Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol).

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.

Hydrolyzable hydrophobic taxanes: synthesis and anti-cancer activities.

A series of taxane prodrugs with 2-bromoacyl chains attached at the 2'-position of the paclitaxel side chain, varying from six, eight, 12, 14 to 16 carbons in length, were synthesized, characterized and evaluated against human breast MCF-7 cancer cell line for their growth inhibitory (GI50) activities. The GI50 is the drug concentration required to inhibit cell growth by 50%. For comparison, hydrophobic taxanes varying in acyl chain lengths from six to 16 carbons were also synthesized and compared for their G050s with taxanes having equivalent bromoacyl chain lengths. The bromoacyl taxanes bearing six, eight and 12 carbon acyl chain lengths had GI50 values very similar to parent paclitaxel. The GI50 was 3 nM for three taxanes versus 1 nM for paclitaxel on the MCF-7 cell line. Increasing the acyl chain length to 14 or 16 carbons resulted in a significant decrease in cytotoxicity and an increase in the GI50 to 20 or 70 nM, respectively. In general, the GI50 values were directly related to the bromoacyl chain lengths in cultured tumor cells. Unlike bromoacyl taxanes, the taxanes lacking bromine in their acyl chain composition were 50- to 250-fold less active, suggesting that the heteroatom facilitated the hydrolysis of acyl chains to yield free paclitaxel. These differences in growth inhibitory activities may indirectly reflect differences in the susceptibility of the acyl chain to bromine-induced hydrolysis after association of the derivative with cell membranes. Liposome formulations of 2-bromoacyl taxanes bearing six, eight, 12 and 16 carbons were prepared and tested in SCID mice against a xenografted human ovcar-3 ovarian tumor. In vivo results showed that bromoacyl taxanes with a longer chain were therapeutically more efficacious than those with a short chain, presumably due to slow hydrolysis of the prodrug followed by sustained delivery of paclitaxel to the tumor.

A pharmacokinetic study of amphotericin B lipid complex injection (Abelcet) in patients with definite or probable systemic fungal infections.

This study describes a pharmacokinetic evaluation of amphotericin B (AMB) lipid complex injection (ABLC or Abelcet) in 17 patients with systemic fungal infection administered 5 mg/kg of body weight/day by infusion for 10 to 17 days. The results showed that AMB exhibited multiexponential disposition with high clearance, large volume of distribution at steady state, and long apparent elimination half-life but no evidence of accumulation in the blood after multiple daily doses. The results confirm previous observations and further reinforce the suggestion that ABLC may exist as a depot in the tissues from which free AMB is slowly released to limit exposure.

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion.

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.

Phospholipid-bound beta 2-glycoprotein I induces the production of anti-phospholipid antibodies.

Apoptotic-cell-bound beta2-glycoprotein I (beta2GPI), but not apoptotic cells or beta2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that beta2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human beta2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with beta2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+beta2GPI induced the highest levels of anti-beta2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while beta2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+beta2GPI was somewhat immunogenic, but less so than PG+beta2GPI. beta2GPI was immunogenic in the presence of native (CL(N)), but not hydrogenated (CL(H)), CL. Circular dichroism analysis demonstrated that the structure of beta2GPI was altered specifically by interaction with CL(N), but not other anionic PL, including CL(H). Similarly, the structure of CL(N)was affected by interaction with beta2GPI, as detected by(31)P nuclear magnetic resonance. These findings demonstrate that beta2GPI complexed with CL(N)is structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on beta2GPI upon interaction with CL(N)require the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.

Novel therapeutic nano-particles (lipocores): trapping poorly water soluble compounds.

