Surface functionalization of metal oxide harmonic nanoparticles for targeted cancer imaging

Cancer is a major health burden worldwide and the leading cause of death in industrialized countries. Early detection is a primary factor of survival and remission rate. Classical imaging methods such as magnetic resonance imaging, ultrasound imaging and microscopy lack in sensitivity for early detection of precancerous lesions. Recent and rapid progresses in nanotechnologies have paved the way for the development of new tools for diagnosis and targeted treatment of cancer. Taking advantage of their surface properties for post-conjugation coupled to their optical characteristics, nanoparticles could provide new level of sensitivity for the detection and imaging of tumors. The investigation of functionalization of nanoparticles with small molecules for the targeting of cancer biomarkers was thus an important aim of this thesis. Secondly, the synthesis of a new multimodal probe coupling magnetic resonance imaging and multiphoton microscopy was also investigated.

This work focuses on the development and application of synthetic pathways for small molecules targeting neoplastic cells and tumor microenvironment. Erlotinib and an inhibitor of fibroblast activation protein Î± were selected for their specific interactions with the epithelial growth factor receptor, present on the surface of lung cancer cells, and cancer-associated fibroblasts respectively. Those molecules where first modified with a biotin residue to assess their biocompatibility and their affinity for the targeted biomarker. The targeting ligands were further equipped with a cyclooctyne moiety for coupling with coated nanoparticles through bioorthogonal reaction.

Nanoparticles based on non-centrosymmetric crystals were selected for their optical properties. Polymeric or inorganic coating, based on poly(ethylene glycol) derivatives and (3-aminopropyl)triethoxysilane respectively, were applied to decorate the nanomaterials with azide moieties. The resulting azides were used to perform copper-free click reaction with the modified targeting ligands. The conjugated nanoparticles where then assessed for their selective association to the targeted biomarker. Nanoparticles decorated with an inhibitor of fibroblast activation protein Î± revealed target-specific association to the enzyme. In vitro investigation of the association of Erlotinib-tagged nanoparticles to epithelial growth factor receptor positive cells is ongoing.

Finally, a gadolinium complex was modified with an alkyne functional group for copper-catalyzed [3 + 2]-cycloaddition to the surface of coated nanoparticles. The functionalized nanoparticles were further assessed for their efficiency as magnetic resonance imaging contrast agent. Results revealed that conjugation of the gadolinium complex to the nanomaterial afforded a potent T1 contrast agent without affecting its optical properties.

To summarize, several nanosystems were prepared through bioorthogonal reactions. The first part of this thesis presents the synthesis of two small molecules and subsequent coupling to coated nanoparticles for targeted tumor imaging. The second part of this work, discusses the preparation of a new multimodal device for magnetic resonance imaging coupled to multiphoton microscopy. Those nanoprobes may provide new sensitivity levels for the study and the early detection of cancers.

Functionalization of Nanoparticles for Targeted Cancer Imaging and Diagnosis

Solène Marcelle Françoise Marie Passemard

At present time, cancer is the leading cause of death in developed countries. The medical imaging tools currently used in clinics such as radiology, nuclear magnetic resonance imaging, microscopy and endoscopy are not able to reach the sensitivity necessary to detect cancers in their earliest stages. The detection and identification of rare circulating individual cells and early cancer metastasis is of the utmost importance to reduce cancer mortality. The development of nanotechnology-based medical diagnostic tools could provide a qualitatively new level of sensitivity and accuracy for the detection of malignant diseases. In this project, the synthesis of functionalized nanoparticles with small targeting molecules that can capture cell entities for the detection of breast, lung and prostate cancers was thus investigated. The research project focused on the chemical development of coated and functionalized second harmonic nanoparticles, displaying unique optical properties. First, the synthesis of hetero-bifunctional poly(ethylene glycol) (PEG) able to covalently coat the nanomaterial and bind to the targeting molecules was performed. The coating of nanoparticles with α-triethoxysilyl-ω-azido and -ω-amino PEG was evaluated on iron oxide nanoparticles (IO NPs) as proof of concept. Coated NPs demonstrated to be non-cytotoxic and stable overtime in biological medium. Similar results were obtained with bismuth iron oxide NPs (BFO NPs) expressing second harmonic generation property. The functionalization of coated NPs was then established with drugs and ligands to specifically target cancer cells biomarkers. Preliminary modification of the selected ligands (e.g. c(RGDfK), Erlotinib, Lapatinib, and an inhibitor of prolyl-endopeptidases) with spacers and a biotin label was undertaken in order to assess their binding specificity to tumor cells. The two best candidates, c(RGDfK) and the inhibitor of prolyl-endopeptidases were modified to contain a simple or strain-cyclic alkyne for the conjugation to the NP's surface through bioorthogonal click reactions. Biological evaluations demonstrated that the ligand-functionalized IO and BFO NPs bind to human cancer cells through target-specific interactions. In summary, chemical modifications of nanoparticles were implemented using two different materials and several targeting ligands, demonstrating the flexibility of this methodology. Functionalized NPs were demonstrated to be biocompatible, stable over-time and suitable for the labelling of cancer cells through specific interactions with extracellular biomarkers overexpressed in tumors.

