CN104587496A - Preparation method and application of polyglutamic acid (PGA)-coated ferric oxide nanoparticles - Google Patents

Preparation method and application of polyglutamic acid (PGA)-coated ferric oxide nanoparticles Download PDF

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CN104587496A
CN104587496A CN201410838190.4A CN201410838190A CN104587496A CN 104587496 A CN104587496 A CN 104587496A CN 201410838190 A CN201410838190 A CN 201410838190A CN 104587496 A CN104587496 A CN 104587496A
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pga
polyglutamic acid
coated
particle
preparation
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史向阳
于智博
罗宇
彭琛
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Donghua University
National Dong Hwa University
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Donghua University
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Abstract

The invention discloses a preparation method of a magnetic resonance contrast agent of polyglutamic acid (PGA)-coated ferric oxide nanoparticles, which is characterized by comprising the following steps: reacting to prepare PGA-coated superparamagnetic ferroferric oxide nanoparticles; and purifying the SPIO-PGA nanoparticle solution. The method has the advantages of simple technique, mild reaction conditions, high safety and no pollution, and is easy to operate. The SPIO-PGA nanoparticles have the advantages of uniform particle size distribution, small particle size, high relaxation rate, obvious contrast effect, favorable water solubility, favorable colloid stability, favorable biocompatibility, favorable compatibility with blood, no adverse effect on organisms and low cost, and is easy for preservation. The abundant active groups coated on the organic matter surface can be used for further modification and deep development, and has potential application value in the field of magnetic resonance imaging diagnosis.

Description

The preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA is coated and application
Technical field
The invention belongs to the preparing technical field of magnetic resonance contrast agent, be specifically related to preparation method and the application of the coated ferric oxide nanometer particle of a kind of polyglutamic acid PGA.
Background technology
Nuclear magnetic resonance (MRI) technology is the imaging technique that a kind of resolution is high, there is higher space and fault imaging ability, MRI does not have radioactive ionization to damage, and can obtain dissection and physiologic information simultaneously, have the unrivaled advantage of other medical imagings.Mr imaging technique plays an increasingly important role in disease surveillance field.But the weakness of MRI is that its sensitivity is lower, and the relaxation time of Different Organs or tumor tissues overlappedly make MRI difficult diagnosis.In recent years, the method by injecting MRI contrast agent effectively can solve the lower problem of MRI sensitivity.Therefore suitable MRI contrast agent is selected just to seem particularly important.
SPIO nano-particle (SPIO) is in an increasingly wide range of applications at biomedical sector in recent years, and the application especially in magnetic resonance imaging contrast receives general concern especially.SPIO nano-particle has the features such as unique magnetic property and higher signal intensity, lower using dosage, good biocompatibility and lower manufacturing cost.Existing commercialization SPIO nano-particle is applied to clinical disease diagnosis as MRI contrast agent at present.But, when these business-like SPIO contrast agent are used for tumor cells image field, also there is lot of challenges.Such as, often relaxation rate is lower for commercialization SPIO contrast agent, can not reach the object to epidemic disease Sensitive Detection.
The result adopting gentle reducing process to prepare Superparamagnetic Iron Oxide nano-particle (SPIO) before this seminar shows, the ferroferric oxide nano granules size prepared by gentle reducing process is less, uniform particle sizes, and shows high r 2(history on the sunny side for Shen Mingwu, Li Jingchao, Hu Yong, Sun Wenjie for relaxation rate.A kind of preparation method of Superparamagnetic Iron Oxide nano-particle of modified with folic acid.Chinese invention patent, application number: 201410182821.1, date of application: 2014-4-30).But said method mainly adopts the polymine (PEI) of carrying positive charge as stabilizing agent, needs further functionalization just to may be used for cell or tumor MR imaging.In order to solve cytotoxicity that PEI positive surface charge produces and cell is non-specific engulfs problem, inventor adopts in the process of reduction method for preparing nanometer granule, polyglutamic acid (PGA) is added in reaction solution, thus the SPIO nano-particle (SPIO-PGA) that preparation PGA is coated.PGA has excellent water solublity, superpower adsorptivity and biodegradability, and degradable product is non-harmful glutamic acid, is a kind of excellent environment-friendly type macromolecule material.-COOH abundant on PGA strand makes it have good biocompatibility, in aqueous in elecrtonegativity, does not need to modify further just can carry out biologic applications, decreases workload, reduce production cost simultaneously.-the COOH enriched also can rhetorical function group further, for further further investigation exploitation of the present invention provides very large space.Characterization data shows, the SPIO-PGA of preparation has good water solublity and colloidal stability, investigates through a series of biological experiment, and SPIO-PGA prepared by the present invention has good biocompatibility and blood compatibility, and its r 2relaxation rate up to 333.7mM -1s -1, there is significant T 2the MRI imaging effect of weighting, is expected to be applied to clinical disease diagnosis as MRI contrast agent.
