Nonlamellar Phases in Cationic Phospholipids, Relevance to Drug and Gene Delivery.

Lipid aggregates have been used as drug carriers for several decades. Recently, nonlamellar liquid crystalline lipid systems have attracted attention as possible drug-delivery vehicles because of their unique nanostructure and physicochemical properties. Here we summarize data on the nonlamellar phase-forming propensity of the cationic phosphatidylcholines (cationic PCs). The class of cationic PCs has been specifically designed and explored for the purpose of nonviral gene delivery. These lipids were found to comprise an attractive cationic lipid class because they are biodegradable, have low toxicities, and in a number of cases, display high transfection activity. Lipids of this class form a variety of polymorphic and mesomorphic phases-lamellar and nonlamellar, depending on the structure of their hydrocarbon chains and especially on the third hydrocarbon chain used to alkylate the PC phosphate group and convert the zwitterionic PC headgroup into a cation. Here we characterize the phase behavior and transfection activity of eight cationic PCs that have been identified as forming nonlamellar phases-inverted hexagonal and cubic. We then demonstrate that those cationic PCs that also form nonlamellar lipoplexes are notably less efficient gene nanocarriers in comparison with the cationic PCs forming lamellar phase lipoplexes.

Fusion peptides promote formation of bilayer cubic phases in lipid dispersions. An x-ray diffraction study.

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the HII phase and shifted the L(α)-H(II) transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q(II), which becomes inserted between the L(α) and H(II) phases on the temperature scale. Peptide-induced Q(II) had strongly reduced lattice constants in comparison to the Q(II) phases that form in pure lipids. Q(II) formation was favored at the expense of both L(α) and H(II) phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L(α) phase and completely abolished the HII phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q(II) phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H(II) phase-like intermediates.

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells.

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH2-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH2-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.

Encapsulation of NF-kappaB decoy oligonucleotides within echogenic liposomes and ultrasound-triggered release.

Echogenic liposomes (ELIP) have additional promise, beyond diagnostic agents, as vehicles for delivering oligonucleotides (ODN), especially if the release of the agent can be triggered and its uptake can be enhanced by ultrasound application at a specific site. The purpose of this study was to co-encapsulate air and NF-kappaB decoy ODN within ELIP allowing ultrasound to release encapsulated ODN from ELIP, and to accurately quantify release of encapsulated ODN from ELIP upon ultrasound application. FITC-labeled sense ODN (2 mM) was incorporated within ELIP using freeze/thaw method. Encapsulation efficiency of FITC-ODN was spectrofluorometrically analyzed by quenching fluorescence of unencapsulated FITC-ODN using a complementary strand tagged with Iowa Black FQ-ODN. Quenching of FITC-ODN (0.05 microM) with Iowa Black FQ-ODN (0.1 microM) was found to be efficient (92.4+/-0.2%), allowing accurate determination of encapsulated ODN. Encapsulation efficiency of ODN was 14.2+/-2.5% in DPPC/DOPC/DPPG/CH liposomes and 29.6+/-1.5% in DPPC/DOPE/DPPG/CH liposomes. Application of ultrasound (1 MHz continuous wave, 0.26 MPa peak-to-peak pressure amplitude, 60s.) to the latter formulation triggered 41.6+/-4.3% release of ODN from ODN-containing ELIP. We have thus demonstrated that ODN can be encapsulated into ELIP and released efficiently upon ultrasound application. These findings suggest potential applications to gene therapy for atherosclerosis as well as a variety of other diseases.

Nitric oxide-loaded echogenic liposomes for nitric oxide delivery and inhibition of intimal hyperplasia.

