Immunomagnetic Particle Induced Lysis of Antibody-Conjugated Liposomes

Abstract

We have prepared liposomes of dioleoylphosphatidylethanolamine (DOPE) which have been stabilized by addition of 9-12 mol% N-biotinyl- phosphatidylethanolamine. Liposomes composed of DOPE/N-biotinyl-PE are quite stable and non-leaky although they exhibit strong temperature-dependent leakage following incorporation of palmitylated murine monoclonal antibodies as a targeting ligand. Addition of magnetic chromium dioxide particles coated with anti-mouse antibody to these immunoliposomes lead to their aggregation and the release of entrapped calcein. The lytic event was biphasic with an initial rapid release of 20% dye within 5 min. followed by a slower rate which reached nearly 40% release after 80 min. The rapid release phase was dependent upon the concentration of the liposomes and that of the multivalent particles. Lysis was immunospecific since no release was observed upon addition of nonspecific immunomagnetic particles to the immunoliposomes or if no antibody was incorporated into the liposome. Lysis could also be blocked by the addition of free murine antibody to the solution. The ability of these liposomes to release their contents in response to binding a multivalent antigen validates their potential for therapeutic or diagnostic applications.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Bilayer stabilization of phosphatidylethanolamine by N-biotinylphosphatidylethanolamine.

We have examined the ability of biotinylated phosphatidylethanolamine and similar lipids to stabilize the bilayer phase of polymorphic dioleoylphosphatidylethanolamine (DOPE). Sonicated lipid mixtures were characterized in terms of their aggregation state, size and ability to encapsulate and retain the fluorescent dye, calcein. Titration of DOPE with N-biotinyl-PE indicated that stable liposomes could be produced by sonication of DOPE based dispersions containing N-biotinyl-PE at concentrations greater than 8 mol%. These liposomes were relatively small, could efficiently encapsulate calcein, and showed minimal leakage upon prolonged storage at 4 degrees C. Maleimido-4-(p-phenylbutyrate)-PE (MPB-PE) was equally effective at stabilizing the bilayer phase of DOPE whereas N-dinitrophenyl-PE and N-(dinitrophenyl-caproyl)-PE were relatively poor stabilizers, requiring at least 15 mol% for stabilization at pH 7.4. Differential scanning calorimetry of dielaidoylphosphatidylethanolamine (DEPE)/N-biotinyl-PE mixtures indicated that stabilizer concentrations as low as 2 mol% could abolish the L alpha/HII phase transition of DEPE.     

Immunomodulation by liposome entrapped allergen

Liposomes are non-toxic, biodegradable and feebly immunogenic lipid vesicles made from natural and synthetic lipids. They are known to act as immunopotentiating agents and can be used to formulate sustained release preparation by encapsulation. In the present study, liposome entrapped allergen and free allergen were used to inject in Balb/C mice at different time intervals and their immune response in terms of specific IgG and specific IgE levels was quantitated by ELISA (Enzyme Linked Immuno sorbent Assay). The results indicated that specific IgE response was significantly higher in mice injected free allergen as compared to that of mice given liposome entrapped allergen. However, the specific IgG response was not statistically significant. Experiments carried out with liposome entrapped allergen and liposome coupled allergen showed no statistically significant difference in specific IgE and specific IgG titre between the two groups of mice. This type of immunomodulatory effect of liposomes in reducing IgE levels and without affecting IgG levels may be useful in Type I allergic disorders.     

Stable target-sensitive immunoliposomes.

Interaction of immunoliposomes composed of dioleoylphosphatidylethanolamine (DOPE) (80%), dioleoylphosphatidic acid (DOPA) (20%), and a small amount of specific antibody with Herpes Simplex virus (HSV) were studied by detecting the immune-dependent lysis of liposomes. DOPA was used as the principal stabilizer of the immunoliposomes. Antibodies conjugated with N-glutarylphosphatidylethanolamine or oxidized GM1 served as the target-specific ligands of immunoliposomes. These immunoliposomes (d = 160-180 nm) were stable for at least one month when stored at 4 degrees C. However, they undergo a rapid aggregation and lysis reaction in the presence of a membrane-bound target such as intact HSV virions. We have also employed epitope peptide-containing liposomes (target liposomes) to mimic the virus and showed that the immunoliposomes could be aggregated and lysed by the target liposomes in an antigen-dependent manner. Immunoliposome lysis could be accelerated by increasing the incubation temperature to 60-70 degrees C. No immunoliposome lysis was observed if the target liposomes were absent, indicating the prolonged stability of the immunoliposomes. Liposome lysis was always accompanied by liposome aggregation. However, the aggregation-induced liposome destabilization is unique to the HII phase-forming lipids such as DOPE. DOPC-containing immunoliposomes did not lyse despite the fact that massive liposome aggregation had taken place.     

