Lipid Bilayer Of Liposomes Research Articles

A Stable Alkylated Cobalt Catalyst for Photocatalytic H2 Generation in Liposomes

AbstractPhotocatalytic proton reduction is a promising way to produce dihydrogen (H2) in a clean and sustainable manner, and mimicking nature by immobilising proton reduction catalysts and photosensitisers on liposomes is an attractive approach for biomimetic solar fuel production in aqueous solvents. Current photocatalytic proton reduction systems on liposomes are, however, limited by the stability of the catalyst. To overcome this problem, a new alkylated cobalt(II) polypyridyl complex (CoC12) was synthesised and immobilised on the lipid bilayer of liposomes, and its performance was studied in a photocatalytic system containing an alkylated ruthenium photosensitiser (RuC12) and a 1 : 1 mixture of sodium ascorbate and tris‐2‐carboxyethylphosphine hydrochloride as sacrificial electron donors. Several parameters (concentration of CoC12 and RuC12, pH, membrane composition) were changed to optimise the turnover number for H2 production. Overall, CoC12 was found to be photostable and the optimised turnover number (161) was limited only by the decomposition of the ruthenium‐based photosensitiser.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

Mechanisms of Influenza Virus HA2 Peptide Interaction with Liposomes Studied by Dual-Wavelength MP-SPR.

A phospholipid-based liposome layer was used as an effective biomimetic membrane model to study the binding of the pH-dependent fusogenic peptide (E4-GGYC) from the influenza virus hemagglutinin HA2 subunit. To this end, a multiparameter surface plasmon resonance approach (MP-SPR) was used for monitoring peptide-liposome interactions at two pH values (4.5 and 8) by means of recording sensorgrams in real time without the need for labeling. Biotinylated liposomes were first immobilized as a monolayer onto the surface of an SPR gold chip coated with a streptavidin layer. Multiple sets of sensorgrams with different HA2 peptide concentrations were generated at both pHs. Dual-wavelength Fresnel layer modeling was applied to calculate the thickness (d) and the refractive index (n) of the liposome layer to monitor the change in its optical parameters upon interaction with the peptide. At acidic pH, the peptide, in its α helix form, entered the lipid bilayer of liposomes, inducing vesicle swelling and increasing membrane robustness. Conversely, a contraction of liposomes was observed at pH 8, associated with noninsertion of the peptide in the double layer of phospholipids. The equilibrium dissociation constant KD = 4.7 × 10-7 M of the peptide/liposome interaction at pH 4.5 was determined by fitting the "OneToOne" model to the experimental sensorgrams using Trace Drawer software. Our experimental approach showed that the HA2 peptide at a concentration up to 100 μM produced no disruption of liposomes at pH 4.5.

Self-quenched liposomal probes for tumor imaging based on cellular on/off system

Near-infrared fluorophores have been widely used in tumor imaging due to superior resolution and sensitivity, high penetration depth and low auto-fluorescence. However, the tumor imaging efficacy of fluorophores in vivo is impeded by the background signals and the delivery efficiency of the fluorophores. To realize sensitive tumor imaging, we developed self-quenched liposomal probes DiR-LP-C and DiR-LP-B as tumor-specific imaging probes with cellular on/off system. To prepare DiR-LP-C probes, hydrophobic DiR was solubilized in DMSO and actively loaded into the aqueous interior of liposomes. Based on the structure, DiR-LP-C showed high self-quenching (46-fold at 1.0% loading ratio) that is ‘off’ state with weak fluorescence signal but recovered fluorescence after cellular uptake. DiR-LP-B was prepared where the hydrophobic DiR molecules were dispersed in the hydrophobic lipid bilayer of liposomes and showed weaker self-quenching (5.4-fold at 1.0% loading ratio). Importantly, DiR-LP-C showed brighter cell imaging than DiR-LP-B. For in vivo imaging, both probes showed excellent tumor targeting with signal to background ratio around 5 at 24 h post-injection, with DiR-LP-C had much lower background. These results proved that the tumor-homing cellular ‘on/off’ nanoparticle system of DiR-LP-C has a great potential for tumor imaging and phototherapeutics.

