Structurally Flexible Triethanolamine Core PAMAM Dendrimers Are

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Structurally Flexible Triethanolamine Core PAMAM Dendrimers Are Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus Xiaoxuan Liu,⊥,† Jiangyu Wu,⊥,† Miriam Yammine,‡ Jiehua Zhou,⊥ Paola Posocco,□ Stephane Viel,§ Cheng Liu,⊥ Fabio Ziarelli,¶ Maurizio Fermeglia,□ Sabrina Pricl,□ Genevieve Victorero,‡ Catherine Nguyen,‡ Patrick Erbacher,∇ Jean-Paul Behr,# and Ling Peng*,† †

Aix-Marseille Université, Centre Interdisciplinaire de Nanoscience de Marseille, CNRS UPR 3118, Département de Chimie, 163 avenue de Luminy, 13288 Marseille cedex 09, France ⊥ State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China ‡ INSERM U928, 163 avenue de Luminy, 13288 Marseille cedex 09, France § Aix-Marseille Université, LCP UMR 6264, Campus de Saint Jérôme, av. Escadrille Normandie Niémen, case 512, 13013 Marseille, France ¶ Aix-Marseille Université, Fédération des Sciences Chimiques, Spectropole, av. Escadrille Normandie Niémen, case 511, 13013 Marseille, France □ Molecular Simulation Engineering (MOSE) Laboratory, Department of Chemical Engineering, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy ∇ Polyplus-transfection SA, Bioparc, Boulevard S. Brandt, BP90018, 67401 Illkirch, France # Laboratoire de Chimie Génétique, Faculté de Pharmacie, CNRS UMR7514, 67401 Illkirch, France S Supporting Information *

ABSTRACT: With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.

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can be used for the treatment of chronic diseases. Other important advantages include their convenient preparation and use, as well as their reasonable manufacturing cost. Their major disadvantage relates to a lower transfection efficiency compared to viral vectors. Therefore, improving the tranfection efficiency of nonviral vectors constitutes the ultimate aim and challenge. Nonviral vectors are usually divided into two main classes cationic lipids and polymers.5 In both cases, the positive charges of the vectors interact with anionic nucleic acids and form stable complexes that protect the nucleic acid from

INTRODUCTION DNA-based gene therapy and the recently developed small interfering RNA (siRNA)-based gene silencing show great promise as therapeutic approaches for treating and controlling inherited as well as acquired diseases. The main obstacle to successful gene therapy is the lack of safe and effective vectors for nucleic acid delivery. To date, the most efficient vectors are viral; however, these do have fatal drawbacks relating to immunogenicity and toxicity, in addition to practical problems concerning large-scale production and quality control. Therefore, developing safe and efficient nonviral vectors for nucleic acid delivery is urgently needed and of paramount importance for gene therapy.1−4 Nonviral vectors are noninfectious and elicit only weak immune responses; thus, repeated injections © 2011 American Chemical Society

Received: June 2, 2011 Revised: October 27, 2011 Published: November 5, 2011 2461

dx.doi.org/10.1021/bc200275g | Bioconjugate Chem. 2011, 22, 2461−2473

Bioconjugate Chemistry

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degradation and promote cell uptake. Among the nonviral vectors, cationic dendrimers, characterized by a unique nanoscale spherical architecture, regular dendritic branching, and radial symmetry, are a special family of polymeric vectors.6−8 The most extensively studied dendrimers for nucleic acid delivery are poly(amidoamine) (PAMAM) dendrimers,9−13 which bear primary amines at the dendrimer surface and tertiary amines at the branching units inside. 14,15 Positively charged at physiological pH, these primary amines yield a high charge density at the dendrimer surface, which is responsible for ionic condensation with nucleic acids and binding to the cell surface. After entering the cells through endocytosis, the dendrimer delivery complexes are entrapped in endosomes where they are reported to release DNA into the cytosol via a “proton sponge” effect.16,17 The released DNA molecules are eventually trapped in the nucleus where transcription can occur. Paradoxically, DNA transfection with partially degraded and structurally fractured PAMAM dendrimers is 2 orders of magnitude higher than that of nondegraded ones.13 This could be ascribed to the fact that partially degraded dendrimers are endowed with a more flexible and open structure compared to their intact counterparts, and are thus more accessible for interaction with DNA via both electrostatic interactions and mutual accommodation in space. In addition, the open and flexible structure allows water molecules to pervade the nanovector interior more easily, consequently increasing the availability of the inner tertiary amines for protonation. This, in turn, may result in an enhanced buffering capacity via a proton sponge effect, ultimately leading to more efficient DNA release and hence a better transfection efficiency. Partially degraded and fractured PAMAM dendrimers are obtained either by thermal degradation or by alkaline hydrolysis of the intact dendrimers. However, these processes cannot offer precise control over structure. In addition, dendrimer synthesis is a time-consuming and painstaking process involving stepwise construction as well as tedious workup for separation and purification. Therefore, deliberately degrading a laboriously synthesized perfect dendrimer is neither satisfactory nor economical, and results in a waste of both manpower and energy. With this in mind, we wished to investigate whether rationally designed genuine (i.e., nondegraded) PAMAM dendrimers with flexible structures might behave similarly to their partially degraded rigid counterparts, thus enabling their use as efficient nanovectors for nucleic acid delivery. We therefore conceived and synthesized structurally flexible PAMAM dendrimers starting with triethanolamine (TEA) as the dendrimer core (Figure 1A).18−21 Our rationale for molecular design of structurally flexible dendrimers is rather straightforward: with TEA as the dendrimer core, the branching units start at a distance of 10 successive bonds away from the central amine, whereas the prototype NH3-core PAMAM dendrimers branch out immediately at the central nitrogen of the NH3 core.14 Consequently, TEA-core dendrimers feature an extended core and are expected to be less congested in space, with their branching units and terminal end groups being less densely packed than those of NH3-core dendrimers. We have previously reported these dendrimers for their efficient interaction with RNA molecules18,19 and their effective delivery of small interfering RNA molecules.20−23 However, their structural flexibility and their DNA delivery ability have not been investigated yet. Here, we report that these dendrimers have indeed flexible structure and are also effective nanovectors for

DNA transfection in vitro and in vivo in the mouse thymus. It is worth mentioning here that the thymus is an attractive organ for immune gene therapy, as it is the primary site of production of functional T lymphocytes playing a crucial role in adaptive immune responses.24 Owing to the potential use of an intrathymic gene transfer strategy for immune modulation, it is of primary importance to develop nanocarriers for effective DNA transduction in thymus.25 Historically, gene transfer in the thymus has been a challenge with respect to efficiency and safety. Intrathymic transfection using viral26−28 or nonviral28 vectors or electroporation29 was previously reported to be either inefficient or technically demanding. The results reported here provide the first experimental evidence that the structurally flexible PAPAM dendrimers are effective nanocarriers for DNA delivery and gene transfer in mouse thymus, which may introduce new perspectives to immune gene therapy via intrathymic dendrimer-mediated DNA delivery.

