Isolation and determination of NPs from face scrubs
The face scrubs (FS) manufactured by two different industries were purchased from the local supermarket and used for NPs extraction by sequential filtration [13]. Since the brand names are not particularly relevant to the study, we presented the samples as FS-1 and FS-2. The FS-1 and FS-2 are used by men and women, respectively. Both products contain common ingredients such as antioxidants, preservatives, and fragrance enhancers. Notably, FS-2 comprises polyquaternium, a cosmetic ingredient designated for several cationic polymers with different properties. Regardless of the ingredient list, we have observed MPs and NPs in both products. The NPs particles isolated from FS-1 and FS-2 were termed as NPs-1 and NPs-2, respectively.
The NPs containing final filtrates obtained from the FS samples via subsequent filtration were examined under the high-resolution scanning electron microscope (HR-SEM) to determine the hydrodynamic size and shape. The electron micrographs showed that the NPs-1 isolated from FS-1 are mostly spherical with a smooth surface and few irregular aggregates (Fig. 1a insert), whereas NPs-2 isolated from the FS-2 are amorphous with sharp edges (Fig. 1c insert). The amorphous nature of NPs might have resulted from the cosmetic production steps such as homogenization, emulsifying, and heating. The size distribution analysis showed a broad size distribution range from 30 to 300 nm and 90 to 230 nm for NPs-1 and NPs-2, respectively. In NPs-1, most of the particles are 100 ± 20 nm in diameter. The observed slight increase in the NPs particle size (>200 nm) could be due to particle agglomeration during the drying process. On the other hand, the PENPs (Fig. 1c insert) prepared from the subsequent breakdown of PE pellets and the commercially procured virgin-PSNPs (Fig. 1d insert) showed irregular particles of about 400 nm or less in diameter and spherical particles of 100 nm in diameter, respectively. More details on the particle size distribution and NPs concentration in FS samples are available in the supplement data.
The dried NPs-1, NPs-2, PENPs, and PSNPs were analysed using Fourier Transform Raman Spectroscopy (FT-Raman). The Raman spectra of NPs-1 (Fig. 1a) and NPs-2 (Fig. 1c) showed a close correlation with the characteristic Raman bands of standard PENPs (Fig. 1e). The bands at 1062, 1132, 1299, and 1444 cm-1 are mainly due to the C-C stretching, CH2 twisting, and CH2 bending vibrations of PE, and the strong bands at 2851 and 2885 cm-1 are due to the CH2 asymmetric and symmetric stretching vibrations of PE [35, 36]. As mentioned above, PE is the most prominently used polymer (about 93%) in cosmetics; hence it could be the highest polymer fraction in the isolated NPs-1 and NPs-2. As a result, the PE Raman peaks might have surpassed the Raman shift of PS and other plastic polymers [7]. On closer examination, the Raman spectra of NPs-1 (Fig. 1b) and NPs-2 (Fig. 1d) showed the characteristic bands at 620, 795, 1002, 1032, 1155, 1450, and 1602 cm-1 corresponding to ring deformation, C-H out of plane deformation, ring breathing, C-H in-plane deformation, C-C stretch, CH2 scissoring, C=C stretch, and the ring-skeletal stretch of PSNPs (Fig. 1f), respectively. This observation indicates that both FS contains different types of nano-sized plastic particles, mainly polyethylene and polystyrene polymers.
