From Phage Display to Magnetophage, a New Tool for Magnetic

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Bioconjugate Chem. 2007, 18, 1251−1258

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From Phage Display to Magnetophage, a New Tool for Magnetic Resonance Molecular Imaging Je´roˆme Segers,§ Catherine Laumonier, Carmen Burtea, Sophie Laurent, Luce Vander Elst, and Robert N. Muller* Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons-Hainaut, 24 Avenue du Champ de Mars, Mons B-7000, Belgium. Received December 7, 2006; Revised Manuscript Received March 23, 2007

Phage display, an extremely promising technology in the context of molecular imaging, allows for the selection of peptides interacting with virtually any target from a heterogeneous mixture of bacteriophages. In this work, we propose the concept of magnetophages, obtained by covalent coupling of ultrasmall particles of iron oxide (USPIO) to the proteins of the phage wall. To validate magnetophages as a magnetic resonance imaging contrast agent (MRI), we have used as a prototype the clone E3 because of its specific affinity for phosphatidylserine, a marker of apoptosis. Enzyme-linked immunosorbent assay showed that E3 magnetophages incubated with phosphatidylserine retained the properties of the nonmagnetically labeled phages. The usefulness of magnetophages as an MRI contrast agent was estimated by incubation with phosphatidylcholine and phosphatidylserine or with apoptotic and control cells. Under these conditions, E3 magnetophages allow the discrimination of phosphatidylserine from phosphatidylcholine and of apoptotic cells from control ones. Injected in ViVo, magnetophages are rapidly cleared from the blood stream and internalized by the phagocytic cells of the liver. To abrogate this problem, USPIO were pegylated to obtain stealthy E3-PEG-magnetophages, invisible to phagocytic cells, which were successfully targeted to apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptides before their synthesis is considered. The concept of the magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).

INTRODUCTION Molecular imaging, a concept that has recently emerged, aims at visualizing molecules expressed in pathological conditions. It has therefore the potential of playing an important role in the early detection of pathologies and understanding their molecular mechanisms (1). Molecular imaging relies on the use of vectorized reporters acting as contrast agents. In magnetic resonance imaging (MRI), contrast agents are made of a magnetic center responsible for the modulation of the recorded signal, which is coupled to a targeting structure. Phage-display technology is a powerful strategy for the selection and identification of peptidic sequences interacting with a target of interest (receptor, protein, etc.). It is based on the use of a phage library which is a heterogeneous mixture of phage clones, each carrying a different foreign DNA insert and therefore displaying a different peptide on its surface (2-8). The identification of binders specific to a target requires several rounds of selection and amplification during which the phage population is gradually enriched in high affinity binders, to finally obtain a population of phages specific for the target. Subsequent coupling of the selected peptide to a magnetic, optical, or radioactive reporter gives specific probes for molecular imaging. We have previously applied this methodology to the design of a MRI contrast agent specific for apoptosis (9). In this study, we propose an alternative strategy: the direct use of the selected phages themselves as vectors of the contrast agent. The conjugation of ultrasmall particles of iron oxide (USPIO) to * Author to whom correspondence should be addressed. E-mail: [email protected]. Phone: +3265373520, Fax: +3265373520. § Present address: INSERM U629, Institut Pasteur de Lille, 1 rue du Prof. Calmette, F-59019 Lille, France.

the phages generates a system that we have called “magnetophages”. USPIO are indeed efficient magnetic reporters for MRI (10, 11), made of nanocrystals containing 2000 to 3000 Fe3+ atoms coated with dextran. The aim of this work was to design, characterize, and evaluate the potential application of magnetophages as markers for magnetic resonance molecular imaging (MRMI). To evaluate this concept, phages displaying the peptide TLVSSL (corresponding to phage clone E3) identified in our previous work as a specific binder for phosphatidylserine (PS) (9), a biomarker of apoptosis (12, 13), were turned into magnetic labeled phages (magnetophages E3). Phages E3 and magnetophages E3 were used as prototypes.

