3D Printer Waste Nanoparticles and Their Internalization in Human Colorectal Cells (HCT15), as in Vitro Cell Model of Nanoplastics Absorption

Rapid globalization and technological advancement have led to increased consumption of plastics due to their versatility, low production costs, and ease of manufacturing [1]. Among these technologies, additive manufacturing (AM) methods such as stereolithography (SLA), and digital light processing (DLP), used in 3D printing (3D-P), have gained popularity. These technologies use UV light to transform liquid photopolymers into solid models, making them useful in various applications, including medical device production [2] and are widely used for domestic purpose [3]. However, the improper handling and storage of 3D printing residues pose significant environmental and health risks [4]. During the printing process, volatile compounds, micro- and nanoplastic particles can be released, and polymers related to 3D printers have been detected in various environments, including beaches, wastewater treatment plants, and even in the food chain [1,5]. Microparticles due to their size, can be accumulated in marine life [6] such as marine algae, and have the potential to be transferred to other organisms [7], reaching humans. This fact has been observed with their presence in human feces [7, 8] and even in the human placenta [9], indicating their widespread distribution in the human body. This highlights the urgent need to understand the health implications of nanoplastic exposure and to develop strategies to mitigate their environmental impact. However, challenges remain in effectively separating and purifying waste to maintain its quality for recycling [10]. While in-vitro studies have focused on the effects of plastic microparticles on cells, research on nanoparticles is limited, particularly those produced by 3D printing technologies [8]. This study aimed to analyze the effects of 3D printer waste nanoparticles, specifically polymethyl methacrylate-polyurethane (PMMA-PU) nanoparticles (NPs), on human colorectal cells (HCT15). The nanoparticles were obtained through a polymerization process involving residual resin and ethanol, followed by exposure to UV radiation [1]. The nanoparticles were characterized using dynamic light scattering (DLS) to determine their hydrodynamic diameter and zeta potential in different mediums, including ultrapure water, PBS, and DMEM. In-vitro toxicity assays were conducted using the HCT15 [11] cell line, which was maintained in DMEM supplemented with fetal bovine serum (FBS) and antibiotic/antimycotic. Cell viability and proliferation were assessed using the AlamarBlue® assay. In addition, Nitrite oxide (NO) concentrations were quantified as a physiological parameter and mitochondrial membrane permeability (MMP) was assessed using Mito-ID® Red staining. The DLS characterization revealed that the PMMA-PU nanoparticles had an average hydrodynamic diameter of 250 nm in ultrapure water (Figure 1A), with a zeta potential of -26.1 mV, while in PBS and DMEM, the nanoparticles showed similar size distributions between them, obtaining 32.64 d.nm (Figure 1B) and 38.11 d.nm (Figure 1C) respectively, with similar peaks indicating no significant differences between media. Cell viability assays showed that the HCT15 cells exhibited varying responses to different concentrations of PMMA-PU nanoparticles. At 1 mg/mL, the cells initially showed increased fluorescence at 24 hours, followed by a decrease at 72 hours. Lower concentrations (1 µg/mL) showed higher fluorescence at 24 hours but lower fluorescence at 48 hours, with a subsequent increase at 72 hours. Other concentrations (100 µg/mL, 10 µg/mL, 100 ng/mL, and 10 ng/mL) did not show significant changes in cell viability compared to the control (Figure 2). At the same time, NO measurements indicated that the presence of PMMA-PU nanoparticles led to a significant increase in nitrite levels at 1 mg/mL after 48 hours. However, at lower concentrations, nitrite levels were lower than control, this suggests that NPs can lead to oxidative stress causing an alteration to the cellular metabolism and contributing to cytotoxicity (Figure 3). Nevertheless, since concentrations between 1 µg/ml and 10 µg/ml have shown the most significant changed during the last assays, we selected the 10 µg/ml for further assays. Mitochondrial staining revealed that cells exposed to 10 µg/mL of PMMA-PU nanoparticles exhibited increased mitochondrial activity and morphological changes (Figure 4). These changes suggest that nanoparticles may induce cellular stress and alter mitochondrial function due to increment in the mitochondria number, which were observed densely packed around the NPs (Figure 4F). These results highlight showed to affect cell viability and cause mitochondrial permeability increment in HCT15 cells. We can conclude that exposure to PMMA-PU nanoparticles can lead to cellular stress, and mitochondrial damage in in-vitro experiments, highlighting the need for further research into health and environmental impacts of 3D printing technologies waste management. In addition, is important to adopt a circular economy approach and improving recycling practices, which are essential steps toward mitigating the risks associated with micro- and nanoplastic pollution [12]. Particle distribution of the PMMA-PU sample by DLS in A) MiliQ water, B) PBS 1X and C) supplemented DMEM medium. Analysis of resazurin reduction vs. cell viability control of HCT15 line by AlamarBlue at 570 nm in the presence of different concentrations of PMMA-PU nanoplastics during the first A) 24, B) 48, and C) 72 hours. The difference is not significant for * P > 0.05; if P values are: ** P ≤ 0.05; there is significant difference. All X and Y axes correspond to that shown in Figure A. Measurement of nitrite concentration released by a cell culture of HCT15 over a 3-day period. The difference is not significant for *(24 hours) ● (48 hours) ▴ (72 hours) P > 0.05. If P values are: **●●▴▴P ≤ 0.05; there is a significant difference. X values correspond to all the concentrations of PMMA-PU NPs. Confocal micrographs of HCT15 incubated with 10 µg/mL of PMMA-PU NPs (D, E, F) and controls (A, B, C) at 24, 48 and 72 hours. All micrographs present the same scale shown in figure A.