When irradiated at its resonance frequency, a metallic nanoparticle efficiently converts the abso... more When irradiated at its resonance frequency, a metallic nanoparticle efficiently converts the absorbed energy into heat which is locally dissipated. This effect can be used in photothermal treatments, e.g., of cancer cells. However, to fully exploit the functionality of metallic nanoparticles as nanoscopic heat transducers, it is essential to know how the photothermal efficiency depends on parameters like size and shape. Here we present the measurements of the temperature profile around single irradiated gold nanorods and nanospheres placed on a biologically relevant matrix, a lipid bilayer. We developed a novel assay based on molecular partitioning between two coexisting phases, the gel and liquid phase, within the bilayer. This assay allows for a direct measurement of local temperature gradients, an assay which does not necessitate any pre-assumptions about this system and is generally applicable to any irradiated nanoparticle system. The nanorods are irradiated with a tightly focused laser beam at a wavelength of 1064 nm where biological matter exhibits a minimum in absorption. By controlling the polarization of the laser light we show that the absorption of light by the nanorod and the corresponding dissipated heat strongly depends on the orientation of the nanorod with respect to the polarization. Finally, by comparing to spherical gold nanopartilces we demonstrate how a change in shape, from spherical to rod like, leads to a dramatic enhancement of heating when using near infrared light.
Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotrans... more Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.
We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while t... more We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while they are not adhesive at pH values higher than 6. This is significant to membrane fusion occurring at low pH, and to membrane experiments using lipid vesicles. We used the experimental method invented by Evans and collaborators in which one flaccid GUV was released to adhere to one tensed GUV. We developed a new analysis method to measure the adhesion energy per unit area. This new method is independent of how the adhesion state was reached. The order of magnitude of the adhesion energy is~0.01 tõ 0.02 erg/cm2 for SOPC. The addition of SOPE slightly decreases the adhesion energy of pure SOPC, while the addition of cholesterol has little effect. The same method of measurement was applied to a case where two lipid bilayers underwent the first step of membrane fusion, called hemifusion. Hemifusion was induced by injecting 5 wt % PEG8000 solution at pH 4. The PEG injection was used to produce a transient osmotic depletion attraction between the two GUVs. The energy of hemifusion is one order of magnitude larger than the adhesion energy, about~0.3 erg/cm2 for DOPC/DOPE/cholesterol (4:4:2). This is the first time the free energy of the membrane fusion intermediate state was experimentally measured.
Obesity has become a major public health concern and represents a predisposition factor for the d... more Obesity has become a major public health concern and represents a predisposition factor for the development of cardiovascular-related diseases and noninsulin dependent diabetes mellitus. Therefore, deciphering the molecular mechanisms of fatty acid (FA) uptake will provide new insights for dietary and other therapeutic interventions for managing diseases associated with obesity. FA uptake into cells occurs by multiple mechanisms, including transport and metabolism, beginning at the plasma membrane. Understanding the contributions of passive diffusion and facilitated transport by plasma membrane proteins, such as FAT/CD36, requires experimental approaches that separate biophysical and metabolic mechanisms. In previous experiments with protein-free lipid bilayers, we have used multiple fluorescence assays to show that FA bind to, and diffuse through, phospholipid bilayers very rapidly but desorb more slowly from the lipid into the aqueous phase. Here we apply these methods to HEK293 cells engineered to stably express CD36. FA movement across the plasma membrane occurred rapidly (within sec) with or without expression of CD36. HEK293 cells without CD36 exhibit very slow conversion of FA into acetylated products. However, incorporation of 14C-labeled oleic acid into triglycerides occurred more rapidly and was significantly increased in HEK293 cells overexpressing CD36. Formation of small lipid droplets was observed after incubation with a fluorescent FA analog BODIPY-FA. Thus, FA transport through plasma membranes occurred by the mechanism of diffusion without a requirement for CD36. It appears that this protein enhanced metabolism by an as yet unknown mechanism. The study of lateral dynamics in corrals has attracted atention since 1983 when Sheetz introduced the corral model. In this project, we propose a simple model consisting of a two-dimensional lattice containing periodically distributed flashing walls. These walls are represented by finite asymmetrical flashing potentials which isolate a portion of the lattice to represent semipermeable corrals. Contigous corrals share a wall creating an arrangement of similar compartments on the lattice. We included the presence of a constant external field to account for the effect of gradients, adding another degree of complexity to the dynamics of a single particle in this medium. We derive general analytical expressions to describe the diffusion coefficient (D) and the current (J) of a single particle moving on this medium. We use a formulation based on a single particle microscopic model and a diffusion relaxation condition to derive our equations as a function of the corral's size (and concentration), the time between flashes, and the strength of the external field. We compare our theory against Monte Carlo simulations.
