Anastasia V. Ryabova

Spectroscopic research of upconversion nanomaterials based on complex oxide compounds doped with rare-earth ion pairs: Benefit for cancer diagnostics by upconversion fluorescence and radio sensitive methods/Spektroskopische Untersuchung von mit Ionenpaaren Seltener Erden dotierten Upconversion-Nanokompositen: Nutzen für die Krebsdiagnostik durch Upconversion-Fluoreszenz und strahlungssensitive Methoden

Abstract Background: Highly photochemically stable nanoparticles, in which upconversion luminescence can be excited – so-called upconversion nanocrystals (UC-NCs) – exhibit widely separated (up to 500 nm) narrow luminescence bands in the visible (VIS) region located far from the excitation near-infrared (NIR) laser radiation, and thus can be more easily identified compared to organic luminophores and semiconductor nanoparticles. Due to a deep penetration of exciting infrared (IR) radiation, the absence of parasitic fluorescence of biomolecules and the absence of phototoxicity and photobleaching upon near IR excitation, UC-NCs can be efficiently used as fluorescent probes in biological studies and fluorescence diagnostics (FD). The doping of such nanoparticles with Gd3+ ions provides the additional possibility of combining fluorescence visualization with magnetic resonance imaging, which will considerably improve the sensitivity of diagnostics of cancer tumors even in the early stages. Materials and methods: We studied the upconversion characteristics of inorganic nanoparticles made of different materials doped with rare-earth ion (REI) pairs Yb3+-Er3+ and Yb3+-Tm3+ as functions of the concentration and composition of a dopant at different excitation intensities. Matrices chosen for doping were complex polycrystalline oxide rare earth compounds Gd2GeMoO8, La4Gd10B6Ge2O34, and Gd11SiP3O26 which permit the introduction of significant concentrations of the activator luminescence ions (Yb3+, Er3+ and Tm3+), synthesized by solid-phase reaction methods from corresponding oxides. The final product was obtained by combined precipitation of initial components from aqueous solutions followed by the annealing of hydroxide mixtures and grinding. The redistribution of the intensity of the 550 nm 2H11/2, 4S3/2→4I15/2 and 650 nm 4F9/2→4I15/2 upconversion luminescence bands in Er3+ was investigated depending on matrices, dopants, and the laser power density. The quantum yield and lifetime of upconversion luminescence were determined for individual electronic transitions, which were used to optimize the composition of dopants in matrices. Results: Based on the results obtained, the matrix La4Gd10B6Ge2O34 is most effective for the upconversion process in the VIS spectrum. Doping nanoparticles by REI pairs Yb3+-Er3+ and Yb3+-Tm3+, each has its advantages. REI pair Yb3+-Er3+ is good for energy transfer in the green and/or red part of the spectrum as well as for the FD and can be used for a further energy transfer to the photosensitizers at photodynamic therapy (PDT). REI pair Yb3+-Tm3+ transform the infrared radiation in the blue region of the spectrum, which is also suitable for FD and PDT and additional intensive energy conversion in NIR will allow for deep tissue imaging. Conclusion: The investigated complex polycrystalline oxide compounds are promising as diagnostic agents for biological tissues