CROSSLINKED POLYSACCHARIDE BEADS COMPRISING AN IMAGING AGENT

Abstract

The present invention relates to a method for preparing beads comprising imaging agent. The present invention further provides beads highly useful for medical imaging.

Claims

1. A method for diagnosing in a subject a pathological condition selected from the group consisting of thrombosis, myocardial ischemia/reperfusion injury, stroke and ischemic brain trauma, neurodegenerative disorders, tumor metastasis and tumor growth, and rheumatoid arthritis, wherein said method comprises the steps of: administering to the subject beads comprising fucoidan and an imaging agent; and detecting the beads within the subject with an imaging technique, wherein the beads are obtained by a method comprising the following steps: i) preparing an alkaline aqueous solution comprising an amount of at least one polysaccharide, an amount of an imaging agent and an amount of a cross linking agent; ii) dispersing said alkaline aqueous solution into an hydrophobic phase comprising a surfactant in order to obtain w/o emulsion; iii) transforming the w/o emulsion into beads by placing said w/o emulsion at a temperature from about 4 C. to about 80 C. for a sufficient time to allow the cross-linking of said amount of polysaccharide; and optionally iv) incubating the beads obtained in step iii) with a solution of fucoidan to graft fucoidan on the surface of the beads, wherein said polysaccharide is selected from the group consisting of dextran, pullulan, fucoidan, agar, alginic acid, hyaluronic acid, inulin, heparin, chitosan and mixtures thereof; and wherein the presence of fucoidan is due to: the presence of fucoidan in the alkaline aqueous solution of step i); and/or the incubation of the beads with a solution of fucoidan during step iv).

2. The method according to claim 1, wherein said imaging agent is chosen among: A) MRI imaging compounds selected from the group consisting of ultrasmall superparamagnetic iron oxide particles (USPIOs), gadolinium III (Gd.sup.3+) chromium III (Cr.sup.3+), dysprosium III (Dy.sup.3+), iron III (Fe.sup.3+), manganese II (Mn.sup.2+), and ytterbium III (Yb.sup.3+), and mixtures thereof; B) radioactive imaging compounds selected from the group consisting of carbon-11 (.sup.11C), nitrogen-13 (.sup.13N), oxygen-15 (.sup.15O) and fluorine-18 (.sup.18F), gallium-68 (.sup.68Ga), yttrium-91 (.sup.91Y), technetium-99m (.sup.99mTc), indium-111 (.sup.111In), iodine-131 (.sup.131I), rhenium-186 (.sup.186Re), and thallium-201 (.sup.201Tl), terbium and mixtures thereof; C) contrast-enhanced ultrasonography imaging compounds, preferably perfluoroctyl bromide (PFOB), acoustically active microbubbles and acoustically active liposomes; D) fluorescence imaging compounds selected from the group consisting of quantum dots, fluorescent dyes such as Texas red, fluorescein isothiocyanate (FITC), phycoerythrin (PE), rhodamine, fluorescein, carbocyanine, Cy-3, Cy-5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes, merocyanine, styryl dye, oxonol dye, BODIPY dye; E) X-ray contrast compounds such as iodine; and F) mixtures thereof.

3. The method according to claim 1, wherein said imaging agent is a contrast-enhanced ultrasonography imaging compound.

4. A method for diagnosing in a subject a pathological condition selected from the group consisting of thrombosis, myocardial ischemia/reperfusion injury, stroke and ischemic brain trauma, neurodegenerative disorders, tumor metastasis and tumor growth, and rheumatoid arthritis, wherein said method comprises the steps of: administering to the subject beads comprising a radioactive imaging compound; and detecting the beads within the subject with a radio-imaging technique, wherein the beads are obtained by a method comprising the following steps: a) preparing an alkaline aqueous solution comprising an amount of fucoidan, an amount of at least one polysaccharide and an amount of a cross linking agent; b) dispersing said alkaline aqueous solution into a hydrophobic phase comprising a surfactant in order to obtain w/o emulsion; c) transforming the w/o emulsion into beads by placing said w/o emulsion at a temperature from about 4 C. to about 80 C. for a sufficient time to allow the cross-linking of said amount of polysaccharide; and d) putting the obtained beads in contact with a radioactive imaging compound, wherein said polysaccharide is selected from the group consisting of dextran, pullulan, agar, alginic acid, hyaluronic acid, inulin, heparin, chitosan and mixtures thereof.

