Design and Characterization of HER-2-Targeted Gold Nanoparticles for Enhanced X-radiation Treatment of Locally Advanced Breast Cancer
Design and Characterization of HER-2-Targeted Gold Nanoparticles for Enhanced X-radiation Treatment of Locally Advanced Breast Cancer
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DOI：
10.1021/mp100207t
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发表时间：
2010-11-01
影响因子：
4.9
通讯作者：
Reilly, Raymond M.
中科院分区：
文献类型：
--
作者：
Chattopadhyay, Niladri;Cai, Zhongli;Reilly, Raymond M.
我们的目的是开发一种以人表皮生长因子受体-2（HER-2）为靶点的纳米放射增敏剂。HER-2在20-30%的乳腺癌中过表达，在局部晚期疾病（LABC）中高出2倍。曲妥珠单抗用聚乙二醇（OPSS-PEG-SVA）交联剂衍生化，生成曲妥珠单抗-PEG-OPSS。通过SDS-PAGE分析这些免疫缀合物，并使用HER-2过表达的SK-BR-3乳腺癌细胞通过流式细胞术评估它们的免疫反应性。曲妥珠单抗与增加比例的PEG反应导致分子量从约148 kDa增加至243 kDa，与PEG取代增加相关（每个曲妥珠单抗0.6至18.9个PEG链）。通过流式细胞术评估，每个曲妥珠单抗连接约7条PEG链导致免疫反应性保留56%。然后将缀合物连接到30 nm AuNP。使用一种新的（123）碘-放射性示踪剂为基础的测定，克服了目前对AuNP上生物分子的分光光度定量的限制，我们估计当2 × 10（11）AuNP与20 μ g曲妥珠单抗-PEG-OPSS反应时，14.3 +/- 2.7个抗体连接到每个AuNP上。曲妥珠单抗-PEG-AuNP对HER-2和SK-BR-3细胞中内化的特异性通过暗视野显微镜比较曲妥珠单抗-PEG-AuNP或PEG-AuNP的摄取来证明。使用γ-H2 AX测定通过免疫荧光评估曲妥珠单抗-PEG-AuNP与300 kVp X射线组合增强SK-BR-3细胞中DNA双链断裂（DSB）的能力。γ-H2 AX测定结果显示，与仅用X辐射处理相比，用曲妥珠单抗-PEG-AuNP和X辐射的DNA-DSB高5.1倍。曲妥珠单抗-PEG-金纳米颗粒是一种有前途的靶向纳米技术为基础的放射增敏剂，用于改善LABC治疗。本研究中描述的用于表面修饰和表征曲妥珠单抗-PEG-AuNP的设计和系统方法将应用于其他用于癌症治疗的分子靶向AuNP。
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 x 10(11) AuNPs were reacted with 20 mu g 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 gamma-H2AX assay. gamma-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.