Application of KU812 cells for assessing complement activation related effects by nano(bio)materials.

Immunocompatibility issues related to nano(bio)materials, particularly liposomal formulations, involving activation of the complement system have been relatively well described however, they highlight the importance of preclinical evaluation of such interactions. These complement-mediated hypersensitivity reactions, in which basophils are implicated, are associated with complement activation-related pseudoallergy (CARPA). Ex vivo investigation of such events using primary basophils is technically challenging due to the relatively limited number of circulating basophils in peripheral blood. In the current work, the KU812 cell line has been applied as an in vitro model for basophil activation to investigate CARPA-related responses following exposure to test materials obtained from the REFINE consortium. To that end, we developed a standard operating procedure measuring a panel of cell-surface markers indicative of basophilic activation. Two laboratories performed the assays, demonstrating a clear difference in responses between liposomal and polymeric nano(bio)materials, while interlaboratory comparison of the standard operating procedure demonstrated reproducibility in results, between the two facilities. These results suggest the potential to use this protocol as a screening method for such responses however, validation using primary basophils is now warranted.

Exposure of the Basophilic Cell Line KU812 to Liposomes Reveals Activation Profiles Associated with Potential Anaphylactic Responses Linked to Physico-Chemical Characteristics.

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1ß secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1ß secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1ß vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted.