Gold Nanorod/Titanium Dioxide Hybrid Nanoparticles for Plasmon-Enhanced Near-Infrared Photoproduction of Hydroxyl Radicals and Photodynamic Therapy.

Gold nanoparticles, such as nanorods (AuNRs), present exceptionally high absorption cross sections that can be tuned to the near-infrared (NIR), the optimal window for light penetration in biological tissues. This makes them valuable photosensitizers for the treatment of cancer using photothermal therapy, where absorbed light energy is converted into heat. In addition, there is a strong interest in using hot electron carriers generated in AuNRs by NIR irradiation to produce cytotoxic radical oxygen species in order to enhance the efficiency of the phototherapy. Here, we show that hybrid nanoparticles composed of AuNRs with TiO2 deposited at their extremities are efficient sensitizers to produce hydroxyl radical species under NIR irradiation. We attribute this phenomenon to the transfer of hot electrons generated from the plasmon excitation in AuNR to the TiO2 tips, followed by reduction of dioxygen. We then functionalize these hybrid AuNR/TiO2 nanoparticles with block poly(ethylene glycol)-phosphonate polymer ligands to stabilize them in a physiological medium. We finally demonstrate that the photodynamic effect induces cell death upon irradiation with a greater efficiency than the photothermal effect alone.

Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells.

Exerting forces on biomolecules inside living cells would allow us to probe their dynamic interactions in their native environment. Magnetic iron oxide nanoparticles represent a unique tool capable of pulling on biomolecules with the application of an external magnetic field gradient; however, their use has been restricted to biomolecules accessible from the extracellular medium. Targeting intracellular biomolecules represents an additional challenge due to potential nonspecific interactions with cytoplasmic or nuclear components. We present the synthesis of sulfobetaine-phosphonate block copolymer ligands, which provide magnetic nanoparticles that are stealthy and targetable in living cells. We demonstrate, for the first time, their efficient targeting in the nucleus and their use for magnetic micromanipulation of a specific genomic locus in living cells. We believe that these stable and sensitive magnetic nanoprobes represent a promising tool to manipulate specific biomolecules in living cells and probe the mechanical properties of living matter at the molecular scale.

Quantum Dots Mediated Imaging and Phototherapy in Cancer Spheroid Models: State of the Art and Perspectives.

Quantum Dots (QDs) are fluorescent nanoparticles known for their exceptional optical properties, i.e., high fluorescence emission, photostability, narrow emission spectrum, and broad excitation wavelength. These properties make QDs an exciting choice for bioimaging applications, notably in cancer imaging. Challenges lie in their ability to specifically label targeted cells. Numerous studies have been carried out with QDs coupled to various ligands like peptides, antibodies, aptamers, etc., to achieve efficient targeting. Most studies were conducted in vitro with two-dimensional cell monolayers (n = 8902) before evolving towards more sophisticated models. Three-dimensional multicellular tumor models better recapitulate in vivo conditions by mimicking cell-to-cell and cell-matrix interactions. To date, only few studies (n = 34) were conducted in 3D in vitro models such as spheroids, whereas these models could better represent QDs behavior in tumors compared to monolayers. Thus, the purpose of this review is to present a state of the art on the studies conducted with Quantum Dots on spheroid models for imaging and phototherapy purposes.

Zwitterionic Polymers toward the Development of Orientation-Sensitive Bioprobes.

Dynamics with an orientational degree of freedom are fundamental in biological events. Probes with polarized luminescence enable a determination of the orientation. Lanthanide-doped nanocrystals can provide more precise analysis than quantum dots due to the nonphotoblinking/bleaching nature and the multiple line-shaped emission. However, the intrinsic polarization property of the original nanocrystals often deteriorates in complex physiological environments because the colloidal stability easily breaks and the probes aggregate in the media with abundant salts and macromolecules. Engineering the surface chemistry of the probes is thus essential to be compatible with biosystems, which has remained a challenging task that should be exclusively addressed for each specific probe. Here, we demonstrate a facile and efficient surface functionalization of lanthanide-doped nanorods by zwitterionic block copolymers. Due to the steric interaction and the intrinsic zwitterionic nature of the polymers, high colloidal stability of the zwitterionic nanorod suspension is achieved over wide ranges of pH and concentration of salts, even giving rise to the lyotropic liquid crystalline behavior of the nanorods in physiological media. The shear-aligned ability is shown to be unaltered by the coated polymers, and thus, the strongly polarized emission of Eu3+ is preserved. Besides, biological experiments reveal good biocompatibility of the zwitterionic nanorods with negligible nonspecific binding. This study is a stepping stone for the use of the nanorods as orientation probes in biofluids and validates the strategy of coupling zwitterions to lanthanide-doped nanocrystals for various bioapplications.