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EXPERIMENTAL PROCEDURES

General. Plasmid DNA containing the luciferase gene was a gift from Prof. Barbara Demeneix (CNRS, Paris, France). EGFP plasmid pEGFP-N1 was purchased from Clontech (Clontech laboratories, Inc. CA, USA). Poly(lysine), ethidium bromide, endocytosis inhibitors (cytochalasin D, genistein, and chlorpromazine), paraformaldehyde, and bovine serum albumin (BSA) were supplied by Sigma (Sigma−Aldrich, Lyon, France). YOYO-1 iodide, endocytosis markers (Alexa-Fluor 647-labeled dextran, transferrin, and cholera toxin B), Alexa-Fluor 647labeled Phalloidin, DAPI, and Hoechst 34580 were purchased from Invitrogen (Invitrogen Ltd., Paisley, UK). HeLa and LMTK− cells were grown in MEM with Earle’s salt (Eurobio, Paris, France) and supplemented with 10% fetal bovine serum (FBS, Perbio, France), 2 mM Glutamax (Eurobio), 100 units/ mL penicillin (Eurobio), and 100 μg/mL streptomycin (Eurobio). Cells were maintained at 37 °C in a 5% CO2 humidified atmosphere. NMR DOSY Experiments. DOSY experiments were performed at 300 K on a Bruker Avance spectrometer operating at 500 MHz for the 1H Larmor frequency with a 5 mm tripleresonance inverse Bruker cryoprobe optimized for 1H detection and equipped with an actively shielded z-gradient coil. D2O solutions were prepared at 1 mg mL−1 by weighing the proper amount of dendrimer sample directly into the NMR sample tube and adding 0.6 mL of deuterated solvent. Note that the D2O viscosity was taken as 1.05 cP. The NMR pulse sequence was based on a simulated echo and incorporated bipolar gradients and a longitudinal eddy current delay.30 Typically, the diffusion time was kept constant at 700 ms and bipolar sine gradient pulses between 0.8 and 2.3 ms were employed. The gradient pulse recovery time and the longitudinal eddy current delay were set to 0.25 and 25 ms, respectively. To determine the weight average molecular weight of the dendrimers, the following procedure was followed. Because the lower generation TEA-core PAMAM dendrimers (G1−G3) have well-defined and fully characterized molecular weights, they were selected and used as references in order to obtain the corresponding scaling law (and hence the calibration curve) between the self-diffusion coefficients and the molecular weight of TEA-core dendrimers: (1) 2462



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Potentiometric pH Titration of Dendrimer. The G5, G6, or G7 dendrimer was diluted to a concentration of primary amine of 1.25 mM. pH titration was carried out with 50 mM HCl using a Mettler Toledo 320-S pH meter. Molecular Modeling. All MD simulations were performed using the sander and pmemd modules of the AMBER 9 suite of programs,31 and the new version of the Dreiding force field recently developed by the Goddard group and specifically optimized for dendrimers in water solutions.32,33 The free energy of binding between the dendrimers and the DNA was calculated according to a previously validated approach34,35 based on the Molecular Mechanics−Poisson−Boltzmann methodology.36 Calculations were carried out in parallel on 128 processors of IBM/BCX calculation cluster of the CINECA supercomputer facility, Bologna, Italy. The mesoscopic simulations were carried out with the DPD modulus of Materials Studio (v 4.4, Accelrys Inc., USA), according to a multiscale procedure developed by our group.35 Preparation of the Plasmid DNA/Dendrimer Complexes. The dendrimers were diluted to an appropriate concentration in 20 mM Tris-HCl buffer (pH 7.6) and 150 mM NaCl, with all solutions stored at 4 °C. The plasmid DNA was diluted to 20 ng/μL in 150 mM NaCl solution. Both solutions were mixed at various N/P (= [total end amines in dendrimer]/[phosphates in DNA]) and incubated at 37 °C for 30 min. The final concentration of plasmid DNA was adjusted to 10 ng/μL. Gel Retardation Assays of the DNA/Dendrimer Complexes. Each complex (4 μL) containing 250 ng plasmid DNA and the corresponding dendrimer was kept at 37 °C in buffer solution for 30 min before loading on a 0.7% agarose gel in standard TAE buffer for electrophoresis. The DNA bands were stained by ethidium bromide and then detected by a Herolab EASY CCD camera (type 429K) (Herolab, Wiesloch, Germany). Stability of DNA/Dendrimer Complex Against DNase. An aliquot of 1.8 μg of DNA and the indicated amounts of dendrimers was kept at 37 °C for 30 min. Then, the complexes were incubated in the presence of 1 unit DNase/μg DNA for 0, 5, 10, 20, 30, 45, 60, 75, and 90 min at 37 °C, and aliquots (4 μL) of the corresponding solution containing 200 ng of DNA were withdrawn, added to 0.44 μL 10% SDS solution on the ice, and then subjected to electrophoresis in 1.2% agarose gel in standard TAE buffer. The DNA bands were stained as described above. Transmission Electron Microscope (TEM) Imaging. Studies were performed with a JEM-1230 electron transmission microscope. Ten microliters of a solution of plasmid DNA (5 ng/μL) were mixed with 10 μL of a solution of dendrimer in 50 mM Tris-HCl buffer (pH 7.4). After equilibration (30 min), 4 μL of this mixture were dropped on a standard carbon-coated copper TEM grid, and then allowed to evaporate (1 h at 30 °C, ambient pressure). The grid was then stained with uranyl acetate (2% in 50% alcoholic solution) for 3 min. Imaging was performed immediately after air−drying for 20 min. Dynamic Light Scattering Measurement. The plasmid DNA solution was mixed with indicated amount of dendrimer solution at different N/P ratios. The final concentration of the plasmid DNA was 50 ng/μL. After incubating at 37 °C for 30 min, dynamic light scattering (DLS) measurements were performed using Zetasizer Nano-ZS (Malvern, Ltd., Malvern, UK) with a He−Ne ion laser of 633 nm.

By using eq 1, the molecular weight of all the TEA-core PAMAM dendrimers (G1−G7), including those used to build the calibration curve (G1−G3), were subsequently estimated (Figure 1B) from their self-diffusion coefficients D, and the

Figure 1. (A) TEA-core PAMAM dendrimer (G2 was drawn for clarity). (B) General information of the synthesized TEA-core PAMAM dendrimers together with their self-diffusion coefficient D and hydrodynamic radius Rh obtained from DOSY NMR experiments. (C) DOSY-derived self-diffusion coefficient D as a function of the calculated molecular weight M for TEA-core PAMAM dendrimers G1−G7 (triangles). The best-fit was obtained as described in the Experimental Section.

results agreed very well (roughly ±5%) with those obtained previously by MS and GPC (Figure 1B). 2463



