Versatile Cellular Uptake Mediated by Catanionic Vesicles - American

Article pubs.acs.org/molecularpharmaceutics

Versatile Cellular Uptake Mediated by Catanionic Vesicles: Simultaneous Spontaneous Membrane Fusion and Endocytosis Chloé Mauroy,†,‡,§ Pauline Castagnos,†,§ Julie Orio,‡ Marie-Claire Blache,‡ Isabelle Rico-Lattes,† Justin Teissié,‡ Marie-Pierre Rols,*,‡ and Muriel Blanzat*,† †

Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique, UMR 5623 Université Paul Sabatier and Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse, France ‡ Institut de Pharmacologie et de Biologie Structurale, UMR 5089 Centre National de la Recherche Scientifique and Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse, France S Supporting Information *

ABSTRACT: Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency. KEYWORDS: catanionic vesicles, membrane fusion, drug delivery, endocytosis, glycolipids

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INTRODUCTION During the last 40 years, a number of drug delivery systems have been developed to control drug release profile, absorption, and distribution, with a view to improving efficacy and safety.1−6 The direct release of active molecules inside the cytoplasm is often considered as the most efficient and the safest mechanism for drug delivery. This promising strategy, which proceeds with drug delivery systems through membrane fusion between suitable carriers and the cell membrane, is not straightforward, due to the competition with endocytotic pathways. It has thus raised important research and developments for the design of synthetic delivery systems. In the early seventies, Papahadjopoulos et al. proposed that liposomes composed of mixtures between phosphatidylethanolamine (PE) and phosphatidylserine (PS) could fuse with plasma membranes and release their contents into the cytoplasm.7 Indeed, vesicles are attractive systems for drug delivery, due in particular to their ability to encapsulate either hydrophilic (in the core) or hydrophobic (in the vesicle membrane) drugs, © XXXX American Chemical Society

controlling their degradation, release, and bioavailability. However, unilamellar vesicles, that are the preferred systems for drug delivery,8,9 remain difficult to obtain since multilayered aggregates are more favorable systems. Therefore, research has been devoted to improve the preparation and stability of vesicles. In the last 20 years, spontaneous unilamellar vesicles formation in the absence of applied external forces has been reported with a new type of surfactants, namely, catanionic systems.10−14 Catanionic vesicles, obtained by spontaneous selfassemblies of oppositely charged amphiphiles, show good stability and dispersion properties in aqueous phase15−19 that make them potential candidates for drug delivery.5 As previously described,20 we managed to form catanionic vesicles made of glycolipids (Figure 1) by a simple and safe preparation Received: July 1, 2014 Revised: September 17, 2014 Accepted: October 13, 2014

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dx.doi.org/10.1021/mp500458f | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Figure 1. (A) Chemical structure of lactose-derived catanionic surfactant (TriCat) used to prepare (B) the glycolipidic catanionic vesicles observed by transmission electron microscopy after freeze-fracturing of the sample.

Figure 2. (A) Confocal microscopy snapshots (merge of the 488 nm channel and the DIC channel) of CHO cells incubated at 37 or 4 °C with (b,e) free surfactants of TriCat/FluoCat at 2 × 10−5 M and (c,f) vesicles of TriCat/FluoCat at 2 × 10−5 M. (a,d) Negative controls (cells alone). These snapshots are representative of what was observed on 30 cells. (B) Flow cytometry results of the interaction of CHO cells incubated at 37 or 4 °C with (b,e) free surfactants of TriCat/FluoCat at 2 × 10−5 M and (c,f) vesicles of TriCat/FluoCat at 2 × 10−5 M. (a,d) Negative controls (cells alone). These experiments have been performed three times, and data were collected on 10 000 cells. The statistical significance of the differences between the means was evaluated by an unpaired Student’s t test. All statistics tests were two sided (NS, not significant; *p < 0.05; **p < 0.01).

