Different drug loading methods and antibiotic structure modulate the efficacy of polydopamine nanoparticles as drug nanocarriers.

The inefficient delivery of antimicrobials to their target is a significant factor contributing to antibiotic resistance. As such, smart nanomaterials that respond to external stimuli are extensively explored for precise drug delivery. Here, we investigate how drug loading methods and the structure of antibiotics impact the effectiveness of photothermally active polydopamine nanoparticles (PDNPs) as a laser-responsive drug delivery system. We examine two loading methods: in-synthesis and post-synthesis, and evaluate how laser irradiation affects drug release. Density functional theory calculations are also performed to gain deeper insights into the drug-PDNP interactions. Our findings point to the critical role of antibiotic structure and drug loading method in the laser-responsive capabilities of PDNPs as drug nanocarriers. Our study offers valuable insights for optimizing the design and efficiency of PDNP-based drug delivery systems.

Laser-responsive sequential delivery of multiple antimicrobials using nanocomposite hydrogels.

Precise control of antimicrobial delivery can prevent the adverse effects of antibiotics. By exploiting the photothermal activity of polydopamine nanoparticles along with the distinct transition temperatures of liposomes, a near-infrared (NIR) laser can be used to control the sequential delivery of an antibiotic and its adjuvant from a nanocomposite hydrogel-preventing bacterial growth.

Buffer Components Incorporate into the Framework of Polyserotonin Nanoparticles and Films during Synthesis.

Polyserotonin nanoparticles (PSeNP) and films are bioinspired nanomaterials that have potential in biomedical applications and surface coatings. As studies on polyserotonin (PSe) nanoparticles and films are still in their infancy, synthetic pathways and material development for this new class of nanomaterial await investigation. Here, we sought to determine how different buffers used during the polymerization of serotonin to form nanoparticles and films impact the physicochemical properties of PSe materials. We show that buffer components are incorporated into the polymer matrix, which is also supported by density functional theory calculations. While we observed no significant differences between the elasticity of nanoparticles synthesized in the different buffers, the nanoscale surface properties of PSe films revealed dissimilarities in surface functional groups influenced by solvent molecules. Overall, the results obtained in this work can be used towards the rational design of PSe nanomaterials with tailored properties and for specific applications.

Immobilization of Polyethyleneimine (PEI) on Flat Surfaces and Nanoparticles Affects Its Ability to Disrupt Bacterial Membranes.

Interactions between a widely used polycationic polymer, polyethyleneimine (PEI), and a Gram-negative bacteria, E. coli, are investigated using atomic force microscopy (AFM) quantitative imaging. The effect of PEI, a known membrane permeabilizer, is characterized by probing both the structure and elasticity of the bacterial cell envelope. At low concentrations, PEI induced nanoscale membrane perturbations all over the bacterial surface. Despite these structural changes, no change in cellular mechanics (Young's modulus) was detected and the growth of E. coli is barely affected. However, at high PEI concentrations, dramatic changes in both structure and cell mechanics are observed. When immobilized on a flat surface, the ability of PEI to alter the membrane structure and reduce bacterial elasticity is diminished. We further probe this immobilization-induced effect by covalently attaching the polymer to the surface of polydopamine nanoparticles (PDNP). The nanoparticle-immobilized PEI (PDNP-PEI), though not able to induce major structural changes on the outer membrane of E. coli (in contrast to the flat surface), was able to bind to and reduce the Young's modulus of the bacteria. Taken together, our data demonstrate that the state of polycationic polymers, whether bound or free-which greatly dictates their overall configuration-plays a major role on how they interact with and disrupt bacterial membranes.

Size-controlled synthesis of bioinspired polyserotonin nanoparticles with free radical scavenging activity.

Polyserotonin-based nanoparticles are a new class of bioinspired nanomaterial with recently demonstrated therapeutic potential for future clinical applications. It is therefore important to establish a robust and rapid method of synthesizing polyserotonin nanoparticles (PSeNP) in the size range ideal for in vivo utilization. Since the formation of PSeNP is base-catalyzed, here we report the influence of solution pH, in the presence of different base systems, on the kinetics of PSeNP formation and physico-chemical properties of the resulting nanoparticles. We show that the rate of formation and the size of PSeNP depend on both the nature of the base and the initial pH of the reaction. We have also improved the kinetics of particle formation by performing the synthesis at an elevated temperature (60 °C), leading to a dramatic reduction in synthesis time from days to hours. This presents a significant advance in the efficiency of PSeNP synthesis and provides a facile approach in tuning the size of nanoparticles to suit various applications. Furthermore, we show that similar to serotonin, PSeNP also exhibits free radical scavenging property. Our results demonstrate that PSeNP has the potential to become a key player in the advancement of nanotechnology-mediated antioxidative therapy.

