Characterization,Stability and In - Vivo Distribution of Asialofetuin Glycopeptide Incorporating DSPC/CHOL Liposomes Prepared by Mild Cholate Incubation

In this study, a small triantennary asialoglycopeptide of fetuin (A-F₂) was used as a ligand to direct liposomes to hepatocytes. A-F₂ was cleaved from asialofetuin, purified, conjugated with fatty acids and incorporated into pre-formed sonicated DSPC/Chol (2:1) liposomes. A mild cholate incubation method for incorporating the A-F₂ ligand on pre-formed vesicles was used. In preliminary in vivo experiments ¹¹In³⁺ encapsulated in A-F₂/palmityl liposomes was seen to accumulate in the liver of mice significantly faster than when encapsulated in non-ligand bearing liposomes of the same lipid composition (studied before), justifying further investigation of this system. The presence of the A-F₂/fatty acid conjugate in a functional form on the vesicle surface was confirmed by their reversible agglutination in the presence of Ricinus communis agglutinin (RCA₁₂₀). Effects of ligand incorporation on the vesicle size distribution, z-potential, membrane integrity and stability were monitored. The results demonstrate that highest ligand incorporation was achieved when liposomes and ligand were co-incubated in the presence of 1mM sodium cholate. Incorporation increased with the length of the fatty acid used for A-F₂ conjugation. Ligand-bearing liposomes were demonstrated to be smaller in diameter (about 30%) with a more positive z-potential in comparison to control vesicles while ligand incorporation did not influence the liposome membrane integrity. The size of the ligand-incorporating vesicles was maintained after 24 hours of incubation in isotonic buffer, proving that the vesicles do not aggregate. Although the preliminary biodistribution results may suggest that ligand bearing liposomes are accumulating in the liver, further cell culture, in vivo distribution and especially liver fractionation studies are required in order to clarify the intrahepatic localization of these liposomes and the ability to target liver hepatocytes in vivo.     

Dexamethasone incorporating liposomes: effect of lipid composition on drug trapping efficiency and vesicle stability

Effect of lipid composition on encapsulation and stability of dexamethasone (DXM) incorporating multilamellar vesicles (MLV) is studied. MLVs composed of phosphatidylcholine (PC) or distearoyl-glycero-PC (DSPC), with or without cholesterol (Chol), are prepared and the release of DXM during vesicle incubation in buffer or plasma proteins is evaluated. Incorporation of DXM is slightly higher in DSPC liposomes compared with PC, whereas the drug is displaced from liposomes, as the Chol content of liposome membranes increases. Plain lipid and Chol-containing liposomes lose similar fractions of vesicle-incorporated DXM during incubation in buffer or serum, whereas DXM release kinetics are similar (for each liposome type studied), implying that drug release is due mainly to dilution of liposome dispersions that leads to repartitioning of DXM.     

Dexamethasone Incorporating Liposomes: Effect of Lipid Composition on Drug Trapping Efficiency and Vesicle Stability

Effect of lipid composition on encapsulation and stability of dexamethasone (DXM) incorporating multilamellar vesicles (MLV) is studied. MLVs composed of phosphatidylcholine (PC) or distearoyl-glycero-PC (DSPC), with or without cholesterol (Chol), are prepared and the release of DXM during vesicle incubation in buffer or plasma proteins is evaluated. Incorporation of DXM is slightly higher in DSPC liposomes compared with PC, whereas the drug is displaced from liposomes, as the Chol content of liposome membranes increases. Plain lipid and Chol-containing liposomes lose similar fractions of vesicle-incorporated DXM during incubation in buffer or serum, whereas DXM release kinetics are similar (for each liposome type studied), implying that drug release is due mainly to dilution of liposome dispersions that leads to repartitioning of DXM.     

Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release.

