DRV liposomes

Grant GJ, Barenholz Y, Piskoun B, Bansinath M, Turndorf H, Bolotin E. DRV liposomal bupivacaine preparation, characterization and in vivo evaluation in mice. Pharm Res 2001 18 336-343. 

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. 

Figure 1 Anti-HA IgG titres ( SD) ( Y-axis) in mice immunized with a single subcutaneous injection of (Alum-adsorbed) DRV liposomes composed of PC, DOPE, and DOTAP (molar ratios 4 2 1) and containing pl.l7/SichHA DNA and killed influenza virus ( ), killed influenza virus only (A), or DNA only ( ). For other details see the text. Abbreviations HA, hemagglutinin DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, 1,2-dioleyloxy-3-(trimethylamonium propane).

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. 

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. 

Diluted PBS or Phosphate Buffer pH 7.40, for preparation of CF (or calcein) solution. This buffer is prepared by diluting PBS buffer 10 times with d.d. H O (see item 5). This buffer is used for preparation of CF (or calcein) solution, prepared for encapsulation in DRV liposomes. 

Sephadex G-50 (medium) (Phase Separations, Pharmacia, Sweden). The powder is dispersed in PBS buffer for swelling and the dispersion is subsequently degassed under vacuum. Gel chromatography columns are packed and used for DRV liposome separation from nonencapsulated molecules (as described in detail in the following section). 

For the preparation of DRV liposomes, empty (see Note 4) SUV liposomes dispersed in d.d. H O with the appropriate lipid composition and concentration are initially prepared (see Note 5). SUV preparation can be performed by several techniques, depending on the specific lipid composition and concentration required, the most convenient and easiest to use being (1) Probe sonication in one step (see Note 6), and (2) Size reduction of MLV liposomes (most applied technique). 

In order to use the DRV liposomes, and/or measure the entrapment or encapsulation efficiency (or yield) they should first be separated or purified from not entrapped solute. Depending on the MW of the entrapped solute and on the final size of the DRV liposomes this can be done by centrifugation or size exclusion chromatography). 

Preparation of DRV Liposomes with Controlled Entrapment Yield and Vesicle Size (as reported in (2))... 

If vaccine-containing DRV liposomes must be converted to smaller vesicles (down to about 100-nm z-average diameter) the following procedure is used ... 

Giant DRV Liposome Entrapped particulate material is separated from nonentrapped Separation from material (bacteria spores, etc.) by sucrose gradient centrifugation. 

For the formation of DRV liposomes entrapping solutes that are not sensitive to the conditions used for MLV and/or SUV preparation, it is possible to prepare drug containing liposomes in the initial step of DRV formation. This is particularly important if amphiphilic/lipophilic or in general substances with low aqueous solubility are to be entrapped. However, when there is interest to have a method that can be easily up-scaled for large batch manufacturing, this approach can be problematic. 

It has been reported that DRV liposomes with comparably high encapsulation eiBciency (compared to plain MLV s) can be produced even by using MLV liposomes for the initial drying step (1). Indeed a EE% of 21% for CF was reported when empty MLV liposomes were used (compared to 1.8% when plain MLV were formed using the same CF hydrating concentration), while a 30% EE% was reached when starting from SUV. 

A column with dimensions 1x35 cm is sufficient to separate 1 ml of liposome or the DRV liposome dispersion. The column is pre-calibrated and at the same time saturated with a dispersion of empty liposomes mixed with a quantity of the encapsulated material (in each case). The void volume of such columns should be between 7 and 13 mL and the bed volume between 17 and 21 mL. 

Ntimenou V, Mourtas S, Christodoulakis EV, Tsilimbaris M, Antimisiaris SG (2006) Stability of protein-encapsulating DRV liposomes after freeze-drying A study with BSA and t-PA. J Liposome Res 16 403-416... 

Monitor the extent of drug entrapment in DRV liposomes by measuring the drug in the suspended pellet and combined supernatants. The easiest way to... 

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