Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: an original drug delivery pathway.

We have designed an amphiphilic prodrug of gemcitabine (dFdC) by its covalent coupling to a derivative of squalene, a natural lipid. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles that displayed a promising in vivo anticancer activity. The aim of the present study was to provide further insight into the in vitro subcellular localization and on the metabolization pathway of the prodrug. Cells treated with radiolabelled squalenoyl gemcitabine (SQdFdC) were studied by differential detergent permeation, and microautography coupled to fluorescent immunolabeling and confocal microscopy. This revealed that the bioconjugate accumulated within cellular membranes, especially in those of the endoplasmic reticulum. Radio-chromatography analysis proved that SQdFdC delivered dFdC directly in the cell cytoplasm. Mass spectrometry studies confirmed that gemcitabine was then either converted into its biologically active triphosphate metabolite or exported from the cells through membrane transporters. To our knowledge, this is the first description of such an intracellular drug delivery pathway. In vitro cytotoxicity assays revealed that SQdFdC was more active than dFdC on a transporter-deficient human resistant leukemia model, which was explained by the subcellular distribution of the drugs and their metabolites. The squalenoylation drug delivery strategy might, therefore, dramatically improve the efficacy of gemcitabine on transporter-deficient resistant cancer in the clinical context.

Specific permeability modulation of intestinal paracellular pathway by chitosan-poly(isobutylcyanoacrylate) core-shell nanoparticles.

This work is focused on the evaluation of the in vitro permeation modulation of chitosan and thiolated chitosan (chitosan-TBA) coated poly(isobutylcyanoacrylate) (PIBCA) nanoparticles as drug carriers for mucosal administration. Core-corona nanoparticles were obtained by radical emulsion polymerisation of isobutylcyanoacrylate (IBCA) with chitosan of different molecular weights and different proportions of chitosan/chitosan-TBA. In this work, the effect of these nanoparticles on the paracellular permeability of intestinal epithelium was investigated using the Ussing chamber technique, by adding nanoparticle suspensions in the mucosal side of rat intestinal mucosa. Results showed that permeation of the tracer [14C]mannitol and the reduction of transepithelial electrical resistance (TEER) in presence of nanoparticles were more pronounced in those formulations prepared with intermediate amounts of thiolated polymer. This effect was explained thanks to the high diffusion capacity of those nanoparticles through the mucus layer that allowed them to reach the tight junctions in higher extent. It was concluded that, although a first contact between nanoparticles and mucus was a mandatory condition for the development of a permeation enhancement effect, the optimal effect depended on the chitosan/chitosan-TBA balance and the conformational structure of the particles shell.

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells.

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN(3) pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC.

Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers.

In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly(iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN.

Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules.

Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

Novel composite core-shell nanoparticles as busulfan carriers.

This study presents a method for the design of novel composite core-shell nanoparticles able to encapsulate busulfan, a crystalline drug. They were obtained by co-precipitation of mixtures of poly(isobutylcyanoacrylate) (PIBCA) and of a diblock copolymer, poly(epsilon-caprolactone)-poly(ethylene glycol) (PCL-PEG), in different mass ratios. The nanoparticle size, morphology and surface charge were assessed. The chemical composition of the top layers was determined by X-ray photo-electron spectroscopy (XPS). (3)H-labelled busulfan was used in order to determine the drug loading efficiency and the in vitro drug release by liquid scintillation counting. Physico-chemical techniques such as Zeta potential determination and XPS analysis provided evidence about a preferential surface distribution of the PCL-PEG polymer. Therefore, composite nanoparticles have a "core-shell"-type structure, where the "core" is essentially formed by the PIBCA polymer and the "shell" by the PCL-PEG copolymer. The use of PIBCA to form the core of the nanoparticles leads to a 2-4 fold drug loading increase, in comparison to the single PCL-PEG nanoparticles. In addition, the complement activation results showed a significant difference between the composite nanoparticles and the single PIBCA nanoparticles, thus demonstrating that PEG at the surface of the nanoparticles reduced the complement consumption. The PIBCA:PCL-PEG composite nanoparticles prepared using the new co-precipitation method here described represent an original approach for busulfan administration.

Skin localization of cow's milk proteins delivered by a new ready-to-use atopy patch test.

