Fully Zwitterionic Nanoparticle Antimicrobial Agents through Tuning of Core Size and Ligand Structure

Transcription

1 Supporting Information Fully Zwitterionic Nanoparticle Antimicrobial Agents through Tuning of Core Size and Ligand Structure Shuaidong Huo,,,, Ying Jiang,, Akash Gupta, Ziwen Jiang, Ryan Landis, Singyuk Hou, Xing-Jie Liang,,, * and Vincent M. Rotello, * Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, Massachusetts 01003, United States Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience; and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, First North Road, Zhongguancun, Beijing , P. R. China University of Chinese Academy of Sciences, Beijing , P. R. China S. H. and Y. J. contributed equally to this work. * Address correspondence to liangxj@nanoctr.cn and rotello@chem.umass.edu

2 Chemicals. Chloroauric acid ( 99.8%) used for gold nanoparticle synthesis was bought from Strem Chemicals Inc. (Newburyport, MA). Sodium borohydride was purchased from Acros (New Jersey, USA). Unless otherwise noted, all chemicals were used as received without further purification. Milli-Q water (18.2 MΩ cm, Millipore System Inc.) was used throughout this study. Synthesis of nanoparticles. The procedure for 2, 4 and 6 nm zwitterionic gold nanoparticles synthesis was based on the Brust-Schiffrin method. 1,2 Briefly, Chloroauric acid (5.0 mg in 500 μl) and zwitterionic ligand 3,4 were dissolved in 700 μl of methanol/acetic acid 6:1 (v/v). 100 μl of sodium borohydrate (10.0 mg) in ice-cold water was added dropwise with rapid stirring. After continuous stirring for 3 h, nanoparticle solution was formed, and the solvent was then removed under vacuum at 40 C. The residues were dissolved in 10 ml de-ionized water, and the solution was then dialyzed (dialysis membrane, Thermo Scientific, MWCO=10000) for 72 h against de-ionized water, which was changed every 8 h. 2, 4, and 6 nm particles were obtained via adjusting the molar ratio of chloroauric acid and zwitterionic ligand (1:3, 1:1 and 3:1, respectively). Characterization of as-prepared gold nanoparticles. The morphology of the AuNPs was examined using a FEI TecaniT12 microscope with an accelerating voltage of 120 kv. Particle hydrodynamic diameter was measured on a Malvern Zetasizer (Nano series, Malvern Instruments Inc, USA) with a He-Ne laser (633 nm) and a backscattering angle of 173º. The molecular peak of ligand was determined by MALDI-MS (Bruker Autoflex III MALDI-TOF MS, 200 shots-20 off-laser 55%, with ion suppression up to 400 Da). S2

3 Mammalian cell viability assay. NIH-3T3 cells (ATCC CRL-1658) were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in Low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic (100 U/mL penicillin and 100 μg/ml streptomycin). Cells were cultured at 37 C under a humidified atmosphere of 5% CO 2 (96-well plate, cells/well). Media was removed and cells were washed with phosphate-buffered saline (PBS) before addition of NPs with each MIC concentration. Then, cells were incubated for another 24 h before cell viability determination using Alamar blue assay according to the manufacturer's protocol (Invitrogen Biosource). After incubation, 200 μl of solution from each wells was transferred in a 96-well black microplate and quantified (excitation/emission: 560 nm/590 nm) on a SpectroMax M5 microplate reader (Molecular Device) to determine the cellular viability. Cells without any NPs treatment were normalized as 100%. Each experiment was performed in triplicate. S3

4 Table S1. Dynamic light scattering analysis and zeta-potential measurements of as-synthesized nanoparticles in 5 mm phosphate buffer (ph = 7.4). S4

5 Figure S1. LDI-MS spectrum of zwitterionic gold nanoparticles with (A) SN (m/z 601.1), and (B) NS (m/z 530.2) ligand modifications. S5

6 Table S2. MIC values (nm) of zwitterionic Au-SN and Au-NS NPs against Gram-negative (P. aeruginosa) and Gram-positive (A. azurea) bacterial strains. Cultures were performed in triplicate, and at least two independent experiments were repeated on different days. S6

7 Figure S2. Visualizing morphological changes in cell membranes using TEM at a low magnification. Gram-negative (P. aeruginosa) and Gram-positive (A. azurea) bacterial strains were treated with 6 nm Au-SN NPs at their MIC concentration for 3 h. S7

8 Figure S3. Propidium iodide staining assay of Gram-negative (P. aeruginosa) control group, and Gram-positive (A. azurea) bacterial strain after 3 h incubation with 6 nm zwitterionic NPs at MIC concentration. S8

9 Figure S4. Qualified leakage percentage of bacteria penetrated by the 6 nm core zwitterionic nanoparticles using ImageJ based on confocal fluorescence images. S9

10 Figure S5. Cell viability assay of gold nanoparticles featuring different size and surface ligand on NIH-3T3 cells. NIH-3T3 cells were treated with each NP s MIC concentration for 24 h. Mean values ± standard deviation, N = 3. S10

11 Figure S6. Hemolytic activity of gold nanoparticles featuring different size and surface ligand at different concentrations on human red blood cells (RBCs) for 30 min at 37 C. The mixture was centrifuged to detect the cell-free hemoglobin in the supernatant. Hemolysis (%) was calculated using water as a positive (+) control. RBCs incubated with PBS were used as negative (-) control. Error bars represent standard deviations (N = 3). S11

12 Figure S7. Hemolytic activity of gold nanoparticles featuring different size and surface ligand at different concentrations on human red blood cells (RBCs) for 24 h at 37 C. The mixture was centrifuged to detect the cell-free hemoglobin in the supernatant. Hemolysis (%) was calculated using water as a positive (+) control. RBCs incubated with PBS were used as negative (-) control. Error bars represent standard deviations (N = 3). S12

13 REFERENCE 1. Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. Synthesis of Thiol-Derivatised Gold Nanoparticles in A Two-Phase Liquid-Liquid System. J. Chem. Soc., Chem. Commun. 1994, Huo, S. D.; Jin, S. B.; Ma, X. W.; Xue, X. D.; Yang, K. N.; Kumar, A.; Wang, P. C.; Zhang, J. C.; Hu, Z. B.; Liang, X. J. Ultrasmall Gold Nanoparticles as Carriers for Nucleus-Based Gene Therapy Due to Size-Dependent Nuclear Entry. ACS Nano 2014, 8, Jiang, Y.; Huo, S. D.; Mizuhara, T.; Das, R.; Lee, Y. W.; Hou, S.; Moyano, D. F.; Duncan, B.; Liang, X. J.; Rotello, V. M. The Interplay of Size and Surface Functionality on the Cellular Uptake of Sub-10 nm Gold Nanoparticles. ACS Nano 2015, 9, Mizuhara, T.; Saha, K.; Moyano, D. F.; Kim, C. S.; Yan, B.; Kim, Y. K.; Rotello, V. M. Acylsulfonamide-Functionalized Zwitterionic Gold Nanoparticles for Enhanced Cellular Uptake at Tumor ph. Angew. Chem. Int. Ed. 2015, 54, S13