NANO LETTERS
Peroxidase Substrate Nanosensors for MR Imaging
2004 Vol. 4, No. 1 119-122
J. Manuel Perez,* F. Joseph Simeone, Andrew Tsourkas, Lee Josephson, and Ralph Weissleder MGH-Center for Molecular Imaging Research, HarVard Medical School, 149 13th Street, Charlestown, Massachusetts 02129 Received November 5, 2003; Revised Manuscript Received November 21, 2003
ABSTRACT Magnetic nanoparticle conjugates that self-assemble in the presence of peroxidases were prepared as having large inducible magnetic resonance signals. When a serotonin−nanoparticle conjugate was incubated with myeloperoxidase, an enzyme implicated with inflammation and coronary diseases, there were significant changes in the T2 relaxation times of the solution, as measured by MRI. These nanoparticle conjugates can potentially serve as an in vivo diagnostic tool to identify peroxidase-induced diseases by MRI.
One of the most immediate applications of nanotechnology in medicine is the development of biocompatible nanomaterials as environmentally sensitive sensors and molecular imaging agents.1,2 In particular, biocompatible nanoparticles with unique optical and/or magnetic properties could have important in vitro and in vivo diagnostic applications. The ability to image specific enzyme activities using such nanoparticles would have far reaching applications for detecting a variety of diseases and evaluating targeted therapies in individual patients. A number of different “sensing” imaging agents have already been described for MR imaging.3,4 These agents, however, use small molecular weight paramagnetic chelates which can have rapid clearance due to glomerular filtration in vivo and exhibit small relaxivity changes upon enzymatic conversion. Recently, superparamagnetic nanoparticle conjugates that reversibly self-assemble in the presence of a target have been described as magnetic nanosensors of molecular interactions.5 Sensing occurs after the target-induced assembly of the nanoparticles causes a decrease in the spin-spin relaxation times (δT2) of the solution, which can be easily detected by NMR or MRI instrumentations. Using this technique, nanoassemblies have been designed to act as substrates for proteases6 and restriction endonucleases.7 In these particular cases, sensing occurs after the peptides or oligonucleotides holding the magnetic nanoparticle together are cleaved by the corresponding enzyme, dispersing the nanoparticles in solution and therefore inducing an increase in T2. This format only allows screening of enzymes that cleave linkers (such as peptides and oligonucleotides) holding together the assembly of nanoparticles. However, for in vivo imaging applications it is desirable to develop configurations where disperse rather than assembled (clustered) nanoparticles serve 10.1021/nl034983k CCC: $27.50 Published on Web 12/18/2003
© 2004 American Chemical Society
as enzyme substrates. Although nanoparticle assemblies can avoid glomerular filtration, larger nanoparticle materials are rapidly cleared by the reticuloendothelial system, thereby limiting their ability to interact with specific molecular targets.8 Here we describe the development of a unique magnetic nanoparticle conjugate that self-assembles in solution by the action of specific peroxidases. This enzyme-mediated magnetic nanoparticle self-assembly would act as an MR signal amplification system, sensitive to the enzymatic activity of the peroxidase. For an initial proof of the concept, we used horseradish peroxidase (HRP), an enzyme widely used in bioassays, while as a clinically relevant target, we used myeloperoxidase (MPO), an enzyme implicated in atherosclerosis and inflammation.9,10 Amino functionalized dextran-caged superparamagnetic iron oxide nanoparticles were used as the starting material.11 In the present studies, either dopamine or serotonin was attached to create two sets of nanoparticle substrates for HRP or MPO, respectively. These phenolic agents (I, Scheme 1) were chosen as electron donors for the peroxidase-catalyzed reduction of hydrogen peroxide that results in tyrosyl radical (II, Scheme 1).12-15 In the specific case of MPO, such radicals have been found to create o,o-dityrosine cross-links between tyrosine in proteins and extracellular free tyrosine within atherosclerotic plaques.12 We hypothesized that peroxidase-induced formation of these tyroxyl radicals would cause o,o-dityrosine cross-links between the magnetic nanoparticles. The resulting self-assembly (III, Scheme 1) would result in detectable changes in MR signal. Dopamine or serotonin was conjugated to the aminated magnetic nanoparticles using suberic acid bis(N-hydroxysuccinimide ester (DSS, Pierce Co) as a linker. On average each nanoparticle
Scheme 1.