Retrieval domestic and foreign literature, does not still find about preparing the relevant report of the coated SPIO nano-particle of PGA as MRI contrast agent research by gentle reducing process.
Summary of the invention
Technical problem to be solved by this invention is that existing SPIO nano-particle can not long-time stable be scattered in aqueous solution, easily occurs agglomeration.
In order to solve the problem, the preparation method of the ferric oxide nanometer particle that a kind of polyglutamic acid PGA is coated, is characterized in that, trivalent iron salt is dissolved in the water, stirs, pass into nitrogen, and dropwise add polyglutamic acid PGA solution, dropwise add sodium sulfite Na while stirring subsequently 2sO 3aqueous solution, obtains mixed solution, then moves in water-bath, drips NH while stirring 3h 2o, reaction 20-30min, then reacts 0.5-1.5h at ambient temperature, centrifugal, and dialysis, obtains the Superparamagnetic Iron Oxide nano-particle that polyglutamic acid PGA is coated.
Preferably, described trivalent iron salt is FeCl 36H 2o, the mass concentration of this solution is not higher than 13mg/mL.
Preferably, the weight average molecular weight of described middle polyglutamic acid PGA is 1,000,000.
Preferably, passing into the nitrogen time described in is 5-15min; Speed of agitator is 1000-5000 rev/min.
Preferably, described FeCl 36H 2o and Na 2sO 3mass ratio be 7: 1; FeCl 36H 2the mass ratio of O and PGA is 7: 1; FeCl 36H 2o and mass fraction are the NH of 91% 3h 2the mass ratio of O is 4: 5.
Preferably, described step 1) middle FeCl 36H 2o solution drips Na 2sO 3rear color becomes to transfer to water-bath after yellow from rufous and drips NH 3h 2o.
Preferably, described bath temperature is 50-65 DEG C.
Preferably, described centrifugal rotational speed is 5000-8000 rev/min.
Preferably, described dialysis is adopt molecular cut off to be the bag filter of 8000-14000, and water for dialysis is distilled water, dialyses three days, changes water every day three times.
Present invention also offers the coated ferric oxide nanometer particle of above-mentioned polyglutamic acid PGA as the T for the preparation of liver and tumor model 2the application of the contrast agent of magnetic resonance image-forming diagnose.
The present invention adopts gentle reducing process to prepare the superparamag-netic iron oxide of PGA parcel, and carries out purification by centrifugal and the nanoparticles solution of dialysis to preparation.
The present invention is easy to operation, and the cost of raw material is low.The nano-particle of preparation has good water solublity, colloidal stability, blood compatibility and biocompatibility.Imaging contrast experiment in nude mouse, SPIO-PGA nano-particle prepared by the present invention has significant contrasting effects, has potential using value in magnetic resonance imaging contrast field.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention adopts gentle reducing process to prepare SPIO-PGA for MR image-forming contrast medium, preparation method is simple, and reaction condition is gentle, is easy to manipulation, stabilizing agent used is cheap and environmentally friendly polyglutamic acid, has the prospect of industrialized implementation.
(2) the SPIO-PGA nano-particle crystal formation prepared of the present invention is good, and size is even, has good water solublity, colloidal stability, cell compatibility, blood compatibility and good T 2contrasting effects, has potential using value at magnetic resonance imaging arts.
