Protease-Specific Nanosensors for Magnetic Resonance Imaging

Nov 14, 2008 - On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles ...
1 downloads 0 Views 980KB Size
Download PDF
2440

Bioconjugate Chem. 2008, 19, 2440–2445

Protease-Specific Nanosensors for Magnetic Resonance Imaging Eyk Schellenberger,* Franziska Rudloff, Carsten Warmuth, Matthias Taupitz, Bernd Hamm, and Jo¨rg Schnorr Department of Radiology, Charite´ - Universita¨tsmedizin Berlin; Charite´platz 1; 10117 Berlin, Germany. Received August 1, 2008; Revised Manuscript Received October 27, 2008

Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in ViVo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in ViVo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity.

INTRODUCTION The advent of fluorescent smart sensor probes activatable by proteases ignited the development of reporter probes (1) and instrumentation (2) establishing optical imaging as the modality of choice for in ViVo detection of enzyme activity (3). To take advantage of the high resolution and deep-tissue imaging capabilities of magnetic resonance imaging (MRI), suitable for whole-body imaging of humans, as well as the absence of radiation exposure, several new probe designs were introduced. Meade and co-workers developed a reporter probe activatable by β-galactosidase on the basis of a paramagnetic contrast agent and demonstrated its successful use for the visualization of gene transfer by MRI (4). A paramagnetic polymerization probe was presented by Bogdanov et al., which allowed imaging of tissue peroxidase activity by MRI (5). Josephson and co-workers introduced magnetic relaxation switches sensing enzymatic activity based on superparamagnetic high-relaxivity probes for in Vitro MRI (6). Upon protease activation, these sterically stabilized crosslinked iron oxide particles (CLIO) switch from large clustered high-relaxivity aggregates to smaller low-relaxivity single particles. This technique is not suitable for in ViVo application, since the nonactivated clustered particles are too large to have a favorable bioavailability and, more importantly, activation by elevated enzyme activity in the target tissue reduces the relaxivity and consequently the contrast effect. Here, we describe a new design of protease-sensitive nanosensors, which are based on electrostatically stabilized, citratecoated very small iron oxide particles (VSOP) with a hydrodynamic diameter of 7.7 ( 2.1 nm (Figure 2a) instead of larger sterically stabilized nanoparticles (over 20 nm). We have previously developed a pharmacologically formulated variant of these superparamagnetic particles (VSOP-C184) for magnetic resonance angiography, which was successfully tested in a clinical phase 1 trial and currently undergoing clinical phase 2 testing (7). As cleavage motif, we chose a recognition site for MMP-9 and MMP-2, which was previously used to prepare a MMP-9activatable fluorescent probe (8, 9). MMPs have been identified * E-mail: [email protected].

as important regulators of pathological remodeling of extracellular matrix in cancer and inflammation (10). Using fluorescent smart probes, Bremer et al. developed a new concept to monitor the success of MMP inhibition in cancer by in ViVo imaging, which may ultimately enable monitoring of therapeutic responses in the large spectrum of diseases with alterations of the extracellular matrix (11).

EXPERIMENTAL PROCEDURES Synthesis. (1) The synthesized fluorescein-labeled MMP-9 peptide NH2-GGPRQITAG-K(FITC)-GGGG-RRRRR-G-RRRRRamide (the italicized amino acids correspond to the MMP-9 substrate (9)) was confirmed by MALDI-TOF (MALDI 2, Shimadzu). The molecular weight was within 1 Da of the expected value. 1 mg of the MMP-9 peptide (in 100 µL DMSO) was reacted with 31 mg NHS-mPEG (O-[(N-succinimidyl)succinyl-aminoethyl]-O′-methyl-poly(ethylene glycol) 5000, Fluka) in 300 µL 0.1 M HEPES, pH 7.5, overnight at 4 °C. The resulting MMP-9-peptide-mPEG copolymer was purified by gel filtration with BioGel P6 (BioRad, in 10 mM HEPES, 140 mM NaCl, pH 7.5). The absence of unreacted MMP-9 peptide was confirmed by HPLC using a reverse-phase C18 column with acetonitrile/water gradient. The concentration of the peptidemPEG copolymers was determined spectrophotometrically by measuring peptide-bound fluorescein absorption (absorption at 494 nm, extinction coefficient of 72 000 M-1 cm-1). (2) VSOP (VSOP-C200) was provided by Ferropharm GmbH. To prepare 6×-MMP-9-PSOP (with six peptide-mPEG copolymers per VSOP), 1.25 nmol VSOP (3.25 µmol Fe assuming 2600 iron atoms per particle, data from Ferropharm GmbH) in 250 µL HEPES buffer (10 mM HEPES, 140 mM NaCl, pH 7.5) were mixed with 7.5 nmol peptide-mPEG in 250 µL HEPES buffer and stirred immediately. Other ratios (3 to 16) were prepared accordingly. Hydrodynamic diameters of MMP-9PSOPs were determined by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments) in the same buffer. Relaxivities were measured with an MR spectrometer at 0.94 T (Bruker, Minispec MQ 40) according to the manufacturer’s instructions. For quenching experiments (Figure 3), 50 pmol VSOP was mixed with different amounts of the peptide-mPEG copolymers (150 pmol to 800 pmol) in 200 µL and subjected to fluorescence

