This work investigated the endocytic pathways taken by poly(isobutylcyanoacrylate) (PIBCA) nanoparticles differing in their surface composition and architecture, assuming that this might determine their efficiency of intracellular drug delivery. Nanoparticles (A0, A25, A100, R0, R25 ) were prepared by anionic or redox radical emulsion polymerization using mixtures of dextran and fucoidan (0, 25, 100 % in fucoidan). Cell uptake was evaluated by incubating J774A.1 macrophages with nanoparticles. Endocytic pathways were studied by incubating cells with endocytic pathway inhibitors (chlorpromazine, genistein, cytochalasin D, methyl-ß-cyclodextrin and nocodazole) and nanoparticle uptake was evaluated by flow cytometry and confocal microscopy. The fucoidan-coated PIBCA nanoparticles A25 were internalized 3-fold more efficiently than R25 due to the different architecture of the fucoidan chains presented on the surface. Different fucoidan density and architecture led to different internalization pathway preferred by the cells. Large A100 nanoparticles with surface was covered with fucoidan chains in a loop and train configuration were internalized the most efficiently, 47-fold compared with A0, and 3-fold compared with R0 and R25 through non-endocytic energy-independent pathways and reached the cell cytoplasm. Internalization pathways of PIBCA nanoparticles by J774A.1 macrophages could be determined by nanoparticle fucoidan surface composition and architecture. In turn, this influenced the extent of internalization and localization of accumulated nanoparticles within cells. The results are of interest for rationalizing the design of nanoparticles for potential cytoplamic drug delivery by controlling the nature of the nanoparticle surface.

The increasing resistance of Neisseria gonorrhoeae to any current antibiotic treatment and the difficulties associated with the use of prevention means such as condom urge the need for alternative methods to prevent this sexually transmitted infection. In this work, a prevention strategy based on the use of a vaginal gel containing Lactobacilli was assessed in vitro. A Lactobacillus crispatus strain (ATCC 33197) was selected based on the published data on its ability to inhibit Neisseria gonorrhoeae. Its probiotic properties were first characterized. Then, a thermo-sensitive hydrogel containing 21.5% of poloxamer 407, 1% of sodium alginate and 9 log(10), CFU of Lactobacillus crispatus per gel sample (5 g) was developed. The gelation temperature and the rheological characteristics of this formulation appeared suitable for a vaginal administration. Lactobacillus crispatus was viable in the gel for six months although a large amount of the bacteria was not culturable. The ability of Lactobacillus crispatus to inhibit Neisseria gonorrhoeae was still observed with the gel. Such system, thus, appeared promising for the prevention of gonorrhea.

By 2030, 43.9% of the US adult population is projected to have some form of cardiovascular disease (CVD) highlighting the need for new therapeutic strategy or an improvement of existing treatments. In many cardiac diseases (i.e. heart failure, myocardial infarction, ischemia/reperfusion), mitochondria can receive harmful signals, dysfunction and then, participate actively in the pathogenesis. They can undergo either a decrease of their bioenergetic capacity, a process called mitochondrial permeability transition which leads to cell death, alterations of mitochondrial protein expression patterns or perturbations in the fusion/fission balance. In a cardioprotective perspective, it is important to evaluate the effect of drugs on mitochondrial structure and function, because this can impact on cardiac function and health at a more general level. In this context, we evaluated the mitochondrial effects of Digoxine and Digitoxigenine, two cardiac glycosides used in clinics for heart failure treatment. We selected these two compounds in a high throughput screening of cell death inhibitors revealing their activity as potent inhibitors of apoptosis and necrosis of cardiomyoblasts. We confirmed their activity in rat neonatal ventricular cardiomyocytes and showed their capacity to induce autophagy. We also analysed their effects on mitochondrial network structure by fluorescent confocal microscopy and on bioenergetics by Seahorse technology. Our study offers new insights into the pharmacological effects of cardiac glycosides that could help to better understand their mechanisms of cardioprotection.

