Film hydration, liposome preparation

In conventional film-shaking liposome preparation, lipids (see Note 2) are dissolved in chloroform and the solvent removed by evaporation to leave a lipid film. Insulin-containing citrate buffer (pH 4.0) is added to hydrate the lipid film. Mechanical shaking of the mixture results in the formation of multilamellar vesicles. 

Yomo, Urabe and coworkers (Yu et al, 2001), for example, reported the expression of a mutant GFP (actually the pET-21-GFPmutl-His6 mutant) in lecithin liposomes. Large GFP-expressing vesicles, prepared by the film hydration method, were analyzed using flow cytometry as well as confocal laser microscopy. 

One of the major drawbacks of liposomes is related to their preparation methods [3,4]. Liposomes for topical delivery are prepared by the same classic methods widely described in the literature for preparation of these vesicles. The majority of the liposome preparation methods are complicated multistep processes. These methods include hydration of a dry lipid film, emulsification, reverse phase evaporation, freeze thaw processes, and solvent injection. Liposome preparation is followed by homogenization and separation of unentrapped drug by centrifugation, gel filtration, or dialysis. These techniques suffer from one or more drawbacks such as the use of solvents (sometimes pharmaceutically unacceptable), an additional sizing process to control the size distribution of final products (sonication, extrusion), multiple-step entrapment procedure for preparing drug-containing liposomes, and the need for special equipment. 

The liposome preparation procedure is based on film formation and hydration. After transferring the mixture of Phospholipon and GDNT solutions into 10 ml round bottom flasks, the chloroform-methanol solution is removed by evaporation at 300 mbar and 45°C to obtain a lipid film. 

The formation of liposomes [or better arsonoliposomes (ARSL)], composed solely of arsonolipids (Ars with R=lauric acid (C12) myristic acid (C14) palmitic acid (C16) and stearic acid (C18) (Fig. 1) have been used for ARSL construction), mixed or not with cholesterol (Choi) (plain ARSL), or composed of mixtures of Ars and phospholipids (as phosphatidylcholine [PC] or l,2-distearoyl- -glyceroyl-PC [DSPC]) and containing or not Choi (mixed ARSL), was not an easy task. Several liposome preparation techniques (thin-film hydration, sonication, reversed phase evaporation, etc.) were initially tested, but were not successful to form vesicles. Thereby a modification of the so called one step or bubble technique (8), in which the lipids (in powder form) are mixed at high temperature with the aqueous medium, for an extended period of time, was developed. This technique was successfiil for the preparation of arsonoliposomes (plain and mixed) (9). If followed by probe sonication, smaller vesicles (compared to those formed without any sonication [non-sonicated]) could be formed [sonicated ARSL] (9). Additionally, sonicated PEGylated ARSL (ARSL that contain polyethyleneglycol [PEG]-conjugated phospholipids in their lipid bilayers) were prepared by the same modified one-step technique followed by sonication (10). 

The pH-sensitive or pH-insensitive, Rh-labeled, TATp-bearing liposomes are prepared by the lipid film hydration method. 

Liposomes are mostly prepared by the thin film hydration method in which a thin fihn is produced by dissolving the phospholipids in suitable organic solvent... 

In most cases, liposomes are prepared by the film hydration method lipid mixtures are dissolved in organic solvent and then evaporated to dryness. Liposomes are then formed by rehydration of the dried film with the aqueous phase. To encapsulate CyDs within the aqueous core of liposomes, preformed complexes are generally dissolved in the aqueous rehydration phase. Another technique is to intro-... 

DNA, coding for the GFP was introduced in liposomes composed by POPC, together with the whole T T machinery (PURESYSTEM). GFP was synthesized inside large vesicles prepared by the film hydration method. 

There are many methods to prepare liposomes. This review will discuss only some of the commonly used methods. These include liposomes made by thin film hydration, reverse phase evaporation, freeze-drying, and proliposome methods. 

Potential of alginate-loaded liposomes as a vehicle for the oral delivery of bioactive proteins. The vesicles were prepared from the phospholipid dipalmatoyl phosphatidylcholine using a simple dry film hydration technique. Alkaline phosphatase (ALP) was used as a model bioactive protein. ALP activity following 2 h of exposure to simulated gastric pH was maintained at a significantly higher level (80%) when encapsulated in the alginate-loaded liposomes as compared to ALP loaded in conventional liposomes (55%)... 

