6-Mercaptopurine and Daunorubicin Double Drug Liposomes—Preparation,Drug-Drug Interaction and Characterization

This article addresses and investigates the dual incorporation of daunorubicin (DR) and 6-mercaptopurine (6-MP) in liposomes for better chemotherapy. These drugs are potential candidates for interaction due to the quinone (H acceptor) and hydroxyl (H donor) groups on DR and 6-MP, respectively. Interactions between the two drugs in solution were monitored by UV/Vis and fluorescence spectroscopy. Interaction between the two drugs inside the liposomes was evaluated by HPLC (for 6-MP) and by fluorescence spectroscopy (for daunorubicin) after phospholipase-mediated liposome lysis. Our results provide evidence for the lack of interaction between the two drugs in solution and in liposomes. The entrapment efficiencies of 6-MP in the neutral Phosphatidyl choline (PC):Cholesterol (Chol):: 2:1 and anionic PC:Chol:Cardiolipin (CL) :: 4:5:1 single and double drug liposomes were found to be 0.4% and 1.5% (on average), respectively. The entrapment efficiencies of DR in the neutral and anionic double drug liposomes were found to be 55% and 31%, respectively. The corresponding entrapment of daunorubicin in the single drug liposomes was found to be 62% on average. Our thin layer chromatography (TLC) and transmission electron microscopy (TEM) results suggest stability of lipid and liposomes, thus pointing plausible existence of double drug liposomes. Cytotoxicity experiments were performed by using both single drug and double drug liposomes. By comparing the results of phase contrast and fluorescence microscopy, it was observed that the double drug liposomes were internalized in the jurkat and Hut78 (highly resistant cell line) leukemia cells as viewed by the fluorescence of daunorubicin. The cytotoxicity was dose dependent and had shown a synergistic effect when double drug liposome was used.     

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.     

Preparation and mucoadhesive test of CSA-loaded liposomes with different characteristics for the intestinal lymphatic delivery

Drug delivery to the lymphatic system may be important in terms of the treatment with lymphatic involvement, such as tumor metastases and immunization. Especially, drug transport via the intestinal lymphatics after oral administration has been attracted lots of interests. The purpose of this study was to prepare cyclosporin A (CSA)-loaded liposomes, with different characteristics, and evaluate their ucoadhesivity. Three liposome preparations were formulated: cationic stearylamine liposomes (SA-Lip), anionic phosphatidylserine liposomes (PS-Lip), polymer (chitosan)-coated liposomes (CS-Lip), and characterized. The liposome preparations were found to be spherical in shape, with PS-Lip being the smallest. The liposome preparations exhibited entrapment efficiencies in the order: PS-Lip (52.5±2.9%)>SA-Lip (48.8±3.3%)> CS-Lip (41.7±4.2%). Finally, mucoadhesive tests were carried out using rat intestine, with SA-Lip (67%) showing the best adhesive rate of the three preparations (PS-Lip: 56%, CS-Lip: 61%). These results suggest that a positive charge on the surface of drug carriers may be an important factor for the intestinal drug delivery.     

脂质体包埋猪血红蛋白的制备及其注入小鼠体内的初步试验

 为探索一条研制猪血红蛋白 (pHb)为基础的血液代用品新途径 ,开发了干膜超声法将猪血红蛋白和别构效应剂、超氧化物歧化酶等联合包埋于脂质体的技术 .考察了氢化大豆卵磷脂、二肉豆蔻酰磷脂酰胆碱、胆固醇、二硬脂酰磷脂酰乙醇胺 甲氧基聚乙二醇和维生素E等在制备脂质体包埋血红蛋白 (LEH)中的作用 .通过控制磷脂与其他成分的配比 ,制得包埋率为 10 3%,pHb浓度达 16 %的稳定的LEH ;进一步将pHb微囊通过小鼠尾静脉多次注入其体内 ,检测受试小鼠血液中红细胞和白细胞数、抗体滴度、血小板聚集率及肾脏组织学等方面的变化 .小鼠体内试验表明了所制备的LEH具有低免疫原性、对肾脏无明显损害等特点 .脂质体包埋pHb是一种极具开发前景的稳定的人工载氧系统     

Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis

The present study aimed to evaluate the potential of liposomes loaded with paromomycin (PA), an aminoglycoside antibiotic associated with poor skin penetration, for the topical treatment of cutaneous leishmaniasis (CL). Fluid liposomes were prepared and characterized for particle size, zeta potential, and drug entrapment. Permeation studies were performed with two in vitro models: intact and stripped skin. The antileishmanial activity of free and liposomal PA was evaluated in BALB/c mice infected by Leishmania (L.) major. Drug entrapment ranged from 10 to 14%, and the type of vesicle had little influence on this parameter. Particle size and polydispersity index of the vesicles composed by phosphatidylcholine (PC) and PC/cholesterol (Chol) ranged from of 516 to 362?nm and 0.7 to 0.4, respectively. PA permeation across intact skin was low, regardless of the formulation tested, while drug penetration into skin (percent of the applied dose) from PC (7.2?±?0.2%) and PC/Chol (4.8?±?0.2%) liposomes was higher than solution (1.9?±?0.1%). PA-loaded liposomes enhanced in vitro drug permeation across stripped skin and improved the in vivo antileishmanial activity in experimentally infected mice. Our findings suggest that the liposomes represent a promising alternative for the topical treatment of CL using PA.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