Cholesterol Microdomain Enhances the Biofilm Eradication of Antibiotic Liposomes.

Resistance and tolerance of biofilms to antibiotics is the greatest challenge in the treatment of bacterial infections. Therefore, developing an effective strategy against biofilms is a top priority. Liposomes are widely used as antibiotic drug carriers; however, common liposomes lack affinity for biofilms. Herein, biofilm-targeted antibiotic liposomes are created by simply adjusting their cholesterol content. The tailored liposomes exhibit significantly enhanced bacterial inhibition and biofilm eradication effects that are positively correlated with the cholesterol content of liposomes. The experiments further demonstrate that this enhanced effect can be ascribed to the effective drug release through the pores, which are formed by the combination of cholesterol microdomains in liposomal lipid bilayers with membrane-damaged toxins in biofilms. Consequently, liposome encapsulation with a high cholesterol concentration improves noticeably the pharmacodynamics and biocompatibility of antibiotics after pulmonary administration. This work may provide a new direction for the development of antibiofilm formulations that can be widely used for the treatment of infections caused by bacterial biofilms.

Engineering Lipusu with lysophosphatidylcholine for improved tumor cellular uptake and anticancer efficacy.

Liposomes have been developed as drug delivery carriers to enhance the antitumor efficiency of therapeutic agents. Lipusu® (Lip), a paclitaxel (PTX) liposome, has been widely used in the treatment of breast cancer. Compared with PTX, Lip could change the biodistribution and reduce the systemic toxicity. However, there was no positive effect on the entry of PTX into tumor cells, and thus the therapeutic effect was not significantly improved. Therefore, it is meaningful to engineer Lip for improving tumor cellular uptake efficiency. Here, lysophosphatidylcholine (LPC)-engineered Lip (LPC-Lip) was constructed via inserting single chain lipid tails into liposomal lipid bilayers, which was realized by simple incubation. Compared with Lip, the better cellular uptake of liposomes modified with LPC resulted in enhanced cytotoxic activity of LPC-Lip in 4T1 cells. Furthermore, stronger tumor growth inhibition was observed in LPC-Lip treated 4T1 tumor-bearing mice without significant side effects. In conclusion, by modulating the lipid composition of Lip, the antitumor efficacy can be improved, and LPC engineered Lip may serve as a promising formulation of PTX for future cancer therapy.

Synthesis and Evaluation of High Functionality and Quality Cell-penetrating Peptide Conjugated Lipid for Octaarginine Modified PEGylated Liposomes In U251 and U87 Glioma Cells

The use of peptide ligand modified PEGylated liposomes has been widely investigated for tumor targeting. Peptides are mainly inserted in the liposomal lipid bilayer using PEG2K-lipid spacer (Peptide-PEG2K-DSPE). However, a lower cellular uptake from longer nonlinear PEG2K spacer was reported, we here synthesized a high functionality and quality (HFQ) lipid with a short, linear serine-glycine repeated peptide [(SG)5] spacer. The objective of the current study is to develop novel octaarginine (R8) peptide-HFQ lipid grafted PEGylated liposomes for glioma cells targeting. In vitro liposomes characterization showed that the mean particle size of all liposomal formulations was in the nano-scale range < 120 nm, with a small PDI value (i.e. ∼0.2) and had a spherical shape under Transmission Electron Microscope, indicating a homogenous particle size distribution. The flow cytometry in vitro cellular association data with U251 MG and U87 cells revealed that 1.5% R8-(SG)5-lipid-PEGylated liposomes exhibited significantly higher cellular association of ∼15.87 and 7.59-fold than the conventional R8-PEG2K-lipid-PEGylated liposomes (10.4 and 6.19-fold), respectively, relative to the unmodified PEGylated liposomes. Moreover, intracellular distribution studies using confocal laser scanning microscopy (CLSM) corroborated the results of the in vitro cell association. The use of ligand-HFQ-lipid liposomes could be a potential alternative to ligand-PEG2K-lipid-modified liposomes as a drug delivery system for tumor targeting.

