Characterization of PECAM Targeting Nanoparticles

Fluorescein isothiocyanate (FITC)-labeled polystyrene spheres (~125 nm effective diameter) were purchased from Polysciences (Warrington, PA). ¹²⁵I-radiolabled poly(4- vinylphenol) nanoparticle spheres (~145 nm effective diameter) were prepared as previously described.^{55 (link),56 (link)} Purified monoclonal antibodies (mAbs) to human PECAM (huPECAM), clone 62 (mouse IgG2a, referred to as Ab_1h), and clone 37 (mouse IgG1, referred to as Ab_2h), were generously provided by Dr. M. Nakada (Centocor, Malvern, PA).^{26 (link)} The antimouse PECAM (muPECAM) monoclonal antibody clones 390 (rat IgG2a, referred to as Ab_1m)^{57 (link)} and MEC13.3 (rat IgG2a, referred to as Ab_2m)^{58 (link)} were purchased from BD Bioscience (Chicago, IL) and BioLegend (San Diego, CA), respectively. Nontargeted IgG Ab was an isotype mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Fluorescent secondary antibody to antihuPECAM were purchased from Invitrogen (Carlsbad, CA). ActinRed 555 reagent was purchased from Life Technologies (Norwalk, CT). Human umbilical vein endothelial cells (HUVECs) endogenously expressing native huPECAM were purchased from Lonza (Walkersville, MD) and maintained in EGM-2 media (Lonza) supplemented with 10% (v/v) fetal bovine serum (FBS).