Adjuvant effects of nonionic block polymer surfactants on liposome-induced humoral immune response

The ability of several surface-active agents to stimulate the humoral immune response in mice against haptenated liposomes was tested. The surfactants were block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) that differed in m.w., percentage of POE, and mode of linkage of POP to POE. The liposomes were haptenated with tripeptide-enlarged dinitrophenyl coupled to phosphatidylethanolamine, which was incorporated into the liposomal membrane. Additional injection of mice with surfactant stimulated serum hemagglutination titers and splenic plaque-forming cell (PFC) numbers to varying extents. Block polymers with POP chains flanking a POE center, as well as polymers with POE chains flanking a POP center, displayed high adjuvant activity. These block polymers stimulated the antibody response in a dose-dependent manner. They stimulated the antibody response with both high and low antigen doses. Furthermore, the addition of one of these adjuvants (25R1) reduced the amount of carrier lipid required in the liposome in order to obtain an optimal antibody response. The surfactants, which displayed high adjuvant activity, did not interfere with liposome stability as measured with a liposome lysis assay. Moreover, in vitro preincubation of liposomes with a block polymer did not affect their immunogenicity. Optimal adjuvant activity was observed when both adjuvant and liposomes were administered by the same route. Simultaneous injection of both components, however, is not a prerequisite. Conclusively, it can be stated that nonionic block polymer surfactants are potent adjuvants for stimulation of the antibody response against haptenated liposomes.     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Effects of bilayer surface charge density on molecular adsorption and transport across liposome bilayers

Second harmonic generation (SHG) was used to study both the adsorption of malachite green (MG), a positively charged organic dye, onto liposomes of different lipid compositions, and the transport kinetics of MG across the liposome bilayer in real time. We found that the dye adsorption increased linearly with the fraction of negatively charged lipids in the bilayer. Similarly, the transport rate constant for crossing the bilayer increased linearly with the fraction of charged lipid in the bilayer.     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Interactions between pH-sensitive liposomes and model membranes

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Structural and functional comparisons of pH-sensitive liposomes composed of phosphatidylethanolamine and three different diacylsuccinylglycerols

The titratable, double-chain amphiphiles 1,2-dipalmitoyl-sn-3-succinylglycerol (1,2-DPSG), 1,2-dioleoyl-sn-3-succinylglycerol (1,2-DOSG) and 1,3-dipalmitoylsuccinylglycerol (1,3-DPSG) have been used in combination with phosphatidylethanolamine (PE) to form pH-sensitive liposomes. The effect of the compounds on dielaidoyl PE bilayer stabilization was examined by differential scanning calorimetry. Only 1,2-DPSG showed bilayer stabilization activity; whereas the other two are destabilizers at pH 7.4. All three amphiphiles became strong destabilizers at pH 5.0. The ability of the amphiphiles to stabilize DOPE liposomes was examined by light scattering and calcein entrapment. In general, 1,2-DPSG is the most potent stabilizer of PE bilayers while 1,3-DPSG is the weakest liposome stabilizer. All three compounds can be combined with DOPE to generate liposomes which are stable at neutral and basic pH. At weakly acidic pH, the liposomes are leaky and exhibit extensive lipid mixing, with protons and calcium showing synergistic effects on lipid mixing. DOPE/1,2-DPSG liposomes are stable in human plasma and remain acid-sensitive even after prolonged plasma incubation. Immunoliposomes prepared from either DOPE/1,2-DPSG or DOPE/1,2-DOSG can deliver diphtheria toxin A fragment to the cytoplasm of cultured cells in a process which involves endocytosis of the liposomes. Immunoliposomes prepared with 1,2-DPSG are more effective drug carriers than those prepared with 1,2-DOSG. These results indicate that the bilayer- and, hence the liposome-stabilization activity of the diacylsuccinylglycerol depends on the structure of the compounds. The potential drug delivery activity of the pH-sensitive liposomes composed of these lipids is discussed.     

Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.     

A Novel Colourimetric Homogeneous Liposomal Immunoassay Using Sulphorhodamine B

Abstract

The dye, Sulphorhodamine B, was entrapped within liposomes prepared by the reverse evaporation technique. Marked differences in absorption spectra were found when free and entrapped dyes were compared, with a shift in the wavelength of maximum absorption. When entrapped dye was released by lysis of the liposomes, for example by detergent, the absorption spectrum reverted to that of free dye. This absorption change was employed in a novel marker system for complement-mediated immunoassay. As a model assay, human serum albumin was measured using this approach. Liposomes which had been coated with albumin were incubated with anti-albumin antibody and complement and the resulting absorption change measured using an automated spectrophotometric analyser. The decrease in absorption change on the addition of albumin formed the basis of a competitive homogeneous immunoassay for human serum albumin. Using purified albumin as a standard, a correlation of 0.96 was obtained when the albumin in human serum was measured in the liposomal assay and the results compared to measurements using a bromocresol green method.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

beta-Galactosidase-induced destabilization of liposome composed of phosphatidylethanolamine and ganglioside GM1

A novel type of liposome bilayer destabilization catalyzed by the enzyme, beta-galactosidase, is described. Unsaturated phosphatidylethanolamine (PE), an HII-phase-forming lipid, does not form stable liposomes at physiological temperature and pH. However, stable unilamellar liposomes can be prepared by mixing PE with a minimum of 5 mol% ganglioside GM1, a micellar-phase-forming lipid. Treatment of these GM1/PE liposomes with beta-galactosidase induces a rapid leakage (3-6 min) of the entrapped fluorescent dye, calcein. The studies indicate that liposome destabilization is the result of catalytic degradation of GM1, rather than a stoichiometric binding of GM1 by beta-galactosidase. Kinetic data indicate that the destabilization takes place via liposome collision. This simple, rapid method of liposome destabilization by beta-galactosidase will be useful in designing a liposome-based signal amplification mechanism for assays involving enzymes.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.     

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.