Effect of magnetic nanoparticles Fe3O4 on viability, attachment, and spreading of isolated fetuses and newborn rats

This study has shown that the toxic effect of nonmodified Fe₃O₄ nanoparticles in vitro depends on the metabolic and morphological conditions of cells from rat fetuses and newborns. In the process of cultivation, cells with magnetic nanoparticles bind to their surface and penetrate the intracellular space. The sorption of nanoparticles on the cell surface hinders their attachment to the substrate and absorption by spread cells can prevent their proliferation. Magnetic nanoparticles are well sorbed by the upper layer of cell aggregates, whereas cells of the inner layer remain intact. As a result, the cell aggregates acquire the property of responding to a constant magnetic field. These aggregates could potentially be used in cell transplantation for directed cell delivery.     

Cellular response to magnetic nanoparticles "PEGylated" via surface-initiated atom transfer radical polymerization

A new method to PEGylate magnetic nanoparticles with a dense layer of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) by surface-initiated atom transfer radical polymerization (ATRP) is reported. In this approach, an initiator for ATRP was first immobilized onto the magnetic nanoparticle surface, and then P(PEGMA) was grafted onto the surface of magnetic nanoparticle via copper-mediated ATRP. The modified nanoparticles were subjected to detailed characterization using FTIR, XPS, and TGA. The P(PEGMA)-immobilized nanoparticles dispersed well in aqueous media. The saturation magnetization values of the P(PEGMA)-immobilized nanoparticles were 19 emu/g and 11 emu/g after 2 and 4 h polymerization respectively, compared to 52 emu/g for the pristine magnetic nanoparticles. The response of macrophage cells to pristine and P(PEGMA)-immobilized nanoparticles was compared. The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles. With the P(PEGMA)-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to <2 pg/cell over a 5 day period. With this amount of nanoparticles uptake, no significant cytotoxicity effects were observed.     

Comparison of the morphologies of a flotable and non-flotable strain of Saccharomyces cerevisiae

In previous paper, Saccharomyces cerevisiae LBG H620 and DAM 2155 were compared regarding their ability to float. LBG H620 did not float at all; cells' surface properties indicated that the yeast LBG H620 has a high surface hydrophilicity and a high electrokinetic potential; yeast DSM 2155 possesses high hydrophobicity and a low electrokinetic potential [Tybussek et al. (1994) J Appl Microbiol Biotechnol 41:13–22]. In the present paper, the morphologies of these two yeast strains are compared. Strain LBG H620 formed only single or dudding cells, strain DSM 2155 formed cell aggregates, their size depending on the cultivation condiotions: in the presence of adequate substrate concentration cell aggregates were formed, and during substrate limitation single cell dominated. During rerspiratory growth rather small spherical aggregates and during respiratory/fermentative growth long-strain aggregates were observed *** DIRECT SUPPORT *** AG903053 00004     

A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic–inorganic hybrid materials in sponge primmorphs

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).     

Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent

The polymeric functionalization of superparamagnetic iron oxides nanoparticles is developed for cancer targeting capability and magnetic resonance imaging. Here the nanoparticles (NP) are decorated through the adsorption of a polymeric layer around the particle surface for the formation of core-shell. The synthesized magnetic nanoparticles (MNPs) are conjugated with fluorescent dye, targeting ligand, and drug molecules for improvement of target specific diagnostic and possible therapeutics applications. In this investigation doxorubicin was loaded into the shell of the MNPs and release study was carried out at different pH. The core-shell structure of magnetic NP coated chitosan matrix was visualized by TEM observation. The cytotoxicity of these magnetic NPs is investigated using MTT assay and receptor mediated internalization by HeLa and NIH3T3 cells are studied by fluorescence microscopy. Moreover, compared with T₂-weighted magnetic resonance imaging (MRI) in the above cells, the synthesized nanoparticles are showed stronger contrast enhancements towards cancer cells.     

Aggregation of normal and transformed cells attached to a substrate]

On the surface of the nirtocellulose membrane filter (pore size 0.3--0.5 mem), normal mouse or hamster embryo fibroblasts formed discrete cell aggregates. Behaviour of transformed fibroblast-like cells of 9 different lines was compared with that of normal cells. Cells of 3 transformed lines grew on this substratum as a uniform monolayer displaying no tendency to aggregation. The following 3 cell lines exposed a slightly "patchy" cell distribution on the 3rd--4th day of cultivation but were unable to form discrete aggregates. The remaining 3 lines did form aggregates but the dynamics of aggregation and the final aggregation pattern for two of them were abnormal. Only one of the 9 investigated transformed lines had the normal aggregation behaviour. Hence, in the course of the neoplastic evolution, cells lose their ability fo form aggregates on the filter surface. Mechanisms of cell aggregation and possible reasons of differencies in the aggregation behaviour between normal and transformed cells, are discussed.     

