Liposomal Formulation of a Cytokine Restraining Agent, (CRA)-HP 228: Encapsulation, Stability, and Release Characteristics

Cytokine restraining agents (CRAs) are a family of compounds having profound anti-inflammatory properties. Among them, HP 228 is a heptapeptide, containing some unnatural amino acids, with the unique ability to restrain the pathological effects of cytokines without interfering with their essential immune functions. A liposomal formulation of HP 228 was developed to protect the drug from proteolytic enzymes and to provide controlled release of the drug. The liposomes were prepared using either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine and cholesterol by the thin-film hydration method. Dicetyl phosphate was added to the lipids to provide negative charge to the liposomes. Homogeneous size distribution of multilamellar vesicles (MLVs) was achieved by extruding them through the 800-nm polycarbonate membranes. The size distribution was characterized using a submicrometer particle size analyzer. Encapsulation efficiency of HP 228 was determined by adding protamine to MLVs and separating the supernatant from the liposome protamine aggregate. Enzymatic stability of the drug was evaluated in phosphate-buffered saline, pH 7.0 and 37°C. The studies were conducted in the presence of trypsin and chymotrypsin. The encapsulation of HP 228 increased with the increase in fatty acid chain length. Encapsulation was further increased in negatively charged liposomes compared with neutral liposomes. The encapsulation efficiency was over 50%. Also, the negative charge helped in preventing the aggregation of the liposomes and promoted the electrostatic interaction between the lipid and the drug. Encapsulating the drug in the aqueous compartment of the liposomes completely protected HP 228 from trypsin and chymotrypsin. The rate of release of HP 228 was measured using a side-by-side diffusion chamber and was found to be drastically lower from the liposomes, as compared with the drug solution, making the formulation suitable for controlled release of HP 228.     

Effect of preparation techniques on the properties of curcumin liposomes: characterization of size, release and cytotoxicity on a squamous oral carcinoma cell line

Curcumin, a chemopreventive agent, was incorporated into liposomes using different preparation techniques and characterized for parameters such as drug loading efficiency, size, in vitro release and in vitro cytotoxicity on a squamous carcinoma cell line. Liposomes were prepared with different methods - thin layer evaporation, ethanol injection and sonication methods, respectively, obtaining, multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs). The preparation techniques influenced the size, encapsulation efficiency, in vitro release and cytotoxicity profiles. Encapsulation efficiency increased with decrease in drug to lipid ratio in the following rank order - MLVs > SUVs > ethanol injection vesicles. In vitro release and in vitro cytotoxicity were a function of the size of vesicle, which varied depending on the preparation technique. Based on these results, it can be concluded that different liposomal formulations can be employed to achieve unique in vivo needs in cancer chemotherapy.     

Effect of bovine serum albumin on the stability of methotrexate-encapsulated liposomes

The effect of bovine serum albumin (BSA) on the encapsulation efficiency and stability of liposomes containing methotrexate (MTX) having different surface charges and cholesterol contents were investigated. The encapsulation efficiency of MTX was lower and the release of MTX was faster by the addition of BSA. The leaking of MTX from lipid bilayer depends upon the BSA concentrations. These results may be derived from the interaction of BSA with lipid bilayers. The dynamic structural changes of BSA were monitored indirectly using circular dichroism spectra. Observed dynamic structural changes of BSA with liposomes are presumed to reflect the interaction of BSA with liposomes. Negatively charged liposomes have more strong interaction with BSA than neutral and positively charged liposomes. BSA attacks lipid bilayers whether it is at the inner or at the outer phase of lipid bilayer and induces leakage of entrapped MTX. Especially, negatively charged liposomes are more sensitive than others. The inclusion of cholesterol in the lipid layers inhibits the interaction of BSA with liposomes and shows protective effect against BSA-induced leakage of MTX. To endure the attacking of BSA, liposomes as drug carriers should be made using cholesterol.     