Peptide-Polydopamine Nanocomposite Hydrogel for a Laser-Controlled Hydrophobic Drug Delivery.

Smart antibacterial systems, delivering antimicrobials in a highly controlled manner, are one strategy toward fighting the rise of antibiotic-resistant pathogens. Here, we engineer a laser-responsive antimicrobial nanocomposite hydrogel combining a peptide amphiphile and a photothermally active polydopamine nanoparticle (PDNP) to entrap the hydrophobic rifampicin within the hydrophilic hydrogel matrix. We show that the ability of the gelator to interact and retain rifampicin within the gel induced structural changes in its nanofiber network and mechanical properties. Furthermore, PDNP inclusion enabled laser-induced drug release, preventing growth of a Gram-negative E. coli. Overall, our work provides a significant advance in designing smart materials for controlled drug delivery applications.

Polyacrylamide hydrogels doped with different shapes of silver nanoparticles: Antibacterial and mechanical properties.

The incorporation of nanoparticles into a hydrogel matrix enables the development of innovative smart materials with enhanced biophysical properties. In this proof-of-concept study, we encapsulated different shapes (spherical, triangular and rod) of silver nanoparticles (AgNPs) within a hydrogel matrix of polyacrylamide (PAA) and N-methylenebisacrylamide (MBA) (PAA-MBA) to investigate whether these hydrogels exhibited shape-dependent antimicrobial and mechanical properties. We examined the mechanism of adsorption of different shapes of AgNPs using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Results showed that the adsorption of AgNPs was primarily occurring on the surface/outer pores of the PAA-MBA hydrogel and that rod AgNPs demonstrated a relatively slower adsorption within the hydrogel matrix. The mechanical properties of AgNP-doped hydrogels were evaluated using rheology and atomic force microscopy (AFM) quantitative imaging. We observed a higher storage and Young's modulus which proved that the incorporation of the various shapes of AgNPs increased the mechanical properties of the hydrogels with no significant differences between the different shapes. While both spherical and triangular AgNP-doped hydrogels showed strong antimicrobial activity, the hydrogel with the rod AgNPs had a relatively lower antimicrobial activity. Overall, our preliminary results demonstrated that nanocomposite hydrogels were promising materials for applications in the future development of wound dressings.

Multi-component peptide hydrogels - a systematic study incorporating biomolecules for the exploration of diverse, tuneable biomaterials.

Peptide-based supramolecular gels can be designed to be functional "smart" materials that have applications in drug delivery, tissue engineering, and supramolecular chemistry. Although many multi-component gel systems have been designed and reported, many of these applications still rely solely on single-component gel systems which limits the functionalities of the materials. Multi-component self-assembly leads to the formation of highly ordered and complex architectures while offering the possibility to generate hydrogels with interesting properties including functional complexity and diverse morphologies. Being able to incorporate various classes of biomolecules can allow for tailoring the materials' functionalities to specific application needs. Here, a novel peptide amphiphile, myristyl-Phe-Phe (C14-FF), was synthesized and explored for hydrogel formation. The hydrogel possesses a nanofiber matrix morphology, composed of ß-sheet aggregates, a record-low gelation concentration for this class of compounds, and a unique solvent-dependent helical switch. The C14-FF hydrogel was then explored with various classes of biomolecules (carbohydrates, vitamins, proteins, building blocks of HA) to generate a multi-component library of gels that have potential to represent the complex natural extracellular matrix. Selected multi-component gels exhibit an excellent compatibility with mesenchymal stem cells showing high cell viability percentages, which holds great promise for applications in regenerative therapy.

Cooperative communication within and between single nanocatalysts.

