Ultrafine nanoparticles as multimodal contrast agent

Abstract

The invention relates to a novel use of ultrafine nanoparticles, of use as a diagnostic, therapeutic or theranostic agent, characterized by their mode of administration via the airways. The invention is also directed toward the applications which follow from this novel mode of administration, in particular for imaging the lungs, and the diagnosis or prognosis of pathological pulmonary conditions. In the therapeutic field, the applications envisioned are those of radiosensitizing or radioactive agents for radiotherapy (and optionally curietherapy), or for neutron therapy, or of agents for PDT (photodynamic therapy), in particular for the treatment of lung tumors.

Claims

1. A method for imaging or treating a brain tumor in a subject in need thereof, comprising administering via the subject's airways, an effective amount of ultrafine nanoparticles as an imaging agent for imaging a brain tumor, as a radiosensitizing agent for treating said tumor, or both, said ultrafine nanoparticles having the following properties: said ultrafine nanoparticles comprise a polyorganosiloxane matrix; said ultrafine nanoparticles also comprise a chelating agent complexing cations M.sup.n+, being a rare earth metal, n being an integer between 2 and 4, and optionally doping cations D.sup.M+, D being a rare earth metal other than M, an actinide or a transition element, m being an integer between 2 and 6, and said ultrafine nanoparticles have a mean diameter having been reduced to a value between 1 and 5 nm by dissolution of all or part of a precursor nanoparticle comprising a core made of a metal oxide or oxyhydroxide of M, and, irradiating said subject, imaging said subject or both.

2. The method of claim 1, wherein said ultrafine nanoparticles comprise at least one imaging agent for T.sub.1 MRI imaging, and at least one imaging agent suitable for one of the following imaging techniques: (i) PET or SPECT scintigraphy, (ii) fluorescence in the near-infrared range, and (iii) X-ray tomodensitometry.

3. The method of claim 1, wherein said M.sup.n+is Gd.sup.3+and said ultrafine nanoparticles have a relaxivity r1 per ultrafine nanoparticle of between 50 and 5000 mM.sup.−1.Math.s.sup.−1 at 1.4T.

4. The method of claim 1, wherein M is a lanthanide.

5. The method of claim 4, wherein said lanthanide is selected from the group consisting of Dy, Lu, Gd, Ho, Eu, Tb, Nd, Er, Yb and mixtures thereof.

6. The method of claim 1, wherein each ultrafine nanoparticle is obtained from a precursor nanoparticle comprising: a core comprising a metal oxide or oxyhydroxide of M, at least partly in cationic form M.sup.n+being an integer between 2 and 4, optionally doped with a doping agent D present at least partly in cationic form D.sup.m+, m being an integer between 2 and 6; at least one coating layer comprising polyorganosiloxanes (POSs); and, optionally, an overcoating comprising a chelating agent C1 capable of complexing the M.sup.n+cations or a hydrophilic molecule capable of suspending the precursor nanoparticle in an aqueous medium; said precursor nanoparticle having been subjected to dissolution of the core using a pH-modifying agent or a chelating agent C2, identical to or different than C1, capable of complexing all or part of the M.sup.n+and D.sup.m+cations, such that a mean diameter of the ultrafine nanoparticle thus obtained is reduced to a value of between 1 and 5 nm.

7. The method of claim 1, wherein said ultrafine nanoparticles further comprise a radioactive isotope that can be used in scintigraphy.

8. The method of claim 7, wherein said radioactive isotope is a radioactive isotope of In, Tc, Ga, Zr, Y, Cu or Lu.

9. The method of claim 7, wherein said radioactive isotope is selected from the group consisting of .sup.111In, .sup.99mTc, .sup.68Ga, .sup.64Cu, .sup.89Zr, .sup.90Y and .sup.177Lu.

10. The method of claim 1, wherein said ultrafine nanoparticles comprise a T.sub.1 contrast agent suitable for magnetic resonance imaging.

11. The method of claim 1, wherein the ultrafine nanoparticles comprise a multimodal contrast agent suitable for T.sub.1 MRI imaging, and at least one imaging technique chosen from the group consisting of: i. PET or SPECT scintigraphy, ii. fluorescence in the near-infrared range, and iii. X-ray tomodensitometry.

12. The method of claim 1, wherein said ultrafine nanoparticles have a Gd weight ratio between 5% and 50%.

13. The method of claim 1, wherein said chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA), 1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), and derivatives thereof.

14. The method of claim 1, wherein said ultrafine nanoparticles are administered intranasally or intratracheally.

15. The method of claim 1, wherein said ultrafine nanoparticles are used both as an imaging agent for MRI of brain tumors and as a radiosensitizing agent for treating said brain tumors.

16. A noninvasive method of imaging of a brain tumor in a human patient or an animal patient, comprising the following steps: (i) administering to said human or animal patient, via the human or animal patient's airways, an effective amount of ultrafine nanoparticles having the following properties: said ultrafine nanoparticles comprise a polyorganosiloxane matrix; said ultrafine nanoparticles comprise a chelating agent complexing cations M.sup.n+, M being a rare earth metal, n being an integer between 2 and 4, and optionally doping cat ions D.sup.m+, D being a rare earth metal other than M, an actinide or a transition element, m being an integer between 2 and 6; and said ultrafine nanoparticles have a mean diameter having been reduced to a value between 1 and 5 nm by dissolution of all or a part of a precursor nanoparticle comprising a core made of a metal oxide or oxyhydroxide of M; and, (ii) capturing MRI images using an appropriate MRI sequence.

