Insights in Cellular Uptake Mechanisms of pDNA-polycationic Amphiphilic Cyclodextrin Nanoparticles (CDplexes)

1 Insights in Cellular Uptake Mechanisms of pDNA―Polycationic Amphiphilic Cyclodextrin Nanoparticles (CDplexes) Alejandro Díaz-Moscoso,†,¶ Dries Vercauteren,‡,¶ Joanna Rejman,‡ Juan M. Benito,† Carmen Ortiz Mellet,*,§ Stefaan C. De Smedt,*,‡ and José M. García Fernández*,† Instituto de Investigaciones Químicas, CSIC – Universidad de Sevilla, Américo Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain, Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Sevilla, Spain, and Laboratory of General Biochemistry and Physical Pharmacy. Department of Pharmaceutics, Gent University, Harelbekestraat 72, B-9000 Gent, Belgium. * Corresponding authors. † Instituto de Investigaciones Químicas. Tel: +34 954 489 559. Fax: 954 460 565. E-mail: [email protected] ‡ Gent University. Tel: +32 9 264 80 76. Fax: +32 9 264 8189. E-mail: [email protected] § Universidad de Sevilla. Tel: +34 954 559 806. Fax: +34 954 624 960. E-mail: [email protected] Running Title: Cellular Uptake of pDNA―Cyclodextrin Nanoparticles ¶ A. D.-M. and D. V. have equally contributed to the reported work. 2 Abstract It is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70 nm). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell uptake and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated β-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol- and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection. Introduction The successful delivery of therapeutic genes into cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. During the last decade, under the advent of nanotechnology , a broad diversity of creative materials featuring promising properties for nonviral gene delivery applications has emerged, (1) . Cationic polymers and lipids, or their combinations, have shown the ability to bind nucleic acids through electrostatic interactions and condense them into complexes that can be readily internalized by cells (2-3) . The gene delivering capabilities of many of these systems have been thoroughly investigated. Yet, drawing firm conclusions that might serve to provide feedback for the design of improved delivery entities is a delicate issue as a consequence of the essentially polydisperse nature of such structures (4) . The control of the architecture of multifunctional macromolecules is a major determinant in the rational design of successful nonviral gene delivery systems. Novel 3 strategies have been implemented to build up well defined constructs featuring selfassembling capabilities in the presence of nucleic acids. In this context, the unique characteristics of dendrimers and other highly ordered clusters, such as uniformity, monodispersity and multivalency, have attracted increasing attention (5-8) . Homogeneous external functionalization of these platforms becomes, however, exponentially more complicated as the dendrimer generation increases (9-10) . The use of preorganized macrocyclic scaffolds, such as calixarenes, to achieve a precise alignment of functional elements represents an interesting alternative (11-14) . The commercially available cyclomaltooligosaccharides (cyclodextrins; CDs) appear particularly attractive on those grounds (15-16) . CDs combine biocompatibility, availability, and a tubular symmetric framework with well differentiated faces that can be selectively modified in order to attain an exquisite control of the presentation and orientation of the assembled elements (17-19) . Chemically modified CDs have been already incorporated into polycationic polymers that can effectively complex and deliver plasmid DNA (pDNA) (20-21) . More recently, monodisperse polycationic CD conjugates with promising pDNA delivery capabilities were prepared by homogeneous functionalization of the CD primary rim (22-24) . Aiming at merging the virtues of both cationic polymers and