CN104225630B - 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, and in particular to a kind of multi-mode suitable for MRI/PA and other imagings is from group
Dress nano-probe and its application.
Background technology
Molecular imaging (Molecular Imaging) is following most one of medical science Disciplinary Frontiers of development potentiality,
It includes plurality of medical image technology, such as：Positron emission computerized tomography (PET), X ray computer fault imaging
(CT), NMR (Nuclear Magnetic Resonance)-imaging (MRI), optical imagery (including bioluminescence, fluorescence, near-infrared spectroscopy), ultra sonic imaging
(US) etc..Different image technologies differ from one another, also each defective, therefore multi-pattern Fusion becomes current research and development heat
Point.With the high speed development of multi-pattern Fusion image documentation equipment, PET/CT, PET/MR, photoacoustic imaging (PA, PAUS) are occurred in that in succession
Deng new technique, by combination among the strong ones, have complementary advantages, be expected to organically combine fine anatomical structure and molecular function information, more
The good morning for realizing disease is anti-, early diagnosis, it is early control, help clinician to suit the remedy to the case, realize individualized treatment, so as to mitigate patient
Pain and financial burden, the health of the mankind is significant.By taking PET/MRI as an example, PET has high sensitivity, function
The characteristic of imaging, and MRI has high-resolution to soft tissue, can accurately provide Anatomical orientation information, the two joint, may be implemented in
Physiology, the noinvasive of pathological process, real-time, reproducible are carried out on molecular level, in diseases such as nervous system, tumor, cardiovascular
There is in terms of sick diagnosis unique value.Photoacoustic imaging (PA) is a kind of image technology for developing rapidly in recent years, and which utilizes has
The tissue of light absorbs can produce local sound source, i.e. PA effects, and different physiological parameters are known so as to according to the situation of light absorbs,
The concentration and oxygen saturation of such as intravital hemoglobin, melanin, water, ion etc..
, up to 5-7cm, spatial resolution is up to submillimeter level for the penetration depth of PA, it is easier to realize clinical conversion.Therefore light
Sound imaging is also the imaging pattern of a kind of potential structure, function and molecule.
If PET/MRI is combined with PA imaging techniques, fine anatomical structure and the PA that PET/MRI can be imaged into
The molecular function information of the deep layer of picture is effectively integrated, and is obtained with space and temporal information, three-dimensional, Crestor in synchronization
The high quality graphic of amount, makes imaging with the high sensitivity and high specific to minimal disease, it is possible to provide comprehensive information.But
It is that, in current medical imaging field, the correlation technique of tri- schema mergings of PET/MRI/PA imaging is rarely reported.
To realize that multi-pattern Fusion is imaged, the successful exploitation of multi-mode probe is one of important prerequisite.Exploitation multi-mode is visited
The purpose of pin is：1) imaging of different mode is capable of achieving using a kind of contrast medium；2) concordance of target area signal is realized, no
There is a problem of because causing signal distributions different using different contrast medium.At present, in multi-mode probe, nano material conduct
There are reports for the probe of carrier.By taking CNT (CNT) as an example, first, CNT itself carries fluorescence, can be used for near-infrared
Optical imagery, while strong absorptions of the CNT near infrared region can be additionally used in optoacoustic imaging；Secondly, metal is needed in CNT preparation process
(such as Co, Fe etc.) catalysis, still has the ferrum of residual after purification, can be used as magnetic resonance contrast agent.In addition, CNT can also carry out various repairing
Decorations, to realize improving its quantum efficiency, strengthen photoacoustic signal, strengthen nuclear magnetic signal, produce PET signal and other effects.Although nanometer
The great prospect of application of the material in multi-mode probe, but due to its process controllability, stability, biocompatibility, degradable
Property, the focus of the always dispute such as toxicity, body absorption and removement performance, therefore, still fail so far to be applied to clinic.
