Liposomes encapsulating polymeric chitosan based vesicles - a vesicle in vesicle system for drug delivery

Drug delivery systems comprising vesicles prepared from one amphiphile encapsulating vesicles prepared from a second amphiphile have not been prepared previously due to a tendency of the bilayer components of the different vesicles to mix during preparation. Recently we have developed polymeric vesicles using the new polymer-palmitoyl glycol chitosan and cholesterol in a 2:1 weight ratio. These polymeric vesicles have now been encapsulated within egg phosphatidylcholine (egg PC), cholesterol (2:1 weight ratio) liposomes yielding a vesicle in vesicle system. The vesicle in vesicle system was visualised by freeze fracture electron microscopy. The mixing of the different bilayer components was studied by monitoring the excimer fluorescence of pyrene-labelled polymeric vesicles after their encapsulation within egg PC liposomes or hexadecyl diglycerol ether niosomes. A minimum degree of lipid mixing was observed with the polymeric vesicle-egg PC liposome system when compared to the polymeric vesicle-hexadecyl diglycerol ether niosome system. The polymeric vesicle-egg PC vesicle in vesicle system was shown to retard the release of encapsulated solutes. 28% of 5(6)-carboxyfluorescein (CF) encapsulated in the polymeric vesicle compartment of the vesicle in vesicle system was released after 4 h compared to the release of 62% of encapsulated CF from plain polymeric vesicles within the same time period

Dynamics of Vesicle Formation from Lipid Droplet: Mechanism and Controllability

A coarse-grained model developed by Marrink et al. [J. Phys. Chem. B 111, 7812 (2007)] is applied to investigate vesiculation of lipid [dipalmitoylphosphatidylcholine (DPPC)] droplets in water. Three kinds of morphologies of micelles are found with increasing lipid droplet size. When the initial lipid droplet is smaller, the equilibrium structure of the droplet is a spherical micelle. When the initial lipid droplet is larger, the lipid ball starts to transform into a disk micelle or vesicle. The mechanism of vesicle formation from a lipid ball is analyzed from the self-assembly of DPPC on the molecular level, and the morphological transition from disk to vesicle with increasing droplet size is demonstrated. Importantly, we discover that the transition point is not very sharp, and for a fixed-size lipid ball, the disk and vesicle appear with certain probabilities. The splitting phenomenon, i.e., the formation of a disk/vesicle structure from a lipid droplet, is explained by applying a hybrid model of the Helfrich membrane theory. The elastic module of the DPPC bilayer and the smallest size of a lipid droplet for certain formation of a vesicle are successfully predicted.Comment: 22 pages, 11 figures Submitted to J. Chem. Phy

Characteristic spatial scale of vesicle pair interactions in a plane linear flow

We report the experimental studies on interaction of two vesicles trapped in a microfluidic analog of four-roll mill, where a plane linear flow is realized. We found that the dynamics of a single vesicle is significantly altered by the presence of another vesicle at separation distances up to about 3.2 \div 3.7 times of effective radius of the vesicles. This is supported by direct measurements of a single vesicle back-reaction on the velocity field. Thus, the experiment provides the lower bound for the interaction scale of vesicles and so the corresponding upper bound for the volume fraction \phi=0.08 \div 0.13 of non-interacting vesicle suspensions.Comment: 5 pages, 8 figures, PRE accepted for publicatio

Activity-dependence of synaptic vesicle dynamics

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENTMechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function

Synapsin selectively controls the mobility of resting pool vesicles at hippocampal terminals

Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals

Soft Confinement for Polymer Solutions

As a model of soft confinement for polymers, we investigated equilibrium shapes of a flexible vesicle that contains a phase-separating polymer solution. To simulate such a system, we combined the phase field theory (PFT) for the vesicle and the self-consistent field theory (SCFT) for the polymer solution. We observed a transition from a symmetric prolate shape of the vesicle to an asymmetric pear shape induced by the domain structure of the enclosed polymer solution. Moreover, when a non-zero spontaneous curvature of the vesicle is introduced, a re-entrant transition between the prolate and the dumbbell shapes of the vesicle is observed. This re-entrant transition is explained by considering the competition between the loss of conformational entropy and that of translational entropy of polymer chains due to the confinement by the deformable vesicle. This finding is in accordance with the recent experimental result reported by Terasawa, et al.Comment: 5 pages, 3 figure

Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion.

The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion

Spontaneous Expulsion of Giant Lipid Vesicles Induced by Laser Tweezers

Irradiation of a giant unilamellar lipid bilayer vesicle with a focused laser spot leads to a tense pressurized state which persists indefinitely after laser shutoff. If the vesicle contains another object it can then be gently and continuously expelled from the tense outer vesicle. Remarkably, the inner object can be almost as large as the parent vesicle; its volume is replaced during the exit process. We offer a qualitative theoretical model to explain these and related phenomena. The main hypothesis is that the laser trap pulls in lipid and ejects it in the form of submicron objects, whose osmotic activity then drives the expulsion.Comment: Plain TeX file; uses harvmac and epsf; .ps available at http://dept.physics.upenn.edu/~nelson/expulsion.p

Vesicle dynamics in confined steady and harmonically modulated Poiseuille flows

We present a numerical study of the time-dependent motion of a two-dimensional vesicle in a channel under an imposed flow. In a Poiseuille flow the shape of the vesicle depends on the flow strength, the mechanical properties of the membrane, and the width of the channel as reported in the past. This study is focused on the centered snaking (CSn) shape, where the vesicle shows an oscillatory motion like a swimmer flagella even though the flow is stationary. We quantify this behavior by the amplitude and frequency of the oscillations of the vesicle's center of mass. We observe regions in parameter space, where the CSn coexists with the parachute or the unconfined slipper. The influence of an amplitude modulation of the imposed flow on the dynamics and shape of the snaking vesicle is also investigated. For large modulation amplitudes transitions to static shapes are observed. A smaller modulation amplitude induces a modulation in amplitude and frequency of the center of mass of the snaking vesicle. In a certain parameter range we find that the center of mass oscillates with a constant envelope indicating the presence of at least two stable states.Comment: 10 pages, 7 figure