EXPERIMENTAL PROCEDURES The in ViVo protocols of the experiments fulfill the prescriptions of the ethics committee of our institution. Phage Display Library, Phage, and Bacterial Strains. A random linear hexapeptide library (fUSE5 vector) was used. The inserts are spliced into the gene corresponding to the minor coat protein pIII (3). In addition, the phages of this library contain a gene that confers resistance to tetracycline (Tc) to the infected bacteria. This library was kindly provided by Dr. Hans Ullrichts (KULAK, Kortrijk, Belgium). Nonselected phages, from the initial library, and a clone (E3), specific for phosphatidylserine (PS), were used to produce the corresponding magnetophages. The clone E3 was selected using the phagedisplay selection procedure carried out in a previous work (9). Bacteria used in this work belong to the enterobacterium Escherichia coli strain K91BluKAN provided by Dr. H. Ullrichts (KULAK, Kortrijk, Belgium). These bacteria contain the kanamycine (Kan) resistance gene. Phage Amplification and Precipitation. Phages were amplified by infecting E. coli host strain K91BluKan. A culture flask

10.1021/bc060377f CCC: $37.00 © 2007 American Chemical Society Published on Web 05/24/2007

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containing LB (Luria-Bertani) medium supplemented with 100 µg/mL Kan was inoculated with a single bacteria colony. Bacteria were allowed to grow overnight at 37 °C with vigorous rotational shaking. The culture was then diluted 100 times in 100 mL LB medium supplemented with 100 µg/mL Kan and allowed to grow until bacteria reached the midlog phase (OD600 ∼ 0.8-0.9). At that time, an amount of 2 × 1011 phages was added to the culture medium that was completed with 10 µg/mL Tc. The culture was allowed to grow overnight. During this phase, phages are secreted in the medium by infected bacteria. Amplified phages were recovered by precipitation with poly(ethylene glycol) (PEG). Briefly, the culture was centrifuged at 10 000 rpm and 4 °C for 15 min. The supernatant was transferred to a new flask and 1/5 volume/volume PEG/NaCl was added (20% PEG 6000, 2.5 M NaCl). Phages were precipitated for 2 h at 4 °C and were then retrieved by centrifugation at 4 °C and 3200 rpm for 20 min. The phage pellet was suspended in 1 mL Tris-buffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5), 200 µL PEG/NaCl was added, and a new precipitation was carried out for 1 h at 4 °C. A final centrifugation at 4 °C and 13 000 rpm for 5 min allowed the isolation of a pellet that was finally suspended in TBS at the suitable phage concentration. Synthesis of Magnetophages. Magnetophages were obtained by reacting phages with activated USPIO obtained by pretreating USPIO with epichlorhydrin (Sigma, Bornem, Belgium). A 1.5 mL amount of USPIO (30 mg of 22 nm Fe particles coated with dextran) was diluted in 5 mL of water and treated with 5 mL of 5 M NaOH and 2 mL of epichlorhydrin. The mixture was stirred for 24 h at 40 °C in the darkness and then dialyzed in a 5 mM citrate solution, pH ) 8 (membrane cutoff: 12000-14000) to remove unreacted epichlorhydrin (14). A 400 µL amount of activated USPIO (150 mM Fe) was added to 600 µL of phage suspension in water at a concentration of 4 × 1012 phages per mL and incubated 24 h at 37 °C. Magnetophages were recovered by PEG-precipitation as described above. This process was repeated three times to ensure total elimination of free USPIO. The magnetophages were finally suspended in 500 µL of water. Estimation of Apparent Affinity Constant Ka,app. The Ka,app of E3 magnetophages for PS was determined by saturation experiments performed by enzyme-linked immunosorbent assay (ELISA). PS (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, Genzyme Pharmaceuticals-Sygena Facility, Liestal, Switzerland) was dissolved in ethanol and immobilized on microplates (Greiner Gmbh, Frichenhausen, Germany) by overnight evaporation of the ethanol at room temperature. Plates were blocked with 200 µL of a blocking solution [4% milk powder in phosphate-buffered saline (PBS, 137 mM NaCl, 3.2 mM KCl, 6.4 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.5)] for 2 h at room temperature. After rinsing three times with washing buffer (PBS completed with 0.1% Tween 20) using a microplate washer (Adil Instruments, Schiltigheim, France), serial magnetophage dilutions ranging from ∼10-8 to ∼10-16 M were prepared in a calcium buffer (2 mM CaCl2, 150 mM NaCl, 10 mM HEPES, 3 mM NaN3, pH 7.4). PS-coated plates were incubated with 200 µL of each dilution for 2 h at 37 °C. Plates were then rinsed six times with washing buffer. Bound phages were detected using the horseradish peroxidase (HRP)conjugated anti-M13 antibody (HRP anti-M13, Amersham Pharmacia Biotech, Roosendaal, The Netherlands). The peroxidase staining reaction was performed with HRP substrate solution containing 100 mM citric acid pH 2, 200 mM Na2HPO4, 30% H2O2 (8 µL for 20 mL of substrate solution) and OPD (o-phenylenediamine dihydrochloride, ICN, Asse-Relegem, Belgium). After incubation with the substrate for 30 min,