Accompanying drawing explanation
Fig. 1 is transmission electron microscope (TEM) figure of SPIO-PGA nano-particle prepared by embodiment 1;
Fig. 2 is the particle size distribution rectangular histogram of SPIO-PGA nano-particle prepared by embodiment 1;
Fig. 3 is SPIO-PGA nano-particle and exposed ferroso-ferric oxide (Fe in embodiment 1 3o 4) comparison diagram (being followed successively by SPIO-PGA nano-particle and exposed ferroferric oxide nano granules from top to bottom) of X ray diffracting spectrum of nano-particle;
Fig. 4 is ferroferric oxide nano granules (Fe exposed in embodiment 1 3o 4), the comparison diagram (being followed successively by exposed ferroferric oxide nano granules, polyglutamic acid, SPIO-PGA nano-particle from top to bottom) of the Fourier transform infrared spectroscopy (FTIR) of polyglutamic acid (PGA), SPIO-PGA nano-particle;
Fig. 5 is ferroferric oxide nano granules (Fe exposed in embodiment 1 3o 4), the comparison diagram (being followed successively by exposed ferroferric oxide nano granules, SPIO-PGA, polyglutamic acid from top to bottom) of thermogravimetric analysis (TGA) collection of illustrative plates of SPIO-PGA, polyglutamic acid (PGA);
Fig. 6 is the MR image of SPIO-PGA nano-particle in embodiment 2;
Fig. 7 is the r of SPIO-PGA nano-particle in embodiment 2 2relaxation rate test curve;
Fig. 8 is the cell viability schematic diagram processing the HeLa cell after 24 hours through exposed ferroferric oxide nano granules (matched group) and SPIO-PGA nano-particle (Fe concentration is 50,150,250,350,450 μ g/mL) of mtt assay test in embodiment 3;
Fig. 9 be in embodiment 3 HeLa cell after normal saline (contrast) and SPIO-PGA nano-particle (Fe concentration is 50,150,250,350,450 μ g/mL) process 24 hours, the comparison diagram of the cell morphology figure that phase contrast microscope is observed (be followed successively by from a to f object of reference normal saline and Fe concentration be respectively 50,150,250,350,450 μ g/mL process after cell morphology);
Figure 10 is the ultraviolet spectrogram of SPIO-PGA nano-particle hemolytic experiment in embodiment 4;
Figure 11 is the schematic diagram processing macrophage phagocytic situation after 4 hours in embodiment 5 through SPIO-PGA nano-particle (Fe concentration is 0,10,100 μ g/mL);
Figure 12 is nude mice tail vein injection SPIO-PGA nano-particle in embodiment 6 (100 μ L, 1000 μ g/mL) comparison diagram that is front and different time points (2,4,6 hours) nuclear magnetic resonance picture after injecting;
Figure 13 is the liver of different time points (2,4,6 hours) nude mice and situation of change (the wherein vertical coordinate representation signal intensity of tumor locus signal intensity after the front and injection of nude mice tail vein injection SPIO-PGA nano-particle in embodiment 6 (100 μ L, 1000 μ g/mL) a () is liver, (b) is tumor);
Figure 14 is the front schematic diagram with injecting the distribution of Fe concentration of element and metabolic condition in rear different time points (2,4,6 hours) each organ of nude mice tail vein injection SPIO-PGA nano-particle (100 μ L, 1000g/L) in embodiment 7.
Detailed description of the invention
For making the present invention become apparent, hereby with preferred embodiment, and accompanying drawing is coordinated to be described in detail below.
Embodiment 1
640mg Iron(III) chloride hexahydrate is dissolved in 50mL ultra-pure water, passes into nitrogen 10 minutes and fully stir.90mg PGA is dissolved in 20mL ultra-pure water, dropwise adds above-mentioned solution.9mg/mL sodium sulfite solution is dropwise added after stirring.Mixed solution is transferred in 60 DEG C of water-baths, adds 1mL ammonia, fully reaction 30 minutes.Mixed solution is moved to room temperature, continues reaction 1.5 hours.Carry out centrifugal to the solution of preparation, after centrifugal 10 minutes with the rotating speed of 8000 revs/min, discard centrifugation, get upper solution.With the bag filter dialysis that molecular cut off is 8000-14000.Water for dialysis is distilled water, dialyses three days, changes water every day three times.
