Role of the fast kinetics of pyroglutamate-modified amyloid-β oligomers in membrane binding and membrane permeability.

Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer's disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1-40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3-42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3-42 and Aβ1-42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3-42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3-42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3-42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability

Lipid and Protein Organizations in Model Membrane Systems- Membrane Curvature, Lipid Structure, Domain Formation, and Membrane Binding Kinetics

The composition and morphology of cellular membranes are highly dynamic. Potential parameters modulating protein and lipid distributions in different organelles include membrane shapes and the structures of lipids and proteins. Moreover, the concept of lipid rafts provides a prevailing view where nanodomains serve as centers for signal transduction, membrane trafficking, and cytoskeletal organization. In this contribution, we first investigated the lipid and protein organizations as a function of membrane curvature. To this end, a system consisting of solid-supported wavy membranes that exhibits a continuous curvature distribution with positive and negative curvature ranges was fabricated. Spatial distributions of ENTH (epsin N-terminal homology) domain and N-BAR (Bin-Amphiphysin-Rvs) domains derived from the proteins Endophilin and BIN-1 were found to vary approximately linearly with membrane curvature. In contrast, streptavidin and fluorescent lipid analogues exhibited homogenous distributions on wavy membranes. Fluorescence recovery after photobleaching and single-molecule tracking experiments revealed that protein domains remain laterally fluid in the curved regions. We next studied the membrane organization with respect to lipid structures, more specifically, the length and degree of saturation of acyl chains of lipids. The ganglioside GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi network (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, GM1 isoforms with alternate ceramide domains were synthesized and their partitioning between liquid-ordered (Lo) and liquid-disordered (Ld) phases in GUVs was imaged. GM1 with differing ceramides showed distinct phase-partitioning behaviors. Furthermore, crosslinking of GM1 by cholera toxin subunit B (CTB) was found to drive phase partitioning shift from less preferential phase preference to exclusively Ld or Lo phases. To shed light on the stability of lipid domains, factors qwhich affect line tension were discussed and potential line-active molecules were examined. We found that the presence of cone-shaped diacylglycerol decreases line tension, while the commonly used fluorescent lipid, Texas-Red DHPE tends to increase line tension. Additionally, to bridge the connection between thermodynamics to highly dynamic cellular environments, we developed a single liposome-based kinetics system which allowed us to examine membrane binding kinetics of proteins as a function of membrane curvature. Overall, these measurements help provide an integrated view of biophysical and structural parameters underlying organizations of lipids and proteins

