The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with ¹⁷⁷Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [¹⁷⁷Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [¹⁷⁷Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [¹⁷⁷Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [¹⁷⁷Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [¹⁷⁷Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [¹⁷⁷Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 μCi (9.25 MBq) and 500 μCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

中文翻译：

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核定位信号增强基于卟啉的分子货物的靶向和治疗功效：体外和离体系统评估

目前工作的目的是评估核定位信号（NLS）在促进细胞内递送和增强分子货物治疗功效方面的潜力。为此，利用内部合成的卟啉衍生物，即 5-羧基亚甲基-氧苯基-10,15,20-三（4-甲氧基苯基）卟啉（UTriMA），与 NLS 序列 [PKKKRKV] 缀合。在本工作过程中合成的三种化合物，即 DOTA-Lys-NLS、DOTA-UTriMA-Lys-NLS 和 DOTA-Lys-UTriMA，在癌细胞系 (HT1080) 中评估了细胞毒性，其中均表现出最小的暗毒性。然而，在同一细胞系中使用 DOTA-Lys-UTriMA 和 DOTA-UTriMA-Lys-NLS 缀合物进行光细胞毒性研究时，与前者相比，后者表现出明显更高的光依赖性毒性。此外，DOTA-UTriMA-Lys-NLS 在健康细胞系 (WI26VA4) 中的光细胞毒性明显低于在癌细胞中观察到的光细胞毒性。在 HT1080 癌细胞中进行的荧光细胞成像研究揭示了 NLS 缀合卟啉 (DOTA-UTriMA-Lys-NLS) 的细胞内积累，而未缀合卟啉 (DOTA-Lys-UTriMA) 则未能做到这一点。为了评估合成缀合物的放射治疗效果，所有三种化合物均用¹⁷⁷ Lu 进行放射性标记，177 Lu 是一种众所周知的治疗放射性核素，具有高放射化学纯度 (> 95%)。在体外研究期间，[ ¹⁷⁷ Lu]Lu-DOTA-UTriMA-Lys-NLS复合物在三种放射性标记复合物中表现出最高的细胞结合和内化。在患有纤维肉瘤的小动物模型中进行了放射性标记化合物的生物分布研究，其中 [ ¹⁷⁷ Lu]Lu-DOTA-UTriMA-Lys-NLS 的积累显着更高，并且保留时间更长（24 小时时为 9.32 ± 1.27% IA/g） pi) 与[ ¹⁷⁷ Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h pi) 和 [ ¹⁷⁷ Lu]Lu-DOTA-Lys-NLS 复合物 (0.26 ± 0.17%观察到 IA/g（注射后 24 小时）。通过记录在不同时间点给予[ ^{177 Lu]Lu-DOTA-UTriMA-Lys-NLS的患有纤维肉瘤的瑞士小鼠的系列SPECT-CT图像，进一步证实了生物分布研究的结果。}使用 [ ¹⁷⁷ Lu]Lu-DOTA-UTriMA-Lys-NLS 在同一动物模型中使用两种不同剂量 [250 μCi (9.25 MBq) 和 500 μCi (18.5 MBq)] 进行的肿瘤消退研究导致肿瘤质量显着减少在接受治疗的动物组中。上述结果表明，与 NLS 缀合后分子货物的靶向能力明显增强，因此表明该策略可能有助于制备作为多模式药物的药物-NLS 缀合物。