Nanoscale Liposomes Research Articles

Near infrared fluorescent chlorophyll nanoscale liposomes for sentinel lymph node mapping.

BackgroundSentinel lymph node (SLN) mapping using in vivo near infrared fluorescence imaging has attracted great attention during the past few years. Here we report on the early use of poorly water-soluble chlorophyll with near infrared fluorescence extracted from the leaf of Chimonanthus salicifolius, for mouse axillary SLN mapping.Methods and resultsTo improve the water solubility and SLN targeting of the chlorophyll, we encapsulated the chlorophyll in nanoscale liposomes. The liposome-coated chlorophyll nanocomposites obtained were spherical in shape and had an average diameter of 21.7 ± 6.0 nm. The nanocomposites dispersed well in water, and in aqueous suspension they exhibited brighter near infrared fluorescence than chlorophyll alone. After incubation of the nanocomposites with normal liver cells (QSG-7701) and macrophage cells (Ana-1) for no more than 48 hours, there was no obvious reduction in cell viability. When the nanocomposites were injected intradermally into the paw of a mouse, the axillary SLN was found to be strongly fluorescent and was easily visualized in real time without a requirement for surgery. The intensity of the near infrared fluorescence emitted by the SLN was obviously brighter than that emitted by the SLN of another mouse that had been intradermally injected with chlorophyll alone.ConclusionOur data show that the liposome-coated chlorophyll nanocomposites could have great potential for clinical SLN mapping due to their lack of toxicity, bright near infrared fluorescence, and small diameter.

Anomalous Freezing Behavior of Nanoscale Liposomes

Anomalous Freezing Behavior of Nanoscale Liposomes

Anomalous freezing behavior of nanoscale liposomes

The effect of the finite size of one-component liposomes on their phase behavior is investigated via simulations of an implicit-solvent model of self-assembled lipid bilayers. We found that the high curvature of nanoscale liposomes has a significant effect on their freezing behavior. While the location of the freezing temperature of liposomes with diameters larger than 30 nm is the same as that of a planar membrane, the transition is broadened as the liposome diameters are decreased. For very small liposomes, with diameters smaller than 30 nm, the transitions of the two leaflets become increasingly decoupled, and their respective freezing temperatures become different. In particular, the outer leaflet gels at a temperature, T(u)g, which is higher than that of the inner leaflet, T(i)g. We argue that this decoupling is due to the difference in configurational entropies of the lipid chains in the two leaflets. At temperatures higher than T(u)g, the liposomes are spherical and the lipids in both leaflets lack both positional and chain order. At temperatures between T(i)g and T(u)g, the lipids in the outer leaflet exhibit chain order but lack positional order. In contrast the lipids in the inner leaflet lack both chain order and positional order in this temperature range. In this range of temperatures, liposomes are essentially spherical. At temperatures lower than T(i)g, lipids in both the inner and outer leaflets exhibit chain order as well as positional order, and the liposomes become faceted. The structure of the outer leaflet for T(i)g < T < T(u)g is surprisingly akin to that of the liquid ordered phase, which is observed in binary mixtures of saturated phospholipids and cholesterol.

Observation of Inhomogeneity in the Lipid Composition of Individual Nanoscale Liposomes

Liposomes are one of the most extensively used model systems for studying the physical and chemical properties of membranes and the majority of these studies have implicitly assumed that all liposomes in an ensemble are identical. However recent measurements performed at the level of single liposomes revealed the existence of intrinsic intra-sample heterogeneities that were otherwise averaged out in ensemble experiments. By employing fluorescently labeled lipids and measuring at the single liposome level we examined the compositional inhomogeneity between individual liposomes within an ensemble. In a recent publication we demonstrated an up to ten-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm.1 This observation is made for both a variety of different lipid-labels and compositions, which suggest that compositional inhomogeneity is a general phenomenon present in liposome systems. As a result, bulk measurement of physical and chemical properties that depend on bilayer composition, e.g. phase-transition temperature, will produce results representing the ensemble average, which will be a convolution of the properties arising from many different bilayer compositions of the individual liposomes. Furthermore, we saw that the choice of liposome preparation method greatly influences the degree of compositional inhomogeneity. This could be particularly important when using liposomes as drug delivery carriers where the monodispersity of, and control over, bilayer properties is instrumental. Since the physicochemical properties of liposomes are directly linked to their composition a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble. (1) Larsen, J.; Hatzakis, N. S.; Stamou, D. J. Am. Chem. Soc. 2011, 133, 10685-10687.

