Linda Swanson

Highly branched poly-(N-isopropylacrylamide)s with arginine–glycine–aspartic acid (RGD)- or COOH-chain ends that form sub-micron stimulus-responsive particles above the critical solution temperature

Highly branched poly(-isopropyl acrylamide)s with peptide-end groups form colloidally stable dispersions of sub-micron particles above the lower critical solution temperature.

The polymer physics and chemistry of microbial cell attachment and adhesion

The attachment of microbial cells to solid substrata is a primary ecological strategy for the survival of species and the development of specific activity and function within communities. An hypothesis arising from a biological sciences perspective may be stated as follows: The attachment of microbes to interfaces is controlled by the macromolecular structure of the cell wall and the functional genes that are induced for its biological synthesis. Following logically from this is the view that diverse attached cell behaviour is mediated by the physical and chemical interactions of these macromolecules in the interfacial region and with other cells. This aspect can be reduced to its simplest form by treating physico-chemical interactions as colloidal forces acting between an isolated cell and a solid or pseudo solid substratum. These forces can be analysed by established methods rooted in DLVO (Derjaguin, Landau, Verwey and Overbeek) theory. Such a methodology provides little insight into what governs changes in the behaviour of the cell wall attached to surfaces, or indeed other cells. Nor does it shed any light on the expulsion of macromolecules that modify the interface such as formation of slime layers. These physical and chemical problems must be treated at the more fundamental level of the structure and behaviour of the individual components of the cell wall, for example biosurfactants and extracellular polysaccharides. This allows us to restate the above hypothesis in physical sciences terms: Cell attachment and related cell growth behaviour is mediated by macromolecular physics and chemistry in the interfacial environment. Ecological success depends on the genetic potential to favourably influence the interface through adaptation of the macromolecular structure, We present research that merges these two perspectives. This is achieved by quantifying attached cell growth for genetically diverse model organisms, building chemical models that capture the variations in interfacial structure and quantifying the resulting physical interactions. Experimental observations combine aqueous chemistry techniques with surface spectroscopy in order to elucidate the cell wall structure. Atomic force microscopy methods quantify the physical interactions between the solid substrata and key components of the cell wall such as macromolecular biosurfactants. Our current approach focuses on considering individually mycolic acids or longer chain polymers harvested from cells, as well as characterised whole cells. This approach allows us to use a multifactorial approach to address the relative impact of the individual components of the cell wall in contact with model surfaces. We then combine these components to increase complexity step-wise, while comparing with the behaviour of entire cells. Eventually, such an approach should allow us to estimate and understand the primary factors governing microbial cell adhesion. Although the work addresses the cell-mineral interface at a fundamental level, the research is driven by a range of technology needs. The initial rationale was improved prediction of contaminant degradation in natural environments (soils, sediments, aquifers) for environmental cleanup. However, this area of research addresses a wide range of biotechnology areas including improved understanding of pathogen survival (e.g., in surgical environments), better process intensification in biomanufacturing (biofilm technologies) and new product development.

Binding bacteria to highly branched poly(N-isopropyl acrylamide) modified with vancomycin induces the coil-to-globule transition.

Binding of highly branched poly(N-isopropylacrylamide) with vancomycin end groups to Staphylococcus aureus induced a coil-to-globule phase transition. The polymers aggregated this gram-positive bacteria (but not gram-negative bacteria) over a wide range of temperatures, but cooling to 24-26 degrees C progressed the polymer-bound bacteria through a globule-to-coil phase transition, after which the bacteria were released.

Time-resolved fluorescence anisotropy studies of the temperature-induced intramolecular conformational transition of poly(N-isopropylacrylamide) in dilute aqueous solution

Abstract Time-resolved fluorescence anisotropy measurements have been performed upon an acenaphthylene-labelled (0.5 mol%) sample of poly( N -isopropylacrylamide), PNIPAM, in dilute solutions in both methanol and water as solvents. In methanol, segmental relaxation of PNIPAM follows an Arrhenius dependence upon temperature over the range investigated (279–323K) characterized by an ‘activation energy’ of 13.4(±0.5) kJ mol −1 . This is only slightly greater (by ca 2.4 kJ mol −1 ) than that of solvent flow and it is likely that specific dipolar interactions between the PNIPAM and the methanol determine the macromolecular dynamics in this solvent. In aqueous solution, the segmental mobility of PNIPAM exhibits a dramatic thermoreversible discontinuity at ca 32°C. This change in conformational behaviour occurs at the polymers lower critical solution temperature in aqueous media. This observation, supports the proposition (Winnik, F. M. Polymer 1990, 31, 2125) that the thermally-induced separation in this system occurs by a ‘dual mode’ mechanism wherein intermolecular aggregation is preceded by intramolecular conformational shrinkage of the polymer coils.

Hyperbranched poly(NIPAM) polymers modified with antibiotics for the reduction of bacterial burden in infected human tissue engineered skin

The escalating global incidence of bacterial infection, particularly in chronic wounds, is a problem that requires significant improvements to existing therapies. We have developed hyperbranched poly(NIPAM) polymers functionalized with the antibiotics Vancomycin and Polymyxin-B that are sensitive to the presence of bacteria in solution. Binding of bacteria to the polymers causes a conformational change, resulting in collapse of the polymers and the formation of insoluble polymer/bacteria complexes. We have applied these novel polymers to our tissue engineered human skin model of a burn wound infected with Pseudomonas aeruginosa and Staphylococcus aureus. When the polymers were removed from the infected skin, either in a polymer gel solution or in the form of hydrogel membranes, they removed bound bacteria, thus reducing the bacterial load in the infected skin model. These bacteria-binding polymers have many potential uses, including coatings for wound dressings.

