FR3071844A1 - EMBOLIZATION PARTICLE COMPRISING NANO PARTICLES - Google Patents

Abstract

The present invention relates to a particle having a diameter of less than 1 mm, usable for marking and embolization, characterized in that it comprises a polymeric matrix encapsulating at least one radio-opaque nano-particle. The present invention also relates to compositions comprising said particle and the uses of the particle according to the invention as a medicament, as an embolus or as a marker.

Description

The present invention relates to a particle having a diameter of less than 1 mm, which can be used equally for marking in the context of numerous imaging techniques (MRI, X-ray, ultrasound, near infrared imaging) and embolization, characterized in that that it comprises a polymer matrix encapsulating at least one nanoparticle The present invention also relates to compositions comprising said particle and the uses of the particle according to the invention as a medicament, as an embolus or as a marker.

Prior art

Tumor embolization using submillimetric particles consists in blocking the blood flow supplying the tumor mass with nutrients and oxygen in order to cause its necrosis by cell death. Many commercial products have been around for a long time using biocompatible or bio-absorbable polymers (Gelfoam®, Curaspon®, Embospheres®, Embozeme® ...). However, nowadays, in order to improve the effects of simple embolization, practitioners often mix anti-cancer agents such as doxorubicin, which will act in synergy to increase the effectiveness of treatments ( Hepasphere®, LC / LD Beads®). For this purpose, hydrophilic embolization spheres called Hepasphere® are known which have the property of being expandable when after drying by lyophilization they are brought into contact with an aqueous medium. During this expansion phenomenon, they then have the property of being able to incorporate molecules

CCNUP1-FR-1_ CONTINUED NOTIFS_SO / ML therapeutic. Once injected into the tumor mass, this therapeutic molecule can be partially released locally.

The synergistic effect of chemotherapy coupled with embolization (chemo-embolization) now seems to be attested [Yi-Xiâng J. Wang & al. Chin J Cancer Res 2015, -27 (2): 96-121], but remains limited and is not without side effects resulting from the intrinsic toxicity of the chemotherapy agents used.

Despite the relevance of the concept, chemo-embolization treatments are not completely satisfactory because it is very difficult to embolize the entire tumor mass completely and homogeneously. Thus, if certain areas are badly embolized, the tumor will recur from these untreated areas. To counteract this negative aspect, the practitioner often uses several embolization lines that span over several weeks. However, despite this iterative, long, costly and traumatic procedure for the patient, the results are not yet completely satisfactory.

Another drawback of polymeric embolization spheres, loaded or not with anti-cancer agents, lies in their invisibility to the main imaging technologies commonly used in the medical field (MRI, X-ray scanner, ultrasound, PET, SPECT etc.). ..). As a result, the practitioner does not see where the product he is injecting is going and he does not observe the effects of the treatment until afterwards.

As a result, research is underway to make embolization spheres visible. We can cite for example the work of [F. Langevin-J & al. Biomedical Science and Engineering, 2010, 3, 1093-1098] who have shown that it is possible to load Hepasphere® with a well-known MRI imaging agent Endorem. Others like

CCNUPl-FR-l_SUfTE NOTIFS_SO / ML almost all porous or [Cilliers R. & Magn Reson Med. 2008 Apr; 59 (4): 898-902] grafted an MRI imaging agent (DTPA-Gd) to the surface of the embolization spheres. These solutions can be used in research, but little in hospital use because MRI is never used to guide the surgical gesture of transarterial embolization (TARE). Easier to use than MRI, TARE is sometimes performed under X-ray guidance. Consequently, it is desirable to make embolization spheres already existing or specially shaped for this use radiopaque.

Finally, since TARE is often performed under ultrasound, it is also desirable to obtain echogenic embolization spheres serving as a contrast agent for ultrasonic exploration and / or as emboli for ultrasonic detection. However, in the particles proposed are a trapped gas and have the disadvantage of being very unstable and having a limited lifespan.

The present invention therefore aims to respond to the problems mentioned above by providing an embolization sphere visible by X-rays, MRI and ultrasound and having a life compatible with their uses.

cases containing

Summary of 1 ¹ invention

Thus the present invention relates to a particle having a diameter of less than 1 mm, usable for marking, X-rays, MRI, ultrasound, near infrared imaging and embolization, characterized in that it comprises a polymer matrix encapsulating at least one radiopaque nanoparticle.

In the context of the present invention, the term

CCNUP1-FR-1_SUITE NOTIFS_SO / ML "polymer matrix" refers to a polymer material which constitutes all or part of the dry weight of said particle Preferably, (excluding encapsulated nanoparticle). said polymer matrix constitutes more than 80%, even more preferably more than 90% and quite preferably more than 99% of the dry weight (excluding encapsulated nanoparticle) of said particle.

In the context of the present invention, the term “nanoparticle” refers to a particle of size between 1 and 1000 nm. For the sake of clarity, it is specified that the term "nanoparticle" does not refer to the free atoms in suspension (for example to the atoms of an iodine solution).

In the context of the present invention, the term “radio-opaque” refers to the materials which interact with X-rays and / or the magnetic field used in MRI and / or the radiation used in ultrasound so that their presence is detectable by these techniques.

According to a preferred embodiment of the invention, said nanoparticle is biocompatible.

In the context of the present invention, the term “biocompatible” is intended to denote materials which are not toxic to human cells or tissues when they are placed in intimate contact with the latter.

