being completely dissolved
Imines (DCC) 0.15g is into mixed solution and continues lucifuge reaction overnight, gained folic acid active ester after next day filters;It is added dropwise
The bovine serum albumin microspheres that 0.005g folic acid active ester has parcel nitrogen to the gained of comparative example 1 and Schiff 's base is contained on surface
In system 2mL, room temperature lucifuge reaction 60min, through dialysis, obtains parcel nitrogen and Schiff 's base and folic acid is contained in surface after post separation
Bovine serum albumin microspheres material, be denoted as sample 12#.
11 particle diameter distribution of embodiment
The particle diameter distribution of sample 1#~sample 10# is have detected, the particle diameter of gained sample is suitable between 0.5 μm~3 μm
As acoustic contrast agent.Using sample 1# as Typical Representative, its grain size distribution as shown in Fig. 2, in 0.8 μm~1.3 μ ms,
Centralized particle diameter is at 1.1 μm or so.
Embodiment 12
Respectively in comparative example 1, bovine serum albumin(BSA), glutaraldehyde and sample 11# in PBS buffer have carried out fluorescence light
Spectrum analysis, the results are shown in Figure 3.(a) is that excitation wavelength is 468nm bovine serum albumin(BSA)s, glutaraldehyde, has parcel nitrogen in Fig. 3
The fluorescence spectra of the bovine serum albumin microspheres of Schiff 's base is contained on gas and surface;(b) it is that excitation wavelength is that 536nm ox bloods are pure
Albumen, glutaraldehyde, have parcel nitrogen and surface contain Schiff 's base bovine serum albumin microspheres fluorescence spectra;(c) it is
Excitation wavelength is 630nm bovine serum albumin(BSA)s, glutaraldehyde, has parcel nitrogen and the bovine serum albumin(BSA) of Schiff 's base is contained on surface
The fluorescence spectra of microballoon.As can be seen that individually bovine serum albumin bletilla glutaraldehyde does not have fluorescent effect, only both combine
After producing Schiff 's base, fluorescence is just produced.
Embodiment 13
Cytotoxicity detection has been carried out to sample 1#~sample 12# respectively, has illustrated low cytotoxicity.Using sample 1# as
Typical Representative, its cytotoxicity analysis figure are as shown in Figure 4.With the rise of concentration, cell survival rate is not under appearance significantly
Drop trend, it was demonstrated that the toxicity of 1#~12# samples is low cytotoxicity.
Embodiment 14
Respectively using sample 1#~sample 10# as contrast agent, ultrasonic contrast detection has been carried out, has illustrated more significant surpass
Sound radiography function.Using sample 1# as Typical Representative, its ultrasonic contrast figure is as shown in Figure 5.Wherein (a) is blank image
(b) Fe of dextran cladding is wrapped up for 1# samples3O4The bovine serum albumin of Schiff 's base is contained on nano-particle and nitrogen and surface
Bai Weiqiu, it can be seen that be shown as white (enhancing of ultrasonic contrast function) in figure (b) blank pipe, illustrate that sample 1#~sample 10# is equal
There is obvious ultrasonic imaging.
Embodiment 15
Respectively using sample 1#~sample 10# as contrast agent, fluoroscopic visualization detection has been carried out, has been illustrated more significant glimmering
Light radiography function.Using sample 1# as Typical Representative, its fluoroscopic visualization figure is as shown in Figure 6.Wherein (a) commonly regards for fluorescence microscope
Image under wild;(b) it is green fluorescence image of the fluorescence microscope in the case where excitation wavelength is 536nm;(c) it is fluorescence microscopy
Red fluorescence image of the mirror in the case where excitation wavelength is 630nm;(d) it is b figures and the merging figure of c figures.It can be seen that swash in difference
Shine under excitation, sample can send the fluorescence such as feux rouges and green light (enhancing of fluoroscopic visualization function), illustrate sample 1#~sample 10#
It is respectively provided with obvious fluorescence imaging function.
Embodiment 16
Respectively using sample 1#~sample 10# as contrast agent, magnetic resonance radiography detection has been carried out, has been illustrated more significant
Magnetic resonance radiography function.Using sample 1#-3# as Typical Representative, its magnetic resonance radiography figure is as shown in Figure 7.It can be seen that the water around
When phase environment is in light gray, black (MRI T are presented in sample 1#-3#2Radiography function strengthens), illustrate sample 1#~sample 10#
It is respectively provided with obvious magnetic resonance imaging function.
Embodiment 17
The relaxation rate of sample 1#~sample 10# is determined, is provided with more typical MRI T2Relaxation behavior.With sample
Product 1# is Typical Representative, its relaxation rate measurement result is as shown in table 1.
Table 1
It is not for limiting claim, any this area skill although the application is disclosed as above with preferred embodiment
Art personnel are not being departed from the application spirit and scope, can make possible variation and modification, therefore the protection of the application
Scope should be subject to the scope that the application claim is defined.