Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Poly(ethylene imine) nanocarriers do not induce mutations nor oxidative DNA damage in vitro in MutaMouse FE1 cells.

Genotoxicity information on polymers used for gene delivery is scant, but of great concern, especially when developing polymeric nanocarriers as nonviral vector systems for cancer treatment. The genotoxicity of some engineered nanomaterials, e.g., metal oxides like ZnO, TiO2, and CuO but also carbon based materials like carbon black or nanotubes, has commonly been related to oxidative stress, and subsequent inflammation. Recent studies of poly(ethylene imine) (PEI)-based polymers, important nonviral vector systems for pDNA and siRNA, might raise concerns because of their toxic effects dominated by cellular oxidative stress and inflammatory responses, similar to the mentioned effects of engineered nanoparticles. In this study, we employed a FE1-MutaMouse lung epithelial cell line based mutation assay to determine the genotoxicity of three PEI-based polymers and nanosized zinc oxide particles (NZO), all of which have previously been shown to trigger oxidative stress and inflammation. In addition, oxidative DNA damage (8-OH-dG) in FE1 cells was assessed by ELISA. The well-known carcinogen benzo[a]pyrene (B[a]P) was used as positive control. FE1 lung epithelial cells were exposed for eight sequential 72 h incubations, and reporter-gene mutation frequency or 8-OH-dG formation was determined to assess mutagenicity and oxidative DNA damage, respectively. No cytotoxic effects were detected at the exposure levels examined, which are representative of PEI concentrations normally used in in vitro transfection studies. In contrast to B[a]P, neither PEI-polymers nor NZO showed any significant mutagenic activity or oxidative DNA damage in the exposed cells, although PEI-based polymers have been shown to generate significant levels of cellular stress and inflammatory responses. We suggest that the lack of any detectable mutagenic/genotoxic activity of the PEI-based polymers studied here is a crucial step toward a safe use of such nanocarriers in clinical trials.

Detrimental effects of microplastic exposure on normal and asthmatic pulmonary physiology.

Concerns that airborne microplastics (MP) may be detrimental to human health are rising. However, research on the effects of MP on the respiratory system are limited. We tested the effect of MP exposure on both normal and asthmatic pulmonary physiology in mice. We show that MP exposure caused pulmonary inflammatory cell infiltration, bronchoalveolar macrophage aggregation, increased TNF-α level in bronchoalveolar lavage fluid (BALF), and increased plasma IgG1 production in normal mice. MP exposure also affected asthma symptoms by increasing mucus production and inflammatory cell infiltration with notable macrophage aggregation. Further, we found co-labeling of macrophage markers with MP incorporating fluorescence, which indicates phagocytosis of the MP by macrophages. A comparative transcriptomic analysis showed that MP exposure altered clusters of genes related to immune response, cellular stress response, and programmed cell death. A bioinformatics analysis further uncovered the molecular mechanism whereby MP stimulated production of tumor necrosis factor and immunoglobulins to activate a group of transmembrane B-cell antigens, leading to the modulation of cellular stress and programmed cell death in the asthma model. In summary, we show that MP exposure had detrimental effects on the respiratory system in both healthy and asthmatic mice, which calls for urgent discourse and action to mitigate environmental microplastic pollutants.

PEGylation affects cytotoxicity and cell-compatibility of poly(ethylene imine) for lung application: structure-function relationships.

Poly(ethylene imine) (PEI) has widely been used as non-viral gene carrier due to its capability to form stable complexes by electrostatic interactions with nucleic acids. To reduce cytotoxicity of PEI, several studies have addressed modified PEIs such as block or graft copolymers containing cationic and hydrophilic non-ionic components. Copolymers of PEI and hydrophilic poly(ethylene glycol) (PEG) with various molecular weights and graft densities were shown to exhibit decreased cytotoxicity and potential for DNA and siRNA delivery. In this study, we evaluated the cytotoxicity and cell-compatibility of different PEGylated PEI polymers in two murine lung cell lines. We found that the degree of PEGylation correlated with both cytotoxicity and oxidative stress, but not with proinflammatory effects. AB type copolymers with long PEG blocks caused high membrane damage and significantly decreased the metabolic activity of lung cells. In addition, they significantly increased the release of two lipid mediators such as 8-isoprostanes (8-IP) and prostaglandin E(2) (PGE(2)) in a dose-dependent manner. In contrast, the cytokine profiles which indicated high levels of acute-phase cytokines such as TNF-alpha, IL-6, and G-CSF did not follow any clear structure-function relationship. In conclusion, we found that modification of PEI 25 kDa with high degree of PEGylation and low PEG chain length reduced cytotoxic and oxidative stress response in lung cells, while the proinflammatory potential remained unaffected. A degree of substitution in the range of 10 to 30 and PEG-chain lengths up to 2000 Da seem to be beneficial and merit further investigations.

Toxicity pathway focused gene expression profiling of PEI-based polymers for pulmonary applications.

