Binding Bacteria to Highly Branched Poly(N-isopropyl acrylamide) Modified with Vancomycin Induces the Coil-to-Globule Transition Joanna Shepherd, †,‡ Prodip Sarker, § Kathryn Swindells, § Ian Douglas, † Sheila MacNeil, ‡ Linda Swanson, § and Stephen Rimmer* ,§ School of Clinical Dentistry, UniVersity of Sheffield, Sheffield S10 2TA, U.K., Department of Engineering Materials, Kroto Research Institute, UniVersity of Sheffield, Sheffield S3 7HQ, U.K., and Department of Chemistry, Brook Hill, UniVersity of Sheffield, Sheffield S3 7HF, U.K. Received September 11, 2009; E-mail: s.rimmer@sheffield.ac.uk It is well-known that at temperatures below the lower critical solution temperature (LCST), stimulus-responsive polymers in water are solvated in an open-coil form. 1-3 However, when the temper- ature is raised to the LCST [32 °C for linear poly(N-isopropylacry- lamide) (PNIPAM)], the polymer becomes desolvated and switches to a more compact conformation. This coil-to-globule transition is the basis of emergent sensing devices, drug delivery systems, and various new tools in biotechnology. 4 In view of current difficulties in clinical management of infections, 5-7 developing responses of devices to biological entities is a highly desirable goal. 8 However, few examples of coil-to-globule transitions in response to binding have been reported. 9 Recently, work on linear PNIPAM targeted at bacteria by Pasparakis and co-workers 10,11 showed that linear PNIPAM func- tionalized along the main chain with sugars that interact with bacteria did not bind or aggregate Escherichia coli above the LCST because the sugar groups were buried within the globule. The linear polymer was bound to the bacteria only in the open-coil state, and an external thermal stimulus was required to drive the polymer through the LCST. This concept, that PNIPAM shields functionality above the LCST, is well-established. On the other hand, Ding et al. 12 showed that the stimulus-responsive polymer poly(N,N- diethylacrylamide), when attached to streptavidin, blocked the binding of biotinylated bovine serum albumin (BSA) below the LCST, although binding did occur when the polymer was in the globular state. Also, Liu et al. 13 recently showed that binding of BSA to anti-BSA conjugated to PNIPAM occurred only above the LCST. From these results, it appears to us that polymer architecture can have a significant effect on the ability of binding sites to interact with their targets: that is, in the case of the work of Pasparakis et al., 11 a receptor on a bacterial cell is prevented from binding to its ligand when a linear polymer is in the globular state, whereas in the case of the work of Ding et al. 12 and Liu et al., 13 the globular form allows easier access to a binding site. In branched polymers, the collapse of the polymer from the open chain to the globule should not prevent the binding groups at the chain ends from interacting with bacteria. In this study, we prepared highly branched polymers with vancomycin end groups (HB- PNIPAM-van) capable of binding to Gram-positive bacteria, and we examined their ability to interact with Gram-positive and Gram- negative bacteria from 4 to 37 °C. HB-PNIPAM with carboxylic acid end groups (HB-PNIPAM- COOH) was prepared by our previously disclosed method. 14 Vancomycin was attached to the chain ends at pH 9.5 by amidation of the end groups activated as N-hydroxysuccinimides, as shown in Scheme 1. Approximately 10% of the end groups were modified with vancomycin. To facilitate visualization, the polymer was also labeled by addition of aminoanthracene to a fraction (1.1 mol %) of the chain ends prior to modification with the binding groups. HB-PNIPAM-COOH passed through a cloud point at 42 °C, and the polymer precipitated from solution to form mainly a coagulated mass. HB-PNIPAM-van showed a cloud point at 55 °C, but the polymer remained suspended as a colloidal dispersion. HB- PNIPAM-van and Staphylococcus aureus bacteria or polymers alone in phosphate buffered saline (PBS) as controls were incubated in U-shaped polystyrene wells at 37 °C for 1 h (Figure 1a). In wells containing polymer alone, there was no visible change after incubation. In wells containing S. aureus alone, the cells rolled † School of Clinical Dentistry. ‡ Department of Engineering Materials. § Department of Chemistry. Scheme 1. Conjugation of Vancomycin to HB-NIPAM Figure 1. Bacteria in contact with polymers: (a) HB-PNIPAM-van in decreasing concentrations (mg cm -3 ) incubated with S. aureus or polymer in PBS (5 + PBS). (b-d) Fluorescence images (bacteria labeled with Dylight-649, polymer labeled with anthracene): (b) S. aureus; (c) S. aureus + HB-NIPAM-van; (d) P. aeruginosa incubated with HB-PNIPAM-van. (e) Fluorescence images of HB-PNIPAM-van + S. aureus at 37 and 4 °C. Scale bars ) 10 μm. Published on Web 01/25/2010 10.1021/ja907466y  2010 American Chemical Society 1736 9 J. AM. CHEM. SOC. 2010, 132, 1736–1737