CN103143041A - 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 characteristics 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.For sensitivity and the specificity that improves the MRI image-forming diagnose, be necessary to select suitable MRI contrast agent.Conventional MRI contrast agent mainly is 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
4Be the metallic iron oxide nano-particles of representative, Superparamagnetic Iron Oxide has unique magnetic property and signal is strong, the low characteristics such as biocompatibility of becoming reconciled of using dosage make it become the good contrast agent of 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 the particle surface band positive charge and functional groups, this is just Fe
3O
4The surperficial multi-functional modification 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 the 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 advantage 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 Fe that the PEI that the present invention adopts identical method to synthesize to have good colloidal stability coats
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, and using for further in-vivo imaging provides guarantee; Also improved simultaneously Fe
3O
4The targeting of nano-particle to tumor cell or tumor locus, thus make the NMR (Nuclear Magnetic Resonance)-imaging diagnosis more accurate, sensitive.
The retrieval domestic and foreign literature does not still have to find about preparing with a step hydrothermal synthesis method Fe that PEI coats and FA modifies
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 stable dispersion for a long time in aqueous solution, agglomeration can not occur.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 stirred under air atmosphere 10～20 minutes, then mixed solution is transferred in autoclave, and the hyperbranched polyethyleneimine PEI aqueous solution of 0.1g/mL is also joined in autoclave, after mixing, in 130～140 ℃ of reactions 2～4 hours; Reaction naturally cools to room temperature after finishing, and after precipitating magnetic separating, washing purification, namely gets 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 being dissolved in FA 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 (is 1000 bag filter with molecular cut off to distill water dialysis three days (6 times, 2L/ time)), remove by-product and impurity, then vacuum lyophilization, namely get COOH-PEG-FA, be stored in-20 ℃ standby;
(3) with the Fe for preparing in step (1)
3O
4-PEI nano-particle is scattered in DMSO after washing with DMSO again, then the FI solution of DMSO dissolving is joined above-mentioned Fe
3O
4In the DMSO solution of-PEI, stirring reaction is after 1～3 day, product Fe
3O
4Then-PEI-FI separating, washing is distributed in DMSO, and be stored in 4 ℃ standby;
(4) after in step (2), the COOH-PEG-FA of preparation and EDC and NHS are dissolved in DMSO, stir-activating 2～4h; Then the COOH-PEG-FA solution of activation is joined the Fe of step (3) preparation
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 for preparing to step (4)
3O
4Add triethylamine in-PEI-FI-PEG-FA nano-particle aqueous solution, and stirred 20～40 minutes; Then add acetic anhydride in mentioned solution, continued stirring reaction 20～30 hours; Separating, washing also is scattered in ultra-pure water, namely 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, preferred 1.25g:7.75mL:6.25mL; Ferrous salt and hyperbranched polyethyleneimine PEI mass ratio are 2～3:1, preferred 2.5:1.
NH in described step (1)
3H
2The O 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, preferred 1:0.9:0.9, FA and NH
2The mol ratio of-PEG-COOH is 1～3:1, preferred 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, preferred 1:50.
COOH-PEG-FA in described step (4) and the mol ratio of EDC, NHS are 1:4～6:4～6, preferred 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, preferred 1:5.
Triethylamine, acetic anhydride and Fe in described step (5)
3O
4The upper primary amino radical mol ratio of the PEI of nano grain surface is 4～6:4～6:1, preferred 5:5:1.
The present invention first utilizes the Fe of the synthetic PEI parcel of one step hydro thermal method
3O
4Then magnetic nanoparticle successively is modified at FI and PEG-FA the surface of nano-particle, at last the residual surface amino of nano-particle is carried out 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.Compare the Fe of modified with folic acid with the control material that there is no modified with folic acid
3O
4Nano-particle has higher targeting to tumor cell or tumor locus.The folate-targeted Fe of the method preparation
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), ultraviolet-visible absorption spectroscopy (UV-Vis), thermogravimetric analysis (TGA), ICP-AES (ICP-AES), Zeta electric potential and the methods such as dynamic light scattering and transmission electron microscope (TEM) characterize the magnetic nanoparticle of preparation, and measure the T of nano-particle by magnetic resonance imager
2Relaxivity, then utilize hemolytic experiment, mtt assay and phase contrast microscope to estimate blood compatibility and the cytotoxicity of nano-particle, recycling flow cytometer, confocal microscopic image and the experiment of in vitro and in vivo NMR (Nuclear Magnetic Resonance)-imaging detect the nano material of modified with folic acid to the targeting diagnosis effect of 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), material and Fe that hydro-thermal method is synthetic
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 COOH-PEG-FA three are at deuterium for the spectrum spectrum peak of the hydrogen in DMSO (as Fig. 2) as can be known, COOH-PEG-FA successfully is connected on PEG at the spectrum peak proof FA that 6-9ppm occurs, and by the integration calculated by peak area as can be known, connected 0.7 FA on each PEG.
