Confirmation of cell encapsulation
To prove the encapsulation of Jurkat T cells in PAH, PSS/PAH, or PSS-GNR/PAH, numerous techniques were utilized.
First, the zeta potentials of different encapsulated Jurkat T cells were measured. Measured cells were dispersed in FBS-free RPMI-1640 for all measurements. The zeta potential value of unencapsulated Jurkat T cells was −14.3 ± 0.2 mV. However, when cells were encapsulated with PAH (PAH@Jurkat cells), the zeta potential value changed to −9.7 ± 0.2 mV. The low negative number of zeta potential value confirms that the cationic polyelectrolyte (PAH) could form a layer on the surface of the Jurkat T cells. When PAH@Jurkat cells were encapsulated with PSS-GNRs (zeta potential ~ −16.6 ± 0.7 mV), the zeta potential of PSS-GNR/PAH@Jurkat cells dropped to have a lower negative value at −11.3 ± 0.4 mV. The change in zeta potential value clearly indicates that Jurkat T cells were encapsulated in PAH or PSS-GNR/PAH. Our results were similar to previous works that investigated the zeta potential values of mammalian cells coated with different polymers (Zhao et al. 2016; Pandey et al. 2013; Bondar et al. 2012). As can be seen, PAH@Jurkat cells had a weak negative value of zeta potential (−9.7 ± 0.2 mV) and the zeta potential value altered to have a further negative value after encapsulating Jurkat T cells with PSS-GNRs. The mechanism underlying the interaction in this case is unclear. We shall not discuss this in further detail, but rather mention that the charge densities of polyelectrolytes and the concentration of the ions could play a major role here. And, available results of this study strongly suggest that PSS-GNRs could modify the surface of PAH@Jurkat cells resulting in changing of zeta potential values. The binding of a weak negative charge on the cell surface to negatively charged polymers was also reported (Zhao et al. 2016). In the case of PSS/PAH@Jurkat cells, the zeta potential was more negative (−21.1 ± 0.4) than that of PSS-GNR/PAH@Jurkat cells (−11.3 ± 0.4). This could be caused by the amount of PSS formed on PSS-GNRs used in cell encapsulation, which should be less than that formed after using PSS alone. As expected, Jurkat T cells directly encapsulated in PSS had a similar zeta potential value (−14.6 ± 0.2) to unencapsulated cells (−14.3 ± 0.2). This implies that PSS could not bind well to Jurkat T cells through the same negative charge interaction of PSS and the cell membrane.
The presence of fluorescent dyes attached on PAH was used to confirm the cell encapsulation. There was no fluorescence signal detected in unencapsulated Jurkat T cells stained with FITC alone (Fig. 1a). However, FITC-positive cells were found in PAH-FITC@Jurkat cells (Fig. 1b). This indicates that cells were encapsulated with PAH. PSS/PAH@Jurkat cells stained with PAH–TRITC showed the red fluorescent signal (Fig. 1c). This confirms that PAH–TRITC could stain the cells through the PSS outer layer shielding on the Jurkat T cells. A similar result was also found in PSS-GNR/PAH@Jurkat cells (Fig. 1d). The negative control was prepared by staining PAH@Jurkat cells with PAH–TRITC. There was no presence of a red fluorescent signal. This result indicates that the same polyelectrolytes could prevent the binding between PAH and PAH-TRTIC on the cell surfaces (Fig. 1e).
We also observed the morphology of Jurkat T cells by SEM. When compared to unencapsulated cells, the appearance of a smooth surface was detected on encapsulated cells (PSS-GNR/PAH@Jurkat cells) (Fig. 2b). However, the surfaces of unencapsulated cells had abundant microvilli (Fig. 2a). Our results are consistent with the previous work published by Zhao et al. (2016) that used chitosan and alginate to conduct a conformal encapsulation of T cells.
All approaches used for confirming the encapsulation of Jurkat T cells showed that the cells were successfully encapsulated through a layer-by-layer technique. The cationic PAH was shielded on the negative charge of the Jurkat T cell membrane. The second layer of PSS or PSS-GNRs was formed surrounding each cell. Overall results from zeta potential measurement, fluorescent observation, and SEM demonstrated the formation of different encapsulated Jurkat T cells (PAH@Jurkat, PSS/PAH@Jurkat, and PSS-GNR/PAH@Jurkat cells).
Investigation of GNRs in PAH/PSS-GNR@Jurkat cells
The ICP-MS approach was used to confirm whether PAH@Jurkat cells could be shielded by PSS-GNRs. The TEM image of PSS-GNRs is shown in Fig. 3. The data from ICP-MS showed a significant presence of gold (64.3 ± 7.7 µg L−1) found in PSS-GNR/PAH@Jurkat cells (Fig. 3). The amount of gold observed provides evidence that PSS-GNRs (negatively charged surface) were shielded on the surface of PAH@Jurkat cells (positively charged cell surface) through the opposite charges of their surfaces. This result strongly indicates that a PSS-GNR layer formed on Jurkat T cells. The very small content of gold detected in PSS-GNR@Jurkat cells (1.8 ± 0.1 µg L−1) could occur due to non-specific binding of PSS-GNRs to cells.
