MR Molecular Imaging of Thrombus:Development and Application of a Gd-bad Novel Contrast Agent Targeting to P-lectin
一建准考证打印
Xue-Feng Wang,MD,Pei-Pei Jin,MS,Tong Zhou,MD,
英文职位名称
Ya-Peng Zhao,MS,Qiu-Lan Ding,PhD,Deng-Bin Wang,MD,Guang-Ming Zhao,PhD,Jing-Dai,MS,
Hong-Li Wang,MD,and Hai-Liang Ge,PhD
dbkMolecular imaging of thrombus formation at initial stage requires a robust thrombus-specific contrast agent with high nsitivity.In this study,we report a novel P-lectin-targeted paramagnetic molecular ima-ging agent and the agent’s potential to nsitively detect occult microthrombi on the intimal surface of endo-thelium.Platelet clots and blood clots targeted in vitro with paramagnetic nanoparticles prented a highly detectable,homogeneous T1-weighted contrast enhancement that was improved with increasing gado-linium level.In vivo contrast enhancement under part of circulation conditions was assd in dogs.The
micro-thrombi around the femoral vein of dog demon-strated higher signal intensities than the control clots and the adjacent muscle.Histology was performed on regions likely to contain thrombus as indicat
英语四级网ed by MRI.The results suggest that molecular imaging of P-lectin-targeted paramagnetic nanoparticles can provide nsitive detection and localization of P-lectin and may allow for early,direct identification of micro-thrombi,leading to early diagnosis.
Keywords:molecular;thrombosis;magnetic reso-nance imaging;P-lectin
Introduction
Venous thromboembolism (VTE),manifested as either deep venous thrombosis (DVT)or pulmonary embolism (PE),is a common medical problem occur-ring either in isolation or as a complication of other dias or medical procedures.1The hemostatic sys-tem is made up of 2distinct but interlocking components:platelets and the coagulation proteins.Multiple factors and events must occur in a highly
regulated fashion for a blood clot to form.P-lectin is one of the factors.2,3Activation of platelets or impaired endothelium leads to the exposure of P-lectin on the surface of platelets,and simultaneously endothelial cells degranulate with the rapid translocation of P-lectin to the plasma membrane.4-7Accumulating lines of evi-dence now invoke a role for P-lectin and its ligand to accumulate tissue factor and subquent generate fibrin within the platelet thrombus.
Although a variety of invasive imaging modalities such as intravascular ultrasound elastography,8x-ray angiography 9have been ud for imaging thrombus,magnetic resonance imaging (MRI)has emerged as the most promising noninvasive imaging technique for the in vivo detection and characterization of thrombogenesis.10,11
Recent advances in the field of molecular imaging have led to the development of novel paramagnetic
From the Ruijin Hospital (X-FW,P-PJ,TZ,Y-PZ,Q-LD,D-BW,G-MZ,J-D,H-LW),and Shanghai Institute of Immunology (H-LG),Shanghai Jiao Tong University School of Medicine,Shanghai,People’s Republic of China.
Address correspondence to:Hai-Liang Ge,Shanghai Institute of Immunology,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,People’s Republic of China;e-mail:gehl@shsmu.edu.177
Clinical and Applied Thrombosis/Hemostasis Volume 16Number 2March/April 2010177-183#2010SAGE Publications 10.1177/1076029608330470
and super-paramagnetic-targeted MR contrast agents that bind exclusively to molecules such as albumin, fibrin,or angiogenesis markers.12,13P-lectin is expresd abundantly in both the activated platelets and stimulated endothelial cells and it is also the molecule marker in this study.P-lectin is a140kd transmembrane glycoprotein,and lectin-epidermal growth factor(L-EGF)domain of P-lectin is the critical unit for leukocyte binding.14-16Therefore, we developed a monoclonal antibody targeting to L-EGF domain and developed a novel MR contrast agent conjugated with the antibody.Becau of the inherently low nsitivity of MR contrast enhance-ment technology,a relatively high target molecule concentration is necessary for adequate signal ampli-fication.17The contrast agent for molecular imaging is required to allow for enhanced,nsitive detection and quantification of occult microthrombi within the intimal surface of endothelium,and provide direct, early,and specific evidence for thrombus diagnosis.
