Introduction: Silver nanoparticles (AgNPs) have widely documented cytotoxic properties that are influenced by size, shape, and surface chemistry. Further, AgNPs interact with proteins and cell membrane receptors, which influence cell uptake, gene expression and cytotoxicity. The relationship between NP physicochemical properties and biological function has not been fully elucidated and further insight will enable better design of AgNPs with a desired biological outcome. The purpose of this study was to investigate the cytotoxicity of AgNPs coated with a thermoresponsive polymer (TRP) with variable composition and aggregation state at 37°C. Here, we used the thermoresponsive copolymer poly(MEO2MA-co-OEGMA) (PMO), which enables manipulation of polymer physicochemical properties by changing the ratio of the hydrophobic monomer (MEO2MA) relative to the more hydrophilic monomer (OEGMA). In this study, PMO-coated AgNPs with varying ratios of MEO2MA:OEGMA were fabricated to investigate the relationship between AgNP surface chemistry, AgNP aggregation, cell uptake, and cytotoxicity.
Methods: PMO was synthesized from random copolymers of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) with MEO2MA:OEGMA molar ratios of 95:5, 90:10 and 80:20, as previously described, and grafted onto citrate stabilized 10nm AgNP via ligand displacement reactions. Murine L-929 fibroblasts were seeded in 96 wells culture plates (20,000 cells/well) and exposed to PMO-coated AgNPs (0.5-5 nM) at 37°C for 8 hours in the presence and absence of serum proteins. In some experiments, cells were treated with endocytosis blockers (wortmannin, dansylcadaverine, nystatin) to inhibit specific endoctytosis pathways. Cell morphology and AgNP aggregates were imaged with phase microscopy. Cytotoxicity was quantified via an LDH-based assay and cell uptake was measured with spectrophotometry.
Results and Discussion: Microscopic images showed that (95:5) PMO coatings elicited substantial AgNP aggregation and accumulation in cells at 37°C relative to (90:10) and (80:20) PMO compositions. This is expected as (95:5) PMO has an LCST of 28°C and; therefore, is hydrophobic at the cell culture temperature of 37°C. Cell uptake and cytotoxicity was correlated to increased MEO2MA content in the PMO coating, as the percent cytotoxicity ranged from 98-60% with PMO compositions of (95:5) to (80:20). The extent of cell uptake and cytoxicity was significantly attenuated in the absence of serum proteins, with a maximum of 70% reduction in cytotoxicity for (95:5) PMO coatings. This suggests that AgNP surface hydrophobicity and serum-protein adsorption is a significant mediator of cell uptake and subsequent cellular responses. Treatment of cells with wortmannin, a PI3K inhibitor, significantly reduced cell uptake and cytotoxicity of all PMO-AgNP formulations. Dansylcadaverine and nyastin, receptor-mediated endocytosis inhibitors, significantly reduced cell uptake and cytotoxicity for (95:5) PMO-AgNP in the presence of serum proteins; however, the inhibitory effect of both molecules was eliminated in the absence of serum proteins.
Conclusion: The results of this study demonstrate a direct relationship between the surface hydrophobicity of PMO-coated AgNPs and cell uptake and cytotoxicity. Serum protein adsorption to PMO-coated AgNP surfaces impacts both cell uptake and cytotoxicity as both were reduced in the absence of serum protein or in the presence of receptor-mediated endocytosis inhibitors. Thus, NP surface hydrophobicity and aggregation may an important consideration in the design of NPs targeted for cellular systems.