Materials
Fetal bovine serum (FBS) was obtained from Biosera (East Sussex, UK); nonessential amino acid solution (NEAA), chlorpromazine (CPZ), sodium phosphotungstate, and 4 % paraformaldehyde phosphate buffer solution (PFA) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Phosphatidylcholine (PC), cholesterol, and phosphatidylethanolamine were kindly provided by Nippon Fine Chemical Co., Ltd. (Tokyo, Japan). Chloroform, methanol, penicillin-streptomycin-L-glutamine solution (×100) (PSG), and propidium iodide (PI) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Methyl-beta-cyclodextrin (MβCD) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ethyl-isopropyl amiloride (EIPA) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Filipin III was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Imject Alum Adjuvant (alum) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals used were of the highest commercially available grade.
Animals
Eight-week-old BALB/c female mice were purchased from Sankyo Labo Service Co., Inc. (Tokyo, Japan) and maintained under specific pathogen-free conditions. The protocols for experiments involving animals were approved by the Institutional Animal Experimentation Committee of the Tokyo University of Science. All experiments involving animals were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines,.
Cell Culture
Murine macrophage-like cell line (RAW264.7), murine colon adenocarcinoma cell line (colon26), and firefly luciferase (fluc) stably expressing-colon26 (colon26/fluc) cells [56] were obtained from Professor Yoshinobu Takakura (Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan), and cultured in RPMI medium supplemented with 10% heat-inactivated FBS and PSG at 37°C in humidified air containing 5% CO2. Murine fibroblast (NIH3T3) cell line (RCB2767) was provided by the Riken BRC through the National BioResource Project of the MEXT/AMED (Ibaraki, Japan) and cultured in DMEM supplemented with 10% heat-inactivated FBS and PSG at 37°C in humidified air containing 5% CO2.
Preparation Of Pc Liposome (Pc-lip)
PC, cholesterol, and phosphatidylethanolamine were mixed at a molar ratio of 10:5:1 and dissolved in 2 mL chloroform in a round-bottom flask. A lipid film was formed on the wall surface of the flask by solvent evaporation under reduced pressure using a vacuum pump in a water bath. Then, 1 mL PBS was added, and crude PC liposomes were prepared by sonication at 70°C for 2 min using an ultrasonic cleaner (Sono Cleaner, Kaijo, Tokyo, Japan) [57]. They were extruded 5 times through Whatman Nuclepore Track-Etched Membrane with 100 nm pore size (Cytiva, Tokyo, Japan) at 70°C. After centrifugation at 10,000 × g for 10 min to remove aggregates, PC-Lip were obtained from the supernatant.
Preparation Of Cnps
Super sweet corn was purchased from a local fresh market and washed thoroughly with distilled water. The edible portion of corn (100 g) was vigorously homogenized with 100 mL of distilled water using a food processor for 2 min. The homogenized juice was sequentially centrifuged at 2,000 ⋅ g for 20 min, 5,000 ⋅ g for 30 min, and 10,000 ⋅ g for 1 h at 4°C. The supernatant was filtered through a 0.45 µm-pore size syringe filter (Minisart NML, Sartorius, Göttingen, Germany) to exclude rough residues. Approximately 38 mL of the filtered sample was added to 2 mL of 60% sucrose solution in a tube, and then ultra-centrifuged at 100,000 ⋅ g for 120 min at 4°C using Optima XL-K with an SW28 rotor (Beckman Coulter, Inc., Brea, CA, USA). The supernatant was carefully removed from the top, and the fraction (approximately 2 mL) containing yellow-colored cNPs that was present just above the 60% sucrose solution was collected carefully without disturbing the sucrose solution.
Characterization Of Cnps
The particle size and zeta potential of cNPs were measured by dynamic light scattering (DLS) using an ELSZ-2000ZS instrument (Otsuka Electronics Co., Ltd, Osaka, Japan). The particle number was measured using a ZetaView Laser Light Scattering Microscope (Particle Matrix GmbH, Microtrac, Meerbusch, Germany). For transmission electron microscopy (TEM) imaging, a drop of cNPs was deposited onto the surface of a carbon-coated copper grid and negatively stained with 1% sodium phosphotungstate for 3 min, and the sample was dried at room temperature (approximately 22°C). Then, the sample was observed using an H-7650 TEM (Hitachi High-Tech Co., Ltd, Tokyo, Japan) operated at 100 kV. The protein concentration of cNPs was measured by the Bradford assay [58], and used to standardize the concentration of cNPs.
Fluorescent Labeling Of Nps
cNPs were labeled with a fluorescent lipophilic dye, 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Thermo Fisher Scientific). In brief, 0.1 mg/mL DiI solution (10 µL) was added to 1000 µg/mL cNPs (1 mL), and the mixture was incubated for 30 min at 37°C. DiI-labeled cNPs were then purified using 100-kDa ultrafiltration (Amicon Ultra-15, Merck KGaA, Darmstadt, Germany). DiI-labeled PC-Lip was prepared in a similar manner.
Cellular Uptake Of Dii-labeled Cnps
Colon26, NIH3T3, and RAW264.7 cells were seeded in 4-well chambered cover glass (IWAKI; AGC Techno Glass Co., Ltd., Chiba, Japan) at a density of 1 ⋅ 105 cells/well and cultured overnight at 37°C. The medium was then replaced with fresh culture medium containing 350 µg/mL DiI-labeled cNPs. After 3 h of incubation, the cells were fixed with 4% PFA for 30 min on ice, and the cells were washed three times with PBS. Subsequently, Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA) was added. The cells were imaged using a Leica SP8 laser scanning confocal microscope (Leica, Wetzlar, Germany) using the LAS X Life Science software. For flow cytometric analysis, the cells fixed with 4% PFA were collected using a cell scraper, and then filtered through a 70 µm cell strainer (Becton Dickinson). Cellular uptake of DiI-cNPs or PC-Lip was quantitatively analyzed using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and FlowJo software ver8.7 (Becton Dickinson).