A comparison of the in vitro antimicrobial activity of liposomes containing meropenem and gentamicin

The antimicrobial activity of eight cationic, two neutral and three anionic liposome compositions containing meropenem and gentamicin was tested in vitro in broth and serum medium. The cationic formulations showed better antibacterial efficacy against both Gram-positive and Gram-negative bacteria than the anionic and neutral ones, regardless of the encapsulated drug. The most effective formulations were the cationic PC/DOPE/DOTAP 3:4:3 and PC/Chol/DOTAP 3:4:3, as the MICs with meropenem were 2 to 4 times lower than those of the free drug.     

Synthesis and characterization of betaine-like diacyl lipids: zwitterionic lipids with the cationic amine at the bilayer interface

We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO(4)(-)) over less polarizable ions (Cl(-)), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol (Chol), these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37°C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.     

Preparation of long-circulating immunoliposomes using PEG-cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics.

Poly(ethylene glycol)-coated liposomes were prepared with two new synthesised pegylated cholesterol (Chol) derivatives linked via carbamate bond. Poly(ethylene glycol) (PEG) was directly linked to Chol (PEG-Chol) or through a space arm of diaminebutane (PEG-L-Chol). In buffer, the physicochemical properties of PC/Chol liposomes (2/1, molar ratio) containing up to 10 mol% of pegylated Chol derivatives did not change significantly and the PEG layer at liposome surface inhibited the agglutination of biotin-liposomes induced by streptavidin. On the other hand, in serum, PEG-L-Chol seemed to reduce the interactions of liposomes with serum proteins, much more than PEG-Chol. The low steric hindrance of PEG-Chol derivative may be due to the slow conformational transition rate of the polymer, since PEG may be deeper located in the membrane. The coupling efficiency of the ligand to the functionalised amino group at the polymer end was also affected, but, its antigen-binding activity was preserved. The basic physical-chemical characteristics studied in this work are relevant to assess the application of pegylated Chol liposomes as drug delivery systems.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Factors affecting the encapsulation of drugs within liposomes.

The reported efficiencies of drug encapsulation into liposomes range from less than 0.1% to more than 10% per micromole phospholipid, depending on the nature of the drug and of the liposome employed. We have sought to investigate some of the factors which control the efficiency of drug encapsulation. We have found that most polar drugs are sequestered within the internal aqueous compartment of the liposomes, while nonpolar drugs can bind to the liposome membrane in addition to being sequestered, thus accounting for their higher efficiencies of encapsulation. The encapsulation of nonpolar drugs, but not of polar drugs, is very sensitive to the physical characteristics of the liposome membrane; in particular, a fluid membrane favors the efficient encapsulation of nonpolar compounds. The drug cytosine arabinoside is anomalous in that this highly polar compound seems to interact with the liposome membrane at physiological conditions of pH and ionic strength, thus allowing it to be encapsulated with high efficiency.     

Effect of phospholipid composition on pharmacokinetics and biodistribution of epirubicin liposomes

The effects of phospholipid composition on the pharmacokinetics (PK) and biodistribution of epirubicin (EPI) liposomes, as well as the in vitro macrophage uptake of various liposome formulations, were investigated. Three liposome formulations were investigated: HSPC:Chol (L-EPI; 5:4 molar ratio), HSPC:Chol:DSPG (D-EPI; 5:4:1 molar ratio), and HSPC:Chol:DSPG:DSPE-mPEG(2000) (S-EPI; 5:4:1:0.3 molar ratio). Small unilamellar liposomes were prepared by the modified thin-film hydration method with extrusion through polycarbonate filters, and EPI was remote loaded into liposomes by the transmembrane ammonium sulfate gradient method. Macrophages were used to evaluate in vitro the cellular uptake of EPI-loaded liposomes. The following decreasing order of uptake amount was observed: L-EPI>D-EPI>S-EPI. D-EPI showed a relatively low level of uptake, probably because of the steric hindrance provided by the glycerol head group on DSPG, protecting it from the direct recognization by cell-membrane receptors. With the presence of serum, uptake values for all liposome formulations were increased for the activation of the complement system. In the PK study, S-EPI showed significantly prolonged circulating time and reduced clearance. The following increasing order of area under the concentration versus time curve was observed among the various liposome formulations: L-EPI相似文献   

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.     

