An Indocyanine Green-Based Nanoprobe for In Vivo Detection of Cellular Senescence.

There is an urgent need to improve conventional cancer-treatments by preventing detrimental side effects, cancer recurrence and metastases. Recent studies have shown that presence of senescent cells in tissues treated with chemo- or radiotherapy can be used to predict the effectiveness of cancer treatment. However, although the accumulation of senescent cells is one of the hallmarks of cancer, surprisingly little progress has been made in development of strategies for their detection in vivo. To address a lack of detection tools, we developed a biocompatible, injectable organic nanoprobe (NanoJagg), which is selectively taken up by senescent cells and accumulates in the lysosomes. The NanoJagg probe is obtained by self-assembly of indocyanine green (ICG) dimers using a scalable manufacturing process and characterized by a unique spectral signature suitable for both photoacoustic tomography (PAT) and fluorescence imaging. In vitro, ex vivo and in vivo studies all indicate that NanoJaggs are a clinically translatable probe for detection of senescence and their PAT signal makes them suitable for longitudinal monitoring of the senescence burden in solid tumors after chemotherapy or radiotherapy.

Biopolymer-based Carriers for DNA Vaccine Design.

Over the last 30 years, genetically engineered DNA has been tested as novel vaccination strategy against various diseases, including human immunodeficiency virus (HIV), hepatitis B, several parasites, and cancers. However, the clinical breakthrough of the technique is confined by the low transfection efficacy and immunogenicity of the employed vaccines. Therefore, carrier materials were designed to prevent the rapid degradation and systemic clearance of DNA in the body. In this context, biopolymers are a particularly promising DNA vaccine carrier platform due to their beneficial biochemical and physical characteristics, including biocompatibility, stability, and low toxicity. This article reviews the applications, fabrication, and modification of biopolymers as carrier medium for genetic vaccines.

Hetero-Diels-Alder Cycloaddition with RAFT Polymers as Bioconjugation Platform.

We introduce the bioconjugation of polymers synthesized by RAFT polymerization, bearing no specific functional end group, by means of hetero-Diels-Alder cycloaddition through their inherent terminal thiocarbonylthio moiety with a diene-modified model protein. Quantitative conjugation occurs over the course of a few hours, at ambient temperature and neutral pH, and in the absence of any catalyst. Our technology platform affords thermoresponsive bioconjugates, whose aggregation is solely controlled by the polymer chains.

Photo-Induced Click Chemistry for DNA Surface Structuring by Direct Laser Writing.

Oligonucleotides containing photo-caged dienes were prepared and shown to react quantitatively in a light-induced Diels-Alder cycloaddition with functional maleimides in aqueous solution within minutes. Due to its high yield and fast rate, the reaction was exploited for DNA surface patterning with sub-micrometer resolution employing direct laser writing (DLW). Functional DNA arrays were written by direct laser writing (DLW) in variable patterns, which were further encoded with fluorophores and proteins through DNA directed immobilization. This mild and efficient light-driven platform technology holds promise for the fabrication of complex bioarrays with sub-micron resolution.

Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

A self-reporting tetrazole-based linker for the biofunctionalization of gold nanorods.

A photochemical approach based on nitrile imine-mediated tetrazole-ene cycloaddition is introduced to functionalize gold nanorods with biomolecules. For this purpose, a bifunctional, photoreactive linker containing thioctic acid as the Au anchoring group and a tetrazole moiety for the light-induced reaction with maleimide-capped DNA was prepared. The tetrazole-based reaction on the nanoparticles' surface results in a fluorescent pyrazoline product allowing for the spectroscopic monitoring of the reaction. This first example of nitrile imine-mediated tetrazole-ene cycloaddition (NITEC)-mediated biofunctionalization of Au nanorods paves the way for the attachment of sensitive biomolecules, such as antibodies and other proteins, under mild conditions and expands the toolbox for the tailoring of nanomaterials.

Biocompatible hydrogel nanocomposite with covalently embedded silver nanoparticles.

