CN110302400A - PET/MRI multi-mode molecular imaging nano-probe and its application for the early diagnosis of atherosclerosis vulnerable plaque - Google Patents
PET/MRI multi-mode molecular imaging nano-probe and its application for the early diagnosis of atherosclerosis vulnerable plaque Download PDFInfo
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- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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Abstract
The invention discloses a kind of PET/MRI multi-mode molecular imaging nano-probe for the early diagnosis of atherosclerosis vulnerable plaque and its applications.Formation and stability in view of matrix metalloproteinase (MMPs) with vulnerable plaque is closely related.PET/MRI multi-mode molecular imaging nano-probe of the present invention, with ferric oxide nanometer particle (abbreviation IONP) for core, passes through NH on the surface of IONP using MMP-2 and MMP-9 as molecular imaging target spot2C-terminus and the NOTA connection of polypeptide MMPsC can be sheared with matrix metalloproteinase (matrix metalloproteinases, MMPs) respectively, the carboxyl of N-terminal amino and PEG that MMPs can shear polypeptide MMPsC connects,64Cu is coupled to the surface IONP by NOTA, has synthesized the novel nano probe for constructing targeting atherosclerosis (AS) vulnerable plaque MMPs and bio-safety.The imaging of joint PET/MRI bimodal and pathological analysis, living body, dynamic, in real-time monitoring AS vulnerable plaque MMPs expression, evaluate the stability of AS patch.A new method is provided for the diagnosis of AS vulnerable plaque.
Description
Technical field
The present invention relates to a kind of diagnosis and treatment new methods of atherosclerosis (AS) vulnerable plaque, in particular to a kind of for moving
The PET/MRI multi-mode molecular imaging nano-probe of pulse atherosclerosis vulnerable plaque early diagnosis and its application.The invention belongs to
Pharmaceutical technology field.
Background technique
Atherosclerosis (atherosclerosis, AS) is that a kind of multifactor driving, the chronic inflammation of concealment are anti-
It answers, often involves the large and medium-sized flesh elastic force artery such as cerebral artery, aorta, coronary artery, and then induce cardiovascular and cerebrovascular disease, seriously
Endanger human life and health.The treatment of AS is not very ideal at present, according to statistics the annual cardiovascular disease death toll in the whole world
Up to more than 1,670 ten thousand.The incidence of China AS related disease and the death rate rise year by year in recent years.The rupture of AS vulnerable plaque is to lead
The main reason for causing acute cardiocerebrovasculaevents events, therefore, EARLY RECOGNITION vulnerable plaque can instruct clinical progress effectively preventing
AS reduces the incidence and the death rate of acute cardiovascular and cerebrovascular disease.Traditional image technology can not reflect that the pathology of patch is raw
Rationality matter, so vulnerable plaque can not be efficiently identified.Although researcher has utilized the means of a variety of iconographies (such as at present
Intravascular ultrasound and optical coherence tomography) to patch carry out load and property component research.However, these detection methods
It is all invasive method, price is also relatively expensive, it is difficult to which the process of the occurrence and development of dynamic monitoring vulnerable plaque is not appropriate for
For vulnerable plaque early screening and diagnosis.Molecular imaging techniques booming at present, can from molecule and cellular level without
The occurrence and development process of the dynamic observation disease of wound, provides strong tool for the early diagnosis of AS vulnerable plaque.With biography
System Imaging Technology main display official occurs organic variation and compares, and Molecular imaging techniques have the characteristics that " morning seen ", energy
Enough detect the anomalous variation that cell and molecular level before organic change not yet occurs in disease, be disease early diagnosis and
The evaluation for occurring, developing and lapsing to provides effective evidence, while being also to promote Protocols in Molecular Biology to clinical medicine application
Conversion construct bridge.
Matrix metalloproteinase (matrix metalloproteinases, MMPs) plays during AS occurrence and development
Key effect, it can degrade extracellular matrix, promote the migration and proliferation of smooth muscle cell, promote atheromatous plaque " fibrous cap "
Degradation, eventually lead to atheromatous plaque rupture, to generate serious complication.Have research confirm, unstable angina pectoris,
Under stable angina cordis and normal condition in patient's internal mammary artery, the expression of MMP-9 is respectively 83%, 25% and 0；Rapid wear spot
The shoulder area of block, especially patch, MMPs activity obviously increase, and more stable patch is 3~5 times high；It is broken with patch in AS patch
Splitting place has MMP-2 and MMP-9 expression.Since effect of the MMPs in AS process is relatively unambiguous, monitoring AS patch part
The expression of MMPs seems particularly significant.The expression of MMPs in AS vulnerable plaque forming process is dynamically detected, it is easy for AS
The judgement of the prevention, early diagnosis and therapy effect of damaging patch has important clinical value.
