br Measurement of IFN and IL secretion
2.6. Measurement of IFN-γ and IL-4 secretion from splenocytes
The cell potential of IFN-γ and IL-4 secretion from mice splenocytes was examined with an Enzyme-Linked ImmunoSpot (ELISpot) kit (Mabtech AB, Sweden) as per the manufacturer’s protocol following immunization with liposomes. In brief, the corresponding detection PCI 32765 were coated onto the surface of ELISpot wells of 96-well plates (PVDF-plate, type MSIP, Mabtech AB, Sweden). Subsequently, the harvested splenocytes were seeded into the wells at 1 × 105, 2 × 105, and 3 × 105 cells/well suspensions and the plates were in-cubated at 37 °C in the cell culture incubator for 24 and 48 h for IFN-γ and IL-4 measurement, respectively. The liposomes were then added to the wells at the total lipid concentrations expressed in Table 2. For positive control, the polyclonal activator of Phytohaemagglutinin (10 µg/ml) was added to a few other wells. After incubation for 24 h, the spots, which are the representative of spot-forming units (SFU), were counted in every well.
2.7. Flow cytometric examination of cell surface markers and generated cytokines
The frequency of cell surface markers and intracellular cytokines were determined in lymphocytes after staining with fluorescent anti-bodies via flow cytometry. In brief, 106 splenocytes/well were treated with liposomes at total lipid dose/concentrations expressed in Table 2 and 1 μl/million cells BD GolgiPlug™ protein transport inhibitor (BD Biosciences, USA) for 24 h in a 24-well plate. Subsequently, the wells were washed twice with PBS containing 2% v/v fetal bovine serum (FBS) washing solution. First, 1 × 106 splenocytes were added to the flow cytometry tubes and surface antigens were stained with anti-mouse CD4-PE-Cy5, anti-mouse CD8-PE-Cy5 and anti-mouse CD25-FITC antibodies (BD Biosciences, California, USA) at 37 °C for 30 min. The stained cells were then fixed using cytofix/cytoperm™ kit (BD Biosciences, USA). In parallel, the intracellular cytokine staining was performed using anti-mouse IFN-γ –FITC, anti-mouse IL-4-PE and anti-mouse FoxP3-PE antibodies (BD Biosciences, USA) at 4 °C for 30 min. The stained cells were washed twice with the washing solution and suspended in 0.3 ml of the staining buﬀer. Finally, the frequency of cell markers and cytokines were measured in the cell population using flowcytometry.
2.8. In-vivo therapeutic studies
The therapeutic eﬀect of liposomes were examined using C26 colon carcinoma BALB/c mice model. Mice (five per each group) were treated with liposomes at total lipid concentrations stated in Table 2 at three subsequent consecutive weeks after tumor inoculation. Mice were monitored regularly by measuring tumor volume and weight and they were euthanized if either the tumor volume exceeded 1000 mm3 or the body weight loss was > 20% or mice were lethargic and unable to feed (Nikpoor et al., 2017). The tumor volume was calculated according to the following formula: [(tumor height × tumor width × tumor length) × 0.5] mm3. Time to reach end point (TTE), i.e. when the tumor size reaches 1000 mm3, increased life span (%ILS) and tumor growth delay (%TGD) indices were also calculated as described pre-viously in (Arabi et al., 2015).
2.9. Statistical analysis
Statistical analysis was conducted using a GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). Descriptive statistics, One-way ANOVA and post hoc test or their alternative nonparametric tests if applicable were used. Moreover, the survival time of mice groups were statistically analyzed using Mantel-Cox method. P values below 0.05 were considered significant.
3.1. Liposomes of diﬀerent net surface charges exhibited similar size distribution
Physicochemical characteristics of liposomes were given in Table 1. Total lipid concentrations of liposomes were, 12, 25, 25 and 24 mM for F1, F2, F3 and F4 formulations. The liposomes exhibited monomodal and similar particle size distribution given the values of Z-average and PdI, which were not significantly diﬀerent between liposomes. How-ever, Z-potential of the liposomes was diﬀerent between the formula-tions; the surface charge of DSPG- and DMPG-containing liposomes were noticeably negative, of DOTAP-containing liposome was positive, and of F4 formulation was nearly neutral as expected (liposome surface charge between −30 and +30 mV is considered as the range of neutral charge).
3.2. Only F1-DOTAP liposome induced apoptosis in splenocytes
The liposomal formulations exerted no cytotoxic eﬀect on the splenocytes, except for F1-DOTAP-containing liposomes that reduced the percent of the live splenocytes and increased the percent of the cells subjected to fully apoptosis (Table 2 and Fig. 1). Other liposome for-mulations did not show any cytotoxicity at the concentrations tested. After 24-h treatment, the highest and lowest percent of cell apoptosis were found in F1 and F4 treated cells, respectively.