visualization by fluorescence, light scattering, and nuclear magnetic resonance imaging. Zusammenfassung Hintergrund: Photochemisch hochstabile Nanopartikel, in denen Upconversion-Lumineszenz angeregt werden kann – die sogenannten Upconversion-Nanokomposite (UC-NCs) – weisen eng begrenzte, schmale Lumineszenz-Banden (bis 500 nm) im sichtbaren Wellenlängenbereich auf, die weit genug von der Anregungs-Laserstrahlung im Nahinfrarot (NIR)-Bereich entfernt sind und somit leichter gegenüber organischen Luminophoren und Halbleiternanopartikeln zu identifizieren sind. Durch die hohe Eindringtiefe des infraroten (IR) Anregungslichtes und das Fehlen parasitärer Fluoreszenz durch Biomoleküle sowie Phototoxizitäts- und Photobleechingeffekte im Bereich der IR-Anregung können UC-NCs effizient als Fluoreszenzsonden für biologische Untersuchungen und die Fluoreszenzdiagnostik (FD) eingesetzt werden. Die Dotierung solcher Nanopartikel mit Gd3+-Ionen bietet zusätzlich die Möglichkeit der Kombination von Fluoreszenz-Visualisierung und Magnetresonanz-Bildgebung, was die Empfindlichkeit der Diagnostik von Tumoren auch in frühen Stadien deutlich verbessert. Material und Methoden: Wir untersuchten die Upconversion-Eigenschaften von anorganischen Nanopartikeln aus unterschiedlichen Materialien, die mit Ionenpaaren Seltener Erden dotiert wurden (Yb3+-Er3+ und Yb3+-Tm3+), und zwar in Abhängigkeit von der Konzentration und Zusammensetzung des Dotands bei unterschiedlichen Anregungsenergien. Als Grundsubstanzen für eine Dotierung wurden komplexe polykristalline Oxidverbindungen Seltener Erden ausgewählt (Gd2GeMoO8, La4Gd10 B6Ge2O34, und Gd11SiP3O26), die die Einführung signifikanter Konzentrationen der Aktivator-Lumineszenz-Ionen (Yb3+, Er3+ und Tm3+) gestatten – synthetisiert durch Festphasenreaktionsmethoden aus korrespondierenden Oxiden. Das Endprodukt wurde durch die kombinierte Ausfällung der anfänglichen Komponenten aus wässriger Lösung und anschließendes Glühen und Mahlen der Hydroxidgemische erzeugt. Die Umverteilung der Intensität der 550 nm 2H11/2, 4S3/2→4I15/2 und 650 nm 4F9/2→4I15/2 Upconversion-Lumineszenz-Banden wurde in Abhängigkeit von den Grundstoffen, Dotanden und der Laserleistungsdichte untersucht. Die Quantenausbeute und die Lebensdauer der Upconversion-Lumineszenz wurden für einzelne elektronische Übergänge bestimmt, die zur Optimierung der Dotanden-Zusammensetzung verwendet wurden. Ergebnisse: Wie sich zeigte, ist die La4Gd10B6Ge2O34-Matrix im sichtbaren Spektralbereich am effektivsten für den Upconversion-Prozess. Beide dotierenden Ionenpaare Seltener Erden (Yb3+-Er3+ und Yb3+-Tm3+) haben ihre Vorteile. Das Yb3+-Er3+-Ionenpaar ist gut für die Energieübertragung im grünen und/oder roten Spektralbereich als auch für die FD und kann für eine weitere Energieübertragung auf den Photosensibilisator während der photodynamischen Therapie (PDT) verwendet werden. Das Yb3+-Tm3+-Ionenpaar verwandelt die IR-Strahlung im blauen Bereich des Spektrums, was ebenfalls für die FD und PDT geeignet ist und eine zusätzliche intensive Energieumwandlung in NIR bewirkt, was ein Imaging tiefer Gewebeschichten erlaubt. Fazit: Die untersuchten komplexen polykristallinen Oxidverbindungen sind als diagnostische Mittel zur Darstellung biologischen Gewebes mittels Fluoreszenz, Lichtstreuung und Magnetresonanz-Bildgebung geeignet.

