Departments of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
Mol Pharm. 2010 Dec 6;7(6):2194-206. doi: 10.1021/mp100207t. Epub 2010 Nov 8.
Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for cancer treatment.
我们的目的是开发一种针对人表皮生长因子受体 2（HER-2）的纳米技术放射增敏剂。HER-2 在所有乳腺癌中的表达率为 20-30%，在局部晚期疾病（LABC）中高达 2 倍。曲妥珠单抗用聚乙二醇（OPSS-PEG-SVA）交联剂衍生化，得到曲妥珠单抗-PEG-OPSS。通过 SDS-PAGE 分析这些免疫偶联物，并使用过表达 HER-2 的 SK-BR-3 乳腺癌细胞通过流式细胞术评估其免疫反应性。曲妥珠单抗与 PEG 的比例增加会导致分子量从约 148 kDa 增加到 243 kDa，与 PEG 取代度（每个曲妥珠单抗 0.6 至 18.9 个 PEG 链）增加相关。每个曲妥珠单抗附着约 7 个 PEG 链会导致流式细胞术评估的免疫反应性保留 56%。然后将这些偶联物与 30nm 的 AuNPs 连接。使用一种新的（123）碘放射性示踪剂测定法，该方法克服了目前在 AuNPs 上定量生物分子的分光光度法的局限性，我们估计当 2×10(11)AuNPs 与 20μg 的曲妥珠单抗-PEG-OPSS 反应时，每个 AuNP 附着 14.3±2.7 个抗体。通过暗场显微镜比较曲妥珠单抗-PEG-AuNPs 或 PEG-AuNPs 的摄取，证明了曲妥珠单抗-PEG-AuNPs 对 HER-2 的特异性和在 SK-BR-3 细胞内的内化。通过使用 γ-H2AX 测定法的免疫荧光法评估曲妥珠单抗-PEG-AuNPs 与 300 kVp X 射线联合增强 SK-BR-3 细胞中 DNA 双链断裂（DSBs）的能力。γ-H2AX 测定结果显示，与单独接受 X 射线治疗相比，曲妥珠单抗-PEG-AuNPs 和 X 射线联合治疗可使 DNA-DSB 增加 5.1 倍。曲妥珠单抗-PEG-AuNPs 是一种有前途的靶向纳米技术放射增敏剂，可改善 LABC 治疗。本研究中采用的表面修饰和表征曲妥珠单抗-PEG-AuNPs 的设计和系统方法可应用于其他用于癌症治疗的分子靶向 AuNPs。
