Au nanostar nanoparticle as a bio-imaging agent and its detection and visualization in biosystems.

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

Phagocytosis and immune response studies of Macrophage-Nanodiamond Interactions in vitro and in vivo.

The applications of nanodiamond as drug delivery and bio-imaging can require the relinquishing ND-drug conjugate via blood flow, where interaction with immune cells may occur. In this work, we investigated the ND penetration in macrophage and the immune response using the tissue-resident murine macrophages (RAW 264.7). Confocal fluorescence imaging, immunofluorescence analysis of nuclear translocation of interferon regulatory factor IRF-3 and transcriptional factor NF-κΒ, analysis of pro-inflammatory cytokines production IL-1ß, IL-6 IL-10 with a reverse transcription-polymerase chain reaction technique were applied. The TNF-α factor production has been studied both in vitro at ND interaction with the macrophage and in vivo after ND injection in the mice blood system using immunoassay. The macrophage antibacterial function was estimated through E. coli bacterial colony formation. ND didn't stimulate the immune response and functionality of the macrophage was not altered. Using MTT test, ND was found negligibly cytotoxic to macrophages. Thus, ND can serve as a biocompatible platform for bio-medical applications. Left: Graphic representation of Nanodiamond internalization in macrophage. Right: (a) Fluorescence images of lysosomes, (b) nanodiamond and (c) merged image of nanodiamond internalization in macrophage.

Nanodiamond for biolabelling and toxicity evaluation in the zebrafish embryo in vivo.

Nanodiamond (ND) has been proposed for various biomedical applications, including bioimaging, biosensing and drug delivery, owing to its physical-chemical properties and biocompatibility. Particularly, ND has been demonstrated as fluorescence- and Raman-detectable labels in many cellular models. Different surface functionalization methods have been developed, varying the ND's surface properties and rendering the possibility to attach biomolecules to provide interaction with biological targets. For this, toxicity is of major concern in animal models. Aside from cellular models, a cost-effective animal test will greatly facilitate the development of applications. In this study, we use the rapid, sensitive and reproducible zebrafish embryo model for in vivo nanotoxicity test. We optimize the conditions for using this animal model and analyze the zebrafish embryonic development in the presence of ND. ND is observed in the embryo in vivo using laser confocal fluorescence microscopy and fluorescence lifetime imaging. Using the zebrafish model for a safety evaluation of ND-based nanolabel is discussed.

Nanodiamonds of Laser Synthesis for Biomedical Applications.

In recent decade detonation nanodiamonds (DND), discovered 50 years ago and used in diverse technological processes, have been actively applied in biomedical research as a drug and gene delivery carrier, a contrast agent for bio-imaging and diagnostics and an adsorbent for protein separation and purification. In this work we report about nanodiamonds of high purity produced by laser assisted technique, compare them with DND and consider the prospect and advantages of their use in the said applications.

Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications.

Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.

Size-dependent surface CO stretching frequency investigations on nanodiamond particles.

In this work, the spectroscopic properties of surface functionalized nanodiamond particles are investigated via Fourier transform infrared spectroscopy. The functionalization of the nanodiamond surface was achieved chemically using strong acid treatment method. The size dependent C=O stretching frequency (between 1680 and 1820 cm(-1)) are studied for particle diameter sizes from the 5 to 500 nm range. The surface C=O stretching frequencies at approximately 1820 cm(-1), for large particle size (500 nm), down shifted to 1725 cm(-1) (5 nm) with decreasing particle sizes. We attributed the shift as a result of hydrogen bond formation between the COOH groups in the carboxylated nanodiamond surfaces. Particle size was characterized with dynamic light scattering method and surface morphology of the particles was investigated with scanning electron microscopy. The influence of pH value on C=O stretching frequency is also analyzed. This finding affords useful information for the studying of surface functionalized nanodiamonds with implications for their interaction with biomolecules.

Second harmonic generation of biotin and biotin ester microcrystals trapped in optical tweezers with a mode-locked Ti:sapphire laser.

Intense second harmonic generation (SHG) was observed from microcrystals of biotin and biotin ester trapped by optical tweezers, formed with a focused near-infrared pulsed laser beam. The intensity of SHG depends strongly on the states of the microcrystals and the excitation wavelength. Microscopic scanning images of biotin and biotin ester were obtained in high contrast with SHG. Simultaneous trapping and excitation of SHG and two-photon autofluorescence of biotin and biotin ester microcrystals allow us to investigate their structure and optical properties. These optically trapped particles (of submicron size) are useful as nonintrusive microscopic probes for high-resolution studies.

Perfluorocarbon emulsions increase transfer of oxygen in plasma from erythrocyte to tissues.

The erythrocyte oxy- and deoxygenation kinetics and the influence of the perfluorocarbon emulsions (PFCE) on the blood were studied using the device modeling blood circulation in the organism. It was found that a small additive of PFCE changed the oxygen transport in the erythrocyte-plasma-tissues. The oxygen-carrying properties of erythrocytes of PFCE-blood system are differ from ones of pure blood.

[Diffusion of glucose through the erythrocyte membrane]. / Issledovanie diffuzii gliukozy cherez membranu éritrotsitov.

The change of volume of human erythrocytes due to diffusion of glucose through their membranes was investigated. The dependence of membrane glucose penetration on functional state of the cells was discovered.

[Magnetic susceptibility and the structure of liposomes]. / Magnitnaia vospriimchivost' i struktura liposom.

Magnetic susceptibility of single liposomes sized 1-10 m was measured. Solid magnetic susceptibility of lipid-egg lecithin equalling chi et = -(5.038 +/- 0.035) X 10(-7) e.m.u./cm3 was determined. The liposome susceptibility was shown to be proportional to the number of bilayers in it and to reflect its packing.