Dehydration-rehydration Method Research Articles

Pharmacokinetics of oligodeoxynucleotides encapsulated in liposomes: effect of lipid composition and preparation method

1. The effect of the method employed to prepare liposomes and their lipid composition were evaluated in terms of the encapsulation efficiency and pharmacokinetic features of two oligodeoxynucleotides of a 21 mer: the normal (N-Odn) and the phosphorothioate (S-Odn) oligodeoxynucleotide. 2. Liposomes were prepared by the classical method of multilamellar vesicles (MV) and by the dehydration-rehydration method (DR). Two lipid mixtures were used to prepare liposomes - the predominant lipid being phosphatidylcholine (PC) and sphingomyelin (SM) respectively. 3. The DR method for liposome preparation provided the highest encapsulation e ciency, regardless of liposome lipid composition and the type of oligodeoxynucleotide involved (N-Odn or S-Odn). 4. The pharmacokinetics of free and liposome encapsulated oligodeoxynucleotides was studied in mouse following i.v. administration. Liposome encapsulated oligodeoxynucleotides exhibited a significantly lower plasma clearance and longer half-life and residence time than free oligodeoxynucleotides. The method used to obtain the liposomes affected plasma clearance, which was lower for liposomes elaborated by the DR method than for liposomes prepared with the MV method. The use of S-Odn in place of N-Odn decreased the plasma clearance of oligodeoxynucleotide when administered encapsulated in liposomes, regardless of the lipid composition and method used to obtain the liposomes.

Drugs, in cyclodextrins, in liposomes: a novel approach to the chemical stability of drugs sensitive to hydrolysis

The entrapment in multilamellar liposomes of the inclusion complexes of cyclodextrins with drugs sensitive to hydrolysis, is being examined. The anti-inflammatory drug indomethacin (IND), rapidly hydrolyzed in alkaline media, can be stabilized 75-fold when its inclusion complex with the hydroxypropylated derivative of β-cyclodextrin (HP βCD) is entrapped in multilamellar liposomes. These liposomes were composed of egg phosphatidylcholine and cholesterol of 1:1 molar ratio and were prepared according to the dehydration-rehydration method. The molar ratio for the IND:HP βCD inclusion complex was estimated to be 1:1 using the continuous variation method based on the 1H NMR data. Furthermore, the binding constant (K 11) of the IND:HP βCD complex was estimated kinetically and by 1H NMR.

Interleukin-15 acts as an immunological co-adjuvant for liposomal antigen in vivo

The recently discovered interleukin-15 (IL-15) is known to bind to the receptor of interleukin-2 (IL-2) and to share several of the latter's immunological properties. In the present study, the first to our knowledge on IL-15 behaviour in vivo, we examined the possibility that IL-15 also shares the ability of IL-2 to enhance the immunological adjuvant property of liposomes by acting as a co-adjuvant. The cytokine and a model antigen (tetanus toxoid) were either co-entrapped by the dehydration rehydration method into, or covalently co-linked by diazotization to the surface of the same liposomes, or entrapped in different liposome populations. Intramuscular immunization of CD-1 mice with a variety of IL-15 and toxoid formulations revealed that IL-15 augments anti-toxoid IgG (IgG 1, IgG 2, IgG 2b) responses well above (up to ten-fold) those achieved with liposomal toxoid alone (or with a mixture of free IL-15 and toxoid) when the cytokine and the antigen are associated with the same vesicles but not when in different vesicle populations that were mixed before injection. Higher responses were observed for all three subclasses studied only with liposomes where IL-15 and antigen were accommodated on their surface.

High-efficiency entrapment of superoxide dismutase into cationic liposomes containing synthetic aminoglycolipid.