Bionanocomposite materials, combining the properties of biopolymers and nanostructured materials, are attracting interest of the wider scientific community due to their potential application in design of implants, drug delivery systems, and tissue design platforms. Herein, we report on the use of maleimide-coated silver nanoparticles (Ag NPs) as cocross-linkers for the preparation of a bionanocomposite gelatin based hydrogel. Diels-Alder cycloaddition of benzotriazole maleimide (BTM) functionalized Ag NPs and furan containing gelatin in combination with additional amide coupling resulted in stable and biocompatible hybrid nanocomposite. The storage moduli values for the hydrogel are nearly three times higher than that of control hydrogel without NPs indicating a stabilizing role of the covalently bound NPs. Finally, the swelling and drug release properties of the materials as well as the biocompatibility and toxicity tests indicate the biomedical potential of this type of material.

DNA from natural sources in design of functional devices.

The role of DNA as structuring or templating agent has become more significant with the development of nanobiotechnology. Although short single and double stranded DNA have extensively been used as immobilization tool, as a template for nanoparticle preparation and in design of various devices such as nanomotors and biosensors, DNA from natural sources has an advantage of being abundant, cheap and readily available. Therefore, it is not surprising that there is a huge interest in making the use of natural DNA properties for both nano- and micro-applications. In this review we attempt to give an overview of the up to date applications of natural DNA, either from viral, marine or mammalian sources, in design of functional devices. This article is meant to be a starting point and a guide to the platforms in which natural DNA is employed such as DNA origami, optoelectronic devices and organic catalysis.

Detection of DNA Hybridization by Methylene Blue Electrochemistry at Activated Nanoelectrode Ensembles.

Nanoelectrode ensembles (NEEs) obtained by electroless gold deposition in track-etched poly-carbonate (PC) membranes are functionalized and applied for DNA hybridization detection, using methylene blue (MB) as electroactive probe. To this aim, an amine terminated (ss)DNA probe is immobilized on the PC surface of the NEE by reaction via carbodiimide and N-hydroxysulfosuccinimide. In order to increase the number of carboxylic groups present on PC and suitable for the functionalization, the surface of NEEs is oxidized with potassium permanganate. The presence of carboxylic functionalities is verified by spectrochemical titration with thionin acetate (THA) and the effect of the activation treatment on the electrode performances is evaluated by cyclic voltammetry (CV). After activation and functionalization with the probes, the NEE-based sensor is hybridized with complementary target sequences. The effect of the functionalization of the NEEs both with the (ss)DNA probe alone and after hybridization with the target, is studied by measuring the changes in the MB reduction signal by square wave voltammetry (SWV), after incubation in a suitable MB solution, rinsing and transfer to the measurement cell. It was observed that this peak signal decreases significantly after hybridization of the probe with the complementary target. Experimental evidences suggest that the interaction between MB and the guanines of (ss)DNA and (ds)DNA is at the basis of the development of the here observed analytical signal. The proposed approach allows the easy preparation and testing of NEE-based sensors for the electrochemical DNA hybridization detection.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

π-conjugated polymer-fullerene covalent hybrids via ambient conditions Diels-Alder ligation.

The established ability of graphitic carbon-nanomaterials to undergo ambient condition Diels-Alder reactions with cyclopentadienyl (Cp) groups is herein employed to prepare fullerene-polythiophene covalent hybrids with improved electron transfer and film forming characteristics. A novel precisely designed polythiophene (M n 9.8 kD, D 1.4) with 17 mol% of Cp-groups bearing repeat unit is prepared via Grignard metathesis polymerization. The UV/Vis absorption and fluorescence (λex 450 nm) characteristics of polythiophene with pendant Cp-groups (λmax 447 nm, λe-max 576 nm) are comparable to the reference poly(3-hexylthiophene) (λmax 450 nm, λe-max 576 nm). The novel polythiophene with pendant Cp-groups is capable of producing solvent-stable free-standing polythiophene films, and non-solvent assisted self-assemblies resulting in solvent-stable nanoporous-microstructures. (1) H-NMR spectroscopy reveals an efficient reaction of the pendant Cp-groups with C60 . The UV/Vis spectroscopic analyses of solution and thin films of the covalent and physical hybrids disclose closer donor-acceptor packing in the case of covalent hybrids. AFM images evidence that the covalent hybrids form smooth films with finer lamellar-organization. The effect is particularly remarkable in the case of poorly soluble C60 . A significant enhancement in photo-voltage is observed for all devices constituted of covalent hybrids, highlighting novel avenues to developing efficient electron donor-acceptor combinations for light harvesting systems.