EPFL2014

New strategies for highly efficient medical MRI contrast agents

Sabrina Laus

During the last two decades, magnetic resonance imaging (MRI) has evolved into one of the most powerful techniques in medical diagnosis. Among the various medical diagnostic modalities, such as X-ray or ultra-sound imaging, MRI is the technique of choice especially because of its non-invasive character, the absence of ionizing radiations and its high spatial resolution. The principle of an MRI examination relies on the relaxation of water protons in the human body placed in a magnetic field after radiofrequency excitations. MRI is sensitive to the nature of the soft tissues, to the proton density and to the relaxation rate of protons to produce a contrast. The development of MRI is closely related to the successful use of paramagnetic contrast agents, essentially gadolinium(III) complexes. Unlike contrast agents used in other clinical imaging techniques, the MRI contrast agents are not themselves imaged but rather enhance the nuclear relaxation rate of water protons in their vicinity. The use of MRI contrast agents induces not only a better image contrast and a better delineating of diseased tissues but also a shortening of the examination time. Since the discovery of GdIII chelates as MRI contrast agents, a lot of effort has been made to increase their relaxivity, i.e. the gauge of efficiency of the contrast agent. In the work presented in this thesis, two groups of paramagnetic compounds have been studied: GdIII poly(amino carboxylates) and GdIII trapped in carbon cage compounds. For traditional monohydrated GdIII poly(amino carboxylate) complexes, the Solomon- Bloembergen-Morgan equations predict maximum proton relaxivities of 100 mM-1 s-1, instead of 4-5 mM-1 s-1 for commercial agents, when the three most important influencing factors, the rotation, the electron spin relaxation and the water exchange rate are simultaneously optimized. On the basis of structural considerations in the inner sphere of nine-coordinate, monohydrated GdIII poly(amino carboxylate) complexes, we succeeded in accelerating the water exchange by inducing steric compression around the water binding site. We modified the common DTPA5- ligand by replacing one ethylene bridge of the amine backbone by a propylene one (EPTPA5--based ligand), leading to a water exchange rate two orders of magnitude higher than that for the commercial [Gd(DTPA)(H2O)]2-. The replacement of two ethylene bridges (DPTPA5-) results in the elimination of the water molecule from the inner sphere. Although the thermodynamic stability of [Gd(EPTPA-bz-NO2)(H2O)]2- is reduced to a slight extent in comparison to [Gd(DTPA)(H2O)]2-, it is stable enough to be used in medical diagnosis. The next step to obtain high relaxivity contrast agents is the covalent coupling of GdIII EPTPA complexes to three generations (5, 7 and 9) of PAMAM dendrimers to ensure fast water exchange rate and slow rotation. These macromolecular GdIII complexes allow high relaxivities with maximum values at magnetic fields presently used in clinical applications thanks to slow molecular tumbling. The relaxivities show a strong and reversible pH dependency for all three dendrimer generations. This smart behaviour originates from the pHdependent rotational dynamics of the dendrimer skeleton. From the studies on the monomeric and dendrimeric GdIII EPTPA complexes, general considerations have been proposed about the crucial need of more rigid macromolecular assemblies to obtain the highest relaxivity values predicted by the Solomon-Bloembergen-Morgan equations. Water-soluble fullerenes and carbon nanotubes have shown a great potential for various biological and medical applications. Gadofullerenes have been proposed not only as high relaxivity contrast agents thanks to the high number of water molecules close to the paramagnetic centre and their slow tumbling arising from aggregation phenomena but also as smart pH-responsive drugs. Proton relaxivity was proposed as an ideal aggregation-state sensor for the disruption of the water-soluble Gd@C60(OH)x (x ≈ 27) and Gd@C60[C(COOH)2]10 aggregates upon salt addition. This phenomenon has important implications for biological or biomedical applications of fullerene-based materials, since real biological fluids present a rather high salt concentration. With the aim of gaining more insight into the paramagnetic relaxation mechanism induced by water-soluble gadofullerenes, an NMR analysis with the common techniques used for GdIII MRI contrast agents allowed a comparison between the aggregated and the disaggregated systems. In analogy to C60 buckyballs, ultra-short single-walled nanotubes have been loaded with Gd3+ aqua-ions clusters. These Gdn3+@US-tubes, which self-organise into bundles-like structures, exhibit relaxivities up to 90 times higher (depending on the magnetic field) than that of any GdIII-based MRI contrast agent in current clinical use.

