Preparation of liposomes containing lysosomal enzymes for therapeutic use

The preparation of fused materials using liposomes has been examined for several decades as a tool for the stabilization of heterogeneous enzymes. We investigated the liposomal encapsulation of lysosomal enzymes extracted from Saccharomyces cerevisiae. Liposomes were formed with L-α-phosphatidylcholine from egg yolk and cholesterol. To encapsulate whole lysosomal enzymes in liposomes made with and without cholesterol, L-α-phosphatidylcholine and cholesterol were added to chloroform at a ratio of 10:0 (L-α-phosphatidylcholine:cholesterol) and then evaporated for 10 min at 4°C. The residue after evaporation was mixed with lysosomal enzymes at the same ratio and then vortexed for 1 min and sonicated for 5 sec to encapsulate the enzymes. Liposome-encapsulated lysosomal enzymes were created using various amounts of lysosomal enzymes and cholesterol. The results indicated that the optimal encapsulation conditions were lipid:cholesterol ratios of 7:3 and 8:2. Liposome formation was confirmed by TEM imaging. After 1 day, two types of liposomes released small amounts of lysosomal enzymes. However, after 6 days, liposomes formed from mixtures of lipid and cholesterol did not exhibit any changes, whereas liposomes formed from only lipids released high amounts of lysosomal enzymes. Lysosomal enzymes encapsulated in liposomes have potential as important drug delivery carriers, as liposomes are able to control drug release and bioavailability.     

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.     

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-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-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 microm. 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.     

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.     

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.     

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.     

Encapsulation of Chicken Egg Yolk Immunoglobulin G (IgY) by Liposomes

Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several μm. The encapsulation efficiency was improved by increasing the rehydration temperature to 60°C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 °C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation

In this article, a hydrophobic (beclomethasone dipropionate; BDP) and a hydrophilic (cytarabine; Ara-C) drugs have been encapsulated in liposomes in order to be administered via the pulmonary route. For this aim, a liposome preparation method, which is easy to scale up, the ethanol injection method, has been selected. The effects of critical process and formulation parameters have been investigated. The drug-loaded liposomes were prepared and characterized in terms of size, zeta potential, encapsulation efficiency, release study, cell uptake, and aerodynamic behavior. Small multilamellar vesicles, with sizes ranging from about 80 to 170?nm, were successfully obtained. Results indicated a significant influence of phospholipid and cholesterol amounts on liposome size and encapsulation efficiency. The higher encapsulation efficiencies were about 100% for the hydrophobic drug (BDP) and about 16% for the hydrophilic one (Ara-C). The in vitro release study showed a prolonged release profile for BDP, in contrast with Ara-C, which was released more rapidly. The cell-uptake test revealed that fluorescent liposomes have been well internalized into the cytoplasm of SW-1573 human lung carcinoma cells, confirming the possibility to use liposomes for lung cell targeting. Nebulized Ara-C and BDP liposomes presented aerodynamic diameters compatible with deep lung deposition. In conclusion, the elaborated liposomes seem to be promising carriers for both Ara-C and BDP pulmonary delivery.     

Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

Optimization on conditions of podophyllotoxin-loaded liposomes using response surface methodology and its activity on PC3 cells

The purpose of this study was to optimize the preparation conditions of podophyllotoxin liposomes (PPT-Lips), and to investigate their effects on PC3 cells. PPT-Lips were prepared by using a thin-film dispersion method. In order to achieve maximum drug encapsulation efficiency (EE), the process and formulation variables were optimized by response surface methodology (RSM). The optimum preparation conditions were cholesterol to lecithin ratio of 3.6:40 (w/w), lipid to drug ratio of 15.8:1 (w/w), and the ultrasonic intensity of 35% (total power of 400?W). The experimental EE of PPT-Lips was 90.425%, which was consistent with the theoretically predicted value. The characterization studies showed that PPT-Lips were well-dispersible spherical particles with an average size of 106?nm and a zeta potential of –10.1?mV. A gradual and time-dependent pattern of PPT from liposomes was found in in vitro drug release with a cumulative release amount up to 70.3% in 24?h. Results of cell viability experiments on PC3 cells demonstrated that PPT-Lips exhibited more effective anticancer activity in comparison with free PPT. Therefore, PPT-Lips represent an efficient and promising drug delivery system for PPT.     

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.     

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.     

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.     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These preclinical observations, suggesting that encapsulation of DOX in PEG-coated liposomes may lead to a significant improvement of the therapeutic index of DOX, have led to the initiation of clinical trials in cancer patients.     

Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Spectrophotometric measurement of intraliposomal trehalose

Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. As mammalian cell membranes are impermeable to trehalose, extensive efforts have been taken to introduce trehalose into mammalian cells. In this study, we report on the characterization of trehalose-containing liposomes, with focus on the entrapment of trehalose inside liposomes, as the first step in establishing liposomes as a delivery system in the biopreservation field. Liposomes were synthesized by hydrating a phospholipid/cholesterol lipid bilayer with 200-400 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles. The trehalose content of the liposomal lysate was determined spectrophotometrically using a commercial kit Megazyme and confirmed with HPLC measurements. The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401+/-8 nm liposome. Based on an intraliposomal trehalose content, the calculated liposomal encapsulation efficiency of 200 mM trehalose liposomes was of 92+/-0.7%. This value was in agreement with the 300 and 400 mM trehalose liposomes (91.1+/-8.2% and 102.1+/-9.4%, respectively). The Megazyme method for trehalose measurement is an inexpensive and sensitive technique that does not require specialized instrumentation or extensive technical expertise. Therefore, it can be used to enhance current efforts in the development of alternative strategies for the cryo- and lyoprotection of mammalian cells.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-γ-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-γ-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-γ-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 μm, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH₄Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug:lipid ratio.