Target spot is imaged in addition to finding AS rapid wear specific molecular, the present invention also optimizes imaging platform.Molecule for AS
Imaging method mainly includes CT, MRI, PET, SPECT, optical imagery and ultrasonic imaging etc..These methods are each in terms of imaging
There are advantage and disadvantage.MRI has many advantages, such as soft tissue resolution height, noninvasive, radiationless, repeatable to check, but the spatial discrimination of MRI
Rate still has certain limit, cannot find more valuable plaque rupture sign, such as in the thickness and patch of plaque fibrous cap
Degree of inflammation etc..MRI molecular imaging can make up this deficiency, using a variety of different MRI probes, can not only effectively divide
Plaque component is analysed, and the AS in different pathological period can be detected, can be accurately positioned and judge patch property, in cell and can be divided
Sub- level carries out living body, dynamic and research in real time to the pathogenesis of AS, for early diagnosing and comprehensive assessment patch
Vulnerability has great help, but the sensibility of MR imaging is low.And SPECT/PET imaging using radioactive tracer have it is very high
Sensibility, use in conjunction can overcome the disadvantages that the deficiency of MR molecular imaging.It is currently combined for two kinds or more of imaging patterns more
Modality can greatly improve the early diagnosis to AS vulnerable plaque.
Signal amplification, surface modification approach abundant and size advantage that nano particle and nanotechnology possess etc., with
Different kinds of molecules imaging technique combines, and the characteristics of passing through itself kernel or marker realize under different imaging modalities at
Picture.Secondly, the large specific surface area of nano particle, can a large amount of chemical group of its surface modification (including amino, carboxyl or
Sulfydryl), the albumen of upper antibody, small peptide or macromolecular is marked using the chemical chelatropic reaction of these groups, becoming has master
The molecular probe of moving-target tropism improves the sensibility of probe, provides new opportunity for the imaging of AS vulnerable plaque living body molecule.
IONP (ferric oxide nanometer particle) is a kind of more popular magnetic resonance enhancing agent of Recent study, its partial size is small, penetration power
By force, T2 relaxation time can be obviously shortened to improve signal-to-noise ratio, and its biological safety is also above gadolinium chelate compound.But it uses
IONP judges AS vulnerable plaque as MRI contrast agent, and there are some urgent problems to be solved: 1) MRI cannot distinguish non-targeted iron
Iron content ferroheme signal caused by phagocytosis declines not after the change and bleeding of signal after contrast agent is swallowed by macrophage
Together；2) IONP is non-targeted iron contrast agent, easily can cause false positive by the macrophage phagocytosis on ground in blood vessel peripheral lymph node.For
The MR image defects of above-mentioned IONP contrast medium are avoided, the present invention carries out surface to IONP particle using polyethylene glycol (PEG) and repairs
Decorations reduce its non-targeted position passively gulping down by macrophage in vivo so as to improve its stability and biocompatibility
It bites.Meanwhile the present invention can shear polypeptide (Ac-GPLGVRGKC-NH2, MMPsC) in IONP particle surface link MMP, pass through enzyme-
The detection to MMPs enzymatic activity in AS is realized in interaction between substrate, to make up asking for living body molecule imaging poor specificity
Topic.The present invention successfully synthesizes the multi-modal nana intelligent probe of novel 64Cu-NOTA-Fe3O4-MMPsC-PEG (referred to as
MMPsCNP is shown in Fig. 1), using this novel nano particle as image platform, special target AS vulnerable plaque MMPs molecule realizes MMPs
The AS vulnerable plaque PET/MRI multi-mode molecule imaging of targeting provides a new method (see figure for the diagnosis of AS vulnerable plaque
2)。
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of atherosclerosis (AS) vulnerable plaque
Diagnosis and treatment new method, specifically a kind of PET/MRI dual-mode molecular imaging method based on nana intelligent probe, passes through targeting
MMPs is used for the early diagnosis of AS vulnerable plaque.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of PET/MRI multi-mode molecular imaging nano-probe for the early diagnosis of AS vulnerable plaque, the PET/
MRI multi-mode molecular imaging nano-probe is to pass through NH on the surface of IONP for core with ferric oxide nanometer particle (simple IONP)2
Can be sheared with matrix metalloproteinase (matrix metalloproteinases, MMPs) respectively the c-terminus of polypeptide MMPsC with
And NOTA connection, the carboxyl of N-terminal amino and PEG that MMPs can shear polypeptide MMPsC connect,64Cu is coupled to by NOTA
The surface IONP, wherein MMPs can shear the amino acid sequence of polypeptide MMPsC are as follows: Ac-GPLGVRGKC-NH2.
Further, a kind of method for preparing the PET/MRI multi-mode molecular imaging nano-probe of the present invention,
It is characterized in that, comprising the following steps:
(1) matrix metalloproteinase (matrix metalloproteinases, MMPs) can shear the preparation of polypeptide
Synthesis MMPs can shear polypeptide MMPsC, amino acid sequence Ac-GPLGVRGKC-NH2；
(2) preparation of MMPsC-PEG
Synthesis is passed through to the carboxyl of acylation reaction and PEG with the N-terminal amino that can shear polypeptide MMPsC of MMPs after purification
Then connection carries out purifying preparation MMPsC-PEG；
(3) preparation of IONP-MMPsC-PEG
Ferric oxide nanometer particle (IONP) is prepared using coprecipitation, the surface IONP is modified, by NH2It is introduced into
The surface IONP utilizes the NH on the surface IONP2The MMPs linked by acylation reaction with PEG can shear the c-terminus of polypeptide MMPsC
Connection, IONP of the preparation with MMPsC-PEG modification, is named as IONP-MMPsC-PEG；
(4) preparation of NOTA-IONP-MMPsC-PEG
Utilize the NH of IONP surface residual2Chelatropic reaction is carried out with NOTA, synthesizes NOTA-IONP-MMPsC-PEG；
(5)64The preparation of Cu-NOTA-IONP-MMPsC-PEG
It will64Cu is coupled to the surface IONP by NOTA, synthesis64The multi-modal nanometer intelligence of Cu-NOTA-IONP-MMPsC-PEG
Energy probe, referred to as MMPsCNP.