17. The method of claim 16, wherein the ultrafine nanoparticles are administered in the form of an aerosol.

18. A method for monitoring the therapeutic efficacy of a therapeutic treatment of a brain tumor in a human or animal, said method comprising the following steps: (i) at the initiation of a treatment of a human or animal, administering an effective amount of ultrafine nanoparticles as an imaging agent, said ultrafine nanoparticles having the following properties: said ultrafine nanoparticles comprise a polyorganosiloxane matrix, said ultrafine nanoparticles also comprise a chelating agent complexing cations M.sup.n+, M being a rare earth metal, n being an integer between 2 and 4, and optionally doping cations D.sup.M+, being a rare earth metal other than M, an actinide or a transition element, m being an integer between 2 and 6, and said ultrafine nanoparticles have a mean diameter having been reduced to a value between 1 and 5 nm by dissolution of all or part of a precursor nanoparticle comprising a core made of a metal oxide or oxyhydroxide of M, (ii) capturing images of the imaging agent using an appropriate imaging technique in order to visualize the brain tumor, (iii) repeating steps (i) and (ii) during the treatment of the human or animal, and (iv) comparing the change in said brain tumor during the treatment, thereby deducing the therapeutic efficacy of the treatment.

Description

FIGURE LEGENDS

(1) FIG. 1: a) MRI of the lungs in the mouse before (left) and 15 minutes after (right) the intratracheal administration of the SRP nanoparticles (40 microliters with a Gd.sup.3+ ion concentration of 20 mM). A 183% enhancement of the intensity of the image is observed after administration of the nanoparticles, b) 3D fluorescence imaging of the mouse, 1 hour after nebulization (50 μl), in the lungs, of SRP nanoparticles grafted with the Cy5.5 fluorescent probe, c) 3D visualization of the lungs before (left) and after (right) nebulization of SRP nanoparticles (50 μl with a Gd.sup.3+ ion concentration of 20 mM), obtained by X-ray microtomography (the “holes” correspond to the presence of nanoparticles), d) enhancement of the MRI signal in the lungs as a function of time (instillation of 40 μl of SRP solution with a Gd.sup.3+ ion concentration of 20 mM), the time constant for detection of the nanoparticles before elimination via the kidneys is approximately two hours. All the nanoparticles used in these examples were synthesized according to the protocol described in example 1.

(2) FIG. 2: images of the lungs of a mouse obtained before (a) by MRI and (c) by optical fluorescence imaging and 3 hours after the intratracheal administration of a solution of SRP nanoparticles obtained according to example 1 (coupled with a Cy 5.5 fluorophore for the fluorescence imaging) (40 microliters with a Gd.sup.3+ ion concentration of 20 mM) (b) by MRI and (d) by fluorescence optical imaging. The images demonstrate the elimination of the nanoparticles via the kidneys (K: kidney; L: lung). No hepatic elimination is detected.

(3) FIG. 3: evolution over time of the enhancement of the MRI signal in the kidneys following the intratracheal administration of a solution of contrast agent (50 mM Gd.sup.3+) obtained according to example 1. The presence of nanoparticles in the kidneys is still detectable 24 hours after the administration of the solution.

(4) FIG. 4: high-resolution MRI image of the biodistribution of the contrast agent in the lungs of a mouse after selective administration of the solution (50 mM Gd.sup.3+) in the left lung (on the right in the image). The nanoparticles used were obtained according to example 1. The main acquisition parameters are echo time 276 μs, section thickness 0.5 mm, field of view 2.5 cm, total acquisition time 4 minutes.

(5) FIG. 5: biodistribution of the SRP Cy 5.5 24 h after intrapulmonary nebulization or intravenous injection of a suspension of particles at 10 mM with respect to Gd.sup.3+ ions. The nanoparticles are obtained according to example 1 with coupling of a fluorophore of Cy 5.5 type to the nanoparticle.

(6) FIG. 6: passive targeting of orthotopic H358-Luc pulmonary tumors after intravenous injection or intrapulmonary nebulization of a suspension of nanoparticles. The nanoparticles are obtained according to example 1 with coupling of a fluorophore of Cy 5.5 type to the nanoparticle.

(7) FIG. 7: survival curves (Kaplan-Meier) obtained on mice carrying H358 orthotopic pulmonary tumors, treated by 10 Gy irradiation alone or treated by 10 Gy irradiation carried out 24 h after intrapulmonary administration of a suspension of nanoparticles at 20 mM with respect to Gd.sup.3+, ions.

EXAMPLES

Chemical Products

(8) The gadolinium chloride hexahydrate ([GdCl.sub.3.6H.sub.2O], 99%), the sodium hydroxide (NaOH, 99.99%), the tetraethoxysilane (Si(OC.sub.2H.sub.5).sub.4, TEOS, 98%), the aminopropyl-triethoxysilane (H.sub.2N(CH.sub.2).sub.3—Si(OC.sub.2H.sub.5).sub.3, APTES, 99%), the triethylamine (TEA, 99.5%), the N-(3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC,>98.0%), the N-hydroxysuccinimide (NHS,>97.0%), the diethylenctriaminepentaacetic dianhydride (DTPADA) and the anhydrous dimethyl sulfoxide (DMSO, 99.5%) were obtained from Aldrich Chemicals (France). The Cy5.5 mono-NHS-ester was ordered from GE Healthcare. The diethylene glycol (DEG, 99%) comes from SDS Carlo Erba (France), while the acetone comes from Sodipro (France). The 1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid (DOTAGA) was provided by CheMatech SAS. The cRGD cyclic tripeptide (Arg-Gly-Asp) comes from Genecust, Luxembourg. The 5-(4-carboxyphenyl)-10, 15,20-triphenylchlorine (TPC) comes from Frontier Scientific (Logan, Utah).