Since the ICP-MS cannot perceive the difference between GNRs deposited on the cell surface and internalized GNRs, TEM was used to evaluate how GNRs were positioned on encapsulated Jurkat T cells. With TEM images, we found that PSS-GNRs were only located on the cell surface of Jurkat T cells encapsulated PAH layers (Fig. 4c, red arrow). This strongly indicates that PSS-GNRs only attached to the layer of PAH and did not internalize inside Jurkat T cells (Fig. 4c). It was reported by Fakhrullin et al. that the polyelectrolyte layer can act as a glue to attach metal nanoparticles and block nanoparticle translocation into the cells (Fakhrullin et al. 2012). As expected, there were no PSS-GNRs located on the cell surface, nor inside Jurkat T cells encapsulated with PSS/PAH (PSS/PAH@Jurkat cells; Fig. 4b) and unencapsulated Jurkat T cells (Fig. 4a). An excellent review article by Dykman and Khlebtsov (2014) has shown the effect of gold nanoparticles and their interaction with mammalian cells through cellular endocytosis. Our results here provide evidence that the PAH layer encapsulating on the cell surface might block the cellular uptake of PSS-GNRs through this endocytic pathway.
Cell viability and cell proliferation of unencapsulated and encapsulated Jurtkat cells
To investigate whether the encapsulating layers affect the cell viability, we measured the cell viability at 0 and 24 h after cultivation of unencapsulated Jurkat T cells, PAH@Jurkat cells, PSS/PAH@Jurkat cells, and PSS-GNR/PAH@Jurkat cells, using the CellTiter-Glo assay. This technique measured viable cells from the content of adenosine-5′-triphosphate (ATP). After encapsulation, the cell viability of PAH@Jurkat cells, PSS/PAH@Jurkat cells, and PSS-GNR/PAH@Jurkat cells was measured immediately (0 h). The relative cell viabilities, based on that of unencapsulated cells of PAH@Jurkat cells, PSS/PAH@Jurkat cells, and PSS-GNR/PAH@Jurkat cells, were ~ 94.4 ± 0.3%, 90.8 ± 1.2%, and 96.7 ± 0.9%, respectively (Fig. 5a). Significant reductions (P < 0.01) in cell viability were observed in encapsulated PAH@Jurkat and PSS/PAH@Jurkat cells when compared with unencapsulated cells. The lowest cell viability was detected in PSS/PAH@Jurkat cells. The cell viability after 24-h cultivation of Jurkat T cells encapsulated with different forms was also measured. It showed that the cell viability of all encapsulated cells significantly decreased (P < 0.01) to around 8–15% depending on the encapsulation method.
Similar to the cell viability at 0 h, PSS/PAH@Jurkat cells at 24 h showed the lowest percentage of cell viability at 85.3 ± 0.7% (Fig. 5b). The cell viabilities of PSS-GNR/PAH@Jurkat and PAH@Jurkat were 87.0 ± 0.5% and 91.5 ± 0.6%, respectively (Fig. 5b). Although the results indicate that the metabolic activity of encapsulated cells was still active after culturing for 24 h, it seems that the number of layers could influence cell viability. Cells encapsulated with a layer of PAH (PAH@Jurkat cells) had a higher cell viability than cells encapsulated with two layers (PSS/PAH@Jurkat and PSS-GNR/PAH@Jurkat cells) after 24-h cultivation. The reason for this could be that there was a higher limitation of nutrient and waste diffusion through cells in cells encapsulated with two layers than that of encapsulation with one layer.
As reported in the review paper (Antipov and Sukhorukov 2004), a higher layer number could lead to a lower amount of penetrated molecules. When compared to the cell viability between PSS/PAH@Jurkat and PSS-GNR/PAH@Jurkat cells, there was no significant difference in cell viability (P ≤ 0.01). Furthermore, cell proliferation was determined gain more information on the metabolic function of cells after encapsulation. Unlike cell viability assay that is normally used to determine the ratio of live and dead cells, it is well known that cell proliferation assay can be used to monitor the growth rate and metabolic activity of cells.
CellTiter 96® AQueous One Solution cell proliferation assay was used to measure cell proliferation by measuring metabolic activity of cells through the reduction of MTS tetrazolium (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium compounds. We found that cell proliferation of PAH@Jurkat, PSS/PAH@Jurkat, PSS-GNR/PAH@Jurkat, and Jurkat T cells was increased when we increased the culturing time from 24 h to 48 and 72 h, respectively (Fig. 6). This indicates that the encapsulation of cells, using our approach here, could influence the growth rate of Jurkat cells. However, cell proliferation of unencapsulated Jurkat T cells was much higher than that of encapsulated cells.
