Liposomal Particles Research Articles

Effect of Laser Irradiation on Reversibility and Drug Release of Light-Activatable Drug-Encapsulated Liposomes.

Although several studies have demonstrated repetitive drug release using light-activatable liposomes, inconsistent drug release at each activation limits widespread usage. Here, we report reversible plasmonic material-coated encapsulated liposomes for proportional controlled delivery of methotrexate (MTX), which is a common drug for cancer and autoimmune diseases, using repetitive laser irradiation. Our results suggest a proportional increase in total drug release after repetitive laser irradiation. We hypothesize that the drug is released via "melted" lipid bilayers when the plasmonic materials on the liposome surface are heated by laser irradiation followed by reversible formation of the liposome. To evaluate our hypothesis, the number density of liposomes after laser irradiation was measured using single-particle (liposome) collision experiments at an ultramicroelectrode. Collisional frequency data suggest that the number density of liposomes remains unaltered even after 60 s of laser irradiation at 1.1 and 1.8 W, indicating that the liposome structure is reversible. The results were further compared with gold nanorod-coated nanodroplets where drug is released via irreversible phase transition. In contrast to what was observed with the liposome particles, the number density of the nanodroplets decreased with increasing laser irradiation duration. The structure reversibility of our liposome particles may be responsible for repetitive drug release with laser heating. We also studied the temperature rise in the lipid bilayer by incorporating polymerized 10,12-pentacosadiynoic acid (PCDA) in the lipid composition. The red shift in the UV-vis spectrum due to the structural change in PCDA lipids after laser irradiation indicates a rise in temperature above 75 °C, which is also above the chain-melting temperature of the main lipid used in the liposomes. All these results indicate that drug is released from the light-activatable liposomes due to reversible nanostructural alteration in the lipid bilayer by plasmonic resonance heating. The liposomes have potential to be a drug carrier for dose-controlled repetitive drug delivery.

Preparation of Barije (Ferula gummosa) Essential Oil–Loaded Liposomes and Evaluation of Physical and Antibacterial Effect on Escherichia coli O157:H7

The aim of this study was to load liposomes with Barije (Ferula gummosa) essential oil (EO) and to evaluate their physical and antibacterial properties. Liposomes were produced with specific ratios of lecithin/cholesterol by thin-film hydration and sonication. The chemical composition of the EO was analyzed by gas chromatography and mass spectroscopy. The physical properties of the liposomes (particle size, polydispersity index, zeta potential, and encapsulation efficiency) were evaluated. The antimicrobial effects of these liposomes against Escherichia coli O157:H7 were determined based on the MIC and disk diffusion results. The effect of subinhibitory concentrations (sub-MICs) of EO against the growth of the bacterium over 24 h was evaluated before and after encapsulation. The major components of EO were β-pinene (60.84%) and α-pinene (9.14%). The mean liposome radius of EO-loaded liposomes was 74.27 to 99.93 nm, which was significantly different from that of the empty liposomes (138.76 nm) (P < 0.05). Addition of cholesterol to the lecithin bilayer increased the particle size and reduced the encapsulation efficiency (P < 0.05). The electrostatic stability of the empty liposomes was improved by adding cholesterol, but when the EO was replaced in the liposomes, there was no significant change in electrostatic stability of liposomes with cholesterol (P < 0.05). MICs were 14.5 μg/mL for the EO-loaded nanoliposomes containing 30 mg of lecithin and 30 mg of cholesterol and 10 μg/mL for nonencapsulated EO. This trend was confirmed by measuring the inhibition zone diameter. Sub-MICs of liposomal EO (containing 60 mg of lecithin) decreased bacterial levels to a greater degree than did free EO, especially at 50 and 75% of the MIC.

