Liposomal Bilayer Research Articles

Calixarene-decorated liposomes for intracellular cargo delivery.

Amphiphilic calix[4]arenes, functionalized with guanidinium groups, are used to decorate the outer surface of liposomes and significantly improve the cellular uptake of a cargo compared to plain liposomes. The improved uptake is elicited and mediated by the interaction between the cationic polar heads of the macrocycle units embedded in the liposome bilayer and anionic heparan-sulfate proteoglycans surrounding the exterior of cells.

MR-labelled liposomes and focused ultrasound for spatiotemporally controlled drug release in triple negative breast cancers in mice

Rationale: Image-guided, triggerable, drug delivery systems allow for precisely placed and highly localised anti-cancer treatment. They contain labels for spatial mapping and tissue uptake tracking, providing key location and timing information for the application of an external stimulus to trigger drug release. High Intensity Focused Ultrasound (HIFU or FUS) is a non-invasive approach for treating small tissue volumes and is particularly effective at inducing drug release from thermosensitive nanocarriers. Here, we present a novel MR-imageable thermosensitive liposome (iTSL) for drug delivery to triple-negative breast cancers (TNBC).Methods: A macrocyclic gadolinium-based Magnetic Resonance Imaging (MRI) contrast agent was covalently linked to a lipid. This was incorporated at 30 mol% into the lipid bilayer of a thermosensitive liposome that was also encapsulating doxorubicin. The resulting iTSL-DOX formulation was assessed for physical and chemical properties, storage stability, leakage of gadolinium or doxorubicin, and thermal- or FUS-induced drug release. Its effect on MRI relaxation time was tested in phantoms. Mice with tumours were used for studies to assess both tumour distribution and contrast enhancement over time. A lipid-conjugated near-infrared fluorescence (NIRF) probe was also included in the liposome to facilitate the real time monitoring of iTSL distribution and drug release in tumours by NIRF bioimaging. TNBC (MDA-MB-231) tumour-bearing mice were then used to demonstrate the efficacy at retarding tumour growth and increasing survival.Results: iTSL-DOX provided rapid FUS-induced drug release that was dependent on the acoustic power applied. It was otherwise found to be stable, with minimum leakage of drug and gadolinium into buffers or under challenging conditions. In contrast to the usually suggested longer FUS treatment we identified that brief (~3 min) FUS significantly enhanced iTSL-DOX uptake to a targeted tumour and triggered near-total release of encapsulated doxorubicin, causing significant growth inhibition in the TNBC mouse model. A distinct reduction in the tumours' average T1 relaxation times was attributed to the iTSL accumulation.Conclusions: We demonstrate that tracking iTSL in tumours using MRI assists the application of FUS for precise drug release and therapy.

Synthesis of Triphenylphosphonium Phospholipid Conjugates for the Preparation of Mitochondriotropic Liposomes.

Surface modification of liposomes with a ligand is facilitated by the conjugation of the ligand to a hydrophobic molecule that serves to anchor the ligand to the liposomal bilayer. We describe here a simple protocol to conjugate a triphenylphosphonium group to several commercially available functionalized phospholipids. The resulting triphenylphosphonium-conjugated lipids can be used to prepare liposomes that preferentially associate with mitochondria when exposed to live mammalian cells in culture.

Systematic Analysis of Phosphatidylinositol-5-phosphate-Interacting Proteins Using Yeast Proteome Microarrays.

We used yeast proteome microarrays (∼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-tagged fluorescent liposomal nanovesicles. Lissamine rhodamine B-dipalmitoyl phosphatidylethanol was incorporated into the liposome bilayer to provide the nanovesicles with fluorescence without any encapsulants, which not only made the liposome fabrication much easier without the need for purification but also improved the chip-probing quality. A special chip assay was washed very gently without the traditional spin-dry step. Forty-five PI5P-interacting proteins were identified in triplicate with this special chip assay. Subsequently, we used flow cytometry to validate these interactions, and a total of 41 PI5P-interacting proteins were confirmed. Enrichment analysis revealed that these proteins have significant functions associated with ribosome biogenesis, rRNA processing, ribosome binding, GTP binding, and hydrolase activity. Their component enrichment is located in the nucleolus. The InterPro domain analysis indicated that PI5P-interacting proteins are enriched in the P-loop containing nucleoside triphosphate hydrolases domain (P-loop). Additionally, using the MEME program, we identified a consensus motif (IVGPAGTGKSTLF) that contains the Walker A sequence, a well-known nucleotide-binding motif. Furthermore, using a quartz crystal microbalance, both the consensus motif and Walker A motif showed strong affinities to PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.

