3.1.1. C22G1 Self-Assembled Dendrimers Have High Heparin Affinity at Physiological Ionic Strength
Since the assays previously employed to monitor aggregation and heparin binding of the selected C22G1 amphiphilic dendron (Figure 3a) were carried out either in pure water or at very low salt concentration (≤5 mM) [36], in the first place we decided to investigate in detail the behavior of this molecule in more biologically relevant media. To the purpose, we initially gained information of the generation of G22G1 self-assembled nanostructures under different ionic strengths using computer-assisted multiscale molecular simulations [21]. In particular, exploiting a combination of atomistic and mesoscale modeling we predicted that at the physiological salt concentration (150 mM NaCl), C22G1 self-organizes into well-defined spherical micelles characterized by an aggregation number (Nagg) of 24 ± 1, a total surface charge (Ntot) of +96 ± 4, and an average micellar diameter (Dm) of 9.3 ± 0.1 nm (Figure 3b). Contextually, when C22G1 was challenged in silico for self-assembling under no-salt conditions, simulations still anticipated the formation of spherical nano-objects, yet with substantially smaller Nagg (11 ± 3), Ntot (44 ± 12), and Dm (6.3 ± 0.5 nm) (Figure 3c). The predicted increase of the micellar aggregates in response to increasing ionic strength was attributed to the salt-mediated screening of the micellar surface charge paralleled by the increasing contribution of the hydrophobic interactions. These two effects, acting in synergy, allow a larger number of individual dendrons to be incorporated into the nanomicelles, ultimately leading to larger nanoassemblies. Interestingly, at variance with the covalent G2 PAMAM molecule discussed above, the non-covalent nature of the G22G1 nanostructures endows them with the ability to respond to this physiologically-relevant environmental stimulus by modifying their characteristic dimensions to an extent covalent would be unable to reach.
The changes in micellar dimensions envisaged by computer simulations were experimentally confirmed by dynamic light scattering (DLS) measurements performed under the same conditions. Indeed, in the presence of 150 mM NaCl the measured average micellar diameter Dm was equal to 9.1 ± 0.1 nm whilst in the absence of salt the Dm value reduced to 5.8 nm, in excellent agreement with both theoretical predictions. Given the established increase in the G22G1 nanomicelle dimensions under physiological salt conditions, and the further evidence—based on Nile Red solubilization assay—that these amphiphilic dendrons could still self-organize in the presence of heparin with a CMC of 14 µM, we surmised that these properties could be beneficial to its heparin binding ability. Therefore, we evaluated the parameters (EC50, CE50, and the related dose) of this self-assembled dendrimer for heparin binding in 150 mM NaCl buffered solution at pH 7.4 (again using our MB displacement assay) and compared the results with those obtained both with protamine and the covalent G2 PAMAM molecule. As seen in the upper part of Table 1, the EC50 values indicate that protamine and the small PAMAM dendrimer apparently are a more effective heparin binder than the C22G1 nanomicelles, for which EC50 is approximately 3 times higher than for the other two compounds.
However, the key parameter to be used for an objective comparison among these three quite dissimilar heparin binders is CE50, which is their charge efficiency. In this case, from Table 1 it is quite evident that the C22G1 self-assembled dendrimer (CE50 = 0.28) substantially outperforms both protamine (0.52) and G2 PAMAM (0.38) in polyanion binding affinity. Also, according to this assay, the C22G1 nanomicelles are active at a dose (0.23 mg/100 heparin IU) significantly lower than protamine (0.32 mg/100 heparin IU) and even slightly lower than that required by the alternative, covalent molecule (0.25 mg/100 heparin IU).
Molecular simulations were again employed to support the experimental binding evidence, and the relevant results are shown in Table 2.
Table 2 indicates that, under physiological salt concentration, C22G1 micelles productively expose only 33% of their global 96 positive charges for effective heparin binding, compared to the 50% and 80% of the charged groups exploited by protamine and the G2 PAMAM dendrimer, respectively. Notwithstanding, the highly flexible nature of the G22G1 self-assembled structures endows them with the ability to adopt the most effective conformation for heparin binding and, in so doing, to present each individual charge for the most efficient polyanion interaction (Figure 3d). Ultimately, this translated in the most favorable heparin effective-charge-normalized free energy binding (∆Gbind,eff/Neff) value (−2.03 kcal/mol) for the C22G1 self-assembled dendrimer along the entire binder series, in agreement with the corresponding experimental data.