Wherein, it is preferred that polypeptide MMPsC can be sheared by synthesizing MMPs by solid-phase synthesis in step (1).
Wherein, it is preferred that PEG described in step (2) is PEG2000.
Wherein, it is preferred that IONP is prepared using coprecipitation in step (3), obtains monodisperse using high temperature thermal decomposition method
IONP.Specific steps are as follows:
I it) synthesizes the third copper and iron of triacetic acid: ferric chloride hexahydrate 270mg being dissolved in 12.5ml distilled water, is stirred continuously simultaneously
Sodium acetate trihydrate 500mg is added, the acetylacetone,2,4-pentanedione of 500ml is added in the above mixed solution, is reacted at room temperature
10min is heated to 80 DEG C, stands 20min and is placed in 4 DEG C of refrigerators, filter and clean after recrystallizing, obtains rose violet
The third copper and iron of triacetic acid of color flaky crystal；
II) the preparation of IONP: being heated to phenylate for the benzophenone of 100ml and be added in triangular flask, reacts in the protection of nitrogen,
It is gradually heated to 110 DEG C of heat preservation 2h under magnetic agitation, is then warming up to 280 DEG C of back flow reaction 1h again, room temperature is cooling, and is added
The ethanol precipitation of 20ml, hexamethylene, oleic acid and oleyl amine, ultrasound, which is added, in sediment makes it be uniformly dispersed, and centrifugation 8000g ×
30min is obtained oiliness ferric oxide nanometer particle (IONP), spare.
Wherein, it is preferred that INOP is modified using the third aminotriacetic acid of 3- base silane (APTS) in step (3), by NH2It introduces
The surface INOP.
Wherein, it is preferred that the MMPs that IONP and PEG is linked in step (3) can shear the connection of polypeptide MMPsC according to following
Step carries out:
EDC and the 1mg NHS of 1mg is added in 1ml deionized water, and 1mg MMPsC-PEG is added, adds 5ml IONP
Solution is vigorously stirred lower reaction 12 hours, recycles deionization washing, and 13000rpm is centrifuged, and obtains IONP-MMPsC-
PEG product is purified using magnetization splitter.
Further, the invention also provides the PET/MRI multi-mode molecular imaging nano-probes to prepare artery
Atherosis vulnerable plaque early diagnoses the application in reagent.
Wherein, it is preferred that the reagent is for PET/MRI multi-mode molecular imaging.
Compared to the prior art, the beneficial effects of the present invention are:
Key science of the present invention for the expression of MMPs in raising AS vulnerable plaque early stage diagnosis and treatment, regulation AS vulnerable plaque
Problem, by means such as smart molecule design, nano-meter characteristic control, targeted molecular modifications, synthesis is for constructing targeting AS rapid wear
The novel nano probe of patch MMPs and bio-safety, this is early diagnosed to AS vulnerable plaque and intervention has great importance.
And academic thinking, material, technology, in terms of have it is significant innovative.Compared with traditional Imaging Technology, point
Sub- imaging technique can detect the anomalous variation that cell and molecular level before organic change not yet occurs in disease, be disease
Early diagnosis and the effective evidence of evaluation offer for occurring, developing and lapsing to.Use in conjunction SPECT/PET imaging and MR molecule
Imaging can overcome the disadvantages that the deficiency being imaged with single method, and this multi-modality imaging, which can greatly improve, examines the early stage of AS vulnerable plaque
It is disconnected.The present invention generates breakthrough progress to the earlier specificity diagnosis of AS vulnerable plaque, by examining for clinic AS vulnerable plaque
It is disconnected to improve important foundation, there is good clinical conversion prospect.
