Phase behavior and aggregate structure in mixtures of dioleoylphosphatidylethanolamine and poly(ethylene glycol)-lipids

Cryo-transmission electron microscopy has been used to investigate the phase behavior and aggregate structure in dilute aqueous mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-phospholipids (PEG-lipids). It is shown that PEG-lipids (micelle-forming lipids) induce a lamellar phase in mixtures with DOPE (inverted hexagonal forming lipid). The amount of PEG-lipid that is needed to induce a pure dispersed lamellar phase, at physiological conditions, depends on the size of the PEG headgroup. In the transition region between the inverted hexagonal phase and the lamellar phase, particles with dense inner textures are formed. It is proposed that these aggregates constitute dispersed cubic phase particles. Above bilayer saturating concentration of PEG-lipid, small disks and spherical micelles are formed. The stability of DOPE/PEG-lipid liposomes, prepared at high pH, against a rapid drop of the pH was also investigated. It is shown that the density of PEG-lipid in the membrane, sufficient to prevent liposome aggregation and subsequent phase transition, depends on the size of the PEG headgroup. Below a certain density of PEG-lipid, aggregation and phase transition occurs, but the processes involved proceed relatively slow, over the time scale of weeks. This allows detailed studies of the aggregate structure during membrane fusion.     

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.     

Temperature dependence of the vesicle-micelle transition of egg phosphatidylcholine and octyl glucoside

The temperature dependence of octyl glucoside micellization was determined and compared to the phase behavior of the octyl glucoside--egg phosphatidylcholine (PC) mixed system in excess water to help elucidate the process of vesicle formation from mixed surfactant-phospholipid micelles. The critical micelle concentration of octyl glucoside (OG) was determined from the sharp increase of ANS fluorescence at micellization in an NaCl buffer at temperatures ranging from 5 to 40 degrees C. The cmc decreased with increasing temperature from 31 mM at 5 degrees C to 16 mM at 40 degrees C. A similar but less steep temperature dependence is observed for the solubilization of egg PC vesicles by OG as monitored by the surfactant-dependent changes in (1) solution turbidity and (2) the resonance energy transfer between NBD-PE and Rho-PE incorporated in the vesicles. These assays identify two breakpoints, most likely the boundaries of the cylindrical micelle and spheroidal micelle coexistence region. The [OG]aq values at these two breakpoints have similar temperature dependencies. However, the cylindrical mixed micelles at the boundary have nearly identical OG:PC ratios over the temperature range studied, whereas the spheroidal mixed micelles have relatively more OG at the higher temperatures (OG:PC ratio increases from 2.92 to 3.72 between 5 and 35 degrees C). Estimation of the acyl volume to surface area ratio for the compositions observed suggests that this parameter remains constant over temperature. The spheroidal mixed micelles, but not the cylindrical PC-OG micelles, exhibit ideal mixing between the two components at all temperatures (5-35 degrees C). This temperature sensitivity may be utilized to improve the efficacy of membrane protein reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol)-lipid.

Surface-grafted polymers, such as poly(ethylene glycol) (PEG), provide an effective steric barrier against surface-surface and surface-macromolecule interactions. In the present work, we have studied the exchange of monooleoylphosphatidylcholine (MOPC) with vesicle membranes containing 750 mol wt surface-grafted PEG (incorporated as PEG-lipid) from 0 to 20 mol % and have analyzed the experimental results in terms of thermodynamic and stationary equilibrium models. Micropipette manipulation was used to expose a single lipid vesicle to a flow of MOPC solution (0.025 microM to 500 microM). MOPC uptake was measured by a direct measure of the vesicle area change. The presence of PEG(750) lipid in the vesicle membrane inhibited the partitioning of MOPC micelles (and to some extent microaggregates) into the membrane, while even up to 20 mol % PEG-lipid, it did not affect the exchange of MOPC monomers both into and out of the membrane. The experimental data and theoretical models show that grafted PEG acts as a very effective molecular scale "filter" and prevents micelle-membrane contact, substantially decreasing the apparent rate and amount of MOPC taken up by the membrane, thereby stabilizing the membrane in a solution of MOPC that would otherwise dissolve it.     

Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Synthesis of multiacyl poly(ethylene glycol) for the conjugation of cytochrome c to phospholipid vesicle

To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Kinetic and structural aspects of reconstitution of phosphatidylcholine vesicles by dilution of phosphatidylcholine-sodium cholate mixed micelles

Dilution of mixed micellar dispersions of egg phosphatidylcholine (PC) and sodium cholate beyond a critical value results in formation of cholate-containing PC vesicles. The structure of the resultant vesicles and some mechanistic aspects of this process have been investigated by the use of light scattering and nuclear magnetic resonance techniques. The main findings and conclusions are the following: Both the state of aggregation (micellar or vesicular) and the apparent equilibrium size distribution of micelles or vesicles obtained by dilution of the PC-cholate mixed micellar dispersions are a function of the cholate to PC molar ratio in the mixed aggregates (micelles or vesicles). When this effective ratio (Re) is higher than 0.4, the dispersion is micellar, and the size of the mixed micelles increases with decreasing Re; when Re less than 0.3, the dispersion is essentially vesicular, and the mean hydrodynamic radius of the vesicles is an increasing function of Re; in dispersions with 0.3 less than Re less than 0.4, mixed micelles and vesicles coexist. Addition of cholate to vesicular dispersions, to Re values below 0.3, results in vesicle size growth through a concentration-independent lipid-exchange mechanism. Addition of cholate to higher Re values results in micellization (solubilization) of the vesicles. On the other hand, dilution of vesicular dispersions does not affect the size of the vesicles. Apparent equilibration of a mixed micellar dispersion following dilution to Re values below 0.3 is slow (many hours). The overall process involves a series of three subsequent categories of steps: (i) a rapid (approximately 1-2 min) prevesiculation equilibration of micellar sizes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of cholesterol solubilized in bile salt micellar aqueous solutions using (13)C nuclear magnetic resonance

In this work, we develop a methodology to quantitatively follow the solubilization of cholesterol on glycodeoxycholic acid (GDCA) micelles using (13)C nuclear magnetic resonance (NMR). The amount of solubilized cholesterol enriched in (13)C at position 4, [4-(13)C]cholesterol, was quantified from the area of its resonance, at 44.5 ppm, using the CH(2) groups from GDCA as an internal reference. The loading of the micelles with cholesterol leads to a quantitative upper field shift of most carbons in the nonpolar surface of GDCA, and this was used to follow the solubilization of unlabeled cholesterol. The solubilization followed a pseudo first-order kinetics with a characteristic time constant of 3.6 h, and the maximum solubility of cholesterol in 50 mM total lipid (GDCA + cholesterol) is 3.0 ± 0.1mM, corresponding to a mean occupation number per micelle ≥1. The solubilization profile indicates that the affinity of cholesterol for the GDCA micelles is unaffected by the presence of the solute, leading essentially to full solubilization up to the saturation limit. The relaxation times of GDCA carbons at 50mM give information regarding its aggregation and indicate that GDCA is associated in small micelles (hydrodynamic [Rh] = 1.1 nm) without any evidence for formation of larger secondary micelles. This was confirmed by dynamic light scattering results.     

Characterization of phospholipid transfer between mixed phospholipid-bile salt micelles

Concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) was used to measure the rate of N-NBD-PE transfer between phosphatidylcholine-bile salt mixed micelles. In a previous study using the same technique, the rate of N-NBD-PE transfer between phosphatidylcholine-taurocholate mixed micelles was found to be several orders of magnitude faster than its transfer between phosphatidylcholine vesicles as a result of an increased rate of transfer through the water at low micelle concentrations and an increased rate of transfer during transient micelle collisions at higher micelle concentrations [Nichols, J. W. (1988) Biochemistry 27, 3925-3931]. In this study we have determined the influence of bile salt structure, incorporation of cholesterol, and temperature on the rate and mechanism of phospholipid transfer between mixed micelles. We found that both transfer pathways were a common property of mixed micelles prepared from a series of different bile salts and that the rates of transfer by both pathways increased as a function of the degree of bile salt hydrophobicity. Cholesterol incorporation into phosphatidylcholine-taurocholate mixed micelles displaced taurocholate from the micelles and resulted in an increased rate of transfer through the water and a decreased rate of transfer during micelle collisions. The temperature dependence of the transfer rates was used to calculate the activation free energy, enthalpy, and entropy for both mechanisms. The activation enthalpy was the major barrier to transfer by both mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Phospholipid transfer between phosphatidylcholine-taurocholate mixed micelles