Designing the Surface Chemistry of Inorganic Nanocrystals for Cancer Imaging and Therapy.

Inorganic nanocrystals, such as gold, iron oxide and semiconductor quantum dots, offer promising prospects for cancer diagnostics, imaging and therapy, due to their specific plasmonic, magnetic or fluorescent properties. The organic coating, or surface ligands, of these nanoparticles ensures their colloidal stability in complex biological fluids and enables their functionalization with targeting functions. It also controls the interactions of the nanoparticle with biomolecules in their environment. It therefore plays a crucial role in determining nanoparticle biodistribution and, ultimately, the imaging or therapeutic efficiency. This review summarizes the various strategies used to develop optimal surface chemistries for the in vivo preclinical and clinical application of inorganic nanocrystals. It discusses the current understanding of the influence of the nanoparticle surface chemistry on its colloidal stability, interaction with proteins, biodistribution and tumor uptake, and the requirements to develop an optimal surface chemistry.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology.

Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

Surface Modification of CdE (E: S, Se, and Te) Nanoplatelets to Reach Thicker Nanoplatelets and Homostructures with Confinement-Induced Intraparticle Type I Energy Level Alignment.

Two-dimensional II-VI semiconductor nanoplatelets (NPLs) present exceptionally narrow optical features due to their thickness defined at the atomic scale. Because thickness drives the band-edge energy, its control is of paramount importance. Here, we demonstrate that native carboxylate ligands can be replaced by halides that partially dissolve cadmium chalcogenide NPLs at the edges. The released monomers then recrystallize on the wide top and bottom facets, leading to an increase in NPL thickness. This dissolution/recrystallization method is used to increase NPL thickness to 9 ML while using 3 ML NPLs as the starting material. We also demonstrate that this method is not limited to CdSe and can be extended to CdS and CdTe to grow thick NPLs. When the metal halide precursor is introduced with a chalcogenide precursor on the NPLs, CdSe/CdSe, CdTe/CdTe, and CdSe/CdTe core/shell homo- and heterostructures are achieved. Finally, when an incomplete layer is grown, NPLs with steps are synthesized. These stress-free homostructures are comparable to type I heterostructures, leading to recombination of the exciton in the thicker area of the NPLs. Following the growth of core/crown and core/shell NPLs, it affords a new degree of freedom for the growth of structured NPLs with designed band engineering, which has so far been only achievable for heteromaterial nanostructures.

Microcavity-Enhanced Fluorescence Energy Transfer from Quantum Dot Excited Whispering Gallery Modes to Acceptor Dye Nanoparticles.

Whispering gallery mode (WGM) microcavities are emerging as potential candidates in the field of biosensing applications, as their resonance wavelengths shift with changes in the refractive index in the region of their evanescent field. Their high-quality resonance modes and accessible surface functionalities make them promising for molecular assays, but their high sensitivity makes them inherently unstable. Here, we demonstrate that WGM resonances also strongly enhance fluorescence energy transfer between donors placed inside the microcavity and acceptors placed outside. We load colloidal quantum dots (QDs) into polymeric microspheres to provide WGMs that benefit from the QD optical features when used as energy-transfer donors. Spectroscopic analysis of the emission from the microcavities shows that the high quality of WGMs enables a very efficient energy transfer to dye-loaded polymer nanoparticle acceptors placed in their vicinity. Compared to Förster resonance energy transfer, WGM-enabled energy transfer (WGET) occurs over a much more extended volume, thanks to the delocalization of the mode over a typically 105 times larger surface and to the extension of the WGM electromagnetic field to larger distances (>100 nm vs a few nm) from the surface of the microcavity. The resulting sensing scheme combines the sensitivity of WGM spectroscopy with the specificity and simple detection schemes of fluorescence energy transfer, thus providing a potentially powerful class of biosensors.

NIR Imaging of the Integrin-Rich Head and Neck Squamous Cell Carcinoma Using Ternary Copper Indium Selenide/Zinc Sulfide-Based Quantum Dots.

The efficient intraoperative identification of cancers requires the development of the bright, minimally-toxic, tumor-specific near-infrared (NIR) probes as contrast agents. Luminescent semiconductor quantum dots (QDs) offer several unique advantages for in vivo cellular imaging by providing bright and photostable fluorescent probes. Here, we present the synthesis of ZnCuInSe/ZnS core/shell QDs emitting in NIR (~750 nm) conjugated to NAVPNLRGDLQVLAQKVART (A20FMDV2) peptide for targeting αvß6 integrin-rich head and neck squamous cell carcinoma (HNSCC). Integrin αvß6 is usually not detectable in nonpathological tissues, but is highly upregulated in HNSCC. QD-A20 showed αvß6 integrin-specific binding in two-dimension (2D) monolayer and three-dimension (3D) spheroid in vitro HNSCC models. QD-A20 exhibit limited penetration (ca. 50 µm) in stroma-rich 3D spheroids. Finally, we demonstrated the potential of these QDs by time-gated fluorescence imaging of stroma-rich 3D spheroids placed onto mm-thick tissue slices to mimic imaging conditions in tissues. Overall, QD-A20 could be considered as highly promising nanoprobes for NIR bioimaging and imaging-guided surgery.