In the nano-carrier reported at present, have using ferritin or apoferritin as the probe of nano-carrier.For example,
In Chinese patent literature CN102462847A, make the carrier of tumor target direction contrast agent using apoferritin, contrast agent is wrapped up
Wherein, targeting is enriched to tumor tissues position, and then carries out NMR (Nuclear Magnetic Resonance)-imaging.Ferritin is natural ferrum storage protein, is gone
Ferrum ferritin (Apoferritin) is the nonferrous existence form of ferritin (ferritin).Ferritin and apoferritin can
Degradation in vivo, safety non-toxic, non-immunogenicity.Simultaneously as many tumor cell surfaces can be special with great expression ferritin
Specific receptor, can produce endocytosis to ferritin and apoferritin by the combination mediation of transferrin receptor-ferritin and make
With, therefore, ferritin and apoferritin also have preferable targeting to tumor cell.Above-mentioned contrast agent is with apoferritin
As carrier, although avoid nano material as carrier probe generally have biocompatibility, degradability, toxicity,
Body absorption and removement performance problem, and there is preferable targeting, but, it is only capable of realizing NMR (Nuclear Magnetic Resonance)-imaging, if it is desired to sharp
It is carrier with ferritin or apoferritin, carries out the imaging of tri- schema mergings of PET/MRI/PA, then face problems with：1) at present
Conventional PET positron radionuclides are the aniones such as C, F, I, and when being marked on ferritin or apoferritin, complex steps are difficult
Degree is high, and low yield is big to operator's irradiation dose；2) ferritin or apoferritin itself do not have optical property, adopt
PA imagings are capable of achieving from the ultraviolet melanin for there are light absorbs near infrared region.Melanin (melanin) is a kind of biochrome,
It is widely present in animal, plant and protista body, is L-Tyrosine or 3,4- dihydroxy phenylpropyl alcohol ammonia through a succession of chemical reaction
Formed, with multiple biological activities such as antioxidation, antitumor, venom, antiviral, hepatoprotective, radioprotectives, commodity at present
Change.But, when melanin is loaded on ferritin or apoferritin carrier, it is larger to be not only due to melanin particle diameter, insoluble
The organic solvent (ethanol, hexane, acetone, stupid, chloroform etc.) of Yu Shui, acid and routine, is dissolved only in alkali, meets oxidant and easily decolourizes, difficult
To load on carrier, and, it is even more important that when the melanin for PA imagings and the common metal for MRI imagings
In the presence of ion is common, as melanic metal chelation abilities are strong, the common metal ion of MRI imagings is run into (such as Fe3+) when
Easily precipitate, even if modifying to melanin, modified for example with Polyethylene Glycol, also can only improve molten to a certain extent
Load capacity of the melanin to metal ion in liquid, it is impossible to reach MRI imagings and the concentration needed for PA imagings, the signal of MRI and PA
Low intensity, sensitivity are also extremely low.
The content of the invention
First technical problem to be solved by this invention is that of the prior art not yet occur being suitable to PET/MRI/PA tri-
The metal ion of the natural material nano-probe of schema merging imaging, the melanin that cannot realize PA imagings and MRI imagings is in water
Stably coexist in solution, melanin is unable to reach sensitive imaging to the load capacity of metal ion, the positron of conventional PET imagings
Nonmetallic nucleic cannot labelling problem, and then provide a kind of imaging of tri- schema mergings of achievable PET/MRI/PA, it is highly sensitive
Degree, high specific, the nano-probe for being capable of achieving imaging comprehensively.
Second technical problem that the present invention is solved is that multi-pattern Fusion Imaging probe of the prior art is controllable in technique
There is technological deficiency in the aspect such as property, stability, biocompatibility, degradability, toxicity, body absorption and removement performance, and then
A kind of process is simple, reproducible, good biocompatibility are provided, safety non-toxic, the imaging of much faster schema merging is removed in vivo
Nano-probe.
The present invention suitable for MRI/PA and the multi-mode self-assembled nanometer probe of other imagings, the nano-probe is going
Ferrum ferritin or ferritin are carrier, are loaded with：By adding in carrier protein solution acid dissociation, and in the carrier egg
White solution be embedded in when recombinating in the carrier protein for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging and for the black of PA imagings
Pigment, and the functional mass being imaged for other.
It is described other be imaged as PET imagings, the functional mass of PET imaging is positron metal nucleic.
Further, the positron metal nucleic is55Co、60Cu、61Cu、62Cu、64Cu、66Ga、68Ga、82Rb、86Y、86One kind in Zr.
Further, water soluble particle of the melanin for particle diameter 3-6nm.Preferably, the melanic water solublity
The preparation method of particle is comprised the following steps：To in the melanin, add alkali to hydrolyze which, then the alkali in acid adding with addition, will
To melanin solution be placed in ultrasound wave and be dissolved to clarification, then ultrafiltration centrifugal filtration again, obtains final product.
Further, the metal ion of the NMR (Nuclear Magnetic Resonance)-imaging is Fe2+、Fe3+、Gd3+、Zn2+、Mn2+、Mn3+、Mn4+、
Mn7+、Pt2+In one kind.
