Quantitation of in <i>vivo</i> IgG SPIO and SPIO tracking using CPMG phase cycled T₂ mapping MRI.

<p>Change in relaxivity (R₂ = 1/T₂) in the spleen (A, B), liver (C, D) and kidney (E, F) after injection of SPIO (red) or IgG-SPIO (green) into the tail vein of the mouse. Injections of 12.5 µg (A, C, E) (n = 6) and 62.5 µg (B, D, F) (n = 5) show higher uptake in circulating monocytes with IgG coating at the lower concentration by 0.5 h post-injection and times thereafter (p<0.05 two way repeated measures ANOVA for effect of time and SPIO type for spleen and liver). At the higher 62.5 µg dosage, no differences in uptake of SPIO vs. IgG-SPIO at any time were observed.</p

NP size and molecular weight on SPIO uptake into monocytes and MDM.

<p>Influence of the size of nanoparticles and the molecular weight of dextran on the uptake of SPIO (A). Uptake by human monocytes of IgG-SPIO with different IgG densities (B) and of native SPIO co-incubated with free IgG (C). Uptake in human monocytes of IgG-SPIO incubated at 4°C and 37°C (D) and comparison between human monocytes and MDM (E). Uptake in human monocytes of IgG-SPIO co-incubated with excess of free IgG (F) and of SPIO conjugated to human serum albumin (HSA) instead of IgG (G). Panel H shows the influence of the oxidation and of the covalent attachment of IgG and HSA on the size and the surface charge of SPIO. Error bars are SEM.</p

Manufacture, column elution and characterization of IgG SPIO.

<p>Top: Sepharose 4B-CL elution profile of IgG-SPIO after incubation of IgG with oxidized SPIO (closed circles). Controls were performed with IgG incubated with non-oxidized SPIO (closed squares) as well as oxidized SPIO blocked by addition of an excess of amine groups (open circles). Bottom: Size and polydispersity index measurements of IgG-SPIO at different time points after the conjugation and storage at 4°C.</p

Schematic diagram for IgG SPIO NP cell tissue delivery.

<p>We posit that NP can be efficiently taken up by circulating blood borne monocyte-macrophages in ways that are not toxic to the cell function or mobility. The NP loaded cells are then transported to areas of the lymphoid and reticuloendothelial system (for example, liver and spleen) and secreted through the kidneys.</p

Mechanisms and cell viability for IgG SPIO monocyte uptake.

<p>Human monocytes were incubated for 1 hour at 37°C with SPIO (A) or IgG-SPIO (B) at a concentration of 0.5 mg iron/ml. Iron was stained by Prussian blue technique and cells were counterstained with nuclear fast red. Toxicity of IgG-SPIO was evaluated after 1-hour incubation with human monocytes. Viability of iron-labeled monocytes was assessed using a live/dead assay with calcein-AM and ethidium homodimer-1 (EthD1) (C). Calcein-AM accumulation and cleavage by cytosolic esterase labeled live cells in green and EthD1 labeled dead cells in red. Trypan blue exclusion was used as a dual control (D). Internalization of IgG-SPIO was observed by TEM. IgG-SPIO labeled monocytes were observed at 4,400× (E) and 10,400× (F) magnifications. The arrowheads show the SPIO within the cell cytoplasm.</p

Intracellular iron levels by MRI phantoms after SPIO and IgG-SPIO monocyte uptake.

<p>Internalization kinetics of IgG-SPIO and SPIO at iron concentrations of 0.5 mg/ml were determined in human monocytes incubated at 37°C. (A) Estimation of the iron loading by ferrozine assay (top) and by measurement of the relaxation (R2) of iron-labeled monocytes (bottom).</p

Fluorescently labeled nanoparticles in spleen and liver.

<p>Mice were injected i.v. with 12.5 µg of fluorescently labeled SPIO or IgG that was covalently conjugated to SPIO (SPIO-IgG). Twenty four hours after injection animals were sacrificed and spleen and liver were recovered, flash frozen, then cut into 10 µ frozen sections. Tissue sections were fixed with methanol/acetone for 5 minutes and examined by fluorescence microscopy. Spleen and liver from untreated mice served as controls for nonspecific fluorescence. Germinal centers (yellow arrows) and perifollicular areas (white arrows) are denoted within spleen. Magnification, ×400.</p