Virosomes: A Novel Strategy for Drug Delivery and Targeting

ABSTRACT

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of macromolecules. The prospect of drug delivery and targeting using virosomes is an interesting field of research and development. Because virosomes are biocompatible, biodegradable, nontoxic, and non-autoimmunogenic, attempts have been made to use them as vaccines or adjuvants as well as delivery systems for drugs, nucleic acids, or genes for therapeutic purposes. Influenza virus is the most common virus of choice. The success of virosomal drug delivery depends on the methods used to prepare the encapsulated bioactive materials and incorporate them into the virosomes, as are characterization and formulation of the finished preparation. Virosome technology could potentially be used to deliver peptides, nucleic acids or genes, and drugs like antibiotics, anticancer agents, and steroids.

Promising drugs are often discontinued during development because they cannot be suitably delivered to target cells, tissues, and organs. The new generation of therapeutics against cancer or neurodegenerative disorders require delivery systems that target drugs to specified cell types and host tissues by receptor-mediated uptake and controlled release. Virosomal technology presents a novel sophisticated delivery system to meet these challenges.

(VETTER PHARMA INTERNATIONAL GMBH)

Virosomes are reconstituted viral envelopes, including membrane lipids and viral spike glycoproteins, but devoid of viral genetic material. The external surface of the virosome resembles that of a virus particle, with spike proteins protruding from the membrane, but their interior compartment is empty. Virosomes were first prepared by Almeida et al., who inserted purified influenza spike proteins into preformed liposomes.¹ Thereafter a range of viral envelopes have been reconstituted, including those of Sendai virus,^2–4 Semliki Forest virus (SFV),^5,6 vesicular stomatitis virus (VSV),^7,8 and Sindbis virus.⁹ Because virosomes display viral envelope glycoproteins, which, in their native conformation stimulate humoral responses, they are highly effective as vaccine antigens and adjuvants.^10–12 Moreover, since the receptor-binding and membrane-fusion properties of the viral envelope glycoprotein can be preserved, virosomes can be used as transport vehicles for cellular delivery of biologically active macromolecules. In this article, we provide a brief overview of virosomal drug delivery.

Overall, virosomes protect pharmaceutically active substances from proteolytic degradation and low pH within endosomes, allowing their contents to remain intact when they reach the cytoplasm. This is a major advantage of virosomal carrier systems over other drug-delivery vehicles, including liposomal and proteoliposomal carrier systems.

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.

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

Targeted Drug Delivery

Ideally one would like to be able to target drug delivery to selected tissues. One can tailor virosomes to targets by incorporating specific molecules (e.g., Fab fragments and ligands) into the virosome's composition. The feasibility of targeted delivery of anticancer drugs by means of virosomal carrier has been demonstrated recently by two independent approaches. In one, a MAb cross-linked to the surface of virosomes mediated specific targeting of the virosomal carrier containing an anticancer drug (e.g., doxorubicin) to human cancer cells. MAbs can bind specifically to cancer-related antigens, providing a means to target systemically administered virosomes to cancerous tissues. Alternatively, ligands that bind surface receptors on the target cells also can be bound to the virosomes to achieve targeted drug delivery. Tumors of mice treated with targeted drug-loaded virosomes failed to grow, and mortality of these animals was significantly reduced. These positive results will definitely open a new field of applications for virosomal technology.^18,19

Administration of Virosomes

Several formulations have been reported. Generally, virosomes are suspended in buffered saline (135–150 mM NaCl), but other suitable vehicles also exist. These compositions should be sterilized by conventional liposomal sterilization techniques, such as membrane filtration. The formulation also generally contains auxiliary substances as required to simulate physiological conditions, such as buffering agents and isotonicity adjusting agents (sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride).^12,17 The concentration of virosomes used in the vehicle ranges from 20–200 mg/mL. These concentrations are varied to optimize treatment with different virosome components or for particular purposes.¹⁹

The virosomes are administered in a variety of parenteral routes, including intravenous, intramuscular, subcutaneous, intra-arterial, and inhalable delivery. In addition, virosomes can be administered topically, orally, or transdermally. The virosomes also can be incorporated into implantable devices for long-term release.^19,21,22

Future Prospects

Virosomes represent an innovative drug-delivery system for various biologically active molecules, but especially nucleic acids or genes, and for numerous indications. The surface of virosomes can be suitably modified to facilitate targeted drug delivery. However, their comprehensive pharmacokinetic profile, bioavailability, clinical effects, and stability should be studied thoroughly to ascertain their long-term reliability as a safe, effective, and affordable means for drug delivery and targeting.

