Vascular Endothelial Growth Factor VEGF

There is preclinical and clinical evidence indicating that angiogenesis plays a major role in the growth and dissemination of malignant tumors.88,89 Inhibition of angiogenesis has yielded promising results in a number of experimental animal tumor models.90,91 The most important molecular targets have been vascular endothelial growth factor (VEGF) and its receptor (VEGFR).92,93 We recently have constructed a human VEGF121 isoform fused to a novel 15-aa cysteine-containing tag designed for site-specific conjugation of therapeutic and diagnostic agents94 (Figure 6.4). A boronated fifth-generation PAMAM dendrimer (BD), tagged with a near-infrared (IR) fluorescent dye Cy5, was conjugated using the heterobifunctional reagent sulfo-LC-SPDP to BD/Cy5 via reactive SH-groups generated in the VEGF fusion protein by mild dithiothreitol (DTT) treatment. The bioconjugate, designated VEGF-BD/Cy5, contained 1050-1100 boron atoms per dimeric VEGF molecule. VEGF-BD/Cy5 retained the in vitro

FIGURE 6.4 Preparation of a vascular endothelial growth factor (VEGF) receptor-targeting nanovehicle (VEGF-BD/Cy5) (A). A fifth-generation (G5) PAMAM dendrimer initially is reacted with SPDP and the near-IR dye, Cy5-NHS, (A) dissolved in dimethylformamide/methanol mixture for 1 h. Following this, it is reacted with the polyisocynato polyhedral borane, Na(CH3)3-NB10H8NCO, in a carbonate buffer (pH 9.0)/9% acetone mixture for 24 h (B). Then a 1.5-fold molar excess of DTT is added to the VEGF monomer in a solution containing 20 mmol/L NaOAc (pH 6.5), 0.5 mol/L urea and 0.5 mol/L NDSB-221 and incubated at 4°C for 16 h (C). Finally, the boronated dendrimer is incubated with the targeting vehicle for 15 min to produce the VEGF-BD/Cy5 (D). (From Backer, M. V. et al., Mol. Cancer Ther., 4, 1423, 2005.)

FIGURE 6.4 Preparation of a vascular endothelial growth factor (VEGF) receptor-targeting nanovehicle (VEGF-BD/Cy5) (A). A fifth-generation (G5) PAMAM dendrimer initially is reacted with SPDP and the near-IR dye, Cy5-NHS, (A) dissolved in dimethylformamide/methanol mixture for 1 h. Following this, it is reacted with the polyisocynato polyhedral borane, Na(CH3)3-NB10H8NCO, in a carbonate buffer (pH 9.0)/9% acetone mixture for 24 h (B). Then a 1.5-fold molar excess of DTT is added to the VEGF monomer in a solution containing 20 mmol/L NaOAc (pH 6.5), 0.5 mol/L urea and 0.5 mol/L NDSB-221 and incubated at 4°C for 16 h (C). Finally, the boronated dendrimer is incubated with the targeting vehicle for 15 min to produce the VEGF-BD/Cy5 (D). (From Backer, M. V. et al., Mol. Cancer Ther., 4, 1423, 2005.)

functional activity of VEGF121, which was similar to that of native VEGF. Tagging the biocon-jugate with Cy5 dye permitted near-IR fluorescence imaging of its in vitro and in vivo uptake. In vitro uptake was determined by incubating VEGF-BD/Cy5 with VEGFR-2 overexpressing PAE/KDR cells and in vivo uptake was evaluated in mice bearing s.c. tumor implants. In vitro, the bioconjugate localized in the perinuclear region and in vivo it primarily localized in regions of active angiogenesis. Furthermore, depletion of VEGFR-2 overexpressing cells from the tumor vasculature with VEGF-toxin fusion protein significantly decreased bioconjugate uptake, indicating that these cells were the primary targets of VEGF-BD/Cy5. These studies demonstrated that VEGF-BD/Cy5 potentially could be used as a diagnostic agent.94 Further studies are planned to evaluate its therapeutic efficacy using the F98 rat glioma model, which we have used extensively to evaluate both LMW and HMW boron delivery agents.95

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