Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids

AbstractLiposomes containing the acidic phospholipid phosphatidylserine (PS) have been shown to avidly interact with proteins involved in blood coagulation and complement activation. Membranes with PS were therefore used to assess the shielding properties of poly(ethylene glycol 2000)-derivatized phosphatidylethanolamine (PE-PEG2000) with various acyl chain lengths on membranes containing reactive lipids. The desorption of PE-PEG2000 from PS containing liposomes was studied using an in vitro assay which involved the transfer of PE-PEG2000 into multilamellar vesicles, and the reactivity of PS containing liposomes was monitored by quantifying interactions with blood coagulation proteins. The percent inhibition of clotting activity of PS liposomes was dependent on the PE-PEG2000 content. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 which transferred out slowly from PS liposomes was able to abolish >80% of clotting activity of PS liposomes at 15 mol%. This level of DSPE-PEG2000 was also able to extend the mean residence time of PS liposomes from 0.2 h to 14 h. However, PE-PEG2000 with shorter acyl chains such as 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-PEG2000 were rapidly transferred out from PS liposomes, which resulted in a 73% decrease in clotting activity inhibition and 45% of administered intravenously liposomes were removed from the blood within 15 min after injection. Thus, PS facilitates the desorption of PE-PEG2000 from PS containing liposomes, thereby providing additional control of PEG release rates from membrane surfaces. These results suggest that membrane reactivity can be selectively regulated by surface grafted PEGs coupled to phosphatidylethanolamine of an appropriate acyl chain length

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

AbstractIn order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG2000-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000). Transfer of DSPE-PEG2000 to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG2000 was transferred efficiently even at 4°C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG2000 to become evenly distributed between the two liposome populations (TEQ) at 4°C and 37°C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

AbstractA liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 °C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled

The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity

<p>Abstract</p> <p>Background</p> <p>HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ.</p> <p>Methods</p> <p>The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. <it>In vivo </it>studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions.</p> <p>Results</p> <p>The gefitinib and RAD001 combination inhibited cell growth <it>in vitro </it>in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated <it>in vitro </it>with cell line dependent increases in cytotoxicity and cytostasis while treatment <it>in vivo </it>promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (<it>in vitro </it>and <it>in vivo</it>) and EGFR signaling <it>in vivo </it>relative to the single drugs.</p> <p>Conclusions</p> <p>The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.</p

Liposomal irinotecan: formulation development and therapeutic assessment in murine xenograft models of colorectal cancer

ABSTRACT Purpose: The purpose is to demonstrate whether an appropriately designed liposomal formulation of irinotecan is effective in treating mice with liver-localized colorectal carcinomas. Experimental Design: Irinotecan was encapsulated in 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol (55:45 molar ratio) liposomes using an ionophore (A23187)-generated transmembrane proton gradient. This formulation was evaluated in vivo by measuring plasma elimination of liposomal lipid and drug after i.v. administration. Therapeutic activity was determined in SCID/Rag-2M mice bearing s.c. LS180 tumors or orthotopic LS174T colorectal metastases. Results: Drug elimination from the plasma was significantly reduced when irinotecan was administered in the liposomal formulation. At 1 hour after i.v. administration, circulating levels of the liposomal drug were 100-fold greater than that of irinotecan given at the same dose. High-performance liquid chromatographic analysis of plasma samples indicated that liposomal irinotecan was protected from inactivating hydrolysis to the carboxylate form. This formulation exhibited substantially improved therapeutic effects. For the LS180 solid tumor model, it was shown that after a single injection of liposomal irinotecan at 50 mg/kg, the time to progress to a 400-mg tumor was 34 days (as compared with 22 days for animals treated with free drug at an equivalent dose). In the model of colorectal liver metastases (LS174T), a median survival time of 79 days was observed after treatment with liposomal irinotecan (50 mg/kg, given every 4 days for a total of three doses). Saline and free drug treated mice survived for 34 and 53 days, respectively. Conclusions: These results illustrate that liposomal encapsulation can substantially enhance the therapeutic activity of irinotecan and emphasize the potential for using liposomal irinotecan to treat liver metastases

Studies on the roles of lipids in membrane structure and function

Lipids in membranes satisfy two basic roles. First they provide a matrix with which membrane proteins are associated and second, they provide a structural bilayer for maintaining a permeability barrier. This thesis investigates certain aspects of the structural and permeability barrier properties of lipids employing simple protein free model membrane systems. It is demonstrated, employing ³¹P and ²H NMR techniques, that cholesterol engendered formation of non-bilayer structures in multilamellar vesicles (MLVs) composed of phosphatidylethanolamine (PE) and either phosphatidylserine (PS) or phosphatidylcholine (PC). Further the presence of cholesterol, in conjunction with Mg²⁺, facilitated Ca²⁺triggered formation of non-bilayer organizations in the PS containing systems. It is indicated that in these complex multicomponent systems where multiple structural phases (ie. bilayer, hexagonal, and "isotropic") coexist, that the phospholipids exhibit ideal mixing behaviour. A basic consequence of the barrier properties of a lipid bilayer is an ability to maintain a membrane potential (Δψ), which is required for a variety of membrane mediated transport processes. To investigate the role of lipids in maintaining Δψ, and the direct effect of Δψ on transport functions, a large unilamellar vesicle (LUV) preparation free of impurities is required. This thesis describes a novel procedure for generating LUVs, by extrusion of MLVs through polycarbonate filters (pore size 100 nm). LUVs can thus be obtained from a wide variety of lipid species and mixtures in the absence of lipid solubilizing agents. Vesicles exhibiting Δψ in response to a Na⁺/K⁺ ion gradient (K⁺ inside) are characterized. It is shown that such a K⁺ diffusion potential can drive the uptake of a variety of biologically active molecules (eg. local anaesthetics, antineoplastic agents, biogenic amines (dopamine)) which have cationic and lipophilic characteristics. The transport process appears to proceed by a antiport cation/lipophilic cation exchange process that is driven by the transmembrane potential.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat