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1 1 Recognition, neutralization and clearance of target peptides in the blood stream of living mice by molecular imprinted polymer nanoparticles: a plastic antibody Yu Hoshino, *, Hiroyuki Koide, Takeo Urakami, Hiroaki Kanazawa, Takashi Kodama, Naoto Oku, & Kenneth J. Shea *, Department of Chemistry, University of California Irvine, Irvine, CA USA, Department of Medical Biochemistry, School of Pharmaceutical Sciences, and Department of Functional Anatomy, School of Nursing, University of Shizuoka, 52-1 Yada, Shizuoka , Japan. University of Shizuoka, 52-1 Yada, Shizuoka , Japan, and Department of Mechanical Engineering, Stanford University, Stanford, CA USA Supporting Information Materials. Following materials were obtained from commercial sources: N-isopropylacrylamide (NIPAm), N,N,N,N - tetramethylethylenediamine, melittin (from honey bee venom), fluorescein o-acrylate and ammonium persulfate were from SIGMA-ALDRICH Inc.; acrylic acid (AAc) and sodium dodecyl sulfate (SDS) were from Aldrich Chemical Company, Inc.; N,N -methylenebisacrylamide (BIS) was from Fluka; N-t-butylacrylamide (TBAm) was from ACRŌS ORGANICS; Acrylamide [1-14 C] was from Amerciam Radiolabeled Chemicals, Inc.; Dulbecco s Modified Eagle Medium was from Wako Chemicals; FBS was from Japan Bioserum Inc., Alamar Blue was from Serotec Ltd.; Cy5-melittin was from CPC Scientific Inc.; penicillin and streptomycin was from MP Biomedicals; BALB/c mice (5 week-old male) and BALB/c nu/nu mice (5 week-old male) were from Japan SLC. NIPAm was recrystalized from hexane before use. Other chemicals were used as received. Water used in polymerization and characterization was distilled and then purified using a Barnstead Nanopure Diamond TM system. Preparation of NPs. MIPNPs and NIPNPs were synthesized as described. 1 Fluorescein labeled MIPNPs was synthesized as described 1 but in the presence of 1 mol% of fluorescein o-acrylate. 14 C labeled MIPNPs was synthesized as described 1 but in the presence of 5 mol% acrylamide [1-14 C] (Supporting Figure 1). Characterization of NPs. The hydrodynamic diameter of NPs was determined in aqueous solution by dynamic light scattering (DLS) (Zetasizer Nano ZS). The temperature of the NP samples was controlled via Peltier device at 25 ± 0.1 C. Yield and concentration of NPs was determined by measuring weight of NP after lyophilization. Here a dilution factor due to dialysis was corrected (Supporting Table 1). AFM imaging. The sample solution was dropped onto freshly cleaved mica. After evaporation of the solution on the surface, the topographic image was acquired in liquid (ph 7.4, 100 mm Tris-HCl buffer, 300 mm KCl) by the tapping measurement mode of the atomic force microscopy (Smena liquid head, NT-MDT). In vitro biocompatibility test. HT-1080 human fibrosarcoma cells were cultured in Dulbecco s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U ml -1 penicillin, and 100 µg ml -1 streptomycin at 37 C in a 5% CO 2 atmosphere. The cells were seeded onto 96-well plate (1 x 10 4 cells/well). After the cells were cultured overnight, various concentrations of the NPs were added to the culture, and incubated for 24 h. Then, 10 µl/well of Alamar Blue was added and incubated for 4 h. Viable cells were determined by the fluorescence (Ex/Em = 550/590 nm) measured with a fluorescence plate reader (ARVOsx, Perkin Elmer Japan, Tokyo, Japan). The neutralization of cytotoxicity was calculated as the percentage of control cell viability without exposure (Supporting Figure 2).

