Liposomal vincristine preparations which exhibit decreased drug toxicity and increased activity against murine L1210 and P388 tumors.

The toxicity and antitumor activity of liposomal vincristine preparations have been examined. Vincristine was encapsulated inside egg phosphatidylcholine (EPC)/cholesterol (55/45, mol/mol) and distearoylphosphatidylcholine (DSPC)/cholesterol (55/45, mol/mol) vesicles utilizing transmembrane pH gradient (inside acidic) drug uptake processes. Trapping efficiencies approaching 100% were achieved for this procedure using drug:lipid ratios as high as 0.2:1 (w/w). Although both EPC/cholesterol and DSPC/cholesterol liposomal systems yielded high trapping efficiencies, DSPC/cholesterol vesicles exhibited superior drug retention properties. This ability to retain entrapped vincristine was related to maintenance of the transmembrane pH gradient as well as the membrane permeability properties. Thirty-day dose-response survival studies in mice indicated that vincristine encapsulated in DSPC/cholesterol liposomes was less toxic than free drug. The 50% lethal dose of 1.9 mg/kg in CD-1 mice observed for free vincristine increased to 4.8 mg/kg upon administration of the drug in liposomal form. Liposome encapsulation of vincristine also enhanced the antitumor activity against murine P388 and L1210 lymphocytic leukemia models. This resulted from increased efficacy for liposomal vincristine at doses equal to free drug (liposomal/free drug median survival times greater than 1.0) as well as the ability to administer increased doses of liposomal vincristine. The combined effects of decreased toxicity and increased antitumor efficacy of liposomal vincristine over free drug suggest significant clinical utility of appropriate liposomal vincristine systems.

Binding of biotinated-liposomes to streptavidin is influenced by liposome composition.

We describe an 'in vitro' assay which allows rapid quantification of the binding of biotinated-vesicles to streptavidin immobilised on microtitre plates by estimating levels of a liposome encapsulated fluorescent molecule, rhodamine 123. It is shown that optimal vesicle binding to streptavidin occurs when a six carbon biotin spacer arm derivative of distearoylphosphatidylethanolamine (biotin-X-DSPE) is incorporated in liposomes. This alleviates steric hindrance arising due to the inclusion of small amounts of large bulky amphiphiles such as monosialoganglioside (GM1, 5 mol%) in vesicles. In contrast the ability of liposomes containing poly(ethylene glycol) derivatives of DSPE (PEG2000-DSPE, 5 mol%) to bind streptavidin was only marginally better when biotin-X-DSPE was substituted for biotin-DSPE in vesicles. It is further shown that amounts of biotinated-vesicles bound to streptavidin were minimally influenced by the fluidity of the liposome preparation when assayed at 4 degrees C. However, at elevated temperatures (37 degrees C) lipid estimates as determined by vesicle entrapped rhodamine 123 were low due to leakage of this marker from vesicles. This was shown by comparing amounts of biotinated-liposomes bound to streptavidin coated plates using the lipid marker [3H]cholesteryl hexadecyl ether to estimates determined by vesicle entrapped rhodamine 123. The 'in vitro' assay protocol described here is a general method applicable in the optimisation of other targeting protocols. In conclusion our work suggests that liposomes containing GM1 and the spacer arm derivative biotin-X-DSPE bind optimally to immobilised streptavidin which should aid in the use of biotinated-liposomes in 'in vivo' targeted delivery applications.

A non-covalent method of attaching antibodies to liposomes.

A novel non-covalent method of attaching antibodies to liposomes which exploits the high affinity of streptavidin for biotin, is described. The two-step coupling protocol involves the initial attachment of streptavidin to liposomes containing biotin PE, followed by the coupling of biotinated antibodies to streptavidin-liposomes. The association of streptavidin with liposomes containing biotinated PE is rapid (less than 5 min), resulting in a maximum association of 40 molecules of streptavidin per 100 nm vesicle. In the presence of equimolar cholesterol, the amount of streptavidin bound is twice that observed when biotin PE/egg PC liposomes are used. Irrespective of the mole ratio of biotin to antibody (e.g. for 1-6 biotins per antibody), or the molar ratio of antibody to streptavidin in the second incubation step, equimolar amounts of antibody bind to streptavidin. It is shown that anti-rat-erythrocyte IgG or F(ab')2 complexed to liposomes via the streptavidin linker bind specifically to rat erythrocytes but not to human erythrocytes. This coupling protocol can be readily extended to other biotinated antibodies.

