Facilitated Monocyte-Macrophage Uptake and Tissue Distribution of Superparmagnetic Iron-Oxide Nanoparticles

BACKGROUND: We posit that the same mononuclear phagocytes (MP) that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP) and tissue delivery. METHODS: Monocytes and monocyte-derived macrophages (MDM) were used as vehicles of superparamagnetic iron oxide (SPIO) NP and immunoglobulin (IgG) or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T(2) measures using magnetic resonance imaging (MRI) were used to monitor tissue distribution in animals. RESULTS: Oxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab')(2) fragments and for Alexa Fluor(R) 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab')(2), and/or Alexa Fluor(R) 488 SPIO demonstrated approximately 50% coupling yield. IgG-SPIO was found stable at 4 degrees C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI. CONCLUSIONS: Facilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy

Cl(−) channels are expressed in human normal monocytes: a functional role in migration, adhesion and volume change

Increased adhesion and diapedesis of monocytes appear to be primary initiating factors in the pathophysiology of occlusive vascular diseases, including atherosclerosis and restenosis. However, the underlying mechanisms of transendothelial migration and invasion of monocytes into the blood vessels are not known. Alterations in ion channels on the cell membrane are generally involved in induced changes in shape and volume. In the present study, we investigated the expression and functional role of chloride channels in freshly isolated human blood monocytes. The Cl(−) currents in whole-cells were measured by the patch-clamp technique. We observed whole cell Cl(−) currents, which were time-independent and outwardly rectifying. The chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy]acetic acid 94 (IAA94) attenuated the Cl(−) currents. NPPB and IAA94 also inhibited chemotaxis of monocytes, as measured in Boyden chemotactic chambers, with the same sensitivity. NPPB but not IAA94, increased the cell volume as measured by shape change, and decreased tumour necrosis factor (TNF)-α-induced monocyte adhesion to endothelial cells. These results suggest that monocytes contain Cl(−) channels which regulate transendothelial migration of monocytes, due presumably to an alteration in cell volume

A novel function for SNAP29 (synaptosomal-associated protein of 29 kDa) in mast cell phagocytosis.

Mast cells play a critical role in the innate immune response to bacterial infection. They internalize and kill a variety of bacteria and process antigen for presentation to T cells via MHC molecules. Although mast cell phagocytosis appears to play a significant role during bacterial infection, little is known about the proteins involved in its regulation. In this study, we demonstrate that the SNARE protein SNAP29 is involved in mast cell phagocytosis. SNAP29 is localized in the endocytic pathway and is transiently recruited to Escherichia coli (E. coli)-containing phagosomes. Interestingly, overexpression of SNAP29 significantly increases the internalization and killing of E. coli, while it does not affect mast cell exocytosis of inflammatory mediators. To our knowledge, these data are the first to demonstrate a novel function of SNAP29 in mast cell phagocytosis and have implications in protection against bacterial infection

Membrane dynamics and organelle biogenesis—lipid pipelines and vesicular carriers

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth