Rapid Nanogram Scale Screening Method of Microarrays to Evaluate Drug-Polymer Blends Using High-Throughput Printing Technology.

A miniaturized, high-throughput assay was optimized to screen polymer-drug solid dispersions using a 2-D Inkjet printer. By simply printing nanoliter amounts of polymer and drug solutions onto an inert surface, drug/polymer microdots of tunable composition were produced in an easily addressable microarray format. The amount of material printed for each dried spot ranged from 25 ng to 650 ng. These arrays were used to assess the stability of drug/polymer dispersions with respect to recrystallization, using polarized light microscopy. One array with a panel of 6 drugs formulated at different ratios with a poly(vinylpyrrolidone-vinyl acetate) (PVPVA) copolymer was developed to estimate a possible bulk (gram-scale) approximation threshold from the final printed nanoamount of formulation. Another array was printed at a fixed final amount of material to establish a literature comparison of one drug formulated with different commercial polymers for validation. This new approach may offer significant efficiency in pharmaceutical formulation screening, with each experiment in the nanomicro-array format requiring from 3 up to 6 orders of magnitude lower amounts of sample than conventional screening methods.

Disruption of diphenylalanine assembly by a Boc-modified variant.

Peptide-based biomaterials are key to the future of diagnostics and therapy, promoting applications such as tissue scaffolds and drug delivery vehicles. To realise the full potential of the peptide systems, control and optimisation of material properties are essential. Here we investigated the co-assembly of the minimal amyloid motif peptide, diphenylalanine (FF), and its tert-butoxycarbonyl (Boc)-modified derivative. Using Atomic Force Microscopy, we demonstrated that the co-assembled fibers are less rigid and show a curvier morphology. We propose that the Boc-modification of FF disrupts the hydrogen bond packing of adjacent N-termini, as supported by Fourier transform infrared and fluorescence spectroscopic data. Such rationally modified co-assemblies offer chemical functionality for after-assembly modification and controllable surface properties for tissue engineering scaffolds, along with tunable morphological vs. mechanical properties.

Permeability effects on the efficiency of antioxidant nanoreactors.

Enzyme-loaded polymeric vesicles, or polymersomes, can be regarded as nanoreactors, which, for example, can be applied as artificial organelles. We implemented a naturally occurring enzymatic cascade reaction in two types of polymersomes, which are known to possess different permeability properties. The selected cascade reaction involved the antioxidant enzymes superoxide dismutase (SOD1) and catalase (CAT) for combating oxidative stress. The activities of both enzymes were investigated by spectrophotometric and electrochemical assays. Whereas the SOD1 substrate (the radical anion superoxide, O2•-) was able to penetrate both membranes equally well, the CAT substrate (H2O2) showed different rates of diffusion. When O2•- was generated inside polymersomes filled with both SOD1 and CAT, the activities of the two systems were comparable again.

Identification of Novel "Inks" for 3D Printing Using High-Throughput Screening: Bioresorbable Photocurable Polymers for Controlled Drug Delivery.

A robust methodology is presented to identify novel biomaterials suitable for three-dimensional (3D) printing. Currently, the application of additive manufacturing is limited by the availability of functional inks, especially in the area of biomaterials; this is the first time when this method is used to tackle this problem, allowing hundreds of formulations to be readily assessed. Several functional properties, including the release of an antidepressive drug (paroxetine), cytotoxicity, and printability, are screened for 253 new ink formulations in a high-throughput format as well as mechanical properties. The selected candidates with the desirable properties are successfully scaled up using 3D printing into a range of object architectures. A full drug release study and degradability and tensile modulus experiments are presented on a simple architecture to validating the suitability of this methodology to identify printable inks for 3D printing devices with bespoke properties.

Hydrazone-modulated peptides for efficient gene transfection.

