Preparation of pigeon egg white (PEW) solution
PEW was collected from fresh market-purchased pigeon eggs, filtered twice through a mesh, diluted with an equal weight of water, stirred for 30 min at 4 °C to homogenize the solution, and centrifuged (10,000g for 10 min). The supernatant was dialyzed twice using a 1000 MW cutoff dialysis bag against water for 12 h to remove small molecular substances and centrifuged (10,000g for 10 min) to remove insoluble aggregates. The clear homogenized supernatant was stored at 4 °C. Protein concentration of the PEW solution was measured by BCA method (BCA Protein Assay Kit, Thermo Fisher Scientific, USA) with bovine serum albumin as the standard.
Synthesis and characterization of surfactants
Polydisperse anionic C12E4.5 was purchased from Sigma-Aldrich (catalog 463256) and purified, polydisperse cationic C12E4.5 was synthesized from polydisperse anionic as previously reported12. Monodisperse anionic and cationic C12E5 surfactants were newly synthesized in this study (the detail of the synthesis are described in supplemental information). ES-TOF MS analyses of surfactants were performed with a Q-TOF Micro spectrometer (Waters Corp., USA) in a positive mode.
Preparation of surfactant solution
Anionic and cationic surfactants were dissolved in MilliQ-grade water. The solutions were neutralized with NaOH and HCl, respectively, and adjusted to 100 mM. The surfactant solutions were stored at room temperature.
Preparation of PC(PEW-C12E5) and PC(PEW-C12E4.5)
Solutions of 100 mM anionic and cationic C12E5 surfactants were mixed in a volume ratio of 15/85, and 100 μL of the mixed solution was added to 1 mL of 10 mg mL−1 PEW solution. The mixed solution was centrifuged (10,000g for 2 min), and PC(PEW-C12E5) was observed at the bottom of the tube as a transparent liquid. The procedure can be scaled up to prepare large quantities. PC(PEW-C12E4.5) was obtained by adding 80 μL of 100 mM mixture of anionic and cationic C12E4.5 surfactants (anionic/cationic = 15/85) to 1 mL of 10 mg mL−1 PEW solution. The PC formation yield was calculated from the protein concentration (measured by BCA method) of the supernatant after PC formation as follows:
$${text{Formation yield }}left( % right) , = { 1}00 times left( {{1 } – , left[ {{text{protein}}} right]_{{{text{sup}}}} /left[ {{text{protein}}} right]_{0} } right),$$
where [protein]0 is protein concentration of PEW solution before the addition of surfactants (10 mg mL−1) and [protein]sup is protein concentration of the supernatant (aqueous layer) after PC formation.
Preparation of PC-gels
PC(PEW-C12E5) and PC(PEW-C12E4.5) were poured into appropriate molds and heated at 50, 60, 70, 80, and 90 °C for 20 min in a water bath or by sandwiching between heat blocks. The prepared gels were stored in water at 4 °C.
Measurement of protein content of PC
An aliquot of PC(PEW-C12E5) and PC(PEW-C12E4.5) was mixed with a ninefold volume of buffer (20 mM Tris–HCl, pH 8.0, containing 200 mM NaCl). Under these electrolyte conditions, the PCs were reconverted to an aqueous protein solution. The protein concentration of the solution was measured by BCA method with bovine serum albumin as the standard. The result was multiplied by ten to determine the protein content of PC.
Measurement of water content of PC
The PC water content was measured as the difference between the mass weight of the wet and dry samples. The dried samples were prepared in a vacuum at 70 °C for 6 h.
Measurement of transparency of PC-gels
The transparency of 1-mm thick PC(PEW-C12E5) and PC(PEW-C12E4.5) gel was measured at 400–800 nm wavelength using a spectrophotometer (Mapada Corp., Shanghai, China).
Measurement of mechanical strength of PC-gels
The compressive stress–strain curve was measured using an INSTRON 5940 (Instron Corp., Norwood, MA, USA). Cylindrical gel samples 8.5 mm diameter and 8.0 mm thick were compressed at a 1.2 mm min−1 strain rate. The tensile stress–strain measurements were performed using an INSTRON 5943. The dumbbell-shaped specimens with a length of 50 mm, narrowest breadth of 5 mm, and tickness of 1 mm were stretched at a 4 mm min−1 strain rate.
Evaluation of the effect of PC(C12E5)-gel washing on cell toxicity
PC(PEW-C12E5)-gel was incubated at room temperature in a mixture of ethanol and phosphate buffer saline (PBS) at 20/80, 30/70, 40/60, and 50/50 (v/v) ratios. The ethanol/PBS mixtures were exchanged with fresh solutions five times every 12 h during the gel-washing process in the 72-h incubation period. The incubated gels were immersed in 75% ethanol for 1 h and PBS for 2 h and then used as washed gels in the following cytotoxicity test.
PC gel cytotoxicity evaluation using HL-60 cells
Human promyelocytic leukemia cells (HL-60) (iCell Bioscience Inc., Shanghai, China) in Iscove’s Modified Dulbecco’s Medium (IMDM) and 20% fetal bovine serum (FBS) medium were cultured in a CO2 incubator at 37 °C with 5% CO2. Two milliliters of 5 × 105 cells mL−1 culture medium cell density were added to 3.5 cm diameter Petri dishes and cultured at 37 °C with 5% CO2 for 48 h in the presence of a 20 mm × 15 mm × 1 mm piece of washed gel. One hundred microliters of the culture were collected every 12 h, mixed with 10 μL of CCK-8 reagent (Cell Counting Kit-8, Dojindo, Inc., Kumamoto, Japan), incubated for 3 h in a CO2 incubator, and the absorbance at 450 nm was measured. All experiments were performed in triplicate and averaged. The same experiment was performed in the absence of a piece of the gel as a positive control group.
Cell culture on gel surface
Cell growth on the surface of the planer-shaped PC(PEW-C12E5)-gel was observed. The planar-shaped PC(PEW-C12E5)-gel (20 × 15 × 1 mm) was washed as described above using a 40/60 ethanol/PBS ratio. The washed gel was placed in a 3.5 cm Petri dish. Three milliliters of NCI-H460 cells suspended in RPMI 1640 and 10% FBS medium 2 × 105 cells mL−1 density was seeded in the dish and cultured in a CO2 incubator at 37 °C with 5% CO2 for 96 h. The culture medium was replaced with fresh medium at the time of 48 h. The same experiment was performed without the gels, and cells were cultured on a tissue culture-treated culture dish (Corning Inc., #430165). Cells were observed at 48 and 96 h using a microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan). 96 h-cultivation cells were stained with -Cellstain- Double Staining Kit (Dojindo, Inc., Kumamoto, Japan), and the living cells were observed using a fluorescent microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan).