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Uptake of DNA/Dendrimer Complexes in HeLa Cells. To evaluate cellular uptake and subsequent intracellular routing of complexes, pCMVLuc was used and labeled with YOYO-1. Briefly, 20 μg of pCMVLuc was mixed with 5 μL of YOYO-1 (80 μM) and incubated at room temperature for 10 min in the dark. One day before use, HeLa cells were seeded at a density of 5 × 104 cells/chamber in 8-well glass chamber slides (Labtek, Nunc, USA). Preparation of the plasmid DNA/dendrimer complexes was performed as described before. Then, the complex containing YOYO-1-labeled plasmid DNA was added to the cells in Opti-MEM transfection medium. Confocal Microscopy. After incubation for 1.5 h at 37 °C, the cells were washed with PBS and staining with Hoechst 34580. Zeiss LSM 510 Meta laser scanning confocal microscope equipped with inverted Zeiss Axiovert 200 M stand (Carl Zeiss GmbH, Jena, Germany) was used for visualization. Images were acquired using LSM 510 software (Carl Zeiss GmbH, Jena, Germany). For cell uptake in the presence of endocytosis markers, the Alexa-Fluor 647-labeled markers were added during the final 15 min incubation of DNA/dendrimer nanaoparticles prior to nuclear staining with Hoechst. For observation of actin rearrangement, after incubation for 15 min at 37 °C with DNA/dendrimer nanaoparticles, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton, incubated with 1% bovine serum albumin (BSA), and stained with Alexa-Fluor 647 phalloidin to label actin fibers, then with DAPI to label the nuclei. Flow Cytometry. The uptake mechanism of the DNA/ dendrimer nanoparticles was examined by means of specific inhibitors of different endocytotic pathways. For inhibition experiments, the cells were seeded at a density of 5 × 105 cells in 3.5 cm dishes (Nunc, USA) one day before, then they were incubated with one of following inhibitors: cytochalasin D (to inhibit macropinocytosis), genistein (to inhibit caveolaemediated endocytosis), or chlorpromazine (to inhibit clathrinmediated endocytosis) for 1 h in completed medium before the DNA/dendrimer complexes were added. Inhibitors were used at concentrations in which they were not cytotoxic for HeLa cells. After incubation for 15 min at 37 °C with YOYO-1labeled DNA/dendrimer complex, the cell uptake efficiency was measured using flow cytometry analysis (Beckman Coulter Epics Elite, Beckman Inc., Miami, FL). Each assay was performed in triplicate. Luciferase Transfection in HeLa Cells. Transfection experiments were carried out using the protocol described previously.37 Briefly, 24 h before transfection, 5 × 104 HeLa cells were seeded in 24-well tissue culture plates. The desired amount of dendrimer reagent or jetPEI (2 μL per μg of DNA) (Polyplus-Transfetion, Illkirch, France) and 1 μg of pCMVLuc were diluted separately in 50 μL of 150 mM NaCl solution. After 10 min, the transfection reagent solution was added to the DNA solution. The solution was homogenized and left 30 min. Before transfection, cells were incubated with 1 mL of MEM without serum. Then, 100 μL aliquots of pCMVLuc/dendrimer complex solution were added per well and the plates were incubated at 37 °C. After 4 h of incubation, 100 μL aliquots of FBS were added per well and the plates were further incubated at 37 °C for 24 h. Each experiment was done in triplicate. Luciferase gene expression was measured using a commercial kit (Promega, France). After removing the complete medium, three washings with 1 mL of PBS solution were made. Then, 100 μL aliquots of 1× lysis buffer were added per well, and the plate was incubated at room temperature for 30 min. The

lysates were collected and centrifuged at 14 000 g for 5 min. The luciferase assay was assessed with 5 μL of lysate after injection of 100 μL of luciferin solution. The luminescence (RLU) was monitored with an integration over 10 s with a luminometer (Berthold, France). Results are expressed as light units integrated over 10 s (RLU), per mg of cell protein using the BCA assay (Pierce, France). EGFP Transfection in HeLa Cells. In vitro transfection efficiency was evaluated on HeLa cell line using a plasmid containing a reporter gene enhanced green fluorescence protein (EGFP). Cells were seeded into a 24-well plates at a density of 1 × 105 cells per well in 500 μL of complete medium 24 h prior to transfection. After the DNA/dendrimer complexes containing 1 μg of DNA were incubated with cells at 37 °C for 4 h, 50 μL FBS was added. After 48 h, cells were washed in PBS and immediately visualized by using an inverted fluorescence microscope Olympus IX70 equipped with an AxioCam HR digital camera from Carl Zeiss. Images were acquired using AxioVision software (Olympus Corp., NY, USA). For quantification, the cells were washed twice with PBS and collected. The fluorescent intensity of positive cells was measured with the flow cytometer (Beckman Coulter Epics Elite, Beckman Inc., Miami, FL). Effect of Bafilomycin A1. The transfection experiments involving bafilomycin A1 were performed as described above except that the HeLa cells were preincubated with 200 nM bafilomycin A1 at 37 °C for 1 h. EGFP Transfection in LMTK− Fibroblasts. Twenty-four hours before transfection, 1 × 104 LMTK− cells were seeded in 96-well tissue culture plates. The desired amount of dendrimers G5−G7 and DNA plasmid pEGFP-N1 (Clontech) were diluted separately in 50 μL of MEM media. Dendrimers were added to DNA and the solution was homogenized and left for 30 min. Then, 100 μL of pEGFP-N1/dendrimer complex solution was added per well and the plate was incubated at 37 °C. After 4 h, 10 μL of FBS was added per well and the plate was left for 48 h at 37 °C. Cells were visualized under fluorescence microscopy for the observation of EGFP positive cells and transfection efficiency was quantified by Image-J software. Cell viability was determined by MTT assay. Each experiment was carried out in triplicate. EGFP Transfection in Mouse Thymus. C57/BL6 Wild Type (WT) mice (Charles River) were bred and maintained under specific-pathogen-free conditions. All experiments were done in agreement with the French and European ethical rules (authorization number: 13−27). Thymic in vivo gene transfer was performed at 5−6 weeks of age. Animals were anesthetized by intraperitoneal injection with a mixture of ketamine (100 mg/kg body weight; Imalgene 500; Rhone-Merieux) and xylazine (10 mg/kg body weight; Rompun 2%; Centravet). A 20 μL formulation containing 10 μg of pEGFP-N1 or pEGFP/ dendrimer complexes prepared in 0.9% NaCl was slowly injected with an insulin syringe in each thymic lobe. Untreated mice were taken as negative control. Animals were kept warm until recovery. After 48 h, mice were sacrificed, and thymi were removed from anaesthetized animals and washed in PBS (1×), then analyzed for EGFP expression by confocal microscopy and flow cytometry. Western Blot. Proteins were extracted by using the Nuclear Protein Extraction Kit (Panomics). Protein concentrations were measured using the Pierce BCA protein assay. Protein samples were run on SDS polyacrylamide gel (Invitrogen) and transferred to nitrocellulose membranes (BioRad). Incubation with the GFP antibody (1:1000, Roche) was performed 2464