Characterizations

(9) The mean sizes of the nanoparticles indicated were measured by PCS (photon correlation spectroscopy) and correspond to their hydrodynamic diameter. These measurements were taken on a Zetasizer NanoS apparatus (the laser used for the PCS is a He-Ne 633 nm laser). The zetametry measurements were carried out on the same equipment, with the particles being diluted beforehand in a 0.01 M NaCl solution. The pH is adjusted a posteriori. Transmission electron microscopy (TEM) is carried out in order to obtain structural and morphological data on the samples. It was carried out using a JEOL 2010 microscope operating at 200 kV. The samples are prepared by deposition on a carbon grid. The relaxometry at 1.4 T (60 MHz) is measured on equipment of Bruker Minispec MQ60 type. The mass spectrometry is carried out using on LTQ spectrometer (Thermo Fisher Scientific, San Jose, Calif.). The elemental analyses were carried out at the analysis center of the CNRS [French National Center for Scientific Research] at Solaize by ICP-MS and made it possible to determine the C, N, Si and Gd contents with a minimum accuracy of 0.3%. The fluorescence analyses were obtained on a Varian Carry Eclipse spectrofluorometer. The lyophilization of the particles is carried on a Christ Alpha 1,2 lyophilizer.

Animals

(10) The animals used for the proof of concept by MRI are 6-week-old Balb/c female mice weighing between 20 and 22 grams, these mice having been purchased from the Janvier breeding center (Le Genest, Saint-Isle, France). The animals used for the proof of concept by fluorescence imaging and X-ray tomography are 6-week-old NMRI nude female mice weighing approximately 25 grams, these mice having been purchased from the Janvier breeding center (Le Genest, Saint-Isle, France). Before the experiment, the mice acclimatized to their new environment in a temperature-controlled room for 1 week. The animal experiments are carried out while adhering to the instructions of INSERM (National Institute for Health and Medical Research) regarding animal welfare.

Imaging Tools

MRI

(11) The MRI images were acquired on a Brucker Biospec 47/50 spectrometer with a field at 4.7 T (Brucker, Ettlingen, Germany), using an emitter/receiver coil with a diameter of less than 25 mm (Rapid Biomedical, Rimpar, Germany). The mice are placed on their stomach on a plastic cradle and kept anesthetized using a gas mask delivering 2% of isofluorane contained in an N.sub.2/O.sub.2 gas mixture (80:20). The body temperature is kept constant by virtue of a circulation of hot water and the respiratory cycle is constantly monitored.

(12) For each animal, 6 axial sections 1 mm thick are acquired. The acquisition is carried out under free breathing without respiratory or cardiac synchronization using a multi-section 2D UTE (ultrashort echo time) sequence. The main parameters are echo time of 276 μs. repetition time of 84 ms, flip angle of 60°,field of view of 3 cm, total acquisition time of 1 minute.

Fluorescence Imaging

(13) In order to carry out the 2D or 3D fluorescence imaging in vivo, the mice are anesthetized (isoflurane/oxygen: 3.5-4% for induction and 1.5-2% for maintenance).

(14) The 2D imaging system is composed of an excitation system composed of diodes emitting at a wavelength of 660 nm. The fluorescence images and also the “black and white” images are taken with a CCD camera cooled to −80° C. (ORCA11-BT-512G, Hamamatsu, Massy, France), equipped with an RG 9 high-pass filter (Schott, Jena, Germany). The image acquisition and also the analysis are carried out with the Wasabi software (Hamamatsu, Massy, France). 24 h after the injection, the mice are sacrificed and the organs are imaged. A semi-quantification of the fluorescence of the organs is obtained by drawing regions of interest (ROIs) around the organs.

(15) The florescence tomography is carried out using an fDOT 3D imaging system. The system is composed of a black box, an excitation light originating from a laser (690 nm, 26 mW, Powertechnology, St Nom La Bretche, France) and a CCD camera (ORCA ER, Hamamatsu) equipped with an RG 9 high-pass filter (Schott, Jena, Germany). The 3D mapping of the fluorescence in the mice is calculated by means of a reconstruction algorithm.

X-Ray Imaging

(16) In order to carry out the X-ray imaging and the 2D or 3D fluorescence imaging in vivo, the mice are anesthetized (isoflurane/oxygen: 3.5-4% for induction and 1.5-2% for maintenance).

(17) The X-ray tomography images are taken with the Scanco viva CT 40 (Scanco Medical, Inc., Bassersdorf, Switzerland) using an energy of 65 keV and an integration time of 200 ms. The 3D reconstructions of the lungs mark the presence of air in the lungs. The presence of the particles is therefore represented as a “hole” on the reconstructions, since the particles partly take the place of the air.

