SELF-ASSEMBLED BIOCOMPATIBLE IMAGING PARTICLES, THEIR SYNTHESIS AND THEIR USE IN IMAGING TECHNIQUES

Abstract

The present invention relates to a biocompatible particle comprising nanoparticles of iron oxide embedded in a polycathecolamine or polyserotonine matrix, a suspension of said particles, a process for preparing said suspension of particles, a conjugate comprising said particle and the use of said particle and said conjugate in imaging techniques.

Claims

1. A particle having a hydrodynamic diameter comprised between 200 and 2000 nm, said particle comprising ultrasmall particles of iron oxide having a diameter between 1 and 50 nm embedded within a polymer matrix selected from polycathecolamines or polyserotonine.

2. The particle according to claim 1, wherein the iron oxide is selected from Fe.sub.2O.sub.3, Fe.sub.3O.sub.4, or a mixture of Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4.

3. The particle according to claim 1, wherein the polymer matrix is from polydopamine (PDA), polynorepinephrine (PNE), polyepinephrine (PEP) and polyserotonine.

4. The particle according to claim 1, wherein the iron concentration is comprised between 50% and 95% in weight with respect to the to total weight of the particle.

5. A suspension of particles according to claim 1.

6. A process for preparing a suspension of particles according to claim 5, comprising the steps of: a) Preparing a suspension of ultrasmall particles of iron oxide having a diameter between 1 and 50 nm; b) Coating of the ultrasmall particles of iron oxide with a catecholamine or serotonine; c) Polymerizing the catecholamine or serotonine in the presence of the ultrasmall particles of iron oxide, d) Terminating said polymerization; and e) Recovering a suspension of particles.

7. Suspension of particles or particle obtained by the process according to claim 6.

8. Conjugate comprising a particle according to claim 1 and a molecule comprising free amine or thiol groups.

9. Conjugate according to claim 8, wherein the molecule comprising free amine or thiol groups is chosen from a protein, a peptide, a nanobody, a monoclonal antibody or a molecule comprising a radiolabeled metal.

10. Conjugate according to claim 9, wherein the monoclonal antibody is chosen from vascular-cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-Selectin, E-Selectin or mucosal addressin cell adhesion molecule 1 (MAdCAM-1).

11. An in vivo method of imaging comprising the step of administering to a patient an effective amount of the particle according to claim 1.

12. The method according to claim 11, wherein the method of imaging is selected from Magnetic Resonance Imaging (MRI), Magnetic Particle Imaging (MPI), photoacoustic imaging and Positron Emission Tomography (PET).

13. A composition comprising a suspension of particles according to claim 5.

14. A method of imaging, comprising the step of administering to a patient an effective amount of the composition of claim 13 and an imaging step.

15. (canceled)

16. Conjugate according to claim 8, wherein the molecule comprising free amine or thiol groups is a monoclonal antibody.

17. Conjugate according to claim 8, wherein the molecule comprising free amine or thiol groups is a protein that can be a tissue plasminogen activator (tPA) or a fragment thereof.

18. Conjugate according to claim 8, wherein the molecule comprising free amine or thiol groups is a recombinant tissue plasminogen activator (rtPA).

19. Conjugate according to claim 8, wherein the molecule comprising free amine or thiol groups is alteplase, reteplase or tenecteplase.

20. Conjugate comprising a suspension of particles according to claim 5 and a molecule comprising free amine or thiol groups.

21. An in vivo method of imaging comprising the step of administering to a patient an effective amount of the conjugate according to claim 8 and an imaging step.

Description

FIGURES

[0110] FIG. 1. Representative phase-microscopy images of USPIO(n)@PDA of different sizes, obtained by using different concentrations of ammonia during synthesis.

[0111] FIG. 2. Size distribution of USPIO(n)@PDA as assessed by dynamic light scattering (representative of 3 independent experiments in three independent batches).

[0112] FIG. 3A-H. Relaxivity measurement of large 700 nm USPIO.sub.(n)@PDA using a 7T preclinical MRI: (a) Hysteresis cycles at 5K of small (left), medium (middle) and large (right) USPIO(n)@PDA clusters; (b) Hysteresis cycles at 300K of small (left), medium (middle) and large (right) USPIO(n)@PDA clusters; (c) Summary of the results. Hc: coercive field. Ms: saturation magnetization.

[0113] FIG. 4A-D. Degradation of large USPIO(n)@PDA clusters in different buffers as assessed by visual inspection and UV-Visible spectroscopy: UV-Vis spectroscopy of the large USPIO(n)@PDA clusters (700 nm) in PBS (a), ALF (v), citrate (c) and citrate with H.sub.2O.sub.2(d) at different time points.

[0114] FIG. 5A-C. Degradation of large USPIO(n)@PDA clusters after 7 days of incubation in citrate buffer with hydrogen peroxide: UV-Vis spectroscopy of the large USPIO.sub.(n) @PDA clusters (700 nm) and USPIO@Dopamine in PBS (b), citrate (c) and citrate with H.sub.2O.sub.2(d).