PURPOSE: Atopy patch tests (APTs) allow the detection of delayed allergies at the skin level. The localization of beta-lactoglobulin delivered into the skin by an innovative ready-to-use APT (E-patch was investigated and the efficacy and safety of this device were assessed. METHODS: The E-patch containing beta-lactoglobulin was placed for 24 h in contact with hairless rat skin mounted in a Franz diffusion cell. Transdermal passage was monitored by measurement of beta-lactoglobulin A-[methyl-(14)C] or by two-site enzyme immunoassay. An iterative skin stripping allowed measurement of the beta-lactoglobulin penetrating the first external skin layers. RESULTS: After 24 h, 92% of beta-lactoglobulin remained on the skin. The iterative skin strippings showed a 135-fold higher concentration of beta-lactoglobulin in the stratum corneum than that found in the epidermis-dermis. Analysis of the solution in the receiver compartment by radioactivity assays or immunoassays indicates that intact protein did not cross the skin. CONCLUSIONS: The E-patch system allows native beta-lactoglobulin to concentrate in the stratum corneum, in the vicinity of immunological cells, but does not lead to its systemic delivery. Therefore, it is suggested that this delivery system creates ideal conditions for promoting a positive topical response with reduced risk of systemic anaphylactic reactions caused by the native form of the beta-lactoglobulin A.

Cationic emulsions improves the delivery of oligonucleotides to leukemic P388/ADR cells in ascite.

The aim of this study was to investigate the in vivo ability of O/W cationic emulsions to deliver oligonucleotides (ON) in leukemic P388/ADR cells in ascite, after intraperitoneal (IP) administration in mice. Cationic emulsions were prepared by microfluidization as previously described by Teixeira et al. [Pharm. Res 16 (1999) 30]. The formulations consisted mainly of medium chain triglycerides, phosphatidylcholine (PC), poloxamer, and either a monocationic lipid stearylamine (PC/SA-emulsion) or a polycationic lipid RPRC(18) (PC/RPRC(18)-emulsion). A model ON (33P-pdT(16)) was associated with cationic emulsions by single addition at the end of the manufacturing process. Seven days after P388/ADR inoculation IP to mice, ON free or associated with PC/SA or PC/RPRC(18) emulsions was injected IP at a dose of 0.5 mg/kg. At different interval times, ascite including cells, blood and the main organs were collected and the radioactivity counted by liquid scintillation. The overall results showed significantly high amounts of ON in the leukemic cell pellet, 24 h after administration of ON associated to either PC/SA (AUC(0-24 h)=13634, %injected dose/min) or PC/RPRC(18) (AUC(0-24 h)=22592, % injected dose/min), contrary to the free ON solution (AUC(0-24 h)=3095, %injected dose/min), which displayed only reduced capture by cancer cells. In conclusion, complexation of ON with cationic emulsions had a beneficial effect in increasing tumor cells uptake in vivo (up to sevenfold for PC/RPRC(18)-emulsion) after IP administration. This could open interesting prospects for the treatment of ovarian cancers.

Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery.

PURPOSE: The aim of this study was to evaluate the ability of long-circulating PEGylated cyanoacrylate nanoparticles to diffuse into the brain tissue. METHODS: Biodistribution profiles and brain concentrations of [14C]-radiolabeled PEG-PHDCA, polysorbate 80 or poloxamine 908-coated PHDCA nanoparticles, and uncoated PHDCA nanoparticles were determined by radioactivity counting after intravenous administration in mice and rats. In addition, the integrity of the blood-brain barrier (BBB) after nanoparticles administration was evaluated by in vivo quantification of the diffusion of [14C]-sucrose into the brain. The location of fluorescent nanoparticles in the brain was also investigated by epi-fluorescent microscopy. RESULTS: Based on their long-circulating characteristics, PEGylated PHDCA nanoparticles penetrated into the brain to a larger extent than all the other tested formulations. Particles were localized in the ependymal cells of the choroid plexuses, in the epithelial cells of pia mater and ventricles, and to a lower extent in the capillary endothelial cells of BBB. These phenomena occurred without any modification of BBB permeability whereas polysorbate 80-coated nanoparticles owed, in part, their efficacy to BBB permeabilization induced by the surfactant. Poloxamine 908-coated nanoparticles failed to increase brain concentration probably because of their inability to interact with cells. CONCLUSIONS: This study proposes PEGylated poly (cyanoacrylate) nanoparticles as a new brain delivery system and highlights two requirements to design adequate delivery systems for such a purpose: a) long-circulating properties of the carrier, and b) appropriate surface characteristics to allow interactions with BBB endothelial cells.

