Disruption of endosomal transport in cyclodextrin-mediated cytotoxicity

Cyclodextrins (CDs) are widely used as pharmaceutical excipients, yet their systemic use is constrained by cytotoxic effects that remain incompletely understood. While β-cyclodextrins are commonly associated with cholesterol extraction, it remains unclear whether this mechanism alone explains their biological activity. Here, we investigate the effects of native and modified CDs in human renal proximal tubule cells using complementary imaging, flow cytometry, and MALDI mass spectrometry. Distinct, host-dependent responses were observed. α-Cyclodextrin induced rapid membrane disruption at elevated concentrations, whereas methylated β-cyclodextrin (Meβ-CD) produced a progressive phenotype characterized by increased membrane permeability, impaired lipid internalization, and reduced cell proliferation under sub-lethal conditions. Despite these differences, both Meβ-CD and γ-cyclodextrin enhanced cholesterol internalization without evidence of immediate depletion, indicating modulation of endogenous transport processes. In contrast, Meβ-CD selectively disrupted membrane lipid uptake and altered extracellular lipid composition, yielding vesicle fractions enriched in cholesterol esters and ceramides but depleted in phosphoinositides. These findings support a model in which cyclodextrins perturb phosphoinositide-dependent lipid trafficking pathways, providing a mechanistic link between altered lipid distribution and cytotoxic response, and informing the design of safer CD-based therapeutics.