3.1. C22G1, the First Example of Performing Heparin-Binding Self-Assembled Dendrimer
In the last decade, our group has been quite active in several international projects dealing with the development of amphiphilic dendrons that, upon self-assembling into micellar spherical pseudo-dendrimers, exhibited unique binding properties towards nucleic acids which entailed them to be exploited as nanocarriers for gene delivery in cancer therapeutics [18,19,20,22,23,25,27,28]. Notably, such systems are synthetically straightforward, with programmed self-assembly of simple building blocks being used as the key nanofabrication step. Since DNA and RNA are also polyanions, we alleged that dendrimer-like micelles originating from the self-organization of amphiphilic dendrons could potentially show high-affinity heparin binding and, as such, act as protamine mimetics.
Therefore, drawing from previous experience, we begin our investigation in the field with C22G1, an amphiphilic dendron featuring two polar head groups constructed from N,N-di(3-aminopropyl)-N-methylamine (DAPMA) and an apolar tail constituted by a C22-long hydrocarbon chain. These molecules readily self-assemble into micellar structures with a critical micelle concentration (CMC) of 4 µM in phosphate buffer saline at physiological pH [36]. Moreover, preliminary heparin binding assays conducted in pure water confirmed the affinity of C22G1 micelles for this anionic polysaccharide. Accordingly, we embarked in a thorough study aimed at fully characterizing these nanostructures in bio-relevant media, investigating their heparin-binding properties in such challenging conditions, and, above all, determining their functional heparin reversal in comparison with the gold-standard protamine and our covalent G2 PAMAM dendrimer alternative.

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.

3.1.2. Human Serum Is a (Moderate) Achille’s Heel in C22-G1 Self-Assembled Dendrimers Heparin Binding
Human serum undoubtedly represents the most biologically relevant and, contextually, the most challenging medium for probing the efficacy of alternative heparin binders. Indeed, with the exception of proteins involved in blood clotting, serum is populated by all other proteins and components (e.g., antibodies, antigens, hormones, and endogenous/exogenous species) which are routinely found in blood. Therefore, the next step in the evaluation of C22G1 self-assembled dendrimers as potential protamine replacers in clinical applications consisted in performing MB displacement assays in the presence of 100% human serum. The lower part of Table 1 illustrates the relevant results, again in comparison with those obtained for protamine and G2 PAMAM under the same experimental conditions. Rather dismayingly, human serum somewhat abated the heparin binding ability of the C22G1 self-assembled dendrimers, resulting in an increase of the relevant CE50 value to 0.96 (from 0.28 in buffered salted solution, upper part of Table 1). On the other hand, the CE50 for protamine was also negatively affected, albeit to a lesser extent (0.79 in serum vs. 0.52 in salted buffer, Table 1) while G2 PAMAM was equally if not even performing more in these challenging conditions (CE50 = 0.32, Table 1 and Section 2).
These results clearly highlight a major difference in the behavior of covalent and self-assembled nanostructures: While in human serum the former can withstand interfering blood components during heparin binding, the latter ones loose (at least in part) their polyanion binding ability when tested in competitive media. We ascribed the reason of this suboptimal performance to a partial disaggregation of the C22G1 self-assembled dendrimers (most likely operated by human albumin, the highly negatively charged, most abundant serum protein with specific binding sites for hydrophobic units) into their individual building blocks. As such the isolated C22G1 molecules, we verified (data not shown), were indeed unable to bind heparin effectively. Nonetheless, since the C22G1 self-assembly was not abrogated but only moderately compromised in the presence of serum with respect to the covalent counterparts, we considered the in-serum heparin binding performance of our systems still effective (CE50 < 1) and that the use of self-assembled dendrimers for this specific application could ultimately bring further advantages, as discussed below.