Preparation of Liposomal Sanguinarine and Study of Its Cytotoxic Effects against Prostate Cancer Cells

Benzophenanthridine alkaloid sanguinarine from Macleaya macrocarpa shows antitumor and anti-angiogenic activity, among others. Some side effects of sanguinarine, which significantly limit the possibilities of its therapeutic use, are described. To increase the effectiveness of its therapeutic effect, sanguinarine was introduced into the pH-sensitive liposomes by remote loading using ammonium dihydrogen phosphate. pH-sensitive liposomes, appearing in the acidic environment of the tumor or endosomal compartment, release sanguinarine, which causes the death of tumor cells. The average diameter of the liposome particles measured by the method of dynamic light scattering was 112.6 ± 1.8 nm; the zeta potential was –13.9 ± 1.7 mV. The effectiveness of the inclusion of sanguinarine in liposomes was 89.54 ± 2.13%. The dynamics of release of sanguinarine from the composition of a liposome preparation was studied, and the prolonged release pattern was demonstrated. The cytotoxic activity (CTA) of liposomal sanguinarine against prostate cancer cell lines LNCaP, DU 145, and PC-3 was studied. Liposomal sanguinarine exhibited dose-dependent CTA against cells of all the studied lines in the micromolar range of concentrations. The highest CTA of liposomal sanguinarine was seen against the hormone-sensitive LNCaP cells (IC50 2.86 μM). For the hormone-independent cells of the DU 145 and PC-3 lines, the cytotoxic activity of liposomal sanguinarine was somewhat lower and amounted to 3.37 μM and 3.63 μM, respectively. A dose-dependent induction of apoptosis of LNCaP, DU 145, and PC-3 cells was also demonstrated. The liposomal sanguinarine with high efficiency induced apoptosis of both hormone-sensitive and hormone-independent cells. The liposomal sanguinarine at a concentration of 8 μM induced apoptosis in 93.14% of LNCaP cells, 90.65% of DU 145 cells, and 98.12% of PC-3 cells. Thus, liposomal sanguinarine can be considered as a promising antitumor agent for the therapy of both hormone-sensitive and hormone-independent tumors.

Mannosylerythritol Lipid B Enhances the Skin Permeability of the Water-Soluble Compound Calcein via OH Stretching Vibration Changes

We confirmed that mannosylerythritol lipid B (MEL-B), a biosurfactant, enhances the skin permeability of the model water-soluble compound calcein. MEL-B liposomes were prepared by the thin-layer evaporation technique, and then applied to the skin. Although we attempted to adjust the size by extrusion, we could not control the particle diameter of the liposomes. However, the MEL-B liposome particle diameter remained the same over the 7-day study period. We observed an endothermic peak, with 74.7 °C as the transition temperature by differential scanning calorimetry. We also performed a fusion experiment with a fluorescence resonance energy transfer. A high amount of fusion of intercellular lipid liposomes and MEL-B liposomes occurred in a short period of time. After applying the MEL-B liposomes containing calcein to the skin, we measured the degree of calcein permeation and the amount of calcein within the skin. The resulting values were higher than those of an aqueous solution. The results obtained using a confocal laser scanning microscope suggested that calcein had been delivered deeply into the skin. Using the attenuation of total reflectance Fourier-transform infrared spectrometry, we observed that the OH stretching vibration had shifted to a higher wavenumber; however, this did not affect the CH stretching vibration. The measurement of transepidermal water loss after four days of continuous application of 1% MEL-B to animals revealed no changes. Our results suggest that MEL-B increases the skin permeability of compounds (calcein) that are difficult to deliver transdermally by changing the OH stretching vibration, which shifts to a higher wavenumber.

Silymarin Spray-Dried Proliposomes: Preparation, Characterization and Cytotoxic Evaluation

