Glutathione conjugation dose-dependently increases brain-specific liposomal drug delivery in vitro and in vivo.

The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology®) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation.

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

Glutathione PEGylated liposomes: pharmacokinetics and delivery of cargo across the blood-brain barrier in rats.

Partly due to poor blood-brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated liposomes (G-Technology®) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p < 0.001). In the microdialysis study 4-fold higher (p < 0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.

Urinary crystallization inhibitors do not prevent crystal binding.

PURPOSE: Renal stone formation requires the persistent retention of crystals in the kidney. Calcium oxalate monohydrate (COM) crystal binding to Madin Darby canine kidney strain I (MDCK-I), a cell line that resembles the epithelium in the renal distal tubule/collecting duct, is developmentally regulated, while LLC-PK1 cells (American Type Tissue Collection), which are widely used as a model of the renal proximal tubule, bind crystals irrespective of their stage of epithelial development. Whereas to our knowledge the binding molecules for COM at the surface of LLC-PK1 cells are still unknown, crystals adhere to the hyaluronan (HA) rich pericellular matrix transiently expressed by mobile MDCK-I cells. In the current study we investigated whether crystal binding to either cell type is influenced by urinary substances, including glycoprotein inhibitors of crystallization MATERIALS AND METHODS: We studied crystal binding to MDCK-I cells during wound repair, to confluent LLC-PK1 cells and to HA immobilized on a solid surface using [14C] COM pretreated or not pretreated with urine from healthy male volunteers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed to assess whether the crystals became coated with urine derived proteins RESULTS: Western blot analysis demonstrated that pretreated COM crystals were covered with protein inhibitors of crystallization. However, this protein coat had no significant effect on the level of crystal binding to either cell type. In contrast, the adherence of urine treated crystals to immobilized HA was significantly reduced CONCLUSIONS: The adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide HA. Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may predominantly depend on the surface properties of the renal tubular epithelium.