Bath sonicator

Ginger extract preparationGinger’s dried rhizomes were purchased from a reputable store and approved by the herbarium of Arak University of Medical Sciences. Dried ginger rhizome (25 gr) was ground and extracted with 250 ml of absolute ethanol (1:10) in the Soxhlet apparatus (Laborota 4000, Heidolph, Germany) for 4 hr at 78.9 ^°C (9 (link)). Then, the ethanol in the extract was evaporated by a rotary evaporator (Laborota 4000, Heidolph, Germany) at 89 ^°C. Finally, the obtained extract was dissolved in dimethyl sulfoxide (DMSO) and kept dark at -20 ^°C until use.
Liposome preparation and characterizationLiposomes containing ginger (LipGin) were prepared by the hydration method. In this way, hydrogenated soybean phosphatidylcholine (HSPC) (Ludwigshafen, Germany), cholesterol (Sigma-Aldrich, Mo), and mPEG2000-DSPE (Ludwigshafen, Germany) were dissolved in chloroform with 50 mM total lipid concentration [HSPC/chol/mPEG2000-DSPE (55:30:5 mol/mol)]. Then, ethanolic ginger extract (Gin) (10% w/w) was added to the lipid phase. The lipid dissolution in chloroform was removed under a rotary evaporator, followed by freeze-drying. In the next step, the lipid film was hydrated with a stabilizing buffer containing sucrose (280 mM) and histidine (10 mM) at pH=6.5 (21 (link)). Then the hydrated liposome was sonicated at 65 ^°C for 40 min in a bath sonicator (Bandelin, Germany). Next, the formulations were extruded under argon gas pressure to decrease the size of liposomes by 400, 200, and 100 nm polycarbonate membranes. Finally, the unencapsulated ginger extract was removed by dialysis against a hydration buffer at 4 ^°C by a 100 kDa dialysis bag (Spectra/Por™ 2 12-14 kD MWCO). A dynamic light scattering device measured particle size, polydispersity index (PDI), and the liposomes’ zeta potential (DLS, Nano-ZS; Malvern, UK). According to the approved protocol, the morphology of the liposomes containing ginger extract was assessed by negative staining using atomic force microscopy (AFM) (Zeiss, Jena, Germany).
Extract encapsulation efficiencyEncapsulation efficiency (EE) was reported as the ratio of encapsulated extract to the total extract in percentage. Hence, the prepared suspension containing liposomal ginger was first centrifuged at 14000 rpm for 10 min to separate the unencapsulated ginger (sample 1). Then, to lyse the liposomes and release the ginger, methanol was added to the residue at a dilution of 1 to 50 and sonicated for 5 min. Then the lysed sample was centrifuged at 14000 rpm for 10 min (sample 2). Finally, the absorption of ginger in samples 1 and 2 was read at a wavelength of 226 nm with a UV-visible spectrophotometer (SPEKOL 1300; Analytik Jena, Germany) compared with the extract’s standard curve. The encapsulation percentage was calculated using the following formula: EE %= encapsulated extract (mg) / amount of primary extract (mg) × 100.
Extract release rate For this purpose, 0.5 ml of the prepared formulation was poured into three dialysis bags with 3.5 kD molecular weight cut off (Pierce, Rockford, IL) and placed in buffers with different pH, including PBS (pH=7.5), sucrose/histidine (pH=6.5) and sucrose/histidine (pH=5.5). Extraction release was measured at other times (0.5, 1, 2, 4, 8, 12, and 24 hr) through optical absorption of all samples at 226 nm by a spectrophotometer.
MTT assayFor the MTT assay, C26 cells were obtained from the Pasture Institute (Tehran, Iran). The cells were cultured in a complete RPMI-1640 medium (Gibco, USA) enriched with 10% FBS (Gibco, USA), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Gibco, USA). The cells were seeded in a 96-well microplate (SPL Life Sciences, Korea) and incubated overnight at 37 ^°C and 5% CO₂. Then, the cultured cells were treated with 50–800 μg/ml of LipGin and Gin for 48 hr in triplicate. After incubation, 5 mg/ml of MTT (Sigma, USA) was added to each well, and the cells were incubated for 4 hr. Then, 200 μl of DMSO (Sigma, USA) was added to each well. After shaking for 45 min in the dark, the formed purple color of formazan crystals in living cells was measured at 560 nm, and IC₅₀ was calculated in Excel software based on the linear equation. Finally, cell viability was calculated using the following formula: Cell Viability%=OD treated cells/ OD control×100 (9 (link)). Also, mouse splenocytes were selected as normal cells and collected according to the previous protocol (22 (link)). Splenocytes, like C26 cells, were treated to similar concentrations of LipGin and Gin for 48 hr in triplicate, and the MTT assay was performed as mentioned above.
Mouse modelIn this study, 4-6 week-old female Balb/c mice were purchased from the Pasteur Institute (Tehran, Iran) and kept in a 12/12-hr light/dark cycle at 22-25 ^°C with free access to water and food. Mice were maintained and examined based on the Animal Ethics Committee of Arak University of Medical Sciences protocol (IR.ARAKMU.REC.1398.336). Colorectal cancer is induced subcutaneously (SC) in mice by injecting 3×10⁵ C26 cells into the body’s right flank. Treatment began in groups one week after tumor induction when the tumors became palpable. Mice-bearing tumors were treated for two weeks with four intravenous injections (twice a week). First, the liposomal ginger (LipGin) group was injected with 100 mg/kg/day of liposomes encapsulated ginger. Second, the ginger extract (Gin) group was injected with 100 mg/kg/day of ginger extract. Third, the Doxil group was injected with 2.5 mg/kg/day of Doxil. Fourth, Doxorubicin (Dox) group was injected with 2.5 mg/kg/day of doxorubicin (23 (link)), and the control group was injected with PBS. During the 14-day treatment period, mice’s tumor size and weight were measured using calipers and scales. Finally, tumor tissues were removed, weighed, and sized using the following equation: Tumor volume=length×(width×2)×0.52.
Toxicity evaluation of the injected liposomesFor this purpose, on the 14^th day of treatment, the mice were anesthetized and blood samples collected for estimation of both liver and renal functions by determining the levels of blood urea nitrogen (BUN), creatinine, serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) (Pars Azmoon, Iran).
Hematoxylin and eosin (H&E) stainingTumor tissues isolated from mice were first fixed in formalin and molded into paraffin blocks for incision. Next, tumor-infiltrating lymphocytes (TILs) were determined in the tumor tissue by H&E staining.
Flow cytometryRemoved tumor tissue and spleen were washed with sterile PBS, and single cells were harvested using a 70 µm cell strainer (SPL Life Sciences, South Korea). For immunophenotyping of cytotoxic CD8 T lymphocytes (CTL), FITC-conjugated mouse anti-CD3 antibody and PerCP-conjugated mouse anti-CD8 antibody were used. Also, PE-conjugated murine anti-CD4 antibody, PerCP-conjugated murine anti-CD25 antibody, and FITC-conjugated murine anti-FoxP3 antibody were used to evaluate T regulatory lymphocytes (Treg cells). All of the compounds were obtained from eBioscience (USA), and flow cytometry was performed by counting 10,000 events based on the manufacturer’s instructions. Finally, the output data were analyzed by Flowjo software (Tree Star Inc., OR, USA).
Real-time PCR assayGene expression of Bax and Bcl-2 in tumor tissue and TNF-α, TGF-β, and IFN-γ in splenocytes were evaluated using real-time PCR. For this purpose, RNA extraction and cDNA synthesis were performed according to the manufacturer’s protocol (Yekta Tajhiz, Iran). Forward and reverse primers of target and reference (Gapdh) genes were designed using AlleleID 6.0 (Premier Biosoft International, USA), and the sequence of the primers was checked by NCBI Blast for specificity (Table 1). The real-time PCR assay was run in duplicate using SYBR Green I 2x Master Mix (Yekta Tajhiz, Iran) in a LightCycler 96 system (Roche, Switzerland). PCR products were evaluated by the melting curve to confirm the absence of nonspecific products. Quantitative determination of target genes was also performed using the Pfaffle method.
Statistical analysisStatistical analyses were performed using GraphPad Prism 6.0 software (San Diego, CA, USA). The assumption of normality was tested using the Kolmogorov-Smirnov test. One-way ANOVA and Tukey’s tests were also performed to evaluate differences in variables between the studied groups. Data are presented as Mean±SEM. A P-value less than 0.05 was considered statistically significant.