Ferroferric oxide nano granules coated for the polyglutamic acid of above-mentioned preparation is passed through transmission electron microscope observation, result shows that the SPIO nano-particle pattern formed is even, mean diameter is 5.3nm, standard deviation is 2.6nm, is distributed in narrower particle size range (as depicted in figs. 1 and 2).The surface potential of Zeta electric potential result display SPIO-PGA nano-particle is-38.6mV, and the hydrodynamic diameter of Dynamic Light Scattering Determination shows: its hydration particle diameter is 217.5 ± 3.75nm.X ray diffracting spectrum proved response has prepared the good ferroso-ferric oxide crystal (as shown in Figure 3) of crystal formation.The enhancing of Fourier transform infrared spectroscopy absworption peak, illustrates that polyglutamic acid is coated on ferroferric oxide nano granules, forms coated nanostructured (as shown in Figure 4).Drawn by thermogravimetric analysis, in SPIO-PGA nano-particle, the content of ferroso-ferric oxide is 75.6%, and the upper carrying capacity of polyglutamic acid reached for 24.4% (as shown in Figure 5).
Comparative example 1
In liquor ferri trichloridi, add sodium sulfite solution, be transferred to 60 DEG C of water bath condition downhill reaction solution and add ammonia, react 30 minutes, be finally placed in room temperature reaction 1.5 hours.After reaction terminates, by obtained black precipitate Magneto separate removing supernatant, add appropriate ultra-pure water ultrasonic disperse again, Magneto separate again, repetition milli-Q water like this three times, to remove impurity, is then scattered in ultra-pure water again, vacuum lyophilization, detects and MTT cytotoxicity experiment for X-ray diffraction.Proved by X-ray diffraction test, reaction has obtained the good ferroso-ferric oxide crystal (as shown in Figure 3) of crystal formation.
Embodiment 2
The concentration of Fe element in the SPIO-PGA nanoparticles solution of embodiment 1 preparation is measured by ICP-OES method of testing.Prepare respectively Fe concentration be 0.004,0.008,0.016,0.032, the SPIO-PGA nano-particle aqueous solution 2mL of 0.064mM, measure the T of material under different Fe concentration by magnetic resonance imaging analysis instrument 2relaxation effect (as shown in Figure 6 and Figure 7).Relaxation rate test result shows that the relaxation time of SPIO nano material reciprocal has good linear relationship along with the increase (in 0.004 ~ 0.064mM concentration range) of concentration of iron.By can be calculated the r of SPIO-PGA prepared by the present invention 2relaxation rate is up to 333.7mM -1s -1.Therefore, the SPIO-PGA prepared by the present invention can as excellent T 2signal attenuation contrast agent.
Embodiment 3
Evaluate the present invention with HeLa cell for model cell and prepare the impact of SPIO-PGA nano-particle on cell viability.With exposed Fe prepared by comparative example 1 3o 4nano-particle in contrast.Solution embodiment 1 obtained prepares the normal saline solution (sodium chloride content 0.9%) that Fe concentration of element is 50,150,250,350,450 μ g/mL SPIO-PGA nano-particle successively.By HeLa cell seeding in 96 orifice plates, often kind of concentration sets 5 Duplicate Samples, and Tissue Culture Plate is placed in CO 2concentration be 5% and temperature be Dual culture 24 hours at 37 DEG C.After MTT process, in microplate reader, detect the light absorption value of each hole at λ=570nm place, and calculate corresponding cell viability accordingly, wherein with the cell of normal saline process for blank, cell viability is designated as 100%.The Fe exposed with matched group 3o 4compare, under each concentration, the cell of SPIO-PGA process all has good cell viability (as shown in Figure 8).Compared with blank group, the vigor of the cell of each concentration SPIO-PGA solution-treated is all better than blank group, shows that the present invention has good biocompatibility further.The cell morphology (as shown in Figure 9) that this result is observed by phase contrast microscope obtains further checking.
Embodiment 4
The SPIO-PGA nano-particle normal saline solution (Fe concentration is 50,150,250,350,450 μ g/mL) embodiment 1 prepared and fresh human red cell were at room temperature hatched through 1 hour, centrifugal observation haemolysis situation.Accordingly, using deionized water as positive controls, normal saline is as negative control group, and the degree of hemolysis of sample carries out quantization signifying by the absorption value of UV, visible light spectrophotometer at 541nm place.Experimental result shows, SPIO-PGA nano-particle has good blood compatibility.Each concentration samples hemolysis rate is all less than 5%, and when iron concentration is 450 μ g/mL, the hemolysis rate of SPIO-PGA nano-particle is 3.42%, along with the reduction hemolysis rate of sample concentration decreases (as shown in Figure 10).As shown in Figure 10, the SPIO-PGA nano-particle that prepared by the present invention has good blood compatibility.
