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

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.