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overnight at 4 °C. Mouse polyclonal anti-β-actin was used as loading control (Santa Cruz Biotechnology). Proteins were visualized using horseradish peroxidase-conjugated secondary antibody (1:1000; Amersham Pharmacia Biotech) and the enhanced chemiluminescence (ECL) detection system (Pierce). Immunofluorescence. To identify the distribution and EGFP cell expression, 12 μm thymic sections were prepared by cryosectioning after embedding the organs in OCT (Sakura Finetech) and mounted on glass slides. Sliced samples were kept in a humidified chamber and were not allowed to dry during staining. Sections were fixed with 4% paraformaldehyde (Sigma Aldrich) in phosphate buffer for 1 h. Frozen sections were stained with anti-CD3 hybridomas (OKT3) (kindly provided by Dr. B. Malissen, CIML, Marseille, France). Alexa 546 goat anti-mouse IgG was used as secondary antibody. As negative control, untreated thymi were used in all experiments. Tissues were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) at 1 mg/mL and mounted with Mowiol fluorescent mounting medium (Calbiochem). Fluorescent images were acquired by a Zeiss LSM 510 confocal microscope with a 515−525 nm bandpass filter set to view EGFP, a 560 nm long-pass filter set to view Alexa-546, and a 420 nm long-pass filter set to view DAPI. All images were exposed using the same exposure time under the same magnification. Flow Cytometry. Mice thymi were minced in FACS buffer (PBS 1×, 3% FCS, 0.02 sodium azide) to obtain thymocytes. Cell suspension was filtered through 200 μm nylon mesh and centrifuged at 1500 rpm for 5 min. Precipitated cells were resuspended in FACS buffer. One to two million thymocytes were first incubated with the 2.4G2 hybridoma supernatant to block nonspecific binding of labeled antibodies. Then, cells were stained with a mixture of PE-labeled CD4 and APClabeled CD8 mAbs (BD Pharmingen). Viable and EGFP positive cells were examined using a FACScalibur flow cytometer and data analyzed with FlowJo software (Tree Star, Inc.). Statistical Analysis. Statistical analysis was performed by a one-way ANOVA test followed by Fisher’s protected least significant difference (PLSD) test (Statview 512, Brain Power Inc., Calabases, CA). p ≤ 0.05 was considered significant (*); p ≤ 0.01 (**); p ≤ 0.001 (***).

where k is Boltzmann’s constant, T is the absolute temperature, η is the solvent viscosity, and Rh is the hydrodynamic radius of the molecule (spherical approximation). In addition, DOSY data can be used to estimate the weight average molecular weight of macromolecules,39 because calibration curves can be established to correlate the self-diffusion coefficient to the molecular weight of macromolecules under specific experimental conditions for a given set of compounds. The self-diffusion coefficients D obtained for TEA-core dendrimers in D2O solutions are reported in Figure 1B, together with the corresponding hydrodynamic radius Rh estimated for G1−G7 by using eq 1. As can be seen, the higher the dendrimer generation, the smaller the self-diffusion coefficient and the greater the hydrodynamic radius (Figure 1B). This is in line with the molecular construction of PAMAM dendrimers, higher-generation dendrimers displaying a greater weight and a larger size. The hydrodynamic radii of TEA-core dendrimers as obtained by DOSY experiments were also compared to those reported in the literature for the NH3-core dendrimers (Table S1 in Supporting Information).41 Although both types of dendrimers exhibit similar chemical composition and molecular weights, these data showed that TEA-core dendrimers systematically had larger hydrodynamic radiihence, larger molecular size than the corresponding NH3-core dendrimers.39 Moreover, the difference in size between these two series of dendrimers increased significantly with increasing dendrimer generation, a clear indication that the TEA-core dendrimers assume a more extended conformation in solution. This can be explained by the larger core of the TEA-core dendrimers and by the branching units not directly connected to the focal point of the core, as opposed to NH3-core dendrimers that exhibit highly compact structures due to the most closely and densely packed branching units. In other words, the molecular structure of TEA-core dendrimers in solution is much less compact than that of NH3-core dendrimers, which in turn suggests that the former may comparatively possess higher structural flexibility. We further studied the evolution of the measured selfdiffusion coefficients as a function of their molecular weights for TEA-core PAMAM dendrimers. On a double-logarithmic D = f(Mw) plot, all data are perfectly aligned (Figure 1C). Using the corresponding scaling law for TEA-core dendrimers (see Experimental section), we could estimate the molecular weights for dendrimers (G1−G7) from their corresponding selfdiffusion coefficients D (Figure 1B). The molecular weights obtained in this way for the TEA-core dendrimers parallel the results previously determined by MS and are even closer to the calculated values than the GPC-derived values (Figure 1B). 19,20 This further confirms the quality of the TEA-core dendrimers synthesized in this work. TEA-Core Dendrimers Have Flexible Structures Favoring Interaction with DNA. We next studied the structural flexibility of the TEA-core dendrimers and their complexes with DNA molecules by computer modeling using atomistic molecular dynamics (MD) and mesoscale simulation techniques. Due to the large size of dendrimers of generation 7 that would lead to extensive calculation and data processing, we performed MD simulations on isolated TEA-core dendrimers of generations 4, 5, and 6 (G4, G5, and G6), NH3-core dendrimers of generations 4, 5, and 6 (G4′, G5′, and G6′) and their respective complexes with DNA. Here, we focus our comments only on the results obtained with dendrimers of generation 6.

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RESULTS AND DISCUSSION DOSY NMR Study of TEA-Core Dendrimers. The TEAcore PAMAM dendrimers of generation 1 to 7 (G1−G7) were synthesized and characterized in terms of structure and molecular weight as previously described (Figure 1). 18−22 In this work, we further studied their behavior in solution and their sizes and molecular weights using diffusion ordered NMR spectroscopy (DOSY).38,39 DOSY is based on the pulsed gradient spin echo (PGSE) experiment,40 which labels the spatial position of the nuclear spins using pulsed magnetic field gradients and allows us to infer the molecular self-diffusion coefficient from their translational displacement over a given time period. Specifically, DOSY38 allows the self-diffusion coefficient D to be measured, which in turn is related at infinite dilution to the molecular size through the well-known Stokes− Einstein equation