According to a preferred embodiment of the invention, said nanoparticles have an average size of between 10 nm and 1000 nm.

According to an even more preferred embodiment of the invention, said nanoparticles have an average size of between 150 nm and 500 nm.

Many techniques are available for measuring the size of nanoparticles. We can notably cite the methods described in The ultimate

CCNUP1-FR-1_SUITE NOT! FS_SO / ML characterization in desktop particle (publishing house of Malvern instruments, 2003) and in Particle Size Measurement (5th Edition, 1997, ISBN 0412729504). Other methods can also be used such as dynamic light scattering '(DLS). The results of the different methods are similar and the differences that may be observed are negligible within the scope of the present invention.

The shape of the nanoparticles can be varied, since it does not have a great influence on the contrasting properties in imagery. However, preferably, substantially spherical nanoparticles are chosen.

According to a preferred embodiment of the invention, the weight of said nanoparticles represents 50 to 97% by weight of said particle in the dry state. For clarity, it is specified that the percentage expressed corresponds to the ratio (weight of nanoparticles) / (weight of the particle excluding nanoparticles + weight of nanoparticles).

According to a preferred embodiment of the invention, said nanoparticles consist of at least 50% by weight of materials with an atomic number greater than 53.

According to an even more preferred embodiment of the invention, said nanoparticles consist of at least 80% by weight of materials with an atomic number greater than 53.

According to a preferred embodiment of the invention, said nanoparticles are associated with a protein facilitating their endocytosis by a cancer cell.

According to a preferred embodiment of the invention, said material with an atomic number greater than 53 is a lanthanide chosen from gadolinium, dysprosium, europium, terbium, erbium, neodymium, thulium and ytterbium .

According to a preferred embodiment of the invention,

CCNUP1FR1_SUITE NOTIFS_SO / ML said nanoparticles are made up in whole or in part and preferably entirely of Gd2O2S, Gd2O2S: Eu or Gd2O2S: Yb, Tm

According to a preferred embodiment of the invention, said nanoparticles are functionalized by a molecule facilitating cell internalization.

According to an even more preferred embodiment of the invention, said molecule facilitating cell internalization is transferrin.

According to another preferred embodiment of the invention, said nanoparticles consist wholly or partly and preferably entirely of Gd2O2S, Gd2O2S: Eu or Gd2O2S: Yb, Tm functionalized with transferrin.

According to a preferred embodiment of the invention, said polymer matrix consists of a biocompatible or bioresorbable polymer.

In the context of the present invention, the term “bioresorbable” is intended to denote the materials which degrade when injected into humans. Preferably, said degradation is greater than 50% even more than the initial weight of the material, preferably greater than 60% by weight and quite preferably greater than 70% by weight within 30 days after injection. Advantageously, the degradation products are assimilated without toxic effect by the body or excreted by natural routes.

According to a preferred embodiment of the invention, said polymer matrix consists of a material chosen from the group comprising polystyrene (PS), poly-di-methylacrylamide (PDMA), gelatin, soluble starches such as pregelified and partially gelled starches, gum arabic, gum tragacanth, polysaccharides such as dextrin and

CCNUP1-FR-1_SUITE NOTIFS_SO / ML sodium alginate, synthetic polymers such as polyvinylpyrrolidone (PVP), polyvinyl ether, polyvinyl alcohol (PVA), carboxyvinyl polymers, polyacrylic polymers, polylactic acid , polyethylene glycol, cellulose ethers such as methylcellulose (MC), carboxymethylcellulose carboxymethylcellulose,

1 hydroxypropylcellulose, Tokyo, Japan), and QuadraSphere ™

Ethylcellulose (EC), (CMC), sodium

1 hydroxyethylcellulose (HEC), (HPC), vinyl acetate, methylacrylate 1 hydroxypropylmethylcellulose (HPMC) and microcrystalline cellulose.

According to a preferred embodiment of the invention, said polymer matrix is made up in whole or in part and even more preferably entirely of gelatin.

The commercially available embolization spheres can also be used in the context of the present invention. Among these, we can notably cite CELPHERE® (Asahi-Kasei Co. Ltd

Embosphere®, EmboGold ™, HepaSphere ™ (Biosphère Medical ™ Inc.).

According to a preferred embodiment of the invention, said particle is a Hepasphere®.

According to a preferred embodiment of the invention, said particle has a diameter between 0.05 and 1 mm.

Said particle is preferably spherical but other shapes can be used. In the latter case, the term "diameter" is intended to denote the diameter of the sphere in which said particle is inscribed. The diameter can be homogeneous in size even if a bimodal distribution (small with larger ones) can prove to be a judicious choice for a more homogeneous tumor marking. Those skilled in the art will be able to choose according to their needs and the observation of the

CCNUP1-FR-1_SUITE NOTIFS_SO / ML vascularization of the tumor mass to mark the right size of embolization sphere to choose.

According to an even more preferred embodiment of the invention, said particle has a diameter between 0.05 and 1 mm and quite preferentially between 0.2 and 0.4 mm.

According to a preferred embodiment of the invention, said particle is expandable. In this case the reported sizes are understood after expansion by physiological saline (Nacl 0.9%)

In the context of the present invention, the term “expandable” is intended to denote particles whose diameter increases, compared to their diameter when they are put in aqueous suspension after drying by lyophilization. According to a preferred embodiment of the invention, said increase is at least 100%, preferably at least 200% and even more preferably at least 400%.

