NANO LETTERS
Presenting Vancomycin on Nanoparticles to Enhance Antimicrobial Activities
2003 Vol. 3, No. 9 1261-1263
Hongwei Gu,† P. L. Ho,‡ Edmond Tong,‡ Ling Wang,† and Bing Xu*,† Department of Chemistry, Hong Kong UniVersity of Science & Technology, Clear Water Bay, Hong Kong, and Center of Infection and Department of Microbiology, Faculty of Medicine, The UniVersity of Hong Kong, Pokfulam Road, Hong Kong Received June 11, 2003; Revised Manuscript Received July 18, 2003
ABSTRACT Here we report the synthesis of vancomycin (Van)-capped Au nanoparticles (Au@Van) and their enhanced in vitro antibacterial activities. Au@Van presumably acts as a rigid polyvalent inhibitor of vancomycin-resistant enterococci (VRE). It also has unexpected activity against an E. coli strain. Our results suggest that gold nanoparticles may serve as a useful model system to explore multi/polyvalent interactions of ligand−receptor pairs.
This paper describes the synthesis and enhanced in vitro antibacterial activities of water-soluble, vancomycin (Van)capped Au nanoparticles, which may act as a polyvalent inhibitor.1 In the fast-developing field of nanotechnology, Au nanoparticles have served as a versatile platform for exploring many facets of basic science. For example, by conjugation with oligosaccharide, DNA, proteins, or small biofunctional molecules,2 Au nanoparticles find applications in biology, and by linking to single molecules or being subjected to an electric field, they assist in fundamental developments in physics and materials science.3 The study of placing antibiotics on Au nanoparticles as a model system of polyvalent inhibitors against antimicrobial drug resistance has received less attention despite their potential importance. We believe that Au nanoparticles, which have a welldeveloped surface chemistry,4 a controllable geometry,5 rigidity, and chemical stability,6 are an excellent tool to evaluate the contribution of conformational entropy in polyvalent binding, as elucidated by Whitesides et al.,1,7 in comparison with the antibiotics attached to flexible polymeric chains.8 Moreover, unlike other polymeric-based nanoparticles, Au nanoparticles not only are smaller in size (4 to 5 nm in diameter, ∼103 times smaller than a bacterium) but also maintain a constant shape and size in solution, which makes Au nanoparticles an ideal model system to understand multivalency. We chose vancomycin as the antibiotic attached to the Au nanoparticle because of its well-established * Corresponding author. E-mail:
[email protected]. Tel: 0852-23587351. Fax: 0852-2358-1594. † Hong Kong University of Science & Technology. ‡ The University of Hong Kong. 10.1021/nl034396z CCC: $25.00 Published on Web 08/22/2003
© 2003 American Chemical Society
Figure 1. Illustration of a possible multivalent interaction between a Van-capped Au nanoparticle (2) and a VanA genotype VRE strain (hexagons: glycosides; ellipses represent the amino acid residues of the glycanpeptidyl precursor with different colors: L-Ala (yellow), D-Glu (orange), L-Lys (green), D-Ala (blue), and D-Lac (purple)).
mechanism and the activities of polyvalent/multivalent Van against vancomycin-resistant enterococci (VRE).9,10 Figure 1 illustrates the possible binding mode of Van-modified Au nanoparticles with a VRE strain. Scheme 1 illustrates the synthetic route for making the Au@Van nanoparticles: Au nanoparticles11 (4 to 5 nm, in toluene) react with bis(vancomycin) cystamide12 (in H2O) under vigorous stirring for 12 h to form Au-S bonds that link Van to Au. A slight excess of Au nanoparticles ensures that all Van molecules are completely consumed. Upon completion of the reaction, Au@Van nanoparticles (2) dissolve in the aqueous phase, which can be easily separated from the organic phase. X-ray photoelectron spectroscopy (XPS) of 2 shows peaks at 83.6 (Au 4f7/2), 87.3 (Au 4f5/2),
Scheme 1. Structure of Vancomycin and the Synthetic Route for Au Nanoparticles Capped with Van and Cysteine