10.1021/bc800330k CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

Protease Nanosensors for MRI

Bioconjugate Chem., Vol. 19, No. 12, 2008 2441

Figure 1. Synthesis and function of MMP-9-PSOP. (a) Synthesis. In step (1), the 25 amino acid peptides consisting of an arginine-rich coupling domain and a cleavage domain with the recognition site for MMP-9 linked by a glycine bridge are reacted with NHS-methyl-poly(ethylene glycol) (NHS-mPEG). The resulting peptide-mPEG copolymers are purified by gel filtration to remove unreacted mPEG and NHS. In step (2), peptidemPEG are mixed with VSOPs, yielding MMP-9-PSOP. (b) Function. When sterically stabilized PSOP with an intact mPEG shell (1) are exposed to a protease specific to the cleavage motif, the peptide-mPEG are cut at the cleavage domain, resulting in a loss of sterical stabilization (2). The remaining particles aggregate due to the magnetic attraction of the iron oxide cores and the electrostatic attraction of the positive (arginine-rich coupling domain) and negative charged surface areas (acid shell).

measurements (excitation/emission 494/521 nm, Hitachi fluorescence spectrophotometer F-7000). Ratios of these quenched fluorescences and corresponding unquenched fluorescences of equal copolymer concentrations without VSOP addition were calculated and compared with the fluorescence of free fluorescein dye mixed with VSOP accordingly. Enzyme Activation. To demonstrate the ability of MMP-9 to activate MMP-9-PSOP (0.125 nmol particles in 50 µL), hydrodynamic size measurements were carried out before and after adding MMP-9 (7.4 U MMP-9, human, Calbiochem) at 37 °C. For experiments with enzyme inhibition, MMP-2/MMP-9 Inhibitor II (30 µM final concentration, Calbiochem) was added before the addition of MMP-9 enzyme. The hydrodynamic diameters (Zetasizer) were monitored for 2 h. The incubation buffer for enzyme experiments was 10 mM HEPES, 140 mM NaCl, 1.3 mM CaCl2, and 50 µM ZnCl2, pH 7.5. MR Imaging. For the MR experiments (Figure 4b), four different particle concentrations of 6×-MMP-9-PSOP (300 nM, 150 nM, 75 nM, and 38 nM particle concentration) were incubated with 1.3 U MMP-9 in 200 µL incubation buffer (see above) at 37 °C. All inhibitor experiments (Figure 4c,d) were done under identical conditions using a particle concentration of 300 nM. All enzyme reactions were imaged with a clinical 1.5-T MR scanner (Siemens Sonata). The gradient echo sequence with 12 echo times (TR ) 100 ms, TE ) 3.1-44.8 ms) was repeated 200 times over 50 min. The images were analyzed with OsiriX DICOM viewer, ImageJ (National Institutes of Health, USA), and Prism 5 (GraphPad Software). Curve

fits for T2 calculation and enzyme inhibition (logarithmic inhibitor response curve, standard slope) were done with Prism 5. Molecular Modeling. The 5 nm octahedral magnetite core of VSOP-C200 with approximately 2600 iron atoms and 75 citrate shell molecules per core (data provided by Ferropharm GmbH) was generated with CrystalMaker (CrystalMaker Software). Molecular modeling of MMP-9-PSOP was done based on the size measurements and the sequence of the MMP-9 peptide and NHS-mPEG (MW 5000) using the PyMOL Molecular Graphics System (12).

