Growth factor choice is critical for successful functionalization of nanoparticles.

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.

Labeling and Tracking of Human Pancreatic Islets Using Carbon Nanotubes.

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.

Evaluation of In-Situ Magnetic Signals from Iron Oxide Nanoparticle-Labeled PC12 Cells by Atomic Force Microscopy.

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.

The effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake.

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.

The orientation of the neuronal growth process can be directed via magnetic nanoparticles under an applied magnetic field.

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields (Ms). The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. In a neuron-like cell line, we have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction. From the clinical editor: This study validates that non-invasive approaches for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields are possible. The hypothesis was confirmed by observing preferential neurite outgrowth in a cell culture system along the direction imposed by an external magnetic field.

Generation of magnetized olfactory ensheathing cells for regenerative studies in the central and peripheral nervous tissue.

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.

Neuronal cells loaded with PEI-coated Fe3O4 nanoparticles for magnetically guided nerve regeneration.

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.

Poly-l-lysine-coated magnetic nanoparticles as intracellular actuators for neural guidance.

PURPOSE: It has been proposed in the literature that Fe(3)O(4) magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons. MNPs could create mechanical tension that stimulates the growth and elongation of axons. Particles suitable for this purpose should possess (1) high saturation magnetization, (2) a negligible cytotoxic profile, and (3) a high capacity to magnetize mammalian cells. Unfortunately, the materials currently available on the market do not satisfy these criteria; therefore, this work attempts to overcome these deficiencies. METHODS: Magnetite particles were synthesized by an oxidative hydrolysis method and characterized based on their external morphology and size distribution (high-resolution transmission electron microscopy [HR-TEM]) as well as their colloidal (Z potential) and magnetic properties (Superconducting QUantum Interference Devices [SQUID]). Cell viability was assessed via Trypan blue dye exclusion assay, cell doubling time, and MTT cell proliferation assay and reactive oxygen species production. Particle uptake was monitored via Prussian blue staining, intracellular iron content quantification via a ferrozine-based assay, and direct visualization by dual-beam (focused ion beam/scanning electron microscopy [FIB/SEM]) analysis. Experiments were performed on human neuroblastoma SH-SY5Y cell line and primary Schwann cell cultures of the peripheral nervous system. RESULTS: This paper reports on the synthesis and characterization of polymer-coated magnetic Fe(3)O(4) nanoparticles with an average diameter of 73 ± 6 nm that are designed as magnetic actuators for neural guidance. The cells were able to incorporate quantities of iron up to 2 pg/cell. The intracellular distribution of MNPs obtained by optical and electronic microscopy showed large structures of MNPs crossing the cell membrane into the cytoplasm, thus rendering them suitable for magnetic manipulation by external magnetic fields. Specifically, migration experiments under external magnetic fields confirmed that these MNPs can effectively actuate the cells, thus inducing measurable migration towards predefined directions more effectively than commercial nanoparticles (fluidMAG-ARA supplied by Chemicell). There were no observable toxic effects from MNPs on cell viability for working concentrations of 10 µg/mL (EC(25) of 20.8 µg/mL, compared to 12 µg/mL in fluidMAG-ARA). Cell proliferation assays performed with primary cell cultures of the peripheral nervous system confirmed moderate cytotoxicity (EC(25) of 10.35 µg/mL). CONCLUSION: These results indicate that loading neural cells with the proposed MNPs is likely to be an effective strategy for promoting non-invasive neural regeneration through cell magnetic actuation.

Carbon nanotube-mediated wireless cell permeabilization: drug and gene uptake.

AIM: This work aims to exploit the 'antenna' properties of multiwalled carbon nanotubes (MWCNTs). They can be used to induce cell permeabilization in order to transfer drugs (normally impermeable to cell membranes) both in in vitro and in vivo models. MATERIAL & METHODS: The performance of the MWCNTs as receiver antenna was modeled by finite element modeling. Once the appropriate field has been identified, the antenna properties of MWCNTs were investigated in sequential experiments involving immortalized fibroblast cell line (drug model: doxorubicin chemotherapeutic agent) and living mice (drug model: bcl-2 antiapoptotic gene) following stereotactic injection in the cerebral motor cortex. RESULTS: Finite element modeling analysis predicts that our MWCNTs irradiated in the radiofrequency field resemble thin-wire dipole antennas. In vitro experiments confirmed that combination of MWCNTs and electromagnetic field treatment dramatically favors intracellular drug uptake and, most importantly, drug nuclear localization. Finally, the brain of each irradiated animal exhibits a significantly higher number of transfected cells compared with the appropriate controls. CONCLUSION: This wireless application has the potential for MWCNT-based intracellular drug delivery and electro-stimulation therapies.