Detailed description of the invention
Fig. 1 is multi-modal nana intelligent probe (MMPsCNP) structural schematic diagram of Novel PET/MRI；
Fig. 2 is that TEM the and Zeta potential of IONP-MMPsC-PEG or IONP-MMPnC-PEG nano particle is detected
As a result；
Fig. 3 is AS vulnerable plaque MMPs multi-modality imaging principle；
Fig. 4 is nano-probe MMPsCNP and control probe MMPsNCNP characterization detection TEM and Zeta potential detection
As a result；
Fig. 5 is a kind of based on Novel PET/MRI multi-modality imaging nana intelligent probe atherosclerosis vulnerable plaque
Diagnosis and treatment schematic diagram；
Fig. 6 is mouse carotid atherosclerosis model MR evaluation；
Wherein, A:FSE；B:2DGRE；C:2DSGRE axial；D:Anatomic image；
Fig. 7 is the PET/CT living body molecule imaging that MMPcNP nano-probe targets MMPs in rat aorta atherosis；
Fig. 8 is the MRI living body molecule imaging that MMPcNP nano-probe targets MMPs in rat aorta atherosis；
Fig. 9 is MMP-2 and MMP-9 fluorescent staining in atherosclerotic plaque, Nanoscale Iron Perls coloration result.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
PET/MRI multi-mode molecular imaging nanometer of the embodiment 1 for the early diagnosis of atherosclerosis vulnerable plaque is visited
The preparation of needle (MMPsCNP)
1. MMPs can shear the preparation of polypeptide
MMPs is synthesized using solid phase lotus methoxycarbonyl group chemistry of peptides synthetic method can shear polypeptide MMPsC (Ac-
GPLGVRGKC-NH2), using the chloro- triphenyl chlorine resin of 2- as carrier, cooperate Fmos amido protecting strategy, according to the amino of MMPsC
Acid sequence adds amino acid, the carboxylic for being by first amino acid of N-terminal first from c-terminus to aminoterminal MMPsC c-terminus in order
Base is connected in the form of covalent bond with resin, then with amino be synthesis starting point, make its with carboxyl occur acylation reaction, formed
Peptide bond.After reaction with remaining active site on suitable pyridine and acetic anhydride closing resin.Subsequent amino-acid is with HOBt/
HBTU/DIEA is sequentially connected for condensing agent.It after reaction, is that main cutting agent splits purpose peptide fragment from resin with trifluoroacetic acid
Solution is got off, and compound is purified by synthetic product using high pressure liquid chromatography (HPLC) technology (HPLC) from reaction system, and is utilized
Mass spectrograph (MS) analyzes and identifies the molecular weight of purified product.
2. the preparation of MMPsC-PEG
Synthesis and MMPs after purification can be sheared into the N-terminal amino of polypeptide MMPsC and pass through acylation reaction and PEG2000
Then carboxyl connection carries out purifying preparation MMPsC-PEG.
3. the preparation of IONP-MMPsC-PEG
Ferric oxide nanometer particle (IONP) is prepared using coprecipitation.It is obtained using high temperature thermal decomposition method monodispersed
IONP, specific steps are as follows:
I it) synthesizes the third copper and iron of triacetic acid: ferric chloride hexahydrate (270mg) being dissolved in 12.5ml distilled water, is stirred continuously
And sodium acetate trihydrate 500mg is added.The acetylacetone,2,4-pentanedione of 500ml is added in the above mixed solution, at room temperature instead
Answer 10min.80 DEG C are heated to, 20min is stood and is placed in 4 DEG C of refrigerators, filter and clean after recrystallizing, obtain rose
The third copper and iron of triacetic acid of purple flaky crystal.
II) the preparation of IONP: the benzophenone of 100ml is heated to phenylate and is added in triangular flask, reacts in the protection of nitrogen, in magnetic
110 DEG C of heat preservation 2h are gradually heated under power stirring, are then warming up to 280 DEG C of back flow reaction 1h again, room temperature is cooling, and 20ml is added
Ethanol precipitation, hexamethylene is added in sediment, oleic acid and oleyl amine, ultrasound make it be uniformly dispersed, is centrifuged 8000g × 30min,
Oiliness IONP is obtained, it is spare.Using the methods of current potential Particle Size Analyzer, infrared spectroscopy, elemental analysis to IONP nanoparticle ruler
Very little, surface charge, surface organic molecule load capacity etc. are characterized.IONP nanoparticle surface is modified, by NH2It introduces
To the surface IONP.
III IONP) is modified using the third aminotriacetic acid of 3- base silane (APTS), by NH2Introduce the surface IONP
IV) MMPs that IONP and PEG2000 is linked can shear the connection of polypeptide MMPsC.
EDC and the 1mg NHS of 1mg is added in 1ml deionized water, and 1mg MMPsC-PEG is added, adds 5ml IONP
Solution is vigorously stirred lower reaction 12 hours, recycles deionization washing, and 13000rpm is centrifuged, and obtains IONP-MMPsC-
PEG product.It is purified using magnetization splitter, measures relaxation rate using mass spectrum of nuclear magnetic resonance instrument (NMR)；Utilize liquid chromatogram
Instrument is analyzed, and measures the Percentage bound of MMPsC and IONP.
Utilize the NH of IONP surface residual2Acylation reaction and MMPs can shear the c-terminus of polypeptide, preparation has
The IONP of MMPsC-PEG modification (see Fig. 1).Polypeptide (MMPnC) modification can not be sheared according to above-mentioned identical method preparation MMPs
IONP (IONP-MMPsNC-PEG) as control, pass through scanning electron microscope (TEM) and Dynamic laser scattering instrument (DLS)
Etc. means the nanoparticle of preparation is characterized.