Complete ablation of resistant tumors with photosensitive black phosphorus quantum dots-based lipid nanocapsules

Treatment of drug-resistant tumors is a great challenge and nanoplatforms combining advanced therapeutic functions have many advantages in offering efficient treatment. However, the reported fabrication of nanoplatforms for multi-modal therapy of tumors is complicated or even uncontrollable since several kinds of therapeutic agents need to be assembled into the nanoparticles. Herein, black phosphorus (BP)-based photosensitive nanocapsules (BP-PNCs) are designed as a highly efficient nanoplatform against resistant tumors by integrating the inherent photothermal and chemotherapeutic effects of BP. BP-PNCs are synthesized by encapsulating BP quantum dots (BPQDs) into the core of liposome and DSPE-PEG2000-cRGD is incorporated into the liposomal bilayers to render BP-PNCs with high cellular uptake and effective in vivo tumor targeting capability after intravenous injection. In vitro and in vivo assessments demonstrate that BP-PNCs in conjunction with near-infrared (NIR) light irradiation lead to complete obliteration of resistant A549R tumors. The excellent anti-cancer efficacy stems from the synergistic therapeutic effects arising from the NIR light induced BP photothermal effects, which result in hyperthermia ablation and triggering the liposome phase transition to release internal BPQDs to perform BP chemotherapeutic effects. The expression inhibition of P-gp by hyperthermia further improves the chemotherapeutic efficiency. BP-PNCs with a simple structure and intrinsic multi-functionalities have great potential in treating resistant tumors and our results provide insights into the design of multifunctional nanoplatforms for biomedicine.

Targeted Drug Delivery Using Tuftsin-bearing Liposomes: Implications in the Treatment of Infectious Diseases and Tumors.

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg), acts as an immunopotentiating molecule with its ability to bind and activate many immune cells, including macrophages or monocytes, neutrophils and dendritic cells. The specific targeting activity of tuftsin has been further increased by its palmitoylation followed by its incorporation into the lipid bilayer of liposomes. Tuftsin-bearing liposomes (Tuft-liposomes) possess several characteristics that enable them to act as a potential drug and vaccine carriers. Tuft-liposomes-loaded anti-microbial drugs have been shown to be highly effective against many infectious diseases, including tuberculosis, leishmaniasis, malaria, candidiasis and cryptococosis. Moreover, Tuft-liposomes also increased the activity of anticancer drug etoposide against fibrosarcoma in mice. Tuft-liposomes showed the immune-potentiating effect and rejuvenated the immune cells in the leukopenic mice. In addition, antigens encapsulated in Tuftsin-bearing liposomes demonstrated greater immunogenicity by increasing the T cell proliferation and antibody secretion. Keeping into consideration their specific targeting and immunopotentiating effects, Tuft-liposomes may potentially be used as promising drug and vaccine delivery systems.

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated Fe3O4 nanoparticles (Fe3O4 NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL−1, making this liposomal system a potential matrix in a virus detection platform. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-020-04656-2.

Phospholipid bilayer responses to ultrasound-induced microbubble cavitation phenomena

Abstract Using liposomal bilayers as a model for bacterial cell membranes, this study investigates the extents to which high-frequency (1.0 and 3.3 MHz) ultrasound-induced microbubble cavitation phenomena are effective for bacterial inactivation. Forster Resonance Energy Transfer (FRET), acoustic scattering and a mathematical model were used to quantify the proximity of microbubbles to liposomes, establish if liposomes affect microbubble cavitation and propose bilayer alterations' mechanisms. Addition of a positive charge on microbubbles increased their proximity to liposomes through electrostatic attraction, however, dampened microbubble's oscillation compared to freely-floating microbubbles. Alleviation of energy transfer due to phospholipid mixing between microbubbles and liposomes established that close-proximity is necessary for bilayer alterations. Approximately 19% mixing of phospholipids was observed at 3.3 MHz due to microstreaming from stable cavitation, while inertial cavitation at 1.0 MHz increased this mixing to 50%. This method can damage bacterial membrane; but, whether bacterial defense mechanisms can attenuate this effect remains to be understood.

