Liposome steps

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

Mixtures of phospholipids in aqueous solution will spontaneously associate to form liposomal structures. To prepare liposomes having morphologies useful for bioconjugate or delivery techniques, it is necessary to control this assemblage to create vesicles of the proper size and shape. Many methods are available to accomplish this goal, however all of them have at least several steps in common (1) dissolving the lipid mixture in organic solvent, (2) dispersion in an aqueous phase, and (3) fractionation to isolate the correct liposomal population. 

Liposomes containing PE residues can be reacted with glutaraldehyde to form an activated surface possessing reactive aldehyde groups. A 2-step glutaraldehyde reaction strategy is probably best when working with liposomes, since precipitated protein would be difficult to remove from a vesicle suspension. 

The following protocol describes the 2-step method wherein the liposome is glutaraldehyde-activated, purified away from excess crosslinker, and then coupled to a protein by reductive amination (Figure 22.23). 

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and crosslinker all in solution at the same time. 

Table 4 Steps in Preparation of Liposomes Having a Transmembrane Ammonium Sulfate Gradient...

Liposome downsizing to desired size (this step is omitted if no defined size is needed)... 

All other steps, which include loading of these liposomes with DOX-HCl, removal of residual free drug, and characterization of the SSL during the various steps of preparation as well as of the final product, are described below and summarized in Table 2. 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

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. ... 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

Cationic lipids cannot be dissolved in water and form aggregates in aqueous solution, such as bilayers. To prepare a homogeneous reagent, in most cases liposomes were made from cationic lipids in a first step. When it is not possible to form stable lipid bilayers (i.e., liposomes) using a single lipid, then it may be necessary to combine the cationic lipid with one or more so-called helper lipids like cholesterol (Choi) (41) or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (42). 

A standard procedure for manufacturing liposomes is the film-forming method where the phospholipids are dissolved in an organic solvent. By rotational evaporation of the solvent a thin, multilayered film of phospholipids arises at the inner wall of the vessel. Redispersion of this film in water or aqueous buffer results in the formation of vesieles. The size of these vesicles and the number of bilayers vary. Henee further manufaeturing steps have to follow to obtain defined vesieular dispersions with a suffieiently long shelf life. 

---