Increase In Size Of Liposomes Research Articles

Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes.

In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze-thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin-BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

Revealing cholesterol effects on PEGylated HSPC liposomes using AF4-MALS and simultaneous small- and wide-angle X-ray scattering.

Liposome development is of great interest owing to increasing requirements for efficient drug carriers. The structural features and thermal stability of such liposomes are crucial in drug transport and delivery. Reported here are the results of the structural characterization of PEGylated liposomes via small- and wide-angle X-ray scattering and an asymmetric flow field-flow fractionation (AF4) system coupled with differential refractive-index detection, multi-angle light scattering (MALS) and dynamic light scattering. This integrated analysis of the exemplar PEGylated liposome formed from hydrogenated soy phosphatid-yl-choline (HSPC) with the addition of cholesterol reveals an average hydro-dynamic radius (R h) of 52 nm with 10% polydispersity, a comparable radius of gyration (R g) and a major liposome particle mass of 118 kDa. The local bilayer structure of the liposome is found to have asymmetric electronic density profiles in the inner and outer leaflets, sandwiched by two PEGylated outer layers ca 5 nm thick. Cholesterol was found to effectively intervene in lipid chain packing, resulting in the thickening of the liposome bilayer, an increase in the area per lipid and an increase in liposome size, especially in the fluid phase of the liposome. These cholesterol effects show signs of saturation at cholesterol concentrations above ca 1:5 cholesterol:lipid molar ratio.

Storage stability of inhalable, controlled-release powder formulations of ciprofloxacin nanocrystal-containing liposomes

Novel inhalable and controlled release powder formulations of ciprofloxacin nanocrystals inside liposomes (CNL) were recently developed. In the present study, the storage stability of CNL powders consisting of lyoprotectant (i.e. sucrose or lactose), lipids, ciprofloxacin (CIP), and magnesium stearate or isoleucine was investigated. These powders were produced by spray drying, collected in a dry box at <15% relative humidity (RH), then stored at room temperature and either 4 or 20 %RH. Liposomal integrity, CIP encapsulation efficiency (EE), in vitro drug release (IVR), aerosol performance, and solid-state properties were examined over six months. Sucrose CNL powder exhibited consistent liposomal integrity, aerosol performance, and controlled release of CIP over six months of storage at 4 %RH. However, storage of the powder at 20 %RH for the same period caused sucrose crystallization and consequently a significant drop in EE and aerosol performance (p-values < 0.05), along with the IVR of CIP becoming similar to that of the non-crystalline CIP liposomal dispersions (f2 > 50). Lactose CNL maintained superior aerosol performance over the six months irrespective of the storage RH. However, liposomal instability occurred at both RHs within the first month of storage with a significant drop in EE and an increase in liposome size (p-values < 0.05). Moreover, the IVR assay of CIP from lactose CNL showed a less controlled release and a substantial difference (f2 < 50) from its initial value after six months regardless of the storage RHs. In conclusion, dry powder inhalers of CNL were physiochemically stable in sucrose lyoprotectant when stored below 4 %RH at room temperature for six months.

Development of hydrophilic gels containing coenzyme Q10-loaded liposomes: characterization, stability and rheology measurements

Objective: The aim of this study was to develop, characterize and evaluate stability of a gel containing coenzyme Q10 (Q10)-loaded liposomes, and enhance the stability of Q10 in the nanocarrier-containing gel compared to the conventional gel.Methods: Q10-loaded liposome dispersions prepared from unsaturated or saturated lecithin, were characterized for particle size, polydispersity index (PDI), zeta-potential, pH value, oxidation index, Q10-content and morphology, and incorporated into carbomer gel. Liposome gels and liposome-free gel were analyzed for flow properties, pH values, Q10-content, and liposomes size and PDI (liposome gels), 48 h after preparation and in predetermined time intervals during 6 months storage at different temperatures in order to predict their long term stability.Results: Liposomes were of small particle size, homogeneous, negatively charged, and their incorporation into gel did not significantly change (p > .05) their particle size and PDI. All gels revealed non-Newtonian, shear-thinning plastic flow behavior during storage with no marked changes in rheological parameters. Storage of gels did not significantly influence the pH value (p > .05), while it significantly decreased Q10-content (p < .05). Q10 was significantly more (p < .05) stable in liposome gel containing unsaturated lecithin liposomes (G1) than in gel containing saturated lecithin liposomes (G2) and liposome-free gel (G3).Conclusions: Q10-loaded liposome gel G1 was the optimal formulation, since during storage at different temperatures, it did not show significant increase in liposome size and PDI, it provided significantly higher stability for Q10 than other gels and its pH value was suitable for skin application. Due to limited Q10-stability it should be stored at 4 °C.

