Ultrasmall Rigid Particles as Multimodal Probes for Agents DOI: 10.1002/anie.201104104 Ultrasmall...

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  • Imaging AgentsDOI: 10.1002/anie.201104104

    Ultrasmall Rigid Particles as Multimodal Probes for MedicalApplicationsFranois Lux, Anna Mignot, Pierre Mowat, Cdric Louis, Sandrine Dufort, Claire Bernhard,Franck Denat, Frdric Boschetti, Claire Brunet, Rodolphe Antoine, Philippe Dugourd,Sophie Laurent, Luce Vander Elst, Robert Muller, Lucie Sancey, Vronique Josserand, Jean-Luc Coll, Vasile Stupar, Emmanuel Barbier, Chantal Rmy, Alexis Broisat, Catherine Ghezzi,Graldine Le Duc, Stphane Roux, Pascal Perriat,* and Olivier Tillement

    Over the past two decades, nanoparticles have been devel-oped in the field of theranostic[1, 2] with the objective ofmeeting three requirements: 1) to exhibit long circulation inbody fluids with major accumulation in tumour tissues due to

    their active or passive targeting properties (enhanced perme-ability and retention (EPR) effect);[3] 2) to be rapidlyeliminated through the renal route to ensure a sufficientdifference in concentration between healthy and diseasedzones; 3) to display therapeutic potential and contrast proper-ties.[4, 5] This latter requirement was reinforced by thesimultaneous development of devices combining imagingtechniques, such as 1) high-sensitive X-ray tomography,positron emission tomography, or single-photon emissioncomputed tomography and 2) magnetic resonance imaging(MRI) with high spatial resolution.[6, 7]

    However, it is still a great challenge to ensure bothappropriate renal elimination (necessarily achieved usingparticles < 5.5 nm in size)[8] and multimodality (requiringmolecules that systematically enlarge the particle size to anon-adequate extent). For instance, quantum dots[9] or goldclusters[10] do not exhibit both features as they requirecoatings that are too prohibitive in size to ensure multi-modality. After reviewing the solutions proposed in scientificliterature,[1113] the most promising strategies rely on theelaboration of silica- or polymeric-based structures thatincorporate different functional entities, such as dyes forfluorescence imaging, magnetic complexes for MRI, radio-active elements for scintigraphy or curie-therapy, heavyelements for interacting with X- or g-rays, neutron absorbersfor neutron-therapy or sensitizers for photodynamic therapy.Concerning multifunctional silica-based particles, even themost investigated technologies (i.e., Stber or reverse emul-sion methods) failed to yield objects smaller than 10 nm insize.

    Here we propose an original top-down method consistingin the fragmentation of sub-10 nm structures already possess-ing all the desired functions (see Supporting Information).Briefly, these starting structures consist of core (gadoliniumoxide)shell (polysiloxane) particles developed by our groupwhich offer several features and functionalities, but are toolarge in size to escape hepatic clearance.[14] Gadolinium wasselected as contrast agent on account of its paramagneticproperties and because of its commercial use in approxi-mately 45% of all MRI analyses.[15] The starting structuresdisplayed an average core size of 3.5 nm and a shell thicknessof 0.5 nm. The fluorophore-encapsulated shell was renderedfunctionally active by modified 1,4,7,10-tetraazacyclodode-cane-1,4,7,10-tetraacetic acid (DOTA) ligands which are ableto chelate core gadolinium ions. In aqueous solutions, the

    [*] Dr. A. Mignot, Prof. P. PerriatMatriaux Ingnierie et Science, INSA-Lyon, UMR 5510 CNRS,Universit de Lyon, 69621 Villeurbanne Cedex (France)andCampus LyonTech La Doua, INSA-Lyon, Btiment Blaise Pascal7 avenue Jean Capelle, 69621 Villeurbanne cedex (France)E-mail: [email protected]

    Dr. F. Lux, Dr. A. Mignot, Dr. P. Mowat, Prof. O. TillementLaboratoire de Physico-Chimie des Matriaux Luminescents, UMR5620 CNRS Universit Claude Bernard Lyon 1, Universit de Lyon,69622 Villeurbanne Cedex (France)

    Dr. A. Mignot, Dr. C. Louis, Dr. S. DufortNano-H SAS, 38070 Saint-Quentin Fallavier (France)

    Dr. C. Bernhard, Prof. F. DenatInstitut de Chimie Molculaire de lUniversit de Bourgogne, UMRCNRS 5260, Universit de Bourgogne, 21078 Dijon Cedex (France)

    Dr. F. BoschettiCheMatech, 21000 Dijon (France)

    Dr. C. Brunet, Prof. R. Antoine, Prof. P. DugourdLaboratoire de Spectromtrie Ionique et Molculaire, UMR CNRS5579, Universit Claude Bernard Lyon 1, 69622 Villeurbanne Cedex(France)

    Dr. S. Laurent, Prof. L. V. Elst, Prof. R. MullerUniv Mons, NMR & Mol Imaging Lab, Dept Gen Organ & BiomedChem, B-7000 Mons (Belgium)

    Dr. L. Sancey, Dr. V. Josserand, Prof. J.-L. CollINSERM, CRI, U823, Inst Albert Bonniot, 38042 Grenoble 9 (France)

