Carbodiimide Conjugation of Latent Transforming Growth Factor ß1 to Superparamagnetic Iron Oxide Nanoparticles for Remote Activation.

Conjugation of latent growth factors to superparamagnetic iron oxide nanoparticles (SPIONs) is potentially useful for magnetically triggered release of bioactive macromolecules. Thus, the goal of this work was to trigger the release of active Transforming Growth-Factor Beta (TGF-ß) via magnetic hyperthermia by binding SPIONs to the latent form of TGF-ß, since heat has been shown to induce release of TGF-ß from the latent complex. Commercially available SPIONS with high specific absorption rates (SAR) were hydrolyzed in 70% ethanol to create surface carboxylic acid conjugation sites for carbodiimide chemistry. Fourier-Transform Infra-Red (FTIR) analysis verified the conversion of maleic anhydride to maleic acid. 1-Ethyl-2-(3-dimethyulaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were used to bind to the open conjugation sites of the SPION in order to graft latent TGF-ß onto the particles. The resulting conjugated particles were imaged with transmission electron microscopy (TEM), and the complexed particles were characterized by dynamic light scattering (DLS) and superconducting quantum interference device (SQUID) magnetometry. Enzyme-linked immunosorbent assay (ELISA) was used to assess the thermally triggered release of active TGF-ß from the latent complex, demonstrating that conjugation did not interfere with release. Results showed that latent TGF-ß was successfully conjugated to the iron oxide nanoparticles, and magnetically triggered release of active TGF-ß was achieved.

Design and characterization of a magnetite/PEI multifunctional nanohybrid as non-viral vector and cell isolation system.

It is described the reproducible formulation and complete physicochemical characterization of nanohybrids based on magnetite (Fe3O4) cores embedded within a polyethylenimine (PEI) matrix. Particle size, surface electrical charge, X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) analyses, and magnetic field-responsive behaviour characterizations defined that the 4:3 (Fe3O4:PEI) weight proportion led to the best production performances of magnetically responsive nanocomposites in which the magnetic nuclei are completely covered by the polymeric shell. Agarose gel electrophoresis assays demonstrated the capacity of the Fe3O4/PEI particles to condense, release, and protect the DNA against enzymatic degradation. In vitro assays were performed to evaluate the transfection efficiency (up to 4.5% of transfected HEK-293 cells at a 10/1 PEI/DNA ratio), iron absorption by D1-mesenchymal stem cells (D1-MSCs, high values after only 15min of magnetic incubation), influence on metabolic activity (negligible effect up to 44µg nanocomposites/105 cells), and cell isolation capacity of the core/shell particles (significant increase in the retention of D1-MSCs transduced with green fluorescent protein). The Fe3O4/PEI nanohybrids hold promising characteristics suggestive of their capacity for transfection and cell isolation applications.

Redox cycling of iron by Abeta42.

The amyloid cascade hypothesis and oxidative damage have been inextricably linked in the neurodegeneration that is characteristic of Alzheimer's disease. We have investigated this link and sought to suggest a mechanism whereby the precipitation of Abeta42 might contribute to the redox cycling of iron and hence the generation of reactive oxygen species via Fenton-like chemistry. We have shown that the critical step in the auto-oxidation of Fe(II) under the near-physiological conditions of our study involved the generation of H2O2 via O2.- and that Abeta42 influenced Fenton chemistry through aggregation state-specific binding of both Fe(II) and Fe(III). The net result of these interactions was the delayed precipitation of kinetically redox-inactive Fe(OH)3(s) such that Fe(II)/Fe(III) were cycled in redox-active forms over a substantially longer time period than if peptide had been absent from preparations. The addition of physiologically significant concentrations of either Cu(II) or Zn(II) reduced the role played by Abeta42 in the Fe(II)/Fe(III) redox cycle whereas a pathophysiologically significant concentration of Al(III) potentiated the redox cycle in favour of Fe(II) whether or not Cu(II) or Zn(II) was additionally present. The results support the notion that oxidative damage in the immediate vicinity of, for example, senile plaques, may be the result of Fenton chemistry catalysed by the codeposition of Abeta42 with metals such as Fe(II)/Fe(III) and Al(III).

Hyperpolarization of human mesenchymal stem cells in response to magnetic force.

Magnetic particle tagging techniques are currently being applied to tissue engineering applications such as controlled differentiation of mesenchymal stem cells (MSC). In order to define key mechanotransducers underpinning these applications, the electrophysiological responses of human MSCs (hMSC) have been investigated. Ferromagnetic microparticles were coated with L-arginyl-glycyl-L-aspartic acid in order to target the application of dynamic force (6 pN) directly to cell surface integrins. Human MSCs demonstrated cell membrane hyperpolarization responses after the application of force, mediated by BK channels and intracellular calcium release.

Controlled differentiation of human bone marrow stromal cells using magnetic nanoparticle technology.

Targeting and differentiating stem cells at sites of injury and repair is an exciting and promising area for disease treatment and reparative medicine. We have investigated remote magnetic field activation of magnetic nanoparticle-tagged mechanosensitive receptors on the cell membrane of human bone marrow stromal cells (HBMSCs) for use in osteoprogenitor cell delivery systems and activation of differentiation in vitro and in vivo toward an osteochondral lineage. HBMSC-labeled with magnetic beads coated with antibodies or peptides to the transmembrane ion channel stretch activated potassium channel (TREK-1) or arginine–glycine–aspartic acid were cultured in monolayer or encapsulated into polysaccharide alginate/chitosan microcapsules. Upregulation in gene expression was measured in magnetic particle-labeled HBMSCs in response to TREK-1 activation over a short period (7 days) with an increase in mRNA levels of Sox9, core binding factor alpha1 (Cbfa1), and osteopontin. Magnetic particle-labeled HBMSCs encapsulated into alginate chitosan capsules were exposed to magnetic forces both in vitro and in vivo intermittently for 21 days. After 21 days the encapsulated, magnetic particle-labeled HBMSCs in vivo were viable as evidenced by extensive cell tracker green fluorescence. The mechanical stimulation of HBMSCs labeled with TREK-1 magnetic nanoparticle receptors enhanced expression of type-1 collagen in vitro with increases in proteoglycan matrix, core binding factor alpha1 (Cbfa1) and collagen synthesis, and extracellular matrix production and elevated the expression of type-1 and type-2 collagen in vivo. Additionally, the magnetically remote stimulation of HBMSCs labeled with magnetic nanoparticle arginine–glycine–aspartic acid considerably enhanced proteoglycan and collagen synthesis and extracellular matrix production and elevated the expression of type-1 and type-2 collagen in vivo and in vitro. Osteogenic mechanosensitive receptor manipulation by magnetic nanotechnology can induce the differentiation of osteoprogenitor cell populations toward an osteogenic lineage. These cell manipulation strategies offer tremendous therapeutic opportunities in soft and hard tissue repair.

Expression of the mechanosensitive 2PK+ channel TREK-1 in human osteoblasts.

TREK-1 is a mechanosensitive member of the two-pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK-1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT-PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK-1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium "background leak" current that shares many similarities to TREK-1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK-1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK-1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK-1 in mechanotransduction, leading to bone remodelling.