Characterization of Virosomes
Protein detection: Virosome preparation should generally result in a relatively uniform protein-to-lipid ratio. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) can confirm the presence of HA protein in the virosomes.17
Structure and size: Negative-stain electron microscopy can generally be used to determine the ultrastructure and size of virosomes. The staining
solutions should preferably be of neutral pH, to avoid acid-induced conformational changes of HA.18
Fusion activity: Generally virosomes exhibit pH-dependent membrane fusion activity similar to native influenza virus. Virosomal fusion with
biological or artificial target membranes can be visualized with a fluorescent resonance energy transfer assay (RET).17 Alternatively, fusion can be assessed in vitro with an excimer assay using pyrene-labeled lipids, where the decrease of
surface density of the pyrene-phosphatidylcholine-label on fusion with an unlabeled membrane corresponds to a reduction of
excimer fluorescence.12
Fusion activity also can be indirectly monitored by determining hemolytic activity, which corresponds closely to fusion activity
and exhibits a pH dependence identical with that of fusion.1
Drug-Delivery Approaches
Bioactive drug compounds can be entrapped in the aqueous interior of the virosome or in the lipid membrane of the virosome
for facilitated entry of the compounds into the cells.19
Virosomes are particularly useful for delivering nucleic acids or genes. These compounds are delivered into the host cell
cytoplasm on fusion of the virosome with the endosome or plasma membrane.20 Nucleic acids or genes encoding a naturally occurring protein can be introduced into host cells and can be expressed, provided
that the expression cassette contains the proper cis-acting regulatory elements.20,21
Drugs or nucleic acids can be incorporated into the virosome at the time of virosome preparation. The bioactive compound is
typically added to the lipid–HA-containing solution following removal of the nucleocapsid. Alternatively, the bioactive compound
is initially incorporated into a liposome, which is then fused with a virosome containing two hemagglutinins with different
pH thresholds to form a virosome–liposome hybrid.22
Proteins also can be delivered to cells via virosome. For example, the gelonin subunit A of diphtheria toxin and ovalbumin
have also been successfully delivered by virosome to target cells.15,22,23 Virosomes carrying peptides derived from the influenza nucleoprotein or intact ovalbumin induced strong cytotoxic T lymphocyte
responses, which suggests that the encapsulated peptides and proteins gained access to the cytoplasm.24,25
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