Short oligoalanine helical peptides for supramolecular nanopore assembly and protein cytosolic delivery

Rúa Jenaro de la Fuente, s/n –Campus Vida – Universidade de Santiago de Compostela -15782 Santiago de Compostela – ciqus.usc.es Short oligoalanine helical peptides for supramolecular nanopore assembly and protein cytosolic delivery Marta Pazo, Giulia Salluce, Irene Lostalé-Seijo, Marisa Juanes, Francisco Gonzalez, Rebeca Garcia-Fandiño and Javier Montenegro Supporting information Copyright information: © 2021 The Author(s). Published by the Royal Society of Chemistry S1 Short Oligoalanine Helical Peptides for Supramolecular Nanopore Assembly and Protein Cytosolic Delivery Marta Pazo, Giulia Salluce, Irene Lostalé-Seijo, Marisa Juanes, Francisco Gonzalez, Rebeca Garcia-Fandiño and Javier Montenegro* e-mail to: [email protected] Supporting Information Table of contents 1. Supporting Figures .................................................................................................................... 2 2. Experimental Procedures ........................................................................................................ 17 2.1. Materials .............................................................................................................................. 17 2.2. Abbreviations ....................................................................................................................... 18 2.3. General protocol for the SPPS ........................................................................................... 18 2.3.1. Synthesis of peptide MP1 ............................................................................................ 19 2.3.2. Synthesis of peptide CF-MP1 ...................................................................................... 19 2.3.3. Synthesis of peptide TM-MP1 ..................................................................................... 19 2.3.4. Synthesis of peptide MP2 ............................................................................................ 19 2.3.5. Synthesis of peptide P3 ................................................................................................ 19 2.3.6. Synthesis of peptide Arg8 ............................................................................................. 19 2.3.7. Synthesis of peptide GALA ......................................................................................... 20 2.3.8. Synthesis of peptide L17E ........................................................................................... 20 2.3.9. Synthesis of Pep-1 ........................................................................................................ 20 2.3.10. Synthesis of dfTAT ..................................................................................................... 20 2.4. Liposome preparation ......................................................................................................... 20 2.5. ANTS/DPX assay ................................................................................................................. 21 2.6. Dextran release in vesicles .................................................................................................. 21 2.7. Circular Dichroism ............................................................................................................. 22 2.8. DLS experiments ................................................................................................................. 22 2.9. FRET and fluoresence experiments ................................................................................... 22 2.10. Computational methods ................................................................................................. 23 2.11. Planar lipid bilayer recordings ...................................................................................... 23 2.12. Cell culture experiments ................................................................................................. 24 2.12.1. Cell lines and culture ................................................................................................ 24 2.12.2. Cellular uptake and endosomal release of dextran ................................................... 24 2.12.3. Cell viability assay (MTT) ....................................................................................... 25 2.12.4. Saporin experiments ................................................................................................. 25 2.12.5. Cre recombinase expression and purification .......................................................... 25 2.12.6. Cre recombinase delivery experiments .................................................................... 25 2.12.7. Antibody delivery in cell culture .............................................................................. 26 2.12.8. CF-labelling and delivery of IgG ............................................................................. 26 2.12.9. Purification and delivery of GST-NLS-GFP ............................................................ 26 2.13. Ex vivo cornea experiments ............................................................................................ 27 2.13.1. Murine corneas culture ............................................................................................. 27 2.13.2. Antibody delivery in cornea ..................................................................................... 