Bovine Serum Albumin Bends Over Backward to Interact with Aged Plastics: A Model for Understanding Protein Attachment to Plastic Debris.

Plastic pollution, a major environmental crisis, has a variety of consequences for various organisms within aquatic systems. Beyond the direct toxicity, plastic pollution has the potential to absorb biological toxins and invasive microbial species. To better understand the capability of environmental plastic debris to adsorb these species, we investigated the binding of the model protein bovine serum albumin (BSA) to polyethylene (PE) films at various stages of photodegradation. Circular dichroism and fluorescence studies revealed that BSA undergoes structural rearrangement to accommodate changes to the polymer's surface characteristics (i.e., crystallinity and oxidation state) that occur as the result of photodegradation. To understand how protein structure may inform docking of whole organisms, we studied biofilm formation of bacteriaShewanella oneidensison the photodegraded PE. Interestingly, biofilms preferentially formed on the photodegraded PE that correlated with the state of weathering that induced the most significant structural rearrangement of BSA. Taken together, our work suggests that there are optimal physical and chemical properties of photodegraded polymers that predict which plastic debris will carry biochemical or microbial hitchhikers.

Size Distributions of Microplastics in the St Louis Estuary and Western Lake Superior.

Identifying the sources and fate of microplastics in natural systems has garnered a great deal of attention because of their implications for ecosystem health. This work characterizes the size fraction, morphology, color, and polymer composition of microplastics in western Lake Superior and its adjacent harbor sampled in August and September 2021. The results reveal that the overall microplastic counts are similar, with the harbor stations ranging from 0.62 to 3.32 microplastics per liter and the lake stations ranged from 0.83 to 1.4 microplastics per liter. However, meaningful differences between the sample locations can be seen in the size fraction trends and polymer composition. Namely, the harbor samples had relatively larger amounts of the largest size fraction and more diversity of polymer types, which can be attributed to the urbanized activity and shorter water residence time. Power law size distribution modeling reveals deviations that help in the understanding of potential sources and removal mechanisms, although it significantly underpredicts microplastic counts for smaller-sized particles (5-45 µm), as determined by comparison with concurrently collected microplastic samples enumerated by Nile Red staining and flow cytometry.

UV Light Degradation of Polylactic Acid Kickstarts Enzymatic Hydrolysis.

Polylactic acid (PLA) and bioplastics alike have a designed degradability to avoid the environmental buildup that petroplastics have created. Yet, this designed biotic-degradation has typically been characterized in ideal conditions. This study seeks to relate the abiotic to the biotic degradation of PLA to accurately represent the degradation pathways bioplastics will encounter, supposing their improper disposal in the environment. Enzymatic hydrolysis was used to study the biodegradation of PLA with varying stages of photoaging. Utilizing a fluorescent tag to follow enzyme hydrolysis, it was determined that increasing the amount of irradiation yielded greater amounts of total enzymatic hydrolysis by proteinase K after 8 h of enzyme incubation. While photoaging of the polymers causes minimal changes in chemistry and increasing amounts of crystallinity, the trends in biotic degradation appear to primarily be driven by photoinduced reduction in molecular weight. The relationship between photoaging and enzyme hydrolysis appears to be independent of enzyme type, though commercial product degradation may be impacted by the presence of additives. Overall, this work reveals the importance of characterizing biodegradation with relevant samples that ultimately can inform optimization of production and disposal.

Quantification of Polymer Surface Degradation Using Fluorescence Spectroscopy.

One solution to minimizing plastic pollution is to improve reuse and recycling strategies. Recycling, however, is limited by the overall degradation of plastics being used, and current techniques for monitoring this plastic degradation fail to observe this in its early stages, which is key for optimizing reusability. This research seeks to develop an inexpensive, reproducible, and nondestructive technique for monitoring degradation of polyethylene (PE) and polypropylene (PP) materials using Nile red as a fluorescent probe. Changes in Nile red's fluorescence spectra were observed upon exposure to stained, aged PE and PP samples. As the surface hydrophobicity of the plastic decreases, Nile red's fluorescence signal undergoes a corresponding signal shift to longer wavelengths (lower energy). The trends seen in the fluorescent profile were related to more commonly used measurements of plastic degradation, namely, the carbonyl index from infrared spectroscopy and bulk crystallinity from calorimetry. Results demonstrate clear trends in fluorescence spectra shifts as related to the chemical and physical changes to the plastics, with trends dependent on the polymer type but independent of polymer film thickness. The strength of this technique is divided into two defined fits of the fluorescence signal; one fit characterizes the degradation throughout the whole range of degradative oxidation and the other is tailored to provide insight into the early stages of degradation. Overall, this work establishes a characterization tool that assesses the extent of plastics' degradation, which may ultimately impact our ability to recover plastics and minimize plastic waste.

Sewage sludge induces changes in the surface chemistry and crystallinity of polylactic acid and polyethylene films.

Plastic pollution is a major threat facing our environment. To understand the full effects, we must first characterize how plastics break down in environmental systems. Heretofore, there has been little work examining how exposure to sewage sludge facilitates the degradation of plastics, particularly of plastics that have been previously weathered. Herein, we characterize how the crystallinity, surface chemistry, and morphology of polylactic acid (PLA) and polyethylene (PE) films change due to sludge exposure. In this work, sludge-induced changes in carbonyl index were found to depend on the level of prior exposure to ultraviolet (UV) irradiation. The carbonyl indices of un-irradiated films increased while those of UV-aged films decreased after 35 days of sludge exposure. In addition, the carbon­oxygen and hydroxyl bond indices of PE films increased with sludge exposure, suggesting the surface oxidation of PE. As for PLA, crystallinity was found to increase with sludge exposure, consistent with a chain scission mechanism. This work will help to predict the behavior of plastic films after transfer from wastewater to sewage sludge.

Photodegradation of polyolefin thin films in simulated freshwater conditions.

Polypropylene (PP) and polyethylene (PE) are commonly used polyolefins in a variety of applications, which have resulted in their accumulation in the environment. Once in the environment, these polymers undergo various chemical and physical transformations as the result of environmental stressors such as sunlight. While photodegradation has been studied for decades, there are key gaps in knowledge on the phototransformations of polyolefins that occur under aqueous conditions. Therefore, the goal of this study is to characterize the phototransformations of PP and PE in simulated freshwater conditions. Polymer thin films were irradiated with 254 nm and 350 nm UV light in air, ultra-pure water, and solutions of dissolved organic matter (DOM) to simulate natural systems. Irradiated plastics were evaluated for oxidation and chain scission. It was observed using Fourier transform infrared spectroscopy (FTIR) that oxidation in aqueous environments happened at a slower rate compared to oxidations in air. However, photo-oxidation was accelerated in the presence of DOM compared to ultrapure water, with singlet oxygen and hydroxyl radical causing varied amounts of degradation depending on the polymer. The vinyl characteristic, a chain scission product, revealed an increased yield but the reaction rate showed that these photoproducts were more likely to occur when oxidation is less favorable. Compared to naturally weathered samples, lab observed transformations were on par with naturally degraded samples and support the importance of the in-lab measurements. This work quantifies the extent and rate of photodegradation pathways in PP and PE to demonstrate the importance of photodegradation in aquatic systems.

The effect of plastic additives on Shewanella oneidensis growth and function.

Plastic waste has the potential for significant consequences on various ecosystems; yet, there are gaps in our understanding of the interaction of bacteria with polymer additives. We studied the impact of representative additive molecules to the viability and cell function of Shewanella oneidensis MR-1. Specifically, we explored the toxicity of three bisphenols (bisphenol A (BPA), bisphenol S (BPS), and tetrabromo bisphenol A (TBBPA)) and two diesters (dibutyl sebacate (DBS) and diisobutyl phthalate (DIBP)) in order to evaluate the generalizability of toxicity based on similar molecular structures. TBBPA caused significant, dose-dependent decreases in viability for acute (4 h) exposures in aerobic and anaerobic conditions. While the other 4 additives showed no significant toxicity upon 4 h exposures, chronic (2 day) anaerobic exposures revealed a significant impact to growth. BPA and BPS cause a significant decrease in growth rates for all exposure doses (8-131 µM) while DBS and DIBP had decreases in growth for the lowest exposure concentrations, though recovered to growth rates similar to the control at the highest concentrations. This highlights that S. oneidensis may have the ability to use the diesters as a carbon source if present in high enough concentrations. Riboflavin secretion was monitored as a marker of cellular health. Most additives stimulated riboflavin secretion as a survival response. Yet, there was no generalizable trend observed for these molecules, indicating the importance of considering the nuances of molecular structure to toxicity responses and the need for further work to understand the consequences of plastic waste in our environment.

Characterizing microplastic size and morphology of photodegraded polymers placed in simulated moving water conditions.