Inclusion complexes of prednisolone (PR) with beta-cyclodextrin (beta-CD) and hydropropyl-beta-cyclodextrin (HPbeta-CD) were formed by the solvation method, and were characterized by DSC, X-ray diffractometry and FT-IR spectroscopy. PC liposomes incorporating PR as plain drug or inclusion complex were prepared using the dehydration-rehydration method and drug entrapment as well as drug release were estimated for all liposome types prepared. The highest PR entrapment value (80% of the starting material) was achieved for PC/Chol liposomes when the HPbeta-CD-PR (2:1, mol/mol) complex was entrapped. The leakage of vesicle encapsulated 5,6-carboxyfluorescein (CF) was used as a measure of the vesicle membrane integrity. As judged from our experimental results liposomes which encapsulate beta-CD-PR complexes are significantly less stable (when their membrane integrity is considered) compared to liposomes of identical lipid compositions which incorporate plain drug or even (in some cases) non-drug incorporating liposomes, which were prepared and studied for comparison. Interestingly, liposomes which encapsulate HPbeta-CD-PR complexes, have very low initial CF latency values, indicating that the leakage of CF is a process of very high initial velocity. Interactions between lipid and cyclodextrin molecules may be possibly resulting in rapid reorganization of the lipid membrane with simultaneous fast release of CF molecules. The release of PR from liposomes was highest when the drug was entrapped in the form of a complex with beta-CD. Nevertheless, the very high entrapment ability of PR in the form of HPbeta-CD-PR complexes in comparison to plain drug is a indubitable advantage of this approach.     

Stability of SUV liposomes in the presence of cholate salts and pancreatic lipases: effect of lipid composition.

The effect of bile salts (sodium cholate and sodium taurocholate), and pancreatic lipases on the structural integrity of SUV liposomes of different lipid compositions was studied. Liposomal membrane integrity was judged by bile salt or pancreatin-induced release of vesicle encapsulated 5,6-carboxyfluorescein, and vesicle size distribution before and after incubations. Bile salt concentration was 10 mM, while a saturated solution of pancreatin (mixed with equal volume of liposomes) was utilized. Results agree with earlier studies, demonstrating the instability of liposomes composed of lipids with low transition temperatures (PC and DMPC) in presence of cholates. Addition of cholesterol (1:1 lipid:chol molar ratio) does not substantially increase the encapsulated molecule retention. Nevertheless, liposomes composed of lipids with high transition temperatures (DPPC, DSPC and SM), retain significantly higher amounts of encapsulated material, under all conditions studied. Furthermore, the vesicles formed by mixing cholesterol with these lipids will possibly be sufficiently stable in the gastrointestinal tract for long periods of time. Sizing results reveal that in most cases release of encapsulated molecules is mainly caused by their leakage through holes formed on the lipid bilayer. However, in stearylamine containing DPPC and DSPC vesicles, the cholate-induced drastic decrease in vesicle size suggests total liposome disruption as the possible mechanism of encapsulated material immediate release.     

Integrity of liposomes in presence of cyclodextrins: effect of liposome type and lipid composition

Liposome stability during incubation in presence of cyclodextrins (CDs) is studied. Dried-rehydrated vesicle (DRV), multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) calcein-encapsulating liposomes, composed of different lipids are formulated, and retention of calcein is followed during vesicle incubation in hydroxypropyl-beta-CD (HP beta-CD), HP gamma-CD or methyl-beta-CD (Me beta-CD), for 24h. Results demonstrate that liposome integrity in cyclodextrins is affected by lipid composition and type. For the same lipid composition calcein release from vesicles is faster in the order: MLV > DRV > SUV. Me beta-CD influences liposome stability most, compared to the other CD's studied. Vesicles composed of saturated phospholipids were found more stable compared to phosphatidyl-choline (PC) liposomes, suggesting that phospholipid saturation and membrane rigidity influences the interaction between liposomal-lipids and CD molecules. Chol (cholesterol) addition in lipid membrane improves PC-liposome integrity, but has opposite or no effect on liposomes consisting of saturated lipids. Decrease of vesicle dispersion turbidity and size distribution in presence of CD, implies that Me beta-CD induces vesicle disruption and solubilization (to micelles). Turbidity measurements confirm that DRV liposomes are affected more than SUV.     

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides.

The paper describes a novel synthesis of cholest-5-en-3 beta-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 micrograms/ml) 84.2 +/- 5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.     

Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]

Bangham et al. (1965) created first the concept of the liposome as a microparticulate lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers. Later Gregoriadis and Ryman (1972) suggested to use liposomes as drug carrier systems. Nowadays liposomes are under extensive investigation for improving the delivery of therapeutic agents, enzymes, vaccines and genetic materials. Liposomes offer an excellent opportunity to selective targeting of drugs which is expected to optimize the pharmacokinetical parameters, the pharmacological effect and to reduce the toxicity of the encapsulated drugs. To understand the system it is important to know the basic properties of these lipoidal vesicles. Our aim was to focus on the lipid composition and the method of liposome preparation what determine the liposomal membrane fluidity, permeability, vesicle size, charge density, steric hindrance and stability of the liposomes as principle factors those influence the fate of liposomes, their interactions with the blood components and other tissues after systemic administration or local use.     

