CN104225630A - Multi-mode self-assembly nanoprobe suitable for MRI (magnetic resonance imaging)/PA (optical activation) and other imaging - Google Patents
Multi-mode self-assembly nanoprobe suitable for MRI (magnetic resonance imaging)/PA (optical activation) and other imaging Download PDFInfo
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Abstract
The invention discloses a multi-mode self-assembly nanoprobe. The multi-mode self-assembly nanoprobe takes apoferritin or ferritin as a carrier, and loaded with metal ions used for magnetic resonance imaging, melanin used for PA imaging, and function substances used for other imaging. The self-assembly nanoprobe disclosed by the invention realizes PET (polyethylene glycol terephthalate)/MRI/PA three-mode fusion development, has high sensitivity and specificity, realizes a good imaging effect, and can be widely applied clinically due to the simple process, good repeatability, good biocompatibility, safety and non-toxicity, fast elimination speed in a body.
Description
Technical field
The invention belongs to bioprobe field, be specifically related to a kind of the multi-mode self-assembled nanometer probe and the application thereof that are applicable to MRI/PA and other imagings.
Background technology
Molecular imaging (Molecular Imaging) is one of following medical science Disciplinary Frontiers most with development potentiality, it comprises plurality of medical image technology, as: positron emission computerized tomography (PET), X ray computer fault imaging (CT), NMR (Nuclear Magnetic Resonance)-imaging (MRI), optical imagery (comprising bioluminescence, fluorescence, near-infrared spectroscopy), ultra sonic imaging (US) etc.Different image technologies differs from one another, also each defectiveness, therefore multi-pattern Fusion imaging becomes current research and development focus.Along with the high speed development of multi-pattern Fusion image documentation equipment, PET/CT, PET/MR, photoacoustic imaging (PA are in succession there is, the new technique such as PAUS), by combination among the strong ones, mutual supplement with each other's advantages, be expected to meticulous anatomical structure and molecular function information to organically combine, the morning better realizing disease prevents, early examine, early control, clinician is helped to suit the remedy to the case, realize individualized treatment, thus alleviate misery and the financial burden of patient, the health of the mankind is significant.For PET/MRI, PET has the characteristic of high sensitivity, functional image, and MRI has high-resolution to soft tissue, can accurately provide Anatomical orientation information, the two associating, can realize carrying out physiology, the noinvasive of pathological process, real-time, reproducible on a molecular scale, there is unique value in the medicals diagnosis on disease such as nervous system, tumor, cardiovascular.Photoacoustic imaging (PA) is a kind of image technology developed rapidly in recent years, it utilizes has the tissue of light absorption can produce local sound source, i.e. PA effect, thus know different physiological parameters according to the situation of light absorption, the concentration of such as intravital hemoglobin, melanin, water, ion etc. and oxygen saturation.
The penetration depth of PA can reach 5-7cm, and spatial resolution can reach submillimeter level, more easily realizes clinical conversion.Therefore optoacoustic video picture is also the imaging pattern of a kind of potential structure, function and molecule.
If PET/MRI and PA imaging technique is combined, then the molecular function information of the deep layer of the meticulous anatomical structure of PET/MRI imaging and PA imaging can be effectively integrated, obtain with having living space and temporal information, three-dimensional, can be quantitative high quality graphic at synchronization, make imaging have high sensitivity to minimal disease and high specific, comprehensive information can be provided.But in current medical imaging field, the correlation technique of PET/MRI/PA tri-schema merging imaging rarely has report.
For realizing multi-pattern Fusion imaging, the successful exploitation of multi-mode probe is one of important prerequisite.The object of exploitation multi-mode probe is: 1) utilize a kind of contrast medium can realize the video picture of different mode; 2) realize the concordance of target area signal, do not exist and cause the different problem of signal distributions because using different contrast medium.At present, in multi-mode probe, there are reports as the probe of carrier for nano material.For CNT (CNT), first, CNT self, with fluorescence, can be used for near-infrared spectroscopy, and CNT also can be used for optoacoustic video picture in the strong absorption of near infrared region simultaneously; Secondly, in CNT preparation process, need metal (as Co, Fe etc.) catalysis, still have residual ferrum after purification, can be used as magnetic resonance contrast agent.In addition, CNT also can carry out multiple modification, to realize improving its quantum efficiency, strengthen photoacoustic signal, strengthen nuclear magnetic signal, to produce the effects such as PET signal.Although the application of nano material in multi-mode probe has prospect, but due to the focus that its process controllability, stability, biocompatibility, degradability, toxicity, body absorption and removing performance etc. are disputes always, therefore, still fail so far to be applied to clinical.
