CN101829341B - Hydroxyapatite nanoparticle radionuclide marked product and preparation method thereof - Google Patents
Hydroxyapatite nanoparticle radionuclide marked product and preparation method thereof Download PDFInfo
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- CN101829341B CN101829341B CN2009100475508A CN200910047550A CN101829341B CN 101829341 B CN101829341 B CN 101829341B CN 2009100475508 A CN2009100475508 A CN 2009100475508A CN 200910047550 A CN200910047550 A CN 200910047550A CN 101829341 B CN101829341 B CN 101829341B
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- hydroxyapatite nanoparticle
- hydroxyapatite
- radionuclide
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- marked product
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Abstract
The invention discloses a hydroxyapatite nanoparticle radionuclide marked product and a preparation method thereof. The hydroxyapatite nanoparticle radionuclide marked product may be prepared by a method comprising: a, performing the amination surface modification of hydroxyapatite nanoparticles by using silane coupling agent; b, preparing a 125I-BH maker; and c, connecting the 125I-BH maker and the modified hydroxyapatite nanoparticles. In the invention, the 125I radionuclide with high chemical activity is used, and the 125I radionuclide has a proper radioactive half-life of 60 days. The 125I marked product obtained by the marking method is stable and can be used in researches on the in-vivo development and distribution of the hydroxyapatite nanoparticles.
Description
Technical field
The present invention relates to a kind of hydroxyapatite nanoparticle radionuclide marked product and labelling technique thereof, specifically, is a kind of radionuclide that is marked with
125Hydroxyapatite nanoparticle of I and preparation method thereof.
Background technology
(Hydroxyapatite is kind of weakly alkaline synthos HA) to hydroxyapatite, and molecular formula is Ca
10(PO4)
6(OH)
2, be the main inorganic composition of human body and sclerous tissueses such as animal skeleton, tooth.Natural hydroxyapatite in the biological hard tissue is kind of a nanostructured that is needle-like six side's prismatics, and length is 200-400nm, and thickness is 15-30nm.The hydroxyapatite of synthetic has good biocompatibility and chemical stability, uses the damaged repairing and treating of bone widely.Hydroxyapatite nanoparticle not only has excellent biological compatibility and biological activity, also has nano material unique small-size effect and skin effect.After the hydroxyapatite nanoparticle of will receiving is implanted in osseous tissue; Can form firm chemical bond with osseous tissue combines; And having bone conductibility and osteoinductive, be widely used in the damaged repairing and treating of various bones, is one of emphasis and focus of the research of present organizational project embedded material.
Because the special performance that hydroxyapatite nanoparticle has nano material shows good characteristic and new functions such as size is little, specific surface area is big, surface activity is high, high adsorption capacity, can act on particular tissues and cell selectively.Have research with hydroxyapatite nanoparticle as carrier, be applied to the exploitation of slow releasing pharmaceutical; The diagnosis of research and utilization hydroxyapatite nanoparticle is also arranged and treat various tumors.
Along with the development of nanotechnology, nano material will be extensive day by day in the application of biomedical sector, also will be paid attention to by people gradually about the biological safety problem of nano material.The biological safety of research nano material just is necessary that spike is carried out in the application in body to nano material, with research nano material metabolic biodistribution and the influence of evaluation nano material to body function in body.Nano material is carried out the important method that radioisotope labeling is a biodistribution in the research nano material body.In addition, through radioisotope labeling, can be that the radiation treatment medicine of carrier provides technical foundation with the hydroxyapatite nanoparticle for exploitation to hydroxyapatite nanoparticle.
At present; The common method of hydroxyapatite nanoparticle being carried out radioisotope labeling mainly is that the phosphonate part that adopts absorption method will be marked with radionuclide respectively is adsorbed in hydroxyapatite surface or adopts ion exchange that the ion in radionuclide and the hydroxyapatite is exchanged; Realization is carried out labelling to hydroxyapatite nanoparticle, and the radioactive nucleus that is used for the hydroxyapatite nanoparticle labelling have
153Sm,
90Y,
99mTc etc.But video picture and biodistribution Application Research aspect there are many deficiencies in existing hydroxyapatite nanoparticle labelling technique in vivo to it, main conclude have following some:
(1) the body internal stability is poor: through the absorption part hydroxyapatite nanoparticle is carried out radioisotope labeling in the existing labeling method, it is relatively poor that this adsorptivity is combined in the body internal stability, and label comes off easily in vivo.In addition, some parts have higher specific binding capacity to some tissue in vivo, like phosphonate osseous tissue are had stronger adsorptivity, and this might influence hydroxyapatite nanoparticle distribution situation in vivo.
