Correlation of mRNA delivery timing and protein expression in lipid-based transfection.

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

A single-cell micro-trench platform for automatic monitoring of cell division and apoptosis after chemotherapeutic drug administration.

Cells vary in their dynamic response to external stimuli, due to stochastic fluctuations and non-uniform progression through the cell cycle. Hence, single-cell studies are required to reveal the range of heterogeneity in their responses to defined perturbations, which provides detailed insight into signaling processes. Here, we present a time-lapse study using arrays of micro-trenches to monitor the timing of cell division and apoptosis in non-adherent cells at the single-cell level. By employing automated cell tracking and division detection, we precisely determine cell cycle duration and sister-cell correlations for hundreds of individual cells in parallel. As a model application we study the response of leukemia cells to the chemostatic drug vincristine as a function of cell cycle phase. The time-to-death after drug addition is found to depend both on drug concentration and cell cycle phase. The resulting timing and dose-response distributions were reproduced in control experiments using synchronized cell populations. Interestingly, in non-synchronized cells, the time-to-death intervals for sister cells appear to be correlated. Our study demonstrates the practical benefits of micro-trench arrays as a platform for high-throughput, single-cell time-lapse studies on cell cycle dependence, correlations and cell fate decisions in general.

Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles.

Non-viral delivery of nucleic acids for therapies based on RNA interference requires a rational design and optimal self-assembly strategies. Nucleic acid particles need to be small, stable and functional in terms of selective cell uptake and controlled release of encapsulated nucleic acids. Here we report on small (∼38 nm) monomolecular nucleic acid/lipid particles (mNALPs) that contain single molecules of short double-stranded oligonucleotides covered by a tight, highly curved lipid bilayer. The particles consist of DOPE, DOTAP, DOPC and DSPE-PEG(2000) and are assembled with 21 bp double-stranded DNA or small interfering RNA by solvent exchange on a hydrodynamic-focusing microfluidic chip. In comparison to vortex mixing by hand this method increases the encapsulation efficiency by 20%, and yields particles with a narrower size distribution, negligible aggregate formation and high stability in blood plasma and serum. Modification of mNALPs with folate-conjugated PEG-lipids results in specific binding and uptake by epithelial carcinoma KB cells overexpressing folate receptors. Binding is significantly reduced by competitive inhibition using free folate and is not observed with non-targeted mNALPs, revealing high specificity. The functionalized mNALPs show gene silencing in the presence of chloroquine, an endosome-destabilizing agent. Together, the robust self-assembly of small-sized mNALPs with their high stability and receptor-specific cell uptake demonstrate that the tight, PEG-grafted lipid-bilayer encapsulation may offer a promising approach towards the delivery of short double-stranded oligonucleotides.

Asymmetric distribution of anionic phospholipids in supported lipid bilayers.

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.

Transport, separation, and accumulation of proteins on supported lipid bilayers.

Transport, separation, and accumulation of proteins in their natural environment are central goals in protein biotechnology. Miniaturized assays of supported lipid bilayers (SLBs) have been proposed as promising candidates to realize such technology on a chip, but a modular system for the controlled transport of membrane proteins does not exist. In this letter, we demonstrate that standing surface acoustic waves drive the in-plane redistribution of proteins on planar SLBs over macroscopic distances (3.5 mm). Accumulation of proteins in periodic patterns of about 10-fold protein concentration difference is accomplished and shown to relax into the homogeneous state by diffusion. Different proteins separate in individual fractions from a homogeneous distribution and are transported and accumulated into clusters using beats. The modular planar setup has the potential of integrating other lab-on-a-chip tools, for monitoring the membrane-protein integrity or adding microfluidic features for blood screening or DNA analysis.

Controlled nanometric phase transitions of phospholipid membranes by plasmonic heating of single gold nanoparticles.

The development of remotely controlled nanoscopic sources of heat is essential for investigating and manipulating temperature sensitive processes at the nanoscale. Here, we use single gold nanoparticles to rapidly deposit controlled amounts of heat in nanoscopic regions of defined size. This allows us to induce and control nanoscale reversible gel-fluid phase transitions in phospholipid membranes. We exploit the optical control over the phase transition to determine the velocity of the fluid phase front into the gel phase membrane and to guide the nanoparticles to specific locations. These results illustrate how single gold nanoparticles enable local thermodynamic investigation and manipulation on nanoscale (bio-) systems.

Microelectrophoresis in a laser trap: a platform for measuring electrokinetic interactions and flow properties within microstructures.

We describe a combination of microelectrophoresis and laser-trap methodology to accurately measure the electric force acting on a charged microsphere which is trapped in an optical tweezer. This field/trap apparatus allows measuring of the zeta potential with submillivolt accuracy and high temporal resolution. The combination with stop-flow techniques in principle provides a mean to observe adsorption or enzyme kinetics with single molecule sensitivity. We show that it is possible to accurately profile the position and frequency dependent hydrodynamic and electro-osmotic flow inside a microchannel structure of dimensions typically used in microfluidic applications without the need of fluorescent markers. We found good agreement to the theory of electrophoretic flow when retardation effects for rapidly alternating electric fields are included.

Wormlike lipid/DNA micelles in a non-polar solvent.

The phase behavior of DOPE/DOTAP-DNA complexes in phase-separated oil(dodecane)/water mixtures was explored using Small Angle X-Ray Scattering (SAXS) and Fluorescence Correlation Spectroscopy (FCS). Inverse micelles of DNA with cationic-lipid coating were found in the oil phase. Varying the ratio between cationic and neutral lipids a transition from wormlike to spherical structures is observed for both long ( approximately 75000bp) and short (30-1246bp) DNA. In contrast to lipid/DNA complexes in the water phase, there is no indication of condensed liquid-crystalline structures in the non-polar phase. In fact, FCS measurements on short DNA oligomers complexed with cationic lipid in alkane give clear evidence for monomeric inverse micelles of DNA. Dilution series revealed a critical lower concentration of lipids and DNA for observing lipid/DNA micelles.

Intramolecular dynamics of linear macromolecules by fluorescence correlation spectroscopy.

A theoretical description of the dynamics of DNA molecules and actin filaments in solution as measured experimentally by fluorescence correlation spectroscopy is provided and compared to recent experimental results. Particular attention is paid to the contribution of the intramolecular dynamics to the fluorescence correlation function. Using a semiflexible chain model, a theoretical expression is presented for the fluorescence correlation spectroscopy correlation function. The dependence of this function on various model parameters, such as chain length, persistence length, and fluorescence label density, is discussed. Our investigations show that the intramolecular dynamics provides a significant contribution or even dominates the correlation function as soon as the longest intramolecular relaxation time significantly exceeds the shortest experimentally accessible time. Correspondingly, the shape of the correlation function changes considerably. Approximate analytical expressions are provided, which are in qualitative agreement with the exact theoretical solutions as well as experimental results, for both DNA and actin filaments. Our approach is in agreement with the predictions of the Zimm model, in the limit of very flexible polymers, as well as the predictions of semiflexible polymer models with respect to the intramolecular dynamics in solution.

Chains, dimers, and sandwiches: melting behavior of DNA nanoassemblies.

We have studied, in experiment and theory, the melting behavior of DNA nanoassemblies of perylenediimide-bis-oligonucleotides (PON), in which two short oligonucleotide chains (16- or 24-mers) are attached to the extremities of a spacer molecule. By varying both the sequence orientation (exchange of 3' and 5' ends neighboring the spacer) and the oligomer composition of the solutions, nanoassemblies of different complexity can be generated. Our results show a subtle dependence of the melting behavior on the supramolecular arrangement of the DNA-based assemblies.

Structural investigations of DNA-polycation complexes.

