A new class of polyphenolic carbosilane dendrimers binds human serum albumin in a structure-dependent fashion

Abstract

The use of dendrimers as drug and nucleic acid delivery systems requires knowledge of their interactions with objects on their way to the target. In the present work, we investigated the interaction of a new class of carbosilane dendrimers functionalized with polyphenolic and caffeic acid residues with human serum albumin, which is the most abundant blood protein. The addition of dendrimers to albumin solution decreased the zeta potential of albumin/dendrimer complexes as compared to free albumin, increased density of the fibrillary form of albumin, shifted fluorescence spectrum towards longer wavelengths, induced quenching of tryptophan fluorescence, and decreased ellipticity of circular dichroism resulting from a reduction in the albumin α-helix for random coil structural form. Isothermal titration calorimetry showed that, on average, one molecule of albumin was bound by 6-10 molecules of dendrimers. The zeta size confirmed the binding of the dendrimers to albumin. The interaction of dendrimers and albumin depended on the number of caffeic acid residues and polyethylene glycol modifications in the dendrimer structure. In conclusion, carbosilane polyphenolic dendrimers interact with human albumin changing its structure and electrical properties. However, the consequences of such interaction for the efficacy and side effects of these dendrimers as drug/nucleic acid delivery system requires further research.

Keywords: Circular dichroism; Isothermal titration calorimetry; Polyphenolic dendrimers; Serum human albumin; Zeta potential.

Figure 1

Structure, chemical formula, and molar mass (MM) of polyphenolic carbosilane dendrimers with ammonium surface groups functionalized with caffeic acid and polyethylene glycol (PEG). (A) compound (1), G₂[(NMe₃Cl)₇(NH-CA)]; (B) compound (2), G₂[(NMe₃Cl)₆(NH-CA)₂]; (C) compound (3), G₂[(NMe₃Cl)₇(PEG-NH-CA)]; (D) compound (4), G₂[(NMe₃Cl)₆(PEG-NH-CA)₂]. Caffeic acid moieties are marked in red.

Figure 2

Dose-dependent effects of polyphenolic dendrimers on zeta potential (A), zeta size (B), and polydispersity index (C). Data points represent mean ± SD obtained from a minimum 3 experiments and each experiment was done in 7 replicates for dendrimer (1) (yellow line open diamonds), (2) (blue line, open squares) (3) (green line, open triangles) and (4) (red line, and open circles). Panels (D) represent the ultrastructure of human serum albumin in the presence of dendrimers. Bars 100 nm and 25 nm, to obtain greater contrast, the color of the micro images has been inverted.

Figure 3

Fluorescence emission spectra of albumin-dendrimer complexes for dendrimers (1–4) at the albumin/dendrimer molar ratios 1–10 (A). Stern–Volmer plots of tryptophan fluorescence quenching in the presence of increasing concentrations of dendrimers (B), double-logarithmic plot of tryptophan fluorescence quenching at albumin concentration 4 µmol/L (C). Data points represent mean ± SD obtained from a minimum 3 separate experiments.

Figure 4

Circular dichroism spectra of free albumin and its complexes with the dendrimers (A). Relative ellipticity of the albumin-dendrimer complexes at its different ratios. Data are presented as means ± SD obtained from a minimum of 3 separate experiments.

Figure 5

Integrated thermal effects of the isothermal titration calorimetry titration of 100 µmol/L HSA solution with 2 mmol/L solution of each dendrimer (red squares) and corresponding effects of the dilution of dendrimer (blue circles) (A). Curves of thermal power as a function of time during titration of 100 µmol/L albumin solution with 2 mmol/L dendrimer solution (red peaks) and dilution of 2 mmol/L dendrimer solution into buffer without albumin (blue peaks). Downward peaks correspond to endothermic heat effects (B). Thermal effects of direct interactions (per mole of injectant) between albumin and polyphenolic carbosilane dendrimers as a function of dendrimer to albumin mole ratio. All titrations were carried out at 25 °C in aqueous 10 mmol/L phosphorous buffer solution pH 7.4.