Heparin Binding of CholSG1 in Buffer at Physiological Ionic Strength and in Human Serum
Both MB displacement assays and ITC measurements were initially conducted in buffer solution at 150 mM NaCl to evaluate heparin binding for the new CholSG1 self-assembled dendrimer and to compare it with that of C22G1 and, especially, protamine. Somewhat disappointedly, these new nanoassemblies were less effective (EC50 = 0.79 ± 0.02) than the other two reference compounds (EC50 = 0.28 and 0.52 for C22G1 and protamine, respectively, Table 1), although TEM images of the CholSG1 micelles in complex with heparin were utterly similar to those seen for C22G1 under the same conditions (Figure 5c), showing no micellar morphology alteration or disaggregation upon polyanion binding. ITC experiments confirmed the results from the MB assays, in that the relevant ∆Gbind values were found to be equal to −30.85 kJ/mol for CholSG1 vs. the slightly more favorable value of −32.73 kJ/mol observed for C22G1. However, for both self-assembled dendrimers the polyanion binding was enthalpically-driven, due to the substantial electrostatic nature of the underlying intermolecular interactions. So, ∆Hbind was equal to −21.18 and −20 71 kJ/mol for C22G1 and CholSG1, respectively. Interestingly, in this case the entropic contribution to ∆Gbind was also substantially favorable (due to the release of water molecules and ions into the bulk solvent upon complex formation), with T∆Sbind values of 11.55 kJ and −10.14 kJ/mol for the dendrons bearing the linear alkyl segment and the Chol segment as the hydrophobic portion, respectively.
Notwithstanding this slight underperformance of the new self-assembled dendrimers CholSG1, we decided to challenge them for heparin binding in 100% serum. Pleasingly, the MB assay returned for them a CE50 value of 0.69 ± 0.07 (corresponding to a dose of 0.63 mg/100 heparin IU), which is sensibly lower than that obtained for both C22G1 and protamine (0.96 and 0.79, respectively, Table 1). These good results led us back to the original hypothesis that the presence of a linear hydrocarbon chain in the C22G1 dendron may results in micelles more prone to degradation by the action of serum proteins (in particular HSA, the most abundant serum protein with high affinity for alkyl-bearing compounds), whilst the nanostructures arising from the self-assembling of CholSG1 might be less subjected to this disruptive protein action and, as such, can perform better in the more physiologically environment of 100% human serum.
To further confirm this concept, we performed two additional investigations, based on ITC experiments and computer simulations, respectively. The former, in which heparin binding to the CholSG1 and C22G1 micelles was carried out in the presence of 500 µM HSA, confirmed the results from the MB assay. In fact, while the serum protein did not significantly interfere with the CholSG1/heparin complexation (∆Gbind = −30.72 kJ/mol, ∆Hbind = −20.35 kJ/mol, and T∆Sbind = 10.37 kJ/mol), the binding of C22G1 to the polyanion was appreciably affected by the presence of HSA in solution, resulting in decidedly less favored thermodynamics parameters (∆Gbind = −29.56 kJ/mol, ∆Hbind = −0.03 kJ/mol, and T∆Sbind = 9.53 kJ/mol). To gain further insights in to the eventual different kinetic stability of the two micelle types, we resorted to computer-based constant-force stirred molecular dynamics (CF-SMD) simulations. In detail, we evaluated the ability of the CholSG1 and C22G1 self-assembled dendrimers to withstand a force applied to pull out one of their respective dendron components. As shown in Figure 15a, the lower stability of the C22G1 micelles was indeed confirmed by these in silico experiments, since even at the smallest pullout force applied (i.e., 1.0 kcal/mol Å) a C22G1 dendron could be extracted from the relevant self-assembled dendrimer already during the early stages of the corresponding CF-SMD simulation. On the contrary, a substantially higher force (1.6 kcal/mol Å) was required to pullout a monomer from the CholSG1 micelles. These quantitative observations are well represented by the corresponding images taken along the CF-SMD trajectories shown in Figure 15b.
As seen from Figure 15b, the C22G1 dendron selected for pullout is completely extracted from its micelle already after only 0.7 ns of CF-SMD simulation (upper panel) while at the same time, the CholSG1 alternative is still well inserted within the corresponding micellar structure. These results clearly indicate that the self-assembled dendrimers based on Chol as the hydrophobic unit are endowed with a substantially greater kinetic stability with respect to those featuring the linear C22- alkyl chain, thereby supporting the initial design hypothesis.
These results, coupled with further data showing that the cholesterol-based amphiphilic dendrons were endowed with significantly lower toxic effects than C22G1 in a wide range of concentrations when assayed in a human hepatoblastoma cell line, make the CholSG1 the new lead compound for further translation study as a potential clinical protamine replacer.