The transfer of fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) between phosphatidylcholine-taurocholate mixed micelles was measured by monitoring the increase in fluorescence as N-NBD-PE, initially contained in mixed micelles at self-quenching concentrations, was diluted into unlabeled mixed micelles. The half-times for transfer of a homologous series of N-NBD-PEs differing in saturated acyl chain length from 11 to 16 carbons increased with acyl chain length from 4 to 35 s. The half-times for transfer of the same N-NBD-PEs between phosphatidylcholine vesicles without taurocholate were 200-6000 times slower than those between the mixed micelles. A kinetic analysis of initial transfer rate data was used to determine the mechanistic model that best described the data. According to this analysis, the increased rate of intermicellar phospholipid transfer relative to that of intervesicular transfer is a result of (1) exchange between micelles during transient micelle collisions which is not observed between vesicles and (2) an increased rate of monomer diffusion due to a faster rate of phospholipid dissociation from mixed micelles into the water phase than from vesicles. The relative significance of dissociation from mixed micelles into the water phase than from vesicles. The relative significance of collision-dependent versus monomer diffusion transfer increases with acyl chain length and hydrophobicity.     

New sterically stabilized vesicles based on nonionic surfactant, cholesterol, and poly(ethylene glycol)-cholesterol conjugates.

Monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-Chol) with polymer chain molecular weights of 1000 (M-PEG1000-Chol) and 2000 (M-PEG2000-Chol) have been newly synthesized and characterized. Their aggregation behavior in mixture with diglycerol hexadecyl ether (C16G2) and cholesterol has been examined by cryotransmission electron microscopy, high-performance gel exclusion chromatography, and quasielastic light scattering. Nonaggregated, stable, unilamellar vesicles were obtained at low polymer levels with optimal shape and size homogeneity at cholesteryl conjugate/ lipids ratios of 10 mol% M-PEG1000-Chol or 5 mol% M-PEG2000-Chol, corresponding to the theoretically predicted brush conformational state of the PEG chains. At 20 mol% M-PEG1000-Chol or 10 mol% M-PEG2000-Chol, the saturation threshold of the C16G2/cholesterol membrane in polymer is exceeded, and open disk-shaped aggregates are seen in coexistence with closed vesicles. Higher levels up to 30 mol% lead to the complete solubilization of the vesicles into disk-like structures of decreasing size with increasing PEG content. This study underlines the bivalent role of M-PEG-Chol derivatives: while behaving as solubilizing surfactants, they provide an efficient steric barrier, preventing the vesicles from aggregation and fusion over a period of at least 2 weeks.     

Micellization phenomena of amphiphilic block copolymers based on methoxy poly(ethylene glycol) and either crystalline or amorphous poly(caprolactone-b-lactide)

This study focuses on the aggregation behavior of the biodegradable amphiphilic block copolymers based on methoxy poly(ethylene glycol) (mPEG) as a hydrophilic block and either crystalline poly(caprolactone-b-l-lactide) (P(CL-LLA)) or amorphous poly(caprolactone-b-D,L-lactide) (P(CL-DLLA)) as a hydrophobic block. These block copolymers have a strong tendency to form micelles in aqueous medium, with very low critical micelle concentrations (CMCs). The CMC of P(CL-LLA)-b-mPEG is higher than that of P(CL-DLLA)-b-mPEG when the mPEG block has the same molecular weight. Furthermore, the partition equilibrium coefficient (K(v)) of pyrene in the micellar solution of P(CL-LLA)-b-mPEG copolymer was lower than that of P(CL-DLLA)-b-mPEG copolymer when the mPEG block was the same length. These differences were believed to be related to the physical state of the core-forming blocks, i.e., the crystalline P(CL-LLA) block and the amorphous P(CL-DLLA) block. The TEM images showed that micelles formed by P(CL-LLA)-b-mPEG assembled in a cylindrical morphology, whereas those formed by P(CL-DLLA)-b-mPEG took a classical spherical shape. In addition, with differential scanning calorimetry (DSC) and wide-angle X-ray diffraction (WAXD) analyses, it is believed that the crystallization tendency of the core-forming blocks is the main factor governing the morphology of micelles in water. A possible mechanism for the cylindrical assembly morphology was discussed.     

Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics

Abstract

The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is – at least to some extent – influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.     

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.