Ivko Valeria, Antipova Olga, Ivanov Boris, Tashlitsky Vadim, Dzarieva Fatima, Samoylenkova Nadezhda, Usachev Dmitry, Pavlova Galina, Kopylov Alexey
Belozersky Research Institute of Physical Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia.
Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.
Int J Mol Sci. 2025 Sep 7;26(17):8712. doi: 10.3390/ijms26178712.
The WHO considers the Epidermal Growth Factor Receptor (EGFR) one of the key biomarkers of glioblastoma (GB). EGFR can be identified and targeted using molecular recognition elements (MoREs), like aptamers and aptamer-drug conjugates (ApDCs). Understanding the kinetics of anti-EGFR ApDC interactions with EGFR as well as the kinetics of their internalization into the cells is a crucial step for the further development of anti-EGFR ApDCs. For the first time, a novel approach was implemented to study real-time kinetics by measuring the cellular index (CI) using impedance (xCELLigence). Doxorubicin (DOX) was used as an indicator drug. Because DOX intercalates into the DNA double helix, aptamer-DOX non-covalent complexes were obtained. For the anti-EGFR DNA aptamer GR20, an additional duplex was constructed by synthesizing the extra region (GR20h) and via hybridization with the complementary oligonucleotide (h') to form a duplex (hh'), thus creating the aptamer construct with complementary oligonucleotide (ACCO) GR20hh'. The original HPLC method quantified the assembly efficiency of an ACCO. The ACCO GR20hh' retained affinity for the recombinant extracellular domain of EGFR, as measured using Biolayer Interferometry (BLI). According to cytofluorimetry, the ACCO GR20hh' interacts with cells of continuous culture from GB patient (CCGBP) surgical samples. The DOX-ACCO GR20hh' complexes are more efficiently internalized by EGFR+ cells lines A-431 and CCGBP 107 than DOX alone.
世界卫生组织认为表皮生长因子受体（EGFR）是胶质母细胞瘤（GB）的关键生物标志物之一。EGFR可以使用分子识别元件（MoREs）进行识别和靶向，如适配体和适配体-药物偶联物（ApDCs）。了解抗EGFR ApDC与EGFR相互作用的动力学以及它们内化进入细胞的动力学是抗EGFR ApDC进一步发展的关键步骤。首次采用了一种新方法，通过使用阻抗（xCELLigence）测量细胞指数（CI）来研究实时动力学。阿霉素（DOX）用作指示药物。由于DOX插入DNA双螺旋中，因此获得了适配体-DOX非共价复合物。对于抗EGFR DNA适配体GR20，通过合成额外区域（GR20h）并与互补寡核苷酸（h'）杂交形成双链体（hh'），构建了一个额外的双链体，从而创建了带有互补寡核苷酸（ACCO）的适配体构建体GR20hh'。原始的高效液相色谱法对ACCO的组装效率进行了定量。使用生物层干涉术（BLI）测量，ACCO GR20hh'对EGFR的重组细胞外结构域保持亲和力。根据细胞荧光测定法，ACCO GR20hh'与胶质母细胞瘤患者手术样本的连续培养细胞（CCGBP）相互作用。与单独的DOX相比，DOX-ACCO GR20hh'复合物更有效地被EGFR+细胞系A-431和CCGBP 107内化。