The development of stable spherical lipid-coated drug particles that are termed 'lipocores' is reported here. Unlike conventional lipid-based particles (i.e. liposomes, emulsions, micelles), these particles are comprised solely of a core of a poorly water soluble drug surrounded by polyethyleneglycol conjugated lipid (PEG-lipid) and are formed via a 'kinetic' trapping process. These lipocore particles were made with the acyl chain of 16 carbon length (C16) acyl-chain derivatives of paclitaxel or vinblastine and with the polyene antifungal hamycin. Formation of the particles occurred regardless of the type of PEG-phospholipid used (i.e. acyl chain length, chain saturation, and polymer length) and could also be formed with the negatively charged lipid N-glutaryl-dioleoyl-phosphatidylethanolamine (DOPE-GA). Images from both freeze-fracture electron microscopy and electron cryo-microscopy revealed solid spherical structures with no internal lamellae for the PEG-lipid particles made with the C16 derivatives of paclitaxel (BrC16-T) or vinblastine (C16-Vin). From a solute distribution study of lipocores made with BrC16-T and distearoyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), the particles were found to have no measurable aqueous captured volume. Fluorescence anisotropy and order parameter measurements revealed the core material of these particles to be highly immobilized. The mole ratio of BrC16-T:lipid in the lipocores was typically > 90: < 10 and as high as 98:2, and the refrigerated lipocores were stable for several months. BrC16-T/DSPE-PEG2000 lipocores of 50-100 nm particle size were far less toxic than paclitaxel (Taxol) after intraperitoneally (i.p.) or intravenously (i.v.) administration in mice and were active against i.p. and subcutaneously (s.c.) planted human (OvCar3) ovarian carcinoma grown in SCID mice. It is believed the high drug:lipid ratio, the stability, and therapeutic efficacy of these novel particles make them a paradigm for delivery of poorly water soluble drugs and/or their hydrophobic derivatives.

A novel N-acyl phosphatidylethanolamine-containing delivery vehicle for spermine-condensed plasmid DNA.

A unique method for formulation of plasmid DNA with phospholipids has been devised for the purpose of producing vehicles that can mediate gene delivery and transfection of living cells. The polycation, spermine, was used to condense plasmid DNA within a water-in-chloroform emulsion stabilized by phospholipids. After organic solvent removal, the particles formed could be extruded to a number average size of about 200 nm and retained DNA that was protected from nuclease digestion. This resulted in a relatively high protected DNA-to-lipid ratio of approximately 1 microg DNA/micromol lipid. The size distribution of the preparation was relatively homogeneous as judged by light microscopy and quasi-elastic light scattering. Electron microscopic studies showed structural heterogeneity, but suggested that at least some of the plasmid DNA in this preparation was in the form of the previously observed spermine-condensed bent rods and toroids and was encapsulated within liposomal membranes. Preparations with the fusogenic phospholipid composition, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3-phosphocholine, showed transfection activity for several cells lines, particularly OVCAR-3 cells. The transfection activity sedimented with the lipid during centrifugation, confirming the association of active plasmid DNA with phospholipids. Transfection efficiency in culture was found to be of the same order of magnitude as cationic lipoplexes but much less toxic to the cells. Significant transfection of OVCAR-3 cells in tissue culture could also be observed, even in the presence of the intraperitoneal fluid from a mouse with an OVCAR-3 ascites tumor. These data indicate a new type of liposomal gene delivery system devoid of cationic lipids, phosphatidylethanolamine, cationic polymers and viral components.

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes.

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC >> DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.

Increased cell-surface receptor expression on U-937 cells induced by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

Association of the ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) with liposomes (ELL-12) reduces acute toxicity while maintaining or enhancing anticancer activity in experimental tumor models. ELL-12 has been shown to induce apoptosis by a cytochrome-c-dependent caspase-mediated pathway, which results in proteolytic cleavage of poly(ADP-ribose) polymerase and lamins, but the antitumor effects of ET-18-OCH3 or ELL-12 could result from tumor cell differentiation or activation. Here we compared the effects of ET-18-OCH3 and ELL-12 on the expression of cell-surface proteins associated with cell differentiation and/or activation in U-937 cells. Phorbol 12-myristate 13-acetate and all-trans-retinoic acid, which induce differentiation in U-937 cells, up-regulated CD11b (MAC1 alpha-integrin) and CD82 and down-regulated CD71 (transferrin receptor) in a time- and dose-dependent manner. In contrast, ET-18-OCH3 and ELL-12 up-regulated both CD71 and CD11b and did not have any effect on expression of CD82 in U-937 cells, suggesting that the ELL-12 may activate these cells rather than induce differentiation. Further evidence of activation was that ET-18-OCH3 and ELL-12 strongly induced tumor necrosis factor alpha production by U-937 cells.