Optimization of the covalent coupling and ionic adsorption of magnetic nanoparticles on Flavobacterium ATCC 27551 using the Taguchi method

Abstract

Flavobacterium ATCC 27551 was used as a model system for the preparation of magnetic biocatalysts. The magnetic modification was carried out by covalently binding carboxylate- and amino-modified magnetic nanoparticles onto cells. Magnetic Fe₃O₄ nanoparticles were also used for ionic adsorption on the cell surface. Magnetically modified cells were concentrated using a magnet and exhibited organophosphate hydrolyzing activity. The Taguchi method was used to optimize the binding of the magnetic nanoparticles on the cell surface. SEM image analyses demonstrated good linkage of the magnetic nanoparticles over the Flavobacterium ATCC 27551 cell surface. Under optimal conditions, the magnetic cells displayed specific activity ratios of 93%, 89% and 95%, compared with untreated cells, after the covalent coupling with carboxylate- and amino-modified magnetic nanoparticles and the ionic adsorption of magnetic Fe₃O₄ nanoparticles, respectively.     

Specific and nonspecific adhesion in the process of retinal cellular aggregation in chick embryos

A study of aggregation of the retinal cells of 8 and 14 day old chick embryos has revealed two phases in this process. The first phase includes the decrease in the concentration of single cells and the increase in the concentration of aggregates. During the second phase the concentration of aggregates falls at the expense of fusion of smaller aggregates into larger ones. The rate of aggregation at both these phases increases with the initial density of cells and decreases with the age of donor embryos and at a suboptimal temperature of cultivation. Aggregation during the first phase does not depend on the presence in the culture medium of divalent cations and colchicine, the level of protein and RNA synthesis in the cells, whereas aggregation during the second phase depends on all these factors. Comparison of these results with the published data suggests that the retinal cell aggregation during the second phase, unlike the first one, is based on the specific adhesiveness of the cells, which is realized via adhesion molecules resynthesized at the cell surface.     

Generation of magnetic nonviral gene transfer agents and magnetofection in vitro

This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3-5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.     

Targeting and cellular trafficking of magnetic nanoparticles for prostate cancer imaging

Antibody-conjugated iron oxide nanoparticles offer a specific and sensitive tool to enhance magnetic resonance (MR) images of both local and metastatic cancer. Prostate-specific membrane antigen (PSMA) is predominantly expressed on the neovasculature of solid tumors and on the surface of prostate cells, with enhanced expression following androgen deprivation therapy. Biotinylated anti-PSMA antibody was conjugated to streptavidin-labeled iron oxide nanoparticles and used in MR imaging and confocal laser scanning microscopic imaging studies using LNCaP prostate cancer cells. Labeled iron oxide nanoparticles are internalized by receptor-mediated endocytosis, which involves the formation of clathrin-coated vesicles. Endocytosed particles are not targeted to the Golgi apparatus for recycling but instead accumulate within lysosomes. In T(1)-weighted MR images, the signal enhancement owing to the magnetic particles was greater for cells with magnetic particles bound to the cell surface than for cells that internalized the particles. However, the location of the particles (surface vs internal) did not significantly alter their effect on T(2)-weighted images. Our findings indicate that targeting prostate cancer cells using PSMA offers a specific and sensitive technique for enhancing MR images.     

Interaction of fibrinogen with magnetite nanoparticles

The interaction between fibrinogen and magnetite nanoparticles in solution has been studied by the methods of spin labeling, ferromagnetic resonance, dynamic and Rayleigh light scattering. It is shown that protein molecules adsorb on the surface of nanoparticles to form multilayer protein covers. The number of molecules adsorbed on one nanoparticle amounts to ∼65 and the thickness of the adsorption layer amounts to ∼27 nm. Separate nanoparticles with fibrinogen covers (clusters) form aggregates due to interactions of the end D domains of fibrinogen. Under the influence of direct magnetic field, nanoparticles with adsorbed proteins form linear aggregates parallel to the force lines. It is shown that the rate of protein coagulation during the formation of fibrin gel under the action of thrombin on fibrinogen decreases ∼2 times in the presence of magnetite nanoparticles, and the magnitude of the average fiber mass/length ratio grows.     

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth. (c) 1996 John Wiley & Sons, Inc.     

Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles

A major limitation in studying molecular interactions between parasitic helminths and their hosts is the lack of suitable in vitro cultivation systems for helminth cells and larvae. Here we present a method for long-term in vitro cultivation of larval cells of the tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Primary cells isolated from cultivated metacestode vesicles in vitro showed a morphology typical of Echinococcus germinal cells, displayed an Echinococcus-specific gene expression profile and a cestode-like DNA content of approximately 300Mbp. When kept under reducing conditions in the presence of Echinococcus vesicle fluid, the primary cells could be maintained in vitro for several months and proliferated. Most interestingly, upon co-cultivation with host hepatocytes in a trans-well system, mitotically active Echinococcus cells formed cell aggregates that subsequently developed central cavities, surrounded by germinal cells. After 4 weeks, the cell aggregates gave rise to young metacestode vesicles lacking an outer laminated layer. This layer was formed after 6 weeks of cultivation indicating the complete in vitro regeneration of metacestode larvae. As an initial step toward the creation of a fully transgenic strain, we carried out transient transfection of Echinococcus primary cells using plasmids and obtained heterologous expression of a reporter gene. Furthermore, we successfully carried out targeted infection of Echinococcus cells with the facultatively intracellular bacterium Listeria monocytogenes, a DNA delivery system for genetic manipulation of mammalian cells. Taken together, the methods presented herein constitute important new tools for molecular investigations on host-parasite interactions in alveolar echinococcosis and on the roles of totipotent germinal cells in parasite regeneration and metastasis formation. Moreover, they enable the development of fully transgenic techniques in this group of helminth parasites for the first time.     

