Evaluating Phospholipid-Functionalized Gold Nanorods for In Vivo Applications.

Gold nanorods (AuNRs) have attracted a great deal of attention due to their potential for use in a wide range of biomedical applications. However, their production typically requires the use of the relatively toxic cationic surfactant cetyltrimethylammonium bromide (CTAB) leading to continued demand for protocols to detoxify them for in vivo applications. In this study, a robust and facile protocol for the displacement of CTAB from the surface of AuNRs using phospholipids is presented. After the displacement, CTAB is not detectable by NMR spectroscopy, surface-enhanced Raman spectroscopy, or using pH-dependent ζ-potential measurements. The phospholipid functionalized AuNRs demonstrated superior stability and biocompatibility (IC50  > 200 µg mL-1 ) compared to both CTAB and polyelectrolyte functionalized AuNRs and are well tolerated in vivo. Furthermore, they have high near-infrared (NIR) absorbance and produce large amounts of heat under NIR illumination, hence such particles are well suited for plasmonic medical applications.

Production of giant unilamellar vesicles and encapsulation of lyotropic nematic liquid crystals.

We describe a modified microfluidic method for making Giant Unilamellar Vesicles (GUVs) via water/octanol-lipid/water double emulsion droplets. At a high enough lipid concentration we show that the de-wetting of the octanol from these droplets occurs spontaneously (off-chip) without the need to use shear to aid the de-wetting process. The resultant mixture of octanol droplets and GUVs can be separated by making use of the buoyancy of the octanol. A simpler microfluidic device and pump system can be employed and, because of the higher flow-rates and much higher rate of formation of the double emulsion droplets (∼1500 s-1 compared to up to ∼75 s-1), it is easier to make larger numbers of GUVs and larger volumes of solution. Because of the potential for using GUVs that incorporate lyotropic nematic liquid crystals in biosensors we have used this method to make GUVs that incorporate the nematic phases of sunset yellow and disodium chromoglycate. However, the phase behaviour of these lyotropic liquid crystals is quite sensitive to concentration and we found that there is an unexpected spread in the concentration of the contents of the GUVs obtained.

Targeted microbubbles carrying lipid-oil-nanodroplets for ultrasound-triggered delivery of the hydrophobic drug, combretastatin A4.

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

Control of Director Fields in Phospholipid-Coated Liquid Crystal Droplets.

In liquid crystal (LC) droplets, small changes in surface anchoring energy can produce large changes in the director field which result in readily detectable optical effects. This makes them attractive for use as biosensors. Coating LC droplets with a phospholipid monolayer provides a bridge between the hydrophobic world of LCs and the water-based world of biology and makes it possible to incorporate naturally occurring biosensor systems. However, phospholipids promote strong perpendicular (homeotropic) anchoring that can inhibit switching of the director field. We show that the tendency for phospholipid layers to promote perpendicular anchoring can be suppressed by using synthetic phospholipids in which the acyl chains are terminated with bulky tert-butyl or ferrocenyl groups; the larger these end-group(s), the less likely the system is to be perpendicular/radial. Additionally, the droplet director field is found to be dependent on the nature of the LC, particularly its intrinsic surface properties, but not (apparently) on the sign of the dielectric anisotropy, the proximity to the melting/isotropic phase transition, the surface tension (in air), or the values of the Frank elastic constants.

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.

Synthesis and organogelating behaviour of amino acid-functionalised triphenylenes.

Four novel amino acid-functionalised triphenylenes have been prepared with glycine, l-alanine, l-phenylalanine and l-tryptophan ethyl ester side-chains. The glycine derivative is a good gelator of chloroform, the alanine derivative gels ethanol and toluene, and the phenylalanine derivative gels benzene and toluene. The tryptophan derivative does not gel any of the solvents tested, most probably due to its more bulky structure, but forms microspheres by evaporation-induced self-assembly. The self-assembly properties of the π-gelators have been investigated using infrared, UV-absorption and fluorescence spectroscopy, concentration- and temperature-dependent NMR, and X-ray scattering experiments on dried xerogel as well as the wet organogel. The latter experiments suggest the glycine gel in chloroform includes columnar aggregates, with an overall disordered columnar oblique mesophase. These compounds are of interest because of the well-known hole-transporting properties of triphenylene liquid crystals: 1-D columnar assemblies of these compounds may find applications in organic electronic devices.