Supported lipid bilayer interactions with nanoparticles, peptides and polymers

Supported lipid bilayers (SLBs) are one of the most common model membranes used in the field of cell membrane biology as they provide a well-defined model membrane platform for determination of molecular-level interactions between different biomolecules (e.g. proteins, peptides) and lipid membrane. Compared to model organisms, the use of SLB is preferable since it mimics cell plasma membrane in a very simple and well-controlled way. Therefore, molecular structure of membrane and experimental conditions (e.g. solution chemistry, temperature, and pH) can be easily adjusted to the required conditions of any systematic research. In addition, SLBs are typically easy to form, cheap and very reproducible and they are compatible with different surface characterization techniques, such as quartz crystal microbalance with dissipation (QCM-D), ellipsometry and atomic force microscopy (AFM). This study demonstrates that QCM-D analysis of SLBs serve as powerful tool to investigate and characterize the mechanisms of interactions between lipid membrane and gold nanoparticles (NPs), environmentally relevant polymers, and disease-inducing peptides. Due to many critical applications of gold NPs in drug delivery and diagnostics, understanding of membrane-NP interactions is crucial especially for determination of NPs cytotoxicity. In this study we focus on membrane disruption as one of the different mechanisms by which metal NPs induce cytotoxicity. The use of SLB is beneficial for this goal as it elucidates the unique mechanism of membrane disruption without interference of other mechanisms taking place simultaneously in biological cells. For NP-membrane interaction studies, a SLB composed of L-α-phosphatidylcholine (egg PC) was formed on a SiO2-coated crystal and QCM-D analysis was performed to obtain information about mass and viscoelastic changes of SLB resulting from interactions with gold NPs. For better understanding of the mechanisms of NP-membrane interactions, we systematically changed the NPs properties and the experimental conditions. In order to understand the effect of NP size, gold NPs with diameters of 2,5,10, and 40 nm were tested and compared to each other. NPs were tested in their citric acid-stabilized state as well as in the presence of poly (methacrylic acid) (PMAA), representing an organic coating that could become associated with NPs in the environment. The results indicated that when dissolved in water, gold NPs with the dimeters of 2, 5, 10, and 40 nm did not perturb the membrane, but in the presence of environmentally relevant polymer, the larger nanoparticles were found to disrupt the membrane. In order to elucidate the effect of surface chemistry, 10 nm - gold NPs with various functionalizations (i.e. anionic, cationic and non-ionic ligands) were tested. Control experiments were designed to test the effect of NPs in the absence of humic substances which means the NPs were dissolved in water. In these cases, regardless of the type of NP functionalization, no substantial bilayer mass changes were observed. This suggests that the charge and chemistry of the ligands had a minor effect on NP-membrane interactions. Furthermore, in both the control and humic acid experiments, there were small dissipation changes (less than 1 unit) indicating that the overall membrane structure was not perturbed. In order to mimic environmentally-relevant conditions, mass and viscoelasticity of SLB was characterized in the presence of four different natural polymers, also known as natural organic materials (NOMs): Fulvic and humic acids extracted from Suwannee River (SRFA and SRHA), which had relatively lower molecular weights and a commercial humic acid (HA) and the humic acid extracted from Elliott soil (ESHA) with higher molecular weight. The results showed that NOMs with lower molecular weights, adsorbed to the bilayer, while higher molecular weight components, did not induce any changes to the bilayers. In addition, the NPs in SRFA and SRHA increased the mass of the bilayer by 20-30 ng, while the NPs in HA and ESHA changed the mass of the bilayer by \u3c 10 ng. It was concluded that the presence of humic substances as well as their physical and chemical properties exert a direct impact on the interactions between cell membrane and the nanoparticles. In addition to the field of NP toxicity, SLBs play a pivotal role in the field of neurodegenerative diseases, such as Alzheimer’s disease (AD), in which the pathological cascade of events starts from interactions of a misfolded peptide with cell membrane. In this thesis, we confirm the validity of QCM-D analysis of SLB as an important platform for investigation of amyloid β (the peptide associated with AD) interactions with lipid membrane. Adsorption of Aβ peptide to cell membrane is known to take place on the so-called “lipid raftâ€� which are membrane microdomains enriched with cholesterol, sphingomyelin and ganglioside. The formation of SLBs containing lipid rafts is not only important for the field of AD research, but also it is important for other in vitro studies of cell biology as the lipid rafts are responsible for a variety of biological functions such as association of some membrane proteins and cellular signaling. However, the presence of lipid raft components such as sphingomyelin and cholesterol makes the formation of the bilayer more challenging which leads to adsorption of intact vesicles on the substrate without formation of the bilayer. In this study, the formation of lipid bilayer composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM) was investigated using QCM-D. A challenge was that the raft-containing vesicles remained intact on the SiO2 crystal. Therefore, different experimental conditions were tested to induce vesicle fusion, such as pH, temperature, osmotic pressure, and vesicle size. The key parameter in forming the bilayer was found to be applying osmotic pressure to the vesicles by having the vesicles exterior concentration of NaCl higher than interior concentration. When this concentration gradient was applied to the vesicles before flowing them on the substrate, vesicle rupture was favored and formation of a complete bilayer could occur. Here, we report the effects of each tested variable on the adsorption and fusion of the raft-containing vesicles, and the results are discussed based on the mechanisms of vesicle-vesicle and vesicle-substrate interactions.After developing the robust method for formation of SLB with lipid rafts, we used that as a template to characterize the mechanism of interactions between Aβ peptide and cell membrane which leads to onset of AD. The mechanism of Aβ toxicity leading to AD has not fully discovered yet, due to the complexity of the process including several steps of Aβ peptide adsorption on membrane, conformational change from disordered in solution to a membrane-bound α-helix structure and then formation of β-sheet aggregates that serve as fibrillation seeds. In this study, we showed that QCM-D technique as a promising tool to conduct systematic studies on the mechanism of interactions between Aβ peptide with lipid membrane. To our knowledge, this was the first time QCM-D was utilized for characterization of Aβ fibrillation starting from monomer states until formation of mature fibrils. The data indicated that peptide-membrane interactions follow a two-step kinetic pathway starting with the adsorption of small (low-n) oligomers until covering all the adsorption sites on the surface. In the second step, the membrane structure is destabilized as the result of interaction with oligomers which leads to lipid loss from the surface. Consistency of the results with the data obtained via other techniques substantiates QCM-D technique as a robust approach to answer the remaining unanswered questions in the field of Alzheimer’s disease

Role of lipids in HIV-1 pathogenesis. Implications in viral infectivity and development of antiretroviral drugs.

