Impact of siRNA Overhangs for Dendrimer-Mediated siRNA Delivery

Article pubs.acs.org/molecularpharmaceutics

Impact of siRNA Overhangs for Dendrimer-Mediated siRNA Delivery and Gene Silencing Paola Posocco,† Xiaoxuan Liu,‡,§,∥,⊥,# Erik Laurini,† Domenico Marson,† Chao Chen,‡,∇ Cheng Liu,‡,■ Maurizio Fermeglia,† Palma Rocchi,§,∥,⊥,# Sabrina Pricl,*,†,○ and Ling Peng*,‡

Mol. Pharmaceutics 2013.10:3262-3273. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/02/18. For personal use only.

†

Molecular Simulation Engineering (MOSE) Laboratory, Department of Engineering and Architecture (DEA), University of Trieste, Via Valerio 10, 34127 Trieste, Italy ‡ Aix-Marseille Université & CNRS, Centre Interdisciplinaire de Nanoscience de Marseille, CNRS UMR 7325, 13288 Marseille, France § Centre de Recherche en Cancérologie de Marseille, Inserm, UMR1068, 13009 Marseille, France ∥ Institut Paoli-Calmettes, 13009 Marseille, France ⊥ Aix-Marseille Université, 13284 Marseille, France # CNRS, UMR7258, 13009 Marseille, France ∇ Aix-Marseille Université & CNRS, Institut de Chimie Radicalaire, UMR 7273, 13390 Marseille, France ■ State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China ○ National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-DEA, University of Trieste, 34127 Trieste, Italy S Supporting Information *

ABSTRACT: Small interfering RNA (siRNA) have attracted considerable attention, as compelling therapeutics providing safe and competent delivery systems are available. Dendrimers are emerging as appealing siRNA delivery vectors thanks to their unique, welldefined architecture and the resulting cooperativity and multivalency confined within a nanostructure. We have recently disclosed the structurally flexible fifth-generation TEA-core PAMAM dendrimer (G5) as an effective nanocarrier for delivery of sticky siRNA bearing long complementary sequence overhangs (dA)n/(dT)n (n = 5 or 7). Here, using combined experimental/computational approaches, we successfully clarified (i) the underlying mechanisms of interaction between the dendrimer nanovector G5 and siRNA molecules bearing either complementary or noncomplementary sequence overhangs of different length and chemistry and (ii) the impact of siRNA overhangs contributing toward the improved delivery potency. Using siRNA with complementary overhangs offer the best action in term of gene silencing through the formation of concatemers, that is, supramolecular structures resulting from synergistic and cooperative binding via (dA)n/(dT)n bridges (n = 5 or 7). On the other hand, although siRNA bearing long, noncomplementary overhangs (dA)n/(dA)n or (dT)n/(dT)n (n = 5 or 7) are endowed with considerably higher gene silencing potency than normal siRNA with (dT)2/(dT)2, they remain less effective than their sticky siRNA counterparts. The observed gene silencing potency depends on length, nature, and flexibility of the overhangs, which behave as a sort of clamps that hold and interact with the dendrimer nanovectors, thus impacting siRNA delivery performance and, ultimately, gene silencing. Our findings can be instrumental in designing siRNA entities with enhanced capability to achieve effective RNA interference for therapeutic applications. KEYWORDS: siRNA delivery, sticky siRNA, siRNA engineering, dendrimer, gene silencing, free energy of binding, steered molecular dynamics

Received: Revised: Accepted: Published: © 2013 American Chemical Society

3262

June 4, 2013 July 6, 2013 July 10, 2013 July 10, 2013 dx.doi.org/10.1021/mp400329g | Mol. Pharmaceutics 2013, 10, 3262−3273