Gene transfection continues to be a major challenge in chemistry, biology and materials sciences. New methodologies and recent breakthroughs have renewed the interest in the discovery and development of new tools for efficient gene transfection. Hydrazone formation between a cationic head and hydrophobic tails has emerged as one of the most promising techniques for nucleotide delivery. In this contribution, we have exploited hydrazone formation to modulate the transfection activity of a parent linear peptide in combination with a plasmid DNA cargo. This strategy allowed the straightforward preparation, under physiologically compatible conditions, of a discrete library of amphiphilic modulated penetrating peptides. Without the requirement of any isolation or purification steps, these modulated amphiphilic peptides were combined with a plasmid DNA and screened in transfection experiments of human HeLa cells. Three of these hydrazone-conjugated peptides were identified as excellent vectors for plasmid delivery with comparable, or even higher, efficiencies and lower toxicity than the commercial reagents employed in routine transfection assays.

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition.

The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.

Multipodal dynamic coordination involving cation-π interactions to control the structure of helical polymers.

A precise tuning of the four possible states of a helix (P/M helical sense and stretched/compressed helical backbone) is attained by controlling the complexation between Li+ and a poly(phenylacetylene) that bears amide, ester and phenyl ring functionalities at the pendant group. Depending on the MeOH ratio that is present as a cosolvent, different coordination sites are involved in interactions leading to complexes I-III, each one with a characteristic structure (tri-, bi-, and unipodal) and an associated helical state. This dynamic coordination allows the selective modification of the helical sense or the stretching/compression backbone of a helical polymer.

The assignment of absolute configuration of cyanohydrins by NMR.

Derivatization of aldehyde cyanohydrins as (R)- and (S)-MPA esters and comparison of the corresponding 1H- and 13C-NMR spectra allows the assignment of their absolute configuration.

Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viability.

Here, we evaluate how cationic gallic acid-triethylene glycol (GATG) dendrimers interact with bacteria and their potential to develop new antimicrobials. We demonstrate that GATG dendrimers functionalised with primary amines in their periphery can induce the formation of clusters in Vibrio harveyi, an opportunistic marine pathogen, in a generation dependent manner. Moreover, these cationic GATG dendrimers demonstrate an improved ability to induce cluster formation when compared to poly(N-[3-(dimethylamino)propyl]methacrylamide) [p(DMAPMAm)], a cationic linear polymer previously shown to cluster bacteria. Viability of the bacteria within the formed clusters and evaluation of quorum sensing controlled phenotypes (i.e. light production in V. harveyi) suggest that GATG dendrimers may be activating microbial responses by maintaining a high concentration of quorum sensing signals inside the clusters while increasing permeability of the microbial outer membranes. Thus, the reported GATG dendrimers constitute a valuable platform for the development of novel antimicrobial materials that can target microbial viability and/or virulence.

Cationic polymer mediated bacterial clustering: Cell-adhesive properties of homo- and copolymers.

New anti-infective materials are needed urgently as alternatives to conventional biocides. It has recently been established that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for anti-infective strategies as they have the potential to inhibit adhesion while at the same time modulating Quorum Sensing (QS) controlled virulence. Here we carefully evaluate the role that charge and catechol moieties in these polymers play on the binding. We investigate the ability of the cationic polymers poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co-pDMAPMAm, P2) and p(3,4-dihydroxy-l-phenylalanine methacrylamide), p(l-DMAm, P3) to cluster a range of bacteria, such as Staphylococcus aureus (Gram-positive), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa (Gram-negative) under conditions of varying pH (6, 7 and 8) and polymer concentration (0.1 and 0.5mg/mL). We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have a positive effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results highlight the subtle interplay between dynamic natural surfaces and synthetic materials, as well as the need to consider synergistic structure-property relationship when designing antimicrobial polymers.

13C NMR as a general tool for the assignment of absolute configuration.

(13)C NMR, alone or in combination with (1)H NMR, allows the assignment of the absolute configuration of chiral alcohols, amines, carboxylic acids, thiols, cyanohydrins, sec,sec-diols and sec,sec-aminoalcohols, derivatized with appropriate chiral auxiliaries. This extends the assignment possibilities of NMR to fully deuterated and to nonproton containing compounds.

Absolute configuration of ketone cyanohydrins by 1H NMR: the special case of polar substituted tertiary alcohols.

The absolute configuration of ketone cyanohydrins can be assigned from analysis of the 1H NMR spectra of the corresponding (R)- and (S)-MPA ester derivatives and use of delta delta(RS) signs. This is an application of the NMR methodology for stereochemical assignment to tertiary alcohols possessing polar groups as substituents.