The effect of ileal brake activators on the oral bioavailability of atenolol in man

The aims of this study were to develop novel liposome formulations for tranexamic acid (TA) from various lipid compositions [neutral (hydrogenated soya phosphatidylcholine and cholesterol), positive (stearylamine) or negative (dicetyl phosphate) charged lipid], and to investigate the effects of concentrations of TA (5 and 10% in DI water) and charges on the physicochemical properties of liposomes. Liposomes were prepared by chloroform film method with sonication. The physical (appearance, pH, size, morphology) and chemical (drug encapsulation efficiency, transition temperature, enthalpy of transition) properties of liposomes were characterized. The TA contents were determined spectrophotometrically at 415 nm, following derivatization with 2,4,6-trinitrobenzosulfonic acid. The charged liposomes demonstrated better physical stability than the neutral liposomes. The percentages of TA entrapped in all liposome formulations varied between 13.2 and 15.6%, and were independent of TA concentrations and charges of liposomes. Charges affected the physical stability, pH and size of liposomes. The particle sizes of negative blank and positive liposomes (with and without the entrapped drug) were approximately 10 times larger than the negative liposome with the entrapped TA. The multilamellar 7:2:1 molar ratio of hydrogenated soy phosphatidylcholine/cholesterol/dicetyl phosphate entrapped with 10% TA liposome (10%TA,-) was selected for further release study, due to its high physical stability, small particle size and relatively high drug encapsulation efficiency.     

Encapsulation of an antivasospastic drug, fasudil, into liposomes, and in vitro stability of the fasudil-loaded liposomes.

The objectives of this work were to develop a liposomal fasudil, an antivasospastic drug, as a possible means to deliver the encapsulated drug to the brain, and to characterize the stability of the liposomal formulation in vitro. Transmembrane electrochemical gradients of H+ or ammonium sulfate were created, and their effect on the uptake of fasudil into preformed hydrogenated soy phosphatidylcholine/cholesterol (HSPC/CHOL) liposomes were examined. Fasudil was successfully loaded into preformed liposomes in response to sulfate ion (SO4(2-)) and, in part, by H+. Encapsulation levels approaching 100% could be achieved up to a drug to lipid ratio of 0.364 (mol/mol). A stability study of the fasudil-loaded liposomes was performed by storage at 4 degrees C in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffer (pH 7.4) and by incubation in human cerebrospinal fluid (CSF) at 37 degrees C. The formulations were stable with respect to drug retention as well as size alteration, for the period studied. A leakage study clearly showed the sustained release properties of the fasudil-loaded liposomes in human CSF. We recently reported that the intrathecal administration of liposomal fasudil significantly decreased ischemia, with no obvious adverse effect in a rat model [Neurol. Med. Chir. 41 (2001) 109]. Taken together, efficient encapsulation of fasudil into preformed liposomes, their long-term stability at 4 degrees C and the sustained release characteristics in CSF indicate that fasudil-loaded liposomes could be potential candidates for further clinical evaluation.     

Encapsulation,Stability and In-vitro Release Characteristics of Liposomal Formulations of Colchicine

The severe toxicity and low therapeutic index of colchicine limit its therapeutic use. Encapsulation in liposomes might reduce these toxic effects. The objective of this study was to determine the factors influencing encapsulation of colchicine in liposomes and to optimize the encapsulation parameters. Colchicine was encapsulated in multilamellar liposomes and large unilamellar liposomes prepared using various phospholipids. The effects of method of preparation, type of vesicle, charge, and concentration of cholesterol on encapsulation of colchicine in liposomes were investigated. Also, stability of colchicine under stress conditions and at various temperatures, and in-vitro release characteristics were determined. A significant difference in encapsulation of colchicine in multilamellar liposomes was observed when prepared by two different methods. Induction of charge on the liposome surface increased encapsulation of colchicine in multilamellar liposomes, but did not affect large unilamellar liposomes. The liposome preparations could withstand simulated transport conditions and frequent changes in temperature. Particle size and concentration of colchicine did not change significantly during storage at various temperatures for six months. In order to retain encapsulated colchicine in liposomes, storage at or below room temperature was found to be suitable. In-vitro release of colchicine from large unilamellar liposomes was biphasic and was influenced by two rate-limiting barriers, the dialysis membrane and the liposome bi-layers. For optimum encapsulation and stability of colchicine liposomes were prepared from a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and either stearylamine or dicetyl phosphate.     

