Isolation and identification of Nano-plastic particles (NPs) from face scrubs
The HR-SEM observation of mNPs and wNPs isolated from major brands of facial scrubs for men and women, respectively, showed the majority of the mNPs are spherical with a smooth surface and few irregular aggregates (Fig. 1a), in contrast, the wNPs are irregular with sharp edges (Fig. 1b). The resulted amorphous nature of the NPs could be due to the homogenization, emulsification, and heating process in cosmetic manufacturing. The particle size measurement displayed a broad size distribution in the mNPs ranging from 30–300 nm (maximum particles are in 100 ± 20 nm size) in diameter and a narrow distribution (90–130 nm in diameter) in the wNPs. Here the increase in mNPs grain size (> 200 nm) was observed as a result of agglomeration during the drying process. The Raman spectra (Fig. 1c) of mNPs and wNPs showed characteristic Raman shifts at 1062.02, 1132.10, 1299.05, and 1444.69 cm-1 corresponding to the C-C stretching, CH2 twist and CH2 bending vibrations of polyethylene particles, respectively.[29, 30] Since, polyethylene particles are the most prominently (i.e., 93%) used polymer type in cosmetics, it could be the highest fraction in the isolated mNPs and wNPs. As a result, the polyethylene peaks surpassed the Raman shift of other polymers such as polypropylene, polyethylene terephthalate, polymethyl methacrylate, nylon, etc.[7] This observation indicates both face scrub contain nano-sized plastic particles, particularly of polyethylene plastic-type. Therefore the effects ensue in this study could be due to the polyethylene NPs.
Impact Of NPs On Keratinocytes Viability
Enormous usage of cosmetics on skin fetches a direct interaction of NPs with keratinocytes; this could provoke the physiological, biochemical, and pathological responses in cells. To unveil this, we exposed HaCaT cells to different concentrations of PSNPs, mNPs, and wNPs for six consecutive days. Resulted viability graph shows an evident cell viability loss at the higher concentrations of mNPs and wNPs (Fig. 2), but no pronounced inhibition by PSNPs and low concentrations of mNPs and wNPs. Remarkably the wNPs exhibited prominent inhibition at higher concentrations, and hence the concentration range was limited to 250 µg/ mL. The observed cell death at high concentrations could be due to the cell damage produced by NPs via physical interaction. Besides, the adsorbed materials and additives used for plastic production may also be augmented the cytotoxic effect[31]. Herein, the pristine PSNPs did not cause acute toxicity in cells as reported by the previous in vitro studies[31, 32, 33]; however, a noticeable increase in the cell viability over control was recorded after 48 hrs of exposure. The cell viability rate increased the overall viability of PSNPs and lower concentrations of mNPs and wNPs exposed cells than the control. The observed growth pattern in the cells exposed to pristine NPs certainly harmonized the assumption of chemically rather inert polymers are not expected to produce pronounced cellular toxicity per se. The sudden increase in overall cell viability in NPs exposed cells will be discussed below.
Oxidative Stress Generation In The HaCaT Cells
In addition to the cell viability, the unspecific stress induced by NPs within the cells was estimated using reactive oxygen species (ROS) assay. Upon observation, the ROS level reached a maximum at 48 and 72 hrs post-NPs exposure, followed by a steady downward stream at 96, 120, and 144 hrs was recorded (Fig. 3). This indicates the NPs interference in cells was high during 48 and 72 hrs, later due to either defensive action against the generated ROS or invaded NPs or destruction of cells, the ROS level was gradually reduced [34, 35]. During metabolism, ROS can be produced naturally in keratinocytes, but it can be rapidly removed by enzymatic (catalase, superoxide dismutase, thioredoxin-reductase, glutathione peroxidase, and GSH-reductase) or non-enzymatic (ascorbic acid, tocopherol, ubiquinol, and GSH) anti-oxidants [36, 37]. As soon as the ROS production overwhelms the anti-oxidant defense, the equilibrium between pro-oxidant and anti-oxidant becomes affected, leading to the oxidative damage in nucleic, lipids, and proteins, followed by the destruction of cells [36, 38, 39, 40]. On the contrary, low level of ROS acts as signaling molecules that promote cell proliferation [41], while the intermediate level of ROS elicits several biological responses such as autophagy, senescence, etc., which leads to apoptosis and inflammation [42, 43]. Because of these phenomena, increased toxicity at the high concentration of mNPs and wNPs, increased viability in the PSNPs as well as low concentration of mNPs and wNPs was observed [42, 43, 44]. We suspect that the PSNPs and low / sub-lethal concentrations of mNPs and wNPs might have stimulated the ROS mediated biological responses in cells. Before perceiving this, there are two queries, how the cells with spectacular differentiation mechanisms recognize and engulf the NPs, and how the internalized NPs interfere with the cellular process at certain time points ought to be answered.