and 163.8 eV (S 2p), corresponding to the binding energies for Au-S bonds. Time-of-flight secondary ion mass spectra (ToF-SIMS) of 2 display mass peaks at 228 (AuS+) and 1506 (Van+), proving the presence of Van on the surface of Au. The aqueous solution of 2 exhibits absorption (λmax ) 280 nm) in its UV-vis spectrum originating from the phenyl groups of Van, also indicating that Van attaches to the Au nanoparticles (Figure 2). The absorption originates from the Au nanoparticles, and its broadness exhibits little change (λmax ) 528 nm), suggesting the preservation of the integrity of the Au nanoparticles and little aggregation. Each Au nanoparticle has approximately 31 Vans on its surface, as calculated according to the calibration curves generated using the UV-vis absorbance of solutions of Van and Au nanoparticles with known concentrations. As a control, Au nanoparticles react with cysteine (Cys) to give Au@Cys (4)13 (Scheme 1), and there are about 1800 molecules of cysteine on an Au particle. The increase in the broadness of the peak at 611 nm indicates the as-prepared Au@Cys aggregates. The different aggregation behaviors of 2 and 4 in aqueous solution likely originate from the different structures of Van and Cys. Transmission electron microscopy (Figure 3) of 2 and 4 indicates that 4 tends to form aggregates easier than 2 does, which agrees with previous observations.14 When the
Figure 2. UV-vis spectra of Au nanoparticles in toluene, the surfactant, 1, 2, and 4 in H2O. 1262
solutions of 2 and 4 are at the minimum inhibition concentration (MIC), the aggregation of 4 decreases dramatically (inset, Figure 3A). The separation of particles of both 2 and 4 at low concentrations (e.g., MIC) suggests that the later observed aggregation of 2 on cell surfaces originates from a biological interaction. Table 1 presents the data from the in vitro studies. Vancomycin and teicoplanin are used as the controls. They exhibit expected activitiesswhereas vancomycin shows little activity against all three types of VRE strains, teicoplanin displays excellent potency against the VanB and VanC strains. With vancomycin attached on its surface, 2 exhibits
Figure 3. Transmission electron micrographs of (A) 4 and (B) 2 in the aggregated state after cryodrying at concentrations of 6.7 and 50 µg/mL. (The insets show TEM images taken at the MIC concentration for 2 and 4.) TEM images of E. coli after being treated by (C) 4 and (D) 2 at minimum inhibition concentrations. Nano Lett., Vol. 3, No. 9, 2003
Table 1. Minimum Concentrations of Vancomycin, Teicoplanin, 2, and 4 Required to Inhibit the Growth of Bacterial Cells in Cation-Adjusted Muller-Hinton Brotha MIC (µg/mL) strain
gene
vancomycin
teicoplanin
2
4
E. faecium E. faecium E. faecalis E. faecium E. faecalis E. faecalis E. faecalis E. faecium E. faecium E. faecium E. faecium E. faecium E. faecium E. GALL E. faecalis E. coli
A A A A A A A A B B B B B C b c
>128 >128 >128 >128 >128 >128 >128 >128 128 128 64 64 64 16 2 >128
64 2 2 32 8 64 64 64 2 1 2 1 1 1 0.5 >128
2 4 4 2 4 4 4 4 4 4 2 4 2 4 4 8
8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 >25
a MIC indicates the total amount of Van and cysteine linked to Au nanoparticles. b The control strain that is sensitive to Van. c Gram-negative strain. The genotype of the strains was confirmed by PCR.
excellent activity against all VRE strains with similar potency. The control, 4, not only is less effective but also shows the same activity against vancomycin-sensitive and VRE strains. Because the composition of the terminal peptides of the vancomycin-sensitive strain and that of the three genotype VREs strains differ, the uniform activity of 4 against VRE indicates that 4 may bind nonspecifically to transpeptidases instead of to terminal peptides of the glycopeptidyl precursors on the cell surfaces of those strains. Considering that surface-bounded cysteines have high effective concentrations and bear a structural resemblance to D-alanine, D-serine, and D-lactate, which correspond to the last residue of the terminal peptides in Van-sensitive, VanC, VanA, and VanB strains, respectively, it would not be inconceivable for cysteine to act as a substrate of transpeptidase.10 This notion is also supported by the fact that 4 exhibits limited activity against Gram-negative E. coli, in which the transpeptidase is inaccessible because