Background Inflammatory processes play a role in vascular disease. Expression of cellular adhesion molecules such as CD44 is up-regulated in inflamed endothelium and therefore may promote atherosclerosis. This may provide a target for delivery of therapeutics specific to reducing or disrupting inflammation. Nonionic surfactant vesicles (niosomes) are potential stable, low-cost drug delivery vehicles that have been shown in other contexts to be useful in lowering systemic doses of drug for efficacy. We addressed the hypothesis that niosomes could be synthesized that could specifically target the CD44 molecule.
Methods Niosomes containing fluorescent green carboxyrhodamine dye were created using a combination of modified surfactants. Through a novel process we developed, we conjugated these to anti-CD44 (IM7) monoclonal antibodies to form “immunoniosomes.” These were incubated with bovine aortic endothelial cells (BAECs) activated with TNF-α to express CD44. To verify and quantify binding, phase contrast and fluorescent images of postincubated cells were captured. Cells were DAPI blue stained to visualize the nuclei. The number of immunoniosomes bound to cells was determined through a custom-made image analysis program. Binding density was defined as a ratio of the number of immunoniosomes per cell nucleus. Controls included cell incubation with unconjugated niosomes and BAECs preincubated with free IM7 to block binding sites.
Results A subgroup of BAECs underwent immunohistochemical staining, which verified expression of CD44 on the cell surface. Comparison of experiments with immunoniosomes versus controls revealed good selectivity and specificity. For a 1-hour incubation of immunoniosomes at a concentration of 3.0 × 107#/mL and a cell density of 2.20 × 105 cells/cm2, binding density was 2.29 ± 0.26 per cell.
Conclusions We have confirmed the capacity to develop monoclonal antibody conjugated niosomes targeted to specific cell receptors, namely CD44. Further exploration of the capacity of vesicle binding and subsequent uptake in endothelial cells of the immunoniosomes to test the potential to target inflammatory disease is warranted.
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