Antimicrobials: An update on new strategies to diversify treatment for bacterial infections.

Ninety-five years after Fleming's discovery of penicillin, a bounty of antibiotic compounds have been discovered, modified, or synthesised. Diversification of target sites, improved stability and altered activity spectra have enabled continued antibiotic efficacy, but overwhelming reliance and misuse has fuelled the global spread of antimicrobial resistance (AMR). An estimated 1.27 million deaths were attributable to antibiotic resistant bacteria in 2019, representing a major threat to modern medicine. Although antibiotics remain at the heart of strategies for treatment and control of bacterial diseases, the threat of AMR has reached catastrophic proportions urgently calling for fresh innovation. The last decade has been peppered with ground-breaking developments in genome sequencing, high throughput screening technologies and machine learning. These advances have opened new doors for bioprospecting for novel antimicrobials. They have also enabled more thorough exploration of complex and polymicrobial infections and interactions with the healthy microbiome. Using models of infection that more closely resemble the infection state in vivo, we are now beginning to measure the impacts of antimicrobial therapy on host/microbiota/pathogen interactions. However new approaches are needed for developing and standardising appropriate methods to measure efficacy of novel antimicrobial combinations in these contexts. A battery of promising new antimicrobials is now in various stages of development including co-administered inhibitors, phages, nanoparticles, immunotherapy, anti-biofilm and anti-virulence agents. These novel therapeutics need multidisciplinary collaboration and new ways of thinking to bring them into large scale clinical use.

Self-assembling ferritin nanoplatform for the development of infectious hematopoietic necrosis virus vaccine.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

In depth characterisation of the biomolecular coronas of polymer coated inorganic nanoparticles with differential centrifugal sedimentation.

Advances in nanofabrication methods have enabled the tailoring of new strategies towards the controlled production of nanoparticles with attractive applications in healthcare. In many cases, their characterisation remains a big challenge, particularly for small-sized functional nanoparticles of 5 nm diameter or smaller, where current particle sizing techniques struggle to provide the required sensitivity and accuracy. There is a clear need for the development of new reliable characterisation approaches for the physico-chemical characterisation of nanoparticles with significant accuracy, particularly for the analysis of the particles in the presence of complex biological fluids. Herein, we show that the Differential Centrifugal Sedimentation can be utilised as a high-precision tool for the reliable characterisation of functional nanoparticles of different materials. We report a method to correlate the sedimentation shift with the polymer and biomolecule adsorption on the nanoparticle surface, validating the developed core-shell model. We also highlight its limit when measuring nanoparticles of smaller size and the need to use several complementary methods when characterising nanoparticle corona complexes.

Influence of Size and Shape on the Anatomical Distribution of Endotoxin-Free Gold Nanoparticles.

The transport and the delivery of drugs through nanocarriers is a great challenge of pharmacology. Since the production of liposomes to reduce the toxicity of doxorubicin in patients, a plethora of nanomaterials have been produced and characterized. Although it is widely known that elementary properties of nanomaterials influence their in vivo kinetics, such interaction is often poorly investigated in many preclinical studies. The present study aims to evaluate the actual effect of size and shape on the biodistribution of a set of gold nanoparticles (GNPs) after intravenous administration in mice. To this goal, quantitative data achieved by inductively coupled plasma mass spectrometry and observational results emerging from histochemistry (autometallography and enhanced dark-field hyperspectral microscopy) were combined. Since the immune system plays a role in bionano-interaction we used healthy immune-competent mice. To keep the immune surveillance on the physiological levels we synthesized endotoxin-free GNPs to be tested in specific pathogen-free animals. Our study mainly reveals that (a) the size and the shape greatly influence the kinetics of accumulation and excretion of GNPs in filter organs; (b) spherical and star-like GNPs showed the same percentage of accumulation, but a different localization in liver; (c) only star-like GNPs are able to accumulate in lung; (d) changes in the geometry did not improve the passage of the blood brain barrier. Overall, this study can be considered as a reliable starting point to drive the synthesis and the functionalization of potential candidates for theranostic purposes in many fields of research.

Mapping protein binding sites on the biomolecular corona of nanoparticles.

Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

Direct surface-enhanced Raman scattering analysis of DNA duplexes.

The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface-enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label-free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5-methylated cytosine and N6-methylated Adenine) in duplexes.

Revealing DNA interactions with exogenous agents by surface-enhanced Raman scattering.