from “green” and cheap raw materials by an acid−base reaction in aqueous solution. Herein, the biomimetic lactose-derived polar head provides sufficient hydrophilicity to catanionic surfactants to ensure great stability of their association into vesicles in aqueous media at equimolarity. Our study on these nanovectors also showed that hydrophilic compounds could be encapsulated in their aqueous core,17 while hydrophobic or amphiphilic drugs could be incorporated inside their bilayer.21−23 According to the cellular uptake mechanism, different pharmacological applications may be considered. Indeed, the mechanisms involved in the nanocarrier cell internalization are significantly influenced by the nanovector’s physicochemical properties. As for a majority of other drug delivery systems,24,25 endocytosis was also hypothesized as the most likely pathway to intervene for our catanionic nanovectors. In a previous study,21 catanionic vesicles were shown to interact with a wide variety of cell types, from human phagocytic primary cells to cancer cell lines. However, vesicle/cell interactions seemed to stem from active and passive pathways. In order to assess the passive processes involved, pure lipidic systems such as giant unilamelar vesicles (GUVs) were recently used as simplified membrane models. In this study, we showed the ability of catanionic vesicles to fuse spontaneously with heterogeneous phospholipid assemblies.20

The comprehension of the cell internalization process of catanionic vesicles is a real challenge to move forward with the drug delivery efficacy of these vectors. The aim of the study described herein is to go deeper in the understanding of the cellular uptake of lactose-derived catanionic vesicles and confirm the simultaneous endocytosis and spontaneous membrane fusion processes. The study of the interaction mechanisms involved is performed with a widely studied mammalian cell line, Chinese Hamster Ovary (CHO),26,27 using confocal microscopy observations28 and flow cytometry fluorescence quantification.29

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RESULTS Monitoring of Catanionic Vesicles Uptake by CHO Cells. In order to visualize the interaction between vesicles of the catanionic surfactant (TriCat) and CHO cells, vesicles’ amphiphilic bilayers were labeled by NBD-derived surfactant (FluoCat), as already described in the literature.21 Nonlabeled cells were incubated at 37 and 4 °C with fluorescent TriCat/ FluoCat vesicles or with corresponding free surfactants at the same concentration. The use of a low incubation temperature was used to block endocytotic processes that are strongly temperature dependent. Vesicle/cell interactions were observed by confocal microscopy, and fluorescence intensities were quantified by flow cytometry (FACS) (Figure 2). Both confocal snapshots and quantitative FACS results (Figure 2) show B



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Figure 3. Confocal microscopy snapshots of CHO cells incubated for 1 h at (A) 37 °C or (B) 4 °C with catanionic vesicles labeled with NBDderived FluoCat amphiphile in their bilayer and with Texas Red hydrophilic probe in their core: (a,e) DIC channel; (b,f) 561 nm channel; (c,g) 488 nm channel; (d,h) merge of the 561 nm, 488 nm, and DIC channels.

Figure 4. (A) Confocal microscopy snapshots at 37 °C (488 nm channel). (B) Fluorescence surface plot of the focused zone. In all cases, cells were incubated with catanionic vesicles labeled with FluoCat probe, depending on incubation duration: (a,d) 2 min, (b,e) 15 min, and (c,f) 60 min.

fluorescence internalization into cytoplasm at 37 °C as well as 4 °C. Compared to the one obtained at 37 °C, the fluorescence increase is lower at 4 °C (Figure 2), when all active processes (cellular energy-dependent processes as endocytosis) are blocked. Active processes are thus shown to play a key role in vesicles’ entry into cells. Nevertheless, the decreased but still significant fluorescence of cells treated with vesicles at 4 °C proves the intervention of a passive process (that is not dependent on the cellular metabolism). Besides, no cell fluorescence was observed on confocal snapshots when CHO was treated with free surfactants at 37 and 4 °C. This was confirmed by FACS quantitative fluorescence detection. One

can thus conclude that interaction occurs only when surfactants are in the autoassembled state. As the CHO cell line is nonphagocytic, the active mechanism classically involved in vesicle/cell interactions is endocytosis. This was further checked with our cationic vesicles by a biochemical approach. The three principal endocytic pathways were then simultaneously inhibited in order to isolate passive processes. On this purpose, cells were simultaneously pretreated with three endocytosis inhibitors classically described in the literature:30 amiloride (A) to block macropinocytosis, chlorpromazine (C) to disrupt a clathrin pathway, and filipin (F) for caveolae-mediated endocytosis inhibition. When blocking the three pathways simultaneously (ACF), fluoresC



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Figure 5. Confocal microscopy snapshots at (A) 37 °C or (B) 4 °C (merge of the 561 nm channel, the 488 nm channel, and the DIC channel). In both cases, cells were incubated with catanionic vesicles labeled simultaneously with FluoCat and Texas Red probes, depending on incubation duration: (a,d) 2 min, (b,e) 15 min, and (c,f) 60 min.

To ascertain that spontaneous membrane fusion is involved in TriCat cellular uptake, mixing of the two bilayers has to be proved. Indeed, even if cellular fusion processes are versatile, some general molecular mechanisms appear in all membrane fusion processes. The fusion of drug delivery systems with the membranes is a process characterized by the mixing of inner contents and the merging of lipid membranes. We already have validated the first criteria, with the cytosolic diffuse red fluorescence. In order to visualize the short and early event of lipid mixing, we analyzed the fluorescence distribution of FluoCat in the first minutes of interaction. Figure 4 shows that during the very first minutes of the interaction between TriCat/FluoCat vesicles with CHO cells, the green fluorescence is localized in the periphery of the cell, while it is found everywhere in the cytoplasm at 60 min. The merging of lipid membranes (cell and vesicle), second criterion validating membrane fusion, could be visualized after only 2 min of interaction. We can conclude that both fusion process and endocytosis contribute to cellular uptake mediated by catanionic vesicles. Membrane Fusion Has Faster Kinetics than Endocytosis Mechanisms. The uptake kinetics of these pathways is now studied. CHO cells were incubated with vesicles containing FluoCat and Texas Red probes simultaneously during various times at 4 and 37 °C (Figure 5). These confocal microscopy snapshots demonstrate that membrane fusion occurs within less than 2 min at 37 °C as well as 4 °C (diffuse red fluorescence). In addition, yellow spots (pointed in yellow) highlighting endocytosis are detected, only after 15 min of incubation. This active phenomenon, nonexisting at 4 °C, is delayed compared to the passive one, which is predictable, and hence endocytosis is a metabolic process.31 This kinetic study also confirms the intervention of a spontaneous membrane fusion process. Indeed, the fact that the diffuse red fluorescence appears prior to the yellow spots confirms that the red diffuse fluorescence is not the result of an endosomal escape of the Texas red following a fusion of the catanionic vesicles with endosomes.

cence is still detected within cells, confirming the intervention of a passive process, as previously noticed by the experiments at 4 °C. Confocal micrographs and mean fluorescence intensity data are reported in Supporting Information (Figure S2). Catanionic Vesicles Mediate Versatile Cellular Uptake. To confirm that both endocytosis and membrane fusion are present in the cellular process of internalization, Texas Red, a hydrophilic probe, was encapsulated inside the aqueous core of catanionic TriCat/FluoCat vesicles,20 while FluoCat amphiphilic probe remains inserted in their bilayers. Figure 3A shows the interaction of CHO cells with such labeled vesicles at 37 °C: (i) a diffuse red fluorescence (pointed in red) highlights content release of the hydrophilic probe from catanionic vesicles to cytoplasm since no spontaneous penetration of free Texas Red probe through cellular membrane is occurring (see Figure S3 in Supporting Information); (ii) punctiform yellow spots (pointed in yellow) correspond to the colocalization of Texas Red and FluoCat. The simultaneous visualization of the two probes (yellow spots) reveals unimpaired catanionic vesicles, assumed to be in endolysosomal compartments as a result of endocytotic mechanisms; (iii) punctiform green labeling (pointed in green) could result from membrane fusion and subsequent cellular uptake of residual fragments of vesicles’ bilayers; and/or they could result from the escape of Texas Red content following the fusion of the catanionic vesicles with endosomes, after an endocytotic internalization. Figure 3B shows the same interaction at 4 °C. A diffuse red fluorescence and a punctiform green labeling were still visualized. However, fluorescence intensities decreased compared to the ones at 37 °C. We previously showed that endocytosis was highly slowed down at 4 °C. The absence of punctiform yellow spots in Figure 2B supports this conclusion of the inhibition of endocytic processes. The punctiform green labeling therefore resulted from a different pathway (independent of the cellular metabolism) linked to a membrane traffic. This implies that the NBD-derived FluoCat was in the cell membrane. Thus, the observed fluorescence at 4 °C results from a fusion mechanism bringing the bilayer components of the vesicle in the cell membrane. D