Peroxidase-Induced Self-Assembly of Magnetic Nanoparticles
had 40 reactive amino groups which were used for conjugation. Serotonin attachment was verified through its fluorescent emission at 345 nm. These nanoparticle conjugates were monodisperse in solution, having a narrow particle size distribution as determined by light scattering with an average particles size of 50 nm (Figure 1A). The water protons spinlattice relaxation (R1) of the nanoparticle conjugates was 25.8 s-1 mM-1 while the spin-spin relaxation (R2) was 67 s-1 mM-1. To test whether incubation of the nanosensors with the corresponding peroxidase would result in cluster formation,
Figure 1. Particle size distribution by light scattering of the dopamine nanoparticles before (A) and after (B) incubation with HRP. Similar results were observed when serotonin nanoparticles were incubated with MPO. 120
the dopamine nanoparticles (10 µg Fe/mL, 0.1 M phosphate pH 6.0) were incubated with HRP (0.9 units/µL) for 2 h at 4 °C. After this incubation period, cluster formation was readily detectable by light scattering (Figure 1B). As expected, no cluster formation occurred in the absence of H2O2. The nanoclusters were stable in aqueous solution, did not continue growing in size, and did not precipitate. Similar results were observed when serotonin nanoparticles were incubated with myeloperoxidase. Next, we investigated whether the peroxidase-mediated clustering would result in T2 relaxation time changes (δT2) of the solution. For these experiments, a solution of the HRP targeting nanosensor (10 µg Fe/mL, 0.1 M phosphate pH 6.0) was incubated with different amounts of HRP (0-0.9 units/µL) for 2 h at 4 °C and the T2 relaxation times were measured at 0.47 T. Increasing δT2 values were observed upon incubation with increasing amount of HRP, reaching saturation at a concentration of 0.9 units/µL in this specific experiment (Figure 2). No changes in T2 were observed in samples incubated with HRP in the absence of H2O2. To further confirm that the detectable changes in T2 are caused by an HRP-mediated mechanism, experiments were performed where increasing the amount of sodium azide, a known inhibitor of peroxi-
Figure 2. Effect of increasing HRP concentration on the δT2 of a solution containing dopamine nanoparticles with (9) and without (1) hydrogen peroxide. Nano Lett., Vol. 4, No. 1, 2004
Figure 3. Effect of increasing amount of sodium azide (inhibitor) on the δT2 of a solution containing dopamine nanoparticles with hydrogen peroxide.
dases, was added to the solution. As expected, sodium azide inhibited HRP activity and reduced the δT2 changes in a concentration-dependent manner (Figure 3). T2 changes did not occur in other control experiment using heat- or SDSdenatured HRP. The above results confirm that the observed changes in δT2 were indeed HRP-specific and that these nanosensors can thus potentially be used as nanosensors for peroxidase activity. To test the above-described nanotechnology as a potentially useful imaging tool to detect a clinically important peroxidase, we examined the ability of our method to image myeloperoxidase (MPO) activity using a 1.5T clinical MRI imaging system. Recent studies have demonstrated the importance of MPO in the development of inflammation and cardiovascular diseases such as atherosclerosis and myocardial infarction.9,10 High levels of intracellular MPO content have been found in plasma samples from patients with coronary heart disease and acute coronary syndromes,9,10 while other studies implicate MPO as one of the pathways for the oxidation of low-density lipoprotein in the artery wall.16-18 Of particular interest is the observation that an increased number of MPO-expressing macrophages can occur in eroded or ruptured plaques causing acute coronary syndromes.19 We therefore designed a serotonin-nanoparticle conjugate, as serotonin is a superior substrate for myeloperoxidase compared to dopamine.20,21 The serotonin-nanoparticles (3 µg Fe/mL, 0.1 M phosphate pH 6.0) were incubated with various amounts of myeloperoxidase with and without H2O2 in a 384 well-plate and imaged by MRI as previously described.22 Similar to HRP, δT2 increased as a function of MPO concentration (Figure 4A). Furthermore, we were able to demonstrate that δT2 changes were of significant magntitude to be detectable using a clinical MR imaging system (Figure 4B). Control samples consisting of MPO-nanosensors incubated with myeloperoxidase in the absence of H2O2 showed no significant increase in δT2 as expected. Likewise, the dopamine nanoparticle described above as a sensor for HRP did not show any δT2 in the presence of MPO (Figure 5). The findings of enzyme-specific selectivity are particularly important as they open up the possibility to develop more selective nanoparticle substrates for other clinically relevant peroxidases. Nano Lett., Vol. 4, No. 1, 2004
Figure 4. (A) δT2 values of the serotonin nanoparticles in the presence of increasing amounts of myeloperoxidase (9) detected using a 1.5T clinical MRI. As a control, samples were incubated in the absence of hydrogen peroxide (1). (B) Corresponding MR image of myeloperoxidase activity at 1.5T.