of the presence of the outer membrane. For the same reason, the outer membrane of Gram-negative bacteria blocks the entry of vancomycin and teicoplanin, which are ineffective against E. coli. Au@Van, however, unexpectedly exhibits notable activity against E. coli, a Gram-negative bacterial strain. We speculate that 2 may bind to the substrates on the outer membrane of E. coli. TEM results support this hypothesis. Followed by the treatment of 2 and 4, E. coli was washed using deionized water before being imaged by TEM. As shown in Figure 3, no particle of 4 is attached to E. coli. On the contrary, particles of 2 clearly bind to the cell membranes of E. coli. Although the exact mechanism of this phenomenon
Nano Lett., Vol. 3, No. 9, 2003
remains to be elucidated in future work, the in vitro activity of 2 agrees with the TEM observation. In summary, we devised a chemical route to produce Au@Van nanoparticles, which exhibit enhanced activities against VRE strains and Gram-negative bacteria. This Au@Van system may provide an advantageous model system for the examination of multi/polyvalency. Acknowledgment. This work was partially supported by RGC (Hong Kong), the DuPont Young Faculty Grant (for BX), HIA (HKUST). and a grant from the University Development Fund (HKU). References (1) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2755. (2) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757; Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078; Lin, C.-C.; Yeh, Y.-C.; Yang, C.Y.; Chen, C.-L.; Chen, G.-F.; Chen, C.-C.; Wu, Y.-C. J. Am. Chem. Soc. 2002, 124, 3508; Welsch, W.; Klein, C.; vonSchickfus, M.; Hunklinger, S. Anal. Chem. 1996, 68, 2000. (3) Chen, S.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098; Wang, Z. L. AdV. Mater. 1998, 10, 13; Jaramillo, T. F.; Baeck, S.-H.; Cuenya, B. R.; McFarland, E. W. J. Am. Chem. Soc. 2003, ASAP; Karical R. Gopidas; Whitesell, J. K.; Fox, a. M. A. J. Am. Chem. Soc. 2003, 125, 6491. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164; Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. (5) Sun, Y. G.; Xia, Y. N. Science 2002, 298, 2176. (6) Boyen, H.-G.; Ka¨stle, G.; Weigl, F.; Koslowski, B.; Dietrich, C.; Ziemann, P.; Spatz, J. P.; Riethmu¨ller, S.; Hartmann, C.; Mo¨ller, M.; Schmid, G.; Garnier, M. G.; Oelhafen, P. Science 2002, 297, 1533. (7) Mammen, M.; Shakhnovich, E. I.; Whitesides, G. M. J. Org. Chem. 1998, 63, 3168. (8) Arimoto, H.; Nishimura, K.; Kinumi, T.; Hayakawa, I.; Uemura, D. Chem. Commun. 1999, 1361. (9) Walsh, C. Science 1999, 284, 442; Walsh, C. T. Science 1993, 262, 164; Walsh, C. Nature 2000, 406, 775; Rao, J. H.; Yan, L.; Lahiri, J.; Whitesides, G. M.; Weis, R. M.; Warren, H. S. Chem. Biol. 1999, 6, 353; Rao, J. H.; Lahiri, J.; Isaacs, L.; Weis, R. M.; Whitesides, G. M. Science 1998, 280, 708; Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, C.; Labischinski, H.; Endermann, R. Angew. Chem., Int. Ed. 2000, 39, 3823; Griffin, J. H.; Linsell, M. S.; Nodwell, M. B.; Chen, Q.; Pace, J. L.; Quast, K. L.; Krause, K. M.; Farrington, L.; Wu, T. X.; Higgins, D. L.; Jenkins, T. E.; Christensen, B. G.; Judice, J. K. J. Am. Chem. Soc. 2003, 125, 6517; Xing, B. G.; Yu, C. W.; Chow, K. H.; Ho, P. L.; Fu, D. G.; Xu, B. J. Am. Chem. Soc. 2002, 124, 14846; Williams, D. H.; Maguire, A. J.; Tsuzuki, W.; Westwell, M. S. Science 1998, 280, 711; Xing, B.; Ho, P. L.; Yu, C.-W.; Chow, K.-H.; Gu, H.; Xu, B. Chem. Commun. 2003, DOI: 10.1039/b305886g. Gu, H.; Ho, P.-L.; Tsang, K. W.; Yu, C.-W.; Xu, B. Chem. Commun. 2003, 1966. (10) Walsh, C. Antibiotics: Actions, Origins, Resistance; 1st ed.; ASM press: Washington, D. C., 2003. (11) Fink, J.; Kiely, C. J.; Bethell, D.; Schiffrin, D. J. Chem. Mater. 1998, 10, 922. (12) Sundram, U. N.; Griffin, J. H.; Nicas, T. I. J. Am. Chem. Soc. 1996, 118, 13107. (13) Zhang, F. X.; Han, L.; Israel, L. B.; Daras, J. G.; Maye, M. M.; Ly, N. K.; Zhong, C. J. Analyst 2002, 127, 462; Wong, M. S.; Cha, J. N.; Choi, K. S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2, 583. (14) Mandal, S.; Gole, A.; Lala, N.; Gonnade, R.; Ganvir, V.; Sastry, M. Langmuir 2001, 17, 6262.
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