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the reaction was stopped by addition of 50 µL/well of 4 M H2SO4. OD450 values were determined with Stat Fax-2100 ELISA plate reader (Awareness Technology, Palm City, FL). Titration curves were obtained by plotting OD450, which reflects the number of phages bound to PS, against the logarithmic concentration of total magnetophages, expressed in mol/L. Curves were fitted according to a sigmoidal model. The phage concentration at half-saturation corresponds to 1/Ka,app (7). Competition Experiments with Annexin V. Annexin V, a protein interacting specifically with PS (15), was used as competitor for E3 magnetophages. Serial dilutions from 6 × 10-4 to 10-10 M of annexin V (33 kDa from human placenta, Sigma-Aldrich, Bornem, Belgium) were prepared in calcium buffer. PS-coated plates were incubated with each dilution of annexin V during 30 min at 37 °C. E3 magnetophages were then added at the concentration yielding half-saturation and incubated for 90 min at 37 °C. The subsequent protocol was identical to that used in the previous section. The competition curve was obtained by plotting OD450 against the logarithmic concentration of total annexin V input, expressed in M. Nuclear Magnetic Resonance Dispersion Profiles. Nuclear magnetic resonance dispersion (NMRD) profiles represent the relaxivity of the contrast agent as a function of the proton Larmor frequencies. Longitudinal relaxivities r1 and transverse relaxivities r2 were obtained respectively from longitudinal relaxation rate R1 and the transverse relaxation rate R2 at each field as described below. R1 and R2 are the reciprocals of the measured longitudinal relaxation time T1 and the transverse relaxation time T2 of water protons respectively, expressed in seconds. The relaxivity reflects the efficiency of a MRI contrast agent: the higher the relaxivity, the lower the amount of contrast agent needed to produce a given contrast enhancement in MRI. Relaxivity (r1 or r2, expressed in s-1 mmol-1 L) is defined as the increase of relaxation rate (R1 and R2) produced by 1 mmol per liter of contrast agent. The r1 and r2 are the main physicochemical parameters that are considered in the development of an effective magnetic label. They depend essentially on the size and chemical structure of the magnetic center as well as on its accessibility to water molecules. The MRI contrast agents are indirect agents because they are not visible by themselves contrary to the situation encountered in other imaging modalities. Indeed, they enhance the difference between healthy and diseased tissues by modifying their intrinsic parameters, mainly the longitudinal, T1, or transverse, T2, relaxation times of the tissues water protons. Relaxivities and observed relaxation rates are linked by the following formula:

Ri(obs) )

1 1 ) + riC; Ti(obs) Ti(diam)

i ) 1 or 2

(1)

where: Ri(obs) and 1/Ti(obs) ) global relaxation rate of the tissue (s-1), Ti(diam) ) relaxation time of the tissue before the addition of the contrast agents, C ) the concentration of the paramagnetic center (mmol L-1), and ri ) the relaxivity (s-1 mmol-1 L). Iron concentration was determined as follows. Magnetophage samples were mineralized in nitric acid/hydrogen peroxide 3:1 volume/volume by microwave treatment (Milestone, Analis, Namur, Belgium). Fe content was then determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Jobin Yvon JY70+, Longjumeau, France). The iron concentration of mineralized samples was also estimated by relaxometry at 20 MHz from the known relaxivity of the Fe3+ ion in acidic solution. R1 and R2 were recorded with different systems. For proton Larmor frequencies ranging between 0.01 and 15 MHz, R1 was measured on a fast field cycling NMR relaxometer (Stelar spa, Mede, Italy). For 20 and 60 MHz, R1 and R2 were measured on