Mechanism Of Cell Uptake
For scrutinizing the NPs internalization, the fluorescently labeled polystyrene NPs (FLPS) was exposed to the cells for 12, 24, 48, 72, 96, 120, and 144 hrs. Fluorescence microscopy examination showed minimal accumulation of FLPS at 12 and 24 hrs and a maximum internalization at 48 and 72 hrs post-exposure, but no further accumulation was observed at 96 and 120 hrs, which indicates the interruption in internalization process. The halt in the internalization process could be due to the commencing of the ROS defense mechanism against oxidative stress. On the other hand, the exclusion experiment displayed a gradual decrease in the FLPS concentration in cells at 96 and 120 hrs and complete exclusion of FLPS at 144 hrs (Fig. 4). It appears that the gradual reduction of FLPS in cells could be due to the ROS mediated decease of cells or the rapid exclusion of internalized FLPS or both. As a result of the increased NPs internalization at 48 and 72 hrs post-exposure (Fig. 4), the elevated ROS level in PSNPs, mNPs, and wNPs exposed cells was observed in the ROS assay. Additionally, the recorded reduction in the ROS level could be due to the exclusion of NPs from cells/ blocking of NPs internalization/ the defensive anti-oxidant activity by the cells/ cell death. The results presented herein suggest that the NPs internalization is not an immediate process; indeed, the NPs required an incubation time to achieve recognition and internalization by cells.
We strongly suspect that the spectacular differentiation mechanisms of keratinocytes recognized the NPs as the external substance and prevented its entry for up to 12 hrs. Later due to the alteration or modification in their surface property, the NPs might have attained cell’s recognition. As described in our previous study, the NPs adsorbs biological macromolecules, especially proteins on its surface known as corona formation, thereby mimic as protein aggregates [15]. To verify the corona formation on the NPs surface, the pristine PSNPs were introduced into the DMEM medium and incubated for 6, 12, and 24 hrs, and then the particles were examined under HR-TEM [15]. Resulted TEM micrographs (Fig. 5) showed the corona formation (Fig. 5a, b) as well as the adsorption of biomolecular aggregates (Fig. 5c-e) on PSNPs surface and mNPs (Fig. 5f). The total number of coronated particles and corona thickness was high in 24 hrs of exposure than in 12 and 6 hrs. Generally, the plastic particles adsorb organic and inorganic substance quite rapidly; herein, the corona formation was delayed due to the limited availability of biomolecules in DMEM, and hence the FLPS internalization commenced after 24 hrs (Fig. 4). The corona formation rate and corona thickness are directly proportional to the biomolecule availability; for instance, the multi-layered protein corona with few 100 nanometers size was achieved in less than 2 hrs in the human serum albumin, human blood, and plasma [15]. Therefore, the corona formation and internalization process can be quite rapid in the human and animal systems. These observations so far evidenced that because of the adsorbed protein and other molecules, the NPs imitate as protein /biological aggregates and succeed the cellular recognition, thereby, trigger the internalization mechanism.
