Ultrafine nanoparticles as multimodal contrast agent

#The invention relates to a novel use of ultrafine nanoparticles, 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 phototherapy or thermotherapy, in particular for the treatment of lung tumors.

#Despite considerable research efforts, cancer remains one of the major causes of death throughout the world (R. Siegel et al.: Cancer Statistics 2012, 2012, 62, 10-29). Among the various types of cancer, lung cancer constitutes the principal cause of cancer-associated death (J. S. Guthi et al., Molecular Pharmaceutics, 2009, 7, 32-40): there are 1.4 million deaths throughout the world each year, the 5-year survival rate is less than 15%.

#The low survival of patients suffering from lung cancer is mainly due to the absence of tools for early diagnosis and for locally targeting therapeutic agents.

#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 Gd3+ 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 Gd3+ 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 Gd3+ 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.

#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 Gd3+ 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.

#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 Gd3+) obtained according to example 1. The presence of nanoparticles in the kidneys is still detectable 24 hours after the administration of the solution.

#The gadolinium chloride hexahydrate ([GdCl3.6H2O], 99%), the sodium hydroxide (NaOH, 99.99%), the tetraethoxysilane (Si(OC2H5)4, TEOS, 98%), the aminopropyl-triethoxysilane (H2N(CH2)3—Si(OC2H5)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).

#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.

#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.