■

Molecular Pharmaceutics

Article

INTRODUCTION Since the discovery of the RNA interference (RNAi) process, small interfering RNA (siRNA, i.e., specific nucleic acid-targeting reagents for gene expression modulation) have attracted tremendous attention as novel therapeutics for treating various diseases.1 However, a successful translational implementation of these highly charged and easily degraded RNA molecules critically requires safe and competent delivery systems able to mask their negative charge and protect them from degradation.2 Over the past few years, endeavors to develop carriers for nucleic acid delivery yielded a myriad of vectors for siRNA delivery.3−6 Among them, a special class of synthetic macromolecules called dendrimers are emerging as compelling nonviral vectors for siRNA delivery by virtue of their unique well-defined threedimensional structure and the resulting cooperativity and multivalency effects.6,7 Among these, the structurally flexible triethanolamine (TEA) core poly(amidoamine) (PAMAM) dendrimers developed in our laboratory8 stand out as excellent nanocarriers for the delivery of siRNA therapeutics, generating effective gene silencing9 in various disease models both in vitro and in vivo.10−12 In parallel, siRNA molecular engineering has been actively pursued to improve and enhance the potency of RNAi for their therapeutic application. Conventional siRNA molecules bear (dT)2/(dT)2 overhangs at the 3′-ends of the oligonucleotides which protect the nucleic acid fragments from RNase degradation and exert a beneficial effect on the RNAi machinery in achieving effective gene silencing.13 Recently, Behr and co-workers demonstrated that sticky siRNA (ssiRNA) with complementary sequence overhangs such as (dA)n/(dT)n (with n ≥ 5) exhibit better gene silencing efficiency compared to normal siRNA when polyethyleneimine (PEI) is used as the delivery agent.14 It is to note that PEI is not efficient in delivering conventional siRNA with (dT)2 overhangs although it is an excellent vector for gene delivery.15 One reason for this could be the self-assembly of sticky siRNA via complementary sequence overhangs into “gene-like” long double-stranded RNA (Scheme 1), which can permit

“gene-like” or “plasmid-like” long double-stranded RNA could be one important factor contributing toward the improved delivery efficiency of our G5 dendrimer. An additional contributing factor might also be the overhangs of the sticky siRNA which, owing to the inherent flexibility of single strand nucleic acid fragment, could behave as a sort of clamps to hold and enwrap the spherical dendrimer nanovector just like protruding molecular arms, enhancing binding and thereby delivery.16 To investigate the impact of the siRNA overhangs with dendrimeric vectors and their putative effect in improving siRNA delivery potency, we designed siRNA carrying overhangs with noncomplementary sequences of different length and nature and compared their gene silencing ability with sticky siRNA bearing complementary overhangs and normal siRNA. In all experiments, our TEA-core G5 dendrimer was used as a standard nanocarrier. Interestingly, we found that the siRNA bearing long, noncomplementary overhangs exhibited considerably higher gene silencing potency than normal siRNA; yet, they were less effective than the sticky siRNA bearing complementary overhangs. The observed potency in gene silencing depends on the length and nature of the siRNA overhangs; importantly, our jointed experimental/computational investigations allowed us to formulate a molecular-based rationale for these findings.

