CN104548145B - A kind of preparation method of the SPIO nano particle of polyglutamic acid PGA claddings - Google Patents
A kind of preparation method of the SPIO nano particle of polyglutamic acid PGA claddings Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of the SPIO nano particle of polyglutamic acid PGA claddings, including：Trivalent iron salt is dissolved in the water, stirs, is passed through nitrogen, and polyglutamic acid PGA solution is added dropwise, sodium sulfite Na is then added dropwise while stirring2SO3Aqueous solution, obtains mixed solution, then moves into water-bath, and NH is added dropwise while stirring3·H2O, reacts 20 30min, then reacts 0.5 1.5h at ambient temperature, centrifuges, dialysis, to obtain the final product.Present invention process very simple, reaction condition is gentle, easily operated, safety non-pollution；SPIO PGA nano particle diameters prepared by the present invention are evenly distributed, particle diameter is smaller, relaxation rate is high, contrasting effects are notable, has good water solubility, colloidal stability, biocompatibility and blood compatibility, has no adverse effects to organism, it is of low cost, it is easy to preserve.
Description
Technical field
The invention belongs to the preparation field of magnetic resonance contrast agent, more particularly to a kind of superparamagnetic of polyglutamic acid PGA claddings
The preparation method of property ferric oxide nanometer particle.
Background technology
Magnetic resonance imaging (MRI) technology is a kind of imaging technique of high resolution, has higher space and fault imaging
Ability, MRI does not have radioactive ionization infringement, while can obtain dissection and physiologic information, has other medical imagings can not
The advantages of analogy.Mr imaging technique plays an increasingly important role in disease surveillance field.But the weakness of MRI is that its is quick
Perception is relatively low, and the relaxation time of Different Organs or tumor tissues overlapped makes MRI difficult diagnosiss.In recent years, note is passed through
Penetrating the method for MRI contrast agent can effectively solve the problems, such as that MRI sensitiveness is relatively low.Therefore suitable MRI contrast agent is selected just to show
It is particularly important that obtaining.
Superparamagnetic iron oxide nano particle (SPIO) has more and more extensive in biomedical sector in recent years
Using the application especially in terms of magnetic resonance imaging contrast is even more to receive universal concern.SPIO nano particles have
Unique magnetic property and higher signal strength, relatively low dosage, good biocompatibility and relatively low manufacture
The features such as cost.Be commercialized SPIO nano particles is applied to clinical disease diagnosis as MRI contrast agent at present.However, this
When a little commercialized SPIO contrast agent are used for tumor cells image field, also there is lot of challenges.For example, commercialization SPIO makes
Often relaxation rate is relatively low for shadow agent, it is impossible to achievees the purpose that to epidemic disease Sensitive Detection.
Using gentle reduction method prepare SPIO nano particle (SPIO) the result shows that, by gently reducing
Ferroferric oxide nano granules size prepared by method is smaller, uniform particle sizes, and shows high r2Relaxation rate (Shen Mingwu,
Li Jing surpasses, Hu Yong, Sun Wenjie, and history faces south.A kind of preparation method of the SPIO nano particle of modified with folic acid.China
Patent of invention, application number：201410182821.1 the date of application：2014-4-30).But the above method is mainly used and carried just
The polyethyleneimine (PEI) of electric charge is used as stabilizer, it is necessary to which further functionalization can be only used for cell or tumour MR imagings.
Domestic and foreign literature is retrieved, still without the SPIO nano particles work found on preparing PGA claddings with gentle reduction method
For the relevant report of MRI contrast agent research.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of Superparamagnetic Iron Oxide nanometer of polyglutamic acid PGA claddings
The preparation method of particle, the present invention prepare SPIO-PGA using gentle reduction method and are used for MR image-forming contrast mediums, and preparation method is simple,
Reaction condition is gentle, easily operated processing, and stabilizer used is cheap and environmentally friendly polyglutamic acid, has industrialization
The prospect of implementation.