Zinc oxide nanoparticles as selective killers of proliferating 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.

Physicochemical properties affecting cellular uptake of carbon nanotubes.

Carbon nanotubes (CNTs) are widely used for biomedical applications as intracellular transporters of biomolecules owing to their ability to cross cell membranes. In this article, we survey the reported literature and results of our published work in an attempt to provide a rational view of the various CNT internalization mechanisms. Essentially three uptake mechanisms (phagocytosis, diffusion and endocytosis) have been reported in the literature. In addressing the subject of cellular internalization of CNTs, the unique physicochemical characteristics of CNTs that influence and drive the cell uptake pathway are considered. According to available evidence, the degree of dispersion, the formation of supramolecular complexes and the nanotube length are crucial factors in determining the exact mechanism of cellular uptake. In conclusion, phagocytosis appears to be the internalization pathway for CNT aggregates, bundles, cluster or single dispersed nanotubes 1 microm or more in length; endocytosis is the internalization mechanism for nanotubes forming supramolecular structures; and diffusion is the internalization mechanism for submicron CNTs that do not form supramolecular complexes. This information may be relevant to the rational design of CNT-based carriers for cell therapy.

A bi-modal approach against cancer: magnetic alginate nanoparticles for combined chemotherapy and hyperthermia.

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles has attracted considerable interest in the medical field. In this paper, we propose an innovative nanotechnological platform for cancer therapy, based on highly magnetized, biodegradable, and biocompatible polymeric nanoparticles. Alginate magnetic nanoparticles were prepared by our group by an efficient emulsion/reticulation technique and tested as drug delivery system. Here, we present a potential application that combines, in a single nanovector, efficient targeting, overcoming of bio-barriers, drug delivery, and physical disruption of tumor tissues.

Combination of polymer technology and carbon nanotube array for the development of an effective drug delivery system at cellular level.

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.

Luminoterapia domiciliaria en el tratamiento de la hiperbilirrubinemia neonatal / Home phototherapy in the treatment of neonatal hyperbilirubinemia

Describir una experiencia de tratamiento de la hiperbilirrubinemia neonatal con luminotrapia realizada en el domicilio de los pacientes. Material y Metodos: Entre los meses de Marzo de 1991 y Diciembre de 1994, 63 recién nacidos huperbilirrubinémicos fueron tratados con luminoterapia domiciliaria. Previamente se fijaron criterios de admisión reltivos a los pacientes y a sus familias. Para el tratamiento se utilizaron unidades de fototerapia Medix de luz halógena de 16 w/cm. 2/nm con producción de 480 nm de longitud de onda, colocadas a una distancia de 50 cm. del paciente. Los pacientes fueron controlados por un equipo conformado por los autores y el pediatra de cabecera. Resultados: El tratamiento fue exitoso en todos los pacientes, disminuyendo sus niveles de vilirrubina con un descenso diario pedio de 1.46 mg/dL. Ningún paciente presentó problemas, complicaciones o interferencias durante el tratamiento, ni requirió reinternación por su hiperbilirrubinemia u otra patología. Todos los pacientes mantuvieron su alimentación al pecho. El costo del tratamiento fue cubierto totalmente por seguros de medicina pre-paga u obras sociales en todos los pacientes que estaban adheridos a algunos de estos seguros. Hubo un alto grado de satisfacción familiar con el procedimiento. Conclusiones: la luminoterapia domiciliaria es un procedimiento seguro, que evita los riesgos inherentes a la prolongación de la internación del neonato en instituciones, preserva el vínculo madre/hijo y protege la alimentación al pecho, permitiendo a la familia participar activamente en el cuidado de su hijo.