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DISCUSSION TriCat catanionic vesicles are easy to formulate sugar-based surfactants assemblies constituting “green” alternatives to more “classic” drug delivery systems. This study reports evidence that catanionic vesicles are able to deliver hydrophilic and amphiphilic substances into cells following various mechanisms. We thus showed that when the three endocytic pathways are inhibited (thermally or chemically), the residual fluorescence is still meaningful and significantly different depending on the inhibition process. Two hypotheses can thus be drawn. First, other active processes than the three ones already mentioned are involved. Second, a passive process is involved. Colocalization experiments (Figure 3) combined to the results obtained at 4 °C (residual fluorescence at 4 °C) allows discriminating these two hypotheses. At 37 °C, colocalization of Texas Red and FluoCat probes with a punctiform shape reveals internalization of TriCat vesicles into cytoplasm, which gives evidence of an endocytic process. However, at 4 °C, such spots were not visualized, highlighting the absence of any kind of endocytosis. Nevertheless, the intracytoplasmic detection of Texas Red demonstrates a spontaneous membrane fusion at low temperatures. This result validates previous observations obtained with giant unilamellar vesicles (GUVs) as cellular membrane models. Indeed, we previously proved that heterogeneous structure on GUVs composition is necessary to create a membrane destabilization inducing fusion process.20 Spontaneous membrane fusion of catanionic vesicles is thus a passive mechanical process, which requires the presence of phospholipidic domains inducing interfacial defaults where lipids are less organized. The results obtained in this study highlight that these vesicles are able to fuse spontaneously with the membrane of living cells, which possess lipid heterogeneities and notably phase interfaces. Indeed, the presence of lipid microdomains rich in sphingomyelin and cholesterol induces inhomogeneous lateral distribution of membranes32−34 enhancing lipid disorganization and destabilization. Not surprisingly, a kinetics study showed that membrane fusion is a much faster process than endocytosis pathways, which are complex cellular processes implying intervention of proteins (indeed Figure 5 shows immediate apparition of green spots either at 37 or 4 °C, contrary to the yellow ones).35

of the cell following the cellular uptake pathway used. Coencapsulation of several drugs can even be foreseen, exploiting the difference of release kinetics depending on their hydrophilicity. It is worth noticing that the composition of such nano-objects could be also adjusted for specific targeting, by optimizing for instance the nature of surfactant sugar-head or by grafting ligands for specific recognition. Such strategies would be of great interest for coadministration of several drugs of complementary efficiency, in cancer treatment for instance.

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MATERIALS AND METHODS Materials. α-Lactose monohydrate, dodecylamine, methanol, hypophosphorous acid, and dodecylaldehyde used to prepare catanionic surfactants were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA) 12-(N-(7-Nitrobenzo-2-oxa-1, 3-diazol-4-yl)amino)dodecanoic acid (NBD) used to synthesize amphiphilic fluorescent probe FluoCat (λex = 488 nm) and hydrophilic fluorescent probe Texas Red (λex = 563 nm) were obtained from Invitrogen Molecular Probes (Carlsbad, CA, USA). Surfactants Syntheses (TriCat and FluoCat). TriCat. 1N-Dodecylammonium-1-deoxylactitol-bis(α-hydroxydodecyl)phosphinate, the tricatenar catanionic surfactant, so-called TriCat, was obtained following protocols described in the literature.17,20 It is an ionic pair composed by an in situ acid− base reaction between two surfactants: N-dodecylamino-1deoxylactitol36 and alpha-bis(hydroxyl) docecylphosphinic acid.37 FluoCat. As previously reported in the literature,21 Nhexadecylammonium-1-deoxylactitol 12-(-N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)dodecanoate, so-called FluoCat, is an amphiphilic fluorescent probe synthesized from commercially available (molecular probes) 12-(N-(7-nitrobenzo-2-oxa1,3-diazol-4-yl)amino)dodecanoic acid (NBD), added to Nhexadecyl amino-1-deoxylactitol. Preparation and Physicochemical Characterization of Catanionic Vesicles (TriCat, TriCat/FluoCat, TriCat/Texas Red, and TriCat/FluoCat/Texas Red). As already described,20,21,36 freeze-dried TriCat or TriCat/FluoCat (95:5) was put in water or in Texas Red solution (500 μM in water) at the concentration of 1 × 10−4 M in TriCat (which is above CAC (3 × 10−5 M)), stirred, and then sonicated (Vibra Cell, Bioblock Scientific, titanium probe, pulse rate 30%, intensity ×3) for 15 min. Sizes of obtained catanionic vesicles were determined using dynamic light scattering (Malvern Instruments, Nano ZS ZEN3600, U.K.). The analysis was performed with a He−Ne laser (633 nm), a scattering angle of 173°, and at a temperature of 25.0 °C ± 0.1 °C. Vesicles’ size and morphology were checked by transmission electron microscopy using a JEOL JEM 1011 electron microscope, operating at 120 kV. Freeze−fracture experiments were performed with a Balzer vacuum chamber BAF (Balzer, Liechtenstein). A small droplet of aqueous solution (5 mM) was sandwiched between two copper specimen holders and was kept at the desired temperature to reach equilibrium. The environment was saturated with water to avoid evaporation. The sandwich was then frozen with liquid propane cooled with liquid nitrogen. The frozen sandwich was additionally fixed to a transport unit under liquid nitrogen and transferred to the fracture replication stage in a chamber that was then pumped down to 10−6 mbar at −120 °C. Immediately after fracturing, replication took place by first shadowing with platinum/carbon at 45 °C and then with carbon deposition at 90 °C. The sample was allowed to warm

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CONCLUSIONS Very few drug delivery systems have been identified to spontaneously fuse with the plasma membrane. Indeed, the fusion of drug delivery systems with the cell membrane is far from being straightforward as different energy barriers have to be overcome to allow membrane fusion. The cell internalization mechanism of lactose-derived catanionic vesicle contents points out that they are better than “another” drug delivery system. These original nanocarriers have proved to be capable of encapsulating active principles of various hydrophilicities and using different cellular uptake pathways to deliver them. These complementary entry pathways highlight the versatility of such potential drug delivery systems. Spontaneous membrane fusion and endocytic processes confer to catanionic vesicles the ability to deliver compounds either directly into cell cytoplasm or into endolysosomal compartments. Therefore, perspectives of such catanionic vesicles are countless. Indeed, these polyvalent drug delivery systems offer a wide variety of applications since one can encapsulate drugs of any hydrophilicity and target any part E



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darkness conditions) before introducing catanionic vesicles, which were then incubated in the same conditions and in the presence of inhibiting agents for 1 h in the case of FACS analyses and 15 min in the case of confocal microscopy study. In the case of inhibition of all active processes under cold temperature, CHO cells were incubated at 4 °C in darkness conditions 15 min before introducing catanionic vesicles, which were added while maintaining this temperature throughout the vesicle/cell interaction experiment. Internalization should not happen. It is indeed well-known that it is a very slow process at 4 °C.38 Confocal Laser Scanning Microscopy. Evaluation of vesicle uptake by cells was performed by confocal laser scanning microscopy. CHO cells were seeded on Lab-Tek plates (Nunc) at 8 × 104 cell/mL for 1 day atmosphere at 37 °C with 80% humidity. Subsequently, cells were extensively washed with PBS at room temperature. Catanionic vesicles were diluted in serum free EMEM at the final concentration of 2 × 10−5 M and incubated for 15 min with the plated CHO cells. Cells were then washed with cold PBS and then observed in fresh PBS. Live cell imaging was performed with a biphoton microscope Zeiss LSM 710 Inv (obj. ×40 in water), using Argon laser at 488 nm and DPSS laser at 561 nm for FluoCat and Texas Red excitations, respectively. Three images of each condition containing up to 30 individual cells were analyzed with ImageJ software (National Institutes of Health) without any quantification. Images are representative of the original data, corresponding to the fluorescence signal of labeled catanionic vesicles since cells were not labeled. A control without vesicles was used to set laser intensity in order to limit cellular autofluorescence. Fluorescence-Activated Cell Sorting (FACS) Analyses. CHO cells were seeded on 24-well plates (Nunc) for 1 day at 7.5 × 10 4 cell/mL in supplemented EMEM in a CO2 atmosphere at 37 °C with 80% humidity. Cells were extensively washed with PBS. Catanionic vesicles were diluted in serum free EMEM at the final concentration of 2 × 10−5 M and incubated for 1 h with the plated CHO cells. Cells were then washed with cold PBS, harvested using trypsin/EDTA, diluted in full serum EMEM, and analyzed by FACS (BD Fascalibur Cell Sorter) by using CellQuest Pro software to quantify the percentage of fluorescent cells. Laser excitation was at 488 nm. Statistical analysis was performed using Prism 4.0 (GraphPad Software) by one-way ANOVA and Tukey test. All data were expressed as the mean ± SEM of three independent sets of experiments on 10,000 cells each. Statistical significance for this study was considered at p < 0.05.