■

EXPERIMENTAL SECTION Materials. The sequence of the siRNA (GenePharm Ltd., Shanghai, China) used corresponded to the human Hsp27 and TCTP sites as follows: Normal Hsp27 siRNA (T2/T2): sense: 5′-GCU GCA AAA UCC GAU GAG AC dTdT-3′, antisense: 5′-GUC UCA UCG GAU UUU GCA GC dTdT-3′. Normal TCTP siRNA (T2/T2): sense: 5′-GGG AGA UCG CGG ACG GGU U dTdT-3′, antisense: 5′-AAC CCG UCC GCG AUC UCC C dTdT-3′. All of the siRNA (An/Tn), (An/An) and (Tn/Tn) (n = 5 or 7) were designed by adding overhangs of (dA)n/(dT)n, (dA)n/(dA)n, and (dT)n/(dT)n at the 3′-end of the sense/antisense, respectively. The triethanolamine-core PAMAM dendrimer of generation 5 (G5) was synthesized as previously described.8−10,17,18 Ethidium bromide, bovine serum albumin (BSA), and heparin were supplied by Sigma-Aldrich (Saint-Quentin Fallacier, France). All other reagents and solvents of analytical grade were used without further purification from commercial sources. Computational Details. All parallel molecular dynamics (MD) simulations and the relevant analyses were performed using the AMBER 11 suites of programs,19 running in parallel on 256 processors of the IBM PLX-GPU supercomputer at the HPC CINECA supercomputer facility (Bologna, Italy) and of the Matrix calculation cluster at the HPC CASPUR supercomputer facility (Rome, Italy). Molecular graphics images were produced using the UCSF Chimera package.20,21 The AMBER f f 03 force field (FF)22 and the new version of the Dreiding FF recently developed by the Goddard group and specifically optimized for dendrimers in water solutions23 were employed for the nucleic acid and the dendrimers, respectively. The free energy of binding between the dendrimers and the siRNA was calculated according to a previously validated approach16,18,24−31 based on the molecular mechanics Poisson−Boltzmann methodology32 (see Supporting Information for full details). The identification of each dendrimer branch involved in siRNA binding (Neff), and the corresponding individual contribution afforded to the overall binding energy, was achieved by

Scheme 1. Self-Assembly of Sticky siRNA into “Gene-Like” Longer Double-Stranded RNA

stronger cooperativity and multivalency in interactions with the PEI vector and, ultimately, improve delivery efficiency. Previous studies using our TEA-core PAMAM dendrimers as siRNA vectors (Scheme 2) also showed that the fifth generation dendrimer, which produced no potent gene silencing effect with normal siRNA, was highly effective at delivering sticky siRNA leading to the down-regulation of the targeted gene at both the mRNA and protein levels.16 Self-assembly of sticky siRNA into 3263

dx.doi.org/10.1021/mp400329g | Mol. Pharmaceutics 2013, 10, 3262−3273


Article

Scheme 2. Chemical Structure of Triethanolamine (TEA) Core PAMAM Dendrimera

a

For clarity, only G3 is presented.

applying a per-residue free energy decomposition technique to each siRNA/G5 complex.28,29,31,33 This analysis was carried out using the MM/GBSA approach34,35 and was based on the same MD snapshots used in the binding free energy calculation. The entire MM/PBSA and MM/GBSA computational procedure was optimized by integrating AMBER 11 in modeFRONTIER,36 a multidisciplinary and multiobjective optimization and design environment. The steered molecular dynamics simulations of the dendrimer/ siRNA unbinding events were performed by adapting the individual pulling SMD scheme originally proposed by Cuendet and Michielin37 (see Supporting Information for full details). The harmonic spring constant kCM was set equal to 2 × 104 kJ/(mol nm2), a value stiff enough to have a good spatial resolution in the free energy

profile while not damping the thermal behavior of the system. A pulling speed ν of 5 × 10−4 nm/ps was chosen as a trade-off between staying as close to equilibrium as possible and keeping the computing time within manageable limits. With these settings, 2 nm were covered in 4 ns of SMD simulations. Size and Zeta Potential Measurement of Various siRNA/G5 Complexes. The various siRNA solutions were mixed with the indicated amount of dendrimer solution at an N/P ratio 10. The final concentration of siRNA was 1 μM. After incubation at 37 °C for 30 min, size and zeta potential measurements were performed using Zetasizer Nano-ZS (Malvern, Ltd., Malvern, U.K.) with a He−Ne ion laser of 633 nm. Cell Culture. Human prostate cancer PC-3 cells and C4-2 cells were purchased from the American Type Culture Collection 3264



