CN102565411B - New method for tracing magnetic nano granules - Google Patents
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Abstract
The invention builds a new internal in-situ marker-free detection method for magnetic nano granules on the basis of the peroxidase activity of the magnetic nano granules. The method comprises the steps as follows: the magnetic nano granules are injected into the body of a rat by using a caudal vein injection method, and according to the peroxidase activity of the magnetic nano granules, catalysis is performed on 3,3'-diaminobenzidine tetrahydrochloride (DAB) to generate tan precipitation at the parts where the magnetic nano granules concentrate, so as to detect the distribution of the magnetic nano granules in different tissues and organs. The new detection method lays an important foundation for building a nano-composite material safety assessment scheme.
Description
Technical field
The present invention relates to a kind of new method of tracing magnetic nano granules, specifically, the present invention has set up a kind of method of new internal in-situ, unmarked tracing magnetic nano granules, can be for the spike of different magnetic nanoparticles, for distribution and metabolism situation in research magnetic nanoparticle body, the biological safety of evaluating magnetic nanoparticle has great importance.
Background technology
Magnetic Nano material refers to have the nano material of magnetic responsiveness, and under the effect of externally-applied magnetic field, these nano materials have strong magnetic response signal.Conventional magnetic Nano material is di-iron trioxide, tri-iron tetroxide, ferrocobalt etc.Magnetic material can be prepared by diverse ways the nanostructured of different scale (1-1000nm) and different-shape, also can utilize organic polymer embedding techniques on the basis of magnetic nanoparticle, to prepare micron-sized magnetic microsphere.
Magnetic Nano material has good magnetic responsiveness, extensive in aspect application prospects such as biomedical and electronics, as (1) is coupled to magnetic Nano material surface by DNA or protein molecule, the magnetic of combining nano material can prepare affinity chromatography for the quick separation of biological sample and efficiently purifying (referring to, J.M.Nam, C.S.Thaxton, C.A.Mirkin, Science 301,1884 (2003); Q.A.Pankhurstl, J.Connolly, S.K.Jones, J.Dobson, J.Phys.D:Appl.Phys.36, R167 (2003); And I.Safarik, M.Safarikova, Biomagn Res Technol.2,7 (2004)); (2) carry out suitable chemical modification by magnetic Nano material surface, the electric charge of material surface is changed, or specific chemical group, can be combined in material surface by drug molecule or gene, utilizes the guiding of externally-applied magnetic field, medicine or foreign gene can be carried to specific position, thereby realize medicine or gene target transportation (referring to, J.Panyam, V.Labhasetwar, Adv Drug DelRev 55,329 (2003); And 5.N.Morishita, et al., Biochem Biophys Res Commun.334,1121 (2005)); (3) magnetic nanoparticle can be for the magnetic resonance imaging of live body or cellular level, set up the live body Real Time Imaging Technology of high spatial resolution, entered clinical for living imaging diagnosis (referring to, M.G.Harishingani, et al., N.Engl.J.Med.348,2491 (2003); A.S.Arbab, et al., Radiology 229,838 (2003); With I.J.de Vries, et al., NatBiotechnol.23,1407 (2005)); (4) magnetic Nano material can be for the treatment of tumour, by drug targeting and magnetic target, magnetic nanoparticle is concentrated on to tumor locus, under the effect of externally-applied magnetic field, magnetic nanoparticle can produce high heat, kills the cell of tumor locus, realize oncotherapy object (referring to, A.Ito, et al., Cancer Lett.212,167 (2004); And F.Sonvico, et al., Bioconjug Chem.16,1181 (2005)).Because magnetic nanoparticle is in the widespread use of biomedical sector, therefore, investigate magnetic nanoparticle distribution and metabolism situation in vivo, set up safety of nano materials evaluation of programme and have great importance.In addition, a lot of nerve degenerative diseases, such as Parkinson's (PD) and senile dementia etc., have all observed deposition of iron (D.Berg, et al.Ultrasound Med Biol, 25,901 (1999)) in patient's brain.Therefore spike iron in the deposition conditions of brain tissue the pathogenesis for research nerve degenerative diseases, and the result for the treatment of of evaluating its medicine has important effect.