to room temperature. Replicas were retrieved and cleaned in water and mounted on 200-mesh copper grids. Observations were made with a cryo-electron microscope FEI EM120 (120 kV), and the images were recorded with an SSCCD 2k Gatan camera. Spectrofluorimetry measurements were performed on vesicles of TriCat/FluoCat (95:5), on a spectrofluorimeter PTI (Photon Technology International) equipped with a EIMAC xenon lamp of 175 W. All the slit widths were set at 2 nm. The excitation wavelength was set at 488 nm, and the emitted intensity was collected from 500 to 600 nm. The emission wavelength has been determined at 549 nm. Physicochemical characteristics (shape, size, and stability) of labeled vesicles with FluoCat and/or Texas Red were checked to remain identical as “empty” TriCat vesicles without any pH dependence, compatible with flow cytometry or confocal laser scanning microscopy detection. To perform vesicles/cells interactions studies, catanionic vesicles were diluted until 2 × 10−5 M. Vesicles of TriCat, labeled or not with FluoCat and/or Texas Red, were checked to be stable under dilution by DLS and tensiometry analyses, even at concentrations inferior to the surfactant CAC.21 Free surfactants were also prepared at the concentration of 2 × 10−5 M. Cell Culture. Chinese Hamster Ovary (CHO) cells (Wild Type Toronto clone) were grown plated in EMEM (Eurobio) supplemented with 8% (v/v) fetal calf serum (Lonza), 1% (v/ v) L-glutamine (2 mM), 1% (v/v) penicillin streptomycin (50 IU/mL) (all from Eurobio), 3.5 g·L−1 glucose, 1% (v/v) vitamins, and 2.95 g·L−1 tryptose phosphate (all from Sigma). Culture medium was renewed every 2 days. Flasks were from Nunc. Culture was operated in a 5% CO2 atmosphere at 37 °C with 80% humidity. Cytotoxicity Test. A 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-arboxanilide (XTT) test was performed to check the cytotolerance of catanionic vesicles depending on the applied concentration. Cells were seeded on 96-well plates for 2 days at 2 × 105 cell/mL. Cells were washed with phosphate buffer solution (PBS) (Sigma) and incubated for 3 h with solutions of catanionic vesicles ranging from 1 × 104 to 2 × 107 M in serum free EMEM. Cells were then washed with PBS, and 50 μL of a Red Phenol free EMEM solution containing XTT (0.5 mg/mL) and coenzyme Q (40 μL/mL) were added. After 3 h of incubation, 100 μL of a sodium dodecyl sulfate solution (10%) was added. The reaction was then read on a Polarstar Galaxy spectrophotometer (BMG Labtech, France) at a wavelength set at 450 nm. The concentration of vesicles applied on cells was chosen according to the cytotoxicity results. About 100% viability of CHO was observed after 3 h of incubation with vesicles at 2 × 10−5 M whatever their composition (TriCat, TriCat/FluoCat, TriCat/Texas Red, and TriCat/FluoCat/Texas Red). Internalization Inhibition. To study the endocytosis phenomenon, endocytosis inhibiting agents widely described in the literature30 were applied on CHO cells: amiloride (A), chlorpromazine (C), and filipin (F). Optimal concentration of each inhibitor was determined by a preliminary study where a range of concentrations was applied on cells. Chosen concentrations showed maximal inhibiting efficiency and lowest cytotoxicity: (A) 1 mg/mL; (C) 1.3 × 10−2mg/mL; (F) 1.5 × 10−3mg/mL; and (ACF) the mixture of the previous amiloride + chlorpromazine + filipin at the same concentrations in culture medium. CHO cells were pretreated with these agents 1 h in an incubator (37 °C, 5% CO2, in

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ASSOCIATED CONTENT

S Supporting Information *

TEM snapshot and size distribution of catanionic vesicles; flow cytometry results; confocal microscopy snapshots; and cytotoxicity of catanionic vesicles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +33 5 61 55 73 75. Fax: +33 5 61 55 81 55. E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

P.C. and C.M. contributed equally to this work.