2 2 In vivo toxicity test. All animal experiments were reviewed, approved, and supervised by the Institutional Animal Care and Use Committee at the University of Shizuoka. MIPNPs (in glucose isotonic solution) were intravenously injected into 5 week-old male, g BALB/c mice at a dose of 30 mg/kg body mass (20 ml kg -1 ). To examine in vivo toxicity, body mass of the mice was monitored every 2 days over a period of 2 weeks and compared with control mice (20 ml kg -1, glucose isotonic solution). The sections of liver, kidney and lung tissues harvested from the mice 2 weeks after injection were stained with haematoxylin and eosin and then examined by a pathologist (Supporting Figure 3). In vivo neutralization assay. Various concentration of melittin solution (10 ml kg -1, glucose isotonic) were injected into 5 week-old male, g BALB/c mice slowly via tail vein. Then, various concentration of NPs solution (glucose isotonic) was injected slowly via tail vein 20 ± 5 second after injection of melittin solution. After injection, all animals were placed into the cage and were observed for the time courses (24 h) indicated in the figures 2a. All animals were included in subsequent statistical analyses. Plots on a figure are always data from same lot of melittin. JMP 6.0 (SAS institute inc.) was used for Willcoxon test of surviving curves. Body mass of the survived mice was measured before and 48 h after melittin injection. Histological analysis of inflammation. To assess the pathological correlates, survived animals after the in vivo neutralization assay were sacrificed 96 h after injection under deep anesthesia by ether. The severity of inflammation observed on the peritoneum was graded into five by the area of inflammations (mostly coverage and high density; 4, partially coverage or medium density; 3, partially and low density; 2, some spots in the restricted area; 1, no spot; 0). The section of an inflamed muscle harvested from the thigh was stained with hematoxylin-eosin and examined by a pathologist (Supporting Figure 4). In vivo fluorescence imaging of Cy5-melittin. 5 week-old male, BALB/c nu/nu mice were fixed in the IVIS 200 imaging system (Cy5.5 filters) under anesthesia. Various concentration of Cy-5 melittin solution (10 ml kg -1, glucose isotonic) were injected into mice slowly via tail vein. Then, various concentration of NPs solution (glucose isotonic) was injected slowly via tail vein 20 ± 5 second after injection of melittin solution. Mice were imaged under anesthesia every 5 min after injection of Cy5 melittin using the Cy5.5 filters. The organs (heart, kidney, liver, lung, small intestine and spleen), harvested 10 or 70 min after injection, were also imaged. In vivo distribution study of 14 C labeled NPs. 5 week-old male, g BALB/c mice were sacrificed at several different times after injection of 14 C NPs (10 mg kg -1, 20 ml kg -1, glucose isotonic) and organs were harvested and weighed. The radioactivity in each organ was determined with a liquid scintillation counter (LSC-3100). Distribution data are presented as injected dose per tissue. Fluorescence imaging of Cy5-melittin and fluorescein-mipnps. 0.3 mg kg -1 of Cy5-melittin (10 ml kg -1, glucose isotonic) were injected into 5week-old male, BALB/c nu/nu mice slowly via tail vein. Then, 10 mg kg -1 of fluorescein-mipnps (10 ml kg -1, glucose isotonic) was injected slowly via tail vein 20 ± 5 second after injection of melittin solution. The section of a liver harvested from the mice 70 min after injection was observed with GFP and Cy5.5 filter using the confocal microscope (LSM 510 META).

3 3 Supporting Table 1. Yield and diameter of NIP/MIP NPs. Yield Diameter (nm) NIPNPs MIPNPs Supporting Figure 1 a. Crystal structure (PDB ID; 2MLT), and amino acid sequence of Melittin and Cy5- melittin. Hydrophobic, positively charged and hydrophilic residues are printed in brown, blue, and green respectively. b. Monomers used for NPs synthesis. Hydrophobic and negatively charged monomers are printed in brown and purple respectively. c. Schematic representatives of melittin imprinting process. Supporting Figure 2 Biocompatibility of NPs in vitro. In vitro cytotoxicity assay of NPs towards HT-1080 human fibrosarcoma cell, determined by the Alamar Blue assay. NIP/MIPNPs (gray/black) at the indicated concentrations were incubated with cells for 24 h. The error bars indicate s.d.

4 4 Supporting Figure 3 Biocompatibility of MIPNPs in vivo. a. Change in body mass of mice injected with MIPNPs (n = 3, dose = 30 mg kg -1 ) compared with control (isotonic glucose solution, n = 3). There is no statistically significant difference in the mass change between control and MIPNPs over a period of 2 weeks. The error bars indicate s.d. b, Liver, spleen and kidney histology. Livers, spleens and kidneys were collected from mice 2 weeks after intravenous injection of MIPNPs (n = 3, dose = 30 mg kg -1 ) or isotonic glucose solution. Organs were stained with haematoxylin and eosin. The scale bar is 30 µm for livers and kidneys and 200 µm for lungs. Supporting Figure 4 a. Cross section of inflamed muscle fibers of the thigh, behind a fascia. The arrow points to the focal areas of inflammation on ballooning striate muscles. b. Vertical sections of damaged muscle fibers of the thigh, behind a fascia. Macrophages (black arrows) and neutrophils (blue arrows) are infiltrated around degenerated striate muscle fibers. Bars: (a) 0.2 cm; (b) 30 µm. c. Level of inflammation of mice quantified by gross pathology (96 hours after poisoning). Left two columns; without melittin with/without 30 mg kg -1 of MIPNPs, middle three columns; with 4.0 mg kg -1 of melittin followed with 0, 9.3, 30 mg kg -1 of MIPNPs, right three columns; with 4.5 mg kg -1 of melittin followed with 0 (no mouse was survived), 9.3, 30 mg kg -1 of MIPNPs.

5 5 Supporting Figure 5 Function of MIPNPs in vivo proposed by this study. REFERENCES (1) Hoshino, Y.; Kodama, T.; Okahata, Y.; Shea, K. J. J. Am. Chem. Soc. 2008, 130,