Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach
Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach
复制标题
DOI：
10.7150/thno.27891
复制
发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Al-Jamal, Khuloud T.
中科院分区：
文献类型：
--
作者：
Faruqu, Farid N.;Wang, Julie Tzu-Wen;Al-Jamal, Khuloud T.
细胞外囊泡，特别是外泌体，由于其生物来源和固有的细胞间生物分子递送能力，最近作为新型药物递送载体获得了关注。因此，深入了解其体内生物分布至关重要。本研究旨在建立一种新的、可靠的、通用的放射性标记exosome的方法来研究exosome在体内的生物分布。使用超离心法将B16 F10衍生的外泌体（Exo（B16））分离到单个蔗糖垫上，并使用纳米颗粒跟踪分析（NTA）、蛋白质测量、流式细胞术和电子显微镜表征大小、产率、纯度、外泌体标志物和形态。使用2种不同的方法放射性标记Exo（B16）-管腔内标记（通过环庚三烯酚酮穿梭捕获（111）铟）;和膜标记（通过共价连接的双官能螯合剂DTPA-酸酐螯合（111）铟）。使用凝胶过滤和薄层色谱法评估标记效率和稳定性。给荷黑色素瘤的免疫活性（C57 BL/6）和免疫缺陷（NSG）小鼠静脉注射放射性标记的Exo（B16）（1 × 10（11）个颗粒/小鼠），然后在注射后1、4和24小时进行代谢笼研究、全身SPECT-CT成像和离体γ计数。膜标记的Exo（B16）显示出上级的放射性标记效率和放射化学稳定性与铝内标记的外泌体（分别为4.73 +/-0.39%和14.21 +/-2.76%）相比，铝内标记的外泌体（分别为19.2 +/-4.53%和80.4 +/-1.6%）。使用膜标记方法，在携带黑色素瘤的C57 Bl/6小鼠中进行了Exo（B16）的体内生物分布，发现主要在肝脏和脾脏中积累（分别类似于56%和类似于38%ID/gT），其次是肾脏（类似于3%ID/gT）。Exo（B16）显示最小肿瘤，即自身组织蓄积（类似于0.7%ID/gT）。膜标记方法也被用于研究Exo（B16）在荷黑色素瘤的免疫受损（NSG）小鼠中的生物分布，以与在免疫活性的C57 B16小鼠中的生物分布进行比较。在C57 BL/6和NSG小鼠中观察到相似的生物分布特征，除了在NSG小鼠中观察到显著较低的肿瘤蓄积外，在肝脏和脾脏中观察到显著蓄积（类似于0.3%ID/gT）。外泌体的膜放射性标记是一种可靠的方法，其允许对潜在的所有外泌体类型进行准确的实时成像和定量生物分布研究，而不需要工程化亲本细胞。
Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin and inherent intercellular biomolecule delivery capability. An in-depth knowledge of their in vivo biodistribution is therefore essential. This work aimed to develop a novel, reliable and universal method to radiolabel exosomes to study their in vivo biodistribution.Methods: Melanoma (B16F10) cells were cultured in bioreactor flasks to increase exosome yield. B16F10-derived exosomes (Exo(B16)) were isolated using ultracentrfugation onto a single sucrose cushion, and were characterised for size, yield, purity, exosomal markers and morphology using Nanoparticle Tracking Analysis (NTA), protein measurements, flow cytometry and electron microscopy. Exo(B16) were radiolabelled using 2 different approaches - intraluminal labelling (entrapment of (111)Indium via tropolone shuttling); and membrane labelling (chelation of (111)Indium via covalently attached bifunctional chelator DTPA-anhydride). Labelling efficiency and stability was assessed using gel filtration and thin layer chromatography. Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled Exo(B16) (1x10(11) particles/mouse) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection.Results: Membrane-labelled Exo(B16) showed superior radiolabelling efficiency and radiochemical stability (19.2 +/- 4.53 % and 80.4 +/- 1.6 % respectively) compared to the intraluminal-labelled exosomes (4.73 +/- 0.39 % and 14.21 +/- 2.76 % respectively). Using the membrane-labelling approach, the in vivo biodistribution of Exo(B16) in melanoma-bearing C57Bl/6 mice was carried out, and was found to accumulate primarily in the liver and spleen (similar to 56% and similar to 38% ID/gT respectively), followed by the kidneys (similar to 3% ID/gT). Exo(B16) showed minimal tumour i.e. self-tissue accumulation (similar to 0.7% ID/gT). The membrane-labelling approach was also used to study Exo(B16) biodistribution in melanoma-bearing immunocompromised (NSG) mice, to compare with that in the immunocompetent C57Bl/6 mice. Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the significantly lower tumour accumulation observed in the NSG mice (similar to 0.3% ID/gT).Conclusion: Membrane radiolabelling of exosomes is a reliable approach that allows for accurate live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells.