Nily Dan

Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery.

Ligand-conjugated polymer (polyplex) gene delivery vectors have strong potential as targeted, in vivo gene transfer vehicles; however, they are currently limited by low delivery efficiency. A number of barriers to polyplex-mediated delivery have been previously identified, including receptor binding, internalization, endosomal escape, and nuclear localization. However, based on understanding of viral gene delivery systems, yet another potential barrier may exist; a limited ability to unpackage the plasmid DNA cargo following localization to the nucleus. We have developed a model system that employs a cationic polymer linked to epidermal growth factor (EGF) as a ligand to target delivery of plasmid DNA encoding the green fluorescent protein to mouse fibroblasts bearing the EGF receptor. Using fluorescence microscopy to simultaneously trace both the plasmid and polymer during gene delivery in combination with an in vitro transcription assay, we provide evidence that plasmid unpackaging can indeed be a limiting step for gene expression for sufficiently large polymer constructs. Short-term expression is significantly enhanced by using short polycations that dissociate from DNA more rapidly both in vitro and in vivo. Finally, we describe a thermodynamic model that supports these data by showing that shorter polycations can have a higher probability of dissociating from DNA. This work demonstrates that vector unpackaging should be added to the list of barriers to receptor-mediated polyplex gene delivery, thus providing an additional design principle for targeted synthetic delivery vehicles.

Effect of Lipid Characteristics on the Structure of Transmembrane Proteins

The activity of embedded proteins is known to vary with lipid characteristics. Indeed, it has been shown that some cell-membrane proteins cannot function unless certain non-bilayer-forming lipids (i.e., nonzero spontaneous curvature) are present. In this paper we show that membranes exert a line tension on transmembrane proteins. The line tension, on the order of 1-100 kT/protein, varies with the lipid properties and the protein configuration. Thus, membranes composed of different lipids favor different protein conformations. Model predictions are in excellent agreement with the data of Keller et al. (Biophys. J. 1993, 65:23-27) regarding the conductance of alamethicin channels.

The Effect of Chain Length on Protein Solubilization in Polymer-Based Vesicles (Polymersomes)

Using a mean-field analysis we derive a consistent model for the perturbation of a symmetric polymeric bilayer due to the incorporation of transmembrane proteins, as a function of the polymer molecular weight and the protein dimensions. We find that the mechanism for the inhibition of protein incorporation in polymeric bilayers differs from that of their inclusion in polymer-carrying lipid vesicles; in polymersomes, the equilibrium concentration of transmembrane proteins decreases as a function of the thickness mismatch between the protein and the bilayer core, whereas in liposomes the presence of polymer chains affects the protein adsorption kinetics. Despite the increased stiffness of polymer bilayers (when compared to lipid ones), their perturbation decay length and range of protein-protein interaction is found to be relatively long. The energetic penalty due to protein adsorption increases relatively slowly as a function of the polymer chain length due to the self-assembled nature of the polymer bilayer. As a result, we predict that transmembrane proteins may be incorporated in significant numbers even in bilayers where the thickness mismatch is large.

The structure of DNA complexes with cationic liposomes-cylindrical or flat bilayers?

DNA complexes with cationic lipids promise to be versatile and effective synthetic transfection agents. Recent experiments identified both flat lamellar structures, where DNA strands are sandwiched between lipid bilayers, and cylindrical ones where the DNA is coated by a curved bilayer. Using a simple model we compare the stability of the two structures, and find that flat-bilayer aggregates are always more stable than the cylindrical ones. The different experimental observations are explained within the framework of the model predictions.

Formation of ordered domains in membrane-bound DNA

The interactions between DNA molecules adsorbed on fluid membranes are calculated. The adsorbing DNA perturbs the equilibrium packing of the lipids, thereby giving rise to membrane-induced, attractive interactions. These balance the direct repulsive interactions between DNA molecules. As a result, DNA adsorbed on membranes is predicted to form ordered domains characterized by a finite spacing, which varies with the membrane characteristics and the solution Debye screening length. Comparing the model predictions to recent experiments (Yang et al. 1996) yields excellent agreement with only one free (i.e., experimentally unknown) parameter.

Multilamellar structures of DNA complexes with cationic liposomes

Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.

Bursting Bubbles and Bilayers

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol) (PEG) - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those - not necessarily involving microubbles - to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly “theranostic” vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.

Effect of liposome charge and PEG polymer layer thickness on cell-liposome electrostatic interactions.

Targeted drug delivery requires binding to (and subsequent uptake by) the carrier and target cell. In this paper, we calculate the work required to bring into contact liposomal carriers and cells as a function of the liposome and cell electrostatic characteristics. We find that cell-liposome adhesion is sensitive to the cell type and optimized at a cell to liposome charge ratio which depends on the degree of cell charge regulation. As a result, uptake (which is dependent on the occurrence of binding) is also optimized. Incorporation of a (poly)ethylene glycol (PEG) layer enhances liposome adhesion in cases where the cell-liposome interactions are repulsive, and suppresses adhesion in systems where the interactions are attractive. Our results, which are in agreement with experimental observations, show that electrostatic interactions may be designed to enable targeted drug delivery by liposomes to a specific cell population.

The effect of charge regulation on cell adhesion to substrates: salt-induced repulsion

Abstract The long-range forces controlling cell or bacteria adsorption onto substrates are governed by electrostatic interactions. In this paper we use a simple mean field model (Debye–Huckel) to examine the interactions between cells and surfaces. We model the cell interface as an ion-penetrable, charge-regulating layer, thereby accounting for the finite thickness of the cells extra-cellular (glycocalyx) layer. We find that charge regulation leads to several non-intuitive trends regarding the repulsion between a cell and similarly charged substrates: (I) instead of increasing monotonically with decreasing cell–substrate separation, the pressure varies non-monotonically, and (II) instead of monotonically decreasing the repulsion (at contact) between the cell and the substrate, there is a regime where adding salt leads to an increase in the repulsion.

Synthesis of hierarchical materials

A major goal of material science is to produce hierarchical materials that are ordered on all length scales, from the molecular (1-100 A) via the nano (10-100 nm) to the meso (1-100 microm). In these materials, the larger-scale properties can be controlled by choosing molecular characteristics. Methods developed to produce three-dimensional, bulk-like hierarchical structures include biomimetic methods, which use polypeptides as building blocks, and amphiphile and colloidal templating, which use amphiphilic or colloidal mesophases as templates for inorganic mesoporous materials. Designing finite mesostructures with a given geometry still remains a challenge.