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Mucosal surfaces contain specialized dendritic cells (DCs) that are able to recognize foreign pathogens and mount protective immunity. We previously demonstrated that intranasal administration of targeted galactosylated liposomes can elicit mucosal and systemic antibody responses. In the present study, we assessed whether galactosylated liposomes could act as an effective DC-targeted mucosal vaccine that would be capable of inducing systemic anti-tumor immunity as well as antibody responses. We show that targeted galactosylated liposomes effectively facilitated antigen uptake by DCs beyond that mediated by unmodified liposomes both in vitro and in vivo. Targeted galactosylated liposomes induced higher levels of pro-inflammatory cytokines than unmodified liposomes in vitro. C57BL/6 mice thrice immunized intranasally with ovalbumin (OVA)-encapsulated galactosylated liposomes produced high levels of OVA-specific IgG antibodies in their serum. Spleen cells from mice receiving galactosylated liposomes were restimulated with OVA and showed significantly augmented levels of IFN-γ, IL-4, IL-5 and IL-6. In addition, intranasal administration of OVA-encapsulated beta-galactosylated liposomes resulted in complete protection against EG7 tumor challenge in C57BL/6 mice. Taken together, these results indicate that nasal administration of a galactosylated liposome vaccine mediates the development of an effective immunity against tumors and might be useful for further clinical anti-tumoral applications., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The mucosal immune system produces secretory IgA (sIgA) as the first line of defense against invasion by foreign pathogens. Our aim was to develop a galactose-modified liposome as a targeted carrier which can be specifically recognized by macrophage, one of the most important antigen presenting cells. First, galactose was covalently conjugated with 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (DLPE) to give a targeted ligand, a galactosyl lipid. The galactosyl lipid was then incorporated into a liposomal bilayer to form a galactosylated liposome carrier. Further, the ovalbumin (OVA) was encapsulated into the galactosylated liposome carriers and mice were intranasally immunized. Confocal laser scanning microscopy and flow cytometry analysis showed that the targeted galactosylated liposome carrier had a higher uptake rate than unmodified liposomes. The targeted galactosylated liposome induced higher levels of tumor necrosis factor-α and interleukin-6 production than unmodified liposomes (P<0.05). Furthermore, 6-week-old BALB/c female mice immunized with the OVA-encapsulated targeted galactosylated liposome had significantly higher OVA-specific s-IgA levels in the nasal and lung wash fluid (P<0.05). In addition, the targeted galactosylated liposome simultaneously augmented the serum IgG antibody response. In summary, the OVA-encapsulated targeted galactosylated liposome induced significantly higher mucosal IgA and systemic IgG antibody titers and is a potential antigen delivery carrier for further clinical applications., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to characterize a nasally delivered bioadhesive liposome using an inactivated H5N3 virus as a model antigen. Bioadhesive liposomes were developed using tremella (T) or xanthan gum (XG) as the bioadhesive polysaccharide. Using chickens as the target animal, we evaluated whether delivery of a bioadhesive liposomal influenza vaccine via a mucosal site of infection could improve vaccine effectiveness. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assays demonstrated that T, XG and liposomes were non toxic to chicken spleen macrophages. Enzyme-linked immunosorbent assay (ELISA) was used to determine the adjuvant effect of the bioadhesive liposomal-vaccines. Chickens immunized with a low dose (200 microL) of bioadhesive liposomal influenza vaccine had significantly higher mucosal and serum antibody levels (P<0.05). In addition, liposomes mixed with a low-viscosity bioadhesive gel used for nasal delivery resulted in superior antibody responses compared with liposomes mixed with a high-viscosity gel (P<0.05). This suggest that a low-viscosity gel mixed with liposomes is more suitable for nasal delivery, and that chickens elicit higher mucosal secretory immunoglobulin A (s-IgA) and serum IgG after two vaccinations.