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trajectories. The structural differences between these two dendrimers are blindingly obvious: while G6 exhibits an open flexible conformation featuring void spaces within its interior (Figure 2A), G6′ is more rigid and compact, with uniformly distributed monomer units and no restricted void spaces in the entire molecule (Figure 2B). The enhanced flexibility of TEAcore dendrimers is well-testified by the wider conformational space visited by G6 (Figure 2C) during the entire MD simulation with respect to the NH3-core G6′ counterpart (Figure 2D). It is noted that the presence of hollow spaces in G6 allows a significantly larger number of water molecules to penetrate into the dendrimer interior with respect to G6′. This property makes the tertiary amine groups in the dendrimer interior more accessible to water molecules, and therefore more readily available for protonation and subsequent ammonium cation hydration in G6. This, in turn, may lead to a higher buffering capacity, which was further confirmed by the results of potentiometric titration experiments, as the dendrimers G5− G7 displayed flat monotonic titration curves (see Figure 5D). Additional insightful structural information on DNA/ dendrimer complexes was again obtained using atomistic molecular dynamics. The conformation of the TEA-core dendrimer G6 is such that the outer branches can readily move toward the phosphate backbone of DNA during complex formation, and the surface amino groups can arrange themselves via “induced-fit” for optimal binding with DNA (Figure 2E). This observation is somehow reminiscent of histones undergoing an induced fit conformational change when binding to DNA to form the nucleosome. In contrast, the more rigid and compact structure of the NH3-core dendrimer G6′ (Figure 2F) prevents this molecule from undergoing significant “induced-fit” conformational readjustment, and consequently, not all terminal amine groups are available to self-orient for optimal DNA binding. Further evidence for the difference in structural flexibility between these two dendrimer series stem from the mesoscale simulations of their complexes with DNA molecules. A typical result of mesoscale simulation is the morphology and the structure of the matter at nanoscale level at the desired conditions of temperature and composition. Figure 2G,H compares the nanoscale morphology of the DNA/G6 and DNA/G6′ systems, whereas Figure 2I,J highlights the different distribution of DNA and water within the self-assembled DNA/ G6 and DNA/G6′ nanoscopic systems. In the DNA/G 6 structure, the dendrimers are able to complex the DNA efficiently and homogeneously (Figure 2G,I). On the contrary, for G6′, some DNA chains are less well enwrapped in the system, and DNA bundles are still present (Figure 2H,J). The water density mapping at the mesoscale level also supports the hypothesis of a higher degree of hydration and a more uniform water molecule distribution in the DNA/G6 system (Figure 2I) with respect to the DNA/G6′ one (Figure 2J). These results are in line with those obtained by MD, demonstrating the easy accessibility of water molecules to the interior of structurally flexible dendrimers. TEA-Core Dendrimers Form Stable Nanoparticles with DNA and Protect DNA from Degradation. Knowledge of cationic dendrimers to self-assemble with anionic DNA via electrostatic interaction is well-established, and the formation of stable and nanoscale DNA/dendrimer complexes is a prerequisite for efficient cell uptake, intracellular delivery, and transgene expression.

Figure 2A,B shows two equilibrium MD snapshots of G6 and G6′ respectively, and Figure 2C,D illustrates the superposition of different snapshots taken from their respective MD

Figure 2. Equilibrium MD snapshots of (A) TEA-core dendrimer G6 and (B) NH3-core dendrimer G6′ (In the MD snapshots, dendrimer atoms are portrayed as colored sticks, except for the terminal units, which are portrayed as yellow sticks-and-balls. Atom color code: white, hydrogen; red, oxygen; blue, nitrogen; gray, carbon. Cl− ions and water molecules have been omitted for clarity). Superposition of different conformations of TEA-core dendrimer G6 (C) and NH3-core dendrimer G6′ (D) taken as snapshot along the entire MD trajectory (The different dendrimer conformations are portrayed with different colors, while water and counterions are omitted for clarity). MD snapshots of (E) TEA-core dendrimer G6 and (F) NH3-core dendrimer G6′, in complex with a small fragment of double-helix DNA (Dendrimers are depicted as in panels (A) and (B), and DNA is highlighted as a magenta ribbon). Mesoscale morphologies of the selfassembled systems between TEA-core dendrimers G6 and DNA (G), and NH3-core G6′ and DNA (H) (The dendrimers are portrayed as gray beads except for the terminal units, which are painted in yellow. The DNA chains are represented as magenta sticks). Panels (I) and (J) highlight the different distribution of DNA and water within the selfassembled DNA/G6 and DNA/G6′ nanoscopic systems (DNA is portrayed in magenta as in panels (G) and (H), while water is represented as colored density fields: according to the scale reported in the lower left corner of the panels, low density values are blue, while high density values are red). 2466



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Figure 3. Formation of plasmid DNA/dendrimer complexes as revealed by agarose gel (A), transmission electron microscopy (B), dynamic light scattering (C), and the resulting complexes protect DNA from DNase degradation (D). (A) Migration of luciferase plasmid DNA (250 ng per well) in the presence of G5−G7 at charge ratios N/P 1/5−10/1 in 50 mM Tris-HCl buffer pH 7.4. (B) Transmission electron microscopic image of DNA/dendrimer complexes prepared in 50 mM Tris-HCl buffer pH 7.4 using 5 ng/μL plasmid DNA and G5−G7 at an N/P ratio of 10. Scale bars represent 200 nm. (C) Light scattering analysis of plasmid DNA/dendrimer complexes prepared using 50 ng/μL plasmid DNA and G5−G7 at an N/P ratio of 10. (D) Compared to the naked DNA (200 ng/well), which was degraded within 10 min in the presence of DNase, DNA complexed with G5−G7 at an N/P ratio of 2.5 was resistant to DNase and remained stable even after 90 min incubation.

We first studied the complex formation between plasmid DNA and TEA-core dendrimers using gel electrophoresis, a widely used technique for assessing complex formation with nucleic acids. Due to the cooperative electrostatic interactions, cationic dendrimers G5−G7 were able to fully condense negatively charged DNA at an N/P ratio above 2.5 (Figure 3A), totally preventing DNA from migration. This suggests the formation of positively charged and stable dendriplexes between dendrimers and plasmid DNA at an N/P ratio ≥2.5. In addition, the complex formation depended on the dendrimer generation: all DNA molecules were complexed with G7 at N/P = 1,whereas free DNA was observed in trace and considerable amounts with G6 and G5, respectively (Figure 3A). This indicates that dendrimers of higher generations form more stable DNA/dendrimer complexes, presumably due to their greater cooperativity and tighter DNA binding domains.42,43 The size of the DNA/dendrimer complex is also critical for gene delivery. Due to their compatibility with both extracellular diffusion and endocytosis, small DNA/dendrimer nanoparticles would be an advantage for in vivo experiments. We examined the size and morphology of the DNA/dendrimer complexes using transmission electron microscopy (TEM). Compact, uniform, and nanoscale spherical particles were observed for the DNA/dendrimer complexes using dendrimers G5−G7 at N/P ratio of 10, with diameters around 100 nm (Figure 3B). This was further confirmed by dynamic light scattering (DLS), a technique which provides the size, size distribution, as well as surface potential (ζ-potential) of DNA/dendrimer complexes in solution. Results from DLS measurement showed that the DNA/dendrimer complexes have sizes around 100 nm using dendrimers G5−G7 at N/P ratios of 10 (Figure 3C). Moreover, ζ-potential measurement gave positive values around +30 mV for the DNA/dendrimer complexes. Taken together, these results demonstrate that the structurally flexible TEA-core PAMAM dendrimers readily condense plasmid DNA into compact and stable colloidal nanoparticles.