5. The method according to claim 4, wherein said method further comprises a step c) after step c) and before step d) of incubating the beads obtained in step c) with a solution of fucoidan to graft fucoidan on the surface of the beads.

6. The method according to claim 4, wherein said radioactive imaging compound is chosen from the group consisting of carbon-11 (.sup.11C), nitrogen-13 (.sup.13N), oxygen-15 (.sup.15O) and fluorine-18 (.sup.18F), gallium-68 (.sup.68Ga), yttrium-91 (.sup.91Y), indium-111 (.sup.111In), rhenium 186 (.sup.186Re), thallium-201 (.sup.291Tl), terbium, and mixtures thereof.

7. The method according to claim 1, wherein said cross-linking agent is selected from the group consisting of trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCE), epichlorohydrin, formaldehydes, hydrosoluble carbodiimides, and glutaraldehydes.

8. The method according to claim 1, wherein said hydrophobic phase is a vegetal oil.

9. The method according to claim 1, wherein said at least one imaging agent is detected by planar scintigraphy (PS), Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), contrast-enhanced ultrasonography (CEUS), Magnetic Resonance Imaging (MRI), fluorescence spectroscopy, Computed Tomography, ultrasonography, or X-ray radiography.

10. The method according to claim 1, wherein said bead has a size comprised from 5 nm to 10 m.

Description

FIGURES LEGEND

[0161] FIG. 1: Fluorescence confocal microscopy of large beads comprising FITC labeled fucoidan.

[0162] FIG. 2: Presentation of the composition of the large beads in percentage of signal detected by Energy-dispersive X-ray spectroscopy analysis for each element C, O, Na, Cl, Fe, F.

[0163] FIG. 3: Distribution of the size of the large beads according to the invention.

[0164] FIG. 4: Mean fluorescence intensity of small MP and MP fucoidan on platelet rich plasma (PRP) not activated, activated and activated then incubated 20 minutes with anti P-Selectin in order to block P-Selectin expression.

[0165] The interaction of FITC fluorescent small MP and MP fucoidan are tested on unactivated platelets rich plasma (PRP), platetets rich plasma activated with TRAP (20 M) and platelets rich plasma activated then P-Selectin blocked with CD62P (100 M). The mean fluorescence intensity was measured in the area of double positivity.

[0166] FIG. 5: Quantification of the number of large beads comprising a fluorescent imaging agent in mice as a function of time after injection; large beads with and without fucoidan associated with the activated endothelium, control group (large beads with fucoidan associated with the non activated endothelium).

[0167] FIG. 6: Quantification of the number of large beads with fucoidan comprising an imaging agent (fluorescent FITC and/or echogenic PFOB and/or MPIO) versus control beads without fucoidan, associated with the activated endothelium (expressed per 100 leukocytes in the region of interest).

[0168] FIG. 7: MRI transversal T.sub.2*-weighted images of the aneurysm of an AAA rat before injection and after injection into the carotid artery of large MPIO comprising fucoidan (200 L of 150 mg/mL in 0.9% NaCl) at 65 minutes, 81 minutes and 115 minutes after injection.

[0169] FIG. 8: Ultrasound in two-dimensional mode of large beads of the invention (250 mg/mL in 0.9% NaCl) without PFOB (a) and with PFOB (b). Mean contrast (in dB) of large beads with and without PFOB (c)

[0170] FIG. 9: Ultrasound in two-dimensional mode of the aneurysm of an AAA rat before injection (a) and after injection into carotid artery of large microparticles comprising PFOB and fucoidan (200 L of 150 mg/mL in 0.9% NaCl) at 5 seconds (b) and 5 minutes (c) after injection.

[0171] FIG. 10: Frontal section of scan images and superimposed (NanoSPECT/CTPLUS) after injection into the penis vein of small .sup.99mTc-MP-fucoidan (200 L, of a 50 mg/mL suspension in NaCl 0.9%) in a healthy rat (a) and in a rat suffering from an AAA (c). Control after injection of non functionalized small .sup.99mTc-MP in a rat suffering from an AAA (b).

[0172] FIG. 11: Average activity measured by autoradiography on sections of healthy rat abdominal aorta (n=17 slices) and rat suffering from AAA (n=35 slices). n=1 rat.

