Molecular Effects of Glycerol on Lipid Monolayers at the Gas-Liquid Interface: Impact on Microbubble Physical and Mechanical Properties.

The production and stability of microbubbles (MBs) is enhanced by increasing the viscosity of both the formation and storage solution, respectively. Glycerol is a good candidate for biomedical applications of MBs, since it is biocompatible, although the exact molecular mechanisms of its action is not fully understood. Here, we investigate the influence glycerol has on lipid-shelled MB properties, using a range of techniques. Population lifetime and single bubble stability were studied using optical microscopy. Bubble stiffness measured by AFM compression is compared with lipid monolayer behavior in a Langmuir-Blodgett trough. We deduce that increasing glycerol concentrations enhances stability of MB populations through a 3-fold mechanism. First, binding of glycerol to lipid headgroups in the interfacial monolayer up to 10% glycerol increases MB stiffness but has limited impact on shell resistance to gas permeation and corresponding MB lifetime. Second, increased solution viscosity above 10% glycerol slows down the kinetics of gas transfer, markedly increasing MB stability. Third, above 10%, glycerol induces water structuring around the lipid monolayer, forming a glassy layer which also increases MB stiffness and resistance to gas loss. At 30% glycerol, the glassy layer is ablated, lowering the MB stiffness, but MB stability is further augmented. Although the molecular interactions of glycerol with the lipid monolayer modulate the MB lipid shell properties, MB lifetime continually increases from 0 to 30% glycerol, indicating that its viscosity is the dominant effect on MB solution stability. This three-fold action and biocompatibility makes glycerol ideal for therapeutic MB formation and storage and gives new insight into the action of glycerol on lipid monolayers at the gas-liquid interface.

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies.

Real Space Imaging of Nanoparticle Assembly at Liquid-Liquid Interfaces with Nanoscale Resolution.

Bottom up self-assembly of functional materials at liquid-liquid interfaces has recently emerged as method to design and produce novel two-dimensional (2D) nanostructured membranes and devices with tailored properties. Liquid-liquid interfaces can be seen as a "factory floor" for nanoparticle (NP) self-assembly, because NPs are driven there by a reduction of interfacial energy. Such 2D assembly can be characterized by reciprocal space techniques, namely X-ray and neutron scattering or reflectivity. These techniques have drawbacks, however, as the structural information is averaged over the finite size of the radiation beam and nonperiodic isolated assemblies in 3D or defects may not be easily detected. Real-space in situ imaging methods are more appropriate in this context, but they often suffer from limited resolution and underperform or fail when applied to challenging liquid-liquid interfaces. Here, we study the surfactant-induced assembly of SiO2 nanoparticle monolayers at a water-oil interface using in situ atomic force microscopy (AFM) achieving nanoscale resolved imaging capabilities. Hitherto, AFM imaging has been restricted to solid-liquid interfaces because applications to liquid interfaces have been hindered by their softness and intrinsic dynamics, requiring accurate sample preparation methods and nonconventional AFM operational schemes. Comparing both AFM and grazing incidence X-ray small angle scattering data, we unambiguously demonstrate correlation between real and reciprocal space structure determination showing that the average interfacial NP density is found to vary with surfactant concentration. Additionally, the interaction between the tip and the interface can be exploited to locally determine the acting interfacial interactions. This work opens up the way to studying complex nanostructure formation and phase behavior in a range of liquid-liquid and complex liquid interfaces.

Poly(ethylene glycol) lipid-shelled microbubbles: abundance, stability, and mechanical properties.

Poly(ethylene glycol) (PEG) is widely used on the outside of biomedical delivery vehicles to impart stealth properties. Encapsulated gas microbubbles (MBs) are being increasingly considered as effective carriers for therapeutic intervention to deliver drug payloads or genetic vectors. MBs have the advantage that they can be imaged and manipulated by ultrasound fields with great potential for targeted therapy and diagnostic purposes. Lipid-shelled MBs are biocompatible and can be functionalized on the outer surface for tissue targeting and new therapeutic methods. As MBs become a key route for drug delivery, exploring the effect of PEG-ylation on the MB properties is important. Here, we systematically investigate the effect of PEG-lipid solution concentration ranging between 0 and 35 mol % on the formation of MBs in a microfluidic flow-focusing device. The abundance of the MBs is correlated with the MB lifetime and the whole MB mechanical response, as measured by AFM compression using a tipless cantilever. The maximal MB concentration and stability (lifetime) occurs at a low concentration of PEG-lipid (∼5 mol %). For higher PEG-lipid concentrations, the lifetime and MB concentration decrease, and are accompanied by a correlation between the predicted surface PEG configuration and the whole MB stiffness, as measured at higher compression loads. These results inform the rationale design and fabrication of lipid-based MBs for therapeutic applications and suggest that only relatively small amounts of PEG incorporation are required for optimizing MB abundance and stability while retaining similar mechanical response at low loads.

Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging.

A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end of linear double-stranded (ds) DNA templates was developed. The loop structure serves as a fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied to study DNA-protein interactions. The nucleic acid end-labels allow discrimination of the polarity of the DNA template in the AFM while limiting non-specific interactions which might occur from non-nucleic acid labels. Homo-polynucleotide ssDNA loops made up of 20 base-pairs (bp) for each of the four bases (A, T, G, C) were investigated to determine the effects of sequence on template labelling. The products were produced with high efficiency and high yield with the loop readily distinguished from the dsDNA template by height and diameter in the AFM. The application of the method to study DNA transcription was investigated by firing Escherichia Coli RNA polymerase (RNAP) from a λPR promoter in the direction of the loop-labelled end. The ssDNA loops captured elongating complexes of RNAP, arresting transcription and preventing dissociation. The dual role of the loop as a polarity marker and retainer of previously active RNAP will allow mechanisms of gene expression to be studied with single molecule sensitivity by AFM. This will enable insight into molecular interactions of RNAP on single DNA templates in convergent or tandem transcription configurations.

Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

Nanomechanics of lipid encapsulated microbubbles with functional coatings.

Microbubbles (MBs) are increasingly being proposed as delivery vehicles for targeted therapeutics, as well as being contrast agents for ultrasound imaging. MBs formed with a lipid shell are promising candidates due to their biocompatibility and the opportunity for surface functionalization, both for specific targeting of tissues and as a means to tune their mechanical response for localized ultrasound induced destruction in vivo. Herein, we acquired force-deformation data on coated lipid MBs using tip-less microcantilevers in an atomic force microscope. Model lipid MBs were designed to test the effects of adding a functional coating on the outside of the lipid leaflet, including a protein coat (streptavidin) or the addition of quantum dots (Q-dots) as optical reporters. MBs (~3 µm diameter) were repeatedly compressed for deformations up to ~50% to obtain a full bubble response. Addition of a coating increased the initial deformation stiffness related to shell bending ~2-fold for streptavidin and ∼3-fold for Q-dots. The presence of a polyethylene glycol (PEG) linker in between the lipid and functional coating, led to enhanced stiffening at high deformations. The plasticity index has been determined and only those MBs that included the PEG linker showed a force dependent short time-scale (<~1s) plasticity. This study demonstrates modulation of the mechanical response of biocompatible MBs through the addition of functional coatings necessary for rationale design of therapeutic lipid MBs for targeted drug delivery.

Single-molecule studies of DNA transcription using atomic force microscopy.

Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA-protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome.

Measuring the true height of water films on surfaces.

Measuring the level of hydrophilicity of heterogeneous surfaces and the true height of water layers that form on them in hydrated conditions has a myriad of applications in a wide range of scientific and technological fields. Here, we describe a true non-contact mode of operation of atomic force microscopy in ambient conditions and a method to establish the source of apparent height. A dependency of the measured water height on operational parameters is identified with water perturbations due to uncontrolled modes of imaging where intermittent contact with the water layer, or even the surface, might occur. In this paper we show how to (1) determine when the water is being perturbed and (2) distinguish between four different interaction regimes. Each of the four types of interaction produces measurements ranging from fractions of the true height in one extreme to values which are as large as four times the real height in the other. We show the dependence of apparent height on the interaction regime both theoretically and empirically. The agreement between theory and experiment on a BaF2(111) sample displaying wet and un-wet regions validates our results.

How localized are energy dissipation processes in nanoscale interactions?

We describe fundamental energy dissipation in dynamic nanoscale processes in terms of the localization of the interactions. In this respect, the areal density of the energy dissipated per cycle and the effective area of interaction in which each process occurs are calculated for four elementary dissipative processes. It is the ratio between these two, which we term M, that provides information about how localized the interactions are. While our results are general, we use concepts from dynamic atomic force microscopy to describe the physical phenomenon. We show that neither the phase lag, nor the magnitude of the energy dissipated alone provide information about how dissipative processes are localized. Instead, M has to be considered.

Bi-stability of amplitude modulation AFM in air: deterministic and stochastic outcomes for imaging biomolecular systems.

