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.