Liposomal IR-780 as a Highly Stable Nanotheranostic Agent for Improved Photothermal/Photodynamic Therapy of Brain Tumors by Convection-Enhanced Delivery.

Simple SummaryTo improve the use of hydrophobic photosensitizer IR-780 in photothermal/photodynamic therapy (PTT/PDT), we entrap IR-780 within the lipid bilayer of liposomes (ILs). Compared to free IR-780, ILs showed well-preserved photothermal response by maintaining the photostability of IR-780 from repeated near infrared (NIR) laser exposure both in vitro and in vivo. Combined with fast endocytosis by human glioblastoma cells, ILs demonstrated enhanced cytotoxicity and induced higher cell apoptosis rate toward human glioblastoma cells over free IR-780, due to PTT with overexpression of heat shock protein and PDT with generation of intracellular reactive oxygen species. To overcome the blood–brain barrier, we used convection enhanced delivery (CED) for specific delivery of ILs to brain tumors in intracranial glioma xenograft. Upon three successive NIR laser irradiations, the liposomal IR-780 could significantly improve the anti-cancer efficacy in glioma treatment, leading to diminished intracranial tumor size and prolonged animal survival time.As a hydrophobic photosensitizer, IR-780 suffers from poor water solubility and low photostability under near infrared (NIR) light, which severely limits its use during successive NIR laser-assisted photothermal/photodynamic therapy (PTT/PDT). To solve this problem, we fabricate cationic IR-780-loaded liposomes (ILs) by entrapping IR-780 within the lipid bilayer of liposomes. We demonstrate enhanced photostability of IR-780 in ILs with well-preserved photothermal response after three repeated NIR laser exposures, in contrast to the rapid decomposition of free IR-780. The cationic nature of ILs promotes fast endocytosis of liposomal IR-780 by U87MG human glioblastoma cells within 30 min. For PTT/PDT in vitro, ILs treatment plus NIR laser irradiation leads to overexpression of heat shock protein 70 and generation of intracellular reactive oxygen species by U87MG cells, resulting in enhanced cytotoxicity and higher cell apoptosis rate. Using intracranial glioma xenograft in nude mice and administration of ILs by convection enhanced delivery (CED) to overcome blood-brain barrier, liposomal IR-780 could be specifically delivered to the brain tumor, as demonstrated from fluorescence imaging. By providing a highly stable liposomal IR-780, ILs significantly improved anti-cancer efficacy in glioma treatment, as revealed from various diagnostic imaging tools and histological examination. Overall, CED of ILs plus successive laser-assisted PTT/PDT may be an alternative approach for treating brain tumor, which can retard glioma growth and prolong animal survival times from orthotopic brain tumor models.

Spatiotemporal Resolved Live Cell Membrane Tracking through Photo-click Reactions Enriched in Lipid Phase.

A set of photo-switchable monopeptides derived from cis-β-dibenzodiazocine-l-alanine (cis-DBDAA) have been designed and synthesized, which are capable of photo-click reacting with diaryltetrazoles or diarylsydnones in a hydrophobic phospholipid bilayer environment. The DBDAA monopeptides include both a hydrophobic tail on C-terminal, providing high affinity toward lipid membrane, and a modularized functional moiety on N-terminal, enabling rapid optimization of the self-assembly strength to form multifunctional supramolecules. With the cis-DBDAA monopeptides photo-switched into trans-configuration, we were able to disrupt the supramolecular assembly through an efficient photo-click reaction across the lipid bilayer of liposomes. We reveal that the performance of the photo-click reactions between the monopeptides and photo-generated nitrile imine intermediates is significantly enhanced by enrichment of both reactants in the hydrophobic membrane lamel of liposomes. Enrichment of the DBDAA monopeptide in lipid phase serves as a convenient method to introduce bioorthogonal chemical handles on live cell membranes, which enables fluorescence labelling of single cell's membrane with high spatiotemporal resolution to facilitate the studies on cell membrane dynamics.

