Detection of Staphylococcus enterotoxin B using fluorescent immunoliposomes as label for immunochromatographic testing

Staphylococcus enterotoxin B (SEB) is one of several toxins produced by the gram positive bacterium Staphylococcus aureus. SEB is a major cause of food poisoning and represents a significant biological threat with regard to bioterrorism. A rapid, sensitive, and specific method is required to monitor food and water in cases of both natural and intentional contamination by this toxin. This report presents an improved immunochromatographic test (ICT) using immunoliposomes as label for the detection of SEB. For the first time in an ICT, the signal generated by the sulforhodamine B encapsulated into immunoliposomes was measured by fluorescence, allowing a 15-fold increase in sensitivity compared with that for visual detection of colored labels. The ICT was completed within 30 min, providing a limit of detection close to 20 pg/ml in buffer and showing no cross-reactivity with the other major toxin of the bacterium, Staphylococcus enterotoxin A. This sensitivity was retained when analyzing SEB spiked in various alimentary matrices, mimicking contaminated foods. Due to the use of fluorescent immunoliposomes as label, the present assay offers the inherent simplicity and speed of a dipstick assay while providing detection of low levels of SEB in real samples.     

INTERFERENCE OF MACROPHAGES WITH IMMUNOTARGETING OF LIPOSOMES

ABSTRACT

We investigated the binding, uptake and intracellular degradation of immunoliposomes by isolated rat liver macrophages in vitro. Immunoliposomes were prepared either by coupling a randomly thiolated anti-CC531 rat colon adenocarcinoma monoclonal antibody to bilayer-incorporated MPB-PE by means of a thioether linkage or by attaching it through its F_c moiety to the distal terminus of hydrazide-modified PEG-DSPE. The two immunoliposome preparations clearly differ in their interaction with the tumor target cells, as well as with the macrophages. At comparable antibody densities both cell types show 1.5–2-fold higher levels of association for the Hz-PEG-immunoliposomes than for the MPB-PEG-immunoliposomes. We provide evidence that immunoliposome macrophage-interaction is both F_c-receptor and scavenger receptor mediated to about equal extents. At low antibody density the hydrazide immunoliposomes favor interaction with the tumor cells to that with macrophages. At higher antibody densities, on the other hand, interaction of these liposomes with the macrophages is increasingly favored, mostly due to enhanced scavenger receptor mediated uptake. The rate of intracellular degradation of (immuno)liposomes internalized by liver macrophages is barely influenced by the presence of either PEG or immunoglobulins on the liposomal surface.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect.

We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with ³H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-oleate as relative measure of degradation.

Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes.

In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface.

These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.     

STUDY OF IN VITRO STABILITY OF LIPOSOMES AND IN VIVO ANTIBODY RESPONSE TO ANTIGEN ASSOCIATED WITH LIPOSOMES CONTAINING GM1 AFTER ORAL AND SUBCUTANEOUS IMMUNIZATION

ABSTRACT

In order to evaluate the usefulness of liposomes as possible vaccine vehicles (oral and subcutaneous), the stability of liposomes in buffer, plasma and saliva at 25 and 37°C was analyzed via fluorescence and enzymatic methodology. The tested mixtures included [EggPC/Chol] 1 : 1 (mixture I), [EggPC/Chol/SM] 1 : 1 : 1 (mixture II), [EggPC/Chol/SM/GM typeIII] 1 : 1 : 1 : 0.14 (mixture III), [EggPC/Chol/SM/GM1] 1 : 1 : 1 : 0.14 (mixture IV) and [DIAPC/DMPC] 1 : 1 non polymerized (mixture V) and polymerized (mixture VI); all mole ratio. Liposome mixtures I and II were more stable in buffer at 25°C. On the other hand, mixtures III and IV were more stable in plasma at 37°C; mixture VI was more stable in plasma at 37°C than in buffer or saliva. Mixtures IV and V liposomes were both stable in saliva for at least one hour. Blood and feces anti-GM1 response to antigen associated liposomes after subcutaneous and oral administration was also examined. After mixture IV mice immunization, no detectable anti-ganglioside GM1 antibody response was detected. Negative stain transmission electron microscopy, shows that liposomes containing SM, GM1, GM typeIII and DIAPC : DMPC were twice the size of those made with EggPC/Chol. The hydrophobicity factor expressed as A(570/500) was obtained using the probe merocynine 540 (MC540). The order of fluidity increased from: mixture II<mixture I<mixture III<mixture IV<mixtureV<mixture VI. Although the high hydrophobicity factor for polymerizable lipids there are other factors like stability must be taken into account according to the administration via selected. Also, the hydrophilicity of the groups protruding from the membrane interphase into the solution in the case of subcutaneous inoculation is very relevant and for oral administration stability is the property to take into account, as long as they have to last through the different fluids of the gastrointestinal tract. The results obtained suggest that liposomes that showed stability in saliva and plasma at 37°C containing GM1, GM typeIII or DIAPC/DMPC would serve effectively as a delivery vehicle for oral and subcutaneous non-viral vaccines.     

Modulating gene expression in stem cells without recombinant DNA and permanent genetic modification

Future therapeutic applications of stem cells in regenerative medicine require efficient techniques for modulating gene expression. Conventionally, this is achieved through the use of recombinant DNA, which invariably leads to permanent genetic alteration to the cell. Overwhelming safety and ethical concerns are likely to preclude the application of genetically modified stem cells in human clinical therapy for the foreseeable near future. An alternative may be to adopt a milieu-based approach to influence gene expression, by exposing stem cells to a cocktail of exogenous cytokines, growth factors, and extracellular matrix. Nevertheless, the non-specific pleiotropic effects exerted by various cytokines, growth factors, and extracellular matrix would make this a relatively inefficient approach. Moreover, a milieu-based approach is likely to require extended durations of in vitro culture, which might delay autologous transplantation of adult stem cells to the patient and might alter their immunogenicity through prolonged exposure to xenogenic proteins within the culture milieu. The obvious solution would be to deliver proteins, RNA, or their synthetic analogs, such as peptide nucleic acid, directly into the cell to modulate gene expression. Currently, two promising delivery platforms are available: (1) protein transduction domains, and (2) immunoliposomes. Because such molecules have a limited active half-life in the cytosol and are obviously not incorporated into the genetic code of the cell, these would only exert a transient modulatory effect on gene expression. Nevertheless, a transient effect may be preferable for clinical therapy, since this would ultimately avoid permanent genetic alteration to the cell.     

Potentiation of Astroglial Nitric Oxide Synthase Type-2 Expression by Lithium Chloride

Abstract: Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.