Ras-transfected human mammary tumour cells are resistant to photodynamic therapy by mechanisms related to cell adhesion.

AIMS: Photodynamic therapy (PDT) is a treatment modality for several cancers involving the administration of a tumour-localising photosensitiser (PS) and its subsequent activation by light, resulting in tumour damage. Ras oncogenes have been strongly associated with chemo- and radio-resistance. Based on the described roles of adhesion and cell morphology on drug resistance, we studied if the differences in shape, cell-extracellular matrix and cell-cell adhesion induced by Ras transfection, play a role in the resistance to PDT. MATERIALS AND METHODS: We employed the human normal breast HB4a cells transfected with H-RAS and a panel of five PSs. KEY FINDINGS: We found that resistance to PDT of the HB4a-Ras cells employing all the PSs, increased between 1.3 and 2.5-fold as compared to the parental cells. There was no correlation between resistance and intracellular PS levels or PS intracellular localisation. Even when Ras-transfected cells present lower adherence to the ECM proteins, this does not make them more sensitive to PDT or chemotherapy. On the contrary, a marked gain of resistance to PDT was observed in floating cells as compared to adhesive cells, accounting for the higher ability conferred by Ras to survive in conditions of decreased cell-extracellular matrix interactions. HB4a-Ras cells displayed disorganisation of actin fibres, mislocalised E-cadherin and vinculin and lower expression of E-cadherin and ß1-integrin as compared to HB4a cells. SIGNIFICANCE: Knowledge of the mechanisms of resistance to photodamage in Ras-overexpressing cells may lead to the optimization of the combination of PDT with other treatments.

Amino-OPE glycosides and blue light: a powerful synergy in photodynamic therapy.

Herein we report the synthesis and biological properties of sugar-conjugated oligophenylene ethynylene (OPE) dyes, used as novel photosensitizers (PSs) for photodynamic treatment (PDT) under blue light. The OPE-bearing glycosides at both ends are successfully prepared by a Pd-catalyzed Sonogashira cross-coupling reaction. The live-cell imaging studies have shown that these OPE glycosides (including glucose, mannose and maltose derivatives) efficiently penetrate the cytoplasm of cultured HeLa cancer cells. No dark toxicity was observed, but upon irradiating the cells under blue light an extraordinary photodynamic effect was observed at low concentrations (10-6-10-8 M). The localization studies indicate that OPE-glucose 1 and OPE-mannose 2 have Golgi patterns, whereas OPE-maltose 3 could be in lysosomes. The PDT and morphological studies in HeLa cells treated with sublethal doses of PS 1-3 revealed that cell death occurs by necrosis.

Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells.

We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic methodology is based on an operationally simple N═N bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cells.

Optical detection of atherosclerosis at molecular level by optical coherence tomography: An in vitro study.

There is an urgent need for contrast agents to detect the first inflammation stage of atherosclerosis by cardiovascular optical coherence tomography (CV-OCT), the imaging technique with the highest spatial resolution and sensitivity of those used during coronary interventions. Gold nanoshells (GNSs) provide the strongest signal by CV-OCT. GNSs are functionalized with the cLABL peptide that binds specifically to the ICAM-1 molecule upregulated in the first stage of atherosclerosis. Dark field microscopy and CV-OCT are used to evaluate the specific adhesion of these functionalized GNSs to activated endothelial cells. This adhesion is investigated under static and dynamic conditions, for shear stresses comparable to those of physiological conditions. An increase in the scattering signal given by the functionalized GNSs attached to activated cells is observed compared to non-activated cells. Thus, cLABL-functionalized GNSs behave as excellent contrast agents for CV-OCT and promise a novel strategy for clinical molecular imaging of atherosclerosis.

Bismuth Selenide Nanostructured Clusters as Optical Coherence Tomography Contrast Agents: Beyond Gold-Based Particles.