cationic lipids in a flexible, well defined framework, we have reported a modular synthetic strategy for the preparation of polycationic amphiphilic CDs (paCDs) that allows the installation of different building blocks onto either face of the truncated-cone structure in a sequential and controlled way, offering a unique opportunity for structure-activity relationship (SAR) studies (25-27) (Figure 1). The pDNA complexing capabilities and in vitro transfection efficiencies of the corresponding pDNA-paCDs complexes (CDplexes) were found to be dependent on the CD decoration pattern. Notably, transfection efficiencies surpassing that of commercial polyethyleneimine (PEI) polymersbased polyplexes for BNL-CL2 and COS-7 cell lines were observed for some paCD representatives, such as 1 (28) (Figure 2). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the resulting pDNA-paCD nanoparticles (CDplexes) might be taken up by the cells and later released from the endosomes by mechanisms earlier described for both lipo- and polyplexes. In eukaryotic cells the following mechanisms of endocytosis can be distinguished: phagocytosis, macropinocytosis (MP), clathrin dependent endocytosis (CDE) and clathrin independent endocytosis (CIE) (29) . Phagocytosis is typically restricted to specialized cells such as macrophages, whereas macropinocytosis is a constitutive bulk internalization process 4 typically characterized by the formation of membrane protrusions and engulfing large volumes (30) . CDE and CIE are the most relevant mechanisms operating in the internalization of pDNA-nonviral gene delivery system complexes. The first route is characterized by the formation of clathrin coated pits (31) . CIE includes several pathways which share a cholesterol dependency (32) , among which caveolin mediated endocytosis Cav-ME is probably the most thoroughly characterized. In this case the membrane of the formed vesicles, which are called cavicles, typically contains caveolin-1 (33,34) . Selective inhibition of the different internalization pathways has been shown to be a powerful tool to investigate uptake mechanisms of nanoparticles and infer the relevance of the blocked pathway in the internalization mechanism (32,35-43) . Previous studies have shown that lipoplexes enter the cells predominantly via CDE (44) . Polyplexes, however, are internalized less uniformly. Their uptake seems to occur via MP and CDE as well as CIE (44) . Nonetheless, it has been observed for PEI complexes that it is predominantly the fraction that is taken up via CIE that leads to gene expression (45-47) . The purpose of this work was to get insight on the uptake mechanisms of pDNA-paCD nanoparticles and study whether the different internalization pathways might influence the intracellular fate of the CDplexes and their transfection efficiency. The dendritic tetradecacationic paCD 1, for which a gram-scale synthetic procedure has been optimized, was selected for this study since it was previously shown to form small (< 70 nm) CDplexes in which pDNA was fully protected from degradation, with transfection efficiencies up to one order of magnitude higher as compared to branched PEI (25 KDa) in BNL-CL2 and COS-7 cells both in serum free and serum-containing (10%) media (28). A set of selective endocytic pathway inhibitors, including chlorpromazine, genistein, dynasore and methylated βcyclodextrin (MbCD), have been used in the present study. In addition, a fluorescently labeled (lissamine-rhodamine) derivative of paCD 1 has been synthesized to study uptake and intracellular trafficking of the CDplexes by confocal laser scanning microscopy. Experimental Section General Methods Reagents and solvents were purchased from commercial sources and used without further purification, except for dichloromethane, which was distilled over CaH2 under N2 stream. Products 2 (48) , 6 (15) and 7 (15) were prepared according to literature procedures. 