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.

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.

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.

Conformational changes and fusion activity of vesicular stomatitis virus glycoprotein: [125I]iodonaphthyl azide photolabeling studies in biological membranes.

The interaction of VSV glycoprotein (VSV G) with biological membranes was studied by photosensitized labeling. The method is based on photosensitized activation by the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine (DiO) of a hydrophobic probe, [125I]iodonaphthyl azide (125INA), that rapidly partitions into the membrane bilayer of virus and cells. 125INA labeling of proteins and lipids can be confined to the site of chromophore localization by photosensitized labeling. Photoactivation using visible light of target membrane labeled with DiO and 125INA, to which unlabeled virions are bound, results in exclusive labeling of envelope glycoproteins inserted into the target membrane [Pak et al. (1994) J. Biol. Chem. 269, 14614]. In this study, we labeled lipid symmetric erythrocyte ghosts with 125INA and DiO. Photosensitized activation of VSV prebound to labeled ghosts with visible light resulted in VSV G labeling under fusogenic conditions. Photoactivation of 125INA by UV light, which is nonspecific, produced labeled VSV G at both acidic and neutral pH. Photosensitized labeling of VSV G by DiO-125INA-ghosts was also observed at pH 5.5, 4 degrees C, in the absence of mixing between viral and cellular lipids, suggesting insertion of the ectodomain of VSV G. Soluble VSV G lacking the transmembrane domain inserted into DiO-125INA-ghosts under the same conditions as intact VSV G. DiO inserted into intact VSV appeared to be a suitable fluorophore for continuous kinetic measurements of membrane fusion by fluorescence dequenching. Our photosensitized labeling results establish biochemical correlates for the three states of VSV G, which we had proposed based on kinetic data [Clague et al., Biochemistry 29, 1303]. In addition, we found that VSV G insertion into the target membrane is reversible, suggesting a "velcro"-like attachment of the fusogenic domain with the target membrane.

Effect of X31 influenza virus fusion on phosphatidylserine asymmetry in erythrocytes.

Influenza virus fusion is mediated by its fusion protein, hemagglutinin (HA). HA undergoes a low pH dependent conformational change that results in insertion into the cell membrane bilayer, formation of a fusion pore, and merging of membrane lipids and establishment of cytoplasmic continuity. Erythrocytes, which can serve as targets of influenza virus fusion, display an asymmetric transbilayer arrangement of their phospholipids. The effect of influenza virus fusion on erythrocyte phosphatidylserine asymmetry was determined. Influenza virus were bound to erythrocytes containing the fluorescent membrane probe NBD-PS in the inner leaflet. Induction of fusion by exposure to a low pH environment resulted in movement of PS to the outer leaflet of the cell as well as hemolysis. Insertion of the fusion protein into erythrocytes and subsequent fusion can be distinguished from hemolysis by examining the interaction of a soluble form of HA (BHA) with cells and by monitoring viral fusion at low temperatures. No hemolysis was observed under either condition. BHA binding and insertion into cells did not affect the asymmetry of PS. Incubation of influenza virus fusion at pH 5, 0 degrees C resulted in complete fusion but no outward movement of PS was observed. These findings suggest the viral fusion pore does not involve a rearrangement of the transbilayer phospholipid organization of the target membrane.

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Detection of influenza hemagglutinin interaction with biological membranes by photosensitized activation of [125I]iodonaphthylazide.

Fusion of influenza virus with cells is triggered by a pH-dependent conformational change in the viral envelope protein, hemagglutinin, which results in exposure of the fusion peptide and its insertion into the target membrane. We have investigated the association of hemagglutinin with erythrocyte membranes by photosensitized labeling with [125I]iodonaphthylazide. This technique relies on the collisional energy transfer from a photosensitizing chromophore to [125I]iodonaphthylazide, which selectively labels proteins in the vicinity of the chromophore. Incubation of influenza virus with erythrocyte membranes containing chromophore and [125I]iodonaphthylazide results in labeling of hemagglutinin under fusogenic conditions (pH 5 and 37 degrees C). We also examined photosensitized labeling of hemagglutinin upon incubation of the X31 strain of influenza virus with labeled erythrocyte membranes in a pre-fusion state (pH 5 and 4 degrees C). There was little hemagglutinin labeling under these conditions, although incubation of bromelain-cleaved hemagglutinin, which lacks the transmembrane region, resulted in rapid labeling. Hemagglutinin was also labeled by [125I]iodonaphthylazide photosensitized by a fluorescent substrate transported through the erythrocyte band 3 sialoglycoprotein. Hemagglutinin labeling decreased after an initial rapid rise, suggesting that the fusion site is close to the sialoglycoprotein and that [125I]iodonaphthylazide photosensitized labeling may be used to assay protein movement during fusion.