The method for preparing above-mentioned nano-probe of the present invention, comprises the following steps：
(1) apoferritin or liquor ferri albuminati are taken, and pH value are adjusted to 1-3, are obtained the protein solution for dissociating；
(2) to step 1) in be separately added into melanin, metal ion in the protein solution of the dissociation that obtains, mix,
Then pH value is adjusted to 7.5-10.0, obtain the protein solution recombinated；
(3) to step 2) in add positron metal nucleic in the protein solution of the restructuring that obtains, mix homogeneously,
React at 18-37 DEG C to positron metal nucleic and be marked on albumen, obtain final product.
Further, step 2) in add melanin, metal ion and the dissociation protein solution in albumen,
The mol ratio of three is (0.5-2)：(500-1500)：1.Preferably, step 2) the middle melanin for adding, metal ion and institute
The albumen in the protein solution of dissociation is stated, 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 can basis
Situation selects the anion being adapted to, including but not limited to SO4 2-, NO3 -, ClO4 -Or the one kind in Cl-.
Further, step 2) the step of also include the protein solution purified concentration by the restructuring.Preferably, it is described pure
Change concentration for one or more in dialysis, column chromatography, ultrafiltration centrifugation.
Further, step 3) also include by it is unmarked on albumen, free positron metal nucleic remove the step of.
Preferably, step 3) it is middle using the free positron metal nucleic of chromatography desalination removing.
Further, the radioactivity of the positron metal radionuclide solution is less than 100mCi.
The nano-probe prepared in said method.
Application of the nano-probe of the present invention in multi-pattern Fusion imaging, particularly in tri- patterns of PET/MRI/PA
Application in fusion of imaging.
Multi-modal imaging agent with nano-probe of the present invention as functional component.
The above-mentioned technical proposal of the present invention, compared to existing technology with advantages below：
(1) nano-probe of multi-pattern Fusion of the invention imaging, with apoferritin or ferritin as carrier, loading
There are the positron radionuclide for PET imagings, the metal ion for NMR (Nuclear Magnetic Resonance)-imaging and the melanin being imaged for PA,
The imaging of tri- schema mergings of PET/MRI/PA is realized, with reference to the advantage of three kinds of imaging techniques, the fine solution that PET/MRI is imaged
The molecular function information for cuing open the deep layer that structure is imaged with PA is effectively integrated, and is obtained with space and temporal information in synchronization
, three-dimensional, can be quantitative high quality graphic, make imaging with the high sensitivity and high specific to minimal disease, it is possible to provide
Comprehensive information.
First, the present invention adopts biological natural materials apoferritin or ferritin in vivo as carrier, biocompatibility
Preferably, and can effectively solving safety problem, on the one hand, apoferritin and ferritin can be by metabolism by biological drop
Solution, it is internal to remove fast, another aspect, even if probe is detained biology in vivo, it is not easy to the toxic and side effects for causing；Meanwhile, with compared with
Good tumor cell targeting, the probe for obtaining have preferable specificity.
Second, apoferritin/ferritin is to encompass hollow spherical, sphere cavity by 24 homologous or heterologous subunits
In have 8 hydrophilic ionic passages and 6 Hydrophobic Ionic passages, can be enriched with a large number metal for NMR (Nuclear Magnetic Resonance)-imaging from
Son, directly embeds metal ion, is not required to add chelating agen；Meanwhile, the positron radionuclide for PET imagings of the present invention, is just
Electronic metal nucleic, also can enter inner chamber by the hydrophilic channel in apoferritin/ferritin nanocages, and stably deposit,
Therefore, on the one hand avoid because of the problem for interfering, affecting protein conformation produced using various chelating agen, do not interfere with
The compatibility of the nano-probe and organism that obtain；Other method, it also avoid using the common nonmetallic positive electricity daughter nucleus such as C, F, I
Element it is cumbersome, difficulty is high, low yield, and the problem big to operator's irradiation dose.
3rd, the hollow chondritic of apoferritin/ferritin so as to which outside, inner chamber, inside and outside intersection can use
Load in each motif, there is provided binding site it is many, while load the positron metal nucleic for PET imagings, for nuclear-magnetism altogether
Shake the metal ion and the melanin for PA imagings of imaging, is still capable of achieving good stability, and three kinds of loadings
Component and apoferritin/ferritin are capable of achieving good ratio control, in particular for the metal ion of NMR (Nuclear Magnetic Resonance)-imaging,
For the melanin of PA imagings, apoferritin/ferritin, accurate quantification is capable of achieving between three, it is to avoid traditional biological idol
Connection method list binding site reverse cyclic loadings and cause poor repeatability, the defect without standard measure.
4th, as melanic metal chelation abilities are strong, some metal ions are run in the solution and is precipitated (such as Fe3 +), this causes greatly obstruction for realizing PA imagings (melanin) and MRI imagings (metal ion) simultaneously, even if by melanin
Water soluble nanometer particles carry out PEG (Polyethylene Glycol) modifications, the load capacity of the melanin in solution and metal ion still compared with
It is low that (ultimate load is only 1：100), too low for the signal that PA is imaged with MRI is imaged, sensitivity is extremely low.And the present invention is to adopt
Melanin and metal ion are wrapped up with apoferritin/ferritin, therefore, the problems referred to above are overcome, the nano-probe tool for obtaining
There is good stability.
(2) nano-probe of the invention, the melanin are the water soluble particle of particle diameter 4nm, can preferably realize and carry
The loading of body, not only substantially increases meltage of the melanin in water, and is more beneficial for realizing for NMR (Nuclear Magnetic Resonance)-imaging
Metal ion, the melanin for PA imagings, apoferritin/ferritin, the accurate quantification between three.
(3) apoferritin/ferritin dissociation is first added thereto to by nano-probe of the invention in acid condition
Metal ion and the melanin for PA imagings for NMR (Nuclear Magnetic Resonance)-imaging, then pH value is adjusted to 7.5-10, that is, recover
Rebuild, the apoferritin/ferritin of above-mentioned metal ion and melanic restructuring is uniformly wrapped up in formation.Using chondritic
Apoferritin/ferritin above-mentioned pH dependencies, the nano-probe for obtaining not only have the advantages that it is homogeneous, stable, its behaviour
Make method also extremely simple, it is reproducible, it is that popularization and application or even the targeted molecular of tri- schema mergings of PET/MRI/PA imaging is aobvious
As fine new page has been opened in field.
Description of the drawings
In order that present disclosure is more likely to be clearly understood, the specific embodiment below according to the present invention is simultaneously combined
Accompanying drawing, the present invention is further detailed explanation.
Fig. 1 is the synthetic route chart of the tri- pattern nano-probes of self assembly PET/MRI/PA of embodiment 1；
Fig. 2 is apoferritin, melanin, FeCl3The abosrption spectrogram of solution, AMF；
Fig. 3 A are the DLS figures of apoferritin (APF)；
Fig. 3 B are the DLS figures of melanin (MNP)；
Fig. 3 C are the DLS figures of the AMF of tri- pattern nano-probes of self assembly PET/MRI/PA；
Fig. 4 is the TEM figures of apoferritin, melanin, AMF；
Wherein, upper left is apoferritin (dyeing)；Upper right is melanin (being unstained)；Lower-left is AMF (being unstained)；It is right
It is down AMF (dyeing)；
Fig. 5 is the PET images of tri- pattern nano-probes of self assembly PET/MRI/PA；
Fig. 6 is the MR images of tri- pattern nano-probes of self assembly PET/MRI/PA；
Fig. 7 is the PA images of tri- pattern nano-probes of self assembly PET/MRI/PA.
Specific embodiment
In the following each embodiments of the present invention, described apoferritin, ferritin, melanin, PD-10 posts can be from existing
The product routinely adopted in having technology carries out the preparation of tri- pattern nano-probes of self assembly PET/MRI/PA, is only given below wherein
A kind of product of model is illustrated the effect of the present invention, effect zero difference between the product of commercially available each model.Wherein：
Apoferritin, purchased from Sigma-Aldrich of the U.S. (Sigma-Aldrich)；
Melanin, purchased from Sigma-Aldrich of the U.S. (Sigma-Aldrich)；
PD-10, purchased from Life Sciences of General Electric (GE Healthcare).
1 self assembly PET/MRI/PA of embodiment, tri- pattern nano-probes
The tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment are：With apoferritin as carrier, it is loaded with
For PET imagings64Cu, for the Fe of NMR (Nuclear Magnetic Resonance)-imaging3+, and for PA imaging melanin.
The synthetic route of the tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment is as shown in figure 1, which is specific
Preparation manipulation is as follows：
First, apoferritin and melanin are done into following process：Apoferritin 90nmol is taken, adds 3.6ml's
Water dilutes, and obtains apoferritin solution, standby；20mg melanin is dissolved in into the concentration of 10ml for 0.1M's (i.e. mol/L)
In NaOH aqueous solutions, the aqueous hydrochloric acid solution of 0.1M is added after dissolving, and adjustment pH value is to 7, then is placed in dissolving in ultrasound, is clarified
Melanin aqueous solution.Above-mentioned melanin aqueous solution is further purified in ultrafiltration centrifugal filter, molecular cut off is
30kDa, deionized water are cleaned 3 times, to remove produced salt and other impurities, finally by its lyophilization, obtain particle diameter
Melanin (abbreviation MNP, similarly hereinafter) solid 15mg for the water soluble particle of 4nm, it is standby.