NanoPaint: A Tool for Rapid and Dynamic Imaging of Membrane Structural Plasticity at the Nanoscale.

Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here, the nano-resolved population dynamics of QDs is exploited to reconstruct the topography and structural changes of the cell membrane surface with high temporal and spatial resolution. For this proof-of-concept study, bright, small, and stable biofunctional QD nanoconstructs are utilized recognizing the endogenous neuronal cannabinoid receptor 1, a highly expressed and fast-diffusing membrane protein, together with a commercial point-localization microscope. Rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allows precise reconstruction of the membrane surface in less than 1 min with a spatial resolution of tens of nanometers. Access of the QD nanoconstructs to the synaptic cleft enables rapid 3D topological reconstruction of the entire presynaptic component. Successful reconstruction of membrane nano-topology and deformation at the second time-scale is also demonstrated for HEK293 cell filopodia and axons. Named "nanoPaint," this super-resolution imaging technique amenable to any endogenous transmembrane target represents a versatile platform to rapidly and accurately reconstruct the cell membrane nano-topography, thereby enabling the study of the rapid dynamic phenomena involved in neuronal membrane plasticity.

Pulsed-laser irradiation of multifunctional gold nanoshells to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

BACKGROUND: HER2-overexpressing metastatic breast cancers are challenging practice in oncology when they become resistant to anti-HER2 therapies such as trastuzumab. In these clinical situations, HER2-overexpression persists in metastatic localizations, and can thus be used for active targeting using innovative therapeutic approaches. Functionalized gold nanoparticles with anti-HER2 antibody can be stimulated by near-infrared light to induce hyperthermia. METHODS: Here, hybrid anti-HER2 gold nanoshells were engineered for photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer xenografts. RESULTS: When gold nanoshells were administered in HER2-tumor xenografts, no toxicity was observed. A detailed pharmacokinetic study showed a time-dependent accumulation of gold nanoshells within the tumors, significantly greater with functionalized gold nanoshells at 72 h. This enabled us to optimize the treatment protocol and irradiate the mice when the anti-HER2 gold nanoshells had accumulated most in the tumors. After weekly injections of anti-HER2 gold nanoshells, and repeated irradiations with a femtosecond-pulsed laser over four weeks, tumor growth was significantly inhibited. Detailed tissue microscopic analyses showed that the tumor growth inhibition was due to an anti-angiogenic effect, coherent with a preferential distribution of the nanoshells in tumor microvessels. We also showed a direct tumor cell effect with apoptosis and inhibition of proliferation, coherent with an immune-mediated targeting of tumor cells by anti-HER2 nanoshells. CONCLUSION: This preclinical study thus supports the use of anti-HER2 gold nanoshells and photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

Zwitterionic polymer ligands: an ideal surface coating to totally suppress protein-nanoparticle corona formation?

In the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC- and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting.

pH-Sensitive Visible or Shortwave Infrared Quantum Dot Nanoprobes Using Conformation-Switchable Copolymeric Ligands.

Intracellular and extracellular pH are key parameters in many physiological processes and diseases. For example, the extracellular pH of the tumor micro-environment is slightly more acidic than in healthy tissue. In vivo mapping of the extracellular pH within the tumor would therefore improve our understanding of the tumor physiology. Fluorescent semiconductor quantum dots (QDs) represent interesting probes for in vivo imaging, in particular in the shortwave infrared (SWIR) range. Here, pH-sensitive QD nanoprobes are developed using a conformation-switchable surface chemistry. The central fluorescent QD is coated with a copolymer ligand and conjugated to gold nanoparticle quenchers. As the pH decreases from physiological (7.5) to slightly acidic (5.5-6), the copolymer reversibly shrinks, which increases the energy transfer between the QD and the gold quenchers and modulates the QD fluorescence signal. This enables the design of ratiometric QD probes for biological pH range emitting in the visible or SWIR range. In addition, these probes can be easily encapsulated and remain functional within ghost erythrocyte membranes, which facilitate their in vivo application.

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence.

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice.

In Vivo Imaging of Single Tumor Cells in Fast-Flowing Bloodstream Using Near-Infrared Quantum Dots and Time-Gated Imaging.