Design and Evaluation of Liposomal Formulation of Pilocarpine Nitrate

Prolonged release drug delivery system of pilocarpine nitrate was made by optimizing thin layer film hydration method. Egg phosphatidylcholine and cholesterol were used to make multilamellar vesicles. Effects of charges over the vesicles were studied by incorporating dicetylphosphate and stearylamine. Various factors, which may affect the size, shape, encapsulation efficiency and release rate, were studied. Liposomes in the size range 0.2 to 1 µm were obtained by optimizing the process. Encapsulation efficiency of neutral, positive and negatively charged liposomes were found to be 32.5, 35.4 and 34.2 percent, respectively. In vitro drug release rate was studied on specially designed model. Biological response in terms of reduction in intraocular pressure was observed on rabbit eyes. Pilocarpine nitrate liposomes were lyophilized and stability studies were conducted.     

Radiopaque liposomes: effect of formulation conditions on encapsulation efficiency

Liposomes containing sodium ioxitalamate were prepared by sonication. Suitable amounts of purified soybean phosphatidylcholine and cholesterol were used at various molar ratios. Stearylamine or dicetylphosphate were added to this lipid composition when charged liposomes were required. After sonication and removal of unencapsulated solute, this manufacturing process yielded small multilamellar vesicles as confirmed by electron microscopy. These liposomes did not exhibit a narrow range of size distribution; the mean particle size varied from 135 to 145 nm. With respect to the efficiency of encapsulation, two parameters were distinguishable: the volume of encapsulated aqueous space per unit of lipid weight and the percentage of the contrast agent added that became encapsulated in the liposomes. Investigation of the preparative parameters revealed that increased molar ratios of cholesterol yielded higher aqueous volume and iodine contents in the liposomes, which were attributed to a reduction of the liposome permeability to the contrast agent. However, the inclusion of cholesterol into the bilayer liposomal membrane was limited, probably by solubility restrictions. Negatively and positively charged liposomes had higher rates of encapsulation than did neutral liposomes. This result was expected since efficient encapsulation of polar compounds requires formation of large aqueous spaces within the vesicles per mole of lipids. Increase of the lipid fractions at a constant, reduced the aqueous volume entrapped per millimole of lipid and, consequently, the iodine content in the liposomes. However, an increase in the initial sodium ioxitalamate concentration diminished the aqueous volume entrapped in the liposomes but increased the iodine content.     

Effect of formulation design and freeze-drying on properties of fluconazole multilamellar liposomes

Fluconazole-entrapped multilamellar liposomes were prepared using the thin-film hydration method. The effects of cholesterol molar ratio, charge-inducing agents, and α-tocopherol acetate on encapsulation efficiency values and in vitro drug release of multilamellar liposomes were studied. Freeze-dried liposomal products were prepared with or without cryoprotectants. Results showed that incorporation of stearylamine resulted in an increased entrapment of fluconazole, whereas incorporation of dicetyl phosphate decreased the drug entrapment efficiency. The incorporation of α-tocopherol acetate into fluconazole multilamellar liposomes resulted in the increase of entrapment efficiency of fluconazole liposomes. In vitro release studies revealed that incorporation of cholesterol into multilamellar liposomal formulations decreased drug permeability from formulations. Positively charged fluconazole multilamellar liposomes gave rise to a slow release rate compared to neutral liposomes whereas negatively charged fluconazole liposomes showed a rapid release rate. Physical stability studies showed that lyophilized cake of liposomes without cryoprotectants was compact and difficult to reconstitute compared to fluffy easily reconstituted cakes upon using cryoprotectants. Fluconazole retained in freeze-dried liposomes without cryoprotectants was 63.452% compared to 91.877% using three grams of trehalose as a cryoprotectant per gram lipid in positively charged multilamellar liposomes. Physical stability studies showed superior potentials of the lyophilized product after reconstitution in comparison with those of a solution product.     