Liposome-Trapped Nystatin in Growth Inhibition of Aspergillus niger

Abstract

Liposome-trapped nystatin caused growth inhibition of the wild type A. niger and its nystatin-resistant mutant at concentrations at which the free drug or free drug plus empty liposomes was significantly less effective. In the case of the mutant, negatively charged liposomes made from phosphatidylcholine (PC)/ phosphatidyiethanolamine (PE)ldicetyl phosphate (DCP) (1:1:0.22) showed the highest efficiency (75%). However, neutral liposomes made from PC/PE (1:1) caused 68% growth inhibition of the wild type and were most effective in this system. Growth inhibition studies as a function of time and increasing concentrations of nystatin entrapped in the above liposome systems showed significantly greater inhibition of both the wild type and the mutant. This indicates that superior efficacy is due to entrapment of nystatin in liposomes. the increased efficiency of negatively charged liposomes in the case of the mutant may be due to electrostatic attraction that favors the interaction between the liposomal membrane and the fungal cell surface in this case.     

Effect of Cholesterol on the Properties of Spray-Dried Lysozyme-Loaded Liposomal Powders

The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respect to morphology, thermal property, and crystallinity using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction, respectively. Most formulations gave slightly aggregated, spherical particles, and percentage yields of the spray-dried powders decreased with increasing Chol content. Degree of particle aggregation depended on the powder composition. The powders spontaneously formed liposomes which efficiently entrapped LSZ after reconstitution with HEPES buffered saline (HBS) at 37°C. Lysozyme entrapment efficiency and size distribution of the reconstituted liposomes were evaluated after the powders were reconstituted with HBS. Increasing Chol content resulted in a decrease in size of the reconstituted liposomes and an increase in entrapment efficiency of LSZ. These results correlated with thermal behaviors of the reconstituted liposomes. Biological activity of LSZ was not affected by the spray-drying process. It was also demonstrated that LSZ-loaded liposomal powders could be produced without the need to preload the LSZ into liposomes prior to spray-drying process.     

Effect of phospholipid composition on pharmacokinetics and biodistribution of epirubicin liposomes

The effects of phospholipid composition on the pharmacokinetics (PK) and biodistribution of epirubicin (EPI) liposomes, as well as the in vitro macrophage uptake of various liposome formulations, were investigated. Three liposome formulations were investigated: HSPC:Chol (L-EPI; 5:4 molar ratio), HSPC:Chol:DSPG (D-EPI; 5:4:1 molar ratio), and HSPC:Chol:DSPG:DSPE-mPEG₂₀₀₀ (S-EPI; 5:4:1:0.3 molar ratio). Small unilamellar liposomes were prepared by the modified thin-film hydration method with extrusion through polycarbonate filters, and EPI was remote loaded into liposomes by the transmembrane ammonium sulfate gradient method. Macrophages were used to evaluate in vitro the cellular uptake of EPI-loaded liposomes. The following decreasing order of uptake amount was observed: L-EPI>D-EPI>S-EPI. D-EPI showed a relatively low level of uptake, probably because of the steric hindrance provided by the glycerol head group on DSPG, protecting it from the direct recognization by cell-membrane receptors. With the presence of serum, uptake values for all liposome formulations were increased for the activation of the complement system. In the PK study, S-EPI showed significantly prolonged circulating time and reduced clearance. The following increasing order of area under the concentration versus time curve was observed among the various liposome formulations: L-EPI<D-EPI<S-EPI. The biodistribution study indicated that S-EPI decreased drug disposition in the liver, spleen, lung, and heart and increased that in the kidney with respect to the other liposomes. The encouraging property of S-EPI, in terms of prolonging circulating time and reducing heart toxicity, might describe a promising perspective toward clinical application, and all the results would support further research into liposome-based drug carriers.     

Physicochemical evaluation of a stability-driven approach to drug entrapment in regular and in surface-modified liposomes

The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10-90% were, excepting sucrose, either similar in the N- and T-liposomes or better in the N- than in the T-liposomes, for both regular and bioadhesive liposomes. (2) For all liposome types and methods of preparation, fluorescein, histidine, and MMC did not adsorb to the liposomal surface. Sucrose and MMC did adsorb to the liposomal surface irrespective of the liposome preparation mode, sucrose favoring bioadhesive over regular liposomes and MMC having the opposite trend. (3) For both regular and bioadhesive liposomes, the mechanism of drug efflux from the N-liposomes was found to be governed by a single rate constant, as previously found for the T-liposomes. The magnitudes obtained, ranging from 3.5(+/-0.2) x 10(-3) to 400(+/-17) x 10(-3) h(-1), were always drug specific and occasionally also liposome type (i.e., regular or bioadhesive) specific. For MMC and CAM, the novel approach rendered liposomes with improved sustained release. The results reported here attest, overall, to the potential of this novel approach, meriting further investigations. Studies currently underway with MMC indicate N-liposomes also have functional advantages.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.