A kind of preparation method of the SPIO nano particle of polyglutamic acid PGA claddings of the present invention, including：
Trivalent iron salt is dissolved in the water, stirs, is passed through nitrogen, and polyglutamic acid PGA solution is added dropwise, is stirred with back
Mix side and sodium sulfite Na is added dropwise2SO3Aqueous solution, obtains mixed solution, then moves into water-bath, and NH is added dropwise while stirring3·
H2O, reacts 20-30min, then reacts 0.5-1.5h at ambient temperature, centrifuges, dialysis, up to polyglutamic acid PGA claddings
SPIO nano particle.
The trivalent iron salt is FeCl3·6H2O；Water is ultra-pure water.
Concentration after the trivalent iron salt is dissolved in the water is not higher than 13mg/mL；The molecular weight of polyglutamic acid PGA is 100
Ten thousand.
It is described that to be passed through the nitrogen time be 5-15min.
The speed of agitator is 1000-5000 revs/min.
The FeCl3·6H2O and Na2SO3Mass ratio be 7:1；FeCl3·6H2The mass ratio of O and PGA is 7:1；
FeCl3·6H2The O and NH that mass fraction is 91%3·H2The mass ratio of O is 4:5.
The FeCl3·6H2Na is added dropwise in O solution2SO3Color is transferred in water-bath after being changed into yellow from rufous and dripped afterwards
Add NH3·H2O。
The bath temperature is 50-65 DEG C.
The centrifugal rotational speed is 5000-8000 revs/min.
To use bag filter of the molecular cut off for 8000-14000, water for dialysis is distilled water for the dialysis, dialysis three
My god, water is changed daily three times.
The SPIO nano particle of the polyglutamic acid PGA claddings is used for liver and tumor model as preparation
T2The application of the contrast agent of magnetic resonance imaging diagnosis.
The present invention adds polyglutamic acid (PGA) into reaction solution using during reduction method for preparing nanometer particle,
So as to prepare the SPIO nano particles (SPIO-PGA) of PGA claddings.PGA has excellent water-soluble, superpower adsorptivity and life
Biodegradable, degradable product are non-harmful glutamic acid, are a kind of excellent environment-friendly type macromolecule materials.PGA strands
Upper abundant-COOH makes it have good biocompatibility, in aqueous in elecrtonegativity, it is not necessary to which further modification can
To carry out biologic applications, reduce workload, while reduce production cost.Abundant-COOH also can further rhetorical function
Change group, very big space is provided for the further further investigation exploitation of the present invention.Characterize data shows, the SPIO- of preparation
PGA has good water solubility and colloidal stability, is investigated by a series of biological experiments, SPIO-PGA prepared by the present invention
With good biocompatibility and blood compatibility, and its r2Relaxation rate be up to 333.7mM-1s-1, there is significant T2
The MRI imaging effects of weighting, are expected to be applied to clinical disease diagnosis as MRI contrast agent.
The present invention is easy to operation, and the cost of raw material is low.The nano particle of preparation is steady with good water solubility, colloid
Qualitative, blood compatibility and biocompatibility.Tested by imaging contrast in nude mouse, SPIO-PGA nanometers prepared by the present invention
Particle has significant contrasting effects, has potential application value in magnetic resonance imaging contrast field.
The present invention uses transmission electron microscope (TEM), X-ray diffraction analysis (XRD), Zeta electric potential and dynamic light scattering
Analyze (DLS), fourier transform infrared spectroscopy analysis (FTIR), thermogravimetric analysis (TGA), inductively coupled plasma atomic emissions
SPIO-PGA nano particles prepared by the means such as spectroscopic methodology (ICP-OES) and magnetic resonance analysis (MR) characterization.Then mtt assay is utilized
The cytotoxicity of nano particle is evaluated, and the pattern of the cell after being co-cultured with material is obtained with phase contrast microscope；Pass through haemolysis
The blood compatibility of the experimental evaluation present invention.The magnetic resonance imaging for finally carrying out cell in vitro and nude mice vivo tumor model is real
Test, investigate the cell in vitro of SPIO-PGA nano particles and the MR imaging effects of vivo tumor model.In addition, pass through Tissue distribution
The metabolic process of experimental study SPIO-PGA nano particles in vivo.Specific test result is as follows：
(1) transmission electron microscope (TEM) test result
The TEM pictures and size distribution histogram of SPIO-PGA nano particles prepared by the present invention show (referring to attached drawing 1)：
The pattern of the SPIO nano particles formed is spherical in shape or torispherical, size uniform, without obvious agglomeration, in the solution
Well dispersed and do not assemble, statistical analysis show that particle diameter size is 5.3 ± 2.6nm, and particle diameter distribution is relatively narrow
In the range of.