Formulation and characterization of azidothymidine-loaded liposomes

Entrapment efficiency (EE%) and in vitro stability of azidothymidine (AZT)-loaded hand-shaken multilamellar vesicles (MLVs), freeze and thaw vesicles (FATMLVs), and reverse phase evaporation vesicles (REVs) were compared. AZT entrapment in FATMLVs was further studied by varying initial lipid concentrations, drug concentration, and lipid composition. The results suggest that AZT entrapment is dependent on the aqueous volume entrapped within liposomes, and the interaction between the drug and liposomal bilayer may not be significant. Increasing the lipid concentration increases the liposomal entrapment of AZT but the encapsulation yield decreases above a lipid concentration of 30 μmol/mL. No significant difference was observed in EE% when the AZT concentration was varied from 5 to 20 mg/mL. The entrapment efficiency was highest (43.2%) for DSPC/CHOL/PS (molar ratio 6:3:3) vesicles but DSPC/CHOL/PS liposome formulations in a molar ratio of 4:3:3 or 4:5:1 and DSPC/CHOL/SA liposome formulations in a molar ratio of 4:5:1 were found to be more stable in vitro. In vitro drug release from liposomes was dependent on bilayer composition and the method of preparation.     

Factors influencing the encapsulation of thioguanine in DRV liposomes

Thioguanine, a chemotherapeutic drug employed in the treatment of leukemia, is, like other amphiphilic drugs, very permeant and presents a high ability to escape from the liposomal membrane. For this reason, it is very important to know which parameters can enhance the relatively low encapsulation of thioguanine. In this way, the influence of four factors (pH, content of cholesterol, charge of lipids, and time of sonication) on the encapsulation of such drug in dehydration-rehydration liposomes was studied using a 2⁴ factorial design. In this study, the maximal encapsulation efficiency obtained was 14.07 mmol/mol of lipid and time of sonication was the unique factor whose influence on the encapsulation was statistically significant (P < 0.05). The energy input concomitant to higher periods of sonication seems to facilitate the entrapment of the drug into the bilayer. Among the two-way interactions, time charge and pH-charge presented levels of significance less than 0.05. In liposomes with negative charge (10% phosphatidic acid), time of sonication barely influenced on the encapsulation, but in stearylamine-containing liposomes, higher times of sonication were necessary to achieve a better yield of encapsulation. More complex was the effect of the pH-charge interaction. At pH 4.7, liposomes with phosphatidic acid favoured the encapsulation, while at pH 7.4, liposomes with stearylamine encapsulated more drug. The different extent of encapsulation in the function of pH and charge only could be explained by the different charge born by the positive and negative liposomes depending on the pH.     

去氢骆驼蓬碱脂质体的制备和体外释放特性

目的：研究去氢骆驼蓬碱（harmine,HM）脂质体的制备工艺和体外释放特性。方法：运用薄膜分散-pH值梯度法制备HM．脂质体以及高速离心法分离脂质体与游离药物,并测定其包封率;借助综合评分法,评价其粒径、多分散系数、包封率、载药量指标;运用正交优化实验法考察磷脂-胆固醇与药-脂比、超声时间、外相pH值对脂质体的影响,述选最优工艺处方,评价脂质体与原料药的体外释放情况。结果：最优处方因素为磷脂-胆固醇比值为4：1,超声时间为300S,药-脂比值为1：5,外相pH值为6,8,即X13X23X32X43,经实验验证其粒径为（155.0±14．5）nm,多分散系数为（0．148±0．011）,包封率为（80．90±0．01）％,载药量为（11．16±0．01）％;其原料药0．5h累积释放百分比大于50％,不到2h已全部释放,而优化后的脂质体在1h内其累积释放百分比大于50％,4h后释放完成。结论：采用薄膜分散-pH值梯度法,以最优处方制得HM-脂质体,其粒径大小适中、形态均匀,包封率和载药量相对较高,体外释放显示具有较好的缓释特性。     

Preparation and properties of poly(vinyl alcohol)-stabilized liposomes

The purpose of this work is to evaluate the improvement in physical stability of poly(vinyl alcohol) (PVA) modified liposomes. Liposomes composed of soya phosphatidylcholile (SPC) and cholesterol (1:1 molar ratio) were prepared by reverse phase evaporation method. Two types of interaction between liposome and PVA were investigated: PVA addition into lipid bilayer during liposome preparation and coating of already formed liposomes with PVA. The microparticles system was morphologically characterized by transmission electron microscopy (TEM) and particles analysis. Changes in particles size and zeta potential confirmed the existence of a thick polymer layer on the surface of liposomes. The amount of PVA adsorbing to liposomes and the encapsulation efficiency increased with increasing polymer concentration. The physical stability was evaluated by measuring the release rate of contents at 20 and 37 degrees C, the PVA modified liposomes were more stable than the conventional liposomes. Comparing with PVA-coated liposomes, the liposomes with PVA addition to the bilayer were more stable, and had higher entrapment efficiency.     

