br F DOTAP liposomes significantly enhanced the Th
3.3. F1-DOTAP liposomes significantly enhanced the Th2- and Th17-related cytokines
As depicted in Fig. 2, the maximum change of cytokines serum level compared to the buﬀer group was related to the F1 formulation, which significantly increased the IL-4 and IL-17 cytokines. The F1 formulation also significantly increased IL-4 cytokine level compared to the other liposomal formulations, with the highest diﬀerences in comparison with the F3 and F4 formulations. In addition, the F1 formulations sig-nificantly induced the serum level of IL-17 compared with the F4 International Journal of Pharmaceutics 567 (2019) 118492
3.4. F1-DOTAP liposome induced the splenocytes to secrete IFN-γ and IL-4
The production of IFN-γ and IL-4 by mice splenocytes is shown in response to stimulation with liposomal formulations in Fig. 3. There was no significant diﬀerence in the secreted cytokines of the spleno-cytes between the non-stimulated cell groups. F1 injection increased significantly the level of the secreted IFN-γ from the splenocytes as compared to the other liposomal formulations as well as control PBS-stimulated 1346242-81-6 (Fig. 3A). Moreover, the results showed that the sti-mulated mice splenocytes with the F1 formulation had the highest IL-4 production among others (Fig. 3B).
3.5. F1-DOTAP- and DOPE-containing liposome was the most eﬀective formulation in inducing CD8+ CTL-IFN-γ-producing cells
The frequencies of CD4+ T and CD8+ T lymphocytes as well as T lymphocytes subtypes including Th1, Th2, and Treg were investigated in splenocytes of treatment groups mice. As shown in Fig. 4, the IFN-γ secretion from CD8+ T lymphocytes was statistically diﬀerent among mice groups which treated with the F1 and F2 with other liposomes treated mice groups, indicating increased cytotoxic T lymphocytes (CTLs) responses in these two groups. However, the CD4+ T, CD8+ T and Treg lymphocytes frequencies showed no statistically significant diﬀerences among treated mice groups. Also, no significant diﬀerences were observed between the IFN-γ or IL-4 secretions by CD4+ T lym-phocytes in the mice groups which treated with the liposomal for-mulations.
3.6. In-vivo therapeutic studies
The liposomes showed neither a noticeable cancer therapy nor side-eﬀect-associated death event in terms of tumor growth and lifespan of mice (Fig. 5A and B). Although TGD% and ILS% were negative for all the liposomes, they were non-significant as opposed to those of the PBS group (Table 3).
Fig. 1. The apoptotic eﬀects of liposomal formulations on BALB/c splenocytes during 24-h culture with liposomes was studied using Annexin V and Propidium iodide (PI). To this aim, the splenocytes were extracted from BALB/c mice spleens and were cultured. The splenocytes were treated for 24 h with liposomes. Then, 105 cells/ 100 μl with 5 µl Annexin V-FITC incubated for 35 min at 37 °C. The PI dye was then added to the samples five minutes before running; apoptosis was evaluated using a BD FACSCalibur flow cytometry. A: The Live cells were gated through FSC-H and SSC-H parameters. B: The frequencies of live cells (lower left side of quadrant), early apoptotic cells (lower right side of quadrant), middle apoptotic cells (upper right side of quadrant) and fully apoptotic cells (upper left side of quadrant) of stained cells were calculated using FL-1 H and FL-2 H channels in a logarithmic mode. r> Fig. 2. Evaluation of the serum cytokines induced by liposomal formulations in mice model of C26 colon cancer. Liposomal formulation injection slightly increased the Th-1 related responses (A, B, and F for IFN-γ, IL-2 and TNF-α, respectively). The injection also slightly decreased the level of the regulatory cytokine, IL-10 (D). F1-L injection significantly increased the level of Th-2-related cytokine of IL-4 (C) and Th-17-related cytokine of IL-17 (E). Data are presented as individual value and mean (n = 5). Statistically significant diﬀerences are shown as follows: *p < 0.05, **p < 0.01.
Fig. 3. In-vitro secretion assay of IFN-γ (A) and IL-4 (B) from mice splenocytes following immunization with liposomal formulations using ELISpot. Phytohaemagglutinin (PHA) with the concentration of 10 µg/mL was also used as a polyclonal activator of splenocytes and as a positive control. The number of spots per well, denoting cytokine-secreting colony-forming units (CFU)/106 splenocytes, were increased significantly in F1-L-treated mice. At the bottom of the figures, as an example of the ELISpot assay wells are shown. 3 × 105 splenocytes was stimulated by PHA as Positive control, liposomal formulation as sample and culture media as negative control. The number of spots were then count after staining. Data are presented as mean ± SD (n = 5). The spots statistically significant diﬀerences are shown as follows: *p < 0.05, ***p < 0.001, ****p < 0.0001.