The objective of the current study was to demon-strate the potential of a P-lectin targeted,paramag-netic nanoparticulate contrast agent to specifically image thrombus deposits both in vitro and in vivo at a clinically relevant magnetic field strength(1.5T). We evaluated the capability of the novel contrast agent to transport high payloads of gadolinium diethylene-triamine-pentaacetic acid(Gd-DTPA) and estimated the T1relaxivities relative to particle number,reflecting their utility as a targeted mole-cular imaging.
Methods
(Gd-DTPA)n-Bovine Serum Albumin
Preparation
Bovine rum albumin(BSA)50mg was dissolved in 2.5mL0.2mol/L NaHCO3/Na2CO3buffer (pH9.6),then Gd-DTPA(prepared by Department of Radiopharmacy,Pharmacy College of Fu Dan University)was added while stirring in ice bath.The reaction reached the end point after stirring for 20hours at room temperature.The(Gd-DTPA)n-BSA was parated and purified by SephadexG-25(Sigma Chemical Co.,St.Louis,MO,USA)with0.01mol/L phosphate buffered saline(PBS;pH7.4)as run-ning buffer.The mixture solution of condend (Gd-DTPA)n-BSA and excess Gd chloride was stirred overnight at room temperature.Unbound Gd was removed by SephadexG-25as above.
Preparation of a PsL-EGFmAb-BSA4-
(Gd-DTPA)n MRI Contrast Agent
Anti-human-P-lectin lectin-EGF domain monoclo-nal antibody(PsL-EGFmAb)was produced and puri-fied by the Department of Nephrology,Ruijin hospital,Shanghai Jiao Tong University School of M
edicine.The antibody was diluted to a final concen-tration of1.5mg/mL in0.1mol/L NaHCO3/Na2CO3 buffer(pH9.6).After adding(Gd-DTPA)n-BSA[0], the solution was left to stir overnight at room temperature.PsL-EGFmAb-BSA4-(Gd-DTPA)n were parated and purified by Sepharo CL-4B(Sigma). It was filtered by0.22m m aptic filtration,con-dend,and stored at4 C.
Properties of PsL-EGFmAb-BSA4-
(Gd-DTPA)n MRI Contrast Agent
The concentration of Gd in contrast agent was measured by inductively coupled plasma-atomic emis-sion spectrometry(ICP-AES),and the purity of con-trast agent was analyzed by high-performance liquid chromatography(HPLC).The number of Gd com-plexes per nanoparticle was calculated from the ratio of the concentrations of Gd and nanoparticles in the emulsion.
The immunocompetence of contrast agent was analyzed by competitive enzyme-linked immuno-sorbent assay(ELISA),using a P-lectin(R&D Systems,Minneapolis,MN,USA)as a coating antigen, PsL-EGFmAb-BSA4-(Gd-DTPA)n,and horradish peroxida–conjugated PsL-EGFmAb as competi-tive antibody.
PsL-EGFmAb-BSA4-(Gd-DTPA)n,PsL-EGFmAb-BSA3-(Gd-DTPA)n,BSA-(Gd-DTPA)n,and Gd-DTPA were imaged in MRI with multiple proportion(double-distilled[dd]H2O as control).
Magnetic Resonance Imaging of Targeted Clots In Vitro
In vitro imaging was carried out by2parate experi-ments.Platelet clots and blood clots were parately produced by combining plasma with100mmol/L CaCl2(3:1vol/vol)and5U thrombin(Dade Behring, Marburg,Germany).In preparing the platelet clots, 80,60,and40m L contrast reagents were added into 500m L platelet-rich plasma;80m L Gd-DTPA and80 m L PBS were added to the same volume plasma as neg-ative control and blank,respectively.Clots werecoating
178Clinical and Applied Thrombosis/Hemostasis/Vol.16,No.2,March/April2010
rially incubated1hour at37 C to complete the binding and were rind3times with sterile saline after incubation to remove any unbound reactants.
In cond in vitro imaging experiment,150m L contrast reagent was added to1mL blood.The con-trol was the same as above.Targeted human blood clots that were suspended in blood in plastic tubes were imaged.Contrast-to-noi(CNR)between the clot and blood was calculated as the difference of
the signal intensity(SI)between a region of interest within the targeted clot and a region of interest in a control clot or the surrounding liquid.