Optically trapped plasmonic nano-heaters are used to mediate efficient and controlled fusion of b... more Optically trapped plasmonic nano-heaters are used to mediate efficient and controlled fusion of biological membranes. The fusion method is demonstrated by optically trapping plasmonic nanoparticles located in between vesicle membranes leading to rapid lipid and content mixing. As an interesting application we show how direct control over fusion can be used for studying diffusion of peripheral membrane proteins and their interactions with membranes and for studying protein reactions. Membrane proteins encapsulated in an inert vesicle can be transferred to a vesicle composed of negative lipids by optically induced fusion. Mixing of the two membranes results in a fused vesicle with a high affinity for the protein and we observe immediate membrane tubulation due to the activity of the protein. Fusion of distinct membrane compartments also has applications in small scale chemistry for realizing pico-liter reactions and offers many exciting applications within biology which are discussed here.
Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We loca... more Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We locally heated the membranes of two adjacent vesicles by laser irradiating gold nanoparticles, thus causing vesicle fusion with associated membrane and cargo mixing. The mixing time scales were consistent with diffusive mixing of the membrane dyes and the aqueous content. This method is useful for nanoscale reactions as demonstrated here by I-BAR protein-mediated membrane tubulation triggered by fusion. T he fusion of the cargos of two selected vesicles allows for controlled nanoscale reactions with femtoliter volumes, thus paving the way for single-or few-molecule reactions. This is highly useful for studying the dynamics of chemical reactions. 1 Fusion of vesicles, or fusion of vesicles with cells is the heart of liposome based targeted drug delivery, a topic of large medical interest. Fusion of one cell with another allows for the creation of hybrid cells that combines the properties of several cell types. Examples include (i) hybridoma technology, 2 which can be used to create monoclonal antibodies, (ii) combination of stem cells with differentiated cells with the potential for novel diabetes treatments through pancreatic islet transplantation, 3 or (iii) the fusion of dendritic cells to a triply negative breast cancer cell, which can be exploited for vaccine creation. 4 As fusion of vesicles and cells is of high interest, considerable effort has been put into developing efficient methods for fusion. There exists an extensive library of biological and chemical molecules that trigger fusion of cells and vesicles, for instance, cellular expressed fusogenic proteins, 5 PEG-polymers incorporated into the membranes, 6 lanthanide salts, 7 viral-based fusion peptides, 8 synaptic SNARE-mediated fusion complexes, 9 or synthetic molecular fusion complexes based on nucleotides. 10 However, fusion can also be mediated by physical means, for instance, via electrofusion 1,3,4,11 or by locally irradiating ultraviolet (UV) light on the contact area between two membranes. Cell-cell fusion can be accomplished by irradiating a cell population in medium with a high powered pulsed UV laser, 12,13 however, with limited control over the system. A more sophisticated implementation of the pulsed UV laser-mediated fusion was demonstrated for immune cells that were brought into contact via an antibody-conjugated nano-particle. 14 The use of intense UV-laser pulses causes generation of highly reactive free radicals, which is an undesirable side-effect when dealing with live cell samples. Although such lasers are focused to a diffraction limited spot, they exhibit high divergence and still illuminate a substantial part of the cells or GUVs (giant unilamellar vesicles) both below and above the focal spot. Here, we report on a novel and efficient method for triggering fusion of two vesicles with one critical benefit being that two vesicles can be specifically chosen among a population. The method is based on optical trapping of a metallic nanoparticle by near-infrared (NIR) light that is essentially harmless to biological material. The metallic nanoparticle will absorb part of the NIR light and the absorbed energy will be dissipated as heat in the surroundings on a length scale comparable to the diameter of the particle, 15,16 and it is this local temperature elevation that triggers membrane heating, expansion, and fusion, possibly by opening a fusion pore. Using the optical trap, two selected GUVs are manipulated and brought into close proximity. 