(1) above-mentioned apoferritin solution is taken, and pH value is adjusted to 2 with HCl, is obtained the protein solution for dissociating；
(2) to step 1) in the above-mentioned standby black of 90nmol is separately added in the protein solution of the dissociation that obtains
Element, the FeCl of 90 μm of ol3, mix, place 15min, then pH value is adjusted to 8 with NaOH, obtain the protein solution recombinated；Will
The protein solution for arriving by PD-10 posts, with PBS (NaCl 137mmol/L, KCl 2.7mmol/L,
Na2HPO410mmol/L, KH2PO42mmol/L, pH=7.2) for eluent, Chromatographic purification is carried out, then will be the albumen after purification molten
During liquid adds ultrafiltration centrifuge, molecular cut off is 30kDa, carries out ultrafiltration, obtains the protein nano granule recombinated, the restructuring
Melanin and Fe are enclosed with albumen3+, hereinafter referred to as AMF, similarly hereinafter；The AMF particles with water is dissolved, i.e., AMF after purification
Solution；
(3) to step 2) in add radioactivity for 1mCi's in the AMF solution that obtains64CuCl2, mixing is
It is even, 30min is reacted at 25 DEG C, by the protein solution for obtaining by PD-10 posts, with PBS as eluent, carry out chromatography and carry
It is pure, then the protein solution after purification is added in ultrafiltration centrifuge, molecular cut off is 30kDa, is concentrated, and is obtained final product.
The structure of tri- pattern nano-probes of above-mentioned PET/MRI/PA is verified using following experiment below：
(1) step 2) in load Fe for NMR (Nuclear Magnetic Resonance)-imaging3+And the melanic loading capacity being imaged for PA
Determine：
Using bovine serum albumin as standard protein, apoferritin in AMF is determined using Bio-Rad albuminometries
Content.Fe is determined using icp mses (ICP-MS)3+Content.Using ultraviolet-visible spectrophotometer
(Cary 60 of Anjelen Sci. ＆ Tech. Inc) determines the absorption spectrum of tri- pattern nano-probe granules of PET/MRI/PA, record
As a result, its result is as shown in Figure 2.Wherein, curve 1 be the absorption spectrum of apoferritin of 25 μ g/mL of concentration, curve 2 be dense
Spend the melanic absorption spectrum of 4 μ g/mL, the FeCl that curve 3 is 10 μ g/mL of concentration3The absorption spectrum of solution, curve 4 are AMF
Absorption spectrum.Abs680 (i.e. light absorption value) with melanin under variable concentrations, sets up standard curve, deducts apoferritin
With the abs680 of Fe, melanic content in AMF is calculated.It is possible thereby in the nano-probe AMF for calculating of the invention, de-iron ferrum egg
In vain：Melanin：Fe3+Mol ratio be 1:1:800.
(2) sign of tri- pattern nano-probe granules of PET/MRI/PA：
It is 250ug/mL, apoferritin that the AMF solution obtained in taking above-mentioned steps 2) adds deionized water to be diluted to containing APF
Solution (APF, 250ug/mL), melanin solution (MNP, 4ug/mL), are placed in measuring cell, using Malvern Zetasizer
Nano ZS90 nanometer particle sizes potentiometric analyzers carry out dynamic light scattering (DLS) measurement, and its result is as shown in Figure 3.By de-iron ferrum egg
In vain, melanin, AMF are added drop-wise on copper sheet after diluting, and are dried, and stain is 1% acetic acid uranium.Shot using perspective electron microscope
Its TME image is obtained, its result is as shown in Figure 4.
From the above results, the addition of melanin and Fe does not change the particle diameter and structure of apoferritin.It is possible thereby to
Illustrate, the present invention tri- pattern nano-probes of PET/MRI/PA wrapped up first particle diameter be 4nm melanin particle, melanin and
Ferrum is wrapped in apoferritin inner chamber.
2 self assembly PET/MRI/PA of embodiment, tri- pattern nano-probes
The tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment are：With ferritin as carrier, be loaded with for
PET imagings68Ga, for the Gd of NMR (Nuclear Magnetic Resonance)-imaging3+, and for PA imaging melanin.
The preparation manipulation of the tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment is as follows：
Ferritin and melanin are carried out into pretreatment according to the method and consumption in embodiment 1 first, it is standby.