(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.We can find out from figure, 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 the 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 the 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 press 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 successfully are connected to Fe
3O
4The surface of nano-particle.
(5) nano-particle Zeta electric potential and dynamic 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
4The surface potential of-PEI-FI-mPEG nano-particle reached respectively+31.0mV and+31.5mV.Through after acetylization reaction, obtain acetylizad Fe
3O
4-PEI-Ac-FI-PEG-FA and Fe
3O
4-PEI-Ac-FI-mPEG nano grain surface electromotive force dropped to respectively+16.3mV and+16.4mV.Result shows, the successful acetylation of the surface amino groups of nano-particle.But after acetylation, the surface potential of nano-particle does not reach neutral, and this may be because surface portion 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 than acetylation slightly; and the hydrodynamic diameter of the nano-particle of preparation can keep almost constant for a long time, thereby the nano-particle that preparation has been described has good colloidal stability.
(6) transmission electron microscope (TEM) test result
Fe by tem observation the present invention preparation
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).The 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.Calculate the Fe of preparation by the diameter of 300 nano-particle of random measurement respectively
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
2The relaxation 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, the MRI signal intensity weakens gradually.Relaxation rate (r
2) reflection Fe
3O
4Nanoparticle is the T2 of unit molar concentration ferrum as the efficient of MRI contrast agent, can be by the relaxation time (T under variable concentrations
2) the Fitting Calculation reciprocal obtain.Fig. 6 is the Fe that the present invention prepares
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 be found out this two kinds of Fe
3O
4The relaxation time inverse of nano material is along with the increase (in the 0-0.04mM concentration range) of concentration of iron has good linear relationship.And by can be calculated the Fe of the present invention's preparation
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
-1Therefore, 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
2The signal attenuation contrast agent.
(8) blood compatibility
Have good blood compatibility the body planted agent of nano-particle be used for saying vital, thus this experimental evaluation the Fe of the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3O
4The blood compatibility of-PEI-Ac-FI-mPEG.Shown Fe in Fig. 7
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 of the supernatant being come the hemolytic of quantitative assessment nano material.As Fig. 7 a and 7b upper right corner uv-spectrogram demonstration, 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 illustrates that all less than 5% these nano materials of preparation have good blood compatibility, thereby can be used for safely MRI imaging in organism.
(9) MTT cell viability and phase contrast microscope test result
Detect the Fe of the present invention's preparation by the vigor of MTT colorimetric method for determining KB cell (a kind of cell strain of human epithelium's cancer)
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).The 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) was cultivated under 37 ℃ 24 hours altogether.Then, after processing, MTT measures light absorption value at 570nm place, and according to the vigor of this value calculating cell.The impact of the material on cell proliferation of variable concentrations compares as contrasting take the cell that buffer PBS processes.Compare Fe with the PBS matched group
3O
4-PEI-Ac-FI-mPEG and Fe
3O
4-PEI-Ac-FI-PEG-FA survival rate to the KB cell in experimental concentration 0 to 100 μ g/mL scope does not have significant difference, and cell survival rate is all more than 80%.This proves absolutely 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 verify further that by phase contrast microscope material is to the toxicity of cell.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
Detect the KB cell to the Fe of the present invention's preparation by flow cytometer
3O
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3O
4-PEI-Ac-FI-mPEG under variable concentrations the amount of engulfing (as Figure 10) and process after the average fluorescent strength (as Figure 11) of cell detect the folate-targeted effect.The 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) cultivated altogether under 37 ℃ 4 hours, and the cell of processing with PBS is as a control group.Then detect the situation of engulfing of cell and the average fluorescent strength of cell by flow cytometer.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 reached 1.0mM, the amount of engulfing almost no longer increased.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 the 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, the KB cell respectively with the Fe of PBS, the present invention's preparation
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) cultivated altogether the fluorescence signal after oily Microscopic observation cytophagy nano-particle then 4 hours under 37 ℃.In Figure 12, there is no fluorescence, Fe in the cell through the PBS processing
3O
4Demonstrate fainter fluorescence signal in the cell that-PEI-Ac-FI-mPEG processes, and Fe
3O
4Demonstrate very strong fluorescence signal in the cell that-PEI-Ac-FI-PEG-FA processes, this nano-particle that further illustrates modified with folic acid has better targeting to the KB cell, thereby in body, the MRI imaging provides reliable foundation for this material success is applied to efficiently.