Summary of the invention
The invention provides a kind of new purposes of magnetic nanoparticle, utilize its activity with peroxidase, the reaction of catalyzing hydrogen peroxide and sedimentation type hydrogen donor, causes hydrogen donor to generate precipitation, by observation precipitate can spike its distribution and metabolism situation in vivo.
Therefore, utilize this principle, the invention provides the new method of utilizing magnetic nanoparticle tracing magnetic nano granules in mammalian body with peroxidase activity, the method comprises:
1) the described magnetic nanoparticle with peroxidase activity is injected in described mammalian body;
2) injection described magnetic nanoparticle after through appropriate time, get described mammiferous organs and tissues, carry out histotomy; With
3) utilize the peroxidase activity catalyzing hydrogen peroxide of magnetic nanoparticle and sedimentation type hydrogen donor to react, cause hydrogen donor to generate precipitation, precipitate distribution and the content of determining magnetic nanoparticle in tissue by observation.
In a preferred embodiment of the invention, described magnetic nanoparticle comprises Fe
3o
4magnetic nanoparticle or γ-Fe
2o
3magnetic nanoparticle.In another preferred embodiment of the present invention, described magnetic nanoparticle is glucan-modified magnetic nanoparticle or coated with silica magnetic nanoparticle.In addition, those skilled in the art should understand that, the present invention's magnetic nanoparticle used is not limited to above-mentioned, usually, the magnetic nanoparticle with peroxidase activity is all suitable for the present invention, for example, coated magnetic nanoparticle of the magnetic nanoparticle of coated with polyethylene glycol, polyacrylic acid etc.
In a preferred embodiment of the invention, described mammal includes, but not limited to mouse, rat, rabbit, dog etc.After biological safety of nano-material checking, described magnetic nanoparticle can further be applied to people.Therefore,, in preferred embodiment of the present invention, described mammal is behaved.
In some embodiments of the present invention, above-mentioned steps 1) in method that magnetic nanoparticle is injected in mammalian body include, but not limited to intravenous injection, lumbar injection, hypodermic injection, intramuscular injection, suction etc.
In a preferred embodiment of the invention, those skilled in the art can determining step 2 through the simple experiment of limited number of time) in after the described magnetic nanoparticle of injection when getting animal viscera and organize elapsed time, in particularly preferred embodiments, the described time is 0.25 hour-72 hours, be preferably 5 hours, it depends on the kind of magnetic nanoparticle used, the factor such as factor, the metabolic rate of magnetic nanoparticle in mammal internal organs of tested mammal itself; Wherein step 2) described in animal tissue's organs and tissues be liver, spleen, lung, kidney, lymph node or thymus gland etc.
In some embodiments of the present invention, above-mentioned steps 3) described in sedimentation type hydrogen donor comprise 3,3 '-diaminobenzidine, four hydrochlorides (DAB), tetramethyl benzidine (TMB) or AEC (AEC).In a preferred embodiment of the invention, above-mentioned steps 3) described in sedimentation type hydrogen donor be 3,3 '-diaminobenzidine, four hydrochlorides (DAB).
In a preferred embodiment of the invention, the new method that described utilization has tracing magnetic nano granules in the magnetic nanoparticle body of peroxidase activity is also included in after animal tissue's section, by histotomy at 0.3%H
2o
2methyl alcohol in hatch 30 minutes, to suppress the activity of endogenous peroxidase, removed the background of tissue itself, improve sensitivity.
According to result of study of the present invention, prove that described magnetic nanoparticle mainly engulfed (referring to Fig. 3) by the mononuclear macrophage in animal tissue.
In a preferred embodiment of the invention, described histotomy can obtain by conventional paraffin section technology, frozen section technique.
In a specific embodiments of the present invention, the invention provides and utilize the magnetic nanoparticle new method of tracing magnetic nano granules in vivo with peroxidase activity, it comprises:
1) the described magnetic nanoparticle with peroxidase activity is injected in mammalian body;
2) injection described magnetic nanoparticle after through appropriate time, get described animal viscera tissue, carry out histotomy; With
3) add 3,3 '-diaminobenzidine, four hydrochlorides (DAB) nitrite ion, the peroxidase activity catalyzing hydrogen peroxide and 3 of magnetic nanoparticle, 3 '-diaminobenzidine, four hydrochlorides (DAB) reaction, cause DAB to form brown color precipitation, determine distribution and the content of magnetic nanoparticle in tissue by observing brown color precipitation.