Embodiment 5
For investigating SPIO-PGA nano-particle by the situation of macrophage phagocytic, SPIO-PGA nano-particle (Fe concentration is respectively 10 and 100 μ g/mL) co-cultivation prepared by Raw 264.7 cell and embodiment 14 hours, the exposed Fe prepared with comparative example 1 3o 4nanoparticle in contrast.After cleaning cell, measured by the Fe concentration of element of the material of macrophage phagocytic by ICP-OES.Compare the exposed ferroferric oxide nano granules that surface free PGA modifies, when Fe concentration is all 100 μ g/mL, the cytophagy amount of SPIO-PGA nano-particle obviously less (as shown in figure 11).Therefore the SPIO-PGA nano-particle prepared of the present invention is expected to avoid by macrophage phagocytic entering after in organism, thus obtains desirable contrasting effects.
Embodiment 6
In order to verify the imaging effect that material of the present invention is actual in animal body, inventor is at nude mice construct in vitro HeLa Transplanted tumor model, organ and tumor locus nuclear magnetic resonance effect is evaluated by tail vein injection SPIO-PGA nano-particle normal saline solution (100 μ L, 1000 μ g/mL).
As shown in Figures 12 and 13, compared with the magnetic resonance image (MRI) of nude mice before injection, injection SPIO-PGA nano-particle is after 2 hours, each major organs of nude mice is dimmed all in various degree, liver and spleen position obviously dimmed, liver region signal intensity has and obviously weakens (as shown in (a) part in Figure 12 and Figure 13, spleen area is too small cannot Accurate Determining signal value), the signal intensity of tumor locus has had certain weakening (as shown in (b) part in Figure 12 and Figure 13), show that the nano-particle injected enters liver first in a large number, reduce the signal intensity of liver region, small part material is engulfed by tumor.After 4 hours, spleen position is dimmed further, and this first enters liver because nano-particle is injected into after in nude mouse, then carries out metabolism through spleen.The signal intensity of tumor locus is the most weak at 2 hours, shows that at this moment nano material reaches maximum in the aggregate amount of tumor locus.Scanogram display after 6 hours, the signal intensity of tumor locus has recovered the level before close to injection, shows that most material is excreted by metabolism.
Embodiment 7
In order to study the distribution and metabolic condition that SPIO-PGA nano-particle of the present invention respectively organizes in vivo, inventor in the nude mouse constructing HeLa Transplanted tumor model by tail vein injection SPIO-PGA nano-particle normal saline solution (100 μ L, 1000 μ g/mL).Then with the content (as Figure 14) of Fe concentration in rear different time points (2,4,6 hours) each vitals of injection and tumor before injecting with ICP-OES measurement, and blank nude mice is set as reference contrast.As can be seen from the figure, compared with matched group, Fe concentration is mainly distributed in spleen place, and along with the change of time, aggregate amount changes.Inject latter 2 hours, in liver, Fe concentration reaches the highest, and along with the prolongation of time, Fe concentration obviously reduces, show Fe element by liver slowly metabolism fall, this is consistent with MR imaging results in body; At 4 hours, in spleen, Fe concentration reached the highest, and higher than Fe concentration in liver, then As time goes on, Fe concentration obviously reduces, show Fe element by spleen slowly metabolism fall, this also with MR imaging results in body coincide.At injection material of the present invention after 6 hours, in each organ of nude mice, Fe concentration has obvious reduction, close to Fe concentration in contrast Mus body.Tumor locus Fe concentration is the highest at 2 hours, and metabolism is gone out gradually subsequently.This result show material can in nude mouse normal metabolite clearance, can tumor locus be entered, allow the MR video picture of tumor.
