Self-association properties of HPMA copolymers containing an amphipathic heptapeptide

Receptor-binding peptides are suitable targeting moieties for macromolecular therapeutics. Binding several targeting peptides to one macromolecule may improve biorecognition due to the multivalency effect. On the other hand, the resulting amphipathic structure of such conjugates may result in the association of side-chains with a concomitant decrease in the accessibility of the side-chain-bound ligands. Using the one-bead one-compound combinatorial method, we have recently identified a heptapeptide (YILIHRN; HP) ligand for the CD21 receptor (Biomacromolecules 7, 3037, 2006). Here, we evaluated the relationship between structure and self-association of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-HP conjugates using fluorescence resonance energy transfer (FRET) to evaluate their conformation in solution. In addition to HP, HPMA copolymers containing side-chains terminating in tryptophan (energy donor) and dansyl (energy acceptor) were synthesized, and solutions were evaluated using an excitation wavelength of 295 nm (ratio of emission intensity 510 nm/370 nm indicated energy transfer efficiency). It was found that higher HP content correlated with higher FRET efficiency, indicating the formation of compact coils. Modification of the HPMA copolymer backbone by the incorporation of acrylic acid (AA) comonomer units resulted in decreased FRET efficiency, presumably due to the expansion of the polymer coils as a result of electrostatic repulsion. The dependence of FRET efficiency on pH was in agreement with the ionization profile of the AA residues. To determine the effect of HP content on enzymatic drug release kinetics, HPMA copolymer-HP conjugates containing GFLG side-chains terminating with doxorubicin (DOX) were incubated with papain and the release of free DOX monitored. When HP content increased above a particular threshold, the rate of DOX release decreased as a result of self-association of HPMA copolymer-GFLG-DOX-HP conjugates. The FRET data correlated well with hydrodynamic volumes determined by size exclusion chromatography (SEC), with molecular weights determined by light scattering, and with the kinetics of drug release.