17 After the two selected GUVs are brought into contact, the trapping of a gold nanoparticle (AuNP) in the contact zone between the GUVs causes the two vesicles to fuse by a thermally triggered mechanism. This fusion causes the membranes and the cargos of the two vesicles to mix. In contrast to fusion methods based on UV lasers, essentially no biological damage is done above nor below the focal volume. Importantly, the process can be followed real-time in a microscope. To demonstrate the general applicability of this method we also prove fusion of GUVs existing in gel and fluid phases, respectively. Finally, as a relevant biophysical application we show how protein-mediated membrane shaping
Plasmonic nanoparticle-based photothermal cancer therapy is a promising new tool to inflict local... more Plasmonic nanoparticle-based photothermal cancer therapy is a promising new tool to inflict localized and irreversible damage to tumor tissue by hyperthermia, without harming surrounding healthy tissue. We developed a single particle and positron emission tomography (PET)-based platform to quantitatively correlate the heat generation of plasmonic nanoparticles with their potential as cancer killing agents. In vitro, the heat generation and absorption cross-section of single irradiated nanoparticles were quantified using a temperature sensitive lipid-based assay and compared to their theoretically predicted photo-absorption. In vivo, the heat generation of irradiated nanoparticles was evaluated in human tumor xenografts in mice using 2-deoxy-2-[F-18]fluoro-D-glucose (18 F-FDG) PET imaging. To validate the use of this platform, we quantified the photothermal efficiency of near infrared resonant silica-gold nanoshells (AuNSs) and benchmarked this against the heating of colloidal spherical, solid gold nanoparticles (AuNPs). As expected, both in vitro and in vivo the heat generation of the resonant AuNSs performed superior compared to the non-resonant AuNPs. Furthermore, the results showed that PET imaging could be reliably used to monitor early treatment response of photothermal treatment. This multidisciplinary approach provides a much needed platform to benchmark the emerging plethora of novel plasmonic nanoparticles for their potential for photothermal cancer therapy. Photothermal therapy, which involves the application of plasmonic nanoparticles as light-triggered thermal transducers 1–5 , has emerged as a promising cancer treatment strategy within the last decade 6–9 , and much effort is being put into developing the most efficient light-to-heat converters 10–15. The temperatures generated upon laser irradiation of plasmonic nanoparticles are so extreme 16 that nearby cancer cells immediately ablate or become irreversibly damaged; meanwhile the heating is sufficiently localized that surrounding tissue is left unharmed 17–20. The strongly enhanced photoabsorption properties of plasmonic nanoparticles are a consequence of a phenomenon known as surface plasmon resonance; the collective oscillations of the plasma electrons when resonant with the excitation light 21. The absorption spectrum of a plasmonic nanoparticle can be tuned by changing the size, shape and composition of the nanoparticle 12,22–25 , and in particular near infrared (NIR) resonant nanoparticles are of interest for photothermal therapy due to the efficient penetration and low absorption of NIR light in tissue 26. A second factor crucial for photothermal therapy is delivery of nanoparticles to the tumor. One of the great advantages of nanomedicine for cancer therapy is that nano-sized drugs and particles, when injected into the bloodstream , passively accumulate in tumor tissue through the leaky tumor vasculature 27,28 ; a phenomenon known as the enhanced permeability and retention (EPR) effect. The EPR effect varies between tumor types 29 , but in general favors accumulation of sub-100 nm structures 30. As a fraction of the nanoparticles will end up in healthy tissue after being injected intravenously, especially in the liver and spleen 31,32 , it is also important to consider the bio-compatibility of the nanoparticles. For these reasons, focus has been put on identifying and developing plasmonic
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Papers by Poul M Bendix