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Biology is dominated by polyanions (cell membranes, nucleic acids, and polysaccharides just to name a few), and achieving selective recognition between biological polyanions and synthetic systems currently constitutes a major challenge in many biomedical applications, nanovectors-assisted gene delivery being a prime example. This review work summarizes some of our recent efforts in this field; in particular, by using a combined experimental/computation approach, we investigated in detail some critical aspects in self-assembled nanomicelles and two major polyanions-DNA and heparin.

The effect of direct or indirect binding of intercalant molecules on DNA structure is of fundamental importance in understanding the biological functioning of DNA. Here we report on self-suspended DNA nanobundles as ultrasensitive nanomechanical resonators for structural studies of DNA-ligand complexes. Such vibrating nanostructures represent the smallest mechanical resonator entirely composed of DNA. A correlative analysis between the mechanical and structural properties is exploited to study the intrinsic changes of double strand DNA, when interacting with different intercalant molecules (YOYO-1 and GelRed) and a chemotherapeutic drug (Cisplatin), at different concentrations. Possible implications of our findings are related to the study of interaction mechanism of a wide category of molecules with DNA, and to further applications in medicine, such as optimal titration of chemotherapeutic drugs and environmental studies for the detection of heavy metals in human serum.

A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C 16 aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C 16 -l-Ala-l-Lys/C 16 -d-Ala-d-Lys) and (C 16 -d-Ala-l-Lys/C 16 -l-Ala-d-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self-assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d-lysine ligands, with a further slight preference for attachment to l-alanine, with the CMC being irrelevant., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper reports a small family of cationic surfactants designed to bind polyanions such as DNA and heparin. Each molecule has the same hydrophilic cationic ligand and a hydrophobic aliphatic group with eighteen carbon atoms with one, two, or three alkene groups within the hydrophobic chain (C18-1, C18-2 and C18-3). Dynamic light scattering indicates that more alkenes lead to geometric distortion, giving rise to larger self-assembled multivalent (SAMul) nanostructures. Mallard Blue and Ethidium Bromide dye displacement assays demonstrate that heparin and DNA have markedly different binding preferences, with heparin binding most effectively to C18-1, and DNA to C18-3, even though the molecular structural differences of these SAMul systems are buried in the hydrophobic core. Multiscale modelling suggests that adaptive heparin maximises enthalpically favourable interactions with C18-1, while shape-persistent DNA forms a similar number of interactions with each ligand display, but with slightly less entropic cost for binding to C18-3-fundamental thermodynamic differences in SAMul binding of heparin or DNA. This study therefore provides unique insight into electrostatic molecular recognition between highly charged nanoscale surfaces in biologically relevant systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This article reports self-assembling dendrons which bind DNA in a multivalent manner. The molecular design directly impacts on self-assembly which subsequently controls the way these multivalent nanostructures bind DNA--this can be simulated by multiscale modelling. Incorporation of an S-S linkage between the multivalent hydrophilic dendron and the hydrophobic units responsible for self-assembly allows these structures to undergo triggered reductive cleavage, with dithiothreitol (DTT) inducing controlled breakdown, enabling the release of bound DNA. As such, the high-affinity self-assembled multivalent binding is temporary. Furthermore, because the multivalent dendrons are constructed from esters, a second slow degradation step causes further breakdown of these structures. This two-step double-degradation mechanism converts a large self-assembling unit with high affinity for DNA into small units with no measurable binding affinity--demonstrating the advantage of self-assembled multivalency (SAMul) in achieving highly responsive nanoscale binding of biological targets.

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.