[0173] FIG. 12: Masson trichrome staining of an AAA cryosection from an AAA rat sacrificed 30 minutes after injection of small microparticles comprising fucoidan and FITC dextran (200 L of a 50 mg/mL suspension in NaCl 0.9%). 3a. P-Selectin immunostaining (left) and control (right). 3b. Fluorescence microscopy. 3c. Alcian blue staining.

[0174] FIG. 13: Development and characterization of beads larger than 10 microns, observed by fluorescence microscopy [0175] (a) The addition of PFOB in the preparation leads to the presence of non-fluorescent droplets, which correspond to the PFOB, observed within the MP [0176] (b) Representative size distribution of the beads, prepared with or without PFOB, with an average size of 49 microns. [0177] (c) The beads were observed by electron microscopy environmental [0178] (d) Cavities were also observed in the MP prepared in the presence of PFOB [0179] (e) The Energy-dispersive X-ray spectroscopy analysis confirmed the presence of fluorine in the beads MP-PFOB, whereas MP alone do not contain [0180] (f). Ultrasound in two-dimensional mode of beads (250 mg/mL in 0.9% NaCl) without PFOB. [0181] (g) Ultrasound in two-dimensional mode of beads (250 mg/mL in 0.9% NaCl) with PFOB

EXAMPLES

IDevelopment of Beads

Reagents

[0182] Pullulan: 9 g (Hayashibara, M=200 000 g/mol); [0183] Dextran: 3 g (Sigma, M=500 000 g/mol); [0184] Dextran-FITC: 100 mg (Sigma, M=500 000 g/mol); [0185] Fucoidan: 1.2 g (Sigma, M=57 000 g/mol); [0186] Trisodium trimetaphosphate, STMP: 150 mg (Sigma, M=305.89 g/mol); [0187] Rapeseed oil: 30 ml (Commercial, HLB=7); [0188] Span 80: 7.5 g (Sigma, M=428.62 g/mol); [0189] Tween 80: 3 g (Fluka Chemika, M=1310 g/mol); and [0190] SnCl.sub.2: 1 mg (Sigma, M=84 g/mol)

Detectable Compounds

[0191] USPIO: 40 mL (Sinerem, Guerbet, 20 mg Fe/mL); [0192] Peifluorooctyl Bromide, PFOB: 240 microL (Sigma, M=498.962 g/mol); [0193] .sup.99mtechnetium: 500 microL (corresponding to an activity of about 10 mCi; and Xavier Bichat Hospital, Department of Nuclear Medicine).

[0194] 1. Preparation of Aqueous Phase

[0195] This aqueous phase is formed by pullulan and dextran, supplemented with one or more detectable compound (FITC fluorophore, USPIO, PFOB). This aqueous phase may be in the form of a solution, suspension or emulsion oil-in-water (O/W).

Preparation of the Solution of Pullulan/Dextran

[0196] 9 g of pullulan (Hayashibara, M=200,000 g/mol) and 3 g of dextran (Sigma, M=500,000 g/mol) are solubilised in 40 mL of purified water in a 100 mL beaker. The solution is stirred with a magnetic stirrer until obtaining a homogeneous solution.

Preparation of the Solution Pullulan/FITC Dextran

[0197] 9 g of pullulan (Hayashibara, 200,000 g/mol), 3 g of dextran (Sigma, 500,000 g/mol) and 100 mg of FITC-dextran (Sigma, 500,000 g/mol) are solubilised in 40 mL of purified water in a 100 mL beaker and the solution is stirred with a magnetic stirrer until obtaining an a homogeneous solution.

Preparation of the Solution Pullulan/Dextran/Fucoidan

[0198] 9 g of pullulan (Hayashibara, M=200,000 g/mol), 3 g of dextran (Sigma, M=500,000 g/mol) and 1.2 g of fucoidan (Sigma, M=57,000 g/mol) are solubilised in 40 mL of purified water in a 100 mL beaker and the solution is stirred with a magnetic stirrer until obtaining an homogeneous solution.

Preparation of the Suspension of Pullulan/Dextran-USPIO

[0199] 9 g of pullulan (Hayashibara, M=200,000 g/mol) and 3 g of dextran (Sigma, M=500,000 g/mol) are solubilised in 40 mL of a suspension of USPIO (Sinerem, Guerbet, 20 mg Fe/mL) in a 100 mL beaker and we stir with a magnetic stirrer until a homogeneous suspension.