Gastrointestinal transit and mucoadhesion of colloidal suspensions of Lycopersicon esculentum L. and Lotus tetragonolobus lectin-PLA microsphere conjugates in rats.

PURPOSE: To investigate in vivo the fate and the behavior of lectin-particle conjugates after oral administration. METHODS: Two plant lectins were selected, namely Lycopersicon esculentum L. and Lotus tetragonolobus lectins, which have been reported to be specific for oligomers of N-acetyl-D-glucosamine and L-fucose, respectively, and conjugated to small poly(lactide) microspheres. Their intestinal transit was investigated in detail using radiolabeled particles. The transport and the distribution of the particles along the intestine, as well as their interactions with the intestinal mucosa, were determined after oral administration in rat. RESULTS: The overall transit of the particles was shown to be strongly delayed when the microspheres were conjugated to the lectins, mainly due to the gastric retention of the particles. A significant fraction of the conjugates adhered to the gastric and intestinal mucosae. No significant differences were observed after a preliminary incubation of lectin-microsphere conjugates with specific sugars. CONCLUSION: Although specific interactions could not be excluded, especially in the stomach, it was likely that adhesion was predominantly due to nonspecific interactions. These results could be attributed both to unfavorable physicochemical characteristics of the conjugates and to premature adsorption of soluble mucin glycoproteins, preventing any further specific adhesion.

Targeting of 3'-azido 3'-deoxythymidine (AZT)-loaded poly(isohexylcyanoacrylate) nanospheres to the gastrointestinal mucosa and associated lymphoid tissues.

PURPOSE: The aim of the studv was to evaluate the capacity of poly(isohexylcyanoacrylate) nanospheres to concentrate 3'-azido 3'-deoxythymidine (AZT) in the intestinal epithelium and associated immunocompetent cells, which are known to be one of the major reservoirs of the human immunodeficiency virus (HIV). METHODS: The tissue concentration of 3H-radiolabeled AZT in the gastrointestinal (GI) tract was obtained 30 and 9() minutes after intragastric administration to rats at a dose of 0.25 mg AZT/100 g of body weight. The distribution along the intestine was determined. AZT concentrations in the lymph were obtained by lymphatic duct cannulation. RESULTS: Unlike the solution. nanoparticles did concentrate AZT very cfficiently in the intestinal mucosa, as well as in the Peyer's patches, and could simultaneously control the release of free AZT. Concentration in Peyer's patches was 4 times higher for nanoparticles, compared with the control solution. The tissue concentration was 30-45 microM, which was much higher than the reported IC50 of AZT (0.06-1.36 microM) and was regularly distributed along the gastrointestinal tract. CONCLUSIONS: Nanoparticles have been shown to be efficient in concentrating AZT in the intestinal epithelium and gut-associated lymphoid tissues, supporting the view that these particles may represent a promising carrier to treat specifically the GI reservoir of HIV.

Spongelike alginate nanoparticles as a new potential system for the delivery of antisense oligonucleotides.

The aim of this study was to design a new antisense oligonucleotide (ON) carrier system based on alginate nanoparticles and to investigate its ability to protect ON from degradation in the presence of serum. Pharmacokinetics and tissue distribution of ON-loaded nanoparticles have been determined after intravenous administration. An original and dynamic process for ON loading into polymeric nanoparticles has been applied. It is based on the diffusion of ON or ON/polylysine complex into the nanoparticle or the alginate gel, respectively. Indeed, the single coincubation of ON with nanoparticles led, within a few days, to an extremely efficient association. The diffusion kinetic of ON was shown to be dependent on several parameters, incubation temperature, ON concentration, presence or absence of polylysine, polylysine molecular weight, and nanoparticle preparation procedure. This new alginate-based system was found to be able to protect [33P]-radiolabeled ON from degradation in bovine serum medium and to modify their biodistribution, as an important accumulation of radioactivity was observed in the lungs, in the liver, and in the spleen after intravenous administration into mice. ON may be associated efficiently with calcium alginate in a colloidal state. Such nanosponges are promising carriers for specific delivery of ON to lungs, liver, and spleen.