A monofatty acid ester of glucosamine (PGlcN) was synthesized to provide liposomal membranes with a positive charge, and the trapping efficiency of negatively charged substances (superoxide dismutases, SODs) into cationic liposomes containing PGlcN or stearylamine (SA) prepared by various methods was compared to find the most efficient trapping methods. We demonstrated that cationic liposomes, which were prepared in a buffer of low ionic strength containing sorbitol by a simple hydration method, could entrap a large amount of negatively charged SODs which retained their activity, as compared with cationic liposomes prepared in a buffer of high ionic strength. We also showed a reverse-phase evaporation method entrapped a large amount of SODs. However, SODs were inactivated during the preparation; therefore, this method was not suitable to entrap the enzyme. Freeze-thaw method induced the formation of cationic liposomes which were smaller than extruded liposomes and could entrap the SODs in a buffer of low ionic strength. Dehydration-rehydration method with a buffer of low ionic strength also entrapped a large amount of SODs, indicating that the integrity of liposomes was lost in the lipid bilayer after freeze-drying and the SODs were entrapped in the reconstruction of liposomes during rehydration. These findings showed that the hydration method based on electrostatic attraction with a buffer of low ionic strength was simple and the most effective for entrapping SODs without loss of their activity.

Effects of Phospholipid Composition on Adjuvant Efficiency of Liposomes

Hampl J., J. Franz, K. Jordanova, J. Stepanek:EffectsojPhospholipidComposition on Adjuvant Efficiency oj Liposomes. Acta vet. Brno 1995, 64: 163-164. Phospholipids with various transition temperatures, including egg phosphatidyl lecithine (EPL), hydrogenated egg phosphatidyl lecithine (HEPL), dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC), and cholesterol (C), stearylamine (SA) and dicetylphosphate (DCP) were used in various molar proportions for the preparation of multilamellar liposomes by the dehydration-rehydration method. BALB/c mice were then immunized and reimmunized with BSA entrapped in the liposomes. No significant effects of transition temperatures of the phospholipids or of surface charge on the intensity of antibody responses were demonstrable. The strongest antibody responses were found in mice immunized with multilamellar liposomes containing in their structures EPL, HEPL or DMPC combined with C, DCP and/or SA, which also showed a high entrapment stability when incubated in vitro in blood serum.

Entrapment of haemoglobin into liposomes by the dehydration-rehydration method: vesicle characterization and in vivo behaviour

Haemoglobin (Hb) was isolated from human erythrocytes under conditions which maintained NADH-cytochrome-b 5 reductase activity and suppressed oxidation of Hb during storage at 4°C (methaemoglobin values < 3% after 29 days). Hb was entrapped into liposomes composed of hydrogenated egg phosphatidylcholine and equimolar cholesterol according to the dehydration/rehydration procedure of Kirby and Gregoriadis ((1984) Biotechnology 2, 979). However, encapsulation of Hb in its intact form was poor (< 5%) as a result of its oxidation and denaturation during freeze-drying. The addition of cryoprotectants and the use of both, higher initial concentrations of Hb and very small void vesicles resulted in Hb-rich dehydration/rehydration vesicles (phospholipid/Hb molar ratio of about 200:1) of the preferred size of 110 nm (mean). Highly homogeneous and small void vesicles as starting material were prepared using the one-step method of Brandl et al. ((1990) Drug Dev. Ind. Pharm. 16, 2167). The cryoprotectants were chosen with respect to their sufficient protection of Hb without affecting its loading into vesicles during freeze-drying and rehydration. 51Cr-labelling of Hb was used for the in vivo monitoring of the fate of Hb-containing vesicles rather than 125I-labelling since the latter induced strong interactions of Hb with liposomes. Upon intravenous administration into rats, liposomal 51Cr-Hb showed greater blood levels and prolonged circulation times in the blood compared to free Hb. The present approach provides high yield entrapment of labile molecules into vesicles of small size known to exhibit long circulation time.

Liposomal palmitoyl-L-asparaginase: characterization and biological activity.

A new derivative of L-asparaginase, palmitoyl-L-asparaginase (palmitoyl-L-ASNase), has been incorporated in liposomes. For this purpose we modified the dehydration-rehydration method and optimized the liposomal composition. The pharmacokinetics, toxicity, and in vivo antitumor activity against P1534 lymphoma of different liposomal palmitoyl-L-ASNase formulations were studied. Liposomal encapsulation of palmitoyl-L-ASNase as compared with free palmitoyl-L-ASNase resulted in a prolongation of the blood half-life (from 2.88 h to longer than 23.7 h), abrogation of acute toxicity, and preservation of in vivo antitumor activity.