Synthesis and luminescent characteristics of submicron powders on the basis of sodium and yttrium fluorides doped with rare earth elements

Submicron powders of solid solutions on the basis of cubic and hexagonal phases existing in a NaF-YF3 system have been synthesized with the precipitation method from aqueous solutions at room temperature. The conditions for the preparation of single-phase samples have been determined. The upconversion luminescence spectra were investigated. The most promising composition is revealed (Na0.414Ca0.183Y0.215Yb0.094Er0.094F1.989), which demonstrates an upconversion luminescence quantum yield of 1.9% with a 3-W pump power at a 974-nm wavelength.

Pulsed periodic laser excitation of upconversion luminescence for deep biotissue visualization

Emission spectral properties and quantum efficiency of upconversion particles NaYF4, SrF2, LaF3, BaF2 и CaF2, doped with rare earth ions pair Yb3+–Er3+ were studied using continuous wave (CW) and pulsed periodic excitation modes in the near infrared (NIR) spectral range. Analysis of the obtained results showed that the intensity ratio of upconversion luminescence in green and red spectral ranges depends on excitation pulse duration. Thus, by changing the pulse duration the spectral properties of upconversion luminescence can be controlled. Crystals with higher phonon energy are more sensitive to the change of pumping mode. Interpretation of results was performed on the rate equation model basis. Using numerical methods for all energy levels involved in the upconversion process the population and depopulation dynamics were obtained with respect to the duration of the excitation pulses. It was shown that about 30 ms was required for the complete population of 4F9/2 state, from which the luminescence in the red spectral range occurs. When the pulse duration was less than 30 ms, the 4F9/2 population did not reach a steady state and the intensity of the luminescence in the red part of the spectrum was reduced. The theoretical dependence of the upconversion luminescence intensity in the green and red ranges of the excitation pulse duration for NaYF4:Yb0.2–Er0.02 composition was obtained and demonstrates good agreement with the experimental results.

Upconversion microparticles as time-resolved luminescent probes for multiphoton microscopy: desired signal extraction from the streaking effect

Abstract. The great interest in upconversion nanoparticles exists due to their high efficiency under multiphoton excitation. However, when these particles are used in scanning microscopy, the upconversion luminescence causes a streaking effect due to the long lifetime. This article describes a method of upconversion microparticle luminescence lifetime determination with help of modified Lucy–Richardson deconvolution of laser scanning microscope (LSM) image obtained under near-IR excitation using nondescanned detectors. Determination of the upconversion luminescence intensity and the decay time of separate microparticles was done by intensity profile along the image fast scan axis approximation. We studied upconversion submicroparticles based on fluoride hosts doped with Yb3+-Er3+ and Yb3+-Tm3+ rare earth ion pairs, and the characteristic decay times were 0.1 to 1.5 ms. We also compared the results of LSM measurements with the photon counting method results; the spread of values was about 13% and was associated with the approximation error. Data obtained from live cells showed the possibility of distinguishing the position of upconversion submicroparticles inside and outside the cells by the difference of their lifetime. The proposed technique allows using the upconversion microparticles without shells as probes for the presence of OH− ions and CO2 molecules.

Chlorin Nanoparticles for Tissue Diagnostics and Photodynamic Therapy

BACKGROUND Organic crystalline nanoparticles (NPs) are not fluorescent due to the crystalline structure of the flat molecules organized in layers. In earlier experiments with Aluminum Phthalocyanine (AlPc)-derived NPs, the preferential uptake and dissolution by macrophages was demonstrated [3]. Therefore, inflamed tissue or cancer tissue with accumulated macrophages may exhibit specific fluorescence in contrast to healthy tissue which does not fluoresce. The present study addresses the photobiological effects of NP generated from Temoporfin (mTHPC), a clinically utilized photosensitizer belonging to the chlorin family. METHODS In-vitro investigations addressing uptake, dissolution and phototoxicity of mTHPC NP vs. the liposomal mTHPC formulation Foslip were performed using J774A.1 macrophages and L929 fibroblasts. For total NP uptake analysis, the cells were lysed, the nanoparticles dissolved and the fluorescence quantified. The intracellular molecular dissolution was measured by flow cytometry. Fluorescence microscopy served for controlling intracellular localization of the dissolved fluorescing molecules. Reaction mechanisms after PDT (mitochondrial activity, apoptosis) were analyzed using fluorescent markers in cell-based assays and flow cytometry. RESULTS Organic crystalline NP of different size were produced from mTHPC raw material. NP were internalized more efficiently in J774A.1 macrophages when compared to L929 fibroblasts, whereas uptake and fluorescence of Foslip was similar between the cell lines. NP dissolution correlated with internalization levels for larger particles in the range of 200-500 nm. Smaller particles (45 nm in diameter) were taken up at high levels in macrophages, but were not dissolved efficiently, resulting in comparatively low intracellular fluorescence. Whereas Foslip was predominantly localized in membranes, NP-mediated fluorescence also co-localized with acidic vesicles, suggesting endocytosis/phagocytosis as a major uptake mechanism. In macrophages, phototoxicity of NPs was stronger than in fibroblasts, even exceeding Foslip when administered in identical amounts. In both cell lines, phototoxicity correlated with mitochondrial depolarization and enhanced activation of caspase 3. CONCLUSIONS Due to their preferential uptake/dissolution in macrophages, mTHPC NP may have potential for the diagnosis and photodynamic treatment of macrophage-associated disorders such as inflammation and cancer.