(18) We also have a bed which fits in the X-ray scanner and the fluorescence tomography, allowing superimposition of the images and visualization of the colocalizations.

Example 1; Synthesis of Hybrid Gadolinium Nanoparticles of Gd-Si-DOTA: SRP (Small Rigid Platforms) Type

(19) The nanoparticles are obtained by means of a three-stage synthesis as described in WO 2011/135101. First of all, the oxide cores are synthesized in diethylene glycol before growth of the polysiloxane layer, followed by the covalent grafting of complexing agents to the surface of the nanoparticle; the gadolinium oxide core dissolves when placed in water, thereby leading to fragmentation of the particles which makes it possible to obtain small particles of less than 5 nanometers composed only of polysiloxane, or organic species and of complexing agents useful in imaging.

(20) The advantage of this top down approach is to make it possible to obtain very small particles which have multimodal characteristics while at the same time having the advantage of being easy to eliminate via the kidneys.

Detailed Description of the Synthesis

Oxide Cores

(21) A first solution is prepared by dissolving 5.58 g of gadolinium chloride hexahydrate in 500 ml of anhydrous diethylene glycol (DEG) at ambient temperature. A second solution of 500 ml of DEG containing 4.95 ml of 10 M sodium hydroxide is added to the first solution. The addition takes place at ambient temperature over the course of 24 H. A transparent colloidal solution is obtained with a mean size for the oxide cores of 3.5 nm in diameter (measurements obtained by PCS and by TEM microscopy).

Encapsulation

(22) The growth of the polysiloxane layer is provided via a sol-gel reaction by adding two silane precursors (APTES and TEOS) in a 60/40 proportion and triethylamine as catalyst. To do this, 1050 μl of APTES and 670 μl of TEOS are added to the previous solution with vigorous stirring at 40° C. For the purpose of obtaining fluorescence in the near-IR, a variable amount of APTES can be coupled to Cy5.5 mono-NHS-ester by creating an amide bond between the APTES and the fluorophore. After 1 H of reaction, 2550 l of a solution of DEG containing triethylamine (0.1 M of TEA, 10 M of water) are added. The sol-gel reaction previously described is repeated three times, each time 24 H apart. At the end of these various steps, the solution is left to stir at 40° C. for 48 H. The final colloidal solution exhibits particles with a mean size of 4.5 nm.

Surface Functionalization

(23) Finally, a considerable excess of DOTAGA (2 DOTAGA per Gd atom) is added to the particles, allowing the formation of a peptide bond between the available amine functions of the surface and a carboxylic acid function of the ligand. 13.76 g of DOTAGA are dispersed in 200 ml of DMSO. After the addition of the particle solution to the solution containing DOTAGA, the mixture is left to stir for a further 48 hours. The nanoparticles are then precipitated from 9 l of acetone. The acetone is removed and the particles are washed with a further 3 l of acetone and recovered by centrifugation. The particles are then redispersed in 200 ml of distilled water, the excess of acetone is evaporated off and then the solution is kept stirring for a further 24 h. The purification is carried out by tangential centrifugation through Vivaspin® membranes (pore size: 5 kDa). Finally, the resulting purified solutions are lyophilized and then stored in a refrigerator for several months without modification of the product. In order to obtain particles which are more fluorescent, it is also possible to add the Cy5.5 post-grafting, i.e. after the particles have been obtained, by direct reaction of the Cy5.5 mono-NHS-ester with the free amine functions of the particle. The fluorescence analysis of the particles made it possible to demonstrate that it is possible to graft approximately one Cy5.5 per particle. The size of these nanoparticles was obtained by fluorescence correlation spectroscopy and made it possible to obtain a hydrodynamic diameter of 4 nm slightly larger than that obtained without fluorophore.

(24) The nanoparticles without fluorophore have a hydrodynamic radius of 3±0.1 nm once rediluted in aqueous solution. The weight of the particles was estimated at 8.5±1 kDa according to a mass spectrometry analysis. The longitudinal relaxivity r.sub.1 is 11.4 mmol.sup.−1s.sup.−1 at 60 MHz. At high field (300 MHz), the relaxivity obtained at 300 MHz is 6 mmol.sup.−1s.sup.−1. The elemental analysis combined with the particle size makes it possible to obtain a total of 10 DOTA, 27 Si and 7 Gd per particle. These data were corroborated by a potentiometric and fluorescence assay of a number of free DOTAs present per particle. Before the use of the particles for the biological applications, the free DOTAs are used to chelate other Gd.sup.3+ ions in order to maximize the r.sub.1 per object (114 mmol.sup.−1.Math.s.sup.−1 per object on average) or to chelate active ions in SPECT or PET scintigraphy. This method is described in the literature (Lux et al., Ange. Chem. Int. Ed., 2011, 50, 12299). A solution for injection of these nanoparticles is prepared by diluting the particles in a saline solution with a HEPES buffer in order to fix the pH. An intravenous injection in the tail of rodents (rats or mice) made it possible to show elimination of the particles via the kidneys, combined with a plasma lifetime twice as long as DOTAREM®.