According to a preferred embodiment of the invention, said particle further encapsulates an anticancer agent.

According to an even more preferred embodiment of the invention, said anti-cancer agent is chosen from the group comprising antimetabolites, alkylating agents, topoisomerase inhibitors, anti-tumor antibiotics and mitotic spindle poisons.

According to an even more preferred embodiment, said anti-tumor molecule is an intercalating agent, topoisomerase inhibitor.

According to an entirely preferred embodiment, said intercalating agent, topoisomerase inhibitor is an anthracycline and even more preferably doxorubicin.

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

According to another entirely preferred embodiment, said anticancer agent is chosen from the group of anticancer agents exhibiting radio-sensitization properties (e.g. temozolomide).

According to another entirely preferred embodiment, said anti-cancer agent is chosen from the group of anti-angiogenic agents (e.g. bevacizumab or sorafenib).

Furthermore, said invention also relates to a pharmaceutical composition comprising a particle according to the invention and a pharmaceutically acceptable carrier fluid.

Those skilled in the art are able to choose the pharmaceutically acceptable carrier fluid suitable for the situation. Among the excipients which can be used, mention may in particular be made of physiological serum and PBS.

Furthermore, said invention also relates to a particle or a composition according to the invention intended to be used as a medicament.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use in the treatment of cancer.

In the context of the present invention, the term “cancer” is intended in particular to designate tumors of the abdominal cavity, for example tumors of the liver, pancreas, kidneys, prostate and tumors of the thoracic cage such as tumors of the lung.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use in the treatment of cancer in a radiotherapy or radiosurgery process.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use in a tumor destruction process.

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- 10 physical requiring guidance such as radiofrequency or microwave ablation, laser ablation, cryodestruction or any other external physical means requiring precise guidance of radiation, or waves, to the tumor target.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended to be used as an embolus.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use as a medicament characterized in that it is administered by micro-catheterization.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use as a medicament characterized in that it is administered at a dose of 10 mg to 1000 mg, preferably from 100 to 400 mg.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use as a medicament characterized in that it is administered at a dose of between 0.1 and 2% by weight, preferably 0, 3-0.8% by weight relative to the tumor mass.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use as a medicament characterized in that it is administered directly into the tumor by percutaneous route.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended for use in a method of "in vivo" diagnosis of cancer.

The invention allows in particular the visualization of so-called mobile tumors which present a difficulty

CCNUP1-FR-1_SUITE NOTIFS_SO / ML specific targeting during radiotherapy treatment guided by imaging or by other technologies using an external physical means of tumor destruction requiring precise targeting.

Preferably, the particles according to the invention are used for detection, localization and possibly dynamic visualization, in fixed time, by medical imaging of mobile cancerous tumors in the context of physical tumor destruction processes requiring guidance such as radiotherapy , radiofrequency or microwave ablation, laser ablation, cryodestruction or any other external physical means requiring precise guidance of radiation, or waves, to the tumor target.

This detection and this localization are preferably carried out using known means, such as tomography or X-ray radiography, but also MRI, ultrasound or fluorescence.

Detection, localization and visualization by medical imaging of the tumor, via the particles according to the invention, allow more precise guidance of radiotherapy technologies guided by imaging, or methods of destroying cancerous tumors also using means external physical such as radiofrequency or microwave ablation, laser ablation, cryotherapy.

For example, coupled with a radiotherapy process guided by imaging, the tumor embolization obtained by the particles according to the invention causes ischemia leading to partial tumor necrosis. Carried out, approximately 10 days after embolization, the above-mentioned imaging techniques will make it possible to determine the correctly embolized areas which necrotize, areas not or badly affected by the particles according to the invention,

CCNUP1-FR-1_SUITE NOTIFS_SO / ML coupled with radiofrequency, techniques allows microwaves or art (radiologist which constitute a possible source of recurrence. It will then be appropriate for the skilled person (radiation oncologist) to determine the zones tumors which must then receive an increased dose of radiation according to a procedure called IMRT (Intensity Modulated Radiation Therapy) or even “dose painting.” The use of the combined effects (synergy) of the embolization caused by the particles according to the invention and “dose painting” in radiotherapy makes it possible to significantly reduce the average dose of ionizing radiation delivered, or to increase the effectiveness at equivalent dose, thus preserving the surrounding healthy tissue and limiting the side effects.

Likewise, these synergistic effects of ablation embolization

The interventional man) to decide to treat only the areas still alive to limit the side effects of thermal heating.

According to a preferred embodiment of the invention, the detection, localization and visualization by medical imaging of the tumor are provided dynamically and resolved in time, thus allowing real-time guidance of the above-mentioned technologies.

Furthermore, said invention also relates to a particle or a composition according to the invention for use as a labeling agent.

The particles according to the invention are detectable by various complementary medical imaging technologies such as radiography or X-ray tomography, MRI, ultrasound or even fluorescence.

Furthermore, said invention also relates to a particle or a composition according to the invention, intended to be used in a method of "in vivo" diagnosis of cancer via an MRI, tomography or

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

- 13 x-ray, ultrasound or fluorescence radiography.

Furthermore, said invention also relates to a process for preparing a particle according to the invention comprising a step consisting in bringing into contact an expandable particle, a nanoparticle and an aqueous solvent.

