A library of FRET-based peptides were prepared and studied as Thrombin substrates. This identified probes that showed selective activation by Thrombin, low fluorescent background signals, stability to Factor Xa, matrix metalloproteases, and primary human inflammatory cell lysates and supernatant. These were selected for further optimization, creating a second generation of fluorogenic probes with improved solubility and Plasmin resistance. The optimised probe allowed the detection of Thrombin activity in ex vivo fibrotic human tissue.

A homogeneous carbene-based palladium catalyst was conjugated to a cell-penetrating peptide, allowing intracellular delivery of catalytically active Pd complexes that demonstrated bioorthogonal activation of a profluorophore within prostate cancer cells.

The fabrication of fluorescence-based pH sensors, embedded into etched pits of an optical fibre via highly controllable and spatially selective photo-polymerisation is described and the sensors validated.

Reactive oxygen species play numerous roles in a number of pathological processes. Monitoring H2O2 is a powerful tool for imaging and therapy of diseases wherein oxidative stress is involved. In particular, we report a specific application of functional microspheres as sensors of H2O2. Reactive oxygen species responsive delivery systems were developed to detect in vitro peroxides thanks to the presence of a boronic ester which is readily cleaved with H2O2. This ROS-sensitive cleavable linker underwent a 1,6-elimination to disrupt fluorescence resonance energy transfer by coupled near-infrared fluorophores such as Cy5.5/Cy7. This technology would allow real-time monitoring of therapeutic regimes (and their success), as well as optical detection of inflammation.

AbstractTransition metals have been successfully applied to catalyze non-natural chemical transformations within living cells, with the highly efficient labeling of subcellular components and the activation of prodrugs. In vivo applications, however, have been scarce, with a need for the specific cellular targeting of the active transition metals. Here, we show the design and application of cancer-targeting palladium catalysts, with their specific uptake in brain cancer (glioblastoma) cells, while maintaining their catalytic activity. In these cells, for the first time, two different anticancer agents were synthesized simultaneously intracellularly, by two totally different mechanisms (in situ synthesis and decaging), enhancing the therapeutic effect of the drugs. Tumor specificity of the catalysts together with their ability to perform simultaneous multiple bioorthogonal transformations will empower the application of in vivo transition metals for drug activation strategies.

AbstractTransition metals have been successfully applied to catalyze non-natural chemical transformations within living cells, with the highly efficient labeling of subcellular components and the activation of prodrugs. In vivo applications, however, have been scarce, with a need for the specific cellular targeting of the active transition metals. Here, we show the design and application of cancer-targeting palladium catalysts, with their specific uptake in brain cancer (glioblastoma) cells, while maintaining their catalytic activity. In these cells, for the first time, two different anticancer agents were synthesized simultaneously intracellularly, by two totally different mechanisms (in situ synthesis and decaging), enhancing the therapeutic effect of the drugs. Tumor specificity of the catalysts together with their ability to perform simultaneous multiple bioorthogonal transformations will empower the application of in vivo transition metals for drug activation strategies.

Infections arising from contaminated medical devices are a serious global issue, contributing to antibiotic resistance and imposing significant strain on healthcare systems. Since the majority of medical device-associated infections are biofilm related, efforts are being made to generate either bacteria-repellent or antibacterial coatings aimed at preventing bacterial colonisation. Here, we utilise a nanocapsule mediated slow release of a natural antimicrobial to improve the performance of a bacteria repellent polymer coating. Poly(lauryl acrylate) nanocapsules containing eugenol (4-allyl-2-methoxyphenol) were prepared and entrapped within a interpenetrating network designed to repel bacteria. When coated on a catheter and an endotracheal tube, this hemocompatible system allowed slow-release of eugenol, resulting in notable reduction in surface-bound Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus.

11 FRET-based fluorogenic substrates were constructed using the pentapeptide template Asp-Glu-X2-Asp-X1′, and evaluated with caspase-3, caspase-7 and cathepsin B. The sequence Asp-Glu-Pro-Asp-Ser was able to selectively quantify caspase-3 activity in vitro without notable caspase-7 and cathepsin B cross-reactivity, while exhibiting low μM KM values and good catalytic efficiencies (7.0–16.9 μM−1 min−1).

A new and highly efficient polymer, postpolymerization, modification platform based on an inverse electron demand Diels–Alder reaction is reported. Well-defined polymers were synthesized from allyl glycidyl ether and glycidol by anionic ring-opening polymerization with postpolymerization modifications conducted with a number of tetrazine derivatives that carried functional groups spanning from carboxylates and esters to primary amines. Analysis of polymerization kinetics by real-time 1H NMR, and GPC revealed a rapid and high degree of side-chain conversion (>99%), with the generation of well-defined functional polymers, in both organic and aqueous solvents, without the need for additives or catalysts.

•We report the optimisation of an electrochemical biosensor for protease activity.•A comparative study of the applicability of Fc and MB as redox reporters is presented.•MB-tagged peptides support enhanced electrochemical performance.•A T-SAM configuration using a PEG-based dithiol improved the analytical performance.Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250 pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis–Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin cleavage. This indicates that a PEG-based peptide, employing methylene blue as redox reporter, and deposited on an electrode as a ternary SAM configuration, is a suitable platform to develop clinically-relevant and quantitative electrochemical peptide-based protease biosensing.

Aberrant fibrogenesis is a feature of many diseases in multiple organ systems. The lysyl oxidase family of enzymes are central to tissue homeostasis and elevated lysyl oxidase activity is implicated in fibroproliferation as well as in cancer stroma. We have synthesised a novel fluorogenic reporter for monitoring lysyl oxidase activity that generates a 3–5 fold increase in fluorescence following probe activation in ventilating fibrotic ex vivo asinine lung and ex vivo human lung tissue. The probe termed “oLOX” can provide real-time measurement of lysyl oxidase activity in a number of biological settings and is tractable from an in vitro setting to man.

The in situ immediate detection of the presence of bacteria in the distal human lung is of significant clinical utility. Herein we describe the development and optimization of a bacterial binding fragment (UBI29–41) of the antimicrobial peptide, ubiquicidin (UBI), conjugated to an environmentally sensitive fluorophore to enable rapid live bacterial imaging within human lung tissue. UBI29–41 was modified for stability in the presence of human lung bronchoalveolar lavage fluid, for affinity to bacterial membranes and functionality in human lung tissue. The optimized cyclic structure yields an optical molecular Smartprobe for bacterial detection in human lung tissue.

A series of monoprotected aliphatic diamines (21 examples) were synthesized via continuous flow methods. The carbamates and enamines were obtained in 45–91% yields using a 0.5 mm diameter PTFE tubular flow reactor. Using readily accessible protecting group precursors, the procedure serves as an attractive alternative to existing batch-mode synthetic routes by providing direct, multigram access to N-Boc-, N-Fmoc-, and N-Ddiv-protected compounds with productivity indexes of 1.2–3.6 g/h.

Peptidomimetics, such as oligo-N-alkylglycines (peptoids), are attractive alternatives to traditional cationic cell-penetrating peptides (such as R9) due to their robust proteolytic stability and reduced cellular toxicity. Here, monomeric N-alkylglycines, incorporating amino-functionalized hexyl or triethylene glycol (TEG) side chains, were synthesized via a three-step continuous-flow reaction sequence, giving the monomers N-Fmoc-(6-Boc-aminohexyl)glycine and N-Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)ethyl)glycine in 49% and 41% overall yields, respectively. These were converted into oligomers (5, 7, and 9-mers) using an Fmoc-based solid-phase protocol and evaluated as cellular transporters. Hybrid oligomers, constructed of alternating units of the aminohexyl and amino-TEG monomers, were non-cytotoxic and exhibited remarkable cellular uptake activity compared to the analogous fully TEG or lysine-like compounds.

A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N′-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a “naïve” state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture.

Nosocomial infections due to bacteria have serious implications on the health and recovery of patients in a variety of medical scenarios. Since bacterial contamination on medical devices contributes to the majority of nosocomical infections, there is a need for redesigning the surfaces of medical devices, such as catheters and tracheal tubes, to resist the binding of bacteria. In this work, polyurethanes and polyacrylates/acrylamides, which resist binding by the major bacterial pathogens underpinning implant-associated infections, were identified using high-throughput polymer microarrays. Subsequently, two ‘hit’ polymers, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)) and PA515 (poly(methoxyethylmethacrylate-co-diethylaminoethylacrylate-co-methylmethacrylate)), were used to coat catheters and substantially shown to decrease binding of a variety of bacteria (including isolates from infected endotracheal tubes and heart valves from intensive care unit patients). Catheters coated with polymer PA13 showed up to 96% reduction in bacteria binding in comparison to uncoated catheters.

Mesenchymal stem cells (MSCs) hold great promise in regenerative medicine due to their wide multilineage potential as well as their ability to suppress/modulate the immune response. Maintaining these cells in vitro and expanding them on a clinically relevant scale remains a challenge that needs to be addressed to realise their full potential. Current culture methods for MSCs typically rely on animal sourced substrates and often result in a heterogeneous population of cells with varying degrees of differentiation capacity. Here, a high-throughput platform was used to identify synthetic substrates for MSC culture that not only facilitated growth but also maintained the MSC phenotype. Two polymers, PU157 (synthesised from poly(butyleneglycol) and 4,4′-methylenediphenyldiisocyanate with 3-(dimethylamino)-1,2-propanediol as a chain extender) and PA338 (N-methylaniline modified poly(methylmethacrylate-co-glycidylmethacrylate)) were able to maintain the growth and phenotype of human embryonic derived mesenchymal progenitors (hES-MPs) and adipose derived MSCs (ADMSCs) for five and ten passages, respectively. Cell phenotype and multipotency were confirmed by flow cytometry analysis of ten MSC markers and differentiation analysis. These new polymer substrates provide a chemically defined synthetic surface for efficient, long-term MSC culture.

Nanoparticles have gained considerable significance in the life sciences due to their ability to be internalised by living cells and the relative ease with which they can be functionalised with cargos ranging from molecular sensors to biomacromolecules. However, the scope of available bioconjugation methods is limited and new bioorthogonal methods are much sought after. Herein, we present dual functionalised (HO)2B/H2N-polymeric nanoparticles which can be conjugated via amide bond formation and/or Pd-mediated Suzuki–Miyaura cross-coupling in a chemoselective and bioorthogonal manner. These dual-functionalised particles were found to be efficiently taken up by mammalian cells without toxicity and were successfully employed in the cellular delivery and intracellular release of a “turn-on” molecular probe thereby demonstrating the potential use of the new particles for chemical biology applications.

Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent reporters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over closely related proteases.

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years.A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

The interaction of the waterborne protozoan parasite, Giardia lamblia, with polymeric materials was investigated by microarray screening of 652 polymers. Polymers were identified which either bound G. lamblia cysts or prevented their binding. Correlation of material properties such as wettability and surface roughness with cyst attachment revealed no influence of these factors upon Giardia adhesion. However, the study of polymer composition allowed the correlation of binding and generation of polymer structure function relationships; glycol and aromatic functionalities appeared to prevent adhesion, whereas secondary amine groups promoted adhesion, in agreement with previous literature. A significant reduction in attachment was observed following both cyst treatments with proteinase K and performing experiments at extremes of pH (2 and 12). It is suggested that proteinase K removes the proteins needed for specific surface interactions, whereas extremes of pH influence either protonation of the polymer or the surface charge of the cysts. The mechanism by which the protozoa attach to polymeric surfaces is proposed to be through ion–pair interactions. Improved understanding of G. lamblia surface interactions could assist in predicting transport and fate behavior in the environment and contribute to better design of water treatment processes, while the polymers identified in this work could find use in sensor applications and membrane filtration.

Polymer microarrays provide an innovative approach to identify materials with novel bacterial binding or repellent properties which could subsequently be used in a variety of practical applications. Here, we report a polymer microarray screen of hundreds of synthetic polymers to identify those which either selectively capture the major food-borne pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium), or prevent its binding. A parallel study with a lab strain of Escherichia coli (E. coli) is also reported; revealing polymers which either display a common binding activity or which exhibit species discrimination. Moreover, substrates were also uncovered which showed no binding of either organism, even when cultured at high density. The correlation between polymer structure and microbial-modulating behaviour was analysed further, while SEM analysis allowed visualization of the detailed interactions between surface and bacteria. Such polymers offer many new opportunities for bacterial enrichment or surface repulsion, in cleaning materials, as surface coatings for use in the food production industry or as a “bacterial scavenger” resin.

The therapeutic use of nucleic acids has long been heralded as a panacea of medicinal opportunity, a vision enhanced by the introduction of RNA interference technology. The Achilles heel of such an approach is the in vivo delivery of the desired nucleic acid into cells, a practice that lacks selectivity, safety and/or efficiency. Herein we report the safe and efficacious in vitro and in vivo delivery of nucleic acids using tripodal biodegradable cationic lipids. Toxicity reduction and transfection potency of these novel amphiphiles were addressed by designing the compounds to undergo complete intracellular degradation thereby enhancing cargo release while minimising toxicity and potential tissue accumulation. Compounds demonstrated high-efficiency in transfecting DNA into cells both in vitro and in vivo with no signs of toxicity, thus potentially offering a safer alternative to viral transfection for gene therapy application.

Cells over-expressing integrins or CCR6 were incubated on a DNA microarray, pre-hybridized with a 10000 member PNA-encoded peptide library allowing novel cell specific ligands for integrins and CCR6 to be identified.

Herein we report a highly-efficient solid-phase strategy for the modular synthesis of 63 double-tailed lipid-peptide conjugates and their application in DNA delivery.

The ability to screen and identify new ligands for cell surface receptors has been a long-standing goal as it might allow targeting of pharmaceutically relevant receptors, such as integrins or G protein coupled receptors. Here, we present a method to amplify hits from a library of PNA-tagged peptides. To this end, human cells, overexpressing either integrins or the CCR6 receptor, were treated with a 10,000 member PNA-encoded peptide library. Extraction of the PNA tags from the surface of the cells was followed by a PNA-tag to DNA translation and amplification enabling decoding of the tags via microarray hybridization. This approach to ligand discovery facilitates screening for differences in surface-receptor ligands and/or receptor expression between different cell types, and opens up a practical approach to PNA-tag amplification.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (439 K)Download as PowerPoint slideHighlights► Screening of a 10,000 member PNA-encoded peptide library with live cells ► Decoding and amplification the PNA-tags via PCR ► Discovery of new peptide ligands for αvβ5 and αvβ3 integrins and CCR6 ► The screening approach is applicable in vivo as well as in vitro

Nano and microparticles are widely used across the life science interface, with applications ranging from chemical probes of biological function to fluorescent particles for flow cytometry and cellular tracking. Increasingly, particles are modified with a variety of chemistries to boost their functionality and broaden their biological applicability. However, although particle modification has become standard laboratory practice, the ability to determine the extent and efficiency of chemical modification is often very limited and empirical in nature. Herein, we report the use of zeta potential analysis as a simple and rapid “direct-on-particle” approach allowing levels of bead modification and derivatization to be evaluated. As a proof-of-concept, aminomethyl-functionalized nano and microparticles were derivatized to display a variety of surface functionalities and their zeta potentials measured, allowing verification of the applicability of the approach for particle analysis. We demonstrate that zeta potential measurement is a convenient approach which allows multistep reaction sequences to be followed, and show that this method can be used to verify and validate successful particle modification.

