Soy Phosphatidylcholine Liposomes Research Articles

Hydrogenated Soy Phosphatidylcholine Liposomes Stimulate Differential Expression of Chemokines And Cytokines by Rat Alveolar Macrophages In Vitro

Liposomes are being developed as inhalable drug delivery systems, but concerns remain about their impact on the lungs. To better understand the impact of liposomes and their physicochemical properties on alveolar macrophages, the cytokine and chemokine expression profile of rat alveolar Nr8383 macrophages exposed to 0.1 and 1 mg/ml hydrogenated soy phosphatidylcholine (HSPC) liposomes was examined. Expression patterns varied considerably between liposomes in a concentration-dependent manner, with both anti- and pro-inflammatory chemokines/cytokines produced. Uncharged liposomes induce the greatest production of cytokines and chemokines, followed by PEGylated liposomes. The most significant increase in cytokine/chemokine expression was seen for IL-2 (up to 24-fold), IL-4 (up to 5-fold), IL-18 and VEGF (up to 10-fold), while liposome exposure significantly reduced MIP1 expression (5-fold). In summary, we demonstrate that liposome surface properties promote variable patterns of cytokine and chemokine secretion by alveolar macrophages. This suggests that the type of liposome employed may influence the type of immune response generated in the lung and by extension, dictate how inhaled liposomal nanomedicines affect the lungs response to inhaled toxicants and local infections.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 μg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

Ophthalmic liposomal formulations using synthetic phospholipids for the treatment of dry eye disease

AbstractPurpose: Liposomes composed of soy phosphatidylcholine (PC) have been widely used in artificial tears for the treatment of dry eye disease (DED) due to their ability to supplement the lipid layer of the ocular surface. The disadvantage of PC extracted from natural sources is the difficulty of standardizing its fatty acid composition. Therefore, the use of synthetic phospholipids such as 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and Dimyristoylphosphatidylcholine (DMPC) allows better characterization and standardization than those from natural sources, being of interest in the preparation of ophthalmic liposomal formulations for DED.Methods: Liposomal formulations comprised of DOPC (F1) and DOPC combined with DMPC (F2) were prepared according to the Bangham technique and dispersed in a borate‐trehalose buffer. Formulations were characterized in terms of pH, size, osmolarity and surface tension. In vitro tolerance was performed in human corneal epithelial cells and compared with soy PC liposomal formulation.Results: Both F1 and F2 synthetic liposomal formulations showed suitable properties for topical ophthalmic administration for DED such as hypotonicity (F1: 244.8 ± 0.74; F2: 252.5 ± 0.08 mOsm/L), neutral pH (F1: 7.52 ± 0.01; F2: 7.54 ± 0.02), low surface tension (F1: 24.53 ± 0.70; F2: 26.93 ± 0.49 mN/m) and a vesicle size of 150.8 ± 59.60 and 181.00 ± 49.83 nm for F1 and F2 respectively. Synthetic phospholipids outperformed soy PC liposomes regarding in vitro tolerance in a very sensitive corneal line, exhibiting cell viability values after 1 hour exposure of 88.71 ± 7.32% and 82.36 ± 8.84% for F1 and F2 respectively compared to soy PC liposomes (73.08 ± 8.74%). After simulated chronic exposure (4 h), cell viability of F1 (78.26 ± 1.65%) and F2 (79.08 ± 1.75%) were higher than the one obtained for PC liposomal formulation (64.53 ± 6.73%).Conclusions: Liposomes composed of synthetic phospholipids are potential candidates for the development of artificial tears for DED treatment.

The Comparison of In Vitro Photosensitizing Efficacy of Curcumin-Loaded Liposomes Following Photodynamic Therapy on Melanoma MUG-Mel2, Squamous Cell Carcinoma SCC-25, and Normal Keratinocyte HaCaT Cells.

