CN103143041B - Preparation method of targeted MRI (magnetic resonance imaging) contrast medium based on folic acid modified iron oxide nanoparticles - Google Patents
Preparation method of targeted MRI (magnetic resonance imaging) contrast medium based on folic acid modified iron oxide nanoparticles Download PDFInfo
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Abstract
The invention relates to a preparation method of a targeted MRI (magnetic resonance imaging) contrast medium based on folic acid modified iron oxide nanoparticles. The method comprises the following steps of: connecting FA to NH2-PEG-COOH to form COOH-PEG-FA; synthesizing PEI coated Fe3O4 nanoparticles (Fe3O4-PEI) by a hydrothermal method; performing surface modification on the Fe3O4-PEI nanoparticles by use of fluorescein isothiocyanate (FI); connecting COOH-PEG-FA to the surface of the Fe3O4 nanoparticles; and finally performing acetylation modification of the residual PEI aminos on the surface of the Fe3O4 nanoparticles to improve the biocompatibility for MRI diagnosis. The preparation method provided by the invention has simple technology and mild reaction conditions, and is easy to operate; and the prepared Fe3O4 magnetic nanoparticles have good colloidal stability, biocompatibility and tumor targeting performance as well as potential application value in the field of in-vivo tumor targeted diagnosis.
Description
Technical field
The invention belongs to the preparation field of NMR contrast agent, particularly a kind of preparation method of targeting MRI contrast agent of the ferroferric oxide nano granules based on modified with folic acid.
Background technology
All the time, malignant tumor is all the No.1 killer of harm humans life, has that mortality rate is high, refractory is treated and worsens the features such as rapid.Therefore, the early diagnosis of tumor and specific treatment seem particularly important.At present, the detection means of tumor is various, mainly contains: ultra sonic imaging, CT imaging, nuclear medicine PET imaging and NMR (Nuclear Magnetic Resonance)-imaging (MRI).Along with the development of nuclear magnetic resonance technique, shorten its sweep time gradually, and resolution improves gradually, also more accurate for the detection of small lesion, the New Type of Diseases detection means that this also makes nmr imaging technique become to grow up in recent years.In order to improve sensitivity and the specificity of MRI image-forming diagnose, be necessary to select suitable MRI contrast agent.Conventional MRI contrast agent is mainly divided into two classes: a class is T
1the MRI contrast agent of weighting, a class is T
2the MRI contrast agent of weighting.T
1the MRI contrast agent of weighting is mainly Gd basigamy compound, yet Gd is heavy metal element, micromolecule Gd reagent in vivo the time of staying too short, its clinical injection dosage is larger, and Gd reagent has very strong nephrotoxicity, particularly to renal insufficiency patient harm more so.And T
2the MRI contrast agent of weighting is mainly superparamagnetic Fe
3o
4for the metallic iron oxide nano-particles of representative, the good contrast agent that Superparamagnetic Iron Oxide has unique magnetic property and signal is strong, the low features such as biocompatibility of becoming reconciled of using dosage become NMR (Nuclear Magnetic Resonance)-imaging.
As MRI contrast agent in organism, Fe
3o
4nano-particle must have good water solublity, stability, biocompatibility and higher T
2relaxation rate.Polymine (polyethyleneimine, PEI) is a kind of water solublity polyamine, has a large amount of amino on its macromolecular chain, is not only magnetic Fe
3o
4nano-particle provides the barrier of stable existence, and makes nanometer Fe
3o
4on particle surface band positive charge and functional groups, this is just Fe
3o
4the multi-functional modification in surface of nano-particle provides feasibility (Cai et al., ACS Appl.Mater.Interfaces2013, DOI:10.1021/am302883m; Patent publication No. 201210277624.9).Polyethylene Glycol (Polyethylene glycol, PEG) be high-hydrophilic polymer, it can improve water solublity and the biocompatibility of nano-particle to the modification of nano-particle, and extend its blood circulation time (Peng et al. in vivo, Biomaterials2012,33,1107-1119; Wen et al., Biomaterials2013,34,1570-1580).Folic acid is as a kind of targeted molecular, have that molecular weight is little, nontoxic, non-immunogenicity, good biocompatibility, stability are high, cheap and easy to get, be easy to the multiple advantages such as modification (Shi et al., Advanced Materials2008,20,1671-1678).This seminar patent (patent publication No. 201210277624.9) achievement in earlier stage shows that the superparamag-netic iron oxide that polymine (PEI) is modified can be synthetic by easy hydro-thermal method.The present invention adopts identical method to synthesize to have the coated Fe of PEI of good colloidal stability
3o
4nano-particle.Subsequently, Fe
3o
4the PEG-FA of nano grain surface modifies water solublity and the biocompatibility that has not only increased nano-particle, for further in-vivo imaging application provides guarantee; Also improved Fe simultaneously
3o
4the targeting of nano-particle to tumor cell or tumor locus, thus make NMR (Nuclear Magnetic Resonance)-imaging diagnosis more accurate, sensitive.
Retrieval domestic and foreign literature, does not still have to find about prepare the Fe that PEI is coated and FA modifies with a step hydrothermal synthesis method
3o
4nano-particle and the relevant report of studying for in-vivo tumour model targeting MRI thereof.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of targeting MRI contrast agent of the ferroferric oxide nano granules based on modified with folic acid, and the method technique is simple, and reaction condition is gentle, easy operating, and cost is lower.The Fe of preparation
3o
4magnetic nanoparticle for a long time stable dispersion, in aqueous solution, there will not be agglomeration.Dressing agent PEI used is cheapness and environment-friendly material, has the prospect of industrialized implementation.
The preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid of the present invention, comprising: (1) is dissolved in ferrous salt in ultra-pure water, adds NH
3h
2o also stirs 10～20 minutes under air atmosphere, then mixed solution is transferred in autoclave, and the hyperbranched polyethyleneimine PEI aqueous solution of 0.1g/mL is also joined in autoclave, after being uniformly mixed, in 130～140 ℃ of reactions 2～4 hours; After reaction finishes, naturally cool to room temperature, will precipitate after magnetic separating, washing purification, obtain the ferroferric oxide nano granules Fe of PEI parcel
3o
4-PEI, product be stored in 4 ℃ standby, get 3mL vacuum lyophilization, desciccate is stored in-20 ℃;
(2), after FA is dissolved in DMSO, first, with EDC and NHS activation 2～4h, then dropwise join NH
2in the DMSO solution of-PEG-COOH, stirring reaction 2～4 days, (bag filter that is 1000 with molecular cut off is to distill water dialysis three days (6 times in dialysis, 2L/ time)), remove by-product and impurity, then vacuum lyophilization, obtain COOH-PEG-FA, be stored in-20 ℃ standby;
(3) by the Fe of preparation in step (1)
3o
4-PEI nano-particle, with after DMSO washing, is scattered in DMSO again, then the FI solution that DMSO is dissolved joins above-mentioned Fe
3o
4in the DMSO solution of-PEI, stirring reaction is after 1～3 day, product Fe
3o
4-PEI-FI separating, washing, is then distributed in DMSO, and be stored in 4 ℃ standby;
(4) in step (2), the COOH-PEG-FA of preparation and EDC and NHS are dissolved in after DMSO, stir-activating 2～4h; Then the COOH-PEG-FA solution of activation is joined to Fe prepared by step (3)
3o
4in-PEI-FI solution, stirring reaction 2～4 days, then separating, washing, and be scattered in ultra-pure water, obtain Fe
3o
4-PEI-FI-PEG-FA nano-particle aqueous solution, and be stored in 4 ℃ standby;
(5) Fe preparing to step (4)
3o
4in-PEI-FI-PEG-FA nano-particle aqueous solution, add triethylamine, and stir 20～40 minutes; Then in above-mentioned solution, add acetic anhydride, continue stirring reaction 20～30 hours; Separating, washing is also scattered in ultra-pure water, makes acetylizad product Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle, is stored in-20 ℃ after getting 1mL vacuum lyophilization, remaining 4mL be stored in 4 ℃ standby.
