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.