Dispersion and stability of NPs in the aqueous medium
The hydrodynamic size of PENPs, PSNPs, NPs-1, and NPs-2 in deionized water, keratin solution (0.2%), and Dulbecco's Modified Eagle's culture medium (DMEM) were measured using the dynamic light scattering (DLS) technique. During this experiment, the samples were vortexed vigorously in respective solutions for 10 min and then incubated for 24 hrs at room temperature or in cell culture conditions (at 37°C with 5% CO2 and 95% humidity). The initial DLS measurements showed moderate stability (ζ ≥ -30 mV) of PENPS and PSNPs and an optimal dispersity and stability (ζ ≥ -40 mV) of NPs-1 and NPs-2 in deionized water (Table S1). But, over a 24 hrs period, the PENPs showed greater instability with a Z-average of 1409±520 nm and PDI of 9.106, followed by a drastic decrease in the zeta potential value (-9±2.6 mV) in deionized water. On the other hand, all the NPs showed significant stability in the keratin solution. In DMEM, the average particle size of NPs-1 and NPs-2 increased 3 to 4-fold than in the deionized water (Table S1). The observed size increase in the NPs under the cell culture medium could be due to the aggregation of particles (Fig. S3d) via non-specific protein-protein attraction and the bridging effect between protein and other biomolecules, as described in our previous study [15]. The above results indicate that the adsorption of biomolecules on the plastic particles plays a significant role in NPs aggregation and stability.
Adherence of NPs particles on keratin coated glass slides
It is a well-known phenomenon that NP/MP particles rapidly adhere to the biological substrate such as protein layers via non-specific attraction forces like Van der Waals force or the surface electric polarity alteration through a strong hydrophobic bond or dipole bonding by hydrogen bond via ─OH, ═O, ─NH, ═NH, ≡N groups or the spontaneous adsorption depending on the amino acid content [15]. The binding stability of NPs may differ depending on the attraction factors and contact/interaction time. To show the rapid attachment of NPs on the skin surface, we have exposed the keratin-coated glass slides to NPs solution (100 µg/mL) for 2-3 min and rinsed twice in ultra-pure water, and then observed under the electron microscope. Since this study uses NPs isolated from face scrubs, we have restricted the contact time for a maximum of 3 min and washed twice with deionized water. The field-emission scanning electron micrographs (FE-SEM) of keratin-coated glass slides showed a significant attachment of PENPs, PSNPs, NPs-1, and NPs-2 within 3 min of exposure (Fig. 2). Although washing removed most of the particles, an ample number of particles remained attached on the keratin-coated glass surface even after two washes (8-10 dipping /wash) (Fig. 2c, f, i, and l). The NPs removal from the keratin layer during washing could be due to the weak adherence of NPs in the given contact time. However, the attached NPs on the protein layer, even after consecutive washes, revealed that washing does not remove all the particles adhered to the skin surface. Doge et al. [30] showed the penetration of 20 and 200 nm-sized PSNPs into the stratum corneum via skin furrows, lipid channels, and vellus hair follicles and accumulation onto the viable epidermis just beneath the stratum corneum as well as within the epidermis cells. Other studies have also reported that the stratum corneum could act as a long-term reservoir for the penetrated polymer particles and facilitate the NPs translocation into viable tissues [37, 38, 39].
Impact of NPs on keratinocytes viability
The constant usage of cosmetics on the skin could lead to the long-term interaction of NPs with keratinocytes, thereby provokes the physiological, biochemical, and pathological responses in cells. To observe the cytotoxic effect on the cell, we have exposed the keratinocytes to different concentrations of PENPs, PSNPs, NPs-1, and NPs-2 for six consecutive days. The MTT assay showed a gradual reduction in cell viability at the high concentrations of PENPs, NPs-1, and NPs-2 with treatment time (Fig. 3). On the contrary, there was no/less inhibition observed in PSNPs and low concentrations of PENPs, NPs-1, and NPs-2 treated cells. The pairwise comparison of cell viability between the NPs dose range and control showed a statistically significant difference (p≤ 0.05), evidencing the NPs concentration- and time-dependent toxicity in cells. Herein, we have limited the NPs-2 concentration range to 250 µg/mL because of the complete growth inhibition of cells at the higher concentrations. The observed cell death at high concentrations of NPs could be due to the cell damage caused by the irregular-shaped NPs with sharp edges during physical interaction. Also, the adsorbed materials and additives in plastics might have enhanced the NPs cytotoxicity [40]. The cells exposed to the pristine PSNPs did not show significant cell death as elsewhere reported in different cell lines [40, 41, 42]. However, we have recorded a remarkable cell viability increase after 48 hrs of exposure compared to control. A similar growth pattern in PSNPs treated cells was observed in the trypan blue assay (discussed below). Likewise, the low concentrations of PENPs, NPs-1, and NPs-2 treatment showed a slight increase in the growth after 48, 72, and 96 hrs than the control (Table S2). The observed increase in the cell viability at the low concentration of NPs strengthens the assumption that chemically inert polymers cannot induce cellular toxicity. This scenario is explained below.