Ferrous salt in described step (1) is FeCl
24H
2o, ferrous salt, ultra-pure water, NH
3h
2the proportioning of O is 1～1.5g:7.5～8.0mL:6.0～6.5mL, preferably 1.25g:7.75mL:6.25mL; Ferrous salt and hyperbranched polyethyleneimine PEI mass ratio are 2～3:1, preferably 2.5:1.
NH in described step (1)
3h
2o mass percent concentration is 25-28%.
The molecular weight of hyperbranched polyethyleneimine PEI in described step (1) is 25000.
In described step (2), the mol ratio of FA, EDC and NHS is 1:0.5～1:0.5～1, preferably 1:0.9:0.9, FA and NH
2the mol ratio of-PEG-COOH is 1～3:1, preferably 2:1.
NH in described step (2)
2the molecular weight of-PEG-COOH is 2000.
FI and Fe in described step (3)
3o
4the mol ratio of-PEI surface amino groups is 1:45～55, preferably 1:50.
COOH-PEG-FA in described step (4) and the mol ratio of EDC, NHS are 1:4～6:4～6, preferably 1:5:5.
COOH-PEG-FA and Fe in described step (4)
3o
4the mol ratio of nano grain surface amino is 1:4～6, preferably 1:5.
Triethylamine, acetic anhydride and Fe in described step (5)
3o
4the upper primary amino radical mol ratio of PEI of nano grain surface is 4～6:4～6:1, preferably 5:5:1.
The present invention first utilizes the Fe of the synthetic PEI parcel of one step hydro thermal method
3o
4magnetic nanoparticle, is then successively modified at FI and PEG-FA the surface of nano-particle, finally the residual surface amino of nano-particle is carried out to acetylation modification.
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 and biocompatibility.Do not compare the Fe of modified with folic acid with there is no the control material of modified with folic acid
3o
4nano-particle has higher targeting to tumor cell or tumor locus.Folate-targeted Fe prepared by the method
3o
4nano-particle has potential application at MRI molecular image diagnostic field.
The present invention use X-ray diffraction (XRD), NMR (Nuclear Magnetic Resonance) spectrum (
1h NMR), the method such as ultraviolet-visible absorption spectroscopy (UV-Vis), thermogravimetric analysis (TGA), ICP-AES (ICP-AES), Zeta electric potential and dynamic light scattering and transmission electron microscope (TEM) characterizes the magnetic nanoparticle of preparation, and by magnetic resonance imager, measures the T of nano-particle
2relaxivity, then utilize hemolytic experiment, mtt assay and phase contrast microscope to evaluate blood compatibility and the cytotoxicity of nano-particle, the targeting diagnosis effect of the nano material of recycling flow cytometer, confocal microscopic image and in vitro and in vivo NMR (Nuclear Magnetic Resonance)-imaging experiment detection modified with folic acid to tumor cell.Concrete test result is as follows:
(1) X-ray diffraction (XRD) test result
By contrast and analysis X-ray diffracting spectrum (as Fig. 1), the material that hydro-thermal method is synthetic and Fe
3o
4collection of illustrative plates (ICSD20-596) consistent, show the Fe that PEI modifies
3o
4crystal structure is ferroso-ferric oxide.
(2) nuclear magnetic resonance spectroscopy (
1h NMR) test result
By contrast NH
2-PEG-COOH, FA and the COOH-PEG-FA three hydrogen spectrum spectrum peak (as Fig. 2) in deuterated DMSO is known, the spectrum peak proof FA that COOH-PEG-FA occurs at 6-9ppm is successfully connected on PEG, and known by integration calculated by peak area, on each PEG, connected 0.7 FA.
(3) uv absorption (UV-Vis) test result
Figure 3 shows that Fe
3o
4-PEI-FI(Fig. 3 a), FI(Fig. 3 b) and Fe
3o
4-PEI(Fig. 3 c) 300 to the ultraviolet absorpting spectrum of 800nm.From figure, we can find out, Fe
3o
4-PEI does not have obvious ultraviolet absorption peak at 400 to 800nm places, and Fe
3o
4-PEI-FI has an obvious ultraviolet absorption peak at 510nm place, thereby explanation FI successfully modifies Fe
3o
4-PEI nano grain surface.
(4) thermogravimetric analysis (TGA) test result
In order to detect in COOH-PEG-FA and matched group mPEG-COOH at Fe
3o
4the upper carrying capacity of nano grain surface, we have carried out TGA test to the nano-particle before and after modifying.As seen from Figure 4, before modifying the weight loss of nano-particle be 8.97%(Fig. 4 a), after PEI surface amino groups and PEG molar ratio 5:1 modification, Fe
3o
4-PEI-FI-PEG-FA and control material Fe
3o
4the weightlessness of-PEI-FI-mPEG is respectively 13.23%(Fig. 4 b) and 14.16%(Fig. 4 c); Through calculating, the rate of uploading of COOH-PEG-FA and mPEG-COOH is respectively 4.26% and 5.19%, shows that thus COOH-PEG-FA and mPEG-COOH are successfully connected to Fe
3o
4the surface of nano-particle.
(5) nano-particle Zeta electric potential and dynamically waterpower footpath test result
The a large amount of amino of nano grain surface can produce cytotoxicity, thereby limited nano-particle application in vivo.So we pass through Fe
3o
4-PEI-FI-PEG-FA and control material Fe
3o
4-PEI-FI-mPEG surface amino groups is carried out acetylation modification, reduces its surface potential, thereby improves the biocompatibility of nano-particle.Potential determination result (table 1) shows, due to the existence of exhibiting high surface amino, Fe
3o
4-PEI-FI-PEG-FA and Fe
3o
4reached respectively+31.0mV of the surface potential of-PEI-FI-mPEG nano-particle and+31.5mV.Through after acetylization reaction, obtain acetylizad Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4dropped to respectively+16.3mV of-PEI-Ac-FI-mPEG nano grain surface electromotive force and+16.4mV.Result shows, the successful acetylation of the surface amino groups of nano-particle.But the surface potential of nano-particle does not reach neutral after acetylation, this may be because surface part is used for stablizing Fe
3o
4the amino of nano-particle can not carry out acetylization reaction.Before and after acetylation; the test result of the hydrodynamic diameter of these nano-particle is as shown in table 1 equally; after acetylation, the hydrodynamic diameter of nano-particle reduces before compared with acetylation slightly; and the hydrodynamic diameter of the nano-particle of preparation can keep almost constant for a long time, thereby illustrated that the nano-particle of preparation has good colloidal stability.
(6) transmission electron microscope (TEM) test result
The Fe preparing by tem observation the present invention
3o
4-PEI-Ac-FI-PEG-FA and matched group material Fe
3o
4the form of-PEI-Ac-FI-mPEG nano-particle and particle diameter (as shown in Figure 5).TEM test result shows Fe
3o
4-PEI-Ac-FI-PEG-FA and control material Fe
3o
4the pattern of-PEI-Ac-FI-mPEG nano-particle is all spherical or torisphericals.By the diameter of 300 nano-particle of random measurement respectively, calculate the Fe of preparation
3o
4-PEI-Ac-FI-PEG-FA and control material Fe
3o
4the diameter of-PEI-Ac-FI-mPEG nano-particle is respectively 15.0nm and 15.3nm.
(7) T
2relaxation rate measurement result
Fe
3o
4nano material can be as the negative contrast medium of NMR (Nuclear Magnetic Resonance)-imaging, and along with the increase of Fe concentration, MRI signal intensity weakens gradually.Relaxation rate (r
2) reflection Fe
3o
4nanoparticle, as the efficiency of MRI contrast agent, is the T2 of unit molar concentration ferrum, can be by the relaxation time (T under variable concentrations
2) the Fitting Calculation reciprocal obtain.Fig. 6 is Fe prepared by the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4the T of-PEI-Ac-FI-mPEG
2the Linear Fit Chart of relaxation time inverse and Fe concentration, can find out this two kinds of Fe
3o
4the relaxation time inverse of nano material is along with the increase (in 0-0.04mM concentration range) of concentration of iron has good linear relationship.And the Fe preparing by can be calculated the present invention
3o
4-PEI-Ac-FI-PEG-FA and control material Fe
3o
4the relaxation rate of-PEI-Ac-FI-mPEG has reached respectively 99.64mM
-1s
-1and 107.30mM
-1s
-1.Therefore, the prepared Fe of the present invention
3o
4-PEI-Ac-FI-PEG-FA and control material Fe
3o
4-PEI-Ac-FI-mPEG all can be used as the good T in the diagnosis of MRI molecular imaging
2signal attenuation contrast agent.
(8) blood compatibility
There is good blood compatibility the body planted agent of nano-particle be used for saying vital, thus this experimental evaluation the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4the blood compatibility of-PEI-Ac-FI-mPEG.In Fig. 7, shown Fe
3o
4-PEI-Ac-FI-mPEG(Fig. 7 a) and Fe
3o
4-PEI-Ac-FI-PEG-FA(Fig. 7 b) the hemolytic test result under variable concentrations (50,100,200,400 μ g/mL).By the extinction spectrum measurement to the supernatant, carry out the hemolytic of quantitative assessment nano material.As Fig. 7 a and the demonstration of 7b upper right corner uv-spectrogram, when concentration reaches 400 μ g/mL, Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4the hemolysis rate of-PEI-Ac-FI-mPEG is all less than 5%, illustrates that these nano materials of preparation have good blood compatibility, thereby can be safely for MRI imaging in organism.
(9) MTT cell viability and phase contrast microscope test result
Vigor by MTT colorimetric method for determining KB cell (a kind of cell strain of human epithelium's cancer) detects Fe prepared by the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4the cytotoxicity of-PEI-Ac-FI-mPEG (as Fig. 8).KB cell respectively with Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (concentration is 10,25,50,75 and 100 μ g/mL) is cultivated altogether 24 hours at 37 ℃.Then, after MTT processes, at 570nm place, measure light absorption value, and according to this value, calculate the vigor of cell.The impact of the material on cell proliferation of variable concentrations be take cell that buffer PBS processes and is compared as contrast.Compare Fe with PBS matched group
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA does not have significant difference to the survival rate of KB cell within the scope of experimental concentration 0 to 100 μ g/mL, and cell survival rate is all more than 80%.This absolutely proves Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA has good biocompatibility, can be applied to MRI image-forming diagnose in organism.We further verify the toxicity of material to cell by phase contrast microscope.As shown in Figure 9, the Fe of variable concentrations
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the cellular morphology that-PEI-Ac-FI-PEG-FA nano material (10,25,50,75 with 100 μ g/mL) was processed after 24 hours is compared with the cell that PBS processes, and does not significantly change, and the good biological that further illustrates synthetic material is compatible.
(10) flow cytometer testing result
By flow cytometer, detect the Fe that KB cell is prepared the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4the engulf amount (as Figure 10) of-PEI-Ac-FI-mPEG under variable concentrations and process after the average fluorescent strength (as Figure 11) of cell detect folate-targeted effect.KB cell respectively with Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4-PEI-Ac-FI-mPEG(Fe concentration be 0.125,0.25,0.5,0.75,1.0,1.25 and 1.5mM) at 37 ℃, cultivate altogether 4 hours, and the cell of processing with PBS is as a control group.Then by flow cytometer, detect the situation of engulfing of cell and the average fluorescent strength of cell.In Figure 10, compare with matched group, along with the increase of Fe concentration, cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-mPEG increases a little, and when Fe concentration reaches 1.0mM, the amount of engulfing almost no longer increases.But along with the increase of Fe concentration, cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-PEG-FA nano-particle but obviously increases always.Equally in Figure 11, along with the increase of Fe concentration, Fe
3o
4after-PEI-Ac-FI-PEG-FA processes, the average fluorescent strength of cell significantly increases, and Fe
3o
4after-PEI-Ac-FI-mPEG processes, the average fluorescent strength of cell increases not obvious.The special target ability of nano-particle to KB cell given in the modification of these presentation of results folic acid.
(11) Laser Scanning Confocal Microscope testing result
The targeting of folic acid verifies by Laser Scanning Confocal Microscope equally, as shown in figure 12, and the Fe that KB cell is prepared with PBS, the present invention respectively
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4-PEI-Ac-FI-mPEG(Fe concentration is 0.3) at 37 ℃, cultivate altogether the fluorescence signal after oily Microscopic observation cytophagy nano-particle then 4 hours.In Figure 12, in the cell of processing through PBS, there is no fluorescence, Fe
3o
4in the cell that-PEI-Ac-FI-mPEG processes, demonstrate fainter fluorescence signal, and Fe
3o
4in the cell that-PEI-Ac-FI-PEG-FA processes, demonstrate very strong fluorescence signal, this nano-particle that further illustrates modified with folic acid has better targeting to KB cell, thereby provides reliable foundation for this material success is applied to MRI imaging in body efficiently.
(12) cell in vitro MRI imaging results
Before experiment, we have evaluated Fe prepared by the present invention in vivo
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4the cell MRI imaging effect (as shown in figure 13) of-PEI-Ac-FI-mPEG, KB cell respectively with Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration be 0.1,0.2,0.4 and 0.8mM) is cultivated altogether 6 hours at 37 ℃, and the cell of processing with PBS as a control group.In Figure 13 a, along with the increase of Fe concentration, Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4cell after-PEI-Ac-FI-PEG-FA nano-particle is processed all shows the trend of MRI signal attenuation, illustrates that cell also increases the amount of engulfing of nano-particle along with the increase of Fe concentration.It is pointed out that under identical Fe concentration Fe
3o
4cell after-PEI-Ac-FI-PEG-FA nano-particle is processed is than control material Fe
3o
4the MRI signal of the rear cell of-PEI-Ac-FI-mPEG processing reduces more obvious, illustrates that cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-PEG-FA nano-particle will be much higher than Fe
3o
4-PEI-Ac-FI-mPEG nano-particle.Figure 13 b is the MRI imaging signal value of cell after the nano-particle of variable concentrations is processed, and from figure, obviously finds out, along with the increase of Fe concentration, the MRI signal value of cell all reduces gradually, and under identical Fe concentration, Fe
3o
4the MRI signal value that-PEI-Ac-FI-PEG-FA nano-particle is processed rear cell will be starkly lower than control material Fe
3o
4cell after-PEI-Ac-FI-mPEG nano-particle is processed.These results not only illustrate that the nano-particle of preparation has good cell MRI imaging effect, and have proved Fe
3o
4the specific target tropism of-PEI-Ac-FI-PEG-FA nano-particle to KB cell.
