Design and Characterization of Luminescent Silica Nanoparticles

The aim of this thesis was to design, synthesize and characterize dye-doped silica nanoparticles (DDSNPs) to be used as chemosensors or labels in bioanalytical applications. DDSNPs represent one of the most versatile and useful components in nanomedicine displaying important features such as high colloid stability in water, low toxicity, one-pot inexpensive synthesis and tunable fluorescence emission. Starting from the one-pot and highly reproducible synthesis of “silica-core/PEG shell” DDSNPs based on the use of micelles of Pluronic F127, in which take place both hydrolysis and condensation of the silica precursor and of the dyes functionalized with a triethoxysilane group, we developed DDSNPs suitable for optical and optoacustic imaging, drug loading and chemical sensing obtaining very interesting results for the further development of nanomedicine

Core–Shell Pluronic-Organosilica Nanoparticles with Controlled Polarity and Oxygen Permeability

Nanostructured systems constitute versatile carriers with multiple functions engineered in a nanometric space. Yet, such multimodality often requires adapting the chemistry of the nanostructure to the properties of the hosted functional molecules. Here, we show the preparation of core-shell Pluronic-organosilica "PluOS" nanoparticles with the use of a library of organosilane precursors. The precursors are obtained via a fast and quantitative click reaction, starting from cost-effective reagents such as diamines and an isocyanate silane derivative, and they condensate in building blocks characterized by a balance between hydrophobic and H-bond-rich domains. As nanoscopic probes for local polarity, oxygen permeability, and solvating properties, we use, respectively, solvatochromic, phosphorescent, and excimer-forming dyes covalently linked to the organosilica matrix during synthesis. The results obtained here clearly show that the use of these organosilane precursors allows for finely tuning polarity, oxygen permeability, and solvating properties of the resulting organosilica core, expanding the toolbox for precise engineering of the particle properties

Tandem Dye-Doped Nanoparticles for NIR Imaging via Cerenkov Resonance Energy Transfer

The detection of the Cerenkov radiation (CR) is an emerging preclinical imaging technique which allows monitoring the in vivo distribution of radionuclides. Among its possible advantages, the most interesting is the simplicity and cost of the required instrumentation compared, e.g., to that required for PET scans. On the other hand, one of its main drawbacks is related to the fact that CR, presenting the most intense component in the UV-vis region, has a very low penetration in biological tissues. To address this issue, we present here multifluorophoric silica nanoparticles properly designed to efficiently absorb the CR radiation and to have a quite high fluorescence quantum yield (0.12) at 826 nm. Thanks to a highly efficient series of energy transfer processes, each nanoparticle can convert part of the CR into NIR light, increasing its detection even under 1.0-cm thickness of muscle

Mapping heterogeneous polarity in multicompartment nanoparticles

Understanding polarity gradients inside nanomaterials is essential to capture their potential as nanoreactors, catalysts or in drug delivery applications. We propose here a method to obtain detailed, quantitative information on heterogeneous polarity in multicompartment nanostructures. The method is based on a 2-steps procedure, (i) deconvolution of complex emission spectra of two solvatochromic probes followed by (ii) spectrally resolved analysis of FRET between the same solvatochromic dyes. While the first step yields a list of polarities probed in the nanomaterial suspension, the second step correlates the polarities in space. Colocalization of polarities falling within few nanometer radius is obtained via FRET, a process called here nanopolarity mapping. Here, Prodan and Nile Red are tested to map the polarity of a water-dispersable, multicompartment nanostructure, named PluS nanoparticle (NPs). PluS NPs are uniform core-shell nanoparticles with silica cores (diameter ~10 nm) and Pluronic F127 shell (thickness ~7 nm). The probes report on a wide range of nanopolarities among which the dyes efficiently exchange energy via FRET, demonstrating the coexistence of a rich variety of environments within nanometer distance. Their use as a FRET couple highlights the proximity of strongly hydrophobic sites and hydrated layers, and quantitatively accounts for the emission component related to external water, which remains unaffected by FRET processes. This method is general and applicable to map nanopolarity in a large variety of nanomaterials

