Small extracellular vesicles (sEVs) such as exosomes are nanocarriers of proteins, RNAs and DNAs. Isolation of pure sEV populations remains challenging, with reports of protein and lipoprotein contaminants in the isolates. Cellular uptake – a cornerstone for understanding exosome and sEV function – is frequently examined using lipophilic dyes such as PKH67 or CellMask to label the vesicles. In this study, we investigated whether contaminants can confound the outcomes from sEV and exosomes uptake experiments. sEVs were isolated from blood plasma of fasted or non-fasted rats as well as from serum-supplemented or serum-free conditioned cell culture medium using size-exclusion chromatography (SEC). Eluent fractions were characterized using nanoparticle tracking, protein and triglyceride assays and immunoassays. SEC fractions were labelled with different lipophilic dyes and cellular uptake was quantified using endothelial cells or primary cardiomyocytes. We report co-isolation of sEVs with apolipoprotein B-containing lipoproteins. Cellular dye transfer did not correspond to sEV content of the SEC fractions, but was severely affected by lipoprotein and protein content. Overnight fasting of rats decreased lipoprotein content and also decreased dye transfer, while late, sEV-poor/protein-rich fractions demonstrated even greater dye transfer. The potential for dye transfer to occur in the complete absence of sEVs was clearly shown by experiments using staining of sEV-depleted serum or pure protein sample. In conclusion, proteins and lipoproteins can make a substantial contribution to transfer of lipophilic dyes to recipient cells. Considering the likelihood of contamination of sEV and exosome isolates, lipophilic dye staining experiments should be carefully controlled, and conclusions interpreted with caution.

外泌体等小细胞外囊泡 (sEV) 是蛋白质、RNA 和 DNA 的纳米载体。纯 sEV 群体的分离仍然具有挑战性，有报道称分离株中存在蛋白质和脂蛋白污染物。细胞摄取是了解外泌体和 sEV 功能的基石，经常使用亲脂性染料（如 PKH67 或 CellMask）来标记囊泡来检查。在这项研究中，我们研究了污染物是否会混淆 sEV 和外泌体摄取实验的结果。使用尺寸排阻色谱法 (SEC) 从禁食或非禁食大鼠的血浆以及添加血清或无血清的条件细胞培养基中分离出 sEV。使用纳米颗粒追踪、蛋白质和甘油三酯测定以及免疫测定来表征洗脱液组分。SEC 级分用不同的亲脂性染料标记，并使用内皮细胞或原代心肌细胞对细胞摄取进行定量。我们报告了 sEV 与含载脂蛋白 B 的脂蛋白的共分离。细胞染料转移与 SEC 级分的 sEV 含量不对应，但受到脂蛋白和蛋白质含量的严重影响。大鼠隔夜禁食降低了脂蛋白含量，也减少了染料转移，而后期，缺乏 sEV/富含蛋白质的部分表现出更大的染料转移。使用 sEV 耗尽的血清或纯蛋白质样品进行染色的实验清楚地表明，在完全不存在 sEV 的情况下发生染料转移的可能性。总之，蛋白质和脂蛋白可以对亲脂性染料向受体细胞的转移做出重大贡献。考虑到 sEV 和外泌体分离物污染的可能性，应仔细控制亲脂性染料染色实验，并谨慎解释结论。