(13) in-vivo tumour MRI imaging results
The Fe preparing by tail vein injection the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3o
4-PEI-Ac-FI-mPEG evaluates the MRI imaging effect (as shown in figure 14) of tumor locus, compares with the matched group before injection, and in latter 30 minutes to 4 hours of injection, injection control material Fe
3o
4-PEI-Ac-FI-mPEG(Fe:500 μ g) mouse tumor position is dimmed a little, and injection Fe
3o
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g) mouse tumor is obviously dimmed, and the nano-particle that shows modified with folic acid has obvious MRI diagnosing tumor effect.In injection latter 24 hours, the mouse tumor position bright-dark degree of two experimental grouies all recovered gradually, illustrate now nano material along with blood circulation from tumor locus gradually metabolism go out that (Figure 14 a).Figure 14 b is that the tumor MRI signal value of corresponding inject time changes, in injection latter 30 minutes to 4 hours, and injection Fe
3o
4the mouse tumor MRI signal value of-PEI-Ac-FI-mPEG changes not obvious, and injection Fe
3o
4the mouse tumor MRI signal value of-PEI-Ac-FI-PEG-FA obviously reduces, and in injection latter 24 hours, the mouse tumor position MRI signal value of two experimental grouies all increased to some extent, and this is consistent with the result of Figure 14 a.Fe prepared by the present invention of these presentation of results
3o
4-PEI-Ac-FI-PEG-FA nano-particle has good cancer target ability, can be successfully applied to the contrast agent of targeting MRI tumor imaging diagnosis in body.
(14) distribution result
In order to study biological tissue's distribution situation of nano-particle, ICP-AES is used for measuring the content (Figure 15) of ferrum in latter 24 hours each vitals of injection.As can be seen from Figure at injection control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g), after nano-particle, in liver, spleen and lung, the content of ferrum all obviously increases before injecting, and at other organ, such as: the heart, kidney and tumor, the gathering of ferrum is less.It is pointed out that injection Fe simultaneously
3o
4the content of the mouse tumor position ferrum of-PEI-Ac-FI-PEG-FA nano-particle is apparently higher than injection Fe
3o
4the situation of-PEI-Ac-FI-mPEG nano-particle.These results have not only proved Fe
3o
4-PEI-Ac-FI-PEG-FA has good targeting to tumor locus, and the nano-particle prepared of explanation the present invention can be in Mice Body normal metabolite clearance.
beneficial effect
(1) the present invention adopts simply " step " hydro-thermal method to prepare the coated Fe of PEI that water solublity is good
3o
4nano-particle, then successively connects FI and PEG-FA molecule at nano grain surface, finally the surface amino groups of nano-particle is carried out to acetylation modification and obtains the Fe for MRI contrast agent
3o
4nano-particle; This method operating procedure is simple, and reaction condition is gentle, and easy operating is separated, is usedly cheap and eco-friendly material, has the business-like prospect of enforcement;
(2) Fe that prepared by the present invention
3o
4in aqueous solution, there is not agglomeration in nano-particle for a long time stable dispersion; The coated Fe that increased of PEI
3o
4the stability of nano-particle, the finishing of PEG-FA has not only increased Fe
3o
4the biocompatibility of nano-particle and hydrophilic, and give the targeting specific of nano-particle to tumor cell or tumor locus; These advantages make the folate-targeted Fe of preparation
3o
4nano-particle can be used as the negative contrast medium of MRI imaging in body effectively.
Accompanying drawing explanation
Fig. 1 is Fe prepared by the present invention
3o
4the x-ray diffraction pattern of-PEI;
Fig. 2 is FA(a), NH
2-PEG-COOH(b) and COOH-PEG-FA(c) the proton nmr spectra spectrogram in deuterated DMSO;
Fig. 3 is Fe prepared by the present invention
3o
4fI(b) and Fe-PEI-FI(a),
3o
4-PEI(c) ultraviolet absorpting spectrum;
Fig. 4 is Fe prepared by the present invention
3o
4-PEI-FI(a), Fe
3o
4-PEI-FI-PEG-FA(b) and control material Fe
3o
4-PEI-FI-mPEG(c) thermogravimetric analysis figure;
Fig. 5 is control material Fe
3o
4-PEI-Ac-FI-mPEG(a, b) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA(c, d) transmission electron microscope picture and distribution of sizes rectangular histogram;
Fig. 6 is control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4the T of-PEI-Ac-FI-PEG-FA nano-particle
2the linear relationship chart of relaxation time inverse and Fe concentration;
Fig. 7 is control material Fe
3o
4-PEI-Ac-FI-mPEG(a) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA(b) the hemolytic experiment uv-spectrogram of nano-particle, what upper right corner illustration showed is the ultraviolet absorpting spectrum amplifying in figure, lower right corner illustration be successively from left to right water, PBS, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL nano-particle process 2 hours and centrifugal after human red cell picture;
Fig. 8 is KB cell process PBS buffer (contrast), the control material Fe of mtt assay test
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (concentration range is at 0-100 μ g/mL) is processed the cell viability after 24 hours;
Fig. 9 is that KB cell is through PBS buffer (contrast, a, g), control material Fe
3o
4-PEI-Ac-FI-mPEG(b:10 μ g/mL, c:25 μ g/mL, d:50 μ g/mL, e:75 μ g/mL, f:100 μ g/mL) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (h:10 μ g/mL, i:25 μ g/mL, j:50 μ g/mL, k:75 μ g/mL, l:100 μ g/mL) is processed the cellular morphology after 24 hours;
Figure 10 is that KB cell is through PBS buffer (contrast, a, i), control material Fe
3o
4-PEI-Ac-FI-mPEG(Fe concentration range b:0.125, c:0.25, d:0.5, e:0.75, f:1.0, g:1.25 and h:1.5mM) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration range j:0.125, k:0.25, l:0.5, m:0.75, n:1.0, o:1.25 and p:1.5mM) is processed flow cytometry figure after 4 hours;
Figure 11 is that KB cell is through PBS buffer, control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration range is at 0.125-1.5mM) is processed the average fluorescent strength of cell after 4 hours;
Figure 12 be KB cell through PBS buffer (contrast, a-d), control material Fe
3o
4-PEI-Ac-FI-mPEG(e-h) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (i-l) (Fe concentration is 0.3mM) is processed the confocal microscopic image picture of cell after 4 hours;
Figure 13 is that KB cell is through PBS buffer, control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration range is at 0.1-0.8mM) is processed the cell T after 6 hours
2mRI imaging picture (a) and corresponding MRI signal value change (b);
Figure 14 is tail vein injection control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4the T of different time points mouse tumor after-PEI-Ac-FI-PEG-FA nano-particle (Fe:500 μ g)
2mRI imaging picture (a) and corresponding MRI signal value change (b);
Figure 15 is tail vein injection control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4latter 24 hours of-PEI-Ac-FI-PEG-FA nano-particle (Fe:500 μ g), Fe element is in the tissue distribution of mice major organs (heart, liver, spleen, lung, kidney and tumor).