Dye-doped silica nanoparticle probes for fluorescence lifetime imaging of reductive environments in living cells

Fluorescence detection sensitivity can be drastically improved by the application of nanoparticles (NPs) because of their superior brightness compared to organic dyes. Here, using dye-doped silica NPs (SiNPs), we developed FRET-based nanoparticle probes for the detection of reductive environments in living cells. To this end, we designed three FRET acceptors based on black hole quenchers (BHQs). Their polarity was tuned by introducing hydroxyl, PEG and sulfate groups. To conjugate them to NPs, we used an original pre-functionalization approach, where the quencher was coupled by a "click" reaction to Pluronic F127 and further used for the preparation of silica NPs. This approach enabled easy preparation of silica NPs functionalized with varying amounts of quenchers by simple mixing of functionalized and parent Pluronic F127 in different mol%. The increase in the quencher concentration at the SiNPs surface produced a rapid drop in the fluorescence intensity with 80% quenching and a 2-fold drop in the emission lifetime for 16 mol% of the quenchers. Then, to obtain turn-ON sensing of reductive environments, the quenchers were coupled to the NPs through a disulfide linker using the same pre-functionalization strategy. The obtained nano-probes showed a >10-fold increase in their fluorescence in the presence of reductive agents, such as tris(2-carboxyethyl)phosphine (TCEP) and glutathione. Remarkably, BHQ quencher bearing sulfate group showed the highest turn-ON response, probably due to its superior capacity to escape from the NP surface after disulfide bond cleavage. The obtained best nanoprobe was successfully applied for detection of reductive environments inside living cells using fluorescence lifetime imaging (FLIM). This work provides insights for FRET acceptor design and its controlled grafting, which enables preparation of the first redox-sensitive silica nanoparticle probe for lifetime imaging

Variable Doping Induces Mechanism Swapping in Electrogenerated Chemiluminescence of Ru(bpy)32+Core-Shell Silica Nanoparticles

The impact of nanotechnology on analytical science is hardly overlooked. In the search for ever-increasing sensitivity in biomedical sensors, nanoparticles have been playing a unique role as, for instance, ultrabright labels, and unravelling the intimate mechanisms which govern their functioning is mandatory for the design of ultrasentitive devices. Herein, we investigated the mechanism of electrogenerated chemiluminescence (ECL) in a family of core shell silica PEG nanoparticles (DDSNs), variously doped with a Ru(bpy)(3)(2+) triethoxysilane derivative, and displaying homogeneous morphological, hydrodynamic, and photophysical properties. ECL experiments, performed in the presence of 2-(dibutylamino)ethanol (DBAE) as coreactant, showed two parallel mechanisms of ECL generation: one mechanism (I) which involves exclusively the radicals deriving from the coreactant oxidation and a second one (II) involving also the direct anodic oxidation of the Ru(II) moieties. The latter mechanism includes electron (hole) hopping between neighboring redox centers as evidenced in our previous studies and supported by a theoretical model we have recently proposed. Quite unexpectedly, however, we found that the efficiency of the two mechanisms varies in opposite directions within the DDSNs series, with mechanism I or mechanism II prevailing at low and high doping levels, respectively. Since mechanism II has an intrinsically lower efficiency, the ECL emission intensity was also found to grow linearly with doping only at relatively low doping levels while it deviates negatively at higher ones. As the (-potential of DDSNs increases with the doping level from negative to slightly positive values, as a likely consequence of the accumulating cationic charge within the silica core, we attributed the observed change in the ECL generation mechanism along the DDSN series to a modulation of the electrostatic and hydrophobic/hydrophilic interactions between the DDSNs and the radical cationic species involved in the ECL generation. The results we report therefore show that the ECL intensity of a nanosized system cannot be merely incremented acting on doping, since other parameters come into play. We think that these results could serve as valuable indications to design more efficient ECL nano- and microsized labels for ultrasensitive bioanalysis