Enzymes often show catalytic allostery in which reactions occurring at different sites communicate cooperatively over distances of up to a few nanometres. Whether such effects can occur with non-biological nanocatalysts remains unclear, even though these nanocatalysts can undergo restructuring and molecules can diffuse over catalyst surfaces. Here we report that phenomenologically similar, but mechanistically distinct, cooperative effects indeed exist for nanocatalysts. Using spatiotemporally resolved single-molecule catalysis imaging, we find that catalytic reactions on a single Pd or Au nanocatalyst can communicate with each other, probably via hopping of positively charged holes on the catalyst surface, over ~102 nanometres and with a temporal memory of ~101 to 102 seconds, giving rise to positive cooperativity among its surface active sites. Similar communication is also observed between individual nanocatalysts, however it operates via a molecular diffusion mechanism involving negatively charged product molecules, and its communication distance is many micrometres. Generalization of these long-range intra- and interparticle catalytic communication mechanisms may introduce a novel conceptual framework for understanding nanoscale catalysis.

Spatiotemporal catalytic dynamics within single nanocatalysts revealed by single-molecule microscopy.

This review discusses the latest advances in using single-molecule microscopy of fluorogenic reactions to examine and understand the spatiotemporal catalytic behaviors of single metal nanoparticles of various shapes including pseudospheres, nanorods, and nanoplates. Real-time single-turnover kinetics reveal size-, catalysis-, and metal-dependent temporal activity fluctuations of single pseudospherical nanoparticles (<20 nm in diameter). These temporal catalytic dynamics can be related to nanoparticles' dynamic surface restructuring whose timescales and energetics can be quantified. Single-molecule super-resolution catalysis imaging further enables the direct quantification of catalytic activities at different surface sites (i.e., ends vs. sides, or corner, edge vs. facet regions) on single pseudo 1-D and 2-D nanocrystals, and uncovers linear and radial activity gradients within the same surface facets. These spatial activity patterns within single nanocrystals can be attributed to the inhomogeneous distributions of low-coordination surface sites, including corner, edge, and defect sites, among which the distribution of defect sites is correlated with the nanocrystals' morphology and growth mechanisms. A brief discussion is given on the extension of the single-molecule imaging approach to catalysis that does not involve fluorescent molecules.

Single-molecule dynamics and mechanisms of metalloregulators and metallochaperones.

Understanding how cells regulate and transport metal ions is an important goal in the field of bioinorganic chemistry, a frontier research area that resides at the interface of chemistry and biology. This Current Topic reviews recent advances from the authors' group in using single-molecule fluorescence imaging techniques to identify the mechanisms of metal homeostatic proteins, including metalloregulators and metallochaperones. It emphasizes the novel mechanistic insights into how dynamic protein-DNA and protein-protein interactions offer efficient pathways via which MerR-family metalloregulators and copper chaperones can fulfill their functions. This work also summarizes other related single-molecule studies of bioinorganic systems and provides an outlook toward single-molecule imaging of metalloprotein functions in living cells.

Single-molecule catalysis mapping quantifies site-specific activity and uncovers radial activity gradient on single 2D nanocrystals.

Shape-controlled metal nanocrystals are a new generation of nanoscale catalysts. Depending on their shapes, these nanocrystals exhibit various surface facets, and the assignments of their surface facets have routinely been used to rationalize or predict their catalytic activity in a variety of chemical transformations. Recently we discovered that for 1-dimensional (1D) nanocrystals (Au nanorods), the catalytic activity is not constant along the same side facets of single nanorods but rather differs significantly and further shows a gradient along its length, which we attributed to an underlying gradient of surface defect density resulting from their linear decay in growth rate during synthesis (Nat. Nanotechnol.2012, 7, 237-241). Here we report that this behavior also extends to 2D nanocrystals, even for a different catalytic reaction. By using super-resolution fluorescence microscopy to map out the locations of catalytic events within individual triangular and hexagonal Au nanoplates in correlation with scanning electron microscopy, we find that the catalytic activity within the flat {111} surface facet of a Au nanoplate exhibits a 2D radial gradient from the center toward the edges. We propose that this activity gradient results from a growth-dependent surface defect distribution. We also quantify the site-specific activity at different regions within a nanoplate: The corner regions have the highest activity, followed by the edge regions and then the flat surface facets. These discoveries highlight the spatial complexity of catalytic activity at the nanoscale as well as the interplay amid nanocrystal growth, morphology, and surface defects in determining nanocatalyst properties.