Oxidative stress generation in the HaCaT cells
In addition to the cell viability assay, we have estimated the unspecific oxidative stress induced by NPs using reactive oxygen species (ROS) assay for six days (Fig. S1). The overall observation showed a difference in the ROS values between the NPs treatments (Fig. 4). The NPs isolated from FS showed significantly low ROS activity compare to the PSNPs and PENPs, which could be due to the increased cytotoxic effect of isolated NPs via physical damage (as mentioned above), denoting high cytotoxicity by direct physical damage over oxidative stress. Though there are similarities among the ROS values of PENPs and PSNPs, only the amorphous PENPs showed cytotoxicity due to the cell damage through physical interaction. On the other hand, the PSNPs with spherical shape and smooth surface did not cause physical damage on the cell, hence showed a minimal cytotoxic effect. It is also possible that the low level of ROS in the cells treated with NPs-1 and NPs-1 could be due to the increased proliferation inhibition as observed in the cell proliferation assay (discussed below). Interestingly, all the NPs showed a maximum ROS level at 48 and 72 hrs of treatment (Fig. 4 a-d), followed by a steady downward trend at 96, 120, and 144 hrs. The increased ROS level at 48 and 72 hrs could be due to the high level of NPs interaction and internalization. The observed gradual reduction in the cellular ROS level after 96 hrs could be due to the defensive action against the ROS or elimination of NPs or cell destruction [43, 44].
The free radicals produced during oxidative stress could damage the cell membrane lipid and fatty acids leading to elevated lipid peroxidation, a primary indicator of cell or organelle damage. Here, the amount of lipid peroxidation in cells was determined from the intracellular malondialdehyde (MDA) level using thiobarbituric acid reactive substances (TBARS) assay. MDA is an end product of lipid peroxidation that reacts with the thiobarbituric acid-trichloroacetic acid (TBA-TCA) complex and produces a detectable coloured substance. The cells exposed to PENPs, PSNPs, NPs-1, and NPs-2 (Fig. 4 e-h) showed a concentration and time-dependent increase (p< 0.05) in the MDA level compare to the control. However, at 96 hrs, there was no significant difference (p> 0.05) observed in MDA level in the cells treated with high concentrations of PENPs, NPs-1, and NPs-2, and all the PSNPs concentrations (Fig. 4 e-h). It is noteworthy to mention that all the tested NPs showed a two-fold increase in MDA production at 48 and 72 hrs compared to control.
Effect of NPs on the antioxidant enzymes
ROS substances are naturally produced in cells during various cellular metabolisms and subsequently eliminated by the enzymatic or non-enzymatic antioxidants [45, 46]. When the ROS production succeeds the antioxidant defence, the equilibrium between prooxidants and antioxidants becomes inadequate, leading to oxidative damage in the nuclei, lipids, and proteins, followed by cell damage [45, 47, 48, 49]. Redox homeostasis is the endogenous capacity of cells to deal with continuous challenges generated by electrophiles. An increased accumulation of superoxide anion (O2•−) and hydrogen peroxide (H2O2) during oxidative stress could disrupt the cellular redox-homeostasis. In order to defend and normalize the ROS stress, cells could produce endogenous antioxidant enzymes. Superoxide dismutase (SOD) is the first line of defence against oxygen‐derived free radicals, dismutates the O2•− into less reactive H2O2, which is split into H2O and O2 by catalase (CAT). Herein, the production of SOD in the cells during ROS generation was assayed spectroscopically at 480 nm by the epinephrine auto-oxidation inhibition method. Similarly, the CAT production was assayed from the depletion of hydrogen peroxide.
