Liposome Membrane Composition Research Articles

INFLUENCE OF THE LIPID COMPOSITION ON THE PROPERTIES, TECHNOLOGY AND QUALITY INDICATORS OF LIPOSOMAL DRUGS

Liposomal drug delivery system is an example of the use of nanodrugs in medical practice. Encapsulation of active pharmaceutical ingredients in liposomal nanoparticles allows increasing their bioavailability and efficacy. Aim. The article is devoted to the analysis of the lipid composition of liposomal drugs developed in Ukraine, its influence on the choice of technology and control parameters. Results. The lipid compositions of liposomal drugs developed in Ukraine in recent years were reviewed. The advantages and disadvantages of natural phosphatidylcholine as the main membrane-forming lipid were analyzed. Data on the influence of anionic phospholipids and cholesterol in the liposomal membrane composition on the stability of liposomal nanoparticles and the level of active pharmaceutical ingredient encapsulation were given. The main technological stages of obtaining liposomes with hydrophilic and hydrophobic active pharmaceutical ingredients were considered. The main groups of quality indicators of liposomal dosage forms have been determined. Conclusions. The lipid composition determines the structure and physicochemical properties of the lipid membrane, the mechanism and level of active pharmaceutical ingredient encapsulation, which significantly influences the pharmacological efficacy of liposomal drug delivery systems.

Optimized thermosensitive liposomes for selective doxorubicin delivery: Formulation development, quality analysis and bioactivity proof

In our study we examined thermosensitive liposomal formulations (TL) from the perspective to minimize the general toxicity drawbacks of chemotherapy. The TL become active in response to local hyperthermia (LH), and remain inactive at physiological conditions. Here, we formulated novel doxorubicin loaded thermoliposomes (Dox-TL) with optimized characteristics and tested their biological activity in vitro. The liposomal membrane composition of Dox-TL and their preparation technology were adjusted for high drug loading and extended formulation stability. The 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol(Chol):1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-2000) in molar ratio 9:1:0.2:0.02:0.2 and drug/lipid weight ratio 0.13–0.20/1 composition has demonstrated best results. The freshly-prepared vesicles contained 94% doxorubicin. The Dox-TL, freeze-dried with 4% sucrose, maintained high level of encapsulated drug, remained stable in serum and prevented premature drug leakage. The Dox-TL proved to be significantly less toxic at 37°C than free Dox. In combination with local hyperthermia of 42.5°C Dox-TL were as effective as free Dox in cell survival, and even outperformed free Dox in proliferation activity suppression, colony proliferation rate, and cellular uptake. These findings represent a solid basis for a safer and more effective antitumor therapy.

Modulation of enzymatic PS synthesis by liposome membrane composition

Phosphatidylserine (PS) is a phospholipid known to exert important physiological roles in humans. However, this phospholipid (PL) is poorly available as a natural source and hardly produced by the chemical route. In this work, PS was obtained by transphosphatidylation using phospholipase D (PLD) and PL self-assembled into liposomes as the substrates. The aim was to better understand how the liposome membrane composition could modulate PS yield. Three lecithins were used as PL substrates, one originated from a marine source providing a high amount of n-3 polyunsaturated fatty acids, and two issued from soya differing in their phosphatidylcholine (PC) content. Different parameters such as Ca2+ content, enzyme and l-serine concentrations modulated PS synthesis. The presence of Ca2+ increased PS conversion yield. The alcohol acceptor (l-serine) concentration positively acted on PL conversion, by governing the equilibrium between transphosphatidylation and hydrolysis. Beside these specific reaction conditions, it was demonstrated that the membrane composition of the liposomes modulated PS synthesis. A direct correlation between PS accumulation and the amount of cholesterol or α-tocopherol incorporated into the soya lecithins was observed. This result was interpreted in terms of “head” spacers promoting PLD transphosphatidylation. On the whole, this work provided key parameters for the formulation of liposomes using enzymatic PLD technology, to produce lecithins enriched in different proportions of PS and esterified with various types of fatty acids depending on the initial lecithin source.

