1. In Vitro and In Vivo Expression of Epidermal Growth Factor Receptor in Cell Lines The expression of EGFR in the U87mg and U251mg cell lines appeared very homogeneous with no detectable differences between the two cell lines. Hence, both cell lines revealed extensive EGFR labeling of the cytoplasm and cellular surfaces without labeling of the nucleus (Figures 1(A) and 1(C)). Substitution of the primary antibody with isotopic nonimmune IgG revealed no immunoreactivity within the cells (Figures 1(B) and 1(D)). Inhibitors,research,lifescience,medical Likewise, no immunoreactivity was observed

when the primary antibody was omitted from the immunoreactions (not shown). When examined in the intracranial xenograft, it was R406 evident that EGFR positive cells were detected in the cells forming a tumor, which contrasted that of neurons and glia of the normal brain tissue (Figures 1(E)–1(G)). When examined

at high magnification, the EGFR-immunoreactive cells exhibited a morphology that corresponded to that of U87mg expressing EGFR in vitro. Inhibitors,research,lifescience,medical In contrast, neurons and glia of the normal brain tissue were devoid of EGFR-immunoreactivity (Figure 1(G)). Figure 1 Representative micrographs showing expression of epidermal growth factor receptor (EGFR) in vitro and in vivo. (A), (C) In vitro expression of EGFR in U87mg (A) and U251mg (C) cell lines using fluorescent antibodies. The cells are labeled … 3.2. Liposome Characterization Fluorescence labeled Inhibitors,research,lifescience,medical liposomes were prepared with anti-EGFR antibodies or isotypic human immunoglobulins coupled with the DSPE-PEG2000-Mal linker. α-hEGFR-ILs were compared to liposomes Inhibitors,research,lifescience,medical conjugated with nonimmune human immunoglobulins and naked liposomes with no antibody conjugation with respect to particle size, polydispersity, and antibody coupling efficiency as illustrated in Table 1. The liposomes were comparable in size and liposomes conjugated with immunoglobulins had similar protein coupling efficiency. Inhibitors,research,lifescience,medical The α-hEGFR-ILs had a mean diameter of 95.2 ± 3nm, whereas liposomes conjugated with nonimmune human immunoglobulins

(hIgG-ILs) had a mean diameter of 119 ± 12nm. The size distribution of all liposomes had a polydispersity index <0.2, indicative of a homogenous size distribution. The charge measured of all liposome preparation was slightly negative (Table 1). Table 1 Characterization of liposomes with respect to particle size, polydispersity, charge, and else protein coupling yield. 3.3. In Vitro Liposomal Targeting in U87mg and U251mg Cell Lines Cellular binding and uptake of the three different DiO-labeled liposomes were evaluated by fluorescent microscopy and flow cytometry in the two cell lines. Liposomes were added at a concentration of 75nmol/105 cells and incubated for two hours at 37°C.The targeting efficiency of α-hEGFR-ILs was considerably higher in both U87mg and U251mg cell lines (Figures 2(A) and 2(I)) compared to that of hIgG-ILs or naked liposomes (Figures 2(D), 2(G), 2(L), and 2(O)).