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Abstract In this article, a carbon nanotube (CNT) array-based system combined with a polymer thin film is proposed as an effective drug release device directly at cellular level. The polymeric film embedded in the CNT array is described and characterized in terms of release kinetics, while in vitro assays on PC12 cell line have been performed in order to assess the efficiency and functionality of the entrapped agent (neural growth factor, NGF). PC12 cell differentiation, following incubation on the CNT array embedding the alginate delivery film, demonstrated the effectiveness of the proposed solution. The achieved results indicate that polymeric technology could be efficiently embedded in CNT array acting as drug delivery system at cellular level. The implication of this study opens several perspectives in particular in the field of neurointerfaces, combining several functions into a single platform.

Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed. [ABSTRACT FROM AUTHOR]

We report a one-step synthesis protocol for obtaining polymer-coated magnetic nanoparticles (MNPs) engineered for uploading neural cells. Polyethyleneimine-coated Fe3O4 nanoparticles (PEI-MNPs) with sizes of 25 ± 5 nm were prepared by oxidation of Fe(OH)2 by nitrate in basic aqueous media and adding PEI in situ during synthesis. The obtained PEI-MNP cores displayed a neat octahedral morphology and high crystallinity. The resulting nanoparticles were coated with a thin polymer layer of about 0.7–0.9 nm, and displayed a saturation magnetization value MS = 58 A m2 kg−1 at 250 K (64 A m2 kg−1 for T = 10 K). Cell uptake experiments on a neuroblastoma-derived SH-SY5Y cell line were undertaken over a wide time and MNP concentration range. The results showed a small decrease in cell viability for 24 h incubation (down to 70% viability for 100 μg ml−1), increasing the toxic effects with incubation time (30% cell survival at 100 μg ml−1 for 7 days of incubation). On the other hand, primary neuronal cells displayed higher sensitivity to PEI-MNPs, with a cell viability reduction of 44% of the control cells after 3 days of incubation with 50 μg ml−1. The amount of PEI-MNPs uploaded by SH-SY5Y cells was found to have a linear dependence on concentration. The intracellular distribution of the PEI-MNPs analyzed at the single-cell level by the dual-beam (FIB/SEM) technique revealed the coexistence of both fully incorporated PEI-MNPs and partially internalized PEI-MNP-clusters crossing the cell membrane. The resulting MNP-cluster distributions open the possibility of using these PEI-MNPs for magnetically driven axonal re-growth in neural cells. [ABSTRACT FROM AUTHOR]

As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries. [ABSTRACT FROM AUTHOR]

Abstract: Nanoparticles engineered for biomedical applications are meant to be in contact with protein-rich physiological fluids. These proteins are usually adsorbed onto the nanoparticle's surface, forming a swaddling layer that has been described as a ‘protein corona’, the nature of which is expected to influence not only the physicochemical properties of the particles but also the internalization into a given cell type. We have investigated the process of protein adsorption onto different magnetic nanoparticles (MNPs) when immersed in cell culture medium, and how these changes affect the cellular uptake. The role of the MNPs surface charge has been assessed by synthesizing two colloids with the same hydrodynamic size and opposite surface charge: magnetite (Fe3O4) cores of 25–30 nm were in situ functionalized with (a) positive polyethyleneimine (PEI-MNPs) and (b) negative poly(acrylic acid) (PAA-MNPs). After few minutes of incubation in cell culture medium the wrapping of the MNPs by protein adsorption resulted in a 5-fold increase of the hydrodynamic size. After 24 h of incubation large MNP-protein aggregates with hydrodynamic sizes of ≈1500 nm (PAA-MNPs) and ≈3000 nm (PEI-MNPs) were observed, each one containing an estimated number of magnetic cores between 450 and 1000. These results are consistent with the formation of large protein-MNPs aggregate units having a ‘plum pudding’ structure of MNPs embedded into a protein network that results in a negative surface charge, irrespective of the MNP-core charge. In spite of the similar negative ζ-potential for both MNPs within cell culture, we demonstrated that PEI-MNPs are incorporated in much larger amounts than the PAA-MNPs units. Quantitative analysis showed that SH-SY5Y cells can incorporate 100% of the added PEI-MNPs up to ≈100 pg/cell, whereas for PAA-MNPs the uptake was less than 50%. The final cellular distribution showed also notable differences regarding partial attachment to the cell membrane. These results highlight the need to characterize the final properties of MNPs after protein adsorption in biological media, and demonstrate the impact of these properties on the internalization mechanisms in neural cells. [Copyright &y& Elsevier]

Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies.