OBJECTIVES: We sought to develop a new bioactive gas-delivery method by the use of echogenic liposomes (ELIP) as the gas carrier. BACKGROUND: Nitric oxide (NO) is a bioactive gas with potent therapeutic effects. The bioavailability of NO by systemic delivery is low with potential systemic effects. METHODS: Liposomes containing phospholipids and cholesterol were prepared by the use of a new method, freezing under pressure. The encapsulation and release profile of NO from NO-containing ELIP (NO-ELIP) or a mixture of NO/argon (NO/Ar-ELIP) was studied. The uptake of NO from NO-ELIP by cultured vascular smooth muscle cells (VSMCs) both in the absence and presence of hemoglobin was determined. The effect of NO-ELIP delivery to attenuate intimal hyperplasia in a balloon-injured artery was determined. RESULTS: Coencapsulation of NO with Ar enabled us to adjust the amount of encapsulated NO. A total of 10 microl of gas can be encapsulated into 1 mg of liposomes. The release profile of NO from NO-ELIP demonstrated an initial rapid release followed by a slower release during the course of 8 h. Sixty-eight percent of cells remained viable when incubated with 80 microg/ml of NO/Ar-ELIP for 4 h. The delivery agent of NO to VSMCs by the use of NO/Ar-ELIP was 7-fold greater than unencapsulated NO. We discovered that NO/Ar-ELIP remained an effective delivery agent of NO to VSMCs even in the presence of hemoglobin. Local NO-ELIP administration to balloon-injured carotid arteries attenuated the development of intimal hyperplasia and reduced arterial wall thickening by 41 +/- 9%. CONCLUSIONS: Liposomes can protect and deliver a bioactive gas to target tissues with the potential for both visualization of gas delivery and controlled therapeutic gas release.

Hydrophobic moiety of cationic lipids strongly modulates their transfection activity.

Synthetic cationic lipids are widely used components of nonviral gene carriers, and the factors regulating their transfection efficiency are the subject of considerable interest. In view of the important role that electrostatic interactions with the polyanionic nucleic acids play in formation of lipoplexes, a common empirical approach to improving transfection has been the synthesis and testing of amphiphiles with new versions of positively charged polar groups, while much less attention has been given to the role of the hydrophobic lipid moieties. On the basis of data for approximately 20 cationic phosphatidylcholine (PC) derivatives, here we demonstrate that hydrocarbon chain variations of these lipids modulate by over 2 orders of magnitude their transfection efficiency. The observed molecular structure-activity relationship manifests in well-expressed dependences of activity on two important molecular characteristics, chain unsaturation and total number of carbon atoms in the lipid chains, which is representative of the lipid hydrophobic volume and hydrophilic-lipophilic ratio. Transfection increases with decrease of chain length and increase of chain unsaturation. Maximum transfection was found for cationic PCs with monounsaturated 14:1 chains. It is of particular importance that the high-transfection lipids strongly promote cubic phase formation in zwitterionic membrane phosphatidylethanolamine (PE). These remarkable correlations point to an alternative, chain-dependent process in transfection, not related to the electrostatic cationic-anionic lipid interactions.

Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: a measure for transfection efficiency.

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by approximately 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, Lalpha-->HII, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct Lalpha-->HII phase transition and promoted formation of an inverted cubic phase between the Lalpha and HII phases. In contrast, moderate and weak transfection agents retained the direct Lalpha-->HII transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.

Synthesis, acoustic stability, and pharmacologic activities of papaverine-loaded echogenic liposomes for ultrasound controlled drug delivery.

BACKGROUND: development of encapsulated therapeutics that could be released upon ultrasound exposure has strong implications for enhancing drug effects at the target site. We have developed echogenic liposomes (ELIP) suitable for ultrasound imaging of blood flow and ultrasound-mediated intravascular drug release. Papaverine was chosen as the test drug because its clinical application requires high concentration in the target vascular bed but low concentration in the systemic circulation. METHODS: the procedure for preparation of standard ELIP was modified by including Papaverine hydrochloride in the lipid hydration solution, followed by three freeze-thaw cycles to increase encapsulation of the drug. Sizing and encapsulation pharmacokinetics were performed using a Coulter counter and a phosphodiesterase activity assay. Stability of Papaverine-loaded ELIP (PELIP) was monitored with a clinical diagnostic ultrasound scanner equipped with a linear array transducer at a center frequency of 4.5 MHz by assessing the mean digital intensity within a region of interest over time. The stability of PELIP was compared to those of standard ELIP and Optison. RESULTS: relative to standard ELIP, PELIP were larger (median diameter = 1.88 +/- 0.10 microm for PELIP vs 1.08 +/- 0.15 microm for ELIP) and had lower Mean Gray Scale Values (MGSV) (92 +/- 24.8 for PELIP compared to 142.3 +/- 10.7 for ELIP at lipid concentrations of 50 microg/ml). The maximum loading efficiency and mean encapsulated concentration were 24% +/- 7% and 2.1 +/- 0.7 mg/ml, respectively. Papaverine retained its phosphodiesterase inhibitory activity when associated with PELIP. Furthermore, a fraction of this activity remained latent until released by dissolution of liposomal membranes with detergent. The stability of both PELIP and standard ELIP were similar, but both are greater than that of Optison. CONCLUSIONS: our results suggest that PELIP have desirable physical, biochemical, biological, and acoustic characteristics for potential in vivo administration and ultrasound-controlled drug delivery.