(2) X-ray diffraction analysis (XRD) test result
The XRD spectrum of the SPIO nano particles of PGA parcels prepared by this method shows (referring to attached drawing 2)：Material prepared
Diffraction maximum at site 220,311,400,422,511 and 440 and ferroferric oxide nano granules diffraction maximum site very
It coincide.Diffraction maximum peak type is sharp, illustrates that the good ferroso-ferric oxide crystal of crystal form has been made in reaction.
(3) Zeta electric potential and dynamic light scattering (DLS) test result
The surface potential of Zeta electric potential the results show SPIO-PGA nano particles is -38.6mV, and hydrodynamics is a diameter of
217.5±3.75nm。
(4) Fourier transform infrared spectroscopy (FTIR) test result
The FTIR collection of illustrative plates of the SPIO nano particles of PGA parcels prepared by this method shows (referring to attached drawing 3)：In 1080cm-1
(C-O)、1410cm-1(O-H)、1637cm-1(C=O) at three, compared to exposed ferroferric oxide nano granules, peak intensity is absorbed
The obvious increase of degree.This explanation PGA has been wrapped on SPIO nano particles well, forms the nanostructured of cladding.
(5) thermogravimetric analyzer (TGA) test result
The TGA analysis results of SPIO-PGA nano particles prepared by this method show SPIO-PGA nanometers (referring to attached drawing 4)
The upper carrying capacity that ferroso-ferric oxide content is about 75.6%, PGA in particle has reached 24.4%.
(6) magnetic resonance (MR) analysis result
r2Relaxation rate reflects imaging efficiency of the SPIO nano-particles as MRI contrast agent, the transverse direction of unit molar concentration iron
In the relaxation time, can pass through the relaxation time (T under various concentrations1Or T2) the Fitting Calculation reciprocal obtain.Prepared by the present invention
SPIO-PGA nano particles T2The relaxation time Linear Fit Chart reciprocal with Fe concentration, it can be seen that this SPIO-PGA nanometers
The relaxation time inverse of grain has good linear relationship with the increase of concentration of iron.With increasing for Fe concentration, its MR signal
Intensity substantially weakens.The magnetic resonance imaging of various concentrations sample can be seen that nano particle has good external imaging effect.
By the r that SPIO-PGA nano particles are calculated2It is worth for 333.7mM-1s-1(referring to attached drawing 5).
(7) MTT cell viabilities and phase contrast microscope observation result
The thin of the SPIO-PGA nano particles of the invention prepared is evaluated by the vigor of MTT colorimetric method for determining HeLa cells
Born of the same parents' compatibility (referring to attached drawing 6).By HeLa cell seedings in 96 orifice plates, every kind of concentration set 5 Duplicate Samples (Fe concentration as
50、150、250、350、450μg/mL).Tissue Culture Plate is placed in CO2Co-cultured in the environment that concentration is 5% and temperature is 37 DEG C
24 it is small when.Light absorption value of each hole at λ=570nm is detected in microplate reader after MTT is handled, and calculates corresponding cell accordingly
Vigor, wherein being denoted as 100% as blank control, cell viability using the cell of physiological saline processing.Compared with blank control group,
It is respectively provided with more through HeLa cells nanoparticle treated SPIO-PGA in the case where Fe concentration is 50,150,250,350,450 μ g/mL
High cell viability, and as the vigor of the increase cell of concentrations of nanoparticles accordingly increases therewith.This is because PGA is a kind of
Boiomacromolecule with good biocompatibility, differentiation and propagation to cell have facilitation.In contrast, as right
For the exposed ferroferric oxide nano granules group of ratio under same concentrations, cell viability is significantly lower than the cell of physiological saline processing
Control group, and as the increase of concentration, cytoactive gradually reduce.It is good that two groups of contrasts show that SPIO-PGA nano particles have
Cell compatibility.Meanwhile we also further demonstrate SPIO-PGA nano particles to thin by phase contrast microscope observation
The influence of born of the same parents' pattern.The result shows that the nano particle (Fe concentration is 50,150,250,350,450 μ g/mL) of various concentrations is 37
At DEG C with cell co-culture 24 it is small when after, cell morphology does not change significantly (referring to attached drawing 7).Further illustrate SPIO-
PGA nano particles have good cell compatibility.