[0115] FIG. 6A-C. T2-weighted imaging after intravenous injection of 4 mg/kg large USPIO.sub.(n)@PDA: (a) Evolution of the signal intensity on T2-weighted imaging in the liver (n=7 per group). Ratio was calculated by taking the signal of the paravertebral muscles as reference; (b) Evolution of the signal intensity on T2-weighted imaging in the spleen (n=7 per group); (c) Evolution of the signal intensity on T2-weighted imaging in the right kidney (n=7 per group). * p<0.05 versus baseline.

[0116] FIG. 7A-D. Cytotoxicity of large USPIO@PDA clusters on human venous endothelial cells (HUVEC): (a-b) Viability of HUVEC cells 3 hours (a) and 24 hours (b) after incubation with different concentrations of large USPIO@PDA clusters as assessed by the lactate dehydrogenase (LDH) assay (n=5 per group); (c-d) Viability of HUVEC cells 3 hours (c) and 24 hours (d) after incubation with different concentrations of large USPIO@PDA clusters as assessed by the Water Soluble Tetrazolium-1 (WST-1) assay (n=5 per group). *p<0.05 compared to the control group (0 ?g/ml).

[0117] FIG. 8A-C. Hemolysis assays in mouse and human blood: (a) Spectroscopy of the supernatant of test tubes containing mouse blood after incubation with different concentrations of large USPIO.sub.(n)@PDA clusters and centrifugation; (b) Spectroscopy of the supernatant of test tubes test tubes containing human blood after incubation with different concentrations of large USPIO.sub.(n)@PDA clusters and centrifugation; (c) Summary of the hemolysis rate for each conditions measured by absorbance at 540 nm (representative of n=3 per group). No condition exceeded 5% of hemolysis.

[0118] FIG. 9A-B. Rotational thromboelastometry (ROTEM) in the presence of large USPIO.sub.(n)@PDA clusters in whole human blood: (a) Representative ROTEM curves using the EXTEM assay with different concentrations of large USPIO@PDA clusters; (b) Corresponding main ROTEM parameters, showing no significant effect of large USPIO@PDA clusters on clot formation.

[0119] FIG. 10A-C. Clot lysis assay using human plasma in the presence of large USPIO.sub.(n)@PDA clusters: (a) Representative clot lysis assay curves showing how 75% clotting time (75% CT) and 50% lysis time (50% LT) were measured; (b) Mean 75% CT of human plasma in the presence of different concentrations of large USPIO.sub.(n)@PDA clusters (n=5 per group); (c) Mean 50% LT of human plasma in the presence of different concentrations of large USPIO.sub.(n)@PDA clusters (n=5 per group). The 50% LT was set to 350 minutes in samples without lysis.

[0120] FIG. 11A-C. Clot lysis assay using mouse plasma in the presence of large USPIO.sub.(n)@PDA clusters: (a) Representative clot lysis assay curves showing how 75% clotting time (75% CT) and 50% lysis time (50% LT) were measured; (b) Mean 75% CT of mouse plasma in the presence of different concentrations of large USPIO.sub.(n)@PDA clusters (n=5 per group); (c) Mean 50% LT of mouse plasma in the presence of different concentrations of large USPIO.sub.(n)@PDA clusters (n=5 per group). The 50% LT was set to 350 minutes in samples without lysis.

[0121] FIG. 12. Body weight of mice at baseline and at different time points after intravenous injection of large USPIO.sub.(n)@PDA clusters (4 mg/kg).

[0122] FIG. 13. Plasma concentrations of liver enzymes at different time points after intravenous injection of large USPIO.sub.(n)@PDA clusters (4 mg/kg) in mice as assessed by ELISA. The negative control (Negative Ctrl) samples were collected in control mice that did not receive USPIO(n)@PDA. The positive control (Positive Ctrl) samples were collected 24 hours after intra-peritoneal injection of 5 mg/kg E. coli lipopolysaccharide (LPS). * p<0.05 versus negative control. ALAT: Alanine transaminase. ASAT: Aspartate aminotransferase.

[0123] FIG. 14A-F. Plasma concentrations of a set of key cytokines and chemokines at different time points after intravenous injection of large USPIO.sub.(n)@PDA clusters (4 mg/kg) in mice as assessed by ELISA. The negative control (Negative Ctrl) samples were collected in control mice that did not receive USPIO(n)@PDA. The positive control (Positive Ctrl) samples were collected 24 hours after intra-peritoneal injection of 5 mg/kg E. coli lipopolysaccharide (LPS). * p<0.05 versus negative control. TNF: tumor necrosis factor. IL: Interleukin. IFN: interferon. CXCL1: chemokine (CXC motif) ligand 1.

[0124] FIG. 15A-C. Complexation of immunoglobulins to USPIO(n)@PDA: (a) UV-Vis spectroscopy of the supernatant of USPIO.sub.(n)@PDA@IgG after complexation to different doses of IgG (from 80 to 400 ?g/mg); (b) SDS-PAGE of the supernatant of USPIO.sub.(n)@PDA@IgG after complexation to different doses of IgG; (c) Flow-cytometry of USPIO.sub.(n)@PDA@IgG after complexation to different doses of IgG (rat) using anti-rat or control (anti-goat) secondary antibodies.

