Crystalline cellulose nanocrystals (CNCs) have emerged as novel materials for a wide variety of important applications such as nanofillers, nanocomposites, surface coatings, regenerative medicine and potential drug delivery. CNCs have a needle-like structure with sizes in the range of 100-200 nm long and 5-20 nm wide and a mean aspect ratio 10-100. Despite the great potential applicability of CNCs, very little is known about their potential immunogenicity. Needle-like materials have been known to evoke an immune response in particular to activate the (NOD-like receptor, pyrin domain-containing 3)-inflammasome/IL-1β (Interleukin 1β) pathway. In this study we evaluated the capacity of unmodified CNC and its cationic derivatives CNC-AEM (aminoethylmethacrylate)1, CNC-AEM2, CNC-AEMA(aminoethylmethacrylamide)1 and CNC-AEMA2 to stimulate NLRP3-inflammasome/IL-1β pathway and enhance the production of mitochondrial reactive oxygen species (ROS). Mouse macrophage cell line (J774A.1) was stimulated for 24 h with 50 µg/mL with unmodified CNC and its cationic derivatives. Alternatively, J774A1 or PBMCs (peripheral blood mononuclear cells) were stimulated with CNC-AEMA2 in presence or absence of LPS (lipopolysaccharide). IL-1β secretion was analyzed by ELISA, mitochondrial function by JC-1 staining and ATP content. Intracellular and mitochondrial reactive oxygen species (ROS) were assessed by DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) and MitoSOX, respectively. Mitochondrial ROS and extracellular ATP were significantly increased in cells treated with CNC-AEMA2, which correlates with the strongest effects on IL-1β secretion in non-primed cells. CNC-AEMA2 also induced IL-1βsecretion in LPS-primed and non-primed PBMCs. Our data suggest that the increases in mitochondrial ROS and ATP release induced by CNC-AEMA2 may be associated with its capability to evoke immune response. We demonstrate the first evidence that newly synthesized cationic cellulose nanocrystal derivative, CNC-AEMA2, has immunogenic properties, which may lead to the development of a potential non-toxic and safe nanomaterial to be used as a novel adjuvant for vaccines.
Keywords: AEM, aminoethylmethacrylate; AEMA, aminoethylmethacrylamide; ASC, apoptosis-associated speck-like protein containing a CARD; CNCs, cellulose nanocrystals; Cationic needle-like nanomaterial; Cellulose nanocrystals; DAMPS, danger-associated molecular pattern molecules; DSL, Dynamic light scattering; ELISA, enzyme-linked immuno assay; H2DCF-DA, 2′, 7′-dichlorodihydrofluorescein diacetate; HRP, horseradish peroxidase-conjugated; HTCFNWs, high-temperature calcined fullerene nanowhiskers; IL-1β; IL-1β, Interleukin 1β; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide; LPS, lipopolysaccharide; MWCNTs, needle-like multi-wall carbon nanotubes; Mitochondrial ROS; Mouse macrophages; NLRP3 inflammasome; NLRP3, NOD-like receptor, pyrin domain-containing 3; PAMPs, pathogen-associated molecular pattern molecules; PBMCs, peripheral blood mononuclear cells; ROS, reactive oxygen species; SAA, serum amyloid A; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel; TMB, 3,3′,5,5′ tetramethylbezidine.