Hydrogen exchange kinetics and the mechanism of reaction B of yeast tryptophan synthase.

It has been shown that yeast tryptophan synthase (L-serine hydro-lyase (adding indoleglycerol-phosphate) EC 4.2.1.20) catalyses tritium exchange reactions between protons on the alpha-carbon of L-serine of L-tryptophan, and water. The absolute rates of these reactions and indole-serine condensation (reaction B), all of which are pyridoxal phosphate-dependent, were measured. L-Serine exchange was resolved into two components, a high-affinity, slow, Michaelian reaction (KmS,H = 0.06 mM, kcats,H 3 X 10(-3) s-1) and a faster reaction (kcat greater than 2.5 S-1) which was not saturated even at 100 mM L-serine. Hydrogen exchange by tryptophan was a Michaelian process (KmT,H = 2.9 mM; kcatT,H = 0.6 s-1). Indole did not inhibit either exchange reaction. A plausible explanation of the results, that reaction B has a ping-pong mechanism with serine as first substrate and water and L-tryptophan as first and second products, respectively, was inadequate because of the observations that L-tryptophan is as first and second products, respectively, was inadequate because of the observations that L-tryptophan is synthesised with less than 1 mol of exchanged proton per mol amino acid, and that the ratio kcat/Km for serine changes between enzyme reactions. A branched modification with two enzyme-serine complexes, only one of which will exchange protons with water, will fit all the results.

Protein-liposome conjugates with defined size distributions.

Conjugation of protein to liposomes by two coupling protocols is shown to result in vesicle aggregation. The degree of aggregation is directly related to the levels of protein conjugated to the liposomes. In an attempt to develop a method of generating stable, homogeneously sized protein-conjugated vesicles, highly aggregated liposome-protein conjugates were extruded through filters of defined pore size distributions, with no loss of protein binding. The extruded samples are relatively stable with respect to size and are easily prepared for various protein to lipid ratios. Liposome size has been shown to be a major factor in determining the in vivo blood circulation times of liposomes. A corresponding, significant enhancement in the blood circulation lifetimes for extruded versus aggregated streptavidin-liposome conjugates is observed. Furthermore, the stability of streptavidin-liposome conjugates in vivo was shown by the binding of biotin to liposomes isolated from plasma 1 and 4 h post-injection. In conclusion, extrusion of the aggregated systems obtained on coupling proteins to liposomes provides a convenient and general method for generating homogeneously sized protein-liposome conjugates.

Characterisation of biotinylated liposomes for in vivo targeting applications.

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.

Optimized procedures for the coupling of proteins to liposomes.

A general, optimized method for coupling proteins to liposomes is presented. This procedure utilizes streptavidin covalently coupled to liposomes to allow the subsequent attachment of a variety of biotinated proteins of interest. In the first part of this study, covalent methods for coupling proteins to liposomes which contain the lipid derivatives MPB-PE and PDP-PE were examined. The maleimide lipid derivative MPB-PE was found to allow more efficient coupling. Thin layer chromatography however revealed that during the standard synthesis of MPB-PE, an impurity was generated which can constitute 40% or more of the derivatized PE. An improved method for the synthesis and isolation of pure MPB-PE is presented here. Subsequently, optimized conditions for the covalent coupling of streptavidin to liposomes containing pure MPB-PE were determined. The flexibility of the streptavidin-liposome system for the preparation of various types of ligand bearing liposomes is demonstrated by the rapid association of a variety of biotinated proteins to streptavidin-liposome systems. The ability of these conjugates to target to specific cell populations in vitro as directed by defined biotinated monoclonal antibodies is demonstrated.

Interactions of liposomes and platelets.