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
By 1.25g FeCl
24H
2o pours in beaker, adds the ultra-pure water of 7.75mL, under gentle agitation, adds 6.25mLNH
3h
2o, by above-mentioned mixed liquor continuous stirring 10 minutes under air atmosphere, makes ferrous iron fully oxidized, then mixed solution is transferred in autoclave.0.5g PEI ultrasonic dissolution, in 5mL aqueous solution, is proceeded in reactor with liquid-transfering gun, fully mixed with solution in reactor, in 134 ℃ of reactions 3 hours.After reaction finishes, naturally cool to room temperature, by resulting black precipitate Fe
3o
4supernatant is removed in the separation of-PEI magnetic, then adds the ultrasonic dispersion of appropriate ultra-pure water, then magnetic is separated, so repeats ultra-pure water washing five times, to remove impurity, is then again scattered in 20mL ultra-pure water, obtains the coated Fe of PEI
3o
4nano-particle (Fe
3o
4-PEI).Get 3mL nanoparticles solution, vacuum lyophilization detects for X-ray diffraction.XRD result has shown Fe
3o
4the crystal structure of-PEI nano-particle is ferroso-ferric oxide (seeing accompanying drawing 1).
Embodiment 2
Get 17.65mg FA, 6.90mg EDC and 4.14mg NHS in a reaction bulb, add 5mL DMSO that it is dissolved completely, and stir-activating 3h.Get 40mg NH
2-PEG-COOH is dissolved in 5mL DMSO.Then the solution of above-mentioned activation (5mL) is dropwise joined to NH
2in the DMSO solution (5mL) of-PEG-COOH, stirring reaction three days.The bag filter that is 1000 with molecular cut off, to distill water dialysis three days (6 times, 2L/ time), is removed by-product and impurity, standby by being stored in-20 ℃ after product C OOH-PEG-FA lyophilization;
FA, NH
2as shown in Figure 2, the spectrum peak explanation FA that COOH-PEG-FA occurs at 6-9ppm is successfully connected on PEG in the nucleus magnetic hydrogen spectrum analysis of-PEG-COOH and synthetic COOH-PEG-FA.Known by integration calculated by peak area, on each PEG, connected 0.7 FA.
Embodiment 3
Get the Fe of embodiment 1 preparation
3o
4-PEI aqueous solution 6.24mL(30mg) with DMSO, wash three times, then by Fe
3o
4-PEI is scattered in 6mL DMSO again, then by the FI(0.84mg being dissolved in 2mL DMSO) solution dropwise joins above-mentioned 6mL Fe
3o
4in the DMSO solution of-PEI, stirring reaction is after one day, and supernatant is removed in magnetic separation, then adds the ultrasonic dispersion of appropriate ultra-pure water, then magnetic is separated, so repeats pure water washing 3 times, to remove impurity, is then again scattered in 10mL DMSO, obtains product Fe
3o
4-PEI-FI.Get respectively 25 μ L Fe
3o
4-PEI(embodiment 1), FI and Fe
3o
4-PEI-FI(embodiment 3) DMSO solution, in 2mL centrifuge tube, then adds 700 μ L ultra-pure waters wherein, ultrasonic evenly, survey uv absorption (seeing accompanying drawing 3).Uv-vis spectra test result shows, Fe
3o
4-PEI does not have obvious ultraviolet absorption peak at 400 to 800nm places, and Fe
3o
4-PEI-FI has an obvious ultraviolet absorption peak at 510nm place, thereby explanation FI successfully modifies Fe
3o
4-PEI nano grain surface.
Embodiment 4
COOH-PEG-FA, the 5.18mg EDC and the 3.11mg NHS that get 12.39mg embodiment 2 preparations are dissolved in respectively 2mLDMSO, and stir-activating 3h after solution is mixed.Then the COOH-PEG-FA solution (6mL) of activation is dropwise joined to the Fe of 5mL embodiment 3 preparations
3o
4in-PEI-FI solution, stirring reaction three days, then use ultrapure hydromagnetic separating, washing 3 times, product Fe
3o
4-PEI-FI-PEG-FA is scattered in 5mL ultra-pure water again, and gets 0.5mL vacuum lyophilization, gets control material Fe simultaneously
3o
4-PEI-FI-mPEG(comparative example 1) 0.5mL and Fe
3o
4-PEI-FI(embodiment 3) 0.5mL vacuum lyophilization, then carries out thermogravimetric analysis (as shown in Figure 4) to three kinds of materials.TG test result shows, Fe before modifying
3o
4the weight loss of-PEI-FI nano-particle be 8.97%(Fig. 4 a), after modifying by PEI surface amino groups and PEG molar ratio 5:1, Fe
3o
4-PEI-FI-PEG-FA and control material Fe
3o
4the weightlessness of-PEI-FI-mPEG is respectively 13.23%(Fig. 4 b) and 14.16%(Fig. 4 c); Through calculating, the rate of uploading of COOH-PEG-FA and mPEG-COOH is respectively 4.26% and 5.19%, shows that thus COOH-PEG-FA and mPEG-COOH are successfully connected to Fe
3o
4the surface of nano-particle.
Embodiment 5
Fe to embodiment 4 preparations
3o
4in-PEI-FI-PEG-FA nano-particle aqueous solution (4mL), add 18 μ L triethylamines (density is 0.726～0.729g/mL, and concentration is 99.0%), and stir 30 minutes.Then in above-mentioned solution, dropwise add 12.2 μ L acetic anhydrides (density is 1.08g/mL, and concentration is 98.5%) (triethylamine, acetic anhydride and Fe
3o
4surface amino groups mol ratio=5:5:1 of-PEI-FI-PEG-FA), continue stirring reaction 24 hours.Ultrapure hydromagnetic separating, washing 3 times for product, and be re-dispersed in 5mL ultra-pure water, make acetylizad Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle.Get Fe prepared by the present invention
3o
4-PEI-FI-PEG-FA(embodiment 4), Fe
3o
4-PEI-Ac-FI-PEG-FA(embodiment 5) the control material Fe and in comparative example 1
3o
4-PEI-FI-mPEG and Fe
3o
4each 0.1mL of-PEI-Ac-FI-mPEG, the aqueous solution that is then mixed with respectively 1.5mL with ultra-pure water is for surveying surface potential and hydrodynamic diameter (as table 1).Potential determination result shows, due to the existence of exhibiting high surface amino, Fe
3o
4-PEI-FI-PEG-FA and Fe
3o
4reached respectively+31.0mV of the surface potential of-PEI-FI-mPEG and+31.5mV.Through after acetylization reaction, the Fe obtaining
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4dropped to respectively+16.3mV of-PEI-Ac-FI-mPEG nano grain surface electromotive force and+16.4mV.Result shows, the successful acetylation of the surface amino groups of nano-particle.But the surface potential of nano-particle does not reach neutral after acetylation, this may be can not carry out acetylization reaction because surperficial part is used for stablizing the amino of ferroferric oxide nano granules.Before and after acetylation; the test result of the hydrodynamic diameter of nano-particle is as shown in table 1 equally; after acetylation, the hydrodynamic diameter of nano-particle diminishes before compared with acetylation slightly; and it is almost constant that the hydrodynamic diameter of nano-particle can keep for a long time, thereby illustrated that the nano-particle of preparation has good colloidal stability.
Embodiment 6
Get respectively the control material Fe after acetylation
3o
4-PEI-Ac-FI-mPEG(comparative example 1) and the Fe for preparing of the present invention
3o
4-PEI-Ac-FI-PEG-FA(embodiment 5) each 5 μ L of nano-particle aqueous solution, are then mixed with the nano granule suspension of 100 μ L with ultra-pure water.Get respectively the Fe of preparation
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4each 5 μ L of-PEI-Ac-FI-PEG-FA nano granule suspension drop in copper mesh surface, and for TEM, test (as shown in Figure 5) after drying in air.TEM result shows Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the pattern of-PEI-Ac-FI-PEG-FA nano-particle is spherical or torispherical.By the diameter of 300 nano-particle of random measurement respectively, calculate Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4the diameter of-PEI-Ac-FI-mPEG nano-particle is respectively 15.0nm and 15.3nm.