Gene expression of a protein, JB70, related to rat alpha1-acid glycoprotein in Euglena gracilis.

Antibodies directed against rat alpha1-acid glycoprotein (AGP) recognize a 70 kDa antigen, designated JB70, present in extracts of achlorophyllous Euglena gracilis cells as well as in their culture medium. By using 2-dimensional electrophoresis, JB70 appears to be composed of two acidic polypeptides. Additionally, Northern blot analysis reveals the presence in E. gracilis cells of a 2.3 kb mRNA hybridizing with a cDNA probe specific for rat AGP mRNA. Moreover, elevated mRNA levels are detected in dexamethasone-treated E. gracilis cells, indicating a response to this inducer similar to that observed for hepatic AGP. These results strongly suggest that polypeptides closely related to hepatic rat AGP are expressed in E. gracilis cells. They also indicate that, like other gene families implicated in natural defense processes such as heat-shock protein and metallothionein genes, the AGP gene appears to be conserved down to this early diverging eucaryote.

Immunodetection of a protein related to mammalian arrestin in Euglena gracilis.

Six monoclonal antibodies directed against different epitopes of bovine retinal arrestin recognized a single polypeptide in extracts of achlorophyllous Euglena gracilis cells. This polypeptide had an apparent molecular weight slightly lower: 45kDa vs. 48 kDa, and a more basic isoelectric point compared to that of bovine visual arrestin. It was located in an insoluble cytoplasmic fraction. Immunofluorescence assays show a cytoplasmic punctuated pattern suggesting a cluster distribution or a linkage to some cell structure. The presence of arrestine-like molecules in achlorophyllous Euglena gracilis cells suggests that these proteins might be involved in a peculiar step of chemical signal transduction processes.

Different heat-shock proteins are constitutively overexpressed in cadmium and pentachlorophenol adapted Euglena gracilis cells.

To determine whether cellular resistance to a given stressor is related to induction of specific stress-proteins, responses of two adapted Euglena gracilis cell lines, one adapted to cadmium, the other adapted to pentachlorophenol, were analyzed. Our experiments showed that two sets of heat-shock proteins (hsps) were constitutively overexpressed in each cell line: while hsp90, hsp70, hsp55, and hsp40 were induced in cadmium-resistant cells, only hsp40 was induced in pentachlorophenol-adapted cells.

Protein synthesis in cadmium- and pentachlorophenol-tolerant Euglena gracilis.

This work is a preliminary characterization of two adapted Euglena gracilis cell lines, one to cadmium and the other to pentachlorophenol. Growth curve analyses indicate that tolerance to one pollutant did not protect against the second pollutant. These suggest that metabolic pathways that are induced by one pollutant are specific for this pollutant. This specificity is detectable at the level of gene expression.

Growth responses of achlorophyllous Euglena gracilis to selected concentrations of cadmium and pentachlorophenol.

The growth response of a wild achlorophyllous Euglena gracilis mutant was studied during exposure to cadmium and pentachlorophenol (PCP). Cadmium gradually reduced the growth rate and terminal cell density; PCP only lengthened the initial lag phase relative to control cultures. Flow cytometry showed that cadmium altered the cell cycle by delaying late S and G2/M phases; PCP did not disturb the cell cycle, but markedly affected DNA staining: the intercalating dyes ethidium bromide and propidium iodide showed little staining compared to controls. However, replication and transcription processes were not altered by PCP, as cell division occurred normally. Cells surviving after PCP treatment apparently developed an adaptative response during the lag phase.

Cadmium resistance of achlorophyllous Euglena gracilis cells: constitutive overexpression of two heat-shock proteins.

The heat-shock response of Euglena gracilis was studied by cell labeling at both the normal growth temperature (23 degrees C) and an elevated temperature (35 degrees C). Analysis of the labeled proteins by two-dimensional polyacrylamide gel electrophoresis indicated that the rate of synthesis of two polypeptides p55 (55 kDa) and p40 (40 kDa) increased in cells labeled at the highest temperature studied. These polypeptides are also overexpressed in Cadmium-resistant Euglena gracilis cells labeled at the normal growth temperature (23 degrees C). On the basis of these results, p55 and p40 appear to be heat-shock proteins involved in some steps of the acquired Cd-resistance process in Euglena gracilis cells.