Pediocin-Loaded Nanoliposomes and Hybrid Alginate–Nanoliposome Delivery Systems for Slow Release of Pediocin

Pediocin is small antilisterial polypeptide bacteriocin. Direct addition of pediocin has been studied so far and is found to be effective only for a limited duration of time. The controlled release or slow release is an efficient method to prolong the effectiveness of pediocin and other such antimicrobials. Encapsulation is one of the numerous strategies that can be employed for achieving controlled release. Diverse wall materials such as phospholipids, proteins and carbohydrate polymers and combinations of these are used to encapsulate active ingredients. One of the aims of this study was to evaluate the effect of process parameters, viz. concentration of phospholipid (lecithin, phophatidylcholine) at the rate of 0.1, 0.2 and 0.3 % (w/v), amplitude for sonication (40, 50 and 60 %) and sonication time (3, 5 and 7 min), on size of nanoliposomes. There was an increase in liposome size with increase in concentration of phospholipids; however, there was decrease in size with increase in amplitude and duration of sonication. The main aim of the study was to compare the controlled release behaviour of pediocin loaded in delivery systems of different wall materials, viz. nanoliposomes (lecithin, phophatidylcholine), capsules of alginate plus guar gum and hybrid capsules of alginate plus guar gum incorporated with nanoliposomes. Encapsulated pediocin was found to be more effective in inhibiting bacterial growth than directly added pediocin. Hybrid capsules of alginate plus guar gum incorporated with pediocin-loaded nanoliposomes of phosphatidylcholine were found to be the best delivery system for controlled release of pediocin.

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.

Heterogeneous Liposome Membranes with pH-Triggered Permeability Enhance the in Vitro Antitumor Activity of Folate-Receptor Targeted Liposomal Doxorubicin

The killing efficacy of doxorubicin from liposome-based delivery carriers has been shown to correlate strongly with its intracellular trafficking and, in particular, its fast and extensive release from the delivery carrier. However, previously explored pH-triggered mechanisms that were designed to become activated during liposome endocytosis have also been shown to interfere with the liposome stability in vivo. We have designed pH-triggered gel-phase liposomes with heterogeneous membranes for the delivery of doxorubicin. These liposomes are triggered to form "leaky" interfacial boundaries between gel-gel phase separated domains on the membrane bilayer with lowering pH. The pH-triggered mechanism does not compromise liposome stability in vivo and results in superior in vitro killing efficacy of delivered doxorubicin when liposomes are endocytosed by a clathrin-mediated pathway. In the present work, we evaluate the general applicability of these liposomes when targeted to the folate receptor (FR) of KB cancer cells in vitro and become endocytosed by a less acidic pathway: the caveolae pathway. FR-targeting liposomes exhibit almost 50% decrease in cell association for increase in liposome size from 120 to 280 nm in diameter after relatively short incubation times (up to 4 h). The fraction of internalized vesicles, however, is approximately 60% of the cell associated vesicles independent of their size. Our findings demonstrate that, for the same doxorubicin uptake per cancer cell, the killing effect of doxorubicin delivered by pH-triggered lipid vesicles is greater (IC(50) = 0.032 mM for a 6 h incubation) than when delivered by a conventional non-pH-responsive composition (IC(50) = 0.194 mM). These findings suggest higher bioexposure of cells to the therapeutic agent possibly via faster and more extensive release from the carrier. Animal studies of FR-targeting non-pH-responsive liposomal doxorubicin report stronger therapeutic potential for the targeted approach relative to nontargeted liposomes and to free doxorubicin. The findings of the present study suggest that the targeted pH-triggered liposomes could potentially further enhance the therapeutic outcomes of doxorubicin in vivo.

Cationic Liposomes in Mixed Didodecyldimethylammonium Bromide and Dioctadecyldimethylammonium Bromide Aqueous Dispersions Studied by Differential Scanning Calorimetry, Nile Red Fluorescence, and Turbidity

The thermotropic phase behavior of cationic liposomes in mixtures of two of the most investigated liposome-forming double-chain lipids, dioctadecyldimethylammonium bromide (DODAB) and didodecyldimethylammonium bromide (DDAB), was investigated by differential scanning calorimetry (DSC), turbidity, and Nile Red fluorescence. The dispersions were investigated at 1.0 mM total surfactant concentration and varying DODAB and DDAB concentrations. The gel to liquid-crystalline phase transition temperatures (Tm) of neat DDAB and DODAB in aqueous dispersions are around 16 and 43 degrees C, respectively, and we aim to investigate the Tm behavior for mixtures of these cationic lipids. Overall, DDAB reduces the Tm of DODAB, the transition temperature depending on the DDAB content, but the Tm of DDAB is roughly independent of the DODAB concentration. Both DSC and fluorescence measurements show that, within the mixture, at room temperature (ca. 22 degrees C), the DDAB-rich liposomes are in the liquid-crystalline state, whereas the DODAB-rich liposomes are in the gel state. DSC results point to a higher affinity of DDAB for DODAB liposomes than the reverse, resulting in two populations of mixed DDAB/DODAB liposomes with distinctive phase behavior. Fluorescence measurements also show that the presence of a small amount of DODAB in DDAB-rich liposomes causes a pronounced effect in Nile Red emission, due to the increase in liposome size, as inferred from turbidity results.