The chronic long-term treatment of NPs showed a concentration-dependent reduction in the total protein levels compared to control and a time-dependent fluctuation in the SOD and CAT activity. All the NPs (25 and 50 µg/mL) treated cells except NP-2 displayed a decreasing trend in SOD activity at 24 and 48 hrs of treatment, followed by a slight increase at 72 and 96 hrs (Fig. 5 a-c). On the other hand, the NPs-2 (Fig. 5d) and high concentration PENPs and PSNPs showed fluctuated SOD levels at different treatment times, while the NPs-1 showed a gradual decrease (Fig. 5c). In CAT activity, all the NPs showed a time- and concentration-dependent fluctuation in CAT levels (Fig. 5 e-h). However, the overall antioxidant enzyme activity remained significantly low in the high concentrations of NPs compared to control. The observed high level of fluctuation in the SOD and CAT activity of the cells under the long-term treatment with NPs signifies the imbalanced redox-homeostasis in cells. A similar negative correlation between the antioxidants and lipid peroxidation was reported in age-related macular degeneration [50] and heart, lung, liver, and testis of rats [51, 52, 53, 54]. It has been postulated that the decrease in the antioxidant activity may be due to the exhaustion of enzymes while detoxifying oxidants [52, 55, 56]. Or else, direct inhibition of antioxidant enzymes by the toxicants [56] and lipid peroxidation [57]. Pigeolet et al. [58] reported that the most important antioxidant enzymes such as SOD, CAT, and GPX (glutathione peroxidase) are susceptible to oxidant metabolites. For instance, SOD is susceptible to H2O2, and CAT and GPX are susceptible to hydroxyl radicals and superoxide anions [58]. Indeed, these enzymes protect each other from inactivation by the antioxidants. The depletion of one enzyme could increase the oxidants that inactivate the other enzyme, which leads to the exponential production of oxidant molecules and high oxidative stress [58, 59]. It is worth mentioning that the reduced antioxidant activity accompanies ROS-mediated cellular responses in the cell.
A low level of ROS acts like signalling molecules that promote cell proliferation [60], while the moderate level of ROS induces biological responses such as autophagy and senescence leading to apoptosis and inflammation [61, 62] whereas, the high level of ROS disrupts the cells [61, 62, 63]. As a result of the above facts, the HaCaT cells showed increased viability in PSNPs and at low concentrations of PENPs, NPs-1 and NPs-2 treatment, and increased cytotoxicity at high concentrations of PENPs, NPs-1, and NPs-2. We believe that the PSNPs and low/ sub-lethal concentrations of PENPs, NPs-1, and NPs-2 could stimulate oxidative stress-mediated biological responses in the cells. Before proceeding with this aspect, two important queries need to be answered: (i) how the cells recognize and engulf the NPs, and (ii) why there is a delay in the cellular response against the NPs.
Mechanism of cell uptake
Cellular internalization of NPs
To examine the internalization of NPs in the cells, we have treated the keratinocytes with fluorescently labelled PSNPs (FLPS) for 12, 24, 48, 72, 96, 120, and 144 hrs, and then examined under a fluorescence microscope. Figure 6 showed a minimum accumulation of FLPS at 12 and 24 hrs and a maximum internalization at 48 and 72 hrs of treatment. There was no further increase in the particle accumulation observed at 96 and 120 hrs, suggesting interruption in the internalization process. Similar ROS-mediated interruption of the endocytosis process was reported in Chinese hamster ovary cells [64] and epidermal cell lines [65]. On the other hand, for the exclusion experiment, the NPs incorporated medium was replaced with NPs free medium after 72 hrs of exposure. After 96 hrs, a gradual decrease in the fluorescence intensity in cells (Fig. 6) followed by a complete absence of fluorescence was observed at 144 hrs (Fig. S2). It appears that the gradual reduction of FLPS in cells could be due to the rapid exclusion of internalized FLPS or ROS-mediated cell death or both. As a result of the increased uptake of NPs at 48 and 72 hrs treatment (Fig. 6), the keratinocytes presented an elevated level of ROS and reduced antioxidant activity. The observed reduction in the ROS level after 72 hrs of treatment could be either due to the rise of defensive antioxidant activity in cells (Fig. 5), NPs removal from the cells, blocking of NPs internalization, or cell death. The result observed in the FLPS experiment signifies that the NPs internalization does not occur immediately but requires some time. However, further investigations are needed for a better understanding of the mechanism of uptake.
