Nanoscale Lipid Research Articles

A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

BackgroundThe reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.ResultsThe styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.ConclusionsWe report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

An in vitro comparative study of the physical and imaging enhancement characteristics between two nano-scale ultrasound contrast agents: PFOB lipid particles and C3F8 lipid microbubbles

Objective To prepare two nano-scale ultrasound contrast agents,lipid particles and microbubbles,and to compare their physical and imaging enhancement characteristics.Methods Lipid nanoparticles were prepared with phospholipid and liquid perfluorocarbon (PFOB).Lipid microbubbles were prepared with phospholipid and perfluoropropane(C3F8).Physical and chemical properties of the two ultrasound contrast agents were measured,including morphology,size,zeta potential,concentration and stability.Both lipid nanoparticles and microbubbles with/without biotin were imaged using a 12.0 MHz linear-array transducer with/without avidin.The imaging data were analyzed by using Matlab software.Comparative data between the two groups were statistically analyzed by two-sample t test.Repeated measures analysis of variance was used among the observations at multiple time points within the same sample and Bonferroni method was used for post hoc analysis.Comparison of the signal intensity with and without avidin of the same sample was performed by paired t test.Results (1) Microscopically,both PFOB lipid particles and C3F8 microbubbles were in form of spheres.Their particle diameters were (152.30 ± 35.99) nm and (774.59 ± 108.59) nm,respectively (t =-24.327,P ＜ 0.001).Their zeta potential was (-40.90 ±6.51) mV and (-14.80 ± 3.97) mV,respectively (t =-15.308,P ＜ 0.001).(2) Concentration and size of PFOB lipid particles remained stable during the whole observation period (C0h =(2.28 ± 0.64) ×1011/ml,C1w =(2.06 ±0.53) × 1011/ml; D0h =(152.30 ±35.99) nm,D1 w =(178.80 ±63.07) nm).Concentrations of lipid microbubbles were stable within 12 h (C0h =(4.08 ± 0.96) × 1010/ml,C12h =(3.25 ± 1.02) × 1010/ml) ; and then reduced significantly after 24 h,2 d,4 d and 1 week (C24h:(2.28 ±0.73) ×1010/ml; C2d:(1.56±0.54) ×1010/ml; C4d:(1.03±0.37) ×1010/ml; C1w:(0.74 ±0.24) ×1010/ml ; F =78.515,P ＜ 0.01).Diameters of the particles remained stable within 2 d (D0h =(774.59 ±108.59) nm,D2d =(1020.68 ± 223.64) nm) ; and then increased after 4 d and 1 week (D4d:(1391.67 ±268.65) nm; D1w:(1532.41 ± 326.25) nm,respectively,F =50.772,P ＜ 0.01).(3) Neither control nor biotinylated PFOB particles were visible without avidin.With the addition of avidin,significantly enhanced images were generated by biotinylated PFOB particles (2.18 ±0.71,82.19 ± 15.68,t =-15.698,P ＜ 0.001) but not with the control PFOB particles.Both control and biotinylated C3F8 microbubbles showed no significant difference in their signal intensity with/without avidin (29.16 ± 6.84 and 31.34 ±7.03,33.69±8.24 and28.75±7.18,t=0.653,-1.393,bothP＞0.05).Conclusion Compared to C3F8 microbubbles,PFOB particles have smaller size,greater stability,higher SNR ratio and better imaging enhancement characteristics,therefore,more suitable for use as the target contrast agent for ultrasound imaging. Key words: Contrast media; Ultrasonography ; Lipid nanoparticles ; Lipid microbubbles ; Comparative study

Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles.

We fabricated a microfluidic device consisting of ciliated micropillars, forming a porous silicon nanowire-on-micropillar structure. We demonstrated that the prototype device can preferentially trap exosome-like lipid vesicles, while simultaneously filtering out proteins and cell debris. Trapped lipid vesicles can be recovered intact by dissolving the porous nanowires in PBS buffer.

