A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(l-lysine iso-phthalamide)

The pH mediated intra-molecular association and inter-molecular aggregation of a range of amphiphilic poly(l-lysine iso-phthalamide) polymers have been investigated in aqueous solution over a range of pH values and concentrations. The desired functionality of these novel bioresponsive amphiphilic polymers was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Incorporation of low levels of bis-functional Cy3 (poly-Cy3) and/or Cy5 dye (poly-Cy3/5 or poly-Cy5) co-monomers in the responsive polymer backbone allowed detailed probing of the pH mediated hydrophobic association using a combination of optical spectroscopic techniques. Both steady-state fluorescence spectroscopy and fluorescence lifetime measurements of poly-Cy3 revealed a conformational transition at pH 4.5. Thus, below a critical pH the polymer collapsed into a compact globular structure (hypercoil) bringing the fluorophore molecules into close proximity with one another. This resulted in a dramatic reduction in fluorescence intensity and fluorescent lifetime in the single fluorophore systems (poly-Cy3) accompanied by a red shift in the maximum emission wavelength. Observed redshifts in the emission maxima and enhancements of fluorescent lifetimes with increasing polymer concentration suggested the formation of polymer aggregates. Fluorescence resonance energy transfer (FRET) was measured in mixtures of single fluorophore containing poly-Cy3 (donor) and poly-Cy5 (acceptor) and dual fluorophore containing poly-Cy3 (donor)/Cy5 (acceptor) in an effort to distinguish between intra-molecular versus inter-molecular association. The relevance of the results with respect to potential in vivo applications (drug delivery and biodiagnostics) is discussed.