Supramolecular Double Helices from Small C3-Symmetrical Molecules Aggregated in Water.

Supramolecular fibers in water, micrometers long and several nanometers in width, are among the most studied nanostructures for biomedical applications. These supramolecular polymers are formed through a spontaneous self-assembly process of small amphiphilic molecules by specific secondary interactions. Although many compounds do not possess a stereocenter, recent studies suggest the (co)existence of helical structures, albeit in racemic form. Here, we disclose a series of supramolecular (co)polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) that form double helices, fibers that were long thought to be chains of single molecules stacked in one dimension (1D). Detailed cryogenic transmission electron microscopy (cryo-TEM) studies and subsequent three-dimensional-volume reconstructions unveiled helical repeats, ranging from 15 to 30 nm. Most remarkable, the pitch can be tuned through the composition of the copolymers, where two different monomers with the same core but different peripheries are mixed in various ratios. Like in lipid bilayers, the hydrophobic shielding in the aggregates of these disc-shaped molecules is proposed to be best obtained by dimer formation, promoting supramolecular double helices. It is anticipated that many of the supramolecular polymers in water will have a thermodynamic stable structure, such as a double helix, although small structural changes can yield single stacks as well. Hence, it is essential to perform detailed analyses prior to sketching a molecular picture of these 1D fibers.

Supramolecular Architectures of Dendritic Amphiphiles in Water.

Dendritic molecules are an exciting research topic because of their highly branched architecture, multiple functional groups on the periphery, and very pertinent features for various applications. Self-assembling dendritic amphiphiles have produced different nanostructures with unique morphologies and properties. Since their self-assembly in water is greatly relevant for biomedical applications, researchers have been looking for a way to rationally design dendritic amphiphiles for the last few decades. We review here some recent developments from investigations on the self-assembly of dendritic amphiphiles into various nanostructures in water on the molecular level. The main content of the review is divided into sections according to the different nanostructure morphologies resulting from the dendritic amphiphiles' self-assembly. Finally, we conclude with some remarks that highlight the self-assembling features of these dendritic amphiphiles.

Supramolecular Copolymerization as a Strategy to Control the Stability of Self-Assembled Nanofibers.

A major challenge in supramolecular polymerization is controlling the stability of the polymers formed, that is, controlling the rate of monomer exchange in the equilibrium between monomer and polymer. The exchange dynamics of supramolecular polymers based on benzene-1,3,5-tricarboxamide (BTA) can be regulated by copolymerizing molecules with dendronized (dBTA) and linear (nBTA) ethylene glycol-based water-soluble side chains. Whereas nBTAs form long nanofibers in water, dBTAs do not polymerize, forming instead small spherical aggregates. The copolymerization of the two BTAs results in long nanofibers. The exchange dynamics of both the BTA monomers in the copolymer are significantly slowed down in the mixed systems, leading to a more stable copolymer, while the morphology and spectroscopic signature of the copolymers are identical to that of nBTA homopolymer. This copolymerization is the supramolecular counterpart of styrene/ maleic anhydride copolymerization.

Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids.

Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein-dendrimer conjugates with the defined architectures. We incorporated Azidohomoalanine (Aha) as methionine substitute in vivo into various protein scaffolds to allow non-invasive dendrimer conjugations (dendronylation). Our approach makes recombinant proteins accessible for the design of multivalent dendrimer conjugates since it enables the preparation of many sequences with various positions for regioselective dendronylation.

Aggregation Behavior of Non-ionic Twinned Amphiphiles and Their Application as Biomedical Nanocarriers.

A new class of twinned amphiphiles was developed by conjugating a pair of hydrophilic head groups from mPEG chains (Mn : 350 or 1000) and a pair of hydrophobic segments from linear alkyl chains (C11 or C18 ) through a novel spacer synthesized from glycerol and p-hydroxybenzoic acid. The aggregation phenomena of the amphiphiles were proven by DLS and fluorescence experiments, whereas size and morphology of the aggregates were evaluated by cryo-TEM. The measurements proved the formation of globular, thread-like or rod-like micelles as well as planar double-layer assemblies, depending on the amphiphile's molecular structure. The applicability of these non-ionic amphiphilic systems as nanocarriers for hydrophobic guest molecules was demonstrated by encapsulating a hydrophobic dye, Nile Red, and a hydrophobic drug, Nimodipine. The transport capacity results for both Nimodipine and Nile Red prove them as a promising candidate for drug delivery.

Crosslinked Redox-Responsive Micelles Based on Lipoic Acid-Derived Amphiphiles for Enhanced siRNA Delivery.