In the nano-carrier reported at present, have using ferritin or apoferritin as the probe of nano-carrier.Such as, in Chinese patent literature CN102462847A, utilize apoferritin to make the carrier of tumor target direction contrast agent, by contrast agent parcel wherein, targeting is enriched to tumor tissues position, and then carries out NMR (Nuclear Magnetic Resonance)-imaging.Ferritin is natural ferrum storage protein, and apoferritin (Apoferritin) is ferritin (ferritin) nonferrous existence form.Ferritin and apoferritin can degradation in vivo, safety non-toxic, non-immunogenicity.Simultaneously, because a lot of tumor cell surface can great expression ferritin specific receptor, can produce endocytosis to ferritin and apoferritin by the combination mediation of transferrin receptor-ferritin, therefore, ferritin and apoferritin also have good targeting to tumor cell.Above-mentioned contrast agent is using apoferritin as carrier, although avoid the biocompatibility that nano material has usually as the probe of carrier, degradability, toxicity, body absorption and removing performance issue, and there is good targeting, but, only NMR (Nuclear Magnetic Resonance)-imaging can be realized, if want to utilize ferritin or apoferritin to be carrier, carry out the imaging of PET/MRI/PA tri-schema merging, then face following problem: 1) conventional at present PET positron radionuclide is C, F, the aniones such as I, when being marked on ferritin or apoferritin, complex steps, difficulty is high, productive rate is low, large to operator's irradiation dose, 2) ferritin or apoferritin itself do not have optical property, adopt and have the melanin of light absorption can realize PA imaging from ultraviolet near infrared region.Melanin (melanin) is a kind of biochrome, extensively be present in animal, plant and protista body, tyrosine or 3,4-dihydroxy phenylpropyl alcohol ammonia formed through a succession of chemical reaction, there is the multiple biological activitys such as antioxidation, antitumor, venom, antiviral, hepatoprotective, radioprotective, commercialization at present.But, when melanin being loaded on ferritin or apoferritin carrier, not only because melanin particle diameter is larger, water insoluble, acid and conventional organic solvent (ethanol, hexane, acetone, stupid, chloroform etc.), only be dissolved in alkali, meet oxidant easily to decolour, be difficult to load on carrier, and, what is more important, when the melanin for PA imaging exists jointly with the common metal ion for MRI imaging, because melanic metal chelation abilities is strong, run into the common metal ion of MRI imaging (as Fe
3+) time easily precipitates, even if modify melanin, Polyethylene Glycol is such as adopted to modify, also melanin can only to be improved in solution to a certain extent to the load capacity of metal ion, MRI imaging and the concentration needed for PA imaging cannot be reached, the signal intensity of MRI and PA is low, sensitivity is also extremely low.
Summary of the invention
First technical problem to be solved by this invention be of the prior artly not yet there is being suitable for the natural material nano-probe of PET/MRI/PA tri-schema merging video picture, the metal ion of the melanin that cannot realize PA imaging and MRI imaging stablely in aqueous coexists, melanin cannot reach sensitive imaging to the load capacity of metal ion, the nonmetal nucleic of positron of conventional PET video picture cannot the problem of labelling, and then provide the video picture of a kind of PET/MRI/PA of realization tri-schema merging, high sensitivity, high specific, the nano-probe of comprehensive video picture can be realized.
Second technical problem that the present invention solves is multi-pattern Fusion Imaging probe of the prior art at process controllability, stability, biocompatibility, degradability, toxicity, body absorption and removes in performance etc. and there is technological deficiency, and then provides that a kind of technique is simple, reproducible, removes the nano-probe of fast multi-pattern Fusion video picture in good biocompatibility, safety non-toxic, body.
The multi-mode self-assembled nanometer probe being applicable to MRI/PA and other imagings of the present invention, described nano-probe with apoferritin or ferritin for carrier, be loaded with: by adding when carrier protein solution acid dissociation and being embedded in the metal ion for NMR (Nuclear Magnetic Resonance)-imaging in described carrier protein and the melanin for PA imaging when described carrier protein solution restructuring, and for the functional mass of other imaging.
Described other is imaged as PET imaging, and the functional mass of described PET imaging is positron metal nucleic.
Further, described positron metal nucleic is
55co,
60cu,
61cu,
62cu,
64cu,
66ga,
68ga,
82rb,
86y,
86one in Zr.