In sum, the invention provides following:
1. utilization has a new method for magnetic nanoparticle tracing magnetic nano granules in mammalian body of peroxidase activity, and it comprises:
1) the described magnetic nanoparticle with peroxidase activity is injected in described mammalian body;
2) injection described magnetic nanoparticle after through appropriate time, get described mammiferous organs and tissues, carry out histotomy; With
3) utilize the peroxidase activity catalyzing hydrogen peroxide of magnetic nanoparticle and sedimentation type hydrogen donor to react, cause hydrogen donor to generate precipitation, precipitate distribution and the content of determining magnetic nanoparticle in tissue by observation.
2. according to the method described in the 1st, wherein said magnetic nanoparticle comprises Fe
3o
4magnetic nanoparticle and γ-Fe
2o
3magnetic nanoparticle.
3. according to the method described in the 1st, it is also included in after animal tissue's section, by histotomy at 0.3%H
2o
2methyl alcohol in hatch 30 minutes.
4. according to the method described in the 2nd, it is characterized in that: described magnetic nanoparticle is glucan-modified magnetic nanoparticle or coated with silica magnetic nanoparticle.
5. according to the method described in the 1st, wherein step 2) described in time be 0.25 hour-72 hours.
6. according to the method described in the 5th, wherein step 2) described in time be 5 hours.
7. according to the method described in the 5th, wherein step 3) described in sedimentation type hydrogen donor comprise 3,3 '-diaminobenzidine, four hydrochlorides, tetramethyl benzidine or AEC.
8. according to the method described in the 1st, wherein said animal viscera is organized as liver, spleen, lung, kidney, lymph node or thymus gland.
9. according to the method described in the 1st, wherein said histotomy can pass through paraffin section technology, frozen section technique obtains.
10. according to the method described in the 1st, wherein said magnetic nanoparticle is mainly engulfed by the mononuclear macrophage in animal tissue.
The method distributing in traditional tracing magnetic nano granules body has some limitations: (1) Radiolabelling method is to utilize
59fe is as raw material, and synthesizing magnetic nano particle, although the method has kept original characteristic of material and highly sensitive, exists radioactive contamination, detection of complex, the shortcoming such as time-consuming; (2) fluorescence labeling method, be by Electrostatic Absorption or covalent coupling, fluorescence molecule to be combined with magnetic nanoparticle, but fluorescence molecule is unstable in vivo, and the metabolism situation of material can not be well indicated in easily cancellation; In addition, the fluorescence molecule of being combined with magnetic nanoparticle by Electrostatic Absorption easily comes off from material in vivo, before not arriving tissue, may discharge from material, what detect may be free fluorescence molecule, well tracer material is in the distribution of each tissue, internal organs, and by the method modifying magnetic nano particle of covalent coupling, may cause the change of material self character, such as causing the crosslinked of magnetic nanoparticle; (3) Prussian blue (PB) decoration method, under acid condition, ferric iron and potassium ferrocyanide effect, the ferric ferrocyanide that forms Prussia's blueness precipitates, although the method does not need material to carry out preliminary making, has the shortcoming that detection sensitivity is low.(4) ICP-AES (ICP-OES), the method is by measuring each internal organs iron content, the distribution in vivo of analyzing magnetic nano particle, but because body contains endogenous ferric ion, ICP cannot distinguish the magnetic nanoparticle of endogenous ferric ion and injection, can cause interference to analyzing.
