The 34A mAb targets pulmonary endothelial cells, and the attachment of >30 34A mAb molecules per 110-nm liposome confers no additional advantage in targeting PEG-liposomes to the lung (12). Using immunoliposomes, an average brain delivery of 0.03 %ID/g brain tissue is possible at 60 min after injection (Fig. half-life in the blood circulation. Liposomes coated with the inert and biocompatible polymer PEG are widely used and are often referred to as sterically stabilized or stealth liposomes (5). PEG covering is believed to prevent acknowledgement of liposomes by macrophages due to reduced binding of plasma proteins (4, 6). Regrettably, it has been difficult Eprosartan to combine steric stabilization of liposomes with efficient immunotargeting. PEG covering of liposomes can create steric hindrances for antibodyCtarget conversation (7, 8). It has therefore been proposed to attach a cell-specific ligand to the distal end of a few lipid-conjugated PEG molecules rather than conjugate the ligand to a lipid headgroup on the surface of a PEG-conjugated liposome. This has been carried out recently with folic acid (9) and monoclonal antibodies (10, 11, 12, 13) to target liposomes to cells in tissue culture and organs (14, 15, 16), because these brokers are not transported through the brain capillary endothelial wall, which makes up the bloodCbrain barrier (BBB) + = 3) was lower than the corresponding value of the commonly used plasma volume marker [125I]mouse IgG2a of 11 l/g (23). The brain = 3), which yielded a logarithmic = ?0.40. RESULTS Synthesis of Immunoliposomes. Liposomes were prepared by repeated extrusion through polycarbonate filter membranes with pore sizes of 100 and 50 nm, respectively. By this method, an average diameter of the liposomes of 85 nm was obtained with a sharp distribution of size (80% of the liposomes experienced a size between 65 and 115 nm; Fig. ?Fig.11= 7). Loading was not affected by lipid composition of the vesicles or the lipid to daunomycin molar ratio (range of ratios tested was from 16:1 to 2500:1). For the synthesis of immunoliposomes, the linker lipid DSPE-PEG-maleimide was incorporated in liposomes. Daunomycin-loaded vesicles were allowed to react with thiolated antibody (Fig. ?(Fig.11product of the [3H]daunomycin-containing liposomes was decreased 8-fold, whereas the plasma AUC was increased 4-fold compared with free daunomycin, and these offsetting effects resulted in no change in brain drug delivery (Fig. ?(Fig.3).3). The use of PEG-conjugated liposomes reduced the BBB value to zero: therefore, no brain uptake of the PEG-liposomes was observed, despite the marked increase in plasma (Fig. ?(Fig.3).3). Conversely, the use of PEG-conjugated OX26 immunoliposomes increased the BBB product, relative to PEG-liposomes, and Eprosartan a brain uptake of 0.03 %ID/g at 60 min was observed (Fig. ?(Fig.3). 3). Open in a separate window Physique 2 %ID/ml of plasma of daunomycin, liposomes, and immunoliposomes is usually plotted versus numerous occasions after intravenous injection. (= 3 rats/point. Table 1 Systemic volume of distribution and plasma clearance of daunomycin, liposomes, and?immunoliposomes = 3 experiments. All immunoliposomes are PEG-conjugated.? ?Statistically significant difference by Students test (= 0.012) when compared to corresponding value of OX26197 immunoliposomes.? ?Statistically significant difference by Students test (< 0.015) when compared to corresponding value of OX2621 immunoliposomes.? Open in a separate window Physique 3 Pharmacokinetic parameters and brain delivery of free daunomycin (column D), not PEG-conjugated liposomes (column L), PEG-conjugated OX2629 immunoliposomes (column 29), and PEG-conjugated liposomes without mAb (column 0) at 60 min after intravenous injection. Data symbolize means SEM (= 3). Titration of OX26. Titration of the amount of OX26 conjugated per liposome revealed an increase in plasma clearance and a decrease in the systemic volume of distribution of immunoliposomes at Eprosartan higher OX26 concentrations (Fig. ?(Fig.22and Table ?Table1).1). The PEG-liposomes are designated as OX260 immunoliposomes in Fig. ?Fig.4.4. Highest product values and brain tissue accumulation were observed for OX2629 immunoliposomes Rabbit Polyclonal to POU4F3 (Fig. ?(Fig.4).4). At higher OX26 densities around the liposome, a saturation effect was observed resulting in reduced product, and brain tissue accumulation of OX26197 immunoliposomes. Open in a separate window Physique 4 Pharmacokinetic parameters and brain delivery of sterically stabilized liposomes (PEG-liposomes) and immunoliposomes at 60 min after intravenous injection. All liposomes are PEG-conjugated. Data symbolize means SEM (= 3). Twenty-four-Hour Pharmacokinetics and Tissue Distribution. To determine if immunoliposomes build up in brain tissue over time, 1-h, 6-h, and 24-h brain uptake experiments were performed using OX2630 immunoliposomes, and the brain of PEG-conjugated immunoliposomes (OX2630). Brain tissue was analyzed 1 h, 6 h, or 24 h after intravenous injection of immunoliposomes packaged with [3H]daunomycin. Data symbolize means SEM (= 3). Coinjection of OX26 and Use of Nonspecific Control Eprosartan IgG Immunoliposomes. To determine if brain delivery of immunoliposomes was mediated by Eprosartan the mAb OX26, two series.