We then verified the ability of the DNA/dendrimer nanoparticles to protect DNA against enzymatic degradation. As shown in Figure 3D, naked DNA was completely digested by DNase within 10 min, whereas DNA complexed with dendrimer G5−G7 remained intact even after 90 min incubation with the DNase. This result demonstrates that the dendrimer nanoparticles effectively protect DNA from enzymatic degradation, which is a basic prerequisite for efficient DNA delivery. Cell Uptake of DNA/Dendrimer Nanoparticles Mainly via Macropinocytosis. Cell uptake of DNA/dendrimer nanoparticles is one of the early events in gene delivery. We studied the internalization of DNA/dendrimer complexes in HeLa cells using live cell confocal microscopy with green fluorescent dye YOYO-1 labeled DNA. The green fluorescent DNA/G6 nanoparticles were effectively internalized in the cells (Figure 4A), whereas no green particles could be observed in the absence of dendrimer under the same experimental conditions (data not shown). Consequently, the effective cell uptake of negatively charged DNA is unambiguously mediated by the cationic dendrimer which acts as nanocarrier. It has been reported that several pathways can drive cell uptake of nanoparticles with sizes around 50−300 nm, such as macropinocytosis, clathrin-mediated endocytosis, and caveolaemediated endocytosis. 44−49 We therefore used specific inhibitors and biomarkers of various endocytotic pathways to distinguish which pathway is taking DNA/dendrimer nanoparticles into cells. As illustrated in Figure 4B, cytochalasin D, a macropinocytosis inhibitor, reduced the cell uptake of DNA/G6 in a dose-dependent manner. Genistein, an inhibitor specifically involved in caveolae-mediated endocytosis, had no notable inhibitory effect on cell uptake; whereas chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, led only to a slight uptake decrease. In addition, the YOYO-1 labeled DNA colocalized with a macropinocytosis marker dextran but not with transferrin or cholera toxin B (Figure 4C), markers of clathrin- and caveolae-mediated endocytosis, respectively. 2467



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Figure 4. Uptake of YOYO-1-labeled DNA/G6 complexes in HeLa cells analyzed by live-cell-confocal microscopy (A), using specific inhibitors (B), and fluorescent endocytosis markers (C) of different uptake pathways and phalloidin probe for actin rearrangement (D). (A) Green channel image of the YOYO-1-labeled DNA/G6 nanoparticles, blue channel image of the nuclei of HeLa cells stained by Hoechst 34580, and merged image of both. (B) Effect of cytochalasin D (to inhibit macropinocytosis), genistein (to inhibit caveolae-mediated endocytosis), and chlorpromazine (to inhibit clathrin-mediated endocytosis) on the cell uptake of the YOYO-1-labeled DNA/G 6 complexes on HeLa cells. (C) Cell uptake of YOYO-1-labeled DNA/G6 complexes in the presence of different endocytosis markers: dextran (marker of macropinocytosis), cholera toxin B (marker of caveolaemediated endocytosis), and transferrin (marker of clathrin-mediated endocytosis). (D) Fluorescent labeling of actin fibers using phalloidin reveals actin depolymerization, a hallmark of macropinocytosis. *, ** and ***, differ from control (p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively) by Student’s t test.

luciferase gene expression in HeLa cells. Significant luciferase expression was obtained with dendrimers G5, G6, and G7 (Figure 5A), whereas the lower-generation dendrimers G1− G4 yielded dramatically reduced expression (data not shown). Moreover, DNA transfection was dependent on the N/P charge ratio. The most efficient gene transfection was observed with G6 at an N/P ratio of 10, with the efficiency being almost

Furthermore, actin depolymerization, a hallmark of macropinocytosis, was observed after the application of the DNA/G 6 particles using phalloidin marker (Figure 4D). Altogether, these results clearly demonstrate that macropinocytosis is the major uptake pathway. Effective DNA Transfections in Vitro. For dendrimermediated DNA transfection in vitro, we first evaluated the 2468



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Figure 5. (A) Luciferase transfection in HeLa cells using TEA-core dendrimers G5−G7 as vectors (at various N/P ratios) and 1 μg of pCMVLuc plasmid. Luciferase activity was determined 24 h after transfection and expressed as RLU/mg protein of cell lysates. (B) Images of EGFP transfected HeLa cells using G5−G7 as vectors and 1 μg pEGFP-N1 plasmid at an N/P ratio of 10, compared to nontreatment control and cells treated with DNA/Lipofectamine complexes. (C) Effects of bafilomycin A1 on dendrimer-mediated and poly(lysine)-mediated transfection of EGFP in HeLa cells as measured by FACS flow cytometry. (D) pH titration curves of the dendrimers G5−G7. (E) EGFP transfection efficiencies of G5−G7 and Lipofectamine in mouse fibroblast LMTK− cells in the absence of serum. (F) MTT assay of the toxicity of DNA/dendrimer complexes in mouse fibroblast LMTK− cells. * and *** differ from control (p ≤ 0.05 and p ≤ 0.001, respectively) by Student’s t test.

In order to further assess the transfection efficacy in individual cells, we carried out assays for enhanced green fluorescent protein (EGFP) expression in HeLa cells using mock-transfected cells as negative control and Lipofectamine as a positive control (Figure 5B). Lipofectamine is one of the most commonly used lipid vectors, characterized also by its well-known cytotoxicity. Under our experimental conditions, most HeLa cells died after treatment with DNA/Lipofectamine; whereas G5−G7 dendrimers led to excellent EGFP expression, particularly G6 being the most efficient vector, with a level of transfection up to 60%. The above transfection results perfectly correlate with those previously obtained with fractured and degraded PAMAM dendrimers,13 namely, that only higher-generation dendrimers mediate effective transfection. These higher-generation dendrimers contain larger DNA binding regions,42,43 thus creating stronger interactions between DNA and dendrimer via

2-fold higher than that of poly(ethylene imine) (PEI), one of the most effective nonviral transfection reagents available to date.16 It should be mentioned here that the thermally degraded fractured PAMAM dendrimers have a transfection activity similar to that of PEI.9 We also compared the transfection efficiency of the TEA core dendrimer with the commercially available structurally rigid ethylenediamine (EDA) core dendrimer (Figure S1 in Supporting Information). Our results confirmed that TEA core dendrimer is more efficient for DNA transfection than the corresponding EDAcore dendrimer. With all the results presented here, we conclude that genuine (i.e., nondegraded), structurally flexible TEA-core dendrimers having precisely controlled structures are effective nanovectors for DNA transfection, with efficiencies similar or superior to PEI and hence to fractured PAMAM dendrimers. 2469