(2) there is not the suitable half-life: because hydroxyapatite is the inorganic substances of kind of stable chemical performance; The degradation in vivo cycle is long; So study hydroxyapatite nanoparticle biodistribution in vivo, have the suitable half-life with regard to the radionuclide that requires labelling.Can carry out in the radionuclide of ion exchange with hydroxyapatite at present, most half-life is too short, as
153The half-life of Sm is 46.3h,
90The half-life of Y is 64.1h,
99mThe half-life of Tc is 6.02h, and the half-life of these radionuclides all is not suitable for studying the interior biodistribution of body of hydroxyapatite nanoparticle.
(3) inconvenience that detects of radioactivity: be biodistribution situation in the body of research hydroxyapatite nanoparticle, need carry out the detection by quantitative of radioactivity to important organ in the body.At present, the radioactivity of radionuclide commonly used adopts the liquid scintillation technology for detection more.In carrying out body during biodistribution research; Need before the radioactivity in the organ-tissue is detected, organ-tissue to be digested; Make its liquefy; If liquid has color also will decolour, simultaneously to carry out quench correction during radiometry, this will greatly increase the difficulty and the cost of organ tissue radiation property activity determination.
125I is kind of a radionuclide commonly used, has the suitable half-life, is widely used in various biomarkers and biological detection.Hydroxyapatite nanoparticle is as a kind of inorganic nanoparticles, and the surface lacks can directly be used for carrying out radionuclide
125The functional group of I labelling is so be difficult to incite somebody to action
125The direct labelling of I is to hydroxyapatite nanoparticle.
Silane coupler is kind of the low molecular organic silicide with special construction, also is kind of an important inorganic nanoparticles surface modifying material.The general formula of silane coupler is RSiX
3, R represents groups such as amino, sulfydryl, vinyl, epoxy radicals, cyanic acid and methyl-prop ethylene acyloxy, and the group that X representative can hydrolysis is like halogen, alkoxyl, acyloxy etc.Silane coupler is mainly realized through forming chemical bond the surface modification of inorganic nano material.
There is hydroxyl in the hydroxyapatite nanoparticle surface, and it can form chemical bond with groups such as alkoxyl in the silane coupler, acyloxy and combines, and groups such as the amino of another section of silane coupler, epoxy radicals can labelling on
125I, thus realize hydroxyapatite nanoparticle is carried out radioisotope labeling.Because silane coupler and hydroxyapatite nanoparticle are bonded through the form of chemical bond,
125I is marked on the groups such as amino in the silane coupler, epoxy radicals.So, design this method hydroxyl lime stone nanometer carried out
125The I labelling can obtain more stable marked product, and the marked product that is obtained can be applied to the interior video picture of body and the biodistribution research of hydroxyapatite nanoparticle.
Summary of the invention
The object of the present invention is to provide the hydroxyapatite nanoparticle of video picture and biodistribution research in a kind of body that can be applied to hydroxyapatite nanoparticle
125The I marked product.
Another object of the present invention is to provide the method for preparing of said marked product.
For realizing above-mentioned purpose, the present invention discloses following technical scheme: at first, the silane coupler dilute solution is handled hydroxyapatite nanoparticle, makes its silane amination.Wherein silane coupler is that (molecular formula is aminopropyltriethoxywerene werene: (CH
3CH
2O)
3SiCH
2CH
2CH
2NH
2); The dilute solution proportioning of its aminopropyltriethoxywerene werene is: coupling agent: ethanol=1: 5V/V; The pH value of its dilute solution is 8.Secondly, preparation
125The I-BH label.Also be most important once more,
125The I-BH label is connected with amidized hydroxyapatite nanoparticle.The following processing of wherein amidized hydroxyapatite nanoparticle process: in the borate buffer of 0.05mol/L pH 8.4, and through 40KHz sonic oscillation 15min; Said being connected under the following condition carried out: stirring reaction 30min in the ice bath adds the glycine solution (0.05mol/L pH 8.4 borate buffer preparation) of 0.2mol/L again.
The present invention has following beneficial effect:
(1) selects the high radionuclide of chemical activity for use
125I, it has the suitable half-life, is 60 days, can be used for video picture and biodistribution research in the hydroxyapatite nanoparticle body, makes radioactive pollution be easy to control and processing.Radionuclide
125I can launch gamma-rays, and penetration power is strong, can be quantitatively and detect biodistribution and video picture in the body of hydroxyapatite nanoparticle intuitively.Utilize
125I launches gamma-ray characteristic, can use gamma counter quickly and easily hydroxyapatite nanoparticle to be carried out video picture and biodistribution research in the body not carrying out tissue digestion, not using under the situation of liquid scintillation technology.