[0125] FIG. 16. Characterization of large USPIO.sub.(n)@PDA clusters after complexation to monoclonal antibodies targeting mouse vascular cell adhesion molecule-1 (VCAM-1): Quantification of particle fluorescence according to the type of fluorescent secondary antibodies (n=25 per group).

[0126] FIG. 17. Quantification of the number of particles per field (n=5 per group) in anti-human USPIO.sub.(n)@PDA@?VCAM-1 or control USPIO.sub.(n)@PDA@IgG after incubation with quiescent (treated with PBS) or activated (treated with TNF) human endothelial cells (HCMECD/3). * p<0.05 versus all other groups.

[0127] FIG. 18A-B. Human brain endothelial cells (HCMECD/3) overexpress VCAM-1 after stimulation with TNF as assessed by flow cytometry: (a) Left: Flow cytometry results using primary anti-VCAM-1 antibody or control immunoglobulin (IgG) after stimulation with TNF (50 ng/ml for 24 hours). Right: corresponding density plot; (b) Same as in (a) in unstimulated cells.

[0128] FIG. 19. Quantification of USPIO.sub.(n)@PDA@?VCAM-1 induced signal void in the right striatum (n=5) after successive intravenous injection of USPIO.sub.(n)@PDA targeted against VCAM-1 using monoclonal antibodies (USPIO.sub.(n)@PDA@?VCAM-1) up to 4 mg/kg (equivalent iron) 24 hours after injection of 1 ?g LPS in the right striatum.

[0129] FIG. 20. Quantification of USPIO.sub.(n)@PDA@?VCAM-1 induced signal void in the right striatum (n=5 per group) after intravenous injection of 4 mg/kg USPIO.sub.(n)@PDA@?VCAM-1 24 hours after injection of different doses of LPS in the right striatum (from 0 to 1.0 ?g).

[0130] FIG. 21. Quantification of signal void in the right striatum (n=5 per group) after intravenous injection of 4 mg/kg USPIO.sub.(n)@PDA@IgG or USPIO.sub.(n)@PDA@?VCAM-1 24 hours after injection of 1 ?g of LPS in the right striatum.

[0131] FIG. 22. USPIO.sub.(n)@PDA@?VCAM-1 accumulate in the inflamed striatum of LPS treated mice: Quantification (n=4 per group) in the striatum of mice at 24 hours after intrastrial LPS injection (1.0 ?g) and 60 minutes after intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1. Three images from three different animals are presented for USPIO.sub.(n) @PDA@?VCAM-1 and for control USPIO.sub.(n)@PDA@IgG.

[0132] FIG. 23. Higher density of anti-VCAM-1 monoclonal antibodies on the surface of USPIO.sub.(n)@PDA improves the sensitivity of molecular MRI: Quantification (n=4 per group) in the brain after intrastriatal injection of LPS (1.0 ?g) and after intravenous injection of USPIO.sub.(n)@PDA (4 mg/kg) with either 100% IgG on their surface (top), 50% control IgG and 50% anti-VCAM-1 antibodies (middle) or 100% anti-VCAM-1 antibodies.

[0133] FIG. 24. Saturation of USPIO.sub.(n)@PDA@?VCAM-1 binding sites with free anti-VCAM-1 monoclonal antibodies prevent USPIO.sub.(n)@PDA@?VCAM-1 binding in the inflamed brain: Quantification of USPIO.sub.(n)@PDA@?VCAM-1 induced signal voids (n=4 per group) in the brain after intrastriatal injection of LPS (1.0 ?g) both before and after intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1 (4 mg/kg) in mice that received an intravenous injection of 100 ?g of either control immunoglobulin (left) or anti-VCAM-1 monoclonal antibodies (right) 15 minutes before imaging.

[0134] FIG. 25A-B. Analysis of the binding kinetic of USPIO.sub.(n)@PDA@IgG and USPIO.sub.(n)@PDA@?VCAM-1 in the LPS model of neuroinflammation: (a) Longitudinal evolution of the MRI signal in the right striatum (orange) and in the ophthalmic vein (blue) after intravenous injection of USPIO.sub.(n)@PDA@IgG 24 hours after intrastriatal injection of LPS (1.0 ?g); (b) Longitudinal evolution of the MRI signal in the right striatum (orange) and in the ophthalmic vein (blue) after intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1 24 hours after intrastriatal injection of LPS (1.0 ?g).

[0135] FIG. 26. Longitudinal follow-up of USPIO.sub.(n)@PDA@?VCAM-1 induced signal voids reveals progressive unbinding of the particles: quantification (n=3 mice at each time points) in the brain after intrastriatal injection of LPS (1.0 ?g) both before and at different time points after a single intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1 (4 mg/kg).

[0136] FIG. 27. Unbound USPIO.sub.(n)@PDA@IgG accumulate in the macrophages of the liver: Quantification (n=4 per group) in the liver of mice 60 minutes after intravenous injection of USPIO.sub.(n)@PDA@IgG (4 mg/kg) or control mice. USPIO.sub.(n)@PDA@IgG are revealed by fluorescent secondary anti-rat antibodies.