Aquatic plastic debris experiences environmental stressors that lead to breakdown into smaller micro-sized plastic particles. This work quantified microplastic formation with the environmental stressors of UV irradiation followed by mechanical strain induced by movement of water with an emphasis on connecting our results to changes in the materials chemical/physical properties. Polypropylene, polyethylene, and polyethylene terephthalate thin films and polypropylene injection-molded sheets were irradiated with 254 nm UV light, placed into aquatic microcosms, collected through sieving, and counted under a microscope. Results showed increasingly more particles in smaller size classes, the smallest being 74-177 µm. Mechanical strain from the turbulent water caused 2.3-3× more microplastics to be formed for the thinnest (∼25 µm) film and 1.4-2× more for thicker films and sheets. The most common morphology of microplastics was fibers, particularly in thicker polypropylene samples, which was attributed to absorbance of the photons and the changes observed in the crystallinity and glass transition as measured with differential scanning calorimetry (DSC). When irradiated for 24, 48, or 72 h, longer irradiation resulted in more microplastics formed by polypropylene films, which correlated with changes in the glass transition temperature as measured by DSC and the extent of oxidation as measured with FTIR. Irradiation at 300 nm produced fewer microplastics due to slower kinetics of phototransformations. Overall, this work evaluates the impact of combined photodegradation and water motion toward microplastic particles formed. It provides quantitative evidence that mechanical strain of water movement exacerbates photo-induced formation of microplastics and shows that the existence of fibers in natural systems can be the result of photodegradation.

Photochemical Transformation of Poly(butylene adipate- co-terephthalate) and Its Effects on Enzymatic Hydrolyzability.

Biodegradable polyesters are being increasingly used to replace conventional, nondegradable polymers in agricultural applications such as plastic film for mulching. For many of these applications, poly(butylene adipate- co-terephthalate) (PBAT) is a promising biodegradable material. However, PBAT is also susceptible to photochemical transformations. To better understand how photochemistry affects the biodegradability of PBAT, we irradiated blown, nonstabilized, transparent PBAT films and studied their enzymatic hydrolysis, which is considered the rate-limiting step in polyester biodegradation. In parallel, we characterized the irradiated PBAT films by dynamic mechanical thermal analysis. The rate of enzymatic PBAT hydrolysis decreased when the density of light-induced cross-links within PBAT exceeded a certain threshold. Mass-spectrometric analysis of the enzymatic hydrolysis products of irradiated PBAT films provided evidence for radical-based cross-linking of two terephthalate units that resulted in the formation of benzophenone-like molecules. In a proof-of-principle experiment, we demonstrated that the addition of photostabilizers to PBAT films mitigated the negative effect of UV irradiation on the enzymatic hydrolyzability of PBAT. This work advances the understanding of light-induced changes on the enzyme-mediated hydrolysis of aliphatic-aromatic polyesters and will therefore have important implications for the development of biodegradable plastics.

Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species.

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

Impact of TiO2 nanoparticles on growth, biofilm formation, and flavin secretion in Shewanella oneidensis.

Understanding of nanoparticle impacts on critical bacteria functions allows us to gain a mechanistic understanding of toxicity and guides us toward design rules for creating safe nanomaterials. Herein, biofilm formation, a general bacteria function, and riboflavin secretion, a species-specific function, were monitored in Shewanella oneidensis, a metal reducing bacterium, following exposure to a variety of TiO2 nanoparticle types (synthesized, Aeroxide P25, and T-Eco). Transmission electron microscopy (TEM) images show that dosed nanoparticles are in close proximity to the bacteria, but they are not internalized. Using quartz crystal microbalance (QCM), it was revealed that S. oneidensis biofilm formation is slowed in the presence of nanoparticles. Though S. oneidensis grows more slowly in the presence of TiO2 nanoparticles, riboflavin secretion, a function related to the S. oneidensis metal reducing capacity, was increased significantly in a nanoparticle dose-dependent manner. Both changes in biofilm formation and riboflavin secretion are supported by changes in gene expression in nanoparticle-exposed S. oneidensis. This broad study of bacterial nanotoxicity, including use of sensitive analytical tools for functional assessments of biofilm formation, riboflavin secretion, and gene expression, has implications for total ecosystem health as the use of engineered nanoparticles grows.

Toxicity of engineered nanoparticles in the environment.

While nanoparticles occur naturally in the environment and have been intentionally used for centuries, the production and use of engineered nanoparticles has seen a recent spike, which makes environmental release almost certain. Therefore, recent efforts to characterize the toxicity of engineered nanoparticles have focused on the environmental implications, including exploration of toxicity to organisms from wide-ranging parts of the ecosystem food webs. Herein, we summarize the current understanding of toxicity of engineered nanoparticles to representatives of various trophic levels, including bacteria, plants, and multicellular aquatic/terrestrial organisms, to highlight important challenges within the field of econanotoxicity, challenges that analytical chemists are expertly poised to address.

Assessing nanoparticle toxicity.

Nanoparticle toxicology, an emergent field, works toward establishing the hazard of nanoparticles, and therefore their potential risk, in light of the increased use and likelihood of exposure. Analytical chemists can provide an essential tool kit for the advancement of this field by exploiting expertise in sample complexity and preparation as well as method and technology development. Herein, we discuss experimental considerations for performing in vitro nanoparticle toxicity studies, with a focus on nanoparticle characterization, relevant model cell systems, and toxicity assay choices. Additionally, we present three case studies (of silver, titanium dioxide, and carbon nanotube toxicity) to highlight the important toxicological considerations of these commonly used nanoparticles.