Optical coherence tomography (OCT) is an imaging technique currently used in clinical practice to obtain optical biopsies of different biological tissues in a minimally invasive way. Among the contrast agents proposed to increase the efficacy of this imaging method, gold nanoshells (GNSs) are the best performing ones. However, their preparation is generally time-consuming, and they are intrinsically costly to produce. Herein, we propose a more affordable alternative to these contrast agents: Bi2Se3 nanostructured clusters with a desert rose-like morphology prepared via a microwave-assisted method. The structures are prepared in a matter of minutes, feature strong near-infrared extinction properties, and are biocompatible. They also boast a photon-to-heat conversion efficiency of close to 50%, making them good candidates as photothermal therapy agents. In vitro studies evidence the prowess of Bi2Se3 clusters as OCT contrast agents and prove that their performance is comparable to that of GNSs.

Eosin Y-Functionalized Upconverting Nanoparticles: Nanophotosensitizers and Deep Tissue Bioimaging Agents for Simultaneous Therapeutic and Diagnostic Applications.

Functionalized upconverting nanoparticles (UCNPs) are promising theragnostic nanomaterials for simultaneous therapeutic and diagnostic purposes. We present two types of non-toxic eosin Y (EY) nanoconjugates derived from UCNPs as novel nanophotosensitizers (nano-PS) and deep-tissue bioimaging agents employing light at 800 nm. This excitation wavelength ensures minimum cell damage, since the absorption of water is negligible, and increases tissue penetration, enhancing the specificity of the photodynamic treatment (PDT). These UCNPs are uniquely qualified to fulfil three important roles: as nanocarriers, as energy-transfer materials, and as contrast agents. First, the UCNPs enable the transport of EY across the cell membrane of living HeLa cells that would not be possible otherwise. This cellular internalization facilitates the use of such EY-functionalized UCNPs as nano-PS and allows the generation of reactive oxygen species (ROS) under 800 nm light inside the cell. This becomes possible due to the upconversion and energy transfer processes within the UCNPs, circumventing the excitation of EY by green light, which is incompatible with deep tissue applications. Moreover, the functionalized UCNPs present deep tissue NIR-II fluorescence under 808 nm excitation, thus demonstrating their potential as bioimaging agents in the NIR-II biological window.

In Vivo Near-Infrared Imaging Using Ternary Selenide Semiconductor Nanoparticles with an Uncommon Crystal Structure.

The implementation of in vivo fluorescence imaging as a reliable diagnostic imaging modality at the clinical level is still far from reality. Plenty of work remains ahead to provide medical practitioners with solid proof of the potential advantages of this imaging technique. To do so, one of the key objectives is to better the optical performance of dedicated contrast agents, thus improving the resolution and penetration depth achievable. This direction is followed here and the use of a novel AgInSe2 nanoparticle-based contrast agent (nanocapsule) is reported for fluorescence imaging. The use of an Ag2 Se seeds-mediated synthesis method allows stabilizing an uncommon orthorhombic crystal structure, which endows the material with emission in the second biological window (1000-1400 nm), where deeper penetration in tissues is achieved. The nanocapsules, obtained via phospholipid-assisted encapsulation of the AgInSe2 nanoparticles, comply with the mandatory requisites for an imaging contrast agent-colloidal stability and negligible toxicity-and show superior brightness compared with widely used Ag2 S nanoparticles. Imaging experiments point to the great potential of the novel AgInSe2 -based nanocapsules for high-resolution, whole-body in vivo imaging. Their extended permanence time within blood vessels make them especially suitable for prolonged imaging of the cardiovascular system.

Bifunctional Tm3+,Yb3+:GdVO4@SiO2 Core-Shell Nanoparticles in HeLa Cells: Upconversion Luminescence Nanothermometry in the First Biological Window and Biolabelling in the Visible.