Intermediates in influenza virus PR/8 haemagglutinin-induced membrane fusion.

The fusion kinetics with erythrocyte ghosts of two influenza A virus strains, A/Aichi/2/68 (X:31) and A/PR/8/34 (PR/8), were compared and correlated with the kinetics of haemagglutinin (HA) conformational change. Previously it had been shown that X:31 fuses with liposomes or erythrocytes at 4 degrees C, pH 5 after a lag time of 5 to 10 min whereas PR/8 displayed no fusion with liposomes at that temperature. We have confirmed the absence of cold fusion by PR/8 with erythrocyte ghosts. In contrast to X:31, PR/8 could not be committed to fuse at neutral pH and 37 degrees C by a preincubation at low pH and 4 degrees C. To examine whether the lack of commitment and cold fusion were due to a failure of PR/8 HA to undergo conformational changes at low temperature and pH, we analysed susceptibility of HA to proteinase K digestion, liposome binding to the virus, and immunoprecipitations of HA with conformation-specific antibodies. Although there was little binding of PR/8 to liposomes at 4 degrees C and pH 5, we did observe exposure of the fusion peptide. This study reveals a low temperature intermediate in membrane fusion exhibited by the HA of influenza virus strain PR/8, which involves low pH-induced conformational changes including exposure of the fusion peptide with little interaction of HA with the target membrane.

Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells.

Human red blood cells (RBC) were separated by density on self-forming Percoll gradients into five distinct populations. The transbilayer movement and equilibrium distribution of 1-oleoyl-2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminocaproyl)phosphatidylserine (NBD-PS) was slower in dense cells and equilibrated in the inner leaflet of these cells to a lesser degree than in light cells. Conversely, the outward movement of the lipid was slower in light cells. Assessment of endogenous PS in the cells' outer leaflet by the prothrombinase activity of externalized PS revealed an increase in its presence at the cell surface with increasing cell density. The presence of PS on the cell surface directly correlated with the propensity of the RBC to bound by autologous monocytes. To determine whether increased cell density is associated with increased cell age, the in vivo clearance of density-separated murine RBC was monitored in syngeneic mice. The T1/2 of circulation of light cells was about twice that of dense cells. The majority of the cleared cells localized in the spleen. Studies carried out in antibody-deficient SCID mice indicated that RBC were cleared via a mechanism that was independent of antibody. These data suggest that cell age is related to cell density and that cells of increasing age and density display higher amounts of PS in their outer leaflet.

Molecular mechanisms for activated macrophage recognition of tumor cells.

The biological heterogeneity of tumor cells requires a therapeutic modality that recognizes and kills resistant as well as susceptible tumor cells but does not harm normal cells. Tumoricidal macrophages appear to be able to fulfill these criteria. The mechanisms by which macrophages recognize tumor cells are not known, but they recognize carbohydrates and proteins that may be relevant for binding to tumor cells. In addition, phospholipids appear to be involved in the macrophage-tumor cell interaction. The abnormal presence of phosphatidylserine (PS) on the outer leaflet of tumor cells correlates with enhanced binding and cytotoxicity by macrophages.

Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages.

We examined the activation to the tumoricidal state of normal mouse peritoneal exudate macrophages, bone marrow macrophages, and human blood monocytes by liposomes containing either lipophilic muramyl tripeptide (CGP 19,835) or a new synthetic analogue of lipoprotein from gram-negative bacteria outer wall, CGP 31,362, or combinations of the two. The superiority of liposomes containing the synthetic lipopeptide over liposomes containing lipophilic muramyl tripeptide for in vitro activation of monocytes and macrophages was demonstrated in several experiments. First, liposome-CGP-19,835 activated monocytes only in the presence of interferon-gamma, whereas activation with liposome-CGP 31,362 was interferon-independent. Second, activation of both mouse macrophages and human blood monocytes by liposome-CGP 31,362 occurred at a lower liposomal concentration than that by liposome-CGP 19,835. Third, monocytes incubated with liposome-CGP 31,362 released both tumor necrosis factor (TNF) and interleukin-1 activities, whereas monocytes treated with liposome-CGP 19,835 (in the absence of interferon-gamma) released only TNF activity. These data suggest that liposomes containing the synthetic lipopeptide CGP 31,362 are superior to liposomes containing CGP 19,835 for systemic activation of macrophages.

Liposomal delivery of biological response modifiers to macrophages.

Macrophages activated to the tumoricidal state can recognize and destroy neoplastic cells and leave normal cells unharmed. Systemic activation of macrophages can be achieved by the intravenous administration of liposomes containing various immunomodulators. Much like any particle, liposomes are cleared from the circulation by phagocytic cells. This passive but specific targeting of immunomodulators to macrophages results in their activation for in vitro and in vivo lysis of tumor cells that can be resistant to conventional therapies.

