Liposomes suspensions

Guiot, P., Baudhuin, P., and Gotfredsen, C. (1980). Morphological characterization of liposome suspensions by stereological analysis of freeze fracture replicas from spray frozen samples, J. Microsc., 120, 159-174. 

Stndies of the antoxidation of carotenoids in liposomal suspensions have also been performed since liposomes can mimic the environment of carotenoids in vivo. Kim et al. stndied the antoxidation of lycopene," P-carotene," and phytofluene" " in liposomal snspensions and identified oxidative cleavage compounds. Stabilities to oxidation at room temperature of various carotenoids incorporated in pig liver microsomes have also been studied." The model took into account membrane dynamics. After 3 hr of reactions, P-carotene and lycopene had completely degraded, whereas xanthophylls tested were shown to be more stable. 

Kim, S.J., Cleavage products formed through autoxidation of zeta-carotene in liposomal suspension. Food Sci. Biotech., 13, 202, 2004. 

Dissolve sodium periodate to a concentration of 0.6 M by adding 128 mg per ml of water. Add 200 pi of this stock periodate solution to each ml of the liposome suspension with stirring. 

Add 25-50 pi of the stock solution of either SPDP or LC-SPDP in DMF to each ml of the liposome suspension to be modified. If sulfo-LC-SPDP is used, add 50-100 pi of the stock solution in water to each ml of liposome suspension. 

Dissolve a sulfhydryl-containing peptide hapten at a concentration of 25 pmol/ml in degassed, nitrogen-purged lOmM HEPES, 0.15M NaCl, pH 7.0. Add the peptide solution to the liposome suspension at a molar ratio necessary to obtain at least a 5 1 excess of thiol groups to the amount of maleimide groups present (as MPB-DPPE). 

Prepare liposomes containing PE by any desired method. For instance, the common recipe mentioned in Section 1 (this chapter) that involves mixing PC cholesterol PG PE in a molar ratio of 8 10 1 1 may be used. Thoroughly emulsify the liposome construction to obtain a good population of SUVs. The final liposome suspension should be in 20mM sodium phosphate, 0.15M NaCl, pH 7.2. Adjust the concentration to about 5 mg lipid/ml buffer. 

Add the protein or peptide to be conjugated to the liposome suspension. The protein may be dissolved first in PBS, pH 7.2, and an aliquot added to the reaction lipid mixture. The amount of protein to be added can vary considerably, depending on the abundance of the protein and the desired final density required. Reacting from 1 mg protein per ml liposome suspension up to about 20 mg protein/ml can be done. 

Prepare a liposome suspension, containing PE, at a total-lipid concentration of 5mg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 6.8. Maintain all lipid-containing solutions under an inert gas atmosphere. Degas all buffers and bubble them with nitrogen or argon prior to use. 

Dissolve the protein or peptide to be conjugated at a concentration of lOmg/ml in 0.5M sodium carbonate, pH 9.5. Mix the activated liposome suspension with the polypeptide solution at the desired molar ratio to effect the conjugation. Mixing the equivalent of 4 mg of protein per mg of total lipid usually results in acceptable conjugates. 

Periodate-oxidize a liposome suspension containing glycolipid components according to Section 2 (this chapter). Adjust the concentration of total lipid to about 5mg/ml. 

Add 0.5 ml of protein solution to each ml of liposome suspension with stirring. 

Prepare a 5mg/ml liposome suspension containing a mixture of PC cholesterol PG PDP-PE in molar ratios of 8 10 1 1. The emulsification may be done by any established method (Section 1, this chapter). Suspend the vesicles in 50mM sodium phosphate, 0.15 M NaCl, 10 mM ethylenediamine triacetic acid EDTA, pH 7.2. 

Mix the protein solution with the liposome suspension in equal volume amounts. 

Liposomes containing PE lipid components may be activated with these crosslinkers to contain iodoacetyl derivatives on their surface (Figure 22.29). The reaction conditions described in Chapter 5, Section 1.5 may be used, substituting a liposome suspension for the initial protein being modified in that protocol. The derivatives are stable enough in aqueous solution to allow purification of the modified vesicles from excess reagent (by dialysis or gel filtration) without... 

Bourel-Bonnet L, GrasMasse H, Melnyk O. A novel family of amphilic alpha-oxo aldehydes for the site-specific modification of peptides by two palmi-toyl groups in solution or in liposome suspensions. Tetrahedron Lett 2001 42 6851. 

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 phospholipid concentration in a (proteo)liposome suspension can be determined by phosphorus analysis (73) and the protein concentration by automated amino acid analysis or by a calibrated colorimetric protein assay... 

The oxidation of can proceed with hydroperoxides at a convenient rate without a catalyst, if the solution is heated to 90 °C, measuring at 348 nm. A modification of the iodometric method was proposed for determination of hydroperoxides in liposomes, using anhydrous EtOH as solvent in all the operations. A sample of liposome suspension is evaporated to dryness in a vacuum, after adding EtOH in a 4 96 sample-to-solvent proportion. 

The peptide subunit was easily incorporated into lipid bilayers of liposome, as confirmed by absorption and fluorescence spectroscopy. Formation of H-bonded transmembrane channel structure was confirmed by FT IR measurement, which suggests the formation of a tight H-bond network in phosphatidylcholine liposomes. Liposomes were first prepared to make the inside pH 6.5 and the outside pH 5.5. Then the addition of the peptide to such liposomal suspensions caused a rapid collapse of the pH gradient. The proton transport activity was comparable to that of antibiotics gramicidin A and amphotericin B. 

Perhaps the simplest solvent dispersion method is that developed by Batzri and Korn (1973). Phospholipids and other lipids to be a part of the liposomal membrane are first dissolved in ethanol. This ethanolic solution is then rapidly injected into an aqueous solution of 0.16 M KC1 using a Hamilton syringe, resulting in a maximum concentration of no more than 7.5% ethanol. Using this method, single bilayer liposomes of about 25-nm diameter can be created that are indistinguishable from those formed by mechanical sonication techniques. The main disadvantages of ethanolic injection are the limited solubility of some lipids in the solvent (about 40 mM for phosphatidyl choline) and the dilute nature of the resultant liposome suspension. However, for the preparation of small quantities of SUVs, this method may be one of the best available. 

---