Dioleoylphosphatidylethanolamine Research Articles

Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

PEtOx-DOPE nanoliposomes functionalized with peptide 563 in targeted BikDDA delivery to prostate cancer.

Nanocarrier-based systems have cultivated significant improvements in prostate cancer therapy. However, the efforts are still limited in clinical applicability, and more research is required for the development of effective strategies. Here, we describe a novel nanoliposomal system for targeted apoptotic gene delivery to prostate cancer. Poly (2-ethyl-2-oxazoline) (PEtOx) dioleoyl phosphatidylethanolamine (DOPE) nanoliposomes were conjugated with the prostate-specific membrane antigen (PSMA)-targeting peptide GRFLTGGTGRLLRIS (P563) and loaded with BikDDA, a mutant form of the proapoptotic Bik. We selected 22Rv1 cells with moderate upregulation of PSMA to test the in vitro uptake, cell death, and in vivo anticancer activity of our formulation, P563-PEtOx-DOPE-BikDDA. BikDDA was upregulated in 22Rv1 cells, inducing cell death, and CD-1 nude mice xenografts administered with the formulation showed significant tumor regression. We suggest that P563-PEtOx-DOPE-BikDDA nanoliposomes can serve as prominent gene carriers against prostate cancer.

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration, and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner one yield less stretchable membranes with higher compressibility modulus compared with their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. In addition, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol %), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such domain formation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques.

Effect of Phosphatidylethanolamine on Pore Formation Induced by the Antimicrobial Peptide PGLa.

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

Preparation and in vitro evaluation of BBG-250 loaded liposomal formulation for anticancer potential

BackgroundLiposome-mediated drug delivery systems have emerged as a promising avenue for enhancing cancer treatment strategies. This study aims to develop and assess liposomal carriers loaded with Brilliant Blue G-250 (BBG-250), a potent P2X7 receptor antagonist that shows potential as an anti-tumor agent. Specifically, two types of liposomal formulations were designed: conventional liposomes composed of hydrogenated soya phosphatidylcholine (HSPC) and cholesterol, and pH-sensitive liposomes consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine-methoxy polyethylene glycol (DSPE-mPEG), dipalmitoylphosphatidylcholine (DPPC), and cholesterol. The investigation focuses on understanding the morphological characteristics, size, stability, drug incorporation efficiency, drug release profiles, blood compatibility, and cytotoxicity of these liposomal formulations.ResultsAdvanced photon correlation spectroscopy using the Nano Plus-3 instrument was employed to evaluate the liposomes. The optimized conventional liposomes (HSPC-cholesterol ratio 7:3) exhibited a size of 125 ± 0.3 nm with a polydispersity index (PDI) of 0.21, indicating uniformity. The pH-sensitive liposomes (DOPE:DPPC:DSPE-PEG2000: Cholesterol 4:3:3:0.3) demonstrated a size of 118 ± 1.2 nm with a PDI of 0.230. Zeta potential measurements confirmed the stability of both formulations under physiological conditions, with values of − 16.93 mV for conventional liposomes and − 25.21 mV for pH-sensitive liposomes. Higher drug-to-lipid ratios were found to enhance drug incorporation efficiency. pH-sensitive liposomes exhibited superior drug release characteristics, with 95% release over 24 h, compared to conventional liposomes, which released 70% of the drug. Blood compatibility assessments revealed the safety of both formulations for intravenous administration. Cytotoxicity studies conducted on A549 cell lines demonstrated the cytocompatibility of both liposomal types across a range of concentrations, with IC50 values surpassing those of the reference drug, docetaxel.ConclusionsThis study underscores the potential of liposomal carriers as effective vehicles for delivering BBG-250, highlighting their stability, biocompatibility, and controlled drug release properties. Despite being slightly less potent than the reference drug, docetaxel, these liposomal formulations hold promise for advancing anticancer strategies. The findings contribute to the evolving landscape of innovative cancer therapy drug delivery systems, offering a novel approach to improving treatment outcomes for cancer patients. The successful development and evaluation of these liposomal carriers pave the way for further investigations and potential clinical applications in the field of cancer therapeutics.

Human breast milk-derived phospholipid DOPE ameliorates intestinal injury associated with NEC by inhibiting ferroptosis.