The bifunctional possibilities of Tm,Yb:GdVO4@SiO2 core-shell nanoparticles for temperature sensing by using the near-infrared (NIR)-excited upconversion emissions in the first biological window, and biolabeling through the visible emissions they generate, were investigated. The two emission lines located at 700 and 800 nm, that arise from the thermally coupled 3F2,3 and 3H4 energy levels of Tm3+, were used to develop a luminescent thermometer, operating through the Fluorescence Intensity Ratio (FIR) technique, with a very high thermal relative sensitivity . Moreover, since the inert shell surrounding the luminescent active core allows for dispersal of the nanoparticles in water and biological compatible fluids, we investigated the penetration depth that can be realized in biological tissues with their emissions in the NIR range, achieving a value of 0.8 mm when excited at powers of 50 mW. After their internalization in HeLa cells, a low toxicity was observed and the potentiality for biolabelling in the visible range was demonstrated, which facilitated the identification of the location of the nanoparticles inside the cells, and the temperature determination.

Plasmonic Copper Sulfide Nanoparticles Enable Dark Contrast in Optical Coherence Tomography.

Optical coherence tomography (OCT) is an imaging technique affording noninvasive optical biopsies. Like for other imaging techniques, the use of dedicated contrast agents helps better discerning biological features of interest during the clinical practice. Although bright OCT contrast agents have been developed, no dark counterpart has been proposed yet. Herein, plasmonic copper sulfide nanoparticles as the first OCT dark contrast agents working in the second optical transparency window are reported. These nanoparticles virtually possess no light scattering capabilities at the OCT working wavelength (≈1300 nm); thus, they exclusively absorb the probing light, which in turn results in dark contrast. The small size of the nanoparticles and the absence of apparent cytotoxicity support the amenability of this system to biomedical applications. Importantly, in the pursuit of systems apt to yield OCT dark contrast, a library of copper sulfide nanoparticles featuring plasmonic resonances spanning the three optical transparency windows is prepared, thus highlighting the versatility and potential of these systems in light-controlled biomedical applications.

Selective miRNA Modulation Fails to Activate HIV Replication in In Vitro Latency Models.

HIV remains incurable because of viral persistence in latent reservoirs that are inaccessible to antiretroviral therapy. A potential curative strategy is to reactivate viral gene expression in latently infected cells. However, no drug so far has proven to be successful in vivo in reducing the reservoir, and therefore new anti-latency compounds are needed. We explored the role of microRNAs (miRNAs) in latency maintenance and their modulation as a potential anti-latency strategy. Latency models based on treating resting CD4 T cells with chemokine (C-C motif) ligand 19 (CCL19) or interleukin-7 (IL7) before HIV infection and next-generation sequencing were used to identify the miRNAs involved in HIV latency. We detected four upregulated miRNAs (miRNA-98, miRNA-4516, miRNA-4488, and miRNA-7974). Individual or combined inhibition of these miRNAs was performed by transfection into cells latently infected with HIV. Viral replication, assessed 72 h after transfection, did not increase after miRNA modulation, despite miRNA inhibition and lack of toxicity. Furthermore, the combined modulation of five miRNAs previously associated with HIV latency was not effective in these models. Our results do not support the modulation of miRNAs as a useful strategy for the reversal of HIV latency. As shown with other drugs, the potential of miRNA modulation as an HIV reactivation strategy could be dependent on the latency model used.

Theiler's murine encephalomyelitis virus infection of astrocytes induces the expression of chemokines which attract activated but not resting T lymphocytes.