[0137] FIG. 28. Clustering USPIO.sub.(n)@PDA into submicrometric particles improves sensitivity of molecular MRI of VCAM-1: Quantification (n=4 per group) after intravenous injection of 4 mg/kg of the particles 24 hours after injection of 1 ?g of LPS in the right striatum.

[0138] FIG. 29A-C. Molecular imaging of endothelial activation in clinically relevant experimental models. (a) Corresponding quantification of signal void in the right hemisphere (n=5 per group) after intravenous injection of 4 mg/kg USPIO.sub.(n)@PDA@?VCAM-1 (left) or USPIO.sub.(n)@PDA@IgG (right) 24 hours after ischemic stroke induction by electrocoagulation of the right middle cerebral artery; (b) Corresponding quantification of signal void in the kidney medulla (n=5 per group) after intravenous injection of 4 mg/kg USPIO.sub.(n)@PDA@?VCAM-1 (left) or USPIO.sub.(n)@PDA@IgG (right) 48 hours after acute kidney injury (rhabdomyolysis) induced by intramuscular injection of 50% glycerol; (c) Corresponding quantification of signal void in the descending colon (n=5 per group) after intravenous injection of 4 mg/kg USPIO.sub.(n)@PDA@?MAdCAM-1 (left) or USPIO.sub.(n) @PDA@IgG (right) in an acute colitis model induced by 5 day treatment with 2.0% of dextran sodium sulfate in the drinking water.

EXAMPLES

Abbreviations

[0139] DSS: dextran sulfate sodium [0140] IgG: immunoglobulin G [0141] LPS: lipopolysaccharide [0142] MAdCAM-1: mucosal addressin cell adhesion molecule 1 [0143] MRI: magnetic resonance imaging [0144] MPIO: microparticles of iron oxide [0145] PBS: phosphate buffered saline [0146] PDA: polydopamine [0147] USPIO: ultrasmall particles of iron oxide [0148] VCAM-1: vascular-cell adhesion molecule 1

Materials and methods

Reagents

[0149] The following reagents were purchased from Sigma-Aldrich: ferric chloride hexahydrate, ferrous chloride tetrahydrate, ammonia solution, dopamine hydrochloride, sodium phosphate monobasic, sodium phosphate dibasic, mannitol. Commercial microparticles of iron oxide (MPIO; diameter 1.08 ?m) with COOH surface groups were purchased from Fisher Technology.

Synthesis of Ultrasmall Particles of Iron Oxide (USPIO)

[0150] USPIO were produced by a co-precipitation method in an alkaline buffer. In a typical synthesis, 540 mg of FeCl.sub.3.Math.6H.sub.2O and 198.8 mg of FeCl.sub.2.Math.4H.sub.2O were dissolved in 5.7 mL of distilled water by vortexing, yielding a homogenous yellow solution. Under continuous agitation at room temperature, 6.3 mL of a 13% (w/v) ammonia solution was progressively added at a rate of 0.2 mL/min. The solution turned from yellow to brown and ultimately to a deep black color, corresponding to the formation of magnetite. The precipitate was washed 5 times with distilled water by magnetic separation and resuspended in 10 mL of distilled water.

Synthesis of Ultrasmall Particles of Iron Oxide Coated with Dopamine (USPIO@Dopamine)

[0151] Eight milliliter of the solution of USPIO was resuspended in 40 mL of a solution containing 2.5 mg/mL of dopamine hydrochloride. The resulting solution was sonicated for 15 minutes at 70% amplitude and 26 KHz using a UP200ST sonicator (Hielscher). The color of the solution slightly changed from black to dark brown. Then, the solution was centrifuged at 3000G for 5 minutes to remove large remaining USPIO aggregates. 30 mL of the supernatant containing USPIO@Dopamine and free dopamine hydrochloride were transferred to a new vial.

Synthesis of Particles of the Invention Comprising Ultrasmall Particles of Iron Oxide Embedded within Polydopamine (USPIO.sub.(n)@PDA)

[0152] The USPIO@Dopamine/Dopamine hydrochloride solution were placed under vigorous steering using an Ultra-Turrax T-25 disperser at 20.500 rpm. To produce large USPIO.sub.(n)@PDA, 67 ?L of a 13% (w/v) ammonia solution was first added to the solution which was left to react for 60 minutes. Then, 203 ?L of a 13% (w/v) ammonia solution was added and the incubation was continued for 30 minutes to allow further polymerization of dopamine. To produce medium sized USPIO(n)@PDA, 270 ?L of a 13% (w/v) ammonia solution was added in one time and the solution was left to react for 60 minutes. To produce small sized USPIO(n)@PDA, 2160 ?L of a 13% (w/v) ammonia solution was added in one time and the solution was left to react for 60 minutes. Then, the solution was centrifuged at 1000G for 3 minutes to remove the largest aggregates, the pellet was discarded and 24 mL of the supernatant were transferred to a new vial. The USPIO.sub.(n)@PDA were then washed five-time with distilled water and finally resuspended in 8 mL of distilled water and stored at 4? C. until further use.