Example 2: Functionalization of the Nanoparticles for Active Targeting of Tumors: SRP-cRGD

(25) The nanoparticles used for the grafting are similar to those described in example 1. They therefore possess a set of free DOTAs (and thus of available carboxylic acid functions) for the grafting of cRGD which is known to be an agent that targets α.sub.vβ.sub.3 integrin. The grafting of cRGD to the particle is carried out by means of peptide coupling between a carboxylic acid function of a DOTA unit and the primary amine function of the cRGD peptide. The nanoparticles obtained in example 1 are diluted in water (Gd.sup.3+ concentration of around 100 mM). A mixture of EDC and NHS (3.4 EDC and 3.4 NHS per Gd) is added to the particles, the pH is adjusted to 5 and the mixture is kept stirring for 30 minutes. The cRGD (2.3 cRGD per Gd) is dissolved separately in anhydrous DMSO. It is then added to the previous solution and the pH of the mixture is adjusted to 7.1 before leaving it to stir for 8 hours. The solution is finally purified by tangential filtration through a 3 kDa membrane, before being lyophilized. The elemental analysis makes it possible to work back to a cRGD content of approximately 2.5 per particle. The same protocol is used to graft cRAD to the nanoparticles; these nanoparticles will serve as a control in the active targeting tests carried out with cRGD.

Example 3: Analysis of the Interaction of SRPcRCd Nanoparticles Obtained According to Example 2 with Cells Expressing α.SUB.v.β.SUB.3 .Integrin by Flow Cytometry and Fluorescence Microscopy

Analysis by Fluorescence Microscopy

(26) HEK293(β3) cells, which overexpress α.sub.vβ.sub.3 integrin, are cultured on coverslips in 12-well plates (100 000 cells per well) overnight at 37° C. They are rinsed once with 1X PBS, then with 1X PBS containing 1 mM of CaCl.sub.2 and 1 mM of MgCl2. They are then incubated for 30 minutes at 4° C. (binding analysis) or 30 minutes at 37° C. (internalization analysis), in the presence of SRP (nanoparticles according to example 1) with a fluorophore of Cy5.5 type, of SRP-cRGD (obtained according to example 2) Cy5.5 or of SRP-cRAD (obtained according to example 2 and acting as a negative control) Cy5.5 at a Gd.sup.3+ ion concentration of 0.1 mM. They are then rinsed with PBS Ca.sup.2+/Mg.sup.2+ (1 mM) and fixed (10 minutes with 0.5% paraformaldehyde). The nuclei are labeled with Hoechst 33342 (5 μM for 10 minutes) (Sigma Aldrich, Saint Quentin Fallavier, France). After washing with 1X PBS, the coverslips are mounted with Mowiol. The images are taken with an Apotome microscope (Carl Zeiss, Jena, Germany).

Analysis by Flow Cytometry

(27) Before the binding analysis, the adherent cells (HEK293(β3)) are trypsinized, then rinsed once with cold (4° C.) 1X PBS, and a second time with cold 1X PBS Ca.sup.+/Mg.sup.2+ (1 mM). One million cells, in a final volume of 200 μl, are resuspended in a solution of SRP Cy5.5, of SRP-RGD Cy5.5 or of SRP-RAD Cy5.5 at a Gd.sup.3+ ion concentration of 0.1 mM and incubated for 30 minutes at 4° C. After two rinses with PBS Ca.sup.2+/Mg.sup.2+ (1 mM), the cells are rapidly analyzed by flow cytometry (LSR II, Becton Dickinson, France).

(28) For the internalization analysis, the protocol is similar, with reagents at 37° C. and an incubation for 30 minutes at 37° C.

(29) The results obtained, by flow cytometry and by microscopy, made it possible to demonstrate specific binding and internalization of the SRP-RGD Cy5.5 on the HEK293(β3) cells, which binding is not observed with the SRP Cy5.5 and the SRP-RAD Cy5.5 (results not shown).

Example 4: MRI Imaging of the Lung Using SRP Nanoparticles Synthesized According to Example 1

(30) A concentration study was carried out in order to determine the injection concentration most suitable for observing the best MRI contrast. The mice were anesthetized by virtue of an intraperitoneal injection by means of 50 μg/g of ketamine (Panpharma, France) and 5 μg/g of xylazine (Sigma-Aldrich, Saint-Quentin Fallavier, France). After the image acquisition without contrast agent, the mice were incubated intratracheally by means of a 22-gauge intravenous Teflon catheter. A volume of 50 μl of the SRP solution was introduced into the lungs by the catheter. 7 different Gd.sup.3+ concentrations were tested (2,5, 10, 20, 33, 50 and 100 mM), while a saline solution was injected into a control mouse. Once the intubation was stopped, the lung image acquisition was carried out at regular intervals over a period of between 5 minutes and several hours after the instillation of the solution. Among all these concentrations, it is the 50 mM concentration which exhibited the best signal enhancement (235±15%), the signal enhancement being a little less in the case of the 100 mM concentration (171±10%). The signal enhancement is defined as the difference between the signal-to-noise ratio in the lungs before and after the administration, normalized with respect to the signal-to-noise ratio in the lungs before the administration of the contrast agent.

(31) The 50 mM concentration was therefore retained for carrying out the subsequent biodistribution studies by MRI. Another study was carried out using this optimum concentration of 50 mM on 3 mice. For these animals, the MRI imaging of the lungs, of the liver, of the kidneys and of the bladder was carried out at regular intervals between 5 minutes and 2 days after the administration of the contrast agent. These manipulations made it possible to demonstrate that the half-lifetime of the contrast agent excreted by the kidneys is 149±51 minutes. This elimination of the nanoparticles by the kidneys is due to their small size and represents a real advantage in terms of toxicity of the contrast agent. It will also be important for an original application described in example 10.