New poly(amino acid methacrylate) brush supports the formation of well-defined lipid membranes.

A novel poly(amino acid methacrylate) brush comprising zwitterionic cysteine groups (PCysMA) was utilized as a support for lipid bilayers. The polymer brush provides a 12-nm-thick cushion between the underlying hard support and the aqueous phase. At neutral pH, the zeta potential of the PCysMA brush was ∼-10 mV. Cationic vesicles containing >25% DOTAP were found to form a homogeneous lipid bilayer, as determined by a combination of surface analytical techniques. The lipid mobility as measured by FRAP (fluorescence recovery after photobleaching) gave diffusion coefficients of ∼1.5 µm(2) s(-1), which are comparable to those observed for lipid bilayers on glass substrates.

Maleimide-Thiol Linkages Alter the Biodistribution of SN38 Therapeutic Microbubbles Compared to Biotin-Avidin While Preserving Parity in Tumoral Drug Delivery.

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome-MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide-thiol linkage, that is currently used in antibody-drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety.

Spectrophotometric Analysis and Optimization of 2D Gold Nanosheet Formation.

Free-standing, 2D gold nanosheets (AuNS) offer broad potential applications from computing to biosensing and healthcare. Such applications, however, require improved control of material growth. We recently reported the synthesis of AuNS only ∼0.47 nm (two atoms) thick, which exhibited very high catalytic activity. The synthesis is a one-pot, seedless procedure in which chloroauric acid is reduced by sodium citrate in the presence of methyl orange (MO). In this study, we use spectrophotometric analysis and TEM imaging to probe AuNS formation and optimize the procedure. Previously, we suggested that MO acted as the confining agent, directing two-dimensional growth of the gold. Here, we provide the first reported analysis of the HAuCl4 and MO reaction. We show that MO is rapidly oxidized to give 4-diazobenzenesulfonic acid, indicating that a complex interplay between HAuCl4, MO, and other reaction products leads to AuNS formation. Time-resolved studies indicate that synthesis time can be significantly reduced from over 12 to 2-3 h. Decreasing the reaction temperature from 20 to 4 °C improved AuNS yield by 16-fold, and the catalytic activity of the optimized material matches that obtained previously. Our elucidation of AuNS formation mechanisms has opened avenues to further improve and tune the synthesis, enhancing the potential applications of ultrathin AuNS.

On-chip alternating current electrophoresis in supported lipid bilayer membranes.

By forming lipid bilayers within SU8 patterns, between interdigitated electrodes, we have demonstrated that it is possible to manipulate charged membrane components using low applied voltages over relatively short time scales. Two distinct patterns were studied: a nested "fish trap" which served as a molecular trap, and a diffusion aided Brownian ratchet which operated as a molecular pump. By reducing the size of the structures we have demonstrated that large applied fields (>200 V/cm) can be achieved on-chip, using low applied potentials (<13 V). By using ac fields applied orthogonal to the direction of desired motion, the molecular pumps provide a voltage independent method for moving charged components within lipid membranes over large distances. The reduced scale of the trap structures compared to those previously used in our laboratory has led to over a 10-fold decrease in the operational time require for charge build-up, from 16 h down to 1.5 h. The observed benefits of scaling means that these systems should be suitable for the on-chip separation and manipulation of charged species within supported lipid membranes.

Improved syntheses of high hole mobility phthalocyanines: A case of steric assistance in the cyclo-oligomerisation of phthalonitriles.

It has been shown that the base-initiated cyclo-oligomerisation of phthalonitriles is favoured by bulky α-substituents making it possible to obtain the metal-free phthalocyanine directly and in high yield. The phthalocyanine with eight α-isoheptyl substituents gives a high time-of-flight hole mobility of 0.14 cm(2)·V(-1)·s(-1) within the temperature range of the columnar hexagonal phase, that is 169-189 °C.