Neonatal necrotizing enterocolitis (NEC) is a severe inflammatory bowel disease that commonly affects premature infants. Breastfeeding has been proven to be one of the most effective methods for preventing NEC. However, the specific role of lipids, the second major nutrient category in human breast milk (HBM), in intestinal development remains unclear. Our preliminary lipidomic analysis of the HBM lipidome revealed that dioleoyl phosphatidylethanolamine (DOPE) is not only abundant but also shows high solubility in lipids, endowing it with significant biological utility. Experimental results confirmed that DOPE significantly reduces the mortality of neonatal rats, ameliorates impairment of intestinal barrier function, and alleviates the expression of intestinal inflammatory factors IL-1β and IL-6. Furthermore, DOPE promotes the migration and proliferation of intestinal epithelial cells, thereby enhancing the integrity of the intestinal barrier function in vitro. The progression of NEC is linked with the onset of ferroptosis. Our cellular-level analysis of lipid peroxide and iron ion concentrations revealed that DOPE significantly reduces the indicators of ferroptosis, while also modulating the expression of pivotal ferroptosis-associated factors, including SLC7A11, GPX4, and ACSL4. Hence, this research on DOPE is expected to provide novel insights into the bioactive lipids present in HBM.

Remodeling tumor microenvironment using pH-sensitive biomimetic co-delivery of TRAIL/R848 liposomes against colorectal cancer.

Despite significant advancements in the development of anticancer therapies over the past few decades, the clinical management of colorectal cancer remains a challenging task. This study aims to investigate the inhibitory effects of cancer-targeting liposomes against colorectal cancer. Liposomes consisting of 3β-[N-(N', N'-dimethylamino ethane)carbamoyl]-cholesterol (DC-CHOL), cholesterol (CHOL), and dioleoylphosphatidylethanolamine (DOPE) at a molar ratio of 1:1:0.5 were created and used as carriers to deliver an apoptosis-inducing plasmid encoding the tumor necrosis factor-related apoptosis-inducing ligand (pTRAIL) gene, along with the toll-like receptor (TLR7) agonist Rsiquimod (R848). The rationale behind this design is that pTRAIL can trigger cancer cell apoptosis by activating the DR4/5 receptor, while R848 can stimulate the immune microenvironment. Experimental results demonstrated the synergistic effects of R848 and pTRAIL encapsulated by liposomes (RTL) in suppressing the proliferation of colorectal cancer cells. Moreover, further in vivo investigations revealed the strong anti-tumor efficacy of RTL in xenograft and orthotropic in situ models of colorectal cancer. These findings collectively highlight the therapeutic potential of R848/pTRAIL-loaded liposomes in the treatment of colorectal cancer.

Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Development of peptide‐18‐targeted nanoliposome formulations with an alternative stealth coating copolymer for targeting breast cancer AU565 cell line

AbstractThe incidence of breast cancer has increased considerably in recent years. Many efforts have been made to develop various nano‐drug delivery systems with specific properties to achieve effective and specific therapy. The stealthy nanoliposomes are the most successful and promising candidates for providing targeted tumor therapy. In this paper, a synthesized PEtOx‐DOPE (poly(2‐Ethyl 2‐Oxazoline)‐Dioleoyl Phosphatidylethanolamine) copolymer was equipped with a breast cancer‐recognizing tumor‐homing peptide to achieve cell‐specific delivery. The prepared liposomes provided stability for 6 months, and their hydrodynamic diameters are around 100 nm. Targeted liposomes remarkably exhibited 8 times higher cellular internalization in comparison with the nontargeted cells in flow cytometry and confocal microscopy. Furthermore, liposome constructs displayed slight toxicity on HaCaT cells when treated with high doses. Hence, Peptide 18‐conjugated PEtOx‐DOPE liposome systems can serve as favorable candidates in breast cancer‐targeted therapy.

Phosphatidylethanolamine and Cholesterol Promote Hemifusion Formation: A Tug of War between Membrane Interfacial Order and Intrinsic Negative Curvature of Lipids.