Physicochemical characterization of liposomes after ultrasound exposure – Mechanisms of drug release

Ultrasound is investigated as a novel drug delivery tool within cancer therapy. Non-thermal ultrasound treatment of solid tumours post i.v.-injection of drug-carrying liposomes may induce local drug release from the carrier followed by enhanced intracellular drug uptake. Recently, ultrasound-mediated drug release of liposomes (sonosensitivity) was shown to strongly depend on liposome membrane composition. In the current study the ultrasound-mediated drug release mechanism of liposomes was investigated. The results showed that differences in ultrasound drug release kinetics obtained for different liposomal compositions were caused by distinctive release mechanisms of the carriers. Two types of liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and hydrogenated soy l-α-phosphatidylcholine (HSPC) as main lipids, respectively, were recently shown to vary in sonosensitivity. Here, these liposomes were analyzed prior to and after a given ultrasound-exposure for their mean size, size distribution and morphology. Cryo-transmission electron microscopy, dynamic light scattering and asymmetric flow field-flow fractionation in combination with multi-angle light scattering revealed a significant change in mean size, size distribution and morphology of DOPE-based liposomes after ultrasound, pointing to an irreversible disruption of the vesicles and concomitant drug release. In contrast, the HSPC-based liposomes remained unchanged in size and structure after ultrasound application, indicating pore-mediated release mechanisms. The results show that the release mechanisms and interactions between ultrasound and liposomes depend on the liposome membrane-composition, explaining their sonosensitive properties.

Multiple Effects of Cholesterol on Influenza Membrane Fusion Kinetics

Influenza and other enveloped viruses differ in their envelope lipid composition from the cellular plasma membrane from which they bud. Viruses also appear to fuse preferentially to specific membrane compartments, suggesting that the lipid environment may play a selective role in permissiveness for fusion. Cholesterol in particular is enriched in the viral membrane and known to exert effects on fusion kinetics and efficiency in a number of model systems for viral infection. Cholesterol can affect membranes in multiple ways, both impacting materials properties such as membrane bending energy and lateral organization of the membrane. To probe the different effects of cholesterol, we have measured viral fusion kinetics as we vary the cholesterol concentration in both live influenza virus and target liposomes. The liposomal membrane composition was selected not to support lateral membrane heterogeneity. We observe an increase in fusion rate with increasing liposomal cholesterol concentration, consistent with a model where cholesterol promotes stalk formation and stalk formation is the rate-limiting step of the fusion reaction we measure. We observe a more complex dose-response relationship as cholesterol is extracted from influenza virions. Since hemagglutinin is known to have cholesterol-dependent lateral organization, we postulate that the altered kinetics with changes to viral sterol content reflect both changes to hemagglutinin organization in the membrane and changes to the material properties of the membranes. We further characterize this dose-response relationship to help resolve these disparate effects of cholesterol in mediating viral entry.

Sonosensitive dioleoylphosphatidylethanolamine-containing liposomes with prolonged blood circulation time of doxorubicin

Ultrasound sensitive (sonosensitive) liposomes represent a drug delivery system designed for releasing a drug load upon exposure to ultrasound (US). Inclusion of dioleoylphosphatidylethanolamine (DOPE) in liposome membranes was previously shown to induce sonosensitivity. Long blood circulation time of the liposomal drug is required for high tumour uptake and efficient US-mediated drug delivery. In this study, blood pharmacokinetics of DOPE-based liposomal doxorubicin (DXR) were evaluated in non-tumoured mice. A markedly faster blood clearance of DXR was observed for DOPE-rich liposomes compared to Caelyx® (standard liposomal DXR). Subsequently, liposome membrane composition was altered to improve drug retention in the bloodstream, whilst maintaining sonosensitivity. Formulations with reduced blood clearance of DXR were obtained by reducing the content of DOPE from 62 to 32 or 25 mol%. These formulations showed long blood circulation time, as approximately 20% of the administered DXR dose was present in the bloodstream 24 h after intravenous injection. The reduction in liposomal DOPE content did not significantly reduce US-mediated DXR release in vitro, indicating that DOPE is a potent modulator of sonosensitivity. The novel liposome formulations, containing moderate amounts of DOPE, displayed similar blood pharmacokinetic profiles as standard liposomal DXR, but a markedly improved sonosensitivity.