(1) above-mentioned liquor ferri albuminati is taken, and pH value is adjusted to 1 with HCl, is obtained the protein solution for dissociating；
(2) to step 1) in the above-mentioned standby black of 90nmol is separately added in the protein solution of the dissociation that obtains
Element, the GdCl3 of 90 μm of ol, mix, and place 15min, then adjust pH value to 10.0 with NaOH, obtain the protein solution recombinated；
By the protein solution for obtaining by PD-10 posts, with PBS (NaCl 137mmol/L, KCl 2.7mmol/L,
Na2HPO410mmol/L, KH2PO42mmol/L, pH=7.2) for eluent, Chromatographic purification is carried out, obtain restructuring after purification
Protein solution；
(3) to step 2) in add radioactivity for 10mCi's in the protein solution of restructuring after purification that obtains68GaCl3, mix homogeneously reacts 30min at 30 DEG C, by the protein solution for obtaining by PD-10 posts, with PBS as eluting
Liquid, carries out Chromatographic purification, then the protein solution after purification is added in ultrafiltration centrifuge, and molecular cut off is 30kDa, is surpassed
Filter, obtains final product.
According to the method in embodiment 1 to step 2) the middle Gd for NMR (Nuclear Magnetic Resonance)-imaging for loading3+And for PA into
The melanic loading capacity of picture is measured, and obtains ferritin：Melanin：Gd3+Mol ratio be 1:1:1000.Melanin, Gd3 +Loading capacity on ferritin has reached the amount of the MRI and PA imaging effects for being capable of achieving ideal.
3 self assembly PET/MRI/PA of embodiment, tri- pattern nano-probes
The tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment are：With apoferritin as carrier, it is loaded with
For PET imagings82Rb, for the Fe of NMR (Nuclear Magnetic Resonance)-imaging2+, and for PA imaging melanin.
The preparation manipulation of the tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment is as follows：
Apoferritin and melanin are carried out into pretreatment according to the method and consumption in embodiment 1 first, it is standby.Its
In, melanic particle diameter is 4nm.
(1) above-mentioned liquor ferri albuminati is taken, and pH value is adjusted to 3 with HCl, is obtained the protein solution for dissociating；
(2) to step 1) in the above-mentioned standby black of 90nmol is separately added in the protein solution of the dissociation that obtains
Element, the FeCl2 of 135 μm of ol, mix, and place 15min, then adjust pH value to 7.5 with NaOH, obtain the protein solution recombinated；
(3) to step 2) in add radioactivity for 30mCi's in the protein solution of restructuring that obtains82Rb Cl, mixing
Uniformly, 30min is reacted at 30 DEG C, by the protein solution for obtaining by PD-10 posts, with PBS as eluent, chromatographed
Purification, then the protein solution after purification is added in ultrafiltration centrifuge, molecular cut off is 30kDa, carries out ultrafiltration, obtains final product.
According to the method in embodiment 1 to step 2) the middle Fe for NMR (Nuclear Magnetic Resonance)-imaging for loading2+And for PA into
The melanic loading capacity of picture is measured, and obtains apoferritin：Melanin：Fe2+Mol ratio be 1:1:1500.Black
Element, Fe2+Loading capacity on apoferritin has reached the amount of the MRI and PA imaging effects for being capable of achieving ideal.
4 self assembly PET/MRI/PA of embodiment, tri- pattern nano-probes
The tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment are：With apoferritin as carrier, it is loaded with
For PET imaging/radionuclide therapies89Zr2+, for the Gd of NMR (Nuclear Magnetic Resonance)-imaging3+, and for PA imaging melanin.
The preparation manipulation of the tri- pattern nano-probes of self assembly PET/MRI/PA of the present embodiment is as follows：
Apoferritin and melanin are carried out into pretreatment according to the method and consumption in embodiment 1 first, it is standby.Its
In, melanic particle diameter is 3nm.
(1) above-mentioned liquor ferri albuminati is taken, and pH value is adjusted to 2 with HCl, is obtained the protein solution for dissociating；
(2) to step 1) in the above-mentioned standby black of 90nmol is separately added in the protein solution of the dissociation that obtains
Element, the GdCl3 of 45 μm of ol, mix, and place 15min, then adjust pH value to 8 with NaOH, obtain the protein solution recombinated；
(3) to step 2) in add radioactivity for 90mCi's in the protein solution of restructuring that obtains89ZrCl2, mixing
Uniformly, 30min is reacted at 30 DEG C, obtain final product.