(8) blood compatibility
SPIO-PGA nano particles prepared by the present invention its blood compatibility of needs assessment before zoopery is carried out.I
Material blood compatibility is evaluated by hemolytic experiment.By fresh human red cell and nano particle physiological saline
Solution (Fe concentration is 50,150,250,350,450 μ g/mL) at room temperature by 1 it is small when be incubated after, centrifugation observation haemolysis feelings
Condition.Using deionized water as positive controls, physiological saline passes through UV, visible light as negative control group, the degree of hemolysis of sample
Absorption value of the absorption spectrum at 541nm carries out quantization signifying.Test result indicates that：Reach 450 μ g/mL in iron concentration
When, the hemolysis rate of SPIO-PGA nano particles is 3.42%, is decreased with the reduction hemolysis rate of sample concentration, each sample is molten
Blood rate is below 5% (referring to attached drawing 8).Illustrate that SPIO-PGA nano particles prepared by the present invention have good blood compatibility
Property.
(9) cell phagocytosis experiment
For biomedical applications, reduction nano particle is swallowed most important by macrophage.Therefore need to SPIO-
PGA nano particles are evaluated by the phagocytosis situation of macrophage.(Fe is dense with SPIO-PGA nano particles for 264.7 cells of Raw
Spend for 10,100 μ g/mL) in CO2When concentration is 5% and temperature is that co-cultivation 4 is small at 37 DEG C, sky is used as using physiological saline culture
White control group.Clean after cell, the Fe concentration of cell phagocytosis is measured by ICP-OES.When Fe concentration is 100 μ g/mL, phase
For exposed ferroferric oxide nano granules than surface free PGA modifications, the cell phagocytosis amount of SPIO-PGA nano particles
Obvious less (referring to attached drawing 9).Therefore material of the present invention is expected to be avoided that and is swallowed by macrophage after entering in organism, from
And obtain preferable contrasting effects.
(10) internal MR imaging results
HeLa Transplanted tumor models are built in nude mouse, it is water-soluble by tail vein injection SPIO-PGA nano particle physiology salts
Liquid (100 μ L, 1000 μ g/mL) evaluates major organs and tumor locus magnetic resonance imaging effect (referring to attached drawing 10 and 11).With
Magnetic resonance image before injection is compared, when injection SPIO-PGA nano particles 2 are small after, nude mice liver and spleen position substantially become
Secretly, signal strength substantially weakens (referring to attached drawing 11a, spleen area is too small can not Accurate Determining signal value).The letter of tumor locus
Number intensity has to be weakened (referring to attached drawing 11b) to a certain degree.This shows that the nano particle of injection can partly escape reticular endothelium system
The phagocytosis of system (liver and spleen position), passes through the high-permeability and retention effect (enhanced of tumor locus
Permeability and retention effect, EPR) nano particle passive target enters tumor locus.With the time
Extend, the MR signal strengths at nude mouse tumor position are gradually recovered, and illustrate that SPIO-PGA nano particles start to be metabolized in vivo.MR into
As the result shows that SPIO-PGA nano particles prepared by the present invention can be used in the MR imagings of internal major organs and tumor model.