The standard protocols for DNA analysis largely involve polymerase chain reaction (PCR). However, DNA structures bound to chemical agents cannot be PCR-amplified, and therefore any sequence changes induced by external agents may be neglected. Thus, the development of analytical tools capable of characterizing the biochemical mechanisms associated with chemically induced DNA damage is demanded for the rational design of more effective chemotherapy drugs, understanding the mode of actions of carcinogenic chemicals, and monitoring the genotypic toxicology of environments. Here we report a fast, high-throughput, low-cost method for the characterization and quantitative recognition of DNA interactions with exogenous agents based on surface-enhanced Raman scattering spectroscopy. As representative chemical agents, we selected a chemotherapeutic drug (cisplatin) which forms covalent adducts with DNA, a duplex intercalating agent (methylene blue), and a cytotoxic metal ion (Hg(II)) which inserts into T:T mismatches. Rich structural information on the DNA complex architecture and properties is provided by the unique changes of their SERS spectra, which also offer an efficient analytical tool to quantify the extent of such binding.

Interactions of gold nanoparticles with a phospholipid monolayer membrane on mercury.

It is demonstrated that a compact monolayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine adsorbed to a hanging mercury drop electrode can serve as a simple electrochemical model system to study biomembrane penetration by gold nanoparticles. The hydrogen redox-chemistry characteristic of ligand-stabilized gold nanoparticles in molecularly close contact with a mercury electrode is used as an indicator of membrane penetration. Results for water-dispersible gold nanoparticles of two different sizes are reported, and comparisons are made with the cellular uptake of the same preparations of nanoparticles by a common human fibroblast cell line. The experimental system described here can be used to study physicochemical aspects of membrane penetration in the absence of complex biological mechanisms, and it could also be a starting point for the development of a test bed for the toxicity of nanomaterials.

Nanomaterials: impact on cells and cell organelles.

Colloidal nanoparticles designed for the interactions with cells are very small, nanoscale objects usually consisting of inorganic cores and organic shells that are dispersed in a buffer or biological medium. By tuning the material properties of the nanoparticles a number of different biological applications of nanomaterials are enabled i.e. targeting, labelling, drug delivery, use as diagnostic tools or therapy. For all biological applications of nanoparticles, it is important to understand their interactions with the surrounding biological environment in order to predict their biological impact, in particular when designing the nanoparticles for diagnostic and therapeutic purpose. Due to the high surface-to-volume ratio, the surface of nanomaterials is very reactive. When exposed to biological fluids, the proteins and biomolecules present therein tend to associate with the nanoparticles' surface. This phenomenon is defined as biomolecular corona formation. The biomolecular corona plays a key role in the interaction between nanoparticles and biological systems, impacting on how these particles interact with biological systems on a cellular and molecular level. This book chapter describes the nature of the interactions at the bio-nano interface, shows the design strategy of nanoparticles for nanomedicine, and defines the concepts of biomolecular corona and biological identity of nanoparticles. Moreover, it describes the interaction of functionalised nanomaterials with cell organelles and intracellular fate of nanoparticles and it shows therapeutic application of gold nanoparticles as dose enhancers in radiotherapy.

Preparation and characterization of Au nanoparticles capped with mercaptocarboranyl clusters.

The preparation of 3-4 nm and 10 nm gold nanoparticles capped with neutral carborane-based mercaptocarboranes, via two different preparative routes, is reported. The resulting boron-enriched nanomaterials exhibit complete dispersibility in water, opening the way for the use of these monolayer protected clusters (MPCs) in medical applications, such as boron neutron capture therapy (BNCT). These newly prepared MPCs have been characterized by FTIR, (1)H and (11)B NMR spectroscopy, UV-visible, centrifugal particle sizing (CPS), and, in some cases, inductively coupled plasma atomic emission spectrometry (ICP-AES). Water dispersibility exhibited by these MPCs allowed the study of the cellular uptake by HeLa cells.

High-resolution sizing of monolayer-protected gold clusters by differential centrifugal sedimentation.