Microwave-enhanced antibacterial activity of polydopamine-silver hybrid nanoparticles.

The ever-increasing risks posed by antibiotic-resistant bacteria have stimulated considerable interest in the development of novel antimicrobial strategies, including the use of nanomaterials that can be activated on demand and result in irreversible damage to pathogens. Microwave electric field-assisted bactericidal effects on representative Gram-negative and Gram-positive bacterial strains were achieved in the presence of hybrid polydopamine-silver nanoparticles (PDA-Ag NPs) under low-power microwave irradiation using a resonant cavity (1.3 W, 2.45 GHz). A 3-log reduction in the viability of bacterial populations was observed within 30 minutes which was attributed to the attachment of PDA-Ag NPs and associated membrane disruption in conjunction with the production of intra-bacterial reactive oxygen species (ROS). A synergistic effect between PDA and Ag has been demonstrated whereby PDA acts both as an Ag NP carrier and a microwave enhancer. These properties together with the remarkable adhesivity of PDA are opening a route to design of antibacterial adhesives and surface coatings for prevention of biofilm formation.

Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

Clickable tyrosine binding bifunctional linkers for preparation of DNA-protein conjugates.

We have prepared bifunctional linkers containing clickable functional groups that enable preparation of protein-DNA conjugates through binding onto tyrosine residues. Mild conjugation strategy was demonstrated using two proteins, streptavidin(STV) and myoglobin (Mb) and it resulted in conjugates with preserved functionality of both the proteins and DNA strands. Furthermore, we show that protein-DNA conjugates can be successfully immobilized onto solid surface containing complementary DNA strands and the enzymatic activity of Mb-DNA conjugates is even higher than that of corresponding conjugates prepared through Lys binding.

Nernst-Planck model of photo-triggered, pH-tunable ionic transport through nanopores functionalized with "caged" lysine chains.

We describe the fabrication of asymmetric nanopores sensitive to ultraviolet (UV) light, and give a detailed account of the divalent ionic transport through these pores using a theoretical model based on the Nernst-Planck equations. The pore surface is decorated with lysine chains having pH-sensitive (amine and carboxylic acid) moieties that are caged with photo-labile 4,5-dimethoxy-2-nitrobenzyl (NVOC) groups. The uncharged hydrophobic NVOC groups are removed using UV irradiation, leading to the generation of hydrophilic "uncaged" amphoteric groups on the pore surface. We demonstrate experimentally that polymer membranes containing single pore and arrays of asymmetric nanopores can be employed for the pH-controlled transport of ionic and molecular analytes. Comparison between theory and experiment allows for understanding the individual properties of the phototriggered nanopores, and provides also useful clues for the design and fabrication of multipore membranes to be used in practical applications.

Activity-enhanced DNAzyme for design of label-free copper(II) biosensor.

Metal ion-driven, DNA-cleaving DNAzymes are characterised by high selectivity and specificity. However, their use for metal ion sensing remains largely unexplored due to long reaction times and poor reaction yields relative to RNA-cleaving DNAzymes and other sensing strategies. Herein we present a study demonstrating a significant rate enhancement of a copper-selective DNA cleaving DNAzyme by both polydopamine (PDA) and gold (Au) nanoparticles (NPs). PDA NPs enhance the reaction through the production of hydrogen peroxide, while for AuNPs the enhancement is aided by the presence of citrate surface moeities, both of which drive the oxidative cleavage of the substrate. A 50-fold enhancement for PDA NPs makes the combination of PDA and DNAzyme suitable for a practical application as a sensitive biosensor for Cu(II) ions. Using DNAzyme deposition onto a gold electrode followed by Polydopamine Assisted DNA Immobilisation (PADI), we achieve a cost-effective, label-free and fast (within 15 min) electrochemical biosensor with a limit of detection of 180 nmol (11 ppm), thus opening a route for the rational design of a new generation of hybrid DNAzyme-based biosensors.

