Abstract:
Daptomycin is a cyclic lipopeptide agent that highly actives against a broad spectrum of fatal gram-positive pathogens. However, initial clinical trials resulted in treatment failure due to the high degree of daptomycin protein binding, rapid renal clearance or inadequate distribution to the target sites. In order to improve therapeutic outcomes of daptomycin, a non-covalent carrier system was developed using polyamidoamine (PAMAM) dendrimers. The study reported herein describes methods for characterization of an interaction between daptomycin and positively charged PAMAM dendrimers using ultrafiltration, UV difference spectroscopy and fluorescence spectroscopy. Binding of daptomycin to dendrimers led to fluorescence enhancement at the kynurenine residue on the daptomycin. These changes were quantitatively described by one- and two-site binding models by using nonlinear regression to estimate binding capacity and affinity constants. The binding of PAMAM dendrimer generation 5 (molecular size of 30 kDa) was described by a single site binding model. The estimated binding dissociation constants in pH range of 4.0-8.0 remained constant whereas the capacity constant was pH dependent. The fluorescence changes due to the binding of daptomycin to the PAMAM dendrimer generation 6 (molecular size of 60 kDa) in a pH range of 4.0 – 7.0 resulted in biphasic binding profiles, explained by a two site binding model. The estimated binding constants at high affinity site were similar to the results observed from the binding of daptomycin to PAMAM generation 5, whereas the binding dissociation and capacity constants of the low affinity sites were both sensitive to pH. The optimum total concentrations of PAMAM dendrimer in different conditions were predicted using the mathematical model in order to gain the optimum condition of complex formation. In addition, the molecular model of interaction between dendrimer and daptomycin was proposed based on the binding parameters and supporting information. The daptomycin appeared to orientate in latitudinal dimension and insert its lipid tail into the PAMAM dendrimer. The results of these investigations can provide an insight into the nature of daptomycin interactions with charged biomolecules.