Comparative Study of Separation of Non-encapsulated Drug from Unilamellar Liposomes by Various Methods

The purpose of this study was to compare the various methods available to separate non-encapsulated drug from large unilamellar liposomes (LUV). Multilamellar liposomes (MLV) were prepared by thin film hydration using distearoylphosphatidylcholine:cholesterol (2:1 molar ratio). MLVs were passed through a 0.2-μm polycarbonate membrane using an extruder to prepare LUVs. Particle size of liposome preparations was characterized using a submicron particle-size analyser. The non-encapsulated drug was separated by: filtering through Centrifree tubes; passing through gel (Sepharose-4B and Sephadex G-25M); passing through minicolumn; ficoll density gradient; protamine aggregation; or dialysis. The dialysis method was found to be unsuitable for separation of non-encapsulated drug due to equilibration of encapsulated drug as the free drug was dialyzed. The upper limit for lipid concentration was 5 mg mL^?1 using the Centrifree method. Separation using gel chromatography led to dilution of liposome preparation. Minicolumn and density gradient techniques did not lead to sample dilution, however the minicolumn method was tedious. The time required for separation of liposomes by protamine aggregation was longer for neutral liposomes. Thus it was concluded that the Centrifree was the fastest method to estimate encapsulation; the density gradient method was ideal to separate non-encapsulated drug; and protamine aggregation was the least expensive method to estimate encapsulation efficiency.     

Mixed micelle proliposomes for preparation of liposomes containing amphotericin B, in-vitro and ex-vivo studies

The aim of this work was to produce a form of injectable liposomes containing amphotericin B derived from mixed micelle proliposomes. Mixed micelles were derived from a mixture of lecithin/sodium cholate in aqueous media. The solubility of amphotericin B in proliposomes was studied as a function of lipid composition (total lipid concentration, molar ratio of lecithin/sodium cholate), and the dispersion media (pH, ionic strength, presence or absence of human serum albumin), and the temperature. The data show that micelle-->liposome transformation occurs during the dilution of proliposomes containing amphotericin B. These transformations could be followed via transmission electron microscopy (TEM). Data related to dilution of proliposomes as well, show that under no circumstance there occurs any precipitation that might be assigned to the decreased solubility of amphotericin B. These indicate that the incorporated drug also participates during the transformation of the proliposomes into liposomes. It is thus concluded that mixed micelle proliposomes are prime candidates for the production of a form of injectable amphotericin B in liposomes.     

Liposomal drug delivery systems: an update review

The discovery of liposome or lipid vesicle emerged from self forming enclosed lipid bi-layer upon hydration; liposome drug delivery systems have played a significant role in formulation of potent drug to improve therapeutics. Recently the liposome formulations are targeted to reduce toxicity and increase accumulation at the target site. There are several new methods of liposome preparation based on lipid drug interaction and liposome disposition mechanism including the inhibition of rapid clearance of liposome by controlling particle size, charge and surface hydration. Most clinical applications of liposomal drug delivery are targeting to tissue with or without expression of target recognition molecules on lipid membrane. The liposomes are characterized with respect to physical, chemical and biological parameters. The sizing of liposome is also critical parameter which helps characterize the liposome which is usually performed by sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). This mode of drug delivery lends more safety and efficacy to administration of several classes of drugs like antiviral, antifungal, antimicrobial, vaccines, anti-tubercular drugs and gene therapeutics. Present applications of the liposomes are in the immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumour therapy. The new developments in this field are the specific binding properties of a drug-carrying liposome to a target cell such as a tumor cell and specific molecules in the body (antibodies, proteins, peptides etc.); stealth liposomes which are especially being used as carriers for hydrophilic (water soluble) anticancer drugs like doxorubicin, mitoxantrone; and bisphosphonate-liposome mediated depletion of macrophages. This review would be a help to the researchers working in the area of liposomal drug delivery.     