Surprisingly, the incorporation of nanoparticles, even relatively large (> 100nm) within the expanding embolization spheres of the Hepasphere® type is easy.

Surprisingly, 100% of nanoparticles, even relatively large (> 100nm) are confined within expansible embolization spheres of the Hepasphere® type if the quantity of physiological saline used is just equivalent to the quantity necessary for expansion Hepasphere®. Under these conditions described above, the final product obtained remains liquid and injectable with ultra-fine needles (27G). It contains 96% (on the dry extract) of contrasting product, which is considerable and surprising.

Experimental part :

Example 1: Hepasphere® loaded with nanoparticles of Gd2O2S: Eu

The following nanoparticles have been used:

- Nanoparticles (NPs) of Gd2O2S of 150 nm of average size, prepared according to the protocol described in FR 2964665 and S.A. Osseni, et al. Nanoscale, 2014, 6, 555-564.

These NPs have contrasting X-ray, MRI and fluorescence properties as described in the two references cited above.

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

The incorporation of these NPs of Gd2O2S: Eu 5% in the microspheres of the Hepasphere® type is carried out in the following manner.

00 mg of Gd2O2S NPs are suspended in 5 ml of 0.9% saline solution by sonication. This solution is then added to the original bottle containing 25 mg of dry Hepasphere® 50 to 100 μm in diameter (200-400 μm after swelling). The Hepasphere® are then left to swell and impregnate with NPs for 20 minutes with constant stirring at room temperature. The loaded Hepasphere® are then recovered by filtration on a 20 micron sieve and washed with 30 ml of 0.9% saline solution, then optionally dried in an oven at 40 ° C for analyzes on dry powders.

A fraction of these spheres charged with NPs of Gd2O2S: Eu is then taken for assay by Differential Thermal Analysis carried out under oxygen.

After combustion (between 100 and 800 ° C) under oxygen of the organic matter (the organic polymer constituting the Hepasphere®), a weight loss of 11% appears, confirming that 100% of the NPs have entered the Hepasphere® and that these these are 89% loaded with mineral NPs.

The incorporation of these NPs of Gd2O2S into Hepasphere® type spheres according to a procedure using 10 ml of saline solution (instead of 5 ml) gave the following results:

After combustion of the organic matter under oxygen (the organic polymer constituting the Hepasphere®), a weight loss of 15% appears, showing that approximately 40% of the NPs have entered the Hepasphere® and that these are loaded at 75%. of mineral NPs.

Example 2: Hepasphere® loaded with nanoparticles

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

Gd2O2S: Eu functionalized with a transfer protein: transferrin (Tf).

The same NPs of Gd2O2S: Eu of 150 nm in average diameter and functionalized with amines were used according to the protocol described below:

Measure 267 mL of propanol and introduce them into a 500 mL single-color flask. Weigh 100 mg of NPs and introduce them into the flask. Sonicate NPs for 1 hour in an ultrasonic bath. Introduce a magnetic bar into the flask and start agitation at a speed of 600 rpm. Heating at 40 ° C. Then take 25 mL of water, take 30 mL of NH4OH and cap the flask and shake it into the flask introduce it into the flask for 15 min at 40 ° C to homogenize. Using a micropipette, take 40 μL of TEOS (Tetra Ethyl Ortho Silicate) and introduce them drop by drop into the flask. Cap the flask and shake for 2 hours at 40 ° C. Using the 100-1000 pL micropipette, take 333 pL of APTMS and introduce them drop by drop into the flask. Cap the flask and shake for another hour at 40 ° C.

Then centrifuge the suspension, remove the supernatant. Put 5 mL of ethanol in the tubes and sonicate for 5 min. Centrifuge the suspension again and recover the nanoparticles. Repeat this washing operation 3 times. Dry the NPs in an oven at 85 ° C overnight.

These amines are then converted into carboxylate according to the following protocol:

Weigh 30 mg of amino NPs and introduce them into a flask. Withdraw 20 ml of DMF using the syringe and introduce it into the flask. Introduce a magnetic bar, close the flask and sonicate the NPs in an ultrasonic bath for 15 min. Shake the suspension for an additional 10 min. Weigh 605 mg of succinic anhydride and the

CCNUP1-FR-1_SUITE NOTIFS_SO / ML introduce into the flask. Purge the balloon with a stream of argon for approximately 30 minutes. Stir the mixture for 8 hours under static argon. Centrifuge the suspension to recover the NPs. Put 5 mL of water in the centrifuge tubes and sonicate them for 5 min. Centrifuge again. Repeat these centrifugation steps 3 times in total.

The carboxylated NPs are now ready for grafting the transferrin according to the following protocol:

Prepare a MES solution by dissolving 1.95 g solid MES in 200 mL of water. Transfer the NPs to a flask and add 15 ml of MES solution. Sonicate for 15 min. Weigh 30 mg of transferrin and place it in a suitable container. Take 15 mL of MES and add them to the transferrin. Shake until completely dissolved. Then add this transferrin solution dropwise to the NP solution. Shake for 10 min at 37 ° C. Then add 30 mg of EDAC (N- (3-Dimethylaminopropyl) -Nethylcarbodiimide hydrochloride and stir for another 2 hours at 37 ° C. Weigh and then add 60 mg of glycine, stir for another 10 min at 37 ° C.

Perform 3 cycles of centrifugation-washing with PBS. Dry the NPs under vacuum or freeze-dry.