Example 5: 2D and 3D Fluorescence and X-Ray Imaging of the Lung Using SRP Nanoparticles Synthesized According to Example 1

(32) The lyophilized SRP Cy5.5 obtained according to example 1 are solublized in an appropriate volume of water to obtain an injectable preparation for at least 30 minutes, in order to obtain a solution at 100 mM with respect to Gd.sup.3+ ions. The suspension of particles is then diluted in a solution containing a HEPES buffer and a saline solution. The preparation obtained has a pH of 7.4 and an osmolarity suitable for administration to animals.

(33) The intrapulmonary nebulization is carried out using a microsprayer® (PennCentury®). Each mouse is anesthetized (Domitor/Ketamine mixture, intraperitoneal injection) and receives 50 μl of nanoparticulate suspension via the intrapulmonary route. The mice used for the proof of concept by fluorescence imaging are 6-week-old NMRI nude mice weighing approximately 25 grams (Janvier, Le Genest Saint Isle, France).

(34) The results obtained by 2D and 3D fluorescence and X-ray imaging showed a distribution of the fluorescent nanoparticles in the two lungs after nebulization. These results presented in FIG. 1 are in perfect agreement with those obtained by MRI.

Example 6: Synthesis of Nanoparticles for Radiosensitizing Effect on Lung Tumors

(35) This example describes the synthesis of nanoparticles employed for use as a radiosensitizing agent in the context of glioblastoma treatment (G. Le Due et al., ACS Nano, 2011, 5, 9566). These nanoparticles are gadolinium oxide nanoparticles covered with a layer of polysiloxane functionalized with DTPA (complex also chelating a gadolinium ion). They are envisioned for treatment of pulmonary tumors by radiosensitization owing to their characteristics which are very similar to the nanoparticles described in example 1 (both in terms of size and in terms of morphology).

(36) The synthesis of the gadolinium oxide core is carried our by dissolving the gadolinium chloride hexahydrate salt (5.576 g) in 100 ml of DEG at ambient temperature and with vigorous stirring. The suspension is heated at 140° C. until complete dissolution of the salt (approximately 1 hour). When the solution has become clear, sodium hydroxide (4 ml, 3.38 M) is added dropwise to the solution while maintaining vigorous stirring. At the end of this addition, the stirring and the heating are maintained for 3 hours. A transparent colloidal solution of gadolinium oxide cores is then obtained, which can be stored at ambient temperature for several weeks without modification.

(37) The functionalization with the polysiloxane layer is carried out in the presence of the silane precursors (APTES (10.1 ml) and TEOS (6.4 ml)) and of a hydrolysis solution (triethylamine in DEG (0.1 M of TEA and 10 M of water)). The solutions are added in several steps to 400 ml of the solution previously prepared containing the gadolinium oxide cores ([Gd.sup.3+]=45 mM) with stirring at 40° C. A silicon content 4 times greater than the gadolinium content is chosen for this synthesis (the precursor mixture is composed of 60% of APTES and 40% of TEOS). The addition of the various precursors is carried out in 6 successive steps. Each step consists of the addition of a part of the solution containing the precursor mixture (5% of the solution for the first step, 15% for the second and 20% for the subsequent steps). The delay between each addition is set at 1 hour. After the final addition, the solution is left to stir for 48 hours at 40° C.

(38) In order to facilitate their colloidal stability in a biological medium, the nanoparticles are functionalized with DTDTPA by virtue of a peptide bond between one of the activated carboxylic acid functions and an amine function resulting from the APTES present at the surface of the nanoparticles. 100 ml of the colloidal solution of nanoparticles previously obtained are added to 4.25 g of DTDTPA dissolved in 20 ml of DMSO.

(39) The nanoparticles are then precipitated from 500 ml of acetone and the supernatant is removed by centrifugation. The white powder obtained is then washed with an ethanol/acetone (85/15) mixture and the supernatant is again removed by centrifugation (the operation is repeated 3 times). The nanoparticles are then dispersed in water and purified by tangential centrifugation (through Vivaspin® 5 kDa membranes).

(40) After a purification by a factor of 1000, the particles are lyophilized. They are then directly injectable by virtue of a HEPES buffer and a saline solution at the correct osmolarity, the pH being adjusted to 7.4. These nanoparticles have a hydrodynamic diameter of 2 nm and an r.sub.1 at 60 MHz of 9.4 mM.sup.−1.Math.s.sup.−1 (the r.sub.1 to r.sub.2 ratio is 1.13). They can therefore be used as T.sub.1 contrast agents in order to visualize their biodistribution and their accumulation in the tumor (G. Le Due et al., ACS Nano, 2011, 5, 9566). This monitoring by imaging is important in order to determine the optimum time at which to initiate the radiotherapy. These therapeutic agents have already proved their radiosensitizing capacity in vitro on human brain cancer cells of U87 type (P. Mowat et al., Journal of Nanoscience and Nanotechnology, 2011, 11, 7833-7839). The use of these nanoparticles or else of those described in examples 1 and 2 therefore appears to be particularly suitable in the context of combating lung cancer by carrying out imaging-guided therapy (in this case radiotherapy).