    Dr. V. Stupar, Dr. E. Barbier, Dr. C. RmyUniversit Grenoble 1, Grenoble Institut des Neurosciences, UMRS836, Grenoble (France)

    Dr. V. Stupar, Dr. E. Barbier, Dr. C. RmyInserm, U826, Grenoble (France)

    Dr. A. Broisat, Prof. C. GhezziLaboratoire radiopharmaceutique bioclinique, INSERM U877, Fac-ult de mdecine de Grenoble, 38700 La Tronche (France)

    Dr. G. Le DucEuropean Synchrotron Radiation Facility, ID 17 Biomedical Beamline,BP220, 38043 Grenoble (France)

    Prof. S. RouxInstitut UTINAM, UMR 6213 CNRS Universit de Franche-Comt,25030 Besanon Cedex (France)

    Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201104104.

    12299Angew. Chem. Int. Ed. 2011, 50, 12299 12303 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


  • ligands strongly accelerate the core dissolution leading to ahollow polysiloxane sphere. This latter collapses and frag-ments into small and rigid platforms (SRPs) of polysiloxane.These SRPs possess all the properties of the initial structure,bearing on their surface DOTA molecules that are partlychelated to dissolved gadolinium cations. DOTA was givenpreference over acyclic chelates, due to its higher complex-ation constant,[16] its lower kinetics that limit transmetallationwith endogen cations such as Ca2+ and reduced toxicity forpatients with severe renal dysfunction.[17,18] Our study aims toconfirm the relevance of this strategy 1) by reporting thechemical and size characterization of the SRPs obtained asdescribed above and 2) by demonstrating that they can bedetected in vivo by four different techniques, namely fluores-cence imaging, MRI, scintigraphy, and X-ray computedtomography.

    Although the starting coreshell particles have an averagesize of only 4.5 0.1 nm, owing to a mean standard deviationof 2 0.2 nm, a significant number of these particles exhibit asize > 5.5 nm, resulting in undesirable hepatic clearance. Inorder to obtain SRPs without any residual Gd-oxide core, asufficient amount of DOTA (at least two DOTA per Gdatom) had to be added. Moreover, the hydrodynamic sizedistribution of the SRPs obtained was entirely below 5.5 nm,with an average size of 3 0.1 nm (see Supporting Informa-tion). Expectedly, the average size using fluorescence corre-lation spectroscopy was slightly larger (about 4 nm), as theparticles measured by this technique are those that encapsu-late the bulky organic Cy5.5 fluorophores. The molecularweight was estimated based on electrospray-mass spectrom-etry (Figure 1c) using a multiplicative correlation algorithm(see Supporting Information).[19] A mass of approximately8.5 1 kDa was obtained which resulted, after correlationwith inductively coupled plasma mass spectrometry (ICP-

    MS) data, in a chemical formula of 10 DOTA, 7 Gd and 27 Siper particle. The scheme of a typical particle displaying thischemistry (Figure 1a) shows that it has effectively a size ofaround 3 nm (see Supporting Information for details). Inaddition, the respective proportions of free and metalchelating ligands were verified by means of luminescencedosages. ICP-MS analysis revealed that 30 % of DOTA arefree whereas luminescence experiments yielded a value ofabout 40 %. This non-negligible proportion of free ligands(3040%) appears to be useful for scintigraphic applications,as it enables the immobilization of numerous radioactiveisotopes onto SRPs. It increases also the relaxation propertiesof SRPs through chelation of supplementary paramagneticatom or provides them with therapeutic properties throughbinding to heavy elements.

    Longitudinal relaxivities per gadolinium were measuredat 310 K, ranging between 0.1 and 60 MHz, following SRPsdispersion in aqueous solutions of NaCl (0.9 %). Thesefeatures were found to be significantly higher compared tothose related to DOTA(Gd) and DTPA(Gd), regardless ofthe frequency used (Figure 1b). At higher field strengths(7 T), the r1 value was equal to 6.0 mm

    1 s1, that is, abouttwice the value of DOTA(Gd), one of the most frequentlyused MRI contrast agent. This increase may certainly beaccounted for by the greater inertia of the rigid structuresupporting the gadolinium cations. Indeed, these structureshave been shown to significantly slow down the rotation rateof the relaxing species, leading to similar r1 increases at highfrequency (e.g., heavy polymers or dendrimers containing Gdcomplexes).[7, 2022]

    Imaging experiments have been performed in vivo onmice and rats using fluorescence imaging, MRI, X-raycomputed tomography (CT) and single photon emission CT(SPECT) in order to validate the efficiency of SRPs as

    multimodal contrastagents, to demonstratethe absence of hepaticclearance, and toassess their appropri-ate biodistribution.For all imaging modal-ities, solutions con-taining GdCl3 or111InCl3 with a highradiochemical purity( 95 %) were addedto SRPs in order toincrease the relaxivityor confer radioactiveproperties throughchelation with freeDOTA. SRPs weretangentially filtratedso as to eliminate theexcess of metal atoms,they were then puri-fied on-column byHPLC in the case ofradiolabeling and

    Figure 1. a) Representation of a typical SRP with a polysiloxane bone and DOTA(Gd) species grafted through amidefunctions