Cationic phospholipids forming cubic phases: lipoplex structure and transfection efficiency.

The transfection activity and the phase behavior of two novel cationic O-alkyl-phosphatidylcholines, 1,2-dioleoyl- sn-glycero-3-hexylphosphocholine (C6-DOPC) and 1,2-dierucoyl- sn-glycero-3-ethylphosphocholine (di22:1-EPC), have been examined with the aim of more completely understanding the mechanism of lipid-mediated DNA delivery. Both lipids form cubic phases: C6-DOPC in the entire temperature range from -10 to 90 degrees C, while di22:1-EPC exhibits an irreversible lamellar-cubic transition between 50 and 70 degrees C on heating. The lipoplexes formed by C6-DOPC arrange into hexagonal phase, while the lipoplexes of di22:1-EPC are lamellar. Both lipids exhibit lower transfection activity than the lamellar-forming 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine (EDOPC). Thus, for the studied cationic phospholipid-DNA systems, the lipoplex phase state is a factor that does not seem to correlate with transfection activity. The parameter that exhibits better correlation with the transfection activity within the present data set is the phase state of the lipid dispersion prior to the addition of DNA. Thus, the lamellar lipid dispersion (EDOPC) produces more efficient lipoplexes than the dispersion with coexisting lamellar and cubic aggregates (diC22:1-EPC), which is even more efficient than the purely cubic dispersions (C6-DOPC; diC22:1-EPC after heating). It could be inferred from these data and from previous research that cubic phase lipid aggregates are unlikely to be beneficial to transfection. The lack of correlation between the phase state of lipoplexes and their transfection activity observed within the present data set does not mean that lipid phase state is generally unimportant for lipofection: a viewpoint now emerging from our previous studies is that the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids.

A method to co-encapsulate gas and drugs in liposomes for ultrasound-controlled drug delivery.

We describe a novel method for the facile production of gas-containing liposomes with simultaneous drug encapsulation. Liposomes of phospholipid and cholesterol were prepared by conventional procedures of hydrating the lipid film, sonicating, freezing and thawing. A single but critical modification of this procedure generates liposomes that contain gas (air, perfluorocarbon, argon); after sonication, the lipid is placed under pressure with the gas of interest. After equilibration, the sample is frozen. The pressure is then reduced to atmospheric and the suspension thawed. This procedure leads to entrapment of air in amounts up to 10% by volume in lipid dispersions at moderate (10 mg/mL) concentrations of lipids. The amount of gas encapsulated increases with gas pressure and lipid concentration. Using 0.32 mol/L mannitol to provide an aqueous phase with physiological osmolarity, 1, 3, 6 or 9 atm of pressure was applied to 4 mg of lipid. This led to encapsulation of 10, 15, 20 and 30 microl of gas in a total of 400 microl of liposome dispersion (10 mg lipids/mL), respectively. The mechanism for gas encapsulation presumably depends on the fact that air (predominantly nitrogen and oxygen), like most solutes, dissolves poorly in ice and is excluded from the ice that forms during freezing. The excluded air then comes out of solution as air pockets that are stabilized in some form by a lipid coating. The presence of air in these preparations sensitizes them to ultrasound (1MHz, 8 W/cm2,10 s) such that up to half of their aqueous contents (which could be a water soluble drug) can be released by short (10 s) applications of ultrasound. Both diagnostic and therapeutic applications of the method are conceivable.