Background: Most liposomes problems are due to stability and consistency. Proliposomes is one of the solutions to overcome the disadvantage of liposomes. They are available in dry powder form, it is easy to distribute, transfer, measure and store. Objective: The aim of the present study was to find a novel method of preparing Silymarin proliposomes and study the effect of cholesterol concentrations and surfactant types on the physicochemical properties of silymarin proliposomes and its in-vitro release. Methods: Silymarin proliposomes were prepared by combining two simple methods ethanol injection method for liposomes preparation followed by the spray drying method to get a dry powder. The physicochemical properties including particle size, TEM, SEM, FTIR, encapsulation efficiency and dissolution studies were studied. Results: The particle size of silymarin liposomes were below 552.36 ± 17.63 nm but after reconstitution of silymarin proliposomes, the particle size was in the micro range due to the influence of the spray drying process. Cholesterol concentration was ranged from 50 to 150 mg per formula. Increasing Cholesterol concentration caused a significant increase in liposomes particle size and reduction in encapsulation efficiency. Three non-ionic surfactants were used to prepare silymarin proliposomes Tween 80, Cremophor RH 40 and Poloxamer 407. Formula F1 prepared with Phosal® 53 MCT (300 mg), Tween 80 (50 mg), cholesterol (50 mg) and Silymarin (140 mg) showed the smallest particle size (2066 ± 164.87 nm) upon reconstitution in water, highest encapsulation efficiency (89.51 ± 0.43%), and fastest in vitro release compared to other formulas. Cytotoxicity of Silymarin, formula F1 and formula F1/blank was assessed using an MTT assay on MCF-7, HepG2 and HBF-4 cells. The cytotoxic effect of silymarin was enhanced by loading it on proliposomes. Conclusion: Silymarin was successfully formulated into proliposomes combining ethanol injection and spray drying methods. The cytotoxicity of silymarin was improved when loaded on proliposomes owing to the formula.

Rapid production of liposomes using high pressure carbon dioxide and direct ultrasonication

Herein, we introduce a protocol for preparing liposomes using high-pressure CO2, water and direct ultrasonication (HPC-D) allowing rapid formation of liposomes in a single-step. In the HPC-D method, phospholipid suspensions were treated at temperatures from 25 °C to 70 °C and pressures from 4 MPa to 6.8 MPa with direct ultrasonication. The liposomes produced from HPC-D had an average particle size of 159 ± 2 nm to 136 ± 8 nm at liposome recovery yields up to 95.3 ± 4.6 %. A mechanism for the HPC-D method is proposed, in which the micro-phase separation between water-CO2 interface increases surface area and phospholipids act as surfactants and reassemble into small liposome particles. Drug loading (DL) and encapsulation efficiency (EE) of liposomes obtained with the HPC-D method for Cyclosporin A gave DL values of 37.4 ± 3.4 % and EE of 79.7 ± 2.5 %, confirming efficient entrapment.

Self-Antigens Displayed on Liposomal Nanoparticles above a Threshold of Epitope Density Elicit Class-Switched Autoreactive Antibodies Independent of T Cell Help.

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qβ virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qβ viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.

Liposomal delivery of hydrophobic RAMBAs provides good bioavailability and significant enhancement of retinoic acid signalling in neuroblastoma tumour cells

Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties. In vivo, retinoids induce CYP26 enzyme production in the liver, enhancing their own rapid metabolic clearance, while retinoid resistance in tumour cells themselves is considered to be due in part to increased CYP26 production. Retinoic acid metabolism blocking agents (RAMBAs), which inhibit CYP26 enzymes, can improve retinoic acid (RA) pharmacokinetics in pre-clinical neuroblastoma models. Here, we demonstrate that in cultured neuroblastoma tumour cells, RAMBAs enhance RA action as seen by morphological differentiation, AKT signalling and suppression of MYCN protein. Although active as retinoid enhancers, these RAMBAs are highly hydrophobic and their effective delivery in humans will be very challenging. Here, we demonstrate that such RAMBAs can be loaded efficiently into cationic liposomal particles, where the RAMBAs achieve good bioavailability and activity in cultured tumour cells. This demonstrates the efficacy of RAMBAs in enhancing retinoid signalling in neuroblastoma cells and shows for the first time that liposomal delivery of hydrophobic RAMBAs is a viable approach, providing novel opportunities for their delivery and application in humans.

Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery.