Toward correlation in in vivo and in vitro nanotoxicology studies.

Nanomaterials have the promise of revolutionizing current treatment and diagnosis of diseases, which has led to 33 nanotherapeutics drugs currently on the market and many more in various stages of clinical trials. With an increasing number of products available and in development, along with the unique, emergent properties of the nanoparticle therapeutics themselves, regulatory agencies are now faced with decisions regarding the regulation of such novel technologies. Regulatory guidance, particularly in pre-clinical stages, has the potential to facilitate quick and safe development of these novel materials, but new regulation beyond what is currently in place must be justified in a clear and distinctive toxic response. Herein, we examine literature that compares and correlates in vivo and in vitro nanotoxicity studies to gain a deeper understanding of the modes of nanoparticle toxicity. Additionally, this comparison aims to identify clear and unique toxicity responses caused by nanoparticles, which informs our perspective on pre-clinical nanotherapeutic oversight.

Functional assessment of metal oxide nanoparticle toxicity in immune cells.

Understanding the nanoparticle-cell interaction is critical for the safe development of nanomaterials. Herein, we explore the impact of three metal oxide nanoparticles, nonporous Stober SiO(2), mesoporous SiO(2), and nonporous anatase TiO(2) nanoparticles, on primary culture mast cells. Using transmission electron microscopy and inductively coupled plasma atomic emission spectroscopy, we demonstrate that each class of nanoparticle is internalized by the mast cells, localizing primarily in the secretory granules, with uptake efficiency increasing in the following order: nonporous SiO(2) < porous SiO(2) < nonporous TiO(2) nanoparticles. The influence of nanoparticle-laden granules was assessed using carbon-fiber microelectrode amperometry measurements that reveal functional changes in chemical messenger secretion from mast cell granules. Both nonporous and porous SiO(2) nanoparticles cause a decrease in the number of molecules released per granule, with nonporous SiO(2) also inducing a decrease in the amperometric spike frequency and, therefore, having a larger impact on cell function. As the two classes of SiO(2) nanoparticles vary only in their porosity, these results suggest that, while the mesoporous SiO(2) has a drastically larger total surface area due to the pores, the cell-contactable surface area, which is higher for the nonporous SiO(2), is more important in determining a nanoparticles' cellular impact. In comparison, exposure to nonporous TiO(2) slows the kinetics of secretion without altering the number of molecules released from the average granule. The varying immune cell response following exposure to nonporous SiO(2) and nonporous TiO(2) indicates that the nanoparticle-cell interactions are also modulated by surface chemistry.

Amperometric assessment of functional changes in nanoparticle-exposed immune cells: varying Au nanoparticle exposure time and concentration.

A mast cell/fibroblast co-culture system is used as a model to assess the toxicity of Au nanoparticles over the course of 72 hours of exposure. Cellular uptake of nanoparticles was found to increase over the 72 hr exposure period and the nanoparticles localized within granular bodies of the primary culture mast cells. These granules were found to increase in volume with the addition of nanoparticles. There was no decrease in viability for 24 hr exposed cells but a slight viability decrease was found after 48 and 72 hr exposure. Carbon-fiber amperometry analysis of exocytosis of serotonin from mast cells revealed changing release profiles over the time course of exposure. In early exposure times, granular secretion of serotonin increased with exposure to Au nanoparticles whereas 72 hr exposure showed decreased secretion of serotonin with nanoparticle exposure. The kinetics of this release was also found to be affected by Au colloid exposure where the rate of serotonin expulsion decreased with increasing nanoparticle exposure. These results illustrate the dynamic nature of nanoparticle-cell interactions and the critical changes in cell behavior even when viability is unaffected.

Toxicity of therapeutic nanoparticles.

A total of six nanotherapeutic formulations are already approved for medical use and more are in the approval pipeline currently. Despite the massive research effort in nanotherapeutic materials, there is relatively little information about the toxicity of these materials or the tools needed to assess this toxicity. Recently, the scientific community has begun to respond to the paucity of information by investing in the field of nanoparticle toxicology. This review is intended to provide an overview of the techniques needed to assess toxicity of these therapeutic nanoparticles and to summarize the current state of the field. We begin with background on the toxicological assessment techniques used currently as well as considerations in nanoparticle dosing. The toxicological research overview is divided into the most common applications of therapeutic nanoparticles: drug delivery, photodynamic therapy and bioimaging. We end with a perspective section discussing the current technological gaps and promising research aimed at addressing those gaps.