(2) adopt silane coupler that hydroxyapatite nanoparticle is carried out surface modification, groups such as silane coupled middle alkoxyl, acyloxy carry out chemical bond with the hydroxyl on hydroxyapatite nanoparticle surface and combine,
125I is then on the group such as the amino of labelling in the silane coupler, epoxy radicals, and is resulting through this labeling method
125The I marked product is stable, can be used for the interior video picture of body and the chorology research of hydroxyapatite nanoparticle.
(3) silane coupler is to combine through the hydroxyl with the hydroxyapatite nanoparticle surface, has the advantages of higher stability of ratio; Silane coupler belongs to low molecular organic silicide, through it hydroxyapatite nanoparticle is carried out
125The I labelling is very little to the size influence of hydroxyapatite nanoparticle.Detect through X-ray diffraction (XRD) and transmission electron microscope (TEM), resulting marked product is that hydroxyapatite is formed phase, and its physical dimension does not change before and after labelling basically, still belongs to the category of nano-particle.
Description of drawings
Fig. 1 is the qualification result figure of silica gel thin-layer paper chromatography (ITLC/SG method) to the hydroxyapatite nanoparticle marked product;
Fig. 2 is the figure as a result of the following 32 days stability change of the room temperature environment of hydroxyapatite nanoparticle marked product;
Fig. 3 is the XRD testing result of hydroxyapatite nanoparticle marked product;
Fig. 4 a is the TEM detection figure before the hydroxyapatite nanoparticle labelling;
Fig. 4 b is the TEM detection figure of hydroxyapatite nanoparticle marked product.
The specific embodiment
Below in conjunction with concrete experiment and interpretation of result and corresponding Figure of description, to further explain of the present invention.
(1) the silane amination of hydroxyapatite nanoparticle
Hydroxyapatite nanoparticle is placed ethanol solution, ultrasonic 30min under the 40KHz frequency, it is subsequent use to clean the back repeatedly with dehydrated alcohol.(molecular formula is: (CH in the 90mL alcohol solvent, to add the 10mL aminopropyltriethoxywerene werene
3CH
2O)
3SiCH
2CH
2CH
2NH
2) dilute solution (coupling agent: ethanol=1: 5, V/V), it is 8 that dropping sodium solution makes the solution pH value, continue to stir hydrolysis 1h under the room temperature.The 200mg hydroxyapatite nanoparticle is joined in the hydrolysis coupling agent solution, 50～60 ℃ stirred in water bath reaction 6h, after reaction finished, room temperature was placed 2 days, filtered, and used washing with alcohol, and vacuum drying obtains amino modified hydroxyapatite nanoparticle.
(2)
125The preparation of I-BH label
With 5mg para hydroxybenzene propanoic acid butanimide fat reagent (Bolton-hunter reagent; BH reagent) be dissolved in the 500 μ l redistillation benzene, get 5 μ g BH reagent benzene solution in little reaction tube, after nitrogen dries up; Add the analytically pure N of 2 μ l successively, N dimethyl formamide (DMF) and 1.5 μ l Na
125I solution (60MBq); Add 5 μ l (20 μ g) toluene-sodium-sulfonchloramide (Ch-T) solution then; Add the sodium metabisulfite of 5 μ l (60 μ g) and the liquor kalii iodide (the phosphate buffer fresh of 0.05mol/L pH7.4) of 50 μ l (200 μ g), mixing cessation reaction behind the reaction 1min under the room temperature at once.In above-mentioned reactant liquor, add 5 μ l N, N dimethyl formamide (DMF), 250 μ l redistillation benzene extraction
125I-BH reagent, careful again sucking-off benzole soln are in little reaction tube, and nitrogen dries up, and promptly get
125The I-BH label.
(3)
125-BH label is connected with amidized hydroxyapatite nanoparticle
With being equipped with in the above-mentioned steps (2)
125The little reaction tube of I-BH label places ice bath; Add the amidized hydroxyapatite nanoparticle solution of silicon (content is 100 μ g, at the borate buffer of 0.05mol/L pH 8.4, and passes through 40KHz sonic oscillation 15min) that makes in the 50 μ l above-mentioned steps (1); Stirring reaction 30min in the ice bath; Add the glycine solution (with the borate buffer preparation of 0.05mol/L pH 8.4) of 150 μ l 0.2mol/L again, continue stirring reaction 15min under the room temperature, excessive to remove
125The I-BH label.
With taking out reactant liquor in the above-mentioned little reaction tube in the centrifuge tube of 5ml, add 2ml 50% ethanol water, behind the sonic oscillation 20min, 4 ℃, the centrifugal 10min of 3000rpm inhales and removes supernatant, and precipitate is marked product.Divide the secondary centrifuging washing precipitate with 4ml 50% ethanol water, behind the reuse 2ml ethanol centrifuge washing precipitate, add 1ml ethanol, sonic oscillation 20min, lyophilizing is subsequent use.
(4) evaluation of marked product
Adopt silica gel thin-layer paper chromatography (ITLC/SG method) to identify.With quick silica gel thin-layer chromatography paper (ITLC/SG) is carrier, launches with the PBS buffer of the 0.01mol/L pH7.4 of 2.5%BSA (W/W), then every chromatographic paper is cut into the equidistant little paper slip of 1cm, puts into the radiometry test tube and measures.
125The I-BH label,
125The I-glycine with
125The I-Rf value is all in 0.80～1.00 scope, and the hydroxyapatite nanoparticle Rf value of labelling is 0.00, and is motionless at paper chromatography strip initial point, identical with the expansion behavior in the paper chromatography strip of unlabelled hydroxyapatite nanoparticle.Identify prepared marked product radio chemistry purity >=95% (seeing accompanying drawing 1) through above method.
(5) Detection of Stability of marked product
It is in 7.4 the PBS solution that marked product is scattered in 0.01mol/LpH; Room temperature preservation; Regularly adopt silica gel thin-layer paper chromatography (ITLC/SG method) to detect the character of radioactive substance in the solution; Analyse the not percentage rate of dissociated marked product of ratio calculation that radiocounting and whole ply of paper on the band origin position analyse the band radiocounting according to ply of paper, thus the stability of evaluation mark product.The result shows that room temperature was placed after 32 days, have good stability (the seeing accompanying drawing 2) of marked product.
(6) sign of marked product
Adopt X-ray diffraction (XRD) to reach the change of size situation mutually with the composition that transmission electron microscope (TEM) detects resulting hydroxyapatite nanoparticle marked product; Its result asks for an interview shown in Fig. 3, Fig. 4 a and Fig. 4 b; Resulting marked product is that hydroxyapatite is formed phase; Its physical dimension does not change before and after labelling basically, still belongs to the category of nano-particle.
Claims (1)
1. the method for preparing of a hydroxyapatite nanoparticle radionuclide marked product, this method may further comprise the steps:
A. the silane coupler dilute solution is handled hydroxyapatite nanoparticle, makes its silane amination, and said silane coupler is an aminopropyltriethoxywerene werene, and its molecular formula is: (CH
3CH
2O)
3SiCH
2CH
2CH
2NH
2, the dilute solution proportioning of described aminopropyltriethoxywerene werene is a coupling agent: ethanol=1: 5V/V, the pH value of its dilute solution are 8;
B.
125The preparation of I-BH label;
C.
125The I-BH label is connected with amidized hydroxyapatite nanoparticle; Said amidized hydroxyapatite nanoparticle is through following processing; In the borate buffer of 0.05mol/L pH 8.4, and through 40KHz sonic oscillation 15min, said being connected under the following condition carried out: stirring reaction 30min in the ice bath; The glycine solution that adds 0.2mol/L again, said glycine solution is with the borate buffer preparation of 0.05mol/L pH 8.4.
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| CN102357261B (en) * | 2011-06-28 | 2014-01-08 | 东华大学 | Surface modification method of nanometer hydroxyapatite mediated by APTS |
| CN105288743B (en) * | 2014-07-23 | 2018-08-21 | 上海交通大学医学院附属第九人民医院 | Mesoporous bioglass nano particle of radioisotope labeling and the preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008008917A2 (en) * | 2006-07-12 | 2008-01-17 | Mayo Foundation For Medical Education And Research | Hydroxyapatite particles |
| CN101301489A (en) * | 2008-06-23 | 2008-11-12 | 中国科学院长春应用化学研究所 | Method for preparing nano hydroxylapatite hybridized material with surface grafting polypeptide |
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2009
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008008917A2 (en) * | 2006-07-12 | 2008-01-17 | Mayo Foundation For Medical Education And Research | Hydroxyapatite particles |
| CN101301489A (en) * | 2008-06-23 | 2008-11-12 | 中国科学院长春应用化学研究所 | Method for preparing nano hydroxylapatite hybridized material with surface grafting polypeptide |
Non-Patent Citations (2)
| Title |
|---|
| 刘长征等.《放射性碘标记物的制备》.《实验核医学与核药学》.人民卫生出版社,1999,第78－82页. * |
| 张中书等.《125I标记消脂素的制备研究》.《标记免疫分析与临床》.2006,第13卷(第4期),第236-237页. * |
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