In this article, we studied the production of the chemokine CXCL9, also termed Mig (monokine induced by gamma interferon) by cultured SJL/J mouse astrocytes infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV). This picornavirus induces demyelination in the SJL/J genetically susceptible strain of mice through an immune process mediated by CD4+ Th1 T cells. Those cells were chemoattracted by chemokines inside the central nervous system (CNS) after blood-brain barrier (BBB) disruption.cRNAs from TMEV- and mock-infected astrocytes cells were hybridized to the Affymetrix murine genome U74v2 DNA microarray. Hybridization data analysis revealed the upregulation of six sequences potentially coding for Mig. We confirmed post infection Mig mRNA increase by quantitative (qPCR) and RT-PCR. The presence of Mig in the supernatants of infected astrocytes was quantified using a specific ELISA. Secreted Mig was biologically active, inducing chemoattraction of mouse activated CD4+ T lymphocytes. Conversely, attracting activity on CD3+ resting T cells that can be attributed to chemokines as CXCL12/SDF-1α could not be demonstrated in these supernatants. No overinduction of the gene coding for this chemokine was assessed by DNA hybridization either. Both recombinant IFN-γ and TNF-α inflammatory cytokines were also strong inducers of Mig in SJL/J astrocyte cultures.

Upconverting Nanorockers for Intracellular Viscosity Measurements During Chemotherapy.

Chemicals capable of producing structural and chemical changes on cells are used to treat diseases (e.g., cancer). Further development and optimization of chemotherapies require thorough knowledge of the effect of the chemical on the cellular structure and dynamics. This involves studying, in a noninvasive way, the properties of individual cells after drug administration. Intracellular viscosity is affected by chemical treatments and it can be reliably used to monitor chemotherapies at the cellular level. Here, cancer cell monitoring during chemotherapeutic treatments is demonstrated using intracellular allocated upconverting nanorockers. A simple analysis of the polarized visible emission of a single particle provides a real-time readout of its rocking dynamics that are directly correlated to the cytoplasmic viscosity. Numerical simulations and immunodetection are used to correlate the measured intracellular viscosity alterations to the changes produced in the cytoskeleton of cancer cells by anticancer drugs (colchicine and Taxol). This study evidences the possibility of monitoring cellular properties under an external chemical stimulus for the study and development of new treatments. Moreover, it provides the biomedical community with new tools to study intracellular dynamics and cell functioning.

Upconverting nanocomposites with combined photothermal and photodynamic effects.

Lanthanide-doped upconverting nanoparticles (UCNPs) have been studied for diverse biomedical applications due to their inherent ability to convert near-infrared (NIR) excitation light to higher energies (spanning the ultraviolet, visible, and NIR regions). To explore additional functionalities, rational combination with other optically active nanostructures may lead to the development of new multimodal nanoplatforms with theranostic (therapy and diagnostic) capabilities. Here, we develop a nanocomposite consisting of NaGdF4:Er3+, Yb3+ UCNPs, mesoporous silica (SiO2), gold nanorods (GNRs) and a photosensitizer, with integrated functionalities including luminescence imaging, photothermal generation, nanothermometry and photodynamic effects. Under 980 nm irradiation, GNRs and UCNPs are simultaneously excited due to the overlap between the surface plasmon resonance of the GNRs and the absorption of the UCNPs leading to plasmonic enhancement of the upconverted luminescence, while concomitantly creating a temperature gradient. The temperature increase can be determined from the intensity ratio of the upconverted green emission of the UCNPs. Finally, a photosensitizer, zinc phthalocyanine, was loaded into the mesoporous SiO2. Upon laser irradiation, the upconverted visible light subsequently activates the photosensitizer to release reactive oxygen species. The multifunctional GNR@SiO2@UCNPs nanocomposites showed strong luminescence signal when incubated in HeLa cervical cancer cells, making them ideal bioprobes for future theranostic applications.

In Vivo Ischemia Detection by Luminescent Nanothermometers.

There is an urgent need to develop new diagnosis tools for real in vivo detection of first stages of ischemia for the early treatment of cardiovascular diseases and accidents. However, traditional approaches show low sensitivity and a limited penetration into tissues, so they are only applicable for the detection of surface lesions. Here, it is shown how the superior thermal sensing capabilities of near infrared-emitting quantum dots (NIR-QDs) can be efficiently used for in vivo detection of subcutaneous ischemic tissues. In particular, NIR-QDs make possible ischemia detection by high penetration transient thermometry studies in a murine ischemic hindlimb model. NIR-QDs nanothermometers are able to identify ischemic tissues by means of their faster thermal dynamics. In addition, they have shown to be capable of monitoring both the revascularization and damage recovery processes of ischemic tissues. This work demonstrates the applicability of fluorescence nanothermometry for ischemia detection and treatment, as well as a tool for early diagnosis of cardiovascular disease.

