Biological Impact of γ-Fe2O3 Magnetic Nanoparticles Obtained by Laser Target Evaporation: Focus on Magnetic Biosensor Applications.

The biological activity of γ-Fe2O3 magnetic nanoparticles (MNPs), obtained by the laser target evaporation technique, was studied, with a focus on their possible use in biosensor applications. The biological effect of the MNPs was investigated in vitro on the primary cultures of human dermal fibroblasts. The effects of the MNPs contained in culture medium or MNPs already uptaken by cells were evaluated for the cases of the fibroblast's proliferation and secretion of cytokines and collagen. For the tests related to the contribution of the constant magnetic field to the biological activity of MNPs, a magnetic system for the creation of the external magnetic field (having no commercial analogues) was designed, calibrated, and used. It was adapted to the size of standard 24-well cell culture plates. At low concentrations of MNPs, uptake by fibroblasts had stimulated their proliferation. Extracellular MNPs stimulated the release of pro-inflammatory cytokines (Interleukin-6 (IL-6) and Interleukin-8 (IL-8) or chemokine (C-X-C motif) ligand 8 (CXCL8)) in a concentration-dependent manner. However, the presence of MNPs did not increase the collagen secretion. The exposure to the uniform constant magnetic field (H ≈ 630 or 320 Oe), oriented in the plane of the well, did not cause considerable changes in fibroblasts proliferation and secretion, regardless of presence of MNPs. Statistically significant differences were detected only in the levels of IL-8/CXCL8 release.

Magnetic Properties of Iron Oxide Nanoparticles Do Not Essentially Contribute to Ferrogel Biocompatibility.

Two series of composite polyacrylamide (PAAm) gels with embedded superparamagnetic Fe2O3 or diamagnetic Al2O3 nanoparticles were synthesized, aiming to study the direct contribution of the magnetic interactions to the ferrogel biocompatibility. The proliferative activity was estimated for the case of human dermal fibroblast culture grown onto the surfaces of these types of substrates. Spherical non-agglomerated nanoparticles (NPs) of 20-40 nm in diameter were prepared by laser target evaporation (LTE) electrophysical technique. The concentration of the NPs in gel was fixed at 0.0, 0.3, 0.6, or 1.2 wt.%. Mechanical, electrical, and magnetic properties of composite gels were characterized by the dependence of Young's modulus, electrical potential, magnetization measurements on the content of embedded NPs. The fibroblast monolayer density grown onto the surface of composite substrates was considered as an indicator of the material biocompatibility after 96 h of incubation. Regardless of the superparamagnetic or diamagnetic nature of nanoparticles, the increase in their concentration in the PAAm composite provided a parallel increase in the cell culture proliferation when grown onto the surface of composite substrates. The effects of cell interaction with the nanostructured surface of composites are discussed in order to explain the results.

Effects of Constant Magnetic Field to the Proliferation Rate of Human Fibroblasts Grown onto Different Substrates: Tissue Culture Polystyrene, Polyacrylamide Hydrogel and Ferrogels γ-Fe2O3 Magnetic Nanoparticles.

The static magnetic field was shown to affect the proliferation, adhesion and differentiation of various types of cells, making it a helpful tool for regenerative medicine, though the mechanism of its impact on cells is not completely understood. In this work, we have designed and tested a magnetic system consisting of an equidistant set of the similar commercial permanent magnets (6 × 4 assay) in order to get insight on the potential of its experimental usage in the biological studies with cells culturing in a magnetic field. Human dermal fibroblasts, which are widely applied in regenerative medicine, were used for the comparative study of their proliferation rate on tissue culture polystyrene (TCPS) and on the polyacrylamide ferrogels with 0.00, 0.63 and 1.19 wt % concentrations of γ-Fe2O3 magnetic nanoparticles obtained by the well-established technique of laser target evaporation. We used either the same batch as in previously performed but different biological experiments or the same fabrication conditions for fabrication of the nanoparticles. This adds special value to the understanding of the mechanisms of nanoparticles contributions to the processes occurring in the living systems in their presence. The magnetic field increased human dermal fibroblast cell proliferation rate on TCPS, but, at the same time, it suppressed the growth of fibroblasts on blank gel and on polyacrylamide ferrogels. However, the proliferation rate of cells on ferrogels positively correlated with the concentration of nanoparticles. Such a dependence was observed both for cell proliferation without the application of the magnetic field and under the exposure to the constant magnetic field.

The Contribution of Magnetic Nanoparticles to Ferrogel Biophysical Properties.

Iron oxide γ-Fe2O3 magnetic nanoparticles (MNPs) were fabricated by laser target evaporation technique (LTE) and their structure and magnetic properties were studied. Polyacrylamide (PAAm) gels with different cross-linking density of the polymer network and polyacrylamide-based ferrogel with embedded LTE MNPs (0.34 wt.%) were synthesized. Their adhesive and proliferative potential with respect to human dermal fibroblasts were studied. At the same value of Young modulus, the adhesive and proliferative activities of the human dermal fibroblasts on the surface of ferrogel were unexpectedly much higher in comparison with the surface of PAAm gel. Properties of PAAm-100 + γ-Fe2O3 MNPs composites were discussed with focus on creation of a new generation of drug delivery systems combined in multifunctional devices, including magnetic field assisted delivery, positioning, and biosensing. Although exact applications are still under development, the obtained results show a high potential of LTE MNPs to be applied for cellular technologies and tissue engineering. PAAm-100 ferrogel with very low concentration of γ-Fe2O3 MNPs results in significant improvement of the cells' compatibility to the gel-based scaffold.

A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells.

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed.

Two clusters of mutations map distinct receptor-binding sites of echovirus 11 for the decay-accelerating factor (CD55) and for canyon-binding receptors.

In this study we present the comparative sequence analysis of the parental haemagglutinating (daf+) and mutant non-haemagglutinating (daf-) clones of echovirus 11 (EV11) isolated from the prototype strain Gregory. The sequence comparison revealed only a single amino acid substitution in the capsid protein VP2 of each mutant clone. These substitutions were located in the area of viral receptor-binding site for DAF. Since daf- mutants of EV11 did not interact with DAF, they used an alternative receptor for the cell entry. To elucidate the nature of the alternative receptor we used subvariant clones of EV11 adapted to human rhabdomyosarcoma (RD), human carcinoma (HEp-2) and African Green monkey kidney (BGM) cell lines. The usage of the subvariant clones with altered host range and the cell cultures of human and simian origin allowed us to map the amino acid substitutions associated with the adaptation of EV11 to the alternative cellular receptors. These amino acid substitutions were located on the surface of the virion in the canyon area. Hence the virus canyon may serve as the receptor-binding site for the alternative (in respect to DAF) cellular receptor(s).