Synthesis of Gemini-Cur nanoparticles (NPs)
Synthesis
Curcumin-loaded NPs were synthesized by a single-step nano-precipitation method as previously described by our group. In brief, 6 mg of Cur (Cat no: Sigma-Aldrich, USA) and 100 mg of methoxyl-poly(ethylene glycol) urethane Gemini surfactant (provided as a gift from Institute for Color Science and Technology, Tehran, Iran) were dissolved in 5 ml of methanol solution (Merck). Then, the methanol was evaporated in a rotary evaporator. The Gemini-Cur NPs solution were filtered using a 0.2 µm size syringe filter (Millex-LG, Millipore Co., USA) to exclude the impurity and sterilize the solution Gemini-Cur NPs, were lyophilized, and stored at 4 °C until use. The characterization of Gemini-Cur NPs was done according to our previous study (Karimpour et al. 2019).
Fourier transforms infrared spectroscopy (FTIR) analysis
The conjugation of Cur with Gemini NPs was assessed using FTIR analysis (Bruker, Tensor 27 spectrophotometer, Germany). Samples from Cur, Gemini NPs, and Cur + Gemini NPs were prepared and mixed with KBr to generate pellets followed by compressing under high pressure. Samples were scanned at the range of 500 to 4500 cm−1.
Scanning electron microscopy (SEM)
The diameters of Gemini-Cur NPs were measured by SEM (Model: MIRA3 FEG-SEM, Tescan) at a voltage of 5 kV. NPs were placed on the SEM stubs and visualized after sputter coating with gold.
Measuring Gemini-Cur NPs uptake using immunofluorescence imaging
To assess the uptake of Gemini-Cur NPs, we exposed HT-29 cells with 80 µM Gemini-Cur NPs for 6 h. To this end, 1 × 104 cells were plated in each well of 96-well plates and allowed to reach 70–80% confluence. Then, cells were incubated with Gemini-Cur NPs for 6 h followed by twice washes in PBS. Cells were fixed using 4% pre-cold paraformaldehyde solution for 20 min and visualized under fluorescence microscopy.
Cell culture protocol
In this study, human colorectal carcinoma cells HT-29 (NCBI Code: C466) were obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). To expand the cells, a high-content glucose DMEM (DMEM/HG; Gibco) culture medium was used. The basal medium was enriched with 10% fetal bovine serum (FBS, Gibco, USA) and 1% Pen-Strep solution (Biochrom GbmH, Berlin, Germany). Cells between passages 3–6 were subjected to different analyses.
MTT assay
Effect of Gemini-Cur NPs on human HT-29 cells viability in a 2D culture system
The viability of HT-29 cells in 2D monolayer culture was evaluated after treatment with Gemini-Cur NPs by using MTT (3-(4, 5-dimethylthiazole-2-yl)-2,5- diphenyltetrazolium bromide) method. For this purpose, an initial number of 1 × 104 cells were suspended in 100 mL of DMEM/HG containing 10% FBS and plated in each well of 96-well plates. Cells were allowed to reach an appropriate confluence. Cells were incubated with different concentrations of Gemini-Cur NPs for 48 h. After completion of the incubation period, supernatants were discarded and replaced with 5 mg/ml of MTT solution. Cells were kept at 37 ˚C for 3–4 h. Then, we added the DMSO solution to dissolve the formazan crystals. The OD of each group was read using a microplate reader and expressed as % of the control group.
Effect of Gemini-Cur NPs on human HT-29 cells spheroid integrity
To this end, cancer cell spheroids were generated using the conventional hanging drop method (Abdolahinia et al. 2019). Briefly, 1 × 104 HT-29 cells were re-suspended in 20 µl of culture medium containing 1% FBS and placed at the inner surface of culture plate lids. The lid and drop containing cells were inverted over the culture plates. The wells were filled with phosphate-buffered saline to inhibit the evaporation of hanging drops. The plates were kept at 37 °C under a humidified condition with 5% CO2. By using the eyepiece of an inverted microscope, the spheroids were monitored. When thick and compact microaggregates with dark edges were generated, the spheroids were transferred into each well of 96-well plates containing 200 µl of culture medium with 1–2% FBS. The spheroids were incubated with different concentrations of Gemini-Cur NPs for 48 h. To evaluate the tumoricidal effect of Gemini-Cur NPs on HT-29 spheroids, we also measured the diameter using Image J software (NIH).
Lactate dehydrogenase (LDH) assay
The release of LDH to the supernatant culture medium exhibits the occurrence of cell membrane injury. To further confirm the cytotoxicity of Gemini-Cur NPs on HT-29 spheroids, supernatants were collected. The concentrations of LDH were measured using the LDH assay kit (Pars Azmun, Tehran, Iran) according to the manufacturer’s instructions.
