André Trouet

Nanocapsules: A new type of lysosomotropic carrier

P. COUVREUR +, P. TULKENS ~, M. ROLAND +, A. TROUET ~ and P. SPEISER* +Laboratoire de Pharmacie Gal~nique, Universit~ Catholique de Louvain, B-1200 Brussels, *Laboratoire de Chimie Physiologique, Universit~ Catholique de Louvain, and International Institute of Cellular and Molecular Pathology, B-1200 Brussels, Belgium and *Pharmazeutische Institut, Eidgen6ssische Technische Hochschule, Zfirich, Switzerland

The uptake and intracellular accumulation of aminoglycoside antibiotics in lysosomes of cultured rat fibroblasts

Abstract The uptake and intracellular localization of aminoglycoside antibiotics: streptomycin, dihydrostreptomycin, gentamicin, kanamycin and amikacin, has been studied in cultured rat embryo fibroblasts, using cell fractionation techniques and microbiological assay. On a volume basis, aminoglycosides are accumulated 2 to 5-fold by fibroblasts, over a wide range of external concentrations. Accumulation proceeds slowly, and stable intracellular contents of drugs are obtained only after 4 days of incubation. After differential or isopycnic centrifugation of cell homogenates, the antibiotics are found consistently associated with lysosomal acid hydrolases, and clearly dissociate from marker constituents of mitochondria, plasma membrane, endoplasmic reticulum or peroxisomes. This indicates that aminoglycosides enter cells, but are localized exclusively in lysosomes. We suggest that the relative inefficiency of aminoglycoside antibiotics to act against intracellular bacteria is due (1) to the acid pH prevailing in lysosomes, which depress the antibacterial activity of these drugs and defeat their accumulation in those organelles; (2) the lack of accumulation of these drugs in other subcellular structures, like phagosomes, which may harbour intracellular bacteria.

Iron uptake and utilization by mammalian cells. I: Cellular uptake of transferrin and iron

The uptake of transferrin-bound iron by cultured mammalian cells involves receptor-mediated endocytosis and internalization of the transferrin-receptor complex, followed by iron release in an intracellular acidic vesicle (which may be the endocytic vesicle itself or a lysosome). The iron is transferred in large part to cytosol ferritin, while the iron-depleted transferrin is recycled back to the plasma membrane and released intact into the extracellular medium. In addition, in some cell types transferrin is also internalized by fluid-phase or adsorptive endocytosis, its iron is released and the protein is degraded in lysosomes.

Effects of antimycin, glucose deprivation, and serum on cultures of neurons, astrocytes, and neuroblastoma cells.

The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.

Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2).

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin. Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation. The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium. Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 microM OTA inhibits MTT reduction by 50%. Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts. At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport. AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption. Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins. Competition experiments showed that carrier systems for large neutral amino acids, H(+)/dipeptides cotransporter, organic anion (p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver. AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp. This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport. Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

Uptake and subcellular localization of daunorubicin and adriamycin in cultured fibroblasts.

Abstract Cellular uptake and subcellular localization of daunorubicin and adriamycin were studied in cultured rat embryo fibroblasts. Both drugs accumulated in the cells manifold above the extracellular concentration. The uptake of daunorubicin was 2–3 time higher than that of adriamycin. Cell fractionation techniques, including isopycnic centrifugation of cytoplasmic extracts, revealed that both drugs were exclusively localized in nuclei and lysosomes, but to quite different proportions. About 20% of accumulated daunorubicin was localized in the nuclei, as compared to 60% of adriamycin, which means that the nuclear concentrations of the drugs were very similar. In the lysosomes, on the other hand, the accumulation of daunorubicin was 4–5 times higher than that of adriamycin. The reason for this difference is so far not clear. The results give an explanation for observations of similar biologic effects of the drugs in spite of a marked difference in cellular accumulation.