5 Optical rotations were measured at 20 ºC in 1-dm tubes. 1H (and 13C) NMR spectra were recorded at 500 (125.7) or 300 (75.5) MHz. 2D COSY and HMQC experiments were used to assist NMR assignments. Thin-layer chromatography (TLC) was carried out on silica-coated aluminum sheets, with visualization by UV light and by charring with 10% H2SO4 or 0.1% ethanolic ninhydrin. Column chromatography was carried out on silica gel (230-400 mesh). Electrospray mass spectra were obtained from samples dissolved in MeOH, MeCN, H2O or their mixtures at low µM concentration. Ampicillin, chlorpromazine, methyl β-cyclodextrin (MbCD), genistein and dynasore were purchased from Sigma Aldrich (Bornem, Belgium). Serumfree medium OptiMEM is purchased from Invitrogen (Merelbeke, Belgium). Heptakis[6-(2- tert -butoxycarbonylaminoethylthio)-6-deoxy]cyclomaltoheptaose (3). To a suspension of heptakis(6-bromo-6-deoxy)cyclomaltoheptaose (48) (2, 2.52 g, 1.59 mmol) and cesium carbonate (5.13 g, 15.6 mmol) in dry DMF (30 mL), tert -butyl N -(2- mercaptoethyl)carbamate (15.9 mmol, 1.4 eq) was added. The suspension was heated, under Ar atmosphere, at 70 ºC for 48 h. The reaction mixture was cooled to room temperature, poured into ice-water, and stirred overnight. The resulting solid was filtered, washed with a large volume of water and finally washed with a small amount of cold Et2O. The residue was purified by column chromatography (40:10:1 → 30:10:1 CH 2Cl2-MeOH-H2O) to give 3 (3.04 g, 85%) having physicochemical properties identical to those previously reported (28) . Heptakis[6-(2- tert -butoxycarbonylaminoethylthio)-6-deoxy-2,3-di- O - hexanoyl]cyclomaltoheptaose (4): To a solution of 3 (2.0 g, 0.89 mmol) in dry pyridine (10 mL) under Ar, DMAP (4.56 g, 37.3 mmol, 3 eq) and hexanoic anhydride (12 mL, 49.8 mmol, 4.0 eq) were added. The mixture was heated at 70 ºC for 4-5 h. Then, MeOH (10 mL) was added and the mixture was further stirred at 70 ºC for 3 h. The solvents were evaporated under reduced pressure, ice-water (50 mL) was added and extracted with CH2Cl2 (4 × 50 mL). The organic phase was washed with diluted sulfuric acid (2 × 50 mL) and cold aqueous saturated NaHCO3 (4 × 50 mL), dried (Na2SO4), filtered, concentrated and purified by column chromatography (1:3 EtOAc-petroleum ether) to give 4 (2.44 g, 76%) having physicochemical properties identical to those previously reported (28) . Heptakis[6-(2-aminoethylthio)-6-deoxy-2,3-di- O -hexanoyl]cyclomaltoheptaose Heptahydrochloride (5): Treatment of carbamate 4 (1.42 g, 0.4 mmol) with 1:2 TFA-CH2Cl2 (20 mL) at RT for 3 h, followed by evaporation of the solvents and freeze-drying from a 6 diluted HCl solution, gave pure 5 (1.20 g, 99%) as a white foam having physicochemical properties identical to those previously reported (28) . 2-[ Bis [2-( tert -butoxycarbonylamino)ethyl]amine]ethyl Isothiocyanate (8). To a solution of 2-azidoethyl-bis[2-( tert -butoxycarbonylamino)ethyl]amine (15) (7, 0.72 g, 1.89 mmol) in dry dioxane (15 mL), triphenylphosphine (0.55 g, 1.1 eq, 2.1 mmol) and carbon disulfide (1.14 mL, 10 eq, 18.9 mmol) were added. The reaction mixture was stirred under N2 atmosphere for 16 h. Then the solvent was removed under reduced pressure and the residue was purified by column chromatography using 1:2 EtOAc-petroleum ether as eluent to give 8 (0.63 g, 86 %) having physicochemical properties identical to those previously reported (28) . Heptakis[6-deoxy-6-(2-( N ’-(2-( N , N -di-(2-( N - tert - butoxycarbonylamino)ethyl)amino)ethyl)-thioureido)ethylthio)-2,3-di- O - hexanoyl]cyclomaltoheptaose (9). To a solution of 5 (0.48 g, 0.15 mmol) and Et3N (0.29 mL, 2.12 mmol, 2.0 eq) in CH2Cl2 (5 mL), a solution of 8 (0.49 g, 1.26 mmol, 1.2 eq) in CH2Cl2 (5 mL) was added and the reaction mixture was stirred at RT for 3 h. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (50:1 → 20:1 CH 2Cl2-MeOH) to give 9 (0.72 g, 85%) having physicochemical properties identical to those previously reported (28) . Heptakis[6-deoxy-6-(2-( N ’-(2-( N , N -di-(2- aminoethyl)amino)ethyl)thioureido)ethylthio)-2,3-di- O -hexanoyl]cyclomaltoheptaose (1). Treatment of carbamate 9 (0.6 g, 0.11 mmol) with 1:2 TFA-CH2Cl2 at RT for 3 h, followed by evaporation of the solvent and freeze-drying from diluted HCl solution, gave 1 (0.50 g) in quantitative yield. Its identity and purity was confirmed by ESI-MS, comparison of the corresponding 1H and 13C NMR spectra with those of an authentic sample (28) and combustion analysis. Lissamine-Rhodamine Labeled paCD 10. To a solution of paCD 1 (83 mg, 17 µmol) and Et3N (50 µL, 0.35 mmol) in freshly distilled CH2Cl2 (10 mL) under N2, lissaminerhodamine sulfonyl chloride (LRSC, 10 mg, 17 µmol) was added. The reaction was stirred at RT for 2 days in the dark. The solvents were then removed under vacuum and the residue was purified by size exclusion chromatography (Sephadex LH-20, MeOH). Lisamine-Rhodamine labeled paCD 10 was obtained as a dark red foam after liophilization from diluted HCl (73 mg, 79%). The corresponding 1H NMR spectrum, recorded in MeOD at 313 K, featured the 7 characteristic aromatic signals for lissamine-rhodamine (LS) at δ 8.75, 8.24, 7.62 and 7.26- 6.97 ppm, with intensities fitting the expected 1:1 LS-paCD average ratio. ESI-MS: m/z 1196.5 [M-Rho + 4 H]4+, 1061.5 [M + 4 H]4+, 957.3 [M-Rho + 5 H]5+, 848.8 [M + 5 H]5+. Plasmid Preparation and Labeling. For transfection, the luciferase encoding plasmid pGL4.13 (Promega, Leiden, The Netherlands) was used. The plasmid was transformed into the competent E. coli DH10b strain (Promega, Leiden, The Netherlands) according to the manufacturer’s instructions. The transformed bacteria were then selected for ampicillin resistance and grown till an optical density of 1.5. The plasmid was purified with the PureLink™ HiPure Plasmid Gigaprep Kit (Invitrogen, Merelbeke, Belgium). The plasmid was re-suspended to a concentration of 1 µg µL-1 in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). For uptake studies, the plasmid was labeled with the nucleic acid stain YOYO®-1 iodide (λex = 491, λem = 509, Molecular Probes, Merelbeke, Belgium). For the labeling, 100 µg of plasmid was added to 15 nmol of YOYO-1 in 20 mM HEPES buffer for 1 h at RT, which should result in a labeling density of 1 YOYO- dye per 10 bp. The YOYO-1-DNA complexes were finally purified by an ethanol precipitation and re-suspended to 1 µg µL-1 in TE buffer. Preparation of CDplexes . To prepare complexes, plasmid DNA was mixed with polycationic amphiphilic cyclodextrins. The paCD derivative was dissolved in DMSO and diluted in water to a final stock solution of 20 mg·mL-1 (4.2 mM) (1:2 DMSO-water). pDNA was diluted with OptiMEM to a concentration of 0.04 µg µL-1 (equal to 124 µM of phosphate) and mixed with paCD derivate to yield the desired N/P ratio. The preparation was vortexed for 12 minutes before diluting it 10 times in OptiMEM. This solution was used for uptake or transfection experiments. Cell Culture Vero (African green monkey kidney epithelial cell line; ATCC number CCL-81) were cultured in Dulbecco’s modified Eagles’ medium (DMEM; Invitrogen, Merelbeke, Belgium) supplemented with 10 % fetal bovine serum (Perbio, Erembodegem, Belgium), 2 mM L- glutamine and 2 % penicillin/streptomycin (Invitrogen, Merelbeke Belgium) and grown at 37 °C in a humidified atmosphere containing 5 % CO2. 