Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.     

Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

Background  

We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.     

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH₂-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH₂-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.     

Optimization of drug loading to improve physical stability of paclitaxel-loaded long-circulating liposomes

Abstract

The effect of formulation and process parameters on drug loading and physical stability of paclitaxel-loaded long-circulating liposomes was evaluated. The liposomes were prepared by hydration–extrusion method. The formulation parameters such as total lipid content, cholesterol content, saturated–unsaturated lipid ratio, drug–lipid ratio and process parameters such as extrusion pressure and number of extrusion cycles were studied and their impact on drug loading and physical stability was evaluated. A proportionate increase in drug loading was observed with increase in the total phospholipid content. Cholesterol content and saturated lipid content in the bilayer showed a negative influence on drug loading. The short-term stability evaluation of liposomes prepared with different drug–lipid ratios demonstrated that 1:60 as the optimum drug–lipid ratio to achieve a loading of 1–1.3?mg/mL without the risk of physical instability. The vesicle size decreased with an increase in the extrusion pressure and number of extrusion cycles, but no significant trends were observed for drug loading with changes in process pressure or number of cycles. The optimization of formulation and process parameters led to a physically stable formulation of paclitaxel-loaded long-circulating liposomes that maintain size, charge and integrity during storage.     

Arsenic trioxide liposomes: encapsulation efficiency and in vitro stability

The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Arsenic Trioxide Liposomes: Encapsulation Efficiency and In Vitro Stability

The use of arsenic‐containing compounds in cancer therapy is currently being re‐considered, after the recent approval of arsenic trioxide (Trisenox®) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy‐dispersive X‐ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37°C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO‐encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Acoustically active liposomes for drug encapsulation and ultrasound-triggered release

Acoustically active liposomes (AAL), previously developed as ultrasound contrast agents, contain small amounts of air. These AAL have potential to carry pharmaceutics and their acoustic activity could enable them to respond to ultrasound stimulation by releasing their contents. Since liposomes can entrap many kinds of drugs, if such entrapment did not affect their echogenicity, then the release of contents could potentially be controlled by ultrasound stimulation. The aim of this research was to investigate the capacity of acoustically active liposomes for hydrophilic molecule encapsulation and to determine their sensitivity to ultrasound-triggered release. Liposomes, composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and cholesterol, were made acoustically active by hydrating a lipid film, sonication, freezing in the presence of mannitol, lyophilization, and rehydration. As a test molecule, calcein was added in the hydration step. The procedure for generating acoustically active liposomes was compatible with an encapsulation efficiency of 15% or more. The presence of mannitol during freeze-drying was essential not only for generation of acoustic activity but also for efficient encapsulation. Ultrasound-triggered release was achieved by applying 1 MHz ultrasound at 2 W/cm2 for 10 s. The inclusion of 4% diheptanolyphosphatidylcholine (DHPC) increased the sensitivity of liposomes to ultrasound stimulation and resulted in very efficient stimulated release of contents (1/3 released in 10 s, 2/3 released in six such applications). Release of contents was highly correlated with the loss of air induced either by ultrasound or rapid pressure reduction. These encapsulation and triggered release techniques are highly efficient, and hence may be applicable to drug delivery.     

Liposomal hydrogel formulation for transdermal delivery of pirfenidone

Context: Pirfenidone (PFD) is an anti-fibrotic and anti-inflammatory agent indicated for the treatment of idiopathic pulmonary fibrosis (IPF). The current oral administration of PFD has several limitations including first pass metabolism and gastrointestinal irritation.

Objective: The aim of this study is to investigate the feasibility of transdermal delivery of PFD using liposomal carrier system.

Materials and methods: PFD-loaded liposomes were prepared using soy phosphatidylcholine (SPC) and sodium cholate (SC). Encapsulation efficiency (EE) of PFD in liposomes was optimized using different preparation techniques including thin film hydration (TFH) method, direct injection method (DIM) and drug encapsulation using freeze–thaw cycles. In vitro drug release study was performed using dialysis membrane method. The skin permeation studies were performed using excised porcine ear skin model in a Franz diffusion cell apparatus.