Interaction of Basic Drugs with Lipid Bilayers Using Liposome Electrokinetic Chromatography

No HeadingPurpose. This study explores factors influencing the interactions of positively charged drugs with liposomes using liposome electrokinetic chromatography (LEKC) for the development of LEKC as a rapid screening method for drug-membrane interactions.Methods. Liposomes were prepared and the retention factors were measured for a series of basic drugs under a variety of buffer conditions, including various buffer types, concentrations, and ionic strengths as well as using different phospholipids and liposome compositions. LEKC retention is compared with octanol-water partitioning.Results. The interaction of ionizable solutes with liposomes decreased with increasing ionic strength of the aqueous buffer. The type of buffer also influences positively charged drug partitioning into liposomes. Varying the surface charge on the liposomes by the selection of phospholipids influences the electrostatic interactions, causing an increase in retention with increasing percentages of anionic lipids in the membrane. Poor correlations are observed between LEKC retention and octanol-water partitioning.Conclusions. These studies demonstrate the overall buffer ionic strength at a given pH is more important than buffer type and concentration. The interaction of positively charged drugs with charged lipid bilayer membranes is selectively influenced by the pK_a of the drug. Liposomes are more biologically relevant in vitro models for cell membranes than octanol, and LEKC provides a unique combination of advantages for rapid screening of drug-membrane interactions.     

Intravenous Pretreatment with Empty pH Gradient Liposomes Alters the Pharmacokinetics and Toxicity of Doxorubicin through In Vivo Active Drug Encapsulation

Liposomes have been used widely to improve the therapeutic activity of pharmaceutical agents. The traditional approach for such applications has been to formulate the pharmaceutical agent in liposomes prior to administration in vivo. In this report we demonstrate that liposomes exhibiting a transmembrane pH gradient injected intravenously (iv) can actively encapsulate doxorubicin in the circulation after iv administration of free drug. Small (110 nm) liposomes composed of phosphatidylcholine (PC)/cholesterol (Chol, 55:45 mol: mol) exhibiting a pH gradient (inside acidic) were administered iv 1 h prior to free doxorubicin, and plasma drug levels as well as toxicity and efficacy were evaluated. Predosing with egg PC/Chol pH gradient liposomes increased the plasma concentration of doxorubicin as much as 200‐fold compared to free drug alone as well as to predosing with dipalmitoyl PC/Chol pH gradient liposomes or EPC/Chol liposomes without a pH gradient. The ability of the liposomes to alter the pharmacokinetics of doxorubicin was dependent on the presence of a transmembrane pH gradient and correlated with the extent of doxorubicin uptake into the liposomes at 37 °C in pH 7.5 buffer, indicating that doxorubicin was being actively accumulated in the circulating liposomes. This in vivo drug loading was achieved over a range of doxorubicin doses (5 mg/kg–40 mg/kg) and was dependent on the dose of EPC/Chol liposomes administered prior to free doxorubicin injection. The altered pharmacokinetic properties of doxorubicin associated with in vivo doxorubicin encapsulation were accompanied by a decrease in drug toxicity and maintained antitumor potency. These results suggest that pretreatment with empty liposomes exhibiting a pH gradient may provide a versatile and straightforward method for enhancing the pharmacological properties of many drugs that can accumulate into such vesicle systems at physiological temperatures.     

The Use of Fluorescence Resonance Energy Transfer to Study the Disintegration Kinetics of Liposomes Containing Lysolecithin and Oleic Acid in Rat Plasma

Purpose. To validate Fluorescence Resonance Energy Transfer (RET) as method to monitor disintegration of fluorescently labeled liposomes varying in lysolecithin/oleic acid (equimolar) content, lysolecithin fatty acid composition and vesicle size in rat blood plasma and buffer. Methods. NBD-PE and Rho-PE were used for RET. The measurements were performed on a Perkin Elmer LS-50 spectrofluorimeter. Liposomes were prepared by the extrusion method. Results. Analysis of the RET data was optimised using a fitting procedure to correct for fluorescence interference by plasma. The disintegration patterns of liposomes could be described by a bi-exponential decay model. Disintegration rate increased at increasing lysolecithin/oleic acid content and decreasing size. In contrast, all liposomes showed no disintegration in buffer. Conclusions. RET is a suitable method to monitor liposome disintegration in non-diluted plasma. Rate and extent of liposome disintegration increases at decreasing liposome size and increasing lysolecithin/oleic acid content.     

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.     

Quantitative <Emphasis Type="BoldItalic">In Vitro</Emphasis> Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4)

Purpose

In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C.

Methods

We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and ¹⁴C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed.

Results

Surprisingly, we observed an enzymatic transfer of ³H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations.

Conclusions

The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled ¹⁴C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.

     

Pre-formulation of liposomes against Helicobacter pylori: Characterization and interaction with the bacteria.