(11) Tissue distribution
In order to study the distribution and metabolism situation that SPIO-PGA nano particles of the present invention are respectively organized in vivo, we are in structure
Built in the nude mouses of HeLa Transplanted tumor models by tail vein injection SPIO-PGA nano particles normal saline solution (100 μ L,
1000μg/mL).With different time points when small (2, the 4,6) content of Fe elements in each vitals after ICP-OES measurement injections
(referring to attached drawing 12), and set blank nude mice to be used as with reference to control.It can be seen from the figure that compared with control group, Fe concentration masters
It is distributed at liver and spleen, and as the change of time, aggregate amount change.When 2 is small, Fe concentration reaches in liver
Highest, over time, Fe concentration substantially reduce, and show that Fe elements are slowly metabolized by liver, this is imaged with internal MR and ties
Fruit is consistent；When 4 is small, Fe concentration reaches highest in spleen, and higher than Fe concentration in liver, as time went on, Fe concentration
It is obvious to reduce, show that Fe elements are slowly metabolized by spleen, this coincide with internal MR imaging results.When injection material 6 of the present invention is small
Afterwards, Fe concentration all significantly decreases in each organ of nude mice.And other organs (heart, lung, kidney), the aggregation of Fe are substantially less,
Fe is finally also slowly metabolized in these organs.This result shows that material can in nude mouse normal metabolite clearance, energy
Into tumor locus, it is allowed to the MR imagings of tumour.
Beneficial effect
(1) present invention is used for MR image-forming contrast mediums using gentle reduction method preparation SPIO-PGA, and preparation method is simple, reaction
Mild condition, easily operated processing, stabilizer used are cheap and environmentally friendly polyglutamic acid, have industrialized implementation
Prospect；
(2) the SPIO-PGA nano particle crystal forms that prepared by the present invention are good, uniform in size, have good water solubility, glue
Body stability, cell compatibility, biocompatibility, blood compatibility and good T2Contrasting effects, the organic matter surface of parcel
There is substantial amounts of active group to can be used for further modifying and deeply developing, have in magnetic resonance imaging arts and potentially apply valency
Value.
Brief description of the drawings
Fig. 1 is transmission electron microscope (TEM) picture (a) and particle diameter point of SPIO-PGA nano particles prepared by the present invention
Cloth histogram (b)；
Fig. 2 is the X ray diffracting spectrum of SPIO-PGA nano particles prepared by the present invention：SPIO- is followed successively by from top to bottom
PGA nano particles and exposed ferroferric oxide nano granules (Fe3O4)；
Fig. 3 is Fourier transform infrared spectroscopy (FTIR), is followed successively by exposed ferroferric oxide nano granules from top to bottom
(Fe3O4), polyglutamic acid (PGA), SPIO-PGA nano particles；
Fig. 4 is thermogravimetric analysis (TGA) collection of illustrative plates, is followed successively by exposed ferroferric oxide nano granules (Fe from top to bottom3O4)、
SPIO-PGA, polyglutamic acid (PGA)；
Fig. 5 is the r of SPIO-PGA nano particles2Relaxation rate test curve and MR images；
Fig. 6 is mtt assay test by exposed ferroferric oxide nano granules (control group) and SPIO-PGA nanometers
The cell viability of HeLa cells after when grain (Fe concentration is 50,150,250,350,450 μ g/mL) processing 24 is small；
Fig. 7 be HeLa cells by physiological saline (control) and SPIO-PGA nano particles (Fe concentration is 50,150,250,
350th, 450 μ g/mL) processing 24 it is small when after, phase contrast microscope observation cell morphology；Picture is followed successively by physiological saline from a to f
(control) and Fe concentration are the cell morphology after 50,150,250,350,450 μ g/mL processing；
Fig. 8 is the ultraviolet spectrogram and hemolytic experiment photo (illustration) of SPIO-PGA nano particle hemolytic experiments；
Fig. 9 be by SPIO-PGA nano particles (Fe concentration be 0,10,100 μ g/mL) handle 4 it is small when after, macrophage
Phagocytosis situation；
When Figure 10 is that nude mice tail vein injection SPIO-PGA nano particles (100 μ L, 1000 μ g/mL) are preceding different with after injection
Between point when small (2,4,6) magnetic resonance imaging picture；
Figure 11 is before nude mice tail vein injection SPIO-PGA nano particles (100 μ L, 1000 μ g/mL) (a) and (b) after injection
Different time points when small (2, the 4,6) liver of nude mice and the situation of change of tumor locus signal strength；Wherein ordinate represents
Figure 12 for nude mice tail vein injection SPIO-PGA nano particles (100 μ L,G/L different time after) preceding and injection
The distribution of Fe concentration of element and metabolic condition in each organ of point when small (2,4,6).
