br Liposome preparation br The nomenclature and lipid
2.2. Liposome preparation
The nomenclature and lipid compositions of the liposomes used in the study are given in Table 1. The liposomes were prepared via thin film hydration and extrusion method (Nikpoor et al., 2015). Briefly, appropriate amounts of phospholipids were added to a round-bottomed flask, previously prepared as chloroform stock solution and the solvent was removed with a rotary evaporator and one-night freeze drying. The resulting film was hydrated and mixed with phosphate-buﬀered saline (PBS, 300 mM, pH: 7.4) at 60 °C for 30 min under argon atmosphere followed by one-night incubation at 4 °C. The resulting multi-lamellar liposomes were then passed through respective 400, 200 and 100 nm pore size polycarbonate membranes. Finally, liposomes were sterilized for further experiments using 0.45 μm sterile syringe filters.
2.2.1. Liposome physicochemical characterization
The liposome properties, including particle size mean (shown as Z-average), particle size distribution (Polydispersity Index or PdI) and particle charge (Z-potential) were measured by a Dynamic Light Scattering (DLS) analyzer (Nano-ZS; Malvern, UK). The particle size and Z-potential measurements were done in PBS (10 mM, pH: 7.4) and 3-N-morpholinopropane sulfonic SR-11302 (MOPS, 10 mM, pH: 7.4) buﬀers, re-spectively at 50-fold dilution of liposomes. Phosphorus content of li-posomes was also measured spectrophotometrically at 800 nm ac-cording to Bartlett phosphate assay (Bartlett, 1959).
2.3. Examination of apoptotic activity of liposomes
The apoptotic eﬀect of the liposomes was tested on primary immune cells using Annexin V and Propidium iodide (PI). To this end, spleno-cytes were harvested from the spleen tissue of BALB/c mice after tissue digestion in collagenase type I solution (0.5% w/v in PBS) supple-mented with 3 mM calcium chloride and cell harvest through 70 µm cell strainer. Subsequently, 1 × 106 cells/ml were cultured in a 24-well culture plate. The splenocytes (106 cells/well) were incubated with varying concentrations of liposomes as shown in Table 2 for 24 h in a humidified 5% CO2 incubator at 37 °C. The cells were then washed twice with PBS and the rate of apoptosis was measured as follows: 1 × 105 cells/100 μl were incubated with 5 µl of Annexin V-FITC (Bio-legned, USA) for 35 min at 37 °C. Subsequently, 10 μl of PI solution was added to the cells. After 5 min incubation, the rate of apoptosis was
Table 2 The apoptotic eﬀects of liposomal formulations on mouse splenocytes during 24-h culture with liposomes.
Formulations Dosea Fully apoptosis % Middle apoptosis% Early apoptosis% Live%
a The liposome dose used is expressed as total lipid µmole/well.
b Data presented as mean ± standard deviation of three independent samples.
c Indicates significant diﬀerence as compared to controlled culture media.
evaluated using a BD FACSCalibur flow cytometry (BD Biosciences, USA) in FL-1 H and FL-2 H channels in a logarithmic mode.
2.4. Tumor inoculation, challenge and treatment
The liposome tumor challenge was conducted in vivo in female C26 tumor-bearing BALB/c mice. The animals were kept in standard con-dition in an animal house under controlled light cycle (12 h light/12 h dark) and fed at libitum. All procedures were performed according to specific national ethical guidelines for biomedical research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education (MOHME) of Iran (Approval code: 91002182). For this purpose, mice aged 6–8 weeks old were inoculated subcutaneously (SC) with C26 colon carcinoma cells (3 × 105 cells/mouse) in the right flank (Nikpoor et al., 2017).
Seven days after tumor induction when the tumor size exceeded 3 mm3, mice were randomly allocated to four cages and treated with liposomes (five mice per group). Mice were received F1 (0.6 µmole/ 50 µl/mouse), F2 (5 µmole/200 µl/mouse), and F3 (5 µmole/200 µl/ mouse) liposomal formulations subcutaneously weekly for three con-secutive weeks. F4 liposome (2.4 µmole/100 µl/mouse) was injected intravenously (i.v.) weekly for three consecutive weeks. After one week, mice were sacrificed with cervical dislocation and their splenocytes were harvested as described previously for the following experiments.
2.5. Measurement of serum cytokines
The level of diﬀerent cytokines, e.g. IFN-γ, IL-2, IL-4, IL-10, IL-17, and TNF-α, were measured in sera of mice treated with liposomes using an Enzyme-Linked ImmunoSorbent Assay (ELISA kit, Hangzhou Eastbiopharm, China) according to the manufacturer's protocol. For this purpose, blood samples were collected into polypropylene tubes at the end of the treatment period via heart puncture and the sera was drawn and stored at −70 °C until cytokine assay.