To sum up, the present invention uses the means such as transmission electron microscope (TEM), X-ray diffraction analysis (XRD), Zeta electric potential and dynamic scattering analysis (DLS), fourier transform infrared spectroscopy analysis (FTIR), thermogravimetric analysis (TGA), ICP-AES (ICP-OES) and magnetic resonance analysis (MR) to characterize the SPIO-PGA nano-particle of preparation.Then utilize mtt assay to evaluate the cytotoxicity of nano-particle, and with phase contrast microscope obtain with material Dual culture after the pattern of cell; Blood compatibility of the present invention is evaluated by hemolytic experiment.Finally carry out the nuclear magnetic resonance experiment of cell in vitro and nude mice vivo tumor model, investigate the cell in vitro of SPIO-PGA nano-particle and the MR imaging effect of vivo tumor model.In addition, by tissue distribution experimentation SPIO-PGA nano-particle metabolic process in vivo.Concrete test result is as follows:
(1) transmission electron microscope (TEM) test result
The TEM picture of SPIO-PGA nano-particle prepared by the present invention and particle size distribution rectangular histogram (see accompanying drawing 1-2) show: the spherical in shape or torispherical of the pattern of the SPIO nano-particle formed, size uniform, there is no obvious agglomeration, good dispersion and not assembling in the solution, statistical analysis show that particle diameter size is 5.3 ± 2.6nm, and particle size distribution is in narrower scope.
(2) X-ray diffraction analysis (XRD) test result
XRD figure spectrum (see accompanying drawing 3) of the SPIO nano-particle of PGA parcel prepared by this method shows: the diffraction maximum of prepared material is very identical in the diffraction maximum site of site 220,311,400,422,511 and 440 place and ferroferric oxide nano granules.Diffraction peak-to-peak type is sharp-pointed, illustrates that reaction has obtained the good ferroso-ferric oxide crystal of crystal formation.
(3) Zeta electric potential and dynamic light scattering (DLS) test result
The surface potential of Zeta electric potential result display SPIO-PGA nano-particle is-38.6mV, and hydrodynamics diameter is 217.5 ± 3.75nm.
(4) Fourier transform infrared spectroscopy (FTIR) test result
The FTIR collection of illustrative plates (see accompanying drawing 4) of the SPIO nano-particle of PGA parcel prepared by this method shows: at 1080cm -1(C-O), 1410cm -1(O-H), 1637cm -1(C=O) three places, compared to exposed ferroferric oxide nano granules, absorption peak strength obviously increases.This illustrates that PGA has well been wrapped on SPIO nano-particle, forms coated nanostructured.
(5) thermogravimetric analyzer (TGA) test result
The TGA analysis result (see accompanying drawing 5) of SPIO-PGA nano-particle prepared by this method shows that the upper carrying capacity that ferroso-ferric oxide content in SPIO-PGA nano-particle is about 75.6%, PGA reaches 24.4%.
(6) magnetic resonance (MR) analysis result
R 2relaxation rate reflection SPIO nanoparticle is as the imaging efficiency of MRI contrast agent, and the T2 of unit molar concentration ferrum, by the relaxation time (T under variable concentrations 1or T 2) the Fitting Calculation reciprocal obtain.The SPIO-PGA nano-particle T prepared by the present invention 2relaxation time inverse and the Linear Fit Chart of Fe concentration, can find out the increase along with concentration of iron reciprocal of the relaxation time of this SPIO-PGA nano-particle, have good linear relationship.Along with increasing of Fe concentration, its MR signal intensity obviously weakens.The nuclear magnetic resonance of variable concentrations sample can find out that nano-particle has good external imaging effect.By calculating the r of SPIO-PGA nano-particle 2value is 333.7mM -1s -1(see accompanying drawing 6-7).
(7) MTT cell viability and phase contrast microscope observed result
The cell compatibility (see accompanying drawing 8) of SPIO-PGA nano-particle prepared by the present invention is evaluated by the vigor of MTT colorimetric method for determining HeLa cell.By HeLa cell seeding in 96 orifice plates, often kind of concentration sets 5 Duplicate Samples (Fe concentration is 50,150,250,350,450 μ g/mL).Tissue Culture Plate is placed in CO 2concentration be 5% and temperature be Dual culture 24 hours in the environment of 37 DEG C.After MTT process, detect the light absorption value of each hole at λ=570nm place in microplate reader, and calculate corresponding cell viability accordingly, wherein using the cell of normal saline process as blank, cell viability is designated as 100%.Compared with blank group, the HeLa cell nanoparticle treated through SPIO-PGA all has higher cell viability under Fe concentration is 50,150,250,350,450 μ g/mL, and along with the vigor corresponding increase thereupon of the increase cell of concentrations of nanoparticles.This is because PGA is a kind of biopolymer with good biocompatibility, there is facilitation to the Differentiation and proliferation of cell.In contrast, exposed ferroferric oxide nano granules group is as a comparison under same concentrations, and cell viability is starkly lower than the cell controls group of normal saline process, and along with the increase of concentration, cytoactive reduces gradually.Two groups of contrasts show that SPIO-PGA nano-particle has good cell compatibility.Meanwhile, inventor also demonstrates the impact of SPIO-PGA nano-particle on cell morphology further by phase contrast microscope observational method.Result shows the nano-particle (Fe concentration is 50,150,250,350,450 μ g/mL) of variable concentrations with co-culture of cells after 24 hours at 37 DEG C, and cell morphology does not significantly change (see accompanying drawing 9).Further illustrate SPIO-PGA nano-particle and there is good cell compatibility.