Membrane fusion is an important process for the survival of eukaryotes. The shape of lipids plays an important role in fusion by stabilizing nonlamellar fusion intermediates. Lipids with intrinsic positive curvature such as lysophosphatidylcholine and others inhibit hemifusion formation, whereas lipids having intrinsic negative curvature, e.g., phosphatidylethanolamine and cholesterol (CH), are known to promote hemifusion formation. In this work, we have measured the effect of dioleoylphosphatidylethanolamine (DOPE) and CH on the depth-dependent organization, dynamics, and fusion of dioleoylphosphatidylcholine membranes. Both DOPE and CH promote hemifusion formation despite their ability to order the interfacial and acyl chain region of the membrane and block water percolation at these regions. Generally, membrane ordering and inhibition of water percolation at the acyl chain region are detrimental to membrane fusion. This clearly emphasizes the importance of intrinsic negative curvature of lipids in membrane fusion. Interestingly, DOPE and CH show differential effects on the rate of hemifusion formation, which might be owing to their ability to induce order at the interfacial region and intrinsic negative curvature. Overall, our result is significant in understanding the role of lipidic shape in membrane fusion.

Influence of the Composition of Cationic Liposomes on the Performance of Cargo Immunostimulatory RNA.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Surface display of functional moieties on extracellular vesicles using lipid anchors

Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs. Notably, DOPE associated with EVs quickly, and the products remained stable under several challenging conditions. Moreover, conjugating fluorophores, receptor-targeting peptides or albumin-binding molecules with DOPE enabled tracking the cellular uptake, enhanceing the cellular uptake or extending the circulation time in mice of engineered EVs , respectively. Taken together, this study reports an efficient lipid anchor for exogenous engineering of EVs and further showcases its versatility for the functionalization of EVs.

HER2/neu Oncogene Silencing in a Breast Cancer Cell Model Using Cationic Lipid-Based Delivery Systems.

The overexpression of the human epidermal growth factor 2 (HER2/neu) oncogene is predictive of adverse breast cancer prognosis. Silencing the HER2/neu overexpression using siRNA may be an effective treatment strategy. Major requirements for siRNA-based therapy are safe, stable, and efficient delivery systems to channel siRNA into target cells. This study assessed the efficacy of cationic lipid-based systems for the delivery of siRNA. Cationic liposomes were formulated with equimolar ratios of the respective cholesteryl cytofectins, 3β-N-(N', N'-dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) or N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), with the neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE), with and without a polyethylene glycol stabilizer. All cationic liposomes efficiently bound, compacted, and protected the therapeutic siRNA against nuclease degradation. Liposomes and siRNA lipoplexes were spherical, <200 nm in size, with moderate particle size distributions (PDI < 0.4). The siRNA lipoplexes exhibited minimal dose-dependent cytotoxicity and effective HER2/neu siRNA transfection in the HER2/neu overexpressing SKBR-3 cells. The non-PEGylated Chol-T-siRNA lipoplexes induced the highest HER2/neu silencing at the mRNA (10000-fold decrease) and protein levels (>111.6-fold decrease), surpassing that of commercially available Lipofectamine 3000 (4.1-fold reduction in mRNA expression). These cationic liposomes are suitable carriers of HER2/neu siRNA for gene silencing in breast cancer.

Dual-Ligand-Functionalized Liposomes Based on Glycyrrhetinic Acid and cRGD for Hepatocellular Carcinoma Targeting and Therapy.

Hepatocellular carcinoma (HCC) is one of the most common cancers, with high mortality. Chemotherapy is one of the main treatment options for HCC. However, the high toxicity and poor specificity of chemotherapeutic drugs have limited their clinical application. In this study, dual-ligand liposomes modified with glycyrrhetinic acid (GA) and cyclic arginine-glycine-aspartic acid (cRGD) (GA/cRGD-LP) were designed to target the GA receptor and αvβ3 integrin, respectively. The aim was to develop a highly selective targeted drug delivery system and further enhance the antitumor efficiency of drugs by targeting both hepatic tumor cells and vasculature. A novel lipid conjugate (mGA-DOPE) by coupling dioleoylphosphatidyl ethanolamine (DOPE) with methyl glycyrrhetinic acid (mGA) was synthesized, and its structure was confirmed. The targeting efficiency of GA/cRGD-LP by in vitro cellular uptake and ex vivo imaging was assessed. GA- and cRGD-modified doxorubicin-loaded liposomes (GA/cRGD-LP-DOX) were prepared, and their cytotoxicity in HepG2 and antitumor activity were evaluated. The results showed that the average particle size of the GA/cRGD-LP-DOX was 114 ± 4.3 nm, and the zeta potential was -32.9 ± 2.0 mV. The transmission electron microscopy images showed that the shapes of our liposomes were spherical. cGA/cRGD-LP-DOX displayed an excellent cellular uptake in both HepG2 and human umbilical vein endothelial cells. In the in vivo study, pharmacokinetic parameters indicated that cGA/cRGD-LP can prolong the circulation time of DOX in the blood. GA/cRGD-LP-DOX showed greater inhibition of tumor growth for HepG2-bearing mice than either the single-ligand-modified liposomes or nontargeted liposomes. GA/cRGD-LP-DOX displayed higher liver tumor localization than that of single-ligand-modified liposomes or free DOX. GA/cRGD-LP is a promising drug delivery system for liver cancer targeting and therapy and is worthy of further study.

DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells

Docetaxel (DTX) is a non-selective antineoplastic agent with low solubility and a series of side effects. The technology of pH-sensitive and anti-epidermal growth factor receptor (anti-EGFR) immunoliposomes aims to increase the selective delivery of the drug in the acidic tumor environment to cells with EFGR overexpression. Thus, the study aimed to develop pH-sensitive liposomes based on DOPE (dioleoylphosphatidylethanolamine) and CHEMS (cholesteryl hemisuccinate), using a Box-Behnken factorial design. Furthermore, we aimed to conjugate the monoclonal antibody cetuximab onto liposomal surface, as well as to thoroughly characterize the nanosystems and evaluate them on prostate cancer cells. The liposomes prepared by hydration of the lipid film and optimized by the Box-Behnken factorial design showed a particle size of 107.2 ± 2.9 nm, a PDI of 0.213 ± 0.005, zeta potential of -21.9 ± 1.8 mV and an encapsulation efficiency of 88.65 ± 20.3%. Together, FTIR, DSC and DRX characterization demonstrated that the drug was properly encapsulated, with reduced drug crystallinity. Drug release was higher in acidic pH. The liposome conjugation with the anti-EGFR antibody cetuximab preserved the physicochemical characteristics and was successful. The liposome containing DTX reached an IC50 at a concentration of 65.74 nM in the PC3 cell line and 28.28 nM in the DU145 cell line. Immunoliposome, in turn, for PC3 cells reached an IC50 of 152.1 nM, and for the DU145 cell line, 12.60 nM, a considerable enhancement of cytotoxicity for the EGFR-positive cell line. Finally, the immunoliposome internalization was faster and greater than that of liposome in the DU145 cell line, with a higher EGFR overexpression. Thus, based on these results, it was possible to obtain a formulation with adequate characteristics of nanometric size, a high encapsulation of DTX and liposomes and particularly immunoliposomes containing DTX, which caused, as expected, a reduction in the viability of prostate cells, with high cellular internalization in EGFR overexpressing cells.

Lipids in Mitochondrial Macroautophagy: Phase Behavior of Bilayers Containing Cardiolipin and Ceramide

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.

Effect of monolayer spontaneous curvature on constant tension-induced pore formation in lipid bilayers.

The methodology of constant tension-induced rupture of giant unilamellar vesicles (GUVs) has provided information on tension-induced pore formation. This method was used to investigate the effect of spontaneous curvature (H0) for a lipid monolayer on the rate constant (kr) for constant tension (σ)-induced rupture, which originates from pore formation in lipid bilayers. Lipids were incorporated with different H0 values into GUV membranes to change the overall H0 value for the GUV monolayer. The dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylethanolamine (DOPE) (4/6, molar ratio, here and elsewhere) monolayer has a negative H0, whereas the DOPG/dioleoylphosphatidylcholine (DOPC) (4/6) monolayer has an essentially zero H0. A higher tension was required to induce the rupture of DOPG/DOPE (4/6)-GUVs compared with DOPG/DOPC (4/6)-GUVs. The line tension (Γ) for a pre-pore in DOPG/DOPE (4/6)-GUVs, determined by the analysis of the tension dependence of kr, was 1.5 times larger than that in DOPG/DOPC (4/6)-GUVs. The kr values for GUVs comprising DOPG/DOPC/18:1 lysophosphatidylcholine (LPC) (40/55/10), which has a positive H0, were larger than those for DOPG/DOPC (4/6)-GUVs under the same tension. The Γ value for DOPG/DOPC/LPC (40/55/10)-GUVs was almost half that for DOPG/DOPC (4/6)-GUVs. These results indicate that Γ decreases with increasing H0, which results in an increase in kr. Based on these results, the effect of H0 on kr and Γ is discussed.

Morphological Behavior of Liposomes and Lipid Nanoparticles.