The relative growth rates of unencapsulated Jurkat T cells at 48 and 72 h were increased ~1.4- and 2.3-fold, respectively, above unencapsulated Jurkat T cells cultured for 24 h. Around a 1.3-fold increase in the proliferation rate of PSS/PAH@Jurkat and PSS-GNR/PAH@Jurkat cells cultured for 48 h was detected as compared to encapsulated Jurkat cells after 24 h culture. The proliferation rate of PAH@Jurkat at 48 h was ~ 1.1-fold increased as compared to at 24 h culture. At 72-h culture, the increase in proliferation rate of all encapsulated Jurkat T cells (~ 1.4–1.6-fold) was lower than that of unencapsulated Jurkat T cells (~ 2.3-fold) when compared to 24-h culture of each cell condition (Fig. 6). The relative proliferating cell percentages of PAH@Jurkat, PSS/PAH@Jurkat, and PSS-GNR/PAH@Jurkat cells at 24 h significantly decreased from 100% (of unencapsulated Jurkat T cells) to ~ 71.9 ± 1.7%, 56.8 ± 1.1%, and 50.2 ± 1.5%, respectively. A significant decrease in proliferation rate of PAH@Jurkat, PSS/PAH@Jurkat, and PSS-GNR/PAH@Jurkat cells cultured for 48 and 72 h was also detected as compared to unencapsulated Jurkat T cells (P < 0.01).
At 72 h, PSS-GNR/PAH@Jurkat cells had the highest increase in growth rate ratio (~ 1.6-fold increase) among all types of encapsulated Jurkat T cells compared to a 24-h culture of encapsulated cells (PSS-GNR/PAH@Jurkat) (Fig. 6). The reason that encapsulated cells could proliferate could be a result of the mild layer-by-layer processing technique. In our case, it appears that the charge density of PAH, PSS, and PSS-GNRs might have no major effect on cell proliferation. This might be due to the low concentration of PAH, PSS, and PSS-GNRs used for cell encapsulation. Although encapsulated cells could proliferate, it seems that the encapsulating layer could reduce the rate of proliferation when compared to unencapsulated Jurkat T cells. This could have occurred due to the effect of the encapsulating layer that is possibly impacting on the diffusion rate of molecules that pass through encapsulated cells (Cook et al. 2013).
Inflammatory cytokine responses of unencapsulated and encapsulated Jurkat T cells
T cells can produce inflammatory mediators such as IL-6 and TNF-α (Wang et al. 2006). However, high secretions of IL-6 and TNF-α (Wang et al. 2006) can lead to several diseases such as cystic fibrosis (Stecenko et al. 2001), autoimmune disease, and chronic inflammatory proliferative disease (Ishihara and Hirano 2002). Hence, we investigated the secretion of these cytokines from encapsulated and unencapsulated Jurkat cells. IL-2 is a lymphokine that plays an important role in maintaining activated T cell proliferation (Pawelec et al. 1982). Therefore, we evaluated whether different encapsulations applied in our study affected the secretion of IL-2 by Jurkat T cells. Besides IL-6, TNF-α, and IL-2, we also investigated the secretion of IL-1β, another cytokine involved in inflammation induction (Tang et al. 2012).
The production of cytokines and lymphokines by encapsulated and unencapsulated Jurkat T cells was investigated at 5 and 24 h after cell encapsulation. Unencapsulated Jurkat cells were used as a control sample in our study (Fig. 7). When compared to unencapsulated Jurkat T cells, the levels of TNF-α and IL-1β at 5 h secreted by PAH@Jurkat, PSS/PAH@Jurkat, and PSS-GNR/PAH@Jurkat cells were similar to unencapsulated Jurkat T cells (Fig. 7a, c). This indicates that the single layer (PAH) or the double layer (PSS/PAH and PSS-GNR/PAH) used for encapsulation of Jurkat T cells had no effect on TNF-α and IL-1β induction. Similar results were also observed in TNF-α production at 24 h. However, in the case of IL-1β, a significant IL-1β reduction was found in PAH@Jurkat cells after treating for 24 h (P < 0.01). The reason for this is uncertain. However, it is a possibility that some IL-1β molecules might not be able to pass through the PAH layer. The zeta potential of PAH@Jurkat cells was less negative than other encapsulating formats, and this could lead to different binding of PAH at the outer layer of cells to other molecules in cell culture media. This binding possibly affected IL-1β release by blocking the diffusion of IL-1β. A decrease in IL-2 levels was found in all encapsulated cells after culturing for 5 h. A significant reduction in IL-2 was detected in all encapsulated cells after cells were encapsulated and cultured for 24 h (P < 0.01). Werner et al. (2015) also found a reduction in IL-2 by Jurkat T cells encapsulated with polyelectrolytes (Fig. 7d).