Preparation of the Solution Pullulan/Dextratz/Fircoiclan USPIO

[0200] 9 g of pullulan (Hayashibara, M=200,000 g/mol), 3 g of dextran (Sigma, M=500,000 g/mol) and 1.2 g of fucoidan (Sigma, M=57,000 g/mol) are solubilised in 40 mL of a suspension of USPIO (Sinerem, Guerbet, 20 mg Fe/mL) in a 100 mL beaker and the solution is stirred with a magnetic stirrer until obtaining an homogeneous suspension.

Preparation of Emulsion Pullulan/PFOB-Dextran

[0201] 240 microL of PFOB (perfluorooctylbromide, Sigma, M=498.962 g/mol), 30 mg of Tween 80 (Sigma, M=428.62 g/mol) and 30 microL of NaOH (10M) were added to 300 mg of pullulan/dextran in a sample tube and we create an emulsion of PFOB in the pullulan/dextran by mixing with a dispenser (Polytron PT 3100, Kinematica) with a rod (5/7, 5 mmREF: PT-07/2EC-B101 DA) for 20 seconds at 28 000 rev/min.

Preparation of Emulsion Pullulan/Dextran/Fucoidan-PFOB

[0202] 240 microL of PFOB (perfluorooctylbromide, Sigma, M=498.962 g/mol), 30 mg of Tween 80 (Sigma, M=428.62 g/mol) and 30 microL of NaOH (10M) were added to 300 mg of pullulan/Dextran-fucoidan in a sample tube and we create an emulsion of PFOB in the pullulan/Dextran-fucoidan by mixing with a dispenser (Polytron PT 3100, Kinematica) with a rod (5/7, 5 mmREF 07/2EC-B101 PT-DA) for 20 seconds at 28 000 rev/min.

[0203] 2. Preparation of Micro-Emulsification and Cross-Linking, Process

Preparation of the Oil Phase (Surfactant HLB 7)

[0204] 7.5 g of Span 80 and 2.7 g of Tween 80 were mixed in a 10 mL beaker and the mixture was homogenised under magnetic stirring. 460 mg of this surfactant was then added to 30 mL of rapeseed oil in a bottle (bottle 3070 PS, VWR Ref 216-2686), for a concentration of 1.5% (by volume) of surfactant. The solution is then put in a bottle bottle at 20 C. for 20 min in order to have a final oil phase at 5 C.

[0205] From this step, two experiments were performed. The first experiment resulted in suspensions of beads with diameters varying from 1 m from 10 m and a mean diameter from 1 to 2 m. For sake of clarity, in the followings, the first experiment is referred to as the large beads experiment, whereas the second experiment is referred to as the small beads experiment. The second experiment indeed resulted in suspensions of smaller beads, with diameters varying from 50 nm to 4 m and a mean diameter from 300 nm to 600 nm.

Emulsification of the Aqueous Phase of Polysaccharides in the Oily Phase

[0206] 30 microL of NaOH (10M) was added to 300 mg of the aqueous phase for the large beads experiment, or to 100 mg of the aqueous phase for the small beads experiment. The resulting solution is mixed then incubated for 10 minutes at room temperature. In order to prepare beads comprising PFOB or beads comprising fucoidan and PFOB, there is no need to add NaOH. Indeed, the emulsions pullulan/dextran-PFOB or pullulan/dextran/fucoidan-PFOB already contain NaOH.

[0207] The rod in the bottle of oily phase is placed just above the level of the oil. 30 l of a solution of the crosslinking agent STMP (Trisodium trimetaphosphate, Sigma, 305.89 g/mol) prepared in 30% (w/v) in water was added to the aqueous phase.

[0208] The resulting solution is stirred with the cone of the pipette. The aqueous phase was then collected with a pipette P5000. The aqueous phase is slowly injected to the oil phase under agitation provided with a disperser running at 28 000 rotations/min. The dispersion is performed until homogenisation.

[0209] The emulsion is then transferred in an oven, at a temperature of 50 C wherein the crosslinking step takes place for 20 minutes.

[0210] Said step results in crosslinked beads. Said beads are placed in PBS and are agitated for 40 minutes at room temperature. The oil phase is eliminated, and the beads are isolated.