Analysis of Gemini-Cur NPs uptake using flow cytometry analysis
For this purpose, the spheroids were incubated with 80 and 300 µM Gemini-Cur NPs for 48 h. After completion of the treatment protocol, the culture medium was discarded. Spheroids were disaggregated by incubating in the Trypsin–EDTA solution. After blocking the enzymatic solution, the spheroids were gently triturated. The cells were washed twice with PBS solution and analyzed by the BD FACSCalibur system. The raw data were analyzed using FlowJo software (ver. 7.6.1). Finally, fluoresce intensity was measured in each group and compared to the non-treated control cells.
Rhodamine-123 (Rh-123) efflux
Rh-123 efflux assay was performed used to detect the functional activity MDR1/P-gp of cells after treatment with Gemini-Cur NPs. HT-29 spheroids were incubated with 80 and 300 µM Gemini-Cur NPs for 48 h. Thereafter, spheroids were incubated with Trypsin–EDTA solution and gently agitated to make a single-cell suspension. Cells were washed twice with PBS and incubated with 10 µg/ml of Rh-123 (Sigma-Aldrich) at 37 °C for 30–40 min. Then, cells were washed with PBS to exclude extra RH-123 solution. The cells were analyzed by flow cytometry and FlowJo software (Ver.7.6.1.).
Transwell migration assay
The migration of HT-29 cells in a 3D condition (spheroids) was evaluated using a Transwell migration assay. To this end, Transwell chambers with 8.0 µm pore polycarbonate membrane inserts (Corning Inc.; Corning, NY, USA) were used. After the treatment of HT-29 spheroids with Gemini-Cur NPs, a single-cell suspension was made using the Trypsin–EDTA solution. Then, 200 µl of serum-free medium containing 2 × 104 cells was transferred into inserted. The basolateral chamber was filled with 700 μl of culture medium enriched with 1–2% FBS. Plates were kept at 37 °C for 24 h. Thereafter, the number of migrated cells at the bottom surface was counted in 5 random fields. To better identify the migrated cells at the bottom of the plates, we fixed the cells with pre-cooled methanol solution for 10 min and stained with Giemsa solution (Sigma-Aldrich; St. Louis, MO).
Real-time PCR assay
Total RNA was extracted from both of the control and treated HT-29 spheroids using TRIzol™ reagent (Bioneer, Korea) according to the manufacturer’s guideline. The quality and concentration of RNAs were determined using a Picodrop spectrophotometer (Thermo) and 1% agarose gel electrophoresis. Easy™cDNA Synthesis Kit (Parstous Company, IRAN) was used to synthesize cDNA by the oligo‐dT method following the manufacturer’s instructions. The expression of genes related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Vimentin, was evaluated using real-time PCR analysis. The reaction was performed by SYBR Green (AMPLIQON, Denmark) and appropriate primers. The primers were designed using Oligo7 software (Table 1). In this study, the total volume for real-time PCR reaction reached 10 μl consisted of 5 μl of SYBR Green PCR master mix, 1 μl of forward and reverse primers, 1 μl of cDNA template, and 3 μl of ddH2O. Relative gene expression was normalized to GAPDH as an internal control, and calculated by using the 2−∆∆CT method.
Western blotting
After treatment with Gemini-Cur NPs, cells were lysed using 500 µl of lysis buffer (Tris–HCl pH 8, 0.08 g NaCl, 0.003 g EDTA, 0.025 g Sodium Deoxycholate, 0.01 g SDS, and 1% NP40 enriched with an anti-protease cocktail). Thereafter, 10 µg of protein from each group was electrophoresed using 10% SDS-PAGE at 120 V for 45 min and then transferred onto polyvinylidene difluoride membranes at 120 V for 1.5 h. Membranes were incubated with appropriate primary antibodies, including E-cadherin (Cat no; sc-21791; Santa Cruz Biotechnology, Inc.), Vimentin (Cat no: 14-9897-82; Thermo Fisher Scientific), MMP-2 (Cat no: sc-10736; Santa Cruz Biotechnology, Inc.), and MMP-9 (Cat no: sc-393859; Santa Cruz Biotechnology, Inc.), at 4 °C overnight. After three-time PBS wash, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Cat no: sc-516102 and sc-2357; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The immunoblots were detected on X-ray films using chemiluminescence ECL solution (Bio-Rad). β-actin (Cat no: sc-47778; Santa Cruz Biotechnology, Inc.) is considered as a housekeeping protein to normalize the protein level of different factors.