Liposomal ET-18-OCH(3) induces cytochrome c-mediated apoptosis independently of CD95 (APO-1/Fas) signaling.

ELL-12, a liposome formulation of the ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH(3)), is a nonmyelosuppressive antiproliferative agent that is more effective and less toxic than the ether lipid itself in tumor model systems. We found that ELL-12 induced apoptosis in Jurkat, H9, and U-937 cells that was preceded by activation of executioner caspases. In addition, ELL-12 triggered release of cytochrome c from mitochondria to the cytoplasm before caspase-9 activation. Apoptosis, activation of caspases, and cytochrome c release were blocked by Bcl-x(L) overexpression in Jurkat T cells, suggesting a critical role for mitochondria in ELL-12-triggered cell death. Furthermore, ELL-12 had no effect on expression of CD95 ligand, and inhibition of the Fas signaling pathway with antagonistic anti-CD95 antibody did not affect apoptosis induced by ELL-12. Hence, ELL-12 could be a promising adjunct for the treatment of tumors in addition to myelosuppressive chemotherapeutic drugs and/or those that use the CD95-ligand/receptor system to trigger apoptosis.

Rigid contact lens fitting relationships in keratoconus. Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study Group.

PURPOSE: Although the influence of flat-fitting contact lenses on corneal scarring in keratoconus is frequently debated, the current standard of care with regard to the apical fitting relationship in keratoconus remains undocumented. METHODS: Patients were examined at baseline in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study (N = 1209). Patients wearing a rigid contact lens in one or both eyes (N = 808) had their habitual rigid contact lenses analyzed, and the fluorescein patterns and base curves were compared to the first definite apical clearance lens (FDACL). The FDACL is the flattest lens in the CLEK Study trial lens set that exhibits an apical clearance fluorescein pattern. For patients wearing a rigid contact lens in both eyes, one eye was selected randomly for analysis. RESULTS: Twelve percent of the rigid contact lens-wearing eyes were wearing lenses fitted with apical clearance based upon the clinician's fluorescein pattern interpretation. The remainder (88%) was wearing lenses fitted with apical touch. For mild (steep keratometric reading <45 D) keratoconus corneas, the mean estimate of the base curve to cornea-fitting relationship was 1.18 D flat (SD +/- 1.84 D); moderate (steep keratometric reading: 45 to 52 D) corneas were fitted on average 2.38 D flat (SD +/- 2.56 D); and severe (steep keratometric reading > 52 D) corneas were fitted an average of 4.01 D flat (SD +/- 4.11 D). CONCLUSIONS: Despite the potential risk for corneal scarring imposed by flat-fitting rigid contact lenses, most CLEK Study patients wear flat-fitting lenses. Overall, rigid lenses were fitted an average of 2.86 D (SD +/- 3.31 D) flatter than the FDACL.

Differential effects of free and liposome-associated 1-O-octadecyl-2-O-methylglycerophosphocholine on protein kinase C.

Incorporation of ET-18-OCH3 into well-characterized liposomes known as ELL-12 has eliminated its gastrointestinal and hemolytic toxicity without loss of growth inhibiting activity. ET-18-OCH3, but not ELL-12, blunted the increase in membrane protein kinase C (PKC) activity induced by 12-O-tetradecanoylphorbol 13-myristate (TPA) and markedly reduced levels of PKC alpha in NIH 3T3 fibroblasts. Furthermore, prolonged treatment with ELL-12 neither inhibited TPA-induced translocations of PKC alpha and PKC delta to the particulate fraction nor caused down-regulation, and did not affect the cellular distribution of TPA-insensitive PKC zeta. In Jurkat T cells, where ELL-12 markedly induced apoptosis that was blocked by an inhibitor of caspase-3-like activities, it had no effect on PKC activity or translocation induced by TPA. Thus, it seems unlikely that PKC is involved in the therapeutic effects of ELL-12.

Elastase activated liposomal delivery to nucleated cells.