High-affinity antigen association to cationic liposomes via coiled coil-forming peptides induces a strong antigen-specific CD4+ T-cell response

Liposomes are widely investigated as vaccine delivery systems, but antigen loading efficiency can be low. Moreover, adsorbed antigen may rapidly desorb under physiological conditions. Encapsulation of antigens overcomes the latter problem but results in significant antigen loss during preparation and purification of the liposomes. Here, we propose an alternative attachment method, based on a complementary heterodimeric coiled coil peptide pair pepK and pepE.PepK was conjugated to cholesterol (yielding CPK) and pepE was covalently linked to model antigen OVA323 (yielding pepE-OVA323). CPK was incorporated in the lipid bilayer of cationic liposomes (180 nm in size). Antigen was associated more efficiently to functionalized liposomes (Kd 166 nM) than to cationic liposomes (Kd not detectable). In vivo co-localization of antigen and liposomes was strongly increased upon CPK-functionalization (35% -> 80%). CPK-functionalized liposomes induced 5-fold stronger CD4+ T-cell proliferation than non-functionalized liposomes in vitro. Both formulations were able to induce strong CD4+ T-cell expansion in mice, but more IFN-y and IL-10 production was observed after immunization with functionalized liposomes. In conclusion, antigen association via coiled coil peptide pair increased co-localization of antigen and liposomes, increased CD4+ T-cell proliferation in vitro and induced a stronger CD4+ T-cell response in vivo.

Comprehensive analysis of liposome formulation parameters and their influence on encapsulation, stability and drug release in glibenclamide liposomes

An understanding of the factors that affect liposome size, drug loading, stability and drug release is critical for the rational design of liposomes with desired pharmacokinetics and biodistribution. This article presents a report on the formulation and characterization of BIIB093 (glibenclamide) liposomes as well as a detailed analysis of the influence of formulation methods and parameters on encapsulation efficiency, liposome size, charge (zeta potential, ZP), polydispersity index (PDI), and drug release. PEGylated liposomes containing BIIB093 were made using ethanol injection and calcium acetate remote loading. The critical formulation parameters investigated include: the effect of lipid chain length, lipid unsaturation, lipid phase transition temperature (Tc) and the amount of cholesterol. Liposomes generated in this study had low average particle size (130 ± 20 nm), PDI (0.15 ± 0.1) and ZP (-2 ± 1 mV). Liposomes made from lipids with long acyl chains showed enhanced drug loading, encapsulation efficiency and drug retention. Similarly, liposomes made from lipids with high degree of unsaturation and low Tc exhibited faster drug release rates. Additionally, increasing the amount of cholesterol in the liposome bilayer improved PDI, decreased drug incorporation and accelerated drug release but had negligible impact on liposome size and ZP. Furthermore, encapsulating the drug in the liposome core enabled sustained drug release.

Redesigning Solvatochromic Probe Laurdan for Imaging Lipid Order Selectively in Cell Plasma Membranes.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

Development of pyrene-based fluorescent ether lipid as inhibitor of SK3 ion channels.

We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3±13.3% at 10μM) and by its inhibition of SK3-dependent cancer cell migration at (-32.5%±4.8% at 1μM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.

Gemcitabine and doxorubicin in immunostimulatory monophosphoryl lipid A liposomes for treating breast cancer.

Cancer therapy is increasingly shifting toward targeting the tumor immune microenvironment and influencing populations of tumor infiltrating lymphocytes. Breast cancer presents a unique challenge as tumors of the triple‐negative breast cancer subtype employ a multitude of immunosilencing mechanisms that promote immune evasion and rapid growth. Treatment of breast cancer with chemotherapeutics has been shown to induce underlying immunostimulatory responses that can be further amplified with the addition of immune‐modulating agents. Here, we investigate the effects of combining doxorubicin (DOX) and gemcitabine (GEM), two commonly used chemotherapeutics, with monophosphoryl lipid A (MPLA), a clinically used TLR4 adjuvant derived from liposaccharides. MPLA was incorporated into the lipid bilayer of liposomes loaded with a 1:1 molar ratio of DOX and GEM to create an intravenously administered treatment. In vivo studies indicated excellent efficacy of both GEM‐DOX liposomes and GEM‐DOX‐MPLA liposomes against 4T1 tumors. In vitro and in vivo results showed increased dendritic cell expression of CD86 in the presence of liposomes containing chemotherapeutics and MPLA. Despite this, a tumor rechallenge study indicated little effect on tumor growth upon rechallenge, indicating the lack of a long‐term immune response. GEM/DOX/MPLA‐L displayed remarkable control of the primary tumor growth and can be further explored for the treatment of triple‐negative breast cancer with other forms of immunotherapy.