Results and discussion: The average particle size and zeta-potential of liposomes were 191?±?4.1?nm and ?40.4?±?4.5?mV, respectively. The liposomes prepared by TFH followed by 10 freeze–thaw cycles showed the greatest EE of 22.7?±?0.63%. The optimized liposome formulation was incorporated in hydroxypropyl methyl cellulose (HPMC) hydrogel containing different permeation enhancers including oleic acid (OA), isopropyl myristate (IPM) and propylene glycol (PG). PFD-loaded liposomes incorporated in hydrogel containing OA and IPM showed the greatest flux of 10.9?±?1.04?μg/cm²/h across skin, which was 5-fold greater compared with free PFD. The cumulative amount of PFD permeated was 344?±?28.8?μg/cm² with a lag time of 2.3?±?1.3?h.

Conclusion: The hydrogel formulation containing PFD-loaded liposomes can be developed as a potential transdermal delivery system.     

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH?-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH?-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.     

Ciprofloxacin as Ocular Liposomal Hydrogel

The purpose of this study was to prepare and characterize an ocular effective prolonged-release liposomal hydrogel formulation containing ciprofloxacin. Reverse-phase evaporation was used for preparation of liposomes consisting of soybean phosphatidylcholine (PC) and cholesterol (CH). The effect of PC/CH molar ratio on the percentage drug encapsulation was investigated. The effect of additives such as stearylamine (SA) or dicetyl phosphate (DP) as positive and negative charge inducers, respectively, were studied. Morphology, mean size, encapsulation efficiency, and in vitro release of ciprofloxacin from liposomes were evaluated. For hydrogel preparation, Carbopol 940 was applied. In vitro transcorneal permeation through excised albino rabbit cornea was also determined. Optimal encapsulation efficiency of 73.04 ± 3.06% was obtained from liposomes formulated with PC/CH at molar ratio of 5:3 and by increasing CH content above this limit, the encapsulation decreased. Positively charged liposomes showed superior entrapment efficiency (82.01 ± 0.52) over the negatively charged and the neutral liposomes. Hydrogel containing liposomes with lipid content PC, CH, and SA in molar ratio 5:3:1, respectively, showed the best release and transcorneal permeation with the percentage permeation of 30.6%. These results suggest that the degree of encapsulation of ciprofloxacin into liposomes and prolonged in vitro release depend on composition of the vesicles. In addition, the polymer hydrogel used in preparation ensure steady and prolonged transcorneal permeation. In conclusion, ciprofloxacin liposomal hydrogel is a suitable delivery system for improving the ocular bioavailability of ciprofloxacin.     

Charge and pH dependent drub binding to model membranes: A 2H-NMR and light absorption study

The binding of the local anesthetics tetracaine and procaine to model membranes of egg phosphatidylcholine and bovine phosphatidylserine has been studied by ²H-NMR and light absorption. Dispersions of drug-lipid mixtures in 0.1 M NaCl were centrifuged and the concentration of drug in the supernatant was measured by ultraviolet light absorption. Several freeze-thaw cycles of the sample were used before centrifugation to facilitate equilibration of the drug between the bilayers. Binding curves for the drug were obtained as a function of pH. The results were simulated by a theoretical model based on the Gouy-Chapman theory, in which both the charged and the uncharged forms of the drug, and the equilibrium between them, were included. Two deuterated forms of the drugs, [²H₆]tetracaine and [²H₄]procaine, were used for the ²H-NMR experiments. In most cases the ²H-NMR spectrum contained a broad central resonance and an underlying quadrupolar pattern. However, after five freeze-thaw cycles only a single broad resonance was observed under most conditions. Particle size measurements showed that freeze-thawing resulted in a more uniform population of liposomes of smaller average diameter than those obtained by simple vortex mixing. The single broad resonance observed in both cases is interpreted as due to rapid exchange of the anesthetic between lipid and bulk solution. In the absence of freeze-thawing, the quadrupolar pattern is attributed to anesthetic species in exchange with only a limited amount of water. The data suggest that a true equilibrium between lipid, water and anesthetic is only attained after freeze-thawing.     