27 2.13.3. Live/Dead Staining ................................................................................................... 27 2.14. Sources for Figure 6 table .............................................................................................. 27 3. Supporting References ............................................................................................................ 28 S2 1. Supporting Figures Figure S1. Circular dichroism spectra of MP1, MP2 and P3 in HKR buffer (gray), Liposomes (red) (L/P ratio: 12) and TFE (orange). Measurements were done at 40 °C. See also Fig. 1d. Figure S2. CF-MP1 aggregates in solution. A) Emission spectra of CF-MP1 (λex = 494 nm) in aqueous solution at different concentrations. B) Maximum fluorescence intensity at different concentrations C) Wavelength of the peak of maximum fluorescence at different concentrations. S3 Figure S3. Vesicle release experiments. A) Kinetic fluorescent traces of LUVsÌANTS/DPX after addition of MP1 (at t = 25 s). Triton X-100 was added at 225 s for complete vesicle lysis and normalization. B) Dose response curves for dextran release in neutral POPC vesicles (LUVsÌDextran) at different MP1 concentrations. The 10 kDa FITC-Dex (green) and 40 kDa TM-Dex (pink) are shown with empty squares (pH = 7.5) and filled circles (pH = 5.5) respectively. C) DLS measurements of vesicles in the presence of MP1. Representation of photon count rate (an indicator of measurement quality) and size (hydrodynamic radius, indicative of vesicle size and stability). Error bars indicate the standard deviation of three replicates calculated by equation S5 for DLS experiments. In all cases the buffer was MES 10 mM with NaCl 150 mM. Experiments were done with neutral POPC or anionic POPC/POPG vesicles at either pH 7.5 or pH 5.5 as indicated at the top of the charts. S4 Figure S4. Molecular dynamics simulation. Top: Snapshot at t = 70 ns of the CG-MD simulation of 31 units of peptide MP1 interacting with a membrane composed by DPPC:DPPE (9:1) and DPPC:DPPE:DPPS (3:5:2), in absence of electric field. Middle: Detail of the insertion of the peptides in the membrane by anchoring and orienting the Leu (orange) enriched hydrophobic N-terminus of peptide MP1 towards the hydrophobic core of the lipid bilayer, and the arginine (blue) cationic residues exposed to the solvent. Down: Detail of the top view of the ensemble of MP1 interacting with the membrane, under an external electric field of 0.03 V/nm applied across the membrane (t = 300 ns, from Figure 5). An initial poration of the membrane can be inferred. S5 A) B) Figure S5. Time lapse of dextran cytosolic release in HeLa cells. A) HeLa cells were incubated with 0.25 mg/mL of Alexa488-Dextran (10 kDa, green) in the presence of 5 µM of TM-MP1 (red) for 1 h in DMEM without FBS. Cells were washed and incubated in DMEM with FBS for 1 hour, 2 hours or 3 hours before imaging. Top rows: 20x magnification; bottom rows: 60x magnification. Each set of images shows: merge of brightfield and TM-MP1 (red; upper left), dextran fluorescence (green; upper right), merge of brightfield and dextran (lower right), merge of TM-MP1, dextran, and brightfield (lower left). Scale bars = 50 µm. B) HeLa cells were incubated with 0.25 mg/mL of FITC-Dextran (10 kDa, green), in the presence of 5 µM TM-MP1 (red) for 1 h in DMEM without FBS. Cells were washed and incubated in DMEM with FBS for 3 h and then stained with LysoTracker Deep Red (100 nM, 30 min, shown in blue), washed and observed at the microscope. Scale bars = 25 µm. S6 Figure S6. Dextran release in the presence of serum or endocytosis inhibitors. A) HeLa cells were incubated for 1 h with a solution of 0.25 mg/mL 10 kDa Alexa488-Dextran (green) and the indicated concentrations of MP1 in DMEM stock (top row) or in DMEM supplemented with 10% FBS (bottom row). Cells were then washed and incubated for 3 hours with DMEM supplemented with 10% FBS before confocal imaging. Cells with diffuse signal were counted and expressed as percentage. Graph shows mean of three experiments ± SD. B) HeLa cells were incubated with 0.25 mg/mL 10 kDa TM-labelled dextran and 30 µM peptide MP1 in the presence of the indicated inhibitors for 1 h. After incubation, the number of cells with cytosolic dextran was counted and normalized to the control with 30 µM peptide in the absence of inhibitors (100%). Data is presented as mean of three independent experiments ± SD. Representative micrographs for each condition are shown at the bottom of the image (top row: dextran fluorescence; bottom row: merge of dextran fluorescence (red) with brightfield). Scale bars = 100 µm. S7 Figure S7. Functional Protein Cytosolic Delivery. A) HeLa cells were incubated with 0 (grey bars) or 10 µg/mL (red bars) saporin in the presence of increasing concentrations of peptides Arg8 or GALA for 1 h; medium was then replaced and cells further incubated for 6 h before measuring viability by MTT assay. B, C) Analogous experiment in HeLa (B) and A549 (C) cells by incubation with MP1 (1 h) and further incubation for 24 h before viability quantification by MTT assay. Figure S8. Cre recombinase delivery efficiency by different peptides. A) and B) Cre-reporter HeLa cells were treated with 1 µM Cre recombinase and 2.5, 5, 10, 20 µM of the indicated peptides for 4 h, and the number of cells that switched from red to green (GFP) fluorescence due to the recombinase activity quantified by flow cytometry 3 days after incubation. The table indicates the