Preparation and experimental study in vitro on nanoscale lipid ultrasound contrast agent targeting to HER2

Objective To prepare targeted nanoscale lipid ultrasound contrast agent and study the targeting function in vitro.Methods After the biotinylated monoclonal antibody Herceptin was prepared,the biotinylated degree and immunological activity were determined.Then biotinylated antibody was attached to the surface of nanoscale lipid ultrasound contrast agents by avidin-biotin system to prepare the targeted nanobubbles.The targeting function was studied by observing the combination ability of the targeted nanobubbles with SKOV3 cells in vitro,non-targeted nanobubbles as controls,and observing ultrasound imaging in vitro.Results About 16 biotin molecules were coupled to each antibody in average,and the immunological activity of the biotinylated antibody didn't decrease compared with the free one(P ＞0.05).SKOV3 cells were combined firmly and surrounded regularly by red dyed targeted nanobubbles,while control groups were negative.Ultrasound imaging could be significantly enhanced by targeted nanobubble binding to SKOV3 cell slides,the other two control groups were negative.Conclusions Nanoscale ultrasound contrast agent and antibodys can be combined firmly by avidin-biotin system to produce the targeted nanobubbles,which have strong targeting function in vitro and significantly enhanced ultrasound signal. Key words: Ultrasonography; Microbubbles ; Nanotechnology

Native Mass Spectrometry Characterization of Intact Nanodisc Lipoprotein Complexes

We describe here the analysis of nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, nanodiscs are native-like lipid bilayers that are well-defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing nanodiscs as a platform for launching membrane proteins into the gas phase.

Mapping the Mechanical Properties of Cholesterol-Containing Supported Lipid Bilayers with Nanoscale Spatial Resolution

It has been demonstrated that many biological processes are influenced by mechanical changes in membranes comprised of a variety of lipid components. As a result, the ability to map physicomechanical properties of surfaces with high temporal and spatial resolution is desirable. Tapping mode atomic force microscopy (AFM) has proven to be a useful technique for imaging biological surfaces due to its ability to operate in solution; however, access to information concerning the mechanical properties of these surfaces can also be obtained by reconstructing the time-resolved tip/sample force interactions during the imaging process. An advantage of such an approach is the direct correlation of topographical features with mechanical properties. Reconstruction of the tip/sample force is achievable by a technique called scanning probe acceleration microscopy (SPAM), which treats the cantilever as an accelerometer. The acceleration, which is directly related to the tip/sample force, of the cantilever is obtained by taking the second derivative of the cantilever deflection signal during a tapping mode AFM experiment in solution with standard cantilevers. Herein, we describe the applicability of SPAM to study mechanical properties of supported lipid bilayers with nanoscale spatial resolution via numerical simulations and experiment. The maximum and minimum tapping forces respond to changes in specific surface mechanical properties. Furthermore, we demonstrate how these changes can be used to map relative changes in the Young's modulus and adhesive properties of supported total brain lipid extract bilayers containing exogenous cholesterol. Finally, the ability of SPAM to distinguish nanoscale lipid raft domains based on changes in local mechanical properties is demonstrated.

The stability and activity of respiratory Complex II is cardiolipin-dependent

Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production.

Photodynamic efficacy of photosensitizers under an attenuated light dose via lipid nano-carrier-mediated nuclear targeting

Photodynamic therapy (PDT) has emerged as a treatment for certain malignant-like skin, head and neck, gastrointestinal, and gynecological cancers. The broader acceptance of PDT treatment for large or deep-seated tumors is still hindered, at least in part, by the low photodynamic efficiency of photosensitizers (PS) in the deep-seated tumor environment where the light energy fluency rate is severely attenuated after propagation via skin and/or tissue barriers. In this report, efficient nuclear-targeted intracellular delivery of PS is achieved using an easily fabricated yet entirely biocompatible and inexpensive polysaccharide-functionalized nanoscale lipid carrier, which triggers the intracellular release of photosensitizers inside cancer cells and targets cell nuclear to achieve a significantly enhanced photocytotoxicity. Cancer cells are killed efficiently even under an extremely low light fluency of 1 mW/cm2 attenuated via an interval meat layer with a thickness of 3 mm. Therefore, this nuclei-targeting system may contribute to the development of a new generation of PS carriers that fight against deep-seated tumors and that exhibit excellent photodynamic efficiency under faint light irradiation.