(12) cell in vitro MRI imaging results
Before the experiment, we have estimated the Fe of the present invention's preparation 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) was cultivated under 37 ℃ 6 hours altogether, 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, and the increase along with Fe concentration is described, cell also increases the amount of engulfing of nano-particle.It is pointed out that under identical Fe concentration Fe
3O
4Cell after-PEI-Ac-FI-PEG-FA nano-particle is processed is than contrast 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 after cell is processed through the nano-particle of variable concentrations, obviously finds out from figure, and along with the increase of Fe concentration, the MRI signal value of cell all reduces gradually, and under identical Fe concentration, Fe
3O
4After-PEI-Ac-FI-PEG-FA nano-particle is processed, the MRI signal value of cell will be starkly lower than control material Fe
3O
4Cell after-PEI-Ac-FI-mPEG nano-particle is processed.These results illustrate that not only 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 the KB cell.
(13) in-vivo tumour MRI imaging results
Fe by tail vein injection the present invention preparation
3O
4-PEI-Ac-FI-PEG-FA nano-particle and matched group material Fe
3O
4-PEI-Ac-FI-mPEG estimates the MRI imaging effect (as shown in figure 14) of tumor locus, compares with the matched group before injection, and in rear 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 rear 24 hours, the mouse tumor position bright-dark degree of two experimental grouies all recovered gradually, illustrate at this moment 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 rear 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 rear 24 hours, the mouse tumor position MRI signal value of two experimental grouies all increased to some extent, and this result with Figure 14 a is consistent.The Fe of these presentation of results the present invention preparation
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 rear 24 hours each vitals of injection.As can be seen from Figure at injection control material Fe
3O
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA(Fe:500 μ g) after nano-particle, all obviously increase before in liver, spleen and lung, the content of ferrum is injected, and at other organ, such as: the heart, kidney and tumor, the gathering of ferrum is less.It is pointed out that simultaneously 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 the nano-particle of explanation the present invention preparation can be in Mice Body normal metabolite clearance.
Beneficial effect
(1) the present invention adopts simply " step " hydro-thermal method to prepare the Fe that the good PEI of water solublity coats
3O
4Then nano-particle successively connects FI and PEG-FA molecule at nano grain surface, at last the surface amino groups of nano-particle is carried out acetylation modification and obtains Fe for the MRI contrast agent
3O
4Nano-particle; This method operating procedure is simple, and reaction condition is gentle, and easy operating separates, and usedly is cheap and eco-friendly material, has the business-like prospect of enforcement;
(2) Fe of the present invention's preparation
3O
4Nano-particle stable dispersion for a long time in aqueous solution, agglomeration do not occur; The coating of PEI has increased Fe
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.