This paper develops a structure-activity relationship understanding of the way in which surfactant-like dendrons with hydrophilic spermine surface groups and a variety of lipophilic units at their focal points can self-assemble and subsequently bind to DNA with high affinity. The choice of functional group at the focal point of the dendron and the high tunability of the molecular structure have a very significant impact on DNA binding. Mesoscale modeling of the mode of dendron self-assembly provides a direct insight into how the mode of self-assembly exerts its effect on the DNA binding process. In particular, the hydrophobic unit controls the number of dendrons in the self-assembled micellar structures, and hence their diameters and surface charge density. The DNA binding affinity correlates with the surface charge density of the dendron aggregates. Furthermore, these structure-activity effects can also be extended to cellular gene delivery, as surface charge density plays a role in controlling the extent of endosomal escape. It is reported that higher generation dendrons, although binding DNA less strongly than the self-assembling lower generation dendrons, are more effective for transfection. The impact of the lipophilic group at the focal point is less significant for the DNA binding ability of these larger dendrons, which is predominantly controlled by the spermine surface groups, but it does modify the levels of gene transfection. Significant synergistic effects on gene delivery were observed when employing combinations of the dendrons and polyethyleneimine (PEI, 25 kDa), with transfection becoming possible at low loading levels where the two components would not transfect individually, giving practically useful levels of gene delivery.

We report the synthesis, DNA binding ability and preliminary gene delivery profiles of dendrons with different amine surface groups, 1,3-diaminopropane (DAP), N,N-di-(3-aminopropyl)-N-(methyl)amine (DAPMA) and spermine (SPM). By using a combination of ethidium bromide displacement, gel electrophoresis and transfection assays, it is shown that the dendrons with SPM groups are the most effective DNA binders, while the DAPMA-functionalised dendrons were the most effective systems for gene delivery (although the gene delivery profiles were still modest). In order to provide deeper insight into the experimental data, we performed a molecular dynamics simulation of the interactions between the dendrons and DNA. The results of these simulations demonstrated that, in general terms, the enthalpic contribution to binding was roughly proportional to the dendron surface charge, but that dendrons with DAP (and DAPMA) surface amines had significant entropic costs of binding to DNA. In the case of DAP, this is a consequence of the fact that the entire dendron structure has to be organised in order for each individual monoamine charge to make effective contact with DNA. For SPM, however, each surface ligand is already a multivalent triamine, therefore, each individual charge has a much lower entropic cost of binding. For DAPMA, we observed that strong binding of the hindered tertiary amine to the DNA double helix led to ligand back-folding and significant geometric distortion of DNA. Although this weakens the overall binding, we suggest that this distortion might be an explanation for the experimentally observed enhanced gene delivery, in which DNA compaction is an important step. Overall, this paper demonstrates how structure-activity relationships can be developed for multivalent dendritic ligands and provides insights into the thermodynamics of multivalent interactions., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper reports the application of molecular dynamics methods to understand the interactions between dendritic molecules with spermine surface groups and double-helical DNA. Importantly, we are able to reproduce the binding effects observed experimentally, indicating that this type of modeling is robust and reliable. The energetic effects were deconvoluted in order to quantify the binding of each spermine unit to the DNA double helix. Importantly, for the first-generation dendron G1, DNA binding was adversely affected by increasing levels of NaCl (>10% of the interaction energy is lost). For second-generation G2 however, we observed a compensation effect, in which some ligands "sacrifice" themselves, losing large amounts of binding energy with DNA. However, these ligands screen the complex, which enables the other spermine residues to bind more effectively to DNA. In this way, the multivalent array is able to maintain its high affinity binding, even as the salt concentration increases (only ca. 1% of the interaction energy is lost). These modeling studies are in agreement with, and provide a unique insight into, the experimental results. Clearly, ligand flexibility and ability to reorganize the interactions with DNA are important, demonstrating that high levels of preorganization and ligand framework rigidity are not always beneficial for multivalent recognition. The concept suggested by this modeling study, in which ligand "sacrifice" and binding site screening combine to enable high-affinity binding, is a new paradigm in multivalency.

Due to their relative easy synthesis and commercial availability, nanovectors based on dendrimers and dendrons are among the most utilized non-viral vectors for gene transfer. Concomitantly, recent advances in molecular simulations and computer architectures not only allow for accurate predictions of many structural, energetical, and eventual self-assembly features of these nanocarriers per se, but are able to yield fundamental information about the interactions of these nanovectors with their nucleic acid cargoes at a molecular level. In this work, we aim at reviewing some of our own efforts in the field of multiscale molecular modeling of these fascinating materials. This review is written by computational scientists for experimental scientists, with the specific purpose of illustrating the potentiality of these methodologies and the usefulness of multiscale molecular modeling as an innovative and complementary tool in their current research. [ABSTRACT FROM AUTHOR]