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cooperative amplification. However, interactions that are too strong between DNA and dendrimer may prevent DNA release from complexes, thereby compromising transfection efficiency. This may explain why G6 has the best transfection efficiency compared to both the lower-generation dendrimer G5 and the higher-generation dendrimer G7. We further examined whether the buffer-based endosomolytic activity plays an important role using bafilomycin A1. Bafilomycin A1 is a proton pump inhibitor and selectively inhibits vacuolar H+-ATPase and prevents acidification of endosomes. Figure 5C shows a significant decrease of EGFP expression level in HeLa cells in the presence of bafilomycin A1 for dendrimer-mediated DNA delivery, suggesting that dendrimer-mediated DNA transfection was dependent on the endosomal acidification process. This is in line with the structural features of TEA-core PAMAM dendrimers, which harbor a large number of tertiary amines in the interior able to produce a robust buffer capacity and proton sponge effect for endosomal release. Additional support for this hypothesis comes from the result obtained from the pH titration experiment (Figre 5D) where indeed the dendrimers G5−G7 displayed flat titration curves, an indication of high buffer capacity. Since we had no NH3-core PAMAM dendrimers available in our hands as a control, we then asked whether poly(lysine), which contains only primary amine groups and has been reported to be devoid of proton-sponge effect, could be used as a negative control. We therefore determined poly(lysine)-mediated EGFP expression in HeLa cells in the presence and absence of bafilomycin A1 (Figure 5C). As expected, there was no notable change of EGFP expression in the presence of bafilomycin A1, suggesting the absence of proton-sponge effect in poly(lysine)-mediated DNA transfection. These results demonstrate unambigously that the “proton-sponge” effect played an important role for our dendrimer mediated DNA transfection. We next performed assays for EGFP expression in mouse fibroblast LMTK− cells. LMTK− fibroblasts are known to be difficult to transfect using nonviral vectors. Gratifyingly, our dendrimers G5−G7 led to effective EGFP expression (Figure 5E). The highest tranfection efficiency was achieved again using G6 at N/P = 10, with a transfection level of up to 40%, better than the commercial transfection reagent lipofectamine (Figure 5E). Under the serumless transfection conditions used, no significant toxicity was observed for G5−G7 (Figure 5F). The transfection efficiency was decreased in the presence of 10% serum in the transfection medium (Figure S2 in Supporting Information). This observation may limit their in vivo applications via systemic administration but much less by direct injection (vide infra). Efficient DNA Transfection in Vivo via Intrathymic Injection. We further evaluated in vivo DNA transfection in the mouse thymus as a model. We used both G6 and G7 for the in vivo delivery of the EGFP plasmid as a proof-of-concept, since G6 showed the best DNA tranfection efficiency in vitro, whereas G7 was successfully used in siRNA delivery in various systems.20−22 In addition, the DNA complexes with G6 and G7 were more stable and retained their size and surface potential values even at high salt concentration (Table S2 in Supporting Information). It should be mentioned that our dendrimers have reduced transfection efficiency in the presence of serum (Figure S2 in Supporting Information); we therefore sought to employ means that do not depend on blood circulation to reach the

target organ. We thus chose direct intrathymic injection of DNA/dendrimer complexes. In vivo DNA transfection experiments were carried out on C57/BL6 WT mice. Mice were first anesthetized, and a volume of 20 μL (containing 10 μg EGFP plasmid complexed with dendrimer at N/P ratio of 10) was then slowly injected into each thymic lobe. All mice survived well during and after the intervention; no signs of toxicity were observed. Strong EGFP expression in the thymi following injection of DNA/dendrimer complexes was revealed by Western blot analysis using an antibody against EGFP (Figure 6A), whereas only weak EGFP levels were detected following naked DNA injection. Therefore, the observed high expression of EGFP in mice thymi is a direct consequence of dendrimer-mediated DNA delivery. We next examined the transfected thymi by confocal microscopy (Figure 6B). Untreated mice exhibited no EGFP signal (control in Figure 6B); mice with naked DNA injection showed a very weak EGFP signal, whereas EGFP expression mediated by G6 was significantly stronger (Figure 6B). In addition, costaining analysis with the CD3 thymocyte marker showed a colocalization with the green EGFP signal within these thymocytes (Figure 6B and enlarged in Figure 6C). Consequently, these results demonstrate an efficient and effective dendrimer-mediated DNA transfection in mouse thymus, specifically in thymocytes. We further analyzed EGFP expression in the different subpopulations of thymocytes. It is known that thymocyte differentiation can be divided into three major stages defined by the expression of CD4 and CD8 markers. The most immature or double-negative stage is characterized by the lack of expression of CD4 and CD8 markers (CD4−CD8−, doublenegative, DN). Double negative cells progress to a stage where there is expression of both CD4 and CD8 markers (CD4+CD8+, double-positive, DP) before lineage commitment to single-positive (SP) cells which express either CD4 (SP CD4+) or CD8 (SP CD8+) marker. We therefore immunophenotyped EGFP expression within the four main thymocyte subpopulations, namely, CD4−CD8− double-negative (DN), CD4+CD8+ double-positive (DP), CD4+ and CD8+ singlepositive (SP) (Figure 6D). We found high percentages of transfection (25.1% of DN, 1.59% of DP, 1.20% of SP CD4 +, and 6.96% of SP CD8+ cells) in mice thymi treated with injection of DNA/dendrimer complexes. As expected, no signal was detected in untreated mice thymi, nor was any notable EGFP expression observed in mice thymi treated with naked DNA injection. This finding strongly confirms transgene expression to be a direct consequence of dendrimer-mediated DNA delivery. Finally, it is to be noted that the DN thymocytes were preferentially transfected. This is because immature DN thymocytes undergo rapid proliferation, and therefore lead to higher transfection with respect to the other three subpopulations. Additionally, notable transfection was also observed with more mature SP CD8+ cells, which are considered relatively quiescent. Collectively, our results demonstrate that the structurally flexible TEA-core PAMAM dendrimers studied in this work are safe and effective nanovectors for intrathymic DNA delivery and gene expression. To our knowledge, this is the first experimental evidence that dendrimers could act as efficient nanovectors for DNA transfection in mouse thymus, opening new avenues for future applications of dendrimermediated gene therapy for both inherited and acquired immunodeficient diseases. 2470



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Figure 6. In vivo EGFP expression in thymi was assessed by Western blot (A), immunofluorescence (B and C) and flow cytometry (D). (A) Western blot analysis of EFGP protein expressed in mice thymi in the presence and absence of dendrimer compared to nontreated control. (B) The EGFP fluoresence was analyzed using confocal microscopy with thymi sections derived from mice that were nontreated (control), injected with naked DNA, or injected with DNA/dendrimer complexes: nuclei appear as blue (DAPI staining) and thymocytes as red (anti-CD3 antibody labeling). (C) Enlarged image showing EGFP transfected thymocytes costained with anti-CD3 antibody (red) and DAPI (blue for nuclei laebling). (D) Thymocyte profile stained with CD4 and CD8 monoclonal antibodies was analyzed by flow cytometry 48 h after injection. The percentage of each population of thymocytes is indicated within each quadrant. The different populations of thymocytes expressing EGFP protein were analyzed for thymi derived from mice that were nontreated (control), injected with naked DNA, and injected with DNA/G 6 complexes, respectively. The percentage of EGFP transfected cells is indicated in red within the quadrant of each population. DN: CD4 −CD8− double-negative cells; DP: CD4+CD8+ double-positive cells; SP CD4+: single-positive cells which express CD4 marker; SP CD8 +: single-positive cells which express CD8 marker.