Fabrication of nano-scale liposomes containing doxorubicin using Shirasu porous glass membrane

Nano-scale liposomes were successfully produced using a Shirasu porous glass (SPG) membrane emulsification technique. Primary liposomes prepared by a film-hydration method were treated using SPG membranes with different pore sizes (2.0, 1.0, 0.7, 0.5, and 0.2 μm) for control over the liposome size. The liposome sizes were evaluated using a dynamic light scattering method and their morphologies were observed by optical microscopy and transmission electron microscopy. As the passage number of liposomes through SPG membrane increased, the size and its distribution of the liposomes gradually decreased. A smaller pore size of the SPG membrane and a higher applied pressure resulted in liposomes with a smaller size. After the preparation of nano-scale liposomes containing ammonium sulfate (AS), doxorubicin (DOX) was encapsulated in the liposomes by a remote loading method, where AS served as a precipitant for DOX. The encapsulation efficiency of the DOX was maximized up to 94% when the concentrations of AS and DOX were 250 and 0.045 mM, respectively. We have obtained the release profiles of the liposomes with different sizes. As shown below, liposomes with smaller size exhibited a faster release profile of drug due to the large surface area. These nano-scale liposomes encapsulating an anti-cancer drug can potentially be employed as drug delivery vehicles for intravenous injection.

Observation of Inhomogeneity in the Lipid Composition of Individual Nanoscale Liposomes

Liposomes, or vesicles, have been studied extensively both as models of biological membranes and as drug delivery vehicles. Typically it is assumed that all liposomes within the same preparation are identical. Here by employing pairs of fluorescently labeled lipids we demonstrated an up to 10-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm. Since the physicochemical properties of liposomes are directly linked to their composition, a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.

Triggered Release of Pharmacophores from [Ni(HAsO3)]-Loaded Polymer-Caged Nanobin Enhances Pro-apoptotic Activity: A Combined Experimental and Theoretical Study

Nanoscale drug delivery platforms can provide an attractive therapeutic strategy for cancer treatments, as they can substantially reduce the adverse side effects associated with toxic small-molecule anticancer agents. For enhanced therapeutic efficacy to be achieved with such platforms, a tumor-specific drug-release trigger is a critical requirement. This article reports the use of a pH-sensitive polymer network that surrounds a nanoscale liposome core to trigger the release of both encapsulated hydrophilic, membrane-impermeable Ni(II) cations and amphipathic, membrane-permeable As(III) anticancer agents under acidic conditions commonly encountered in hypoxic tumor tissues and late endosomes. Computational modeling studies provide clear evidence that the acid-triggered drug-release mechanism for this polymer-caged nanobin (PCN) platform arises from a pH- and temperature-responsive conformation change of the cross-linked polymer cage. As a result, the simultaneous release of both of the active agents in this multicomponent therapeutic enhances the pro-apoptotic activity of As(III) while diminishing its acute toxicity, potentially reducing the undesirable side effects commonly associated with this free drug. The ability to engender acid-triggered release of drugs co-encapsulated inside a liposomal template makes drug delivery using PCN an attractive strategy for triggered drug release.