Compared with traditional tracing method, utilize its distribution and metabolism situation in vivo of peroxidase activity spike of magnetic nanoparticle, there is more advantage: after (1) histotomy, only need add DAB chromogenic substrate, can be observed magnetic nanoparticle distribution in vivo, simple, convenient; (2), without material is carried out to preliminary making, well kept the original characteristic of material; (3) because magnetic nanoparticle has the catalytic activity higher than horseradish peroxidase, can well form brown color precipitation by catalysis DAB, detection sensitivity is high; (4) will organize and use in advance hydrogen peroxide treatment, can suppress the activity of endogenous peroxidase, remove the background of tissue itself.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1. paraffin section is analyzed the distribution of glucan-modified magnetic nanoparticle at Mouse Liver, spleen, lung, kidney, lymph node and thymus gland: "+" represents to add DAB colour developing, and "-" represents not add DAB colour developing;
Fig. 2. frozen section is analyzed the distribution of glucan-modified magnetic nanoparticle at Mouse Liver, spleen, lung, kidney, lymph node and thymus gland;
Fig. 3. glucan-modified magnetic nanoparticle is by each internal organs macrophage phagocytic;
Fig. 4. glucan-modified magnetic nanoparticle and the metabolism of coated with silica magnetic nanoparticle in liver;
Fig. 5. glucan-modified magnetic nanoparticle and the metabolism of coated with silica magnetic nanoparticle in spleen;
Fig. 6. glucan-modified magnetic nanoparticle and the metabolism of coated with silica magnetic nanoparticle in lungs;
Fig. 7. glucan-modified magnetic nanoparticle catalysis DAB dyeing is dyeed remolding sensitivity with Prussian blue (PB);
Fig. 8. coated with silica magnetic nanoparticle catalysis DAB dyeing and prussian blue staining remolding sensitivity are.
Embodiment
Carry out by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
The preparation of magnetic nanoparticle
1. glucan-modified Fe
3o
4magnetic nanoparticle
Preparation 0.1mol/L FeCl
36H
2the ethylene glycol solution of O (purchased from Beijing chemical reagents corporation), adds anhydrous sodium acetate, and making its final concentration is 0.05mol/L, then to add molecular weight be 10000 glucosan (purchased from Sigma), and making its final concentration is 0.05mg/ml.Said mixture is placed in airtight 50ml reactor (model: HZ50, purchased from Zhengzhou He Zhong Instrument Ltd.), reacts 25 hours at 100 ℃, obtaining glucan-modified magnetic nanoparticle (is glucan-modified Fe
3o
4magnetic nanoparticle).
2. coated with silica Fe
3o
4magnetic nanoparticle
2.1Fe
3o
4the preparation of magnetic nanoparticle
By FeCl
24H
2o (purchased from Beijing chemical reagents corporation) and FeCl
36H
2o (purchased from Beijing chemical reagents corporation) solution adds in there-necked flask by the mol ratio of 2: 1, N
2under protection, be warming up to 55 ℃; the NaOH solution that stirring adds 10 times of ferrous iron molal quantitys (; the concentration of NaOH solution is 10 times of ferrous iron volumetric molar concentration) and be warming up to 65 ℃ reaction half an hour; continue to be warming up to 90 ℃; react half an hour; stop reaction, under stirring, be cooled to room temperature, product is Fe
3o
4magnetic Nano material, vacuum freeze drying.
Get 3g Fe
3o
4powder, joins logical N
2in trisodium citrate (purchased from Beijing chemical reagents corporation) solution of the 200ml 0.5mol/L of deoxygenation, under ultrasonic, make Fe
3o
4fully disperse, then at 60 ℃, logical N
2stir 12 hours, then after system is cooled to room temperature, utilize magnet by product separation out, wash and be dried with 95% ethanol, deionized water successively, obtain Fe
3o
4powder.
The Fe of 2.2 coated with silica
3o
4magnetic nanoparticle
Get the Fe that 0.2g obtains in 2.1
3o
480ml ethanol and 16ml distilled water diluting for powder, ultrasonic 20 minutes, add 2ml 25% ammoniacal liquor (purchased from Beijing chemical reagents corporation), drip 1ml ethyl orthosilicate (TEOS) (purchased from Sigma), 40 ℃ ultrasonic 5 hours, with magnet separation, with 95% ethanol, washed with de-ionized water several.
The magnetic nanoparticle of preparation is as stated above used for to following embodiment.Above-mentioned glucan-modified magnetic nanoparticle and coated with silica magnetic nanoparticle also can obtain by being purchased approach, for example, glucan-modified magnetic nanoparticle can be buied from Physical Chemistry Technology Inst., Chinese Academy of Sciences, and coated with silica magnetic nanoparticle can be buied from Shanghai Communications University.