Growing attention has been given to the potential of a pulmonary route as a non-invasive administration for systemic delivery of therapeutic agents (mainly peptides and proteins). The lungs provide a large absorptive surface area, extremely thin absorptive mucosal membrane, and good blood supply. The non-invasive nature of this pathway makes it especially valuable for the delivery of large molecular protein. However, pulmonary delivery of peptides and proteins is complicated by the complexity of the anatomic structure of the human respiratory system and the effect of disposition exerted by the respiration process. In this study, novel nebulizer-compatible liposomal carrier for aerosol pulmonary drug delivery of insulin was developed and characterized. Experimental results showed that insulin could be efficiently encapsulated into liposomes by preformed vesicles and detergent dialyzing method. The optimal encapsulation efficiency was achieved when 40% ethanol was used. The particle size of liposomal aerosols from ultrasonic nebulizer approximated to 1 mum. Insulin was stable in the liposomal solution. Animal studies showed that plasma glucose level was effectively reduced when liposomal insulin was delivered by inhalation route of using aerosolized insulin-encapsulated liposomes. Including fluorescent probe (phosphatidylethanolamine-rhodamine) into liposome, we found that the liposomal carriers were effectively and homogeneously distributed in the lung aveolar. Liposome-mediated pulmonary drug delivery promotes an increase in drug retention-time in the lungs, and more importantly, a reduction in extrapulmonary side-effects which invariably results in enhanced therapeutic efficacies.

This study describes a highly efficient method for encapsulating protein drugs into liposomes without using toxic solvents such as chloroform. Large unilamellar vesicles (LUVs) were formed by the ethanol injection method. The effects of composition of phospholipid, buffer concentration, incubation time, incubation temperature, drug loading, ethanol content, and the presence of poly(ethylene) glycol (PEG) lipids on the entrapment efficiency of protein were investigated. It was shown that these preformed LUVs could be induced to entrap protein drugs in the presence of ethanol. Protein could be efficiently encapsulated into liposomes. The interaction of the liposomes with proteins leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. Protein drugs were stable in the liposomal solution. There is no significant activity loss during the encapsulation process. The optimal encapsulation efficiency was achieved when 30% approximately 40% ethanol was used in encapsulating protein drugs. Due to the steric hindrance, LUVs containing a PEG coating will dramatically reduce the encapsulation efficiency, even in liposomes containing very low amount of PEG.

We propose the adjuvant effects of phospholipid liposome compositions using intranasal inoculation of a liposomal‐Newcastle disease virus (NDV) vaccine in chickens. The immunogenicity of three liposome formulations was determined in chickens using the hemagglutination‐inhibition (HI) test, nasal secretory immunoglobulin A and serum immunoglobulin A (IgG) antibody titers using the enzyme‐linked immunosorbent assay. The immune response against NDV antigens was determined after immunization with neutral charged liposomes composed of egg phosphatidylcholine (EPC) (60 μmol), cholesterol (Chol) (15 μmol), and EPC‐liposomes (EPC‐Lip), which elicited strong systemic (serum) and local (nasal) humoral responses. However, the intranasal administration with cationic charged liposomes composed of EPC (30 μmol), stearylamine (SA) (15 μmol), Chol (15 μmol), and SA‐liposomes (SA‐Lip) induced poor humoral immune responses. Only the vaccine formulated with anionic charged liposomes composed of EPC (30 μmol), dipalmitoylphosphatidylserine (15 μmol), Chol (15 μmol), and phosphatidylserine‐liposomes (PS‐Lip) elicited the highest titers of HI antibodies. These are the first results to suggest that antigen delivery using EPC‐Lip is very useful in enhancing antibody production at the mucosal site and in serum. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [ABSTRACT FROM AUTHOR]

Abstract: In order to potentiate the low immunogenicity of the inactivated Newcastle disease virus immunized into chickens by mucosal route, liposomes as a drug delivery system and LPS (lipopolysaccharide) as an immuno-stimulator were evaluated. Here, we report a new nasal delivery system of inactivated Newcastle disease virus (NDV) vaccine. The intranasal vaccine was based on different lipids to form MLV (multi-lamellar vehicles) liposomes. The liposomes had combined carrier and adjuvant activities, which induced strong systemic (serum) and local (lung and nasal) humoral responses in SPF (specific-pathogen-free) chickens, and provided protective immunity. PC-Lip (phosphatidylcholine-liposome) elicited significant mucosal secretary immunoglobulin A (s-IgA) levels (p <0.05) in tracheal lavage fluid and serum IgG levels (p <0.05). In response to virulent viral challenge, birds treated with PBS (phosphate buffered saline) as control group died, whereas 80% of chickens which received PC-Lip, PC-Lip-LPS, PS-Lip (phosphatidylserine-liposome), and PS-Lip-LPS survived. HAI titers were 1:2560 in the PS-Lip-LPS group and 1:1280 in the PC-Lip, PC-Lip-LPS, and PS-Lip groups after two vaccinations. The results suggest that PC-Lip or PS-Lip might thus be suitable as a potential adjuvant for mucosal vaccination against NDV in chickens. [Copyright &y& Elsevier]