ENHANCED PROTEIN LOADING INTO LIPOSOMES BY THE MULTIPLE CROSSFLOW INJECTION TECHNIQUE

ABSTRACT

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

Preparation of liposomal doxorubicin-graphene nanosheet and evaluation of its in vitro anti-cancer effects

In recent years there has been much interest in development of multifunctional drug delivery systems. In this work, liposomes that contain doxorubicin (Dox), a potent anticancer drug, and graphene nanosheets (GNS) were prepared. The GNSs have excellent optical properties, such as photoluminescence which enables tracking of the liposomes, high absorption in ultra violet region of electromagnetic spectrum which can be exploited in photodynamic and photothermal therapy, and low toxicity to mammalian cells. Nanoliposomes were prepared using the thin film hydration method. Dox and GNSs were loaded to the liposomes during the hydration of the lipid film. Liposomes were characterized and the profile of in vitro drug release, cellular uptake, and cytotoxicity of the prepared liposomes on MCF-7 cells were determined. Despite the earlier reports, the liposomes have kept their spherical structures in the presence of GNSs. The cytotoxicity of liposomal Dox and GNSs were shown to be higher than the free forms of them. Novel nanoliposomes that contain GNSs have provided a multi-functional system with the potential of tracking, photodynamic and photothermal therapy. Further improvements of this versatile nanosystem would be promising for treatment of cancer.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Synthesis,Acoustic Stability,and Pharmacologic Activities of Papaverine-Loaded Echogenic Liposomes for Ultrasound Controlled Drug Delivery

Background: development of encapsulated therapeutics that could be released upon ultrasound exposure has strong implications for enhancing drug effects at the target site. We have developed echogenic liposomes (ELIP) suitable for ultrasound imaging of blood flow and ultrasound-mediated intravascular drug release. Papaverine was chosen as the test drug because its clinical application requires high concentration in the target vascular bed but low concentration in the systemic circulation. Methods: the procedure for preparation of standard ELIP was modified by including Papaverine hydrochloride in the lipid hydration solution, followed by three freeze-thaw cycles to increase encapsulation of the drug. Sizing and encapsulation pharmacokinetics were performed using a Coulter counter and a phosphodiesterase activity assay. Stability of Papaverine-loaded ELIP (PELIP) was monitored with a clinical diagnostic ultrasound scanner equipped with a linear array transducer at a center frequency of 4.5 MHz by assessing the mean digital intensity within a region of interest over time. The stability of PELIP was compared to those of standard ELIP and Optison?. Results: relative to standard ELIP, PELIP were larger (median diameter?=?1.88?±?0.10 μm for PELIP vs 1.08?±?0.15 μm for ELIP) and had lower Mean Gray Scale Values (MGSV) (92?±?24.8 for PELIP compared to 142.3?±?10.7 for ELIP at lipid concentrations of 50 μg/ml). The maximum loading efficiency and mean encapsulated concentration were 24%?±?7% and 2.1?±?0.7 mg/ml, respectively. Papaverine retained its phosphodiesterase inhibitory activity when associated with PELIP. Furthermore, a fraction of this activity remained latent until released by dissolution of liposomal membranes with detergent. The stability of both PELIP and standard ELIP were similar, but both are greater than that of Optison?. Conclusions: our results suggest that PELIP have desirable physical, biochemical, biological, and acoustic characteristics for potential in vivo administration and ultrasound-controlled drug delivery.     

Vincristine-sulphate–loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system

Vincristine-sulfate–loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate–coated liposomes show higher cell uptake than uncoated liposomes.     

Enhanced protein loading into liposomes by the multiple crossflow injection technique

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000     

Encapsulation of Enrofloxacin in Liposomes I: Preparation and In Vitro Characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL‐α‐phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of α‐tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 µm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.