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2018 年制药生物技术大会：用聚苯乙烯纳米粒子瞬时加载 CD34+ 造血祖细胞 - Bart Wathiong - 佛兰德技术研究所

巴特·瓦西翁

介绍

造血干细胞和造血祖细胞为开发多种恶性和非恶性疾病的新疗法提供了绝佳的机会。

最近的研究集中在将 NP 导向 HSC；然而，NP 与 HSC 的结合使用尚未得到充分利用。HSC 已用超顺磁性氧化铁纳米颗粒和氧化钆纳米颗粒标记，用于磁共振成像对比度增强和细胞跟踪。

在本研究中，我们研究了这种相互作用，包括与细胞膜和摄取的关联，以及人脐带血来源的 CD34 ^{+的动力学} HPC 具有明确的 40 nm 大小黄绿色荧光标记的羧化聚苯乙烯 NP。PS NP 引起了医学兴趣，因为它们被用作治疗的多功能载体。“由于它们的商业可用性、高质量以及广泛的尺寸和表面化学性质，这些也常被用作模型纳米颗粒来研究它们与生物系统的相互作用。观察到独特的瞬态加载行为，表明 HPC 主动加载和释放 YG-PS NP。在 YG-PS NP 与骨髓型树突状细胞相互作用过程中比较了这种行为。^{DCs是由CD34 +}分化而来 HPCs 并在发起中发挥着关键作用；指导和控制先天性和适应性免疫反应。CD34-DC 没有表现出瞬态加载行为。

 

材料与方法

CD34 ⁺  HPC 和 CD34-DC的分离和培养 纳米颗粒的表征 NP 的透析 流式细胞仪 共焦显微镜 细胞活力和增殖

结果：

纳米粒子表征

Fluorescently labeled, carboxylated YG-PS NPs with the nominal diameter of 40 nm were used in present study. Before interaction with the cells, proteins and electrolytes present with in the cell culture medium may influence the intrinsic physicochemical parameters of the NP’s used. Therefore, YG-PS NP’s dispersions in both the water and CCM after different incubation periods were characterized by means of NTA. The resulting YG-PS NP’s dispersion’s remained to be stable over the duration of the experiment.

Transient NP loading in HPCs The interaction of HPC’s with NP’s was evaluated by exposing the cells to fluorescently labelled with the concentration range. After 24 hours of the continuous exposure, the cellular NP load was evaluated by measuring the GMFI by using the flow cytometry. However, compared to the differentiated CD34-DCs, HPCs had a smaller NP loading capacity. As HPC’s are non-phagocytic cells and have a lower cytoplasmic-to-nuclear ratio, they are expected to be less efficient in engulfing the large amounts of extracellular material, such as NPs, than DCs with a prominent phagocytic activity. In order to confirm the processing of NPs is different by both cell types, the interaction of HPC’s and CD34-DCs with NPs was studied as a function of time. The NP load was to be measured at after 1, 2, 3, 4, 5, 6 and 24 hours of exposure to 50 µg mL⁻¹ of YG-PS NPs Energy-dependent loading of HPCs and CD34-DCs with NPs

The NP load of the cells could be accumulating in several ways. Discrimination between passive and active processes can be made by performing the NP exposure of the cells at 4°C. This is the common way to assess the involved mechanisms as cooling down the cells inhibits all energy-dependent cellular processes. Pre-incubation at 4°C for 1 hour and exposure of HPC’s, CD34-DCs to 50 µg mL⁻¹ of YG-PS NPs also at 4°C strongly abolished the loading with YG-PS NP’s. This was even more obvious when compared to acquire load with that accumulated by their cellular counterparts obtained from the same donors but exposed at 37°C.

The observed transient loading cannot be ascribed to the fluorescent staining of the NPs

NP’s are recognized to be enter the mammalian cells through the endocytosis mechanism. NPs’ journey passes through the endocytic compartment corresponds to an acidification of the NPs’ environment. The endosomal acidification was mimicked here by dispersing the YG-PS NPs in citric acid – phosphate buffer having a pH of 5, 6 or 7, which corresponds to the pH within the endolysosomal compartment. Thereafter, YG-PS NPs dispersions were dialyzed against citric acid – phosphate buffers with different acidity. YG-PS NPs stock dispersions were used as purchased or pre-dialyzed for 48 hours to remove all labile dye already present in the NPs dispersions .The time kinetics of the dialyses was measured by fluorescence spectroscopy.

Discussion

In the present study, the interaction kinetics of carboxylate YG-PS NPs in HPCs and CD34-DCs were observed to respond differently & to identical NP’s, under identical conditions. HPC’s was showed a transient association with these YG-PS NPs, whereas the CD34-DCs displayed a monotonic increase of the NP load over time. It can partially be explained by the difference in their cell physiology. CD34-DCs are more confined to and involved in antigen presentation and immune responses, whereas the main ability of HPCs is to self-renew or multiply. The differences in cell physiology are coupled with the morphological differences. HPC’s was derived from the cord blood having a high nucleus–cytoplasm ratio, with the cytoplasm poor in organelles, although a few mitochondria and endoplasmic reticulum cisternae can be seen. In this contrast, CD34-DCs have been relatively lower nucleus–cytoplasm ratio, with the cytoplasm containing significantly more organelles, including endosomal vesicles. 

 Conclusion

Therapeutic strategies and transplantations were using the HSCs and HPCs may benefit from their remarkable response to YG-PS NPs. The observed transient loading mechanisms can open the new opportunities for the safe delivery of drugs or molecules of interest with limited bio-accumulation. However, the further investigation was required to be explore whether the observed phenomenon is specific for the NPs used. Effects of NP species, size, functionalization, administered dose, incubation time and intracellular fate should be the carefully investigated. Moreover, further research is needed to reveal how HPC’s are capable of mediating the release and which cellular functions and associated dynamics are involved in this process. The resulting knowledge will allow us to exploit the full potential of NP applications in medicine.

注：这项工作的部分内容将在 5 月 16 日至 17 日于新加坡举行的 2018 年制药生物技术年度大会上展示