Preparation and evaluation of a liposomal formulation of sodium cromoglicate

Sodium cromoglicate (SCG) is given by inhalation in prophylactic control of asthma. It was encapsulated in liposomes with a view to improve utilization of the drug when given via pulmonary route. The liposomes were characterized for encapsulation efficiency, shape, size and release rate. Liposomal dispersions were freeze-dried using a cryoprotectant. Freeze-dried liposomal dispersion retained 60% of drug upon reconstitution but increase in size of liposomes was noted. Liposomes exhibited good keeping properties when stored at 4 °C. In vivo performance of liposomal SCG was evaluated in sensitized guinea pigs. In one of the studies, differential leukocyte count and total leukocyte count in bronchoalveolar lavage fluid was measured. Liposomal dispersion showed significant inhibition of influx of neutrophils as compared with drug solution at 24 h. However, in the second study, when recovery period required by animal to revert back to normal respiratory pattern from the onset or preconvulsion time was measured, no significant difference was found between drug solution and liposomal dispersion when administered 2 h before allergen challenge.

Steam sterilisation of vesicular phospholipid gels

Vesicular phospholipid gels (VPGs), highly concentrated phospholipid dispersions of semisolid consistency and vesicular morphology are under investigation as potential implantable depots for sustained release of drugs and as intermediates for subsequent dilution into ‘conventional’ liposome dispersions. It was investigated here if VPGs can be steam sterilised. VPGs prepared from 400 mg/g egg-phosphatidylcholine by high-pressure homogenisation retained their vesicular structure but showed a slight increase in vesicle size (freeze-fracture electron microscopy). However, autoclaving slowed down both, the in vitro release of the hydrophilic marker carboxyfluorescein and vesicles from VPGs. This was assumed to be due to bigger vesicle sizes and corresponding increase in packing density of the vesicular matrix. Upon dilution into a liposome dispersion both negative staining electron microscopy and dynamic laser light scattering analysis confirmed a distinct increase in liposome size, mainly due to fusion of small (20 nm) vesicles with unfavourable curvature. This was consistent with the observed increase in encapsulation efficiency of carboxyfluorescein. Phospholipid hydrolysis during autoclaving was negligible with lysophosphatidylcholine formation of less than 2% (thin layer chromatography). Despite significant change of their morphological and functional properties during autoclaving VPGs retained their main characteristics, such as vesicular structure, sustained release and dilutability to liposome dispersions, and are, therefore, considered as autoclavable.

Poly(ethylene glycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes.

Liposome aggregation is a major problem associated with the covalent attachment of proteins to liposomes. This report describes a procedure for coupling proteins to liposomes that results in little or no change in liposome size. This is achieved by incorporating appropriate levels of poly(ethylene glycol)-modified lipids into the liposomes. The studies employed thiolated avidin-D coupled to liposomes containing the thio-reactive lipid N-(4-(p-maleimidophenyl)butyryl)dipalmitoyl phosphatidylethanolamine (1 mol % of total lipid) and various amounts of MePEG-S-POPE (monomethoxypoly(ethylene glycol) linked to phosphatidylethanolamine via a succinate linkage). The influence of PEG chain length and density was also assessed. The presence of PEG on the surface of liposomes is shown to provide an effective method of inhibiting aggregation and the corresponding increase in liposome size during the covalent coupling of avidin-D. A balance between the size of the PEG used and the amount of PEG-lipid incorporated into the liposome had to be achieved in order to maintain efficient coupling. Optimal coupling efficiencies in combination with minimal aggregation effects were achieved using 2 mol % MePEG2000-S-POPE (PEG of 2000 MW) or 0.8 mol % MePEG5000-S-POPE (PEG of 5000 MW). At these levels, the presence of PEG did not affect the biotin binding activity of the covalently attached avidin. The ability of the resulting liposomes to specifically target to biotinylated cells is demonstrated.

Encapsulation of drugs by lyophilized empty dipalmitoylphosphatidylcholine liposomes: effect of calcium ion.

The effects of divalent cation (Ca2+) on the characteristics of dipalmitoylphosphatidylcholine (DPPC) liposomes regenerated from lyophilized empty liposomes by rehydration and warming were investigated. The results showed that the volume (ml) of internal aqueous compartment per g lipid (captured volume; Vcap) has a maximum at a certain concentration range of calcium chloride and the maximal value is more than ten times the minimal value. This phenomenon can be explained by considering that binding of Ca2+ to phosphate groups in DPPC molecules induces an increase in the distance (r) between adjacent bilayer membranes in multilamellar liposomes through electrostatic force and causes an increase in Vcap. The dynamic properties of lyophilized liposomes in the rehydration process were examined using a multilamellar vesicle model. The results of simulation suggested that a repulsive force induced between the adjacent bilayer membranes causes rearrangement of the constituent lipid molecules in a liposome followed by an increase in the distance r, a decrease in the internal lamellar number, a smaller increase in liposome size and finally a significant increase in Vcap.