Analysis of liposomal membrane composition using Raman tweezers.

We have developed a methodology for the analysis of liposomal membranes and their contents using near-IR Raman spectroscopy on liposomes held in an optical trap. We were able to detect a variety of membrane components including lipids, cholesterol, and small molecule solutes such as ethanol, DMSO and hexafluoroisopropanol. The methodology is able to distinguish between solutes that equilibrate across the liposomal membrane from those that partition selectively into the lipid bilayer.

Influence of cationic lipids on the stability and membrane properties of paclitaxel‐containing liposomes

Paclitaxel (taxol) is a poorly soluble anticancer agent that is in widespread clinical use. Liposomes provide a less toxic vehicle for solubilizing the drug and increasing the therapeutic index of paclitaxel in model tumor systems. The role of liposome membrane composition in the stability of paclitaxel‐containing formulations is understood partially for neutral and anionic liposomes, but poorly for other compositions. We investigated the effect of dialkyl cationic lipids on the stability and physical properties of paclitaxel‐containing liposomes, using circular dichroism (CD), fluorescence spectroscopy, and differential interference contrast microscopy (DIC). DOTAP (1,2‐dioleoyl‐3‐trimethylammonium propane), a cationic lipid used frequently for gene delivery, was combined at various ratios with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or distearoylphosphatidylcholine (DSPC). In the absence of DOTAP, the stability of liposomes containing ≥3 mol% paclitaxel was observed to follow the following rank order: DPPC >DSPC > DMPC. Increasing concentrations of DOTAP increased the physical stability of all compositions, and maximal stabilization was achieved at 30–50 mol% DOTAP, depending on the paclitaxel concentration and the acyl chain length of the phosphatidylcholine. The relationship between stability and mole fraction of DOTAP was complex for some compositions. DOTAP exerted a major fluidizing effect on DMPC, DPPC, and DSPC membranes, and the addition of paclitaxel at 3–8 mol% did not increase fluidity further. Studies of membrane phase domain behavior using the probe Laurdan (6‐dodecanoyl‐2‐dimethylaminonaphthalene) indicated that both paclitaxel and DOTAP were miscible with the phosphatidylcholine phase. The physical events leading to destabilization of formulations are hypothesized to arise from concentration‐dependent paclitaxel self‐association rather than immiscibility of the membrane lipids. Given the increased incorporation and stability of paclitaxel in DOTAP‐containing membranes and the potential for enhanced interaction with cells, cationic liposomes may provide a therapeutic advantage over previously described liposome formulations. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1091–1105, 2001

Tertiary Structural Changes of the α-Hemolysin fromStaphylococcus aureuson Association with Liposome Membranes

The interaction of α-hemolysin (also called α-toxin) fromStaphylococcus aureuswith mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of α-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the α-hemolysin on association with the liposome membrane. The second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition.

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.

Parameters affecting the liposome-mediated insertion of RNA into eucaryotic cells in vitro

