David S. Dean

What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues

Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells.

Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

Langevin equation for the density of a system of interacting Langevin processes

We present a simple derivation of the stochastic equation obeyed by the density function for a system of Langevin processes interacting via a pairwise potential. The resulting equation is considerably different from the phenomenological equations usually used to describe the dynamics of non-conserved (model A) and conserved (model B) particle systems. The major feature is that the spatial white noise for this system appears not additively but multiplicatively. This simply expresses the fact that the density cannot fluctuate in regions devoid of particles. The steady state for the density function may, however, still be recovered formally as a functional integral over the coursed grained free energy of the system as in models A and B.

Aging on Parisi's Tree

We present a detailed study of simple «tree» models for off equilibrium dynamics and aging in glassy systems. The simplest tree describes the landscape of a random energy model, whereas multifurcating trees occur in the solution of the Sherrington-Kirkpatrick model. An important ingredient taken from these models is the exponential distribution of deep free-energies, which translate into a power-law distribution of the residence time within metastable «valleys». These power law distributions have infinite mean in the spin-glass phase and this leads to the aging phenomenon. To each level of the tree is associated an overlap and the exponent of the time distribution. We solve these models for a finite (but arbitrary) number of levels and show that a two-level tree accounts very well for many experimental observations (thermoremanent magnetisation, a.c. susceptibility, second noise spectrum....). We introduce the idea that the deepest levels of the tree correspond to equilibrium dynamics whereas the upper levels correspond to aging. Temperature cycling experiments suggest that the borderline between the two is temperature dependent. The spin-glass transition corresponds to the temperature at which the uppermost level is put out of equilibrium but is subsequently followed by a sequence of (dynamical) phase transitions corresponding to non equilibrium dynamics within deeper and deeper levels. We tentatively try to relate this «tree» picture to the real space «droplet» model, and speculate on how the final description of spin-glasses might look like

Electrotransfer as a Non Viral Method of Gene Delivery

Over the last few decades, various vectors have been developed in the field of gene therapy. There still exist a number of important unresolved problems associated with the use of viral as well as non viral vectors. These techniques can suffer from secondary toxicity or low gene transfer efficiency. Therefore an efficient and safe method of DNA delivery still needs to be found for medical applications. DNA electrotransfer is a physical method that consists of the local application of electric pulses after the introduction of DNA into the extra cellular medium. As electrotransfer has proven to be one of the most efficient and simple non viral methods of delivery, it may provide an important alternative technique in the field of gene therapy. The present review focuses on questions related to the mechanism of DNA electrotransfer, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses the current limitations of the method as applied to DNA transfer, in particular its efficiency in achieving in vitro gene expression in cells and also its potential use for in vivo gene delivery.

Extreme Value Statistics of Eigenvalues of Gaussian Random Matrices

We compute exact asymptotic results for the probability of the occurrence of large deviations of the largest (smallest) eigenvalue of random matrices belonging to the Gaussian orthogonal, unitary, and symplectic ensembles. In particular, we show that the probability that all the eigenvalues of an (NxN) random matrix are positive (negative) decreases for large N as approximately exp [-beta theta(0)N2] where the Dyson index beta characterizes the ensemble and the exponent theta(0)=(ln 3)/4=0.274653... is universal. We compute the probability that the eigenvalues lie in the interval [zeta1,zeta2] which allows us to calculate the joint probability distribution of the minimum and the maximum eigenvalue. As a by-product, we also obtain exactly the average density of states in Gaussian ensembles whose eigenvalues are restricted to lie in the interval [zeta1,zeta2] , thus generalizing the celebrated Wigner semi-circle law to these restricted ensembles. It is found that the density of states generically exhibits an inverse square-root singularity at the location of the barriers. These results are confirmed by numerical simulations. Some of the results presented in detail here were announced in a previous paper [D. S. Dean and S. N. Majumdar, Phys. Rev. Lett. 97, 160201 (2006)].

Large Deviations of Extreme Eigenvalues of Random Matrices

We calculate analytically the probability of large deviations from its mean of the largest (smallest) eigenvalue of random matrices belonging to the Gaussian orthogonal, unitary, and symplectic ensembles. In particular, we show that the probability that all the eigenvalues of an (N x N) random matrix are positive (negative) decreases for large N as approximately exp[-betatheta(0)N2] where the parameter beta characterizes the ensemble and the exponent theta(0)=(ln3)/4=0.274 653... is universal. We also calculate exactly the average density of states in matrices whose eigenvalues are restricted to be larger than a fixed number zeta, thus generalizing the celebrated Wigner semicircle law. The density of states generically exhibits an inverse square-root singularity at zeta.

Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery

Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2 ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is proposed to explain the appearance of distinct interaction sites, the quantitative increase in DNA and also their immobility leading to a tentative explanation for the success of electro-mediated gene delivery.

Visualization of Membrane Loss during the Shrinkage of Giant Vesicles under Electropulsation

We study the effect of permeabilizing electric fields applied to two different types of giant unilamellar vesicles, the first formed from EggPC lipids and the second formed from DOPC lipids. Experiments on vesicles of both lipid types show a decrease in vesicle radius, which is interpreted as being due to lipid loss during the permeabilization process. We show that the decrease in size can be qualitatively explained as a loss of lipid area, which is proportional to the area of the vesicle that is permeabilized. Three possible modes of membrane loss were directly observed: pore formation, vesicle formation, and tubule formation.

Full dynamical solution for a spherical spin-glass model

We present a detailed analysis for the Langevin dynamics of a spherical spin-glass model (the spherical Sherrington-Kirkpatrick model). The effects of initial conditions on the ultimate dynamical behaviour are closely examined. In addition, the effects of temperature variations in the model are studied. Somewhat surprisingly, this simple model captures some of the effects seen in laboratory spin-glasses.