Induction of anti-tumor immunity and T-cell responses using nanodelivery systems engrafting TLR-5 ligand

Evaluation of: Faham A, Altin JG. Antigen-containing liposomes engrafted with flagellin-related peptides are effective vaccines that can induce potent antitumor immunity and immunotherapeutic effect. J. Immunol. 185(3), 1744–1754 (2010).Induction of activated T-cell responses is a prerequisite for the development of vaccine against intracellular infection and for the control of cancer. Particulate nanoscale delivery systems deliver antigens intracellularly and help in inducing T-cell responses. T-cell responses can be further augmented by targeting these particles to dendritic cells, which have the ability to induce both innate and adaptive immune responses. Flagellin, which acts as a TLR-5 ligand, has been extensively explored for its adjuvant activity. The paper under evaluation reports a novel vaccine delivery platform technology for induction of a T-cell response using a nanoscale liposome containing antigen and a small synthetic peptide representing TLR-5-binding motifs of flagellin. Vaccination using this nanodelivery system activated dendritic cells through TLR-5 activation and induced both innate and adaptive immune responses. Such novel delivery systems can improve modern vaccine formulation, particularly for the generation of activated T-cell responses and anti-tumor immunity.

Leukemia Cell–Selective Uptake of Cytarabine and Daunorubicin in the Bone Marrow Compartment Mediated by CPX-351 Liposome Injection

Abstract Background Anticancer drug combinations such as cytarabine plus daunorubicin can act synergistically, additively or antagonistically depending on the ratio of the agents being combined. We have shown that delivering synergistic cytarabine-to-daunorubicin drug ratios in vivo using nanoscale liposomes (CPX-351 liposome injection) provides dramatic efficacy improvements compared to the free drug cocktail in a wide range of preclinical leukemia models without exacerbating myelosuppressive effects. This enhanced efficacy is associated with increased and prolonged delivery of the synergistic drug ratio to bone marrow. Promising signs of efficacy have been observed with CPX-351 in previously treated patients with AML with poor prognosis. We aim to investigate the pharmacodynamic basis for the potent therapeutic activity of CPX-351. Patients and Methods A bone marrow–engrafting CCRF-CEM human leukemia xenograft model was developed. CPX-351 was injected intravenously (I.V.), and the bone marrows of femurs from tumor bearing mice were subsequently harvested. Cells were analyzed by flow cytometry, and leukemia cells were separated from normal bone marrow cells using anti-CD45 coated magnetic nanoparticles. Cells were analyzed for intracellular cytarabine-to-daunorubicin and liposome content using 3 H-Cyt, HPLC, and 14 C-lipid, respectively. Results Twenty-eight days after I.V. tumor inoculation, femurs contained approximately equal numbers of CCRF-CEM and normal bone marrow cell populations that could be readily separated and quantitatively harvested by antibody-coated magnetic nanoparticle-mediated isolation. Eighteen hours after CPX-351 I.V. injection, bone marrows of the tumor bearing mice contained 82.6 ng cytarabine, 63.0 ng daunorubicin, and 1.2 μg liposomal lipid per total femur aspirate. Within the separated cell populations, leukemia cells contained 5.9 ng cytarabine, 8.2 ng daunorubicin, and 0.16 μg liposomal lipid per 10 6 cells. These intracellular levels were 9.5-, 2.2-, and 1.9-fold higher than those observed in the normal bone marrow cell population. Confocal microscopy on cells incubated with CPX-351 in vitro demonstrated that CPX-351 liposomes are taken up intact by human leukemia cells and subsequently release the drugs intracellularly. Conclusion CPX-351 was designed to enhance the antitumor efficacy of cytarabine-to-daunorubicin combination therapy by encapsulating both agents within a drug carrier that maintains the synergistic 5:1 molar ratio for extended times after injection. The potent anti-leukemic activity obtained in the absence of significant nonhematologic toxicity with this formulation may be due, in part, to the selective accumulation of CPX-351 into leukemia cells.

Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts

The objective of this study was to examine the pharmacodynamic basis for the potent preclinical and clinical anti-leukemic activity of CPX-351, a nano-scale liposome formulation of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio. A bone marrow-engrafting CCRF-CEM leukemia model in Rag2-M mice was utilized to correlate the therapeutic and myelosuppressive properties of CPX-351 with bone marrow delivery and drug uptake in leukemia cells relative to normal bone marrow cell populations. When administered to mice bearing CCRF-CEM human leukemia xenografts, CPX-351 ablated bone marrow (BM) leukemic cells to below detectable levels for multiple weeks, whereas the free-drug cocktail only transiently suppressed leukemia growth. In contrast to the activity against leukemia cells, CPX-351 and free-drug cocktail induced similar myelosuppression in non-tumor-bearing BM. In leukemia-laden BM, drug concentrations were markedly elevated for CPX-351 over free-drug cocktail and the first dose of CPX-351, but not free-drug cocktail, potentiated BM drug accumulation for subsequent doses. Confocal fluorescence microscopy revealed that CPX-351 liposomes are taken up by CCRF-CEM cells and subsequently release drugs intracellularly. The improved in vivo efficacy of CPX-351 appears related to increased and prolonged exposure of synergistic cytarabine:daunorubicin ratios in BM, and the selective killing of leukemia may arise from direct liposome-leukemia cell interactions. These features may also have broader applicability in the treatment of other haematological malignancies.

Fabricating a New Stabilized Lipid-Based Platform for Handling and Presenting GPCRs

G protein-coupled receptors (GPCRs) are members of large family of signaling molecules. They have a key function in neuronal biology and play a critical rule in transmitting extracellular signals in eukaryotic cells. GPCRs include receptors for many neurotransmitters like serotonin. The serotonin receptor 5HT1A has an important role in mental disorders like anxiety, and is the target of some anti anxiety drugs. Here we constructed a novel biomimetic lipid membrane-based platform to be used for screening molecules that interact with GPCRs. Since fragility and short life time limit the applicability of liposomes that are normally used to present membrane proteins, we have developed a platform based on nanoscale liposomes with enhanced longevity and stability. We developed two approaches to make stable liposomes: liposomes containing a UV-initiated poly(ethylene glycol) (PEG) hydrogel and conjugated hydrogel liposomes made from lipids covalently anchored to the hydrogel network. Stability of nanoscale liposomes was confirmed by addition of high concentration of sodium dodecyl sulfate (SDS) to the liposome suspension. The liposome/detergent micelle mixture was passed over a size exclusion chromatography (SEC) column, separating intact liposomes from micelles. Using dynamic light scattering (DLS), we could confirm the existence of intact 160 nm liposomes. This result was in good agreement with the SEC result for polymerized liposomes without detergent. Since our goal is making GPCR-bearing liposomes, serotonin receptor 5HT1A was incorporated into liposomes using a detergent-mediated method. GPCR incorporation was confirmed by binding labeled HTR1A antibody to liposomes containing biotinylated lipids and subsequently separating them from bulk with streptavidin-coated magnetic beads. We are currently undertaking further studies on antibody-liposome binding by fluorescence anisotropy to validate our approach.

Accurate Optical Analysis of Single-Molecule Entrapment in Nanoscale Vesicles

We present a nondestructive method to accurately characterize low analyte concentrations (0-10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device. A confocal microscope was used to detect a laser-induced fluorescence signal resulting from both encapsulated and unencapsulated SRB molecules. The first two cumulants of this signal along with the autocorrelation function (ACF) were used to quantify liposome entrapment efficiency. Our analysis moves beyond typical, nonphysical assumptions of equal liposome size and brightness. These advances are essential for characterizing liposomes in the single-molecule encapsulation regime. Our work has further analytical impact because it could increase the interrogation time of free-solution molecular analysis by an order of magnitude and form the basis for the development of liposome standard reference materials.