Small-molecule microarrays are often limited by the requirement for each compound undergoing immobilization to contain a common functional group or by the need to prepare glass slides containing photo-reactive groups. Herein, we present a generic strategy that allows any compound library to be immobilized. This was achieved by printing a fluorous-tagged photo-reactive 3-aryl-3-trifluoromethyldiazirine, which undergoes non-selective insertion into compounds following UV-activation, onto fluorous-functionalized glass slides. The arrays could be reused following aqueous stripping and re-assessment of the compounds with the same protein or another target of interest.The 3-aryl-3-trifluoromethyldiazirine photoreactive group conjugated to a fluoroalkyl chain allows the non-selective immobilization of compounds onto fluorous-functionalized glass slides and offers an approach for the generic fabrication of small-molecule microarrays.

Innate immune cell ingress into the site of inflammation is central for the efficient clearance of pathogens. However, in some circumstances the inflammatory response may become pathogenic to the host. Understanding the temporal ingress of innate immune cells is thus essential to predict both the defensive and adverse effects mediated by these cells in an infectious process and for developing novel therapeutic strategies. In this context, optical imaging has emerged as a powerful technique for the visualization of specific cellular events in preclinical models. Herein we describe a non-covalent tagging strategy to stably label innate immune cells using in vivo-traceable cell penetrating peptoids and their application in real-time imaging of cell migration in murine models, thereby providing a sensitive and cost effective way to visualize cellular recruitment in preclinical models of inflammation.

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease origins and differences in drug responses. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or alternatively by the hybridization of DNA probes. MALDI-TOF mass spectrometry (MS) has been widely used as a read-out tool. We developed a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping. In a blind trial, peptide nucleic acid (PNA) probes and aldehyde-modified nucleobases were employed to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. MALDI-TOF MS reported the resulting allele-specific products. Enzyme-free dynamic chemistry permitted the genotyping of twelve individuals for the ΔF508 (indel) and G551D (SNP) mutations. No false positives or negatives were observed, and analysis could be performed in both singleplex and duplex formats. Dynamic chemistry provides a distinct method of allele discrimination with certain advantages over those reported previously. Although PCR amplification is required, the analysis stage is performed without enzymes, and there is no need for the stringent optimization and control of conditions associated with hybridization-based methods of allele discrimination.

Fluorescent particles are used for a diverse number of biochemical assays including intracellular imaging, cellular tracking, as well as detection of a variety of biomolecules. They are typically prepared by postpolymerization conjugations of dyes onto preformed particles. Herein we report the synthesis of aminomethyl-functionalized fluorescent particles via the synthesis and application of polymerizable fluorescein monomers. These monomers allowed high and controllable fluorophore loading into the particles, resulting in enhanced fluorescence properties in comparison with more commonly used carboxyfluorescein conjugated particles. Furthermore, the particles were rapidly taken up by cells with enhanced fluorescence. The herein presented results demonstrate the advantages of dye polymerization in contrast with more conventional conjugation strategies for fluorescent particle generation with applications in the life sciences.

Amino functionalised cross-linked polystyrene microspheres of well defined sizes (0.2–2 μm) have been prepared and shown to be efficient and controllable delivery devices, capable of transporting anything from small dye molecules to bulky proteins into cells. However, the specific mechanism of cellular entry is largely unknown and widely variant from study to study. As such, chemical, biological and microscopic methods are used to elucidate the mechanism of cellular uptake for polystyrene microspheres of 0.2, 0.5 and 2 μm in mouse melanoma cells. Uptake is found to be wholly unreliant upon energetic processes, while lysosomal and endosomal tracking agents failed to show co-localisation with lysosomes/endosomes, suggesting a non-endocytic uptake pathway. To further explore the consequences of microsphere uptake, gene expression profiling is used to determine if there is a transcriptional response to “beadfection” in both murine and human cells. None of the common transcriptional responses to enhanced endocytosis are observed in beadfected cells, further supporting a non-endocytic uptake mechanism. Furthermore, the microspheres are noted to have a limited interaction with cells at a transcriptional level, supporting them as a non-toxic delivery vehicle.

Amide bond formation is a fundamentally important reaction in organic synthesis, and is typically mediated by one of a myriad of so-called coupling reagents. This critical review is focussed on the most recently developed coupling reagents with particular attention paid to the pros and cons of the plethora of “acronym” based reagents. It aims to demystify the process allowing the chemist to make a sensible and educated choice when carrying out an amide coupling reaction (179 references).

An approach for complex cell patterning, using laser printing, is described allowing essentially any cellular image or pattern to be rapidly fabricated.

Polymer microarrays, consisting of either discrete features or a matrix of inter-crossed lines were directly fabricated in situ by inkjet printing individual monomers and initiator solutions in organic solvents through a film of oil, thereby allowing the rapid generation of a broad range of co-polymers, while solving the problem of selective monomer evaporation.

In this study, polymer microarrays were used for the rapid identification of polymer substrates upon which a suspension cell line would both adhere and proliferate giving a detailed and rapid understanding of cell-biomaterial interactions. Analysis demonstrated that suspension K562 human erythroleukemic cells, which normally grow in suspension, adhered and proliferated on several different polymers. Phenotypic and transcriptomic analysis techniques allowed examination of the interaction between cells and polymers permitting the elucidation of putative links between phenotypic responses to cell-biomaterial interactions and global gene expression.

