Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, 50670-901, Brazil; Departamento de Biofísica e Radiobiologia, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, 50670-901, Brazil.
Departamento de Química Fundamental, Centro de Ciências Exatas e da Natureza, Universidade Federal de Pernambuco, Recife, PE, 50740-560, Brazil.
Int J Biol Macromol. 2023 Dec 1;252:126453. doi: 10.1016/j.ijbiomac.2023.126453. Epub 2023 Aug 22.
Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.
丝氨酸蛋白酶在生物学中发挥着至关重要的作用，其活性受到抑制剂的调控，如 EcTI，这是一种从扭黄檀种子中分离纯化得到的丝氨酸蛋白酶抑制剂，具有抗癌活性。本研究旨在将 EcTI 与量子点（QDs）相偶联，后者是一类具有优异光学性质的荧光染料，并研究 QDs-EcTI 纳米探针与癌细胞的相互作用。通过荧光相关光谱（FCS）和荧光微孔板检测（FMA）评估了偶联物的相互作用。还分析了 QDs-EcTI 纳米探针与癌细胞相互作用后 EcTI 的抑制活性。通过 FCS 发现，与 bare QDs 相比，偶联物的水动力直径约增加了 4 倍，表明成功偶联。FMA 也支持了这一结果，该结果显示纳米系统的相对荧光强度约为 bare QDs 或单独 EcTI 的 3815%。在与 QDs 相互作用后，EcTI 的抑制活性保持不变。通过流式细胞术分析，约 62%的 MDA-MB-231 和 90%的 HeLa 细胞被 QD-EcTI 缀合物标记，这表明它们的细胞膜上具有不同的蛋白酶水平，而 EcTI 对这些蛋白酶水平具有亲和力。综上所述，QD-EcTI 是一种很有价值的纳米工具，可用于使用荧光技术研究该抑制剂与癌细胞的相互作用，其潜在用途是揭示癌症生物学中蛋白酶和抑制剂之间相互作用的复杂动态。