Embodiment 7
Fe prepared by the present invention
3o
4-PEI-Ac-FI-PEG-FA(embodiment 5) nano-particle and control material Fe
3o
4-PEI-Ac-FI-mPEG(comparative example 1) by ICP-AES method of testing, record the concentration of Fe element in solution, then by ultra-pure water preparation Fe concentration, be followed successively by 0.0025,0.005,0.01,0.02 and the aqueous solution 2mL of 0.04mM in EP pipe, pass through T
2the T of material under different Fe concentration measured in nuclear magnetic resonance
2relaxation effect (as shown in Figure 6).Relaxation rate test result shows Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4the T of-PEI-Ac-FI-mPEG nano-particle
2relaxation time inverse is that within the scope of 0.0025-0.04mM, the increase along with concentration of iron has good linear relationship in Fe concentration.And by calculating known Fe
3o
4-PEI-Ac-FI-PEG-FA(99.64mM
-1s
-1) and Fe
3o
4-PEI-Ac-FI-mPEG(107.30mM
-1s
-1) all there is good T
2relaxation effect and r
2relaxation rate.Therefore, the prepared Fe of the present invention
3o
4-PEI-Ac-FI-PEG-FA and control material Fe
3o
4-PEI-Ac-FI-mPEG all can be used as the good T in the diagnosis of MRI molecular imaging
2signal attenuation contrast agent.
Embodiment 8
For make the nano-particle of preparation can be safely for bio-imaging diagnosis in body, this experimental evaluation the Fe of preparation
3o
4-PEI-Ac-FI-PEG-FA nano-particle and control material Fe
3o
4the blood compatibility of-PEI-Ac-FI-mPEG.Take the Fe of lyophilizing
3o
4-PEI-Ac-FI-PEG-FA nano-particle (embodiment 5) and control material Fe
3o
4each 1mg of-PEI-Ac-FI-mPEG (comparative example 1), being scattered in respectively the concentration that is mixed with 1mg/mL in PBS is mother solution, with PBS successively compound concentration, is then the nano granule suspension of 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL.Get appropriate people's fresh blood, first centrifugal (2000rpm/min, 5min) removes supernatant, then by PBS washing for erythrocyte 5 times, collects healthy erythrocyte and with 10 times of PBS dilutions.Again by Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4-PEI-Ac-FI-mPEG nano material (50-400 μ g/mL) is mixed with erythrocyte after standing 2 hours, and the centrifugal 1min of 10000rpm takes pictures and surveys the ultraviolet light absorption spectrum of supernatant.This process is usingd PBS as negative control, and ultra-pure water is as positive control.In Fig. 7, shown Fe
3o
4-PEI-Ac-FI-mPEG(Fig. 7 a) and Fe
3o
4-PEI-Ac-FI-PEG-FA(Fig. 7 b) the hemolytic test result under concentration 50,100,200,400 μ g/mL.By measuring the hemolytic of the absorbance quantitative assessment nano material of the supernatant.As Fig. 7 a and the demonstration of 7b upper right corner uv-spectrogram, when concentration reaches 400 μ g/mL, Fe
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4the hemolysis rate of-PEI-Ac-FI-mPEG is all less than 5%, illustrates that these nano materials of preparation have good blood compatibility, thereby can be safely for MRI imaging in organism.
Embodiment 9
The Fe that the KB cell of take is prepared as model cell evaluation the present invention
3o
4-PEI-Ac-FI-PEG-FA nano-particle and control material Fe
3o
4the impact of-PEI-Ac-FI-mPEG on cell proliferation.Take the Fe of lyophilizing
3o
4-PEI-Ac-FI-PEG-FA nano-particle (embodiment 5) and control material Fe
3o
4-PEI-Ac-FI-mPEG(comparative example 1) each 1mg, is then scattered in respectively the PBS solution that is mixed with 1mg/mL in aseptic PBS, and with the ultra-vioket radiation sterilization of spending the night.Then at superclean bench, with aseptic PBS compound concentration, be the aseptic Fe of 10,25,50,75 and 100 μ g/mL
3o
4-PEI-Ac-FI-PEG-FA and Fe
3o
4-PEI-Ac-FI-mPEG nano granule suspension.KB cell seeding after 96 orifice plates respectively with Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (concentration is 10,25,50,75 and 100 μ g/mL) is cultivated altogether 24 hours at 37 ℃.Then, to cultivating in plate hole, add 20 μ L MTT, continue to cultivate after 4 hours at 37 ℃, discard culture fluid, and add 150 μ L DMSO, vibrate and after 15 minutes, at 570nm place, measure light absorption value, and be worth the vigor (as Fig. 8) of calculating cell according to this.The impact of the material on cell proliferation of variable concentrations take buffer PBS as contrast compare.Compare Fe with matched group
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA does not have significant difference to the survival rate of KB cell within the scope of experimental concentration 0 to 100 μ g/mL, and cell survival rate is all more than 80%.This absolutely proves synthetic Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA all has good biocompatibility, can be applied to MRI imaging in organism and detect.We have further verified the toxicity of material to cell by phase contrast microscope.As shown in Figure 9, the Fe of variable concentrations
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the cellular morphology that-PEI-Ac-FI-PEG-FA nano material (10,25,50,75 with 100 μ g/mL) was processed after 24 hours is compared with the cell that PBS processes, and does not significantly change, and does not further illustrate the good biocompatibility of synthetic material.
Embodiment 10
By flow cytometer, detect the Fe of variable concentrations
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4after-PEI-Ac-FI-PEG-FA processes, KB cell is evaluated folate-targeted effect to the average fluorescent strength (as Figure 11) of the amount of engulfing of material (as Figure 10) and cell.By ICP-AES, measure the aseptic Fe of 1mg/mL in embodiment 9
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the concentration of Fe in-PEI-Ac-FI-PEG-FA PBS solution, then with aseptic PBS, being mixed with respectively Fe concentration is 0.125,0.25,0.5,0.75,1.0,1.25 and two kinds of nano granule suspensions of 1.5mM.KB cell is with 2 * 10
5plant in 12 orifice plates in/hole, after incubated overnight more respectively with Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration be 0.125,0.25,0.5,0.75,1.0,1.25 and 1.5mM) is cultivated altogether 4 hours at 37 ℃, and the cell of processing with PBS as a control group.After cultivating altogether, for cell, PBS cleans three times, then with trypsinization centrifugal, abandons supernatant, by cell suspension in 1mL PBS.By flow cytometer, detect the average fluorescent strength of cell to the situation of engulfing of nano-particle and the rear cell of processing.In Figure 10, compare with matched group (PBS processes cell), along with the increase of Fe concentration, cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-mPEG increases a little, and when Fe concentration reaches 1.0mM, the amount of engulfing almost no longer increases.But along with the increase of Fe concentration, cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-PEG-FA but obviously increases always.Same in Figure 11 along with the increase of Fe concentration, Fe
3o
4after-PEI-Ac-FI-PEG-FA processes, the average fluorescent strength of cell significantly increases, and control material Fe
3o
4after-PEI-Ac-FI-mPEG processes, the average fluorescent strength of cell increases not obvious.The modification of this explanation folic acid has been given nano-particle to expressing the special target ability of the KB cell of folacin receptor.
