Membrane restructuring by phospholipase A2 is regulated by the presence of lipid domains.

Secretory phospholipase A(2) (sPLA(2)) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L(α)/L(ß)) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA(2). Specifically, we show that the presence of L(α)/L(ß) and L(α)/P(ß') phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L(α) regions, the L(ß) and P(ß') regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA(2) hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.

Liposomes containing alkylated methotrexate analogues for phospholipase A(2) mediated tumor targeted drug delivery.

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C16-alkyl chain attached to the gamma-carboxylic acid and one of the analogues had an additional benzyl group attached to the alpha-carboxylic acid. The cytotoxicity of the gamma-alkylated compound towards KATO III (IC(50) = 55 nM) and HT-29 (IC(50) = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the alpha-carboxyl group made the compound nontoxic. The gamma-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A(2) (sPLA(2)), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA(2) hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA(2). However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA(2) hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.

Molecular basis of phospholipase A2 activity toward phospholipids with sn-1 substitutions.

We studied secretory phospholipase A(2) type IIA (sPLA(2)) activity toward phospholipids that are derivatized in the sn-1 position of the glycerol backbone. We explored what type of side group (small versus bulky groups, hydrophobic versus polar groups) can be introduced at the sn-1 position of the glycerol backbone of glycerophospholipids and at the same time be hydrolyzed by sPLA(2). The biophysical characterization revealed that the modified phospholipids can form multilamellar vesicles, and several of the synthesized sn-1 functionalized phospholipids were hydrolyzed by sPLA(2). Molecular dynamics simulations provided detailed insight on an atomic level that can explain the observed sPLA(2) activity toward the different phospholipid analogs. The simulations revealed that, depending on the nature of the side chain located at the sn-1 position, the group may interfere with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. The simulation results are in agreement with the experimentally observed sPLA(2) activity toward the different phospholipid analogs.

Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue.

Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level in the periphery of the tumors, compared to central tumor regions. Increased levels of sPLA2 IIA protein correlated with a two-fold increase in sPLA2 enzymatic activity in the peripheral regions compared to central regions. Nineteen out of 22 tumors showed high levels of sPLA2 IIA, whereas 7 out of the 22 tumors showed sPLA2 type X. These data demonstrate that both sPLA2 type IIA and X are present in human colon cancer and suggest a role for sPLA2 in colon cancer tumor immunology and tumorigenesis.

Secretory phospholipase A2 hydrolysis of phospholipid analogues is dependent on water accessibility to the active site.

A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A2 (sPLA2). To investigate the unexpected sPLA2 activity, we have compared three different phospholipids by using fluorescence techniques and HPLC, namely: (R)-1,2-dipalmitoyl-glycero-3-phosphocholine (hereafter referred to as 1R), (R)-1-O-hexadecyl-2-palmitoyl-glycero-3-phoshocholine (2R), and (S)-1-O-hexadecyl-3-palmitoyl-glycero-2-phosphocholine (3S). Furthermore, to understand the underlying mechanisms for the observed differences, we have performed molecular dynamics simulations to clarify on a structural level the substrate specificity of sPLA2 toward phospholipid analogues with their head groups in the 2-position of the glycerol backbone. We have studied the lipids above 1R, 2R, and 3S as well as their enantiomers 1S, 2S, and 3R. In the simulations of sPLA2-1S and sPLA2-3R, structural distortion in the binding cleft induced by the phospholipids showed that these are not substrates for sPLA2. In the case of the phospholipids 1R, 2R, and 3S, our simulations revealed that the difference observed experimentally in sPLA2 activity might be caused by reduced access of water molecules to the active site. We have monitored the number of water molecules that enter the active site region for the different sPLA2-phospholipid complexes and found that the probability of a water molecule reaching the correct position such that hydrolysis can occur is reduced for the unnatural lipids. The relative water count follows 1R > 2R > 3S. This is in good agreement with experimental data that indicate the same trend for sPLA2 activity: 1R > 2R > 3S.

Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2.

Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and 2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water molecules to the active site.

Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production.

Methoxy(polyethylene glycol), mPEG, -grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non-IgE-mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid-mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid-mPEG conjugate responsible for PEGylated liposome-induced complement activation in normal as well as C1q-deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid-mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid-mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b-9, C4d, and C3a-desarg levels in normal human sera as well as SC5b-9 in EGTA-chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid-mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated phospholipid-mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid-mPEG micelles had no effect on complement activation, thus suggesting a possible role for vesicular zwitterionic phospholipid head-groups as an additional factor contributing to PEGylated liposome-mediated complement activation. Our findings provide a rational conceptual basis for development of safer vesicles for site-specific drug delivery and controlled release at pathological sites.

Activation of the human complement system by cholesterol-rich and PEGylated liposomes-modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels.

Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed liposome-mediated SC5b-9 generation considerably. While intravenous injection of cholesterol-rich liposomes into pigs was associated with an immediate circulatory collapse, the drop in systemic arterial pressure following injection of liposomes preincubated with human lipoproteins was slow and extended. Therefore, surface-associated lipoprotein particles (or apolipoproteins) seem to lessen liposome-induced adverse haemodynamic changes, possibly as a consequence of suppressed complement activation in vivo. PEGylated liposomes were also capable of activating the human complement system, and the presence of surface projected methoxypoly(ethylene glycol) chains did not interfere with generation of C3 opsonic fragments. We also show that poly(ethylene glycol) is not responsible for PEGylated liposome-mediated complement activation. The net anionic charge on the phosphate moiety of the phospholipid-mPEG conjugate seemed to play a critical role in activation of both the classical and alternative pathways of the complement system.

Domain-induced activation of human phospholipase A2 type IIA: local versus global lipid composition.

Secretory human phospholipase A2 type IIA (PLA2-IIA) catalyzes the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. The enzyme is coupled to the inflammatory response, and its specificity toward anionic membrane interfaces suggests a role as a bactericidal agent. PLA2-IIA may also target perturbed native cell membranes that expose anionic lipids to the extracellular face. However, anionic lipid contents in native cells appear lower than the threshold levels necessary for activation. By using phosphatidylcholine/phosphatidylglycerol model systems, we show that local enrichment of anionic lipids into fluid domains triggers PLA2-IIA activity. In addition, the compositional range of enzyme activity is shown to be related to the underlying lipid phase diagram. A comparison is done between PLA2-IIA and snake venom PLA2, which in contrast to PLA2-IIA hydrolyzes both anionic and zwitterionic membranes. In general, this work shows that PLA2-IIA activation can be accomplished through local enrichment of anionic lipids into domains, indicating a mechanism for PLA2-IIA to target perturbed native membranes with low global anionic lipid contents. The results also show that the underlying lipid phase diagram, which determines the lipid composition at a local level, can be used to predict PLA2-IIA activity.

Activation of interfacial enzymes at membrane surfaces.

A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A(2) (sPLA(2)), are only activated at the interface between water and membrane surfaces, where they lead to a break-down of the lipid molecules into lysolipids and free fatty acids. The activation is critically dependent on the physical properties of the lipid-membrane substrate. A topical review is given of our current understanding of the physical mechanisms responsible for activation of sPLA(2) as derived from a range of different experimental and theoretical investigations.

Synthesis and biological activity of anticancer ether lipids that are specifically released by phospholipase A2 in tumor tissue.

The clinical use of anticancer lipids is severely limited by their ability to cause lysis of red blood cells prohibiting intravenous injection. Novel delivery systems are therefore required in order to develop anticancer ether lipids (AELs) into clinically useful anticancer drugs. In a recent article (J. Med. Chem. 2004, 47, 1694) we showed that it is possible to construct liposome systems composed of masked AELs that are activated by secretory phospholipase A2 in cancerous tissue. We present here the synthesis of six AELs and evaluate the biological activity of these bioactive lipids. The synthesized AEL 1-6 were tested against three different cancer cell lines. It was found that the stereochemistry of the glycerol headgroup in AEL-2 and 3 has a dramatic effect on the cytotoxicity of the lipids. AEL 1-4 were furthermore evaluated for their ability to prevent phosphorylation of the apoptosis regulating kinase Akt, and a correlation was found between their cytotoxic activity and their ability to inhibit Akt phosphorylation.

Triggered activation and release of liposomal prodrugs and drugs in cancer tissue by secretory phospholipase A2.

The selectivity of anticancer drugs in targeting the tumour tissue presents a major problem in cancer treatment. In this article we review a new generation of smart liposomal nanocarriers that can be used for enhanced anticancer drug and prodrug delivery to tumours. The liposomes are engineered to be particularly degradable to secretory phospholipase A2 (sPLA2), which is a lipid hydrolyzing enzyme that is significantly upregulated in the extracellular microenvironment of cancer tumours. Thus, when the long circulatory liposomal nanocarriers extravasate and accumulate in the interstitial tumour space, sPLA2 will act as an active trigger resulting in the release of cytotoxic drugs in close vicinity of the target cancer cells. The sPLA2 generated lysolipid and fatty acid hydrolysis products will furthermore be locally released and function as membrane permeability promoters facilitating the intracellular drug uptake. In addition, the liposomal membrane can be composed of a novel class of prodrug lipids that can be converted selectively to active anticancer agents by sPLA2 in the tumour. The integrated drug discovery and delivery technology offers a promising way to rationally design novel tumour activated liposomal nanocarriers for better cancer treatment.

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides.

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.

Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers.

A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.

Advanced strategies in liposomal cancer therapy: problems and prospects of active and tumor specific drug release.

