Stimuli responsive polymer/quantum dot hybrid platforms modified at the nanoscale

Quantum dots, QDs, receive growing attention from many research disciplines owing\ud to their advantages as fluorescent probes including their nanoscale size (similar to\ud biomolecules), high quantum yield and molar extinction coefficients, versatility in surface\ud modification, broad excitation spectra (for multicolor imaging) and narrow band emission\ud and tunable optical properties. Fabricating QD/polymer hybrid nanostructures enables\ud realization of many potential applications as optoelectronic devices, biological sensors, and\ud photonic structures because encaging QDs within polymer matrices not only enables the\ud control over optical and spectroscopic properties of QDs but also introduces a strong\ud resistance to chemical and photodegradation. The research described in this thesis aims at\ud synthesis and characterization of CdSe/ZnS core/shell QDs, synthesis and characterization of\ud temperature-responsive polymer matrices made of poly(N-isopropylacryl amide), PNIPAM,\ud as carriers of QDs, and fabrication of QD/PNIPAM assemblies with potential applications as\ud sensing devices to be used in bio-nanotechnology

MT1-MMP directs force-producing proteolytic contacts that drive tumor cell invasion

International audienceUnraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers

Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity

Biological Sciences; Molecular Biology; Cell BiologyEndogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved

Colloidal Nanoparticles for Signal Enhancement in Optical Diagnostic Assays

International audienceThe use of nanotechnologies for the development of highly sensitive and affordable diagnostic assays has significantly improved the ability to detect and characterize multiple types of biomarkers. Semiconductor and metal nanoparticles with unique optical properties have been successfully integrated within biomarker detection schemes for the generation and enhancement of optical signals in label-based and label-free assays. Highly sensitive label-based diagnostics has been realized particularly via using quantum dots (QDs) as labeling probes. Similarly, many label-free techniques that are emerging as potential complements to label-based approaches benefit from signal enhancement strategies using e.g., metal nanoparticles. This review presents a concise overview of recent advances in diagnostic assays that utilize nanoparticles for the generation and enhancement of optical signals in fluorescence- and surface plasmon resonance-based techniques. Advanced diagnostic assays that utilize nanoparticles provide major improvements in detection sensitivity, which can potentially meet the challenging requirements of clinical diagnostics

Sensitivity Enhancement of Förster Resonance Energy Transfer Immunoassays by Multiple Antibody Conjugation on Quantum Dots

International audienceQuantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (AB) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many AB per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the AB on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of AB per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both AB and QDs can significantly influence the assay performance

Surface Engineering of Quantum Dots with Designer Ligands

Semiconductor nanocrystal particles (quantum dots, QDs) exhibit unique electronic and optical properties, which depend on their size and composition, and can be tuned by a controlled variation of the particle dimensions. Due to this tunability, applications of QDs in optoelectronics, sensing, biolabeling and biodiagnostics have attracted great interest. For these applications it is essential to consider the particular structure of the nanoparticles, as well as the surrounding, usually organic, shells. In addition to the size and composition dependence, the optical and electronic properties of QDs are modulated by the chemical composition, attachment, and molecular conformation of the organic (ligand) shell. Control of the ligand‐shell attachment to QD surfaces, and understanding of its influence on the physicochemical, optical, and electronic properties of isolated QDs, and QDs incorporated in particle ensemble of molecularly controlled matrices are essential to successfully achieve most of the promising applications. For example, chemical derivatization of the ligands improves the emission characteristics of the QDs, can render the QDs water soluble and biocompatible, enables one to incorporate the QDs into composite materials, promotes their assembly into hierarchical structures, makes coupling of the QDs to surfaces possible, and finally, opens the possibility to introduce biorecognition functions onto their surface. Modulation of the photophysical properties of QDs through molecular engineering of the ligand shell unfolds new avenues in chemical sensing based on energy‐ or electron‐transfer processes. This chapter briefly reviews the progress and current status of chemical functionalization of the surface of QDs