There is increasing evidence that the chaperone-like protein CDC48 (cell division cycle 48) plays a role in plant immunity. Cytosolic ascorbate peroxidase (cAPX), which is a major regulator of the redox status of plant cells, has previously been shown to interact with CDC48. In this study, we examined the regulation of cAPX by the ATPase NtCDC48 during the cryptogein-induced immune response in tobacco cells. Our results not only confirmed the interaction between the proteins but also showed that it occurs in the cytosol. cAPX accumulation was modified in cells overexpressing NtCDC48, a process that was shown to involve post-translational modification of cAPX. In addition, cryptogein-induced increases in cAPX activity were suppressed in cells overexpressing NtCDC48 and the abundance of the cAPX dimer was below the level of detection. Furthermore, the levels of both reduced (GSH) and oxidized glutathione (GSSG) and the GSH/GSSG ratio decreased more rapidly in response to the elicitor in these cells than in controls. A decrease in cAPX activity was also observed in response to heat shock in the cells overexpressing NtCDC48, indicating that the regulation of cAPX by NtCDC48 is not specific to the immune response.

Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies.

Nanoparticles made of metal-organic frameworks (nanoMOFs) attract a growing interest in gas storage, separation, catalysis, sensing and more recently, biomedicine. Achieving stable, versatile coatings on highly porous nanoMOFs without altering their ability to adsorb molecules of interest represents today a major challenge. Here we bring the proof of concept that the outer surface of porous nanoMOFs can be specifically functionalized in a rapid, biofriendly and non-covalent manner, leading to stable and versatile coatings. Cyclodextrin molecules bearing strong iron complexing groups (phosphates) were firmly anchored to the nanoMOFs' surface, within only a few minutes, simply by incubation with aqueous nanoMOF suspensions. The coating procedure did not affect the nanoMOF porosity, crystallinity, adsorption and release abilities. The stable cyclodextrin-based coating was further functionalized with: i) targeting moieties to increase the nanoMOF interaction with specific receptors and ii) poly(ethylene glycol) chains to escape the immune system. These results pave the way towards the design of surface-engineered nanoMOFs of interest for applications in the field of targeted drug delivery, catalysis, separation and sensing.