The research focused on the investigation of curcumin encapsulated in hydrogenated soy phosphatidylcholine liposomes and its increased photoactive properties in photodynamic therapy (PDT). The goal of this study was two-fold: to emphasize the role of a natural photoactive plant-based derivative in the liposomal formulation as an easily bioavailable, alternative photosensitizer (PS) for the use in PDT of skin malignancies. Furthermore, the goal includes to prove the decreased cytotoxicity of phototoxic agents loaded in liposomes toward normal skin cells. Research was conducted on melanoma (MugMel2), squamous cell carcinoma (SCC-25), and normal human keratinocytes (HaCaT) cell lines. The assessment of viability with MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) evaluated cell death after exposure to blue light irradiation after 4 h of pre-incubation with free and encapsulated curcumin. Additionally, the wound healing assay, flow cytometry, and immunocytochemistry to detect apoptosis were performed. The malignant cells revealed increased phototoxicity after the therapy in comparison to normal cells. Moreover, liposome curcumin-based photodynamic therapy showed an increased ratio of apoptotic and necrotic cells. The study also demonstrated that nanocurcumin significantly decreased malignant cell motility following PDT treatment. Acquired results suggest that liposomal formulation of a poor soluble natural compound may improve photosensitizing properties of curcumin-mediated PDT treatment in skin cancers and reduce toxicity in normal keratinocytes.

Ascorbyl palmitate effects on the stability of curcumin-loaded soybean phosphatidylcholine liposomes

The effect of ascorbyl palmitate (AP) on the physical stability of soy phosphatidylcholine (SPC) liposomes and the retention rate of curcumin loaded into SPC liposomes enriched with different concentrations of AP was studied. Results showed that the transmittance, stability coefficient and particle size of liposomes were all influenced by the addition of AP. The highest stability of AP-stabilized liposomes was achieved when the mass ratio of SPC to AP was at 50:5 by increasing the surface charge, as indicated by an increase in ζ-potential from −15.1 mV (pure liposome) to −35.4 mV (AP-enriched SPC). Beyond this ratio, excessive AP addition was unfavorable to the stability of liposomes. Also at this 10% ratio, small angle X-ray scattering and differential scanning calorimetry results showed the strongest interaction between AP and the SPC liposomes, with the altering of the bilayer structure and the main phase transition temperature. Atomic force microscopy results showed that the addition of AP did not obviously change the vesicle microstructure but increased the vesicle height and contact angle. When curcumin was loaded into AP-enriched liposomes as a model bioactive, the best storage stability was observed in the liposome formulation with 10% added AP, which correlated well with the physical stability of the system. These results open up the possibility of AP-modified liposomes being developed as a multifunctional formulation for encapsulating and delivering nutraceuticals.

Direct Synthesis of Rhenium and Technetium-99m Metallosurfactants by a Transmetallation Reaction of Lipophilic Groups: Potential Applications in the Radiolabeling of Liposomes.

A new zinc dithiocarbamate functionalized with palmitoyl groups is described as a useful tool for the preparation of metallosurfactants through a transmetallation reaction with the transition metals rhenium and technetium. An amphiphilic rhenium complex is synthesized by a transmetallation reaction with the zinc complex in presence of the polar phosphine sodium triphenylphosphine trisulfonate, which leads to a rhenium complex with a lipophilic dithiocarbamate and a polar phosphine ligand. The study of this rhenium complex has shown that it self-aggregates, leading to the formation of aggregates that have been analyzed by dynamic light scattering and cryotransmission electron microscopy (cryo-TEM). In addition, this amphiphilic rhenium complex is incorporated into soy phosphatidylcholine liposomes, whether liposomes are prepared by mixing phospholipid and the rhenium complex or by the incorporation of the rhenium complex into preformed liposomes. The one-pot reaction of the radiocompound [99mTc(H2O)3(CO)3]+ with the above-mentioned zinc dithiocarbamate, the phosphine sodium triphenylphosphine trisulfonate and the phospholipid soy phosphatidylcholine, leads to liposomes labeled with a Tc-99m homologous complex of the rhenium complex, in accordance with the high-performance liquid chromatography (HPLC) data.