Protein-corona formation on the NPs
We assume that the keratinocytes might have recognized the NPs as a foreign substance and prevented its entry for about 12 hrs. Later due to the surface modification of NPs, the HaCaT cells might have recognized and internalized it. As described in our previous study, the biological macromolecules, especially proteins, tend to adsorb on NPs surface called protein corona, which in turn mimic protein aggregates [15]. To validate the corona formation on NPs under DMEM, the NPs were introduced into the medium and incubated for 6, 12, and 24 hrs and then examined under HR-TEM [15]. The TEM micrographs (Fig. S3) showed about 10 to 200 nm-sized corona formation by protein and smaller biomolecular aggregates on PENPs (Fig. S3a) and PSNPs (Fig. S3b). On the other hand, the NPs-1 (Fig. S3c) and NPs-2 (Fig. S3d) showed corona-mediated aggregation in correlation with the DLS results, where we observed a 3 to 4-fold increase in the particle size. After 24 hrs, the total number of aggregated particles was high compared to 12 and 6 hrs of exposure, but the average size remained <700 nm.
Protein-corona-mediated internalization of NPs
Generally, plastic particles tend to adsorb organic and inorganic substances quite rapidly, and the rate of corona formation and corona thickness is directly proportional to the availability of biomolecules. For example, in human serum albumin solution and human blood plasma, a multi-layered protein-corona of a few hundred-nanometre radii was formed on PSNPs within 2 hrs [15]. We strongly suspect that the observed delay in the protein-corona formation in DMEM could be due to the solution properties, competitive exchange of media components, or shortage of macromolecules. As a result of the delayed corona formation, we observed a limited FLPS uptake by keratinocytes for up to 12 hrs (Fig. 6 and Fig. S2). As mentioned above, under the in vivo system, corona formation and cellular uptake could occur within few hours of exposure. To demonstrate the significant role of protein-corona in NPs recognition and internalization, we have prepared fluorescent PENPs using Nile red stain (Supplement data) and added it into the keratin solution (0.2%) for 1 hr to produce keratin-corona (Fig. S4). Likewise, the keratin-coronated green fluorescent FLPS was prepared, purified, dispersed in DMEM, and exposed to the HaCaT cells (Supplement data). After 30 min and 60 min of incubation, the cells were harvested and examined under a fluorescent microscope. Figure 7 showed a rapid internalization of fluorescent-PENPs and -PSNPs compared to the cells treated with corona-free-NPs (Fig. 6), where the uptake occurred only after 12 hrs. This observation certainly proved that the adsorption of protein and other molecules on the NPs provided a cloaked identity, subsequently triggered the cell recognition and internalization of NPs. To maintain uniformity in the corona formation on NPs and corona-mediated effect on cells, we preferred the protein-corona formation under DMEM throughout this study.