Nanodiscs and SILAC-Based Mass Spectrometry to Identify a Membrane Protein Interactome

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.

Nanomechanical detection of cholera toxin using microcantilevers functionalized withganglioside nanodiscs

The label-free detection of cholera toxin is demonstrated using microcantileversfunctionalized with ganglioside nanodiscs. The cholera toxin molecules bind specifically tothe active membrane protein encased in nanodiscs, nanoscale lipid bilayers surrounded byan amphipathic protein belt, immobilized on the cantilever surface. The specific molecularbinding results in cantilever deflection via the formation of a surface stress-induced bendingmoment. The nanomechanical cantilever response is quantitatively monitored byoptical interference. The consistent and reproducible nanomechanical detection ofcholera toxin in nanomolar range concentrations is demonstrated. The resultsvalidated with such a model system suggest that the combination of a microcantileverplatform with receptor nanodiscs is a promising approach for monitoring invasivepathogens and other types of biomolecular detection relevant to drug discovery.

Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers

High-density lipoproteins (HDLs) play an important role in human health through the metabolism and trafficking of cholesterol as well as providing the feedstocks for steroid hormone biosynthesis. These particles contain proteins, primarily Apo-AI and phospholipid and progress through various structural forms including 'lipid-poor', 'discoidal' and 'spherical' entities as cholesterol esters and lipid are incorporated. The discoidal form of HDL is stabilized in solution by two encircling belts of Apo-AI. Previous protein engineering of the Apo-AI sequence has led to a series of amphipathic helical proteins, termed membrane scaffold proteins (MSPs), which have shown great value in assembling nanoscale soluble membrane bilayers, termed Nanodiscs, of homogeneous size and composition and in the assembly of numerous integral membrane proteins for biophysical and biochemical investigations. In this communication we document a protein engineering approach to generate and optimize an extended polypeptide MSP, which will self-assemble phospholipids into larger Nanodiscs with diameters of 16-17 nm. We extensively characterize these structures by size exclusion chromatography and solution X-ray scattering.

Microfluidic Directed Self-Assembly of Liposome−Hydrogel Hybrid Nanoparticles

We present a microfluidic method to direct the self-assembly of temperature-sensitive liposome-hydrogel hybrid nanoparticles. Our approach yields nanoparticles with structural properties and highly monodisperse size distributions precisely controlled across a broad range relevant to the targeted delivery and controlled release of encapsulated therapeutic agents. We used microfluidic hydrodynamic focusing to control the convective-diffusive mixing of two miscible nanoparticle precursor solutions (a DPPC:cholesterol:DCP phospholipid formulation in isopropanol and a photopolymerizable N-isopropylacrylamide mixture in aqueous buffer) to form nanoscale lipid vesicles with encapsulated hydrogel precursors. These precursor nanoparticles were collected off-chip and were irradiated with ultraviolet (UV) light in bulk to polymerize the nanoparticle interiors into hydrogel cores. Multiangle laser light scattering in conjunction with asymmetric flow field-flow fractionation was used to characterize nanoparticle size distributions, which spanned the approximately 150 to approximately 300 nm diameter range as controlled by microfluidic mixing conditions, with a polydispersity of approximately 3% to approximately 5% (relative standard deviation). Transmission electron microscopy was then used to confirm the spherical shape and core-shell composition of the hybrid nanoparticles. This method may be extended to the directed self-assembly of other similar cross-linked hybrid nanoparticle systems with engineered size/structure-function relationships for practical use in healthcare and life science applications.