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minispecs (PC-20 and mq-60, Bruker, Karlsruhe, Germany). At 200 MHz, R1 and R2 were estimated by image analysis on an Avance-200 vertical magnet spectrometer (Bruker, Karlsruhe, Germany). For R1 determination, an inversion-recovery sequence was used [repetition time (TR)/echo time (TE) ) 2004/ 8.1 ms, slice thickness ) 1.5 mm, field of view (FOV) ) 5 cm, matrix ) 64]. R1 was calculated by fitting the intensities observed for different inversion times (TI) with the equation

Mz(T1) ) Mz(0)[1 - (1 - R) × expTI/T1]

(2)

R2 was obtained by a spin-echo T2 sequence (TR/TE ) 2062/ 6.6 ms, echo number (NE) ) 24, slice thickness ) 1 mm, FOV ) 5 cm, matrix ) 64). R2 was calculated by fitting the curve representing the image intensities as a function of echo time according to the equation

Mxy(TE) ) A + C × expTE/T2

(3)

MaLISA and C-MaLISA. MaLISA (magnetic-linked immunosorbent assay) is the MRI equivalent of ELISA (16). PS or phosphatidylcholine (PC, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-choline, Genzyme Pharmaceuticals-Sygena Facility, Liestal, Switzerland) were adsorbed on a microtiter plate by overnight evaporation of a phospholipid solution (concentration: 25 mg/30 mL ethanol). Phospholipid-coated plates were then blocked by incubation with 200 µL of blocking solution (5 mg/mL BSA in PBS) for 90 min at 4 °C. After washing three times with 0.1% Tween 20 in PBS, serial magnetophage dilutions ranging from ∼10-8 to ∼10-16 M were prepared in calcium buffer. A 100 µL amount of each magnetophage dilution was added to the wells and incubated at 37 °C during 90 min. After washing six times with 0.1% Tween 20 in PBS, 100 µL of 0.2 M glycine-HCl (pH2.2) was added and a 10 min incubation was carried out in order to eluate bound magnetophages. This resuspension is necessary to obtain a homogeneous solution of magnetophage required for MRI measurements. As acidic solution can alter phages and digest iron particles, the pH was neutralized with 25 µL of 1 M Tris pH 8. Magnetophages were then detected by MRI using an Avance200 imaging system equipped with a vertical magnet (Bruker, Karlsruhe, Germany). Images were acquired using the following parameters: TR/TE ) 3000/20 ms, NE ) 40, number of acquisitions (NA) ) 2, slice thickness ) 1.5 mm, FOV ) 5 cm, matrix 256 × 256. C-MaLISA, for cellular magnetic-linked immunosorbent assay, was performed with apoptotic and control Jurkat cells. Jurkat cells (contribution from Prof. Oberdan Leo, Free University of Brussels, IBMM, Belgium) were grown in RPMI1640 medium (Sigma-Adrich, Bornem, Belgium) supplemented with 10% FCS (Fetal calf-serum, Gibco, Paisley, UK), and 1% antimycotic-antibiotic solution (Gibco, Paisley, UK). Apoptosis was induced with 1 mM campthotecin (ICN, Asse-Relegem, Belgium) (17). Cells were fixed in 0.5% formalin (37% formaldehyde solution, Sigma-Adrich, Bornem, Belgium) in PBS at a final concentration of 107 cells/mL. Wells were coated by evaporating 200 µL of the cell suspension at 37 °C (18). After blocking for 90 min at 4 °C with 200 µL/well of 5 mg/ mL BSA in PBS, plates were washed three times with 0.1% Tween 20 in PBS. A 100 µL amount of each magnetophage dilution was incubated during 90 min at 37 °C. The subsequent steps of the protocol are the same as described above. Competition with annexin V was carried out at a final concentration of the competitor equal to 0.3 mM. Stealthy Magnetophages. Stealthy magnetophages were obtained by covalent attachment of pegylated USPIO to the proteins of the phage wall using the protocol already described.