Synthetic nanoparticle-based drug delivery system is widely known for its ability to increase the efficacy and specificity of loaded drugs, but it often suffers from relatively higher immunotoxicity and higher costs as compared to traditional drug formulations. Contrarily, plant-derived nanoparticles appear to be free from these limitations of synthetic nanoparticles; they are naturally occurring biocompatible vesicles that do not generate immunotoxicity and are easy to obtain. Additionally, lipids isolated from plant-derived nanoparticles have shown the capability of assembling themselves to spherical nano-sized liposomal particles. Herein, we employ lipids extracted from ginger-derived nanoparticles and load them with therapeutic siRNA (CD98-siRNA) to create CD98-siRNA/ginger-lipid nanoparticles. Characterization of the CD98-siRNA/ginger-lipid nanoparticles showed that they present a spherical shape, with a diameter of around 189.5 nm. The surface zeta potential of the nanoparticles varies from -18.1 to -18.4 mV. Furthermore, in recent research, the CD98-siRNA/ginger-lipid nanoparticles have shown specific colon targeting capability and excellent anti-inflammatory efficacy in a Dextran Sodium Sulfate (DSS) induced mouse model of colitis.

The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics.

The aim of this work was to assess the impact of solvent selection on the microfluidic production of liposomes. To achieve this, liposomes were manufactured using small-scale and bench-scale microfluidics systems using three aqueous miscible solvents (methanol, ethanol or isopropanol, alone or in combination). Liposomes composed of different lipid compositions were manufactured using these different solvents and characterised to investigate the influence of solvents on liposome attributes. Our studies demonstrate that solvent selection is a key consideration during the microfluidics manufacturing process, not only when considering lipid solubility but also with regard to the resultant liposome critical quality attributes. In general, reducing the polarity of the solvent (from methanol to isopropanol) increased the liposome particle size without impacting liposome short-term stability or release characteristics. Furthermore, solvent combinations such as methanol/isopropanol mixtures can be used to modify solvent polarity and the resultant liposome particle size. However, the impact of solvent choice on the liposome product is also influenced by the liposome formulation; liposomes containing charged lipids tended to show more sensitivity to solvent selection and formulations containing increased concentrations of cholesterol or pegylated-lipids were less influenced by the choice of solvent. Indeed, incorporation of 14 wt% or more of pegylated-lipid was shown to negate the impact of solvent selection.

A Liposomal Platform for Delivery of a Protein Antigen to Langerin-Expressing Cells.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Critical process parameters in manufacturing of liposomal formulations of amphotericin B

Identifying the critical process parameters (CPPs) of a complex drug product manufacture and the associated impact on critical quality attributes (CQAs) is essential to the development and quality control of both new and generic drugs. AmBisome, a liposomal amphotericin B (AMB) macrolide antibiotic widely adopted as an important antifungal drug product, was used as a model complex drug product in the current study. This study investigated how multi-step production approaches and related manufacturing conditions may affect essential physico-chemical and toxicological properties of the final drug product. A key challenge in the manufacture and analysis of liposomal AMB was the drug substance’s propensity to aggregate, with associated poor solubility in water and organic solvents. This study identified three key CPPs in a four step manufacturing process: (i) proper acidification during formation of the drug-lipid complexes (Step 1), (ii) liposome heat curing following liposomal particle sizing (Step 3), and (iii) flash-freezing at the initial stages of the lyophilization cycle (Step 4). Over-acidification led to rapid degradation of the drug, whereas under-acidification hampered full solubilization and formation of the soluble drug-lipid complexes. Extended heat treatment of the formed liposomes at 65 °C, just above the lipid phase transition temperature, brought dramatic changes in the aggregated state and/or packing of the drug in the liposomal bilayer, as followed by the complex changes in the UV/Vis spectra. Such thermal conditioning resulted in a five- to ten-fold reduction in the in-vitro toxicity of the drug product, bringing it close to the values for AmBisome used as control and measured by the RBC assay. Finally, flash-freezing conditions during lyophilization was critical to prevent aggregation and maintaining the 80–120 nm liposome size when reconstituted. Our research found that changes in the amphotericin’s UV/Vis spectra were a sensitive CQA measure and provided a set of quantitative parameters for a facile non-destructive process monitoring in-situ, as well as for comparison of the quality of final formulations.