Polyethylene imine (PEI) based polycations, successfully used for gene therapy or RNA interference in vitro as well as in vivo, have been shown to cause well-known adverse side effects, especially high cytotoxicity. Therefore, various modifications have been developed to improve safety and efficiency of these nonviral vector systems, but profound knowledge about the underlying mechanisms responsible for the high cytotoxicity of PEI is still missing. In this in vitro study, we focused on stress and toxicity pathways triggered by PEI-based vector systems to be used for pulmonary application and two well-known lung toxic particles: fine crystalline silica (CS) and nanosized ZnO (NZO). The cytotoxicity profiles of all stressors were investigated in alveolar epithelial-like type II cells (LA4) to define concentrations with matching toxicity levels (cell viability >60% and LDH release <10%) for subsequent qRT-PCR-based gene array analysis. Within the first 6 h pathway analysis revealed for CS an extrinsic apoptotic signaling (TNF pathway) in contrast to the intrinsic apoptotic pathway (mitochondrial signaling) which was induced by PEI 25 kDa after 24 h treatment. The following causative chain of events seems conceivable: reactive oxygen species derived from particle surface toxicity triggers TNF signaling in the case of CS, whereby endosomal swelling and rupture upon endocytotic PEI 25 kDa uptake causes intracellular stress and mitochondrial alterations, finally leading to apoptotic cell death at higher doses. PEG modification most notably reduced the cytotoxicity of PEI 25 kDa but increased proinflammatory signaling on mRNA and even protein level. Hence in view of the lung as a sensitive target organ this inflammatory stimulation might cause unwanted side effects related to respiratory and cardiovascular disorders. Thus further optimization of the PEI-based vector systems is still needed for pulmonary application.

In vitro cytotoxic and immunomodulatory profiling of low molecular weight polyethylenimines for pulmonary application.

Polyethylenimines (PEI) are potent non-viral nucleic acid delivery vehicles used for gene delivery and RNA interference (RNAi). For non-invasive pulmonary RNAi therapy the respiratory tissue is an attractive application route, but offers particularly unwanted side-effects like cytotoxicity as well as inflammatory and immune responses. In the current study, we determined the most crucial issues of pulmonary applications for two low molecular weight PEIs in comparison to the well-known lung toxic crystalline silica. Cytotoxic effects and inflammatory responses were evaluated in three murine pulmonary target cell lines, the alveolar epithelial (LA4), the alveolar macrophage (MH-S) and the macrophage-monocyte-like (RAW 264.7) cell line. For both PEIs, cytotoxicity was detected most prominently in the alveolar epithelial cells and only at high doses. Cytokine responses, in contrast were observed already at low PEI concentrations and could be divided into three groups, induced (i) by free PEI (IL-6, TNF-alpha, G-CSF), (ii) by PEI/siRNA complexes (CCL2, -5, CXCL1, -10), or (iii) unaffected by either treatment (IL-2, -4,-7, -9, and CCL3). We conclude that even for the respiratory tissue both PEIs represent powerful siRNA delivery tools with reduced cytotoxicity and minor proinflammatory potency. However, in relation to response levels observed upon crystalline silica exposures, some PEI induced proapoptotic and proinflammatory responses might not be considered completely harmless, therefore further in vivo investigations are advisable.

Surface modification and size dependence in particle translocation during early embryonic development.

Since the mid-1990 s, the number of studies linking air pollutants to preterm and low birth weight, as well as to cardiac birth defects, has grown steadily each year. The critical period in the development of mouse embryos begins with the commencement of gastrulation at day 7.5 of gestation. Our aim is to examine the role of particles size and surface modification in particle translocation during this early embryonic development. Fluorescent polystyrene particles (PS) were employed because they offer an efficient and safe tracking method. Pregnant female mice were sacrificed at 7.5 days of gestation. After cutting open the deciduas, the parietal endoderm was carefully separated and removed. Different sizes of amine- and carboxyl-modified PS beads were injected via the extraembryonic tissue. The embryos were incubated for 12 h, and were investigated under fluorescent microscopy, confocal microscopy, and mesoscopic fluorescence tomography. The results show that 20-nm carboxylic PS distribute in the embryonic and extraembryonic germ layers of ectoderm, mesoderm, and endoderm. Moreover, when the particles are bigger than 100 nm, PS accumulate in extraembryonic tissue, but nevertheless 200-nm amine-modified particles can pass into the embryos. Interestingly, a growth inhibition was observed in the embryos containing nanoparticles. Finally, the stronger translocation effect is associated with amine-modified PS beads (200 nm) instead of the smaller (20 nm, 100 nm) carboxyl ones.

RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing.

Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ∼80% reduction in tumour size without associated inflammation.

Comparative in vivo study of poly(ethylene imine)/siRNA complexes for pulmonary delivery in mice.

Pulmonary siRNA delivery offers a new way to treat various lung diseases. Poly(ethylene imines) (PEIs) are promising cationic nanocarriers and various modifications are still under investigations to improve their cytotoxicity and efficacy for siRNA delivery. In this study, we analyzed two different types of PEI-based nanocomplexes in mice after intratracheal administration regarding their toxicity and efficacy in the lungs. Ubiquitously enhanced green fluorescent protein (EGFP) expressing transgenic and BALB/c mice were intratracheally instilled with 35µg siRNA complexed with the different types of PEI nanocarriers. Lung toxicity and inflammation were investigated after 24h, 3d and 7d treatment and knockdown of EGFP expression was analyzed by flow cytometry and fluorescence microscopy five days post instillation. Three different polyplexes caused more than 60% knockdown of EGFP expression, but only the fatty acid modified low molecular weight PEI 8.3kDa (C16-C18-EO25)1.4 specifically reduced EGFP expression in CD45+ leucocytes (25±12%) and CD11b-/CD11c+ lung macrophages (36±14%). Hydrophobic and hydrophilic PEG modifications on PEI caused severe inflammatory response and elevated levels of IgM in broncho-alveolar fluid (BALF). Thus, the PEG modification reduced cytotoxicity, but elevated the immune response and proinflammatory effects. Further investigations of the proinflammatory and immunomodulatory effects of the PEI-modified carriers are necessary to clarify the highly unspecific knockdown effects in the lung in more detail. Nevertheless, the more hydrophobic modification of PEI based non-viral vector system appeared to be a promising approach for improved siRNA therapeutics offering successful pulmonary siRNA delivery.