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator.

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

Quantitative super-resolution imaging uncovers reactivity patterns on single nanocatalysts.

Metal nanoparticles are used as catalysts in a variety of important chemical reactions, and can have a range of different shapes, with facets and sites that differ in catalytic reactivity. To develop better catalysts it is necessary to determine where catalysis occurs on such nanoparticles and what structures are more reactive. Surface science experiments or theory can be used to predict the reactivity of surfaces with a known structure, and the reactivity of nanocatalysts can often be rationalized from a knowledge of their well-defined surface facets. Here, we show that a knowledge of the surface facets of a gold nanorod catalyst is insufficient to predict its reactivity, and we must also consider defects on the surface of the nanorod. We use super-resolution fluorescence microscopy to quantify the catalysis of the nanorods at a temporal resolution of a single catalytic reaction and a spatial resolution of ∼40 nm. We find that within the same surface facets on the sides of a single nanorod, the reactivity is not constant and exhibits a gradient from the centre of the nanorod towards its two ends. Furthermore, the ratio of the reactivity at the ends of the nanorod to the reactivity at the sides varies significantly from nanorod to nanorod, even though they all have the same surface facets.

Single-molecule fluorescence imaging of nanocatalytic processes.

This tutorial review covers recent developments in using single-molecule fluorescence microscopy to study nanoscale catalysis. The single-molecule approach enables following catalytic and electrocatalytic reactions on nanocatalysts, including metal nanoparticles and carbon nanotubes, at single-reaction temporal resolution and nanometer spatial precision. Real-time, in situ, multiplexed measurements are readily achievable under ambient solution conditions. These studies provide unprecedented insights into catalytic mechanism, reactivity, selectivity, and dynamics in spite of the inhomogeneity and temporal variations of catalyst structures. Prospects, generality, and limitations of the single-molecule fluorescence approach for studying nanocatalysis are also discussed.

Tackling metal regulation and transport at the single-molecule level.

To maintain normal metal metabolism, organisms utilize dynamic cooperation of many biomacromolecules for regulating metal ion concentrations and bioavailability. How these biomacromolecules work together to achieve their functions is largely unclear. For example, how do metalloregulators and DNA interact dynamically to control gene expression to maintain healthy cellular metal level? And how do metal transporters collaborate dynamically to deliver metal ions? Here we review recent advances in studying the dynamic interactions of macromolecular machineries for metal regulation and transport at the single-molecule level: (1) The development of engineered DNA Holliday junctions as single-molecule reporters for metalloregulator-DNA interactions, focusing onMerR-family regulators. And (2) The development of nanovesicle trapping coupled with single molecule fluorescence resonance energy transfer (smFRET) for studying weak, transient interactions between the copper chaperone Hah1 and the Wilson disease protein. We describe the methodologies,the information content of the single-molecule results, and the insights into the biological functions of the involved biomacromolecules for metal regulation and transport. We also discuss remaining challenges from our perspective.

Single-molecule study of metalloregulator CueR-DNA interactions using engineered Holliday junctions.

To maintain normal metal metabolism, bacteria use metal-sensing metalloregulators to control transcription of metal resistance genes. Depending on their metal-binding states, the MerR-family metalloregulators change their interactions with DNA to suppress or activate transcription. To understand their functions fundamentally, we study how CueR, a Cu(1+)-responsive MerR-family metalloregulator, interacts with DNA, using an engineered DNA Holliday junction (HJ) as a protein-DNA interaction reporter in single-molecule fluorescence resonance energy transfer measurements. By analyzing the single-molecule structural dynamics of the engineered HJ in the presence of various concentrations of both apo- and holo-CueR, we show how CueR interacts with the two conformers of the engineered HJ, forming variable protein-DNA complexes at different protein concentrations and changing the HJ structures. We also show how apo- and holo-CueR differ in their interactions with DNA, and discuss their similarities and differences with other MerR-family metalloregulators. The surprising finding that holo-CueR binds more strongly to DNA than to apo-CueR suggests functional differences among MerR-family metalloregulators, in particular in their mechanisms of switching off gene transcription after activation. The study also corroborates the general applicability of engineered HJs as single-molecule reporters for protein-DNA interactions, which are fundamental processes in gene replication, transcription, recombination, and regulation.

Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.