Carboxymethyl cellulose films containing nanoliposomes loaded with an angiotensin-converting enzyme inhibitory collagen hydrolysate

A collagen hydrolysate (HC) obtained from giant squid (Dosidicus gigas) tunics was encapsulated in soy phosphatidylcholine liposomes in the presence and absence of glycerol. The z-average and ζ-potential of empty and loaded liposomes ranged from 92.4 to 96.0 nm and from −30.4 to −34.5 mV, respectively. The HC was encapsulated with an entrapment efficiency of 80–83%, and the resulting liposomal dispersions showed ≈50% of angiotensin-converting enzyme (ACE) inhibitory capacity. An in vitro process of gastrointestinal digestion of the liposomes increased the antihypertensive potential, regardless of the presence or absence of glycerol. The liposomal dispersions were incorporated into carboxymethyl cellulose films, in which two ways of adding glycerol were compared: adding it directly to the film-forming dispersion or incorporating it previously into the liposomes. The presence of intact liposomes in the films, which was corroborated by transmission electronic microscopy at cryogenic temperature, decreased the water solubility and increased adhesiveness. The results demonstrated that glycerol-containing liposomes were less affected by the film drying step and also the subsequent simulated gastrointestinal digestion, with greater preservation of vesicle size and morphology, than if glycerol was added directly to the film-forming solution.

Encapsulation of antioxidant sea fennel (Crithmum maritimum) aqueous and ethanolic extracts in freeze-dried soy phosphatidylcholine liposomes

Soy phosphatidylcholine liposomes encapsulating increasing concentrations of two sea fennel extracts (aqueous and ethanolic) prepared by ultrasonication were freeze-dried, using glycerol as lyoprotectant. Particle properties, water dispersibility, colour, thermal properties and antioxidant capacity (radical scavenging capacity, ferric ion reducing power, Folin-reactive substances) of the liposomal preparations were determined. The freeze-drying process caused an overall increase in particle size and polydispersity index, while the zeta-potential became more electronegative. Both sea fennel extracts were rich in chlorogenic acid (42.61 and 58.48 mg/g for the aqueous and ethanolic extracts, respectively) and showed great antioxidant activity. Vitamin C was identified in the aqueous extract, whereas rutin and rosmarinic acid in the ethanolic one. The entrapment efficiency, determined in the liposomes prepared at the highest extract concentration, was 65.6% and 49.1% for the aqueous extract and the ethanolic extract, respectively. The liposomal antioxidant activity and total phenolic content followed a linear increasing tendency as a result of increasing the extract concentration, irrespective of the type of extract. Higher antioxidant activity was found in the liposomes loaded with the ethanolic extract, in a clear relationship to the greater amount of highly antioxidant phenolic compounds extracted, and also to their lower entrapment efficiency, which caused a greater amount of extract to remain outside the liposome. Both extracts were suitable for producing liposomes with antioxidant properties which could be dried and used to design functional foods.

Gelling properties of hake muscle with addition of freeze-thawed and freeze-dried soy phosphatidylcholine liposomes protected with trehalose

Soy phosphatidylcholine liposomes made with addition of trehalose as cryoprotectant were subjected to freeze-thawing and freeze-drying treatments, and subsequently incorporated in salt-ground hake (M. merluccius) muscle to study their effects on protein aggregation, water binding and thermal gelation. Both liposomal preparations presented similar particle size (≈215 nm, expressed as z-average) and strong electronegative zeta potential (−46 mV). The addition of both types of liposomal preparations led to more water trapped within the myofibrillar protein in the salt-ground muscle, as observed by water holding capacity (WHC) and low field nuclear magnetic resonance (LF-NMR). However, the liposomes interfered strongly with the thermal gelation ability of the muscle protein. Differential scanning calorimetry (DSC) analysis of the salt-ground muscle showed that the liposomes caused an increase in the main transition temperature associated with the actin molecule, with a concomitant reduction in total enthalpy change. The hydration state of the trehalose-containing liposomes did not play a significant role in textural properties of the resulting gels. The detrimental role of liposomes in the texture of fish gels should be considered in the design of functional fish products.