Example 7: Protocol Envisioned for a Radiosensitization Application of these Nanoparticles Injected Via the Airways

(41) The nanoparticles synthesized according to example 1 are injected via the airways. Their accumulation in the tumor zone is then pinpointed by means of MRI (example 4), of florescence imaging coupled to X-ray tomography (example 5) or else of scintigraphy after chelation of a radioactive isotope used in PET or in SPECT. Once the nanoparticle concentration is pinpointed as optimal in the tumor zone (ratio of the contrast between the healthy zone and the tumor zone), the treatment by radiotherapy can be activated. The nanoparticles are subsequently eliminated by the kidneys after they have passed into the blood. The administration via the airways makes it possible to inject a smaller amount of nanoparticles into the patient. Since treatment by radiotherapy is divided up into sessions under clinical conditions, it will then be possible to carry out a further administration of particles before each of these sessions because of the possibility of elimination of these particles and the relatively small amounts injected. The various trials previously carried out on radioresistant tumors of U87 type lead one to predict advantageous results in lung radiosensitization.

Example 8: Theranostic for Asthma

(42) No noninvasive imaging technique currently makes it possible to evaluate the severity and the extent of bronchial remodeling in severe asthmatics. Bronchial remodeling corresponds to an abnormal change in the bronchial and peribronchial tissues following repeated bronchial inflammation. This remodeling results in various histological changes: thickening of the subepithelial membrane, increase in extracellular matrix deposits, neoangiogenesis, mucus gland hypertrophy and increase in bronchial smooth muscle mass. Bronchial remodeling in severe asthmatics results in an unfavorable prognosis, significant morbidity and a marked degradation of respiratory function; furthermore, patients do not respond to the usual therapies. Severe asthmatics represent 10% of the asthmatic population, i.e. 350 000 patients in France, and more than half the costs associated with this pathological condition. For the development of effective treatments, it is essential to have available a noninvasive imaging technique which makes it possible to accurately evaluate the severity of the remodeling and the response to treatments. The use of aerosols of nanoparticles grafted with targeting molecules (for example the cRGD tripeptide for α.sub.vβ.sub.3 integrin as described in example 2) should allow the imaging of the angiogenesis associated with bronchial remodeling for the diagnosis and for the evaluation of the therapeutic efficacy of the treatment of severe asthma. With this objective, it will be possible to set up a murin modeling of chronic asthma. These mice (6-week-old female Balb/c mice) will receive intraperitoneal injections of ovalbumin (100 μg) or of Dermatophagoides pteronyssinus (D. pter) (100 μg) for sensitization and then intranasal instillations of the same compounds at regular intervals (days 14,27,28,29,47,61,73,74 and 75) for the appearance of bronchial remodeling 15 weeks after the beginning of the sensitization.

Example 9: Synthesis of Nanoparticles for Dynamic Phototherapy Effect on Lung Tumors

(43) The addition of a photosensitizer to the nanoparticles makes it possible to give them a toxic effect under the action of light. Nevertheless, light cannot penetrate into the body beyond a few centimeters, even with the most appropriate chromophores (absorption in the tissue transparency window, i.e. the near-infrared zone). The main advantage of the approach via the airways is to be able to obtain a concentration in pulmonary tumors. The latter can then be illuminated by means of the optical fiber of an endoscope (thus avoiding the problems of penetration of the tissues by light).

(44) The synthesis of nanoparticles which have an action by PDT and which can be injected via the airways is set up in the following way:

(45) Gadolinium chloride hexahydrate (3.346 g) is placed in 60 ml of DEG at ambient temperature. The suspension is then heated at 140° C. with vigorous stirring in order to ensure total dissolution of the gadolinium salt. 4 ml of a 2.03 M sodium hydroxide solution are then added dropwise while maintaining the vigorous stirring. The solution is then left to stir at 180° C. for 3 hours. A colloidal solution of gadolinium oxide cores is then obtained, which can be stored at ambient temperature for several weeks without risk of degradation.

(46) The addition of a chlorine-derived photosensitizer (TPC) can be carried out by virtue of the activation of the carboxylic acid function of the TPC with an EDC/NHS mixture so as to obtain the 5,10,15-tri-(p-tolyl)-20-(p-carboxylphenyl)chlorinesuccinidyl ester (TPC-NHS) according to the protocol described by C. Frochot et al. (Bioorganic Chemistry, 2007, 35, 205-220). 20 mg of TPC-NHS are coupled by peptide bonding with 12.3 μl of APTES in 4.2 ml of anhydrous DMSO overnight.

(47) The silane precursors (APTES (1.5 ml) and TEOS (1.0 ml)) and also the hydrolysis solution (aqueous solution of triethylamine in DEG (0.015 M of TEA and 1.5 M of water)) are added stepwise to the 60 ml of DEG containing the particles, with stirring at 40° C. The total addition is carried out in 6 steps. Each step consists of the addition of a part of the precursor solution to the colloidal solution in DEG (5% for the first step, 15% for the next step and 20% for the final steps). The solution containing the TPC coupled to the APTES is added during the first step at the same time as the other precursors. The time between each addition is one hour. After the final addition, the mixture is kept stirring for 48 hours at 40° C.