(8) blood compatibility
SPIO-PGA nano-particle prepared by the present invention is its blood compatibility of needs assessment before carrying out zoopery.Inventor is evaluated material blood compatibility by hemolytic experiment.By fresh human red cell and nano-particle normal saline solution (Fe concentration is 50,150,250,350,450 μ g/mL) at room temperature after 1 hour hatches, centrifugal observation haemolysis situation.Using deionized water as positive controls, normal saline is as negative control group, and the degree of hemolysis of sample carries out quantization signifying by the absorption value of ultraviolet-visible absorption spectroscopy at 541nm place.Experimental result shows: when iron concentration reaches 450 μ g/mL, and the hemolysis rate of SPIO-PGA nano-particle is 3.42%, and along with the reduction hemolysis rate of sample concentration decreases, each sample hemolysis rate is all lower than 5% (see accompanying drawing 10-11).Illustrate that SPIO-PGA nano-particle prepared by the present invention has good blood compatibility.
(9) cytophagy experiment
For biomedical applications, reduce nano-particle most important by macrophage phagocytic.Therefore need to be evaluated by the situation of engulfing of macrophage SPIO-PGA nano-particle.Raw 264.7 cell and SPIO-PGA nano-particle (Fe concentration is 10,100 μ g/mL) are at CO 2concentration be 5% and temperature be Dual culture 4 hours at 37 DEG C, cultivate as blank group using normal saline.After cleaning cell, measure cytophagic Fe concentration by ICP-OES.When Fe concentration is 100 μ g/mL, compare the exposed ferroferric oxide nano granules that surface free PGA modifies, the cytophagy amount of SPIO-PGA nano-particle obviously less (see accompanying drawing 11).Therefore material of the present invention is expected to avoid by macrophage phagocytic entering after in organism, thus obtains desirable contrasting effects.
(10) MR imaging results in body
At nude mice construct in vitro HeLa Transplanted tumor model, evaluate major organs and tumor locus nuclear magnetic resonance effect (see accompanying drawing 12-14) by tail vein injection SPIO-PGA nano-particle normal saline solution (100 μ L 1000 μ g/mL).Compared with the magnetic resonance image (MRI) before injection, injection SPIO-PGA nano-particle is after 2 hours, nude mice liver and spleen position obviously dimmed, signal intensity obviously weakens (see accompanying drawing 13a, spleen area is too small cannot Accurate Determining signal value).The signal intensity of tumor locus has and to a certain degree weakens (see accompanying drawing 13b).This shows that the nano-particle injected partly can escape engulfing of reticuloendothelial system (liver and spleen position), tumor locus is entered by the high-permeability of tumor locus and retention effect (enhanced permeability and retention effect, EPR) nano-particle passive target.Along with the prolongation of time, the MR signal intensity at nude mouse tumor position recovers gradually, illustrates that SPIO-PGA nano-particle starts metabolism in vivo.MR imaging results shows that SPIO-PGA nano-particle prepared by the present invention can be used in the MR imaging of major organs and tumor model in body.