Effects on interactions of oppositely charged phospholipid vesicles of covalent attachment of polyethylene glycol oligomers to their surfaces: adhesion, hemifusion, full fusion and "endocytosis".

Oppositely charged giant vesicles are known to adhere, hemifuse and fuse, all of which depend upon the nature of surface contacts. To further understand such interactions, vesicles were surface-modified with polyethylene glycol (PEG), a moiety that reduces surface-surface interactions. Positively charged vesicles were composed of O-ethyldioleoylphosphocholine (EDOPC), dioleoylphosphatidylcholine (DOPC) and a carbocyanine dye (DiO), with and without DPPE-PEG (dipalmitoylphosphatidylethanolamine-N-PEG MW of the PEG portion = 2000). Negatively charged vesicles were composed of dioleoylphosphatidylglycerol (DOPG), DOPC and a rhodamine B dye (Rh-PE), with as well as without DPPE-PEG (MW 2,000). A microscope-mounted electrophoresis chamber allowed selected pairs of vesicles to be brought into contact while color images were collected at video rates (30 frames/s). Data collection focused on effects of PEG on vesicle interactions as a function of the surface charge density. Relative to PEG-free preparations, vesicles containing DPPE-PEG (1) formed larger contact zones, (2) underwent adhesion and fusion processes more slowly (by two to four times) and (3) at high charge density were less susceptible to rupture upon contact. Unexpectedly, PEG-containing vesicles exhibited engulfment of a smaller by a larger vesicle, a process topologically similar to cellular endocytosis. These observations are interpreted to mean that, although initial surface-surface interactions are weakened by the intervening layer of PEG chains, eventual and strong bilayer-bilayer contact is still possible, evidently because the lipid anchors of these chains can diffuse away from the contact zone.

Echogenic liposome compositions for increased retention of ultrasound reflectivity at physiologic temperature.

Targetable echogenic liposomes (ELIP) for ultrasound enhancement of atheroma have recently been developed; however, their retention of echogenicity at physiological temperature is less than desirable. The purpose of this study was to improve ELIP stability and increase clinical potential. The approach utilized the original procedures but involved manipulation of the lipid composition by reducing the level of unsaturation of the phospholipids components to minimize the rate of loss of echogenicity. Echogenicity was measured using a 20 MHz intravascular ultrasound (IVUS) catheter and quantified (as mean gray scale values) using computer-assisted videodensitometry. The optimal preparation for retention of echogenicity stability at physiologic temperature was egg phosphatidylcholine/dipalmitoylphosphatidylcholine/dipalmitoylphos-phatidylethanolamine/dipalmitoylphosphatidylglycerol/cholesterol (27:42:8:8:15, molar percent). This preparation retained 51 +/- 3.5% of its echogenicity after 1 h at 37 degrees C, more than 5x that retained by the previously descried preparation. In this composition nearly 2/3 of the phosphosphatidylcholine is fully saturated. Such an increase in saturation is anticipated to stiffen the lipid acyl chains. The air pockets that are responsible for reflection of ultrasound waves can be assumed to be stabilized by a lipid monolayer at the interface between the air and bulk water. The increased rigidity of that monolayer is presumed to be responsible for reducing the loss of air and extending the duration of echogenic activity. The stability of this improved formulation now appears to be more than adequate for clinical applications.

Single lipoplex study of cationic lipoid-DNA, self-assembled complexes.