200 and 500 nm polymeric microspheres have been conjugated to siRNA targeted against EGFP expressed in human cervical cancer (HeLa) cells and shown to efficiently silence protein expression over 72 h, without detrimental cytotoxicity. Furthermore, with use of an independent Cy5 tracking label on the siRNA-laden microsphere, silencing of EGFP could be assessed by selecting only those cells that contained the delivery vehicle (and thus the siRNA) generating a more accurate picture of microsphere-induced gene silencing.

The long chain saturated fatty acids, arachidic (C20) and lignoceric (C24), are found as components of phospholipids within mammalian cellular membranes. Although these lipids have rarely been used as components of transfection reagents, we recently demonstrated that elongation of the fatty tail beyond C18 provide a means of increasing the transfection efficiency of cationic lipids. To investigate this effect further, a new library of single-chained cationic lipids consisting of mono-, di- or tri-arginine residues, a range of amino acid spacers and these long-chain saturated fatty tails were synthesised using an Fmoc solid-phase strategy, which allowed the preparation of 18 compounds, some with remarkable transfection abilities.

Unsymmetrical functionalised cyanine dyes, covering the whole colour range, were readily synthesised (in 100 mg amounts) by a combination of microwave and solid-phase methodologies.

The search for novel, generally applicable and highly efficient delivery tools is a major activity in the biotechnology arena. Using highly optimized microwave based solid-phase chemistry a series of fluorescein-labelled cationic peptoid conjugates were synthesized within 24 h and cellular uptake into HeLa, L929 and K562 cells examined via flow cytometry. As expected, analysis revealed that longer oligomers achieved greater cellular penetration (7e (9 mer) > 7d (7 mer) > 7c (5 mer) > 7b (3 mer) > 7a (1 mer)) with the nonamer 7e proving to be a remarkable vehicle for all the cell lines, showing excellent penetrability into K562 and L929 cells and extraordinary cell delivery into HeLa cells. Confocal microscopy showed that the hybrid peptoid-nuclear localizing sequence (PKKKRKV from the simian virus 40 large T antigen) resulted in very high levels of nuclei delivery after 3 h, opening up a range of applications such as nuclei staining of living cells with non-DNA-intercalating fluorescent probes.A cell-penetrating peptoid nonamer conjugated to the SV40 nuclear localization signal (NLS) permitted rapid and specific intracellular trafficking and targeting to the cell nucleus, facilitating nuclei staining of live cells with a non-DNA-intercalating fluorescent probe.

The synthesis and detailed enzymatic analysis of fluorescence resonance energy transfer (FRET)-based peptides as substrates for chymopapain are reported. The design of these substrates arose from a massively parallel high-throughput microarray screening process using peptide nucleic acid (PNA) encoding technology, allowing the identification of detailed substrate specificities of any protease. Two peptides so identified with chymopapain were observed to be excellent substrates with low micromolar Km values and turnover numbers on the order of hundreds per second. Mass spectroscopy studies showed unequivocally the specificity of chymopapain toward Ala, Pro, Val, and Lys for positions P4 to P1 while not presenting high specificity for residues in position P1′.

Polymer hydrogel microarrays were fabricated by inkjet printing of monomers and initiator, allowing up to 1800 individual polymer features to be printed on a single glass slide.

Micron-sized polymeric “doughnuts” prepared viadispersion polymerization were found to be highly selective in their cellular translocation abilities.

An innovative family of tridentate−cationic “single-chained lipids” designed to enhance DNA compaction and to promote endosomal escape was synthesized by coupling various lipids to a multibranched scaffold. DNA retardation assays confirmed the ability of the most members of the library to complex DNA. Classical molecular dynamics simulations performed on the lauryl derivative, bound to a short strand of DNA in aqueous solution supported these observations. These showed that two “arms” of the tripodal molecule are ideally suited to forming strong Coulombic interactions with two contiguous phosphate groups from the DNA backbone while the lipophilic tail stays perpendicular to the DNA helix. Gene transfer abilities of the library were assessed in multiple cell lines (CHO, Cos7, and 16HBE14o-) with some library members giving excellent transfection abilities and low cytotoxicity, supporting the use of this tripodal approach for the development of efficient gene delivery agents.

Solid-phase synthesis of a generation 3.0 polyamidourea 1→3 C-branched bis-dendron followed by capping of the peripheral amino groups with L-lysine gave an efficient transfection reagent.

In this manuscript, we report how transfection efficiencies vary as a function of the substrate upon which cells adhere using a polymer microarray platform to allow rapid analysis of a large number of substrates. During these studies, traditional transfection protocols were nonsatisfactory, and thus we developed an approach in which an ultrasonic nebulizer was used to dispense lipoplexes onto cell-based microarrays in the absence of liquid. Under these conditions, droplets were directly deposited onto the cells thereby enhancing transfection. This approach was successfully applied to the transfection of various cell lines immobilized on a library of polyacrylates and permitted the identification of highly efficient transfection/polymer combinations, while showing that specific polymer–cell interactions may promote the efficacy of chemical transfection.

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.

Palladium nanoparticles were entrapped within resin plugs and used in a range of ligand-free cross-coupling reactions; the convenient modular format of the resin plug enhanced resin handling and allowed the catalysts to be easily recovered and multiply reused.