As a result of false identity, the cells might have internalized the coronated-PSNPs, -mNPs and -wNPs through macropinocytosis, an ideal mechanism that internalizes protein aggregates [45, 46, 47, 48]. Yet the macropinocytes activation mechanism by protein aggregates remains unidentified [49]. The HR-TEM micrograph (Fig. 6) clearly illustrates the binding of coronated NPs on keratinocytes surface (Fig. 6a, b) triggers the pinocytic cups/ large membrane ruffles production (Fig. 6c, d), which then folds back on the cell surface along with the coronated NPs and extracellular fluid (Fig. 6e) and finally produces a membrane surrounded intracellular compartment (Fig. 5f) [50]. Having settled with the internalization process, first, the fusion of the lysosome with macropinocytes was examined under CLSM (Fig. 7). For this experiment, FLPS that emits green fluoresce (at 485 nm) while exciting at 441 nm, and neutral red (a lysosomal probe) [51] which emits red fluoresce (at 610 nm) while at 541 nm excitation [52] were used. Under CLSM, the intense green and red fluorescence were observed respectively from the FLPS containing macropinosomes (Fig. 7f) and neutral red stained lysosomes (Fig. 7g). The stratified images of red and green channels depicted macropinolysosomes (yellow color) generated through the fusion of lysosomes with macropinosomes (Fig. 7h). Further, the accumulation of lysosomes was noticed around macropinosomes, which indicates the fusion attempt. The formation of macropinolysosomes represents the commencement of degradation of protein covered NPs.
The Fate Of NPs During Macropinolysosomal Activity
Generally, the toxic substances and undigested materials from the macropinolysosomes are expelled from cells through exosomes or naked or by cell death in an extreme situation. To determine the fate of PSNPs subjected to macropinolysosomal action, the culture medium was collected and examined under HR-TEM (Fig. 8) during 72 and 96 hrs post-exposure (based on the FLPS exclusion assay). It is essential to mention that the culture medium (with PSNPs) was removed and replaced with the fresh medium (without PSNPs) at 48 hrs post-exposure. Resulted electron micrographs presented partly damaged (Fig. 8b-f), disintegrated (Fig. 8g-j), and enlarged PSNPs (Fig. 8m-o) due to various enzymatic actions in the macropinolysosomes. The corrosion of PSNPs within the macropinolysosomes may lead to leaching of styrene molecules and additives, which can be subsequently released into the cells. The release of styrene molecules from PSNPs treated cells was determined using gas chromatography along with the styrene standard [53] (data not presented). Further, the corrosion and release of styrene molecules into the cell raises concerns about the easily dischargeable endocrine-disrupting compounds such as bisphenol A, nonylphenol, and octylphenol used in plastics production [54, 55, 56, 57]. The enlargement in PSNPs could be due to the protein-mediated coalescence effect between the coronated PSNPs under the protein-rich environment [15]. All the above observations clearly confirm the surface modifications of NPs eventually triggers the internalization followed by the lysosomal activity, ROS generation, and subsequent elicitation of oxidative stress-mediated cellular responses.
The Cytoprotective Response Of NPs Exposed Cells
The absence of significant cytotoxicity in the cell exposed to PSNPs and low concentration of mNPs and wNPs clearly indicates the exertion of cytoprotective mechanisms, especially inhibition of cell proliferation, senescence, and autophagy. For perceiving this, the cells were exposed to the biological effective single dose of lethal and sub-lethal concentrations of NPs. Primarily, the cell proliferation inhibition in the NPs internalized cells was determined using the cumulative population from succeeding cultures. Viable cell count indicated a gradual and concentration-dependent decrease in the population of PSNPs, mNPs, and wNPs internalized cells after 72 hrs (Fig. 9a). However, during the microscopic examination, the trypan blue uptake in the NPs internalized cells was measly observed. This clearly signifies that the cells are certainly seized proliferation but metabolically active and yet remain viable. Among the NPs, significant growth inhibition was observed in wNPs, followed by mNPs and PSNPs. The relative difference of proliferation index calculated from the NPs internalized cells and control cells exhibited a concentration and NPs physical properties dependant inhibition in the proliferation index (Fig. 9b). Additionally, the observed effects so far from styrene molecules justify the possible contribution of leachates from macropinolysosomes in the cellular responses. Further, the results are in close correlation with the MTT assay signifying a single dose of NPs can cause the cytostatic effect in cells.