IONP-MMPsC-PEG and IONP-MMPsNC-PEG nano particle characterization:
I) the form and partial size of transmission electron microscope tem observation IONP: IONP-MMPsC-PEG or IONP-MMPnC-PEG are received
The sample deionized water dilution of rice grain is adjusted to suitable concentration, and ultrasonic-microwave makes it be uniformly dispersed.By IONP-MMPsC-
1 drop of PEG or IONP-MMPnC-PEG nanoparticles solution drop is on transmission electron microscope copper mesh, vacuum drying 6-8h at 40 DEG C, then
It observes and takes pictures under transmission electron microscope.
II) Malvern particle size analyzer (3000HS) is to IONP-MMPsC-PEG or IONP-MMPnC-PEG nano particle
It carries out dynamic light scattering (DLS) analysis: with the concentration that deionized water is diluted to Fe being 0.5mg/ml by sample, detect at room temperature
The particle diameter distribution of its hydrodynamics.
III) the saturation magnetic of vibrating specimen magnetometer analysis IONP-MMPsC-PEG or IONP-MMPnC-PEG nano particle
Change intensity and take 1ml IONP-MMPsC-PEG or IONP-MMPnC-PEG nanoparticle sample, is modulated with the HCL solution of 6mol/L
Black solid is precipitated in isoelectric point, and is washed repeatedly with deionized water 3 times, and 40 DEG C of vacuum drying obtain black powder, to be measured.
As a result as shown in Figure 2.
4. the preparation of NOTA-IONP-MMPsC-PEG
Utilize the NH of IONP surface residual2Chelatropic reaction is carried out with NOTA, synthesizes NOTA-IONP-MMPsC-PEG.Specifically
Method is as follows:
It weighs 50mg NOTA to be dissolved in 5mL water, 9.5mg EDC is dissolved in 1ml water, and the two is mixed.Use 0.1mol/L
PH value is adjusted to 5 by NaOH, reacts 10min.Reaction flask is moved in ice bath, 9mgNHS is added, then with 0.1mol/L NaOH by pH
Value is adjusted to 5.5, the reaction was continued 30min.NOTA:EDC:NHS weighs 25mg IONP-PEG-MMPsC for 1:1:0.8. and is dissolved in 3ml
It in water, is added in above-mentioned reaction solution, then reacting liquid pH value is adjusted to 7 with 0.1mol/L NaOH, reaction is overnight.After reaction,
It dialysed (MWCO=10000), be lyophilized, black solid NOTA-IONP-MMPsC-PEG is obtained, after bottling sealing at -20 DEG C
It saves.
⑤64The preparation of Cu-NOTA-IONP-MMPsC-PEG
It will64Cu is coupled to nano grain surface by NOTA, synthesis64Cu-NOTA-IONP-MMPsC-PEG is multi-modal to be received
Rice intelligent probe, referred to as MMPsCNP.The specific method is as follows:
It takes the NOTA-IONP-MMPsC-PEG of 5mg to be dissolved in 500 μ L ammonium acetate buffer solutions (pH=6.5), is added to 2ml
In EP pipe, add64CuCl2About 300MBq), it is vortexed and mixes, be incubated for 45min at 60 DEG C.Using be concentrated by ultrafiltration centrifuge tube from
The method of the heart is purified.Marker is moved to, centrifuge tube upper layer is concentrated by ultrafiltration, is centrifuged 10min under conditions of 5000r/min,
Discard lower liquid, upper layer residue washed respectively with 500 μ L 10mmol/L DTPA and 1mmol/L DTPA solution it is each primary,
Centrifugation, until the substantially "dead" activity detection of lower layer's filtrate, then collects upper layer residue.It is surveyed using instant thin-layer chromatography method
It is fixed64The mark rate and radiochemicsl purity of Cu-NOTA-IONP-MMPsC-PEG.It will64Cu is coupled to the surface IONP (Fig. 3 by NOTA
It is shown), product after purification by PD-10, by TEM, as a result be shown in using ZETA potential measurement surface charge by DLS measurement partial size
Fig. 4.It is saved after measurement, for testing in next step.
2 PET/MRI multi-mode molecular imaging nano-probe of embodiment is in the early diagnosis of atherosclerosis vulnerable plaque
Application
Method:
1, mouse carotid atherosclerosis model is established
C57BL/6J male mice (20-30g, 8-10 week old), after being adapted to environment 7 days in animal housing.The edible height of mouse
Rouge diet (35% fat, 1.25% cholesterol, 0.5% cholic acid, 63.25% basal feed) one month, is then subcutaneously injected chain
Urea mycin (150mg/kg) 5 days.Hereafter, give mouse ligation of carotid, continue to make for High-fat diet 3 months AS model (see
Fig. 5).Specific steps are as follows: mouse is anaesthetized with 1.5% yellow Jackets (0.1ml/20g).It is intubated and connects using No. PE-90
Mouse breathing machine.Mouse is fixed, and neck median incision is taken, and is hindered in left common carotid artery proximal part, internal carotid distal end 5-0 line
Clinopodium polycephalum ligatures external carotid artery distal end, opens an osculum in ligation of external carotid artery line proximal part with fine needle, then with elasticity seal wire
External carotid artery is punctured, seal wire pushes the ligation of seal wire across the through arteria carotis communis proximal part of aortic bifurcation after being sent into external carotid artery again
At line, rotate and push back and forth and exit seal wire seal wire four times, ligature the puncture orifice proximal part of external carotid artery, release arteria carotis communis and
The slip-knot of internal carotid sutures cervical incision.Gradin-echo (3D-SPGR) imaging technique is damaged using nuclear magnetic resonance, 3D,
Whether dissection and analysis and immunohistochemistry verifying mouse AS model foundation succeed, as a result as shown in fig. 6, illustrating mouse AS model structure
Build up function.