Figure 5. MR image of myeloperoxidase activity using serotonin nanoparticles and dopamine nanoparticles. Note that no difference in signal intensity is observed when dopamine nanoparticles are incubated with myeloperoxidase.
In summary, we demonstrate that magnetic nanoparticle conjugates can act as sensitive and selective nanosensors for peroxidase enzyme activity. Immobilization of dopamine or serotonin to the magnetic nanoparticles has allowed for sensitive and direct detection by MRI of horseradish peroxidase and myeloperoxidase activity, respectively. Distinct features of the developed nanoparticle conjugates are that they remain monodisperse in solution until they self-assemble in response to a specific peroxidase enzymatic activity and that the process is detectable by MR imaging. Furthermore, by changing the ligand substrates covalently attached to the magnetic nanoparticle, other peroxidase associated with disease could be targeted. We envision the use of libraries of phenolic substrates attached to such nanoparticles to screen by high throughput NMR methods23-24 for numerous per121
oxidases. Such materials may also have biotechnology applications for in vitro assays as well as for clinical in vivo imaging. More importantly, related iron oxide nanoparticles have been used in recent clinical trials as MRI contrast agents25 and are biocompatible. While additional feasibility studies will have to be carried out to validate in vivo uses, we believe that the described properties make the proposed magnetic nanosensors a potentially attractive in vivo diagnostic tool to identify peroxidase-induced diseases by MRI. Acknowledgment. We gratefully acknowledge partial support through the National Cancer Institute by a P50 Center Grant to R.W. (CA86355) and a Career Award to J.M.P. (CA101781). References (1) Emerich, D. F.; Thanos, C. G. Expert Opin. Biol. Ther. 2003, 3, 655663. (2) Bogunia-Kubik, K.; Sugisaka, M. Biosystems 2002, 65, 123-138. (3) Bogdanov, A., Jr.; Matuszewski, L.; Bremer, C.; Petrovsky, A.; Weissleder, R. Mol. Imag. 2002, 1, 16-23. (4) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats, R.; Jacobs, R. E.; Fraser, S. E.; Meade, T. J. Nature Biotechnol. 2000, 18, 321-325. (5) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.; Weissleder, R. Nature Biotechnol. 2002, 20, 816-820. (6) Zhao, M.; Josephson, L.; Tang, Y.; Weissleder, R. Angew. Chem. Int. Ed. Engl. 2003, 42, 1375-1378. (7) Perez, J. M.; O’Loughin, T.; Simeone, F. J.; Weissleder, R.; Josephson, L. J. Am. Chem. Soc. 2002, 124, 2856-2857. (8) Iannone, A.; Magin, R. L.; Walczak, T.; Federico, M.; Swartz, H. M.; Tomasi, A.; Vannini, V. Magn. Reson. Med. 1991, 22, 435442.
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NL034983K
Nano Lett., Vol. 4, No. 1, 2004