Liposomal l-asparaginase: in vitro evaluation

The purpose of this work was the development of liposomal formulations of l-asparaginase ( l-ASNase) with the following characteristics: preservation of active enzyme, high entrapment efficiency, prolonged serum half-life and reduced toxicity compared with the free enzyme. Several liposome formulations were developed using simplified dehydration-rehydration vesicles (sDRV) or extruded vesicles (VET). The effect of lipid composition, vesicle size, ionic strength and osmolarity on enzyme encapsulation was investigated. Using a simplified dehydration-rehydration method (sDRV) we were able to achieve encapsulation efficiencies of up to 100% with full preservation (99%) of the specific activity of the encapsulated enzyme. The protein to lipid ratios of the liposomal formulations ranged from 5 to 27 μg/μmol, depending on the lipid composition. Extruded vesicles ranging from 85 to 250 nm in diameter were also tested. The encapsulation efficiency of extruded vesicles was lower than that of large vesicles and the range of preservation of specific activity was dependent on the lipid composition. Lipid combinations of phosphatidylcholine and cholesterol and either stearylamine, phosphatidylinositol or monosialoganglioside resulted in a high encapsulation efficiency (40 and 98% in VET and sDRV, respectively), high stability in saline and human serum (65–90% after 48 h) and considerable preservation of enzymatic activity (74–98%). The liposomal formulations were significantly less toxic than the free enzyme against normal CHO cells. In vivo toxicity, pharmacokinetics, biodistribution and antitumour activity studies are planned with the best formulations described in this paper.

Lipase activity in vesicular systems: Characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation.

Encapsulation of chicken egg yolk immunoglobulin G (IgY) by liposomes.

Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several microns. The encapsulation efficiency was improved by increasing the rehydration temperature to 60 degrees C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.

Efficient entrapment of amikacin and teicoplanin in liposomes.

A higher encapsulation rate was obtained using the dehydration-rehydration method compared with the reverse-phase evaporation technique in negative multilamellar vesicles with amikacin (AMK) (45% versus 15%; P < 0.05) and teicoplanin (TCP) (34% versus 25%; P < 0.05). The addition of 250 mM sucrose to AMK- or TCP-containing liposomes without prior drying prevented a significant decrease in antibiotic content in unilamellar and multilamellar vesicles over a 3-month period at -70 degrees C.

The effect of liposomal cefoperazone against Pseudomonas aeruginosa in a granulocytopenic mouse model of acute lung infection.

The therapeutic efficacy of liposomal cefoperazone against Pseudomonas aeruginosa was investigated in a granulocytopenic mouse model of acute lung infection. Granulocytopenia was induced in mice by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide. Mice were challenged by exposure to an aerosol containing P. aeruginosa and were treated i.p. with liposomal cefoperazone prepared by the dehydration-rehydration method. The half-life of free cefoperazone in the lungs following i.p. administration of the liposomal drug was significantly lengthened (13 min vs. 261 min), and the cefoperazone activity in the lungs remained above the MIC longer after administration of liposomal cefoperazone than after treatment with cefoperazone. Liposomal cefoperazone was more effective than cefoperazone alone in preventing death of granulocytopenic mice from lethal pulmonary challenge with P. aeruginosa (75% vs. 38% survival, p = 0.031). Finally, P. aeruginosa was cleared faster from the lungs of mice treated with liposomal cefoperazone when compared with those treated with cefoperazone. This study shows that incorporation of cefoperazone into liposomes enhances the activity of the antibiotic against P. aeruginosa in a granulocytopenic host.

Recombinant Human Tumor Necrosis Factor-β Entrapped in Liposomes Formed by a Modification of the Dehydration-Rehydration Method Retains Potent Cytotoxic Activity on L929 Cells In Vitro

Recombinant human tumor necrosis factor-β (rhTNF-β) may be encapsulated with high efficiency in phosphatidylcholine and distearoylphosphatidylcholine liposomes, with entrapment values of 93.4% and 92.3%, respectively, by first entrapping the substance in multilamellar vesicles using a high solute-to-phospholipid ratio followed by freeze-drying and then rehydration. The entrapped cytokine retains potent cytotoxic activity on L929 cells in vitro, causing 100% cytotoxicity, equal to that of free rhTNF-β at a concentration of about 5 × 10−8 g/ml.