Pilocarpine hydrochloride liposomes: characterization in vitro and preliminary evaluation in vivo in rabbit eye

Liposomes containing either pilocarpine hydrochloride or pilocarpine free base were prepared by the sonication method. This manufacturing process yielded after removal of non-encapsulated solute, small multilamellar vesicles (MLV) as was confirmed by electron microscopy examinations. For an identical liposomal composition, the encapsulation capacity and the drug content of the liposomes were drastically higher for pilocarpine hydrochloride than for pilocarpine free base. Investigation of the preparative parameters revealed that increasing the initial amount of drug decreased the drug content and the encapsulation efficiency of the liposomes formed. Since fixed amounts of lipids were used, the volume sequestration rate decrease was attributed to a moderate viscosity increase of the dispersion medium. Increase of phospholipid concentration at a constant ratio of cholesterol and dicetylphosphate to phosphatidylcholine reduced the aqueous volume entrapped per mg of lipid and subsequently the pilocarpine content in the liposomes. Negatively charged liposomes gave larger rates of pilocarpine hydrochloride and aqueous volume encapsulation than neutral liposomes but, on the contrary, positively charged liposomes gave the lowest rates of pilocarpine hydrochloride and aqueous volume encapsulation. Thus, for drug carrying the same net charge as the phospholipids an increase in the surface charge density of the liposome was not only ineffective, but actually resulted in a lower drug encapsulation due to electrostatic repulsion. Preliminary in vivo results on rabbit eyes suggested that the liposomal vehicle was probably unable to improve sufficiently the corneal penetration of pilocarpine to reach satisfactory therapeutic levels when administered at lower concentrations than commonly used.     

Preparation and physicochemical characterization of atovaquone‐containing liposomes

This work describes the preparation and the physicochemical properties of atovaquone‐loaded liposomes. It also describes drug release from the liposomes. As many factors can influence liposome stability, we studied several formulations, including different concentrations of atovaquone, phospholipids, and cholesterol. The effect of atovaquone (ATV) concentration was also evaluated. The highest binding percentage (100±2.5%) was obtained under alkaline conditions for a 2 mg/ml concentration of ATV. The percentage of encapsulation decreased significantly when drug concentrations increased. Drug uptake (expressed per unit mass of phospholipids) was nonlinearly related to equilibrium ATV concentration. A Langmuir‐type sorption was suggested (r = 0.978). In acidic or neutral buffer, the binding percentage reached 42.1 ± 0.02%. The increase of phospholipids and cholesterol concentrations did not significantly improve the ATV binding yield for the lowest ATV concentration. Conversely, ATV binding was significantly increased for the highest ATV concentration. All the formulations tested gave monodispersed liposomes with a mean diameter around 260 nm. The dilution (1/5–1/20) of liposomes in alkaline conditions induced a significant release of ATV (74% release). In acidic or neutral buffer no release was observed, suggesting an encapsulation process. Drug Dev. Res. 47:155–161, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of preparation technique on the properties of liposomes encapsulating ketoprofen-cyclodextrin complexes aimed for transdermal delivery