Nanodelivery of the Gramicidin Peptide for Enhancing Antimicrobial Activity

AbstractGramicidin S (GraS) is an amphiphilic peptide that has emerged as an effective alternative antibiotic. However, its applicability is restricted for clinical uses due to its effect on eukaryotic cells and low aqueous solubility. In this work, a novel water‐soluble peptide formulation with bactericidal activity is developed by the incorporation of Gramicidin S in the lipid bilayer of liposomes (L‐GraS). As a result, GraS included in the lipid vesicles is stabilized in aqueous medium and showed antimicrobial activity against Staphylococcus aureus. In addition, L‐GraS reveals enhanced biocompatibility with mammalian cells in comparison with the free peptide. Molecular dynamics simulations shed light on the collective behavior between GraS peptides and liposomes from a molecular approach. The molecular dynamic simulations are in agreement with experimental results and further confirm the effective incorporation of GraS in the liposomes, letting understand the best formulation procedure of the vesicles. Therefore, the L‐GraS nanosystem presented here is an interesting alternative to conventional antibiotics, with less restrictions and promising features to improve current therapies.Practical Applications: Incorporation of GraS in liposomes is an interesting approach to increase the solubility of the peptide, thus expanding its therapeutic horizon as a promising alternative to combat bacterial colonization especially in wound infections.

PD1 blockade potentiates the therapeutic efficacy of photothermally-activated and MRI-guided low temperature-sensitive magnetoliposomes

This study investigates the effect of PD1 blockade on the therapeutic efficacy of novel doxorubicin-loaded temperature-sensitive liposomes. Herein, we report photothermally-activated, low temperature-sensitive magnetoliposomes (mLTSL) for efficient drug delivery and magnetic resonance imaging (MRI). The mLTSL were prepared by embedding small nitrodopamine palmitate (NDPM)-coated iron oxide nanoparticles (IO NPs) in the lipid bilayer of low temperature-sensitive liposomes (LTSL), using lipid film hydration and extrusion. Doxorubicin (DOX)-loaded mLTSL were characterized using dynamic light scattering, differential scanning calorimetry, electron microscopy, spectrofluorimetry, and atomic absorption spectroscopy. Photothermal experiments using 808 nm laser irradiation were conducted. In vitro photothermal DOX release studies and cytotoxicity was assessed using flow cytometry and resazurin viability assay, respectively. In vivo DOX release and tumor accumulation of mLTSL(DOX) were assessed using fluorescence and MR imaging, respectively. Finally, the therapeutic efficacy of PD1 blockade in combination with photothermally-activated mLTSL(DOX) in CT26-tumor model was evaluated by monitoring tumor growth, cytokine release and immune cell infiltration in the tumor tissue. Interestingly, efficient photothermal heating was obtained by varying the IO NPs content and the laser power, where on-demand burst DOX release was achievable in vitro and in vivo. Moreover, our mLTSL exhibited promising MR imaging properties with high transverse r2 relaxivity (333 mM−1 s−1), resulting in superior MR imaging in vivo. Furthermore, mLTSL(DOX) therapeutic efficacy was potentiated in combination with anti-PD1 mAb, resulting in a significant reduction in CT26 tumor growth via immune cell activation. Our study highlights the potential of combining PD1 blockade with mLTSL(DOX), where the latter could facilitate chemo/photothermal therapy and MRI-guided drug delivery.

Liposome co-encapsulation of anti-cancer agents for pharmacological optimization of nanomedicine-based combination chemotherapy.