Voltage-dependent InsP3- insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.

Topical delivery of liposomally encapsulated interferon evaluated by in vitro diffusion studies

The topical delivery of several liposomal interferon formulations was evaluated by in vitro diffusion experiments in an effort to understand the effects of liposomal composition and method of preparation on the deposition of interferon into the stratum corneum and deeper strata of the skin. Application of liposomes prepared from lipids with a composition similar to that of the stratum corneum resulted in almost twice the amount of interferon being deposited in the deeper skin layers than did application of liposomes prepared from phospholipids. Topical application of "skin lipid" liposomes prepared by the dehydration-rehydration method was twice as effective as was topical application of liposomes prepared by the reverse-phase evaporation method with respect to their ability to deposit interferon into the skin strata where the basal cell layers reside. These results are consistent with the effects of liposomal composition and method of preparation on the ability of the formulation to reduce lesion scores in the cutaneous herpes simplex virus type 1 guinea pig model.

Topical delivery of liposomally encapsulated interferon evaluated in a cutaneous herpes guinea pig model.

The topical delivery of liposomally encapsulated interferon was evaluated in the cutaneous herpes simplex virus guinea pig model. Application of liposomally entrapped interferon caused a reduction of lesion scores, whereas application of interferon formulated as a solution or as an emulsion was ineffective. The method of liposomal preparation rather than the lipid composition of the bilayers appeared to be the most important factor for reducing lesion scores. Only liposomes prepared by the dehydration-rehydration method were effective. This finding implied that the dehydration and subsequent rehydration of the liposomes facilitate partitioning of the interferon into liposomal bilayers, where the drug is positioned for transfer into the lipid compartment of the stratum corneum. Liposomes do not appear to function as permeation enhancers but seem to provide the needed physicochemical environment for transfer of interferon into the skin.

Covalent coupling of sugars to liposomes

A procedure is described for the covalent coupling of p- aminophenyl-α- d- mannopyranoside , in the presence of carbodiimide, to a derivative of phosphatidylethanolamine ( N-glutarylphosphatidylethanolamine) incorporated into the bilayers of multilamellar liposomes prepared by the dehydration-rehydration method. It appears that much of the phospholipid derivative expoed on the surface of the outer liposomal bilayer interacts with the aminosugar. The procedure is simple, does not destabilize liposomes and could be applied to other receptor-specific aminosugars.

Evaluation of the dehydration-rehydration method for production of contrast-carrying liposomes.

We measured the amounts of three types of radiographic contrast media (RCM) that could be entrapped in liposomes prepared by the dehydration-rehydration vesicle (DRV) technique. To make DRVs, one initially makes water-containing, small unilamellar vesicles, adds contrast media and lyophilizes the mixture. Upon rehydration, the DRVs re-form, passively entrapping RCM. Diatrizoate, iohexol and iotrolan proved to be entrappable in similar amounts (diatrizoate was best), but all of these amounts were less than for other small molecules, such as carboxyfluorescein (P less than 0.05). Entrapment was directly proportional to lipid concentration (r = 0.76; P less than 0.002), and inversely related to iodine concentration (r = 0.86; P less than 0.002). Under ideal conditions with neutral lipids, 19.45 +/- 9.9% of diatrizoate was entrapped, corresponding to 1.05 +/- 0.50 g I per g lipid. These values are close to those achievable for large unilamellar vesicles. Use of an automated mixing device (the Microfluidizer) in place of sonication, facilitated production of large liposome batches and improved entrapment (P less than 0.05). Computed tomography (CT) scans of rats showed 30 and 218 HU of liver and spleen enhancement, respectively, per g I/kg injected DRVs. These studies showed this method (possible augmented by the Microfluidizer) allows efficient production of contrast-carrying liposomes.