Macropinocytosis and lysosomal action
As a result of protein-corona formation on the NPs, the cells might have recognized the coronated-NPs as protein aggregates and internalized it via macropinocytosis, an ideal mechanism towards protein aggregates [66, 67, 68, 69]. Yet, the macropinocyte activation mechanism by protein aggregates remains unidentified [70]. To observe the internalization process, we have treated the cells with PSNPs for 48 hrs, fixed, harvested, embedded, and sectioned before the HR-TEM analysis (Fig. 8). Herein, the spherical NPs were applied to avoid ambiguity in the visual sorting of NPs and cell structures under electron microscopy. The electron micrographs showed an attachment of coronated-NPs on the cell surface (Fig. 8a, b) and the formation of pinocytic cups, or large membrane ruffles (Fig. 8c, d). Additionally, the folding back of membrane ruffle onto the cell surface with the coronated NPs (Fig. 8e) and the formation of a membrane surrounded intracellular compartment were seen (Fig. 8f) [71]. After understanding the process of NPs uptake, we have studied the lysosome fusion with macropinocytes and macropinolysosomal activity under the confocal laser scanning microscope (CLSM). For this experiment, we used the FLPS emitting green fluorescence (λex 458 nm and λem 485 nm), and neutral red (a lysosomal probe) [72] that emits red fluorescence (λex 541 nm and λem 610 nm) [73]. Under CLSM, the cells treated with NPs for 48 hrs showed the intense green and red fluorescence from the FLPS within the macropinosomes (Fig. 9f) and neutral red stained lysosomes, respectively (Fig. 9g). The stratified images (Fig. 9h) of red and green channels depicted the formation of macropinolysosomes (yellow colour). Additionally, the accumulated lysosomes (red fluorescence) around macropinosomes indicate a possible fusion attempt. The formation of macropinolysosomes represents the commencement of degradation of protein-covered NPs.
The fate of NPs in macropinolysosomal activity
Generally, cells eliminate the undigested or toxic substances through exosomes. But during adverse conditions or cell death, these substances are released nakedly. To examine the morphological alterations in the NPs by lysosomal action, we have treated the cells with PSNPs for 48 hrs, washed twice with PBS (phosphate-buffered saline), and then incubated with the fresh NPs-free medium. After 24 and 48 hrs of incubation, the NPs released in the culture medium were separated and examined under HR-TEM (Fig. S5). The electron micrographs showed partly damaged (Fig. S5c-f), disintegrated (Fig. S5g-l), and enlarged NPs (Fig. S5m-o) due to various enzymatic actions in the macropinolysosomes. The observed corrosion in PSNPs (Fig. S5g-l) under HR-TEM suggests that the NPs might have disintegrated during the digestion process. It could pave the way for the leaching and release of styrene molecules and additives into the cells. The release of styrene molecules from PSNPs treated cells was detected using gas chromatography along with the styrene standard (Fig. S6) [73] yet, it has to be studied further to quantify the molecular release with other NPs as well. The observed corrosion of PSNPs and release of styrene molecules within the cells raises concerns about the emission of endocrine-disrupting additives, such as bisphenol A, nonylphenol, and octylphenol present in plastic products [74, 75, 76, 77]. All the above results revealed that the surface modification of NPs (protein-corona formation) eventually triggers the engulfment process followed by lysosomal action, particle degradation, and oxidative stress.
The cytoprotective activity in keratinocytes post-NPs internalization
Inhibition of cell proliferation
The absence of significant cytotoxic effect in cells at the low concentration of NPs and all PSNPs concentrations indicates the possible activation of cytoprotective mechanisms especially, inhibition of cell proliferation, senescence, and autophagy. To demonstrate the cytoprotective events in keratinocytes, we have treated the cells with lethal- and sub-lethal-doses of NPs for 48 hrs. After achieving the optimum internalization of NPs, the medium containing NPs was removed and replaced with a fresh, NPs-free medium. Then, the cell proliferation inhibition in the NPs internalized cells were determined from the total number of viable cells at every 24 hrs interval for four consecutive days compared to the control. The NPs internalized cells showed a concentration-dependent decrease in the cell viability (Figure 10a). However, under microscopic examination, minimal accumulation of trypan blue in the cells was observed, denoting that the cells are at the early stage of proliferation inhibition but metabolically active and viable. Among the tested NPs, NPs-2 showed significant growth inhibition, followed by NPs-1, PENPs, and PSNPs. The cell proliferation index calculated from the relative difference between the pre- and post-internalization of NPs exhibited NPs concentrations- and physicochemical properties-dependant inhibition in the cell proliferation (Fig. 10b). The results are correlated with the cytotoxic assay and signified a single dose of NPs could cause a cytostatic effect in cells.