Before starting, the vat was filled with the resin and positioned in the printer under the build plate. Then, the build plate was lowered into the vat to a predetermined height, and the DLP projected the first slice of the design for a predetermined amount of time. Next, the build plate rose for a few seconds and then returned to the vat, and the DLP projected the next slice of the design. Subsequently, the build plate rose again, and this process continued until the entire design was printed. Next, the printed object was removed from the build plate, rinsed with isopropyl for 3 min in a sonicator bath (BANDELIN, Germany), dried using air pressure and cured in a UV oven for 5 min (PCU LED, Dreve, Germany).

Freeze-dried cyanobacteria material was used for the analysis of intracellular amount of cyanometabolites. The extraction of microcystins (MCs) and other NRPs was performed using 75% methanol in MiliQ water. The saxitoxin (STX) was extracted with a mixture containing 4 mM ammonium formate buffer (pH 3.5) and acetonitrile (95:5, v/v) at a ratio of 2:3. All samples were disrupted by vortexing for 5 min and maintained for 5 min in a bath sonicator (Bandelin, Berlin, Germany), centrifuged at 10,000× g for 20 min, and the supernatant was transferred to chromatographic vials. The analysis was performed using liquid chromatography tandem with mass spectrometer LC-MS/MS (AB Sciex. Concorde, ON, Canada) equipped with a turbo ion spray ionization, operating in positive mode using the information-dependent acquisition method (IDA) for the detection of NRPs as described in Grabowska et al. [22 (link)]. The identification of NRPs (aeruginosins, anabaenopeptins, cyanopeptolins) was performed based on the enhanced ion product spectra. The relative amount of the NRPs was estimated and provided as the peak area in the extracted ion chromatogram. MCs quantitative analysis was performed by MRM (Multiple Reaction Monitoring) using standards for MC-LR, MC-RR, dmMC-LR, dmMC-RR, MC-YR, MC-LA, MC-LY, MC-LW, MC-LF variants (Alexis Biochemicals, San Diego, CA, USA). Detailed methodology presented in Khomutovska et al. [69 (link)]. Saxitoxin detection and quantitative analysis was conducted in MRM as described in Karosienė et al. [30 (link)] using STX standard (National Research Council, Halifax, Canada). Data were analyzed using Analyst QS^® 1.5.1 software.

Prior to printing, the vat was filled with resin and positioned under the build platform. The printing process is then carried out as follows: First, the platform surface lowers into the vat to a predetermined height and the DLP projects the first layer of the design for a predetermined time. The platform then elevates above the surface of the vats for a few seconds before dipping again into the vat for the formation of the next layer based on the CAD design. This sequence is repeated until the entire design is printed. The printed object is removed from the platform, the uncured resin is removed by washing the model with isopropyl for 3 min in a bath sonicator (Bandelin, Germany), dried using air pressure, and cured in an UV oven for 5 min (PCU Led, Dreve, Germany).