percent of GFP-expressing cells ± SD (n = 3) for each peptide and concentration. C) TM-MP1 and Cre form aggregates. HeLa-Cre reporter cells were incubated with 1 µM Cre recombinase and 20 µM TAMRA-labelled MP1 (TM-MP1) for 4 h, and cells imaged 3 days later. Both TAMRA (peptide) and dsRED (expressed by cells) are shown in red, GFP in green and Hoechst-stained nuclei in blue. Arrows indicate fluorescent peptide aggregates as detected by the TAMRA fluorescence, which may be responsible of the lower uptake at high concentrations. S8 Figure S9. Delivery of GST-NLS-GFP and IgG-CF into HeLa cells. A) HeLa cells were incubated in DMEM with GST-NLS-GFP (10 µM; shown in green) in the absence or presence of 30 µM MP1 for 1 h, washed and further incubated for 90 min in DMEM supplemented with 10% FBS before confocal imaging. Nuclei were stained with Hoechst (blue). Around 15% of the cells showed nuclear distribution of GST-NLS-GFP. B) HeLa cells were incubated with IgG-CF (1 mg/mL, green) in the presence or absence of 30 µM MP1 for 1 h, in the same conditions as in A. Scale bars = 50 µm. IC50 HeLa VERO A549 ARPE-19 MP1 126.6 µM 116.2 µM 137.5 µM 133.4 µM Figure S10 MTT viability assays. Viability was determined in HeLa (A), Vero (B), A549 (C) and ARPE-19 (D) cells. Cells were incubated with different concentrations of MP1 (0, 1, 2, 4, 8, 16, 32, 62.5, 125, 250, 500, 1000 µM) in DMEM for 1 h, then the solutions were removed and replaced with complete medium. After 24 hours, cells were incubated for 3 hours with medium supplemented with 0.5 mg/mL MTT tetrazolium salt, and the medium was removed before solubilizing the formazan salt with DMSO. Cells viability was then quantified by absorbance. Data was normalized to the untreated control (100 %). Data is shown as mean of five replicates ± SD. S15 Figure S23. dfTAT characterization. RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)] (Rt 11.5 min) absorbance at 222 nm and ESI-MS for dfTAT. S16 Video S1. Detail of a CG-MD simulations trajectory (5 ns in total) highlighting the initial interaction of MP1 with a lipid bilayer composed by DPPC:DPPE (9:1) and DPPC:DPPE:DPPS (3:5:2). As it can be noticed, although the preliminary interaction of MP1 with the membrane is led by electrostatic interactions between the Arg residues and the polar heads of the lipids, the peptide quickly orients the Leu enriched hydrophobic N-terminus of MP1 towards the hydrophobic core of the lipid bilayer, and the arginine cationic residues remain exposed to the solvent. CG residues are represented in yellow (Ala), orange (Leu) and blue (Arg). S17 2. Experimental Procedures 2.1. Materials Commercially available Rink Amide-resin ChemMatrix, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L- Arg(pbf)-OH, triisopropylsilane (TIS), Diisopropylethyl amine (DIEA), diisopropylcarbodiimide (DIC), and 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) were obtained from Sigma-Aldrich. Trifluoroethanol, 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and chlorpromazine were purchased from TCI. Egg yolk L-α- phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in chloroform (POPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol sodium salt in chloroform (POPG), 16:0-12:0 1- palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3- phosphoethanolamine (NBD-PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3- benzoxadiazol-4-yl) (PE-NBD) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) were purchased from Avanti Polar Lipids. Ethyl(hydroxyimino)cyanoacetate (Oxyma), methyl-β-cyclodextrin, and 5-carboxytetramethyl rhodamine (TAMRA) were available from Carbosynth. N-HATU was provided by Glentham life sciences. N-HBTU was obtained from Iris. Peptide synthesis grade N,N- dimethylformamide was purchased from Scharlau. Dynasore was from EMD Millipore Corporation. All the other solvents were HPLC grade, purchased from Sigma-Aldrich® or Fisher Scientific®, and used without further purification. Dulbecco’s Modified Eagle’s Medium (4500 mg/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate) was obtained from Gibco. Puromycin was purchased from Enzo Life Sciences. Ames’ medium, Alexa Fluor 488 – Dextran (10 kDa), Tetramethylrhodamine – Dextran (10 kDa), fluorescein – Dextran (70 kDa), and LysoTracker Deep Red were acquired from Thermo Fisher. FITC – Dextran (40 kDa), Saporin, Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (4500 mg/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate), human IgG (#I4506) and anti-nuclear pore complex proteins antibody Mab414 (Mouse monoclonal, clone 414, #N8786) were purchased from Sigma-Aldrich. A microwave assisted peptide synthesiser (Liberty Lite, CEM) was used to prepare the peptide according to standard methods developed by the manufacturers involving diisopropylcarbodiimide (DIC) 0.5 M in DMF as activator and ethyl(hydroxyimino)cyanoacetate (Oxyma) 1 M in DMF as activator base. High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analyses were carried out on Agilent Technologies 1260 Infinity II associated with a 6120 Quadrupole LC-MS using an Agilent SB-C18 column or on DIONEX Ultimate 3000 U-HPLC+ (Thermo Scientific) with an Acclaim RSLC 120-C18 column with Solvent A:Solvent B gradients between 5:95 (Solvent A: H2O with 0.1% TFA; Solvent B: CH3CN with 0.1% TFA). High-performance liquid chromatography (HPLC) semipreparative purification was carried out on Jasco LC-4000 with an Agilent Eclipse XDB-C18 column. Accurate mass determinations (HR-MS) using ESI-MS were performed on a Bruker MicroTof mass spectrometer. Circular Dichroism (CD) measurements were performed with a Jasco J-1100 CD Spectrometer equipped with a Jasco MCB-100 Mini Circulation Bath for temperature control. Vesicles were prepared with a Mini-Extruder from Avanti Polar Lipids (pore size 100 nm). Sample incubation was performed in 500 µL eppendorf vials. Fluorescence measurements were performed with a FluoroMax-2 spectrofluorometer (Jobin-Yvon Spex) equipped with a stirrer and a temperature controller. DLS measurements were performed in a Malvern Zetasizer Nano ZSP using standard disposable cuvettes. For the acquisition of cell microscopy images a Nikon Eclipse Ti-E epifluorescence microscope with an Andor Zyla 4.2 digital camera, or a Dragonfly confocal spinning-disk on a Nikon Eclipse Ti-E equipped with a Andor Zyla 4.2 PLUS sCMOS digital camera were used. Images were processed with FIJI.S1 3D images were reconstructed from the different individual confocal planes with Imaris 9.0.0 software (Oxford instruments). A Tecan Infinite F200Pro microplate reader was used to directly measure in Costar cell culture 96- well plates UV-Vis absorbance for the MTT viability assays. Flow cytometry was performed on a Guava easyCyteTM cytometer and data analysed with InCyte software included in GuavaSoft 3.2 (Millipore). S18 2.2. Abbreviations Aa: Amino acid; Arg: Arginine; BSA: bovine serum albumin; Calcd: Calculated; CF: Carboxyfluorescein; CF-MP1: oligoalanine MP1 labelled with carboxyfluorescein at N-terminus; CPZ: Chlorpromazine; DCM: Dichloromethane; Dex: dextran; DIC: diisopropylcarbodiimide; DIEA: N,N- Diisopropylethylamine; DMF: N,N-Dimethylformamide; DMEM: Dulbecco’s Modified Eagle’s Medium; DPPC: dipalmitoyl phosphatidylcholine; DPhPC: 1,2-diphytanoyl-sn-glycero-3-phosphocholine; DPPE: dipalmitoyl phosphatidylethanolamine; DPPS: dipalmitoyl phosphatidylserine; Dyn: Dynasore; EGFP: enhanced green fluorescent protein; EIPA: 5-(N-Ethyl-N-isopropyl)amiloride; FDA: fluorescein diacetate; GST: glutathione-S-transferase; HFIP: 1,1,1,3,3,3-Hexafluoro-2-propanol; HRMS (ESI): High resolution mass spectrometry (electrospray ionization); IPTG: Isopropyl β-D-1-thiogalactopyranoside; LB: Lysogeny broth; Leu: Leucine; Lys: Lysine; LUV: large unilamellar vesicles; MbCD: methyl-β- cyclodextrin; MES: 4-Morpholineethanesulfonic acid; Leu: Leucine; Lys: Lysine; MP1: minimal peptide 1; MP2: peptide 2 with aligned leucines; MOPS: 3-(N-Morpholino)propanesulfonic acid; Mtt: 4- Methyltrytil; MTT: 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl tetrazolium bromide; NBD-PE: 16:0-12:0 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3- phosphoethanolamine; N-HATU: N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1ylmethylene]- N-methylmethanaminium-hexafluorophosphate N-oxide; N-HBTU: N-[(1HBenzotriazol-1-yl)- (dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; NLS: Nuclear Localization Signal; P3: longer oligoalanine peptide; PE-NBD: 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); Pbf: 2,2,4,6,7- Pentamethyldihydrobenzofuran-5-sulfonyl; PBS: Phosphate-Buffered Saline; POPC: 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine; PI: propidium iodide; POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol; SPPS: solid phase peptide synthesis; SV40: Simian Virus 40; TAMRA: 5- carboxytetramethyl rhodamine; TFE: Trifluoroethanol; TIS: Triisopropylsilane; TM-MP1: oligoalanine 1 labelled with TAMRA at N-terminus; TNBS: 2,4,6-Trinitrobenzenesulfonic acid;. 2.3. General protocol for the SPPS All peptides were synthesized by automated or manual Fmoc solid-phase peptide synthesisS2 using Rink Amide ChemMatrix resin (loading 0.5 mmol/g). For manual synthesis, the resin (0.5 mmol) was swelled in DMF (peptide synthesis grade, 2 mL) for 20 min in a peptide synthesis vessel prior synthesis. Coupling cycle consisted of the removal of Fmoc protecting group with a solution of piperidine in DMF (20%, 2 mL) for 10 min and then the mixture was filtered and the resin was washed with DMF (3 x 2 mL, 1 min). The amino acid coupling was carried out by treatment with a solution of α-amino acids (4 equiv), N-HBTU (3.95 equiv) in DMF (2 mL), which was mixed with DIEA (0.195 M solution in DMF, 1.2 equiv) 1 min before the addition and the resulting mixture was shaken by bubbling Ar for 15 min. Finally, the resin was washed with DMF (3 x 2 mL, 1 min). The efficiency of each amino acid coupling and deprotection was monitored employing the TNBS test.S3 For automated synthesis, a variant of the previous protocol was used instead, according to manufacturer’s recommendations. 0.05 mmol of Rink Amide resin was placed into the peptide synthesiser reaction vessel, swollen in DMF, followed by cycles of Fmoc cleavage with piperidine 20% in DMF, washings (3 x 5 mL), then amino acid (5 equiv 2 M amino acid solution in DMF), DIC (10 equiv) and Oxyma (10 equiv) were added into the reaction vessel and microwaved for 5 min under temperature control followed by washings (3 x 5 mL). All steps were performed under nitrogen atmosphere. After the linear peptide was finished the resin was transferred to a different reaction vessel to perform the peptide modification manually. Fluorophore coupling: the Fmoc-protecting group of the initial amino acid was removed by using a solution of piperidine in DMF (20%, 4 mL) for 15 min and the resin was washed with DMF (3 x 3 mL). The coupling was carried out by the addition of a solution of 