Liposome vaccine entrapment

DNA and/or protein vaccine entrapment in DRV liposomes is monitored by measuring the vaccine in the suspended pellet and combined supernatants. The most convenient way to monitor DNA entrapment is by using radio-labelled or DNA. For protein entrapment, the use of I-labelled protein tracer is recommended. If a radiolabel is not available or cannot be used, appropriate quantitative techniques should be employed. To determine DNA or protein by such techniques, a sample of the liposome suspension is mixed with Triton X-100 (up to 5% final concentration) or, preferably, with isopropanol (1 1 volume ratio) so as to liberate the entrapped materials. However, if Triton X-100 or the solubilized liposomal lipids interfere with the assay of the materials, liposomal lipids or the DNA must be extracted using appropriate techniques (6). Entrapment values for protein and DNA, whether alone or coentrapped, range between about 20% to 80% (protein) and 30%i to 100%i (DNA) of the initial material depending on the DNA or protein used and, in the case of DNA, the presence or absence of cationic charge. Values are highest for DNA when it is entrapped into cationic DRV (typical values in Table 1). 

The content of vaccine within the small liposomes is estimated as in the section Estimation of Vaccine Entrapment in Dehydration-Rehydration Vesicles Liposomes for both microfluidized and sucrose liposomes and expressed as percentage of DNA and/or protein in the mixture subjected to freeze drying as in the section Preparation of Vaccine-Containing Small Liposomes by the Sucrose Method in the case of sucrose small liposomes or in the original DRV preparation (obtained in the section Estimation of Vaccine Entrapment in DRV Liposomes ) for microfluidized liposomes. Vesicle size measurements are carried out by PCS as described elsewhere (6,8,17). Liposomes can also be subjected to microelectrophoresis in a Zetasizer to determine their zeta potential. This is often required to determine the net surface charge of DNA-containing cationic liposomes. 

From 1984, when they were first developed, DRV liposomes have been used for liposomal encapsulation of various active substances which may be divided into three main categories (1) Low MW drug molecules (mainly hydrophilic drugs) (3-20) (2) Proteins or peptides and enzymes (21-26), and (3) DNA or oligonucleotides (26-32). From these categories, the last two are primarily used as liposomal vaccines. Some examples of substances entrapped in DRV liposomes from the last 10 year literature are presented in Table 1. 

Gregoriadis G, McCormack B, Obrenovic M, Roghieh S, Zadi B, Perrie Y (1999) Vaccine entrapment in liposomes. Methods 19 156-162... 

Rhalem, A., Bourdieu, C., Luffau, G., and Pery, P. (1988). Vaccination of mice with liposome-entrapped adult antigens of Nippo-strongylus brasiliensis. Ann. Inst. Pasteur/Immunol.. 139, 157-166. 

Liposome-Based DNA/Protein Vaccines Procedures for Entrapment and Immunization Studies... 

ENTRAPMENT OF PLASMID DNA AND PROTEIN VACCINES INTO LIPOSOMES BY THE DEHYDRATION-REHYDRATION PROCEDURE... 

Up to 500 pg of plasmid DNA (for the amount of PC shown above) is dissolved in 2mL distilled water, or lOmM sodium phosphate buffer (PB) of pH 7.2 if needed. For liposomes containing both the plasmid DNA and the vaccine protein it encodes (or only the protein), up to 1 mg of the protein is included. The nature of buffer with respect to composition, pH, and molarity can be varied as long as this does not interfere with liposome formation or DNA and protein entrapment yield. Amounts of added DNA and protein can be increased proportionally to the total amount of lipid used. For cationic liposomes, the amount of added DNA can also be increased by employing more cationic lipid. 

Entrapment of plasmid DNA and/or protein into liposomes entails the preparation of a lipid film from which multilamellar vesicles and, eventually, small unilamellar vesicles (SUVs) are produced. SUVs are then mixed with the plasmid DNA and/or protein destined for entrapment and dehydrated. The dry cake is subsequently broken up and rehydrated to generate multilamellar dehydration-rehydration vesicles (DRV) containing the plasmid DNA and/or protein. On centrifugation, liposome-entrapped vaccines are separated from nonentrapped materials. When required, the DRV are reduced in size by microfluidization in the presence or absence of nonentrapped materials or by employing an alternative method (7) of DRV production, which utilizes sucrose (see below). 

Quantitative entrapment of vaccines into small (up to about 200 nm diameter) liposomes in the absence of microfluidization (which can damage DNA and other labile materials when extensive) can be carried out by a novel one-step method (7) as follows SUVs (e.g., cationic) prepared as in section Preparation of Small Unilamellar Vesicles are mixed with sucrose to give a range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appropriate amount of plasmid DNA (e.g., 10-500 pg) and/or protein (e.g., up to 1 mg). The mixture is then rapidly frozen and subjected to dehydration by freeze-drying, followed by rehydration as in section Preparation of Vaccine-Containing Dehydration-Rehydration Vesicles. ... 

It can therefore be concluded that immunization with liposomes containing both DNA and the encoded antigen leads to superior immune responses when compared with liposomes entrapping the DNA or protein vaccine alone. 

Antigen delivery through liposomes, hollow membrane-bound spheres, can be achieved by entrapping the molecule in the lipid membrane or inside the hollow cavity. Modified liposomes have been able to induce mucosal IgA responses compared to free antigen (Ann Clark et al. 2001 Aziz et al. 2007). Liposomes containing pertussis toxin (Guzman et al. 1993), Streptococcus mutans (Childers et al. 2002), or bovine serum albumin (Therien et al. 1990) as vaccine antigens have been tested in experimental models and induced effective antibody- and cell-mediated immune responses. 

Dried reconstituted vesicles (DRV) are liposomes that are formulated under mild conditions and have the capability to entrap substantially high amounts of hydrophilic solutes (compared with other types of liposomes). These characteristics make this liposome type ideal for entrapment of labile substances, as peptide, protein or DNA vaccines and sensitive drugs. In this chapter, we initially introduce all possible types of DRV liposomes (in respect to the encapsulated molecule characteristics and/or their applications in therapeutics) and discuss in detail the preparation methodologies for each type. 

To determine the vaccine by such techniques a sample of the liposome suspension is mixed with Triton X-100 (up to 5% final concentration) or 2-propanol (1 1 volume ratio) in order to liberate the entrapped material. 

Dilute the liposomal suspension obtained in step 6 (of the general procedure described in Subheading 3.1.1) prior to separation of the entrapped from the nonentrapped vaccine, to 10 ml with H O. 

When the required volume has been reached, the sample is treated for the separation of entrapped from nonentrapped vaccine, by molecular sieve chromatography using a Sepharose CL-4B column, in which case vaccine-containing liposomes are eluted at the end of the void volume (see Note 12). 

Liposomes are microstructures composed of one or more concentric spheres of (phospho)lipid bilayer, separated by water or aqueous buffer compartments. Those particles can encapsulate and dehver both hydrophilic and lipophilic substances. Water soluble substances can be entrapped in the central aqueous core, lipid soluble substances in the membrane and peptide and small proteins at the hquid aqueous interface. The size of such a particle can differ from 20 nm to 10 pm. Liposomes are in general made synthetically e.g. by the lipid hydration method. Liposomal medicines are on the market for the treatment of systemic fungal infections, tumours and for vaccination. 

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