The specific activation of liposomes for delivery has been explored by enzyme mediated cleavage of a peptide substrate covalently conjugated to a fusogenic lipid. We have previously shown an elastase sensitive peptide conjugated to 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [corrected] (DOPE) could be activated by enzymatic cleavage, triggering liposome-liposome lipid mixing and fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13-27). Further optimization of this system has been aimed at obtaining substrate cleavage at or below physiological elastase levels and to demonstrate triggered delivery to living cells. Therefore a new peptide-lipid, MeO-suc-AAPV-DOPE (N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE), has been developed that exhibits greater sensitivity and selectivity for elastase cleavage and subsequent conversion to DOPE. This peptide-lipid was used with DODAP (dioleoyl dimethylammonium propane, a pH dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposome with an overall positive charge if enzymatic cleavage had occurred. Elastase treated liposomes displayed pH dependent enhancement of binding, lipid mixing, and delivery of 10000 MW dextrans, relative to untreated liposomes, when incubated with HL60 human leukemic cells. Heat denatured elastase did not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating enzymatic activity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be activated by elastase to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm. These results suggest enzyme substrate peptides linked to a fusogenic lipid may be used to elicit specific delivery from liposomes to cells.

Inhibition of cell division but not nuclear division by 1-O- octadecyl-2-O-methyl-Sn-glycero-3-phosphocholine.

1-O-Octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH(3)) selectively inhibits the growth of cancer cells. Here we show that in some cell types ET-18-OCH(3)and liposome-associated ET-18-OCH(3)inhibit cell division without concurrent inhibition of nuclear division, leading to multinucleate cell formation, and cell death through apoptosis. Cell cycle analysis revealed that ET-18-OCH(3)-treated U-937 cells continued to move through the cell cycle, but many cells were not able to divide and instead accumulated as tetraploid cells or octaploid cells in the G0/G1 phase of the cell cycle. Inhibition of cytokinesis has been shown to be paralleled by activation of U-937 cells, including upregulation of some cell-surface markers, acquisition of phagocytic activity, and secretion of tumor necrosis factor (TNF)-alpha (Pushkareva et al., 2000). Furthermore, treatment of cells with ET-18-OCH(3)results in the accumulation of apoptotic cells in time- and dose-dependent manner. It is possible that inhibition of cytokinesis may be related to cytoskeletal effects.

Growth inhibition of a human ovarian tumor by a novel paclitaxel derivative in SCID mice.

We report here the toxicity and therapeutic effects of 2'-alpha-bromohexadecanoyl paclitaxel (BrC16HT), a prodrug form of paclitaxel, in mice. Paclitaxel is the active ingredient of Taxol. The maximum tolerated dose, at a one dose per day for 5-day schedule, was 37.5 mg/kg for BrC16HT compared to 12.5 for Taxol administered IP, and was 12.5-25 mg/kg for either agent administered IV. Dose-dependent therapeutic effects were found for BrC16HT against a human ovarian tumor (OVCAR-3) grown in SCID mice. IP treatments with BrC16HT against early or established IP-implanted OVCAR-3 tumor increased mean survival times more than treatment with Taxol. Long-term survivors were found only in groups treated with BrC16HT. Intravenously administered BrC16HT was more effective than Taxol against SC OVCAR-3 tumor. Early treatment (25 mg/kg x 5) completely inhibited tumor growth through 120 days after tumor implantation. Pharmacokinetic studies suggest that BrC16HT is slowly hydrolyzed to paclitaxel and circulates longer than paclitaxel from Taxol. Thus, BrC16HT may provide sustained levels of paclitaxel, which may contribute to the increased efficacy of BrC16HT compared to Taxol.

Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient.

We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.

Triggerable liposomal fusion by enzyme cleavage of a novel peptide-lipid conjugate.

A novel peptide-lipid sensitive to enzyme cleavage was designed to generate liposomes that could be triggered to fuse by enzymatic activation. Covalent linkage of dioleoyl phosphatidylethanolamine (DOPE) to an elastase substrate, N-acetyl-ala-ala-, resulted in a cleavable peptide-lipid (N-Ac-AA-DOPE) with no intrinsic fusogenic activity. Cleavage of N-Ac-AA-DOPE and concomitant conversion to the fusogenic lipid DOPE could be detected after treatment with human leukocyte elastase or proteinase K, two proteases with similar substrate specificities. A strategy to utilize this cleavage to trigger fusogenicity was tested by modeling the fusion of liposomes containing the expected product of complete cleavage. Based on these results liposomes were designed to contain N-Ac-AA-DOPE, DOTAP, and PE in the ratio of 15/15/70. These liposomes exhibited lipid mixing with acceptor liposomes after elastase or proteinase K protease treatment. Activation of fusion, as monitored by a lipid mixing assay, appeared to be dependent on protease activity, as (1) heat inactivated enzyme did not activate liposomal fusion, and (2) the time and concentration dependence of proteinase K mediated cleavage of N-Ac-AA-DOPE correlated with membrane mixing. Liposomes could also be formulated that exhibited lipid mixing and transfer of aqueous fluorescent probe with erythrocyte ghosts. These observations demonstrate fusogenic lipids conjugated to enzyme substrates serve as triggerable fusion systems that may be useful for gene and drug delivery.