RESULTS To add enzyme-sensing capability, we designed a construct shown in Figure 1a consisting of a peptide and a methylpoly(ethylene glycol) polymer (mPEG, molecular weight 5000). The peptide consists of a cleavage domain with the enzyme recognition motif and a highly positively charged, arginine-rich coupling domain, which are connected by a linker sequence. For analysis by fluorescence methods, a fluorescine is coupled to the peptide. In a first step, this peptide was reacted with NHSmPEG at the end of the cleavage domain. In a second step, after purification by gel filtration and HPLC control for purity, these peptide-mPEG copolymers were mixed in different ratios with the VSOP. A ratio of 6 peptide-mPEG per VSOP yielded MMP-9-PSOP with a hydrodynamic diameter of 24.9 ( 7.0 nm (Figure 2b). Mixtures with ratios between 6 and 16 peptidemPEG consistently yielded particles with sizes around 24 nm,

2442 Bioconjugate Chem., Vol. 19, No. 12, 2008

Schellenberger et al.

Figure 2. Size and model of MMP-9-PSOP. The hydrodynamic size distribution of the parent VSOP with 7.7 ( 2.1 nm is shown in (a). With the copolymers attached to the surface, the PSOP are still small at 25 nm with a narrow size distribution (b). For illustration, a model of MMP-9-PSOP based on the peptide sequence, the structure of mPEG, and the size measurements is shown in (c). The parent VSOP consists of a 5 nm magnetite core (gray) covered by a negatively charged citrate shell (red). The peptide-mPEG copolymers are electrostatically bound by the positively charged coupling domains (blue). The mPEG polymers (light blue) are linked to the coupling domain via the cleavage domain (yellow) and a linker peptide. A model of MMP-9 (22) (brown) is shown for size comparison at the cleavage domain. Fluorescein dyes (green).

Figure 3. Dependence of size and fluorescence quenching on the ratio of peptide-mPEG copolymers to VSOP. Left axis: With an increasing number of peptide-mPEG copolymers per VSOP, the hydrodynamic size (mean of distribution by number, SD) of MMP-9-PSOP decreases slightly up to a ratio of 6 peptide-mPEG per VSOP and stays constant around 24 nm up to a ratio of 16. An average ratio of 6 peptide-mPEG per VSOP seems to be necessary for good steric stabilization. Below that ratio, the particles most likely form more dimers or multimers. Right axis: Due to the proximity of the copolymer-coupled fluorescein dyes to each other and to the nanoparticle cores, strong quenching of fluorescence can be observed for PSOP with ratios up to 8, proving nearly complete binding of the copolymers to the VSOP. With higher ratios, the quenching effect decreases, indicating incomplete coupling of copolymers due to saturation of the VSOP surface with peptidemPEG copolymers.

whereas ratios below 6 resulted in particles over 30 nm in size, probably due to insufficient steric stabilization (Figure 3 and Table 1). To evaluate the level of copolymer coupling to the VSOP surface, we measured the fluorescence quenching effect of the peptide-bound fluorescein, which is caused by nonradiative energy transfer due to the proximity of the dyes to each other and to the iron oxide cores (13). Compared with the fluorescence of the peptide-mPEG complexes without the particles, the fluorescence was strongly quenched up to ratios of 8 complexes per VSOP (Figure 3). PSOP with 6 complexes per VSOP had a quenched relative fluorescence of 0.02, indicating nearly

complete coupling of the complexes to the VSOP surface. Higher ratios of complexes to particle cores showed a decreasing quenching effect, revealing incomplete coupling, most likely due to saturation of the VSOP surface. When free fluorescein was mixed with the particles (6 fluorescein dyes per VSOP) as control, the fluorescence was only reduced to 0.78, confirming that the reduction of copolymer fluorescence was not only caused by light absorption by the particles. Therefore, 6 peptidemPEG copolymers per particle was considered a good ratio for further experiments. For illustration, a model of MMP-9-PSOP (6 peptide-mPEG per particle), based on the sequence of the peptide-mPEG complexes and particle size measurements, is shown together with a crystal structure of MMP-9 in Figure 2c. The function of the particles is explained in Figure 1b. When the sterically stabilized MMP-9-PSOP (1) are exposed to MMP9, the protease cleaves the peptide at the recognition site (2), resulting in loss of the sterically stabilizing mPEG shell. Due to the superparamagnetic properties of the iron oxide cores and the ambivalent surface of the remaining particles with positively (coupling domains) and negatively (citrate coat of VSOP) charged areas, the particles aggregate driven by magnetic and electrostatic attraction (3). This process has two important consequences: First, clustering of the superparamagnetic nanoparticles causes a substantial increase in R2-relaxivity, called magnetic switch (6). Second, the particles are converted from mPEG-covered stealth particles into highly aggregative particles with strongly charged surfaces (14). Conveniently, mPEG-5000 has been shown to be optimal for achieving stealth properties for nanoparticles (15). Consequently, once injected, the intact PSOP should remain for a long time in the blood circulation until they reach an MMP-9 expressing target tissue, where they are converted into aggregative particles and accumulate. In contrast to mPEG-5000, peptide-mPEG copolymers made of mPEG-2000 (molecular weight 2000) or of branched mPEG2000 did not yield stable particles but precipitating aggregates, presumably due to insufficient steric stabilization (data not