5-carboxytetramethylrhodamine or 5,6- carboxyfluorescein (1 equiv), HATU (1 equiv), and DIEA (0.195 M, 1 equiv) in DMF (2 mL) and the mixture was stirred by bubbling Ar for 4 hours. Finally, the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). Acetylation in the N-terminus: Once the lineal peptide was finished the acetylation capping of N- terminal group was performed by standard Fmoc deprotection conditions (20% piperidine in DMF (2 x 5 mL, 15 min) followed by treatment with a solution of 2,6-lutidine/acetic anhydride (1:1, 3 mL). The resin suspension was mechanically shaken for 40 min and washed with DMF (3 x 3 mL, 1 min) and DCM (3 x 3 mL, 5 min). S19 General protocol for peptide cleavage and purification: Finally, peptides were deprotected and cleaved from the resin by standard TFA cleavage procedure at rt by using the TFA/DCM/H2O/TIS (90:5:2.5:2.5, 3 mL per 70 mg of resin) for 2 h. Then, the mixture was filtered, washed with TFA (1 mL) and the peptide was precipitated with ice-cold Et2O (50 mL). The precipitate was centrifuged and dissolved in H2O (5 mL) and purified by RP-HPLC. Finally, the corresponding fractions were lyophilised to afford the pure solid peptides. 2.3.1. Synthesis of peptide MP1 Following the general protocol of the SPPS, MP1 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 30%. Rt 14.7 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→21 min)]. MS (ESI, H2O): 1578.0 (6, [M+H]+), 789.6 (100, [M+2H]2+), 526.9 (71, [M+3H]3+). HRMS (ESI): Calcd for C68H125N26O17 [M+H]+: 1577.9702; found: 1577.9711. See Figure S15. 2.3.2. Synthesis of peptide CF-MP1 Following the general protocol of the SPPS followed by fluorophore coupling, CF-MP1 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 9%. Rt 14.9 min [RPHPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 947.8 (37, [M+2H]2+), 632.2 (100, [M+3H]3+), 474.4 (27, [M+4H]4+). HRMS (ESI): Calcd for C87H134N22O22 [M+2H]2+: 947.5100; found: 947.5078. See Figure S16. 2.3.3. Synthesis of peptide TM-MP1 Following the general protocol of the SPPS followed by fluorophore coupling, TM-MP1 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 12%. Rt 15.2 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. Synthesis and characterization had been previously described as P8 in Pazo et al.S4 2.3.4. Synthesis of peptide MP2 Following the general protocol of the SPPS, MP2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 10%. Rt 14.4 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1578.9 (5, [M+H]+), 789.6 (100, [M+2H]2+), 526.9 (77, [M+3H]3+). HRMS (ESI): Calcd for C68H124N26O17 [M+2H]2+: 1577.9716; found: 1577.9711. See Figure S17. 2.3.5. Synthesis of peptide P3 Following the general protocol of the SPPS, P3 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 16%. Rt 15.4 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1081.1 (33, [M+2H]2+), 721.1 (100, [M+3H]3+), 541.1 (39, [M+4H]4+). HRMS (ESI): Calcd for C92H168N36O24 [M+2H]2+: 1080.6515; found: 1080.6511. See Figure S18. 2.3.6. Synthesis of peptide Arg8 Following the general protocol of the SPPS, Arg8 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 58%. Rt 9.5 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 882.9 (11, [M+2H+4TFA]2+), 826.2 (22, [M+2H+3TFA]2+), 769.0 (26, [M+2H+2TFA]2+), 712.3 (17, [M+2H+TFA]2+), 589.2 (30.3, [M+3H+4TFA]3+), 551.2 (78, [M+3H+3TFA]3+), 513.1 (100, S20 [M+3H+2TFA]3+), 475.1 (77, [M+3H+TFA]3+), 437.1 (48, [M+3H+TFA]3+). HRMS (ESI): Calcd for C50H103N33O9 [M+2H]2+: 654.9304; found: 654.9303. See Figure S19. 2.3.7. Synthesis of peptide GALA Following the general protocol of the SPPS, GALA was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 0.5%. Rt 20.6 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1528.8 (46, [M+2H-OH]2+), 1537.2 (17, [M+2H]2+), 1025.3 (100, [M+3H]3+), 769.2 (16, [M+4H]4+). HRMS (ESI): Calcd for C138H220N34O45 [M+2H]2+: 1536.7981; found: 1536.7980. See Figure S20. 2.3.8. Synthesis of peptide L17E L17E was synthesized and purified as previously described,S5 with an overall yield of 23.3%. Rt 10.6 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1430.2 (23, [M+2H]2+), 953.9 (100, [M+3H]3+), 715.8 (26, [M+4H]4+), 572.95 (7, [M+5H]5+). See Figure S21. 2.3.9. Synthesis of Pep-1 Pep-1 was synthesized and purified as previously described,S6 with an overall yield of 3.5%. Rt 11.6 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1424.2 (15, [M+2H]2+), 949.9 (100, [M+3H]3+), 712.7 (20, [M+4H]4+), 570.8 (13, [M+5H]5+). See Figure S22. 2.3.10. Synthesis of dfTAT dfTAT was synthesized and purified as previously described,S7 with an overall yield of 8%. Rt 11.5 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1588.0 (10, [M+3H+6TFA]3+) 1550.2 (10, [M+3H+5TFA]3+), 1162.9 (75, [M+4H+5TFA]4+), 1134.4 (100, [M+4H+4TFA]4+), 1105.8 (61, [M+4H+3TFA]4+) 884.9 (72, [M+5H+3TFA]5+), 862.1 (55, [M+5H+2TFA]5+), 699.5 (50, [M+6H+TFA]6+), 680.75 (45, [M+6H]6+), 599.75 (16, [M+7H+TFA]7+). See Figure S23. 