2, mouse carotid artery atherosclerosis plaques living body micro-PET/CT is imaged
Experiment mice is deprived of food but not water 12 hours before Micro-PET/CT scanning.Experimental rat is put into anesthesia case,
After several seconds of 5% short isoflurane induced anesthesia, scanning bed upper (the different fluorine of 1.5-2% of Micro-PET/CT is fixed in prone position
Alkane sucking maintains anesthesia), keep rat head long axis parallel with scanning bed long axis and is located in the scanner visual field.Wait anaesthetize completely
Afterwards, leading CT positioning determines scan position and range, scanning bed merging scan position as scanning (Scout).Through tail vein after
Tracer injection64About 100 μm of Ci/0.3ml of Cu-NOTA-IONP-MMPsC-PEG (MMPsCNP) are shown in that injection is shown rapidly when blood back
Track agent starts simultaneously at acquisition image.PET data acquisition energy window 250-700keV, list mode record storage, continuous collecting 1h,
2h, 4h, 12h, for 24 hours.Subsequent conventional Christmas CT image 5min.It keeps mouse to fix in entire scanning process, monitors mouse
Heartbeat and breathing avoid animal dead or position chanP occur because anaesthetizing deficiency.Dynamic image reconstruction: it is wanted according to quantitative analysis
It asks, the data of 1 hour type collection of PET is divided into 17 frames: 2 frames 30 seconds, 3 frame 60 seconds, 2 frame 180 seconds and 10 frames 300 seconds.17
Frame data use 3D-OSEM method reconstruction image after various corrections.Image array size 175 × 175 × 61, image voxel ruler
Very little 0.39 × 0.39 × 0.77mm.Image storage and output format are DICOM3.0.CT image reconstruction: method for reconstructing is
FeldKamp method, image array size 532 × 532 × 349,0.13 × 0.13 × 0.13mm of image voxel size.Image storage
And output format is DICOM3.0.CT image is converted to image array identical with PET image and voxel size when exporting,
MMPcNP nano-probe target rat aorta atherosis in MMPs PET/CT living body molecule imaging results as shown in fig. 7, from
When the result can be seen that 12h, there is novel nano probe to assemble in mouse carotid plaques, and the normal carotid artery vascular in opposite side
It is interior to assemble without obvious probe.
3, mouse carotid artery atherosclerosis plaques living body MRI is imaged
By 200 μ64Cu-NOTA-IONP-MMPsC-PEG (MMPsCNP) (1mg/ml Fe) probe enters neck through tail vein injection
In atherosclerosis Mice Body, respectively before the injection, MRI imaging is carried out respectively for 24 hours after injection.Mouse takes dorsal position,
It is advanced, the head of mouse and neck are placed in scanning area.At the heart body surface projection of mouse anterior pectorial region connect electrocardio to
It measures (VCG) and gates conducting wire, place respiration gate control in middle upper abdomen and detect respiratory movement.Scanner is Siemens Company 3.0T MR
Imaging system, mouse coil are unenhanced to use 3D-SPGR, T1-3D axial scan, T2-TIRM axial scan, SET2WI sagittal plain
Scanning, dynamic contrast enhancement use T1-3D axle position dynamic contrast enhancement.T1-3D axial scan sequence, TE/TR=4.7/8.6,
Thickness 1.2mm, the visual field (FOV) 340mm.T2 turbo inversion recovery magnitude sequence (TIRM), TE/TR=68/5500, thickness 4.0mm, depending on
Open country (FOV) 350mm.SET2WI uses fast spin echo (TSE) sequence row sagittal bit scan, TE/TR=68/3210, thickness
4.0mm, the visual field (FOV) 200mm.Analysis is further processed to image processing software after scanning and obtains dynamic enhancing curve.
Image is further processed and is analyzed by image processing software after scanning and obtains imaging data.It is weighted using T2 as 3D
Flash sequence, sweep parameter: TR:14.7ms, TE:4.6ms, FOV:2.5 × 2.5cm2, matrix: 256 × 256, flipangel
=20 °, slice thickness/slice spacing 0.5mm/0.5mm.
MRI image processing and data acquisition utilize the image processing workstations of Bruker 3.0T MRI system, to acquisition
Mouse arteria carotis MRI image is handled.And 20 width are respectively selected in the image of each group, arteria carotis blood is successively measured on the image
Signal strength (SI (wall) value), signal strength SI (muscle) value of vascular wall peripheral muscle tissue of tube wall, and tissue
Ambient noise signal SI (noise) value of outside.CNR (contrast to noise) value of every tomographic image is calculated, is calculated public
Formula is as follows: CNR=(SI (wall)-SI (muscle))/SI (noise) is successively compared and is visited according to resulting above-mentioned CNR value is calculated
The variation of the CNR value of needle injection front and back image, calculates the change rate of CNR value, and carry out statistical analysis, calculation formula is such as
Under: CNR changes%=(CNR36h post-CNR pre)/CNR pre × 100%.