Protease Nanosensors for MRI

Bioconjugate Chem., Vol. 19, No. 12, 2008 2443

Figure 4. Function of PSOP. (a) Hydrodynamic size measurements. Time course of aggregation-induced size increase of the MMP-9-PSOP probe (0.125 nmol particles) induced by enzyme activation with 7.4 U of MMP-9. There was no probe activation when the experiments were done in the presence of 30 µM MMP-9-inhibitor. (b-c) MRI experiments. (b) Time course of activation of MMP-9-PSOP at four different particle concentrations. As aggregates form, the T2*-relaxivity of these particles increases, causing a drop in signal intensities on the gradient echo sequences to a minimum until no further decrease occurs because continuing aggregation and precipitation reduce the amount of water protons affected. MR image obtained at the time point indicated by the arrow. (c) Time course of signal intensities of MMP-9-PSOP (300 nM particle concentration) incubated with different concentrations of a MMP-9-inhibitor. (d) Inhibition of the T2*-time-shortening at the time point indicated by the arrow in (c). The 50% inhibitory concentration in this assay is approximately 10 nM. MR experiments were done with 1.3 U MMP-9 in 200 µL buffer. Table 1. Properties of MMP-9-PSOP size [nm]

24.9 ( 7.0

Peptide-mPEG copolymers per particle R1 relaxivity [mM Fe1-s-1] R2 relaxivity [mM Fe1-s-1] Fe atoms per particle*

6 8.9 41 2600

* Data provided by Ferropharm GmbH. Relaxivities at 0.94 T.

shown). Another control experiment with uncoupled peptide and mPEG-5000 did not result in stable particles either (data not shown). To demonstrate the function of MMP-9-PSOP, we measured the changes in size and T2* contrast in MRI following activation by MMP-9. Results are shown in Figure 4. First, we tested the activation of MMP-9-PSOP by monitoring hydrodynamic diameters (Figure 4a). Incubation of MMP-9-PSOP (6 peptidemPEG per particle) with MMP-9 caused dramatic aggregation already after a few minutes leading to particle sizes exceeding the micrometer range. When the experiment was repeated in the presence of a MMP-9 inhibitor, the size remained almost unchanged over a period of 2 h. To monitor the activation using MRI, we prepared 200 µL reactions with different concentrations of MMP-9-PSOP (300 nm, 150 nM, 75 nM, and 38 nM) and 1.3 U MMP-9 each. Approximately every 15 s, T2*-weighted images (gradient echo sequences with 12 echo times (TE) between 3.1 and 44.8 ms and a repetition time (TR) of 100 ms) were acquired over a period of 50 min. Figure 4b shows the time course of the signal intensities (for a TE of 10.6 ms). Depending on the concentra-

tion, the signal intensities decrease and reach a minimum at about 15 min for a concentration of 75 nM concentration and at about 27 min for 300 nM. The signal decrease was about 79% for 300 nM and 24% for 38 nM. The differences in the time it took to reach the signal minimum can be explained by the different amounts of peptide substrates to be cleaved by the MMP-9. The MR image in Figure 4b represents a snapshot taken at 17.6 min. Figure 4c shows the time course of signal intensities measured under the same conditions for 300 µMMMP-9-PSOP incubated with different concentrations of a MMP-9-inhibitor. The signal drop is delayed at a concentration of 10 nM and nearly completely suppressed for the observation period of 50 min at a MMP-9-inhibitor concentration of 1 µM. T2* relaxation times for the different inhibitor concentrations were calculated from the MR images obtained at 32 min and analyzed using an inhibition function of Prism 5 software (log(inhibitor) vs response curve with standard slope, R2 ) 0.996). The 50% inhibitory concentration in this assay was 9.3 nm (95% confidence interval 4.2 nM to 21 nM).

