3.3. “Less Is More” Further Investigated
We found the concept that small spherical micelles retain their shape while forming mesoscopic aggregate structure upon heparin binding quite intriguing. Accordingly, to investigate this aspect in more detail, we designed and optimized synthetically-simple, minimal self-assembling dendrons that could serve this purpose, ultimately ending up with “reduced versions” of C22G1, which is two molecules still featuring a single DAPMA polar head and a single apolar chain of 14 (C14G0) and 16 (C16G0) carbon atoms, respectively [24]. As per molecular design, in buffered solutions at physiologically ionic strength these two amphiphilic dendrons self-assembled into small, spherical nanostructures with high and positive ζ potentials (Dm = 5.8 ± 1.6 nm and ζ = + 41.3 ± 1.6 mV for C14G0 and 6.2 ± 1.3 nm and + 51.7 ± 2.2 mV for C16G0, respectively), and showed good heparin binding ability (CE50 = 0.88 and 0.46 for C14G0 and C16G0, respectively) yet inferior to those of C22G1, indicating that the structure this latter amphiphile was indeed optimized for binding the polyanion. However, given that both these simpler dendrons were endowed with the required characteristics (spherical micelle formation and heparin binding), we employed them to investigate the structure of the relevant heparin complexes starting from TEM imaging, as shown in Figure 8a,b for C16G0 as an example (analogue images were obtained for the alternative C14G0-based system). These TEM observations, beside confirming the spherical nature of these self-assembled dendrimers, suggest that their stability is preserved, without disruption or reorganization, upon heparin binding with which they form strong electrostatic interactions. However, since we were aware that TEM images were collected on dried samples, we wondered whether the drying process might have somewhat forced the systems (and, by extensions, also the other self-assembled dendrimers previously discussed) to assume such morphology. Therefore, we first resorted again to molecular simulations to predict the self-assembly and spatial organization of these two amphiphiles in the presence of heparin in solution. The output of these simulations (Figure 8c,d) shows that not only both molecules self-assemble in small, stable spherical micelles in the presence of the polyanion but, perhaps even more important, these are not randomly dispersed into the heparin solution but adopt a highly-ordered, hierarchical nanoscale structure matching a face-centered (fcc) organization, with lattice constant value a equal to 8.1 nm and 8.6 nm for the C14G0 and C16G0 self-assembled dendrimers, respectively. The corresponding center-to-center distance (a2) is equal to 5.7 nm in the case of the C14G0 micelles and to 6.1 nm for the self-assembled C16G0, in excellent agreement with the Dm values measured by DLS (5.8 and 6.2 nm, respectively, see above).
With these comforting in silico indications at hand, we proceeded to an experimental verification via small angle X-ray scattering (SAXS). The relevant results are shown in Figure 9. In particular, for both self-assembled dendrimers/heparin supermolecular complexes, the bi-dimensional (2D) SAXS diffraction patterns are characterized by a Debye ring with a diffuse symmetric halo that does not present intensity differences (inserts in Figure 9a)—a distinctive feature of polycrystalline samples with isotropic orientation of multiple crystals [40].
As noted in Figure 9a, the positions of the diffraction peak for the C14G0 micelles bound to heparin locate at values of the momentum transfer (aka scattering vector) q equal to 0.129 and 0.259 Å−1 which, assuming a fcc morphology, in terms of crystal plane reflections with Miller indices correspond to (hkl) = (111) and (222). The alternative, self-assembled dendrimers C16G0 in complex with heparin showed 3 diffraction peaks, at q = 0.122, 0.138, and 0.246 Å−1, respectively. These, again using Miller indices notation for an fcc structure, correspond to (hkl) = (111), (200), and (222). By plotting the quadratic Miller indices against the measured q(hkl) values for both systems, the experimental lattice constant a =2πh2+k2+l2/q(hkl) could be estimated by data linear fitting (Figure 9b) as 8.5 and 8.9 nm for the C14G0 and C16G0 systems, respectively. The corresponding experimental center-to-center distances a2 were also calculated as 6.0 nm for the C14G0 micelles and 6.3 nm for the G16G0 assemblies, and all these values were in excellent agreement with those predicted by computer simulations (see above).
In a conclusive effort, experimental (SAXS) and computational data for both self-assembled dendrimers in complex with heparin were compared with the corresponding TEM images, as shown in Figure 9c. The top left panel in this Figure illustrates the crystal projection view along the [110] zone axis. The analysis of the linear profile over the crystal projection (red double pointed arrow) yielded an average period (ap) of 4.5 nm, corresponding to fcc lattice constant values of 7.8 and 8.0 nm for C14G0 and C16G0 nanoassemblies, respectively. These values agree with those derived from simulation and SAXS reported above, the minor reduction in the cell unit size being ascribable to the drying effect on the TEM grid. The Fast Fourier (FF) transforms of the crystalline area and the subsequent selected filtered inverse FF transforms yielded the representative image of the crystal cell (Figure 9c, bottom row, left). The fcc arrangement of the micelles was confirmed by superposing this image with the corresponding unit cell model (Figure 9c, bottom row, center). Finally, the micelle center-to-center distance values of 5.5 nm and 5.6 nm estimated for C14G0 and C16G0, respectively (Figure 9c, bottom row, right), are in good agreement both with those obtained by computer simulations and with the micellar diameters estimated by DLS.
In summary, these results contributed for the first time to verify that even the “reduced version” of the amphiphilic dendron C22G1, that is the single-tail, small DAPMA-based C14G0 and C16G0 amphiphiles, are able to form very stable spherical micelles even when electrostatically bound to heparin, and that the presence of the polyanion induces the adoption of a highly regular, nanoscale hierarchical structure.