SUMMARY To overcome poor diffusion of antibiotics such as β– lactam, we developed and compared antibacterial activity of new terpene-based nanoparticles (NPs) coupled to Penicillin G (PNG). Squalene-penicillin G with or without a pH-sensitive linker (SqPNG-pH or SqPNG) have been synthesized and characterized. An in vitro comparative efficacy study has been performed and SqPNG-pH NPs have proved to increase the intracellular antibacterial activity on the intracellular pathogen Staphylococcus aureus compared to the free drug. INTRODUCTION In the field of antibiotherapy, it is now well established that the poor intracellular penetration of antibiotics reduces the effectiveness of many treatments. During infections, some bacteria such as Staphyloccocus aureus are able to hide from host defense and/or action of antibiotics. They survive and replicate into the host cells, thus establishing a cell-niche causing persistent or recurrent infections. Moreover, sub-lethal concentrations of antibiotics provide to bacteria a suitable hostile environment for the emergence of antibiotic resistance. To overcome it, a drug targeting strategy has proved be useful in conveying antibiotics directly into host cells and reaching therapeutically active concentrations. In our laboratory we have developed the “squalenoylation” technology 1 which has already shown remarkable pharmacological activity with drugs like gemcitabine (anti-cancer drug) or other nucleosides analogues (antiviral drugs). 2-3 This concept is based on a covalent chemical conjugation of a natural and biodegradable lipid, the squalene (or others terpenic moieties), to an active drug in order to prepare stable nanoparticles (NPs). In this context, we synthesized two prodrugs derived from the bioconjugation of the historical β-lactam antibiotic, Penicillin G (PNG) to the squalene (Sq) moiety with and without a pH sensitive linker (SqPNGpH and SqPNG). All these analogs self-assembled in water to form stable nanoparticles ( 145 nm). Moreover, all NPs have shown similar morphology and surface charge, but those with pH-sensitive linker induced a better intracellular antibacterial activity on the facultative intracellular pathogen S. aureus. EXPERIMENTAL METHODS SqPNG and SqPNG-pH nanoparticles were obtained using nanoprecipitation procedure. Briefly, 4 mg of compounds were dissolved in 500 μL of ethanol before being added drop-wide under stirring into 1 mL MilliQ water. Then, ethanol was completely evaporated using a Rotavapor® to obtain an aqueous suspension of SqPenG or SqPenGpH NPs. When needed, the green dye cholesteryl Bodipy FLC12 (at 0.5 % molar), was used to prepare florescentlabeled NPs by adding the probe to the organic phase before nanoprecipitation. The diameter-average of the nanoparticles was determined by dynamic light scattering (DLS), using a Zetasizer Nano 6.12, (Malvern Instrument Ltd, Worcestershire, UK). The zeta potential (ζ) was measured using the same equipment. Nanoparticles’s morphology was observed by cryogenic transmission electron microscopy. The experiments utilized penicillin-sensitive strain, Staphylococcus aureus (ATCC 55585) which was obtained from the ATCC and growth in Brain Heart Infusion (BHI) media (Invitrogen) at 37°C. Murine macrophage cell line (J774) were chose and cultured in RPMI 1640 (Roswell Park Memorial Institute) medium supplemented with 10% of inactivated fetal bovine serum (FBSd) (Gibco) at 37 °C in humidified atmosphere containing 5% CO2. Intracellular antimicrobial activity of NPs was determined using the following method. J774 cells were seeded in 24-well plates at a concentration of 2 × 10 5 cells per well and allowed to adhere overnight. After washing with PBS, cells were pre-treated with NPs or free PNG during 6 h. J774 cells were washed and allowed to engulf S. aureus at a multiplicity of infection (MOI) of 10 for 2 h. To remove the extracellular bacteria, J774 macrophages were then washed again and incubated with gentamicin (50 μg/mL) during 6 or 24 hours. Cells were lysis and viable bacteria plated onto BHI/agar for colony forming unit (CFU) enumeration after 24 h. For confocal microscopy studies, J774 cells were treated then infected in similar conditions explained previously. Then LIVE/DEAD BacLight bacterial viability kit (Molecular Probes) were used to visualize viable/dead bacteria, intracellularly. Fluorescent-NPs helped to explore and compare macrophage capture and intracellular localization of NPs using confocal microscopy and flow cytometry analysis. RESULTS AND DISCUSSION SqPNG and SqPNG-pH compounds showed the capability to spontaneously self-assemble as stable NPs in water. The SqPNG and SqPNG-pH NPs' average diameter was 140 and 150nm, respectively, with a very low polydispersity. Their Z-potential was about -50 mV. Similar results (size, zeta potential and stability) were obtained when fluorescent probe was added to the formulation process. Cryo transmission electron microscopy (CryoTEM) analysis of SqPNG-pH NPs revealed a spherical and regular shape (Figure 1). Figure 1. Cryogenic transmission electron microscopy of a suspension of SqPNGpH nanoparticles. Scale bars = 100 nm The ability of SqPNG and SqPNG-pH NPs to be internalized by macrophages was studied by flow cytometry and confocal microscopy. We observed that both types of NPs were internalized (and not adsorbed on the cells surface) differently by macrophages, depending on the linkage. SqPNG NPs reached a maximum of internalization at 6 h, while fluorescence of SqPNG-pH NPs continued to increase during 24 h. Figure 2. Confocal fluorescence microscopic images of viable bacteria in J774 cells, after 2h of pretreatment with SqPenGpH NPs or free PNG and following by S. aureus infection. Viable S. aureus cells are stained using LIVE/DEAD BacLight kit. In green: viable bacteria and in red: dead bacteria. Intracellular survival assay were carried out in infected J774 cell line in order to determine the efficiency of our systems in delivering antibiotic intracellularly. Practically, cells were preincubated with different NP formulations maintaining similar PNG concentrations (20 μg/mL) and then infected with S. aureus bacteria. The control PNG-unloaded NPs as well as free PNG had no antimicrobial activity on intracellular bacteria while SqPNG and SqPNG-pH NPs decrease the number of intracellular bacteria after only 6h. 24h after infection this effect was even more pronounced. However, in both case (6 and 24h post treatment), the presence of pH-sensitive linker improved the antibacterial effect suggesting that PNG was more efficiently released from SqPNG-pH NPs, intracellularly. Moreover, further investigation using the LIVE/DEAD BacLight kit, which allowed to differentiate between viable and dead S. aureus, clearly shown that free PNG did not induce any significant mortality of intracellular S. aureus, unlike SqPNG-pH NPs, which raise the number of red/dead bacteria (Figure 2). Finally, NPs and bacteria were found to localize in different intracellular compartments suggesting that intracellular released PNG diffused and crossed the endolysosmal membranes in order to reach intracellular bacterial vacuole and kill bacteria. We further investigated the modification of terpene moieties in order to improve even more the antibacterial activity of penicillin G. Preliminary results shown remarkable antibacterial effect (with 3-log reduction of viability) on intracellular S. aureus. CONCLUSION In brief, two bioconjugates of penicillin G with squalene, using either a pH-sensitive (SqPNG-pH) or a pH-insensitive linker (SqPNG), were synthesized and found to spontaneously form stable nanoparticles which were able to enter and release Penicillin G intracellularly. The SqPNG-pH nanoparticles have proved to induce fast and significant killing of S. aureus-infected J774 cells. This approach opens interesting perspectives to combat invasive bacterial infections such as S. aureus, which represents a worldwide public health concern and a major burden for the dairy industry. REFERENCES 1. Couvreur P, Vauthier C. Nanotechnology: intelligent design to treat complex disease. Pharm Res 2006;23:1417-50. 2. Bildstein L, Dubernet C, Couvreur P. Prodrugbased intracellular delivery of anticancer agents. Advanced Drug Delivery Reviews 2011;63:3-23. 3. Maksimenko A, Mougin J, Mura S, et al. Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy. Cancer Lett 2013;334:346-53. ACKNOWLEDGMENTS Thanks to Ghislaine Frébourg for technical help with cryo-TEM. The research leading to this work has received funding from European Research Council under the European Community’s Seventh Framework Program FP7/2007-2013 (grant agreement no. 249835) but also from the CNRS and Université Paris-Sud.