DISCUSSION After mixing with the VSOP, the peptide-mPEG copolymers adsorb at the strongly negatively charged VSOP citrate surface with their positively charged coupling domains. The resulting protease-specific iron oxide particles (PSOP) with the copolymers at the surface are no longer electrostatically but sterically stabilized, i.e., the natural tendency of superparamagnetic magnetite nanocores to aggregate is countered by the thick mPEG coat and not by the negatively charged surface of the

2444 Bioconjugate Chem., Vol. 19, No. 12, 2008

VSOP as before. Instead of PEG, other macromolecules such as dextran could provide sterical stabilization as well. The results of the size measurements and in Vitro MRI experiments presented here confirm the specific protease-sensing function of these newly developed PSOP. Upon incubation with MMP-9, the nanosensors start clustering after a few minutes, as can be shown by dynamic laser light scattering and MRI. This activation by protease activity was largely suppressed by preincubation with an MMP-2/MMP-9 inhibitor. Inhibition occurred in a typical, concentration-dependent manner (Figure 4d), proving that activation was due to the protease activity of MMP-9. The PSOP comprise several key characteristics making them a good candidate for an enzyme reporter probe for in ViVo MRI: Synthesis of the particles is fairly straightforward. They can be adapted to any proteases or possibly other cleaving enzymes. Poly(ethylene glycol)s have been proven to be biocompatible (listed in the pharmacopeia) and provide stealth properties, which is a prerequisite for a sufficiently long circulation time in ViVo (15). Together with the nanosensors’ small diameter of about 25 nm, these properties ensure good bioavailability in the desired target tissues. Unlike the caspase-3-sensitive nanosensors presented by Josephson and co-workers (6), which are clusters of approximately 45 nm sized CLIO monomers before activation and are cleaved into monomers by caspase-3, leading to a decrease in relaxivity and contrast effect, PSOP become larger upon activation and relaxivity increases. This property allows the in ViVo application of the nanoparticles. The parent VSOP has been shown to be safe in preclinical (16) and clinical trials (7). In contrast to near-infrared fluorescence imaging, MRI with PSOP is a potential candidate for whole-body imaging of protease activity in humans. Moreover, the particles could be used to deliver therapeutics, or the accumulating magnetic cores themselves could serve as “theranostics” in clinical magnetic thermotherapy of cancer, which currently requires direct injection of magnetic nanoparticles into the tumor (17). The in Vitro experiments performed in this study have some limitations: First, the size measurements yielded meaningful results only until the aggregates started to precipitate, thereby dropping out of the measurement field of the zetasizer. In the MR experiments, the T2* contrast effect also started to decrease at a certain point in time, which is partially attributable to precipitation as well. Additionally, the fraction of water protons influenced by the magnetic field of the particles also decreases from a certain time onward as the aggregates continue to increase in size while their number decreases (18). Although precipitation due to aggregation is problematic for these in Vitro settings, it is an intended and important mode of function for in ViVo application. The accumulation of the particles in protease-expressing target tissues following the switch from a PEGylated stealth state to a highly surface-charged, aggregative state cannot be demonstrated with the types of experiments used here and remains to be shown in ViVo. Although the aggregation effect and consequently the magnetic switch would be desirable for an enhanced contrast effect in the in ViVo situation, it is not a necessity, since a particle binding to the target tissue due to the exposed charged surface is sufficient to produce a strong contrast effect (19-21). We are convinced that the nanosensors presented here have great potential as reporter probes for assessing enzyme activity of proteases by in ViVo MRI and can thus help diagnose diseases with pathologically altered protease activity or monitor protease inhibitor therapies.

ACKNOWLEDGMENT VSOP was provided by Ferropharm GmbH. We thank Bettina Herwig for language editing.

Schellenberger et al.