8 Transfection experiments Cells were seeded into 12-well plates (100.000 cells per well) and allowed to attach overnight. Subsequently, the culture medium was removed and CDplexes were added to the well (2 µg of DNA, N/P 10). The complexes were removed after 4 h. Luciferase activity, as well as the total cellular protein concentration, was measured after 48 h. To determine the luciferase activity, the cells were washed with PBS and lysed with 200 µL of cell culture lysis buffer (Promega, Leiden, The Netherlands) (30 min, RT). Samples were centrifuged (4 ºC, 14000 rpm, 5 min). Subsequently, luciferase activity was determined in the supernatants with the Promega luciferase assay kit (Promega, Leiden, The Netherlands) according to the manufacturer's instructions. Briefly, 100 µL of substrate solution were added to 40 µL sample in a white 96-microtiter plate and after a 2 s delay, the luminescence was measured during 10 s with the GloMax™ 96 luminometer (Promega, Leiden, The Netherlands). The protein concentration was determined with the Biorad Bradford Protein assay (Biorad, Eke, Belgium). The Biorad reagent (50 µL) was mixed with cell lysate (2 µL) and water (198 µL) in a transparent 96-microtiter plate and the absorbance was measured on a Wallac Victor2 absorbance plate reader (Perkin Elmer-Cetus Life Sciences) at 590 nm. The luciferase activity, expressed as relative light units (RLU) per µg of protein, was used as a measure for the efficiency of transfection with the CDplexes. Mean values were obtained from at least two separate experiments carried out in triplicate. Cell Viability Assay Cell viability was assessed with the CellTiterGlo Proliferation Assay (Promega, Leiden, The Netherlands) according to the manufacturer's instructions. Briefly, 10.000 Vero cells were seeded in a white 96-well microtiter plate with transparent bottom (Greiner Bio- One, Wemmel, Belgium). The cells were treated with 100 µL of inhibitors and/or complexes for the indicated times. Subsequently, CellTiterGlo reagent was added and incubated with the cells for 5 min till complete lysis. Cell debris was spun down and luminescence was measured in the supernatants on a GloMax™ 96 luminometer with 1s integration time. Each experiment was performed in quadruplicate. CDplexes Uptake Studies Vero cells were seeded into 6-well plates (300.000 cells per well) and allowed to attach overnight. The cells were then incubated with the CDplexes, prepared with YOYO-1 9 labeled pDNA, in OptiMEM. The fluorescence of the non-internalized complexes, attached to the plasma membrane was quenched by a 5-minute-washing with 0.2% trypan blue (Sigma, Bornem, Belgium) in PBS solution. After washing with PBS, the cells were detached with trypsin, centrifuged and re-suspended in ice cold flow buffer. The samples were analyzed with a 5-color FC500 flow cytometer (Beckman Coulter, Nyon, Switzerland) equipped with an argon laser (λex = 488 nm). CXP software (Beckman Coulter, Nyon, Switzerland) was used for the analysis. The internalization of CDplexes was calculated as the mean fluorescence value (average of three independent experiments). The 0% uptake was determined from cells incubated at 4 °C. The 100% uptake was determined from cells that had been incubated with the complexes without being exposed to any inhibitor. Inhibitor Studies Prior to incubation with CDplexes, Vero cells were pre-incubated with chlorpromazine, MbCD, genistein or dynasore prepared in OptiMEM (1 h, 37 °C). Subsequently this medium was removed and substitute with OptiMEM containing fresh inhibitor and complexes. The following stock solutions of inhibitors were used: freshly prepared 10 mg mL-1 chlorpromazine in water, freshly prepared 5 mM (6.6 mg mL-1) MbCD in water, 50 mM genistein in DMSO (stored in the freezer), and 1 µg µL-1 dynasore in DMSO (stored in the freezer). Live Cell Imaging of Fluorescently Labeled CDplexes Vero cells were seeded at a concentration of 400.000 cells per well on sterile MatTek coverslips (1.5)-bottom dishes (MatTek Corporation, MA, USA). The cells were incubated with 2 mL of CDplexes (N/P 10, 12 µg DNA). The fluorescent CDplexes were visualized with a Nikon C1 confocal laser scanning microscope (CLSM) (Nikon Benelux, Brussels, Belgium) equipped with a Plan Apo VC 60x 1.4 NA oil immersion objective lens. Lissaminerhodamine labeled compound (10) was excited with the solid state 561 nm laser line and the emitted light was collected using a 580-630 nm band pass filter. Cells were kept in a stage top incubator with controlled temperature, humidity and CO2 pressure (Tokai Hit, Shizuoka, Japan). Representative 2D confocal images were acquired approximately through the central plane of the cell. 16 Receptors: Design, synthesis and biological evaluation of glycodendrimer-cyclodextrin conjugates. J. Am. Chem. Soc. 126 , 10355-10363. (16) Gómez-García, M., Benito, J. M., Rodríguez-Lucena, D., Yu, J.