*4.3. Dermal Exposure*

Health and beauty products are another key source of nanoplastics, particularly in the body and facial scrubs that are used topically on the skin [11]. Nanocarriers for drug delivery via dermal application is another important exposure route. Although there are no conclusive data showing the effects of nanocarriers, small particle size and stressed skin conditions are critical factors to skin penetration [51]. There is no current research that has specifically looked at the ability of nanoplastics to penetrate the surface of the skin. Only one study reported the likelihood of engineered nanoparticles from textiles in permeating the skin barrier at very minute quantity [80].

The skin is protected by the stratum corneum, the outermost layer, which forms a barrier against injuries, chemicals and microbial agents. The stratum corneum consists of corneocytes that are surrounded by lamellae of hydrophilic lipids including ceramide, longchain free fatty acids and cholesterol [81]. Plastic particles may be introduced on the skin through health and beauty products, or through contact with nanoplastic-contaminated water. As micro- and nanoplastics are hydrophobic, it is predicted that absorption through the stratum corneum through contaminated water is unlikely, though plastic particles could enter the body via sweat glands, skin wounds or hair follicles [51].

Alvarez-Roman et al. looked at how plastic particles enter the body and how they are then distributed throughout the skin tissue. They used fluorescent polystyrene particles between 20 and 200 nm in diameter and skin tissue from a pig to conduct their experiment [82]. A confocal laser scanning micrograph of the skin revealed that a greater number of 20 nm polystyrene nanoplastics concentrated in the hair follicles than those of 200 nm nanoplastics. However, neither particles were able to permeate the stratum corneum in order to embed themselves into the deeper skin tissue. Campbell et al. supported these findings and established that polystyrene particles with diameters of 20–200 nm can infiltrate only the top layers of the skin at a depth of 2–3 μm [83]. Vogt et al. were able to

distinguish 40 nm-diameter fluorescent polystyrene nanoparticles in the perifollicular tissue of skin explants that had been treated with cyanoacrylate follicular stripping. This work ascertained that when particles were applied transcutaneously, they were then absorbed by the Langerhans cells [84].

The mechanical production method used to manufacture the microbeads of the health and beauty products, including facial and body scrubs, increases the likelihood of the breakdown of microbeads into even more harmful nanoplastics. Hernandez et al. investigated the amount of nanoplastics present in facial scrubs containing 200 μm polyethene microbeads. The results from scanning electron microscopy confirmed that nanoparticles were observed at sizes between 24 ± 6 nm and 52 ± 14 nm. Then, they examined the chemical composition of these nanoparticles by using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy and established that they consisted of polyethylene [11].

Data from these previous studies can be used to determine the likelihood of nanoplastics penetrating the stratum corneum. Exposure to UV rays causes skin damage, which means that the skin barrier becomes weaker [85]. A study on the effects of UV irradiation on murine skin found an increase in the skin permeation by carboxylated quantum dots. The intercellular adhesion in the irradiated skin was compromised by the perturbed expression of tight-junction-related proteins: Zonula occludens-1, claudin-1, and occludin [86]. There are a number of compounds, e.g., short chain- and long chain-alcohols, cyclic amides, esters, fatty acids, glycols, pyrrolidones, sulphoxides, surfactants and terpenes, that are used to enhance the chemical permeation of drugs and formulations through the skin barrier [87]. Widely used ingredients in body lotions, such as urea, glycerol and α-hydroxyl acids also enhanced the ability of nanoparticles to permeate the skin barrier [88].

Kuo et al. highlighted how oleic acid, ethanol and oleic acid-ethanol enhancers impact the transdermal delivery of 10 nm zinc oxide nanoparticles. They determined that each chemical had the potential to improve the effectiveness of zinc oxide nanoparticles in penetrating the skin barrier, through the multilamellar lipid regions between corneocytes [89]. Through crystalline structure analysis of various compositions of lipid lamellae in stratum corneum samples taken from human and porcine sources, Bouwstra et al. presented a three-layer "sandwich model", which likely prevents large nanoparticles from permeating undamaged skin [81].

To summarize, both in vitro and in vivo studies, discussed above, have established that micro- and nanoplastics can be absorbed into the human body through the skin barrier. That being said, these studies all rely on polystyrene particle models. Further studies conducted with samples collected from the environment would be helpful to fully understand the permeation qualities of micro- and nanoplastics. Such samples would include a variety of plastic particles with wide-ranging characteristics.

### **5. Cellular Uptake and Intracellular Fate of Microplastic and Nanoplastic Particles**

After permeating into the stratum corneum and absorbed into the human body, microplastics and nanoplastics are then able to interact with numerous target cells. The quantity of nanoparticles that are absorbed and subsequently react with cells depends on a number of factors, such as their size, surface chemistry or charge of the biological elements they encounter, including proteins, phospholipids and carbohydrates [90]. As nanoparticles absorb proteins from the human body, they create 'protein coronas' around themselves [91]. This means that nanoparticles that interact with organs or skin cells will usually already be surrounded with protein corona as opposed to an exposed nanoparticle. The protein coating will lead to modified characteristics of the nanoparticle. Previous in vitro experiments have determined that protein coronas will surround polystyrene nanoparticles and that this enables the nanoparticles to translocate at greater rates [62]. These studies also showed that the protein coronas would alter their form according to their environment [67] and that there were more occurences of cell interactions and

increased toxicity [92]. Finally, it was shown that protein coronas around the polystyrene nanoparticles led to them being accumulated in the gut.