2.4. Liposome preparation The procedure for liposome preparation has been previously reported.S8 Briefly, a thin lipid film was prepared using 1 mL of 25 mg/mL of lipid in chloroform and evaporating the chloroform on a rotary evaporator and then in vacuo overnight. The lipids were resuspended with one of the intravesicular solutions described below for 45 min. The resulting vesicles were extruded 11 times through a 0.1 µm Nucleopore polycarbonate filter. Extravesicular components were removed by desalting column (Sephadex G-50) pre-equilibrated with extravesicular buffer. The final concentration of vesicles is 5 mM. In the case of ANTS/DPX – encapsulated LUVs (LUVsÌANTS/DPX) the extravesicular buffer is composed by 10 mM MES and 150 mM NaCl, at pH 7.4 or 5.5, and the intravesicular buffer is composed by 10 mM MES, 70 mM NaCl, 12.5mM ANTS and 45 mM DPX at pH 7.5 or 5.5 respectively. The lipid composition is 100% POPC or POPC/POPG at 3:1 molar ratio. In order to make NBD-labelled lipid vesicles, lipids labelled in the polar head (PE-NBD) or in the hydrophobic tail (NBD-PE) are added to the lipid mixture as 0.5%. The remaining lipid composition is 99.5% POPC or POPC/POPG molar ratio 3:1. The intra and extravesicular buffer are the same (10 mM MES and 150 mM NaCl, at pH 7.5 or 5.5). In this case, the desalting column (Sephadex G-50) step is not necessary.S9 In the case of non-fluorescent LUVs, the intra and extravesicular buffers are composed by 10 mM MES and 150 mM NaCl, at pH 7.5 or 5.5 respectively, and the lipid composition is 100% POPC or POPC/POPG molar ratio 3:1. In this case, the desalting column (Sephadex G-50) step is omitted. S21 Dextran loaded liposomes (LUVsÌDextran) were prepared following the protocol described by Sani et al.S10 First, the film lipid was formed with POPC or POPC/POPG 3:1 by rotary evaporation and then in vacuo overnight. Thus, the lipid was rehydrated with a solution of 20 mM of 10 kDa FITC-dextran and 20 mM of 40 kDa TM-Dextran, 10 mM MES, 5 mM NaCl at two different pH (7.5 and 5.5). After that, the resulting mixture was extruded 11 times through a 0.2 µm Nucleopore polycarbonate filter. In order to remove the extravesicular components, the samples were centrifuged three times for 40 min, 1500 rpm at 10 °C. After each centrifugation the supernatant was removed and replaced by fresh extravesicular buffer (10 mM MES, 7 mM NaCl) at the corresponding pH. 2.5. ANTS/DPX assay LUVsÌANTS/DPX stock solutions (50 µL) were diluted with a buffer (10 mM MES and 150 mM NaCl, at pH 7.4 and 5.5), placed in a thermostated fluorescence cuvette (25 ºC) and gently stirred (total volume ~2000 µL; final lipid concentration ~125 µM). ANTS efflux was monitored at λem 510 nm (λex 353 nm) as a function of time after addition of MP1 at t = 50 s and aqueous Triton X-100 (1.2%, 20 µL, 185 µM final concentration) at t = 225 s; each measurement was made in triplicate. Fluorescence intensities were normalized to fractional emission intensity I(t) using equation (S1). I(t) = (It – I0) / (I∞ – I0) (S1) where I0 is the initial intensity of the vesicles alone, I∞ = It at saturation after lysis. Effective concentration for peptides (EC50) and Hill coefficient (n) were determined by plotting the fractional activity Y (= I(t) at saturation just before lysis, t = ~200 s) as a function of MP1 cPeptide and fitting them to the Hill equation (S2). Y = Y0 + (YMAX – Y0) / {1 + (EC50 / cPeptide)n} (S2) where Y0 is Y before the addition of the peptide, YMAX is Y with an excess of peptide at saturation. For reversible pore gating experiments, LUVsÌANTS/DPX (3:1 molar ratio POPC:POPG) stock solutions (50 µL) were diluted with a buffer (10 mM MES and 150 mM NaCl, at pH 7.4 and 5.5) and placed in a thermostated fluorescence cuvette (25 ˚C) and gently stirred (total volume ~2000 µL; final lipid concentration ~125 µM). ANTS efflux was monitored at λem 510 nm (λex 353 nm) as a function of time after addition of MP1 (10 µM) at t = 25 s peptide addition, t = 50 s lipid addition (POPG, 50 µM), t = 150 s peptide addition, t = 175 s lipid addition, t = 275 s peptide addition, t = 300 s lipid addition. Finally, an aqueous Triton X-100 solution (1.2%, 20 µL, 185 µM final concentration) was added at t = 475 s. 2.6. Dextran release in vesicles LUVsÌDextran stock solutions (25 µL) were diluted with a buffer (10 mM MES and 7 mM NaCl, at either pH 7.4 or 5.5) and placed in an eppendorf. Then the minimal peptide MP1 was added at different concentrations and stirred for 30 min (total volume ~1000 µL; final lipid concentration ~125 µM). The mixture was centrifuged for 35 min at 10 ºC and 1500 rpm. The supernatant was collected and measured by spectrofluorometry: FITC-dextran (10 kDa) was monitored at λem 500 - 650 nm (λex 492 nm), TM- dextran (40 kDa) was monitored at λem 560 - 700 nm (λex 555 nm). Fluorescence intensities were normalized to fractional emission intensity I(t) using equation (S1) I(t) = (It – I0) / (I∞ – I0) (S1) S22 where I0 is It of vesicles alone, I∞ = It at saturation after lysis. Effective concentration for peptides (EC50) and Hill coefficient (n) were determined by plotting the fractional activity Y (= I(t) at saturation just before lysis) as a function of peptide concentration cPeptide and fitting them to the Hill equation (S2). Y = Y0 + (YMAX – Y0) / {1 + (EC50 / cPeptide)n} (S2) where Y0 is Y without peptide, YMAX is Y with an excess of MP1 at saturation. 