Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin

Abstract

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review

“…Otherwise, FNs can be purified from the horse spleen, where the ratio between Hc and Lc subunits was found to be ~1/10 [67]. To ensure that purified FNs are not contaminated by endotoxins that could impact both in vitro and in vivo experiments, additional procedures to remove endotoxins might be required [68].…”

Vibrio cholerae toxin coregulated pilus provokes inflammatory responses in Coculture model of Caco‐2 and peripheral blood mononuclear cells (PBMC) leading to increased colonization

“…The amount of LPS in TcpA recombinant protein was calculated as less than 0.25 Eu/mL, considering the fact that the sensitivity of the endotoxin assay kit was 0.25 EU/mL. It is worth noting that the acceptable level of LPS in recombinant protein is less than 1 EU/mL 15,16 …”

Ferritin: A Multifunctional Nanoplatform for Biological Detection, Imaging Diagnosis, and Drug Delivery

“…Escherichia coli is a well-known host organism, by the use of which the recombinant production of ferritins begins at the genetic level, followed by the expression of ferritin proteins and purification processes . It is worth noting that to avoid nanocage immunogenicity and pyrogenicity, endotoxin contaminations should be carefully removed in the process of ferritin purification by the use of affinity chromatography, detergents, or other protocols . To date, human HFn, human LFn, rat HFn, and RGD-modified human HFn have been recombinantly expressed in sufficient quantity and quality for broad application (Table ).…”

“…1 It is worth noting that to avoid nanocage immunogenicity and pyrogenicity, endotoxin contaminations should be carefully removed in the process of ferritin purification by the use of affinity chromatography, detergents, or other protocols. 14 To date, human HFn, human LFn, rat HFn, and RGD-modified human HFn have been recombinantly expressed in sufficient quantity and quality for broad application (Table 1).…”