El sumario, el resumen y el capítulo 5 están sujetos a confidencialidad por el autor. 283 p.En esta tesis doctoral se ha estudiado el papel de los lípidos en distintos pasos del ciclo infectivo del VIH-1, y su potencial uso para el desarrollo de agentes antiretrovirales. Los resultados de este trabajo demuestran que la proteína gp41 del VIH-1 interacciona con colesterol tanto en la membrana plasmática como en la membrana viral, y que las secuencias LLP de la cola citoplásmatica de la proteína están involucradas en esta interacción.También se ha estudiado el mecanismo de acción de compuestos antirretrovirales sintéticos similares a lípidos naturales, conocidos como lipidomiméticos. Se ha descubierto que éstos llevan a cabo su acción antirretroviral inhibiendo el paso de entrada al alterar la estructura de la membrana viral y el estado de empaquetamiento de los lípidos. En esta tesis se desarrolló también un sistema de nanopartículas con gangliósidos que pueden ser específicamente focalizadas a células dendríticas maduras y transferidas a células latentemente infectadas mediante la ruta gangliósido/Siglec-1 para el transporte de agentes reactivadores de latencia, con el fin de purgar los reservorios virales que establecen una infección latente

Anti-GD2 Etoposide-Loaded Immunoliposomes for the Treatment of GD2 Positive Tumors

Systemic chemotherapeutics remain the standard of care for most malignancies even though they frequently suffer from narrow therapeutic index, poor serum solubility, and off-target effects. Monoclonal antibodies that specifically bind antigens overexpressed on many tumors such as the ganglioside, GD2, can be conjugated to drug-loaded liposomes to create a targeted drug delivery system. In this study, we have encapsulated etoposide, a topoisomerase inhibitor effective against a wide range of cancers, in surface modified liposomes decorated with anti-GD2 antibodies. We characterized the properties of the liposomes using a variety of methods including dynamic light scattering, electron microscopy, and Fourier transformed infrared spectroscopy. We examined whether these immunoliposomes were able to target cell lines expressing varying levels of surface GD2 and affect cellular proliferation. Anti-GD2 liposomes were generally targeted in a manner that correlated with GD2 expression and inhibited proliferation in cell lines to which they were efficiently targeted. The mechanism by which the immunoliposomes entered targeted cells appeared to be via clathrin-dependent uptake as demonstrated using flow cytometry and confocal microscopy. We initiated pilot studies in animals to examine the biodistribution and antitumor effects of immunoliposome mediated etoposide therapy. Fluorescent liposomes were observed at the tumor site with in vivo imaging, warranting further animals studies for antitumor efficacy. These studies suggest that anti-GD2-targeted, etoposide-loaded liposomes represent a potential strategy for more effective delivery of anti-cancer drugs that could be used for GD2 positive tumors

In Situ Synthesis of Artificial Lipids

This review comes from a themed issue on Synthetic Biomolecules (2022); Edited by Neal K. Devaraj and Shinya TsukijiFinanciado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] Lipids constitute one of the most enigmatic family of biological molecules. Although the importance of lipids as basic units of compartmental structure and energy storage is well-acknowledged, deciphering the biosynthesis and precise roles of specific lipid species has been challenging. To better understand the structure and function of these biomolecules, there is a burgeoning interest in developing strategies to produce noncanonical lipids in a controlled manner. This review covers recent advances in the area of in situ generation of synthetic lipids. Specifically, we report several approaches that constitute a powerful toolbox for achieving noncanonical lipid synthesis. We describe how these methodologies enable the direct construction of synthetic lipids, helping to address fundamental questions related to the cell biology of lipid biosynthesis, trafficking, and signaling. We envision that highlighting the current advances in artificial lipid synthesis will pave the way for broader interest into this emerging class of biomimetic molecules.Roberto J. Brea acknowledges support from Xunta de Galicia through the “Atracción de talento investigador” programme (ED431H2020/19). Roberto J. Brea also thanks the Agencia Estatal de Investigación (AEI) and the Ministerio de Ciencia e Innovación (MICINN) for his Ramón y Cajal contract (RYC2020-030065-I). The authors acknowledge funding for open access charge from Universidade da Coruña/Consorcio Interuniversitario de Galicia (CISUG)Xunta de Galicia; ED431H2020/1

Fabrication of Functionalized Double-Lamellar Multifunctional Envelope-Type Nanodevices Using a Microfluidic Chip with a Chaotic Mixer Array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>105) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency

Effect of Glycolipid Incorporation on Liposome Uptake by Antigen-Presenting Cells.