Photodynamic effect of iron(III) oxide nanoparticles coated with zinc phthalocyanine

The possibility of using iron oxide(III) nanoparticles covered with photoactive derivative of zinc phthalocyanine for diagnostics and treatment of malignant tumors was studied experimentally.

Use of optical-spectral methods for in vivo noninvasive assessment of nanoparticles accumulation in biological tissues

Methods for noninvasive estimation of the nanoparticles concentration in biological tissues in vivo were developed with a view to laying the scientific groundwork for diagnosis and treatment of malignant tumors. The methods were tested in experimental animals (mice inoculated with Ehrlich carcinoma). Luminescent spectroscopy and diffuse reflectance spectroscopy were applied for examination of biotissues. A special algorithm of spectral data analysis with the use of the model solution simulating the optical properties of biotissues was devised. It was shown that the optical-spectral methods developed are effective in high-accuracy noninvasive real-time quantitative analysis of the nanoparticles accumulation in biotissues.

Raman and fluorescence microscopy to study the internalization and dissolution of photosensitizer nanoparticles into living cells

In this present study we applied Raman and fluorescence microscopy to investigate the internalisation, cellular distribution and effects on cell metabolism of photosensitizer nanoparticles for photodynamic therapy in fibroblasts and macrophages.

TAM identification by fluorescence lifetime on different models

Nowadays problem of cell differentiation in vivo is the topical in oncology. Laser time-resolved spectroscopy allows to evaluate the activity of different types of cells in a tumor microenvironment, in particular tumor associated macrophages (TAM), considering specific cell’s features of photosensitizer (PS) accumulation. The technique is based on the fluorescence lifetime estimation, which allows one to judge the degree of PS interaction, thereby distinguishing the type of cells.

Bioimaging with controlled depth using upconversion nanoparticles

The study of bioimaging with controlled depth using upconversion nanoparticles under near-infrared excitation was performed in this work. Monte Carlo simulation was performed to determine optimal distance between the fiber - source of laser radiation, and the receiving fiber for obtaining the signal from maximal depth in biological tissue. Also theoretical modeling of the spatial distribution of diffusely scattered radiation inside the tissue depending on wavelength is presented. Penetration depth for wavelengths corresponding to the upconversion luminescence was calculated. Experimental modeling was carried out on phantoms of biological tissues simulating their scattering properties as well as accumulation of the investigated nanoparticles doped with rare earth ions. Measurements were performed using NaGdF4 nanoparticles doped with Yb3+, Er3+ and Tm3+ rare earth ions, which demonstrated several luminescence bands from the blue (475nm) to the near-infrared (800 nm) regions of the spectrum under 980 nm excitation. The different penetration depth of various wavelengths in biotissue allows us to estimate the depth from which the signal was obtained using luminescence intensity ratio (LIR). Due to non-linearity of upconversion process, pumping power dependences of luminescence intensity was taken into account. The number of involved photons for each spectral band was estimated and intensity ratio of emission bands was calculated. Based on calculations and experimental measurements, the theoretical and experimental luminescence intensity ratio for different depths was estimated. The experimental study was performed on biological tissue phantoms containing Lipofundin® with red blood cells and has shown good agreement with calculations. The use of theoretically calculated LIR allows us to solve the inverse problem and estimate the depth from which the signal was obtained.