Rats were injected intravenously with liposomes of various compositions and sizes and blood platelet count measured. It was found that negatively-charged liposomal systems produced a transient reduction in platelet count in the first 5 minutes after injection which recovered by 60 minutes post-injection. This effect was most striking for multilamellar vesicles (MLV's) containing phosphatidylglycerol (PG). Dose levels of 25 mg/kg of MLV's containing 10 mole% PG caused the platelet count to drop from a control value of 1,086 +/- 21 X 10(9)/l to 193 +/- 14 X 10(9)/l by 2 minutes post-injection, an 82% decline. This thrombocytopenic effect was observed to diminish as vesicle size or vesicle dose was decreased. Positively-charged liposomes produced a less pronounced transient reduction in platelet count while neutral liposomes caused only a mild, transient platelet decline. This transient thrombocytopenic effect was not blocked by common anticoagulants and fibrinolytic agents but was prevented by liposomal pretreatment. Radiolabeled platelet studies revealed that transient sequestration of platelets occurs in the liver and spleen 2 minutes after PG:EPC:CHOL MLV injection with a normalization of platelet distribution by 60 minutes post-injection. In vitro studies, using an automated blood counter, suggest a transient association of liposomes and platelets occurring following injection. Liposomally-induced transient thrombocytopenia suggests a role for platelets in the biodistribution of liposomes.

The binding of phosphatidylglycerol liposomes to rat platelets is mediated by complement.

Previous work has shown that intravenous administration of phosphatidylglycercol (PG) containing liposomes to rats results in a rapid transient decline in platelet count (1). Here the interactions of PG liposomes with rat platelets in vitro have been examined with the aim of characterizing factors associated with the decline. It is shown that PG liposomes induce formation of rat (but not human) platelet-liposome microaggregates in vitro. The PG liposome dependent thrombocytopenia observed in vivo can therefore be attributed to sequestration of PG liposome-platelet aggregates. Further, the aggregation of platelets with PG liposomes, which can be monitored as a reduction in platelet count using a coulter counter, is shown to be mediated by a serum complement factor, likely C3b. This is indicated by a requirement of plasma for the in vitro reduction in platelet count induced by PG liposomes, and the inhibition of this effect by heat treatment of plasma, by incubation of plasma with purified cobra venom factor, or by removal of C3 from plasma.

Inhibition of HIV-1 replication in cultured cells with phosphorylated dideoxyuridine derivatives encapsulated in immunoliposomes.

Among the 2',3'-dideoxynucleoside 5'-triphosphates containing a physiological base, 2',3'-dideoxyuridine 5'-triphosphate (ddUTP) has been reported to be among the most powerful inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) in cell-free systems. However, in contrast to other dideoxynucleosides, 2',3'-dideoxyuridine (ddU) is inactive in treatment of HIV-infected cells in culture, since it is a poor substrate for cellular nucleoside kinases. This problem cannot be overcome by the use of phosphorylated ddU because such compounds are unable to cross cell membranes. To promote entry and thus bypass the limiting steps of intracellular phosphorylation, we have encapsulated mono- and tri-phosphorylated ddU in liposomes coupled to monoclonal antibodies (immunoliposomes). We investigated antiviral effects in two human T cell lines (MT-4, CEM). We observed that ddU nucleotides remain phosphorylated for several weeks after encapsulation in immunoliposomes, and potent antiviral activity is obtained when these drugs are delivered into infected cells by cell-specific antibodies (ED50 < or = 1 microM on CEM). In contrast, no inhibition was observed with non-targeted liposomes containing phosphorylated ddU, or with empty liposomes, whether targeted or not.

Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients.

Transmembrane movement of dopamine in response to K+ or H+ ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K+ diffusion potential (delta psi; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30-40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting delta pH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K+ potential is more complex. Although generation of a K+ diffusion potential results in acidification of the vesicle interior, the magnitude of the induced delta pH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (greater than 200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium.

The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey.

We have shown previously that transmembrane proton gradients can be used to efficiently accumulate biogenic amines [M.B. Bally et al. (1988) Chem. Phys. Lipids 47, 97-107] and doxorubicin [L.D. Mayer, M.B. Bally and P.R. Cullis (1986) Biochim. Biophys. Acta 857, 123-126] to high concentrations within liposomes. To determine the generality of this loading procedure, representative drugs from a variety of different classes (antineoplastics, local anaesthetics, antihistamines, etc.) were examined as to their ability to redistribute in response to a proton gradient. While the majority of drugs examined, all of which are weak bases, were accumulated by large unilamellar vesicles exhibiting a pH gradient (interior acid) the extent of uptake varied considerably between different pharmaceuticals. These differences are discussed in the context of various factors which will likely influence drug accumulation including its membrane/water partition coefficient and its solubility in the intravesicular medium.

