ovalbumin and 1-2-distearoyllecithin

Technology such as the use of microfluidics to generate liposomes has been well researched, yet the stabilisation of liposomal formulations is a major challenge to their greater implementation. To the best of our knowledge, this is the first study investigating the use of 96 well plates to freeze-dry ovalbumin (OVA) loaded neutral (DMPC:Chol and DSPC:Chol), anionic (DSPC:Chol:PS) and cationic (DSPC:Chol:DOTAP) liposomes. Through the use of high throughput screening, a freeze drying cycle was optimised; ramp freezing from from 4 °C to -45 °C, followed by primary drying at -30 °C and secondary drying at 30 °C under a vacuum of 0.1 mBar. These parameters maintained liposome physicochemical properties, with the liposomes remaining below 100 nm and were homogenous (polydispersity index of less than 0.2 post rehydration). Minimal leakage of the OVA protein was observed, with almost 100% OVA remaining encapsulated post rehydration of the formulations. Here we have identified a simple method that allows for the rapid screening and freeze-drying of a range of liposomal formulations.

Topics: Cholesterol; Dimyristoylphosphatidylcholine; Drug Delivery Systems; Fatty Acids, Monounsaturated; Freeze Drying; High-Throughput Screening Assays; Liposomes; Microfluidics; Ovalbumin; Phosphatidylcholines; Proteins; Quaternary Ammonium Compounds; Technology, Pharmaceutical

One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) is a versatile adjuvant that has shown potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic distearoylphosphatidylcholine (DSPC). The liposomes were tested with the model protein antigen ovalbumin for the mucosal deposition, the effect on cellular viability and the epithelial integrity by using the two cell lines A549 and Calu-3, representing cells from the alveolar and the bronchiolar epithelium, respectively. The Calu-3 cells were cultured under different conditions, resulting in epithelia with a low and a high mucus secretion, respectively. A significantly larger amount of lipid and ovalbumin was deposited in the epithelial cell layer and in the mucus after incubation with the cationic liposomes, as compared to incubation with the neutral liposomes, which suggests that the cationic charge is important for the delivery. The integrity and the viability of the cells without a surface-lining mucus layer were decreased upon incubation with the cationic formulations, whereas the mucus appeared to retain the integrity and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways.

Topics: Adjuvants, Immunologic; Adjuvants, Pharmaceutic; Cations; Cell Line, Tumor; Cell Survival; Epithelial Cells; Glycolipids; Humans; Lipids; Liposomes; Lung; Mucus; Ovalbumin; Phosphatidylcholines; Quaternary Ammonium Compounds; Respiratory Mucosa; Vaccines

A specific antigen-sensitized animal has antigen-specific immune cells that recognize the antigen. Therefore, an antigen-modified drug carrier would be recognized by the immune cells. When such a carrier encapsulates certain drugs, these drugs should be specifically delivered to the immune cells. To examine this strategy, ovalbumin (OVA) was used as model antigen, and mice were presensitized with 100 μg of OVA with Alum. For preparing OVA-modified liposomes (OVA-lipo), OVA was incubated with DSPE-PEG-NHS and resulting DSPE-PEG-OVA was inserted into liposomes. OVA-specific IgG was produced 6-fold higher by intravenous injection of OVA-lipo thrice (10 μg as OVA in each injection) in OVA-sensitized mice, than that by the injection of control liposomes, suggesting that OVA-lipo was recognized by the antigen-specific immune cells. Moreover, intra-splenic accumulation of OVA-lipo was observed in OVA-sensitized mice, but not in naive mice. To achieve the delivery of a drug to specific immune cells, OVA-lipo encapsulated low dose of doxorubicin (DOX) as a model drug (20 μg DOX/mouse, Ca. 1 mg/kg) was injected in the sensitized mice. The injection of OVA-lipo encapsulating DOX suppressed the production of IgE against OVA, suggesting that the specific delivery of the drug to immune cells responsible for OVA recognition was achieved and that these immune cells were removed by the drug treatment. This strategy would be useful for the fundamental treatment of allergy by the use of immunosuppressing agents.