Cellular senescence and autophagy
The senescence is the permanent halt in cell growth, which could resist apoptotic death for a long period [78, 79]. The abnormal accumulation of β-galactosidase has widely been reported in senescent cells, which allows the senescence-associated β-galactosidase (SA-β-gal) to be an important biomarker for cellular senescence. However, the underlying mechanism in the origin of SA-β-gal activity and its role in senescence and aging are still unknown. The HaCaT cells treated with PENPs (10, 100 & 500 µg/mL), PSNPs (10, 100 & 500 µg/mL), NPs-1 (10, 50 & 100 µg/mL), NPs-2 (10, 50 & 100 µg/mL) and H2O2 (10, 50 & 100 µM) for 48 hrs (for maximum NPs uptake) were washed and incubated in NPs-free medium for 24 hrs and then stained for SA-β-gal activity (Fig. S7). The microscopic observations showed a concentration-dependent increase in the SA-β-gal-activity in the PENPs, PSNPs, NPs-1, NPs-2, and H2O2 treated cells compared to the untreated cells (Fig. 11 and Table. S3). The mean percentage of SA-β-gal positive cells showed a significant difference (p < 0.05) within the NPs concentrations and with control. Additionally, the NPs and H2O2 treated cells showed typical senescent morphologies such as enlarged, flattened cells and increased accumulation of cytoplasmic granules [80, 81].
Besides, the cellular senescence may lead to chromatin modification, metabolic refinement and high autophagy activity, and pro-inflammatory-secretome production in cells [78]. Among the phenotypes, autophagy, a genetically regulated bulk degradation process, was detected in the NPs treated cells (Fig. 12). Autophagy is a cell survival mechanism that degrades the damaged cytoplasmic organelles and long-lived proteins using lysosomes. [82, 83]. Autophagy could facilitate senescence or limit cell damage, or delay apoptosis, which allows the cell to recover the normal functions [84, 85, 86]. The keratinocytes treated with NPs-1 (Fig. 12a-d) and NPs-2 (Fig. 12e-h) showed a series of autophagy structures under HR-TEM. These structures revealed an active engulfment of damaged organelles by phagophore that becomes an autophagosome. After maturation, the autophagosome fuses with the lysosome (termed autophagolysosome/ autolysosomes) for active digestion. The observed proliferation inhibition, senescence, and autophagy activity in the NPs treated cells envisages the homeostasis against a low level of oxidative stress. The results further emphasized that all the ROS-mediated molecular pathways may be interconnected [84, 87]. However, a high level of cytoprotective events can trigger inflammation and apoptosis [61, 62, 85, 87].
Recent studies on the cytotoxicity of MPs and NPs in human and animal models presented that low concentration plastic particles produce oxidative stress in cells, and high concentration plastic particles cause cytotoxic [88, 89]. Additionally, in this study, we have demonstrated the NPs concentration-dependent regulatory activity and cytoprotective and cytotoxic effects in HaCaT cells. This study presents three lines of evidence that are essential to close the existing knowledge gap in the cell uptake and response against NPs; firstly, the plastic particles adsorb protein molecules and mimic protein aggregates, thereby triggers and accelerate the internalization process. Secondly, the internalized NPs undergo lysosomal activity, which damages the NPs and leads to the release of plastic molecules and additives within the cells, subsequently accelerates oxidative stress. Finally, the ROS stress down-regulates cell proliferation and inhibits cell growth leading to premature aging, autophagy, or ROS-induced cytotoxic effect. To conclude, the continuous use of NPs and MPs particles incorporated cosmetics over a prolonged time may result in premature aging of skin cells.