Regulation of Properties of Lipid Membranes by Interaction with 2-Hydroxypropyl β-Cyclodextrin: Molecular Details

The interaction between 2-hydroxypropyl β-cyclodextrin (HPCD) and a liposomal bilayer has been studied. The main binding sites of HPCD on the surface of neutral liposomes based on dipalmitoylphosphatidylcholine (DPPC) are the phosphate groups of lipids. The complex formation with HPСD leads to the stabilization of the gel state of monocomponent liposomes. The inclusion of the anionic component, cardiolipin (CL, 20%), in the bilayer changes the nature of the liposome-HPCD interaction. The observed lipid disorder disturbs membrane integrity, which is revealed in the release of the included dye (phenolphthalein). The effect of HPCD on the process and parameters of the phase transition of anionic liposomes has been studied using thermograms, which show the change in the position of the absorption bands of lipid acyl chains in the FTIR spectrum of liposomes. A stratification of DPPC/CL (80/20%) bilayer into two microphases with different cardiolipin content has been detected. HPCD causes the more pronounced stratification in the bilayer, i.e., membrane destabilization near the melting point of CL-rich microphase and, vice versa, a decrease in the lipid mobility in regions with a low CL content. We have studied the effect of HPCD on the interaction of an antibacterial drug, levofloxacin (LV), with the lipid bilayer. It has been found that the complexation of the drug molecules with HPCD leads to an increase in the efficiency of drug adsorption on the surface of the bilayer. This may facilitate the transport of the drug through the bilayer due to the formation of defects in the membrane. LV in the complex with HPCD shows a high antibacterial efficiency in vitro against E. coli, which is not lower as compared with free LV.

Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage

In the present work, we investigated the interaction of flavonoids (quercetin, naringenin and catechin) with cellular and artificial membranes. The flavonoids considerably inhibited membrane lipid peroxidation in rat erythrocytes treated with tert-butyl hydroperoxide (700 μM), and the IC50 values for prevention of this process were equal to 9.7 ± 0.8 μM, 8.8 ± 0.7 μM, and 37.8 ± 4.4 μM in the case of quercetin, catechin and naringenin, respectively, and slightly decreased glutathione oxidation. In isolated rat liver mitochondria, quercetin, catechin and naringenin (10–50 μM) dose-dependently increased the sensitivity to Ca2+ ions - induced mitochondrial permeability transition. Using the probes TMA-DPH and DPH we showed that quercetin rather than catechin and naringenin strongly decreased the microfluidity of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomal membrane bilayer at different depths. On the contrary, using the probe Laurdan we observed that naringenin transfer the bilayer to a more ordered state, whereas quercetin dose-dependently decreased the order of lipid molecule packing and increased hydration in the region of polar head groups. The incorporation of the flavonoids, quercetin and naringenin and not catechin, into the liposomes induced an increase in the zeta potential of the membrane and enlarged the area of the bilayer as well as lowered the temperature and the enthalpy of the membrane phase transition. The effects of the flavonoids were connected with modification of membrane fluidity, packing, stability, electrokinetic properties, size and permeability, prevention of oxidative stress, which depended on the nature of the flavonoid molecule and the nature of the membrane.

Light-induced lipid mixing implies a causal role of lipid splay in membrane fusion

The fusion of lipid membranes is central to many biological processes and requires substantial structural reorganization of lipids brought about by the action of fusogenic proteins. Previous molecular dynamics simulations have suggested that splayed lipids, whose tails transiently contact the headgroup region of the bilayer, initiate lipid mixing. Here, we explore the lipid splay hypothesis experimentally. We show that the light-induced trans/cis conversion of the azobenzene-based tail of a model lipid molecule enhances the probability by which its own acyl chains, or the acyl chains of the host lipid, transiently contact the lipid headgroup in a liposomal bilayer. At the same time, the trans/cis conversion triggers lipid mixing of sonicated or extruded liposomes, without requiring fusogenic proteins. This establishes a causal relationship between lipid splay and membrane fusion.

Lipid Composition of Liposomal Membrane Largely Affects Its Transport and Uptake through Small Intestinal Epithelial Cell Models.

Lipid composition of liposomal bilayer should alter the cell response for permeability, transport, and uptake in small intestine. This work was done to investigate the transport and uptake of liposomes composed of docosahexaenoic acid-enriched phosphatidylcholine (PtdCho), phosphatidylserine (PtdSer), and sulfoquinovosyl diacylglycerol (SQDG) derived from marine products on multilamellar vesicles (MLV) in small intestinal epithelial cell models. The results showed that addition of PtdSer and SQDG as liposomal bilayer could improve the efficiency entrapment of liposomes. The liposomes containing PtdSer showed higher transport and uptake through both Caco-2 cell and M cell monolayers as compared to PtdCho-MLV. SQDG-containing liposomes exhibited only higher transport through M cell monolayer, while its uptake effect was higher both in Caco-2 cell and M cell monolayers. The results of experiments done with endocytosis inhibitors indicated that PtdCho-MLV must be transported via macropinocytosis and uptaken by phagocytosis in M cell monolayer model. PtdCho/PtdSer-MLV and PtdCho/SQDG-MLV might be transported and uptaken through M cell monolayer by phagocytosis. The result also indicated that PtdCho/SQDG-MLV could open the tight junction of small intestinal epithelial cell monolayers. Furthermore, our findings demonstrated that the surface status of cholesterol-containing liposomes were smooth, but they did not affect their transport and uptake through Caco-2 cell and M cell monolayers.

