被称为多核苷酸激酶 3'-磷酸酶 (PNKP) 的 DNA 修复酶抑制剂与拓扑异构酶 I (TOP1) 抑制剂联合使用有望在癌症中表现出协同细胞毒性。在这项研究中，研究了新型 PNKP 抑制剂（即 A83B4C63）与强效 TOP1 抑制剂（即 SN-38）对结直肠癌细胞的协同细胞毒性。用于优选肿瘤递送 A83B4C63 的聚合物胶束（PM）是通过将该化合物物理封装在甲氧基聚（环氧乙烷）-聚（α-羧酸苄酯-ε-己内酯）（mPEO-b-PBCL）胶束中而开发的，与SN-38 有免费或 PM 形式。 SN-38 的 PM 形式是通过将 SN-38 化学缀合到 mPEO-b-PBCL 的官能端基上并在水中进一步组装 mPEO-b-PBCL-SN-38 来制备的。此外，由 mPEO-b-PBCL 和 mPEO-b-PBCL-SN-38 组成的混合胶束用于在同一纳米制剂中共同负载 A83B4C63 和 SN-38。共负载制剂中 SN-38 和 A83B4C63 对 mPEO-b-PBCL 的负载含量 (% w/w) 分别为 7.91 ± 0.66 和 16.13 ± 0.11% (w/w)，相比之下为 15.67 ± 0.34 (% w/w) 和 23.06 ± 0.63 (% w/w) 负载单个药物的 mPEO-b-PBCL 胶束。值得注意的是，共封装 SN-38 和 A83B4C63 的 PM 的平均直径大于单独封装这两种化合物的 PM 的平均直径，但仍低于 60 nm。 24小时内，共包封两种药物的PM中A83B4C63的释放率为76.36±1.41%，显着高于包封A83B4C63的胶束（42.70±0.72%）。相比之下，共封装两种药物的 PM 中 SN-38 的释放量在 24 小时时为 44.15 ± 2.61%，显着低于 SN-38 结合的 PM 的释放量 (74.16 ± 3.65%)。 Combenefit 软件分析的 MTS 测定的细胞毒性评估表明 PM/A83B4C63（浓度范围为 10-40 μM）和游离 SN-38（浓度范围为 0.001-1 μM）之间存在明显的协同作用。当与 10 或 20-40 μM 的 PM/A83B4C63 组合时，SN-38 的协同细胞毒性浓度范围分别缩小至 0.1-1 或 0.01-1 μM。一般来说，在协同范围内的药物浓度（A83B4C63 为 10 μM，SN-38 为 0.05-1 μM）共封装 A83B4C63 和 SN-38 的 PM 表现出的 SN-38 抗癌活性增强程度略低于单个胶束的组合，即相同摩尔浓度的 A83B4C63 PM SN-38 PM。这是由于与仅封装 SN-38 的 PM 相比，SN-38 和 A83B4C63 共封装的 PM 中 SN-38 的释放速度较慢。在大多数情况下，用 TOP1 抑制剂和 A83B4C63 制剂共同处理细胞可增强 γ-HA2X、裂解的 PARP、caspase-3 和 caspase-7 的表达水平。对于同时封装 A83B4C63 和 SN-38 的 PM，这种趋势更加一致和显着。研究的总体结果表明，SN-38 和 A83B4C63 的 PM 之间作为单独药物的两种 PM 的混合物或共同封装两种药物的 PM 之间具有协同作用。

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 μM) and free SN-38 (at a concentration range of 0.001-1 μM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 μM when combined with PM/A83B4C63 at 10 or 20-40 μM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 μM for A83B4C63 and 0.05-1 μM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.