Embodiment
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Scope.
Embodiment 1
640mg Iron(III) chloride hexahydrates are dissolved in 50mL ultra-pure waters, nitrogen is passed through 10 minutes and is sufficiently stirred.Will
90mgPGA is dissolved in 20mL ultra-pure waters, and above-mentioned solution is added dropwise.It is molten that 9mg/mL sodium sulfites are added dropwise after stirring evenly
Liquid.Mixed solution is transferred in 60 DEG C of water-baths, adds 1mL ammonium hydroxide, fully reaction 30 minutes.Mixed solution is moved into room temperature,
The reaction was continued 1.5 it is small when.The solution of preparation is centrifuged, with 8000 revs/min of rotating speed centrifugation after ten minutes, discards centrifugation
Precipitation, takes upper solution.The bag filter for being 8000-14000 with molecular cut off is dialysed.Water for dialysis is distilled water, dialysis three
My god, water is changed daily three times.The ferroferric oxide nano granules coated to polyglutamic acid prepared by the present invention are shown by transmitted electron
Micro mirror is observed, the results showed that and the SPIO nano particle patterns of formation are uniform, average grain diameter 5.3nm, standard deviation 2.6nm,
It is distributed in relatively narrow particle size range (referring to attached drawing 1).The surface potential of Zeta electric potential the results show SPIO-PGA nano particles
For -38.6mV, the hydrodynamic diameter of dynamic light scattering measure shows：It is 217.5 ± 3.75nm that it, which is hydrated particle diameter,.X-ray diffraction
The good ferroso-ferric oxide crystal of crystal form has been prepared in collection of illustrative plates proved response (referring to attached drawing 2).Fourier transform infrared spectroscopy
The enhancing of absworption peak, illustrates that polyglutamic acid is coated on ferroferric oxide nano granules, forms the nanostructured of cladding (referring to attached
Fig. 3).Drawn by thermogravimetric analysis, in SPIO-PGA nano particles the content of ferroso-ferric oxide be 75.6%, polyglutamic acid it is upper
Carrying capacity has reached 24.4% (referring to attached drawing 4).
Embodiment 2
The concentration of Fe elements in the SPIO-PGA nanoparticles solutions prepared by the ICP-OES methods of testing measure present invention.
Prepare respectively Fe concentration for 0.004,0.008,0.016,0.032, the SPIO-PGA nano particle aqueous solution 2mL of 0.064mM, lead to
Cross T of the magnetic resonance imaging analysis instrument measure material under different Fe concentration2Relaxation effect (referring to attached drawing 5).Relaxation rate test knot
Fruit shows the relaxation time inverse of SPIO nano materials with the increase of concentration of iron (in 0.004~0.064mM concentration ranges)
With good linear relationship.The r of the SPIO-PGA prepared by can be calculated the present invention2Relaxation rate is up to 333.7mM-1s-1。
Therefore, the SPIO-PGA prepared by the present invention can be used as excellent T2Signal attenuation contrast agent.
Embodiment 3
Influence of the SPIO-PGA nano particles to cell viability is prepared by the model cell evaluation present invention of HeLa cells.With
Exposed Fe prepared by comparative example 13O4Nano particle is as control.The solution that embodiment 1 is obtained prepares Fe concentration of element successively
For the normal saline solution (sodium chloride content 0.9%) of 50,150,250,350,450 μ g/mL SPIO-PGA nano particles.Will
For HeLa cell seedings in 96 orifice plates, every kind of concentration sets 5 Duplicate Samples, and Tissue Culture Plate is placed in CO2Concentration is 5% and temperature
Spend for co-culture at 37 DEG C 24 it is small when.Detect light absorption value of each hole at λ=570nm in microplate reader after MTT is handled, and according to
This calculates corresponding cell viability, wherein the cell handled using physiological saline is blank control, cell viability is denoted as 100%.With
The exposed Fe of control group3O4Compare, under each concentration, SPIO-PGA processing cell be respectively provided with good cell viability (referring to
Attached drawing 6).Compared with blank control group, the vigor of the cell of each concentration SPIO-PGA solution treatments is superior to blank control group,
Further demonstrate that the present invention has good biocompatibility.Cell morphology (the ginseng that this result is observed by phase contrast microscope
See attached drawing 7) obtain further verification.
