Supporting Information

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1 Supporting Information Internalization of High-density Lipoproteins Bearing Arginine-rich Peptides Tatsuya Murakami,* Haruki Okamoto, and Hyungjin Kim Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Sakyo-ku, Kyoto (Received October 29, 2014; CL ; Copyright The Chemical Society of Japan

2 MATERIALS AND METHODS Materials. General reagents were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was obtained from NOF Corp. (Tokyo, Japan). Amberlite XAD-2 was from Sigma-Aldrich, Inc. (St. Louis, MO, USA). NAP-5 and Ni Sepharose column columns were from GE Healthcare UK Ltd. (Little Chalfont, UK). Spectra/Por dialysis membranes (molecular weight cut-off, MWCO, 50 kda) were from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). Eagle s minimal essential medium (MEM), fetal bovine serum (FBS), and Alexa Fluor 546 succinimidyl ester (Alexa546) were from Invitrogen Corp. (Carlsbad, CA, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4- yl, NBD-PE) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Accumax was from Innovative Cell Technologies, Inc. (San Diego, CA, USA). Cell culture dishes were obtained from BD Biosciences Co., (San Jose, CA, USA). Triple-well glass-bottom dishes were purchased from Asahi Glass Co., Ltd. (Tokyo, Japan). HDL preparation. An apoa-i mutant missing 43 N-terminal amino acids was genetically fused with either of two derivatives of TAT peptide, YGRKKRRQKKR and YGRKKRRQSSS, at the C-terminus, according to a similar procedure previously reported for TAT peptide (YGRKKRRQRRR) fusion. 1 These apoa-i mutants containing TAT peptide, YGRKKRRQKKR, or YGRKKRRQSSS were expressed in BL21 and purified on a Ni Sepharose column. Lyophilized apoa-i mutants were solubilized in phosphate-buffered saline (PBS) containing 4 M urea and then mixed with POPC in PBS containing 30 mg/ml sodium cholate at a lipid/protein molar ratio of 250/1. 2 The

3 mixtures were dialyzed against PBS at 4 C for 24 h. Reconstituted HDLs were centrifuged at 20,400 g at 4 C for 30 min and further treated with Amberlite XAD2 at 4 C for 4 h to remove residual cholate. 3 HDL mutants containing YGRKKRRQRRR, YGRKKRRQKKR, or YGRKKRRQSSS were designated HDL RRR, HDL KKR, or HDL SSS, respectively. Fluorescence labeling. Amine-reactive Alexa546 was covalently attached to the protein moiety of HDL mutants according to the manufacturer s protocol. Briefly, HDL mutants were reacted with Alexa546 at room temperature for 4 h at a dye/protein weight ratio of 15/1. The resulting Alexa546-labeled HDL mutants were purified by NAP-5 column chromatography followed by dialysis against PBS at 4 C for overnight. For lipid-bilayer labeling, HDL reconstitution was performed with mixtures of NBD-PE (1 mol%) and POPC. Doxorubicin (DXR) incorporation and release. HDL RRR, HDL KKR, or HDL SSS (100 µg/ml on a protein basis) was incubated with DXR (200, 50, or 50 µg/ml) at 50 C for 0.5, 1, or 1 h, respectively. 2 These different conditions were designed to prepare similar-sized DXR HDL mutant complexes by controlling the amounts of incorporated DXR. The release profiles of DXR from DXR HDL mutant complexes were examined by a dialysis method. 1 Briefly, DXR HDL in 0.9% NaCl (10 µg/ml DXR, 900 µl) was placed into a dialysis tube with a MWCO of 50,000 and dialyzed against 0.9% NaCl containing 0.1% bovine serum albumin with a gentle rocking at 37 C. The analysis for DXR HDL solubilized in cell culture medium containing serum, instead of 0.9% NaCl, in a dialysis tube was not possible because DXR released and then bound to serum proteins with molecular weights greater than 50,000 could not be filtered out, leading to underestimates in DXR release results. The amounts of DXR

4 incorporated and released were analyzed by a spectrofluorometer FluoroMax-4 (Horiba Jobin Yvon Ltd., Kyoto, Japan) and fluorescently detected (Ex/Em, 467/555 nm). Cell culture. HeLa cells were purchased from JCRB Cell Bank (Osaka, Japan). The cells were maintained in Eagle s MEM supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin, nonessential amino acids, and 10% (by vol) FBS at 37 C in a humidified 5/95% CO 2 /air incubator. Cell cultures were passaged every 3 4 days. Dynamic light scattering (DLS) analysis. The hydrodynamic diameters of HDLs were determined on a volume basis using a Nanotrac UPA-EX250 particle size analyzer (Nikkiso Co., Ltd., Tokyo, Japan). The zeta potential of HDLs in 10% PBS were determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Confocal and fluorescence-activated cell sorter (FACS) analyses. HeLa cells were seeded at a density of cells/ml and cultured for 1 d. The cells were then treated with HDL mutants or apoa-i mutants (10 µg protein/ml) or DXR-loaded HDL mutants (3 µg DXR/mL) at 37 C for 1 h in the presence of serum. Confocal images were acquired using an Olympus confocal laser scanning microscope (FV10i-LIV, Olympus Corp., Tokyo, Japan). For FACS analysis, treated cells were detached from their dishes using Accumax and subjected to FACS analysis using a guava easycyte flow cytometer (EMD Millipore, Merck KGaA, Darmstadt, Germany). Cytotoxicity assay. HeLa cells were treated with DXR HDL complexes ( µg DXR/mL) in the presence of serum for 1 h at 37 C. After washing, the cells were cultured for 2 d. Cell viabilities were evaluated using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Gaithersburg, MD, USA) and a SpectraMax M2 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA), and normalized (OD 450 OD 620 ) with respect to untreated cells. Assays were performed in triplicate.

5 Figure S1. Size distribution of three HDLs determined by dynamic light scattering analysis. (a) Three non-labeled HDLs. Data from each HDL derived from three independent preparations. (b) NBD-PE- and Alexa546-labeled HDLs (solid and broken lines, respectively). A representative size distribution for each labeled HDL was shown. The mean size is also indicated in each panel.

6 Figure S2. Size distribution of three DXR HDL complexes determined by dynamic light scattering analysis. Data from each HDL derived from three independent preparations. The mean size is also indicated in each panel.

7 Figure S3. Cytotoxicity of three DXR HDL complexes. Free DXR, DXR-HDL RRR, DXR-HDL KKR, and DXR-HDL SSS (closed circle, open circle, closed triangle, and open square, respectively). HeLa cells treated with an HDL for 1 h and then cultured in fresh medium for 2 d prior to Cell Counting Kit-8 assays performed in triplicate. Even DXR-HDL RRR showed much lower cytotoxicity than free DXR, most likely due to endosomal entrapment of the complex. 1

8 References 1 T. Murakami, W. Wijagkanalan, M. Hashida, K. Tsuchida, Nanomedicine (London, U. K.) 2010, 5, T. Murakami, K. Tsuchida, M. Hashida, H. Imahori, Mol. BioSyst. 2010, 6, A. Das, S. G. Sligar, Biochemistry 2009, 48,