A flow fluorometric analysis (using a commercial flow cytometer) of individual cationic lipoid-DNA complexes is presented. Such single lipoplex studies have the advantage of providing detailed characterization of heterogeneous ensembles of lipoplex preparations that cannot be obtained with methods that provide only population averages. Specifically, the composition (amount of lipoid and the lipoid-DNA ratio) was determined for statistically large ensembles (10 (3)-10 (4) particles) under a variety of conditions, including DNA:lipoid mixing ratio, lipoid dispersion method (extruded, vortexed), DNA morphology (linear, supercoiled), and concentration. In addition, the kinetics of formation were assessed for several conditions. Under essentially all conditions, two distinct regimes were observed, and on the basis of present and past data, these were identified as (1) coexistence of multilamellar lipoplexes and DNA-coated vesicles and (2) highly fused multilamellar complexes. The former outcome is favored by excess of DNA, reduced vesicle size, linear DNA, high concentration, and short incubation times. Fused multilamellar complexes represent the structures of lipoplexes usually used for DNA transfection; these were formed by interaction and breakdown of DNA-coated vesicles. Because the composition of individual lipoplexes could be determined, it was possible to assess how much of the bulk sample heterogeneity originates within individual vesicles and how much is due to differences between lipoplexes.

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently.

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, approximately 40-45 degrees C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer "frustration" which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a "frustrated" bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.

Synergy in lipofection by cationic lipid mixtures: superior activity at the gel-liquid crystalline phase transition.

Some mixtures of two cationic lipids including phospholipid compounds (O-ethylphosphatidylcholines) as well as common, commercially available cationic lipids, such as dimethylammonium bromides and trimethylammonium propanes, deliver therapeutic DNA considerably more efficiently than do the separate molecules. In an effort to rationalize this widespread "mixture synergism", we examined the phase behavior of the cationic lipid mixtures and constructed their binary phase diagrams. Among a group of more than 50 formulations, the compositions with maximum delivery activity resided unambiguously in the solid-liquid crystalline two-phase region at physiological temperature. Thus, the transfection efficacy of formulations exhibiting solid-liquid crystalline phase coexistence is more than 5 times higher than that of formulations in the gel (solid) phase and over twice that of liquid crystalline formulations; phase coexistence occurring at physiological temperature thus appears to contribute significantly to mixture synergism. This relationship between delivery activity and physical property can be rationalized on the basis of the known consequences of lipid-phase transitions, namely, the accumulation of defects and increased disorder at solid-liquid crystalline phase boundaries. Packing defects at the borders of coexisting solid and liquid crystalline domains, as well as large local density fluctuations, could be responsible for the enhanced fusogenicity of mixtures. This study leads to the important conclusion that manipulating the composition of the lipid carriers so that their phase transition takes place at physiological temperature can enhance their delivery efficacy.

Destruction thresholds of echogenic liposomes with clinical diagnostic ultrasound.

Echogenic liposomes (ELIP) are submicron-sized phospholipid vesicles that contain both gas and fluid. With antibody conjugation and drug incorporation, these liposomes can be used as novel targeted diagnostic and therapeutic ultrasound contrast agents. The utility of liposomes for contrast depends upon their stability in an acoustic field, whereas the use of liposomes for drug delivery requires the liberation of encapsulated gas and drug payload at the desired treatment site. The objective of this study was twofold: (1) to characterize the stability of liposome echogenicity after reconstitution and (2) to quantitate the acoustic destruction thresholds of liposomes as a function of peak rarefactional pressure (P(r)), pulse duration (PD) and pulse repetition frequency (PRF). The liposomes were insonified in an anechoic sample chamber using a Philips HDI 5000 diagnostic ultrasound scanner with a L12-5 linear array. Liposome stability was evaluated with 6.9-MHz fundamental and 4.5-MHz harmonic B-mode pulses at various P(r) at a fixed PRF. Liposome destruction thresholds were determined using 6.0-MHz Doppler pulses, by varying the PD with a fixed PRF of 1.25 kHz and by varying the PRF with a fixed PD of 3.33 micros. Videos or freeze-captured images were acquired during each insonation experiment and analyzed for echogenicity in a fixed region of interest as a function of time. An initial increase in echogenicity was observed for fundamental and harmonic B-mode imaging pulses. The threshold for acoustically driven diffusion of gas out of the liposomes using 6.0-MHz Doppler pulses was weakly dependent upon PRF and PD. The rapid fragmentation thresholds, however, were highly dependent upon PRF and PD. The quantification of acoustic destruction thresholds of ELIP is an important first step in their development as diagnostic and drug delivery agents.

Synergistic effect between components of mixtures of cationic amphipaths in transfection of primary endothelial cells.