Photophysics of carbazole‐containing systems. 1. Dilute solution behavior of poly(N‐vinyl carbazole) and N‐vinyl carbazole/methyl acrylate copolymers

Steady-state and time-resolved fluorescence techniques have been used to study the photophysical behaviors of poly(N-vinyl carbazole), PNVCz and a series of N-vinyl carbazole-methyl acrylate (NVCz-co-MA) copolymers in dilute solution as a function of both NVCz composition and temperature. A kinetic scheme, intended to describe intramolecular excimer formation across the entire NVCz composition range, is proposed. In low aromatic content copolymers, two monomer species (unquenched and quenched monomer) and two excimer species (the sandwich-like excimer and a higher energy excimer) exist. The contribution from monomer emission to the overall fluorescence decreases with increasing NVCz content through increased excimer formation: this is likely to be consequent upon (1) an increase in the number of excimer forming sites, and (2) increasing efficiency of energy transfer from the excited monomers to the excimer forming sites. In the homopolymer, PNVCz, the only emission that can be observed on a nanosecond timescale is excimeric. This fluorescence appears to originate from three excimer species (the sandwich-like excimer, and two higher energy forms). For the homopolymer, the current observations are consistent with the model proposed by Vandendriessche and De Schryver [Polym. Photochem. 7, 153 (1986)].

Time-resolved fluorescence anisotropy studies of the cononsolvency of poly(N-isopropyl acrylamide) in mixtures of methanol and water

Time-resolved anisotropy measurements (TRAMS) have provided invaluable information concerning the molecular interactions responsible for cononsolvency in the poly(N-isopropylacrylamide), PNIPAM, H2O, and methanol ternary system. TRAMS have successfully monitored the intramolecular segmental dynamics of an acenaphthylene labelled sample, ACE–PNIPAM, revealing details about the conformation adopted by the polymer constituent as a function of both alcohol composition and temperature of the system. In pure aqueous solution, ACE–PNIPAM undergoes a conformational transition from an expanded solvent swollen structure to a compact globule at the lower critical solution temperature (LCST). With increasing alcohol content up to 55% v/v of methanol there is both a reduction in the magnitude and an onset temperature of the LCST of ACE–PNIPAM. From 57.5–65% v/v methanol, ACE–PNIPAM forms an extended solvent swollen structure: observation of an LCST at higher polymer concentrations (e.g., 0.1 wt%) is a consequence of intermolecular aggregation between expanded chains.

Highly Branched Polymers with Polymyxin End Groups Responsive to Pseudomonas aeruginosa

Polymyxin peptide conjugated to the end groups of highly branched poly(N-isopropyl acrylamide) was shown to bind to a Gram negative bacterium, Pseudomonas aeruginosa . The nonbound polymer had a lower critical solution temperature (LCST) above 60 °C. However, binding caused aggregation, which was disrupted on cooling of the bacteria and polymer mixture. The data indicate that polymer binding of bacteria occurred by interaction of the end groups with lipopolysaccharide and that the binding decreased the LCST to below 37 °C. Cooling then progressed the polymer/bacteria aggregate through a bound LCST into an open polymer coil conformation that was not adhesive to P. aeruginosa .

Temperature-dependent phagocytosis of highly branched poly(N-isopropyl acrylamide-co-1,2 propandiol-3-methacrylate)s prepared by RAFT polymerization

Highly branched poly(N-isopropyl acrylamide-co-1,2 propandiol-3-methacrylate)s with imidazole end groups and containing anthramethyl methacrylate (AMMA) were prepared. The branch points were produced by incorporating a styryl dithioate ester (a RAFT monomer). The inclusion of AMMA ensures that the polymers fluoresce in the blue region so that they can be visualized in cells in culture. The feed composition was designed to provide lower critical solution temperatures (LCST) between 30 and 37 °C, and therefore the polymers are above the LCST at the usual temperature for culture of human cells. Inclusion of 1,2 propandiol-3-methacrylate (GMA) results in the formation of stable aggregates above the LCST rather than flocculated masses of polymer, and these colloidally stable sub-micron particles can undergo phagocytosis into human dermal fibroblasts. The phagocytosis is temperature dependant and does not occur below the LCST (at 30 °C) when the polymers are in the open-chain fully solvated and non-aggregated state.

Manipulating the thermoresponsive behaviour of poly(N-isopropylacrylamide) 3. On the conformational behaviour of N-isopropylacrylamide graft copolymers

The thermally triggered conformational change of poly(N-isopropyl acrylamide), PNIPAM, in aqueous media occurs at the lower critical solution temperature, LCST. Manipulation of the switch can be achieved via simple free radical copolymerisation, for example. However, the magnitude of the transition is reduced which has a detrimental effect on the solubilisation and controlled release properties of the polymer. In an attempt to over come these limitations the effect of architecture on the thermal response has been examined through syntheses of a range of fluorescently labelled graft copolymers. To examine the effect of topography, samples containing a NIPAM-based backbone and NIPAM branches have been prepared. Simultaneous variation of the entropic and enthalpic contribution to the thermal response has been achieved through syntheses of macromolecules containing a dimethylacrylamide-based backbone and NIPAM grafts. Time-resolved fluorescence anisotropy (TRFA) measurements have been successful in determining the onset, magnitude and dispersion of the LCST of these samples and, by selective labelling of sites, have provided information concerning the role of backbone and graft on the resultant thermorespsonive behaviour. TRFA measurements confirm that the temperature of the conformational switch can be varied through simultaneous manipulation of the entropic and enthalpic contribution to the thermal response of the graft copolymers, whilst maintaining the magnitude of the transition.