USPIO were modified by monoamine polyethylene glycol 2000 (PEG, Shearwater Corporation, Huntsville, AL) as follows: 100 mg of PEG 2000 was incubated with 0.8 mL of activated USPIO (100 mM Fe) for 24 h at room temperature. Particles were dialyzed (membrane cut off: 12000-14000) and the resulting PEG-derivatized USPIO were again treated with epichlorydrin. These particles were finally incubated with phages to obtain stealthy magnetophages. In ViWo Evaluation of Magnetophages. Magnetophages (stealthy or not) were injected to Balb/c mice (5-6 weeks old, Universal Limited Hull, UK) in the caudal vein. The injected amount was 65 µL at an iron concentration of 2.4 mM. Animals were anesthetized by intraperitoneal injection of 60 mg/kg Nembutal (sodium pentobarbital, CEVA, Sanofi Sante´ Animale, Benelux). Apoptosis of hepatic cells was induced by intravenous injection of 10 µg anti-Fas antibody (Becton-Dickinson, Erembodeghem, Belgium) (19). TUNEL staining (terminal deoxynucleotidyl-transferase-mediated UTP end labeling) demonstrated that 70% of hepatocytes were apoptotic 2 h after treatment (results not shown). Magnetic resonance images were acquired on a 4.7 T Avance200 imaging system. Acquisition parameters were: TR ) 2000 ms, TE ) 20 ms, matrix ) 128, slice thickness ) 2.5 mm, FOV ) 6 cm. As explained above, the MRI contrast agent enhances the relaxations rates R1 and R2 of the water protons. The modification of the relaxation rates are reflected on the MR image by a local modification of the image intensity. The MRI signal was expressed as relative postcontrast enhancement (RE%), i.e., the signal modification related to the precontrast image. RE% is calculated by the following formula:

Ii RE(%) )

Iref,i

-

I0 Iref,0

I0

× 100

(4)

Iref,0 where RE(%) is the relative enhancement expressed as a percentage, I0 and Iref,0 are respectively the image intensity measured in the liver and in the reference (tube containing an USPIO solution of 0.1 mM Fe) before administration of the contrast agent, and Ii and Iref,i are respectively the signal intensity in the liver and in the reference after the contrast agent administration. A decrease of the image intensity (negative enhancement, darkening of the image) or the increase of the image intensity (positive enhancement, brightening of the image) are respectively reflected by negative or positive RE(%). Intensities were measured in circular ROI (region of interest) drawn in the liver or in the external reference. Statistics. Data are given as the mean ( SEM (standard error of the mean). Comparison between groups was performed with the Student t test and differences were considered to be significant for p e 0.05 and highly significant for p e 0.01.

RESULTS Synthesis of Magnetophages. Magnetophages were obtained by linking of iron particles pretreated with epichlorhydrin directly to the amino functions of the phage proteins. We used previously similar experimental procedure to graft small peptides on USPIO, and we could estimate that less than five peptides were grafted on each particle (see ref 16, Burtea et al.). We can thus expect that each USPIO activated with epichlorydrin has only a few amine reactive sites and that the risk of crosslinking virions is low. Moreover, the water solubility of the synthesized magnetophages confirms the absence of considerable cross-linking of the virions.

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Figure 1. Estimation of Ka,app. OD450% is the percentage ratio of OD450 related to OD450 measured for the maximal concentration. OD450 measured for magnetophages at maximal concentration was 1.3.

Figure 2. Competition between annexin V and E3 magnetophages for PS. Each value represents the mean of three measurements.

From the magnetophage concentration and the iron concentration (determined by ICP-AES), we evaluate the average number of USPIO per phage at 80 (160 000 to 240 000 iron atoms per virion) assuming that each USPIO contains 20003000 iron atoms. Determination of Ka,app. The apparent association constant Ka,app of E3 magnetophages for PS was compared to that of the nonmagnetically labeled E3 clone, previously determined (9), in order to evaluate the capacity of magnetophages to recognize their target. Figure 1 shows that the titration curve of magnetophages is shifted to the right, meaning that their Ka,app is slightly decreased as compared to that of the nonmagnetically labeled phages. Ka,app is equal to 1.6 × 1011 and 6.55 × 1010 M for phages E3 and magnetophages E3, respectively. Specificity of the Interaction with Phosphatidylserine. To confirm the specific interaction between E3 magnetophages and PS, annexin V, known to bind PS with high affinity (15), was used in a competitive binding assay with E3 magnetophages. The amount of magnetophages bound to PS, as expressed by the OD450 value, decreased when the concentration of competitor was increased (Figure 2). These results confirmed that E3 magnetophages selectively interact with PS and not with other structures exposed during the ELISA experiments, such as polystyrene or blocking buffer. Determination of Relaxivity. NMRD profiles reflect the relaxation rate as a function of field strength and allow for the

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Figure 3. NMRD relaxivity profiles of magnetophages and USPIO. Black squares: USPIO. Black, gray, and white triangles: different samples of magnetophages. r1 relaxivity: plain lines; r2 relaxivity: dashed lines.

