LIPOSOME

1.- BACTERIAL CAPSULAR EXOPOLYSACCHARIDES

2.- BACTERIAL BIOFILMS: THE ROLE OF EXOPOLYSACCHARIDES IN CHRONIC PROCESSES

3.- LIPOSOMES: ANTIGEN VEHICLES

4.- LATEST GENERATION AUTOVACCINES

3.- LIPOSOMES: ANTIGEN VEHICLES

The following chapters discuss the use of liposomes to induce an adequate immune response to the presence of exopolysaccharides.

In the two previous articles of this series dedicated to the development of autovaccines for veterinary use, the role of exopolysaccharides in bacterial virulence was explained. This third article will deal with the use of liposomes for preparing vaccines, particularly those including purified exopolysaccharides.

Current applications of liposomes in medicine

Fig. 1: Liposome with details of the wall composed of a double phospholipid layer. A representation of the exopolysaccharides is shown in the interior.

Liposomes are hollow microspheres composed of one or more double lipid layers (Fig. 1). Liposomes were first used more than 30 years ago as vehicles for drug substances, and since then knowledge of their behavior in vitro has allowed a more rational design focused on the specific treatment of certain diseases. At present, a number of biotechnological companies work exclusively with liposomes for the development of different therapies: antibiotics, antitumor drugs, allergic sensitization formulas, gene therapy, and so on. In this sense, vehicle function for vaccinal antigens is what interests us here. Many of these antifungal, antitumor and vaccinal products have already been registered for use in human patients, while some are still in the phase III clinical trial stage (conducted in patients).

Interesting characteristics of liposomes

The interesting aspect of liposomes lies in their membranes, which are composed of cholesterol and phospholipids (such as phosphatidylcholine and dicetylphosphate) — the structure, composition and proportion being practically the same as in the host cell membranes. The phospholipids possess a hydrophobic tail structure and a hydrophilic head component, and organize in the following manner when dissolved in water: the hydrophobic tails mutually attract, while the hydrophilic heads contact with the aqueous medium external and internal to the liposome surface. In this way, double lipid layers are formed which seal off to form small vesicles similar to the body cells and their organelles (Fig. 1).

These spheres or liposomes constitute small deposits (Fig. 1) that can be made to contain an antigen, an antibiotic, an allergen, a drug substance or a gene (as in gene therapy). The liposomes can in turn be introduced in the body without triggering immune rejection reactions. In fact, 70 years after approval of the use of aluminum hydroxide for inclusion in vaccines, liposomes have been authorized for human use (Gregoradis 1998).

Depending on the requirements of each drug substance, the liposome characteristics can be modified. Thus, to reduce the rate of liposome degradation and therefore slow down the release of the contents, the composition and size of the spheres can be modified. Liposome affinity for a given tissue can also be incremented by varying vesicle composition, electrical charge or by adding receptors or adhesion factors — thereby contributing to increase drug presence in the target tissues or organ.

An example of this effect is provided by doxorubicin, an expensive and toxic antitumor drug that is used in human medicine. When included in liposomes of a given size and electrical charge, slow diffusion of the drug is ensured, thereby contributing to prolong its half-life and making it possible to administer much smaller amounts (with the resulting cost savings and treatment toxicity reduction).

The liposomes as vaccinal adjuvants

One of the most important characteristics of liposomes is that they are avidly phagocytosed by macrophages and other cells of the reticuloendothelial system. As a result, they make excellent adjuvants for many purified antigens. An example that can be used to explain this principle is represented by a bacterial exopolysaccharide or a recombinant protein. These elements are expensive to produce and purify, though when inoculated into liposomes in small amounts, an adequate immune response can be achieved.

Fig. 2: Resuspension of the lyophilized product.

In producing vaccines, we can make use of all the abovementioned liposome modifications, though it is also possible to insert cytokines or adjuvants such as muramyldipeptide or lipid A, which allow us to modulate cellular and humoral responses according to our needs, or to introduce certain antigens into the membrane. In the case of the bacterial exopolysaccharides, the lipidic (hydrophobic) portion becomes embedded in the thickness of the membrane, while the hydrophilic exopolysaccharide component emerges from the membrane and protrudes external or internal from the liposome surface.

With certain antigens, the combination of liposomes and aluminum hydroxide affords excellent results, since aluminum hydroxide acts as a reservoir that slowly releases the liposomes after administration.

An alternative in the production of vaccines and autovaccines: dry reconstituted vesicles (DRVs)

The greatest problem posed by liposomes is that once they have been manufactured they have only limited stability. This is because their constituting membranes tend to aggregate, forming large vesicles that are less effective in vivo. Moreover, in this autoaggregation process, much of the encapsulated material destined for delivery is lost to the surrounding environment.

In order to minimize autoaggregation, the liposomes must be kept refrigerated — although even so, stability is prolonged only a few weeks, in the best of cases. This clearly poses difficulties for the practical use of liposomes. An alternative would be lyophilization, though it is not easy to lyophilize liposomes, for despite the use of cryoprotectors in this process, the vesicles ultimately rupture.

Liposome fragmentation during lyophilization is precisely the phenomenon used for the production of dry reconstituted vesicles (DRVs). In the course of lyophilization, the frozen water sublimates, i.e., it evaporates and transforms directly into gas without first passing through a liquid water phase. On subjecting liposomes to lyophilization, the water contained in the interior of the vesicles sublimates first, thus increasing the concentration of salts and consequently the osmotic pressure, which finally leads to liposome rupture.

In most liposome-encapsulated products the substances destined for delivery are introduced within the vesicles in the course of liposome manufacture, for once the latter have been created it is no longer possible to insert anything into them. In contrast, in the DRV technique the liposomes are produced without concomitant antigen insertion, and the latter therefore remains external to the vesicles. When lyophilization is performed, the liposome membranes rupture - though when reconstitution in water is carried out the liposomes again seal off into small vesicles and trap the antigen in their interior (Fig. 3). This technique makes it possible to achieve antigen encapsulation yields of over 80%.

The great advantage offered by DRVs in the production of autovaccines and vaccines for veterinary use is that they allow the utilization of liposomes as vehicles for vaccinal antigens and adjuvants while also affording a lyophilized product that is very easy to store and transport. The use of aluminum hydroxide for resuspending the liposomes in turn improves the liposome-induced immune response and minimizes the risk of side effects at the injection site.

Bibliografia

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