Animal Model of Venous Thrombosis
The concept of the P-lectin–targeted,paramag-netic nanoparticles to enhance the detect ability of clots in a flowing intravascular environment was evaluated in dogs.Six dogs(%12kg)were pretreated with tranexamic acid(0.25g/h)to inhibit endogen-ous thrombolysis.Each animal was anesthetized (sodium pentobarbitone)and prepared for surgery. The left femoral vein of animal was expod.Injury was performed at the femoral vein with a variable length of0.5to1.5cm in a random manner.Nylon monofilament(4-0)was ud to destroy the endo-thelium.Approximately5minutes after repeated rial vesl damage,flow was reestablished.Injury was always performed with the same Nylon mono-filament and by the same operator to minimize variability.
Magnetic Resonance Imaging of Targeted Clots In Vivo
After operation,2mL 1.5mg/mL(Gd-DTPA)n-BSA4-PsL-EGFmAb or Gd-DTPA(as control)was infud into the blood vesl of each dog.Becau the P-lectin was expresd abundantly on the acti-vated platelets and stimulated endothelial cells,the contrast agent was acquired bolus injection a
s soon as the operation finished.Dogs were dated with sodium pentobarbitone(as described earlier)and were imaged in a supine position with a1.5-T MRI system.The injured femoral vein and the contralat-eral normal femoral vein were imaged30minutes after bolus injection.Two additional dogs were ud to compare P-lectin–targeted contrast agent and a conventional Gd-DTPA for thrombus detection.
Magnetic Resonance Imaging
Experiments
The imaging parameters of MRI in vitro were the same as in vivo.All imaging experiments were performed with a1.5-T clinical MR imager(Echo Twin-speed by General Electric Inc,Fairfield,CT,USA)with a standard surface coil(3inch).Canine thrombi created within the femoral vein underwent MR imaging using 3-dimensional fat-suppresd with T1-weighted fast spoiled gradient echo(3D-FSPGRE)quence and 3D-pha-contrast FSPGRE angiogram.Parameters for3D-FSPGRE were repetition time(TR):500ms, echo time(TE):20ms,field of view(FOV): 120mm,matrix:256Â256,slab thickness:40mm, number of partitions:40,and number of excitation (NEX):2.Parameters for3D-pha-contrast FGRE angiogram were TR:15ms,TE:5.3ms,flip angle: 15 ,FOV:200mm,matrix:192Â256,slab thick-ness:36mm,number of partitions:40,and NEX:1.
Histopathology
Thrombus detection was performed by gross histolo-gical analysis of the ctioning and immunohistologi-cal analysis.All ctions(slice thickness¼5m m, every3mm,cryostat ctioned)of the femoral vein were stained with hematoxylin and eosin and assd visually for the prence or abnce of thrombus.
Results
Properties of PsL-EGFmAb-BSA4-(Gd-
DTPA)n MRI Contrast Agent
The concentration of Gd in(Gd-DTPA)n-BSA4-PsL-EGFmAb is44mg/kg(molecular weight of Gd is 157.25),detected by ICP-AES.We calculate that1 PsL-EGFmAb binds with70to100Gd.The competi-tive ELISA results show that the immunocompetence of contrast agent is consistent when PsL-EGFmAb binds with4(Gd-DTPA)n-BSA(Figure1).Through the BSA,PsL-EGFmAb can bind280to400Gd,which do not affect the immunocompetence of PsL-EGFmAb. The purity level of(Gd-DTPA)n-BSA4-PsL-EGFmAb was98%,determined by HPLC.The capacity of the nanoparticl
es to support high paramagnetic payloads is of paramount importance to the efficacy of the agents when they are ud for molecular imaging of biochemical epitopes.
MR Molecular Imaging of Thrombus/Wang et al179
法律英语Relaxivities of the Paramagnetic Nanoparticles
T1relaxivities of the PsL-EGFmAb-BSA 4-(Gd-DTPA)n paramagnetic formulation determined at the 3fixed magnetic field strengths clearly reveal the superior per-formance of the PsL-EGFmAb-BSA 4-(Gd-DTPA)n nanoparticles.At 1.5T,a typical medical imaging field strength,PsL-EGFmAb-BSA 4-(Gd-DTPA)n ,PsL-EGFmAb-BSA 3-(Gd-DTPA)n ,BSA-(Gd-DT-PA)n ,and Gd-DTPA were imaged in 12points (dd H 2O as control).PsL-EGFmAb-BSA 4-(Gd-DTPA)n showed a stronger signal at the same concentration of the anti-body (Figure 2).From the Figure 2,we found that high concentration of contrast agent has no signal.However,as the solution incread in concentration,the strength
of SI incread to a certain level before dropping off.The 1:64diluted contrast agent has the most SI,and the SI starts to decrea from 1:128dilution.
