Hans Schreier

Liposomes and niosomes as topical drug carriers: dermal and transdermal drug delivery

Abstract A critical analysis of (trans) dermal delivery of substances encapsulated within liposomes and niosomes is presented. Topical liposomes or niosomes may serve as solubilization matrix, as a local depot for sustained release of dermally active compounds, as penetration enhancers, or as rate-limiting membrane barrier for the modulation of systemic absorption of drugs. The mechanism(s) of vesicle-skin interaction and drug delivery are being extensively investigated using radioactive- or fluorescence-labeled marker molecules and drugs, and various electron and (laser) light microscopic visualization techniques, and different models describing the interaction with and fate of vesicles in the skin have been proposed. With the current experimental data base on hand, most investigators agree that direct contact between vesicles and skin is essential for efficient delivery, although phospholipids per se apparently do not penetrate into deeper skin layers. Investigators have mostly focused on dermal corticosteroid liposome products. However, localized effects of liposome-associated proteins such as superoxide dismutase, tissue growth factors and interferons appear also to be enhanced. The delivery of liposome-encapsulated proteins and enzymes into deeper skin layers has been reported, although the mechanism of delivery remains to be elucidated. An objective assessment of the performance of topical liposome formulations vs. conventional dosage forms is frequently obscured by investigators comparing equal concentrations, rather than equivalent thermodynamic activities of their respective formulations. We conclude that liposomes and niosomes may become a useful dosage form for a variety of dermally active compounds, specifically due to their ability to modulate drug transfer and serve as nontoxic penetration enhancers.

Pulmonary delivery of liposomes

Abstract An overview of current data on pulmonary delivery of liposomes is provided, entailing fate of aerosols in the respiratory tract, physicochemical characterization of liposome aerosols, their therapeutic applications, pulmonary fate and kinetics, and pulmonary safety. Drugs that have been investigated for pulmonary delivery via liposomes include anticancer agents (ara-C), antimicrobials (enviroxime, amikacin, pentamidine), peptides (glutathione), enzymes (superoxide dismutase), antiasthmatic and antiallergic compounds (metaproterenol, salbutamol, cromolyn sodium, corticosteroids). Promising developments including pulmonary delivery of immunomodulators, antiviral agents and gene constructs (cystic fibrosis, α 1 -antitrypsin gene) are also discussed. Finally, pulmonary deposition and kinetics of drugs delivered via liposome aerosols, and targeting strategies to deliver drugs selectively to infected or impaired phagocytic (alveolar macrophages) and nonphagocytic (epithelial) cells in the lung are outlined. Based on the data on therapeutic efficacy and pulmonary safety currently available, we conclude that liposome aerosols may play an important future role in the therapy of pulmonary diseases including intracellular infections, immunologie disorders, and gene defects.

Nebulization of Liposomes. I. Effects of Lipid Composition

A series of multilamellar liposome dispersions was prepared from lipids of soy phosphatidylcholine or hydrogenated soy phosphatidylcholine containing from 0 to 30 mol% of either cholesterol, steary-lamine, or dipalmitoyl phosphatidylglycerol. The liposome dispersions were aerosolized with a Collison nebulizer for 80 min at an output flow rate of 4.7 liters of air/min. The effects of nebulization on the vesicles were determined by monitoring the release of encapsulated 5,6-carboxyfluorescein (CF) from dispersions containing ≈200 µg of total CF, of which 93.1 ± 2.4% (N = 18) was initially encapsulated. In all experiments CF was released from the liposomes while being aerosolized, and this ranged from a mean of 12.7 ± 3.8 to 60.9 ± 1.9% of the encapsulated CF, depending upon the lipid composition. The lipid concentration in the dispersions did not affect the rate or percentage release of CF over a range of ≈0.5 to 50 mg per nebulized dispersion. If liposomes are to be used as drug carriers in an inhalation aerosol a lipid composition should be employed which will minimize the release of encapsulated drug caused by nebulization.