Comparative study on gene transfer efficiency of homemade nanoscale ultrasound microbubbles contrast agent and liposome

Objective To evaluate the availability of homemade nanoscale ultrasound microbub-bles contrast agent as a new gene carrier. Methods MTT was used to measure the cell survival rate to chose the safe concentration of the contrast agent. The mixture of Pshuttle-IRES-hrGFP-1 plasmid and mi-crobubbles contrast agent/LipofectamineTM 2000 was transfected into HepG2 cells. Twenty-four h later, the expression of GFP in the cells was observed by fluorescence microscopy, then flow cytomertry was used to evaluate the gene transfer efficiency. Results (1) ≤5% microbubbles contrast agent had no significant influence on HepG2 growth (P ＜ 0.05). (2) Homemade microbubbles contrast agent could delivery GFP into HepG2 and the genes could be expressed efficiently. The gene transfer efficiency of homemade micro-bubbles contrast agent was (32.61 ± 3.42) %, and that of LipofectamineTM 2000 was (34.12 ± 8.06) % with the difference being not statistically significant between them (P ＞ 0.05). Conclusion Homemade nanoscale microbubbles contrast agent could delivery exogenous DNA into HepG2 cells, and its gene trans-fer efficiency had no statistically significant difference compared with LipofectamineTM2000. Key words: Contrast agent; Nanometer; Liposeme; Gene transfer efficiency

Regulatory and Scientific Barriers to The Safety Evaluation of Medical Nanotechnologies

Nanotechnology has permeated several key markets, with its fastest and highest rate of incorporation found in the development of medicinal products, medical device and other healthcare products. This has been achieved without reform of regulatory testing guidelines that were developed before nanotechnologies were even conceptualized. A number of medicinal products containing nanomaterials have already been authorized both in the EU and the USA. These nanotechnology-based products incorporate nanoscale liposomes (e.g., Caelyx/Doxil and MyocetTM), lipid micelles (e.g., Estrasorb), virosomes (e.g., Epaxal Berna) and pegylated proteins (e.g., Neulasta, Pegasys, PegIntron, Macugen and Somavert). Other products include CALAA-01 with its proprietary siRNApolymer delivery system, the synthetic amino acid polymer in Copaxone, protein conjugates (e.g., Abraxane), microemulsions (e.g., of cyclosporine) and nanocrystalline suspensions (e.g., Rapamune, Emend and Megace ES). Additional organic nanomaterials being investigated for drug delivery include carbon fullerenes and nanotubes, whilst inorganic nanoparticles are already incorporated into a number of in vivo diagnostic products (iron nanoparticles), wound care products (nanosilver) or dental and bone restoratives (calcium nanoparticles and composites) [1]. The fact that novel characteristics of nanomaterials, such as SilcrystTM and NanoOss, are conferred by their size, shape, surface and core chemistry have been long since recognized by organizations such as the US and European Patent and Trademark Offices and regulatory agencies such as the European Medicines Agency (EMEA) and the US FDA. These novel characteristics have met important medical needs, such as the ability to deliver therapeutic agents across biological membranes, improve their solubility, reduce their toxicity or allow their localized concentration at tumor sites. However, in spite of a growing body of evidence that many of the desirable properties of nanomaterials can also translate to risks, to date there is no specific guidance pertaining to the safety evaluation of nanotechnology-based products. One such risk is the ability of nanomaterials to cross biological barriers impervious to larger particles. In some cases, this may trigger neurotoxicity [2]. Furthermore, chemical composition does not necessarily correlate with risk. Indeed, last year it was reported that engineered carbon nanotubes, once thought to be no more toxic than graphite, trigger the formation of lesions akin to those seen in asbestosis in mice [3]. In light of such evidence, in April 2009, the EU Parliament called the Commission to task over its decision to allow single registration of nano and bulk equivalent materials under the EU Chemicals Policy after having already come to the conclusion that existing test methods are not adequate for assessing the risks associated with nanomaterials in foods and having earlier this year called upon the Commission to compile a database of nanomaterials used in the EU by 2011 and to review legislative measures to ensure the safety of nanomaterials for all uses.