[0211] In the large beads experiment, after centrifugation at 4100 rpm for 10 minutes, a pellet of beads is obtained. In the small beads experiment, after a first centrifugation at 4100 rpm for 10 minutes, the supernatant is taken carefully (avoiding that any bead from the pellet is taking with) and after a second centrifugation at 8000 rpm for 10 minutes, a pellet of beads is obtained. In both experiments, the supernatant is removed and PBS in added before mixing by vortexing the beads.

Rinsing Beads

[0212] Centrifugation is performed again, at 4100 rpm for the large beads experiment or at 8000 rpm for the small beads experiment for 10 minutes, and the pellet of beads is resuspended in 0.04% SDS. This step is repeated two times.

[0213] After centrifugation at 4100 rpm/8000 rpm for 10 minutes, the pellet of beads is resuspended in 0.9% NaCl. This step is repeated six times.

Filtration of Beads

[0214] In the large beads experiment, beads are sorted in solution in 0.9% NaCl through a sieve (AS 200, Retsch) combined with a filter paper nylon mesh opening 5 m (SEFAR NITEX, 03-5/1 115 cm).

Storage of Beads

[0215] After filtration, the suspension of beads in 0.9% NaCl was stored at 4 C.

[0216] 3. Radiolabelling Beads with Technetium (.sup.99mTc)

[0217] In order to obtain beads comprising .sup.99mTc, the addition of .sup.99mTc on beads is made in an extemporaneous manner.

[0218] For this purpose, 30 l of a solution of SnCl, (1 mg/mL, Sigma, M=84 g/mol), were added to 500 L of .sup.99mTcO.sub.4.sup. and 500 L of 0.9% NaCl to a pellet of 120 mg of beads in a 1.5 mL eppendorf.

[0219] Homogenisation is briefly performed with a vortex and the suspension is incubated 1 h at room temperature. The particles were then centrifuged and the supernatant was removed.

[0220] The labelling yield was calculated after measuring the radioactivity associated with particles and that found in the supernatant.

[0221] 4. Grafting of Fucoidan on Polysaccharide Beads

[0222] The large beads of the invention (100 mg) were incubated with a solution of fucoidan (10 mg) in water (1 mL) followed by the addition of 10 L NaOH 10M under magnetic stirring for 10 min. Grafting of fucoidan was performed by adding 3 mg of sodium trimetaphosphate (STMP) and by incubating the suspension for 20 min at 50 C. Beads were then washed by centrifugation 3 times with PBS and 3 times with purified water to remove any free reagents. When fluorescein-labeled fucoidan was grafted on rhodamine-labeled pullulan, fluorescence observations confirmed the presence of green fucoidan on red beads.

IIPhysicochemical Characterization of Beads

[0223] 1. Form and Composition of Beads

[0224] Energy-dispersive X-ray spectroscopy analysis on small and large beads suspension confirmed the presence of sulphur at the surface of the beads comprising fucoidan and the absence of sulphur at the surface of the beads not comprising fucoidan. This demonstrates the presence of fucoidan at the surface of beads comprising fucoidan. Additionally, the inventors performed confocal imaging on beads prepared with fluorescent fucoidan. FITC-labeled fucoidan was observed on the surface of beads and also inside the bead structure (FIG. 1)

[0225] Energy-dispersive X-ray spectroscopy analysis on sections of large beads of different formulations has allowed us to characterize their composition. The beads were included in blocks (50 L of solutions at 250 mg/mL in 0.9% NaCl included in Cryomatrix) frozen in contact with liquid nitrogen, then sectioned (8 microns) using a microtome and finally analyzed.

[0226] In each sample, the elements carbon and oxygen were found in large proportions (FIG. 2), which is quite normal since they are the key elements present in the polysaccharides used in the beads.

[0227] Significant amounts of sodium and chlorine were also found, the beads being suspended in 0.9% NaCl when analyzed. This basic analysis shows the presence of iron in the large beads comprising USPIO, and no iron in suspensions containing other types of large beads.

[0228] The presence of fluoride in suspensions of large beads comprising PFOB, while not in suspensions containing other types of large beads, demonstrates the presence of PFOB in the large beads comprising PFOB.

[0229] Atomic absorbtion spectroscopy measurement revealed that a suspension of large beads comprising USPIO (150 mg/mL in NaCl 0.9%) has an iron concentration of 11 mmol/L.