Embodiment 1, utilize the activity of the peroxidase of magnetic nanoparticle, its distribution in tissue of spike
By glucan-modified magnetic nanoparticle (purchased from Physical Chemistry Technology Inst., Chinese Academy of Sciences, or by the preparation of previously described method) dosage of pressing 40mg/kg body weight through tail vein injection BALB/c mouse (purchased from Beijing Experimental Animal Center, weigh 20 ± 2 grams, age in 4-6 week), after injecting glucan-modified magnetic nanoparticle, after 5 hours, put to death mouse, get respectively liver, spleen, lung, kidney, lymph node and thymus gland.Utilize the peroxidase activity of magnetic nanoparticle, catalysis DAB forms brown color precipitation where at magnetic nanoparticle, detects the distribution of magnetic nanoparticle at each tissue, internal organs.
Concrete grammar is as follows: the fixing 12-24 hour of 4% paraformaldehyde for mice organs tissue (purchased from Beijing chemical reagents corporation) getting, with 70% ethanolic solution flushing, dewatered step by step 35 minutes by 70-80-90-95-95-100% ethanol again, immerse 1: 1 ethanol/dimethylbenzene (purchased from Beijing chemical reagents corporation) solution 5 minutes, take out and immerse again dimethylbenzene 20 minutes transparent 2 times; Drop into rapidly in the paraffin of hot melt (molten some 56-58 ℃) (purchased from Leica) 4 hours, take out waxdip tissue and move into containing in the carton of dewaxing, down, embedding after paraffin surface is condensed, solidifies in immersion cold water tangent plane.With Leica paraffin slicing machine (model: RM2125, purchased from Leica), paraffin-embedded tissue is cut into 4 μ m and cuts into slices, 42 ℃ of exhibition sheets on microslide, natural drying rear 50 ℃ of roasting sheets 6 hours.Enter 100% dimethylbenzene (purchased from Beijing reagent company) dewaxing three times, each 5 minutes, reenter aquation in 100-95-80-70-50% ethanol and distilled water.Then containing 0.3%H
2o
2in the methyl alcohol of (purchased from Beijing chemical reagents corporation), hatch the interference of removing endogenous peroxydase for 30 minutes, PBS washing three times, (DAB chromogenic reagent box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge finally to use the DAB nitrite ion of fresh configuration, color operation is undertaken by kit instructions) colour developing, develops the color after complete and redyes nucleus with haematoxylin (purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge).
As shown in Figure 1, by magnetic nanoparticle catalysis DAB colour developing, can clearly observe magnetic nanoparticle and mainly be distributed in liver, spleen and lung, and not distribute in kidney, lymph node and thymus gland.The result shows, utilize the activity of the peroxidase of magnetic nanoparticle, can indicate its distribution situation in tissue.
Because magnetic nanoparticle has than the higher stability of horseradish peroxidase (HRP), there is more wide range of application.Except paraffin section, frozen section is also one of important means of analyzing changes in microstructure and damage.In order to confirm that ultralow temperature does not affect the enzymatic activity of magnetic nanoparticle, we are by BALB/c mouse tissue through OCT (optimum cutting temperature compound) embedding, and-80 ℃ freezing, section.Section is fixed 5 minutes the acetone (purchased from Beijing chemical reagents corporation) of precooling is inner, and then contains 0.3%H
2o
2methyl alcohol in hatch and within 30 minutes, remove the interference of endogenous peroxydase, PBS washing three times, developing the color with the DAB nitrite ion of fresh configuration, (DAB chromogenic reagent box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, color operation is undertaken by kit instructions), develop the color after complete and redye nucleus with haematoxylin.Result as shown in Figure 2, process and still keeping good catalytic activity through ultralow temperature, can well its distribution at each tissue of spike by magnetic nanoparticle.