Embodiment 11
Further by Laser Scanning Confocal Microscope, verify the targeting effect of folic acid, first coverslip is positioned in 12 porocyte culture plates and by 1640 culture medium and soaks 12h, then every hole supplements 1.0mL culture medium and inoculates 5 * 10
4individual KB cell, after spending the night respectively with PBS, Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration is 0.5mM) is cultivated altogether 4 hours at 37 ℃, then with PBS, clean three times, then use successively that 2.5% glutaraldehyde (0.5mL) is fixed 15 minutes, hochest33343(0.8mL) dyeing 30 minutes, finally coverslip is positioned on microscope slide, by the pattern of oily sem observation cell.As shown in figure 12, in the cell of processing through PBS, there is no fluorescence, control material Fe
3o
4in the cell that-PEI-Ac-FI-mPEG processes, demonstrate fainter fluorescence signal, and Fe
3o
4in the cell that-PEI-Ac-FI-PEG-FA processes, demonstrate very strong fluorescence signal, this nano-particle that further illustrates modified with folic acid has targeting specific to expressing the KB cell of folacin receptor, thereby provides reliable foundation for this material success is applied to MRI imaging in body efficiently.
Embodiment 12
Before in-vivo imaging experiment, we have evaluated the cell MRI imaging effect of nano-particle, measure the aseptic Fe of 1mg/mL in embodiment 9 by ICP-AES
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the concentration of Fe in-PEI-Ac-FI-PEG-FA PBS solution, then with aseptic PBS, being mixed with respectively Fe concentration is 0.1,0.2,0.4 and two kinds of nano granule suspensions of 0.8mM.KB cell is with 3 * 10
6plant in 6 orifice plates in/hole, after overnight incubation, respectively with PBS, Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle (Fe concentration be 0.1,0.2,0.4 and 0.8mM) is cultivated altogether 6 hours at 37 ℃, after cultivation finishes, cell cleans 5 times with PBS, trypsinization again, centrifugal, filtration, be finally dispersed in 1mL PBS(containing 0.5% agarose) in.With nuclear magnetic resonance imaging instrument, survey the T of each cell sample
2relaxation effect (as Figure 13).In Figure 13 a, along with the increase of Fe concentration, Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4cell after-PEI-Ac-FI-PEG-FA nano-particle is processed all shows the trend of MRI signal attenuation, illustrates that cell also increases the amount of engulfing of nano-particle along with the increase of Fe concentration.It is pointed out that under same Fe concentration Fe
3o
4cell after-PEI-Ac-FI-PEG-FA nano-particle is processed compares Fe
3o
4the MRI signal of the rear cell of-PEI-Ac-FI-mPEG processing reduces more obvious, illustrates that cell is to Fe
3o
4the amount of engulfing of-PEI-Ac-FI-PEG-FA nano-particle will be much higher than Fe
3o
4-PEI-Ac-FI-mPEG nano-particle.Figure 13 b is the MRI imaging signal value after cell is processed by the nano-particle of variable concentrations, from figure, obviously finds out, along with the increase of Fe concentration, the MRI signal value of cell all reduces gradually, and under identical Fe concentration, Fe
3o
4the MRI signal value that-PEI-Ac-FI-PEG-FA nano-particle is processed rear cell will be starkly lower than Fe
3o
4cell after-PEI-Ac-FI-mPEG nano-particle is processed.These results not only illustrate that the nano-particle of preparation has good cell MRI imaging effect, and have proved Fe
3o
4the specific target tropism of-PEI-Ac-FI-PEG-FA nano-particle to tumor cell.
Embodiment 13
Fe prepared by the present invention
3o
4-PEI-Ac-FI-PEG-FA(embodiment 5) and control material Fe
3o
4-PEI-Ac-FI-mPEG(comparative example 1) aqueous solution is scattered in 2mL PBS again with after PBS centrifuge washing 3 times, and with ICP-AES, measures the concentration of Fe in each solution.Then the Fe for preparing same volume and contain identical Fe quality with aseptic PBS
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4the PBS solution of-PEI-Ac-FI-PEG-FA.KB cell is inoculated in nude mouse, after three weeks when diameter of tumor reaches 1.0-1.2cm, by tail vein injection Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA nano-particle PBS solution is evaluated the MRI imaging effect (as shown in figure 14) of tumor locus.Compare with the matched group before injection, in latter 30 minutes to 4 hours of injection, injection 0.5mL control material Fe
3o
4-PEI-Ac-FI-mPEG(Fe:500 μ g) mouse tumor position is dimmed a little, and injection 0.5mLFe
3o
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g) mouse tumor is obviously dimmed, and the nano-particle that shows modified with folic acid has obvious MRI diagnosing tumor effect.In injection latter 24 hours, the mouse tumor position bright-dark degree of two experimental grouies all recovered gradually, illustrate now nano material along with blood circulation from tumor locus gradually metabolism go out that (Figure 14 a).Figure 14 b is that the tumor MRI signal value of corresponding inject time changes, in injection latter 30 minutes to 4 hours, and injection control material Fe
3o
4the mouse tumor MRI signal value of-PEI-Ac-FI-mPEG changes not obvious, and injection Fe
3o
4the mouse tumor MRI signal value of-PEI-Ac-FI-PEG-FA obviously reduces.In injection latter 24 hours, the mouse tumor position MRI signal value of two experimental grouies all increased to some extent, and this is consistent with the result of Figure 14 a.This Fe preparing of these presentation of results
3o
4-PEI-Ac-FI-PEG-FA nano-particle has good cancer target ability, can be successfully applied to the contrast agent of in-vivo tumour targeting MRI image-forming diagnose.
Embodiment 14
Take to plant and have the nude mice (embodiment 13) of tumor as animal pattern detection control material Fe
3o
4fe prepared by-PEI-Ac-FI-mPEG and the present invention
3o
4the tissue distribution (Figure 15) of-PEI-Ac-FI-PEG-FA nano-particle tail vein injection ferrum after 24 hours.Fe to nude mice tail vein injection 0.5mL embodiment 13 preparations
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g) PBS solution is after 24 hours, puts to death mice, and take out each organ and weigh, the section of cutting into pieces then, and add 3mL chloroazotic acid (hydrochloric acid/nitric acid; Volume ratio 3:1) soak 2 days, with ICP-AES, measure the concentration of ferrum in each histoorgan.As can be seen from Figure 15 at injection Fe
3o
4-PEI-Ac-FI-mPEG and Fe
3o
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g), after nano-particle, in liver, spleen and lung, the content of ferrum all obviously increases before injecting, and at other organ, such as: the heart, kidney and tumor, the gathering of ferrum is less.But it is pointed out that injection Fe
3o
4the content of the mouse tumor position ferrum of-PEI-Ac-FI-PEG-FA nano-particle is apparently higher than injection Fe
3o
4the situation of-PEI-Ac-FI-mPEG nano-particle.These results have not only proved Fe
3o
4-PEI-Ac-FI-PEG-FA has good targeting to tumor locus, and illustrate the nano-particle of this invention preparation can be in Mice Body normal metabolite clearance.
