Madeleine Ramstedt

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

Biofunctionalized Protein Resistant Oligo(ethylene glycol)-Derived Polymer Brushes as Selective Immobilization and Sensing Platforms

Poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes are extremely protein resistant polymer coatings that can reduce nonspecific adsorption of proteins from complex mixtures such as blood, sera and plasma. These coatings can be prepared via atom transfer radical polymerization with excellent control of their thickness and grafting density. We studied their direct functionalization with streptavidin and developed an assay for determining which coupling conditions afford the highest streptavidin loading efficiency. Disuccinimidyl carbonate was found to be the most efficient activating agent for covalent capture of the receptor. Using infrared and X-ray photoelectron spectroscopy, fluorescence microscopy, surface plasmon resonance, and ellipsometry, we examined how structural parameters such as the length of the oligo(ethylene glycol) side chain affect streptavidin functionalization, but also immobilization of biotinylated antibodies, subsequent selective secondary recognition and nonspecific binding of proteins. We found evidence that large macromolecules cannot infiltrate dense polymer brushes and that bulky antibody recognition occurs in the upper part of these coatings.

Bacterial and mammalian cell response to poly(3-sulfopropyl methacrylate) brushes loaded with silver halide salts

This study investigates the antibacterial and cytotoxic effect of surfaces with sulphonate brushes containing silver salts. By using the same type of samples for both cytotoxicity and antibacterial studies, these two parameters could be compared in a controlled way. The silver was incorporated into the brush in four different forms to enable release of silver ions at different concentrations and different rates. It was found that although the surfaces displayed very good antibacterial properties in buffer solutions, this effect disappeared in systems with high protein content. Similarly, the silver-containing surfaces displayed cytotoxic effects in the absence of serum proteins but this effect was reduced in the presence of serum. The speciation of silver in the different solutions is discussed. Cytotoxic and antibacterial effects are compared at the different silver concentrations released. The implications of a concentration range where silver could be used to kill bacterial without harmful effects on mammalian cells are also discussed and questioned.

Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis.

Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.

The Antibacterial Activity of Ga3+ Is Influenced by Ligand Complexation as Well as the Bacterial Carbon Source

ABSTRACT Gallium ions have previously been shown to exhibit antibacterial and antibiofilm properties. In this study, we report differential bactericidal activities of two gallium complexes, gallium desferrioxamine B (Ga-DFOB) and gallium citrate (Ga-Cit). Modeling of gallium speciation in growth medium showed that DFOB and citrate both can prevent precipitation of Ga(OH)3, but some precipitation can occur above pH 7 with citrate. Despite this, Ga-Cit 90% inhibitory concentrations (IC90) were lower than those of Ga-DFOB for clinical isolates of Pseudomonas aeruginosa and several reference strains of other bacterial species. Treatment with Ga compounds mitigated damage inflicted on murine J774 macrophage-like cells infected with P. aeruginosa PAO1. Again, Ga-Cit showed more potent mitigation than did Ga-DFOB. Ga was also taken up more efficiently by P. aeruginosa in the form of Ga-Cit than in the form of Ga-DFOB. Neither Ga-Cit nor Ga-DFOB was toxic to several human cell lines tested, and no proinflammatory activity was detected in human lung epithelial cells after exposure in vitro. Metabolomic analysis was used to delineate the effects of Ga-Cit on the bacterial cell. Exposure to Ga resulted in lower concentrations of glutamate, a key metabolite for P. aeruginosa, and of many amino acids, indicating that Ga affects various biosynthesis pathways. An altered protein expression profile in the presence of Ga-Cit suggested that some compensatory mechanisms were activated in the bacterium. Furthermore, the antibacterial effect of Ga was shown to vary depending on the carbon source, which has importance in the context of medical applications of gallium.

Aqueous geochemistry in the Udden pit lake, northern Sweden

The Udden pit lake in northern Sweden was studied from June 1998 to February 1999 in order to increase knowledge of the geochemistry in lakes created as a result of decommissioning open pit mines. ...