Progesterone receptor A and B isoform expression in human osteoblasts.

We have previously shown that human progesterone receptors (PR) are expressed in human osteosarcoma cells and in primary human osteoblast cultures. The aim of this study was to examine PRa and PRb isoform expression in human osteosarcoma cells. In addition, the effect of beta-estradiol on PR promoter activity in three human osteosarcoma cell lines was analyzed. Rapid amplification of 5'cDNA ends (5'RACE) were used to detect PR mRNA transcripts coding for both PR isoforms in HOS-TE85, an early progenitor human osteosarcoma cell line. Analogous 5'RACE products were detected in the PR-positive breast-cancer cell line MCF-7. Southern blot analysis confirmed that the amplified products were PR specific. It was shown that the larger of two RACE products coded specifically for B isoform mRNA and that of the smaller product corresponded to a PRa specific transcript. No RACE products were detected in the PR-negative HeLa cell line. To determine if both PR promoters were active in osteoblasts, chimeric recombinants bearing the PRa (+464, +1105) and PRb (-711, +31) promoter regions subcloned into minimal pBLCAT vectors, were transiently expressed in three human osteosarcoma cell lines-HOS-TE85, MG-63, and SAOS-2. It was shown that beta-estradiol induced both PRa and PRb promoter activity in all of the osteosarcoma cell lines examined. The finding that PRa and PRb mRNA transcripts are expressed in human osteoblasts, and that promoters for both isoforms are estrogen responsive provides further evidence that bone-forming cells are physiologically influenced by progesterone.

Progesterone receptors are expressed in human osteoblast-like cell lines and in primary human osteoblast cultures.

Expression of progesterone receptors (PR) was studied in human osteoblast-like cell lines and primary human osteoblast cultures at the molecular level. Using the sensitive reverse transcriptase polymerase chain reaction (RT-PCR) and oligonucleotide primers which flank the progesterone-binding domain of human PR, progesterone receptor (PR) mRNA was detected in three osteoblast-like cell lines--HOS-TE85, MG-63, and SAOS-2. When compared with beta-actin gene expression, levels of PRmRNA transcripts varied between cell lines (PRmRNA in HOS-TE85 > MG-63 >> SAOS-2). In addition, RT-PCR confirmed the presence of PRmRNA transcripts in primary human osteoblast cells cultured from collagenase-treated bone. Immunostaining was used to visualize PR protein in cells. All osteoblast-like cell lines showed specific staining for PR. Immunoreactivity was distributed equally in the nucleus and cytoplasm. The level of staining was significantly lower than that detected in PR-positive MCF-7 breast cancer cells though well above background levels obtained for PR-negative HeLa cells. The finding that PR is expressed at both the level of mRNA and protein in several osteoblast-like cell lines as well as in human primary osteoblast cultures indicates that bone-forming osteoblast cells are direct targets for progesterone action.

Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy.

P-glycoprotein (Pgp) functions as an ATP-dependent drug efflux pump to confer multidrug resistance to tumor cells. In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pgp structure and function remain poorly understood. Fluorescence spectroscopy of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at high resolution. We have used site-directed mutagenesis to modify the wild-type (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replacement of all 11 tryptophan residues individually with phenylalanine. None of the 11 tryptophans were found to be absolutely essential for Pgp activity, because Chinese hamster ovary cells transfected and overexpressing this mutant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can carry out active transport of vinblastine, colchicine, and Calcein-AM. The mdr3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a unique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally complement ste6 in yeast. Studies with Pgp mutants containing either single or double tryptophan residues or with chimeric molecules constructed between wild-type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was responsible for the reduced activity of the mdr3F(1-11) mutant. Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the mdr3F(1-11) mutant. Altogether, we show that a Trp-less Pgp is functionally active and can be used as a molecular backbone for insertion of tryptophans in strategic locations to probe various aspects of Pgp function.