Whereas in vivo fluorescence imaging of cells immobilized within tissues provides a valuable tool to a broad range of biological studies, it still lacks the sensitivity required to visualize isolated cells circulating fast in the bloodstream due, in particular, to the autofluorescence from endogenous fluorophores. Time-gated imaging of near-infrared emitting ZnCuInSe/ZnS quantum dots (QDs) with fluorescence lifetimes in the range of 150-300 ns enables the efficient rejection of fast autofluorescence photons and the selection of QD fluorescence photons, thus significantly increasing sensitivity. We labeled model erythrocytes as well as lymphoma cells using these QDs coated with a stable zwitterionic polymer surface chemistry. After reinjection in the bloodstream, we were able to image and count individual QD-labeled cells circulating at mm·s-1 velocities in blood vessels.

The targeting ability of fluorescent quantum dots to the folate receptor rich tumors.

BACKGROUND: Quantum dots (QDs) bring new insights in cancer theranostics. Exceptional brightness together with the simple possibility to modify surface with targeting molecules make QDs attractive agents in fluorescence guided surgery and photodynamic therapy. Currently, many targeted QDs have been developed for theranostic purpose. However, their targeting ability was tested mainly in two dimensional monolayer tumor cell models, while our study includes 3D tumor model reflecting the specificity of in vivo tumor environment. METHODS: Core/multilayer shell CdSe/CdS/ZnS QDs were conjugated with folic acid (FA) and characterized spectroscopically. Cytotoxicity of QDs on KB and A549 cells lines were evaluated using the MTT assay. Cellular uptake of QDs was assessed by epifluorescent microscopy. To study the distribution of QDs in tumor tissue, KB spheroids were prepared by means of the liquid overlay technique and then frozen cut of spheroids treated with QDs were imaged by epifluorescence microscopy. RESULTS: We confirmed the specificity of QD-FA for the folic acid receptor positive KB cells. In 3D tumor spheroid model we demonstrated uptake enhancement of QD-FA compared with non-targeted QD. It was demonstrated that penetration profiles were similar for both QDs with penetration depth never exceeding 100 µm. CONCLUSIONS: We have demonstrated the effectiveness of FA conjugated QDs to target tumor spheroids thus confirming the crucial role of FRα receptor as a target. Further improvement of QD-FA targeting ability could be performed using dual targeting different targeting agents, such as FA and cyclic RGD.

Doping as a Strategy to Tune Color of 2D Colloidal Nanoplatelets.

Among colloidal nanocrystals, 2D nanoplatelets (NPLs) made of II-VI compounds appear as a special class of emitters with an especially narrow photoluminescence signal. However, the PL signal in the case of NPLs is only tunable by a discrete step. Here, we demonstrate that doping is a viable path to finely tune the color of these NPLs from green to red, making them extremely interesting as phosphors for wide-gamut display. In addition, using a combination of luminescence spectroscopy, tight-binding simulation, transport, and photoemission, we provide a consistent picture for the Ag+-doped CdSe NPLs. The Ag-activated state is strongly bound and located 340 meV above the valence band of the bulk material. The Ag dopant induces a relative shift of the Fermi level toward the valence band by up to 400 meV but preserves the n-type nature of the material.

Fluorescent Nanoparticles for the Guided Surgery of Ovarian Peritoneal Carcinomatosis.

Complete surgical resection is the ideal cure for ovarian peritoneal carcinomatosis, but remains challenging. Fluorescent guided surgery can be a promising approach for precise cytoreduction when appropriate fluorophore is used. In the presence paper, we review already developed near- and short-wave infrared fluorescent nanoparticles, which are currently under investigation for peritoneal carcinomatosis fluorescence imaging. We also highlight the main ways to improve the safety of nanoparticles, for fulfilling prerequisites of clinical application.

Clickable-Zwitterionic Copolymer Capped-Quantum Dots for in Vivo Fluorescence Tumor Imaging.

In the last decades, fluorescent quantum dots (QDs) have appeared as high-performance biological fluorescent nanoprobes and have been explored for a variety of biomedical optical imaging applications. However, many central challenges still exist concerning the control of the surface chemistry to ensure high biocompatibility, low toxicity, antifouling, and specific active targeting properties. Regarding in vivo applications, circulation time and clearance of the nanoprobe are also key parameters to control the design and characterization of new optical imaging agents. Herein, the complete design and characterization of a peptide-near-infrared-QD-based nanoprobe for biomedical optical imaging is presented from the synthesis of the QDs and the zwitterionic-azide copolymer ligand, enabling a bio-orthogonal coupling, till the final in vivo test through all the characterization steps. The developed nanoprobes show high fluorescence emission, controlled grafting rate, low toxicity, in vitro active specific targeting, and in vivo long circulating blood time. This is, to our knowledge, the first report characterizing the in vivo circulation kinetics and tumor accumulation of targeted zwitterionic QDs.