Embodiment 4
It is prepared by embodiment 1 SPIO-PGA nano particles normal saline solution (Fe concentration is 50,150,250,350,
450 μ g/mL) with fresh human red cell at room temperature by 1 it is small when be incubated, centrifugation observation haemolysis situation.Correspondingly, to go
Ionized water is divided light as negative control group, the degree of hemolysis of sample as positive controls, physiological saline by UV, visible light
Spend absorption value of the instrument at 541nm and carry out quantization signifying.Test result indicates that SPIO-PGA nano particles have good blood
Compatibility.Each concentration samples hemolysis rate is respectively less than 5%, when iron concentration is 450 μ g/mL, SPIO-PGA nano particles it is molten
Blood rate is 3.42%, is decreased (referring to attached drawing 8) with the reduction hemolysis rate of sample concentration.Illustrate that the present invention prepares
SPIO-PGA nano particles have good blood compatibility.
Embodiment 5
To investigate the situation that SPIO-PGA nano particles are swallowed by macrophage, 264.7 cells of Raw are made with embodiment 1
When standby SPIO-PGA nano particles (Fe concentration is respectively 10 and 100 μ g/mL) co-incubation 4 is small, prepared with comparative example 1 naked
The Fe of dew3O4Nanoparticle is as control.Clean after cell, the Fe members of the material swallowed by ICP-OES measurements by macrophage
Plain concentration.It is all 100 μ g/mL in Fe concentration for exposed ferroferric oxide nano granules compared to surface free PGA modifications
When, the cell phagocytosis amount of SPIO-PGA nano particles is obvious less (referring to attached drawing 9).Therefore the SPIO-PGA that prepared by the present invention receives
Rice grain is expected to avoid being swallowed by macrophage after entering in organism, so as to obtain preferable contrasting effects.
Embodiment 6
In order to verify material of the present invention actual imaging effect in animal body, HeLa transplantable tumor moulds are built in nude mouse
Type, is evaluated organ and is swollen by tail vein injection SPIO-PGA nano particles normal saline solution (100 μ L, 1000 μ g/mL)
Knurl position magnetic resonance imaging effect.As shown in FIG. 10 and 11, compared with the magnetic resonance image of nude mice before injection, SPIO-PGA is injected
After when nano particle 2 is small, each major organs of nude mice are dimmed in various degree, and liver and spleen position are substantially dimmed, liver region
Signal strength has obvious decrease (referring to attached drawing 10 and attached drawing 11a, spleen area is too small can not Accurate Determining signal value), tumour portion
Position signal strength have certain decrease (referring to attached drawing 10 and attached drawing 11b), show injection nano particle first largely into
Liver is entered, has reduced the signal strength of liver region, small part material is swallowed by tumour.4 it is small when after, spleen position is further
It is dimmed, this is because nano particle initially enters liver after being injected into nude mouse, then it is metabolized by spleen.Tumour portion
The signal strength of position is most weak when 2 is small, shows that at this moment nano material reaches maximum in the aggregate amount of tumor locus.6 it is small when after sweep
Image display is retouched, the recovered level close to before injection of signal strength of tumor locus, shows that most materials have been metabolized
Excrete.
