1. Introduction
In healthy individuals, blood freely circulates along arteries and veins throughout the entire human body, in which the normal vascular endothelium acts as an overall antithrombotic surface. Yet, it becomes immediately active if the hemostatic system is triggered by the coagulation cascade. As we know, when a blood vessel is damaged platelets and fibrin quickly engage in an aggregation process leading to hemorrhage prevention. This is beneficial in avoiding blood loss, at the same time excessive clotting can lead to life-threatening thrombotic complications. Such a physiological situation might disgracefully happen during different clinical practices, such as large surgeries, blood transfusions, or dialysis treatments; accordingly, a delicate balance between thromboembolism risk and excessive bleeding prevention must be carefully optimized for each patient. Heparin, a linear polysaccharide consisting of repeating units of 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine (IdoA(2S)-GlcNS(6S) (Figure 1a), is one of the most charged dense naturally occurring polyanion in biological systems [1] and, equally, one of the most widely used clinical anticoagulants worldwide [2]. Once the clinical treatment requiring coagulation control is over, reversal of the administered heparin anticoagulant activity is obviously required. Protamine, a small, nuclear, basic, arginine-rich protein (Figure 1b), is the only FDA (Food and Drug Administration) approved molecule employed to that purpose. In fact, by virtue of its high positive charge, protamine binds to heparin via strong electrostatic interaction thereby removing the polysaccharide from the bloodstream and ultimately re-enabling clotting. Unfortunately, protamine is associated with several, important side-effects, including immunological and inflammatory alterations, and anaphylactic responses characterized by hypotension, bradycardia, pulmonary vasoconstriction, and allergy if not exactly administered [3]. Hence, the development of new, protamine-alternative heparin-rescue agents, which could stably bind the polyanion so that excretion of their intact complexes can easily and quantitatively occur, but at the same time, could safely degrade into non-toxic components if administered in excess, is a current hot need in medical practice.
DNA is indisputably another archetypal charge-dense polyanion that dominates biology. Being the guardian of the genetic information in humans and almost all other organisms, it is likely the most studied biological macromolecule. And, since upon unlocking the secrets of the human genome it was discovered that nearly all diseases have a genetic component, the biomedical interest in DNA for gene therapy has dramatically increased in recent years [4]. The Food and Drug Administration (FDA) defines gene-based therapeutics as “products that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered micro-organisms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient [5]”. Thus, the opportunity to treat such disorders by replacing the defective gene(s) with normal (healthy) one(s) offers a novel therapeutic approach for patients who suffer from such diseases. However, to be successful in the clinics gene, therapy largely relies on the adoption of safe, effective, and efficient viral/non-viral nanovectors to transfer DNA to mammalian cells both ex vivo and in vivo [6]. In the plethora of currently available nanocarriers for gene delivery dendrimers, i.e., nanosized, hyperbranched, and self-similar molecules represent prototypical vectors with fine-tunable properties for optimal DNA delivery [7]. Their peculiar structure is constituted by three distinct domains (Figure 1c): (1) A fundamental atom, or most frequently, a group of atoms defined as the core; (2) the branching units, which, emanating from the core through diverse chemical reactions, allow the dendrimeric molecule to grow in geometrically organized radial layers known as generations (G); and (3) an exponentially increasing number peripheral surface groups, which constitute a multivalent nanoscale array, and can therefore form high-affinity interactions with a variety of biological targets, including nucleic acids [8,9,10]. However, high generation dendrimer synthesis is extremely laborious and time-consuming, since the final product purification is difficult and hampered by the presence of highly similar side products. Thus, notwithstanding the highly promising results achieved with these molecules, the difficulties inherent in large-scale good manufacturing practice (GMP) production of high generation dendrimers are hampering their way to the clinics [11]. One way to circumvent these obstacles and to make multivalent systems which are synthetically simpler and more responsive is to design low molecular weight amphiphilic dendrons which, upon spontaneous self-assembly into dendrimer-like nanovectors, can mimic (and eventually outperform) their covalent counterparts in size, shape, and functions [12,13,14,15,16,17,18,19,20].
In the panorama described above, during the last 5 years our laboratory, in collaboration with different international groups, has designed and produced a series of amphiphilic molecules bearing dendritic portions as polar heads and various hydrocarbon chains as hydrophobic moieties, able to self-organize into supramolecular nanostructures of different size and shape with the unique capability of selectively binding the two major polyanions, heparin and DNA [21,22,23,24,25,26,27,28,29]. This, with the goal of employing the resulting self-assembled dendrimers as protamine replacer and gene delivery nanovectors, respectively. This short review summarizes our efforts focused on the first, less popular but certainly not less important field in the hope of paving the way for new heparin antidots to reach the stage of clinical nanomedicine.