Protein aggregation, water binding and thermal gelation of salt-ground hake muscle in the presence of wet and dried soy phosphatidylcholine liposomes

Different soy phosphatidylcholine liposomal preparations (fresh, high-pressure-treated, frozen-thawed, freeze-dried and spray-dried) were incorporated in salt-ground hake (M. merluccius) muscle and their effects on protein aggregation, water binding and thermal gelation were studied. Hydrodynamic properties of liposomes varied within the range of 123–507 nm for particle size and −40 to −49.5 mV for zeta potential. Addition of liposomes to the salt-ground muscle decreased protein solubility and increased water holding capacity, regardless of the vesicle particle size or membrane surface charge. Liposomes caused an increase in protein thermal stability, as observed by DSC, and also increased the spacing between myofibrils, leading to more water trapped within the myofibrillar protein network, as revealed by the LF-NMR-1H study. The presence of liposomes slightly modified the viscoelastic behaviour and interfered with the thermal aggregation of muscle proteins, the mechanisms for this interference being different depending on the type of liposome preparation (wet or dry form). The present work suggests the possible use of a highly appreciated fish species, which could be subjected to landing obligation under Total Allowable Catch regulations (EU), for the development of a high-added-value fish product functionalized by the addition of liposomes.

Encapsulation of food waste compounds in soy phosphatidylcholine liposomes: Effect of freeze-drying, storage stability and functional aptitude

Liposomes made from soy phosphatidylcholine entrapping food waste compounds (collagen hydrolysate, L-HC; pomegranate peel extract, L-PG; and shrimp lipid extract, L-SL) were freeze-dried and stored for seven months. The freeze-drying process increased the particle size and decreased water solubility. The freeze-dried L-HC and L-PG preparations presented large multivesicular vesicles with spherical and unilamellar morphology. Large multilamellar vesicles were observed in L-SL, coinciding with greater structural changes in the membrane bilayer and increased thermal stability, as observed by ATR-FTIR and DSC. Dynamic oscillatory rheology revealed a slight hardening in the dried liposomes, induced by storage time. A sharp rigidifying effect in the temperature range from 40 to 90 °C was observed in L-SL. The loading with antioxidant compounds prevented freeze-drying-induced lipid oxidation. The storage stability of freeze-dried liposomes and their technological aptitude as a food ingredient varied depending on the chemical nature of the entrapped compounds.

Freeze-dried phosphatidylcholine liposomes encapsulating various antioxidant extracts from natural waste as functional ingredients in surimi gels

Three antioxidant extracts (collagen hydrolysate, pomegranate peel extract, shrimp lipid extract) were encapsulated in soy phosphatidylcholine liposomes with the addition of glycerol. The particle size of the fresh liposomes ranged from 75.7 to 81.0 nm and zeta potential from -64.6 to −88.2 mV. Freeze-drying increased particle size (199–283 nm), and slightly decreased zeta potential. The lyophilized liposomes were incorporated in squid surimi gels at 10.5% concentration. An alternative functional formulation was also prepared by adding 2% of non-encapsulated bioactive extract. The gels were characterized in terms of colour, texture and oxidative stability (TBARS) after processing and also after frozen storage. The incorporation of the freeze-dried liposomes caused a slight decrease in gel strength and contributed to maintaining the stability of the gels during long-term frozen storage. The antioxidant properties of the bioactive extracts, liposomes and in vitro digested surimi gels were determined.

Dynamical regimes of lipids in additivated liposomes with enhanced elasticity: A field-cycling NMR relaxometry approach

We study the molecular dynamics of lipids in binary large unilamellar liposomes suspended in D2O composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or soy phosphatidylcholine (SPC) additivated with different percentiles of sodium deoxycholate (SDC). We use the fast field-cycling proton NMR relaxometry technique over a wide timescale and at diverse temperatures. A model previously validated in different formulations is here employed for the relaxometric analysis of elastic vesicles. A new dynamical regime is observed for the first time in additivated DMPC and additivated/non-additivated SPC liposomes. This surprising feature is discussed in terms of vesicle shape fluctuations, enhanced elasticity and lipid & additive diffusion within the membrane. The continuum elastic theory is revisited for a better understanding of recent experiments and those here presented. We address the point of deformability measurements across rigid permeable barriers versus measurements of the bending elastic modulus in free-standing vesicles.

Soy PC liposomes as CLA carriers for food applications: Preparation and physicochemical characterization

Soy phosphatidylcholine (PC) liposomes added with conjugated linoleic acid isomers, (CLA, 9c, 11t, and 10t, 12c) at two PC/CLA ratios, 2:0.4 and 2:1, were prepared by ethanolic injection, and followed during 30 days of storage. These systems were investigated in encapsulation efficiency and fatty acid composition by gas chromatography, size by dynamic light scattering, morphology by TEM images, and membrane fluidity by electron paramagnetic resonance (EPR). Both type of formulations showed highly significant stability and protective effect on CLA isomers with encapsulation efficiencies over 80% during storage. Liposome sizes increased at increasing CLA content. TEM showed evidence of oligolamellar vesicles formation. Regarding membrane fluidity, two behaviors were distinguished in the bilayer: CLA at both formulations disordered the outer membrane zone increasing its fluidity, but the formulation with higher CLA content caused a decreased fluidity near the center of the membrane. The method is safe and easily scalable, and formulations are suitable for future applications in food industry.

Interactions of lipid-based liquid crystalline nanoparticles with model and cell membranes

Lipid-based liquid crystalline nanoparticles (LCNPs) are interesting candidates for drug delivery applications, for instance as solubilizing or encapsulating carriers for intravenous (i.v.) drugs. Here it is important that the carriers are safe and tolerable and do not have, e.g. hemolytic activity. In the present study we have studied LCNP particles of different compositions with respect to their mixing behavior and membrane destabilizing effects in model and cell membrane systems. Different types of non-lamellar LCNPs were studied including cubic phase nanoparticles (Cubosome ®) based on glycerol monooleate (GMO), hexagonal phase nanoparticles (Hexosome ®) based on diglycerol monooleate (DGMO) and glycerol dioleate (GDO), sponge phase nanoparticles based on DGMO/GDO/polysorbate 80 (P80) and non-lamellar nanoparticles based on soy phosphatidylcholine (SPC)/GDO. Importantly, the LCNPs based on the long-chain monoacyl lipid, GMO, were shown to display a very fast and complete lipid mixing with model membranes composed of multilamellar SPC liposomes as assessed by a fluorescence energy transfer (FRET) assay. The result correlated well with pronounced hemolytic properties observed when the GMO-based LCNPs were mixed with rat whole blood. In sharp contrast, LCNPs based on mixtures of the long-chain diacyl lipids, SPC and GDO, were found to be practically inert towards both hemolysis in rat whole blood as well as lipid mixing with SPC model membranes. The LCNP dispersions based on a mixture of long-chain monoacyl and diacyl lipids, DGMO/GDO, displayed an intermediate behavior compared to the GMO and SPC/GDO-based systems with respect to both hemolysis and lipid mixing. It is concluded that GMO-based LCNPs are unsuitable for parenteral drug delivery applications (e.g. i.v. administration) while the SPC/GDO-based LCNPs exhibit good properties with limited lipid mixing and hemolytic activity. The correlation between results from lipid mixing or FRET experiments and the in vitro hemolysis data indicates that FRET assays can be one useful screening tool for parenteral drug delivery systems. It is argued that the hemolytic potential is correlated with chemical activity of the monomers in the mixtures.

UVB induced photooxidation of vitamin E.

