Molecular Logistics using Cytocleavable Polyrotaxanes for the Reactivation of Enzymes Delivered in Living Cells

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1 Supplementary Information Molecular Logistics using Cytocleavable Polyrotaxanes for the Reactivation of Enzymes Delivered in Living Cells Atsushi Tamura 1, Go Ikeda 1,2, Ji-Hun Seo 1, Koji Tsuchiya 2, Hirofumi Yajima 2, Yoshihiro Sasaki 1,3, Kazunari Akiyoshi 3, and Nobuhiko Yui 1 1 Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda, Tokyo , Japan. 2 Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo , Japan. 3 Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, A3-317 Katsura, Nishikyo-ku, Kyoto , Japan. Correspondence and requests for materials should be addressed to N.Y. ( yui.org@tmd.ac.jp) Synthesis and characterization of polyrotaxanes. Poly(ethylene glycol) (PEG) (M n = 9,810, M w /M n = 1.02) (Sigma-Aldrich, St. Louis, MO, USA) was used for the polymer axle of PRX. The cytocleavable PRX was prepared from cystamine-terminated PEG (PEG-SS-NH 2 ) [S1]. Similarly, non-cleavable PRX was prepared from α-,ω-bisamino-peg (PEG-NH 2 ) [S1]. The polypseudorotaxanes were capped by condensation reaction with N-benzyloxycarbonyl-L-tyrosine (Z-Tyr-OH) in the presence of 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in methanol [S1]. The DMAE-SS-PRX and the DMAE-PRX were synthesized by the reaction with N,N -carbonyldiimidazole (CDI), followed by nucleophilic reaction with N,N-dimethylaminoethyl amine in anhydrous DMSO [S1]. 1 H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany) in D 2 O containing 0.1 M sodium deuteroxide (NaOD) and 0.05 wt% sodium 3-(trimethylsilyl)propionic acid-2,2,3,3-d 4 (Sigma-Aldrich) or D 2 O. The number of threading α-cds and the number of modified DMAE groups on the PRXs were determined by 1 H NMR spectra. 1

2 Figure S1. (A, B) 1 H NMR spectra of cleavable PRX (A) and non-cleavabn ble PRX (B) in 0.1 M NaOD/D 2 O. (C, D) 1 H NMR spectra of DMAE-SS-PRX (C) and DMAE-PRX (D) in D2O. 2

3 Kinetics study of enzymatic reaction The ONPG solution (10 mm HEPES buffer, ph 7.4) (225 μl) was added the DMAE-SS-PRX/β-gal complexes, and a time-course of the increment in to an equal volume of absorbance at 410 nm was recorded on a V-550 UV-VIS spectrophotometer (Jasco, Tokyo, Japan) for 1 min. The relativee enzymatic activity was determined from thee initial slope of the absorbance a variation derived from the product. The apparent maximum velocity (V max,app p) of the enzymatic reaction and the t apparent Michaelis constant (K M,app) were determined from the Hanes-Woolf f plot as follows [S2,S3]; [S] v [S] = V max,app K + V M,app max,app (1) where [S] and v represent the concentration of substrate (mm) and initial reaction velocity (μm/sec), respectively. Table S1. Kinetic parameters of DMAE-SS-PRX/β-gal complexes at various N/C ratios. Sample β-gal DMAE-SS-PRX/β-gal DMAE-SS-PRX/β-gal N/C ratio V max,app ( M/sec) 1.01 ± ± ± K M,app p (M) 8.06 ± ± ± Figure S2. Hanes-Woolf plots of the β-gal and DMAE-SS-P PRX/β-gal complexes.. Symbols represent β-gal (open squares) and DMAE-SS-PRX/β-gal complexes prepared at an N/C ratio of 1 (closed circles) and 2 (closed triangles). Data are expressed as thee means ± SD (n = 3). 3

4 Figure S3. (A) Mean fluorescence intensity of HeLa cells after 24 h incubation with DMAE-SS-PRX/FITC-β-gal at various N/CC ratios. (B) Time-course of the fluorescence intensity change of TG-β-gal in HeLa cells after a 24 h incubation with cleavable DMAE-SS-PRX/β-gal at various N/C ratios. In these experiments, the initial concentrationn of the FITC-β-gal or β-gal in the medium was adjusted to 20 nm. Data are expressed as the means ± SD (n = 3). Figure S4. Relative enzymatic activity of free β-galactosidase after a 1 h incubation with various concentrations of glutathione ( GSH) as determined by colorimetry using ONPG. Data are expressed as the means ± SD (n = 3). 4

5 Figure S5. Relative enzymatic activity of the Xfect/β-gal after 1 h incubation with various concentrations of glutathione ( GSH) as determined by colorimetry using ONPG. Data are expressed as the means ± SD (n = 3). References [S1] Tamura, A. & Yui, N. Cellular internalization and gene silencing s off sirna polyplexes by cytocleavablee cationic polyrotaxanes with tailored rigid backbones. Biomaterials 34, (2013) [S2] Copeland, R. A. Enzymes:a practical introduction to structure, mechanism, and dataa analysis (John Wiley & Sons, New York, 2004) [S3] Marangoni, A. G. Enzymatic kinetics:a modern approach (John Wiley & Sons, New York, 2003) 5