The internal structure of DNA-polycation complexes is investigated by synchrotron small-angle X-ray scattering (SAXS). Hexagonal packing of DNA is observed for DNA complexed with poly-L-lysine (PL), poly-L-arginine (PA), spermine (Sp), and linear and branched polyethyleneimine (lPEI and bPEI, respectively). Variations in the internal spacings and degree of long-range ordering are dependent on both polycation type and concentration of added salt. With increasing concentration of monovalent salt, a discontinuous phase transition is observed from compact to loose bundles and finally to an isotropic network phase. This salt-induced melting transition was found to be universal for all polyplexes studied and is in quantitative agreement with a simple free energy model based solely on electrostatic and entropic contributions. Using the osmotic stress method, bulk modulus (K) is measured for PL-DNA and PA-DNA polyplexes at various salt concentrations. With increasing osmotic force, we show that the salt-induced melting transition is shifted and compression in the loose bundle regime is in qualitative agreement with our model.

Flow profiling of a surface-acoustic-wave nanopump.

The flow profile in a capillary gap and the pumping efficiency of an acoustic micropump employing surface acoustic waves is investigated both experimentally and theoretically. Ultrasonic surface waves on a piezoelectric substrate strongly couple to a thin liquid layer and generate a quadrupolar streaming pattern within the fluid. We use fluorescence correlation spectroscopy and fluorescence microscopy as complementary tools to investigate the resulting flow profile. The velocity was found to depend on the applied power approximately linearly and to decrease with the inverse third power of the distance from the ultrasound generator on the chip. The found properties reveal acoustic streaming as a promising tool for the controlled agitation during microarray hybridization.

Flow profile near a wall measured by double-focus fluorescence cross-correlation.

We present an experimental approach to flow profiling within femtoliter sample volumes, which allows the high-precision measurements at the solid interface. The method is based on the spatial cross-correlation of the fluorescence response from labeled tracer particles (latex nanospheres or single dye molecules). Two excitation volumes, separated by a few micrometers, are created by two laser foci under a confocal microscope. The velocity of tracer particles is measured in a channel about 100 microm wide within a typical accuracy of 0.1%, and the positions of the walls are estimated independently of any hydrodynamic data. The underlying theory for the optical method is given for an arbitrary velocity profile, explicitly presenting the numerical convolutions necessary for a quantitative analysis. It is illustrated by using the Poiseuille flow of a Newtonian liquid with slip as an example. Our analysis yields a large apparent fluid velocity at the wall, which is mostly due to the impact of the colloidal (electrostatic) forces. This colloidal lift is crucially important in accelerating the transport processes of molecules and nanoparticles in microfluidic devices.

Dynamics of large semiflexible chains probed by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy was used to probe the dynamics of lambda-phage DNA in aqueous solution labeled with the randomly intercalating dye TOTO. The linear macromolecules (i). carry more than one chromophore and (ii). are larger than the waist of the focal volume. The correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in good agreement with the dynamic model of semiflexible chains including hydrodynamic interactions. As the chromophore density is lowered the correlation time decreases in accordance with this model.

Interface dynamics of lipid membrane spreading on solid surfaces.

As a model system for two-dimensional interface dynamics, we study the wetting front of a lipid membrane moving over a solid substrate that is structured with regularly spaced pinning centers. By analyzing the contour of the front, we derive the normal growth rate and the relaxation coefficient. Both exhibit a 1/t(1/2) time dependence. Moreover, the friction coefficient and the line tension can be determined. Randomly distributed pinning centers cause a fractal contour line, whereas on surfaces that are artificially roughened, self-affine contour lines are observed. The latter exhibit an anomalous roughness exponent of zeta = 0.81+/-0.05.

Microelectrophoresis of a bilayer-coated silica bead in an optical trap: application to enzymology.

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-delta1 (PLC-delta) on a single bilayer-coated silica bead with a time resolution of approximately 1 s. A 1-microm-diameter bead was coated with a phospholipid bilayer composed of electrically neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP2) and captured in a laser trap. When an AC field was applied (160 Hz, 20 V/cm), the electrophoretic force produced a displacement of the bead, Delta(x), from its equilibrium position in the trap; Delta(x), which was measured using a fast quadrant diode detector, is proportional to the zeta potential and thus to the number of PIP2 molecules on the outer leaflet (initially, approximately 10(5)). When a solution containing PLC-delta flows past the bead, the enzyme adsorbs to the surface and hydrolyzes PIP2 to form the neutral lipid diacylglycerol. We observed a nonexponential decay of PIP2 on the bead with time that is consistent with a model based on the known structural properties of PLC-delta.

