The Immunotoxic Effects of Environmentally Relevant Micro- and Nanoplastics: An In Vitro Approach to Human Health Hazard Assessment

Micro- and nanoplastics (MNPs), polymeric fragments of < 5 mm and < 1 µm, respectively, are increasingly recognized as emergent human-health hazards. After inhalation or ingestion, these particles may cross epithelial barriers, disseminate via the circulatory system, accumulate in secondary organs, and interact with innate immune cells such as macrophages and dendritic cells. This thesis investigates how physicochemical parameters shape immunomodulatory outcomes. More than 20 diverse particles (PS, PVC, PP, PA6.6, PE; 50 nm–10 µm; spherical or fragmented; pristine, oxidized and/or environmentally weathered) were subjected to physicochemical characterization and evaluated in complementary human in vitro systems, including Caco-2/HT29-MTX intestinal co-cultures, THP-1-derived macrophages with various reporters, and human monocyte-derived dendritic cells. In Chapter 2, responses of human MoDCs to environmentally weathered PS particles (0.2, 1, 10 µm) were assessed. Weathering was achieved by shaking virgin beads in river water under natural sunlight for four weeks. All particle sizes were internalized by MoDCs, and heat-inactivated plasma modified uptake in a size-dependent manner. Cell viability remained above 90% for all treatments. Weathered 0.2 µm PS induced up-regulation of maturation markers CD83 and CD86 and promoted allogeneic CD8+ T-cell activation, whereas pristine PS and larger weathered particles were largely inert. These findings indicate that nano-scale, weathered PS can act as potent immune adjuvants. Chapter 3 examined mechanically fragmented PVC, PP and PA6.6 (<1 µm and 1–5 µm) and spherical PS beads (50–1000 nm) in THP-1 macrophages. All MNPs increased lysosomal activity and were taken up. Fragmented PP/talc caused loss of mitochondrial activity and LDH release at 100 µg/ml, while PVC and PA6.6 were cytotoxic already at 1–10 µg/ml. Distorted Grid dosimetry modelling revealed rapid settling and nearly full dose delivery for PVC and PA6.6, while PP/talc delivered only ~45% of the nominal dose. Despite cytotoxicity, none of the particles activated NF-κB or induced pro-inflammatory cytokines, indicating that top-down MNPs impair viability but do not directly elicit inflammatory responses under short-term exposure. This highlights the need to move beyond uniform PS beads in hazard assessment. Chapter 4 compared oxidized, fragmented nanoPE (350 nm) and nanoPP (180 nm) with spherical 200 nm pristine nanoPS. NanoPE and nanoPP possessed rough, oxidized surfaces, in contrast to smooth nanoPS. At 1–10 µg/ml, nanoPE and nanoPP triggered NF-κB activation, cytokine release (TNF-α, IL-6, IL-1β), membrane damage and NLRP3 inflammasome activation, whereas nanoPS remained inert. Partial polymyxin B inhibition and low detected LPS levels implicated surface oxidation as the main driver of pro-inflammatory signalling. Chapter 5 showed that neither oxidized nor pristine nanoplastics induced direct epithelial toxicity. However, oxidized nanoPE and nanoPP amplified IL-8 and CCL2 release when combined with a low-dose cytomix, indicating that weathered nanoplastics can potentiate pre-existing inflammation. Overall, the thesis demonstrates that MNP immunotoxicity is governed predominantly by size, morphology, and weathering-related surface chemistry rather than polymer type. Incorporating dosimetry, co-stimulation scenarios and exposure-realistic materials is essential for accurate human-health hazard assessment. A tiered risk-assessment approach is recommended, alongside improved exposure metrics, delivered-dose estimation and integration with epidemiological data, to support future regulatory decision-making.