Figure 4. Titration curves obtained by MaLISA. They were obtained after incubation of E3 magnetophages (Mφ) with phosphatidylserine (PS) and phosphatidylcholine (PC).

determination of relaxivity at the magnetic field of interest. These profiles are largely used to characterize contrast agents because they reflect the efficacy of a MRI contrast agent and are very sensitive to the agents’ structural modifications (20). They can be seen as a fingerprint of the MRI contrast agents. The NMRD profiles of the three magnetophage samples (Figure 3), synthesized under identical experimental conditions, were compared to those of the original USPIO (represented by black squares). Although small differences exist at lower magnetic fields, the three profiles corresponding to the three magnetophage samples mainly overlap above 5 MHz, confirming the reproducibility of our protocol. The low field r1 and the high field r2 are higher than those observed for the USPIO not conjugated to virion and are characteristic of some clustering of the magnetic centers as shown by Roch et al. (21). As compared to that of free USPIO, the high field r2 of magnetophages (about 160 s-1mM-1 at 60 MHz) is indeed four times higher than that of unconjugated USPIO (about 40 s-1mM-1 at 60 MHz). As a result, a similar MRI contrast enhancement will be obtained with a four times lower amount of iron than with nonconjugated USPIO. MaLISA. Titration curves like those obtained by ELISA, which represent the amount of magnetophages bound to the target, can be obtained by MaLISA where the colorimetric evaluation is replaced by the measurement of signal intensity by MRI. The amount of bound magnetophages was determined from a calibration curve obtained from the MRI measurement of the transverse relaxation time T2 as a function of iron

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Magnetophage, a New MRI Contrast Agent Table 1. T2 (ms) Obtained in C-MaLISA after Incubation of E3 Magnetophages (Initial Concentration ∼ 10-8 M) with Apoptotic and Control Jurkat Cells in the Absence and in the Presence of the Competitor Annexin Va [magnetophages], M

JURKAT apoptotic

JURKAT control

competition with annexin V

10-8 M 5 × 10-9 M 2.5 × 10-9 M 1.25 × 10-9 M

296 295 316 326

335 329 362 353

353 336 361 367

a E3 magnetophages (10-8 M corresponding to 6 × 1012 magnetophages/ mL) were diluted, and each dilution was incubated with the cells.

concentration. The fixation curves for PS and PC obtained by MaLISA are represented in Figure 4. The fixation curve for PC is shifted to the right, suggesting that the affinity for PS of E3 magnetophages is much higher than for PC. The fitting of the fixation curve for PS gave a Ka,app equal to 8.3 × 109 M. The same MaLISA protocol was used to validate the potential of magnetophages as an MRI contrast agent. The acquired image is represented in Figure 5. Signal intensities are lower and therefore T2 values shorter in the wells coated with PS than in those coated with PC, confirming the selective binding of the E3 magnetophages. These results confirm that magnetophages are able to discriminate between PS and PC. C-MaLISA. E3 magnetophages were incubated with apoptotic and control Jurkat cells by a C-MaLISA protocol. The T2 values are given in Table 1. In the case of apoptotic Jurkat cells, E3 magnetophages induced a higher decrease of intensity than in the case of control Jurkat cells. Competition with annexin V weakened this interaction, confirming a specific interaction of E3 magnetophages with phosphatidylserine. These results corroborate the conclusion that magnetophages are able to discriminate apoptotic and normal structures at the cellular level. In ViWo MRI Evaluation of Magnetophages: Stealthy Magnetophages. Magnetophages, specific or not to PS, are rapidly sequestered by the liver (healthy or apoptotic) when they are injected in mice and induce a decrease of the relative enhancement (RE%) (Figure 6). As shown on Figure 7, stealthy nonspecific magnetophages, produced from the native phage display library, are not sequestrated by the liver, contrary to their nonstealthy analogs. Figure 7 shows that stealthy magnetophages specific to PS induce a decrease of RE% only in apoptotic livers, and not in healthy ones. These results are conceivably correlated with the specific accumulation of stealthy magnetophages specific to PS in apoptotic livers. The reasons for the increase of the signal intensity in the case of healthy mice treated with specific or nonspecific stealthy magnetophages remains to be explored. It could be attributed to a very low concentration of magnetic nanoparticles in the liver caused by an absence of immobilization and a rapid diffusion in the hepatic vascular tree (22).