A 10,000 member peptide nucleic acid (PNA) encoded peptide library was prepared, treated with the Abelson tyrosine kinase (Abl), and decoded using a DNA microarray and a fluorescently labeled secondary antiphosphotyrosine antibody. A dual-color approach ensured internal referencing for each and every member of the library and the generation of robust data sets. Analysis identified 155 peptides (out of 10,000) that were strongly phosphorylated by Abl in full agreement with known Abl specificities. BLAST analysis identified known cellular Abl substrates such as c-Jun amino-terminal kinase as well as new potential target proteins such as the G-protein coupled receptor kinase 6 and diacylglycerol kinase gamma. To illustrate the generalization of this approach, two other tyrosine kinases, human epidermal growth factor 2 (Her2) and vascular endothelial growth factor receptor 2/kinase insert domain protein receptor (VEGFR2/KDR), were profiled allowing characterization of specific peptide sequences known to interact with these kinases; under these conditions Her2 was demonstrated to have a marked preference for d-proline perhaps offering a unique means of targeting and inhibiting this kinase.

High throughput (HT) techniques are now extensively used for the synthesis of libraries of several thousands of compounds. More recently, HT methods began to be applied to other areas, such as physical organic chemistry. This has allowed for instance the development of tools for HT reaction assessment, HT kinetic and thermodynamic measurements, and physicochemical property profiling, using a broad set of analytical tools, ranging from mass spectrometry to image analysis based techniques. This article provides an overview of recent HT physical organic chemistry techniques. Special attention is given to the application of quantitative analytical constructs for HT monomer reactivity profiling and HT evaluation of Hammett parameters.

A 10000 member PNA-encoded library of FRET based peptides was synthesised for global analysis of protease cleavage specificity; analysis was achieved using a DNA microarray and consumed minimal quantities of enzyme (60 pmole) and library (3.5 nmole).

Microarray screening of polymer libraries for cellular adhesion was developed utilising a thin film of agarose to allow unsurpassed localisation of cell binding onto the array substrate and the discovery of cell specific polymers.

A polymer microarray of 120 polyurethanes was used to identify polymers that promoted the adhesion of bone marrow dendritic cells (BMDC). Identified polymers were coated onto glass cover slips and shown to be efficient substrates for the immobilisation of these primary cells, which underwent efficient phagocytosis while still presumably maintaining their immature state.

The synthesis and cellular uptake of fluorescently labelled PNA–peptide conjugates is described; Dde/Mmt protected PNA monomers, fully orthogonal to Fmoc chemistry, were used to develop a flexible strategy to give Peptide Nucleic Acids conjugated to tri- and hepta-arginine and the short basic Tat48–57 peptide as examples of cellular penetrating peptides, thereby allowing efficient cellular delivery of PNA into cells.

Here, we detail the major developments in methods and techniques that are applicable to high-throughput synthesis that have evolved over the past five years, with an emphasis on the combination of microwave-based synthesis with techniques such as polymer-assisted purification and immobilized reagents and catalysts. Other aspects, such as automation, miniaturization and flow-based synthesis, are also presented as approaches that can be used for the rapid discovery and optimization of small organic molecules.

Constant advancements in printing technology, informatics, surface modification strategies and peptide chemistries mean that peptide arrays have, like DNA arrays, become even more miniaturised and complex in terms of not only the numbers of peptides immobilised but also their lengths. As a result peptide-based arrays have become a powerful tool in the interrogation, examination and perturbation of a host of biological systems.

The ability to target specific cell types to achieve optimal distribution of therapeutic entities into diseased tissues, while limiting possible adverse off-target effects, has long been a goal of many research groups and pharmaceutical organizations. This review focuses on peptidic tissue-specific biomarkers that allow peptides to act as homing devices for specific tissue types or organs, with a focus on homing peptides (HPs) and cell-penetrating homing peptides (CPHPs). These HPs, in addition to promoting cellular uptake, can deliver a variety of cargos (drugs, oligonucleotides and nanoparticles) into cells. Two such peptides that have entered clinical trials are the tumor-homing peptide asparagine-glycine-arginine (NGR) (fused to human tumor necrosis factor), which selectively binds CD13, an aminopeptidase that is overexpressed on tumor blood vessels, and cyclo[Arg-Gly-Asp-D-Phe-(NMeVal)] (cRGD, cilengitide), an anti-angiogenic agent that targets the αvβ3 and αvβ5 integrins.

A library of FRET-based peptides were prepared and studied as Thrombin substrates. This identified probes that showed selective activation by Thrombin, low fluorescent background signals, stability to Factor Xa, matrix metalloproteases, and primary human inflammatory cell lysates and supernatant. These were selected for further optimization, creating a second generation of fluorogenic probes with improved solubility and Plasmin resistance. The optimised probe allowed the detection of Thrombin activity in ex vivo fibrotic human tissue.

As a novel prodrug activation strategy Pd(0) nanoparticles, entrapped within a modular polymeric support, were used in cell culture, to synthesise the anticancer agent PP-121 from two non-toxic precursors, thereby inducing cell death in the first example of in situ mediated drug synthesis.

