Figure 5. PL and PLE spectra in the solid state of the two modified hydrogels, VGALG (Green), measured with excitation wavelength at 475 nm and emission wavelength at 653 nm; and VGCB8ALG (Red), measured with excitation wavelength at 454 nm and emission wavelength at 675 nm.
The maxima shift from 475 to 540 nm and 653 to 675 nm in the excitation and emissions spectra, respectively, with the complexation of VG to CB8. The literature reveals entirely different PL and PLE spectra for VG or VGCB7[21,25] in the solid state, and ALG is known to be non-emissive. The results confirm that the chemical engineering of ALG conceived novel solid PL/PLE properties of the new hydrogels enabling us to monitor their cellular uptake utilizing the flow cytometry technique. Moreover, the excited-state average lifetime values of the hydrogels in the solid state (see Methods section, Figure S5, and Table S4 in the Supporting Information) increased from 0.28 to 0.85 ns with the addition of CB8 because of the confinement effect.[21,25–27] A more rigid biopolymer helps interpret the results of cellular internalization below.
Immunomodulatory Properties (Cellular Internalization)
The present study explored the potential effects of newly designed and prepared hydrogels on immunocompetent cells. Thus, the effects of VGALG and VGCB8ALG on the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors using the WST-1 test (Figure S6 in the Supporting Information) were first evaluated. It was shown that the percentage of viable cells decreased only when cells were treated with VGALG, and not VGCB8ALG, at the maximum 1.8 mg/ml concentration used. We also evaluated the concentration of LDH in the culture medium after treatment with hydrogels to determine a possible cytotoxic effect (Figure S7 in the Supporting Information). LDH is released into the cell culture supernatant when the plasma membrane is damaged. Treatment of cells with both hydrogels resulted in cell damage only at high concentrations of 1.8 mg/ml. Therefore, hydrogels at concentrations of 0.18 mg/ml and below do not have a cytotoxic effect on PBMCs. That allows us to conclude that hydrogels under study have high biocompatibility characteristics and low toxic effects on immune cells. Following the literature, we expected a high safety profile from VGALG and VGCB8ALG since each component of the hydrogels, CB8,[31] alginate, [32,33]and viologen, [34] is safe, biocompatible and has already been used separately to create similar hydrogels. The hydrogels (0.18 mg/mL) were then used for investigating the drug internalization to the PBMCs (CD3-and CD3+T-lymphocytes) by flow cytometry (Figure S8 in the Supporting Information). PBMCs uptake of VGALG has no significant differences from the control (non-treated cells). In the case of VGCB8ALG, internalization into CD3- cells was low and was measured at about 6%, while the median uptake by T cells was 62%, which is ten times higher. Noticeably, CB8 is a critical factor for internalization into the T cells. In the case of CD14+monocytes, internalization was carried out efficiently by both VGALG and VGCB8ALG (0.18 mg/mL), and no significant differences were observed between hydrogels (Figure S9 in the Supporting Information). The observed differences in internalization are associated with the mechanism of particle capture by various subpopulations of PBMCs; monocytes are capable of active phagocytosis of particles from the surrounding space, while T-lymphocytes, like most other body cells, do not have such a function.
Motivated by our results on the high level of internalization of the VGCB8ALG hydrogel into T-lymphocytes of healthy donors, we decided to evaluate the effect of hydrogels on human T-cell lymphoma 1301 cells. It was found that at all concentrations used, both hydrogels did not reduce the viability of 1301 cells at 24 and 72 hours of treatment (Figures S10 and S11 in the Supporting Information). Specifically, after 24 hours of treatment with 1301 hydrogels (Figure S10 in the Supporting Information), cell viability increased under the influence of VGCB8ALG compared to VGALG at a concentration of 1.8 mg/mL. Thus, VGCB8ALG at a high concentration can increase the number of viable cells in the culture, increasing the proliferation of the cells. Moreover, after 72 hours of treatment with 1301 hydrogels, the percentage of viable 1301 cells differed when treated with different concentrations of VGALG and VGCB8ALG (Figure S11 in the Supporting Information). For example, at a concentration of 1.8 mg/ml, there was an increase in viability in the VGCB8ALG group compared to VGALG. In contrast, treatment with VGALG increased the number of viable cells compared to treatment with VGCB8ALG at concentrations of 0.18 mg/ml and 0.018 mg/ml. Nonetheless, the two hydrogels do not have a toxic effect on 1301 human T-cell lymphoma cells with such long-term treatment for 72 hours. In summary, VGALG can enhance the proliferative activity of cells at average concentrations of 0.18 mg/ml and 0.018 mg/ml with long-term treatment for 72 h. Contrarily, VGCB8ALG increased the proliferative activity of these cells only at high concentrations (1.8 mg/ml) in culture for 24 and 72 hours. Following the viability results, we decided to evaluate the internalization of hydrogels at a concentration of 1.8 mg/ml by T-cell lymphoma cells at different treatment times (Figure S12 in the Supporting Information). The VGCB8ALG hydrogel was shown to be rapidly internalized by cells; after 1 hour, more than 80 percent of 1301 cells had absorbed the gel. After 24 and 72 hours, the percentage of internalized VGCB8ALG cells increased to more than 90%. Interestingly, VGALG was practically not absorbed by the cells at all times of treatment. To better explain this result, we evaluated the effect of hydrogel concentration on the uptake of T-cell lymphoma by the cell line (Figure 6). VGCB8ALG was efficiently taken up by cells at all concentrations used. The relative number of cells that took up VGCB8ALG varied with concentration, ranging from 99% at the highest to 15% at the lowest concentration. VGALG was poorly taken up by cells at all concentrations used.