The combined approach of cyclodextrin complexation and entrapment in liposomes was investigated in order to develop an effective topical formulation of ketoprofen. Equimolar complex of drug and hydroxypropyl-beta-cyclodextrin (HPbetaCyd) was added at different concentrations to the aqueous phase of liposomes consisting of phosphatidylcholine and cholesterol (60%/40%, w/w). Liposomes were prepared with different techniques, such as thin layer evaporation, freezing and thawing, extrusion through microporous membrane, and reverse phase evaporation method, obtaining, respectively, multi-lamellar vesicles (MLV), frozen and thawed MLV (FATMLV), small uni-lamellar vesicles (SUV) and large uni-lamellar vesicles (LUV). Size and morphology of the different types of liposomes were investigated by light scattering analysis, transmission electron microscopy, and confocal laser scanning microscopy, whereas drug entrapment efficiency was determined by dialysis experiments. Cyclodextrin complexation improved drug solubilization and allowed a strong improvement of its entrapment into the aqueous liposomal phase. Liposome preparation method and operating conditions clearly affected both liposome size and drug loading capacity. Encapsulation efficiency increased with increasing the complex concentration up to 10 mM, and was in the order MLV>LUV>SUV. An opposite behaviour was observed for FATMLV, probably due to the freezing phase required by such a preparation method, which reduced the complex solubility. Moreover, it was not possible to use higher complex concentrations, due to the destabilizing effect of cyclodextrins toward the liposomal membrane. Permeability studies of drug-HPbetaCyd complexes, directly in solution or incorporated in liposomes, performed across artificial membranes simulating the skin behaviour, highlighted, as expected, a prolonged release effect of liposomal formulations. Furthermore, the drug permeation rate depended on the vesicle characteristics and varied in the order: SUV>MLV=FATMLV>LUV. Therefore, the most suitable liposome preparation method can be suitably selected on the basis of drug encapsulation efficiency and/or desired drug release rate.     

Interaction of colistin and colistin methanesulfonate with liposomes: colloidal aspects and implications for formulation

Interaction of colistin and colistin methanesulfonate (CMS) with liposomes has been studied with the view to understanding the limitations to the use of liposomes as a more effective delivery system for pulmonary inhalation of this important class of antibiotic. Thus, in this study, liposomes containing colistin or CMS were prepared and characterized with respect to colloidal behavior and drug encapsulation and release. Association of anionic CMS with liposomes induced negative charge on the particles. However, degradation of the CMS to form cationic colistin over time was directly correlated with charge reversal and particle aggregation. The rate of degradation of CMS was significantly more rapid when associated with the liposome bilayer than when compared with the same concentration in aqueous solution. Colistin liposomes carried positive charge and were stable. Encapsulation efficiency for colistin was approximately 50%, decreasing with increasing concentration of colistin. Colistin was rapidly released from liposomes on dilution. Although the studies indicate limited utility of colistin or CMS liposomes for long duration controlled-release applications, colistin liposomes were highly stable and may present a potential opportunity for coformulation of colistin with a second antibiotic to colocalize the two drugs after pulmonary delivery.     

5-Fluorouracil in topical liposome gels for anticancer treatment--formulation and evaluation

Liposome gels bearing an antineoplastic agent, 5-fluorouracil, intended for topical application have been prepared and drug release properties in vitro have been evaluated. Different formulations of liposomes were prepared by the film hydration method by varying the lipid phase composition (PL 90H/cholesterol mass ratio) and hydration conditions of dry lipid film (drug/aqueous phase mass ratio). Topical liposome gels were prepared by incorporation of lyophilized liposomes into a structured vehicle (1%, m/m, chitosan gel base). Also, hydrogels containing different concentrations of 5-fluorouracil were prepared and drug release properties were investigated. The rate of drug release from liposome gels was found to be dependent on the bilayer composition and the dry lipid film hydration conditions. Also, liposomes embedded into a structured vehicle of chitosan showed significantly slower release than hydrogels. The drug release obeyed the Higuchi diffusion model, while liposomes acted as reservoir systems for continuous delivery of the encapsulated drug.     

A novel method to prepare liposomes containing amikacin.

This work describes a novel method to prepare liposomal amikacin composed of soyabean lecithin and cholesterol; these were also prepared using two other methods (cast film method and proliposome method). Encapsulation efficiency was evaluated. Liposomes prepared by the new method, which combines the method of preparing proliposomes with freeze-drying, had the highest encapsulation efficiency. The influence of drug to lipid ratio on the encapsulation efficiency was investigated. The in vitro efflux of amikacin from liposomes with different lecithin: cholesterol ratios was also investigated.     

Liposome formulation of poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 micromol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 micromol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 micromol:4 micromol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 micromol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome.