BackgroundThe use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders. However, the stability of siRNA and other small molecule therapeutics is challenged by their intrinsic instability and limited passage across the blood–brain barrier (BBB). Based on these premises, our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB. For this purpose, 5(6)-carboxyfluorescein-conjugated OL and scramble peptide were synthesized, and then their BBB cellular internalization/trafficking and stability were characterized versus temperature, pH and serum content in the media in hCMEC/D3 cells as a model of BBB endothelium. Specifically, integrity of the internalized peptide in cell lysates was analyzed by LC/MS while cellular distribution and intracellular trafficking of OL was examined by fluorescence microscopy with early-late endosome (pHRodo Red®) and lysosome (Lysotracker®) markers.ResultsOur data show that cellular uptake of OL increased linearly with the concentrations tested in this study at 37 °C and the uptake was two to threefolds higher when compared to scramble peptide. While there were no differences for scramble peptide, the uptake of OL decreased by 50% at 4 °C incubation (vs. 37 °C). No effects of pH were observed on endothelial uptake of OL. Immunofluorescence studies also indicated a significant cellular internalization of OL that remained intact (as evaluated by LC–MS/MS) and co-localized with endosomal, but not lysosome marker. Importantly, OL was found non-toxic to cells at all concentrations tested.ConclusionsIn summary, our data suggest the existence of a receptor-mediated transcytosis pathway for cellular uptake of OL at the BBB endothelium. However, in vivo studies will be needed to assess the siRNA loading capacity of OL and its trans-BBB transport efficiency for targeted delivery in the brain.

中文翻译：

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用于跨血脑屏障的基于肽的药物递送的气味凝集素的体外表征

背景 最近强调使用基于 siRNA 的基因沉默作为治疗神经系统疾病的潜在治疗策略。然而，siRNA 和其他小分子疗法的稳定性受到其内在不稳定性和穿过血脑屏障 (BBB) 的限制的挑战。基于这些前提，我们的目标是表征/优化气味凝集素 (OL)，一种小的非免疫原性凝集素样肽，作为跨 BBB 靶向递送的载体。为此，合成了 5(6)-羧基荧光素偶联的 OL 和扰乱肽，然后将它们的 BBB 细胞内化/运输和稳定性表征为 hCMEC/D3 细胞培养基中的温度、pH 和血清含量作为模型BBB 内皮。具体来说，细胞裂解物中内化肽的完整性通过 LC/MS 进行分析，而 OL 的细胞分布和细胞内运输通过具有早期内体 (pHRodo Red®) 和溶酶体 (Lysotracker®) 标记的荧光显微镜检查。结果我们的数据显示细胞在 37 °C 下，OL 的吸收随本研究中测试的浓度线性增加，与加扰肽相比，吸收高出两到三倍。虽然 scramble 肽没有差异，但在 4°C 孵育时 OL 的吸收降低了 50%（与 37°C 相比）。没有观察到 pH 值对 OL 的内皮摄取有影响。免疫荧光研究还表明 OL 的显着细胞内化保持完整（如通过 LC-MS/MS 评估）并与内体共定位，但不是溶酶体标记。重要的，发现在所有测试浓度下 OL 对细胞均无毒。结论 总之，我们的数据表明存在受体介导的胞吞作用通路，用于 BBB 内皮细胞摄取 OL。然而，需要体内研究来评估 OL 的 siRNA 负载能力及其在大脑中靶向递送的跨 BBB 转运效率。