Macrophages and cancer.

The uncontrolled growth of metastases resistant to conventional therapeutic modalities is a major cause of death from cancer. Data from our laboratory and others indicate that metastases arise from the nonrandom spread of specialized malignant cells that preexist within a primary neoplasm. These metastases can be clonal in their origin, and different metastases can originate from different progenitor cells. In addition, metastatic cells can exhibit an increased rate of spontaneous mutation compared with benign nonmetastatic cells. These data provide an explanation for the clinical observation that multiple metastases can exhibit different sensitivities to the same therapeutic modalities. These findings suggest that the successful therapy of disseminated metastases will have to circumvent the problems of neoplastic heterogeneity and the development of resistance. Appropriately activated macrophages can fulfill these demanding criteria. Macrophages can be activated to become tumoricidal by interaction with phospholipid vesicles (liposomes) containing immunomodulators. Tumoricidal macrophages can recognize and destroy neoplastic cells in vitro and in vivo, leaving nonneoplastic cells uninjured. Although the exact mechanism(s) by which macrophages discriminate between tumorigenic and normal cells is unknown, it is independent of tumor cell characteristics such as immunogenicity, metastatic potential, and sensitivity to cytotoxic drugs. Moreover, macrophage destruction of tumor cells apparently is not associated with the development of tumor cell resistance. Macrophages are found in association with malignant tumors in a definable pattern, suggesting that the most direct way to achieve macrophage-mediated tumor regression is in situ macrophage activation. Intravenously administered liposomes are cleared from the circulation by phagocytic cells, including macrophages, so when liposomes containing immunomodulators are endocytosed, cytotoxic macrophages are generated in situ. The administration of such liposomes in certain protocols has been shown to bring about eradication of cancer metastases. Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at the start of treatment is minimal. For this reason, we have been investigating various methods to achieve maximal cytoreduction in metastases by modalities such as chemotherapy or radiotherapy prior to macrophage-directed therapy. It is important to note that even the destruction of 99.9% of cells in a metastasis measuring 1 cm2 would leave 10(6) cells to proliferate and kill the host. The ability of tumoricidal macrophages to distinguish neoplastic from bystander nonneoplastic cells presents an attractive possibility for treatment of the few tumor cells which escape destruction by conventional treatments. Macrophage-directed therapy has been studied in several human protocols, yielding important biological information about the use of liposome-encapsulated macrophage activators in cancer patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Activated macrophages distinguish undifferentiated-tumorigenic from differentiated-nontumorigenic murine erythroleukemia cells.

The interaction between macrophages and differentiating cells was examined using murine erythroleukemia cells (MELC). Inflammatory macrophages activated with recombinant murine interferon-gamma (rMuIFN-gamma) and lipopolysaccharide (LPS) first specifically recognized and bound tumorigenic-undifferentiated MELC and then produced their lysis. MELC that were induced to differentiate by a 5-day treatment with 5 mM N,N'-hexamethylene-bis-acetamide (HMBA) accumulated hemoglobin (benzidine positive) and were not recognized by the macrophages. Qualitative examination by light and electron microscopy confirmed the specific nature of the macrophage-MELC interaction. Quantitative assessment showed that the binding was dependent on the temperature and divalent cations and independent of serum components. A 24-h treatment of MELC with HMBA resulted in decreased binding, prior to hemoglobin accumulation and commitment to differentiation. The lack of binding of nontumorigenic-differentiated cells by macrophages was not due to residual HMBA. It thus appears that macrophages can distinguish MELC at different stages of differentiation.

Inhaled atropine sulfate: dose-response characteristics in adult patients with chronic airflow obstruction.

Dose-response characteristics of inhaled atropine sulfate were examined in ten patients with chronic airflow obstruction using spirometric and plethysmographic measurements. Inhaled atropine in doses of 0.005, 0.01, 0.25, and 0.05 mg2kg of body weight and placebo were delivered by means of a precision metering device. All pulmonary function tests (FEV1, V50, and SGaw) improved progressively with increasing dose. There was a high degree of linear correlation between the peak response of each test and the logarithm of dose (r greater than or equal to 0.98). The highest dose studied (0.05 mg/kg) was found to have marginal benefit over 0.025 mg/kg, and had the highest incidence of adverse reactions. Duration of effect was dependent on dose. These results suggest that for adult patients with chronic airflow obstruction, 0.025 mg/kg delivered by a dosimeter approximates the optimally effective dose of inhaled atropine sulfate that can be given without unacceptable side effects.