Determination of the Hydrodynamic Diameter of the Particles of the Invention

[0153] Dynamic light scattering (DLS) was used to determine the average hydrodynamic diameter, the polydispersity index (PDI) and the diameter distribution by volume of the USPIO.sub.(n)@PDA particles with a NanoZS@ apparatus (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser at a fixed scattering angle of 173?. The temperature of the cell was kept constant at 25? C. and all dilutions were performed in pure water. The particles are for this measure put in suspension in water at a concentration of 20 ?g to 200 ?g of iron per mL of water. Measurements were performed in triplicate.

Determination of Iron Concentration in the Particles of the Invention

[0154] Iron content of USPIO.sub.(n)@PDA suspension was measured with FerroZine method. Particles were degraded overnight at room temperature in HCl 1M, releasing ferric (Fe.sup.3+) and ferrous (Fe.sup.2+) ions in solution. Samples were incubated 30 min with ascorbic acid 0.65% (w/v) to reduce ferric ions in ferrous ions. Samples pH was adjusted with ammonium acetate 12% (w/v). 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p-disulfonic acid monosodium salt hydrate was added (FerroZine Iron Reagent, Sigma-Aldrich, 1 mM) and absorbance was measured at 562 nm with a spectrophotometer (ELx808 Absorbance reader, BioTeK) indicating the amount of complexes formed with ferrous ions. Iron content was determined against standard curves obtained from iron chloride dilutions.

Coating of Particles of the Invention (USPIO.sub.(n)@PDA) with Antibodies

[0155] In a typical coating procedure, 2.8 mg of USPIO(n)@PDA were washed one time with purified water and resuspended in 5 mL of 10 mM phosphate buffer (pH 8.5). Then, 400 ?g of monoclonal antibodies (or another appropriate amount) were incubated with USPIO(n)@PDA at room temperature for 24 hours. The resulting solution was sonicated for 5 minutes at 20% amplitude and 26 KHz using a UP200ST sonicator to break any aggregates. The coated USPIO(n)@PDA were then washed three times with a 0.3 M mannitol solution and finally resuspended in 5 mL of a 0.3 M mannitol solution and stored at 4? C. until further use.

Measurements of the Relaxivities by 1H 7T MRI.

[0156] Large 700 nm USPIO(n)@PDA were dispersed in agarose gels (2%) in Tris-Acetate-EDTA buffer at different concentrations. The sample were then imaged using a BioSpec 7 T TEP-MRI and the following sequences were performed: T1 mapping using Flow-sensitive Alternating Inversion Recovery (FAIR)RARE sequence with repetition time (TR)=3000 ms and inversion Time (TI) ranging from 6.5 ms to 2000 ms; T2 mapping using Multislice Multiecho (MSME) sequence with TR=4000 ms and echo time (TE) ranging from 3.65 to 51.11 ms; T2* mapping using multi gradient echo (MGE) sequence with TR=4000 ms and TE ranging from 2 ms to 17.47 ms. The corresponding R1, R2 and R2* relaxivities were calculated as described above.

Animals

[0157] All experiments were performed on 8 to 16-week-old male Swiss mice (Janvier, France). Animals were maintained under specific pathogen-free conditions at the Centre Universitaire de Ressources Biologiques (CURB, Basse-Normandie, France) and all had free access to food and tap water.

Magnetic Resonance Imaging (MRI) In Vivo

[0158] Experiments were carried out on a Pharmascan 7 T/12 cm system using surface coils (Bruker, Germany). Mice were anesthetized with isoflurane (1.5%-2.0%), maintained at 37? C. by the integrated heat animal holder and the breathing rate was monitored during the imaging procedure. T2-weighted images were acquired using a MSME sequence: TE/TR 51 ms/2500 ms with 70 ?m*70 ?m*500 ?m spatial resolution. T2*-weighted 3D fast low angle shot gradient echo imaging with flow compensation (FLASH, spatial resolution of 78 ?m*78 ?m*150 ?m) with TE/TR 8.6 ms/50 ms and a flip angle (FA) of 200 was performed to reveal USPIO.sub.(n)@PDA clusters and USPIO (acquisition time=17 min). High resolution T2*-weighted images presented in this study are minimum intensity projections of 3 consecutive slices (yielding a Z resolution of 450 ?m).

Statistical Analysis

[0159] Results are presented as the mean?SD. Statistical analyses were performed using Mann-Whitney's U-test. When more than two groups were compared, statistical analyses were performed using Kruskal-Wallis (for multiple comparisons) followed by post-hoc Mann-Whitney's U-test. When comparing two groups, a p-value <0.05 was considered significant (two sided).

Results

Synthesis of USPIO.SUB.(n).@PDA Submicrometric Clusters

[0160] In alkaline buffers, dopamine oxidation induces the formation of submicrometric particles of PDA. To obtain submicrometric clusters of USPIO, we hypothesized that USPIO coated with dopamine (USPIO@Dopamine) would be incorporated as building blocks during the formation of PDA particles. Thus, we synthetized USPIO by a classical co-precipitation method using a 2:1 FeCl.sub.3:FeCl.sub.2 ratio. After extensive washing steps, purified USPIO were incubated with dopamine in pure water for 15 minutes under continuous sonication to obtain USPIO@dopamine. The resulting solution containing USPIO@Dopamine and free dopamine was stirred using a mechanical disperser at room temperature and ammonia was added to start dopamine polymerization. This led to the self-assembly of USPIO.sub.(n)@PDA submicrometric clusters. The mean hydrodynamic diameters of the USPIO.sub.(n)@PDA ranged from 300 nm to 700 nm depending on the concentration of ammonia during cluster formation as measured by dynamic light scattering, with polydispersity indexes <0.2 (FIGS. 1 and 2) and zeta-potentials ranging between ?37 and ?42 mV. The particles produced were large enough to be rapidly separated from an aqueous solution using a bench magnet.