Liposomes, which consist of bilayer lipids surrounding interior aqueous compartment(s), were first characterized nearly 60 years ago. Remarkably, many fundamental properties of liposomes and their micellar-like "solid core" counterparts (a lipid monolayer surrounding a hydrophobic core) and transitions between these structures remain poorly understood. In this work, we examine the effects of basic variables on the morphology adopted by lipid-based systems produced by rapid mixing of lipids in ethanol with aqueous media. We show that, for lipids such as distearolyphosphatidylcholine (DSPC)-cholesterol mixtures that form bilayer vesicles on hydration, osmotic stress can induce regions of high positive membrane curvature, leading to fusion between unilamellar vesicles to produce bilamellar vesicles. Addition of lyso PC, an "inverted cone"-shaped lipid that supports regions of high positive curvature, can inhibit the formation of these bilamellar vesicles by stabilizing a hemifused intermediate structure. Conversely, the presence of "cone"-shaped lipids such as dioleoylphosphatidylethanolamine (DOPE) that results in negative membrane curvature promotes fusion events subsequent to vesicle formation (during the ethanol dialysis stage), leading to bilamellar and multilamellar systems even in the absence of osmotic stress. Alternatively, the presence of increasing amounts of triolein, a lipid that is insoluble in lipid bilayers, results in increasing internal solid core structures until micellar-like systems with a hydrophobic core of triolein are achieved. These results are interpreted in terms of the intrinsic membrane curvature that bilayer vesicles can stably maintain as well as the ability of bilayer lipids to first form a monolayer around a solid core of hydrophobic material such as triolein and then, as the proportion of bilayer lipids is increased, progressively form bilayer structures that can eventually form a complete bilayer encapsulating both a hydrophobic core and an aqueous compartment. These hybrid intermediate structures may have utility as novel drug delivery systems.

Biodegradable Sucrose Ester-Based Cationic Lipids as Novel Vectors for Efficient and Safe Delivery of IGF-1R siRNA

The key to the success of siRNA therapy depends on the use of siRNA delivery agents that effectively penetrate tumor cells and simultaneously protect siRNA from RNase attacks. Sucrose esters (SEs) are found to have tremendous potential applications in siRNA delivery because of their biocompatibility and low toxicity. However, the esterification of sucrose yields complex mixtures that greatly limit their progress in this field. We synthesized a series of novel sucrose monoester-based cationic lipids (SECLs) by using 1′,4,6′-trichlorogalactosucrose (TGS) to achieve monoesterification of sucrose. SECL-based liposomes formed from these lipids and colipids [dioleoylphosphatidylethanolamine (DOPE) or cholesterol (Chol)] could effectively condense genes into nanocomplexes, which exhibited significantly higher uptake rates and gene silencing efficiency than 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposome. Furthermore, SECL-based liposomes containing cholesterol-mediated IGF-1R siRNA delivery could inhibit tumor growth by the downregulation of Bcl-2 and the upregulation of Bax. Most importantly, these liposomes displayed low cytotoxicity in vitro and in vivo because they could be degraded under tumor acidic pH environments. Our study establishes a novel method for the synthesis of SEs from TGS and demonstrates the applicability of SECLs for gene delivery with the potential for clinical therapy.

Temperature and oxidation-sensitive dioleoylphosphatidylethanolamine liposome stabilized with poly(ethyleneimine)/(phenylthio)acetic acid ion pair

Temperature and oxidation-sensitive liposomes were prepared by stabilizing dioleoylphosphatidylethanolamine (DOPE) bilayers with the ion pair of poly(ethyleneimine)/(phenylthio)acetic acid (PEI/PTA). An upper critical solution temperature (UCST) behavior was observed when PEI/PTA ion pair was suspended in an aqueous solution. It was observed that the UCST increased with increasing PTA content. The ion pair was self-assembled into nanospheres owing to its amphiphilic property which was confirmed by transmission electron microscopy. The FT-IR spectroscopic spectrum showed that the ion pair formed a salt bridge between the amino group and the carboxyl group and the PTA content in the ion pair was readily oxidized by H2O2. Further, DOPE liposomal membranes could be stabilized with PEI/PTA ion pair. Due to the amphiphilic property, the ion pair played a role as a stabilizer for the formation of DOPE liposomes. The liposome released its payload in a temperature-responsive manner, possibly because when the temperature is raised, the ion pair loses its amphiphilic property and can be detached from the liposomal membrane. The liposome was also oxidation-responsive in terms of release, possibly because the amphiphilic property of the ion pair disappears when the PTA is oxidized.