[0230] Gas chromatographymass spectrometry analyses on a suspension of large beads comprising PFOB (150 mg/mL in NaCl 0.9%) indicated a concentration in PFOB of 8.47 mg/mL.

[0231] 2. Diameter Size Distribution of Beads

[0232] Large beads were prepared with dextran-FITC and were observed with optical fluorescence microscope. From digital photos, with the help of an image processing software, the size of the beads was measured. The distribution of particle size (in percentage) is shown in FIG. 3.

[0233] Small beads suspension was analyzed by dynamic light scattering method (Nano-ZS). A mean hydrodynamic diameter of 360 nm for beads not comprising fucoidan and 500 nm for small beads comprising fucoidan were found. Zeta potential were also measured and the beads comprising fucoidan had a higher electronegativity than the beads not comprising fucoidan (16.2 mV vs 9.1 mV).

IIIAffinity of Beads Functionalized with Fucoidan

[0234] 1. Affinity for Activated Platelets

[0235] In a first step, the inventors studied the in vitro interaction of small beads with activated platelets. Using flow cytometry, they showed the affinity of small beads functionalized with fucoidan for P-selectin expressed on the surface of activated platelets.

[0236] The beads are detected by green fluorescence (FITC) and platelets in red fluorescence (marking CD41-PE-Cy5). A double fluorescent element corresponds to the pair beads/platelets and the area of double positivity thus reflects the affinity between platelets and beads. The highest affinity (Mean Fluorescence Intensity (MFI) of 67329) is obtained with beads functionalized with fucoidan which were incubated with platelets activated with TRAP (20 M) (FIG. 4).

[0237] The inventors noticed a weak affinity for these same beads when incubated with unactivated platelets (MFI of 7982) or with activated platelets incubated 20 minutes with anti P-Selectin in order to block P-Selectin expression (MFI of 10691). Finally, the inventors showed also a weak affinity for non-functionalized beads, whether they were incubated with activated, unactivated, or activated then blocked platelets (MFI of 8537, 8206 and 8833 respectively).

[0238] 2. Affinity for Activated Endothelium

[0239] The inventors then assessed the affinity of the large beads for an activated endothelium. They used a model of inflammation of the calcium ionophore in the mouse mesentery. For this purpose, leukocytes were successfully labelled in red fluorescence by retro-orbital injection of rhodamine B (30 l of a 0.3% solution). The inventors have then activated the endothelial wall by direct application of calcium ionophore (10 l to 18 mM). Suspensions of beads or beads comprising fucoidan and FITC were then injected.

[0240] The inventors observed interactions at the activated site by intra-vital microscopy fluorescence.

[0241] An accumulation of leukocytes was observed at the area of interest, confirming the activation of the endothelium. A significant accumulation of large beads was also observed when they are functionalized with fucoidan. On the contrary, during an injection of non-functionalized beads, very few of them were found at the activated endothelial wall.

[0242] To characterize this high affinity, change in the number of beads found in the area of interest was measured. The inventors have thus shown that in the case of large beads prepared without fucoidan, the number of beads found in the activated endothelium is low and does not increase. On the contrary, in the case of injected large beads prepared with fucoidan, the number of beads comprising fucoidan found is higher and increases with time (FIG. 5).

[0243] The inventors further observed the lack of affinity for large beads comprising fucoidan for unactivated endothelium, since these particles are circulating and are not found at the area of interest. They also quantified the total number of beads accumulated at the activated endothelium at the end of the experiment, this number being expressed over the number of leukocytes found in the area of interest (FIG. 6).

[0244] The functionalized large beads have an affinity for an activated endothelium over 18 times greater than non-functionalized large beads (187 beads comprising fucoidan on average per 100 leukocytes versus 10 beads per 100 leukocytes). They further demonstrated that functionalized beads prepared in the presence of an imaging compound have a high affinity for activated endothelium, whatever the imaging agent encapsulated (206 beads with fucoidan and USPIO and 176 beads with fucoidan and PFOB, with results expressed per 100 leukocytes in the area of interest).

[0245] It is therefore possible to incorporate an imaging compound in the large beads without affecting their affinity for the activated endothelium. The inventors therefore have shown that the large beads of the invention prepared in the presence of fucoidan, with imaging agent or not, have a strong affinity for an activated endothelium.