Further, described melanin is the water soluble particle of particle diameter 3-6nm.Preferably, the preparation method of described melanic water soluble particle comprises the following steps: in described melanin, add alkali make it be hydrolyzed, then the alkali that acid neutralization adds, and the melanin solution obtained is placed in ultrasound wave and is dissolved to clarification, and then ultrafiltration centrifugal filtration, to obtain final product.
Further, the metal ion of described NMR (Nuclear Magnetic Resonance)-imaging is Fe
2+, Fe
3+, Gd
3+, Zn
2+, Mn
2+, Mn
3+, Mn
4+, Mn
7+, Pt
2+in one.
The method of the above-mentioned nano-probe of preparation of the present invention, comprises the following steps:
(1) get apoferritin or liquor ferri albuminati, adjust ph, to 1-3, obtains the protein solution dissociated;
(2) to step 1) in obtain described in add melanin, metal ion respectively, mixing in the protein solution that dissociates, then adjust ph is to 7.5-10.0, obtains the protein solution of recombinating;
(3) to step 2) in add positron metal nucleic, mix homogeneously in the protein solution of described restructuring that obtains, react at 18-37 DEG C and be marked on albumen to positron metal nucleic, to obtain final product.
Further, step 2) in add melanin, metal ion and described in albumen in the protein solution that dissociates, the mol ratio of three is (0.5-2): (500-1500): 1.Preferably, step 2) in add melanin, metal ion and described in albumen in the protein solution that dissociates, the mol ratio of three is 1:1000:1.
Further, step 2) in add metal ion by adding metal chlorination salt.Those skilled in the art according to circumstances can select the anion be applicable to, and include but not limited to SO
4 2-, NO
3 -, ClO
4 -or the one in Cl-.
Further, step 2) also comprise the step of the protein solution purified concentration of described restructuring.Preferably, described purified concentration be dialysis, column chromatography, ultrafiltration centrifugal in one or more.
Further, step 3) also comprise by unmarked on albumen, freely positron metal nucleic removing step.Preferably, step 3) in adopt chromatography desalination to remove positron metal nucleic freely.
Further, the radioactivity of described positron metal core cellulose solution is for being less than 100mCi.
The nano-probe prepared in said method.
The application of nano-probe of the present invention in multi-pattern Fusion imaging, the application particularly in the imaging of PET/MRI/PA tri-schema merging.
The multi-modal imaging agent being functional component with nano-probe of the present invention.
Technique scheme of the present invention, has the following advantages compared to existing technology:
(1) nano-probe of multi-pattern Fusion video picture of the present invention, with apoferritin or ferritin for carrier, be loaded with the positron radionuclide for PET imaging, for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging, and for the melanin of PA imaging, achieve the imaging of PET/MRI/PA tri-schema merging, in conjunction with the advantage of three kinds of imaging techniques, the molecular function information of the meticulous anatomical structure of PET/MRI imaging and the deep layer of PA imaging is effectively integrated, obtain with having living space with temporal information at synchronization, three-dimensional, high quality graphic that can be quantitative, imaging is made to have high sensitivity to minimal disease and high specific, comprehensive information can be provided.
First, the present invention to adopt in organism natural materials apoferritin or ferritin as carrier, biocompatibility is better, and effectively can solve the problem of safety, on the one hand, apoferritin and ferritin are biodegradable by metabolism, remove fast in body, on the other hand, even if probe is detained in organism, the toxic and side effects also not easily caused; Meanwhile, have good tumor cell targeting, the probe obtained has good specificity.
Second, apoferritin/ferritin be encompassed by 24 homologies or allos subunit hollow spherical, 8 hydrophilic ionic passages and 6 Hydrophobic Ionic passages are had in sphere cavity, enrichment can be used for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging in a large number, direct embedding metal ion, does not need to add chelating agen; Simultaneously, positron radionuclide for PET imaging of the present invention, for positron metal nucleic, also inner chamber is entered by the hydrophilic channel in apoferritin/ferritin nanocages, and stable to deposit, therefore, avoiding the mutual interference produced because using multiple chelating agen, the problem affecting protein conformation on the one hand, the compatibility of nano-probe and the organism obtained can not be affected; Other method, it also avoid the complex operation of the common nonmetal positron radionuclides such as use C, F, I, difficulty is high, productive rate is low, and the problem large to operator's irradiation dose.