EPFL2005

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Flavio Maurizio Mor

Life sciences have a constantly growing need for novel methodological approaches suitable to investigate the intracellular environment with increased temporal and spatial resolutions. Recently, optical near-field probes, such as laser-irradiated pointed metal, uncoated/metal-coated tapered optical fibers, as well as nano-emitters, such as single molecules or nanoparticles, have attracted increasing attention as key components of high-resolution microscopes. In parallel, super-resolution fluorescence microscopy, operating in far-field regime and overcoming the light diffraction limit, has markedly improved imaging resolution. Although both near- and far-field optical approaches drastically improve image resolution, they still represent numerous drawbacks, such as, technical difficulties concerning probe preparation (near-field) or potential damaging through very high light intensities (far-field). The aforementioned shortcomings of high-resolution optical microscopy can be overcome to a great extent by photonic force microscopy (PFM). PFM employs a strongly focused near-infrared (NIR) laser light to hold a dielectric or metallic particle as a local probe. Such an optical trap enables one to ‘cage’ a mesoscopic particle and track its three-dimensional (3D) thermal fluctuations in the surrounding environment, e.g. a viscous liquid. Therefore, besides offering near-field 3D imaging, PFM reports also on other important quantities concerning local mechanical properties, including force and viscosity as well as dynamical properties of the surrounding medium. This additional information is derived from the careful analysis of the jittery motion (so-called Brownian motion) of the trapped single-particle probe that collides with thermally-activated surrounding molecules. In this thesis, we first study in detail the Brownian motion of a single spherical particle that is confined within the harmonic potential of the optical trap. To this end, we optimize the NIR light path and electronic noise floor of our custom-built PFM set-up for detecting and quantifying resonances in the Brownian motion. These resonances arise from the coupling between the hydrodynamic memory and strong strength of the optical trap. Due to the high sensitivity of the short-time dynamics, the size of the particle can be measured by simultaneous fitting of the velocity autocorrelation function and power spectral density of its thermal fluctuations. In order to choose the best-suitable spherical probe for a given experiment in PFM, computational modeling based on a Matlab toolbox is performed. The generalized Lorenz-Mie theory is computed using the T -matrix method for various experimental conditions, including changes in the size and refractive index of the sphere, as well as different laser polarization states. The axial equilibrium position is examined to predict its location compared with the position of the laser focus. Optical forces acting on the sphere are investigated in 3D to highlight, in particular, the limits of the perfectly harmonic potential assumption. These limits are quantified and discussed. Subsequently, we identify different types of inorganic nano/micro-sized particles that can be trapped by the NIR laser of the PFM and used simultaneously as mechanical and near-field light sources. To this end, we take advantage of the visible light emission from single probes confined in the optical trap and excited by the NIR laser light. The emphasis is on particles revealing nonlinear optical properties. In particular, nonlinear optical field enhancement resulting from excitation of surface plasmon resonance (SPR) on trapped gold particles of 200 nm is demonstrated by measuring the emission of that second-harmonic generation (SHG). Furthermore, we show, under optical trapping conditions, SHG emission from single potassium niobate (KNbO3) nano/micro-sized particles. We also report on the multicolor upconversion luminescence (UCL), at the single particle level, for randomly shaped crystals of sodium yttrium fluoride codoped with ytterbium and erbium, NaYF4:Yb,Er (UCC), when they are held in the optical trap. In parallel, 3D Brownian fluctuations of a single trapped particle are quantified. Moreover, luminescence resonance energy transfer (LRET) between a KNbO3 particle or an UCC and molecules of an organic dye (rose bengal) adsorbed on their surface is highlighted and analyzed. Finally, randomly shaped UCCs and hexagonal nanoplates are characterized in detail at the single particle level. The hexagonal β phase in the polycrystalline and monocrystalline crystals is identified, in both randomly and hexagonally shaped particles by electron microscopy. UCL emission from the trapped crystal is quantified in terms of power and found to be dependent on the laser power density, size and shape of the particle as well as its orientation within the trap. Most importantly, the different colors in the UCL spectrum do not vary with the same trend for different orientations of the randomly or hexagonally shaped crystal upon trapping. This suggests the existence of crystalline anisotropy-dependent UCL. Thereby, we should be able to design the best-suited particle probe for LRET experiments with subwavelength resolution.

EPFL2013