Physical Characterization of USPIO.SUB.(n).@PDA Submicrometric Clusters

[0161] Using a preclinical MRI, at room temperature and 7T (300 MHz), the relaxivity values were, for r1, r2 and r2* were respectively 0.35, 139.9 and 301.7 mM.sup.?1.Math.s.sup.?1. All these parameters are within the range of previously reported value using USPIO with similar crystallite sizes, supporting that clustering USPIO using PDA preserves their favorable superparamagnetic properties.

Biodegradability of USPIO.SUB.(n).@PDA

[0162] Biodegradability of 700 nm USPIO.sub.(n)@PDA was investigated both in vitro and in vivo. First, the particles were incubated at 37? C. in phosphate buffered saline (PBS), artificial lysosomal fluid (ALF), citrate buffer or citrate buffer with hydrogen peroxide. These buffers mimic the lysosomal environment where nanoparticles accumulate after intravenous injection. Degradation was monitored by direct visual inspection and ultraviolet-visible spectroscopy (UV-Vis) during 1 week at 37? C. under mild agitation FIG. 4). Visual aspect and UV-vis spectroscopy of USPIO@Dopamine and USPIO.sub.(n)@PDA in PBS did not change significantly during the monitoring. In ALF and citrate buffers, the brown color of the USPIO@Dopamine solution turned with time into the expected yellow color of free Fe(III) ions. The same phenomenon seemed to occur for USPIO.sub.(n)@PDA solutions but an additional dark precipitate remained present at every time points. This precipitate appeared dark-brown on bright field microscopy and was not attracted by a magnet. This appearance was compatible with free PDA remaining after degradation of the USPIO. In line with this hypothesis, this precipitate progressively solubilized in samples containing hydrogen peroxide, which generates hydroxyl radicals that are able to degrade PDA. UV-vis spectroscopy further supported these findings by showing progressive degradation of USPIO.sub.(n)@PDA in ALF and citrate, with persistence of a chemical specie absorbing in the near infrared region (650 nm), compatible with PDA (FIG. 5). In citrate with hydrogen peroxide buffer, the absorbance of the solution initially containing USPIO.sub.(n)@PDA progressively diminished in the near-infrared region until reaching zero after 7 days of incubation. At day 7, the UV-vis curve was almost undistinguishable between solutions initially containing USPIO@Dopamine or USPIO.sub.(n)@PDA, supporting complete degradation of PDA in this buffer.

[0163] Second, the degradation of USPIO.sub.(n)@PDA was investigated in cell culture of macrophages, the cell type in which large particles accumulate after intravenous injection in vivo. Macrophages were obtained by activation of a human monocytic cell line (THP-1) and were incubated with either USPIO(n)@PDA or non-biodegradable commercial MPIO (Dynabeads MyOne) made of USPIO embedded in a polystyrene matrix. After 96 hours, the cells and particles were observed by transmission electronic microscopy. Both types of particles were internalized at this time point. Whereas commercial MPIO remained morphologically intact, USPIO(n)@PDA fragmented into smaller particles, demonstrating that the PDA matrix is rapidly degraded and releases USPIO once internalized in macrophages.

[0164] In vivo, the degradation of USPIO.sub.(n)@PDA was investigated in mice at different time points after intravenous injection (from 1 hour to 6 months) by MRI (FIG. 6) and histochemistry with Prussian blue staining. All the methods concur in showing that USPIO.sub.(n)@PDA first accumulate in the liver and spleen (reticulo-endothelial system) and that their iron oxide and superparamagnetic components are subsequently degraded. No residual particles were detected more than 30 days after intravenous injection. Importantly, there was no significant accumulation of USPIO.sub.(n)@PDA in the kidneys, lungs or heart at any time points. No microvascular plugging was observed.

Biocompatibility of USPIO.SUB.(n).@PDA

[0165] The biocompatibility of USPIO.sub.(n)@PDA was investigated both in vitro and in vivo. No significant cytotoxicity on endothelial cells (HUVEC) was detected at doses up to 320 g/ml for 3 hours (FIG. 7). At 24 hours, the viability of endothelial cells was slightly reduced in the high-dose groups. Hemolysis (FIG. 8), coagulation (FIG. 9) and fibrinolysis (FIGS. 10 and 11) were all unaffected by USPIO.sub.(n)@PDA using either human or murine blood. After intravenous injection of 4 mg/kg (equivalent iron) of USPIO.sub.(n)@PDA, body weight remained within normal ranges (FIG. 12) and no liver enzymes (FIG. 13), inflammatory cytokines (FIG. 14) nor histological findings were of toxicological significance.

