Polymers that transport cells and detect bacteria

coil_globule_transitionIn 2010-11 we reported major breakthroughs in two key areas of enormous importance in regenerative medicine and healthcare. Based on work originally reported in 20051 and 20072 from Steve Rimmer’s group our consortium (Steve Rimmer, Sheila MacNeil, Ian Douglas, Linda Swanson) have shown that stimulus responsive highly branched polymers with appropriate end groups can lift human cells from culture substrates3 and similar polymers (with different end groups can respond to bacteria4. The work is based on the behaviour of water soluble polymers, which have lower critical solution temperatures (LCST) at which point they pass from an open coil to a more compact globule: as shown schematically for both linear and branched polymers in figure 1.

Figure 1 (above right) Schematic of the coil-to-globule transition at the LCST for a branched or linear polymer in aqueous solution. Note in the branched case the end groups (red) remain on the outside whereas in the linear polymer they are shielded within the globule. Shielded end groups are not available for binding.

Both technologies use three important ground breaking discoveries.

LIFT- Ligand functionalised temperature sensitive polymers for cell binding and release

Based on the work described in patent: “Transfer of cells using smart polymers” S. Rimmer, S. MacNeil, S.A. Hopkins, S. R. Carter P121539GB

The transportation of human cells to other substrates or wound beds is of key importance during many strategies aimed at regenerating tissue and in laboratory-based research in cell biology. With these aspects in mind the thermally responsive polymer, poly(N-isopropyl acrylamide) (PNIPAM), has been used to culture and release cells without employing animal derived trypsin for both research and clinical purposes. Cell culture plasticware grafted with PNIPAM is now available and has been used to culture sheets of cells which are then released from the culture dishes with a decrease in temperature to below the lower critical solution temperature (LCST). The cooling process causes the degree of swelling of the film to increase, which decreases the cell-adhesive nature of the material by producing a substrate that interacts poorly with adsorbed cell–adhesion promoting proteins.

However, a major drawback with these polymers is that although cell sheets can be produced and detached the cultures can be difficult to handle since it is essential that they are kept above the LCST until they are harvested. The cell adhesive state (above the LCST) of these systems can be improved by the anchoring of cell adhesion peptides, such as peptides based on the RGD sequence).

LIFT_cell_transfer

 

Fgure 2 LIFT technology for cell transfer compared to conventional thermal responsive surfaces. LIFT allows the cell biologist more freedom to optimise the cell culture steps without considering temperature fluctuations.

However, we recently discovered another general strategy that can be used to release cells that are cultured in the conventional manner on commercially available substrates. This is an important advance in responsive smart polymer cell-transfer technology because it allows the cell technologist to optimise the culture of the cells without considering the cell adhesive nature of the cell releasing polymer. This then produces much more robust protocols that are much less sensitive to small aberrations of temperature. In figure 1 we compare the conventional “cell-sheet” strategy with this new approach, which we have termed LIFT ( ligand functionalised temperature sensitive polymers).

Firstly a highly-branched poly(N-isopropyl acrylamide) (HB-PNIPAM) with an RGD-containing peptide at the chain ends is prepared. As the temperature is raised through LCST the polymer chain desolvates and collapses into nanoglobules, which then aggregate to form sub-micron particles (Topologically open sub-micron particles) functionalised with a cell adhesive peptide and which provide a substrate that is generally amenable to cell spreading. Cells cultured on tissue culture plastic prefer to migrate to these particles because the GRGDS sequence binds to specific species on the cell surface, integrins. Thus, the particles lift the cells from the original substrate and they can be transferred, as a dispersion, to another substrate. Decreasing the temperature releases the cells as the particles re-dissolve and the aggregates break up. Anchorage dependant cells are then reseeded onto the new substrate where they proliferate and grow.

Read more about LIFT in reference 3.

Stimulus responsive polymers for detecting bacteria

Based on the work described in patent: “Bacteria Binding Polymer” S.Rimmer, L. Swanson, S. MacNeil P124848GB

Stimulus responsive polymers pass from an open coil, which is fully solvated by water, to more compact globule in aqueous media as the environment changes. Well known environmental factors include pH, temperature and ionic strength. Other factors are important such as adsorption of proteins and surfactants and recently we showed that polymer chain branching has a strong controlling effect on the temperature of the transition (Lower critical solution temperature (LCST)). In fact any perturbation of the structure of water around the dissolved can induce the transition and we considered that binding of biological entity to specific ligands attached to the chain could induce the transition. This induced transition would be important for many applications because it can be detected by a number of physical measurements. However, another important feature of the globular state is that the polymers are generally cell adhesive whereas the open chain form repeals cells and is non-fouling. Recent work in the field has shown that linear polymers containing groups that bind to bacteria do not pass through the coil-t0-globule transition on binding. In our view this is because if the a local transition occurs as the ligand binds the collapse process shield the ligand and the ligand-receptor complex breaks down. This shielding does not occur with branched polymers if the ligand is located at the chain ends. Therefore binding perturbs the solvation of the end groups which in turn causes the polymer to pass through the transition. The process is shown in figure 3 and an example of a polymer interacting and aggregating bacteria is shown in figure 4.

bacteria_binding_polymer

Figure 3 Stimulus responsive highly branched polymers with end groups that bind bacteria and aggregate them

bound_bacteria

Figure 4 S. aureus dyed red (A) and S. aureus + polymer dyed blue (B)

Read more about this work in reference 4.

References

  1. “Highly Branched Poly(N-isopropylacrylamide)s with Imidazole End Groups Prepared by Radical Polymerization in the Presence of a Styryl Monomer Containing a Dithioester Group” S. Carter, B. Hunt, S. Rimmer, Macromolecules 38 4595 (2005)
  2. “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” S. Rimmer, S. Carter, R. Rutkaite, J. W.Haycock, L. Swanson Soft Matter, 3 971 (2007)
  3. “Sub-micron poly(N-isopropyl acrylamide) particles as temperature responsive vehicles for the detachment and delivery of human cells” S. Hopkins, S.R. Carter, J.W. Haycock, N.J. Fullwood, S. MacNeil, S. Rimmer Soft Matter 5, 4928, (2009)
  4. “Binding bacteria to highly branched poly(N-isopropyl acrylamide) modified with vancomycin induces the coil-to-globule transition” J. Shepherd, P. Sarker, K. Swindells, I. Douglas, S. MacNeil, L. Swanson, S. Rimmer J. Am. Chem. Soc. 132, 1736, (2010)