Cytoskeletal stiffening in synthetic hydrogels

Although common in biology, controlled stiffening of hydrogels in vitro is difficult to achieve; the required stimuli are commonly large and/or the stiffening amplitudes small. Here, we describe the hierarchical mechanics of ultra-responsive hybrid hydrogels composed of two synthetic networks, one semi-flexible and stress-responsive, the other flexible and thermoresponsive. Heating collapses the flexible network, which generates internal stress that causes the hybrid gel to stiffen up to 50 times its original modulus; an effect that is instantaneous and fully reversible. The average generated forces amount to ~1 pN per network fibre, which are similar to values found for stiffening resulting from myosin molecular motors in actin. The excellent control, reversible nature and large response gives access to many biological and bio-like applications, including tissue engineering with truly dynamic mechanics and life-like matter

Temperature-modulated quenching of quantum dots covalently coupled to chain ends of poly(N-isopropyl acrylamide) brushes on gold

A thermo-responsive polymer/quantum dot platform based on poly(N-isopropyl acrylamide) (PNIPAM) brushes 'grafted from' a gold substrate and quantum dots (QDs) covalently attached to the PNIPAM layer is presented. The PNIPAM brushes are grafted from the gold surface using an iniferter-initiated controlled radical polymerization. The PNIPAM chain ends are functionalized with amine groups for coupling to water-dispersible COOH-functionalized QDs. Upon increasing the temperature above the lower critical solution temperature (LCST) of PNIPAM the QD luminescence is quenched. The luminescence was observed to recover upon decreasing the temperature below the LCTS. The data obtained are consistent with temperature-modulated thickness changes of the PNIPAM layer and quenching of the QDs by the gold surface via nonradiative energy transfer

Industrial Scale Manufacturing and Downstream Processing of PLGA-Based Nanomedicines Suitable for Fully Continuous Operation

Despite the efficacy and potential therapeutic benefits that poly(lactic-co-glycolic acid) (PLGA) nanomedicine formulations can offer, challenges related to large-scale processing hamper their clinical and commercial development. Major hurdles for the launch of a polymeric nanocarrier product on the market are batch-to-batch variations and lack of product consistency in scale-up manufacturing. Therefore, a scalable and robust manufacturing technique that allows for the transfer of nanomedicine production from the benchtop to an industrial scale is highly desirable. Downstream processes for purification, concentration, and storage of the nanomedicine formulations are equally indispensable. Here, we develop an inline sonication process for the production of polymeric PLGA nanomedicines at the industrial scale. The process and formulation parameters are optimized to obtain PLGA nanoparticles with a mean diameter of 150 ± 50 nm and a small polydispersity index (PDI < 0.2). Downstream processes based on tangential flow filtration (TFF) technology and lyophilization for the washing, concentration, and storage of formulations are also established and discussed. Using the developed manufacturing and downstream processing technologies, production of two PLGA nanoformulations encasing ritonavir and celecoxib was achieved at 84 g/h rate. As a measure of actual drug content, encapsulation efficiencies of 49.5 ± 3.2% and 80.3 ± 0.9% were achieved for ritonavir and celecoxib, respectively. When operated in-series, inline sonication and TFF can be adapted for fully continuous, industrial-scale processing of PLGA-based nanomedicines

Surface Engineering of Quantum Dots with Designer Ligands

Semiconductor nanocrystal particles (quantum dots, QDs) exhibit unique electronic and optical properties, which depend on their size and composition, and can be tuned by a controlled variation of the particle dimensions. Due to this tunability, applications of QDs in optoelectronics, sensing, biolabeling and biodiagnostics have attracted great interest. For these applications it is essential to consider the particular structure of the nanoparticles, as well as the surrounding, usually organic, shells. In addition to the size and composition dependence, the optical and electronic properties of QDs are modulated by the chemical composition, attachment, and molecular conformation of the organic (ligand) shell. Control of the ligand‐shell attachment to QD surfaces, and understanding of its influence on the physicochemical, optical, and electronic properties of isolated QDs, and QDs incorporated in particle ensemble of molecularly controlled matrices are essential to successfully achieve most of the promising applications. For example, chemical derivatization of the ligands improves the emission characteristics of the QDs, can render the QDs water soluble and biocompatible, enables one to incorporate the QDs into composite materials, promotes their assembly into hierarchical structures, makes coupling of the QDs to surfaces possible, and finally, opens the possibility to introduce biorecognition functions onto their surface. Modulation of the photophysical properties of QDs through molecular engineering of the ligand shell unfolds new avenues in chemical sensing based on energy‐ or electron‐transfer processes. This chapter briefly reviews the progress and current status of chemical functionalization of the surface of QDs