Description of drawings
Fig. 1 is the Fe that the present invention prepares
3O
4The x-ray diffraction pattern of-PEI;
Fig. 2 is FA(a), NH
2-PEG-COOH(b) and COOH-PEG-FA(c) at deuterium for the proton nmr spectra spectrogram in DMSO;
Fig. 3 is the Fe that the present invention prepares
3O
4-PEI-FI(a), FI(b) and Fe
3O
4-PEI(c) ultraviolet absorpting spectrum;
Fig. 4 is the Fe that the present invention prepares
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 of the present invention preparation
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
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
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 of the present invention preparation
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 that amplifies 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 processed 2 hours and centrifugal after the human red cell picture;
Fig. 8 is KB cell process PBS buffer (contrast), the control material Fe of mtt assay test
3O
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
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 the 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 of the present invention's preparation
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 the 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 of the present invention's preparation
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 the KB cell is through PBS buffer, control material Fe
3O
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA nano-particle (the Fe concentration range is at 0.125-1.5mM) is processed the average fluorescent strength of cell after 4 hours;
Figure 12 be the KB cell through the PBS buffer (contrast, a-d), control material Fe
3O
4-PEI-Ac-FI-mPEG(e-h) and the Fe of the present invention preparation
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 the KB cell is through PBS buffer, control material Fe
3O
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA nano-particle (the 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
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
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
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
3O
4Rear 24 hours of-PEI-Ac-FI-PEG-FA nano-particle (Fe:500 μ g), the 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 only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
With 1.25g FeCl
24H
2O pours in beaker, adds the ultra-pure water of 7.75mL, under gentle agitation, adds 6.25mLNH
3H
2O with 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 the 5mL aqueous solution, is changed over to it in reactor with liquid-transfering gun, with the abundant mixing of solution in reactor, in 134 ℃ of reactions 3 hours.Reaction naturally cools to room temperature, with resulting black precipitate Fe after finishing
3O
4-PEI magnetic separates removes supernatant, then adds appropriate ultra-pure water ultra-sonic dispersion, then the magnetic separation, so repeats ultra-pure water washing five times, to remove impurity, then again is scattered in the 20mL ultra-pure water, namely gets the Fe that PEI coats
3O
4Nano-particle (Fe
3O
4-PEI).Get the 3mL nanoparticles solution, vacuum lyophilization is used for X-ray diffraction and detects.The XRD result has shown Fe
3O
4The crystal structure of-PEI nano-particle is ferroso-ferric oxide (seeing accompanying drawing 1).
Get 17.65mg FA, 6.90mg EDC and 4.14mg NHS in a reaction bulb, add 5mL DMSO that it is dissolved fully, and stir-activating 3h.Get 40mg NH
2-PEG-COOH is dissolved in 5mL DMSO.Then the solution (5mL) with above-mentioned activation dropwise joins NH
2In the DMSO solution (5mL) of-PEG-COOH, stirring reaction three days.With molecular cut off be 1000 bag filter to distill water dialysis three days (6 times, 2L/ time), remove by-product and impurity, standby with being stored in-20 ℃ after product C OOH-PEG-FA lyophilization;
FA, NH
2The nucleus magnetic hydrogen spectrum analysis of-PEG-COOH and synthetic COOH-PEG-FA as shown in Figure 2, COOH-PEG-FA successfully is connected on PEG at the spectrum peak explanation FA that 6-9ppm occurs.By the integration calculated by peak area as can be known, connected 0.7 FA on each PEG.
Get the Fe of embodiment 1 preparation
3O
4-PEI aqueous solution 6.24mL(30mg) wash three times with DMSO, then with Fe
3O
4-PEI is scattered in 6mL DMSO again, then will be dissolved in the FI(0.84mg in 2mL DMSO) solution dropwise joins above-mentioned 6mL Fe
3O
4In the DMSO solution of-PEI, stirring reaction is after one day, and magnetic separates removes supernatant, then adds appropriate ultra-pure water ultra-sonic dispersion, then the magnetic separation, so repeats pure water washing 3 times, to remove impurity, then again is scattered in 10mL DMSO, namely 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 then adds 700 μ L ultra-pure waters wherein in the 2mL centrifuge tube, ultrasonic evenly, survey uv absorption (seeing accompanying drawing 3).The 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 the 510nm place, thereby explanation FI successfully modifies Fe
3O
4-PEI nano grain surface.
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 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 the 5mL ultra-pure water again, and gets 0.5mL vacuum lyophilization, gets simultaneously control material Fe
3O
4-PEI-FI-mPEG(Comparative Examples 1) 0.5mL and Fe
3O
4-PEI-FI(embodiment 3) then 0.5mL vacuum lyophilization carry out thermogravimetric analysis (as shown in Figure 4) to three kinds of materials.The TG test result shows, Fe before modifying
3O
4The weight loss of-PEI-FI nano-particle be 8.97%(Fig. 4 a), after pressing PEI surface amino groups and PEG molar ratio 5:1 and modifying, 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 successfully are connected to Fe
3O
4The surface of nano-particle.