Monitoring Surface Chemical Changes in the Bacterial Cell Wall MULTIVARIATE ANALYSIS OF CRYO-X-RAY PHOTOELECTRON SPECTROSCOPY DATA

Gram-negative bacteria can alter the composition of the lipopolysaccharide (LPS) layer of the outer membrane as a response to different growth conditions and external stimuli. These alterations can, for example, promote attachment to surfaces and biofilm formation. The changes occur in the outermost layer of the cell and may consequently influence interactions between bacterial cells and surrounding host tissue, as well as other surfaces. Microscopic analyses, fractionation of bacterial cells, or other traditional microbiological assays have previously been used to study these alterations. These methods can, however, be time consuming and do not always give detailed chemical information about the bacterial cell surface. We here present an analytical method that provides chemical information on the outermost portion of bacterial cells with respect to protein, peptidoglycan, lipid, and polysaccharide content. The method involves cryo-x-ray photoelectron spectroscopy analyses of the outermost portion (within ∼10 nm of the surface) of intact bacterial cells followed by a multivariate curve resolution analysis of carbon spectra. It can be used as a tool for characterizing and monitoring variations in the chemical composition of bacterial cell walls or of isolated outer membrane vesicles, variations that result from e.g. mutations or external stimuli. The method enabled us to predict accurately the alterations in polysaccharide content and surface chemistries of a set of well characterized Escherichia coli LPS mutants. The described approach may moreover be applied to monitor surface chemical composition of other biological samples.

Natural Green Coating Inhibits Adhesion of Clinically Important Bacteria

Despite many advances, biomaterial-associated infections continue to be a major clinical problem. In order to minimize bacterial adhesion, material surface modifications are currently being investigated and natural products possess large potential for the design of innovative surface coatings. We report the bioguided phytochemical investigation of Pityrocarpa moniliformis and the characterization of tannins by mass spectrometry. It was demonstrated that B-type linked proanthocyanidins-coated surfaces, here termed Green coatings, reduced Gram-positive bacterial adhesion and supported mammalian cell spreading. The proposed mechanism of bacterial attachment inhibition is based on electrostatic repulsion, high hydrophilicity and the steric hindrance provided by the coating that blocks bacterium-substratum interactions. This work shows the applicability of a prototype Green-coated surface that aims to promote necessary mammalian tissue compatibility, while reducing bacterial colonization.

Decoupling geometrical and chemical cues directing epidermal stem cell fate on polymer brush-based cell micro-patterns.

The intricacy of the different parameters involved in cell adhesion to biomaterials and fate decision (e.g. proliferation, differentiation, apoptosis) makes the decoupling of the respective effects of surface properties, extra-cellular matrix protein adsorption and ultimately cell behaviour difficult. This work presents a micro-patterned polymer brush platform to control the adsorption of extra-cellular matrix (ECM) proteins to well defined micron-size areas and consequently control cell adhesion, spreading and shape independently of other chemical and physical surface properties. Protein patterns can be readily generated with brushes presenting a range of hydrophilicity and surface charge density. The surface properties of the selected brushes are fully characterised using a combination of FTIR, XPS, ellipsometry, atomic force microscopy, water contact goniometry, dynamic light scattering and ζ-potential measurements. Interactions of proteins relevant to cell patterning and culture with these brushes are studied by surface plasmon resonance, dynamic light scattering, ellipsometry and immuno-fluorescence microscopy. Finally this platform is used in an assay investigating the relative contributions of matrix geometry and surface chemistry on epidermal stem cell differentiation. It is found that moderate hydrophobicity does not impact stem cell commitment, whereas strongly negative surface potential increases the incidence of differentiation. This correlates with a marked decrease in the formation of focal adhesions (but not cell spreading).

Surface modification of PDMS via self-organization of vinyl-terminated small molecules

Polydimethylsiloxane (PDMS) elastomers are widely used in soft lithography, microfluidics and biomedical applications as they combine a range of desirable chemical and physical properties. We studied the surface of PDMS modified using surface enrichment with long-chain alkenes. These alkenes were immobilized during crosslinking of the PDMS pre-polymer mixture cast against a template of matching surface energy. A range of different functional groups was introduced, including perfluorinated and oligoethylene glycol groups, alkyl chains and initiators for controlled radical polymerization. A detailed analysis of the composition of the modified PDMS surfaces was carried out using contact angle measurements, AFM, SIMS and XPS. The results demonstrate that the PDMS was enriched with small molecules near the surface and show that the functional molecules follow the template surface energy on patterned surfaces with edge resolutions equal to the template. By introducing hydrophilic alkenes receding contact angles on PDMS can be lowered below 5°, and we investigated how these surfaces rearrange in air due to the chain mobility of the PDMS backbone and PDMS short-chain fragments.