Embodiment 2, magnetic nanoparticle are mainly by each internal organs macrophage phagocytic
Magnetic nanoparticle enters in body by tail vein, mainly engulfed by mononuclear phagocyte system, in order to confirm that glucan-modified magnetic nanoparticle is really by each internal organs macrophage phagocytic, we carry out SABC with anti-macrophage specific antibody anti-F4/80 (purchased from Abcam company, F4/80 can specific recognition macrophage).Concrete grammar is as follows: BALB/c mouse tissue, through OCT embedding, carries out frozen section.Section is fixed 5 minutes in the acetone of precooling, and then contains 0.3%H
2o
2methyl alcohol in hatch and within 30 minutes, remove the interference of endogenous peroxydase.PBS washing three times, with 5% sheep blood serum (purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) sealing mounting, then adds anti-F4/80 antibody, 4 ℃ of overnight incubation.Add afterwards the goat Chinese People's Anti-Japanese Military and Political College mouse two anti-(Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of HRP mark, hatch histotomy, finally with the DAB nitrite ion colour developing (DAB chromogenic reagent box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, and color operation is undertaken by kit instructions) of fresh configuration.Develop the color after complete and redye nucleus with haematoxylin.As shown in Figure 3, glucan-modified magnetic nanoparticle is mainly distributed in the macrophage interior (DAB is coloured to the cell of brown color) of antibody specific recognition, illustrates that magnetic nanoparticle is mainly by monokaryon macrophage phagocytic.
The metabolism in vivo of embodiment 3, magnetic nanoparticle
We utilize the peroxidase activity of the magnetic nanoparticle (purchased from Shanghai Communications University) of glucan-modified magnetic nanoparticle and coated with silica, have analyzed their situation of metabolism in vivo.By the magnetic nanoparticle of glucan-modified magnetic nanoparticle and coated with silica respectively with the dosage of 40mg/kg body weight through tail vein injection mouse, in injection latter 0.25 hour respectively, after 5 hours and 72 hours, put to death mouse, get liver, spleen and lungs, analyzing magnetic nano particle is in the metabolism situation of each internal organs.
After the glucan-modified magnetic nanoparticle of mouse tail vein injection 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in liver, finding after 5 hours that material has in liver is dispersed in distribution in a large number, and tail vein injection after 72 hours glucan-modified magnetic nanoparticle reduce (on Fig. 4) in the distribution of liver, illustrate that glucan-modified magnetic nanoparticle cleared up by liver after 72 hours.
After mouse tail vein injection coated with silica magnetic nanoparticle 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in liver, finding after 5 hours that material has in liver is dispersed in distribution in a large number, and it is different from glucan-modified magnetic nanoparticle, after tail vein injection 72 hours, coated with silica magnetic material still has a large amount of distributions (under Fig. 4) in liver, illustrates that coated with silica magnetic nanoparticle can not be cleared up by liver after 72 hours.
After the glucan-modified magnetic nanoparticle of mouse tail vein injection 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in spleen, find that it is marginarium (marginal zone) that material after 5 hours is assembled the intersection that is distributed in white pulp and red pulp in spleen, and tail vein injection after 72 hours the distribution of glucan-modified magnetic nanoparticle in spleen reduce (on Fig. 5), illustrate that glucan-modified magnetic nanoparticle cleared up by spleen after 72 hours.
After mouse tail vein injection coated with silica magnetic nanoparticle 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in spleen, find that it is marginarium (marginal zone) that material after 5 hours is assembled the intersection that is distributed in white pulp and red pulp in spleen, and it is different from glucan-modified magnetic nanoparticle, after tail vein injection 72 hours, coated with silica magnetic nanoparticle still has a large amount of distributions (under Fig. 5) in spleen, illustrate that coated with silica magnetic nanoparticle can not be cleared up by spleen after 72 hours.
After the glucan-modified magnetic nanoparticle of mouse tail vein injection 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in lungs, find that after 5 hours, material becomes spot distribution in lungs, and tail vein injection after 72 hours the distribution of glucan-modified magnetic nanoparticle in lungs reduce (on Fig. 6), illustrate that glucan-modified magnetic nanoparticle cleared up by lungs after 72 hours.
After mouse tail vein injection coated with silica magnetic nanoparticle 0.25 hour, after 5 hours and 72 hours, put to death mouse, analyze the distribution of magnetic nanoparticle in lungs, find that after 5 hours, coated with silica magnetic nanoparticle becomes spot distribution in lungs, and it is different from glucan-modified magnetic nanoparticle, after tail vein injection 72 hours, coated with silica magnetic material still has a large amount of distributions (under Fig. 6) in lungs, illustrates that coated with silica magnetic material can not be cleared up by lungs after 72 hours.