The incorporation of these NPs of Gd2O2S: Eu functionalized with transferrin in microspheres of the Hepasphere® type according to the protocol already described in example 1 gave the following results:

After combustion under oxygen of the organic material (the polymer organic component the Hepasphere®) he appears a loss of weight of 12% confirming than virtually 100% of NPs are returned in the Hepasphere® and than those -are loaded about 90% of

Mineral NPs.

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

Example 3: Release kinetics of NPs encapsulated in Hepasphere®.

The release kinetics of the NPs incorporated in the Hepasphere® are then observed in a physiological saline solution according to the following protocol:

5 mg of Hepasphere® loaded with 200 mg of NPs (example 1 and 2 with 5 ml of physiological saline) are resuspended in 20 ml of physiological saline with slow stirring. After 6h, 24h and 1 week a 2ml sample is taken. The Hepasphere® are separated from the supernatant containing the released NPs by filtration and washing and then dried in an oven at 40 ° C. The quantity of NPs still present in the Hepasphere® is again measured by Differential Thermal Analysis as already explained and reported in the following table.

Time T = 0 6h 24 7d % of NPs in Hepasphere® 89 82 70 63 % of NPs-Tf in Hepasphere® 90 75 52 43

Table 2: Release kinetics of NPs incorporated into Hepasphere®.

It can be seen from this table that the NPs released Hepasphere® slowly and partially. The release kinetics are significantly faster with NPs functionalized with transferrin

Example 4: Internalization by endocytosis of NPs Gd2O2S: Eu, functionalized or not, by cancer cells.

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In order to guarantee a localization of the Gd2O2S NPs: Eu after release by the Hepasphere® within the tumor mass without risking their dissemination outside it is important to evaluate their capacity to be quickly captured and internalized (by endocytosis) by cancer cells.

Consequently, the following experimental protocol has been tested.

NIH3T3 type cancer cells were incubated at a concentration of 0.1 mg / ml of Gd2O2S: Eu NPs, naked or functionalized with transferrin. The cells are then rinsed to remove the non-internalized NPs, detached from the plates, then recovered by centrifugation. The gadolinium present inside the cells is then assayed by ICP. The main results are grouped in the following table.

Functionalization of NPs naked transferrin Incubation time at 0.1 mg / ml 24 2 hours Concentration in mg / l of Gd in the cell ground material (100,000 cells) 14.8 36.7

Table 3: Internalization kinetics of NPsGd2O2S: Eu.

It emerges from these trials that NPs functionalized with transferrins internalize themselves much more easily and more quickly in tumor cells.

EXAMPLE 5 Gelatin Embolization Spheres Loaded with NPs Gd2O2S: Eu

According to another possible embodiment, the embolization microspheres are non-porous and non-expandable and the mineral nanoparticles are incorporated directly during the process of synthesis of

CCNUP1-FR-1_SUITE NOTIFS_SO / ML the latter. Advantageously, these embolization microspheres consist of a resorbable biopolymer capable of releasing the NPs during their degradation.

Among the many possible choices, gelatin is an interesting solution because gelatin is a by-product from the food industry, inexpensive, biocompatible, non-irritant, non-allergenic, non-immunogenic, widely available and already widely used by the pharmaceutical industry. [KM-Gattaz-Asfura 2005 and AI Van de Bulke 2000]. Gelatin can also be crosslinked using different crosslinking agents (formaldehyde, glutaraldehyde, polyepoxy, carbodiimide, UV light, etc.) [ref 21 to 29 of the article Curcio 2010]. By controlling this degree of crosslinking, the rate of degradation in vivo can be controlled, which ranges from a few hours for uncrosslinked gelatin to completely nonabsorbable gel, for example for the trisacryl gelatin spheres [A. Laurent 1996].

The following example shows the preparation of a partially crosslinked gelatin sphere loaded with Gd2O2S NPs: Eu nude. The experimental protocol used is derived from the work of [Tabata 1989]

250 mg of Span 80 (sorbital monoleate) are added to a flask already containing 2.5 ml of toluene, 2.5 ml of chloroform and 50 mg of NPs. 100 mg of gelatin previously dissolved in 2.5 ml of water are then added to the solution with very vigorous stirring in order to produce the emulsion. Stirring is continued for 30 minutes at 30 ° C. The emulsion thus obtained is then quickly added to a 40ml solution composed of 75% toluene (30 ml) and 25% chloroform (10 ml) containing 2g of Span 80.

The gelatin emulsion is then crosslinked (if necessary) with a solution of glutaraldehyde in toluene (10 mg / 10 ml; quantity adjustable depending on the

CCNUP1-FR-1_SUITE NOTIFS_SO / ML desired degree of polymerization). Crosslinking takes place in an ice bath for 4 hours (time adjustable depending on the degree of polymerization desired).

The microspheres are then recovered by filtration then washed with a toluene and chloroform solution (25/75) then with a PBS solution. They are finally stored in a PBS solution.

Under the conditions described above, the charged gelatin microspheres contain only 10% by mass of NPs G2O2S: Eu (ATG assay). Their size is variable (between 30 and 300 μπι). However, it is quite easy to sort them by size by sieving if the need arises.

Example 6: Hepasphere® loaded with Gd2O2S: Eu nanoparticles for radiography and X-ray tomography imaging on phantom (model).