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(17) Soussan, E.; Mille, C.; Blanzat, M.; Bordat, P.; Rico-Lattes, I. Sugar-derived tricatenar catanionic surfactant: Synthesis, self-assembly properties, and hydrophilic probe encapsulation by vesicles. Langmuir 2008, 24 (6), 2326−2330. (18) Ghosh, S.; Ambade, B.; Ray, A. Stable catanionic vesicles as drug delivery vehicle. Sci. Adv. Mater. 2013, 5 (12), 1837−1846. (19) Qu, W.; Xia, Z.; Guo, H.; Li, X.; Dou, T. Vesicle Formation with an Anionic Surfactant and a Conventional Cationic Surfactant in Mixed Systems. J. Dispersion Sci. Technol. 2013, 34 (2), 240−243. (20) Mauroy, C.; Castagnos, P.; Blache, M.-C.; Teissié, J.; RicoLattes, I.; Rols, M.-P.; Blanzat, M. Interaction between GUVs and catanionic nanocontainers: new insight into spontaneous membrane fusion. Chem. Commun. 2012, 48, 6648−6650. (21) Boudier, A.; Castagnos, P.; Soussan, E.; Beaune, G.; Belkhelfa, H.; Ménager, C.; Cabuil, V.; Haddioui, L.; Roques, C.; Rico-Lattes, I.; Blanzat, M. Polyvalent catanionic vesicles: Exploring the drug delivery mechanisms. Int. J. Pharm. 2011, 403 (1−2), 230−236. (22) Consola, S.; Blanzat, M.; Perez, E.; Garrigues, J.-C.; Bordat, P.; Rico-Lattes, I. Design of original bioactive formulations based on sugar-surfactant/non-steroidal anti-inflammatory catanionic self-assemblies: A new way of dermal drug delivery. Chem.Eur. J. 2007, 13 (11), 3039−3047. (23) Castagnos, P.; Siqueira-Moura, M. P.; Leme Goto, P.; Perez, E.; Franceschi, S.; Rico-Lattes, I.; Tedesco, A. C.; Blanzat, M. Catanionic vesicles charged with chloroaluminium phthalocyanine for topical photodynamic therapy. In vitro phototoxicity towards human carcinoma and melanoma cell lines. RSC Adv. 2014, 4, 39372−39377. (24) Bareford, L. M.; Swaan, P. W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Delivery Rev. 2007, 59 (8), 748−758. (25) Marianecci, C.; Di Marzio, L.; Rinaldi, F.; Celia, C.; Paolino, D.; Alhaique, F.; Esposito, S.; Carafa, M. Niosomes from 80s to present: the state of the art. Adv. Colloid Interface Sci. 2014, 205, 187−206. (26) Cezanne, L.; Navarro, L.; Tocanne, J.-F. Isolation of the plasma membrane and organelles from Chinese hamster ovary cells. Biochim. Biophys. Acta, Biomembr. 1992, 1112 (2), 205−214. (27) Gottesman, M. M. Molecular Cell Genetics, 1st ed.; John Wiley & Sons, Inc.: New York, 1985. (28) Torchilin, V. P. Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate. Adv. Drug Delivery Rev. 2005, 57 (1), 95−109. (29) Huth, U. S.; Schubert, R.; Peschka-Süss, R. Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J. Controlled Release 2006, 110 (3), 490−504. (30) Khalil, I. A.; Kogure, K.; Alkita, H.; Harashima, H. Uptake pathways and subsequent intracellular trafficking in non viral gene delivery. Pharmacol. Rev. 2006, 58, 32−45. (31) Grant, B. D.; Donaldson, J. G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol. 2009, 10 (9), 597−608. (32) Edidin, M. The state of lipid rafts: From model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 2003, 32 (1), 257−283. (33) Tocanne, J.-F.; Cézanne, L.; Lopez, A.; Piknova, B.; Schram, V.; Tournier, J.-F.; Welby, M. Lipid domains and lipid/protein interactions in biological membranes. Chem. Phys. Lipids 1994, 73 (1−2), 139−158. (34) Tocanne, J.-F.; Dupou-Cézanne, L.; Lopez, A.; Tournier, J.-F. Lipid lateral diffusion and membrane organization. FEBS Lett. 1989, 257 (1), 10−16. (35) Marsh, M.; McMahon, H. T. The structural era of endocytosis. Science 1999, 285 (5425), 215−220. (36) Castagnos, P. Vésicules catanioniques: Design et mécanismes de délivrance de principes actifs. Ph.D. thesis, Toulouse III, Toulouse, France, 2011. (37) Brun, A.; Etemad-Moghadam, G. New double-chain and aromatic (alpha-hydroxyalkyl)phosphorus amphiphiles. Synthesis 2002, 10, 1385−1390. (38) Di Marzio, L.; Marianecci, C.; Cinque, B.; Nazzarri, M.; Cimini, A. M.; Cristiano, L.; Cifone, M. G.; Alhaique, F.; Carafa, M. pHsensitive non-phospholipid vesicle and macrophage-like cells: Binding,

