Layered Double Hydroxide Nanoparticles Induce Composition-Dependent Cytotoxic and Phenotypic Effects in Mammalian Cells

Layered double hydroxides (LDHs) are increasingly explored for agricultural, environmental, and biodelivery applications, but their composition-dependent effects on mammalian cells remain insufficiently defined. Here, we synthesized Al–Ni, Al–Co, and Al–Cu LDH nanoparticles and evaluated their physicochemical properties and biological responses across exposure-relevant mammalian cell models. The formulations showed hydrodynamic diameters of approximately 200–300 nm, moderate dispersity, strongly positive surface charge, and characteristic lamellar LDH features. Cytotoxicity was assessed using MTT, Calcein-AM, and Hoechst–PI assays in HaCaT, A549, and HT-29 cells, representing dermal, pulmonary, and intestinal exposure contexts, together with NIH/3T3 fibroblasts as a sensitive comparative model. LDH toxicity was strongly dependent on metal composition and cell type, following a general trend where Al–Cu showed the highest overall cytotoxic activity, while Al–Ni and Al–Co produced responses dependent on the formulation and cell type. To detect cellular perturbations beyond overt viability loss, we applied high-content imaging using Live Cell Painting. Multiparametric single-cell profiling revealed composition- and dose-dependent alterations in acidic vesicle organization, nuclear texture, and cytoplasmic granularity. Notably, phenotypic deviations were detected at concentrations below those producing measurable effects in conventional viability assays, and linear discriminant analysis separated the phenotypic signatures induced by the three LDH formulations. Together, these findings show that LDH biological activity cannot be generalized across metal compositions and that high-content phenotypic profiling provides added sensitivity for detecting early cellular perturbations. This integrated approach supports composition-aware nanosafety evaluation and may inform the safer development of LDH-based technologies for agricultural and biotechnological applications.