Carboxymethylated Cellulose Nanofibrils with Controlled Substitution: Physicochemical Properties and In Vitro Biosafety Assessment

Abstract

Cellulose nanofibrils (CNF) are increasingly explored for biomedical applications; however, the relationship between surface functionalization and biological responses remains incompletely understood. In this study, CNF were carboxymethylated to controlled degrees of substitution (DS) using kraft pulp as a starting material, and their physicochemical properties and in vitro biosafety were evaluated. Increasing DS altered crystallinity and surface charge, resulting in measurable changes in zeta potential and structural characteristics. Cell viability was assessed in HepG2, HEK293, and RAW 264.7 cells across a concentration range of 100 to 1000 µg/mL using complementary assays. All tested materials exhibited concentration-dependent trends in cell viability. However, under the tested conditions, the observed effects remained within the non-cytotoxic range. Among the tested samples, CM CNF with DS 0.2 showed stable cell viability and limited apoptosis-related responses comparable to those of hyaluronic acid (HA), while samples with lower or higher substitution levels showed modest reductions in viability at higher concentrations. Apoptosis-related gene and protein expression analyses further indicated limited transcriptional and translational changes relative to the vehicle-treated control. Overall, the findings suggest that the degree of carboxymethylation influences cell–material interactions in a concentration-dependent manner, while maintaining biosafety within the tested DS range.