Ultrasonicator

A chromatin immunoprecipitation (ChIP) assay was performed with a ChIP kit (#9005, Cell Signaling Technology, Danvers, MA, USA). Cells cultured in 15-cm plates were crosslinked with 1% formaldehyde, quenched with 0.125 mmol/L glycine, and then lysed. DNA was fragmented using an ultrasonicator (SONICS, Tokyo, Japan). Protein-DNA complexes were immunoprecipitated with anti-Sp1 or IgG antibodies at 4 °C overnight. The crosslinked complexes were washed and decrosslinked at 65 °C for 30 min. The pulled-down DNA fragments and input DNA were purified and used for qRT-PCR analysis with primers designed to amplify the claudin-1 and claudin-3 promoter regions containing the putative Sp1 binding sites. The primer sequences are listed in Tables S6 and S7.

SLNp have been fabricated by the solvent emulsification evaporation method, such as BSE-loaded with chia seed phospholipid (containing 3:1 ratio of omega 3:omega 6), phosphatidylethanolamine, palmitic acid, and stearic acid according to the method of Xue et al. (2015) (link), with minor modifications. Briefly, BSE (5, 10, and 15 mg, respectively), solvent-free CSE-phospholipids (30 mg), phosphatidylethanolamine (0.25% w/w), palmitic acid (0.25% w/w), and stearic acid (0.25 mg) were dissolved in 5 ml of chloroform and methanol (2:1) and kept in a magnetic stirrer at 65°C for complete dissolving. The warm organic lipid phase has been emulsified with the aqueous phase containing Tween 80 (30 mg dissolved in 15 ml of water). The whole process was carried out at 70°C (above the melting point of the lipid) using a hot plate magnetic stirrer with constant stirring for 30 min. The coarse oil-in-water dispersion was sonicated at a frequency of 0.5 cycles and 60% amplitude using a probe-type Ultrasonicator (Sonics, U.S.) in an ice bath. Approximately 20 ml of the formulation contained 0.05% of the drug, 50 mg of the lipid phase and 15 ml of the aqueous phase. The obtained dispersion was collected and stored in a brown glass container and stored at 2–4°C in the freezer until further use.

The reverse evaporation method was used to produce PHP-DPS@INS NPs. First, 20 mg of DPPC, cholesterol, and DSPE-PEG2000-SS31 (mass ratio = 3:1:1) were dissolved in 10 ml chloroform, and then a 2 ml INS solution (4 mg/ml) was added. The W/O colostrum was formed through ultrasonic shock treatment for 5 min. Subsequently, the round bottom flask was placed on a rotary evaporator, and CHCl₃ was removed through vacuum evaporation (50 rpm, 30 °C, 1 h) until the liposome gel formed. Next, the liposome gel was detached by adding normal saline. It was then re-fixed in a rotary evaporator (50 rpm, 30 °C, 30 min) to obtain the DPS@INS NP suspension. Subsequently, the suspension was slowly shaken and incubated for 1 h after adding 4 mg PEG2000-Hyd-PEI. It was then sonicated for 5 min in an ice water bath (35 W, on and off intervals of 5 s each, for 5 min) using an ultrasonicator (Sonics & Materials Inc., USA). Subsequently, PHP-DPS@INS NPs were obtained by extruding through a polycarbonate membrane using a 220 nm aperture. On the other hand, PHP-DPS NPs were prepared using the same protocol as PHP-DPS@INS NPs but without INS loading. Additionally, DPS NPs were obtained following the same scheme as DPS@INS NPs but without loading INS. Through the same scheme as DPS@INS NPs, DP@INS NPs were obtained by replacing DSPE-PEG2000-SS31 with DSPE-PEG2000.

Rat liver samples from the same group were pooled, frozen in liquid nitrogen, and ground using a mortar. Lysis buffer containing 1% SDS (Merck, Germany), 1% triton X-100 (Merck) and 1% nacl (Merck) was then added and lysates were prepared using an ultrasonicator (Sonics & Materials, USA). The lysates were then centrifuged at 12,000×g for 5 min at 4 °C, the supernatants were collected, and their protein concentrations were measured using a Quick Start Bradford Protein Assay (Bio-Rad, USA). Lysate aliquots containing 30 µg of protein were separated by 12% SDS-PAGE (Bio-Rad) and the gels generated were stained using Coomassie G-250 solution (Bio-Rad). Each lane was then cut into 13 pieces and the proteins within subjected to tryptic in-gel digestion^{16 (link)}. The trypsin-digested fractions were separated using an Ultimate 3000 Nano-LC system (Dionex; Surrey, UK) at a flow rate of 300 nl/min. Mobile phase A was 2% (v/v) acetonitrile and 0.1% (v/v) formic acid in HPLC-grade water and mobile phase B was 0.1% (v/v) formic acid in HPLC-grade acetonitrile. The peptides were then analyzed using a micrOTOF QII mass spectrometer (MS) (Bruker; Bremen, Germany) and Hystar software. The MS and MS/MS spectra were acquired in m/z ranges of 400–2000 and 50–1500, respectively.

Escherichia coli BL21 (DE3) cells carrying the nanosensor construct were grown on Luria–Bertani (LB) medium containing ampicillin at 20°C for 24 h. Induction of the nanosensor protein was carried out by adding 0.5 mM Isopropyl β-D-1-thiogalactopyranoside, Isopropyl β-Dthiogalactoside (IPTG) (Fermentas, Germany). After induction, cells were kept for 48 h in the dark at 16°C with continuous shaking. The cells were then harvested and centrifuged at 6,500 × g at 4°C for 30 min, for the isolation of the nanosensor protein. The cell pellet was resuspended in chilled Tris-HCl buffer (20 mM, pH 7.2). An ultrasonicator (Sonics, USA) was used for bacterial cells lysis, followed by centrifugation and filtration of the lysate, to remove cell debris. The isolated protein fraction was collected and applied to nickel-aminotriacetic acid (Ni-NTA) column (BioRad, CA, USA) for His-tag-mediated purification of the nanosensor protein. After washing of the column, elution of the sensor protein was carried out by applying elution buffer (20 mMTris-Cl, pH 8.0, 250 mM imidazole). The eluted protein was incubated at 4°C for 12 h, for proper folding.