In general, the cells under permanent growth arrest can eventually turn into senescent cells [58, 59]. The HaCaT cells exposed to PSNPs (10, 100 & 500 µg/mL), styrene (10, 100 & 500 µg/mL), mNPs (1, 50 & 100 µg/mL), wNPs (1, 25 & 50 µg/mL) and H2O2 (25, 50 & 100 µM) for 48 hrs were washed and then incubated with NPs free medium. At 72 hrs post-exposure, the cells were stained for SA–β–gal activity and examined under a microscope (Fig. 10). Microscopic analysis revealed the concentration-dependent increase in the percentage of SA–β –gal–positive cells in PSNPs (37.5 ± 1.0, 44.5 ± 0.4 & 68.9 ± 1.0%), mNPs (15.5 ± 0.6, 43.4 ± 0.3 & 55.3 ± 1.2%), wNPs (37.9 ± 1.1, 59.2 ± 0.5 & 73.5 ± 0.0%), styrene (34.2 ± 0.1, 67.6 ± 0.4 & 72.2 ± 0.5%) and H2O2 (71.7 ± 1.5, 85.4 ± 1.0 & 86.0 ± 1.3%) treatment, respectively. Additionally, the typical senescent morphology, i.e., enlarged and flattened cells, as well as cytoplasmic granules accumulation, was detected in the NPs, styrene, and H2O2 treated cells [60, 61]. Certainly, the cellular senescence can be accompanied by the following phenotypic alterations such as chromatin modification, metabolic refinement, high autophagy activity, and production of proinflammatory secretome [58]. Among the phenotypes, autophagy, a genetically regulated bulk degradation process, was detected in the NPs treated cells (Fig. 11). Autophagy is a cell survival mechanism where the damaged cytoplasmic organelles and long-lived proteins can be degraded with the help of lysosomes [62, 63]. Recent studies on keratinocytes indicated that autophagy is associated with the increased anti-apoptotic effects [64, 65, 66] and plays a crucial role in keratinocytes' biology and pathology. The keratinocytes exposed to mNPs (Fig. 11a-d) and wNPs (Fig. 11e-h) displayed a series of autophagy structures namely, autophagosomes (Fig. 11a,f), autophagolysosome (Fig. 11b,c,g) and autolysosome (Fig. 11a,d,e,h). These structures evidence that the damaged intracellular organelles are actively engulfed by phagophores eventually turns into autophagosomes; after maturation, the autophagosomes are fused with lysosomes and produced autophagolysosomes/autolysosomes where the damaged organelles are actively digested. The observed proliferation inhibition, senescence, and autophagy certainly prove the cell’s determination to maintain cellular homeostasis via cytoplasmic and organelle turnover against a low level of ROS stress. The results further emphasize that all those ROS mediated molecular pathways may be interconnected [64, 67]. However, the increased level of cytoprotective events could also trigger inflammation and apoptosis [42, 43, 65, 67].
Recent immense attention paid on the cytotoxicity of micro/nano-plastics in human and animal models evidenced that plastic particles produce oxidative stress in cells at low concentration and cytotoxic at higher concentrations [68, 69]. In the present study, we have provided insights into the concentration-dependent regulatory, cytoprotective, and cytotoxic effects in HaCaT cells produced by smaller plastic particles. Herein, we present three lines of evidence that may help to close the existing knowledge gap on the NPs internalization and cell response. First, plastic particles mimic as protein aggregates via protein corona formation on their surface that triggers and accelerates the internalization process. Second, the internalized NPs are subjected to the lysosomal activity, which damages the NPs leading to the plastic molecules and additives release into the cells, thereby accelerate oxidative stress. Third, the ROS mediated down-regulation of cell growth and proliferation inhibition induces autophagy, followed by premature aging of cells. Therefore the increased use of nano/micro- plastic particles containing cosmetics may lead to premature aging in the skin cells, which may result in pigmentary stains and deep wrinkles, and further increase the risk of skin cancer [70, 71].