NEBULIZATION OF LIPOSOMES. II, THE EFFECTS OF SIZE AND MODELING OF SOLUTE RELEASE PROFILES

A series of carboxyfluorescein (CF)-containing multilamellar vesicle (MLV) dispersions was prepared and extruded through polycarbonate membranes ranging in size from 0.2 to 5 µm. Vesicle dispersions were nebulized for 80 min using a Collison nebulizer, and the release of CF was monitored during nebulization. Solute retention was dependent upon the size of the vesicles and leakage ranged from 7.9 ± 0.4% (N = 3) for vesicles extruded through 0.2-µm filters to 76.8 ± 5.9% (N = 3) for liposomes that were not filtered. Solute release profiles obtained over ≥420-min nebulization periods conformed to a two-compartment kinetic model and exhibited a “fast” initial phase (kl = 0.052 ± 0.0043) followed by a “slow” terminal phase (k2 = 0.0034 ± 0.00018). The results show that CF retention can be increased by nebulizing small vesicles and modeling suggests that the rate of CF leakage from the bilayers is faster than from the core of the liposomes.

Nebulization of liposomes iii. the effects of operating conditions and local environment

Multilamellar liposomes (MLV) of saturated phosphatidylcholine and dipalmitoyl phosphatidylglycerol (DPPG) (9:1 mole ratio) containing 5,6-carboxyfluorescein (CF) were prepared and extruded through 1.0-µm polycarbonate membranes. Diluted aqueous dispersions were aerosolized for a total of 80 min using a Collison nebulizer under a variety of conditions. The effects of air pressure, temperature, buffer osmotic strength, and pH on nebulized liposome dispersions were studied. Changes in air pressure produced large changes in the percentage release of CF and ranged from 1.3% (4 psig) to 88.2% (50 psig) after 80 min of nebulization. The temperature of the nebulizer dispersions dropped during experiments. The extent of the temperature drop varied according to the air pressure used and ranged from 5°C (4 psig) to 11°C (≥30 psig). The temperature of dispersions caused no increase in CF release until the gel-to-liquid crystalline transition temperature was exceeded (54.6°C), whereupon a 20% increase in leakage was observed after 80 min of nebulization. Aerosol mass output was relatively unaffected by the starting temperature of experiments when conducted within the ambient temperature range. Leakage from the liposomes was increased in hypotonic solution but decreased in hypertonic solutions. At a buffer pH of 2.85 the percentage leakage of CF was increased ≈18% compared to that at pH 7.2 and pH 10.75. Results show that the stability of liposomes composed of saturated phosphatidylcholine and DPPG (9:1 mole ratio) is affected by the operating and environmental conditions under which aerosolization takes place, with air pressure having the greatest effect.

Pulmonary Effects of Chronic Exposure to Liposome Aerosols in Mice

Administering liposome-encapsulated drugs by aerosols could be a feasible way of targeting drugs to the lung, specifically to pulmonary alveolar macrophages (AM). In the mouse model, we characterized uptake of carboxyfluorescein- (CF-) labeled liposomes by AM in vivo after acute inhalation of liposome aerosols, and the effects of chronic exposure to liposome aerosols on lung histology and AM function. Mice were placed in a nose-only exposure module and exposed to liposome or saline aerosols for 1 h per day, 5 days per week, for 4 weeks. Five mice of both the experimental and control groups were removed weekly and their lungs examined. Liposomes were made from hydrogenated soy phosphatidylcholine (HSPC) at 50 mg/mL. In vivo uptake of liposomes by AM was documented by fluorescence microscopy and flow cytometry of bronchoalveolar lavage (BAL). A consistent amount of 1-3 micrograms of lipid inhaled per dosing per mouse was estimated from fluorescence measurements. Addition of Triton X-100 to BAL caused a significant increase in fluorescence intensity, indicating that liposomes remained intact in the lung for a period of time. The chronic inhalation study showed no histologic changes of the lung or untoward effects on the general health or survival of animals. AM phagocytic function, intracellular killing, and fatty acid composition were not affected. Transmission electron microscopy and morphometry (computerized image analysis) of AM likewise showed no alterations as a result of the treatment. It was concluded that AM uptake of liposomes delivered by aerosol was operant in vivo. This finding validates the concept of alveolar macrophage-directed delivery of liposome-encapsulated agents to the lung via inhalation. It was also concluded that chronic liposome aerosol inhalation in mice produced no untoward effects on survival, histopathology, and macrophage function. These data confirm and extend prior findings regarding the functional and morphologic interactions of liposomes with AM in vitro (Gonzalez-Rothi et al., Exp. Lung Res. 17:687-705, 1991).

