Brain drug delivery of small molecules using immunoliposomes

Abstract

Immunoliposomes (antibody-directed liposomes) were used in the present study for delivery of the antineoplastic agent daunomycin to the rat brain. A coupling procedure was introduced, which allows conjugation of a thiolated antibody to maleimide-grafted 85-nm liposomes sterically stabilized with PEG. Antibody was thereby coupled to the terminal end of a PEG-conjugated linker lipid. No brain uptake of PEG-conjugated liposomes carrying [3H]daunomycin was observed. However, brain targeting of immunoliposomes carrying [3H]daunomycin was mediated by the OX26 monoclonal antibody to the rat transferrin receptor, which is selectively enriched at the brain microvascular endothelium that comprises the blood-brain barrier in vivo. Coupling of 30 OX26 antibodies per liposome resulted in optimal brain delivery. Saturation of delivery was observed at higher antibody densities. Determination of brain levels of immunoliposomes over 24 h revealed that immunoliposomes accumulate in brain tissue. Brain targeting of immunoliposomes was not observed in immunoliposomes conjugated with a mouse IgG2a isotype control. In addition, coinjection of free OX26 saturated plasma clearance of immunoliposomes. Since a single liposome may carry > or = 10,000 drug molecules, the use of PEG-conjugated immunoliposomes increases the drug carrying capacity of the monoclonal antibody by up to 4 logarithmic orders in magnitude. In summary, specific OX26-mediated targeting of daunomycin to the rat brain was achieved by the use of an immunoliposome-based drug delivery system.

Figure 1

Preparation of PEG-conjugated immunoliposomes. (A) Schematic diagram of coupling of the thiolated monoclonal antibody OX26 (OX26-SH) with sterically stabilized liposomes containing DSPE-PEG-maleimide. (B) Size distribution of sterically stabilized liposomes prepared by rapid extrusion. (C) Elution profile of the separation of [³H]daunomycin-loaded immunoliposomes from unencapsulated [³H]daunomycin and unbound ¹²⁵I-labeled mAb OX26 by gel filtration.

Figure 2

%ID/ml of plasma of daunomycin, liposomes, and immunoliposomes is plotted versus various times after intravenous injection. (A) Comparison of free daunomycin, liposomes, sterically stabilized liposomes (PEG-liposomes), and PEG-conjugated immunoliposomes (OX26₂₉). (B) Titration of OX26 bound to PEG-conjugated liposomes. The liposomes designated as PEG-liposomes have no mAb attached. Data are means ± SEM of n = 3 rats/point.

Figure 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 represent means ± SEM (n = 3).

Figure 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 represent means ± SEM (n = 3).

Figure 5

Brain V_D 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 represent means ± SEM (n = 3).