# Toxicity of High-Density Polyethylene Nanoparticles in Combination with Silver Nanoparticles to Caco-2 and HT29MTX Cells Growing in 2D or 3D Culture

## TL;DR

This study examines how high-density polyethylene and silver nanoparticles together affect intestinal cells in different culture conditions.

## Contribution

The study reveals that combined nanoparticle exposure shows reduced toxicity in mixed cell cultures compared to monocultures.

## Key findings

- HDPE and AgNPcit nanoparticles mutually inhibit each other's cellular uptake.
- Combined nanoparticle toxicity is less than the sum of individual effects in monocultures.
- Triple-culture cells show reduced toxicity compared to monocultures.

## Abstract

The enormous applications of various nanoparticles (NPs) have raised the possibility that humans may be simultaneously exposed to mixtures of them in real life. Realistically, this situation may apply to plastic NPs, mainly derived from the breakdown of larger plastics, and to silver NPs, both of which are among the most frequently detected NPs in the envirnment due to their applications in healthcare, consumer products, and water purification. Although numerous studies have examined the toxicity of plastic and silver NPs individually, knowledge of their combined toxicity remains limited. Hence, the main objective of our study was to investigate the toxicity of high-density polyethene nanoparticles (HDPE NPs), thermally isolated from food-cooking bags, in combination with citrate-stabilised silver nanoparticles (AgNPcit) to Caco-2 and HT29MTX cells growing in 2D monoculture or in 3D triple-culture with Raji cells. Cellular uptake of NPs was quantified from the side-scatter (SSC) signal in flow cytometry; toxicity was evaluated by the neutral red assay; apoptosis was evaluated by the Annexin V method; and induction of oxidative stress was evaluated by a fluorescent method using DCFDA and DHR probes. Both cell lines took up both types of NPs; however, HT29MTX cells were more effective in the NPs’ uptake. Interestingly, HDPE NPs and AgNPcit mutually inhibited each other’s uptake, which suggests a similar mechanism of entry. Both types of NPs were toxic to both cell lines growing in monoculture; Caco-2 cells were more susceptible than HT29MTX. The toxicity was attributed to the induction of oxidative stress and associated apoptosis. In line with the mutual inhibition of the NPs’ uptake, the toxic effect of both NPs in the mixture was less than that expected as the sum of individual treatments. The toxic effects of both NPs or their mixture were less pronounced in the triple-culture Caco-2/HT29MTX/Raji, than in Caco-2 and HT29MTX growing in monoculture.