Embodiment 7
In order to study distribution and the metabolic condition that SPIO-PGA nano particles of the present invention are respectively organized in vivo, construct
In the nude mouses of HeLa Transplanted tumor models by tail vein injection SPIO-PGA nano particles normal saline solution (100 μ L, 1000
μg/mL).Then with each vitals of different time points when small (2,4,6) and tumour before ICP-OES measurement injections and after injection
The content (referring to attached drawing 12) of middle Fe concentration, and set blank nude mice to be used as with reference to control.It can be seen from the figure that and control group
To compare, Fe concentration is mainly distributed at spleen, and as the change of time, aggregate amount change.After injection 2 it is small when, liver
Middle Fe concentration reaches highest, with the extension of time, Fe concentration substantially reduces, shows that Fe elements are slowly metabolized by liver, this
It is consistent with internal MR imaging results；When 4 is small, Fe concentration reaches highest in spleen, and higher than Fe concentration in liver, Ran Housui
The passage of time, Fe concentration substantially reduces, and shows that Fe elements are slowly metabolized by spleen, this is also kissed with internal MR imaging results
Close.After when injection material 6 of the present invention is small, Fe concentration has obvious reduction in each organ of nude mice, close in control mouse body
Fe concentration.Tumor locus Fe concentration highest when 2 is small, is then gradually metabolized away.This is the result shows that material can be in nude mouse
Interior normal metabolite clearance, can enter tumor locus, it is allowed to the MR imagings of tumour.
Comparative example 1
In liquor ferri trichloridi, sodium sulfite solution is added, is transferred under 60 DEG C of water bath conditions to reaction solution and adds
Ammonium hydroxide, react 30 minutes, be finally placed in room temperature reaction 1.5 it is small when.After reaction, obtained black precipitate Magneto separate is removed
Supernatant is removed, then adds appropriate ultra-pure water ultrasonic disperse, then Magneto separate, so repeat milli-Q water three times, to remove impurity, so
It is scattered in again in ultra-pure water afterwards, vacuum freeze drying, for X-ray diffraction detection and MTT cytotoxicity experiments.Penetrated by X
The test of line diffraction proves that the good ferroso-ferric oxide crystal of crystal form has been made in reaction (referring to attached drawing 2).
Claims (10)
1. a kind of preparation method of the SPIO nano particle of polyglutamic acid PGA claddings, including：
Trivalent iron salt is dissolved in the water, stirs, is passed through nitrogen, and polyglutamic acid PGA solution is added dropwise, then while stirring
Sodium sulfite Na is added dropwise2SO3Aqueous solution, obtains mixed solution, then moves into water-bath, and NH is added dropwise while stirring3·H2O,
20-30min is reacted, then reacts 0.5-1.5h at ambient temperature, is centrifuged, dialysis, up to the super suitable of polyglutamic acid PGA claddings
Superparamag-netic iron oxide；Wherein the molecular weight of polyglutamic acid PGA is 1,000,000, the superparamagnetism oxygen of polyglutamic acid PGA claddings
The particle diameter size for changing iron nano-particle is 5.3 ± 2.6nm；The trivalent iron salt is FeCl3·6H2O；The FeCl3·
6H2O and Na2SO3Mass ratio be 7:1；FeCl3·6H2The mass ratio of O and PGA is 7:1.
A kind of 2. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The water is ultra-pure water.
A kind of 3. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：Concentration after the trivalent iron salt is dissolved in the water is not higher than 13mg/mL.
A kind of 4. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：It is described that to be passed through the nitrogen time be 5-15min；Speed of agitator is 1000-5000 revs/min.
A kind of 5. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The FeCl3·6H2The O and NH that mass fraction is 91%3·H2The mass ratio of O is 4:5.
A kind of 6. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The FeCl3·6H2Na is added dropwise in O solution2SO3Color transfers to water after being changed into yellow from rufous afterwards
NH is added dropwise in bath3·H2O。
A kind of 7. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The bath temperature is 50-65 DEG C.
A kind of 8. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The centrifugal rotational speed is 5000-8000 revs/min.
A kind of 9. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：To use bag filter of the molecular cut off for 8000-14000, water for dialysis is distilled water for the dialysis,
Dialysis three days, changes water three times daily.
A kind of 10. preparation side of the SPIO nano particle of polyglutamic acid PGA claddings according to claim 1
Method, it is characterised in that：The SPIO nano particle of the polyglutamic acid PGA claddings is used to prepare liver and tumour mould
The T of type2The contrast agent of magnetic resonance imaging diagnosis.
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