Photohyperthermal therapy using liposomally formulated indocyanine green for feline nasal lymphoma: A case report.

Our previous research has focused on the development of a novel cancer therapy by using photohyperthermal therapy (PHT) with indocyanine green (ICG) as an optical sensitizer. ICG-Lipo is a liposomally formulated ICG derivative in which ICG is tagged with an octadeca-alkyl chain to incorporate into liposome bilayers, and contains antitumor drugs such as carboplatin and paclitaxel within the inner membrane space. The present study reported a case of feline nasal lymphoma that was treated with combination therapy of PHT with ICG-Lipo. An antitumour effect was observed, and the patient entered remission. Complications from the radiation treatment included skin burns and bleeding from the irradiated hard palate. Serious side effects related to the drugs were not observed. This report suggested that PHT using ICG-Lipo enabled efficient and safe treatment of lymphoma, and that treatment with a liposomal drug delivery system was enhanced by PHT.

Modulating Lipoprotein Transcellular Transport and Atherosclerotic Plaque Formation in ApoE-/- Mice via Nanoformulated Lipid-Methotrexate Conjugates.

Macrophage inflammation and maturation into foam cells, following the engulfment of oxidized low-density lipoproteins (oxLDL), are major hallmarks in the onset and progression of atherosclerosis. Yet, chronic treatments with anti-inflammatory agents, such as methotrexate (MTX), failed to modulate disease progression, possibly for the limited drug bioavailability and plaque deposition. Here, MTX–lipid conjugates, based on 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were integrated in the structure of spherical polymeric nanoparticles (MTX-SPNs) or intercalated in the lipid bilayer of liposomes (MTX-LIP). Although, both nanoparticles were colloidally stable with an average diameter of ∼200 nm, MTX-LIP exhibited a higher encapsulation efficiency (>70%) and slower release rate (∼50% at 10 h) compared to MTX-SPN. In primary bone marrow derived macrophages (BMDMs), MTX-LIP modulated the transcellular transport of oxLDL more efficiently than free MTX mostly by inducing a 2-fold overexpression of ABCA1 (regulating oxLDL efflux), while the effect on CD36 and SRA-1 (regulating oxLDL influx) was minimal. Furthermore, in BMDMs, MTX-LIP showed a stronger anti-inflammatory activity than free MTX, reducing the expression of IL-1β by 3-fold, IL-6 by 2-fold, and also moderately of TNF-α. In 28 days high-fat-diet-fed apoE–/– mice, MTX-LIP reduced the mean plaque area by 2-fold and the hematic amounts of RANTES by half as compared to free MTX. These results would suggest that the nanoenhanced delivery to vascular plaques of the anti-inflammatory DSPE-MTX conjugate could effectively modulate the disease progression by halting monocytes’ maturation and recruitment already at the onset of atherosclerosis.

Systematic Quantification of Electron Transfer in a Bare Phospholipid Membrane Using Nitroxide-Labeled Stearic Acids: Distance Dependence, Kinetics, and Activation Parameters.

In this report, we present a method to characterize the kinetics of electron transfer across the bilayer of a unilamellar liposome composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The method utilizes synthetic phospholipids containing noninvasive nitroxide spin labels having the >N–O• moiety at well-defined distances from the outer surface of the liposome to serve as reporters for their local environment and, at the same time, permit measurement of the kinetics of electron transfer. We used 5-doxyl and 16-doxyl stearic acids. The paramagnetic >N–O• moiety is photo-oxidized to the corresponding diamagnetic oxoammonium cation by a ruthenium electron acceptor formed in the solution. Electron transfer is monitored by three independent spectroscopic methods: by both steady-state and time-resolved electron paramagnetic resonance and by optical spectroscopy. These techniques allowed us to differentiate between the electron transfer rates of nitroxides located in the outer leaflet of the phospholipid bilayer and of those located in the inner leaflet. Measurement of electron transfer rates as a function of temperature revealed a low-activation barrier (ΔG‡ ∼ 40 kJ/mol) that supports a tunneling mechanism.