PsL-EGFmAb-BSA 4-(Gd-DTPA)n MRI for Thrombus Detection In Vitro
admonishMagnetic resonance imaging results demonstrated the significant contrast enhancement produced by the P-lectin–specific nanoparticles targeted to human platelet clots in vitro at clinically relevant field strength (1.5T).With a typical low-resolution clinical imaging protocol,the platelet clots targeted with nanoparticles prented a homogeneous T1-weighted contrast enhancement,which improved by increasing Gd level (Figure 3).The amount of Gd-DTPA was not associated with a significant improvement in throm-bus detection,as confirmed by CNR values.Relative SI and normalized CNR were significantly incread at different quantity of contrast reagent (P <0.01).The SI of P-lectin–specific nanoparticles targeted to human blood clots is higher than control,and the appearance of human blood clots can be easily en (Figure 4).
PsL-EGFmAb-BSA 4-(Gd-DTPA)n MRI for Thrombus Detection In Vivo
The magnitude of contrast enhancement expected in vivo with part of circulation conditions was evaluated in dogs.Control or 2mL 1.5mg/mL PsL-EGFmAb-BSA 4-(Gd-DTPA)n was targeted in situ as a single-step system to thrombus created within the femoral vein.Thrombus was imaged with 3D T1-weighted,fat-suppresd,fast-gradient echo quence,and the results showed that the targeted clot was markedly enhanced by the
P-lectin–specific
Figure 2.Magnetic resonance imaging of contrast
media.
Figure 3.
Magnetic resonance imaging of the platelet clots (cross ction).A,Blank:phosphate buffered saline solution;B,Control:Gd-DTPA solution;C-E,80,60,and 40m L 1.5mg/mL (Gd-DTPA)n -BSA 4-PsL-EGF mAb.
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paramagnetic nanoparticles compared with control thrombus (Figures 5A and B).
Corresponding gradient-echo images revealed a lective enhancement of the treated clot,yielding higher SI than the control clot and the adjacent muscles,but lower than the bright fat signal.On T1-weighted gradient-recalled-echo images with fat suppression,the targeted clot showed the higher SI.
Thirty minutes after the administration of PsL-EGFmAb-BSA 4-(Gd-DTPA)n ,signal enhancement in the thrombus around the femoral vein was obrved (Figure 5A),with good differentiation from arterial blood and thrombus-free vesl wall.As expected,no change was en in the uninjured veins (controls).
Visible agreement in location was found with histopathology (Figure 6).P-lectin immunostaining of the excid vesl and clot confirmed the abun-dance and localization of P-lectin corresponding to the contrast enhancement in vivo.
Discussion
In this study,we demonstrated the novelty and superiority of contrast-enhanced MRI with a P-lectin
ltp–targeted contrast agent in vitro and in vivo for thrombus detection at the early stage of coagula-tion.This unique agent can carry high Gd-DTPA pay-loads for high detection nsitivity and is different from the fibrin-target contrast agent that was reported.18
The latter aimed directly at the fibrin,which is the product of blood clotting;however,PsL-EGFmAb-BSA4-(Gd-DTPA)n aimed at the P-lectin,which was expresd abundantly on the surface of activated platelets and stimulated endothelial cells.The fibrin deposition in venous thrombi is associated with the expresd P-lectin,19,20and P-lectin plays a signif-icant role of thrombus formulation.21Therefore,the PsL-EGFmAb-BSA4-(Gd-DTPA)n can be ud for thrombus detection at an early stage.