When liposomes are prepared from a molecular mixture of lipid components it is important that all lipids be homogeneously dissolved in an organic solvent in order to obteiin bilayers with evenly distributed lipids after hydration. For example, the solubilities of phosphatidylcholine and cholesterol in chloroform are similar their solubility in benzene differs. Upon removal of benzene from the lipid solution an inhomogeneous lipid film is formed on the glass wall and... 

In Step 2 After hydration of the thin film (step 2), the MLV suspension prepared is filtered (Whatmman no. 5) to remove precipitated (nonincorporated in liposomes) drug. 

To prepare Rhodamine-labeled liposomes, add 0.5% Rhodamine-PE in standard formulation (Subheading 3.1, item 1) and hydrate lipid film with 40 mM FlTC-dextran in K-H buffer. Remove non-encapsulated FlTC-dextran with by gel-filtration on NAP column. 

One of the most popular methods for preparation of liposomes is the hydration of thin lipid films, combined with freeze/thaw and extrusion cycles. 

Lipids are mixed together and solvent is removed using freeze-drying. Then an aqueous solution is introduced and the hpid cake around the vessel wall is reconstituted. This method works best for manufacture of neutral liposomes, as the hydro-phobic lipids readily dissolve in solvents such as chloroform and are deposited dry on the wall of the rotavapor vessel. Then the material to be encapsulated is dissolved in an aqueous solution and the dry film on the vessel wall is hydrated with this solution. The exact steps involved in the preparation of hydrogenated soy phosphatidylcholine (PHSPC)- and cholesterol-containing vesicles at a molar ratio of 60 to 40% are as listed here ... 

Deuterated liposomes were prepared from a dried lipid film cast from a chloroform solution of deuterated l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-dyo Avanti Polar Lipids, Inc., Alabaster, AL, USA Figure 5.11b) [37, 38]. This film was hydrated and vortexed with HEPES-buffered saline (HBS), pH 7.4, at a DSPC concentration of 1.6mM. The resulting Uposomes were sized through double-stacked 200nm pore size polycarbonate membranes (Nucleopore), after which the... 

Thermotropic Properties. Glycophorin can be incorporated into multilamellar liposomes by hydration of a glycophorin-lipid film prepared by rotary evaporation of a mixture of glycophorin and lipid in an azeotropic solvent (chloroform/methanol/water, 81 15 14% v/v bpt 52.6 C). VVe found that the azeotropic solvent mixture facilitated uniform dispersion of the glycophorin in the lipid (28). 

The preparation methods of these vesicles are various. In some cases, the liposomal suspension is firstly prepared and then amphiphilic CyDs are inserted inside the phospholipid-bilayer, for example by sonication [93]. Another method mixes phospholipid (+/— cholesterol) and amphiphilic CyD solutions, to form hposomes by conventional methods [92, 94]. Lastly, vesicles consisting of bilayers of purely amphiphilic CyDs as raw material can be prepared applying a technique used to prepare hposomes, i.e. hydration of lipidic film followed by sonication [99-101]. 

Zhang and Zhu [60] have combined two methods in preparing liposomes, by hydrating particulate-based proliposomes followed by freeze-drying. This approach has been reported to enhance the entrapment of amikacin compared to the same vesicles before freeze-drying or vesicles prepared by the thin film method. 

Liposomes are prepared in a pear-shaped flask in a rotating evaporator at 37° as previously described (Matsuoka et ah, 1998). Briefly, 100 p of a 3 M lipid stock mixture resembling the Golgi lipid composition (43 mol% phosphatidylcholine (PC), 19 mol% phosphatidylethanolamine (PE), 5 mol % phosphatidylserine (PS), 10 mol% phosphatidylinositol (PI), 7 mol% sphingomyelin (SM), 16 mol% cholesterol (CL)) are diluted in 2 ml chloroform and evaporated under an argon stream. The obtained lipid film is hydrated in 900 p of hydration buffer. Lipid suspensions are subjected to... 

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