The therapeutic activity of selective serotonin (5-HT) reuptake inhibitors (SSRIs) relies on long-term adaptation at pre- and post-synaptic levels. The sustained administration of SSRIs increases the serotonergic neurotransmission in response to a functional desensitization of the inhibitory 5-HT1A autoreceptor in the dorsal raphe. At nerve terminal such as the hippocampus, the enhancement of 5-HT availability increases brain-derived neurotrophic factor (BDNF) synthesis and signaling, a major event in the stimulation of adult neurogenesis. In physiological conditions, BDNF would be expressed at functionally relevant levels in neurons. However, the recent observation that SSRIs upregulate BDNF mRNA in primary cultures of astrocytes strongly suggest that the therapeutic activity of antidepressant drugs might result from an increase in BDNF synthesis in this cell type. In this study, by overexpressing BDNF in astrocytes, we balanced the ratio between astrocytic and neuronal BDNF raising the possibility that such manipulation could positively reverberate on anxiolytic-/antidepressant-like activities in transfected mice. Our results indicate that BDNF overexpression in hippocampal astrocytes produced anxiolytic-/antidepressant-like activity in the novelty suppressed feeding in relation with the stimulation of hippocampal neurogenesis whereas it did not potentiate the effects of the SSRI fluoxetine on these parameters. Moreover, overexpressing BDNF revealed the anxiolytic-like activity of fluoxetine in the elevated plus maze while attenuating 5-HT neurotransmission in response to a blunted downregulation of the 5-HT1A autoreceptor. These results emphasize an original role of hippocampal astrocytes in the synthesis of BDNF, which can act through neurogenesis-dependent and -independent mechanisms to regulate different facets of anxiolytic-like responses.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed ‘mammospheres’. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1+) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

The purpose of this work was to investigate the potential of α-cyclodextrin combined to soybean oil-based formulations to modulate the release of a model drug, indomethacin. Dry emulsion, naked and coated beads were prepared from the same initial formulation using the same manufacturing process. Dry emulsion was selected to accelerate drug release while beads coated with α-cyclodextrin were designed to sustain it. Indomethacin-loaded systems were prepared, characterised and evaluated in vitro. Pharmacokinetic studies were performed in fasted and fed rats. The presence of the α-cyclodextrin coat was confirmed by confocal microscopy, and an increase of the mass and diameter of the beads. The layer of α-cyclodextrin improved their resistance in simulated gastro-intestinal fluids. In vitro, the dissolution of indomethacin was slower with coated beads than with emulsion and naked beads. Lipid-based formulations showed an increase of relative bioavailability of IND versus Indocid®. Whatever the formulation, greater and faster release of indomethacin was noticed in sodium taurocholate-rich medium and in fed rats. Compared to naked beads, an increased Cpmax with a shorter Tmax was observed with the emulsion while Tmax and MRT were increased and Cpmax reduced with the coated beads. Interestingly, formulations based on alpha cyclodextrin and soybean oil can modify the release of a lipophilic drug depending on the system formed.