Optical Torques on Upconverting Particles for Intracellular Microrheometry.

Precise knowledge and control over the orientation of individual upconverting particles is extremely important for full exploiting their capabilities as multifunctional bioprobes for interdisciplinary applications. In this work, we report on how time-resolved, single particle polarized spectroscopy can be used to determine the orientation dynamics of a single upconverting particle when entering into an optical trap. Experimental results have unequivocally evidenced the existence of a unique stable configuration. Numerical simulations and simple numerical calculations have demonstrated that the dipole magnetic interactions between the upconverting particle and trapping radiation are the main mechanisms responsible of the optical torques that drive the upconverting particle to its stable orientation. Finally, how a proper analysis of the rotation dynamics of a single upconverting particle within an optical trap can provide valuable information about the properties of the medium in which it is suspended is demonstrated. A proof of concept is given in which the laser driven intracellular rotation of upconverting particles is used to successfully determine the intracellular dynamic viscosity by a passive and an active method.

Overcoming Autofluorescence: Long-Lifetime Infrared Nanoparticles for Time-Gated In Vivo Imaging.

The always present and undesired contribution of autofluorescence is here completely avoided by combining a simple time gating technology with long lifetime neodymium doped infrared-emitting nanoparticles.

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response.

Endoglin is an auxiliary receptor for members of the TGF-ß superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-ß receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng(fl/fl)LysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng(fl/fl)LysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-ß1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.

Unveiling in Vivo Subcutaneous Thermal Dynamics by Infrared Luminescent Nanothermometers.

The recent development of core/shell engineering of rare earth doped luminescent nanoparticles has ushered a new era in fluorescence thermal biosensing, allowing for the performance of minimally invasive experiments, not only in living cells but also in more challenging small animal models. Here, the potential use of active-core/active-shell Nd(3+)- and Yb(3+)-doped nanoparticles as subcutaneous thermal probes has been evaluated. These temperature nanoprobes operate in the infrared transparency window of biological tissues, enabling deep temperature sensing into animal bodies thanks to the temperature dependence of their emission spectra that leads to a ratiometric temperature readout. The ability of active-core/active-shell Nd(3+)- and Yb(3+)-doped nanoparticles for unveiling fundamental tissue properties in in vivo conditions was demonstrated by subcutaneous thermal relaxation monitoring through the injected core/shell nanoparticles. The reported results evidence the potential of infrared luminescence nanothermometry as a diagnosis tool at the small animal level.

Thermal Scanning at the Cellular Level by an Optically Trapped Upconverting Fluorescent Particle.

3D optical manipulation of a thermal-sensing upconverting particle allows for the determination of the extension of the thermal gradient created in the surroundings of a plasmonic-mediated photothermal-treated HeLa cancer cell.

In vivo autofluorescence in the biological windows: the role of pigmentation.

Small animal deep-tissue fluorescence imaging in the second Biological Window (II-BW, 1000-1350 nm) is limited by the presence of undesirable infrared-excited, infrared-emitted (900-1700 nm) autofluorescence whose origin, spectral properties and dependence on strains is still unknown. In this work, the infrared autofluorescence and laser-induced whole body heating of five different mouse strains with distinct coat colors (black, grey, agouti, white and nude) has been systematically investigated. While neither the spectral properties nor the magnitude of organ autofluorescence vary significantly between mouse strains, the coat color has been found to strongly determine both the autofluorescence intensity as well as the laser-induced whole body heating. Results included in this work reveal mouse strain as a critical parameter that has to be seriously considered in the design and performance of small animal imaging experiments based on infrared-emitting fluorescent markers.