-X., Chmurski, K., Ortiz Mellet, C., Gutiérrez-Gallego, R., Maestre, A., Defaye, J., and García Fernández, J. M. (2005) Probing secondary carbohydrate-protein interactions with highly dense cyclodextrin-centered heteroglycoclusters: The Heterocluster Effect . J. Am. Chem. Soc. 127 , 7970-7971. (17) Vargas-Berenguel, A., Ortega-Caballero, F., and Casas-Solva, J. M. (2007) Supramolecular chemistry of carbohydrate clusters with cores having guest binding abilities. Mini-Rev. Org. Chem . 4 , 1-14. (18) Villalonga, R., Cao, R., and Fragoso, A. (2007) Supramolecular chemistry of cyclodextrins in enzyme technology. Chem. Rev. 107 , 3088-3116. (19) García Fernández, J. M., Ortiz Mellet, C., and Defaye, J. (2006) Glyconanocavities: Cyclodextrins and beyond. J. Incl. Phenom. Macrocycl. Chem. 56 , 149-159. (20) Davis, M. E., and Brewster, M. E. (2004) Cyclodextrin-based pharmaceutics: past, present and future. Nat. Rev. Drug. Discov . 3 , 1023-1035. (21) Heidel, J. D., Yu, Z., Liu, J. Y.-C., Rele, S. M., Liang, Y., Zeidan, R. K., Kornbrust, D. J., and Davis, M. E. (2007) Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA. Proc. Nat. Acad. Sci. USA 104 , 5715-5721. (22) Mourtzis, N., Paravatou, M., Mavridis, I. M., Roberts, M. L., and Yannakopoulou, K. (2008) Synthesis, characterization, and remarkable biological properties of cyclodextrins bearing guanidinoalkylamino and aminoalkylamino groups on their primary side. Chem. Eur. J. 14 , 4188-4200. (23) Mourtzis, N., Eliadou, K., Aggelidou, C., Sophianopoulou, V., Mavridis, I. M., and Yannakopoulou, K. (2007) Per(6-guanidino-6-deoxy)cyclodextrins: synthesis, characterisation and binding behaviour toward selected small molecules and DNA. Org. Biomol. Chem. 5 , 125-131. (24) Srinivasacchari, S., Fichter, K. M., and Reineke, T. M. (2008) Polycationic βcyclodextrin “click clusters”: monodisperse and versatile scaffolds for nucleic acid delivery. J. Am. Chem. Soc. , 130 , 4618-4627. (25) Díaz-Moscoso, A., Balbuena, P., Gómez-García, M., Ortiz Mellet, C., Benito, J. M., Le Gourriérec, L., Di Giorgio, C., Vierling, P., Mazzaglia, A., Micalli, N., Defaye, J., and García Fernández, J. M. (2008) Rational design of cationic cyclooligosaccharides as efficient gene delivery systems. Chem. Commun. 2001-2003. (26) Ortega-Caballero, F., Ortiz Mellet, C., Le Gourriérec, L., Guilloteau, N., Di Giorgio, C., Pierre Vierling, P., Defaye, J., and García Fernández, J. M. (2008) Tailoring β-cyclodextrin for DNA complexation and delivery by homogeneous functionalization at the secondary face. Org. Lett. 10 , 5143-5146. (27) Méndez-Ardoy, A., Gómez-García, M., Ortiz Mellet, C., Girón-González, M. D., Sevillano-Tripero, M., Salto-González, R., Santoyo-González, F., and García Fernández, J. 17 M. (2009) Design of macromolecular gene delivery systems by preorganization of functional elements onto a cyclodextrin scaffold: amphiphilic β-cyclodextrin “click clusters”. Org. Biomol. Chem. 7 , 2681-2684. (28) Díaz-Moscoso, A., Gómez-García, M., Balbuena, P., Ortiz Mellet, C., Benito, J. M., Le Gourriérec, L., Di Giorgio, C., Vierling, P., Defaye, J., and García Fernández, J. M. (2009) Polycationic amphiphilic cyclodextrin-scaffolded monodisperse gene delivery systems. Chem. Eur. J. in press. (29) Conner, S. D., and Schmid S. L. (2003) Regulated portals of entry into the cell. Nature 422 , 37-44. (30) Kerr, M. C., and Teasdale, R. D. (2009) Defining macropinocytosis. Traffic 10 , 