According to the method in embodiment 1 to step 2) the middle Gd for NMR (Nuclear Magnetic Resonance)-imaging for loading3+And for PA into
The melanic loading capacity of picture is measured, and obtains apoferritin：Melanin：Gd3+Mol ratio be 1:1:500.Melanin,
Gd3+Loading capacity on apoferritin has reached the amount of the MRI and PA imaging effects for being capable of achieving ideal.
The preparation of tri- mode imaging agent of embodiment 5PET/MRI/PA
The tri- pattern nano-probes of self assembly PET/MRI/PA prepared in Example 1, are dissolved in normal saline,
The concentration for making the nano-probe is 1mCi/mL, is obtained final product.
Used as the substitute mode of the embodiment, tri- pattern nano-probes of self assembly PET/MRI/PA that can be described are also replaceable
For 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,
Normal saline also can be replaced glucose for injection solution.
The imaging of tri- pattern nano-probe of embodiment 6PET/MRI/PA
Take mouse bare subcutaneous injection 2-5 × 106 colon cancer HT29 cell (the high expression of TfR) or hepatocarcinoma HepG2 is thin
Born of the same parents' (TfR low expression), after inoculation, 2-5 is all, obtains gross tumor volume and reaches 150-500mm3Tumor model.
PET is imaged：The tri- pattern nano-probes of PET/MRI/PA prepared in Example 1, are formulated as64The radiation of Cu
Property activity be 100 μ Ci solution, Jing tail vein injections enter in nude mice body respectively to take 0.2ml, injection preparation after carry out
MicroPET (SIEMENS INVEON) is imaged, and scans different time dot image, IAW software analysis tumor uptakes (ID%/g).
Its result is as shown in Figure 5.
NMR (Nuclear Magnetic Resonance)-imaging：The tri- pattern nano-probes of PET/MRI/PA prepared in Example 1, are formulated as by Fe
Concentration is calculated as the solution of 2mg/mL.Jing tail vein injections enter in nude mice body respectively to take 0.2ml, carry out 1T after injection preparation
MR (Bruker) T1 is imaged, and scans different time dot image, and sweep parameter is set to the T1-flash MRI sequence (repetition times
(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 tri- pattern nano-probes of PET/MRI/PA prepared in Example 1, are formulated as dense by MNP
Degree is calculated as the solution of 2mg/mL.Jing tail vein injections enter in nude mice body respectively to take 0.2ml, carry out PA after injection preparation
(VevoLAZR；VisualSonics) image, parameter is set to：Light acoustic gain 45dB, dynamic range 20dB, mid frequency
21MHz.Scanning different time dot image, its result are as shown in Figure 7.
Knowable to PET image results, HT29 tumor uptakes after injection 1,2,4,18,26 and 48h respectively up to 4.82 ±
0.59,6.14 ± 0.77,7.34 ± 0.93,7.26 ± 1.32,6.74 ± 0.51,6.54 ± 0.79%ID/g, and transferrinss
The hepatocarcinoma HepG2 tumor uptake of receptor low expression is then relatively low, and after injection, 1,2,4,18,26 and 48h respectively up to 2.95 ± 0.40,
3.55 ± 0.58,4.33 ± 1.16and 4.61 ± 1.58,4.77 ± 0.47,3.81 ± 0.54%ID/g this explanation, APF can target
To the tumor of the high expression of TfR, intake is high and is detained length.The tumor uptake of TfR low expression is because
Enhancing infiltration retention effect (EPR) effect of nanoparticle, then absorb low and remove fast.From MR image results, AMF is injected
Tumor uptake is high-visible afterwards, MR signal enhancings, and 1h after the injection of HT29 colon cancer Mus, 2h, 4h and 24h are taken the photograph with injection pre-neoplastic
Value ratio (T/T0) for 1.58,1.73,2.02,1.30, HepG2 liver cancer murines are then 1.21,1.44,1.03,0.99, aobvious with PET
As result is consistent, the targeting and EPR effects of apoferritin are again demonstrated.From after the visible injection AMF of PA image results,
HT29 tumor photoacoustic signals are gradually strengthened, and after injection, 4h reaches peak.This result fully proves that AMF is an effective PET/MR/
Tri- mode probes of PA.
In sum, tri- pattern nano-probes of self assembly PET/MRI/PA of the invention realize tri- patterns of PET/MRI/PA
Fusion imaging, and with good sensitivity and specificity, realize good imaging effect, its process is simple, repeatability in addition
Good, good biocompatibility, safety non-toxic, in vivo removing are fast, can be widely used in clinic.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need to be exhaustive to all of embodiment.And thus it is extended obvious change or
Among changing still in the protection domain of the invention.