The long chain saturated fatty acids, arachidic (C20) and lignoceric (C24), are found as components of phospholipids within mammalian cellular membranes. Although these lipids have rarely been used as components of transfection reagents, we recently demonstrated that elongation of the fatty tail beyond C18 provide a means of increasing the transfection efficiency of cationic lipids. To investigate this effect further, a new library of single-chained cationic lipids consisting of mono-, di- or tri-arginine residues, a range of amino acid spacers and these long-chain saturated fatty tails were synthesised using an Fmoc solid-phase strategy, which allowed the preparation of 18 compounds, some with remarkable transfection abilities.

The in situ immediate detection of the presence of bacteria in the distal human lung is of significant clinical utility. Herein we describe the development and optimization of a bacterial binding fragment (UBI29–41) of the antimicrobial peptide, ubiquicidin (UBI), conjugated to an environmentally sensitive fluorophore to enable rapid live bacterial imaging within human lung tissue. UBI29–41 was modified for stability in the presence of human lung bronchoalveolar lavage fluid, for affinity to bacterial membranes and functionality in human lung tissue. The optimized cyclic structure yields an optical molecular Smartprobe for bacterial detection in human lung tissue.

Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent reporters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over closely related proteases.

Solid-phase synthesis of a generation 3.0 polyamidourea 1→3 C-branched bis-dendron followed by capping of the peripheral amino groups with L-lysine gave an efficient transfection reagent.

High throughput (HT) techniques are now extensively used for the synthesis of libraries of several thousands of compounds. More recently, HT methods began to be applied to other areas, such as physical organic chemistry. This has allowed for instance the development of tools for HT reaction assessment, HT kinetic and thermodynamic measurements, and physicochemical property profiling, using a broad set of analytical tools, ranging from mass spectrometry to image analysis based techniques. This article provides an overview of recent HT physical organic chemistry techniques. Special attention is given to the application of quantitative analytical constructs for HT monomer reactivity profiling and HT evaluation of Hammett parameters.

Unsymmetrical functionalised cyanine dyes, covering the whole colour range, were readily synthesised (in 100 mg amounts) by a combination of microwave and solid-phase methodologies.

Aberrant fibrogenesis is a feature of many diseases in multiple organ systems. The lysyl oxidase family of enzymes are central to tissue homeostasis and elevated lysyl oxidase activity is implicated in fibroproliferation as well as in cancer stroma. We have synthesised a novel fluorogenic reporter for monitoring lysyl oxidase activity that generates a 3–5 fold increase in fluorescence following probe activation in ventilating fibrotic ex vivo asinine lung and ex vivo human lung tissue. The probe termed “oLOX” can provide real-time measurement of lysyl oxidase activity in a number of biological settings and is tractable from an in vitro setting to man.

A homogeneous carbene-based palladium catalyst was conjugated to a cell-penetrating peptide, allowing intracellular delivery of catalytically active Pd complexes that demonstrated bioorthogonal activation of a profluorophore within prostate cancer cells.

Palladium nanoparticles were entrapped within resin plugs and used in a range of ligand-free cross-coupling reactions; the convenient modular format of the resin plug enhanced resin handling and allowed the catalysts to be easily recovered and multiply reused.

Polymer microarrays, consisting of either discrete features or a matrix of inter-crossed lines were directly fabricated in situ by inkjet printing individual monomers and initiator solutions in organic solvents through a film of oil, thereby allowing the rapid generation of a broad range of co-polymers, while solving the problem of selective monomer evaporation.

An approach for complex cell patterning, using laser printing, is described allowing essentially any cellular image or pattern to be rapidly fabricated.

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.

Amide bond formation is a fundamentally important reaction in organic synthesis, and is typically mediated by one of a myriad of so-called coupling reagents. This critical review is focussed on the most recently developed coupling reagents with particular attention paid to the pros and cons of the plethora of “acronym” based reagents. It aims to demystify the process allowing the chemist to make a sensible and educated choice when carrying out an amide coupling reaction (179 references).

Polymer hydrogel microarrays were fabricated by inkjet printing of monomers and initiator, allowing up to 1800 individual polymer features to be printed on a single glass slide.

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease origins and differences in drug responses. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or alternatively by the hybridization of DNA probes. MALDI-TOF mass spectrometry (MS) has been widely used as a read-out tool. We developed a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping. In a blind trial, peptide nucleic acid (PNA) probes and aldehyde-modified nucleobases were employed to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. MALDI-TOF MS reported the resulting allele-specific products. Enzyme-free dynamic chemistry permitted the genotyping of twelve individuals for the ΔF508 (indel) and G551D (SNP) mutations. No false positives or negatives were observed, and analysis could be performed in both singleplex and duplex formats. Dynamic chemistry provides a distinct method of allele discrimination with certain advantages over those reported previously. Although PCR amplification is required, the analysis stage is performed without enzymes, and there is no need for the stringent optimization and control of conditions associated with hybridization-based methods of allele discrimination.

