Design and Applications of Responsive Polymers in Diagnostics, Separations, Bioprocesses, and Drug Delivery
Allan S. Hoffman, Universty of Washington, Box 352255, Seattle, WA 98195 and Patrick S. Stayton, Bioengineering, Universty of Washington, Box 352255, Seattle, WA 98195.
We have been designing and synthesizing a wide variety of stimuli-responsive polymers (which are sometimes called "smart" polymers) for many different applications in biotechnology and medicine. In many cases we have conjugated or complexed the responsive polymers to biomolecules, such as proteins and nucleic acids. Random conjugation to proteins is usually carried out by reaction of the polymer end group with a protein lysine amino group. Thus, a conjugate of a smart polymer and an affinity protein may recognize and bind to the affinity binding partner of the protein in a complex mixture, and then the polymer may be phase-separated by a specific stimulus, selectively removing the affinity binding partner from the complex mixture. In this way, a solution-based affinity chromatography operation may be carried out, rather than having to use a packed chromatographic column. If a second, labeled affinity protein is added to the complex mixture, then the three, affinity-linked proteins may be removed by the phase-separating smart polymer. This is like an immunoassay carried out in solution rather than in a well on a plate reader (ELISA). We have extended this technology to site-specific protein-polymer conjugates, usually by cloning mutant proteins with cysteine -SH groups in specific sites, permitting us to block and unblock the active, recognition site of the protein by different stimuli. The different stimuli we have used include temperature, pH and light (visible-UV), and we have synthesized a wide variety of polymers with responsivities to those stimuli. In some cases the polymers are random, block or graft copolymers of two monomers with different responsivities, such that the copolymers are responsive to two different stimuli. We have extended our applications to microfluidic devices for point-of-care diagnostics, including affinity separations as a first step. The reponsive polymers may also remove the protein and bind it by hydrophobic interactions to the channel wall of the device when stimulated. Site-specific conjugation of dual-sensitivity polymers to selected proteins are especially useful for these purposes. More recently we are making RAFT-polymerized block copolymers and conjugating them to specific sites on proteins. We have also demonstrated control of an enzyme bioprocess using both light- and temperature-sensitive copolymer-enzyme conjugates. We have applied pH-sensitive polymers to enhance intracellular drug delivery, especially to facilitate endosomal escape of protein drugs or nucleic acid drugs such as plasmid DNA, antisense ODNs and siRNA. Selected examples of these smart, responsive polymer-biomolecule hybrid systems will be presented.