Silicon-Gold Nanoparticles Affect Wharton's Jelly Phenotype and Secretome during Tri-Lineage Differentiation.

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton's Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon-gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4-9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.

An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography.

In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical microspectroscopy (nanoscale chemical mapping and optical properties) and allowing simultaneous 3D measurements.

Cell Staining by Photo-activated Dye and Its Conjugate with Chitosan.

Photo-activated or "Caged" rhodamine dyes are the most useful for microscopic investigation of biological tissue by various fluorescent techniques. Novel precursor of the fluorescent dye (PFD813) has been studied for photosensitive staining of numerous animal cells. The functional rhodamine dye (Rho813) with intensive fluorescence has been obtained after photoactivation of its precursor PFD813 inside cells. The dye Rho813 has been successfully used for the optical detection of particular features in biological objects (HaCaT cells, HBL-100, MDCK, lymphocytes). Moreover, the chitosan conjugate with PFD molecules ("Chitosan-PFD813″) has been obtained and studied for the first time. The developed procedures and obtained data are important for further applications of novel precursors of fluorescent dyes ("caged" dyes) for microscopic probing of biological objects. As example, the synthesized "Chitosan-PFD813″ has been successfully applied in this study for intracellular transport visualization by fluorescent microscopy.

Multifunctional membranes based on photosensitive crown-ether derivatives with advanced properties.

This review discusses recent works on monolayer, multilayer and polymer films of various crown-ether derivatives. Preparation and investigation of such membrane nanostructures based on photosensitive and surface-active crown-ethers is a rapidly growing field at the "junction" of colloids and polymers, materials sciences and nanotechnology. These membranes can serve as convenient models for studying the self-organization and molecular recognition processes at interfaces that are typical for biomembranes. The results obtained for such structures by absorption and fluorescence spectroscopy, atomic force and Brewster-angle microscopy, surface pressure and surface potential isotherm measurements have been described. The possibility of developing multifunctional materials possessing advanced properties has been demonstrated.

Conjugates of a photoactivated rhodamine with biopolymers for cell staining.

Conjugates of the photoactivated rhodamine dyes with biopolymers (proteins, polysaccharides, and nucleic acids) are important tools for microscopic investigation of biological tissue. In this study, a precursor of the photoactivated fluorescent dye (PFD) has been successfully used for staining of numerous mammalian cells lines and for conjugate formation with chitosan ("Chitosan-PFD") and histone H1 ("Histone H1.3-PFD"). The intensive fluorescence has been observed after photoactivation of these conjugates inside cells (A431, HaCaT, HEK239, HBL-100, and MDCK). Developed procedures and obtained data are important for further application of novel precursors of fluorescent dyes ("caged" dyes) for microscopic probing of biological objects. Thus, the synthesized "Chitosan-PFD" and "Histone H1-PFD" have been successfully applied in this study for intracellular transport visualization by fluorescent microscopy.

Controlled influence of quantum dots on purple membranes at interfaces.

The development of bio-sensitized nanofilms engineered from biomembrane components and inorganic nanoparticles is a promising field of colloid and interface science and technologies. Recent nano-bioengineering approaches employing quantum dots (QDs) permit the enhancement of the purple membrane (PM) "light-harvesting capacity" compared to native PMs. The influence of QDs on the PM properties, especially the bacteriorhodopsin (bR) photocycle, has been found that has both fundamental (mechanisms of photoreception) and applied implications (including the fabrication of hybrid bionanomaterials). Samples of PM-QD complexes capable of energy transfer and characterized by increased rates of M-intermediate formation and decay have been obtained. The modified bR photocycle kinetic parameters may be explained by changes in the PM interface upon QD adsorption. The increase and decrease in absorption at 410 nm (or photopotential) for PM-QD complexes are, on average, several times more rapid than for PM suspensions or PM dry films. These results provide a strong impetus for the development of nanomaterials with advanced properties.

Novel precursors of fluorescent dyes. 1. Interaction of the dyes with model phospholipid in monolayers.

Photoactivated ("caged") fluorescent dyes are modern tools for structure and function studies of cell membranes and subcellular organelles. Recently synthesized precursors of rhodamine fluorescent dyes (abbreviations PFD813 and PFD814) important for microscopic probing of biological objects have been studied in solution. In order to characterize the behavior at interfaces, monolayers of PFD813 and PFD814 on water have been formed and investigated. The interactions of these precursors with the biomembrane component dimyristoylphosphatidylethanolamine in monolayers at the air-water interface and after transfer to glass plates have been studied by measuring monolayer parameters and spectroscopic properties before and after photo-chemical formation of the fluorescent rhodamine dyes Rho813 and Rho814, respectively.

Thin films and assemblies of photosensitive membrane proteins and colloidal nanocrystals for engineering of hybrid materials with advanced properties.

The development and study of nano-bio hybrid materials engineered from membrane proteins (the key functional elements of various biomembranes) and nanoheterostructures (inorganic colloidal nanoparticles, transparent electrodes, and films) is a rapidly growing field at the interface of materials and life sciences. The mainspring of the development of bioinspired materials and devices is the fact that biological evolution has solved many problems similar to those that humans are attempting to solve in the field of light-harvesting and energy-transferring inorganic compounds. Along this way, bioelectronics and biophotonics have shown considerable promise. A number of proteins have been explored in terms of bioelectronic device applications, but bacteriorhodopsin (bR, a photosensitive membrane protein from purple membranes of the bacterium Halobacterium salinarum) and bacterial photosynthetic reaction centres have received the most attention. The energy harvesting in plants has a maximum efficiency of 5%, whereas bR, in the absence of a specific light-harvesting system, allows bacteria to utilize only 0.1-0.5% of the solar light. Recent nano-bioengineering approaches employing colloidal semiconductor and metal nanoparticles conjugated with biosystems permit the enhancement of the light-harvesting capacity of photosensitive proteins, thus providing a strong impetus to protein-based device optimisation. Fabrication of ultrathin and highly oriented films from biological membranes and photosensitive proteins is the key task for prospective bioelectronic and biophotonic applications. In this review, the main advances in techniques of preparation of such films are analyzed. Comparison of the techniques for obtaining thin films leads to the conclusion that the homogeneity and orientation of biomembrane fragments or proteins in these films depend on the method of their fabrication and increase in the following order: electrophoretic sedimentation < Langmuir-Blodgett and Langmuir-Schaefer methods < self-assembly and layer-by-layer methods. The key advances in the techniques of preparation of the assemblies or complexes of colloidal nanocrystals with bR, purple membranes, or photosynthetic reaction centres are also reviewed. Approaches to the fabrication of the prototype photosensitive nano-bio hybrid materials with advanced photovoltaic, energy transfer, and optical switching properties and future prospects in this field are analyzed in the concluding part of the review.