Comparative example 1
1.25g Fe (II) salt is dissolved in 7.75mL ultra-pure water, then adds 6.25mL NH
3h
2o also stirs 10 minutes under air atmosphere, then mixed solution is transferred in autoclave.Get 0.5g PEI and be dissolved in 5mL ultra-pure water, and this 5mL PEI aqueous solution is also joined in autoclave, after being uniformly mixed, in 134 ℃ of reactions 3 hours.After reaction finishes, naturally cool to room temperature, will precipitate after magnetic separating, washing purification, be again scattered in 20mL ultra-pure water, obtain the nano-particle Fe of PEI parcel
3o
4-PEI, product be stored in 4 ℃ standby.Characterization of The Products is shown in Fig. 1;
Get Fe prepared by 6.24mL above-mentioned steps
3o
4-PEI nano-particle (30mg), with after DMSO washing three times, is scattered in 6mL DMSO again.Get 0.84mg FI, and be dissolved in 2mL DMSO.Then the FI solution (2mL) DMSO being dissolved dropwise joins above-mentioned 6mL Fe
3o
4in the DMSO solution of-PEI, stirring reaction is after one day, product Fe
3o
4ultrapure hydromagnetic separating, washing 3 times for-PEI-FI, is then distributed in 10mL DMSO, and be stored in 4 ℃ standby.Characterization of The Products is shown in Fig. 3;
Get 10.80mg mPEG-COOH, 5.18mg EDC and 3.11mg NHS and be dissolved in respectively 2mL DMSO, and stir-activating 3h after solution is mixed.Then the mPEG-COOH solution (6mL) of activation is dropwise joined to the Fe of the above-mentioned preparation of 5mL
3o
4in-PEI-FI solution (5mL), stirring reaction three days, then use ultrapure hydromagnetic separating, washing 3 times, product Fe
3o
4-PEI-FI-mPEG after being again scattered in 5mL ultra-pure water, be stored in 4 ℃ standby.Characterization of The Products is shown in Fig. 4 and table 1; The Fe preparing to above-mentioned steps
3o
4in-PEI-FI-mPEG nano-particle aqueous solution (4mL), add 18 μ L triethylamines, and stir 30 minutes.Then in above-mentioned solution, dropwise add 12.2 μ L acetic anhydrides, continue stirring reaction 24 hours.Ultrapure hydromagnetic separating, washing 3 times for product, and be re-dispersed in 5mL ultra-pure water, make the acetylizad Fe of control material
3o
4-PEI-AC-FI-mPEG nano-particle, is stored in-20 ℃ after getting 2mL vacuum lyophilization, remaining 3mL be stored in 4 ℃ standby.Characterization of The Products is shown in Fig. 5-15 and table 1.
Table 1.Fe
3o
4-PEI-FI-mPEG, Fe
3o
4-PEI-Ac-FI-mPEG, Fe
3o
4-PEI-FI-PEG-FA and Fe
3o
4the electromotive force of-PEI-Ac-FI-PEG-FA nano-particle and hydrodynamic diameter.
Claims (8)
1. a preparation method for the targeting MRI contrast agent of the ferroferric oxide nano granules based on modified with folic acid, comprising:
(1) ferrous salt is dissolved in ultra-pure water, adds NH
3h
2o also stirs under air atmosphere, then mixed solution is transferred in autoclave, and the hyperbranched polyethyleneimine PEI aqueous solution of 0.1g/mL is also joined in autoclave, after being uniformly mixed, in 130～140 ℃ of reactions 2～4 hours; After reaction finishes, naturally cool to room temperature, precipitate and separate is washed after purification, obtain the ferroferric oxide nano granules Fe of PEI parcel
3o
4-PEI;
(2), after FA is dissolved in DMSO, first, with EDC and NHS activation 2～4h, then dropwise join NH
2in the DMSO solution of-PEG-COOH, stirring reaction 2～4 days, dialysis, then vacuum lyophilization, obtains COOH-PEG-FA;
(3) by the Fe of preparation in step (1)
3o
4-PEI nano-particle, with after DMSO washing, is scattered in DMSO again, then the FI solution that DMSO is dissolved joins above-mentioned Fe
3o
4in the DMSO solution of-PEI, stirring reaction is after 1～3 day, product Fe
3o
4-PEI-FI separating, washing, is then distributed in DMSO;
(4) in step (2), the COOH-PEG-FA of preparation and EDC and NHS are dissolved in after DMSO, stir-activating 2～4h; Then the COOH-PEG-FA solution of activation is joined to Fe prepared by step (3)
3o
4in-PEI-FI solution, stirring reaction 2～4 days, then separating, washing, and be scattered in ultra-pure water, obtain Fe
3o
4-PEI-FI-PEG-FA nano-particle aqueous solution; Wherein, the mol ratio of COOH-PEG-FA and EDC, NHS is 1:4～6:4～6; COOH-PEG-FA and Fe
3o
4the mol ratio of nano grain surface amino is 1:4～6;
(5) Fe preparing to step (4)
3o
4in-PEI-FI-PEG-FA nano-particle aqueous solution, add triethylamine, and stir 20～40 minutes; Then in above-mentioned solution, add acetic anhydride, continue stirring reaction 20～30 hours; Separating, washing is also scattered in ultra-pure water, obtains.
2. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: the ferrous salt in described step (1) is FeCl
24H
2o, ferrous salt, ultra-pure water, NH
3h
2the proportioning of O is 1～1.5g:7.5～8.0mL:6.0～6.5mL, and ferrous salt and hyperbranched polyethyleneimine PEI mass ratio are 2～3:1.
3. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: the NH in described step (1)
3h
2o mass percent concentration is 25-28%.
4. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: the molecular weight of the hyperbranched polyethyleneimine PEI in described step (1) is 25000.
5. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: in described step (2), the mol ratio of FA, EDC and NHS is 1:0.5～1:0.5～1, FA and NH
2the mol ratio of-PEG-COOH is 1～3:1.
6. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: NH in described step (2)
2the molecular weight of-PEG-COOH is 2000.
7. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: FI and Fe in described step (3)
3o
4the mol ratio of-PEI surface amino groups is 1:45～55.
8. the preparation method of the targeting MRI contrast agent of a kind of ferroferric oxide nano granules based on modified with folic acid according to claim 1, is characterized in that: triethylamine, acetic anhydride and Fe in described step (5)
3o
4the upper primary amino radical mol ratio of PEI of nano grain surface is 4～6:4～6:1.
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| CN103432100B (en) * | 2013-08-22 | 2014-12-10 | 东华大学 | Method for preparing polyethyleneimine modified PLGA (poly(lactic-co-glycolic acid)) loading hollow micro-capsules based on polyethylene glycol and folic acid grafting |
| CN103965905A (en) * | 2014-04-25 | 2014-08-06 | 上海大学 | Synthetic method for folic acid-modified water-soluble rare-earth up-conversion luminescence nanocrystalline |
| CN103933584B (en) * | 2014-04-30 | 2016-08-24 | 东华大学 | A kind of preparation method of the superparamagnetic iron oxide nano-particle of modified with folic acid |
| CN104274842B (en) * | 2014-10-13 | 2016-11-16 | 东华大学 | A kind of preparation method of the multi-functional trimanganese tetroxide nano granular core magnetic resonance contrast agent of polyethyleneimine |
| CN106075459A (en) * | 2016-06-14 | 2016-11-09 | 东华大学 | A kind of method of the multi-functional hyperbranched polyethyleneimine load amycin of folate-targeted |
| CN106581697A (en) * | 2016-12-22 | 2017-04-26 | 宜兴市人民医院 | Nanometer contrast agent with lung-cancer-targeting magnetic resonance imaging capacity and preparation method of nanometer contrast agent |
| CN108514642B (en) * | 2018-04-02 | 2019-12-10 | 东华大学 | preparation method of tree-shaped macromolecule stable ultra-small ferroferric oxide/gold nanoflowers |
| CN108635595B (en) * | 2018-07-04 | 2019-11-08 | 东华大学 | Extra small ferric oxide nano-probe and its preparation and application based on photoresponse polymerization |
| CN109395103B (en) * | 2018-12-05 | 2021-04-02 | 中国人民解放军军事科学院军事医学研究院 | Magnetic nano-composite with antioxidant function and preparation method thereof |
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