Successful application of gene silencing approaches critically depends on systems that are able to safely and efficiently deliver genetic material such as small interfering RNA (siRNA). Due to their beneficial well-defined dendritic nanostructure, self-assembling dendrimers are emerging as promising nanovectors for siRNA delivery. However, these kinds of vectors are plagued with stability issues, especially when considered for in vivo applications. Therefore, in the present study, disulfide-based temporarily fixed micelles are developed that can degrade upon reductive conditions, and thus lead to efficient cargo release. In detail, lipoic acid-derived crosslinked micelles are synthesized based on small polymerizable dendritic amphiphiles. Particularly, one candidate out of this series is able to efficiently release siRNA due to its redox-responsive biodegradable profile when exposed to simulated intracellular environments. As a result, the reduction-triggered disassembly leads to potent gene silencing. In contrast, noncrosslinkable, structurally related constructs fails under the tested assay conditions, thereby confirming the applied rational design approach and demonstrating its large potential for future in vivo applications.

Dendritic core-shell systems as soft drug delivery nanocarriers.

Unimolecular micelles are covalently bound molecular architectures and therefore highly stable which makes them particularly attractive for drug delivery. Accordingly, many reports in the literature emphasize the importance of these molecular architectures for nanomedicine. This conceptual review will present some of the recent advances in the application of these dendritic core-shell systems for drug delivery. Unimolecular micelles based on hyperbranched and dendritic cores will be discussed and sorted by the nature of their core and structure.

Towards engineering of self-assembled nanostructures using non-ionic dendritic amphiphiles.

Engineering nanostructures of defined size and morphology is a great challenge in the field of self-assembly. Herein we report on the formation of supramolecular nanostructures of defined morphologies with subtle structural changes for a new series of dendritic amphiphiles. Subsequently, we studied their application as nanocarriers for guest molecules.

Systematic adjustment of charge densities and size of polyglycerol amines reduces cytotoxic effects and enhances cellular uptake.

Excessive cationic charge density of polyplexes during cellular uptake is still a major hurdle in the field of non-viral gene delivery. The most efficient cationic vectors such as polyethylene imine (PEI) or polyamidoamine (PAMAM) can be highly toxic and may induce strong side effects due to their high cationic charge densities. Alternatives like polyethylene glycol (PEG) are used to 'shield' these charges and thus to reduce the cytotoxic effects known for PEI/PEG-core-shell architectures. In this study, we compared the ability of hyperbranched polyglycerol amines (hPG amines) with different amine densities and molecular weights as non-viral cationic vectors for DNA delivery. By adjusting the hydroxyl to amine group ratio on varying molecular weights, we were able to perform a systematic study on the cytotoxic effects caused by the effective charge density in correlation to size. We could demonstrate that carriers with moderate charge density have a higher potential for effective DNA delivery as compared to high/low charged ones independent of their size, but the final efficiency can be optimized by the molecular weight. We analyzed the physicochemical properties and cellular uptake capacity as well as the cytotoxicity and transfection efficiency of these new vector systems.

Optimized effective charge density and size of polyglycerol amines leads to strong knockdown efficacy in vivo.

RNA interference (RNAi)-based therapy extends the range of "druggable" targets beyond existing pharmacological drugs and enables the development of new treatment strategies for various diseases. A prerequisite are non-viral polyvalent gene delivery vectors capable for safe and effective siRNA delivery to cells in vivo allowing a broad clinical application. We synthesized hyperbranched polyglycerol amines (hPG amines) which varied in their charge density, multiplicity (absolute frequency of amine groups) and core size to successfully develop potent and safe siRNA transfer vectors. The characterization of hyperbranched polyglycerol amines with an invariable core size (8 kDa) but different amine loading revealed a correlation between the effective charge density and the transfection efficacy without impacting the cell viability in vitro. However, this correlation was not seen in tumor bearing mice in vivo treated with 8 kDa hPG amine-siRNA complexes. Improving the effective charge density and the multiplicity of amine functionalities by increasing the molecular weight (43 kDa) revealed comparable transfection efficacy in vitro but less toxic side effects after systemic administration in vivo compared to the respective hPG amine (8 kDa). In addition, in vivo delivery of 43 kDa hPG amine-siRNA-polyplexes in tumors resulted in a highly specific and significant knockdown effect. These findings demonstrate that hyperbranched polyglycerol amines with a balanced effective charge density, multiplicity and core size are promising gene delivery vectors for siRNA therapy which enable to address so far "undruggable" targets due to high tolerability and effective siRNA delivery.

Carbon-based cores with polyglycerol shells - the importance of core flexibility for encapsulation of hydrophobic guests.

Two core-shell nanoparticles with polyglycerol shells and sp3 carbon cores with different flexibilities (soft dendritic polyethylene and hard nanodiamond) were synthesized, their encapsulation capacities were compared, and their ability to transport into tumor cells was investigated. The nanocarrier with a soft core was superior to the hard one.

Online monitoring the isomerization of an azobenzene-based dendritic bolaamphiphile using ion mobility-mass spectrometry.

Ion mobility-mass spectrometry was used to obtain detailed information about the kinetics of the light-induced cis/trans isomerization process of a new supramolecular azobenzene-based bolaamphiphile. Further experiments revealed that the investigated light-induced structural transition dramatically influences the aggregation behaviour of the molecule.