MMPcNP nano-probe targets MRI living body molecule imaging results such as Fig. 8 institute of MMPs in rat aorta atherosis
Show, before can be seen that injection probe from the result, Ipsilateral carotid plaques cause obliteration, and magnetic resonance imaging is without Faxian
It is existing, after the novel and multifunctional probe MMPsCNP for injecting our synthesis, it is seen that T2 imaging is obvious in carotid plaques, prompt office
Portion has a large amount of Nanoscale Iron probe to assemble.In addition, control probe MMPsNCNP can not achieve carotid plaques T2 magnetic resonance imaging,
Illustrate characteristic of the probe with MMPs targeted imaging that the present invention synthesizes.
4, vascular tissue's immunofluorescence dyeing
Sodium citrate antigen retrieval buffers (pH=6.0) are prepared: 0.1M citric acid solution: 21.01g citric acid+1L distilled water；
0.1M citric acid three sodium solution: 29.41g citric acid three sodium solution+1L distilled water；0.1M citric acid solution 810ml+0.1M lemon
It is spare that three sodium solution 190ml of acid are made into 1000ml 0.1M citric acid antigen reparation liquid.Tissue paraffin section de is by dewaxing, gradient
After dehydration of alcohol, it is placed in sodium citrate antigen retrieval buffers and boils 30 minutes, then rinse 5min × 3 time with 0.01M PBS.It cuts
2%BSA is added dropwise on piece, is placed in 37 DEG C of closing 30min, filter paper in wet box and wipes extra BSA, do not wash.It is added dropwise on sample suitable
It measures mouse α-SMA primary antibody (1:200 dilution), places 4 DEG C of overnight incubations in wet box, 0.01M PBS rinses 5min × 3 time.It is marking
The goat anti-mouse secondary antibody (1:100 dilution) of appropriate TRITC label is added dropwise in sheet, places 37 DEG C of incubation 30min in wet box,
0.01M PBS rinses 5min × 3 time, is protected from light.Appropriate rabbit anti-mouse MMP-2 or MMP-9 primary antibody (1:200 is added dropwise on sample
Dilution), 4 DEG C of overnight incubations in wet box are placed, 0.01M PBS rinses 5min × 3 time, is protected from light.Appropriate FITC is added dropwise on sample
The goat antirabbit fluorescence secondary antibody (1:100 dilution) of label, places 37 DEG C of incubations 30min in wet box, and 0.01M PBS rinsing 5min ×
It 3 times, is protected from light.DAPI (1:1000 dilution) is added dropwise on sample and is incubated for 3-5min at room temperature, 0.01M PBS rinses 5min × 3 time,
It is protected from light.Drip few drops of anti-fluorescent quenching liquid on sample, covered is observed under Laser Scanning Confocal Microscope.As a result as shown in figure 9, from
The result can be seen that macrophage height in AS group patch and express, and the expression quantity of MMP-2 and MMP-9 are apparently higher than control group.
5, vascular tissue's prussian blue staining
The principle reacted according to iron ion prussian blue staining: iron ion, which encounters ferrous cyanogen root, can generate the blue of specificity
Ferric ferrocyanide precipitating.Therefore, have in the patch in order to verify vascular wall64Cu-NOTA-IONP-MMPsC-PEG
(MMPsCNP) assemble, we take out the vascular wall of mouse after injecting probe, carry out in vitro Prussian blue dyeing, specific to walk
It is rapid as follows: to prepare vascular tissue section, then carry out dewaxing treatment, slice is placed in I 10min of dimethylbenzene, then successively add again
Enter II 10min of dimethylbenzene, 95% ethyl alcohol, I 2min, 95% ethyl alcohol, II 2min, distilled water washs 10min × 2 time；Prepare Perl ' s
Liquid mixes isometric 2% hydrochloric acid and 10% potassium ferrocyanide, spare；Perl ' s liquid is added dropwise on sample, acts at room temperature
30min；5min × 3 time are washed with distilled water；Sample 3-5min is redyed with core fast red dyeing liquor, then washs 5 points with distilled water
Clock × 3 time；It is carried out dehydrating: sequentially adding 95% ethyl alcohol, I 1-2min, 95% ethyl alcohol, II 1-2min, 100% ethyl alcohol, I 1-
2min, 100% ethyl alcohol, II 2-5min；Sequentially add I 1min of dimethylbenzene, II 1min of dimethylbenzene；Neutral gum mounting, under microscope
Observation, takes pictures.As a result as shown in figure 9, can be seen that the aggregation for having a large amount of Nanoscale Iron in AS group patch from the result, with work
The result of body PET/CT imaging and MRI molecular imaging is coincide, and is synthesized from living body and the in vitro horizontal confirmation present invention novel
Nano-probe MMPsCNP has the expression for identifying MMPs in the athero- vulnerable plaque of artery in real time, dynamically, at many levels.