Embodiment 4, magnetic nanoparticle catalysis DAB dyeing and prussian blue staining remolding sensitivity are
With the prussian blue staining comparison that traditional detection magnetic nanoparticle distributes in vivo, utilize the sensitivity of its distribution of catalytic activity spike of magnetic nanoparticle higher.Concrete grammar is as follows: by glucan-modified magnetic nanoparticle with the dosage of 40mg/kg body weight through tail vein injection BALB/c mouse, after latter 0.25 hour and 5 hours, put to death mouse in injection respectively, get liver, through OCT embedding, carry out frozen section.Get two serial section and fix 5 minutes in the acetone of precooling, and then contain 0.3%H
2o
2methyl alcohol in hatch and within 30 minutes, remove the interference of endogenous peroxydase.PBS washing three times, wherein (DAB chromogenic reagent box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge in a freshly prepared DAB nitrite ion colour developing of use, color operation is undertaken by kit instructions), the Prussian blue colour developing of another use (purchased from Yuan Ye bio tech ltd, Shanghai).Result as shown in Figure 7, is compared with Prussian blue colour developing, and DAB colour developing has higher sensitivity.
Utilize the remolding sensitivity of the catalytic activity of magnetic nanoparticle and the magnetic nanoparticle of two kinds of method spike coated with silica of prussian blue staining, concrete grammar is as follows: by the magnetic nanoparticle of coated with silica with the dosage of 40mg/kg body weight respectively through tail vein injection BALB/c mouse, after latter 0.25 hour and 5 hours, put to death mouse in injection respectively, get liver, through OCT embedding, carry out frozen section.Get two serial section and fix 5 minutes in the acetone of precooling, and then contain 0.3%H
2o
2methyl alcohol in hatch and within 30 minutes, remove the interference of endogenous peroxydase.PBS washing three times, wherein (DAB chromogenic reagent box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge for the colour developing of the DAB nitrite ion of a fresh configuration of use, color operation is undertaken by kit instructions), the Prussian blue colour developing of another use (purchased from Yuan Ye bio tech ltd, Shanghai).Result as shown in Figure 8, is compared with Prussian blue colour developing, and DAB colour developing has higher sensitivity.
Claims (9)
1. utilization has a new method for magnetic nanoparticle tracing magnetic nano granules in mammalian body of peroxidase activity, and it comprises:
1) the described magnetic nanoparticle with peroxidase activity is injected in described mammalian body;
2) injection described magnetic nanoparticle after through 0.25 hour-72 hours, get described mammiferous organs and tissues, carry out histotomy; With
3) utilize the peroxidase activity catalyzing hydrogen peroxide of magnetic nanoparticle and sedimentation type hydrogen donor to react, cause hydrogen donor to generate precipitation, precipitate distribution and the content of determining magnetic nanoparticle in tissue by observation.
2. method according to claim 1, wherein said magnetic nanoparticle comprises Fe
3o
4magnetic nanoparticle and γ-Fe
2o
3magnetic nanoparticle.
3. method according to claim 1, it is also included in after animal tissue section, by histotomy at 0.3%H
2o
2methyl alcohol in hatch 30 minutes.
4. method according to claim 2, is characterized in that: described magnetic nanoparticle is glucan-modified magnetic nanoparticle or coated with silica magnetic nanoparticle.
5. method according to claim 1, wherein step 2) in institute elapsed time be 5 hours.
6. method according to claim 1, wherein step 3) described in sedimentation type hydrogen donor be 3,3 '-diaminobenzidine, four hydrochlorides, tetramethyl benzidine or AEC.
7. method according to claim 1, wherein said animal viscera is organized as liver, spleen, lung, kidney, lymph node or thymus gland.
8. method according to claim 1, wherein said histotomy obtains by paraffin section technology or frozen section technique.
9. method according to claim 1, wherein said magnetic nanoparticle is engulfed by the mononuclear macrophage in animal tissue.
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