LITERATURE CITED (1) Weissleder, R., Tung, C. H., Mahmood, U., and Bogdanov, A. J. (1999) In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375–378. (2) Ntziachristos, V., Ripoll, J., Wang, L. V., and Weissleder, R. (2005) Looking and listening to light: the evolution of wholebody photonic imaging. Nat. Biotechnol. 23, 313–320. (3) Weissleder, R., and Pittet, M. J. (2008) Imaging in the era of molecular oncology. Nature 452, 580–589. (4) Louie, A. Y., Hu¨ber, M. M., Ahrens, E. T., Rothba¨cher, U., Moats, R., Jacobs, R. E., Fraser, S. E., and Meade, T. J. (2000) In vivo visualization of gene expression using magnetic resonance imaging. Nat. Biotechnol. 18, 321–325. (5) Bogdanov, A., Matuszewski, L., Bremer, C., Petrovsky, A., and Weissleder, R. (2002) Oligomerization of paramagnetic substrates result in signal amplification and can be used for MR imaging of molecular targets. Mol. Imaging 1, 16–23. (6) Perez, J. M., Josephson, L., O’Loughlin, T., Hogemann, D., and Weissleder, R. (2002) Magnetic relaxation switches capable of sensing molecular interactions. Nat. Biotechnol. 20, 816–820. (7) Taupitz, M., Wagner, S., Schnorr, J., Kravec, I., Pilgrimm, H., Bergmann-Fritsch, H., and Hamm, B. (2004) Phase I clinical evaluation of citrate-coated monocrystalline very small superparamagnetic iron oxide particles as a new contrast medium for magnetic resonance imaging. InVest. Radiol. 39, 394–405. (8) Chen, J., Tung, C. H., Allport, J. R., Chen, S., Weissleder, R., and Huang, P. L. (2005) Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction. Circulation 111, 1800–1805. (9) Kridel, S. J., Chen, E., Kotra, L. P., Howard, E. W., Mobashery, S., and Smith, J. W. (2001) Substrate hydrolysis by matrix metalloproteinase-9. J. Biol. Chem. 276, 20572–20578. (10) Hu, J., Van den Steen, P. E., Sang, Q. X., and Opdenakker, G. (2007) Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat. ReV. Drug DiscoVery 6, 480–498. (11) Bremer, C., Tung, C. H., and Weissleder, R. (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743–748. (12) DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, USA. (13) Josephson, L., Kircher, M. F., Mahmood, U., Tang, Y., and Weissleder, R. (2002) Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes. Bioconjugate Chem. 13, 554–60. (14) Moghimi, S. M., and Szebeni, J. (2003) Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog. Lipid Res. 42, 463–478. (15) Shi, B., Fang, C., and Pei, Y. (2006) Stealth PEG-PHDCA niosomes: effects of chain length of PEG and particle size on niosomes surface properties, in vitro drug release, phagocytic uptake, in vivo pharmacokinetics and antitumor activity. J. Pharm. Sci. 95, 1873–1887. (16) Wagner, S., Schnorr, J., Pilgrimm, H., Hamm, B., and Taupitz, M. (2002) Monomer-coated very small superparamagnetic iron oxide particles as contrast medium for magnetic resonance imaging: preclinical in vivo characterization. InVest. Radiol. 37, 167–177. (17) Thiesen, B., and Jordan, A. (2008) Clinical applications of magnetic nanoparticles for hyperthermia. Int. J. Hyperthermia 1–8. (18) Koh, I., Hong, R., Weissleder, R., and Josephson, L. (2008) Sensitive NMR sensors detect antibodies to influenza. Angew. Chem., Int. Ed. Engl. 47, 4119–4121. (19) Kaufels, N., Korn, R., Wagner, S., Schink, T., Hamm, B., Taupitz, M., and Schnorr, J. (2008) Magnetic resonance imaging

Protease Nanosensors for MRI of liver metastases: experimental comparison of anionic and conventional superparamagnetic iron oxide particles with a hepatobiliary contrast medium during dynamic and uptake phases. InVest. Radiol. 43, 496–503. (20) Schnorr, J., Wagner, S., Abramjuk, C., Drees, R., Schink, T., Schellenberger, E. A., Pilgrimm, H., Hamm, B., and Taupitz, M. (2006) Focal liver lesions: SPIO-, gadolinium-, and ferucarbotran-enhanced dynamic T1-weighted and delayed T2-weighted MR imaging in rabbits. Radiology.

Bioconjugate Chem., Vol. 19, No. 12, 2008 2445 (21) Schellenberger, E., Schnorr, J., Reutelingsperger, C., Ungethum, L., Meyer, W., Taupitz, M., and Hamm, B. (2008) Linking proteins with anionic nanoparticles via protamine: ultrasmall protein-coupled probes for magnetic resonance imaging of apoptosis. Small 4, 225–230. (22) Browner, M. F., Smith, W. W., and Castelhano, A. L. (1995) Matrilysin-inhibitor complexes: common themes among metalloproteases. Biochemistry 34, 6602–6610. BC800330K