Microplastics and nanoplastics can be absorbed by cells via a number of routes [93]. The primary route is via endocytotic nanoparticle uptake where adhesive interaction of nanoparticles (or inactive permeation of the cell membrane) with channel- or transportprotein occurs. Several endocytotic pathways have been identified, such as phagocytosis and macropinocytosis, along with clathrin- and caveolae-mediated endocytosis (Figure 3) [94–96].

**Figure 3.** Routes of cellular uptake of plastic particles. Phagocytosis, macropinocytosis, clathrin- and caveolae-mediated endocytosis are the common endocytotic pathways that have been identified for cellular uptake of plastic particles. Microand nanoplastics can be absorbed by cells through different routes, of which endocytotic nanoparticle uptake is the primary route where adhesive interaction of nanoparticles (or inactive permeation of the cell membrane) with channel- or transport-protein occurs.

> The initial barrier to nanoparticle incursion into the skin is the outer cell membrane. Coarse-grained molecular simulations of polystyrene (PS) particles in interacting with biological membranes revealed that polystyrene nanoparticles effortlessly penetrated the lipid bilayer membranes, causing changes to the structure of the cell membrane, ultimately disrupting the cell function [97]. Uptake inhibition studies on the absorption rate of 44 nm polystyrene particles to human colon fibroblasts and bovine oviductal epithelial cells indicated that polystyrene nanoparticles were primarily absorbed through a clathrinindependent uptake mechanism [98].

> The cellular absorption of carboxylated polystyrene nanoparticles of 40 nm and 200 nm was studied using several tumor cell lines, including human cervical HeLa cells, human glial astrocytoma 1321N1, and adenocarcinomic human alveolar basal epithelial A549 in the presence of various transport inhibitors [99]. The results indicated that the nanoparticles were always absorbed by cells through an active and energy-dependent method, suggesting that the nanoparticle uptake for different cell types utilized different pathways. Actin depolymerization significantly influenced the nanoparticle uptake in HeLa and 1321N1 cell lines, while clathrin-mediated endocytosis of the nanoparticles in 1321N1 was significantly

reduced by the inhibitor chlorpromazine. Nanoparticle uptake in A549 cell line via caveolaemediated endocytosis was notably diminished due to disruption of microtubule formation by the inhibitor genistein [99].

The routes of nanoparticle absorption in human cells are dependent on the size and surface chemistry of the particles, but also vary according to the type of cell penetrated. This is seen when 120 nm polystyrene nanoparticles altered with amidine groups were seen to permeate rat alveolar epithelial monolayers using non-endocytic pathways, whilst MDCK-II cells use energy-dependent processes to absorb nanoparticles [76,100]. Macrophages and epithelial cells were found to use various combinations of endocytotic uptake mechanisms to absorb 40 nm carboxylated polystyrene nanoparticles.

Using several endocytotic pathway inhibitors, J774A.1 macrophages were shown to absorb nanoplastics via macropinocytosis, phagocytosis, and clathrin-mediated endocytosis pathways, whilst absorption in A549 cells relied on caveolae- and clathrin-mediated endocytosis pathways [101]. If nanoplastic particles are entering the human body via non-vesicular pathways, then they may be able to interact with intracellular molecules or discharge persistent organic pollutants (POPs) straight into the cytoplasm. This, in turn, implies that POPs may be stored in human cells which could have a negative toxicological impact [102].

Plastic particles enter cells via the intracellular endocytotic pathway engaging with early and late endosomes before combining with lysosomes. Polystyrene nanoparticles have been reported to accumulate in the lysosome [103], including the observance of intracellular localization of 40–50 nm polystyrene nanoparticles in A549 cells [77]. No lysosomal leakage or any fragmentation of the nanoparticles were reported when acidic conditions were applied [104].

A comparison of two types of nanoparticles made of polystyrene and mesoporous silica demonstrated clear differences in cellular uptake mechanisms in ovarian cancer cells. Data gathered demonstrated that ovarian cancer cells absorbed both types of nanoparticles with different endocytotic pathways [105]. Caveola-mediated endocytosis was the pathway used by mesoporous silica particles to permeate the cells and these then, according to the size of the particle, either remained in the lysosome (50 nm) or translocated into the cytoplasm (10 nm). On the other hand, polystyrene nanoparticles were absorbed through a caveola-independent pathway. Localized amine-modified 50 nm polystyrene particles showed toxicity to the lysosome after 4–8 h, whereas 30 nm carboxyl-modified polystyrene particles showed no signs of toxicity and did not enter cells via the standard acidic endocytotic route.

Previous research has also determined that cytotoxicity is greater with a positive surface charge and this also leads to increased absorption of nanoparticles via non-specific binding, where they end up on the negatively charged sugar moieties on cell surfaces. In contrast, the repellent interactions of negatively charged particles will impede endocytosis [103,106]. Studies have determined that there are several cellular absorption pathways and intracellular localization of polystyrene nanoparticles depending on their physicochemical characteristics. Despite this, there is little quantitative data concerning how nanoplastics enter cells and their eventual fates.

The size of plastic particles also affects their interaction with human cells [107]. Due to their high specific surface areas, the nanoparticle-cell interaction is vastly different compared to larger particles. Furthermore, the charge of the particle can also affect its interaction with the cell and its structure [107]. Since most of the in vitro studies described here have used polystyrene particles, it is difficult to extrapolate the results to other kinds of plastic particles. It is important that future research looks at cellular uptake and behaviours of other types of plastic particles.

### **6. Potential Toxic Effects of Microplastics and Nanoplastics on Human Health**

Several in vitro and in vivo studies have shown that micro- and nanoplastics were able to cause serious impacts on the human body, including physical stress and damage, apoptosis, necrosis, inflammation, oxidative stress and immune responses (Table 1) [108–111].