Conjugation of USPIO.SUB.(n).@PDA to Monoclonal Antibodies for Targeted Imaging

[0166] Having demonstrated favorable biocompatibility and biodegradability profiles, we investigated the feasibility of using 700 nm USPIO.sub.(n)@PDA submicrometric clusters as a platform for molecular imaging. To this aim, we coated USPIO.sub.(n)@PDA with antibodies in phosphate buffer at pH 8.5 since alkaline buffer favors reactive quinone over catechol groups on the PDA coating. We performed a dose-response experiment by varying the concentration of antibodies in the solution during coupling. Then, we measured the concentration of bound antibodies on USPIO.sub.(n)@PDA by flow cytometry and the concentration of remaining antibodies in the solution by SDS-PAGE and UV-Vis (FIG. 15). We selected the dose of 160 ?g of antibodies for 1 mg of USPIO.sub.(n)@PDA for further experiments, since higher doses did not significantly increase the concentration of bound antibodies. Using this ratio, we coated USPIO.sub.(n)@PDA with monoclonal antibodies targeting mouse vascular-cell adhesion molecule 1 (VCAM-1), mucosal addressin cell adhesion molecule 1 (MAdCAM-1) or control immunoglobulin G (USPIO.sub.(n)@PDA@?VCAM-1, USPIO.sub.(n)@PDA@?MAdCAM-1 and USPIO.sub.(n)@PDA@IgG, respectively). Using fluorescent secondary antibodies, the conjugated USPIO.sub.(n)@PDA clusters can be detected by immunofluorescence by revealing the primary antibodies on their surface (FIG. 16).

[0167] Then, we investigated the binding of targeted USPIO@PDA in vitro. To this aim, anti-human USPIO.sub.(n)@PDA@?VCAM-1 or control USPIO.sub.(n)@PDA@IgG were incubated with either quiescent or activated cerebral endothelial cells (hCMECD/3). The number of bound particles was evaluated by immunofluorescence microscopy. USPIO.sub.(n)@PDA@?VCAM-1 bound significantly more to activated endothelial cells than control USPIO.sub.(n)@PDA@IgG (FIG. 17). Moreover, USPIO.sub.(n)@PDA@?VCAM-1 bound significantly more to activated than quiescent endothelial cells, in line with a higher expression of VCAM-1 by the formers (FIG. 18). These results demonstrate that USPIO@PDA coated with anti-VCAM-1 monoclonal antibodies are able to bind selectively activated endothelial cells.

USPIO.SUB.(n).@PDA@?VCAM-1 Reveal Neuroinflammation at High Sensibility

[0168] To determine the feasibility of molecular MRI using targeted USPIO(n)@PDA, we used an experimental model of neuroinflammation, induced by intrastriatal injection of E. coli lipopolysaccharide (LPS). Twenty-four hours after intrastriatal injection of LPS (1.0 ?g), brain MRI was performed both before and 3 minutes after iterative injections of USPIO.sub.(n)@PDA@?VCAM-1 corresponding to doses from 1.33 to 4 mg/kg of iron (FIG. 19). On pre-contrast images, no significant difference between the right and left hemispheres was observed in these mice on neither T2 nor T2* weighted images. After intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1, numerous signal void were observed predominantly in the right striatum. Iterative injection demonstrated that higher doses led to more signal voids. We selected the 4 mg/kg dose for further experiments to achieve high sensitivity while keeping within clinically tolerated doses of iron. Notably, USPIO.sub.(n)@PDA@?VCAM-1 were detectable at clinically relevant spatial resolution in this model and at this dose.

[0169] Thereafter, to investigate whether the signal voids of USPIO.sub.(n)@PDA@?VCAM-1 correlate with the severity of neuroinflammation, we administered different doses of LPS (0, 0.25, 0.5 or 1.0 ?g) in the right striatum of naive mice. Twenty-four hours thereafter, we injected 4 mg/kg of USPIO.sub.(n)@PDA@?VCAM-1 intravenously and performed post-contrast T2*-weighted MRI of the brain. Consistent with a higher expression of VCAM-1, more signal voids were observed in the right hemisphere of the mice that received the highest doses of LPS (FIG. 20). Importantly, almost no signal void was observed in the brain of control mice, in line with a low basal expression of VCAM-1. Moreover, by comparing post to pre-injection images, we were able to generate 3D maps of VCAM-1 expression in the brain in vivo and at high spatial resolution.

USPIO.sub.(n)@PDA@?VCAM-1 Combines High Sensitivity with High Specificity

[0170] To investigate the specificity of our method, we compared USPIO.sub.(n)@PDA@?VCAM-1 to control USPIO.sub.(n)@PDA@IgG. Whereas USPIO.sub.(n)@PDA@?VCAM-1 induced numerous signal voids in the right striatum 24 hours after intrastriatal injection of LPS (1 ?g), no signal void was visible in mice that received USPIO.sub.(n)@PDA@IgG (FIG. 21). Histological analyses confirmed the MRI findings by revealing significantly more USPIO.sub.(n)@PDA@?VCAM-1 than USPIO.sub.(n)@PDA@IgG in the right hemisphere of LPS treated mice (FIG. 22). By varying the ratio of IgG and ?VCAM-1 on the surface of particles, we observed that the higher the concentration of targeted antibodies, the higher the binding of USPIO.sub.(n)@PDA@?VCAM-1 in the inflamed striatum (FIG. 23). Moreover, when the mice were pre-treated with ?VCAM-1 monoclonal antibodies to saturate USPIO.sub.(n)@PDA@?VCAM-1 binding sites, no significant binding of USPIO.sub.(n)@PDA@?VCAM-1 was observed, confirming the specificity of the contrast agent (FIG. 24).

