A new
cysteine-based methacrylic monomer (CysMA) was conveniently
synthesized via selective thia-Michael addition of a commercially
available methacrylate-acrylate precursor in aqueous solution without
recourse to protecting group chemistry. Poly(cysteine methacrylate)
(PCysMA) brushes were grown from the surface of silicon wafers by
atom-transfer radical polymerization. Brush thicknesses of ca. 27
nm were achieved within 270 min at 20 °C. Each CysMA residue
comprises a primary amine and a carboxylic acid. Surface zeta potential
and atomic force microscopy (AFM) studies of the pH-responsive PCysMA
brushes confirm that they are highly extended either below pH 2 or
above pH 9.5, since they possess either cationic or anionic character,
respectively. At intermediate pH, PCysMA brushes are zwitterionic.
At physiological pH, they exhibit excellent resistance to biofouling
and negligible cytotoxicity. PCysMA brushes undergo photodegradation:
AFM topographical imaging indicates significant mass loss from the
brush layer, while XPS studies confirm that exposure to UV radiation
produces surface aldehyde sites that can be subsequently derivatized
with amines. UV exposure using a photomask yielded sharp, well-defined
micropatterned PCysMA brushes functionalized with aldehyde groups
that enable conjugation to green fluorescent protein (GFP). Nanopatterned
PCysMA brushes were obtained using interference lithography, and confocal
microscopy again confirmed the selective conjugation of GFP. Finally,
PCysMA undergoes complex base-catalyzed degradation in alkaline solution,
leading to the elimination of several small molecules. However, good
long-term chemical stability was observed when PCysMA brushes were
immersed in aqueous solution at physiological pH.
Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one method by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterize the feedback between bacteria clustering and quorum sensing signalling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population level.
We report the synthesis of thermo-responsive polymer brushes with Upper Critical Solution Temperature (UCST)-type behaviour on glass to provide a new means to control cell attachment. Thermoresponsive poly(N-acryloyl glycinamide)-statpoly(N-phenylacrylamide) (PNAGAm-PNPhAm) brushes with three different monomer ratios were synthesized to give tunable phase transition temperatures (Tp) in solution. Surface energies of surface-grafted brushes of these polymers at 25, 32, 37 and 50 C were calculated from contact angle measurements and atomic force microscopy (AFM) studies confirmed that these polymers were highly extended at temperatures close to Tp in physiologically-relevant media. Importantly, NIH-3T3 cells were attached on the collapsed PNAGAm-PNPhAm brush surface at 30 C after 20 h incubation, while release of cells from the extended brushes was observed within 2 h after the culture temperature was switched to 37 C. Furthermore, the changes in cell attachment followed changes in the Lewis base component of surface energy. The results indicate that, in contrast to the established paradigm of enhanced cell attachment to surfaces where polymers are above a Lower Critical Solution Temperature (LCST), these novel substrates enable detachment of cells from surfaces at temperatures above a UCST. In turn these responsive materials open new avenues for the use of polymer-modified surfaces to control cell attachment for applications in cell manufacture and regenerative medicine.
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