In the case of IL-6 (Fig. 7b), at 24 h post-encapsulation, we found that cells encapsulated with PAH, PSS/PAH, and PSS-GNR/PAH secreted IL-6 at a significantly higher level than unencapsulated cells (P < 0.01). It was reported that the IL-6 secretion in encapsulated cells could be enhanced by polyelectrolytes used for encapsulation (Mooranian et al. 2016). Based on our results here, an induction in IL-6 levels was significantly detected in PAH@Jurkat cells at 5 h post-encapsulation. This implies that the PAH layer could first impact IL-6 induction. After cells were encapsulated with the second layer, higher levels of IL-6 were detected. Thus, the second layer of PSS or PSS-GNRs could also be involved in triggering an immune response related to IL-6 expression. Furthermore, the second layer of encapsulation might lead to a decrease in porosity that could cause an accumulation of metabolic by-products related to IL-6 production (Mooranian et al. 2016; Schmidt et al. 2008).
Biological activity in co-culture between macrophage cells and Jurkat T cells encapsulated with polyelectrolytes and PSS-coated GNRs
It is well known that macrophage cells play an important role in the immune system. Because of the lack of information on biological activities between encapsulated cells and macrophages, we were therefore interested in the biological activities of these two cells in co-culture. The co-culture system of macrophages and T cells can be used to acquire more information on immune responses between encapsulated T cells and macrophages.
We used non-activated Jurkat T cells, with and without encapsulation, to investigate whether the encapsulation of cells could influence inflammatory cytokine enhancement in the co-culture system between human macrophages and human T cells. Here, we only focused on the investigation of the double layer encapsulation (PSS/PAH@Jurkat & PSS-GNR/PAH@Jurkat cells) because we aim to use PSS-GNR/PAH@Jurkat cells in a future study for therapeutic applications. The ratio of THP-1 macrophage per Jurkat T cell used in the co-culture system was 1:1. The results showed that there were no significant differences in IL-2 and IL-1β expression in THP-1 macrophage and Jurkat T cell co-cultures when both encapsulated and unencapsulated Jurkat T cells were used (Fig. 8c, d). This implies that there was no cell-contact-mediated activation of THP-1 macrophages by encapsulated and unencapsulated Jurkat T cells. As stated previously, TNF-α and IL-6 cytokines are inflammatory cytokines and they may be involved in some disease pathogenesis (Rossol et al. 2005). Therefore, it was also worthwhile to test whether encapsulated cells could lead to induction of IL-6 and TNF-α expression.
Our results showed that there was a significant induction of TNF-α in THP-1 macrophages/unencapsulated Jurkat T cells after co-culturing for 24 h when compared with THP-1 macrophages cultured alone. However, there was no increase in TNF-α level when THP-1 macrophages were cultured with encapsulated Jurkat T cells (PSS/PAH@Jurkat or PSS-GNR/PAH@Jurkat) (Fig. 8b). These results indicate that the encapsulation of Jurkat T cells could help prevent the induction of TNF-α production through cell-contact-mediated activation of THP-1 macrophages by Jurkat T cells. The layer of PSS/PAH or PSS-GNR/PAH could block the ligand interactions involved in TNF-α production by THP-1 macrophages resulting in no activation of the THP-1 macrophages to enhance TNF-α production. The TNF-α levels in co-cultures of THP-1 macrophages with PSS/PAH@Jurkat or PSS-GNR/PAH@Jurkat cells were similar. This implies that GNRs had no impact on inducing TNF-α. These two types of cell encapsulation resulted in different zeta potential values (−21.1 ± 0.4 for PSS/PAH@Jurkat and −11.3 ± 0.4 for PSS-GNR/PAH@Jurkat cells), but it seems that the different degree of zeta potential values did not impact on the induction of TNF-α in our system.
In the case of IL-6 expression, we found that there was no significant difference in IL-6 production of THP-1 macrophage/Jurkat T cell (both unencapsulated and encapsulated cells) co-cultures. These results also revealed that there was no cell-contact-mediated activation of THP-1 macrophages through ligand interactions by encapsulated and unencapsulated Jurkat T cells (Fig. 8a). However, it is worth noting that though, there was an induction of IL-6 in encapsulated Jurkat T cells. Nonetheless, the induction amount of IL-6 was much lower than Jurkat cells treated with a positive control chemical (phorbol 12-myristate 13-acetate; PMA) (Supplementary Material, Fig. S1) and there was no significant production of IL-6 in THP-1 macrophage/Jurkat T cell co-culture. This implies that the induction amount of IL-6 in encapsulated Jurkat T cells might be at a level that does not lead to an adverse effect.