Claims (10)
1. a kind of PET/MRI multi-mode molecular imaging nano-probe for the early diagnosis of atherosclerosis vulnerable plaque,
It is characterized in that, the PET/MRI multi-mode molecular imaging nano-probe is with ferric oxide nanometer particle (abbreviation IONP) for core
The heart passes through NH on the surface of IONP2It respectively can with matrix metalloproteinase (matrix metalloproteinases, MMPs)
C-terminus and the NOTA connection of polypeptide MMPsC are sheared, the carboxyl of N-terminal amino and PEG that MMPs can shear polypeptide MMPsC connects
It connects,64Cu is coupled to the surface IONP by NOTA, and it is Ac- that wherein MMPs, which can shear the amino acid sequence of polypeptide MMPsC,
GPLGVRGKC-NH2。
2. a kind of method for preparing PET/MRI multi-mode molecular imaging nano-probe as described in claim 1, feature exist
In, comprising the following steps:
(1) matrix metalloproteinase (matrix metalloproteinases, MMPs) can shear the preparation synthesis MMPs of polypeptide
Polypeptide MMPsC, amino acid sequence can be sheared are as follows: Ac-GPLGVRGKC-NH2；
(2) preparation of MMPsC-PEG
Synthesis and the N-terminal amino that can shear polypeptide MMPsC of MMPs after purification are connected by the carboxyl of acylation reaction and PEG
It connects, then carries out purifying preparation MMPsC-PEG；
(3) preparation of IONP-MMPsC-PEG
Ferric oxide nanometer particle (IONP) is prepared using coprecipitation, the surface IONP is modified, by NH2It is introduced into IONP table
Face utilizes the NH on the surface IONP2It is connect, is made with the MMPs that PEG the is linked c-terminus that can shear polypeptide MMPsC by acylation reaction
The standby IONP with MMPsC-PEG modification, is named as IONP-MMPsC-PEG；
(4) preparation of NOTA-IONP-MMPsC-PEG
Utilize the NH of IONP surface residual2Chelatropic reaction is carried out with NOTA, synthesizes NOTA-IONP-MMPsC-PEG；
(5)64The preparation of Cu-NOTA-IONP-MMPsC-PEG
It will64Cu is coupled to the surface IONP by NOTA, synthesis64The multi-modal nana intelligent of Cu-NOTA-IONP-MMPsC-PEG is visited
Needle, referred to as MMPsCNP.
3. method according to claim 2, which is characterized in that synthesizing MMPs by solid-phase synthesis in step (1) can shear
Polypeptide MMPsC.
4. method according to claim 2, which is characterized in that PEG described in step (2) is PEG2000.
5. method according to claim 2, which is characterized in that prepare IONP using coprecipitation in step (3), utilize high temperature
Thermal decomposition method obtains monodispersed IONP.
6. method as claimed in claim 5, which is characterized in that specific steps are as follows:
I it) synthesizes the third copper and iron of triacetic acid: ferric chloride hexahydrate 270mg being dissolved in 12.5ml distilled water, is stirred continuously and is added
The acetylacetone,2,4-pentanedione of 500ml is added in the above mixed solution, reacts at room temperature by sodium acetate trihydrate 500mg
10min is heated to 80 DEG C, stands 20min and is placed in 4 DEG C of refrigerators, filter and clean after recrystallizing, obtains rose violet
The third copper and iron of triacetic acid of color flaky crystal；
II) the preparation of IONP: being heated to phenylate for the benzophenone of 100ml and be added in triangular flask, reacts in the protection of nitrogen, in magnetic force
It is gradually heated to 110 DEG C of heat preservation 2h under stirring, is then warming up to 280 DEG C of back flow reaction 1h again, room temperature is cooling, and is added 20ml's
Ethanol precipitation, hexamethylene, oleic acid and oleyl amine, ultrasound, which is added, in sediment makes it be uniformly dispersed, and is centrifuged 8000g × 30min, obtains
It obtains oiliness ferric oxide nanometer particle (IONP), it is spare.
7. method according to claim 2, which is characterized in that utilize the third aminotriacetic acid of 3- base silane in step (3)
(APTS) IONP is modified, by NH2Introduce the surface IONP.
8. method according to claim 2, which is characterized in that the MMPs that IONP and PEG is linked in step (3) can shear polypeptide
The connection of MMPsC follows the steps below:
EDC and the 1mg NHS of 1mg is added in 1ml deionized water, and 1mg MMPsC-PEG is added, it is molten to add 5ml IONP
Liquid is vigorously stirred lower reaction 12 hours, recycles deionization washing, and 13000rpm is centrifuged, and obtains IONP-MMPsC-
PEG product is purified using magnetization splitter.
9. PET/MRI multi-mode molecular imaging nano-probe described in claim 1 is in preparation atherosclerosis vulnerable plaque
Early diagnose the application in reagent.
10. application as claimed in claim 9, which is characterized in that the reagent be for PET/MRI multi-mode molecule at
Picture.
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