2.7. Circular Dichroism Circular dichroism measurements were carried out with the following settings: acquisition range: 300- 190 nm; bandwidth: 1.0 nm; accumulation: 3 scans; data pitch: 1 nm; CD scale 200 mdeg/1.0 dOD; D.I.T. (Data Integration Time): 1 s; scanning mode: continuous; scanning speed: 200 nm/min. Measurements were done at 10 ºC and 40 ºC in a quartz cell of 0.2 cm path length at a final volume of 0.5 mL (buffer 10 mM MES, 150 mM NaCl at two pH: 7.4 and 5.5) with a final peptide concentration of 100 µM. The results are expressed as the mean residue molar ellipticity [θ]MRt with units of degrees·cm2·dmol-1 and calculated using the equation S3. The percentage of helicity was calculated using the equation S4.S11 As this value is used for comparisons among similar peptides in different environments, no correction for the number of aromatic aminoacids is required. For the measurements in presence of non-fluorescent LUVs (100% POPC or POPC/POPG at molar ratio 3:1), samples were prepared by mixing the peptides at 100 µM and different amounts of liposomes at different lipid:peptide ratios (1.25, 5, 12, 50). For each sample, a blank measurement was done prior addition of the peptide, and subtracted to the other measurements before analysis. [Ɵ]MRt =!.#$%$Ɵ '·$)·$*+,-./$01$/.234+.2 (deg · cm2 · dmol -1) (S3) Equation S3: Formula to calculate the ellipticity. Ɵ is the ellipticity (mdeg), C is the peptide concentration (M) and l is the cell path length (cm). [Ɵ]% =$ | Ɵ678!!! | $9$:;<! ;!;!! x 100 (S4) Equation S4: Formula to calculate the percent of helicity in which the molar ellipticity at 222 nm is an absolute value.S11 2.8. DLS experiments Non-fluorescent LUVs (50 µL, 125 µM) (POPC or POPC/POPG 3:1) were added to a buffer solution (10 mM MES and 150 mM NaCl, at pH 7.5 and 5.5) to a final volume of 200 µL (20 ⁰C, duration = 70 s). The best parameters were found using the automatic mode, after which the attenuator index was fixed at 6. Measurements were done after the addition of increasing amounts of the peptide (50, 100, 200, 400, 800 µM) to the sample, and ended after vesicle lysis with Triton X-100, when the equipment could not measure, as the counted photons are 0. The standard deviation is given by equation S5.S12 PdI = $ %$=> 6.?* & : (S5) Equation S5: Formula to calculate the standard deviation using the mean and polydispersity index (PdI). 2.9. FRET and fluoresence experiments The minimal peptide MP1 was modified at the N-terminus with either 5-TAMRA or 5- carboxyfluorescein, which constitute a FRET pair, to achieve the peptides TM-MP1 and CF-MP1 respectively. The emission of the donor carboxyfluorescein (at λex = 494 nm, λem = 518 nm) was measured in a final volume of 2 mL containing a mixture of 50 µL, 125 µM vesicles and 2.5 µM CFMP1. The peptide FRET partner TM-MP1 2.5 µM (5µM final concentration of both CF-MP1 and TM- S23 MP1) was added at t = 25 s and the fluorescence monitored until t = 200 s. CF-MP1 emission was determined at t = 10 s and t = 150 s and FRET efficiency calculated according equation S6. E = 1 – (FDA/FD) (S6) Equation S6: Formula to calculate the Efficiency (E), FDA is the fluorescence of the donor in the presence of the acceptor; FD fluorescence of the donor alone. The second FRET experiment was performed using the same peptide labelled at the N-terminus with TAMRA (TM-MP1). Anionic and neutral vesicles containing 0.5% NBD-PE or PE-NBD were diluted (50 µL in 2 mL) in MES buffer at two different pH (7.5 and 5.5). The emission of the donor NBD (λex = 470 nm , λem = 531 nm) was measured at t = 0 s and after the addition of peptide acceptor pair (0.05, 0.1, 0.5, 1 µM) from t = 25 s until t = 200 s. NBD emission values at t = 10 s and t = 150 s were used for FRET efficiency calculation using equation S6. Emission fluorescence spectra was also measured at different concentrations of CF-MP1 (λex = 494 nm) in aqueous solution (MES buffer, pH 7.5). Quenching effect at increasing concentrations suggests a micelle-like aggregation at concentrations over 1.5µM.S13 The shift of the peak of maximum fluorescence intensity indicates a complete aqueous solvating effect in peptide molecules at concentration lower than 1.5µM.S14 2.10. Computational methods Since it is generally believed that early endosomes share the same overall lipid composition as the plasma membrane, as they are both part of the same recycling territory,S15 this study used an asymmetric human erythrocyte membrane model, following the study of Tian et al.S16, which involves three lipid types, including dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), and dipalmitoyl phosphatidylserine (DPPS). In the outer leaflet of the membrane, the lipid ratio is 9:1 for DPPC and DPPE; in the inner membrane leaflet, the lipid ratio is 3:5:2 for DPPC, DPPE, and DPPS. Mapping of lipid molecules was based on the Martini CG force field (Martini_v2.2).S11,S12 Choline groups of lipids are represented by the CG beads Qo, Qd, and P5 for DPPC, DPPE, and DPPS, respectively. The phosphate groups of all lipids are represented by Qa. The glycerol groups and carbon tails of all lipids are represented by Na and C1, respectively. The peptide MP1 model was built following the extended Martini force field for proteins using the martine.py script.S11,S13 Peptide MP1 contains 3 Leu residues, 3 Arg residues and 10 Ala residues, so that the total charge of each unit is +3. The terminal edges of the peptides were not charged. A random distribution of 31 units of peptide MP1 interaction of an asymmetric membrane composed by 1309 CG lipids was investigated, with an initial size of the box of 20 x 20 x 15 nm. After a short equilibration, long MD simulations of 1000 ns for each case were performed in absence and presence of an external electric field of 0.03 V/nm applied across the bilayer. The system temperature was kept at 310 K using a V-rescale thermostat,S20 and periodic boundary conditions were used in all simulations. The Parrinello-Rahman barostatS21 with semisotropic pressure was applied. The van der Waals interaction cutoff was 1.1 nm. In the case