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.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Cholesterol and ergosterol influence nystatin surface aggregation: relation to pore formation

Nystatin interaction with liposomes mimicking fungal and mammalian membranes (ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles, respectively) was studied by fluorescence spectroscopy. The activity of this antibiotic was also measured using a pyranine fluorescence detected K+/H+ exchange assay. Nystatin mean fluorescence lifetime varied with the antibiotic concentration and ergosterol content (0-30 mol%) of the lipid vesicles. It sharply increased from 5 to 37 ns upon reaching 100 molecules per liposome, reporting nystatin oligomerization in the membrane. Concomitantly, spectral alterations typical of excitonic coupling were detected and there was a pronounced increase in the initial rate of pore formation by nystatin. These findings suggest that nystatin exerts its antibiotic activity via a two-stage mechanism: at low antibiotic concentrations, surface-adsorbed monomeric antibiotic molecules perturb the lipid packing, changing the permeability properties of the ergosterol-rich liposomes. Upon reaching a critical threshold, nystatin mode of action switches to the classical model of transmembrane aqueous channel formation. In the presence of cholesterol-containing POPC liposomes, neither nystatin spectroscopic properties, nor the kinetics of K+ efflux varied with the antibiotic concentration suggesting that in this case the first stage of antibiotic mode of action always prevails or the assemblies formed by nystatin and cholesterol are very loose.     

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.     

Bilayer permeability-based substrate selectivity of an enzyme in liposomes

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.     

Size distribution of spontaneously formed liposomes by the alcohol injection method

A dynamic light scattering study of the size distribution of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes formed by the injection method is presented. By this method, an aliquot of methanol stock solution containing the surfactant is injected into water. The main aim of the present work was to determine under which conditions a monomodal and narrow size distribution could be obtained. The influence of several parameters on the size distribution was investigated. Firstly, we examined the influence of the POPC concentration in the initial stock methanol solution, when the POPC concentration in the final aqueous solution remains constant; secondly, the influence of POPC concentration in the aqueous phase, while the lipid concentration in the stock methanol remains constant. In both cases narrow monomodal size distributions of liposomes, centered between 40 and 70 nm, are obtained at low concentrations of POPC, in the stock methanol solution (相似文献   

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Fully detachable molecular umbrellas as peptide delivery agents

A persulfated molecular umbrella, derived from cholic acid and spermidine, has been covalently attached to H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) by use of an o-dithiobenzyl carbamate linkage. Treatment of the resulting conjugate (1) with glutathione in solution resulted in the liberation of the free form of the peptide. Addition of 1 to glutathione-entrapped liposomes, prepared from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and cholesterol [POPC/POPG/cholesterol, 72/4/24 (mol/mol/mol)], resulted in the delivery of DADLE into their aqueous interior.     

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulated release of cholesterol from liposomal membranes by a PEGylated phospholipid

PEGylated phospholipids are commonly used to increase the blood-circulation time of liposomes by providing a steric barrier around them. This paper documents a fundamentally new property of these lipids-an ability to stimulate the release of cholesterol from phospholipid membranes. Evidence for such stimulation has been obtained by measuring the transport of dehydroergosterol (DHE), a fluorescent simulant of cholesterol, from donor liposomes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG(2000)), and DHE to acceptor liposomes made from POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cholesterol. The potential of PEGylated lipids to serve as novel cholesterol-lowering agents is briefly discussed.     

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state ²H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.     

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-microm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.     

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-μm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.     

31P and 2H relaxation studies of helix VII and the cytoplasmic helix of the human cannabinoid receptors utilizing solid-state NMR techniques

Cannabinoid receptors are G-protein-coupled receptors comprised of seven transmembrane helices. We hypothesized that the extended helix of the receptor interacts differently with POPC bilayers due to the differing distribution of charged amino acid residues. To test this, hCB1(T377-E416) and hCB2(K278-H316) peptides were studied with 31P and 2H solid-state NMR spectroscopy by incorporating them into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine bilayers. Lipid affinities of the 40- and 39-residue peptides were analyzed on the basis of 31P and 2H spectral line shapes, order parameters, and T1 relaxation measurements of the POPC bilayers. Lipid headgroup perturbations were noticed in the 31P NMR spectra in the lipid/peptide mixtures when compared with the pure lipids. 2H order parameters were calculated from the quadrupolar splitting of the de-Paked 2H NMR spectra. At the top of the acyl chain, pure lipids had an average S(CD) approximately = 0.20, whereas S(CD) approximately = 0.16 and S(CD) approximately = 0.18 were found in the presence of hCB1(T377-E416) and hCB2(K278-H316), respectively. S(CD) values decreased in the central part of the acyl chains when compared to the pure POPC lipids, indicating a change in the dynamic properties of the lipid membrane in the presence of the cannabinoid peptides. R(1Z) vs S2(CD) plots exhibited a linear dependency with and without the peptides, with an increase in slope upon addition of the peptides to the POPC, indicating that the dynamics of the lipid bilayer is dominated by fast axially symmetric motion. This study provides insights into the interaction of cannabinoid peptides with the membrane bilayer by investigating the headgroup and acyl chain dynamics.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Spontaneous phosphatidylcholine transfer by collision between vesicles at high lipid concentration

The transfer kinetics of [3H]-1-palmitoyl-2-oleoylphosphatidylcholine ([3H]POPC) and 1-palmitoyl-2-(pyrenyldecanoyl)phosphatidylcholine (PyrPC) from POPC small unilamellar vesicles were examined at 37 degrees C with lipid concentrations ranging from 0.1 to 40 mM. The rate of [3H]POPC transfer was determined by analyzing the movement of this lipid from charged donor to neutral acceptor vesicles. The rate of decay of the ratio of the intensity of pyrene excimer fluorescence to that from the pyrene monomer (E/M) upon addition of an unlabeled vesicle population to a population containing PyrPC was used to evaluate PyrPC transfer. For both lipids, the kinetic data are best described by a model which assumes that transfer occurs by vesicle collisions as well as by desorption from the bilayer. For [3H]POPC, the off-rate constant is 0.014 h-1 while the collisional rate constant is 0.0016 mM-1 h-1. PyrPC has an off-rate constant of 0.023 h-1 and a collisional constant of 0.0015 mM-1 h-1. These numbers were calculated by assuming the rate of interbilayer transfer to be negligible relative to that of intervesicular transfer. The large transfer fluxes in the high vesicle concentration range where the collisional process dominates suggest that spontaneous transfer may be of importance in membrane biogenesis.