The photochemistry of alpha-tocopherol (alpha-TH, vitamin E) may contribute to its inhibition of UVB (290-320 nm) photocarcinogenesis. Photochemical reactions of alpha-TH were studied by monitoring the fate of alpha-TH in UVB irradiated liposomes and solution. Soy phosphatidylcholine (SPC) and dioleoylphosphatidylcholine (DOPC) liposomes were supplemented with alpha-TH (1.0 mol % alpha-TH/phospholipid) and irradiated with UVB at a dose rate of 6.0 J m-2s-1 for up to 90 min. alpha-TH was rapidly depleted in UVB irradiated liposomes. Oxidative damage, assessed by monitoring lipid peroxidation, was suppressed in SPC liposomes until alpha-TH was depleted to 20% of initial levels. alpha-TH also was rapidly depleted by UVB irradiation in acetonitrile/H2O (4:1 v/v) solution. In SPC liposomes, products previously identified as marker products for peroxyl radical scavenging by alpha-TH were observed, including alpha-tocopherol quinone, 5,6-epoxy-alpha-tocopherol quinone, and 2,3-epoxy-alpha-tocopherol quinone. These products also were formed in DOPC liposomes, which are resistant to lipid peroxyl radical formation. In addition, an alpha-tocopherol dihydroxy dimer and several 8a-(hydroperoxy)epoxytocopherones were identified by HPLC and HPLC-MS. The dimer appears to result from recombination of photoinduced tocopheroxyl radicals. Products associated with peroxyl radical scavenging (quinones, epoxyquinones, 8a-(hydroperoxy)epoxytocopherones) and with UVB dependent production of tocopheroxyl radicals (dihydroxy dimer) also were found when alpha-TH was oxidized by UVB in acetonitrile. Because the acetonitrile contained no autoxidizable substrate, formation of peroxyl radical derived products may occur via intermediate tocopherone peroxyl radicals. These results indicate that alpha-TH photooxidation proceeds via competing reactions of UVB induced tocopheroxyl radicals.

Amikacin liposomes: characterization, aerosolization, and in vitro activity against Mycobacterium avium-intracellulare in alveolar macrophages

A feasible way to treat pulmonary Myobacterium avium-intracellulare (MAI) infections is inhalation of liposome-encapsulated antimycobacterial agents aimed at infected alveolar macrophages (AM). To this end, amikacin-containing liposomes consisting of soy phosphatidylcholine (SPC), hydrogenated SPC (HSPC) and phosphatidylglycerol (PG) or cholesterol (CH) were prepared by extrusion and characterized with respect to drug content, encapsulation efficiency, drug retention in lung lavage fluid, stability during aerosolization, and in vitro efficacy against MAI in murine AM. Unencapsulated amikacin was separated by dialysis and ion-exchanged (Amberlite IRC50) adsorption which was found to be fast, complete and less cumbersome than dialysis. Drug content increased linearly with drug concentration for a fixed lipid concentration from 1 to 12% for SPC, and from 5 to 21% for SPC/PG (7:3 molar ratio) liposomes, while the amikacin content remained constant at 0.5–1% (SPC) and 1.5–2% (SPC/PG) over a lipid concentration range of 20–160 mg/ml for a fixed amikacin concentration. With increasing lipid concentration (10–160 mg/ml), the encapsulation efficiency increased linearly for SPC liposomes (2–20%), and in a saturable fashion for SPC/PG liposomes (1–35%). Aerosolization (Collison nebulizer) over 80 min resulted in loss of content of approx. 20% (SPC and HSPC) and 30–40% (SPC/PG and SPC/CH), respectively. Incubation with fresh lung lavage fluid at 37°C resulted in poor retention of amikacin in SPC/PG liposomes, whereas SPC and SPC/CH liposomes essentially retained the drug over 24 h. A dose of 20 μg/ml amikacin when encapsulated within SPC/PG liposomes was approx. 100-times efficacious against intracellular MAI in the infected alveolar macrophage model in vitro, than an equivalent concentration of free amikacin, indicating that uptake of drug-carrying liposomes by infected macrophages is operative.