3rd, the hollow chondritic of apoferritin/ferritin, make outside it, inner chamber, inside and outside intersection all can be used for each motif and loads, the binding site provided is many, load the positron metal nucleic being used for PET imaging simultaneously, for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging, and for the melanin of PA imaging, still good stability can be realized, and three kinds of components loaded and apoferritin/ferritin can realize good ratio and control, in particular for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging, for the melanin of PA imaging, apoferritin/ferritin, accurate quantification can be realized between three, avoid traditional biological coupling method list binding site reverse cyclic loadings and the poor repeatability caused, defect that cannot be quantitative.
4th, because melanic metal chelation abilities is strong, runs into some metal ion in the solution and namely precipitate (as Fe
3+), this causes great obstruction for realizing PA imaging (melanin) simultaneously with MRI imaging (metal ion), even if melanic water soluble nanometer particles is carried out PEG (Polyethylene Glycol) to modify, melanin in solution and the load capacity of metal ion be lower (ultimate load is only 1:100) still, signal for PA imaging and MRI imaging is too low, and sensitivity is extremely low.And the present invention adopts apoferritin/ferritin parcel melanin and metal ion, therefore, overcome the problems referred to above, the nano-probe obtained has good stability.
(2) nano-probe of the present invention, described melanin is the water soluble particle of particle diameter 4nm, better can realize the loading with carrier, not only substantially increase the meltage of melanin in water, and be more conducive to realizing the metal ion for NMR (Nuclear Magnetic Resonance)-imaging, the melanin for PA imaging, apoferritin/ferritin, the accurate quantification between three.
(3) nano-probe of the present invention, first in acid condition apoferritin/ferritin is dissociated, add for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging and the melanin for PA imaging wherein, again pH value is adjusted to 7.5-10, i.e. restoration and reconstruction, form the apoferritin/ferritin of the above-mentioned metal ion of uniform parcel and melanic restructuring.Utilize the above-mentioned pH dependency of the apoferritin/ferritin of chondritic, the nano-probe obtained not only has homogeneous, stable advantage, its operational approach is also very simple, reproducible, for the video picture of PET/MRI/PA tri-schema merging apply so that targeted molecular imaging art has opened fine new page.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation.
Fig. 1 is the synthetic route chart of the self assembly PET/MRI/PA tri-pattern nano-probe of embodiment 1;
Fig. 2 is apoferritin, melanin, FeCl
3the abosrption spectrogram of solution, AMF;
Fig. 3 A is the DLS figure of apoferritin (APF);
Fig. 3 B is the DLS figure of melanin (MNP);
Fig. 3 C is the DLS figure of the AMF of self assembly PET/MRI/PA tri-pattern nano-probe;
Fig. 4 is the TEM figure of apoferritin, melanin, AMF;
Wherein, upper left is apoferritin (dyeing); Upper right is melanin (being unstained); Lower-left is AMF (being unstained); Bottom right is AMF (dyeing);
Fig. 5 is the PET image of self assembly PET/MRI/PA tri-pattern nano-probe;
Fig. 6 is the MR image of self assembly PET/MRI/PA tri-pattern nano-probe;
Fig. 7 is the PA image of self assembly PET/MRI/PA tri-pattern nano-probe.
Detailed description of the invention
In the following each embodiment of the present invention, described apoferritin, ferritin, melanin, PD-10 post can select the conventional product adopted in prior art to carry out the preparation of self assembly PET/MRI/PA tri-pattern nano-probe, below the product only providing wherein a kind of model is set forth effect of the present invention, effect between the product of commercially available each model zero difference.Wherein:
Apoferritin, purchased from American Sigma-Aldrich (Sigma-Aldrich);
Melanin, purchased from American Sigma-Aldrich (Sigma-Aldrich);
PD-10, purchased from Life Sciences of General Electric (GE Healthcare).
Embodiment 1 self assembly PET/MRI/PA tri-pattern nano-probe
The self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is: be carrier with apoferritin, is loaded with for PET imaging
64cu, Fe for NMR (Nuclear Magnetic Resonance)-imaging
3+, and for the melanin of PA imaging.