Flurbiprofen-loaded ethanolic liposome particles for biomedical applications

Present study deals with the preparation, characterization and in-vivo evaluation of flurbiprofen loaded ethanolic liposome which provides predetermined and controlled release of drug through a transdermal drug delivery system. Ethanolic liposomes were prepared by using flurbiprofen, phospholipon 90-G, and ethanol in varied concentration ratio. The prepared ethanolic liposomes were optimized and characterized for particle size, zeta potential, polydispersive index and % entrapment efficiency. FTIR study was performed to analyze the interaction between drug and excipient. To study the thermal behavior of the formulation DSC and TGA were carried out. The surface morphology of ethanolic liposome was performed with the help of SEM, TEM, and AFM. In-vitro drug permeation study of the optimized formulation was carried out using the albino rat skin model and peripheral nociceptive activity was evaluated by writhing assay. In addition, formulations were also inspected for stability study for three months at a different temperature. The optimized formulation EF5 exhibited a particle size of 167.2 ± 3.7 nm with a zeta potential of −51.6 ± 0.2 mV and PDI of 0.209. The optimized formulation showed an ideal surface morphology with a maximum % entrapment efficiency i.e. 93.51 ± 2.1. In-vitro permeation study shows a release of 70.23% in 24 h and transdermal flux was found as 238.2 μg/cm2/h. Writhing assay demonstrate that the optimized formulation decreases the number of writhes and thus shows the peripheral analgesic activity. In stability study, optimized formulation showed maximum stability at 4 °C. These results suggest that transdermal system mediated application of flurbiprofen loaded ethanolic liposome can be considered as an effective way to afford consistent and predictable release of flurbiprofen which could provide beneficial effects in the management of various inflammatory diseases.

Fabrication of novel PHB-liposome nanoparticles and study of their toxicity in vitro

Liposomes represent a tool used in cosmetics and for drug delivery due to their small size, biocompatibility, and ability to house both hydrophobic and hydrophilic molecules. However, under storage, there is a tendency for liposomes to release encapsulated components. In order to enhance their stability, combination with biodegradable polymers may be a promising option. Bacterial polymer poly(3-hydroxybutyrate) (PHB) is one such material that may contribute liposome structure, as it is also biocompatible and biodegradable and promotes controlled release of active agents in the skin. Nanoparticles consisting of PHB and phospholipids can be very attractive as biomaterials for application in cosmetics. In this work, novel combined PHB-liposome nanoparticles were synthesized and characterized, and their biocompatability with mammalian cells was assessed in vitro. Incorporation of PHB into liposome particles led to preservation of small size (123.8 ± 0.9 nm; evaluated by DLS) and colloidal stability of particles for 2 months in water environment. Cytotoxicity studies using MTT test with two different skin keratinocytes cell lines (HEK and HaCaT) showed significant dose-dependent differences in cell viability after exposure to liposome and PHB liposome particles in vitro. However, these PHB-liposomes were not found toxic both for HEK and HaCaT cells. Regarding to natural UV-scattering effect of PHB, we suggest that newly fabricated combined liposome-PHB particles could exhibit added value as carriers of active substances for topical applications. However, further study is necessary in order to reveal interactions between this new type of particle and human cells.

Native Nano-electrospray Differential Mobility Analyzer (nES GEMMA) Enables Size Selection of Liposomal Nanocarriers Combined with Subsequent Direct Spectroscopic Analysis.