IVDetection of Beads by MRI Imaging, Ultrasound and Scintigraphy

[0246] 1. MRI

[0247] Ex vivo, the inventors tested the large MPIO by injecting them into an aneurysm of the abdominal aorta, which is a rat model developed in the laboratory.

[0248] After injection of the suspension of large MPIO, the inventors observed interactions by MRI. The beads comprising fucoidan and USPIO show a strong MRI contrast and a high affinity for the inner wall of an aneurysm.

[0249] The inventors then performed histological sections of the aneurysm in which functionalized MPIO were injected. For this purpose, the inventors performed an immunolabeling of P-selectin. They observed that the beads are preferentially localized at the wall of the aneurysm and the fragments of thrombus. In addition, areas where the beads are adsorbed on the wall correspond to areas where P-selectin is expressed and we find much iron in the same places that our beads.

[0250] In vivo, the inventors injected the large MPIO comprising fucoidan, prepared with first example protocol, into the carotid artery of a rat suffering from an abdominal aortic aneurysm (AAA) and a strong MRI contrast were observed at the inner wall, 80 minutes after the injection (FIG. 7).

[0251] 2.

[0252] Ultrasound

[0253] In vitro, using a device for assessing the echogenic particle flow, the inventors demonstrated the echogenicity of the large beads PFOB of the invention (FIG. 8). They also showed the echogenicity of beads of average size 49 microns (FIG. 13).

[0254] Therefore, the inventors have demonstrated the echogenic characteristic of the PFOB large beads of the invention. Those results therefore corroborate that said beads are highly adapted for use as contrasting agents in vivo.

[0255] In vivo, the inventors tested the large beads comprising PFOB and fucoidan by injecting them (200 microL of 150 mg/mL beads in 0.9% NaCl) into the carotid artery of a rat AAA. Ultrasound imaging showed circulating echogenic beads in the abdominal aorta and accumulation of an echogenic signal in the aneurysmal area, 5 seconds after the injection (FIG. 9). As a control, the inventors tested the large beads comprising PFOB but not fucoidan and they showed that these circulating echogenic beads in the abdominal aorta did not accumulate in the aneurysmal area for up to 5 minutes after the injection.

[0256] 3. Scintigraphy

[0257] To detect small and large beads by scintigraphy, the inventors have coupled them to .sup.99mtechnetium. The stability of this coupling in vitro in 0.9% NaCl was tested.

[0258] In plasma, the grafting of the technetium is considered stable for 1 hour. The inventors therefore studied the distribution of organic beads 30 minutes after the injection into the penis vein. The manipulation was performed after injection of small beads comprising or not fucoidan and radiolabeled with .sup.99mTc in a healthy rat and in a rat AAA. Results are presented as percentage of radioactivity in each organ, compared to the total radioactivity injected. At the rat aorta suffering from abdominal aortic aneurysm, radioactivity was found 4 times greater than that found in the rat aorta of healthy rat (8.2% vs. 1.9%). The results indicate that the small beads of the invention are accumulated at the aneurysm.

[0259] The inventors further injected small beads comprising fucoidan and .sup.99mTc (200 microL of 50 mg/mL beads in 0.9% NaCl) into the penis vein in order to image by in vivo scintigraphy the presence of these beads in the rat AAA (FIG. 10).

[0260] On the frontal section of a rat AAA, there is an obvious contrast enhancement in the aneurysm, compared with the abdominal aorta of a healthy rat and with a rat AAA injected with small beads comprising .sup.99mTc but not fucoidan. These results indisputably show that small .sup.99mTc-MP-fucoidan can be used to detect in vivo by scintigraphy, the presence of AAA in a rat. To quantify this signal, the inventors measured by autoradiography, the radioactivity found on activated cross-sections of 20 microns, produced by a microtome from abdominal aorta of healthy rats and rats bearing AAA (FIG. 11). The inventors find an average activity by cutting more than four times the level of the abdominal aorta of rat AAA compared with healthy cell of the rat (3575 counts versus 752 counts, respectively).

[0261] The inventors then performed histological sections of the aneurysm of the rat that was injected with functionalized radiolabeled small beads. For this purpose, the inventors performed an immunolabeling of P-selectin and a polysaccharide staining with alcian blue (FIG. 12). They observed that the beads are preferentially localized inside the wall of the aneurysm and the fragments of thrombus. In addition, areas where the beads are adsorbed inside the wall correspond to areas where P-selectin is expressed. CLAIMS