Chiral nematic liquid crystal droplets as a basis for sensor systems.

For a series of phospholipid coated calamitic nematic liquid crystal droplets (5CB, 6CB, 7CB, E7 and MLC7023) of diameter ∼18 µm, the addition of chiral dopant leaves the sign of surface anchoring unchanged. Herein we report that for these chiral nematic droplets an analyte induced transition from a Frank-Pryce structure (with planar anchoring) to a nested-cup structure (with perpendicular anchoring) is accompanied by changes in the intensity of reflected light. We propose this system as both a general scheme for understanding director fields in chiral nematic liquid crystal droplets with perpendicular anchoring and as an ideal candidate to be utilised as the basis for developing cheap, single use LC-based sensor devices.

A study of cytochrome bo3 in a tethered bilayer lipid membrane.

An assay has been developed in which the activity of an ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), is determined as a function of the hydrophobic substrate ubiquinol-10 (UQ-10) in tethered bilayer lipid membranes (tBLMs). UQ-10 was added in situ, while the enzyme activity and the UQ-10 concentration in the membrane have been determined by cyclic voltammetry. Cbo(3) is inhibited by UQ-10 at concentrations above 5-10 pmol/cm(2), while product inhibition is absent. Cyclic voltammetry has also been used to characterise the effects of three inhibitors; cyanide, inhibiting oxygen reduction; 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibiting the quinone oxidation and Zn(II), thought to block the proton channels required for oxygen reduction and proton pumping activity. The electrochemical behaviour of cbo(3) inhibited with HQNO and Zn(II) is almost identical, suggesting that Zn(II) ions inhibit the enzyme reduction by quinol, rather than oxygen reduction. This suggests that at Zn(II) concentration below 50µM the proton release of cbo(3) is inhibited, but not the proton uptake required to reduce oxygen to water.

Concentrating membrane proteins using asymmetric traps and AC electric fields.

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.

Effect of the structure of cholesterol-based tethered bilayer lipid membranes on ionophore activity.

Tethered bilayer lipid membranes (tBLM) are formed on 1) pure tether lipid triethyleneoxythiol cholesterol (EO(3)C) or on 2) mixed self-assembled monolayers (SAMs) of EO(3)C and 6-mercaptohexanol (6MH). While EO(3)C is required to form a tBLM with high resistivity, 6MH dilutes the cholesterol content in the lower leaflet of the bilayer forming ionic reservoirs required for submembrane hydration. Here we show that these ionic reservoirs are required for ion transport through gramicidin or valinomycin, most likely due to the thermodynamic requirements of ions to be solvated once transported through the membrane. Unexpectedly, electrochemical impedance spectroscopy (EIS) shows an increase of capacitance upon addition of gramicidin, while addition of valinomycin decreases the membrane resistance in the presence of K(+) ions. We hypothesise that this is due to previously reported phase separation of EO(3)C and 6MH on the surface. This results in ionic reservoirs on the nanometre scale, which are not fully accounted for by the equivalent circuits used to describe the system.

Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane.

We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo(3) (cytochrome bo(3)), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo(3) substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo(3) activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo(3) mediated by UQ-10. The apparent K(m) for oxygen with this system is 1.1+/-0.4 microM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo(3). Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo(3) activity. The activity of cbo(3) with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis-Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.

Freeze-Dried Therapeutic Microbubbles: Stability and Gas Exchange.