In the following experiment, a 32 cm x 24 cm x 15 cm plastic box is filled with gelatin, thereby mimicking the conditions of absorption of X-rays by a human abdomen. In this mass of gelatin are introduced four simulated tumors of growing size of 1.7 respectively; 3.3; 5.1 and 8.3 cm in diameter. Each of these simulated tumors, cylindrical in shape (length equal to diameter), consists of gelatin loaded with Hepasphere® loaded with nanoparticles Gd2O2S: Eu as described in Example 1 (200 mg of NPs for 25 mg Hepasphere®).

The characteristics of this phantom (model) are shown in the following table.

Tumor Diameter Volume Load in Concentration Concentration simulated and tumor NPs volume in area in length cm3 mg NPs mg / cm3 and NPs *

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in cm (% p) Mg / cm2 1 1.7 3.85 66.2 17.2 (1.72) 29.3 or 23 2 3.3 28.2 251 8.9 (0.89) 2 9.8 or 2 3 3 5.1 104 613 5.9 (0.59) 30 or 23.4 4 8.3 449 1616 3.6 (0.36) 29.9 or 23.3

* The surface concentration of NPs is equal to the concentration of NPs relative to the projected area of the tumor which intercepts ray beam X. The projected area is equal to: π xd ^2/4 or 1 xd following is observed along the axis of the cylinder or perpendicular to it. The surface concentration of NPs is adjusted to a constant value of 30 mg / cm2 to the nearest measurement uncertainties.

Table 4: Synthetic data characteristic of the simulated tumors tested on X-ray and X-ray tomography.

The imaging results performed in X-ray tomography (CB-CT 110 kv) and plane KV radiography (85 Kv; 15 mAs are reported in the following table:

Tumor simulated Diameter and length in cm Concentration volume in Concentration area in NPs * mg / cm2 CB-CT Unit Hounsfield (difference type) KV Units of Grey arbitrary (difference type) NPs and mg / cm3 (% P) 1 1.7 17.2 (1.72) 29.3 403 (70) 27850 (460) 2 3.3 8.9 (0.89) 29.8 206 (61) 27890 (661) 3 5.1 5.9 (0.59) 30 119 (36) 28063 (705) 4 8.3 3.6 (0.36) 29.9 62 (12) 28566 (726)

Table 5: Quantified results of the contrast obtained on the simulated tumors tested on radiography and X-ray tomography.

These results show that by using a plane KV radiography device the contrast obtained on the images is substantially constant with the surface concentration in NPs whatever the size of the simulated tumor. With a concentration of NPs set at 30 mg / cm2 of projected surface, the contrast is sufficient to achieve

CCNUP1-FR-1_SUITE NOTIFS_SO / ML tumor tracking by X-ray radiography.

On the other hand, in X-ray tomography, which produces sectional images, and no longer in projection, the contrast expressed in Hounsfield units is proportional to the volume (or weight) concentration in NPs.

Those skilled in the art consider that in X-ray tomography a contrast of the order of 30 to 50 Hounsfield units is sufficient to detect a pathological process or a marked tumor which refers to inserted quantities of weak NPs, of the order of 3mg / cm3 (0.3% by weight). However, the majority of guidance methods use planar imaging systems which are much less sensitive than tomography devices. Consequently, we will retain the value of 30 mg / cm2 of projected area as the lower limit value. For tumor sizes ranging between 1.5 and 8cm, this amounts to incorporating at least between 2% and 0.3% by mass of NPs.

On the CB-CT tomography images obtained, a less significant contrast is observed for large simulated tumors because the volume concentration of nanoparticles decreases.

On the flat radiography (KV) images obtained, a much less overall contrast is clearly observed than in CB-CT tomography. However, this contrast is substantially constant regardless of the size of the tumors because the surface concentration of nanoparticles is also constant.

Example 7: Hepasphere® loaded with Gd2O2S nanoparticles: Eu for guiding radiotherapy on mobile phantom (model).

The experiment consisted in using a mobile abdominal phantom mimicking a patient's breathing movements in order to perform a tracking experiment

CCNUP1-FR-1_SUITE NOTIFS_SO / ML dynamic tumor in real conditions of use on a Cyberknife radiotherapy device guided by two crossed KV plane imagers.

In this experiment, the simulated gelatin tumor has a cylindrical shape 1.5 cm in diameter and 1.5 cm in length representing a tumor volume of 2.64 cm3 (projected area 1.76 cm2). This tumor is loaded with 53 mg of NPs (the mass of Hepasphere® is neglected considering that their contrasting property is zero). This quantity represents a surface concentration of 30 mg / cm2.

This simulated tumor is then introduced into a mobile plastic cylinder with a density exactly equal to that of water. This plastic cylinder is itself introduced into a mass made of the same plastic material of size and shape simulating the absorption of a human abdomen. This cylinder moves (using a jack) in a movement that simulates a human respiratory cycle (range of motion 3cm).

Under these conditions, the Cyberknife radiotherapy device managed to follow the movements of the simulated tumor without difficulty.

The experiment was repeated with a simulated gelatin tumor with a cubic shape of 3.2 cm on the side representing a tumor volume of 32 cm3 (projected area 10.24 cm2). This tumor is loaded with 324 mg of NPs. This quantity represents a surface concentration of 31 mg / cm2. Under these conditions, the Cyberknife radiotherapy device managed to follow the movements of the simulated tumor without difficulty.

Example 8: Hepasphere® loaded with Gd2O2S nanoparticles: Eu for ultrasound.