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It has been previously reported that the thermally degraded fractured PAMAM dendrimers have an activity similar to PEI. 9 Our results demonstrate that genuine (i.e., nondegraded), structurally flexible dendrimers with precise structural features also are effective nanovectors for DNA transfection, with efficiencies similar or superior to PEI and hence to fractured PAMAM dendrimers. The experimental results presented here, coupled with computer-aided molecular simulations, provide evidence for the development of functional dendrimers for DNA transfection using rational design of structurally flexible dendrimers.50−52 Compared to random thermal or alkaline degradation, a process which does not allow precise control over the structure, rational design based synthesis allows the development of dendrimer nanovectors with a defined yet flexible structure. Consequently, these dendrimers have a higher potential of becoming vectors for nucleic acid-based therapies in general. It is important to note that these dendrimers are particularly effective in delivering and expressing a gene in the thymus. The thymus is an attractive organ for immune gene therapy, and yet,

CONCLUSION

We have developed triethanolamine (TEA) core PAMAM dendrimers as structurally flexible nanovectors for nucleic acid delivery. As shown in the present work, these dendrimers have greater structural flexibility and a more open conformation with void spaces in the interior. They self-assembled with DNA molecules into nanoparticles via an “induced fit” process, resembling histone/DNA interaction in nature. The resulting DNA/dendrimer nanoparticles could effectively protect DNA from degradation and facilitate cell entry mainly via macropinocytosis. Furthermore, due to their open and void structure, these dendrimers are well-adapted to act as 3D protons sponges per se with easier access of water molecules inside the dendrimer interior and leading to increased protonation of interior tertiary amines. This enhances the buffering capacity thus benefiting DNA endosome release. Fulfilling our expectations, these dendrimers were efficient nanovectors for gene delivery in vitro to transfection-resistant fibroblasts as well as in vivo to the mouse thymus, an extremely challenging organ for DNA transfection. 2471



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(9) Navarro, G., and Tros de Ilarduya, C. (2009) Activated and nonactivated PAMAM dendrimers for gene delivery in vitro and in vivo. Nanomedicine 5, 287−297. (10) Dennig, J., and Duncan, E. (2002) Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers. J. Biotechnol. 90, 339−347. (11) Haensler, J., and Szoka, F. C. Jr. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 372−379. (12) Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spindler, R., Tomalia, D. A., and Baker, J. R. Jr. (1996) Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. U. S. A. 93, 4897−4902. (13) Tang, M. X., Redemann, C. T., and Szoka, F. C. Jr. (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703−714. (14) Tomalia, D. A., Baker, H., Dewald, J., Hall, M., Kallos, G., Martin, S., Roeck, J., Ryder, J., and Smith, P. (1985) A new class of polymers: starburst-dendritic macromolecules. Polym. J. 17, 117−132. (15) Tomalia, D. A., Naylor, A. M., and Goddard, W. A. III (1990) Starburst dendrimers: molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem., Int. Ed. Engl. 29, 138−175. (16) 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 oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 92, 7297−7301. (17) Sonawane, N. D., Szoka, F. C. Jr., and Verkman, A. S. (2003) Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J. Biol. Chem. 278, 44826− 44831. (18) Wu, J., Zhou, J., Qu, F., Bao, P., Zhang, Y., and Peng, L. (2005) Polycationic dendrimers interact with RNA molecules: polyamine dendrimers inhibit the catalytic activity of Candida ribozymes. Chem. Commun. (Camb.), 313−315. (19) Shen, X. C., Zhou, J., Liu, X., Wu, J., Qu, F., Zhang, Z. L., Pang, D. W., Quelever, G., Zhang, C. C., and Peng, L. (2007) Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer complexes. Org. Biomol. Chem. 5, 3674−3681. (20) Zhou, J., Wu, J., Hafdi, N., Behr, J. P., Erbacher, P., and Peng, L. (2006) PAMAM dendrimers for efficient siRNA delivery and potent gene silencing. Chem. Commun. (Camb.), 2362−2364. (21) Liu, X. X., Rocchi, P., Qu, F. Q., Zheng, S. Q., Liang, Z. C., Gleave, M., Iovanna, J., and Peng, L. (2009) PAMAM dendrimers mediate siRNA delivery to target Hsp27 and produce potent antiproliferative effects on prostate cancer cells. ChemMedChem 4, 1302−1310. (22) Zhou, J., Neff, C. P., Liu, X., Zhang, J., Li, H., Smith, D. D., Swiderski, P., Aboellail, T., Huang, Y., Du, Q., Liang, Z., Peng, L., Akkina, R., and Rossi, J. J. (2011) Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice. Mol. Ther. doi:10.1038/mt.2011.1207. (23) Liu, X. X., Rocchi, P., and Peng, L. (2012) Dendrimers as nonviral vectors for siRNA delivery. New J. Chem., doi: 10.1039/ C1031NJ20408D. (24) Cournoyer, D., and Caskey, C. T. (1993) Gene therapy of the immune system. Annu. Rev. Immunol. 11, 297−329. (25) Adjali, O., Montel-Hagen, A., Swainson, L., Marty, S., Vicente, R., Mongellaz, C., Jacquet, C., Zimmermann, V., and Taylor, N. (2009) In vivo and ex vivo gene transfer in thymocytes and thymocyte precursors. Methods Mol. Biol. 506, 171−190. (26) Moreau, A., Vicente, R., Dubreil, L., Adjali, O., Podevin, G., Jacquet, C., Deschamps, J. Y., Klatzmann, D., Cherel, Y., Taylor, N., Moullier, P., and Zimmermann, V. S. (2009) Efficient intrathymic gene transfer following in situ administration of a rAAV serotype 8 vector in mice and nonhuman primates. Mol. Ther. 17, 472−479. (27) Adjali, O., Marodon, G., Steinberg, M., Mongellaz, C., ThomasVaslin, V., Jacquet, C., Taylor, N., and Klatzmann, D. (2005) In vivo

gene transfer in the thymus is exceedingly challenging with respect to efficiency and safety. Previous work on intrathymic transfection using viral26−28 and nonviral28 vectors as well as electroporation29 was either inefficient or technically demanding. We demonstrate here for the first time that the structurally flexible PAPAM dendrimers could effectively deliver DNA and reach a high level gene expression in the mouse thymus. Therefore, structurally flexible dendrimer-mediated gene transfer is a promising tool for further development of strategies aimed at immune gene therapy via intrathymic DNA delivery. In addition, as these dendrimers are also efficient nanovectors for siRNA delivery in vitro and in vivo,20−22 they hold great potential for both in vitro (functional genomics) and in vivo (therapeutic) applications of nucleic acid delivery in general. Last but not least, structure-controlled, size-tailored dendrimers should provide useful tools for use in various applications in biotechnology such as targeted drug delivery systems and drugs based on mimicking the functions of certain natural biomacromolecules.53

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1, S2, Tables S1, S2 and related experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author *Tel: (33) 4 91829154, Fax: (33) 4 91829301, e-mail: ling.peng@ univmed.fr.

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ACKNOWLEDGMENTS This research was funded by Association Française contre les Myopathies (No. 13074, 10793), the international ERA-Net EURONANOMED European Research project DENANORNA, National Natural Science Foundation of China (No. 20572081), National Mega Project on Major Drug Development (No. 2009ZX0930-014), Wuhan University, CNRS, INSERM and under the auspice of European COST Action TD0802 “Dendrimers in Biomedical Applications”. Liu Xiaoxuan is supported by China Scholarship Council. Miriam Yammine is supported by a grant from the INSERM.

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