Synthesis of photoactive polymer colloids by polymerization in aqueous dispersed media to product singlet oxygen

International audienceAPME 2019: The 13thInternational Conference on Advanced Polymers via Macromolecular Engineering, 15-18April 2019, Stellenbosch, SASYNTHESIS OF PHOTOACTIVE POLYMER COLLOIDS BY POLYMERIZATION IN AQUEOUS DISPERSEDMEDIA TO PRODUCE SINGLET OXYGENCharlène Boussiron, Luca Petrizza, Mickaël Le Bechec, Sylvie Lacombe, Maud Save*CNRS/ Univ Pau & Pays Adour/ E2S UPPA, IPREM, Institut des Sciences Analytiqueset de Physicochimie pour l'Environnement et les Matériaux (IPREM), UMR5254, 64000, Pau, France.* email: [email protected] design of photoactive polymer substrates producing singlet oxygen under visible light irradiation has great technological potential. Production of singlet oxygen (1O2), a selective reactive oxygen species (ROS) produced by irradiation under visible light of organic photosensitizers, has attracted increasing interest in the fields of fine chemistry,1photo-decontamination of air/water,2antimicrobial materials,3or Photodynamic Therapy (PDT).4Immobilization of photosensitizers on solid substrates improves their handling, recyclability, stability and facilitates purification steps to remove photocatalyst from reactants in fine chemistry. In that context, functional waterborne latex synthesized by the scalable emulsion polymerization process, are versatile to prepare photosensitizer-supported materials either as stable colloidal particles or as polymer film. We will present two strategies to design photosensitizer-grafted polymer colloids. First, film forming latex particle with a decorated shell will be synthesized by polymerization-induced self-assembly (PISA, Fig. 1 left).5Secondly, crosslinked particles will be synthesized by miniemulsion copolymerization to produce core-functionalized particles(PISA, Fig. 1 right). The organic photosensitizer chosen to synthesize the functional monomer is the Rose Bengal molecule

Proper design of silica nanoparticles combines high brightness, lack of cytotoxicity and efficient cell endocytosis

Silica-based luminescent nanoparticles (SiNPs) show promising perspectives in nanomedicine in light of their chemical properties and versatilities. In this study, we have characterized silica core/PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in an animal model. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2 leukemic cell line and primary normal peripheral blood mononuclear cells) or in adherence (human hepatocarcinoma Huh7 and umbilical vein endothelial cells). Moreover, by multiparametric flow cytometry, we could demonstrate that the highest efficiency of cell uptake and entry was observed with the NP-PEG-amino, with a stable persistence of the fluorescence signal associated to SiNPs in the loaded cell populations both in vitro and in vivo settings suggesting this as an innovative method for cell traceability and detection in whole organisms. Finally, experiments performed with the endocytosis inhibitor Genistein clearly suggested the involvement of a caveolae-mediated pathway in SiNP endocytosis. Overall, these data support the safe use of these SiNPs for diagnostic and therapeutic applications

Proper design of silica nanoparticles combines high brightness, lack of cytotoxicity and efficient cell endocytosis

Silica-based luminescent nanoparticles (SiNPs) show promising prospects in nanomedicine in light of their chemical properties and versatility. In this study, we have characterized silica core-PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in animal models. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2 leukemic cell line and primary normal peripheral blood mononuclear cells) or in adherence (human hepatocarcinoma Huh7 and umbilical vein endothelial cells). Moreover, by multiparametric flow cytometry, we could demonstrate that the highest efficiency of cell uptake and entry was observed with NP-PEG-amino, with a stable persistence of the fluorescence signal associated with SiNPs in the loaded cell populations both in vitro and in vivo settings suggesting this as an innovative method for cell traceability and detection in whole organisms. Finally, experiments performed with the endocytosis inhibitor Genistein clearly suggested the involvement of a caveolae-mediated pathway in SiNP endocytosis. Overall, these data support the safe use of these SiNPs for diagnostic and therapeutic applications