Sensitive MR molecular imaging of micro-thrombi requires a high-avidity,thrombus-specific MR contrast agent with high paramagnetic impact.Using the BSA to formulate the Gd polymer,22,23we reported that the contrast agent can bear 280to 400Gd molecules.In molecular imaging applica-tions,the paramagnetic influence of each binding complex is critical.Magnetic resonance imaging of PsL-EGFmAb-BSA4-(Gd-DTPA)n showed that remarkable SI and platelets clots or blood clots imaged at 1.5T are detectable despite the expected resolution limitation.The SI was not affected by the accumulation of fibrin but was enhanced with the increasing amount of P-lectin–targeted contr
ast agent.PsL-EGFmAb-BSA 4-(Gd-DTPA)n produced information on thrombus formulation at early time,highlighting the P-lectin–rich areas,activation pla-telets,and stimulated endothelial cells.
To confirm the conjugation with activated platelets and stimulated endothelial cells in vivo,we injected PsL-EGFmAb-BSA 4-(Gd-DTPA)n to parts of the femoral vein in the dog.The microthrombi around the femoral vein of canine demonstrated higher signal intensities than the normal control and the adjacent muscle,but lower than the bright fat signals.It may be due to the insufficient level of PsL-EGFmAb-BSA 4-(Gd-DTPA)n injection.How-ever,the experiment results demonstrated that PsL-EGFmAb-BSA 4-(Gd-DTPA)n can efficiently combine with activation platelets and stimulated endothelial cells in vivo,showing the specificity of imaging.
The development of a stable,targeted MRI contrast agent prents a wide variety of the opportu-nities for improving diagnosis.Unlike PsL-EGF-mAb-BSA 4-(Gd-DTPA)n ,Gd-DTPA is a nonspecific contrast agent that dispers to the intravascular and interstitial spaces by diffusion.24It does not allow for thrombus enhancement.Interestingly,no uptake of Gd-DTPA within the thrombus was noted at any imaging time in our study,in contrast to the peri-vascular muscle enhancement due to
Gd-DTPA
Figure 4.
Magnetic resonance imaging of human blood clots (longitudinal ction).
MR Molecular Imaging of Thrombus /Wang et al 181
diffusion.Therefore,PsL-EGFmAb-BSA 4-(Gd-DTPA)n provides an effective way to diagno early throm-bus formations.
Becau thrombosis is a dynamic process,with thrombus material of different ages forming a layered structure due to successive mural deposition,P-lectin–targeted,contrast-enhanced MRI may be ud to diagno thrombi only at the stage of plate-lets activated.Acute coronary syndromes,stroke,deep vein thrombosis complicated by PE,and
arterial fibrillation with a high incidence of various cardiac manifestations result from thrombus forma-tion in different vascular beds after either plaque rupture/erosion or incread blood thrombogeni-city.25,26Therefore,a noninvasive modality with a P-lectin–targeted contrast agent might be ideal for in vivo detection of all thrombi formation at an early-stage.In addition,this novel co
ntrast may benefit patients with tendency of DVT and facilitate detec-tion of hypercoagulabale state at an early stage.The enhancement of P-lectin expression in the rup-tured blood vesls with targeted paramagnetic nanoparticles was readily apparent,in contradistinc-tion to the control specimens.Similar early diagnosis of ruptured intimal and/or activated platelet P-lectin expression in a patient with thrombosis may someday prompt early anticoagulant therapy.
In summary,we reported a novel P-lectin–targeted paramagnetic molecular imaging contrast agent that enhances the nsitivity of in vitro and in vivo clots detected by MRI.This unique agent,becau of its strong MR contrast impact,has the ability to enhance the detection of intravascular clots.Emerging molecular MR imaging technolo-gies,such as tho with paramagnetic nanoparticles,may provide early direct diagnosis of thrombus in patients with heralding symptoms and facilitate early therapeutic intervention.However,the agent we reported here may have a limitation in detecting thrombosis which is not mediated by
P-lectin.
Figure 5.
A,Axial magnetic resonance imaging of thrombi in femoral vein targeted with P-lectin–specific paramagnetic nanoparticles demonstrating dramatic T1-weighted contrast enhancement in gradient-echo image (arrow)on left without flow deficit (arrow)of thrombus.Note the benefit of using the P-lectin–targeted MR contrast agent for thrombus detection.Thrombus around the vesl wall has higher signal than the adjacent muscle.Accurate detection and localization of the thrombus were suc
unesco是什么意思cessful in nonocculsive thrombi.B,Control thrombus in contralateral femoral vein imaged as in
A.
Figure 6.Results of immunohistological analysis.
182Clinical and Applied Thrombosis/Hemostasis /Vol.16,No.2,March/April 2010sleek