(11) tissue distribution
In order to study the distribution and metabolism situation that SPIO-PGA nano-particle of the present invention is respectively organized in vivo, inventor in the nude mouse constructing HeLa Transplanted tumor model by tail vein injection SPIO-PGA nano-particle normal saline solution (100 μ L, 1000 μ g/mL).Measure the rear content of different time points (2,4,6 hours) the Fe element in each vitals (see accompanying drawing 14) of injection with ICP-OES, and blank nude mice is set as reference contrast.As can be seen from the figure, compared with matched group, Fe concentration is mainly distributed in liver and spleen place, and along with the change of time, aggregate amount changes.At 2 hours, in liver, Fe concentration reached the highest, and As time goes on, Fe concentration obviously reduces, and show that Fe element is by liver slowly metabolism, this is consistent with MR imaging results in body; At 4 hours, in spleen, Fe concentration reached the highest, and higher than Fe concentration in liver, along with time lengthening, Fe concentration obviously reduces, and showed that Fe element is by the slow metabolism of spleen, and in this and body, MR imaging results is coincide.Inject material of the present invention after 6 hours, in each organ of nude mice, Fe concentration all significantly decreases.And other organs (heart, lung, kidney), the gathering of Fe is obviously less, and in these organs, Fe is last also by slowly metabolism.This result show material can in nude mouse normal metabolite clearance, can tumor locus be entered, allow the MR video picture of tumor.

Claims (10)

1. a preparation method for the ferric oxide nanometer particle that polyglutamic acid PGA is coated, is characterized in that, is dissolved in the water by trivalent iron salt, stirs, passes into nitrogen, and dropwise add polyglutamic acid PGA solution, dropwise add sodium sulfite Na while stirring subsequently 2sO 3aqueous solution, obtains mixed solution, then moves in water-bath, drips NH while stirring 3h 2o, reaction 20-30min, then reacts 0.5-1.5h at ambient temperature, centrifugal, and dialysis, obtains the Superparamagnetic Iron Oxide nano-particle that polyglutamic acid PGA is coated.
2. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, it is characterized in that, described trivalent iron salt is FeCl 36H 2o, the mass concentration of this solution is not higher than 13mg/mL.
3. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, it is characterized in that, the weight average molecular weight of described middle polyglutamic acid PGA is 1,000,000.
4. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, is characterized in that, described in pass into the nitrogen time be 5-15min; Speed of agitator is 1000-5000 rev/min.
5. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, is characterized in that, described FeCl 36H 2o and Na 2sO 3mass ratio be 7: 1; FeCl 36H 2the mass ratio of O and PGA is 7: 1; FeCl 36H 2o and mass fraction are the NH of 91% 3h 2the mass ratio of O is 4: 5.
6. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, is characterized in that, described step 1) middle FeCl 36H 2o solution drips Na 2sO 3rear color becomes to transfer to water-bath after yellow from rufous and drips NH 3h 2o.
7. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, it is characterized in that, described bath temperature is 50-65 DEG C.
8. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, it is characterized in that, described centrifugal rotational speed is 5000-8000 rev/min.
9. the preparation method of the ferric oxide nanometer particle that polyglutamic acid PGA as claimed in claim 1 is coated, is characterized in that, described dialysis is adopt molecular cut off to be the bag filter of 8000-14000, and water for dialysis is distilled water, dialyses three days, changes water every day three times.
10. the ferric oxide nanometer particle that a polyglutamic acid PGA according to claim 1 is coated is as the T for the preparation of liver and tumor model 2the application of the contrast agent of magnetic resonance image-forming diagnose.
CN201410838190.4A 2014-12-25 2014-12-25 Preparation method and application of polyglutamic acid (PGA)-coated ferric oxide nanoparticles Pending CN104587496A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870499A (en) * 2010-06-03 2010-10-27 四川大学 Ultrasonic vibration and in-situ coating preparation method of superparamagnetic ferroferric oxide nanoparticle
CN104027823A (en) * 2014-06-26 2014-09-10 吉林大学 Method for preparing polypeptide modified superparamagnetic iron oxide nano particle aggregate by virtue of ne-pot method
CN104548145A (en) * 2014-12-19 2015-04-29 东华大学 Preparation method of polyglutamic acid PGA-coated superparamagnetic iron oxide nanoparticle

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870499A (en) * 2010-06-03 2010-10-27 四川大学 Ultrasonic vibration and in-situ coating preparation method of superparamagnetic ferroferric oxide nanoparticle
CN104027823A (en) * 2014-06-26 2014-09-10 吉林大学 Method for preparing polypeptide modified superparamagnetic iron oxide nano particle aggregate by virtue of ne-pot method
CN104548145A (en) * 2014-12-19 2015-04-29 东华大学 Preparation method of polyglutamic acid PGA-coated superparamagnetic iron oxide nanoparticle

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