An immobilized nanoparticle-based platform for efficient gene knockdown of targeted cells in the circulation

It is well established that specific interaction between adhesion molecules of endothelial cells and receptors on leukocytes can separate and recruit leukocytes from the bloodstream to sites of inflammation and coagulation. Previously, we showed that P-selectin can be absorbed onto the surface of a blood-compatible microrenathane tube, and the P-selectin-coated surface could successfully capture P-selectin receptor-positive stem cells from physiological shear flow in vitro and from the bloodstream in vivo. In this paper, P-selectin was covalently attached to the surface of nanoscale liposomes to create targeting nanoparticles (NPs). Small interfering RNA (siRNA) was encapsulated by these nanoscale liposomes, and the liposomes were stabilized by PEGylation with DSPE-PEG2000. Experiments showed that these P-selectin-, PEGylated-, nanoscale-liposomes (PS-DSPE-PEG NPs) could be absorbed onto the inner surface of microrenathane tubing. The coated surface could specifically capture targeted cells from physiological shear flow, efficiently deliver encapsulated siRNA into adherent cells and dramatically silence the targeted gene neutrophil elastase. With this device, we create a high localized concentration for siRNA delivery in the circulatory system, providing circulating target cells adequate time to interact with therapeutic materials. SiRNA is efficaciously delivered into specific target cells, thereby providing a powerful tool for highly efficient siRNA transfection and other therapeutic materials delivery in circulation. The method should prove especially useful for diseases derived from disorders of blood cells.

Liposome-coated quantum dots targeting the sentinel lymph node

Sentinel lymph node (SLN) mapping with near-infrared (NIR) quantum dot (QDs) have many advantages over traditional methods. However, as an inorganic nanomaterial, QDs have low biocompatibility and low affinity to the lymphatic system. Here, we encapsulated QDs into nanoscale liposomes and then used these liposome-coated QDs for SLN mapping. The results showed that the liposome-coated QDs exhibited core–shell characterization, and their fluorescence emission did not decrease but slightly increased after being continuously excited by a xenon lamp source (150 W) at 488 nm at 37 °C for 1 h. After storing at 4 °C for more than one and half years, the liposome-coated QDs were found to have retained their spherical structure containing a large amount of QDs. When liposome-coated QDs with average size of 55.43 nm were injected intradermally into the paw of a mouse, the SLN was strongly fluorescent within only a few seconds and visualized easily in real time. Moreover, the fluorescence of the QDs trapped in the SLN could be observed for at least 24 h. Compared with the SLN mapping of QDs absent of liposomes and liposome-coated QDs with a larger average size (100.3 and 153.6 nm), more QDs migrated into the SLN when the liposome-coated QDs with smaller average size (55.43 nm) were injected. This technique may make a great contribution to the improvement of the biocompatibility of QDs and the targeting delivery capacity of QDs into the SLN.

Liposome-Templated Supramolecular Assembly of Responsive Alginate Nanogels

Nanosized gel particles (nanogels) are of interest for a variety of applications, including drug delivery and single-molecule encapsulation. Here, we employ the cores of nanoscale liposomes as reaction vessels to template the assembly of calcium alginate nanogels. For our experiments, a liposome formulation with a high bilayer melting temperature (Tm) is selected, and sodium alginate is encapsulated in the liposomal core. The liposomes are then placed in an aqueous buffer containing calcium chloride, and the temperature is raised up to Tm. This allows permeation of Ca2+ ions through the bilayer and into the core, whereupon these ions gel the encapsulated alginate. Subsequently, the lipid bilayer covering the gelled core is removed by the addition of a detergent. The resulting alginate nanogels have a size distribution consistent with that of the template liposomes (ca. 120-200 nm), as confirmed by transmission electron microscopy and light scattering. Nanogels of different average sizes can be synthesized by varying the template dimensions, and the gel size can be further tuned after synthesis by the addition of monovalent salt to the solution.