Aim: Co-encapsulation of anti-cancer agents in pegylated liposomes may provide an effective tool to maximize efficacy of combined drug therapy by taking advantage of the long circulation time, passive targeting, and reduced toxicity of liposome formulations.Methods: We have developed several liposome formulations of co-encapsulated drugs using various permutations of three active agents: doxorubicin (Dox), mitomycin-C lipidic prodrug (MLP), and alendronate (Ald). Dox and MLP are available in single drug liposomal formulations: pegylated liposomal Dox (PLD, Doxil®), clinically approved, and pegylated liposomal MLP (PL-MLP, Promitil®), in phase 1-2 clinical testing. We have previously shown that co-encapsulation of Dox and Ald in pegylated liposomes (PLAD) results in a formulation with valuable immuno-pharmacologic properties and superior antitumor properties over PLD in immunocompetent animal models. Building on the PLAD and PL-MLP platforms, we developed a new pegylated liposomal formulation of co-entrapped Dox and MLP (PLAD-MLP), with the former localized in the liposome water phase via remote loading with an ammonium alendronate and the latter passively loaded into the liposome lipid bilayer. An alternative formulation of co-entrapped MLP and Dox in which ammonium Ald was replaced with ammonium sulfate (PLD-MLP) was also tested for comparative purposes.Results: PLAD-MLP displays high loading efficiency of Dox and MLP nearing 100%, and a mean vesicle diameter of 110 nm. Cryo-transmission electron microscopy (cryo-TEM) of PLAD-MLP reveals round vesicles with an intra-vesicle Dox-alendronate precipitate. PLAD-MLP was tested in an in vitro MLP activation assay with the reducing agent dithiothreitol and found to be significantly less susceptible to thiolytic activation than PL-MLP. Alongside thiolytic activation of MLP, a significant fraction of encapsulated Dox was released from liposomes. PLAD-MLP is stable upon in vitro incubation in human plasma with nearly 100% drug retention. In mouse pharmacokinetic studies, PLAD-MLP extended MLP half-life in circulation when compared to that of MLP delivered as PL-MLP. In addition, the MLP levels in tissues were greater than those obtained with PL-MLP, indicating that PLAD-MLP slows down the cleavage of the prodrug MLP to MMC, thus resulting in a more sustained and prolonged exposure. The circulation half-life of Dox in PLAD-MLP was similar to the PLD Dox half-life. The pattern of tissue distribution was similar for the co-encapsulated drugs, although Dox levels were generally higher than those of MLP, as expected from cleavage of MLP to its active metabolite MMC. In mouse tumor models, the therapeutic activity of PLAD-MLP was superior to PL-MLP and PLD with a convenient safety dose window. The Ald-free formulation, PLD-MLP, displayed similar pharmacokinetic properties to PLAD-MLP, but its therapeutic activity was lower.Conclusion: PLAD-MLP is a novel multi-drug liposome formulation with attractive pharmacological properties and powerful antitumor activity and is a promising therapeutic tool for combination cancer chemotherapy.

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.

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.

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.

Co-Delivery of Hispolon and Doxorubicin Liposomes Improves Efficacy Against Melanoma Cells.

Hispolon is a small molecular weight polyphenol that has antioxidant, anti-inflammatory, and anti-proliferative activities. Our recent study has demonstrated hispolon as a potent apoptosis inducer in melanoma cell lines. Doxorubicin is a broad spectrum first-line treatment for various kinds of cancers. In this study, co-delivery of doxorubicin and hispolon using a liposomal system in B16BL6 melanoma cell lines for synergistic cytotoxic effects was investigated. Liposomes were prepared using a lipid film hydration method and loaded with doxorubicin or hispolon. The formulations were characterized for particle size distribution, release profile, and encapsulation efficiency (EE). In addition, in vitro cytotoxicity, in vitro cell apoptosis, and cellular uptake were evaluated. Liposomes exhibited small particle size (mean diameter ~ 100nm) and narrow size distribution (polydispersity index (< 0.2) and high drug EE% (> 90%). The release from liposomes showed slower release compared to free drug solution as an additional time required for the release of drug from the liposome lipid bilayer. Liposome loaded with doxorubicin or hispolon exhibited significantly higher cytotoxicity against B16BL6 melanoma cells as compared to doxorubicin solution or hispolon solution. Likewise, co-delivery of hispolon and doxorubicin liposomes showed two-fold and three-fold higher cytotoxicity, as compared to hispolon liposomes or doxorubicin liposomes, respectively. In addition, co-delivery of doxorubicin and hispolon in liposomes enhanced apoptosis more than the individual drugs in the liposome formulation. In conclusion, the co-delivery of hispolon and doxorubicin could be a promising therapeutic approach to improve clinical outcomes against melanoma.

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.

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.

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.