364-371. (31) Maxfield, F. R., and McGraw T. E. (2004) Endocytic recycling. Nature Rev. Mol. Cell Biol. 5 , 121-132. (32) Mayor, S., and Pagano, R. E. (2007) Pathways of clathrin-independent endocytosis. Nature Rev. Mol. Cell Biol. 8 , 603-612. (33) Pelkmans, L., Kartenbeck, J., and Helenius. A, (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nature Cell Biol. 3 , 473-483. (34) Sharma, D. K., Choudhury, A., Singh, R. D., Wheatley, C. L., Marks, D. L., and Pagano, R. E. (2003) Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem. 278 , 7564-7572. (35) Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127 , 915-934. (36) Liu, S. H., Marks, M. S., and Brodsky, F. M. (1998) A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway. J. Cell Biol. 140 , 1023-1037. (37) Benmerah, A., Bayrou, M., Cerf-Bensussan, N., and Dautry-Varsat, A. (1999) Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112 , 1303-1311. (38) Nichols, B. J. (2002) A distinct class of endosome mediates clathrin-independent endocytosis to the golgi complex. Nature Cell Biol. 4 , 374-378. (39) Hinrichsen, L., Harborth, J., Andrees, L., Weber, K., and Ungewickell, E. J. (2003) Effect of clathrin heavy chain- and alpha -adaptin specific small interfering RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 278 , 45160-45170. (40) Huang, F. T., Khvorova, A., Marshall, W., and Sorkin, A. (2004) Analysis of clathrinmediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 279 , 16657-16661. 18 (41) Vanden Broeck, D., and De Wolf, M. J. S. (2006) Selective blocking of clathrinmediated endocytosis by RNA interference: Epsin as target protein. Biotechniques 41 , 475- 484. (42) Ivanov, A. I. (2008) Pharmacological inhibition of endocytic pathways: Is it specific enough to be useful?. Exocytosis and endocytosis (Ivanov, A. I., Ed.) pp 15-33, Humana Press, Totowa. (43) Vercauteren, D. N. E., Vandenbroucke, R. E., Demeester, J., De Smedt, S. C., Braeckmans, K., and Sanders, N. N. (2008) The use of endocytic inhibitors to study uptake of gene garriers. J. Control. Rel. 132 , e17-e18. (44) Midoux, P., Breuzard, G., Gomez, J. P., and Pichon, C. (2008) Polymer-based gene delivery: A current review on the uptake and intracellular trafficking of polyplexes. Current Gene Ther. 8 , 335-352. (45) Gonçalves, C., Mennesson, E., Fuchs, R., Gorvel, J. P., Midoux, P., and Pichon, C. (2004) Macropinocytosis of polyplexes and recycling of plasmid via the clathrin-dependent pathway impair the transfection efficiency of human hepatocarcinoma cells[Ast]. Mol. Ther. 10 , 373-385. (46) Rejman, J., Bragonzi, A., and Conese, M. (2005) Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol. Ther. 12 , 468-474. (47) von Gersdorff, K., Sanders, N. N., Vandenbroucke, R., De Smedt, S. C., Wagner, E., and Ogris, M. (2006) The internalization route resulting in successful gene expression depends on polyethylenimine both cell line and polyplex type. Mol. Ther. 14 , 745-753. (48) Gadelle, A., and Defaye, J. (1991) Selective halogenation at primary positions of cyclomaltooligosaccharides and a synthesis of per-3,6-anhydro cyclomaltooligosaccharide. Angew. Chem. Int. Ed. 30 , 78-80. (49) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oliginucleotide transfer into cells in culture and in-vivo – polyethylenimine. Proc. Nat. Acad. Sci. USA 92 , 7297-7301. (50) Parton, R. G., Joggerst, B., and Simons, K. (1994) Regulated internalization of caveolae. J. Cell Biol. 127 , 1199-1215. (51) Orlandi, P. A., and Fishman, P. H. (1998) Filipin-dependent inhibition of cholera toxin: Evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol. 141 , 905-915. (52) Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen, T. (2006) Dynasore, a cell-permeable inhibitor of dynamin. Developmental Cell 10 , 839-850. (53) Wang, L. H., Rothberg, K. G., and Anderson, R. G. (1993) Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123 , 1107-1117. 