Gas-phase electrophoresis employing a nano-electrospray differential mobility analyzer (nES DMA), aka gas-phase electrophoretic mobility molecular analyzer (nES GEMMA), enables nanoparticle separation in the gas-phase according to their surface-dry diameter with number-based concentration detection. Moreover, particles in the nanometer size range can be collected after size selection on supporting materials. It has been shown by subsequent analyses employing orthogonal methods, for instance, microscopic or antibody-based techniques, that the surface integrity of collected analytes remains intact. Additionally, native nES GEMMA demonstrated its applicability for liposome characterization. Liposomes are nanometer-sized, biodegradable, and rather labile carriers (nanoobjects) consisting of a lipid bilayer encapsulating an aqueous lumen. In nutritional and pharmaceutical applications, these vesicles allow shielded, targeted transport and sustained release of bioactive cargo material. To date, cargo quantification is based on bulk measurements after bilayer rupture. In this context, we now compare capillary electrophoresis and spectroscopic characterization of vesicles in solution (bulk measurements) to the possibility of spectroscopic investigation of individual, size-separated/collected liposomes after nES GEMMA. Surface-dried, size-selected vesicles were collected intact on calcium fluoride (CaF2) substrates and zinc selenide (ZnSe) prisms, respectively, for subsequent spectroscopic investigation. Our proof-of-principle study demonstrates that the off-line hyphenation of gas-phase electrophoresis and confocal Raman spectroscopy allows detection of isolated, nanometer-sized soft material/objects. Additionally, atomic force microscopy-infrared spectroscopy (AFM-IR) as an advanced spectroscopic system was employed to access molecule-specific information with nanoscale lateral resolution. The off-line hyphenation of nES GEMMA and AFM-IR is introduced to enable chemical imaging of single, i.e., individual, liposome particles.

Liposome-embedded, polymeric scaffold for extended delivery of galantamine

The purpose of this study was to construct a liposome-embedded, polymeric scaffold (LEPS) that is capable of delivering an alkaloid drug, galantamine. The LEPS was engineered by embedding drug-loaded functionalized liposomes (particle size: 100–158 nm, zeta potential: −34 mV) into a depot chitosan, Eudragit® RSPO, and polyvinyl alcohol (CEP) scaffold. Swelling, erosional dynamics, porosity/diffusion and particle size influenced the release behaviour of the drug-loaded functionalized liposomes post-embedment into the depot CEP scaffold of the LEPS. High accumulation of galantamine (GAL; ∼50–90%) validated that drug-loaded functionalized liposomes were still intact following release from the LEPS in simulated physiological conditions over 50 days. PC12 neuronal cell proliferation was supported on the surface of the LEPS. The Lactate Dehydrogenase (LDH) level, which is associated with cell membrane damage/injury induced by the drug-loaded functionalized liposomes, for CEP scaffold and LEPS, were ±15%, ±30% and ±32%, respectively, confirming no significant cytotoxic effects over 28 days. High accumulation of the GAL and FITC intracellularly validated that synthetic peptide potency was maintained following delivery of the drug-loaded functionalized liposomes post-escape from the LEPS.

Macrophage Response to Particulates Plays a Pivotal Role in Development of Allergic Immune Response to Airborne Pollens

Innate immune responses in respiratory mucosa, such as those mediated by airway epithelial cells, likely play pivotal roles in development of type 2 immunity to inhaled allergens. The goal of this project was to investigate the roles for another innate immune cell type, namely airway macrophages, to induce allergic sensitization to pollens by using mouse models. Naïve BALB/c mice were exposed intranasally (i.n.) to ragweed pollen particles or ragweed extract twice a week for up to 3 weeks. Development of allergic immune responses was examined by analyzing sera and by challenging the animals i.n. with ragweed extract. The roles for macrophages were examined by in vitro culture and by depleting them in CD169-DTR transgenic mice. Clodronate liposome particles were used to characterize macrophage responses. Mice developed allergic immune responses to ragweed, including increased serum levels of IgE antibodies and airway eosinophilia, when exposed to ragweed pollen particles but not to ragweed extract. Pollen particles showed adjuvant activities to promote development of follicular helper T cells and allergen-specific IgE antibodies. Isolated airway macrophages produced IL-1α in response to pollen particles or clodronate liposome particles, but not to ragweed extract, in vitro. IL-1α and clodronate liposomes showed similar adjuvant activities as ragweed pollen particles to promote allergic immune responses in vivo. Finally, depletion of airway macrophages in CD169-DTR mice abolished IL-1α production and development of allergic immune response to ragweed pollens. Airway macrophages play a pivotal role to promote development of allergic immune responses to inhaled allergen particles.

Impact of the Environment upon the Candida albicans Cell Wall and Resultant Effects upon Immune Surveillance.