A catch-and-release nano-based gene delivery system.

The design of nanomaterial-based nucleic acid formulations is one of the biggest endeavours in the search for clinically applicable gene delivery systems. Biopolymers represent a promising subclass of gene carriers due to their physicochemical properties, biodegradability and biocompatibility. By modifying melanin-like polydopamine nanoparticles with poly-L-arginine and poly-L-histidine blends, we obtained a novel catch-and-release gene delivery system for efficient trafficking of pDNA to human cells. A synergistic interplay of nanoparticle-bound poly-L-arginine and poly-L-histidine was observed and evaluated for pDNA binding affinity, cell viability, gene release and transfection. Although the functionalisation with poly-L-arginine was crucial for pDNA binding, the resulting nanocarriers failed to release pDNA intracellularly, resulting in limited protein expression. However, optimal pDNA release was achieved through the co-formulation with poly-L-histidine, essential for pDNA release. This effect enabled the design of gene delivery systems, which were comparable to Lipofectamine in terms of transfection efficacy and the catch-and-release surface modification strategy can be translated to other nanocarriers and surfaces.

The future of early cancer detection.

A proactive approach to detecting cancer at an early stage can make treatments more effective, with fewer side effects and improved long-term survival. However, as detection methods become increasingly sensitive, it can be difficult to distinguish inconsequential changes from lesions that will lead to life-threatening cancer. Progress relies on a detailed understanding of individualized risk, clear delineation of cancer development stages, a range of testing methods with optimal performance characteristics, and robust evaluation of the implications for individuals and society. In the future, advances in sensors, contrast agents, molecular methods, and artificial intelligence will help detect cancer-specific signals in real time. To reduce the burden of cancer on society, risk-based detection and prevention needs to be cost effective and widely accessible.

Kinetics and Mechanism of In Situ Metallization of Bulk DNA Films.

DNA-templated metallization is broadly investigated in the fabrication of metallic structures by virtue of the unique DNA-metal ion interaction. However, current DNA-templated synthesis is primarily carried out based on pure DNA in an aqueous solution. In this study, we present in situ synthesis of metallic structures in a natural DNA complex bulk film by UV light irradiation, where the growth of silver particles is resolved by in situ time-resolved small-angle X-ray scattering and dielectric spectroscopy. Our studies provide physical insights into the kinetics and mechanisms of natural DNA metallization, in correlation with the multi-stage switching operations in the bulk phase, paving the way towards the development of versatile biomaterial composites with tunable physical properties for optical storage, plasmonics, and catalytic applications.

Tuning riboflavin derivatives for photodynamic inactivation of pathogens.

The development of effective pathogen reduction strategies is required due to the rise in antibiotic-resistant bacteria and zoonotic viral pandemics. Photodynamic inactivation (PDI) of bacteria and viruses is a potent reduction strategy that bypasses typical resistance mechanisms. Naturally occurring riboflavin has been widely used in PDI applications due to efficient light-induced reactive oxygen species (ROS) release. By rational design of its core structure to alter (photo)physical properties, we obtained derivatives capable of outperforming riboflavin's visible light-induced PDI against E. coli and a SARS-CoV-2 surrogate, revealing functional group dependency for each pathogen. Bacterial PDI was influenced mainly by guanidino substitution, whereas viral PDI increased through bromination of the flavin. These observations were related to enhanced uptake and ROS-specific nucleic acid cleavage mechanisms. Trends in the derivatives' toxicity towards human fibroblast cells were also investigated to assess viable therapeutic derivatives and help guide further design of PDI agents to combat pathogenic organisms.