Figure 5. Image obtained after incubation of successive two-fold dilutions of E3 magnetophages with PS (1 to 4) and with PC (5 to 8). 9 and 10: PS and PC, respectively, without magnetophages.

DISCUSSION The coupling of target-specific peptides identified by phage display technology with an appropriate reporter is a powerful strategy in the context of molecular imaging. This approach defines the new rationale in the design of novel MRI contrast agents pursued by our laboratory. However, the behavior of the peptide isolated from its phage carrier cannot be predicted after in ViVo injection, since it could potentially interact less with its target and show unspecific binding with other structures. This is a consequence of a different spatial conformation of the target when immobilized on a solid support or from lower target concentration in an in ViVo environment (2). To avoid these problems, and because phage production is more economical than peptide synthesis, we propose an alternative system, designated with the generic name “magnetophages”, obtained

Figure 6. Relative enhancement of the NMR signal in apoptotic (A) or healthy (H) mouse liver after intravenous injection of nonspecific magnetophages (NS) or magnetophage E3 (E3). A + NS: injection of nonspecific magnetophage to apoptotic liver bearing mouse, H + NS: injection of nonspecific magnetophages to healthy liver bearing mouse, H + E3: injection of magnetophages carrying the peptide E3 specific for PS to mice with normal liver.

by conjugation of USPIO to the phage clone presenting the highest affinity for the target. Magnetophages would be inoperative if the linkage of USPIO to the phage proteins would alter their capacity to interact with their target, an attribute conferred by the displayed peptide. To characterize magnetophages, and subsequently to evaluate them as a contrast agent, we synthesized magnetophages from the

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Figure 7. Relative enhancement of the NMR signal in healthy (H) or apoptotic (A) mouse liver (A) after intravenous injection of stealthy magnetophages carrying the peptide specific to PS (stealthy E3) or nonspecific stealthy magnetophages (NS). H + stealthy E3: injection of magnetophage E3 to mice with healthy liver, A + stealthy E3: injection of magnetophages E3 to mice with apoptotic liver, H + stealthy NS: injection of nonspecific magnetophages to mice with healthy liver. *: statistically significant (p < 0.05); **: highly statistically significant (p < 0.01).

phage clone E3 obtained in our laboratory to target PS. The titration curve of E3 magnetophages showed that the peptide ability to link to PS is maintained after the conjugation with USPIO to create magnetophages, although with a slightly decreased Ka. The specific interaction was further demonstrated by a competition experiment with annexin V, where the extent of PS binding decreased as a function of the competitor’s concentration. These results suggest that USPIO do not significantly alter the potential of peptide displayed by magnetophages to interact with the target. Relaxivity is a measure of the contrast agent efficiency and a way of characterization. NMRD profiles (Figure 3) showed that our protocol of synthesis gives magnetophages with reproducible magnetic properties. The relaxivity r1 is similar for the three magnetophage batches and approaches 0 with increasing magnetic fields. As usual, this suggests that the T1 effect of magnetophages at 200 MHz (4.7 T) would be very weak. However, at 20, 60, and 200 MHz, the r2 relaxivity is constant and four times higher than that of USPIO. This suggests a strong T2 effect at 200 MHz. Thus, magnetophages would act as a superparamagnetic contrast agent locally, disturbing the homogeneity of the magnetic field and inducing a subsequent suppression of the NMR signal. It has to be emphasized that these modifications of the NMRD profiles as compared to USPIO are characteristic of clustered systems and confirms the multiple USPIO attachment to each virion. This conclusion agrees with the determination of the number of USPIO particles per virion, based on the determination of the concentration of phages and of iron (calculation of 80 USPIO per virion, see above). Results obtained from the in Vitro characterization of magnetophages encourage their use as a new kind of contrast agent for MRMI. In this context, two different in Vitro protocols were used to evaluate magnetophages. These protocols basically rely on the ELISA technique and were adapted as an MRI application for biomedical research and clinical diagnosis. Under these conditions, immobilized targets (MaLISA) or intact cells (C-MaLISA) are detected with specifically targeted contrast agents. Hence, the ligand itself (e.g., phages) is labeled with a superparamagnetic moiety (USPIO), which directly reports on the interaction with the specific reporter. MaLISA and C-