[0171] Using high temporal resolution imaging, we also investigated the kinetic of USPIO.sub.(n)@PDA@?VCAM-1 binding on activated endothelial cells in the LPS model. As shown on FIG. 25, the targeted particles rapidly accumulate in the right striatum, with near maximal contrast reached less than 60 seconds after intravenous injection. Control USPIO(n)@PDA@IgG only induced a short transient drop of T2*-weighted signal in the right striatum, consistent with a lack of binding. Importantly, both particles were rapidly cleared from the circulation with an estimated primary half-life of 50-70 seconds. At later time-points, T2*-weighted imaging revealed progressive unbinding of the particles from the right striatum with near complete disappearance of USPIO.sub.(n)@PDA@?VCAM-1 induced signal voids 24 hours after injection (FIG. 26). Histological analyses revealed that unbound USPIO(n)@PDA@IgG accumulated in macrophages in the liver (FIG. 27).

[0172] Altogether, these experiments demonstrate that USPIO.sub.(n)@PDA@?VCAM-1 are both sensitive and specific to reveal VCAM-1 overexpression in the brain vasculature.

Clustering USPIO into Large Submicrometric Clusters Improves the Sensitivity of Molecular Imaging

[0173] To illustrate the gain in sensitivity provided by clustering USPIO into submicrometric particles, we compared the sensitivity of unclustered USPIO, small (300 nm) and large (700 nm) USPIO.sub.(n)@PDA clusters conjugated to anti-VCAM-1 monoclonal antibodies to reveal endothelial activation. In the LPS model of neuroinflammation, mice received 4 mg/kg of either particles and MRI was performed 20 minutes thereafter (to allow clearance of the smallest particles). Quantitative analysis revealed significantly more signal void in the mice that received the largest particles (FIG. 28). Almost no signal void was observed in mice that received control large USPIO.sub.(n)@PDA@IgG. These results support the improved sensibility of large submicrometric particles compared to unclustered USPIO for molecular imaging of VCAM-1.

USPIO.SUB.(n).@PDA Conjugated to Antibodies Targeting Activated Endothelial Cells Reveal Inflammation in Clinically Relevant Experimental Models

[0174] Then, we performed molecular imaging of endothelial activation in more clinically relevant experimental models. First, in a model of ischemic stroke induced by permanent occlusion of the middle cerebral artery (pMCAo). In this model, aseptic inflammation develops in the subacute phase (from 24 hours to 7 days after pMCAo), which is thought to play a key role in stroke pathophysiology. At 24 hours after pMCAo, intravenous injection of USPIO.sub.(n)@PDA@?VCAM-1 induced numerous signal voids in the right hemisphere, in the periphery of the ischemic lesion (FIG. 29a). In contrast, no significant signal void was observed after injection of control USPIO.sub.(n)@PDA@IgG. These data demonstrate that USPIO.sub.(n)@PDA@?VCAM-1 can unmask the neuroinflammatory reaction taking place in the subacute phase of ischemic stroke.

[0175] Second, in a model of acute kidney injury induced by rhabdomyolysis. In this model, an intramuscular injection of glycerol is performed in the two limbs to induce rhabdomyolysis, thereby releasing myoglobin from the muscles into the bloodstream and leading to subsequent acute kidney injury related to hypovolemia and direct toxicity of myoglobin on renal tubules. In this model, USPIO.sub.(n)@PDA@?VCAM-1 revealed endothelial inflammation mainly in the kidney medulla, in line with anatomical repartition of renal tubules (FIG. 29b). Again, USPIO.sub.(n)@PDA@IgG did not induce any significant signal void.

[0176] Lastly, in a model of inflammatory bowel disease. Mice were fed during 5 days with dextran sulfate sodium (DSS) in the drinking water. DSS induces intestinal inflammation by disrupting the intestinal epithelial monolayer lining, leading to the entry of luminal bacteria and associated antigens into the mucosa, triggering an immune response. After 2 days without DSS, we performed molecular imaging of the descending colon both before and after injection of USPIO.sub.(n)@PDA@MAdCAM-1, targeted to an adhesion molecule overexpressed by activated endothelial cells in mucosal tissues. As shown on FIG. 29c, intravenous injection of USPIO.sub.(n)@PDA@MAdCAM-1 induced numerous signal void in the inflamed mucosa of DSS-treated mice, whereas USPIO@PDA@IgG did not.

[0177] Altogether, these results demonstrate that immuno-MRI using targeted USPIO.sub.(n)@PDA can reveal inflammation in clinically relevant experimental models, as shown in three different organs (brain, kidney and intestines) and with two different targets (VCAM-1 and MAdCAM-1).