(48) The nanoparticles are then functionalized with DTDTPA by means of peptide coupling between the amines of the APTES and the activated carboxylic acid function of the complexing agent. 2.5 g of DTDTPA in 12 ml of anhydrous DMSO are added to the previous solution. The resulting mixture is then stirred for 1 hour. The nanoparticles are then precipitated from 300 ml of acetone and the supernatant is removed by centrifugation. The powder obtained is washed 3 times with an ethanol/acetone (85/15) mixture. The powder is finally redispersed in water and purified by tangential centrifugation on a 5 kDa membrane (Vivaspin®). This procedure is repeated several times in order for a degree of purification of at least 100 to be reached. The purified solution of colloids is then lyophilized.

Example 10: Passive Targeting of a Subcutaneous Tumor After Intrapulmonary Administration of Nanoparticles

(49) The lyophilized cyanine 5.5 particles are solubilized in an appropriate volume of injection-grade water for at least 30 minutes, in order to obtain a solution at 100 mM with respect to Gd.sup.3+ ions. The suspension of particles is then diluted in a solution containing a HEPES buffer and a saline solution. The preparation obtained has a pH of 7.4 and an osmolarity which is suitable for administration to animals.

(50) The mice received, beforehand, a subcutaneous graft of tumor cells on the flank. When the tumor reaches a size of 5×5 mm, the mice receive an intrapulmonary injection of nanoparticles (50 μL per mice). The nebulization is carried out using a microsprayer® (Penncentury®).

(51) The results obtained showed a very rapid passage of the particles into the blood stream (15-20 minutes) from the lungs, combined with elimination via the kidneys.

(52) A signal is detected in the subcutaneous tumor starting from 5 h after the nebulization. The signal increases up to 24 h after the administration.

(53) The biodistribution is comparable to that obtained for the administration of an equivalent amount of Gd.sup.3+ ions intravenously (injection in the caudal vein), but with a slightly lower passive tumor accumulation (FIG. 5). The nanoparticles injected via the airways have the advantage of ensuring a lower renal uptake.

Example 11: Passive Targeting of an Orthotopic Pulmonary Tumor After Intravenous or Intrapulmonary Administration of Nanoparticles

(54) The lyophilized cyanine 5.5 particles are solubilized in an appropriate volume of injection-grade water for at least 30 minutes, in order to obtain a solution at 100 mM with respect to Gd.sup.3+ ions. The suspension of particles is then diluted in a solution containing a HEPES buffer and a saline solution. The preparation obtained has a pH of 7.4 and an osmolarity appropriate for administration to animals.

(55) The mice received, beforehand, an orthotopic graft of pulmonary tumor cells (H358) in the lung. The tumor cells stably express the luciferase gene, thereby making it possible to monitor the tumor growth by bioluminescence imaging in vivo. When the tumor is well established and detectable by bioluminescence imaging in vivo, the mice receive an intravenous injection (200 μl per mouse) or an intrapulmonary injection (50 μl per mouse) of nanoparticles. The nebulization is carried out using a microsprayer® (Penncentury®).

(56) The results obtained show that, after intravenous administration, the particles are distributed throughout the body of the mouse and are eliminated via the kidneys. Starting from 5 h after the administration, a signal is detectable by 3D fluorescence imaging in the H358 pulmonary tumor implanted orthotopically in the lung. The signal is stable up to 24 h post-administration.

(57) As regards the intrapulmonary administration, the results show that, after administration, the particles pass very rapidly into the blood stream (15-20 minutes) from the lungs, and are eliminated via the kidneys. Starting from 5 h after the nebulization, most of the particles are eliminated from the lung and a signal is detectable in the pulmonary tumor orthotopically implanted in the lung. The signal is stable up to 24 h post-administration.

(58) The in vivo fluorescence imagings show good colocalization of the fluorescence with the presence of the tumor (X-ray tomography carried out before the administration of the particles, see FIG. 6).

(59) The fluorescence and bioluminescence imagings carried out ex vivo on the lungs show a colocalization of the bioluminescence signal (corresponding to the tumor cells) and of the fluorescence (corresponding to the Cy5.5-particles) (see FIG. 6).

(60) The particles are therefore capable of passively accumulating in orthotopically implanted pulmonary tumors, irrespective of the route of administration (intravenous or intrapulmonary).

Example 12: Radiosensitizing Effect of the Nanoparticles on Pulmonary Tumors After Intrapulmonary Administration

(61) The lyophilized particles are solubilized in an appropriate volume of injection-grade water for at least 30 minutes, in order to obtain a solution at 100 mM with respect to Gd.sup.3+ ions. The suspension of particles is then diluted in a solution containing a HEPES buffer and a saline solution. The preparation obtained has a pH of 7.4 and an osmolarity appropriate for administration to animals.

(62) The mice received, beforehand, an orthotopic graft of pulmonary tumor cells (H358) in the lung. The tumor cells stably express the luciferase gene, thereby making it possible to monitor the tumor growth by bioluminescence imaging in vivo. When the tumor is well established and detectable by bioluminescence imaging in vivo, the mice receive an intrapulmonary injection (50 μl per mouse) of nanoparticles. The nebulization is carried out using a microsprayer® (Penncentury®). Twenty-four hours post-administration, the mice are irradiated (X-rays) in a single dose in a conventional irradiator.

(63) The results obtained show that the intrapulmonary administration of the nanoparticles before irradiation improves the survival of the mice, in comparison with a single irradiation (see FIG. 7). The particles therefore exhibit a radiosensitizing effect for the pulmonary tumors.