Supporting Information A ph-dependent Charge Reversal Peptide for Cancer Targeting Naoko Wakabayashi 1, Yoshiaki Yano 1, Kenichi Kawano 1, and Katsumi Matsuzaki 1 1 Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
835.9 [M+3H] 3+ 690.7 [M+4H] 4+ [M+4H] 4+ 627.2 MG Exact mass: 2504.3 HE Exact mass: 2757.3 [M+5H] 5+ 552.6 920.3 [M+3H] 3+ Fig. S1 ESI-MS analysis of MG and HE peptides (Mariner ESI-TOF-MS).
LUVs PC and PS were purchased from Sigma (St. Louis, MO) and Avanti (Alabaster, AL), respectively. A lipid film, after drying under vacuum overnight, was hydrated with a buffer (10 mm MES/150 mm NaCl/1 mm EDTA, ph 7.4 or 5.5) and vortex-mixed to produce multilamellar vesicles. The suspension was subjected to five freeze-thaw cycles and then extruded through polycarbonate filters (100 nm pore size filter, 31 times). The lipid concentration was determined in triplicate by phosphorus analysis (Bartlett 1959). Membrane binding Peptide solutions (2 µm) were titrated with PC/PS or PC liposomes at ph 7.4 or ph 5.5 at 37 C while the fluorescence spectra of the Trp residue were recorded at an excitation wavelength of 280 nm on a Shimadzu RF-5300 spectrofluorometer (Kyoto, Japan). Blank spectra (LUVs) were subtracted and volume correction for dilution (up to 5 %) was performed. Experiments were carried out in duplicate. Similarly, the binding of
HE to PC/PS bilayers were determined at various ph values. A peptide solution (2 µm) was mixed with PC/PS liposomes (400 µm) and ph was changed by adding 1 M NaOH while fluorescence spectra were measured. CD Spectra CD spectra of 20 µm HE in the absence and the presence of LUVs were measured on a Jasco J-820 apparatus at 37 ºC using a 1-mm path length quartz cell to minimize the absorbance due to buffer components. We confirmed that the light scattering due to a high concentration of LUVs did not distort the spectrum (Yano, et al. 2010). The instrumental outputs were calibrated with nonhygroscopic ammonium d-camphor-10-sulfonete (Takakuwa, et al. 1985). Eight scans were averaged for each sample. The blank spectra (LUV suspension or buffer) were subtracted. ANTS/DPX Assay
ANTS and DPX were obtained from Molecular Probes (Eugene, OR). The release of ANTS and/or DPX from LUVs was fluorometrically monitored according to our previous report (Kobayashi, et al. 2000). In the preparation of LUVs, dried lipid film (6 µmol) composed of PS/PC (7/3) was hydrated with 500 µl of MES buffer (ph 5.5) including ANTS (6 mm) and DPX (24 mm). Immediately after the extrusion, vesicles containing ANTS/DPX were separated from free dyes on a Bio-Gel A-1.5m column, and incubated with MG or HE (8 µm) for 10 min 37 C. Fluorescence was monitored on a Shimadzu RF-5300 spectrofluorometer at an excitation wavelength of 353 nm and an emission wavelength of 520 nm. The maximum fluorescence intensity corresponding to 100% leakage was determined by the addition of 10% (w/v) Triton X-100 (20 µl) to 2 ml of the sample. The apparent percent leakage value was calculated as 100(F F 0 )/(F t F 0 ), where F and F t denote fluorescence intensities before and after detergent addition, respectively. F 0 represents fluorescence intensity of intact vesicles. Measurement of proton permeability
MEM (Gibco) and the ph-indicator dye SNARF-4F AM acetate were purchased from Thermo Fisher Scientific (Waltham, Massachusetts). FBS was bought from Nichirei Biosciences Inc. (Tokyo, Japan). ACHN cells were seeded at a density of 2.5 10 5 in 2 ml antibiotic-free MEM containing 10% FBS (v v) on a 35-mm (ϕ) advanced-tc-treated glass bottom dish (Kremsmünster, Austria). Twenty four hrs after cell seeding, the ph-indicator dye solution [10 µm in PBS(+) buffer (ph 7.3)] was loaded into cells and incubated for 20 min. Before substitution to ph-controlled PBS(+) buffer (ph 6.0), the spectra of SNARF-4F excited at 514 nm were acquired in the range of 555 745 nm (resolution: 10 nm) for the measurement of initial intracellular ph at t = 0 sec. The HE peptide at a final concentration of 100 µm was dissolved in 1 ml of the replacement PBS(+) (ph 6.0) buffer. After buffer exchange, the spectra of the same cells were successively recorded at 20-sec intervals for 1200 sec. The apparent proton permeability, k (s 1 ), was calculated according to our previous report (Kawano, et al. 2014). All imaging experiments were performed using a confocal microscope (Nikon C1) under a water-immersed 60 objective (Plan Apo VC) with a 514 nm laser. Spectral
images for the proton permeation experiment were obtained with a spectrum detector at the range of 555 745 nm (resolution: 10 nm). Results To evaluate the proton permeabilizing ability of the HE peptide, proton influx across the plasma membrane was estimated based on the change in the spectrum of the ph-indicator dye SNARF-4F loaded into ACHN cells. In the presence of 100 µm HE at ph 6.0, changes in both spectra of the SNARF-4F and the cellular morphologies were observed upon buffer replacement (data not shown). The apparent proton permeability parameter k (s 1 ) value was slightly, but significantly, higher than that in the absence of HE (Fig. S2). Slight changes in the spectra and the cellular morphologies were observed even without treatment with HE (data not shown).
Fig. S2 HE proton permeability in the absence or presence of HE at ph 6.0. The k (s 1 ) values were determined according to our previous report (Kawano, et al. 2014). Error bars represent the mean ± SD, n = 7 cells for HE( ) at ph 6.0, and n = 8 cells for HE(+) at ph 6.0, respectively. Significant difference was determined based on the parametric two-sample t test. The single asterisk indicates that the differences are significant at P < 0.05. Reference Bartlett GR (1959) Phosphorus assay in column chromatography. J Biol Chem 234:466 468 Kawano K, Yano Y, Matsuzaki K (2014) A dimer is the minimal proton-conducting unit of the influenza a virus M2 channel. J Mol Biol
426:2679 2691 Kobayashi S, Takeshima K, Park CB, Kim SC, Matsuzaki K (2000) Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor. Biochemistry 39:8648 8654 Takakuwa T, Konno T, Meguro H (1985) A new standard substance for calibration of circular dichroism: Ammonium d-10-camphorsulfonate. Anal Sci 1:215 218 Yano Y, Shimai N, Matsuzaki K (2010) Design of a soluble transmembrane helix for measurements of water-membrane partitioning. J Phys Chem B 114:1925 1931