MaLISA revealed that E3 magnetophages interacted with apoptotic Jurkat cells at a higher level than with control cells and that this interaction was specific for phosphatidylserine, as confirmed by the competition experiment. In addition, it was possible to estimate the constant describing the interaction with the phospholipid by MaLISA. In these experiments, E3 magnetophages were able to discriminate PS from PC and apoptotic cells from nonapoptotic ones. They suggest that magnetophages can be used as contrast agents in Vitro, playing the role of a reporter for the expression of cellular receptors in any type of biomedical screening. In our protocol of C-MaLISA, we have assumed that PS was not exposed (or was exposed at a lesser extent) in control samples as suggested by the lower level of magnetophage binding on control cells compared to apoptotic cells. Still, binding on controls could have been lower if cells would not have been fixed. However, our rationale was to propose a protocol for high-throughput MRI screening by using magnetophages instead of more complicated, expensive, and time-consuming ELISA protocols which use several antibodies to measure ligand-receptor interaction. In the vascular system of a mammal, magnetophages are processed by the host’s defense system, i.e., the immunologic response and the pathway of endocytosis by the phagocytic cells of the reticuloendothelial system (RES), such as Kupffer cells of the liver. It has been shown that circulating bacteriophages are rapidly sequestered by the RES (23). On the other hand, USPIO used to produce magnetophages are coated by dextran that adsorbs plasma proteins such as albumin, transferrin, and immunoglobulins. These proteins act as opsonins, facilitating endocytosis by Kupffer cells (22, 24, 25). Parameters like the size or the charge of USPIO favor the internalization by the cells of RES (10). The accumulation of magnetophages in the RES with a subsequent reduction of their bioavailability was confirmed by experiments summarized in Figure 6, where nonspecific magnetophages and E3 magnetophages (carrying a PS-specific peptide) were rapidly and nonspecifically taken up by healthy or apoptotic livers. Stealthy nanoparticles have two characteristics: they are invisible to the RES, meaning a decrease of their capture by phagocytic cells, and they have a long vascular circulating time,

Magnetophage, a New MRI Contrast Agent

making them available to interact with the target (26, 27). As a consequence, these particles can act efficiently as biocarriers and are used in molecular targeting for both therapy and molecular imaging (27, 28). These properties are conferred by grafting PEG onto particles to prevent the absorption of the proteins and to reduce the phagocytosis. This is because of the fact that PEG is neutral and allows a relatively strong binding of water molecules around the polymer chain that induces high steric exclusion. In addition, pegylated surfaces are biocompatible and not immunogenic nor antigenic (26, 29-32). This approach has already been applied to liposomes and superparamagnetic nanoparticles, reducing their capture by Kupffer cells (33, 34). We employed the same procedure with magnetophages to fashion stealthy magnetophages. Stealthy E3 magnetophages (carrying a peptide specific for PS) were injected into healthy mice and into mice with apoptotic liver. The absence of signal depression in the liver on MR images of healthy mice and the negative signal enhancement of apoptotic livers (decrease of RE(%), Figure 7) confirm that these magnetophages were not internalized by the RES of healthy mice but are retained by apoptotic livers. These results are convincing arguments that stealthy E3 magnetophages can discriminate between apoptotic and normal livers.

CONCLUSIONS E3 magnetophages selected for their affinity for apoptotic structures were able to discriminate apoptotic from normal cells and between PS and PC in Vitro, while stealthy E3 magnetophages targeted in ViVo apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptide before their synthesis is considered. This means that the targeting peptide discovered by phage display can be investigated without having to incorporate those peptides into more conventional molecular imaging probes. Furthermore, magnetophages themselves can be used as an MRI contrast agent. Finally, the concept of magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).

ACKNOWLEDGMENT The financial supports of the FNRS (Fonds National de la Recherche Scientifique), of the French Community of Belgium ARC (Concerted Research Action), of Region of Wallonia, of the NOMADE program, and of EU (EMIL Network of Excellency, FP6) are acknowledged. Mrs. Patricia De Francisco is thanked for her help in preparing the manuscript.

NOTE ADDED AFTER ASAP PUBLICATION This manuscript was released ASAP on May 24, 2007 with a typographical error in Figure 5. The corrected version was posted on July 2, 2007.

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