生物纳米颗粒，如蛋白质和细胞外囊泡，由于其生物相容性、高载荷效率和生物利用度，正在快速成为纳米药物输送剂。然而，大多数出现在临床前的候选者在进入临床试验时很少证实其潜力。其中，原因之一是合成过程的低控制和其潜在免疫反应性特征的有限表征。在这里，我们提出了一种联合方法，可以在生产、纯化、内毒素去除和药物装载过程中完全表征 H-铁蛋白纳米颗粒的免疫反应性。H-铁蛋白是一种非常有趣的纳米笼，由于其固有的癌症靶向性、令人满意的尺寸和高稳定性而受到评估，其是一种重组蛋白，因此，其免疫反应性应在临床前进行仔细评估，以实现进一步的临床转化。令人惊讶的是，这个方面常常被科学界低估。通过以内毒素含量为函数的促炎性细胞因子释放测量，我们发现即使从纳米笼中去除所有致热原污染物，仍然留下轻微的免疫反应性。当我们通过高度标准化的装载方法进一步纯化了 H-铁蛋白，通过负载阿霉素，促炎性细胞因子释放被消除。这证实了 H-铁蛋白纳米笼在癌症治疗的药物输送中的安全性。我们的方法表明，在评估纳米药物的安全性时，需要进行急性毒性和免疫反应性的联合分析，以确保新开发产品的安全性并揭示其真实的转化潜力。

Biological nanoparticles, such as proteins and extracellular vesicles, are rapidly growing as nanobased drug-delivery agents due to their biocompatibility, high loading efficiency, and bioavailability. However, most of the candidates emerging preclinically hardly confirm their potential when entering clinical trials. Among other reasons, this is due to the low control of synthesis processes and the limited characterization of their potential immunoreactivity profiles. Here, we propose a combined method that allow us to fully characterize H-ferritin nanoparticles' immunoreactivity during their production, purification, endotoxin removal, and drug loading. H-Ferritin is an extremely interesting nanocage that is being under evaluation for cancer therapy due to its innate cancer tropism, favorable size, and high stability. However, being a recombinant protein, its immunoreactivity should be carefully evaluated preclinically to enable further clinical translation. Surprisingly, this aspect is often underestimated by the scientific community. By measuring proinflammatory cytokine release as a function of endotoxin content, we found that even removing all pyrogenic contaminants from the nanocage, a mild immunoreactivity was still left. When we further purified H-ferritin by loading doxorubicin through a highly standardized loading method, proinflammatory cytokine release was eliminated. This confirmed the safety of H-ferritin nanocages to be used for drug delivery in cancer therapy. Our approach demonstrated that when evaluating the safety of nanodrugs, a combined analysis of acute toxicity and immunoreactivity is necessary to guarantee the safety of newly developed products and to unveil their real translational potential.