The magnetic signals from magnetite nanoparticle-labeled PC12 cells were assessed by magnetic force microscopy by deploying a localized external magnetic field to magnetize the nanoparticles and the magnetic tip simultaneously so that the interaction between the tip and PC12 cell-associated Fe3O4 nanoparticles could be detected at lift heights (the distance between the tip and the sample) larger than 100 nm. The use of large lift heights during the raster scanning of the probe eliminates the non-magnetic interference from the complex and rugged cell surface and yet maintains the sufficient sensitivity for magnetic detection. The magnetic signals of the cell-bound nanoparticles were semi-quantified by analyzing cell surface roughness upon three-dimensional reconstruction generated by the phase shift of the cantilever oscillation. The obtained data can be used for the evaluation of the overall cellular magnetization as well as the maximum magnetic forces from magnetic nanoparticle-labeled cells which is crucial for the biomedical application of these nanomaterials.

We report a one-step synthesis protocol for obtaining polymer-coated magnetic nanoparticles (MNPs) engineered for uploading neural cells. Polyethyleneimine-coated Fe 3 O 4 nanoparticles (PEI-MNPs) with sizes of 25 ± 5 nm were prepared by oxidation of Fe(OH) 2 by nitrate in basic aqueous media and adding PEI in situ during synthesis. The obtained PEI-MNP cores displayed a neat octahedral morphology and high crystallinity. The resulting nanoparticles were coated with a thin polymer layer of about 0.7-0.9 nm, and displayed a saturation magnetization value M S = 58 A m 2 kg -1 at 250 K (64 A m 2 kg -1 for T = 10 K). Cell uptake experiments on a neuroblastoma-derived SH-SY5Y cell line were undertaken over a wide time and MNP concentration range. The results showed a small decrease in cell viability for 24 h incubation (down to 70% viability for 100 μg ml -1 ), increasing the toxic effects with incubation time (30% cell survival at 100 μg ml -1 for 7 days of incubation). On the other hand, primary neuronal cells displayed higher sensitivity to PEI-MNPs, with a cell viability reduction of 44% of the control cells after 3 days of incubation with 50 μg ml -1 . The amount of PEI-MNPs uploaded by SH-SY5Y cells was found to have a linear dependence on concentration. The intracellular distribution of the PEI-MNPs analyzed at the single-cell level by the dual-beam (FIB/SEM) technique revealed the coexistence of both fully incorporated PEI-MNPs and partially internalized PEI-MNP-clusters crossing the cell membrane. The resulting MNP-cluster distributions open the possibility of using these PEI-MNPs for magnetically driven axonal re-growth in neural cells.

Background: It has recently been demonstrated that zinc oxide nanoparticles (ZnO NPs) induce death of cancerous cells whilst having no cytotoxic effect on normal cells. However, there are several issues which need to be resolved before translation of zinc oxide nanoparticles into medical use, including lack of suitable biocompatible dispersion protocols and a better understanding being needed of the mechanism of their selective cytotoxic action., Methods: Nanoparticle dose affecting cell viability was evaluated in a model of proliferating cells both experimentally and mathematically. The key issue of selective toxicity of ZnO NPs toward proliferating cells was addressed by experiments using a biological model of noncancerous cells, ie, mesenchymal stem cells before and after cell differentiation to the osteogenic lineage., Results: In this paper, we report a biocompatible protocol for preparation of stable aqueous solutions of monodispersed zinc oxide nanoparticles. We found that the threshold of intracellular ZnO NP concentration required to induce cell death in proliferating cells is 0.4 ± 0.02 mM. Finally, flow cytometry analysis revealed that the threshold dose of zinc oxide nanoparticles was lethal to proliferating pluripotent mesenchymal stem cells but exhibited negligible cytotoxic effects to osteogenically differentiated mesenchymal stem cells., Conclusion: Results confirm the ZnO NP selective cytotoxic action on rapidly proliferating cells, whether benign or malignant.