Anna Barnard

Degradable Self-Assembling Dendrons for Gene Delivery – Experimental and Theoretical Insights into the Barriers to Cellular Uptake

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery.

Self-assembled multivalency: dynamic ligand arrays for high-affinity binding.

Multivalency is a powerful strategy for achieving high-affinity molecular recognition in biological systems. Recently, attention has begun to focus on using self-assembly rather than covalent scaffold synthesis to organize multiple ligands. This approach has a number of advantages, including ease of synthesis/assembly, tunability of nanostructure morphology and ligands, potential to incorporate multiple active units, and the responsive nature of self-assembly. We suggest that self-assembled multivalency is a strategy of fundamental importance in the design of synthetic nanosystems to intervene in biological pathways and has potential applications in nanomedicine.

Mallard Blue: A High-Affinity Selective Heparin Sensor That Operates in Highly Competitive Media

We report the simple synthesis and full investigation of a novel heparin binding dye, mallard blue, an arginine-functionalized thionine. This dye binds heparin in highly competitive media, including water with high levels of competitive electrolyte, buffered aqueous solution and human serum. The dye reports on heparin levels by a significant change in its UV-vis spectroscopic profile. Molecular dynamics modeling provides detailed insight into the binding mode. Heparin binding is shown to be selective over other glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate. Importantly, we demonstrate that, in the most competitive conditions, mallard blue outperforms standard dyes used for heparin sensing such as azure A.

Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions

Inhibition of protein–protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed “proteomimetics”, which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.

Controlled Release of DNA From Photoresponsive Hyperbranched Polyglycerols with Oligoamine Shells

Two photo-responsive core/shell nanoparticles based on hyperbranched polyglycerol (hPG) are synthesized for controlled release of DNA. The shell is composed either of bis-(3-aminopropyl)methylamine (AMPA) or pentaethylenehexamine (PEHA) derivatives and is attached to the hPG core with a photo-responsive o-nitrobenzyl linker. Ethidium bromide displacement assay, gel electrophoresis, DLS, and ζ-potential measurements are performed with these nanoparticles. Photo-responsive changes within the carrier scaffold are investigated by irradiating the polymer solution with 350 nm monochromatic light. Fully covered APMA-shelled carriers are found to complex the DNA at an N/P ratio of 10 with an average size ranging from 54 to 78 nm depending on the degree of functionalization of the core.

Polyglycerol-based amphiphilic dendrons as potential siRNA carriers for in vivo applications

The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA transfection technology is of particular importance for treatment of human diseases, which is yet an unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined, low-molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were introduced in the structural motif relate to dendron generation and the type of linker between the hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their aggregation behaviour and further evaluated with respect to their siRNA transfection potential by comparing their physico-chemical and biological features. Our findings demonstrated that all four synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds (G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA induced inflammatory side effects after systemic administration in vivo, which is noteworthy because such highly positively charged compounds are typically associated with toxicity concerns which in turn supports their prospective application for in vivo purposes. Therefore, we believe that these structures may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for further synthetic modifications.

Enantioselective lactate binding by chiral tripodal anion hosts derived from amino acids

Chiral, tripodal anion hosts derived from either S-phenylalanine or S-leucine bind D-lactate enantioselectively. The nature of the host-guest interaction has been probed by solution NMR methods and by DFT calculations. The calculations suggest that the D-lactate may form an additional hydrogen bond in the host-guest complex while the L-lactate complex contains an intramolecular hydrogen bond. Anion binding is in competition with host dimerisation, as demonstrated by DOSY and (1)H NMR spectroscopy, and DFT calculations.

Probing dendron structure and nanoscale self-assembly using computer-aided analysis of EPR spectra

The self-assembly of dendritic molecules is an effective way of generating functionalised nanoscale architectures. This study reports a computer-aided analysis of the EPR spectra of 5 doxyl-stearic acid (5DSA) and Cu(II) probes interacting with self-assembling dendrons in water. The dendrons investigated have previously been reported as potential gene delivery vehicles, and possess amine surface groups, different dendritic architectures based on ether-amide or ester branching, and hydrophobic groups at the focal point which can encourage self-assembly in aqueous solution. The parameters extracted from computation provide information about both the structure and dynamics in solution and the interacting ability of the dendrons to be used in gene therapy. The hydrophobic 5DSA probe is able to effectively probe the hydrophobic core of self-assembled dendron nanostructures. It reports on the polarity of its local environment and is most affected by dendrons with two cholesterol units at the focal point, partly affected by dendrons with cholesterol groups at their focal point, but is unaffected by dendrons with a simple phenyl group at the focal point. This reflects the different modes of self-assembly observed for these dendrons. When Cu(II) is used as an EPR probe of the branched environment, it was found that at pH7, much of the Cu(II) was ‘external’ to the dendritic structure, presumably due to protonation of the peripheral amine groups. On gradually increasing the Cu(II) loading, and using computer-aided analysis, it was possible to quantify the levels of ‘internal’ (dendron-bound) and ‘external’ Cu(II) and it was found that this was strongly dependent on the structure of the dendritic branching and the ability of the dendron to self-assemble, with self-assembling ester dendrons being best able to bind the Cu(II). It was also possible to propose the nature of the copper binding sites associated with the ‘internal’ signal as either Cu–NO3 and Cu–N2O2 distorted square-planar coordination sites. The self-assembling ester based dendrons which also contain 1,2,3-triazole units, had higher levels of ‘internal’ Cu(II) and showed the latter form of coordination, while the other dendrons, with lower levels of Cu(II) uptake showed the former. In summary, this paper demonstrates that two complementary EPR probes can be used to provide information about different regions of a self-assembled dendritic architecture.

A high throughput screen for next-generation leads targeting malaria parasite transmission

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2–1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male–female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.Sexual forms of malaria parasites are responsible for transmission to the mosquito. Anti-malarial drug resistance remains a serious problem and requires advent of new drug therapies. Here, the authors present a high-throughput screen of potential antimalarial compounds, identifying seventeen drug-like molecules specifically targeting transmission.

Probing Protein Surfaces: QSAR Analysis with Helix Mimetics.

α‐Helix‐mediated protein–protein interactions (PPIs) are important targets for small‐molecule inhibition; however, generic approaches to inhibitor design are in their infancy and would benefit from QSAR analyses to rationalise the noncovalent basis of molecular recognition by designed ligands. Using a helix mimetic based on an oligoamide scaffold, we have exploited the power of a modular synthesis to access compounds that can readily be used to understand the noncovalent determinants of hDM2 recognition by this series of cell‐active p53/hDM2 inhibitors.