Differential centrifugal sedimentation (DCS) has been applied to accurately size ligand-protected gold hydrosols in the 10 to 50 nm range. A simple protocol is presented to correct for particle density variations due to the presence of the ligand shell, which is formed here by either polyethylene glycol-substituted alkane thiols (PEG-alkane thiols) of different chain length or oligopeptides. The method gives reliable data for all particle sizes investigated and lends itself to rapid routine sizing of nanoparticles. Unlike TEM, DCS is highly sensitive to small changes in the thickness of the organic ligand shell and can be applied to monitor shell thickness variations of as little as 0.1 nm on particles of a given core size.

Positively charged silver nanoparticles and their effect on surface-enhanced Raman scattering of dye-labelled oligonucleotides.

Improved positively charged nanoparticles are described to provide a simplified SERS substrate for DNA detection. Complete flocculation of the nanoparticles is prevented due to the controlled analyte induced aggregation. This provides a stable aggregation state which significantly extends the analysis window simplifying DNA detection by SERS.

Directed assembly of DNA-functionalized gold nanoparticles using pyrrole-imidazole polyamides.

Traditional methods for the construction of nanoparticle arrays and lattices exploit Watson-Crick base pairing of single-stranded DNA sequences as a proxy for self-assembly. Although this approach has been utilized in a variety of applications in nanoassembly, diagnostics, and biomedicine, the diversity of this recognition lexicon could be considerably increased by developing strategies that recognize the base-pairing landscape of double-stranded DNA (dsDNA) sequences. Herein we describe the first report of programmed gold nanoparticle (GNP) aggregation directed by the recognition of dsDNA sequences using pyrrole-imidazole polyamide-GNP (PA-GNP) conjugates. We demonstrate the reversibility and selectivity of this strategy for forming GNP aggregates in the presence of fully matched dsDNA sequences relative to dsDNA sequences containing one- and two-base-pair mismatches.

Importance of nanoparticle size in colorimetric and SERS-based multimodal trace detection of Ni(II) ions with functional gold nanoparticles.

Colorimetric detection of analytes using gold nanoparticles along with surface-enhanced Raman spectroscopy (SERS) are areas of intense research activity since they both offer sensing of very low concentrations of target species. Multimodal detection promotes the simultaneous detection of a sample by a combination of different techniques; consequently, surface chemistry design in the development of multimodal nanosensors is important for rapid and sensitive evaluation of the analytes by diverse analytical methods. Herein it is shown that nanoparticle size plays an important role in the design of functional nanoparticles for colorimetric and SERS-based sensing applications, allowing controlled nanoparticle assembly and tunable sensor response. The design and preparation of robust nanoparticle systems and their assembly is reported for trace detection of Ni(II) ions as a model system in an aqueous solution. The combination of covalently attached nitrilotriacetic acid moieties along with the L-carnosine dipeptide on the nanoparticle surface represents a highly sensitive platform for rapid and selective detection of Ni(II) ions. This systematic study demonstrates that significantly lower detection limits can be achieved by finely tuning the assembly of gold nanoparticles of different core sizes. The results clearly demonstrate the feasibility and usefulness of a multimodal approach.

Mercaptocarborane-capped gold nanoparticles: electron pools and ion traps with switchable hydrophilicity.

A simple single-phase method for the preparation of ca. 2 nm gold nanoparticles capped with mercaptocarborane ligands is introduced. The resultant monolayer protected clusters (MPCs) exhibit redox-dependent solubility and readily phase transfer between water and nonpolar solvents depending on the electronic and ionic charge stored in the metal core and in the ligand shell, respectively. The particles and their properties have been characterized by high angle annular dark field imaging in a scanning transmission electron microscope, elemental analysis, centrifugal particle sizing, UV-vis and FTIR spectroscopy, and thermogravimetric analysis and by (1)H, (11)B, and (7)Li NMR spectroscopy. Cellular uptake of the MPCs by HeLa cells has been studied by TEM, and the subsequent generation of reactive oxygen species inside the cells has been evaluated by confocal fluorescence microscopy. These MPCs qualitatively showed significant toxicity and the ability to penetrate into most cell compartments with a strong tendency of finally residing inside membranes. Applications in catalysis, electrocatalysis, and biomedicine are envisaged.

Intracellular mapping with SERS-encoded gold nanostars.

Here we report on the design, synthesis and application of small, highly bright, star-shaped SERS encoded single nanoparticles with the ability of providing an optical signal upon excitation with near infrared light. These particles are colloidally stable, fully biocompatible and can be internalized into living cells for intracellular imaging.

Negotiation of intracellular membrane barriers by TAT-modified gold nanoparticles.

This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.