Fe to embodiment 4 preparations
3O
4Add 18 μ L triethylamines (density is 0.726～0.729g/mL, and concentration is 99.0%) in-PEI-FI-PEG-FA nano-particle aqueous solution (4mL), and stirred 30 minutes.Then dropwise add 12.2 μ L acetic anhydrides (density is 1.08g/mL, and concentration is 98.5%) (triethylamine, acetic anhydride and Fe in mentioned solution
3O
4The surface amino groups mol ratio of-PEI-FI-PEG-FA=5:5:1), continued stirring reaction 24 hours.Product is with ultrapure hydromagnetic separating, washing 3 times, and is re-dispersed in the 5mL ultra-pure water, namely makes acetylizad Fe
3O
4-PEI-Ac-FI-PEG-FA nano-particle.Get the Fe of the present invention's preparation
3O
4-PEI-FI-PEG-FA(embodiment 4), Fe
3O
4-PEI-Ac-FI-PEG-FA(embodiment 5) and the control material Fe in Comparative Examples 1
3O
4-PEI-FI-mPEG and Fe
3O
4Then each 0.1mL of-PEI-Ac-FI-mPEG is mixed with respectively the aqueous solution of 1.5mL for surveying surface potential and hydrodynamic diameter (as table 1) with ultra-pure water.The potential determination result shows, due to the existence of exhibiting high surface amino, Fe
3O
4-PEI-FI-PEG-FA and Fe
3O
4The surface potential of-PEI-FI-mPEG reached respectively+31.0mV and+31.5mV.Through after acetylization reaction, the Fe that obtains
3O
4-PEI-Ac-FI-PEG-FA and Fe
3O
4-PEI-Ac-FI-mPEG nano grain surface electromotive force dropped to respectively+16.3mV and+16.4mV.Result shows, the successful acetylation of the surface amino groups of nano-particle.But after acetylation, the surface potential of nano-particle does not reach neutral, and this may be can not carry out acetylization reaction because the part on surface 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 than acetylation slightly; and it is almost constant that the hydrodynamic diameter of nano-particle can keep for a long time, thereby the nano-particle that preparation has been described has good colloidal stability.
Get respectively the control material Fe after acetylation
3O
4-PEI-Ac-FI-mPEG(Comparative Examples 1) and the Fe of the present invention preparation
3O
4-PEI-Ac-FI-PEG-FA(embodiment 5) then each 5 μ L of nano-particle aqueous solution are 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 the copper mesh surface, and are used for TEM test (as shown in Figure 5) after drying in air.The 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.Calculate Fe by the diameter of 300 nano-particle of random measurement respectively
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.
Fe with the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA(embodiment 5) nano-particle and control material Fe
3O
4-PEI-Ac-FI-mPEG(Comparative Examples 1) record the concentration of Fe element in solution by the ICP-AES method of testing, then be followed successively by 0.0025,0.005,0.01,0.02 and the aqueous solution 2mL of 0.04mM with ultra-pure water preparation Fe concentration in the 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).The 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
2The increase along with concentration of iron in Fe concentration is the 0.0025-0.04mM scope of relaxation time inverse has good linear relationship.And by calculating Fe as can be known
3O
4-PEI-Ac-FI-PEG-FA(99.64mM
-1s
-1) and Fe
3O
4-PEI-Ac-FI-mPEG(107.30mM
-1s
-1) all have a 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
2The signal attenuation contrast agent.
For the nano-particle that makes preparation can be used for bio-imaging diagnosis in body safely, 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 Examples 1), being scattered in respectively the concentration that is mixed with 1mg/mL in PBS is mother solution, is then the nano granule suspension of 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL with PBS compound concentration successively.Get appropriate people's fresh blood, at first centrifugal (2000rpm/min, 5min) removes supernatant, then erythrocyte washed 5 times with PBS, collects healthy erythrocyte also with 10 times of PBS dilutions.Again with 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 standing 2 hours with erythrocyte after, the centrifugal 1min of 10000rpm takes pictures and surveys the ultraviolet light absorption spectrum of supernatant.As negative control, ultra-pure water is as positive control with PBS for this process.Shown Fe in Fig. 7
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.The hemolytic of the absorbance quantitative assessment nano material by measuring the supernatant.As Fig. 7 a and 7b upper right corner uv-spectrogram demonstration, 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 illustrates that all less than 5% these nano materials of preparation have good blood compatibility, thereby can be used for safely MRI imaging in organism.
The Fe of preparation take the KB cell 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 Examples 1) then each 1mg is 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 be the aseptic Fe of 10,25,50,75 and 100 μ g/mL with aseptic PBS compound concentration at superclean bench
3O
4-PEI-Ac-FI-PEG-FA and Fe
3O
4-PEI-Ac-FI-mPEG nano granule suspension.The 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) was cultivated under 37 ℃ 24 hours altogether.Then, add 20 μ L MTT to cultivating in plate hole, continue to discard culture fluid, and add 150 μ L DMSO after cultivating 4 hours under 37 ℃, vibrate after 15 minutes and to measure light absorption value at 570nm place, 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 compares take buffer PBS as contrast.Compare Fe with matched group
3O
4-PEI-Ac-FI-mPEG and Fe
3O
4-PEI-Ac-FI-PEG-FA survival rate to the KB cell in experimental concentration 0 to 100 μ g/mL scope does not have significant difference, and cell survival rate is all more than 80%.This proves absolutely 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 detection in organism.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.