of atomistic resolution, AT-MD simulations were carried out using the Gromos54a7 force field.S22 The edges of peptide MP1 were modelized as ACE and NH2, respectively. The box was solvated with water or TFE, respectively. The system temperature was kept at 310 K using a V-rescale thermostat,S20 and periodic boundary conditions were used in all simulations. The Parrinello- Rahman barostatS21 with isotropic pressure was applied. The van der Waals interaction cutoff was 1.0 nm. Particle mesh Ewald summation (PME) methodS23 was used to determine electrostatic interactions. All simulations were performed by the GROMACS 4.5.4 packageS24 and results were represented by Visual Molecular Dynamics (VMD) 1.9 software.S25 2.11. Planar lipid bilayer recordings Experiments were performed in planar lipid bilayer membranes (BLMs) formed by the Montal and Rudin technique.S26 The recordings were carried out by using bilayers of 1,2-diphytanoyl-sn-glycero-3- phosphocholine (DPhPC) across a 80-100 µm diameter hole of a 25 µm thick PTFE (Goodfellow Corporation, Malvern, PA, USA). Bilayers were formed by first pretreating the aperture with a drop of 1% (v/v) hexadecane in n-pentane. The buffer solution (0.5 M of KCl and 10 mM of MOPS) was added to both 1 mL chambers, then, DPhPC in n-pentane (5 µL, 5 mg/mL) was added to both wells; after that, the solvent evaporated. A bilayer was formed when the electrolyte was lowered and raised, bringing the S24 two lipid surface monolayers together at the aperture. The capacitance of the bilayer was checked and the stability of the bilayer was monitored for 5 min at +200 mV, observing no fluctuations in the current. After planar membrane formation the peptide dissolved in MQ water was added to both chambers at 25 µM. The whole BLM conductance was measured at voltage-clamp-mode-holding potential from trans to cis chamber. Raw ion current recording was acquired at 1 kHz at a sampling frequence of 5 kHz. The current was amplified by using an Axopatch 200B amplifier (Molecular Devices), digitized with a Digidata 1440 A (Molecular Devices) converter, while data collection was done by the Clampex 10.7 software (Molecular Devices). Data processing and analysis was carried out with Clampfit 10.7 (the resulting current was filtered using Low pass Bessel (8-pole)). All Axons instruments and software are products of Molecular Devices (Sunnyvale, CA). According to the pore conductance, the peptide pore size was calculated following the Hille equation (S7).S27 The conductivity of 0.5 M KCl 10 mM MOPS at 25 °C is 5.1 S·m-1, r the radius and l the length of the pore (4.7 nm) (theoretical thickness of DPhPC membrane). From the single channel electrical recordings, the current of the open state is around 1,100 pA and the estimated diameter is around 3.16 nm; for long opened states is around 1,900 pA and the diameter around 4.4 nm. 1 (=% $ ) +%+, 2 & ·% $ / +,: & Equation S7. Hille equation. g is the pore conductance; l the length of the pore, ρ the resistivity of the recording solution and r the inner radius of the pore. 2.12. Cell culture experiments 2.12.1. Cell lines and culture HeLa, Vero, and A549 cells were grown in the same conditions: at 37 ºC, 5% CO2, in Dulbecco’s Modified Eagle’s Medium (DMEM; 4500 mg/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate), supplemented with 10% fetal bovine serum and 1% of Penicillin-Streptomycin-Glutamine Mix. Human retinal pigmentary epithelium ARPE-19 cell line was maintained on Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (4500 mg/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate) supplemented with 10% FBS and 1% Penicillin-Streptomycin-Glutamine Mix (Fisher) at 37 ºC, 5% CO2 in an INCO 108 incubator (Memmert). To generate a Cre Reporter-expressing HeLa cell line, HeLa cells were transfected with the plasmid Cre Reporter (a gift from Niels Geijsen; Addgene plasmid # 62732).S28 In this plasmid, the gene for dsRED is flanked by two loxP sites and followed by EGFP gene. In the absence of recombination, the stop codon from dsRED prevents EGFP translation, while Cre-mediated recombination removes the dsRED gene, switching the expression from the red fluorescent protein to the green fluorescent protein. Stable transfectants were selected by maintaining the cell culture in presence of 0.5 µg/mL puromycin (Enzo Life Sciences). 2.12.2. Cellular uptake and endosomal release of dextran For live cell imaging, cells grown on glass bottom dishes were washed with DMEM stock and incubated for 1 h with a solution of peptide and dextran, using different concentrations of MP1 and 0.25 mg/mL 10 kDa Alexa488-Dextran, 0.25 mg/mL 10 kDa TM-Dextran, 1 mg/mL of 40 kDa FITC-Dextran, or 0.25 mg/mL of 70 kDa FITC-Dextran. In co-incubation experiments, 0.25 mg/mL 10 kDa TM- Dextran and 1.25 mg/mL 70 kDa FITC-Dextran were used. After that, cells were washed with DMEM twice and incubated for 3 h with DMEM supplemented with 10% FBS. Finally, cells were washed with DMEM without phenol red and observed with a spinning disk confocal microscope. For lysosomal labelling, cells were incubated for 30 min with 100 nM of LysoTracker Deep Red after the 3 h incubation, and washed before imaging. For the experiments in the presence of endocytosis inhibitors, HeLa cells were seeded the day before in 8-well micro-slides (ibiTreat, Ibidi) at 30000 cells per well, washed and treated with chlorpromazine (30 µM), dynasore (80 µM), methyl-β-cyclodextrin (5 mM) or EIPA (50 µM) diluted in DMEM without (S7)