Microbubbles (MBs) are widely used as contrast enhancement agents for ultrasound imaging and have the potential to enhance therapeutic delivery to diseases such as cancer. Yet, they are only stable in solution for a few hours to days after production, which limits their potential application. Freeze-drying provides long-term storage, ease of transport, and consistency in structure and composition, thereby facilitating their use in clinical settings. Therapeutic microbubbles (thMBs) consisting of MBs with attached therapeutic payload potentially face even greater issues for production, stability, and well-defined drug delivery. The ability to freeze-dry thMBs represents an important step for their translation to the clinic. Here, we show that it is possible to freeze-dry and reconstitute thMBs that consist of lipid-coated MBs with an attached liposomal payload. The thMBs were produced microfluidically, and the liposomes contained either calcein, as a model drug, or gemcitabine. The results show that drug-loaded thMBs can be freeze-dried and stored for at least 6 months. Upon reconstitution, they maintain their structural integrity and drug loading. Furthermore, we show that their in vivo echogenicity is maintained post-freeze-drying. Depending on the gas used in the original bubbles, we also demonstrate that the approach provides a method to exchange the gas core to allow the formulation of thMBs with different gases for combination therapies or improved drug efficacy. Importantly, this work provides an important route for the facile off-site production of thMBs that can be reformulated at the point of care.

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.

A cholesterol-based tether for creating photopatterned lipid membrane arrays on both a silica and gold surface.

Surfing gold and silica! A new cholesterol-based tether was attached to both amine-functionalised silica and gold surfaces. The resultant self-assembled monolayers, which can be deep-UV-patterned, were used in attaching essentially equivalent patterned supported lipid bilayers both on silica for fluorescence studies (left figure) and on gold for impedance studies (right figure).We report a new cholesterol-based self-assembled monolayer (SAM) for use in attaching lipid membranes to both gold and silica surfaces that can be patterned by deep UV (254 nm) photolysis. It allows essentially equivalent patterned supported bilayers to be created both on gold for impedance studies and on silica for fluorescence studies. On either surface an amine-functionalised SAM is reacted with an N-hydroxysuccinimidylcarbonyl-functionalised EO3-cholesteryl derivative. The formation of the amine-functionalised SAM and its reaction with the EO3-cholesteryl derivative were followed by contact-angle measurements, ellipsometry and X-ray photoelectron spectroscopy. The resultant layer of cholesterol tethers was patterned by deep UV photolysis, which regenerates the original SiO(2) surface in exposed regions on a silica substrate and oxidises thiol groups on a gold substrate. This patterned surface containing hydrophilic SiO(2) (or -OH groups) and hydrophobic cholesterol tether regions can be converted to a surface patterned with supported lipid bi- and monolayers (respectively) by immersing in a solution of small unilamellar vesicles of egg yolk phosphatidycholine. The formation of the lipid bi- and monolayer regions on the silica surface was evidenced by fluorescence microscopy. Crucially the bilayer regions remain fully fluid yielding lipid mobilities comparable to those found in physisorbed bilayers. Furthermore charged fluorescent lipids are shown to migrate in an applied field thus providing a platform for the studying the electrophoresis (potentially) for a wide range of charged membrane components, such as membrane proteins. The formation of the patterned lipid membrane on the gold surface was confirmed by electrochemical impedance measurements.

Lipid coated liquid crystal droplets for the on-chip detection of antimicrobial peptides.

We describe a novel biosensor based on phospholipid-coated nematic liquid crystal (LC) droplets and demonstrate the detection of Smp43, a model antimicrobial peptide (AMP) from the venom of North African scorpion Scorpio maurus palmatus. Mono-disperse lipid-coated LC droplets of diameter 16.7 ± 0.2 µm were generated using PDMS microfluidic devices with a flow-focusing configuration and were the target for AMPs. The droplets were trapped in a bespoke microfluidic trap structure and were simultaneously treated with Smp43 at gradient concentrations in six different chambers. The disruption of the lipid monolayer by the Smp43 was detected (<6 µM) at concentrations well within its biologically active range, indicated by a dramatic change in the appearance of the droplets associated with the transition from a typical radial configuration to a bipolar configuration, which is readily observed by polarizing microscopy. This suggests the system has feasibility as a drug-discovery screening tool. Further, compared to previously reported LC droplet biosensors, this LC droplet biosensor with a lipid coating is more biologically relevant and its ease of use in detecting membrane-related biological processes and interactions has the potential for development as a reliable, low-cost and disposable point of care diagnostic tool.