Claims (13)
1. a kind of tri- pattern nano-probes of self assembly PET/MRI/PA, it is characterised in that tri- moulds of self assembly PET/MRI/PA
Formula nano-probe is loaded with apoferritin or ferritin as carrier：
By adding in carrier protein solution acid dissociation, and the carrier egg is embedded in when the carrier protein solution is recombinated
Metal ion and the melanin for PA imagings for NMR (Nuclear Magnetic Resonance)-imaging in white, and for the positron of PET imagings
Metal nucleic；
The metal ion of the NMR (Nuclear Magnetic Resonance)-imaging is Fe2+、Fe3+、Gd3+、Zn2+、Mn2+、Mn3+、Mn4+、Mn7+、Pt2+In one
Kind；
Water soluble particle of the melanin for particle diameter 3-6nm；
The positron metal nucleic is55Co、60Cu、61Cu、62Cu、64Cu、66Ga、68Ga、82Rb、86Y、86One kind in Zr；
The method for preparing described tri- pattern nano-probes of self assembly PET/MRI/PA, comprises the following steps：
(1) apoferritin or liquor ferri albuminati are taken, and pH value are adjusted to 1-3, are obtained the protein solution for dissociating；
(2) melanin, metal ion are separately added in the protein solution of the dissociation obtained in step (1), are mixed, then
PH value is adjusted to 7.5-10.0, the protein solution recombinated is obtained；
(3) positron metal nucleic, mix homogeneously, 18-37 are added in the protein solution of the restructuring obtained in step (2)
React at DEG C to positron metal nucleic and be marked on albumen, obtain final product；
Albumen in the protein solution of the melanin, metal ion and the dissociation that add in step (2), the mol ratio of three
For (0.5-2)：(500-1500)：1.
2. tri- pattern nano-probes of self assembly PET/MRI/PA according to claim 1, it is characterised in that the melanin
The preparation method of water soluble particle comprise the following steps：To in the melanin, add alkali to hydrolyze which, then in acid adding and add
Alkali, the melanin solution for obtaining is placed in ultrasound wave and is dissolved to clarification, then ultrafiltration centrifugal filtration again, obtains final product.
3. a kind of method of the tri- pattern nano-probes of self assembly PET/MRI/PA prepared described in claim 1 or 2, its feature exist
In comprising the following steps：
(1) apoferritin or liquor ferri albuminati are taken, and pH value are adjusted to 1-3, are obtained the protein solution for dissociating；
(2) melanin, metal ion are separately added in the protein solution of the dissociation obtained in step (1), are mixed, then
PH value is adjusted to 7.5-10.0, the protein solution recombinated is obtained；
(3) positron metal nucleic, mix homogeneously, 18-37 are added in the protein solution of the restructuring obtained in step (2)
React at DEG C to positron metal nucleic and be marked on albumen, obtain final product.
4. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to claim 3, it is characterised in that
Albumen in the protein solution of the melanin, metal ion and the dissociation that add in step (2), the mol ratio of three is
(0.5-2)：(500-1500)：1.
5. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to claim 4, it is characterised in that
Albumen in the protein solution of the melanin, metal ion and the dissociation that add in step (2), the mol ratio of three is 1：
1000：1.
6. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to any one of claim 3-5, its
Be characterised by, in step (2), metal ion is added by adding metal salt solution.
7. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to claim 6, it is characterised in that
The step of step (2) also includes the protein solution purified concentration by the restructuring.
8. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to claim 7, it is characterised in that
The purified concentration be dialysis, column chromatography, ultrafiltration centrifugation in one or more.
9. the side for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to any one of claim 3-5 or 7 or 8
Method, it is characterised in that step (3) also include by it is unmarked on albumen, free positron metal nucleic remove the step of.
10. the method for preparing tri- pattern nano-probe of self assembly PET/MRI/PA according to claim 9, its feature exist
In using the free positron metal nucleic of chromatography desalination removing in step (3).
The 11. tri- pattern nanometers of preparation self assembly PET/MRI/PA according to any one of claim 3-5 or 7 or 8 or 10 are visited
The method of pin, it is characterised in that the radioactivity of the positron metal radionuclide solution is less than 100mCi.
Tri- pattern nano-probes of self assembly PET/MRI/PA described in 12. claim 1 or 2 are preparing multi-pattern Fusion preparation
In application.
A kind of multimode of the 13. tri- pattern nano-probes of self assembly PET/MRI/PA with described in claim 1 or 2 as functional component
Formula fusion of imaging agent.
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