19 (54) Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., van Deurs, B., and Sandvig, K. (1999) Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrincoated endocytic vesicles. Mol. Biol. Cell 10 , 961-974. 20 Figures Scheme 1. Convergent Synthesis of Polyaminothioureido Amphiphilic βCD (paCD) 1. O O HO HO 7 R Cs 2 CO 3, DMF 70ºC, 85% 2, R = Br 3, R = S(CH 2 ) 2 NHBoc HS NHBoc DMAP, Py 70ºC, 76% 2 N NHBoc NHBoc X HN NHBocBocHN O O O O 7 SNHR OO O O O O 7 SN H OO N H SN RHN NHR 9, R = Boc 1, R = H·HCl 4, R = Boc 5, R = H·HCl H + , 99% O O 7, X = N 3 8, X = NCS TPP, CS 2 dioxane, 86% 6 K 2 CO 3, MeCN reflux, 54% N 3 OTs Et 3 N, CH 2 Cl 2 85% H + , 99% Scheme 2. Statistical Labeling of paCD 1 with Lissamine-Rhodamine Sulfonyl Chloride (LRSC). O O RO RO S NH NH S N ClH 3 N NH 3 Cl 1 O O RO RO SNH NH SN HN NH 3 Cl O N N S O O SO 3 n m n + m = 7 R = O10 LRSC,Et 3 N CH 2 Cl 2, 82% 21 Figure 1. Schematic Representation of (a) “skirt” and (b) “jelly-fish” type Polycationic Amphiphilic CDs (paCDs). The central light-blue platform represents the cyclodextrin core, the blue weaving lines account for hydrophobic chains, the orange rectangular boxes indicate the presence of spacer elements and the encircled + signs stand for the positively charged groups. O OO O S HN HN OO O O O OS HN HN O O N N O O OSH NH N O O O O O OO S NH NH O ON N O S S S S NH 3 H 3 N H 3 N H 3 N OO O S NH NH O O N O O O S N H N HO O O O O O S HN HN O O N N O S S S NH 3 NH 3 NH 3 NH 3 NH 3 H 3 N H 3 N NH 3 NH 3 H 3 N 1 Figure 2. Chemical structure of Polyaminothioureido Amphiphilic βCD 1 (colors were selected to match Figure 1 encoding). a b + + + + + + + O O O O O O O O O O O O + + + + + + + + + + O O O O O 22 Figure 3. In vitro transfection efficiency of pDNA:paCD nanoparticles in HeLa cells (a) in the presence and in the absence of serum (10%), and (b) over the time. 0 20 40 60 80 100 120 140 160 015 30 45 60 90 120 180 240 Time (min) Mean fluorescence (A.U.) 0 20 40 60 80 100 120 Fluorescent cells (%) Figure 4. Uptake kinetics of YOYO-1 labeled pDNA:1 CDplexes in Vero cells. Average cell fluorescence (A.U., bars) and amount of significantly fluorescent cells (%, dots) at the indicated time-point. The internalization was quantified with flow cytometry. 23 Figure 5. Confocal micrographs of the internalization of CDplexes in living Vero cells during 48 h post–transfection period (A, 40 min; B, 120 min; C, 150 min; D, 2 days). 0 20 40 60 80 100 120 Pos. Control 100 200 300 400 [genistein] (µM) Cell Viability (%) A 0 20 40 60 80 100 120 Pos. Control 20 30 40 50 [dynasore] (µM) Cell Viability (%) B 24 0 20 40 60 80 100 120 Pos. Control 510 20 25 [chlorpromazine] (µg·mL -1 ) Cell viability (%) C 0 20 40 60 80 100 120 Pos. Control 2.5 57.5 10 [MbCD] (mM) Cell Viability (%) D Figure 6. Viability of Vero cells incubated for 5 h with the inhibitors (white bars) or a mixture of the inhibitors and CDplexes at N/P 5 (dotted bars) and 10 (hatched bars). A, genistein; B, dynasore; C, chlorproazine; D, MbCD. 0 20 40 60 80 100 Pos. Control Inhib. Neg. Control Mean fluorescence (A.U.) 0 25 50 75 100 125 % Fluorescent cells Figure 7. Effect of inhibitors on internalization of CDplexes. Vero cells were pretreated with the inhibitors for 1 h. Subsequently, fluorescently labeled CDplexes were added and incubated with the cells in the presence of inhibitors for 2 h (400 µM genistein, white bars and ∆; 50 µM dynasore, dotted bars and ○; 10 µg mL-1 chlorpromazine, hatched bars and +; 5 mM MbCD, cross-hatched bars and ×. 25 0 20 40 60 80 100 120 140 Pos. Control 100 200 300 400 Neg. Control [genistein] (µM) Transfection (%) Pos. Control 20 30 40 50 Neg. Control [dynasore] (µM) 0 20 40 60 80 100 120 140 Pos. Control 510 20 25 Neg. Control [chlorpromazine] (µg·mL -1 ) Transfection (%) Pos. Control 2.5 57.5 10 Neg. Control [MbCD] (mM) Figure 8. In vitro transfection efficiencies of CDplexes (N/P 10) in Vero cells in the presence of the indicated inhibitor.