As shown in Figure 1, its concrete preparation manipulation is as follows for the synthetic route of the self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment:
First, apoferritin and melanin are done following process: get apoferritin 90nmol, add the water dilution of 3.6ml, obtain apoferritin solution, for subsequent use; Concentration 20mg melanin being dissolved in 10ml is in the NaOH aqueous solution of 0.1M (i.e. mol/L), adds the aqueous hydrochloric acid solution of 0.1M, adjusted to ph to 7, then be placed in ultrasonic dissolving after dissolving, obtains the melanin aqueous solution clarified.Above-mentioned melanin aqueous solution is further purified in ultrafiltration centrifugal filter, molecular cut off is 30kDa, by washed with de-ionized water 3 times, to remove the salt and other impurity that produce, finally by its lyophilization, obtain melanin (being called for short MNP, lower same) the solid 15mg that particle diameter is the water soluble particle of 4nm, for subsequent use.
(1) get above-mentioned apoferritin solution, by HCl adjust ph to 2, obtain the protein solution dissociated;
(2) to step 1) in obtain described in add the melanin above-mentioned for subsequent use of 90nmol, the FeCl of 90 μm of ol in the protein solution that dissociates respectively
3, mixing, places 15min, then uses NaOH adjust ph to 8, obtains the protein solution of recombinating; By the protein solution that obtains by PD-10 post, with PBS buffer (NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO
42mmol/L, pH=7.2) be eluent, carry out Chromatographic purification, then add in ultrafiltration centrifuge by the protein solution after purifying, molecular cut off is 30kDa, carries out ultrafiltration, obtains the protein nano granule of recombinating, is enclosed with melanin and Fe in the albumen of this restructuring
3+, hereinafter referred to as AMF, lower same; Described AMF particles with water is dissolved, the AMF solution namely after purification;
(3) to step 2) in add radioactivity in the described AMF solution that obtains be 1mCi's
64cuCl
2, mix homogeneously, reacts 30min at 25 DEG C, by the protein solution that obtains by PD-10 post, with PBS buffer for eluent, carries out Chromatographic purification, then adds in ultrafiltration centrifuge by the protein solution after purifying, and molecular cut off is 30kDa, concentrates, and to obtain final product.
Following experiment is adopted to verify the structure of above-mentioned PET/MRI/PA tri-pattern nano-probe below:
(1) step 2) the middle Fe loaded for NMR (Nuclear Magnetic Resonance)-imaging
3+and the mensuration of melanic heap(ed) capacity for PA imaging:
Using bovine serum albumin as standard protein, Bio-Rad albuminometry is adopted to determine the content of apoferritin in AMF.Icp ms (ICP-MS) is adopted to measure Fe
3+content.Adopt ultraviolet-visible spectrophotometer (Cary 60 of Anjelen Sci. & Tech. Inc) to measure the absorption spectrum of PET/MRI/PA tri-pattern nano-probe granule, record result, its result as shown in Figure 2.Wherein, curve 1 be the absorption spectrum of the apoferritin of concentration 25 μ g/mL, the curve 2 melanic absorption spectrum that is concentration 4 μ g/mL, curve 3 FeCl that is concentration 10 μ g/mL
3absorption spectrum, the curve 4 of solution are the absorption spectrum of AMF.With the abs680 of melanin under variable concentrations (i.e. light absorption value), Criterion curve, the abs680 of deduction apoferritin and Fe, calculates melanic content in AMF.Can calculate thus in nano-probe AMF of the present invention, apoferritin: melanin: Fe
3+mol ratio be 1:1:800.
(2) sign of PET/MRI/PA tri-pattern nano-probe granule:
Get above-mentioned steps 2) in the AMF solution that obtains add deionized water to be diluted to containing APF be 250ug/mL, apoferritin solution (APF, 250ug/mL), melanin solution (MNP, 4ug/mL), be placed in measuring cell, adopt Malvern Zetasizer Nano ZS90 nanometer particle size potentiometric analyzer to carry out dynamic light scattering (DLS) to measure, its result as shown in Figure 3.To be added drop-wise on copper sheet after apoferritin, melanin, AMF dilution, dry, stain be 1% acetic acid uranium.Adopt perspective electron microscope shooting to obtain its TME image, its result as shown in Figure 4.
From the above results, melanin and Fe add the particle diameter and structure that do not change apoferritin.It can be said that bright, PET/MRI/PA tri-pattern nano-probe of the present invention has wrapped up the melanin particle that particle diameter is 4nm first, and melanin and ferrum are wrapped in apoferritin inner chamber.
Embodiment 2 self assembly PET/MRI/PA tri-pattern nano-probe
The self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is: be carrier with ferritin, is loaded with for PET imaging
68ga, Gd for NMR (Nuclear Magnetic Resonance)-imaging
3+, and for the melanin of PA imaging.