The fungal cell wall is an essential organelle that maintains cellular morphology and protects the fungus from environmental insults. For fungal pathogens such as Candida albicans, it provides a degree of protection against attack by host immune defences. However, the cell wall also presents key epitopes that trigger host immunity and attractive targets for antifungal drugs. Rather than being a rigid shield, it has become clear that the fungal cell wall is an elastic organelle that permits rapid changes in cell volume and the transit of large liposomal particles such as extracellular vesicles. The fungal cell wall is also flexible in that it adapts to local environmental inputs, thereby enhancing the fitness of the fungus in these microenvironments. Recent evidence indicates that this cell wall adaptation affects host-fungus interactions by altering the exposure of major cell wall epitopes that are recognised by innate immune cells. Therefore, we discuss the impact of environmental adaptation upon fungal cell wall structure, and how this affects immune recognition, focussing on C. albicans and drawing parallels with other fungal pathogens.

Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells

PurposeCancer stem cells (CSCs), also known as tumor-initiating cells, are involved in tumor progression, metastasis, and drug resistance. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the inhibitory effects of HL on the growth of CSC subpopulations in liver cancer cells (HepG2) in vitro. MethodsHLs composed of 90 mol% L-α-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23) dodecyl ether were prepared by sonication. Cell viability was determined by the trypan blue exclusion assay. In liver cancer cells, CSCs were identified by the presence of the cell surface marker proteins CD133 and EpCAM by flow cytometry. A soft agar colony formation assay was performed using HepG2 cells pretreated with HLs. ResultsHLs selectively inhibited liver cancer cell growth without affecting normal hepatocytes. Additionally, HLs induced apoptosis of HepG2 cells by a"ctivating caspase-3. Notably, the CD133(+)/EpCAM(+) CSC sub-population of liver cancer cells treated with HLs was reduced. Furthermore, HLs markedly decreased the number of colony-forming cells. Finally, we confirmed the fusion and accumulation of HLs into the cell membranes of CSCs using a fluorescently labeled lipid (NBDPC). Significant accumulation of HL/NBDPC into the CSCs (particularly EpCAM(+) cells) occurred in a dose-dependent manner. ConclusionThese results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy.

Development of a flow-through USP 4 apparatus drug release assay for the evaluation of amphotericin B liposome

AmBisome® is a liposomal formulation of amphotericin B (Amp B), a complex parenteral antifungal product with no US FDA approved generic version available to date. For generic Amp B liposomal product development, examination of the drug release profile is important for product quality control and analytical comparability evaluation with the reference listed drug. Yet, there is no standardized in vitro drug release (IVR) assay currently available for Amp B liposomes. In this study, we describe the development of a USP-4 apparatus-based IVR assay capable of discriminating liposomal Amp B formulations based on the drug release profile. The goal of the IVR assay development was to identify release media compositions and assay temperatures capable of facilitating 70–100% of drug release from AmBisome® in 24 h without Amp B precipitation or disruption of liposome structure. We found that an addition of 5% w/v of γ-cyclodextrin to the release media of 5% sucrose, 10 mM HEPES, and 0.01% NaN3 (pH = 7.4) prevented Amp B precipitation and facilitated drug release. Increased IVR assay temperature led to increased drug release rate, and 55 °C was selected as the highest temperature that induced drug release close to our target without causing product precipitation. The developed IVR assay was used to discriminate between drug release rates from AmBisome® and micellar Amp B products like Fungizone® and Fungcosome. The IVR assay was also capable of discriminating between Amp B liposomes with the same composition as AmBisome® but prepared by either extrusion or homogenization processes, both of which resulted in measurable liposomal particle size heterogeneity and Amp B concentration differences. Finally, the USP-4 IVR assay was used to compare Amp B release profiles between AmBisome® and two generic products approved in India, Amphonex® (Bharat Serums and Vaccines Ltd.) (f2 = 66.3) and Phosome® (Cipla Ltd.) (f2 = 55.4). Taken together, the developed USP-4 IVR assay can be a useful tool for drug release profile characterization in generic liposomal Amp B formulation development.