Nanoparticles have gained considerable significance in the life sciences due to their ability to be internalised by living cells and the relative ease with which they can be functionalised with cargos ranging from molecular sensors to biomacromolecules. However, the scope of available bioconjugation methods is limited and new bioorthogonal methods are much sought after. Herein, we present dual functionalised (HO)2B/H2N-polymeric nanoparticles which can be conjugated via amide bond formation and/or Pd-mediated Suzuki–Miyaura cross-coupling in a chemoselective and bioorthogonal manner. These dual-functionalised particles were found to be efficiently taken up by mammalian cells without toxicity and were successfully employed in the cellular delivery and intracellular release of a “turn-on” molecular probe thereby demonstrating the potential use of the new particles for chemical biology applications.

Nosocomial infections due to bacteria have serious implications on the health and recovery of patients in a variety of medical scenarios. Since bacterial contamination on medical devices contributes to the majority of nosocomical infections, there is a need for redesigning the surfaces of medical devices, such as catheters and tracheal tubes, to resist the binding of bacteria. In this work, polyurethanes and polyacrylates/acrylamides, which resist binding by the major bacterial pathogens underpinning implant-associated infections, were identified using high-throughput polymer microarrays. Subsequently, two ‘hit’ polymers, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)) and PA515 (poly(methoxyethylmethacrylate-co-diethylaminoethylacrylate-co-methylmethacrylate)), were used to coat catheters and substantially shown to decrease binding of a variety of bacteria (including isolates from infected endotracheal tubes and heart valves from intensive care unit patients). Catheters coated with polymer PA13 showed up to 96% reduction in bacteria binding in comparison to uncoated catheters.

Infections arising from contaminated medical devices are a serious global issue, contributing to antibiotic resistance and imposing significant strain on healthcare systems. Since the majority of medical device-associated infections are biofilm related, efforts are being made to generate either bacteria-repellent or antibacterial coatings aimed at preventing bacterial colonisation. Here, we utilise a nanocapsule mediated slow release of a natural antimicrobial to improve the performance of a bacteria repellent polymer coating. Poly(lauryl acrylate) nanocapsules containing eugenol (4-allyl-2-methoxyphenol) were prepared and entrapped within a interpenetrating network designed to repel bacteria. When coated on a catheter and an endotracheal tube, this hemocompatible system allowed slow-release of eugenol, resulting in notable reduction in surface-bound Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus.

The therapeutic use of nucleic acids has long been heralded as a panacea of medicinal opportunity, a vision enhanced by the introduction of RNA interference technology. The Achilles heel of such an approach is the in vivo delivery of the desired nucleic acid into cells, a practice that lacks selectivity, safety and/or efficiency. Herein we report the safe and efficacious in vitro and in vivo delivery of nucleic acids using tripodal biodegradable cationic lipids. Toxicity reduction and transfection potency of these novel amphiphiles were addressed by designing the compounds to undergo complete intracellular degradation thereby enhancing cargo release while minimising toxicity and potential tissue accumulation. Compounds demonstrated high-efficiency in transfecting DNA into cells both in vitro and in vivo with no signs of toxicity, thus potentially offering a safer alternative to viral transfection for gene therapy application.

Polymer microarrays provide an innovative approach to identify materials with novel bacterial binding or repellent properties which could subsequently be used in a variety of practical applications. Here, we report a polymer microarray screen of hundreds of synthetic polymers to identify those which either selectively capture the major food-borne pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium), or prevent its binding. A parallel study with a lab strain of Escherichia coli (E. coli) is also reported; revealing polymers which either display a common binding activity or which exhibit species discrimination. Moreover, substrates were also uncovered which showed no binding of either organism, even when cultured at high density. The correlation between polymer structure and microbial-modulating behaviour was analysed further, while SEM analysis allowed visualization of the detailed interactions between surface and bacteria. Such polymers offer many new opportunities for bacterial enrichment or surface repulsion, in cleaning materials, as surface coatings for use in the food production industry or as a “bacterial scavenger” resin.

Herein we report a highly-efficient solid-phase strategy for the modular synthesis of 63 double-tailed lipid-peptide conjugates and their application in DNA delivery.

A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N′-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a “naïve” state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture.

Cells over-expressing integrins or CCR6 were incubated on a DNA microarray, pre-hybridized with a 10000 member PNA-encoded peptide library allowing novel cell specific ligands for integrins and CCR6 to be identified.

Mesenchymal stem cells (MSCs) hold great promise in regenerative medicine due to their wide multilineage potential as well as their ability to suppress/modulate the immune response. Maintaining these cells in vitro and expanding them on a clinically relevant scale remains a challenge that needs to be addressed to realise their full potential. Current culture methods for MSCs typically rely on animal sourced substrates and often result in a heterogeneous population of cells with varying degrees of differentiation capacity. Here, a high-throughput platform was used to identify synthetic substrates for MSC culture that not only facilitated growth but also maintained the MSC phenotype. Two polymers, PU157 (synthesised from poly(butyleneglycol) and 4,4′-methylenediphenyldiisocyanate with 3-(dimethylamino)-1,2-propanediol as a chain extender) and PA338 (N-methylaniline modified poly(methylmethacrylate-co-glycidylmethacrylate)) were able to maintain the growth and phenotype of human embryonic derived mesenchymal progenitors (hES-MPs) and adipose derived MSCs (ADMSCs) for five and ten passages, respectively. Cell phenotype and multipotency were confirmed by flow cytometry analysis of ten MSC markers and differentiation analysis. These new polymer substrates provide a chemically defined synthetic surface for efficient, long-term MSC culture.