Tumor specific drug delivery has become increasingly interesting in cancer therapy, as the use of chemotherapeutics is often limited due to severe side effects. Conventional drug delivery systems have shown low efficiency and a continuous search for more advanced drug delivery principles is therefore of great importance. In the first part of this review, we present current strategies in the drug delivery field, focusing on site-specific triggered drug release from liposomes in cancerous tissue. Currently marketed drug delivery systems lack the ability to actively release the carried drug and rely on passive diffusion or slow non-specific degradation of the liposomal carrier. To obtain elevated tumor-to-normal tissue drug ratios, it is important to develop drug delivery strategies where the liposomal carriers are actively degraded specifically in the tumor tissue. Many promising strategies have emerged ranging from externally triggered light- and thermosensitive liposomes to receptor targeted, pH- and enzymatically triggered liposomes relying on an endogenous trigger mechanism in the cancerous tissue. However, even though several of these strategies were introduced three decades ago, none of them have yet led to marketed drugs and are still far from achieving this goal. The most advanced and prospective technologies are probably the prodrug strategies where non-toxic drugs are carried and activated specifically in the malignant tissue by overexpressed enzymes. In the second part of this paper, we review our own work, exploiting secretory phospholipase A2 as a site-specific trigger and prodrug activator in cancer therapy. We present novel prodrug lipids together with biophysical investigations of liposome systems, constituted by these new lipids and demonstrate their degradability by secretory phospholipase A2. We furthermore give examples of the biological performance of the enzymatically degradable liposomes as advanced drug delivery systems.

Secretory phospholipase A2 as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids.

The use of many common clinically relevant chemotherapeutics is often limited due to insufficient delivery to the tumor and dose-limiting systemic toxicities. Therefore, therapeutics that specifically target tumor cells and are nontoxic to normal cells are required. Here, we report the development of a novel class of liposomes composed of lipid prodrugs, which use the increased secretory phospholipase A2 type IIA (sPLA2) activity of the tumor microenvironment as a trigger for the release of anticancer etherlipids (AEL). Treatment of sPLA2-secreting tumor cells in vitro with liposomes consisting of proAELs resulted in growth inhibition comparable with addition of the AELs alone. Using a specific sPLA2 inhibitor, we showed the low cytotoxicity of the nonhydrolyzed proAEL liposomes and have proven the sPLA2 dependency of the activation of proAELs to cytotoxic AELs. In addition, we showed that our proAEL liposomes circumvent the inherent hemolytic toxicities associated with the use of etherlipids, thereby allowing i.v. administration of such therapeutics as nontoxic prodrug liposomes. Furthermore, using a sPLA2-secreting human colon cancer xenograft model, we showed that the proAEL liposomes are capable of inducing a tumor growth delay in vivo. Taken together, these data support the validity of this novel tumor-selective liposomal prodrug delivery strategy. This new approach also provides a promising system for tumor-selective delivery and release of conventional chemotherapeutics encapsulated in the sPLA2-degradable prodrug liposomes.

Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by time-resolved AFM.

The sensitivity of phospholipase A(2) (PLA(2)) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA(2) is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA(2) preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA(2) for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.

Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug diffusion.

Freeze/thaw effects on lipid-bilayer vesicles investigated by differential scanning calorimetry.

Differential scanning calorimetry (DSC) has been used to study the effects of repeated freezing and thawing on dipalmitoylphosphatidylcholine (DPPC) vesicles. Aqueous suspensions of both multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) were cycled between -37 and 8 degrees C, and for each thawing event, the enthalpy of ice-melting was measured. In the case of MLVs, the enthalpy increased each time the vesicles were thawed until a steady state was attained. In contrast, the enthalpies measured for LUV suspensions were independent of the number of previous thawing events. It was concluded that MLVs in terms of freezing characteristics contain two pools of water, namely bulk water and interlamellar water. Interlamellar water does not freeze under the conditions employed in the present study, and the MLVs therefore experience freeze-induced dehydration, which is the reason for the observed increase in ice-melting enthalpy. Furthermore, the thermodynamic results suggest that the osmotic stress resulting from the freeze-induced dehydration changes the lamellarity of the MLVs.

Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers.

Temperature-controlled atomic force microscopy (AFM) has been used to visualize and study the structure and kinetics of ripple phases in one-component dipalmitoylphosphatidylcholine (DPPC) and two-component dimyristoylphosphatidylcholine-distearoylphosphatidylcholine (DMPC-DSPC) lipid bilayers. The lipid bilayers are mica-supported double bilayers in which ripple-phase formation occurs in the top bilayer. In one-component DPPC lipid bilayers, the stable and metastable ripple phases were observed. In addition, a third ripple structure with approximately twice the wavelength of the metastable ripples was seen. From height profiles of the AFM images, estimates of the amplitudes of the different ripple phases are reported. To elucidate the processes of ripple formation and disappearance, a ripple-phase DPPC lipid bilayer was taken through the pretransition in the cooling and the heating direction and the disappearance and formation of ripples was visualized. It was found that both the disappearance and formation of ripples take place virtually one ripple at a time, thereby demonstrating the highly anisotropic nature of the ripple phase. Furthermore, when a two-component DMPC-DSPC mixture was heated from the ripple phase and into the ripple-phase/fluid-phase coexistence temperature region, the AFM images revealed that several dynamic properties of the ripple phase are important for the melting behavior of the lipid mixture. Onset of melting is observed at grain boundaries between different ripple types and different ripple orientations, and the longer-wavelength metastable ripple phase melts before the shorter-wavelength stable ripple phase. Moreover, it was observed that the ripple phase favors domain growth along the ripple direction and is responsible for creating straight-edged domains with 60 degrees and 120 degrees angles, as reported previously.