被称为多核苷酸激酶3'-磷酸酶(PNKP)的DNA修复酶的抑制剂预期在癌症中与拓扑异构酶I(TOP1)抑制剂组合显示协同细胞毒性。在这项研究中,一种新型PNKP抑制剂的协同细胞毒性,即,A83B4C63,具有有效的TOP1抑制剂,即,研究了针对结肠直肠癌细胞的SN-38。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,用于A83B4C63的首选肿瘤递送的聚合物胶束(PM),与SN-38以自由或PM形式组合。SN-38的PM形式通过SN-38与mPEO-b-PBCL的官能端基的化学缀合以及mPEO-b-PBCL-SN-38在水中的进一步组装来制备。此外,由mPEO-b-PBCL和mPEO-b-PBCL-SN-38组成的混合胶束用于将A83B4C63和SN-38共负载在相同的纳米制剂中。共同负载制剂中SN-38和A83B4C63对mPEO-b-PBCL的负载含量(%w/w)为7.91±0.66和16.13±0.11%(w/w),分别,与负载单个药物的mPEO-b-PBCL胶束的15.67±0.34(%w/w)和23.06±0.63(%w/w)相比。值得注意的是,共包封SN-38和A83B4C63的PMs的平均直径大于单独包封这些化合物的PMs的平均直径,但仍低于60nm。A83B4C63在24小时内从共封装两种药物的PM中释放为76.36±1.41%,显著高于A83B4C63包封的胶束(42.70±0.72%)。相比之下,SN-38从PM共封装两种药物的释放在24小时为44.15±2.61%,显著低于SN-38缀合的PMs(74.16±3.65%)。通过Combleefy软件分析的MTS测定的细胞毒性评估表明PM/A83B4C63(浓度范围为10-40μM)和游离SN-38(浓度范围为0.001-1μM)之间有明显的协同作用。当与PM/A83B4C63在10或20-40μM下组合时,SN-38的协同细胞毒性浓度范围缩小到0.1-1或0.01-1μM,分别。总的来说,在协同范围内的药物浓度下(A83B4C63为10μM,SN-38为0.05-1μM)的PM共封装A83B4C63和SN-38,其SN-38抗癌活性的增强略低于单个胶束的组合,即,相同摩尔浓度下的A83B4C63PMs+SN-38PMs。这归因于与仅封装SN-38的PM相比,SN-38从SN-38和A83B4C63共封装的PM释放较慢。用TOP1抑制剂和A83B4C63制剂共处理细胞可增强γ-HA2X的表达水平,裂开的PARP,在大多数情况下,caspase-3和caspase-7。对于共封装A83B4C63和SN-38两者的PM,这种趋势更加一致和显著。来自该研究的总体结果显示SN-38和A83B4C63的PM之间的协同作用,作为单独药物的两种PM的混合物或共包封两种药物的PM。
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.