One-Step Synthesis of Biocompatible NaY0.65Gd0.15F4:Yb,Er Upconverting Nanoparticles for In Vitro Cell Imaging

There is a great technological interest in synthesis of lanthanide doped upconverting nanoparticles (UCNPs) with controlled crystal phase, morphology and intense luminescence properties suitable for biomedical use. A conventional approach for synthesis of such particles comprises decomposition of organometallic compounds in an oxygen-free environment, followed either with a ligand exchange, or biocompatible layer coating. Biocompatible NaY0.65Gd0.15F4:Yb,Er nanoparticles used in this study were synthesized through chitosan assisted one-pot hydrothermal synthesis and were characterized by X-ray powder diffraction (XRPD), Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDS) and photoluminescence measurement (PL). Due to the presence of the amino groups at their surface, excellent biocompatibility and notably low cytotoxicity against MRC-5 cells (line of normal human fibroblasts) and A549 cells (human lung cancer cells) were detected using MTT assay. Furthermore, upon 980 nm laser irradiation, particles were successfully used in vitro for labeling of both, MRC-5 and A549 cells

Biocompatible Up-converting Yb/Er Doped Ln-fluoride Mesocrystals for Cell Labeling

Imaging of biological processes are of the great importance for early diagnostic of diseases and for understanding the disease pathway in the human body [1]. In order to improve image contrast and resolution, different optical fluorescent probes, such as florescent dyes, fluorescent proteins and quantum dots, have been developed. However, their application is somewhat restricted by photobleaching, cell autofluorescence, and small tissue penetration depth [2]. Lanthanide doped up-conversion nanoparticles (UCNPs) owing to their unique optical properties and high photochemical stability, represents a promising material for superior optical diagnostic and drug delivery systems [3]. Due to large anti-Stocks shift, UCNPs have converse excitation and emission profiles: under excitation by near-infrared light (NIR), they are able to emit visible or UV photons whit a shorter wavelength [4,5]. Hexagonal-phase NaYF4:Yb,Er nanocrystals are the best NIR-to-visible up-converting materials to date, but YF3 phase due to its wider optical transmission window, and the minimization of the excited state quenching of doped ions, is considered to be promising material for application in biolabeling as well. In this work, biocompatible YF3:YbEr and GdF3:YbEr mesocrystals were prepared by one-pot solvothermal synthesis in the presence of chitosan, and are used successfully as biolabeling agents. The crystallinity and phase purity of the obtained powders were confirmed by X-ray powder diffraction which revealed that both samples are with the orthorhombic Pnma crystal structure, and monophase. Homogeneous distribution of dopant ions in the host matrix was confirmed by STEM/EDS mapping (not shown). SEM and TEM analysis implied that particles possess morphology similar to the unshelled peanuts, which are built from smaller monocrystals (Figs 1a and b). It was shown that their capacity toward self-organization is determined by the amount of surface-preserved chitosan ligands, which presence is confirmed by FTIR spectroscopy. Chitosan presence provides biocompatibility, thus the viability of both, MRC- 5 lung fibroblast, and A549 lung carcinoma cells was highly preserved (>80%), after 24 h of incubation with the synthetized samples (50μg mL-1). To monitor the intracellular uptake, cells incubated with particles were further analyzed by laser scanning microscopy under 980nm excitation. A strong up-conversion emission (green spots at Figs 1c and d), indicated that particles (green spots) are positioned in the cells cytoplasmic area adjacent to the plasma membrane, in both cell lines. Such positioning, without disturbing cells nuclei confirms their capacity to be used as NIR cell labeling agents

One-Step Synthesis of Biocompatible NaY0.65Gd0.15F4:Yb,Er Upconverting Nanoparticles for In Vitro Cell Imaging

There is a great technological interest in synthesis of lanthanide doped upconverting nanoparticles (UCNPs) with controlled crystal phase, morphology and intense luminescence properties suitable for biomedical use. A conventional approach for synthesis of such particles comprises decomposition of organometallic compounds in an oxygen-free environment, followed either with a ligand exchange, or biocompatible layer coating. Biocompatible NaY0.65Gd0.15F4:Yb,Er nanoparticles used in this study were synthesized through chitosan assisted one-pot hydrothermal synthesis and were characterized by X-ray powder diffraction (XRPD), Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDS) and photoluminescence measurement (PL). Due to the presence of the amino groups at their surface, excellent biocompatibility and notably low cytotoxicity against MRC-5 cells (line of normal human fibroblasts) and A549 cells (human lung cancer cells) were detected using MTT assay. Furthermore, upon 980 nm laser irradiation, particles were successfully used in vitro for labeling of both, MRC-5 and A549 cells

Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology

Adaptive changes in mitochondrial morphology are associated with changes in the mitochondrial function and metabolic fitness of eukaryotic cells. We previously described in young hyphae of the filamentous fungus Phycomyces blakesleeanus a dramatic effect of an increase in ambient temperature during growth: a 3°C warmer environment compared with a control temperature of 22°C resulted in the appearance of long elongated (“tubular”) mitochondria accompanied by an increase in lipid droplet density. Here, we examined how cooler ambient temperature (18°C) during growth affects mitochondrial morphology in P. blakesleeanus compared with the control grown at 22°C. We used two-photon fluorescence imaging (TPEF) of live hyphae stained with the vital mitochondrial dye rhodamine 123. Extraction of relevant parameters (number, size, and shape of mitochondria) from TPEF images was performed using the Ilastik machine learning-based software. The suitability of the Ilastik analysis was compared with the Particle Analysis (ImageJ). Cold treatment resulted in the appearance of tubular mitochondrial morphology that was absent in the control group. Tubular mitochondrial morphology appears to be an adaptive feature that occurs in both warmer and colder conditions and is likely part of the stress response

