Advantages of Virosomal Drug Delivery
- Virosomal technology is approved by the FDA for use in humans, and has a high safety profile
- virosomes are biodegradable, biocompatible, and non-toxic12
- no disease-transmission risk
- no autoimmunogenity or anaphylaxis10
- broadly applicable with almost all important drugs (anticancer drugs, proteins, peptides, nucleic acids, antibiotics, fungicides)
- enables drug delivery into the cytoplasm of target cell
- promotes fusion activity in the endolysosomal pathway
- protects drugs against degradation.
Virosomal Structure and Modifications
Virosomes are spherical unilamellar vesicles with a mean diameter of around 150 nm. Influenza virus is most commonly used
for virosome production. Virosomes cannot replicate but are pure fusion-active vesicles. In contrast to liposomes, virosomes
contain functional viral envelope glycoproteins: influenza virus hemagglutinin (HA) and neuraminidase (NA) are intercalated
within the phospholipid bilayer membrane (Figure 1). Further characteristics of virosomes depend on the choice of bilayer
components. Virosomes can be optimized for maximal incorporation of the drug, or for the best physiological effect by modifying
the content or type of membrane lipids used. It is even possible to generate carriers for antisense-oligonucleotides or other
genetic molecules, depending on whether positively or negatively loaded phospholipids are incorporated into the membrane.
Various ligands, such as cytokines, peptides, and monoclonal antibodies (MAbs) can be incorporated into the virosome and displayed
on the virosomal surface. Even tumor-specific monoclonal antibody fragments (Fab) can be linked to virosomes to direct the
carrier to selected tumor cells.1,11
Figure 1. Virosomes are reconstituted infl uenza virus envelopes devoid of inner core and genetic information
Difference From Liposomes
Liposomes have been considered promising vehicles for targeting and delivery of biologically active molecules to living cells
both in vitro and in vivo. However, liposomes have little potential to fuse with cells and thus, generally fail to provide appreciable delivery of
encapsulated molecules to the cell cytoplasm. In contrast, virosomes contain functional viral envelope glycoproteins
with receptor-binding and membrane-fusion properties that enable the cellular delivery of encapsulated molecules.13
Fusion Activity of Virosomal Carriers
Virosomes have unique fusion properties because of the presence of influenza HA in their membranes. HA not only confers structural
stability and homogeneity to virosomal formulations, but it also significantly contributes to the fusion activity of virosomes.
Virosomal HA promotes binding at the target cell surface followed by receptor-mediated endocytosis. The acidic environment
of the endosome triggers HA-mediated membrane fusion, and the therapeutically active substance escapes from the endosome into
the cytoplasm of the target cell. Thus, virosomal HA significantly enhances cytosolic delivery. Overall, virosomes protect
pharmaceutically active substances from proteolytic degradation and low pH within the endosomes before they reach the cytoplasm.
This is a major advantage of the virosomal carrier system over liposomal and proteoliposomal carrier systems, which provide
less protection for therapeutic macromolecules from harsh compartmental microenvironments.12,13
Methods of Preparation
To prepare virosomes, a viral membrane-fusion protein such as HA—the generally preferred fusion protein for virosomes—is either
purified from the corresponding virus or produced recombinantly. The success of virosomes as a vaccine or delivery vehicle
requires that reconstituted membrane proteins retain their immunogenic properties as well as their receptor-binding and membrane-fusion
activities. This involves functional reconstitution of influenza virus membranes, which is based on solubilizing viral membranes
by nondenaturing detergents. Influenza virus envelopes incorporated with HA can be solubilized with nonionic detergents having
a relatively low critical micellar concentration (CMC). Octaethylene glycol mono (n-dodecyl) ether (C12E8) is the most commonly
used detergent. Triton X-100 is a frequently used alternative detergent. Other nonionic detergents also can be used.14
Following solubilization, the viral nucleocapsid, which contains the endogenous viral genes, is removed by ultracentrifugation.
The viral membranes are reconstituted when C12E8 is removed by adsorption onto a hydrophobic resin. Virosomes produced by
this method fuse in a pH-dependent manner similar to native influenza virus.
The C12E8 method has certain inherent drawbacks. This method involves batch processing, often in open systems. This is a challenging
situation for industrial processing, particularly to maintain sterility. Furthermore the compounds to be encapsulated within
the virosomes could be adsorbed or inactivated by the hydrophobic resin. It is also difficult to remove low-CMC detergents
like C12E8 for solubilization from the system. Detergent removal by dialysis can circumvent these complications.15
Dialysis requires the use of detergents with relatively high CMCs, such as N-octyl- -D-glucopyranoside (octyl glucoside),
that can effectively solubilize influenza virus envelopes. However, fusogenic virosomes are not readily prepared by subsequent
removal of the octyl glucoside detergent. During dialysis, the HA concentrates primarily in lipid-poor aggregates with a very
limited aqueous space, while the viral lipid is recovered in protein-poor vesicles. Although these vesicles exhibit some HA-mediated
membrane fusion activity, only a small fraction of the HA is recovered in these vesicles. Researchers are in pursuit of novel
detergents and detergent-like compounds that can be almost completely removed by dialysis. These will be crucial for refining
an effective dialysis procedure to reconstitute influenza virus membranes for industrial purposes.14
Other lipids also can be added to the membranes during preparation. These lipids include cholesterol and phospholipids such
as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine. Cationic lipids also are added to
concentrate nucleic acids in the virosomes or to facilitate virosome-mediated cellular delivery of nucleic acids or genes.
These include, DOTAP: (N-[1-(2,3-dioleoyloxy) propyl] - N,N,N-trimethylammonium chloride), DODAC: (N,N-dioleyl-N,N, dimethylammonium
chloride), stearylamine, etc. DODAC is the preferred cationic lipid for complexing nucleic acids to the virosome to ensure
cellular delivery of nucleic acids. Concentrations of DODAC in the range of 25–45% are particularly good to ensure cellular
delivery of nucleic acids.12,16
Additional components can be added to the virosomes to target them to specific cell types. For example, virosomes can be conjugated
to MAbs that bind cellular epitopes present on the surfaces of specific cell types.