Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

Gullapalli, Ramachandra R.; Tabouillot, Tristan; Mathura, Rishi A.; Dangaria, Jhanvi H.; Butler, Peter J.

doi:10.1117/1.2673245

Cited by 39 publications

(38 citation statements)

References 57 publications

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“…This method has been described in details and validated elsewhere. 39,46,47 Briefly, a lipophilic dye (e.g., DiI) is used to stain cell membrane, which is embedded into the hydrophobic tails of phospholipids due to the hydrophobic interactions. The lifetime of the fluorescent dye after being excited by a laser is an indicator of the membrane tension level because its nonradiative decay is correlated to the extent of the dye molecule exposure to water, that is, the free area occupied by the dye molecule.…”

Section: Resultsmentioning

confidence: 99%

The role of substrate topography on the cellular uptake of nanoparticles

Huang

Özdemir

et al. 2015

J Biomed Mater Res

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Improving targeting efficacy has been a central focus of the studies on nanoparticle (NP)-based drug delivery nanocarriers over the past decades. As cells actively sense and respond to the local physical environments, not only the NP design (e.g., size, shape, ligand density, etc.) but also the cell mechanics (e.g., stiffness, spreading, expressed receptors, etc.) affect the cellular uptake efficiency. While much work has been done to elucidate the roles of NP design for cells seeded on a flat tissue culture surface, how the local physical environments of cells mediate uptake of NPs remains unexplored, despite the widely known effect of local physical environments on cellular responses in vitro and disease states in vivo. Here, we report the active responses of human osteosarcoma cells to fibrous substrate topographies and the subsequent changes in the cellular uptake of NPs. Our experiments demonstrate that surface topography modulates cellular uptake efficacy by mediating cell spreading and membrane mechanics. The findings provide a concrete example of the regulative role of the physical environments of cells on cellular uptake of NPs, therefore advancing the rational design of NPs for enhanced drug delivery in targeted cancer therapy.

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Section: Resultsmentioning

confidence: 99%

The role of substrate topography on the cellular uptake of nanoparticles

Huang

Özdemir

et al. 2015

J Biomed Mater Res

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“…32 In brief, the excitation beam from a PicoTRAIN water-cooled 532-nm, 80-MHz, 5.4-ps pulsed laser (High-Q Laser, Hohenems, Austria) entered the side port of an IX-71 microscope (Olympus, Tokyo, Japan) and slightly underfilled (80%) the back aperture of an Olympus 60×/1.2-NA water-immersion objective. 35 Emitted light passed a polarizer positioned at the magic angle, and was focused onto a 50-µm, 0.22-NA optical fiber—acting as confocal pinhole.…”

Section: Methodsmentioning

confidence: 99%

Endothelial Cell Membrane Sensitivity to Shear Stress is Lipid Domain Dependent

2010

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Blood flow-associated shear stress causes physiological and pathophysiological biochemical processes in endothelial cells that may be initiated by alterations in plasma membrane lipid domains characterized as liquid-ordered (lo), such as rafts or caveolae, or liquid-disordered (ld). To test for domain–dependent shear sensitivity, we used time-correlated single photon counting instrumentation to assess the photophysics and dynamics of the domain-selective lipid analogues DiI-C12 and DiI-C18 in endothelial cells subjected to physiological fluid shear stress. Under static conditions, DiI-C12 fluorescence lifetime was less than DiI-C18 lifetime and the diffusion coefficient of DiI-C12 was greater than the DiI-C18 diffusion coefficient, confirming that DiI-C12 probes ld, a more fluid membrane environment, and DiI-C18 probes the lo phase. Domains probed by DiI-C12 exhibited an early (10 s) and transient decrease of fluorescence lifetime after the onset of shear while domains probed by DiI-C18 exhibited a delayed decrease of fluorescence lifetime that was sustained for the 2 min the cells were subjected to flow. The diffusion coefficient of DiI-C18 increased after shear imposition, while that of DiI-C12 remained constant. Determination of the number of molecules (N) in the control volume suggested that DiI-C12-labeled domains increased in N immediately after step-shear, while N for DiI-C18-stained membrane transiently decreased. These results demonstrate that membrane microdomains are differentially sensitive to fluid shear stress.

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“…The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35,36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36,37).…”

Section: Significancementioning

confidence: 99%

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Shen

Wen

Erbakan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar [5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10 −14 cm 3 /s or 3.5(±1.0) × 10 8 water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10 8 water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10 8 water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10 7 water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10 5 pores per μm 2 ) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10 3 pores per μm 2 ). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.artificial aquaporins | artificial water channels | peptide-appended pillar [5]arene | single-channel water permeability | two-dimensional arrays T he discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1-9). Areas of application include liquid and gas separations (10-13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen-Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The largescale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water chan...

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Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

Cited by 39 publications

References 57 publications

The role of substrate topography on the cellular uptake of nanoparticles

The role of substrate topography on the cellular uptake of nanoparticles

Endothelial Cell Membrane Sensitivity to Shear Stress is Lipid Domain Dependent

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

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