Elucidation Of Cellular Uptake Pathway Of Cnps
To elucidate the cellular uptake pathways of cNPs, endocytosis inhibitors (10 mM MβCD, 30 µM CPZ, 40 µM EIPA, and 0.5 µg/mL Filipin III) were added to colon26 cells and incubated for 40 min at 37°C. DiI-labeled cNPs were then added to the cells and incubated for 3 h at 37°C. The cells were fixed on a glass chamber slide using 4% PFA for fluorescence imaging under a confocal microscope. Cellular uptake of DiI-cNPs was quantitatively analyzed using a BD FACSCalibur flow cytometer and FlowJo software ver8.7, as described above.
Cell Number And Proliferation Assay
Colon26, NIH3T3, and RAW264.7 cells were seeded in 96-well culture plate at a density of 5 ⋅ 103 cells/well and incubated for 24 h at 37°C. Culture medium was then replaced with fresh medium containing various concentrations of cNPs. After 24 h of incubation, cell number was measured using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). The experiment was repeated except the medium was replaced with fresh medium containing 1000 µg/mL cNPs. The cell number was measured using the Cell Counting Kit-8 after 6, 12, 24, and 48 h of incubation.
Cell Cycle Analysis
Colon26 cells were seeded in 6-well culture plate at a density of 1 ⋅ 105 cells and incubated for 24 h. The cells were washed with PBS, and cNPs at concentrations of 250, 500, and 1000 µg/mL were added to them. After 24 h incubation, the cells were washed with PBS and fixed with 70% ethanol at 4°C for 3 h. PI (50 µg/mL) was added to the cells, which were then incubated for 30 min at 4°C in the dark. The cells were analyzed using a BD FACSCalibur flow cytometer, and cell cycle analysis was performed using the FlowJo software ver8.7.
Apoptosis Assay
Colon26 cells were seeded in 6-well culture plate at a density of 1 ⋅ 105 cells and incubated for 24 h. The cells were washed with PBS, and cNPs at a concentration of 1000 µg/mL were added to them. The cells were collected using a cell scraper after 1, 3, and 6 h of incubation. Immediately, PI solution (50 µg/mL) was added to the cells, which were then incubated for 5 min at 4°C in the dark. The cells were analyzed using a BD FACSCalibur flow cytometer, and the proportion of dead cells was calculated using the FlowJo software ver8.7.
Cytokine Measurement By Elisa
RAW264.7 cells were seeded in 96-well culture plate at a density of 1 ⋅ 104 cells/well and incubated for 24 h. The medium was replaced with fresh medium with or without cNPs at various concentrations. After 24 h incubation, the concentration of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in the supernatant were measured using Mouse TNF-α ELISA MAX Deluxe Set (BioLegend, San Diego, CA, USA) and Mouse IL-10 Uncoated ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively.
Effect of cNPs and RAW264.7 cells on the proliferation of colon26/fluc cells
Colon26/fluc cells were seeded in 12-well culture plate at a density of 1 ⋅ 105 cells/well and incubated for 24 h. RAW264.7 cells (with varying numbers) and 1000 µg/mL cNPs were added to colon26/fluc cells. After 24 h incubation, cells were washed three times with PBS. Subsequently, the cells were lysed using 100 µL cell lysis buffer and incubated for 3 h on ice for complete lysis. Luciferase activity in cell lysates was measured using Picagene luciferase assay kit (Toyo B-Net Co., Ltd., Tokyo, Japan). In addition to this, colon26/fluc cells were seeded in the lower chamber of Transwell cell culture plates at a density of 1 ⋅ 105 cells/well. After 24 h incubation, RAW264.7 cells (1 ⋅ 105 in number) and cNPs at a concentration of 1000 µg/mL were added to the insert (upper chamber). After 24 h of incubation, colon26/fluc cells in the lower chamber were washed three times with PBS and lysed using 100 µL of cell lysis buffer. Luciferase activity in cell lysates was measured as described above.
Anti-tumor effect of cNPs in a subcutaneous xenograft mouse model
To prepare a subcutaneous tumor xenograft mouse model, colon26 cells suspended in PBS (1 ⋅ 106 cells/100 µL) were subcutaneously injected into the back area of BALB/c mice. When the tumor size reached 50 mm3, the mice were randomly assigned to three different treatment groups, and then saline or cNPs (50 or 150 µg/shot) was injected daily into the tumor for 21 days. Tumor size and body weight of the mice were measured at the time of the sample injection. Tumor volume was calculated as follows: volume = 1/2 LW2, where L is the long diameter and W is the short diameter of the tumor [59]. At the end point of the experiment, mice were euthanized under isoflurane anesthesia.
Evaluation of the skin inflammation in mouse after cNPs injection
The hair on the back area of BALB/c mice was removed using a clipper, and then 50 µL of PBS (vehicle), alum (40 mg/mL aluminum hydroxide and 40 mg/mL magnesium hydroxide), or cNPs (1000 or 3000 µg/mL) was subcutaneously injected. The injection site was then photographed after 24 h. After the experiment, mice were euthanized under isoflurane anesthesia.
Statistical analysis
Statistical differences were evaluated by one-way analysis of variance (ANOVA) followed by the Dunnett’s test for multiple comparisons or Student’s t-test for comparison between two groups. Statistical significance was set at p < 0.05.