According to an advantageous and surprising mode of the invention

CCNUP1-FR-1_SUITE NOTIFS_SO / ML NPs confined in the embolization microspheres have very marked contrasting properties in ultrasound.

Thus the experiment consisted in using Hepasphère® loaded with NPs according to Example 1 (25mg Hepasphere®, 200mg NPs-150nm, 5ml of physiological saline). Simulated tumors were then produced according to the same method as previously described in a mass of gelatin for increasing concentrations of NPs: 1, 2, 5 and 10 mg / ml.

The images obtained by ultrasound make it possible to observe that the simulated tumors comprising an amount of lmg / ml (0.1 l% p) of NPs are easily detectable (the amount present of Hepasphere® is considered negligible because not echogenic).

For low NP content (<2 mg / ml) the tumor / medium interface is more strongly contrasted.

This finding is corroborated by another series of photographs taken on other phantoms comprising a constant quantity of NPs (53 mg) distributed in simulated tumors of increasing size.

It is found that for an NPs content of less than 0.5 mg / ml the interface appears visibly.

In order to get even closer to the physiological conditions, 1 ml of physiological serum loaded with an increasing amount of Hepasphere® loaded with NPs (still produced according to example 1) was injected into a pig liver.

Under these conditions, the spot formed by a nanoparticle load of less than 1 mg / ml becomes very difficult to detect.

Nevertheless, very surprisingly, the sensitivity of detection of the embolization spheres of the present invention, by ultrasound

CCNUP1-FR-1_SUITE NOTIFS_SO / ML is remarkable. The sensitivity of the ultrasound (lmg / ml or 0.1 l% p) is at least as good as for X-ray tomography (0.3% p) or MRI (0.2%, see example 9) and indeed better than plane radiography.

Finally, we carried out a similar experiment in a recently euthanized dog liver. We injected 2ml (2cm ³ ) of Hepasphere® loaded with 40mg / ml of NPs (prepared according to Example 1: 200mg of NPs for 25 mg Hepasphere®, 5ml physiological saline). The spot obtained is clearly visible in both ultrasound and X-ray tomography or on flat radiography images.

Example 9: Hepasphere® loaded with Gd2O2S nanoparticles: Eu for MRI.

The same injected pig liver used for the ultrasound experiments of Example 8 was visualized by MRI (T2 imaging).

The spots formed by a nanoparticle load greater than 2 mg / ml are easily detectable.

Phantoms (1cm x 1cm cylinder) were prepared for a concentration of 0.5 mg / ml NPs in gelatin. In the first phantom, the NPs are free and well dispersed throughout the mass of gelatin. In the second phantom, these NPs were previously incorporated into Hepasphere® according to Example 1 (25 mg Hepasphere®, 200 mg NPs, 5 ml of physiological saline).

Analysis by MRI imaging shows that the contrast is significantly improved for the phantom containing the Hepasphere® loaded with NPs. The MRI contrast effect of NPs incorporated into Hepasphere® is increased compared to free NPs.

Example 10: Hepasphere® loaded with Gd2O2S nanoparticles: Yb, Tm for near infrared imaging.

CCNUP1-FR-1_SUITE NOTIFS_SO / ML

Near infrared optical imaging is hardly used for human applications because near infrared light (650-1000nm) hardly penetrates more than a centimeter of biological tissue. On the other hand, it is widely used for preclinical studies on small animals.

The following example demonstrates that embolization spheres loaded with NPs emitting in the near infrared could be very useful for studying embolization phenomena by photonic imaging. The NPs previously used based on Gd2O2S: Eu5%, although fluorescent, are not well suited for in vivo imaging because it is excited in the UV which does not penetrate the skin of an animal. Consequently, we changed the fluorescent dopant Eu by the couple Yb, Tm more suitable for imaging in the infrared.

Nanoparticles (NPs) of Gd2O2S: Yb8% Erl%, of 130 nm of average size, are prepared according to the protocol described in FR 2964665 and S. A. Osseni, et al. (Nanoscale, 2014, 6, 555-564). Only the doping agent Eu of example 1 is replaced by two other lanthanides, ytterbium and erbium in order to exhibit remarkable fluorescence properties (emission 650-670 nm) after an excitation located in the near infra -red (excitation 980nm). Those skilled in the art know the fluorescence properties of this type of material. The X-ray absorption and contrast agent properties for MRI are substantially equivalent to the NPs of neighboring composition Gd2O2S: Eu5%.

The incorporation of these Gd2O2S NPs: Yb8% Erl% is done in the same way as in Example 1. However, in order to test the sensitivity of this imaging technique, the amount of NPs incorporated into the Hepasphere® has been very greatly reduced. (10 mg NPs, 25 mg Hepasphere®, 10 ml of physiological saline). 10ml of physiological saline were

CCNUP1-FR-1_SUITE NOTIFS_SO / ML used (instead of 5) in order to guarantee the obtaining of a very fluid suspension for injection with the micro-syringes used for this experiment. Obtaining a suspension loaded with 0.5 mg / ml of NPs is then done by diluting the mother suspension.

microliters of Hepasphere® suspension loaded with NPs are then injected directly into the kidney of a rat. The kidney was chosen as the test organ for this test because it is an organ that is particularly well vascularized and very loaded with hemoglobin, strongly absorbing light. Consequently, this constitutes a difficult case for the visualization of luminescent compounds.