In vitro and in vivo antifungal activity of amphotericin B lipid complex: are phospholipases important?

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids L-alpha-dimyristoylphosphatidylcholine (DMPC) and L-alpha-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.

Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine.

N-acyl phosphatidylethanolamines (NAPEs) are natural lipid components of many organisms. N-acylation of unsaturated phosphatidylethanolamines with a saturated fatty acid converts them from non-lamellar organizing lipids into lamellar organizing, acidic lipids which can interact with cations and potentially return to non-lamellar structures. These special properties make NAPEs candidates for fusogens. We tested the fusogenicity of one of the NAPEs, N-dodecanoyl-di-oleoylphosphatidylethanolamine (N-C12-DOPE) mixed with dioleoylphosphatidylcholine (DOPC) in liposomes. Binding and fusion to erythrocyte ghosts in the presence of 3 mM Ca2+ required at least 60 mol% of N-C12-DOPE. Fusion was not observed when phosphatidylglycerol or phosphatidylserine was substituted for N-C12-DOPE, indicating specificity for properties of this lipid. Binding of N-C12-DOPE/DOPC (70:30) liposomes required 1 mM Ca2+ while 1.25 mM Ca2+ and Mg2+ were sufficient for lipid mixing and delivery of encapsulated dextrans to erythrocyte ghosts. These liposomes also bound and possibly mixed lipid with nucleated U-937 cells in a Ca2+ -and endocytosis-dependent manner. Low pH-dependent fusion with ghosts was observed in the absence of any divalent cation, indicating that fusion with U-937 cells could result after endocytosis into the acidic endosomes. The possible mechanisms for N-C12-DOPE mediated binding and fusion and the potential application of these liposomes as delivery vehicles for therapeutic agents are discussed.

Enhancement of the in vivo circulation lifetime of L-alpha-distearoylphosphatidylcholine liposomes: importance of liposomal aggregation versus complement opsonization.

Incorporation of N-(omega-carboxy)acylamido-phosphatidylethanolamines (-PEs) into large unilamellar vesicles (LUVs) of L-alpha-distearoylphosphatidylcholine (DSPC) was found to dramatically increase the in vivo liposomal circulation lifetime in rats, reaching a maximal effect at 10 mol.% of the total phospholipid. Neither pure DSPC liposomes nor those with the longest circulating derivative, N-glutaryl-dipalmitoylphosphatidylethanolamine (-DPPE), were found to significantly bind complement from serum. Therefore, the relatively short circulation time of pure DSPC liposomes did not appear to be related to greater complement opsonization leading to uptake by the reticuloendothelial system. However, N-(omega-carboxy)acylamido-PEs were particularly efficient inhibitors of a limited aggregation detected for pure DSPC liposomes. The aggregation tendency of DSPC liposomes incorporating various structural analogs of N-glutaryl-DPPE correlated inversely with the circulation lifetimes. Therefore, it is concluded that such PE derivatives enhance the circulation time by preventing liposomal aggregation and avoiding a poorly understood mechanism of clearance that is dependent on size but is independent of complement opsonization. At high concentrations of N-glutaryl-DPPE (above 10 mol.%), the liposomes exhibited strong complement opsonization and were cleared from circulation rapidly, as were other highly negatively charged liposomes. These data demonstrate that both the lack of opsonization and the lack of a tendency to aggregate are required for long circulation. Liposomal disaggregation via N-(omega-carboxy)acylamido-PEs yields a new class of large unilamellar DSPC liposomes with circulation lifetimes that are comparable to those of sterically stabilized liposomes.