3.1.3. C22-G1 Nanomicelles Can Be Degraded and Disassembled at Physiological pH but Are Stable in the Presence of Heparin and Can Reverse its Anti-Coagulant Effect
As mentioned in Section 1, once a given medical procedure in which heparin is used for clotting prevention is concluded, there is the immediate need to neutralize the polyanion anti-coagulant effect to allow blood clotting and recovery to begin. This is achieved by treating the patient with the only FDA approved heparin reversal compound, protamine, which requires a rigorous and personalized administration to avoid, or at least, minimize the deleterious side-effects. Therefore, a protamine replacer which, in addition to the required heparin binding characteristics, can safely degrade into non-toxic components if administered in excess could represent an ideal alternative heparin antidote. As it can be noticed from Figure 3a, the C22G1 molecular structure features one ester group in its central, linker part, which was incorporated by design with the idea of making this dendritic scaffold degradable via hydrolysis of this moiety in the presence of biological triggers (e.g., pH or esterases). We also surmised that, should our G22G1 dendron break down over time in a controllable and predictable way into smaller subunits, this could ultimately enhance its biocompatibility, lower its toxicity, and limit its persistence in cells. Moreover, dendron degradation would also disassemble the multivalent heparin binding array, thereby acting as an effective way of “switching off” its biological activity. To verify whether our molecular design and the underlying hypotheses were correct, we conducted an electrospray mass spectrometry (ESMS)-based assay to probe the pathways of dendron degradation which were actually taking place at pH = 7.4 [15,21]. While at the initial stages of the experiment the molecular ions associated with the intact C22G1 molecule (Figure 4a) were clearly visible after 24 h they completely disappeared, the dominant peaks in the spectra being those corresponding to the ester hydrolysis products (see Figure 4b).
Having ascertained the feasibility of our dendron degradation, we next verified that this process ultimately leads to the relevant self-assembled dendrimer disaggregation. To the scope, C22G1 was dissolved into a buffer solution (pH = 7.4) at a final concentration above its CMC and in the presence of Nile Red. This dye is almost non-fluorescent in water and other polar solvents but undergoes fluorescence enhancement and large absorption and emission blue shifts in nonpolar environments. Accordingly, we measured the Nile Red fluorescence intensity over a 24-h time interval, obtaining the curve shown in red in Figure 5a. As it can be seen from this Figure, at the end of the assay the dye fluorescence has dropped to the same value measured in absence of the self-assembled dendrimers, allowing us to conclude that the degradation of the C22G1 building blocks indeed resulted in the corresponding micelles disassembly. The same experiment performed with the additional presence of heparin led to the green curve in Figure 5a, from which the high fluorescence intensity still detected after 24 h suggested that, once bound to the polyanion, the self-assembled dendrimers are much more resistant to degradation and retain their stability. This indirect evidence was further supported by transmission electron microscopy (TEM) imaging, from which it is clear that, although obviously clustered around the heparin surface (Figure 5b), the spherical C22G1 self-assembled nanostructures remain almost identical to those observed in the absence of the polyanion (Figure 5c) [21,36].
Therefore, under the perspective presented above, these results support the potential therapeutic use of the self-assembled dendrimers generated by C22G1 in heparin rescue, in that excess nanomicelles with degrade and disaggregate over a relatively short time frame—a property that does not pertain to their covalent counterparts. Yet, when bound to heparin the C22G1 nanoassemblies are more stable, hopefully allowing for the excretion of their intact heparin complexes.
The final step in the characterization of C22G1 self-assembled dendrimers as potential protamine replacers was the determination of their ability to reverse the coagulation inhibition induced by heparin. As we know, blood coagulation is an exceedingly complex process [37] which, for the sake of brevity, can be simplified and condensed as follows. After any blood vessel endothelial injury, platelets (aka thrombocytes) quickly gather at the lesion site and create a cross-linked plug (primary hemostasis). This, in turn, activates the so-called coagulation cascade, with resultant fibrin deposition and linking (secondary hemostasis). Platelets retraction and inhibition, followed by wound repair, complete the process. For diagnostic/prognostic purposes, the clotting cascade is assumed to consist of two separate yet interactive pathways: The intrinsic pathway, activated by the external trauma that causes blood to leave the vascular system and involves factor VII; and the extrinsic pathway, which originates by the trauma within the vascular system, is activated by platelets, exposed endothelium, chemicals, or collagen, and involves factors XII, XI, IX, and VIII. Typically, the intrinsic route is quantified by the activated partial thromboplastin time (aPTT) assay, which measures the time required by the complex formed among various plasma clotting factors (called thromboplastin) to convert prothrombin to thrombin and, hence, generate the fibrin clot. The extrinsic pathway, on the other hand, is routinely monitored by the prothrombin clotting time (PT) assay, a one-stage test based upon on the time required for a fibrin clot to form after the addition of tissue factor (TF, aka called platelet tissue factor, factor III, or CD142, an integral transmembrane receptor for Factor VII/VIIa), phospholipid, and calcium to decalcified, platelet-poor plasma.
Accordingly, both aPTT and PT tests were performed to check the capacity of our C22G1 self-assembled dendrimers to reverse the effect of heparin (Figure 5d). The experimental clotting time of pure plasma was equal to 35.7 s according to the intrinsic (aPTT) assay and to 12.8 s when measured by the PT assay. When heparin was added to plasma, the clotting time reduced to ~0, as expected from the anticoagulant activity exerted by the polyanion. On further addition of C22G1 nanomicelles, however, although for some reasons still unknown the clotting time measured by the aPTT assay increased to 81.8 s, the PT assay yielded a clotting time of 13.1, very close to that of native plasma, supporting the concept that the self-assembled C22G1 dendrimers are endowed of functional heparin reversal in the most biologically relevant environment.
In aggregate, all positive and innovative results obtained with the C22G1 pseudo-dendrimers as potential protamine replacers in heparin-induced anti-coagulation inhibition strongly encouraged us to embark on further studies focused on further design/optimization of self-assembling amphiphilic dendron structures and related structure-activity relationships aimed at gaining a more fundamental understanding of heparin binding phenomena with the ultimate goal of contributing in paving the way of these nanotechnologically-driven compounds to pre-clinical investigations.