The aim of this work was to investigate the stability in vitro, in simulated gastro-intestinal fluids, of beads, made of α-cyclodextrin and soybean oil, and to study the release of progesterone, a model of lipophilic drug. This was evaluated over time by the monitoring of the proportion of intact beads, their volume and the percentage of progesterone dissolved. Their incubation in the simulated gastric fluid provoked a moderate reduction of their number (20%) and a decrease of their volume (50%) after 55 min. Whatever the intestinal medium subsequently introduced, bead number and volume decreased more until bead disintegration that appeared faster in sodium taurocholate rich-medium. In such fluid, the amount of progesterone dissolved increased rapidly between 65 and 180 min, with both beads and emulsion to be equal after 85 min. With soft capsules, the increase was more gradual. In sodium taurocholate free-medium, more progesterone was dissolved from the emulsion than from beads or soft capsules. The release of progesterone from beads resulted from the erosion of their matrix and its partition equilibrium between oily micro-droplets and aqueous phase. The original structure of beads confers to this multiparticulate system interesting properties for the oral delivery of lipophilic drugs.

The aim was to synthesize and characterize fucoidan-coated poly(isobutylcyanoacrylate) nanoparticles. The nanoparticles were prepared by anionic emulsion polymerization (AEP) and by redox radical emulsion polymerization (RREP) of isobutylcyanoacrylate using fucoidan as a new coating material. The nanoparticles were characterized, and their cytotoxicity was evaluated in vitro on J774 macrophage and NIH-3T3 fibroblast cell lines. Cellular uptake of labeled nanoparticles was investigated by confocal fluorescence microscopy. Results showed that both methods were suitable to prepare stable formulations of fucoidan-coated PIBCA nanoparticles. Stable dispersions of nanoparticles were obtained by AEP with up to 100% fucoidan as coating material. By the RREP method, stable suspensions of nanoparticles were obtained with only up to 25% fucoidan in a blend of polysaccharide composed of dextran and fucoidan. The zeta potential of fucoidan-coated nanoparticles was decreased depending on the percentage of fucoidan. It reached the value of -44 mV for nanoparticles prepared by AEP with 100% of fucoidan. Nanoparticles made by AEP appeared more than four times more cytotoxic (IC50 below 2 mu g/mL) on macrophages J774 than nanoparticles made by RREP (IC50 above 9 mu g/mL). In contrast, no significant difference in cytotoxicity was highlighted by incubation of the nanoparticles with a fibroblast cell line. On fibroblasts, both types of nanoparticles showed similar cytotoxicity. Confocal fluorescence microscopy observations revealed that all types of nanoparticles were taken up by both cell lines. The distribution of the fluorescence in the cells varied greatly with the type of nanoparticles. (C) 2011 Elsevier B.V. All rights reserved.

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.

Although numerous drugs are used to treat HIV infection with increasing efficacy, the patient's brain is often infected by the virus and acts as a sanctuary where drugs cannot penetrate due to their low passage through the blood brain barrier. Therefore, the design of new medicine able to reach the brain is extremely challenging. An approach based on prodrug synthesis and encapsulation into PEGylated nanocarriers was proposed and applied to didanosine, a nucleosidic analogue used to treat HIV-1 associated dementia. In this study, appropriate formulations of PEGylated liposomes were designed to incorporate two glycerolipidic prodrugs of didanosine. Preparation methods based on Bangham's or emulsion/evaporation techniques were optimized for each prodrug formulation according to the influence of critical parameters on vesicle size distribution. The obtained formulations exhibited particle size under 300 nm with high incorporation of prodrugs as shown by light scattering, optical microscopy experiments and differential scanning calorimetry. Finally the uptake of fluorescently labeled PEGylated formulations by rat brain immortalized endothelial cells modeling the BBB was evidenced by confocal laser scanning microscopy. All the results suggest that the encapsulation of didanosine prodrugs into PEGylated liposomes is a promising approach in the goal of increasing didanosine concentration in the brain and treating HIV-1-associated dementia.

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.