Glycolaldehyde and maleyl conjugated human serum albumin as potential macrophage-targeting carriers for molecular imaging purposes

Abstract

Maleylated bovine serum albumin is a known ligand for targeting macrophages and has potential as a carrier for molecular imaging purposes. We present a novel synthesis of glycolaldehyde-conjugated human serum albumin (GA-HSA) and maleylated human serum albumin (Mal-HSA). Seventeen modifications of fluorescently tagged GA-HSA and Mal-HSA molecules with different degrees of conjugation were prepared. The comparative uptake studies, using 12 of these modifications, were done in vitro on mouse monocytes/macrophages (RAW264.7), and evaluated qualitatively by confocal microscopy and quantitatively by flow cytometry. The GA modifications are taken up by the macrophages approximately 40% better than the maleyl modifications at low concentrations (≤ 3 μM), while at higher concentrations it appears that the maleyl modifications are taken up around 25-44% better than the GA-modified HSA. However, high uptake at low concentrations will be beneficial for in vivo localizing inflammation in areas with low penetration of the probe as in an atherosclerotic plaque. Further, another advantage of GA-HSA is that GA competes less than the maleyl group for the free reactive amine sites that are to be used for conjugation of metal chelating ligands (e.g. tetraazacyclododecanetetraacetic acid and triazacyclononanetriacetic acid). Metal ions such as Gd(3+) and Mn(2+) can be chelated for positive Magnetic Resonance (MR) contrast and positron emitting ions such as (64) Cu(2+) and (68) Ga(3+) for Positron Emission Tomography (PET) imaging. These are important properties, especially, when considering the MR contrast possibilities owing to the low sensitivity of the technique, and would motivate the use of GA-HSA before Mal-HSA.

Keywords: MRI; PET; atherosclerosis; cellular targeting; contrast agent; fluorescent probes; glycolaldehyde; human serum albumin; molecular imaging.

Figure 1

Confocal microscopy images of RAW264.7 cells at 189× magnification showing (a) 10 μm GA₉-HSA-FITC, (b) 50 μm GA₁₆-HSA-FITC, (c) 50 μm Mal₄₁-HSA-FITC and (d) 50 μm Mal₅₄-HSA-FITC incubated for 15 min at 37 °C. The green colour represents the FITC-labelled compound and the blue colour represents the 4′,6-diamidine-2′-phenylindole, dihydrochloride (DAPI) nucleus stain.

Figure 2

Diagram, obtained from the flow cytometry analysis, showing the cellular uptake as median fluorescence intensity of HSA-fluorescein isothiocyanate (FITC), maleylated human serum albumin (Mal-HSA)-FITC, glycolaldehyde conjugated human serum albumin (GA-HSA)-FITC and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose as a function of concentration. Incubations on RAW264.7-cells were performed for 15 min at 37 °C.

Figure 3

Diagram, obtained from the flow cytometry analysis, showing the simultaneous, competitive cellular uptake as median fluorescence intensity of Mal-HSA-FITC and GA-HSA-Pacific Blue™ succinimidyl ester as a function of concentration. Incubations on RAW264.7 cells were performed for 15 min at 37 °C.