The preparation manipulation of the self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is as follows:
First ferritin and melanin are carried out pretreatment according to the method in embodiment 1 and consumption, for subsequent use.
(1) get above-mentioned liquor ferri albuminati, by HCl adjust ph to 1, obtain the protein solution dissociated;
(2) to step 1) in obtain described in add the melanin above-mentioned for subsequent use of 90nmol, the GdCl3 of 90 μm of ol in the protein solution that dissociates respectively, mixing, places 15min, then uses NaOH adjust ph to 10.0, obtains the protein solution of recombinating; By the protein solution that obtains by PD-10 post, with PBS buffer (NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO
42mmol/L, pH=7.2) be eluent, carry out Chromatographic purification, obtain the protein solution of the restructuring after purification;
(3) to step 2) in restructuring after the purification that obtains protein solution in add radioactivity be 10mCi's
68gaCl
3, mix homogeneously, reacts 30min at 30 DEG C, by the protein solution that obtains by PD-10 post, with PBS buffer for eluent, carries out Chromatographic purification, then adds in ultrafiltration centrifuge by the protein solution after purifying, and molecular cut off is 30kDa, carries out ultrafiltration, to obtain final product.
According to the method in embodiment 1 to step 2) the middle Gd for NMR (Nuclear Magnetic Resonance)-imaging loaded
3+and measure for the melanic heap(ed) capacity of PA imaging, obtain ferritin: melanin: Gd
3+mol ratio be 1:1:1000.Melanin, Gd
3+heap(ed) capacity on ferritin reaches the amount that can realize ideal MRI and PA imaging effect.
Embodiment 3 self assembly PET/MRI/PA tri-pattern nano-probe
The self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is: be carrier with apoferritin, is loaded with for PET imaging
82rb, Fe for NMR (Nuclear Magnetic Resonance)-imaging
2+, and for the melanin of PA imaging.
The preparation manipulation of the self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is as follows:
First apoferritin and melanin are carried out pretreatment according to the method in embodiment 1 and consumption, for subsequent use.Wherein, melanic particle diameter is 4nm.
(1) get above-mentioned liquor ferri albuminati, by HCl adjust ph to 3, obtain the protein solution dissociated;
(2) to step 1) in obtain described in add the melanin above-mentioned for subsequent use of 90nmol, the FeCl2 of 135 μm of ol in the protein solution that dissociates respectively, mixing, places 15min, then uses NaOH adjust ph to 7.5, obtains the protein solution of recombinating;
(3) to step 2) in add radioactivity in the protein solution of restructuring that obtains be 30mCi's
82rb Cl, mix homogeneously, reacts 30min at 30 DEG C, by the protein solution that obtains by PD-10 post, with PBS buffer for eluent, carry out Chromatographic purification, then the protein solution after purifying is added in ultrafiltration centrifuge, molecular cut off is 30kDa, carries out ultrafiltration, to obtain final product.
According to the method in embodiment 1 to step 2) the middle Fe for NMR (Nuclear Magnetic Resonance)-imaging loaded
2+and measure for the melanic heap(ed) capacity of PA imaging, obtain apoferritin: melanin: Fe
2+mol ratio be 1:1:1500.Melanin, Fe
2+heap(ed) capacity on apoferritin reaches the amount that can realize ideal MRI and PA imaging effect.
Embodiment 4 self assembly PET/MRI/PA tri-pattern nano-probe
The self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is: be carrier with apoferritin, is loaded with for PET imaging/radionuclide therapy
89zr
2+, for the Gd of NMR (Nuclear Magnetic Resonance)-imaging
3+, and for the melanin of PA imaging.
The preparation manipulation of the self assembly PET/MRI/PA tri-pattern nano-probe of the present embodiment is as follows:
First apoferritin and melanin are carried out pretreatment according to the method in embodiment 1 and consumption, for subsequent use.Wherein, melanic particle diameter is 3nm.
(1) get above-mentioned liquor ferri albuminati, by HCl adjust ph to 2, obtain the protein solution dissociated;
(2) to step 1) in obtain described in add the melanin above-mentioned for subsequent use of 90nmol, the GdCl3 of 45 μm of ol in the protein solution that dissociates respectively, mixing, places 15min, then uses NaOH adjust ph to 8, obtains the protein solution of recombinating;
(3) to step 2) in add radioactivity in the protein solution of restructuring that obtains be 90mCi's
89zrCl
2, mix homogeneously, reacts 30min at 30 DEG C, to obtain final product.