This paper deals with the formulation of targeted liposome against Helicobacter pylori. We describe the characterization of liposomes loaded with antimicrobial agents (ampicillin and metronidazole) and the quantification of the interactions between such formulations and bacteria. If the encapsulation rate of ampicillin seems not strongly affected by the change of phospholipidic composition, the encapsulation of metronidazole drastically decreased in epikuron 170 liposomes compared to DPPC ones. Furthermore, as observed with X-ray diffraction measurements, the presence of metronidazole results in the disorganisation of the phospholipid bilayers. Concerning the liposome-bacteria interactions, it has been observed that the incorporation of fucosyled glycolipids in the vesicle membrane leads to liposomes that are able to interact with the bacteria either in their spiral or in their coccoid forms. Since coccoid forms are occasionally found in vivo, their recognition by the liposomes we have formulated seems promising in the fight against Helicobacter pylori.     

Drug Release from ß-Cyclodextrin Complexes and Drug Transfer into Model Membranes Studied by Affinity Capillary Electrophoresis

Purpose

Is to characterize the drug release from the ß-cyclodextrin (ß-CD) cavity and the drug transfer into model membranes by affinity capillary electrophoresis. Phospholipid liposomes with and without cholesterol were used to mimic the natural biological membrane.

Methods

The interaction of cationic and anionic drugs with ß-CD and the interaction of the drugs with liposomes were detected separately by measuring the drug mobility in ß-CD containing buffer and liposome containing buffer; respectively. Moreover, the kinetics of drug release from ß-CD and its transfer into liposomes with or without cholesterol was studied by investigation of changes in the migration behaviours of the drugs in samples, contained drug, ß-CD and liposome, at 1:1:1 molar ratio at different time intervals; zero time, 30 min, 1, 2, 4, 6, 8, 10 and 24 h. Lipophilic drugs such as propranolol and ibuprofen were chosen for this study, because they form complexes with ß-CD.

Results

The mobility of the both drug liposome mixtures changed with time to a final state. For samples of liposomal membranes with cholesterol the final state was faster reached than without cholesterol.

Conclusions

The study confirmed that the drug release from the CD cavity and its transfer into the model membrane was more enhanced by the competitive displacement of the drug from the ß-CD cavity by cholesterol, the membrane component. The ACE method here developed can be used to optimize the drug release from CD complexes and the drug transfer into model membranes.

     

Plasma Factor Triggering Alternative Complement Pathway Activation by Liposomes

Several plasma components, such as complement (C) components, play a role in the clearance of liposomes from the circulation. The interactions between liposomes and the C system were investigated in this study. Multilamellar vesicle (MLV) liposomes, which were damaged by activation of the complement, became susceptible depending on the density of cetylmannoside (Man) on the liposome membrane, and activation proceeded through the alternative C pathway as observed for liposomes without Man (PC-MLV) (K. Funato et al, Biochim. Biophys. Acta 1103:198–204, 1992). In addition, the capacity of Man-modified liposomes (Man-MLV) to activate the alternative C pathway was abolished by preadsorption of plasma with Man-MLV but not with PC-MLV. The results suggest that a specific plasma factor adsorbed with Man-MLV was responsible for the augmentation of the C activation and, further, that the rapid clearance of Man-MLV from the circulation is caused by both enhanced C-mediated liposome permeability and enhanced C-mediated phagocytosis of liposomes.     

Interactions of Liposome Carriers with Infectious Fungal Hyphae Reveals the Role of β-Glucans

Relatively little is known about how liposomal formulations modulate drug delivery to fungal pathogens. We compared patterns of hyphal cell wall binding for empty rhodmine-labeled liposomes and the clinically available amphotericin B-containing liposomal formulation (AmBisome) in Aspergillus fumigatus and Candida albicans. Following 0.5 h of coincubation with A. fumigatus , empty liposomes concentrated primarily in fungal septae along at the surface of the cell wall, suggesting that liposome uptake is concentrated in areas of the cell wall where linear glucan is exposed on the cell surface, which was confirmed by aniline blue staining. Consistent with this hypothesis, pretreatment of liposomes with soluble linear glucan (laminarin) decreased liposome binding in both Aspergillus and Candida fungal hyphae, while growth of Aspergillus hyphae in the presence of an agent that increases fungal cell wall surface exposure of linear β-glucans without cell death (caspofungin) increased liposome uptake throughout the Aspergillus fungal cell wall. Increasing the polyethylene glycol (PEG) concentration in liposomes from 0 to 30% significantly increased fungal uptake of liposomes that was only modestly attenuated when fungal cells were incubated in serum concentrations ranging from 10 to 100%. The presence of β-glucans on the fungal hyphae cell walls of Aspergillus fumigatus is one of the factors responsible for mediating the binding of liposome carriers to the hyphae and could explain possible synergy reported between liposomal amphotericin B and echinocanins.