The effect of various parameters on the liposome-mediated insertion of RNA into eucaryotic cells in vitro has been studied. Maximization of the insertion of liposome-encapsulated RNA into cells was approached at three levels: (1) alteration of liposome membrane composition, (2) alteration of the recipient cell membrane, and (3) manipulation of the conditions of liposome-cell cocultivation. (1) Changes in liposome membrane composition failed to affect the amount of RNA sequestered within liposomes but did alter the efficiency and mode of liposome uptake by human epithelial carcinoma cells, rabbit spleen lymphocytes, and carrot protoplasts. Addition of lysolecithin to the liposome membrane enhanced the cellular uptake of liposome-sequestered RNA by a “fusion” mechanism (uptake in the presence of cytochalasin B), while addition of cholesterol was inhibitory. (2) Uptake of liposome-sequestered RNA was enhanced when (a) cells were in the mitotic phase of the cell cycle; (b) cells were pretreated with cholesterol-free liposomes; and (c) cells were treated with Piracetam (2-oxo-1-pyrrolidine acetamide). The increased cellular uptake of liposomes appeared in most cases to be due to enhanced cell membrane fluidity. (3) Liposome uptake by cells was directly proportional to the time of liposome-cell cocultivation and to cell number. Increasing doses of liposomes resulted in a reduction of the percentage of RNA uptake, possibly due to a saturation phenomenon. When several of the investigated parameters were simultaneously maximized, as high as 20% of the liposome-sequestered RNA was inserted into human epithelial carcinoma cells.

Interactions of C-Reactive Protein and Complement with Liposomes

Abstract We found previously that interaction of C-reactive protein (CRP) with liposomal model membranes resulted in complement(C)-dependent membrane damage. In the present study, we investigated the influence of membrane composition on the interactions of CRP and C with liposomes. Adsorption experiments showed that binding of CRP was greatest to strongly positive liposomes. A lesser, but still substantial, extent of CRP binding also was observed with negative liposomes, but negligible amounts of CRP bound to neutral or weakly positive liposomes. CRP-mediated consumption of hemolytic C, and C-dependent glucose release from liposomes both were strongly influenced by liposomal charge, positive being superior to negative. Glucose release and, to a lesser extent, consumption of hemolytic C were inversely related to phospholipid fatty acyl chain length. Phospholipid fatty acyl unsaturation and liposomal cholesterol concentration both had strong influences on C consumption and glucose release. The data suggest that CRP-mediated C consumption and membrane damage require an optimum membrane fluidity. Complement damage in the presence of CRP was enhanced by certain sphingolipids and also by digalactosyl diglyceride, but not by sphingomyelin. Our results thus demonstrate that CRP-mediated C consumption and C-dependent membrane damage both are influenced by the liposomal membrane composition.

Cholesterol-Dependent Human Complement Activation Resulting in Damage to Liposomal Model Membranes

Human (but not guinea pig) complement-mediated damage. It was concluded that human complement was activated spontaneously by liposomes containing a high concentration (71 mol %) of cholesterol. This occurred in the absence of any recognizable antigen or antibody, and did not occur at a low concentration (43 mol %) of cholesterol. Activation of complement resulted in membrane damage and release of trapped liposomal glucose. The complement activity was inhibited by preheating (56 degrees C, 30 min), 10 mM Mg2EDTA3 or EGTA, and by prior adsorption with insoluble immune complexes. Almost all human sera had some reactivity, but it ranged from very low levels (less than 7% liposomal glucose release) to very high levels (greater than 50% glucose release). Complement activation appeared to be mediated by a serum factor which could be removed by adsorption and which was partially heat labile. The factor was transferred by adding heated high reacting human serum to unheated low reacting human serum, or to guinea pig serum. The serum factor, although quantitatively diminished in potency due to heat lability, caused equal activation of each of these two latter complement sources in the presence of high cholesterol liposomes. It did not cause activation of C4-deficient guinea pig complement. These data suggested that the classical complement pathway was activated. The liposomal membrane composition had an influence on this phenomenon. Activities of about half of the human sera were enhanced when galactosyl ceramide, or ceramide alone, was present in the liposomes. Activity was enhanced by longer fatty acyl chain lengths of lecithin when dimyristoyl-, dipalmitoyl-, or distearoyllecithin was employed in the liposomes. Liposomes containing sphingomyelin as the only phospholipid were not sensitive to cholesterol-dependent complement-mediated damage. It was concluded that human complement was activated in the presence of high concentrations of membrane cholesterol and that this was caused by an uncharacterized serum factor and was influenced by the lipid composition of the membrane.