Microfluidic Mixing and the Formation of Nanoscale Lipid Vesicles

We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process. Microfluidic device geometry in conjunction with hydrodynamic flow focusing strongly influences vesicle size distributions, providing a coarse method to control liposome size, while total flow rate allows fine-tuning the vesicle size in certain focusing regimes. Although microfluidic liposome synthesis is relatively simple to implement experimentally, numerical simulations of the mixing process reveal a complex system of fluid flow and mass transfer determining the formation of nonequilibrium vesicles. These results expand our understanding of the microfluidic environment that controls liposome self-assembly and yield several technological advances for the on-chip synthesis of nanoscale lipid vesicles.

All Trans Retinoic Acid Nanodisks Enhance Retinoic Acid Receptor-Mediated Apoptosis and Cell Cycle Arrest in Mantle Cell Lymphoma.

Abstract 3722Poster Board III-658 BackgroundMantle cell lymphoma (MCL) is characterized by the translocation t (11; 14)(q13; q32), aggressive clinical behavior, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel pathways. All-trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. We hypothesized that since MCL cells express retinoid receptors, ATRA will exert anti-proliferative effects and thus may have a role in treatment. In the present study, we pursued a novel approach to deliver an ATRA payload to MCL cells in culture. This water insoluble bioactive lipid was stably incorporated into nanoscale lipid particles, termed nanodisks (ND). ND are comprised of a disk-shaped phospholipid bilayer whose periphery is stabilized by amphipathic apolipoproteins. It was hypothesized that following cellular uptake and/or liberation of ATRA from ND, this retinoid will interact with intracellular binding proteins and, ultimately, nuclear hormone receptors, leading to target gene transactivation, cell growth arrest and/or apoptosis. MethodsWe studied the mantle cell lymphoma cell lines Granta, NCEB and JEKO. ATRA-ND were prepared using recombinant human apolipoprotein A-I as the scaffold protein. Empty ND, lacking ATRA, were prepared in the same manner except that ATRA was omitted from the formulation contents. In various experiments ATRA was presented to cells using dimethylsulfoxide (DMSO) as vehicle (naked ATRA). Reactive oxygen species (ROS) were measured by oxidation of 2'7'dichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF) and quantified by fluorescence intensity using FACS. Apoptosis was quantified by Annexin V-FITC and PI using flow cytometry and FACS. Cell cycle analysis was measured by flow cytometry using FACS. PARP cleavage, caspase activation and analysis of cell cycle regulator proteins was measured by Western blotting, and retinoic acid receptor activity was measured by RT-PCR. ResultsWe found that ATRA-ND induced significantly more cell death than naked ATRA (in dimethylsulfoxide) or empty ND. In all three cell lines, ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. Incubation of cells with the antioxidant, N-acetylcysteine, inhibited ATRA-ND-induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 cell cycle arrest. Cyclin dependent kinases (CDK) regulate checkpoints that integrate mitogenic and growth inhibitory signals during cell cycle transitions. CDK inhibitors bind to CDK and inhibit kinase activity, leading to cell cycle arrest. P21 and p27 negatively regulate CDKs, and since p21 is induced by the tumor suppressor p53 in response to DNA damage, the expression level of these proteins was examined in Granta cells as a function of ATRA exposure. Compared to untreated control cells, no changes in the level of these proteins were seen following incubation with empty-ND. Whereas naked ATRA induced a modest increase in p21, p27 and p53, much larger increases were induced by ATRA-ND. Further ATRA-ND resulted in striking decrease in cyclin-D1. At ATRA concentrations that induce apoptosis, expression levels of the retinoic acid receptor- α (RAR α) and retinoid X receptor- γ (RXR γ) were increased. Furthermore, the RAR antagonist, Ro41-5253, inhibited ATRA-ND-induced ROS generation and abrogated ATRA-ND-induced cell growth arrest and apoptosis. Taken together, we found that ATRA-ND-induced apoptosis is dependent on ROS generation and RAR activation. We do not know if ATRA in ND is more stable or resists degradation during the course of cell incubations or whether ATRA-ND are taken up by the cells via receptor mediated endocytosis. Further work is required to elucidate the molecular basis of the enhanced biological activity of ATRA when presented to cells as a component of ND. ConclusionATRA-ND greatly enhanced apoptosis and cell cycle arrest in MCL cell lines, and resulted in increase in p21, p27 and p53 with a decrease in cyclin D1. It is conceivable that solubilization of ATRA in the ND hydrophobic milieu effectively concentrates this bioactive lipid and provides a means for more efficient delivery to target cells. Nonetheless, results obtained in this study suggest ATRA-ND represent a potentially effective approach to the treatment of MCL and should be explored further. Disclosures:No relevant conflicts of interest to declare.

Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer

Nanometer-scale intermembrane contact areas (CAs) formed between single small unilamellar lipid vesicles (SUVs) and planar supported lipid bilayers are quantified by measuring fluorescence resonance energy transfer (FRET) between a homogenous layer of donor fluorophores labeling the supported bilayer and acceptor fluorophores labeling the SUVs. The smallest CAs detected in our setup between biotinylated SUVs and dense monolayers of streptavidin were approximately 20 nm in radius. Deformation of SUVs is revealed by comparing the quenching of the donors to calculations of FRET between a perfectly spherical shell and a flat surface containing complementary fluorophores. These results confirmed the theoretical prediction that the degree of deformation scales with the SUV diameter. The size of the CA can be controlled experimentally by conjugating polyethylene glycol polymers to the SUV or the surface and thereby modulating the interfacial energy of adhesion. In this manner, we could achieve secure immobilization of SUVs under conditions of minimal deformation. Finally, we demonstrate that kinetic measurements of CA, at constant adhesion, can be used to record in real-time quantitative changes in the bilayer tension of a nano-scale lipid membrane system.

MONTE CARLO STUDY OF LIPID NANOSCALE ORGANIZATION IN CELL MEMBRANES: TUNING DOMAIN SIZE AND STABILITY NEAR THE PHASE BOUNDARY

Lipids in cell membranes are organized in cholesterol-rich domains that are involved in many important cellular functions. Depending on the cell state, such structures are believed to develop covering a wide range of submicrometric sizes and different levels of stability. Using a simple Monte Carlo approach I demonstrate that when the membrane lipid mixture approaches a phase boundary, the cell has several mechanisms to tune both the size and stability of these domains. Cholesterol levels variation, presence of ceramides and insertion of proteins provide plausible mechanisms for the control of the nanoscale lipid organization in cell membranes.

Interactions of oritavancin, a new lipoglycopeptide derived from vancomycin, with phospholipid bilayers: Effect on membrane permeability and nanoscale lipid membrane organization

Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (< 5 min) and complete (> 95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0–600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 μM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin.

Development of Lipidoid–siRNA Formulations for Systemic Delivery to the Liver

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Phase Transitions in Single Nano-Vesicles

Phase transitions in nano-scale lipid vesicles are believed to be influenced by the morphology as well as the finite size of the system. Both the effect of finite size and the asymmetric lateral stresses in the bilayer are believed to broaden the phase transition in small vesicles which has been confirmed by bulk studies. However, it still remains to be resolved whether the measured phase transition of the ensemble results from a broadening of the phase transition in individual vesicles or to heterogeneities among single vesicles exhibiting sharp phase transitions. We investigate the transition from gel (ordered) to fluid (disordered) state in single nano-sized lipid vesicles, composed of only ∼104 molecules, by measuring the anomalous permeability which peaks at the phase transition. We study single vesicles by immobilizing them on functionalized substrates. The vesicle size and efflux/influx for each vesicle is accurately quantified which allows us to study the effect of curvature on phase transitions. To assess the width of the phase transition we probe the pore size at different temperatures using chromophores of different molecular radii. Our data suggest a revaluation of the currently accepted concepts about the nature of permeation at Tc and of phase transitions in nano-scale systems.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.