Detect the Fe of variable concentrations by flow cytometer
3O
4-PEI-Ac-FI-mPEG and Fe
3O
4After-PEI-Ac-FI-PEG-FA processes, the KB cell is estimated the folate-targeted effect to the amount of engulfing (as Figure 10) of material and the average fluorescent strength (as Figure 11) of cell.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 being mixed with respectively Fe concentration with aseptic PBS is 0.125,0.25,0.5,0.75,1.0,1.25 and two kinds of nano granule suspensions of 1.5mM.The 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) was cultivated under 37 ℃ 4 hours altogether, and the cell of processing with PBS as a control group.After cultivating altogether, cell cleans three times with PBS, then with trypsinization and centrifugal, abandons supernatant, with cell suspension in 1mL PBS.Detect cell to the average fluorescent strength of the situation of engulfing of nano-particle and the rear cell of processing by flow cytometer.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 reached 1.0mM, the amount of engulfing almost no longer increased.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 special target ability of nano-particle to the KB cell of expression folacin receptor given in the modification of this explanation folic acid.
Embodiment 11
Further verify the targeting effect of folic acid by Laser Scanning Confocal Microscope, first coverslip is positioned in 12 porocyte culture plates and with 1640 culture medium and soaks 12h, then every hole replenishes the 1.0mL culture medium and inoculates 5 * 10
4Individual KB cell, spend the night rear 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) was cultivated under 37 ℃ 4 hours altogether, then clean three times with PBS, then use successively that 2.5% glutaraldehyde (0.5mL) is fixed 15 minutes, hochest33343(0.8mL) dyeing 30 minutes, at last coverslip is positioned on microscope slide, by the pattern of oily sem observation cell.As shown in figure 12, there is no fluorescence, control material Fe in the cell through the PBS processing
3O
4Demonstrate fainter fluorescence signal in the cell that-PEI-Ac-FI-mPEG processes, and Fe
3O
4Demonstrate very strong fluorescence signal in the cell that-PEI-Ac-FI-PEG-FA processes, this nano-particle that further illustrates modified with folic acid has targeting specific to the KB cell of expressing folacin receptor, thereby in body, the MRI imaging provides reliable foundation for this material success is applied to efficiently.
Before the in-vivo imaging experiment, we have estimated 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 being mixed with respectively Fe concentration with aseptic PBS is 0.1,0.2,0.4 and two kinds of nano granule suspensions of 0.8mM.The 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) was cultivated under 37 ℃ 6 hours altogether, cell cleans 5 times with PBS after cultivating end, trypsinization again, centrifugal, filtration are dispersed at last 1mL PBS(and contain 0.5% agarose) in.Survey the T of each cell sample with nuclear magnetic resonance imaging instrument
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, and the increase along with Fe concentration is described, cell also increases the amount of engulfing of nano-particle.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, obviously finds out from figure, and along with the increase of Fe concentration, the MRI signal value of cell all reduces gradually, and under identical Fe concentration, Fe
3O
4After-PEI-Ac-FI-PEG-FA nano-particle is processed, the MRI signal value of cell will be starkly lower than Fe
3O
4Cell after-PEI-Ac-FI-mPEG nano-particle is processed.These results illustrate that not only 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 with the present invention's preparation
3O
4-PEI-Ac-FI-PEG-FA(embodiment 5) and control material Fe
3O
4-PEI-Ac-FI-mPEG(Comparative Examples 1) aqueous solution is scattered in 2mL PBS again with after PBS centrifuge washing 3 times, and measures the concentration of Fe in each solution with ICP-AES.Then with aseptic PBS preparation equal volume and contain the Fe of identical Fe quality
3O
4-PEI-Ac-FI-mPEG and Fe
3O
4The PBS solution of-PEI-Ac-FI-PEG-FA.The 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 estimated the MRI imaging effect (as shown in figure 14) of tumor locus.Compare with the matched group before injection, in rear 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 rear 24 hours, the mouse tumor position bright-dark degree of two experimental grouies all recovered gradually, illustrate at this moment 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 rear 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 rear 24 hours, the mouse tumor position MRI signal value of two experimental grouies all increased to some extent, and this result with Figure 14 a is consistent.The Fe that these presentation of results should prepare