According to the method in embodiment 1 to step 2) the middle Gd for NMR (Nuclear Magnetic Resonance)-imaging loaded
3+and measure for the melanic heap(ed) capacity of PA imaging, obtain apoferritin: melanin: Gd
3+mol ratio be 1:1:500.Melanin, Gd
3+heap(ed) capacity on apoferritin reaches the amount that can realize ideal MRI and PA imaging effect.
The preparation of embodiment 5PET/MRI/PA tri-mode imaging agent
The self assembly PET/MRI/PA tri-pattern nano-probe prepared in Example 1, be dissolved in normal saline, the concentration making described nano-probe is 1mCi/mL, to obtain final product.
As the substitute mode of this embodiment, self assembly PET/MRI/PA tri-pattern nano-probe that can be described also can be replaced the nano-probe prepared in embodiment 2-4, the concentration of nano-probe can also be the arbitrary value in 20 μ Ci/mL to 2Ci/mL, and normal saline also can be replaced glucose for injection solution.
The video picture of embodiment 6PET/MRI/PA tri-pattern nano-probe
Get mouse bare subcutaneous injection 2-5 × 106 colon cancer HT29 cell (TfR high expressed) or hepatoma Hep G 2 cells (the low expression of TfR), after inoculation, in 2-5 week, obtain gross tumor volume and reach 150-500mm
3tumor model.
PET imaging: the PET/MRI/PA tri-pattern nano-probe prepared in Example 1, is formulated as
64the radioactivity of Cu is the solution of 100 μ Ci, getting 0.2ml enters in nude mouse through tail vein injection respectively, MicroPET (SIEMENS INVEON) video picture is carried out, scanning different time dot image, IAW software analysis tumor uptake (ID%/g) after injection preparation.Its result as shown in Figure 5.
NMR (Nuclear Magnetic Resonance)-imaging: the PET/MRI/PA tri-pattern nano-probe prepared in Example 1, is formulated as the solution being calculated as 2mg/mL by Fe concentration.Getting 0.2ml enters in nude mouse through tail vein injection respectively, 1T MR (Bruker) T1 video picture is carried out after injection preparation, scanning different time dot image, sweep parameter is set to T1-flash MRI sequence (repetition time (TR)/echo time (TE)=300/6.1ms; Experiment number (NEX)=16; Matrix: 256 × 256; Slice thickness: 1.25cm; FOV:3.0cm; 15), its result is as shown in Figure 6.
Photoacoustic imaging: the PET/MRI/PA tri-pattern nano-probe prepared in Example 1, is formulated as the solution being calculated as 2mg/mL by MNP concentration.Getting 0.2ml enters in nude mouse through tail vein injection respectively, carries out PA (VevoLAZR after injection preparation; VisualSonics) video picture, optimum configurations is: optoacoustic gain 45dB, dynamic range 20dB, mid frequency 21MHz.Scanning different time dot image, its result as shown in Figure 7.
From PET image results, HT29 tumor uptake is in injection rear 1,2,4,18,26 and 48h reach 4.82 ± 0.59 respectively, 6.14 ± 0.77,7.34 ± 0.93,7.26 ± 1.32,6.74 ± 0.51,6.54 ± 0.79%ID/g, the hepatocarcinoma HepG2 tumor uptake of the low expression of TfR is then lower, in injection rear 1,2,4,18,26 and 48h reach 2.95 ± 0.40 respectively, 3.55 ± 0.58, this explanation of 4.33 ± 1.16and 4.61 ± 1.58,4.77 ± 0.47,3.81 ± 0.54%ID/g, APF can the tumor of targeting TfR high expressed, absorbs high and is detained long.The tumor uptake of the low expression of TfR is because enhancing infiltration retention effect (EPR) effect of nanoparticle, then absorb low and remove fast.From MR image results, after injection AMF, tumor uptake is high-visible, and MR signal strengthens, the uptake values ratio (T/T of 1h, 2h, 4h and 24h and injection pre-neoplastic after the injection of HT29 colon cancer Mus
0) be 1.58,1.73,2.02,1.30, HepG2 liver cancer murine be then 1.21,1.44,1.03,0.99, consistent with PET image results, again demonstrate targeting and the EPR effect of apoferritin.After injecting AMF as seen from PA image results, HT29 tumor photoacoustic signal is strengthened gradually, and after injection, 4h reaches peak.This result sufficient proof AMF is effective PET/MR/PA tri-mode probe.