Experimental leukemia chemotherapy with a “lysosomotropic” adriamycin-DNA complex

Abstract The complex formed between adriamycin and DNA possesses the physicochemical and in vitro biological characteristics of a lysosomotropic drug. Adriamycin is resistant to lysosomal hydrolases and binds firmly to high molecular weight DNA. When linked to DNA, adriamycin looses its properties of fluorescence, of dialysis and its antibiotic activity. Adriamycin can however be released from the complex and recovers all its properties after digestion of the DNA by lysosomal desoxyribonucleases. The adriamycin-DNA complex is two times less toxic than free adriamycin when given intraperitoneally or intravenously on five consecutive days in NMRI mice. While two times less toxic than free adriamycin, adriamycin-DNA possesses higher chemotherapic efficiency against the L 1210 Leukemia in DBA 2 mice. If one takes advantage of these two properties, adriamycin-DNA induces very high percentages of increase in life span in DBA 2 mice inoculated intravenously with L 1210 cells and allows a great proportion of mice to survive longer than 30 days. All these data are in favor of a lysosomotropic mode of action of adriamycin-DNA. According to this hypothesis, adriamycin-DNA penetrates only the cells possessing a significant endocytic activity. After degradation of DNA inside the lysosomes, adriamycin becomes free and exerts its toxic activity mostly on those cells, which like some tumor cells, possess besides an high endocytic activity an high mitotic rate. Many normal cells with an high mitotic rate or susceptible to the action of adriamycin but with a low endocytic activity, are in this way protected.

GABA induces down-regulation of the benzodiazepine-GABA receptor complex in the rat cultured neurons.

Cultured neurons from embryonic rat brain display central type benzodiazepine receptors characterized by high-affinity binding of [3H]flunitrazepam which is allosterically enhanced in the presence of gamma-aminobutyric acid (GABA). A 48 h treatment of the cultured neurons with 1 microM diazepam, 0.1 microM clonazepam or 0.1 microM beta-carboline ester derivatives did not change either Bmax or KD values of the [3H]flunitrazepam specific binding. A 48 h incubation in the presence of GABA (1 mM) or muscimol (0.1 mM) induced a 30% decrease of the Bmax value of [3H]flunitrazepam specific binding without change of the KD value. The down-regulation was dependent on GABA concentrations and temperature, and was partially inhibited by bicuculline but not by the benzodiazepine antagonist Ro 15-1788. The other subunits of the benzodiazepine-GABA-chloride channel receptor complex also seemed to be down-regulated by GABA since there was a decrease of the specific binding of [3H]muscimol and [35S]t-butylbicyclophosphorothionate (TBPS) to the GABAA and chloride channel sites respectively. The GABA-induced down-regulation of the GABA-benzodiazepine receptor seems to be selective since the specific binding of ligands to other receptors was not affected. Our results suggests that activation of the low-affinity GABA subunit which is involved in cellular electrophysiological responses, induced the receptor down-regulation.

Cellular pharmacology of deferrioxamine B and derivatives in cultured rat hepatocytes in relation to iron mobilization

Two radiolabelled derivatives of deferrioxamine B (DF) have been synthesized: methyl-DF and acetyl-DF. Both derivatives are non cytotoxic and stable in cell culture but they are degraded in human plasma and more extensively in rat plasma. Methyl-DF, acetyl-DF and DF mobilize radioiron to the same extent from hepatocytes loaded with 59Fe citrate in the same range of extracellular concentrations. The uptake and release of the 3H-labelled derivatives and their corresponding iron complexes have been measured and appear to represent a passive phenomenon resulting from the gradient of concentration between the cellular compartment and the extracellular medium. The results indicate that only a limited pool of cellular iron is accessible for chelation and that neither the permeability of the cellular membrane, nor the intracellular concentration of the chelators are the limiting factors for iron mobilization. On the basis of the subcellular distribution of the 3H-DF analogues, methylamine inhibition of iron chelation by siderophores in cell cultures and the positive effect of acidic pH and hydrolysis by lysosomal enzymes on in vitro iron mobilization from radiolabelled ferritin, we suggest that iron mobilization by DF and its derivatives occurs in lysosomes where they complex iron released from ferritin under the conjugate actions of acidic pH and lysosomal enzymes.

Transferrin Protein and Iron Uptake By Cultured-hepatocytes

The binding and uptake of 59Fe‐loaded 3H‐labelled rat transferrin by cultured rat hepatocytes was investigated. At 4°C, there is no evidence for a specific binding of transferrin which could be related to the association of neo‐synthesized transferrin with plasma membrane receptors. At 37°C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.