Topics: Animals; Antibiotics, Antineoplastic; Antigens; Cholesterol; Doxorubicin; Female; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Kidney; Liposomes; Liver; Lung; Mice; Mice, Inbred BALB C; Myocardium; Ovalbumin; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Spleen

In dendritic cell (DC)-based cancer immunotherapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I, and activate tumor-specific cytotoxic T lymphocytes (CTLs). However, MHC class I generally present endogenous antigens expressed in the cytosol. We therefore developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs; i.e., a MHC class I-presenting pathway. In this study, we investigated the effect of antigen delivery using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure on MHC class I presentation levels in DCs, as well as the feasibility of using this antigen delivery system in DC-based cancer immunotherapy. DCs were treated with ovalbumin (OVA) as a model antigen, BLs and US exposure. OVA was directly delivered into the cytosol but not via the endocytosis pathway, and OVA-derived peptides were presented on MHC class I. This result indicates that exogenous antigens can be recognized as endogenous antigens when delivered into the cytosol. Immunization with DCs treated with OVA, BLs and US exposure efficiently induced OVA-specific CTLs and resulted in the complete rejection of E.G7-OVA tumors. These data indicate that the combination of BLs and US exposure is a promising antigen delivery system in DC-based cancer immunotherapy.

Topics: Animals; Antigen Presentation; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Survival; Cytotoxicity Tests, Immunologic; Dendritic Cells; Endocytosis; Fluorocarbons; Immunotherapy, Active; Interleukin-2; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Sodium Azide; Survival Analysis; T-Lymphocytes, Cytotoxic; Ultrasonics

Since liposomes are known as strong adjuvants, we attempted to use liposomes in immunotherapy as adjuvants, and to achieve desensitization in pre-sensitized mice. At first, we sensitized mice with intraperitoneal injection of model antigen, 100 microg ovalbumin (OVA), with Alum and treated them with liposome composed of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 as a molar ratio), which was coupled with a small amount of OVA (10 microg OVA in 400 nmol DSPC and 200 nmol cholesterol-liposome was injected into 20 g mouse). It is well known that antigen-specific immunotherapy increases IgG blocking antibodies and decreases in IgE antibodies. The treatment with i.v. injection of OVA-liposome at days 8, 10, and 12 after sensitization strongly suppressed OVA-specific IgE production without affecting IgG level after the boost (100 microg OVA with Alum). Moreover, the treatment with high-density OVA-liposome (10 microg OVA in 80 nmol DSPC and 40 nmol cholesterol-liposome/20 g mouse) not only strongly suppressed IgE levels but also reduced IgG production after the boost of OVA-sensitized mice suggesting the importance of liposomal characteristic in desensitization immunotherapy. Next we reduced the dose of OVA-liposome and the desensitization effect was also observed at the dose of as low as 1 microg OVA on OVA-liposome/mouse. On the contrary, free OVA did not affect the production of both IgG and IgE levels. Biodistribution study indicated that OVA-liposome was highly accumulated in spleen of OVA-sensitized mice compared to control liposome at 3 h after i.v. injection. These results suggest that the liposomal OVA effectively interacts with and desensitizes immune cells, therefore, liposomes coupling with a certain antigen may be effective in allergy immunotherapy.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens; Cholesterol; Desensitization, Immunologic; Dose-Response Relationship, Immunologic; Drug Administration Schedule; Enzyme-Linked Immunosorbent Assay; Female; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Liposomes; Mice; Mice, Inbred BALB C; Ovalbumin; Phosphatidylcholines; Spleen; Tissue Distribution

The aim of this work was to develop a liposomal formulation which could act as a carrier for allergens during oral desensitization therapy. A model protein, ovalbumin, was associated with negatively charged, multilamellar vesicles of various compositions and their stability in the presence of synthetic intestinal media (bile salt, pancreatic enzymes and their combination) was investigated. Liposomes containing soya phosphatidylcholine as the main lipid, regardless of their cholesterol content (20-40%), were unable to protect ovalbumin against the combined action of pancreatic enzymes and bile salt. In contrast, liposomes prepared from distearoylphosphatidylcholine and cholesterol (6:3.5 molar ratio) were more stable: about 50% of the lipid remained as liposomes after a 4-h incubation at 37 degrees C and intact ovalbumin could be demonstrated therein by immunoblotting. The immunomodulating properties of liposomes were tested by following changes in serum IgE levels (by passive cutaneous anaphylaxis) in Balb/C mice sensitized to ovalbumin, after feeding various preparations. In this model, free ovalbumin was able to provoke a premature fall in IgE levels, and liposomes, whatever their composition, contributed no further effect.

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Desensitization, Immunologic; Drug Carriers; Female; Immunoblotting; Immunoglobulin E; Liposomes; Mice; Mice, Inbred BALB C; Ovalbumin; Phosphatidylcholines