In sum, self assembly PET/MRI/PA tri-pattern nano-probe of the present invention achieves the video picture of PET/MRI/PA tri-schema merging, and there is the good imaging effect of good sensitivity and specificity, realization, in addition its technique is simple, reproducible, good biocompatibility, safety non-toxic, body are interior removes soon, can be widely used in clinical.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (17)
1. be applicable to the multi-mode self-assembled nanometer probe of MRI/PA and other imagings, it is characterized in that, described nano-probe, is loaded with for carrier with apoferritin or ferritin:
By adding when carrier protein solution acid dissociation and being embedded in the metal ion for NMR (Nuclear Magnetic Resonance)-imaging in described carrier protein and the melanin for PA imaging when described carrier protein solution restructuring, and for the functional mass of other imaging.
2. nano-probe according to claim 1, is characterized in that, described other is imaged as PET imaging, and the functional mass of described PET imaging is positron metal nucleic.
3. nano-probe according to claim 2, is characterized in that, described positron metal nucleic is
55co,
60cu,
61cu,
62cu,
64cu,
66ga,
68ga,
82rb,
86y,
86one in Zr.
4., according to the arbitrary described nano-probe of claim 1-3, it is characterized in that, described melanin is the water soluble particle of particle diameter 3-6nm.
5. nano-probe according to claim 4, it is characterized in that, the preparation method of described melanic water soluble particle comprises the following steps: in described melanin, add alkali make it be hydrolyzed, the alkali that adds of acid neutralization again, the melanin solution obtained is placed in ultrasound wave and is dissolved to clarification, and then ultrafiltration centrifugal filtration, to obtain final product.
6., according to described nano-probe arbitrary in claim 1-5, it is characterized in that, the metal ion of described NMR (Nuclear Magnetic Resonance)-imaging is Fe
2+, Fe
3+, Gd
3+, Zn
2+, Mn
2+, Mn
3+, Mn
4+, Mn
7+, Pt
2+in one.
7. prepare a method for the nano-probe in claim 1-6 described in any one, it is characterized in that, comprise the following steps:
(1) get apoferritin or liquor ferri albuminati, adjust ph, to 1-3, obtains the protein solution dissociated;
(2) to step 1) in obtain described in add melanin, metal ion respectively, mixing in the protein solution that dissociates, then adjust ph is to 7.5-10.0, obtains the protein solution of recombinating;
(3) to step 2) in add positron metal nucleic, mix homogeneously in the protein solution of described restructuring that obtains, react at 18-37 DEG C and be marked on albumen to positron metal nucleic, to obtain final product.
8. according to the method described in claim 7, it is characterized in that, step 2) in add melanin, metal ion and described in albumen in the protein solution that dissociates, the mol ratio of three is (0.5-2): (500-1500): 1.
9. according to Claim 8 described in method, it is characterized in that, step 2) in add melanin, metal ion and described in albumen in the protein solution that dissociates, the mol ratio of three is 1:1000:1.
10., according to described method arbitrary in claim 7-9, it is characterized in that, step 2) in add metal ion by adding metal salt solution.
11., according to described method arbitrary in claim 7-10, is characterized in that, step 2) also comprise the step of the protein solution purified concentration of described restructuring.
12., according to the method described in claim 11, is characterized in that, described purified concentration be dialysis, column chromatography, ultrafiltration centrifugal in one or more.
13., according to described method arbitrary in claim 7-12, is characterized in that, step 3) also comprise by unmarked on albumen, the step of positron metal nucleic removing freely.
14. methods according to claim 13, is characterized in that, step 3) in adopt chromatography desalination to remove positron metal nucleic freely.
15. according to described method arbitrary in claim 7-14, and it is characterized in that, the radioactivity of described positron metal core cellulose solution is for being less than 100mCi.
The arbitrary described application of nano-probe in multi-pattern Fusion imaging in 16. claim 1-6.
17. 1 kinds of multi-pattern Fusion preparations that are functional component with the nano-probe according to any one of claim 1-6.
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| CN105833302A (en) * | 2016-06-06 | 2016-08-10 | 重庆医科大学附属儿童医院 | Contrast medium and preparation method thereof |
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| CN109589408A (en) * | 2018-12-04 | 2019-04-09 | 江苏省原子医学研究所 | A kind of meter of shape liquid metal nanoparticle and its synthetic method and application |
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| CN110208236A (en) * | 2019-06-28 | 2019-09-06 | 郑州大学 | Ratio-type fluorescent pH nano-probe based on apoferritin, preparation method and applications |
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