To date, the primary approach to improving the transfection properties of cationic lipids has been the synthesis of new kinds of cationic amphipaths. Recently, however, it was found that combining two cationic lipid derivatives having the same head group but tails of different chain lengths can provide another, and often superior, approach to higher transfection efficiency. For example, the combination of medium-chain and long-chain homologues of O-ethylphosphatidylcholine transfected DNA into primary human umbilical artery endothelial cells (HUAECs) more than 30-fold more efficiently than did either compound separately. Here it is reported that this synergism of mixtures is not limited to O-ethylphosphatidylcholine homologues, but is also exhibited by other common cationic amphipathic transfection reagents; for example, combining DC-Chol (3beta-[N',N'-dimethylaminoethane)-carbamol] cholesterol), dimethylditetradecylammonium bromide, or DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane) with EDOPC increased transfection significantly both in the absence and in the presence of serum. Furthermore, combining a poorer transfection agent-dimethyldioctadecylammonium bromide-with dimethylditetradecylammonium bromide increased transfection by about an order of magnitude with a maximum at an intermediate composition. Lack of synergy occurred with some mixtures, such as DMTAP and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), in which case transfection activity was a linear function of composition both in the absence and presence of serum. Although the mechanism of enhanced transfection by mixtures is not fully understood, the existence of a number of optimal mixtures with diverse cationic compounds indicates that attention to mixture formulations can lead to greatly improved transfection by cationic amphipathic carriers.

Fibrin targeting of tissue plasminogen activator-loaded echogenic liposomes.

We recently reported entrapment of tissue-plasminogen activator (tPA) into echogenic liposomes (ELIP) with retention of echogenicity and thrombolytic effect. Integral to the potential of this agent for ultrasound-detectable local drug delivery is the specific binding of tPA-ELIP to clots. tPA contains fibrin-binding sites; we hypothesized that tPA when associated with ELIP, will maintain fibrin binding properties, rendering further manipulation for targeting of the tPA-ELIP unnecessary. We demonstrated strong fibrin binding of the ELIP-associated tPA. Fibrin binding for ELIP-associated tPA was twice that of free tPA. This strong affinity for fibrin was confirmed using echogenicity analysis of porcine clots in vitro. Both objective (mean gray scale analysis) and subjective (visual estimation by two experienced echocardiographers) evaluation of the clots showed enhanced highlighting of clots treated with tPA-ELIP when compared to control. The findings in this study represent new approaches for fibrin-targeted, ultrasound-directed and enhanced local delivery of a thrombolytic agent.

Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and readily releases DNA.

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.

Lipid encapsulation of arsenic trioxide attenuates cytotoxicity and allows for controlled anticancer drug release.

Arsenic trioxide (ATO, As2O3) is emerging as a front line agent for treatment of acute promyelocytic leukemia with giving a complete remission rate of 83-95%. ATO also shows significant activity in relapsed/refactory multiple myeloma; however, efforts to expand clinical utility to other cancers have been limited by its toxicity profile at higher doses. New bioavailable, liposome encapsulated As(III) materials exhibit a significantly attenuated cytotoxicity that undergoes pH-triggered release of an active drug. The arsenic drugs are loaded into 100-nm-scale liposomes at high concentration (>270 mM) and excellent retention (shelf life > 6 months at 4 degrees C), as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), transmission electron microscopy (TEM), and energy-dispersive X-ray (EDX) diffraction. In the loading mechanism, arsenous acid crosses the bilayer membrane in exchange for acetic acid and an insoluble transitional metal (e.g., Ni2+, Co2+) arsenite salt is formed. The resultant liposomal arsenic nanoparticles appear to be stable in physiological situations but release the drug cargo in a lower pH environment, as encountered in intracellular endosomes. These drugs exhibit attenuated cytotoxicities against human lymphoma tumor cells compared with that of free As2O3. Controlled release of arsenic drugs, and hence control of toxicity, is feasible with this system. The results demonstrate that cytotoxicity can be controlled via transitions of the inorganic drug between solid and solution phases and suggest a mechanism for further improvement of the risk/benefit ratio of As2O3 in treatment of a variety of cancers.