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
There is the nude mice (embodiment 13) of tumor to be animal pattern detection control material Fe take kind
3O
4The Fe of-PEI-Ac-FI-mPEG and the present invention's preparation
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) soaked 2 days, measure the concentration of ferrum in each histoorgan with ICP-AES.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, all obviously increase before in liver, spleen and lung, the content of ferrum is injected, 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 Examples 1
1.25g Fe (II) salt is dissolved in the 7.75mL ultra-pure water, then adds 6.25mL NH
3H
2O also stirred 10 minutes under air atmosphere, then mixed solution was transferred in autoclave.Get 0.5g PEI and be dissolved in the 5mL ultra-pure water, and this 5mL PEI aqueous solution is also joined in autoclave, after mixing, in 134 ℃ of reactions 3 hours.Reaction naturally cools to room temperature after finishing, and after precipitating magnetic separating, washing purification, again is scattered in the 20mL ultra-pure water, namely gets the nano-particle Fe of PEI parcel
3O
4-PEI, product be stored in 4 ℃ standby.Characterization of The Products is seen Fig. 1;
Get the Fe of 6.24mL above-mentioned steps preparation
3O
4-PEI nano-particle (30mg) is scattered in 6mL DMSO after washing three times with DMSO again.Get 0.84mg FI, and it is dissolved in 2mL DMSO.Then the FI solution (2mL) with the DMSO dissolving dropwise joins above-mentioned 6mL Fe
3O
4In the DMSO solution of-PEI, stirring reaction is after one day, product Fe
3O
4Then-PEI-FI is distributed in 10mL DMSO with ultrapure hydromagnetic separating, washing 3 times, and be stored in 4 ℃ standby.Characterization of The Products is seen 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 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
4Be stored in after-PEI-FI-mPEG is scattered in the 5mL ultra-pure water again 4 ℃ standby.Characterization of The Products is seen Fig. 4 and table 1; Fe to the above-mentioned steps preparation
3O
4Add 18 μ L triethylamines in-PEI-FI-mPEG nano-particle aqueous solution (4mL), and stirred 30 minutes.Then dropwise add 12.2 μ L acetic anhydrides in mentioned solution, continued stirring reaction 24 hours.Product is with ultrapure hydromagnetic separating, washing 3 times, and is re-dispersed in the 5mL ultra-pure water, namely makes 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 seen 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 (10)
1. preparation method based on the targeting MRI contrast agent of the ferroferric oxide nano granules of modified with folic acid comprises:
(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 mixing, in 130～140 ℃ of reactions 2～4 hours; Reaction naturally cools to room temperature after finishing, and after precipitate and separate is washed purification, namely gets the ferroferric oxide nano granules Fe of PEI parcel
3O
4-PEI;
(2) after being dissolved in FA 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 namely gets COOH-PEG-FA;
(3) with the Fe for preparing in step (1)
3O
4-PEI nano-particle is scattered in DMSO after washing with DMSO again, then the FI solution of DMSO dissolving is joined above-mentioned Fe
3O
4In the DMSO solution of-PEI, stirring reaction is after 1～3 day, product Fe
3O
4Then-PEI-FI separating, washing is distributed in DMSO;
(4) after in step (2), the COOH-PEG-FA of preparation and EDC and NHS are dissolved in DMSO, stir-activating 2～4h; Then the COOH-PEG-FA solution of activation is joined the Fe of step (3) preparation
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;
(5) Fe for preparing to step (4)
3O
4Add triethylamine in-PEI-FI-PEG-FA nano-particle aqueous solution, and stirred 20～40 minutes; Then add acetic anhydride in mentioned solution, continued stirring reaction 20～30 hours; Separating, washing also is scattered in ultra-pure water, and get final product.
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, it 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
2The O 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, it 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, it 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, it is characterized in that: the COOH-PEG-FA in described step (4) and the mol ratio of EDC, NHS are 1:4～6:4～6.
9. 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: COOH-PEG-FA and Fe in described step (4)
3O
4The mol ratio of nano grain surface amino is 1:4～6.
10. 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 the PEI of nano grain surface is 4～6:4～6:1.
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