Robust cell-mediated immune responses are considered vital for successful vaccination against intracellular pathogens and cancer. Enhancing antigen uptake by dendritic cells (DCs) is a proposed way to stimulate cell-mediated immune responses when using typically low immunogenic protein antigens. Formulating antigen into a nanoparticle carrier can increase antigen uptake by two major antigen-presenting cells (APCs), DCs and macrophages. However, macrophage uptake is generally accepted as the main route of nanoparticle clearance due to macrophages’ high phagocytic capability. To skew the antigen uptake by DC, previous approaches have relied on attaching complex targeting moieties to antigen itself or onto nanoparticle antigen carriers such as liposomes. We hypothesized that by retarding high macrophage uptake of liposomes, we could facilitate enhanced uptake of liposomes by DCs. Simple liposome formulations containing phosphatidylinositol (PI) or monosialotetrahexosylganglioside (GM1) are well documented to deter rapid uptake by macrophages. In vitro uptake studies showed that incorporating 10mol% PI promoted uptake by DCs, while having minimal effect on uptake by macrophages. This trend was not observed with 10mol% GM1 incorporation. In vivo uptake studies upon subcutaneous injection confirmed PI-liposomes are indeed internalized more by DCs than GM1-liposomes, however, this result was also observed in macrophages. These liposomal formulations are of interest as vaccine carriers to stimulate enhanced cell-mediated immune responses and the generation of CD8+ T cells via enhanced uptake by DCs. Extending on our previous work using liposomes co-encapsulating the model antigen (OVA) and a hemolysin, listeriolysin-O (LLO), we show, herein, that PI- and GM1-liposomes encapsulating OVA and LLO can deliver OVA to the cytosol of DCs and macrophages in cell culture, resulting in efficient antigen presentation to CD8+ T cells. Mice immunized with PI-liposomes or GM1-liposomes co-encapsulating OVA and LLO generated similar CD8+ T cell-mediated immune responses as determined by MHC I tetramer staining and IFN−gamma ELISPOT analysis. Vaccination with either liposome formulation resulted in enhanced antigen-specific serum IgG2a titers indicative of better Th1 helper T cell activation. Both PI-liposomes and GM1-liposomes represent simple, inexpensive vaccine carriers for effectively stimulating cell-mediated immune responses utilizing subunit protein antigens.PhDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133467/1/amatyas_1.pd

Development and Evaluation of Paclitaxel-Loaded Liposomal Formulations for Targeted Drug Delivery to Breast Cancer

The objective of this work was to develop and evaluate paclitaxel-loaded liposomal formulations for targeted drug delivery to breast cancer. The liposomal formulation was optimized to maximize drug loading and physical stability. Cholesterol and saturated lipid content showed a negative influence on paclitaxel loading. Short-term stability studies showed that optimum drug-lipid ratio is necessary for adequate physical stability. Biodistribution studies in mouse xenografts bearing MDA-MB-231 breast cancer using near infrared fluorescence imaging showed that the accumulation of tumor vasculature targeted long-circulating liposomes (LCL) in the tumor was significantly less than non-targeted LCL at 48 h. The accumulation of these liposomes in the peritoneal cavity was higher suggesting that they were cleared rapidly by the reticuloendothelial system. The antitumor efficacy of paclitaxel-loaded tumor vasculature targeted LCL was compared with paclitaxel-loaded tumor cell targeted LCL. The antitumor efficacy was comparable for tumor vasculature targeted LCL, non-targeted LCL, and paclitaxel solution formulation with tumor volumes of ~ 60-70% of the control treatment on 39 days post tumor inoculation. In contrast, tumor cell targeted LCL showed a significantly higher antitumor efficacy compared to all other treatments with tumor volumes of ~ 30% of the control treatment. To improve the long-term stability, these liposomes were lyophilized. The leakage and vesicle size increase during lyophilization was minimized by using lyoprotectant sucrose in the formulation. Feasibility of developing gas-filled liposomes for ultrasound mediated drug delivery was evaluated using freeze drying gas entrapment method for the preparation. The in vitro measurements of echogenicity showed that these paclitaxel-loaded tumor vasculature targeted gas-filled liposomes were acoustically active and can be disintegrated by high intensity ultrasound pulses