Calcein

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Diastearoyl-sn-glycero-3-phosphoethanolamine-n-[biotinyl(polyethylene glycol)-2000] (DSPE–PEG (2000) Biotin), Cholesterol and 1,2-diastearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (DSPE–PEG (2000) and 200-nm polycarbonate filters were purchased from Avanti Polar Lipids, Inc., (Alabaster, AL, USA). Recombinant cTnI antigen and cTnI Antibodies [Anti-h cTnI 9705 SPTN-1, Anti-h cTnI 9703 SPTN-5 and Anti-h cTnI 9701 SPTN-5] from Medix Biochemica (Espoo, Finland). NHS-dPEG 4-biotin (Quanta Biodesign), Streptavidin from Streptomyces avidinii, H4522-Human serum type AB (Male) Maxisorp-NUNC immune plate (Thermo scientific, Waltham, MA, USA), PD-10 Sephadex TMG-25 M (GE Healthcare, Chicago, IL, USA), Calcein, SephadexTM-G-100 (GE-Healthcare), Dialysis tubing (CAROLINA Dialsis tubing, Molecular cut off 13,000, Burlington, NC, USA), Triton ^TM X-100, n,n-Dimethylformamide, PBS Tween ^TM-20, phosphate buffered saline tablet, borate buffer (pH 8.5), sodium bicarbonate buffer (pH 9.4), and all other chemicals used for the study were of high analytical grade and were purchased from Sigma-Aldrich/Merck (Baden-Württemberg, Germany).

The phospholipids we used were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, MW = 734.04), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, MW = 790.15). Besides, we used DPPC incorporating cholesterol (MW = 386.65) (DPPC:cholesterol = 66 mol%:33 mol%). DPPC, DSPC, and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Target proteins we used were bovine carbonic anhydrase (CAB, MW = 28,400) and lysozyme (MW = 17,307), which were purchased from Sigma Aldrich (St. Louis, MO, USA). The fluorescent molecule encapsulated in liposomes was calcein (MW = 622.53), which was also purchased from Sigma Aldrich. Silpot184 and Silpot184 CAT (hardener) purchased from Toray Dow Corning (Tokyo, Japan) were used for the fabrication of polydimethylsiloxane (PDMS)-based chips. The gel beads, Sepharose 4B, which are used for filtering out the free calcein during liposome preparation, were purchased from GE Healthcare (Uppsala, Sweden). The filtration buffer, phosphate buffered saline (PBS), were purchased from Thermo Fisher Scientific (Yokohama, Japan). All the chemical reagents were of analytical grade.

Lipid stock solution (50 mg/ml) was prepared by mixing soya bean, type II S Phospholipid powder, L-α- Phosphatidylcholine (Sigma) with hexane (BHD, VWR, UK) and few drops of ethanol, and stored at −20 °C. For experiments, 2 ml (100 mg) of the lipid solution were dried to a film in a round bottom flask. calcein solution (20 mM) was prepared by dissolving calcein (Sigma) in 100 mM sodium phosphate buffer and filtering through a 0.2 μm membrane. To create calcein-trapped multilamelar (MLV) vesicles, 2 ml of the calcein solution was added into the lipid film and this shaken with clean glass beds (2.5–3.5 mm) for 20 min at room temperature. Unilamellar vesicles (ULV) were prepared by passing the MLV through a miniextruder (Avanti) with 100 nm polycarbonated membrane at least 11 times. Untrapped (free) calcein molecules were removed using a PD-10 desalting column (GE Healthcare). calcein leakage from the ULV liposomes was monitored using a fluorescent spectrophotometer (Varian) and Cary Eclipse software, Excitation (493 nm) and emission (505-600 nm). TritonX-100 (1% final concentration) was used as a 100% leakage positive control. Leakage was calculated using the equation: Leakage % = (Fp-F0)/ (Ft-F0) *100%, when Fp is the measurement of fluorescent leakage by the peptides, Ft is the complete leakage by TritonX and F0 is intact vesicles before adding peptide or TritonX.

LUVs were prepared using 1,2-dioleoyl-sn-gylcero-3-phosphocholine (DOPC; Avanti Polar Lipids)
alone or in combination with either 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS; Avanti Polar Lipids)
or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol
(PI; Avanti Polar Lipids) in a 70:30 molar ratio, as previously described.
Stocks of DOPC, DOPS, or PI were mixed to reach final concentrations
of 10 mM total lipid. DOPC, DOPC/DOPS, or DOPC/PI solutions were evaporated
under dry N₂ and dried in a desiccator. Lipid films were
resuspended in 10 mM Tris–HCl, 50 mM NaCl buffer (pH 7.4),
supplemented with or without 70 mM calcein (Millipore Sigma). To reduce
vesicle size, LUVs were subject to five freeze–thaw cycles
from −80 to 40 °C. LUVs were bath sonicated for 30 min
or until the solutions were entirely cleared. LUVs were filtered 20
times through a 0.2 μm pore filter (Anatop 10, Whatman). Vesicles
hydrated in calcein-containing buffer were purified twice through
PD-10 Desalting Columns (GE Healthcare) containing Sephadex G-25 resin
to liberate excess calcein. LUVs were eluted from columns with 10
mM Tris–HCl, 50 mM NaCl buffer (pH 7.4). Final concentrations
of vesicles were calculated using a colorimetric phospholipid quantification
kit as described by the manufacturer (Millipore Sigma). LUVs were
stored at 4 °C up to one week.