Functionalization of Nanoparticles for Targeted Cancer Imaging and Diagnosis

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.

Surface functionalization of metal oxide harmonic nanoparticles for targeted cancer imaging

Raphaël Jovita De Matos

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.

EPFL2020

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

Functionalized bismuth ferrite harmonic nanoparticles for cancer cells labelling and imaging

Luigi Bonacina, Sandrine Gerber, Solène Marcelle Françoise Marie Passemard, Giona Sonego

Bismuth ferrite (BFO) harmonic nanoparticles (NPs) display high nonlinear optical efficiency and excellent biocompatibility profile which make them attractive for the development of diagnostic applications as contrast agents. In this study, we present a general method for the functionalization of this material with chemical ligands targeting cancer molecular biomarkers. In particular, a conjugation protocol based on click reaction between alkynyl containing targeting ligands and poly(ethylene glycol) coated BFO NPs (67.7 nm) displaying surface reactive azido groups was developed. Copper-free click reaction allowed fast and efficient conjugation of a covalent inhibitor of prolyl-specific endopeptidases to coated BFO NPs. The ability of these functionalized nanomaterials (134.2 nm) to act as imaging probes for cancer cells was demonstrated by the selective labelling of human lung cancer cells.