SANJIB BHATTACHARYA is a lecturer at the Bengal School of Technology (A College of Pharmacy), Hooghly, West Bengal, India, and BHASKAR MAZUMDER, PhD, is a senior lecturer at the Deptartment of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India, +91 9435256182, bhmaz@yahoo.co.in

References

1. Almeida JD, Brand CM, Edwards DC, Heath TD. Formation of virosomes from influenza subunits and liposomes. Lancet. 1975;2:899–901.

2. Bagai S, Puri A, Blumenthal R, Sarkar DP. Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells. J Virol. 1993;67:3312–18.

3. Uchida T, Kim J, Yamaizumi M, Miyake Y, Okada Y. Reconstitution of lipid vesicles associated with HVJ (Sendai virus) spikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin. J Cell Biol. 1979;80:10–20.

4. Vainstein A, Hershkovitz M, Israel S, Rabin S, Loyter A. A new method for reconstitution of highly fusogenic Sendai virus envelopes. Biochim Biophys Acta. 1984;773:181–8.

5. Helenius A, Sarvas M, Simons K. Asymmetric and symmetric membrane reconstitution by detergent elimination. Studies with Semliki-Forest-virus spike glycoprotein and penicillinase from the membrane of Bacillus licheniformis. Eur J Biochem. 1981;116:27–35.

6. Helenius A, Fries E, Kartenbeck J. Reconstitution of Semliki Forest virus membrane. J Cell Biol. 1977;75: 866–80.

7. Metsikkö K, van-Meer G, Simons K. Reconstitution of the fusogenic activity of vesicular stomatitis virus. EMBO J. 1986;5:3429–35.

8. Petri WA, Wagner RR. Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis. J Biol Chem. 1979;254:4313–16.

9. Scheule RK. Novel preparation of functional Sindbis virosomes. Biochem, 1986;25:4223–32.

10. Glück R, Mischler R, Finkel B, Que JU, Scarpa B, Cryz SJ. Immunogenicity of new virosome influenza vaccine in elderly people. Lancet. 1994;344:160–3.

11. Huckriede A, Bungener L, Veer W, Holtrop M, Daemen T, Palache AM, Wilschut J. Influenza virosomes: combining optimal presentation of hemagglutinin with immunopotentiating activity. Vaccine. 2003;21:925–31.

12. Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM, Wilschut J. The virosome concept for influenza vaccines. Vaccine 2005;23(S1):S26–38.

13. Kaneda Y. Virosomes: evolution of the liposome as a targeted drug delivery system. Adv Drug Deliv Rev. 2000;43:197–205.

14. Stegmann T, Morselt HWM, Booy FP, Van Breemen JFL, Scherphof G, Wilschut J. Functional reconstitution of influenza virus envelopes. EMBO J. 1987;6:2651–9.

15. Bron R, Ortiz A, Wilschut J. Cellular cytoplasmic delivery of a polypeptide toxin by reconstituted influenza-virus envelopes (virosomes). Biochem. 1994;33:9110–17.

16. Schoen P, Bron R, Wilschut J. Delivery of foreign substances to cells mediated by fusion-active reconstituted influenza virus envelopes (virosomes). J Liposome Res. 1993;3:767–92.

17. Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM. The virosome concept for influenza vaccines. Vaccine. 2005;23:S26–38.

18. Felnerova D, Viret JF, Glück R, Moser C. Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs. Curr Opin Biotechnol. 2004;15:518–29.

19. Cusi MG. Applications of influenza virosomes as a delivery system. Human Vaccines. 2006;2:1–7.

20. Daemen T, de Mare A, Bungener L, de Jonge J, Huckriede A, Wilschut J. Virosomes for antigen and DNA delivery. Adv Drug Deliv Rev. 2005;57:451–63.

21. Sarkar DP, Ramani K, Tyagi SK. Targeted gene delivery by virosomes. Methods Mol Biol. 2002;199: 163–73.

22. Schoen P, Chonn A, Cullis PR, Wilschut J, Scherrer P. Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles. Gene Ther. 1999;6:823–32.

23. Bungener L, Serre K, Bijl L, Leserman L, Wilschut J, Daemen T, Machy P. Virosome-mediated delivery of protein antigens to dendritic cells. Vaccine. 2002;20:2287–95.

24. Arkema A, Huckriede A, Schoen P, Wilschut J, Daemen T. Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes. Vaccine. 2000;18: 1327–33.

25. Bungener L, Huckriede A, de Mare A, de Vries-Idema J, Wilschut J, Daemen T. Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity. Vaccine. 2005;23:1232–41.