Temperature Sensing Properties of Biocompatible Yb/Er-Doped GdF3 and YF3 Mesocrystals

Y0.8−xGdxF3:Yb/Er mesocrystals with a biocompatible surface and diverse morphological characteristics were successfully synthesized using chitosan-assisted solvothermal processing. Their structural properties, studied using X-ray powder diffraction, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy and energy dispersive X-ray analysis, were further correlated with the up-conversion emission (λexc = 976 nm) recorded in function of temperature. Based on the change in the visible green emissions originating from the thermally coupled 2H11/2 and 4S3/2 levels of Er3+, the corresponding LIR was acquired in the physiologically relevant range of temperatures (25–50 °C). The detected absolute sensitivity of about 0.0042 °C−1, along with the low cytotoxicity toward both normal human lung fibroblasts (MRC-5) and cancerous lung epithelial (A549) cells, indicate a potential for use in temperature sensing in biomedicine. Additionally, their enhanced internalization in cells, without suppression of cell viability, enabled in vitro labeling of cancer and healthy cells upon 976 nm laser irradiation

Lanthanide doped up-converting nanoparticles for bioimaging and thermal sensing

Bioimaging and thermal sensing refer to techniques related to the observation of biological structure and function of cells, which are of paramount importance for early diagnostics of diseases. Due to their unique ability to convert NIR to Vis light through a nonlinear multiphonon anti-Stokes process, lanthanide doped up-converting nanoparticles (Ln-UCNP) have an important role in this field. The work will present the synthesis procedures applied for the in-situ obtaining of various biocompatible Ln-UCNP and their application for cell labelling (Figure 1), and for temperature sensing in the physiologically relevant range of temperatures. Additional selectivity toward target cancer cell labelling was enabled through their conjugation with the anti-human CD44 antibodies. The Ln-UCNP structural and morphological properties, revealed through XRPD, FTIR, XPS, SEM/TEM-EDS, and HRTEM analysis, are correlated with their upconversion emission efficiency, and capacity to be used in medicine. For assessing of biological safety of their use, viability of commercially available cell lines, as well as human cells, was additionally evaluated by a colorimetric MTT assay. Laser scanning microscopy imaging under excitation of 976 nm verified their feasibility to be used as new fluorescence imaging probes

Selective in vitro labeling of cancer cells using NaGd0.8Yb0.17Er0.03F4 nanoparticles

Cancer represents one of the leading problems of today, with clinical detection oftentimes being difficult, currently based on imaging techniques, such as X-ray, computed tomography (CT) and magnetic resonance imaging (MRI). However, mortality rate is often reduced by early detection, therefore much focus has been directed towards developing methods for early detection of the disease. Recent research in the field of nanotechnology is focused on the use of nanoparticles, particularly Lanthanide-doped up-conversion nanoparticles (UCNPs), for the detection of cancer cells using near infrared (NIR) fluorescence microscopy. The reason for this is that in long-term tracking tests, nearinfrared (NIR) light, has lower phototoxicity and higher tissue penetration depth in living systems as compared with UV/VIS light. In this research, NaGd0.8Yb0.17Er0.03F4 UCNPs were prepared by solvothermal synthesis in the presence of chitosan, a ligand which enables UCNPs biocompatibility and the specific antibody conjugation. Morphological and structural characterization of synthetized UCNPs were performed based on X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and photoluminescence spectroscopy (PL). Results confirmed the presence of the cubic phase with a minor portion of hexagonal phase in nanoparticles. Synthesized nanoparticles were conjugated further with anti-human CD44 antibodies, labeled with fluorescein isothiocyanate (FITC), which signal is used for confirmation of nanoparticles positioning in cells. Such obtained conjugates were successfully used for selective in vitro biolabeling of oral squamous cell carcinoma cells

Lanthanide-doped bioglass for non-specific cell labelling

Bioglass is glassy ceramic biocompatible material, usually composed of silicon, calcium, phosphorus and sodium oxides. As such, it has a great potential for application in bone tissue engineering. If it possessed optically and magnetic features too, it would allow unambiguous and non-invasive tracing of bone defects reparation process. For this purpose, we prepared bioglass doped with different combinations of rare earth elements that should enable multimodal imaging in biomedicine: ytterbium/erbium/gadolinium and europium/gadolinium. After confirmation of phase composition of synthetised bioglass, luminescent measurements were performed. As expected, ytterbium/erbium/gadolinium-doped sample showed up-conversion properties, emitting visible blue, green and red light when excited by NIR laser, while europium/gadolinium-doped sample showed down-conversion features, by emitting red visible response induced by excitation with UV laser. Cytotoxicity tests implied that lanthanide-doped bioglass particles are non-toxic, and safe to use in hard tissue engineering, while their successful visualization in cells by laser scanning and inverted-fluorescence microscopy confirmed their cell labelling capacity