Liposomes and pulmonary alveolar macrophages : functional and morphologic interactions

In vitro toxicity of liposomes and their functional and morphologic interactions with rat pulmonary alveolar macrophage (AMs) were investigated using viability (trypan blue exclusion), phagocytic and killing activity (uptake and digestion of live S. cerevisiae), surface adherence, respiratory burst (nitro-blue tetrazolium reduction), and morphometry (computerized image analysis) as indicators. Liposome stability in physiologic solutions and uptake of liposome-encapsulated carboxyfluorescein (CF) by AMs was assessed by fluorescence spectroscopy and microscopy. Liposomes made from saturated phospholipids and cholesterol were stable, whereas liposomes consisting of unsaturated phospholipids without cholesterol lost 30% to 40% of their content over 24 h. However, CF uptake was highest with unsaturated phospholipid preparations, whereas uptake of the three other formulations was comparable. Although liposome exposure did not affect macrophage viability, a reduction in the number of phagocytizing macrophages to 73% of control was noted after 24-h incubation with the highest lipid concentration tested (10 mumol/ml). Phagocytic killing was similar under all circumstances observed. The fraction of intracellularly killed yeast ranged from 32% to 42% for both control and experimental samples. An increase in cell surface area from 166.1 +/- 39.9 microns 2 on day O (n = 709) to 196.3 +/- 57.6 microns 2 on day 1 (n = 516) and 211.2 +/- 48.0 microns 2 on day 4 (n = 834) was observed after liposome treatment. The corresponding average cell areas of control samples did not change during the observation period. There was no net cell loss of adherence from monolayers as determined by protein assay. The respiratory burst, indicating generation of intracellular superoxide, was also similar--84% to 92% of experimental and control cells under all conditions showed a strong nitro-blue tetrazolium reduction. In summary, in vitro exposure of AMs to large concentrations of liposomes, although producing an increase in macrophage size, was not associated with aberrant macrophage morphologic features, function, or toxicity for the parameters examined.

The protective effect of free and membrane-bound cryoprotectants during freezing and freeze-drying of liposomes

Liposomes were prepared from natural (EPC) and hydrogenated (HEPC) egg phosphatidylcholine, with and without cholesterol (CHOL), from sucrose fatty acid ester (SPS7; sucrose-palmitate/stearate) with CHOL and dicetylphosphate (DCP) or from EPC and HEPC with the mono-, di- and tri-ester of SPS7. The cryoprotective activity of sucrose or membrane-bound sucrose fatty esters was assessed. Vesicles were frozen and thawed, or freeze-dried and reconstituted, and retention of the encapsulated marker 5,6-carboxyfluorescein (CF) was monitored. CF retentio n decreased with decreasing freezing temperature, while increasing concentrations of sucrose provided increasing cryoprotection during freezing and thawing. SPS7 vesicles were fully protected by 0.6 M sucrose, whereas equimolar mixtures of EPC and HEPC with SPS7 required 1 M sucrose for complete protection. EPC/CHOL liposomes retained maximally 85% and HEPC/CHOL liposomes 95% marker at the highest sucrose concentration. Lyophilized liposomes without sucrose or in mixture with the SPS mono- or diester retained <10% CF. Lyophilization of EPC and HEPC liposomes in the presence of 0.4 M sucrose resulted in 75% retention of originally encapsulated marker. Differential scanning calorimetry showed a significant reduction of the transition temperature (Tc) of lyophilized HEPC liposomes in the presence of sucrose and the SPS monoester. Infrared spectroscopy indicated sucrose and the SPS monoester forming strong hydrogen bonds with phosphate head groups which supports the water replacement of ‘pseudohydration’ hypothesis.

Pulmonary targeting of liposomal triamcinolone acetonide phosphate.