Lipid-DNA and lipid-polyelectrolyte mesophases: structure and exchange kinetics.

Cationic lipid-DNA complexes are used as gene transfer vehicles in molecular biology and potentially in human gene therapy. In recent synchrotron X-ray scattering studies the molecular structure of such self-assembling aggregates was elucidated. A rich polymorphism of lamellar, hexagonal, lamellar-columnar and micellar mesophases was found. In this article we describe composite phases of cationic lipid mixed with hyaluronic acid and dextran sulfate which likewise form intercalated lamellar complexes. Heterogeneous phases of lipid/dextran sulfate mixed with lipid/DNA exhibit macroscopic phase separation. When dextran sulfate is added to preformed cationic lipid DNA complexes the latter are dissolved in favor of the lipid-polyelectrolyte phases. We investigated the kinetics of the DNA replacement by dextran sulfate. The experiments are intended to mimic the interaction of cationic lipid gene delivery complexes with highly charged extracellular matrix components.

An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery.

A two-dimensional columnar phase in mixtures of DNA complexed with cationic liposomes has been found in the lipid composition regime known to be significantly more efficient at transfecting mammalian cells in culture compared to the lamellar (LalphaC) structure of cationic liposome-DNA complexes. The structure, derived from synchrotron x-ray diffraction, consists of DNA coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice (HIIC). Two membrane-altering pathways induce the LalphaC --> HIIC transition: one where the spontaneous curvature of the lipid monolayer is driven negative, and another where the membrane bending rigidity is lowered with a new class of helper-lipids. Optical microscopy revealed that the LalphaC complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HIIC complexes are unstable and rapidly fuse and release DNA upon adhering to anionic vesicles.

Collective membrane motions of high and low amplitude, studied by dynamic light scattering and micro-interferometry.

Undulations of lipid bilayers were experimentally studied for the two limiting cases of high and weak lateral tension using two well established model systems: freely suspended planar lipid bilayers, so-called black lipid membranes (BLM) for high-tension studies and large unilamellar vesicles (LUV) for measurements at weak tension. This variation in tension results in changes of undulation amplitudes from several hundred nm (LUV) down to 1 nm (BLM), thus requiring different physical methods for their detection. We have employed microinterferometric techniques (RICM) for studying the regime of weak tension and dynamic light scattering (DLS) for that of high tension. The dedicated DLS set-up allowed the measurements of undulations over a wide wave vector range of 250 < q/cm-1 < 35,000 cm-1. This enabled the observation of collective membrane modes in two regimes, the oscillating one at low q and the overdamped regime at high q. The transition between both regimes at the bifurcation point is rather abrupt and depends on the lateral tension of the bilayer, as is demonstrated by comparing the dispersion curves of pure lipid and of lipid-cholestrol BLMs over the same q-range. The DLS measurements allowed a critical test of a hydrodynamic theory of the dispersion behaviour of membrane collective modes under tension. The DLS measurements are compared with RICM results of undulatory excitations of giant vesicles weakly adhering to substrates in the 10(-6)-2.5 x 10(-7) m wavelength regime and at low frequencies (0.1-25 Hz). Experimental evidence for the strong decrease in the relaxation rate by the hydrodynamic coupling of the membrane with the wall is established.

Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes.

Cationic liposomes complexed with DNA (CL-DNA) are promising synthetically based nonviral carriers of DNA vectors for gene therapy. The solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron x-ray diffraction and optical microscopy. The addition of either linear lambda-phage or plasmid DNA to CLs resulted in an unexpected topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. X-ray diffraction of the globules revealed a novel multilamellar structure with alternating lipid bilayer and DNA monolayers. The lambda-DNA chains form a one-dimensional lattice with distinct interhelical packing regimes. Remarkably, in the isoelectric point regime, the lambda-DNA interaxial spacing expands between 24.5 and 57.1 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced repulsion that is possibly enhanced by chain undulations.