Antireflection TiO x Coating with Plasmonic Metal Nanoparticles for Silicon Solar Cells

It is known that the light scattering from the metal particles deposited on the surfaces of cells can be used for increasing light trapping in the solar cells. In this work, plasmonic structures are composite materials that consisted of silver nanoparticles embedded in dielectric films of TiO _x —used as cell antireflection coating. The films are deposited by sol–gel method using spin-on technique. Microstructure of prepared samples is analyzed by SEM observation. Good homogenity and particles density was obtained by this simple, cheap, and short time-demanding method. We demonstrate that due to light scattering by metal particles, the plasmonic-ARC layer is more effective than TiO _x layer without Ag nanoparticles. Implementation of nanoparticles on bare cell surface was carried out too. The influence of the plasmonic structures on the silicon solar cells parameters is presented as well. We announce about 5 % additional growth in short circuit current for cells with nanoparticles.     

Cell–surface interactions involving immobilized magnetite nanoparticles on flat magnetic substrates

A new method to affect cells by cell–surface interaction is introduced. Biocompatible magnetic nanobeads are deposited onto a biocompatible magnetic thin layer. The particles are composed of small magnetite crystals embedded in a matrix which can be functionalized by different molecules, proteins or growth factors. The magnetic interaction between surface and beads prevents endocytosis if the setup is utilized for cell culturing. The force acting between particles and magnetic layer is calculated by a magnetostatic approach. Biocompatibility is ensured by using garnet layers which turned out to be nontoxic and stable under culturing conditions. The garnet thin films exhibit spatially and temporally variable magnetic domain configurations in changing external magnetic fields and depending on their thermal pretreatment. Several patterns and bead deposition methods as well as the cell–surface interactions were analyzed. In some cases the cells show directed growth. Theoretical considerations explaining particular cell behavior on this magnetic material involve calculations of cell growth on elastic substrates and bending of cell membranes.     

Magnetic nanoparticle-based hyperthermia for cancer treatment

Nanotechnology involves the study of nature at a very small scale, searching new properties and applications. The development of this area of knowledge affects greatly both biotechnology and medicine disciplines. The use of materials at the nanoscale, in particular magnetic nanoparticles, is currently a prominent topic in healthcare and life science. Due to their size-tunable physical and chemical properties, magnetic nanoparticles have demonstrated a wide range of applications ranging from medical diagnosis to treatment. Combining a high saturation magnetization with a properly functionalized surface, magnetic nanoparticles are provided with enhanced functionality that allows them to selectively attach to target cells or tissues and play their therapeutic role in them. In particular, iron oxide nanoparticles are being actively investigated to achieve highly efficient carcinogenic cell destruction through magnetic hyperthermia treatments. Hyperthermia in different approaches has been used combined with radiotherapy during the last decades, however, serious harmful secondary effects have been found in healthy tissues to be associated with these treatments. In this framework, nanotechnology provides a novel and original solution with magnetic hyperthermia, which is based on the use of magnetic nanoparticles to remotely induce local heat when a radiofrequency magnetic field is applied, provoking a temperature increase in those tissues and organs where the tumoral cells are present. Therefore, one important factor that determines the efficiency of this technique is the ability of magnetic nanoparticles to be driven and accumulated in the desired area inside the body. With this aim, magnetic nanoparticles must be strategically surface functionalized to selectively target the injured cells and tissues.     

Development of a cell surface display system in a magnetotactic bacterium, "Magnetospirillum magneticum" AMB-1

Bacterial cell surface display is a widely used technology for bioadsorption and for the development of a variety of screening systems. Magnetotactic bacteria are unique species of bacteria due to the presence of magnetic nanoparticles within them. These intracellular, nanosized (50 to 100 nm) magnetic nanoparticles enable the cells to migrate and be manipulated by magnetic force. In this work, using this unique characteristic and based on whole-genomic and comprehensive proteomic analyses of these bacteria, a cell surface display system has been developed by expressing hexahistidine residues within the outer coiled loop of the membrane-specific protein (Msp1) of the "Magnetospirillum magneticum" (proposed name) AMB-1 bacterium. The optimal display site of the hexahistidine residues was successfully identified via secondary structure prediction, immunofluorescence microscopy, and heavy metal binding assay. The established AMB-1 transformant showed high immunofluorescence response, high Cd(2+) binding, and high recovery efficiency in comparison to those of the negative control when manipulated by magnetic force.     

Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.     

Modeling cell apoptosis for simulating three-dimensional multicellular morphogenesis based on a reversible network reconnection framework

Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.