AbstractPurpose. To explore the use of triamcinolone acetonide phosphate liposomes as a pulmonary targeted drug delivery system. Methods. Triamcinolone acetonide phosphate liposomes composed of 1,2-distearoyl phosphatidylcholine and 1,2-distearoyl phosphatidyl glycerol and triamcinolone acetonide 21-phosphate dipotassium salt were prepared by dispersion and extruded through polycarbonate membranes. Encapsulation efficiency and in vitro stability at 37°C were assessed after size exclusion chromatography. TAP liposomes (TAP-lip) or TAP in solution (TAP-sol) were delivered to rats either by intratracheal instillation (IT) or intravenous (IV) administration. Pulmonary targeting was assessed by simultaneous monitoring of glucocorticoid receptor occupancy over time in lung (local organ) and liver (systemic organ) using an ex vivo receptor binding assay as a pharmacodynamic measure of glucocorticoid action. Results.In vitro studies in different fluids over 24 hours, showed that more than 75% of the TAP remained encapsulated in liposomes. Cumulative pulmonary effects after IT administration of TAP-lip were 1.6 times higher than liver receptor occupancy. In contrast, there was no difference in the pulmonary and hepatic receptor occupancy time profiles when TAP was administered intratracheally as a solution. No preferential lung targeting was observed when TAP-lip was administered IV. As indicated by the mean effect times, lung receptor occupancy was sustained only when TAP-lip was administered IT. Conclusions. Intratracheal administration of TAP-lip provided sustained receptor occupancy, and increased pulmonary targeting which was superior to IT administration of TAP-sol or IV administration of TAP-lip. The use of liposomes may represent a valuable approach to optimize sustained delivery of glucocorticoids to the lungs via topical administration.

Effect of Dose and Release Rate on Pulmonary Targeting of Liposomal Triamcinolone Acetonide Phosphate

AbstractPurpose. To demonstrate the importance of dose and drug release rate for pulmonary targeting of inhaled glucocorticoids using an animal model of intrapulmonary drug deposition. Methods. Liposomes composed of 1,2-distearoyl phosphatidylcholine (DSPC), 1,2-distearoyl phosphatidylglycerol (DSPG) and triamcinolone acetonide phosphate (TAP) or liposomes containing triamcinolone acetonide (TA) were prepared by a mechanical dispersion method followed by extrusion through polycarbonate membranes. Encapsulation efficiency was assessed after size exclusion gel chromatography by reverse phase HPLC. The effect of liposome size (200 nm and 800 nm) on the release kinetics of water-soluble encapsulated material was determined in vitro at 37°C using 6-carboxyfluorescein as a marker and Triton X-100 (0.03%) as a leakage inducer. To investigate the relationship between drug release and pulmonary targeting, 100 μg/kg of TAP in 800 nm liposomes was delivered to male rats by intratracheal instillation (IT) and the results compared to data for 100 μg/kg TA liposomes (recently shown to exhibit a rapid drug release under sink conditions) and to previous studies reported for an equal dose of TAP in solution and TAP in 200 nm (1). Pulmonary targeting was assessed by simultaneously monitoring glucocorticoid receptor occupancy over time in lung and liver using an ex vivo receptor binding assay as a pharmacodynamic measure of glucocorticoid action. To assess the effect of dose on pulmonary targeting experiments were performed using 2.5, 7.5, 25, 100, and 450 μg/kg of TAP in 800 nm liposomes. Results. The in vitro efflux of 6-carboxyfluorescein from (DSPC:DSPG) liposomes after exposure to Triton-X was biexponential. The terminal half-lives of 3.7 h and 9.0 h for the 200 nm and 800 nm liposomes, respectively, demonstrated that larger liposomes promote slower release of encapsulated water-soluble solute while previous results already indicated that encapsulation of lipophilic TA does not result in sustained release. Pulmonary targeting, defined as the difference between cumulative lung and liver receptor occupancies was most pronounced for the 800 nm liposomes (370%*h), followed by the 200 nm preparation (150%*h). No targeting was observed for TAP in solution (30%*h) or the rapid releasing TA liposome preparation. Correspondingly, the mean pulmonary effect time (MET) increased from 2.4−3.0 hr for TA liposomes or TAP in solution to 5.7 h and >6.2 h for TAP in 200 nm and in 800 nm liposomes, respectively. Escalating doses of TAP encapsulated in 800 nm liposomes revealed a distinct bell shaped relationship between the TAP dose and pulmonary targeting with a maximum occurring at 100 μg/kg (370%*h). Conclusions. The in vivo data presented here confirm that pulmonary residence time and dose affect the extent of lung targeting of glucocorticoids delivered via the lung.