The dynamics of the oscillating microcantilever for amplitude modulation atomic force microscopy (AM AFM) operating in air is well understood theoretically but the experimental outcomes are still emerging. We use double-stranded DNA on mica as a model biomolecular system for investigating the connection between theory and experiment. A demonstration that the switching between the two cantilever oscillation states is stochastic in nature is achieved, and it can be induced by means of topographical anomalies on the surface. Whether one or the other attractor basin is accessed depends on the tip-sample separation history used to achieve the imaging conditions, and we show that the behaviour is reproducible when the tip is stable and well characterized. Emergence of background noise occurs in certain regions of parameter space regardless of whether two cantilever oscillation states coexist. The low state has been explored in detail and we note that at low to intermediate values of the free amplitude, noise-free imaging is achieved. The outcomes shown here are general and demonstrate that a thorough and systematic experimental approach in conjunction with standard modelling gives insight into the mechanisms behind image contrast formation in AM AFM in air.

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules.

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.

Ultrasound-triggered therapeutic microbubbles enhance the efficacy of cytotoxic drugs by increasing circulation and tumor drug accumulation and limiting bioavailability and toxicity in normal tissues.

Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.

Tuning the elastic modulus of hydrated collagen fibrils.

Systematic variation of solution conditions reveals that the elastic modulus (E) of individual collagen fibrils can be varied over a range of 2-200 MPa. Nanoindentation of reconstituted bovine Achilles tendon fibrils by atomic force microscopy (AFM) under different aqueous and ethanol environments was carried out. Titration of monovalent salts up to a concentration of 1 M at pH 7 causes E to increase from 2 to 5 MPa. This stiffening effect is more pronounced at lower pH where, at pH 5, e.g., there is an approximately 7-fold increase in modulus on addition of 1 M KCl. An even larger increase in modulus, up to approximately 200 MPa, can be achieved by using increasing concentrations of ethanol. Taken together, these results indicate that there are a number of intermolecular forces between tropocollagen monomers that govern the elastic response. These include hydration forces and hydrogen bonding, ion pairs, and possibly the hydrophobic effect. Tuning of the relative strengths of these forces allows rational tuning of the elastic modulus of the fibrils.

Sub-Nanometer Thick Gold Nanosheets as Highly Efficient Catalysts.

2D metal nanomaterials offer exciting prospects in terms of their properties and functions. However, the ambient aqueous synthesis of atomically-thin, 2D metallic nanomaterials represents a significant challenge. Herein, freestanding and atomically-thin gold nanosheets with a thickness of only 0.47 nm (two atomic layers thick) are synthesized via a one-step aqueous approach at 20 °C, using methyl orange as a confining agent. Owing to the high surface-area-to-volume ratio, abundance of unsaturated atoms exposed on the surface and large interfacial areas arising from their ultrathin 2D nature, the as-prepared Au nanosheets demonstrate excellent catalysis performance in the model reaction of 4-nitrophenol reduction, and remarkable peroxidase-mimicking activity, which enables a highly sensitive colorimetric sensing of H2O2 with a detection limit of 0.11 × 10-6 m. This work represents the first fabrication of freestanding 2D gold with a sub-nanometer thickness, opens up an innovative pathway toward atomically-thin metal nanomaterials that can serve as model systems for inspiring fundamental advances in materials science, and holds potential across a wide region of applications.

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid.

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.

Amyloid under the atomic force microscope.

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.

Studying silane mobility on hydrated mica using ambient AFM.

The mobility of the mono-functional aminosilane, aminopropyldimethylmethoxysilane (APDMMS), on mica has been investigated under ambient relative humidity (RH) using tapping-mode (TM) AFM. Silane layers were formed from vapour phase at various, controlled humidities and then imaged at ambient laboratory conditions (typically 30-40% RH). At low RH of formation (<25%) films without any resolvable sub-structure were formed, and these were stable to the imaging probe. At high RH of formation (>25%), where islands of phase II water are known to exist on mica, a two-phase domain structure was seen as two height levels and the surface area of the higher domains correlated with the RH. Sequential images taken over a 30-60 min time period show that these domains are mobile and at intermediate to high RH the domains coalesce under the influence of the scanning AFM tip. The results suggest the APDMMS resides at the air-water interface (rather than interacting with the mica) and that it preferentially interacts with the mobile phase II water as opposed to the phase I water tightly bound to the mica.

Nested genes: biological implications and use of AFM for analysis.

A "nested" gene is located within the boundaries of a larger gene, often within an intron and in the opposite orientation. Such structures are common in bacteria and viruses, but have also been described in higher species as diverse as Drosophila and humans. Expression of nested and host genes may be simultaneously up-regulated due to use of common enhancers, or down-regulated through steric hindrance or interference caused by annealing of the complementary RNAs, leading to degradation. Methods for RNA analysis such as RT-PCR and in situ hybridization reveal the presence of specific mRNAs, but do not address regulation of expression within a single cell at a single genetic locus. Atomic force microscopy is a relatively new technology, which allows visualization of the movement of an RNA polymerase along a DNA template. The potential of this technology includes a greater molecular understanding of cellular decision making processes, leading to enhanced opportunities to intervene in disease progression through use of novel treatment modalities.