Figure 4

Synthesis of rhodamine B dye (RB) conjugated Gd-dendrimers and fluorescence microscopy of rhodamine B conjugated Gd-dendrimer uptake in cultured RG-2 glioma cells versus in RG-2 glioma cells of harvested RG-2 glioma tumor specimens. A) Synthetic scheme for production of rhodamine B dye conjugated Gd-dendrimers. Rhodamine B and DTPA are conjugated to the naked dendrimer terminal amines via stable covalent bonds. In functionalized dendrimers, approximately 35% of the terminal amines are occupied by Gd-DTPA, and approximately 7% of the terminal amines are occupied by rhodamine B. B) In vitro fluorescence microscopy of cultured RG-2 glioma cells incubated for 4 hours in media containing either rhodamine B conjugated Gd-G2 dendrimers (left), rhodamine B conjugated Gd-G5 dendrimers (middle), or rhodamine B conjugated Gd-G8 dendrimers (right) at a concentration of 7.2 μM with respect to rhodamine B. Scale bars = 20 μm. Rhodamine B conjugated Gd-G2 dendrimers enter RG-2 glioma cells, and in some cases, the cell nuclei (left). Rhodamine B conjugated Gd-G5 dendrimers (middle) and rhodamine B conjugated Gd-G8 dendrimers (right) enter the cytoplasm of RG-2 glioma cells, but do not localize within the nuclei. C) Dynamic contrast-enhanced MRI-based Gd concentration curves of RG-2 glioma tumor tissue over time following the intravenous bolus of 0.06 mmol Gd/kg of rhodamine B conjugated Gd-G5 dendrimers (n = 6) and rhodamine B conjugated Gd-G8 dendrimers (n = 2). There is substantial extravasation of rhodamine B conjugated Gd-G5 dendrimers across the BBTB, which is more pronounced than that of Gd-G5 dendrimers across the BBTB. There is also some extravasation of rhodamine B conjugated Gd-G8 dendrimers across the BBTB, which is not the case for Gd-G8 dendrimers. D) Ex vivo low power fluorescence microscopy of RG-2 glioma tumor and surrounding brain tissue harvested at 2 hours following the intravenous bolus of rhodamine B conjugated Gd-G5 dendrimers. There is substantial accumulation of rhodamine B conjugated Gd-G5 dendrimers within tumor tissue, and some in surrounding normal brain tissue (left, T = tumor, N = normal, scale bar = 100 μm). High power image of RG-glioma tumor shows subcellular localization of rhodamine B conjugated Gd-G5 dendrimers within individual RG-2 malignant glioma cells (upper right, scale bar = 20 μm). H&E stain of tumor and surrounding brain (lower right, scale bar = 100 μm). Tumor volume is 31 mm³. E) Ex vivo low power fluorescence microscopy of RG-2 glioma tumor and surrounding brain tissue harvested at 2 hours following the intravenous bolus of rhodamine B conjugated Gd-G8 dendrimers. There is some minimal accumulation of rhodamine B conjugated Gd-G8 dendrimers within brain tumor tissue (left, T = tumor, N = normal, scale bar = 100 μm). High power confirms there is some minimal subcellular localization of rhodamine B conjugated Gd-G8 dendrimers within individual RG-2 glioma cells (upper right, scale bar = 20 μm). H&E stain of tumor and surrounding brain (lower right, scale bar = 100 μm). Tumor volume is 30 mm³. Rhodamine B conjugated Gd-G5 dendrimers and rhodamine B conjugated Gd-G8 dendrimers administered intravenously over 1 minute at a Gd dose of 0.06 mmol Gd/kg animal body weight. Adapted from reference[73].