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The imaging platform Toulouse Réseau Imagerie is acknowledged for providing the technical support in microscopy and FACS. TRI is funded by the region Midi Pyrenees, the grand Toulouse cluster, and the EU FEDER. FONDEREPHAR at the Pharmacy Faculty of Toulouse is acknowledged for their expertise in cytotoxicity experiments. P.C. and C.M. were supported by fellowships from the MENESR. We acknowledge financial support from the Association Française contre les Myopathies, the ANR contracts Cemirbio and CMIDT, COPOPDT, the Direction Générale de l′Armement, the Ministère des Affaires Etrangères et Européennes, and the Région Midi-Pyrénées (réseau thérapie génique).

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REFERENCES

(1) Allen, T. M.; Cullis, P. R. Drug delivery systems: entering the mainstream. Science 2004, 303, 1818−1824. (2) Faraji, A. H.; Wipf, P. Nanoparticles in cellular drug delivery. Bioorg. Med. Chem. 2009, 17 (8), 2950−2962. (3) Moses, M. A.; Brem, H.; Langer, R. Advancing the field of drug delivery: Taking aim at cancer. Cancer Cell 2003, 4 (5), 337−341. (4) Singh, R.; Lillard, J. W., Jr. Nanoparticle-based targeted drug delivery. Exp. Mol. Pathol. 2009, 86 (3), 215−223. (5) Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I. Drug delivery by soft matter: Matrix and vesicular carriers. Angew. Chem., Int. Ed. 2009, 48 (2), 274−288. (6) Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discovery 2005, 4 (2), 145− 160. (7) Papahadjopoulos, D.; Poste, G.; Schaeffer, B. E. Fusion of mammalian cells by unilamellar lipid vesicles: Influence of lipid surface charge, fluidity and cholesterol. Biochim. Biophys. Acta, Biomembr. 1973, 323, 23−42. (8) Gregoriadis, G.; Allison, A. C. Liposomes in Biological Systems; John Wileys and Sons: New York, 1990. (9) Malmsten, M. Surfactants and Polymers in Drug Delivery. Marcel Dekker: New York, 2002. (10) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants. Science 1989, 245 (4924), 1371−1374. (11) Marques, E.; Khan, A.; da Graca Miguel, M.; Lindman, B. Selfassembly in mixtures of a cationic and an anionic surfactant: the sodium dodecyl sulfate-didodecyldimethylammonium bromide-water system. J. Phys. Chem. 1993, 97 (18), 4729−4736. (12) Menger, F. M.; Binder, W. H.; Keiper, J. S. Cationic surfactants with counterions of glucuronate glycosides. Langmuir 1997, 13 (12), 3247−3250. (13) Blanzat, M.; Perez, E.; Rico-Lattes, I.; Prome, D.; Prome, J. C.; Lattes, A. New catanionic glycolipids. 1. Synthesis, characterization, and biological activity of double-chain and gemini catanionic analogues of galactosylceramide (gal beta(1)cer). Langmuir 1999, 15 (19), 6163−6169. (14) Blanzat, M.; Perez, E.; Rico-Lattes, I.; Lattes, A.; Gulik, A. Correlation between structure, aggregation behaviour and cellular toxicity of anti-HIV catanionic analogues of galactosylceramide. Chem. Commun. 2003, 2, 244−245. (15) Marques, E. F.; Regev, O.; Khan, A.; Lindman, B. Selforganization of double-chained and pseudodouble-chained surfactants: counterion and geometry effects. Adv. Colloid Interface Sci. 2003, 100− 102, 83−104. (16) Vivares, D.; Soussan, E.; Blanzat, M.; Rico-Lattes, I. Sugarderived tricatenar catanionic surfactant: Self-assembly and aggregation behavior in the cationic-rich side of the system. Langmuir 2008, 24, 9260−9267. G



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uptake and endocytotic pathway. Biochim. Biophys. Acta 2008, 1778 (12), 2749−2756.

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Versatile Cellular Uptake Mediated by Catanionic Vesicles - American

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