Research Program
Cell Surface Interactions

Objectives
Synthesis and characterization of DNP functionalized polymers (both conductive and non-conductive) that are specific to antibodies and immune receptors are being carried out. These polymers are expected to be useful for controlling receptor binding and cell activation, with eventual application in molecular (nanoscale) therapeutics and biodiagnostics. During the last reporting period, we reported the successful synthesis of a water soluble a,w-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), that binds specifically to anti-DNP IgE receptors on RBL mast cells.  We showed that one of these water soluble polymers (BS5-25, 16.1 kDa) is a potent inhibitor of receptor-mediated degranulation responses in mast cells. We began to investigate the mechanism of inhibition by this family of polymers. This motivated our preparation of several additional water soluble polymers with different molecular weights and further structure/function analyses. Also during this reporting period, we began to develop a method for preparing a DNP functionalized biodegradable polymers based on polylactide and poly(ethylene glycol).

Methods
Synthesis of the water soluble CDNP-PEO-P2MS-PEO-CDNP was carried out by the method developed during the last reporting period. The synthesis of DNP functionalized biodegradable DNP polymers (see figure 1) was carried out by bulk polymerization. A tetrahydroxy functionalized poly(ethylene glycol) was used as the initiating system coupled with Sn(Oct)₂ as the catalyst for polymerizing the lactide monomer. Characterizing of the polymers and cellular interaction studies were carried out by established methods.

Summary
During the current reporting period four different water soluble CDNP-PEO-P2MS-PEO-CDNP ligands of molecular weights (BS5-25, 16.1 kDa; BS5-14, 32.6 kDa; BS9-3, 26.6 kDa; BS5-20, 59.5 kDa) were synthesized and characterized. We tested these for their IgE binding as well as their capacity to stimulate and inhibit IgE-receptor-mediated cellular responses. All bind to IgE on cells with Kd ranging over 80 – 450 nM (see figure 2).  In tests so far we find that these ligands do not stimulate cellular responses and inhibit Ca²⁺ mobilization (see figure 3) and degranulation that is stimulated by a potent antigen, consistent with long term applications in  nanoscale therapeutics based on these polymers.  During this period, we also successfully developed a method for preparing DNP functionalized biodegradable polymers (see structure figure 1). During the next several months, the method developed will be used to prepare both bivalent and tetravalent DNP functionalized biodegradable polymers, and these will be evaluated for their capacity to bind to anti-DNP IgE receptors on RBL mast cells and affect cellular responses.

CREST CFNM and NBTC Collaborative work:  The groups of Baird, Khan and Craighead work together to develop application of polymeric nanostructures for therapeutics and biodiagnostics. In work relevant to the overall NBTC mission, we have electrospun fibers from a 30% THF solution of CDNP-PEO-P2MS-PEO-CDNP with different single  wall carbon nanotube (SWCNT) contents. Fibers containing 3.3% SWCNT and decorated with DNP functionalized groups and were electrospun and confirmed to be conductive.

Accomplishments

• Synthetic method for preparing DNP functionalized biodegradable polymers
• Specific binding of four DNP functionalized water soluble polymers to anti-DNP IgE on cells
• Inhibition of receptor-mediated cell responses by DNP functionalized water soluble polymers