The results obtained show that under these conditions, with an excitation at 980nm, the red fluorescence of Hepasphere® charged with NPs is very easily detectable through the entire body of the animal.

microliter of a suspension injected at 0.5 mg / ml of NPs represents only 10 micrograms of NPs (25 micrograms of dry Hepasphere®). Consequently, and surprisingly, the sensitivity of this near infrared imaging technique using Hepasphere® loaded with NPs is about three orders of magnitude higher than the other imaging techniques reported in this document (quantities injected of the order of ten mg).

Claims (28)

1. Particle having a diameter of less than 1 mm, for its use for marking, X-rays, MRI, ultrasound, near infrared imaging and embolization, characterized in that it comprises a matrix polymer encapsulating at least one radiopaque nanoparticle. 2. Particle according to the preceding claim characterized in that said nanoparticles have an average size between 10 nm and lOOOnm. 3. Particle according to the preceding claim characterized in that said nanoparticles have an average size between 150 nm and 500 nm. 4. Particle according to one of the preceding claims, characterized in that the weight of said nanoparticles represents 50 to 97% by weight of said particle in the dry state. 5. Particle according to one of the preceding claims, characterized in that the said nanoparticles consist of at least 50% by weight of materials with an atomic number greater than 53. 6. Particle according to the preceding claim, characterized in that the said nanoparticles consist of at least 80% by weight of materials with an atomic number greater than 53. 7. Particle according to one of the preceding claims, characterized in that said material number CCNUP1-FR-1_ CONTINUED NOTIFS_SO / ML atomic greater than 53 is a lanthanide preferentially chosen from gadolinium, dysprosium, europium, terbium, erbium, neodymium, thulium and ytterbium. 8. Particle according to one of the preceding claims, characterized in that said nanoparticles consist in whole or in part of Gd2O2S: Eu or of Gd2O2S: Yb, Tm. 9. Particle according to one of the preceding claims, characterized in that said nanoparticles consist in whole or in part of Gd2O2S: Eu or of Gd2O2S: Yb, Tm functionalized with a molecule facilitating cellular internalization. 10. Particle according to the preceding claim characterized in that said polymer matrix consists of a biocompatible or bioresorbable polymer. 11. Particle according to the preceding claim, characterized in that the polymer matrix consists of a material chosen from the group comprising polystyrene (PS), poly-di-methylacrylamide (PDMA), gelatin, soluble starches such as pregelified and partially gelled starches, gum arabic, tragacanth, polysaccharides such as dextrin and sodium alginate, synthetic polymers such as polyvinylpyrrolidone (PVP), polyvinyl ether, polyvinyl alcohol (PVA) , carboxyvinyl polymers, polyacrylic polymers, polylactic acid, polyethylene cellulose such as glycol, methylcellulose (MC) ethers, Ethylcellulose (EC), carboxymethylcellulose (CMC), CCNUP1-FR-1_SUITE NOTIFS_SO / ML carboxymethylcellulose sodium, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), vinyl acetate, methylacrylate and hydroxypropylmethylcellulose (HPMC) and microcrystalline cellulose. 12. Particle according to characterized in that it consists of gelatin. the preceding claim the polymer matrix is 13. Particle according to the preceding characterized in 14. Particle according to previous characterized in between 0.05 and 1 mm 1 a that ^one of claims it is a Hepasphere®. one of what it claims has a diameter 15. Particle according to the preceding claim characterized in that it has a diameter between 0.2 and 0.4 mm. 16. Particle according to one of the preceding claims, characterized in that it is expandable. 17. Particle according to one of the preceding claims, characterized in that it also encapsulates an anti-cancer agent. 18. Pharmaceutical composition comprising a particle according to one of claims 1 to 17 and a pharmaceutically acceptable excipient. 19. Particle according to one of claims 1 to 17 or composition according to claim 18 intended for use as a medicament. CCNUP1-FR-1_SUITE NOTIFS_SO / ML 20. Particle according to one of claims 1 to 17 or composition according to claim 18 for use in the treatment of cancer. 21. Particle according to one of claims 1 to 17 or composition according to claim 18 intended to be used as an embolus. 22. Particle according to one of claims 1 to 17 or composition according to claim 18 intended to be used as a medicament characterized in that it is in a form suitable for its administration by microcatheterization. 23. Particle according to one of claims 1 to 17 or composition according to claim 18 intended for use as a medicament characterized in that it is in a form suitable for its administration at a dose of 10 mg to 1000 mg, preferably of 100 to 400mg. 24. Particle according to one of claims 1 to 17 or composition according to claim 18 intended for use as a medicament characterized in that it is in a form suitable for its administration directly into the tumor by percutaneous route. 25. Particle according to one of claims 1 to 17 or composition according to claim 18 intended for use in a method of "in vivo" diagnosis of cancer. 26. Particle according to one of claims 1 to 17 or composition according to claim 18 for use CCNUP1-FR-1_SUITE NOTIFS_SO / ML as a marking agent. 27. Particle according to one of claims 1 to 17 or composition according to claim 18 intended to be 5 used in a method of “in vivo” diagnosis of cancer via an MRI, tomography or X-ray radiography, ultrasound or fluorescence method. 10 28. A method of preparing a particle according to one of claims 1 to 17 or a composition according to claim 18 comprising a step of contacting an expandable particle, a nanoparticle and an aqueous solvent.