Article

were normalized to wells containing only siRNA and ethidium bromide. Experiments were performed in triplicate. RNA Dissociation Assay. In a 96 well plate, 1.00 μg of ethidium bromide and 0.95 μg of siRNA were mixed in phosphate buffer (12 mM phosphate, 10 mM NaCl, pH 7.4) to achieve a total volume of 30 μL at pH 7.4. The plate was incubated at 25 °C for 15 min; then 6.8 μg of dendrimer (N/P = 10 to siRNA) in buffer solution and additional buffer solution was added to achieve a total volume of 60 μL. This plate was further incubated at 25 °C for 30 min. A portion of 40 μL of different concentrations of heparin diluted in buffer solution were added to siRNA complexes to achieve the total volume of 100 μL with the final concentration of ethidium bromide at 25.4 μM, siRNA at 15 μM per base pair, and the dendrimer at 3.1 μM. The mixture was incubated for a further 30 min. This plate was excited at 560 nm. The fluorescence emission was recorded at 590 nm (Fluostar OPTMA, BMG LABTECH, Germany). The fluorescence values were normalized to wells containing only siRNA/ethidium bromide complexes. Experiments were performed in triplicate. Statistical Analysis. Statistical analysis was performed using 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 (***).

(Manassas, VA). PC-3 cells were maintained in DMEM (Invitrogen Ltd., Paisley, United Kingdom), supplemented with 10% fetal bovine serum (FBS). C4-2 cells were maintained in RPMI 1640 (Invitrogen Ltd.), supplemented with 10% fetal bovine serum (FBS). Human breast cancer MDA-MB-231 and MCF-7 cells were kindly offered from Mr. Julien Wicinski (Institute Paoli Calmette-CRCM, Marseille, France). MDA-MB-231 cells were maintained in RPMI 1640 (Invitrogen Ltd.), supplemented with 10% fetal calf serum (FCS), non-essential amino acid solution (NEAA), HEPES, and antibiotic-antimycotic (ANTI−ANTI). MCF-7 cells were maintained in RPMI 1640 (Invitrogen Ltd.), supplemented with 10% FCS, NEAA, HEPES, ANTI−ANTI, and insulin humalog. Cells were maintained at 37 °C in a 5% CO2 humidified atmosphere. In Vitro Transfection Experiments. One day before transfection, 1.5 × 105 prostate cancer PC-3 and C4-2 cells or breast cancer MDA-MB-231 and MCF-7 cells were seeded in 6 cm dishes in 4 mL of fresh complete medium containing 10% FBS. Before transfection, complexes of various siRNA/ dendrimer reagents were prepared. The desired amount of siRNA, scramble siRNA, and dendrimer reagents were diluted in 200 μL of Opti-MEM transfection medium. The solutions were mixed with a vortex for 10 s and then left for 10 min at room temperature. The dendrimer reagent was added to the siRNA or scramble siRNA solution, homogenized for 10 s with a vortex, and left for 30 min at room temperature. Then, 1.6 mL of serumfree medium was added to the complex solution, making up a final volume of 2 mL. Before adding the transfection complexes, the complete medium with serum was removed, and the cell was washed once with PBS. Then, 2 mL of complex solution at an N/P ratio 10 was added and incubated at 37 °C in the absence of 10% FBS or FCS. After 8 h of incubation, the transfection mixture was replaced with the complete culture medium and maintained under normal growth conditions for a further incubation period of 72 h. Western Blot Analysis. Samples containing equal amounts of protein (15 μg) from lysates of cultured prostate cancer PC-3 or C4-2 cells or breast cancer MDA-MB-231 or MCF-7 cells were analyzed by Western blot analysis as described previously10,38 with 1:5000-diluted antihuman Hsp27 rabbit polyclonal antibody (Stressgen Assay Designs Inc., Michigan, USA) or 1:2500-diluted anti-TCTP antibody (Abcam, Paris, France) or 1:2000-diluted antihuman Vinculin mouse monoclonal antibody (Sigma Chemical Co., St. Louis, MO). Filters were then incubated for 1 h at room temperature with 1:5000diluted horseradish peroxidase conjugate antirabbit or mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific proteins were detected using an enhanced chemiluminescence Western blotting analysis system (Amersham Life Science, Arlington Heights, IL). Ethidium Bromide Exclusion Assay. In a 96 well plate, 1.00 μg of ethidium bromide and 0.95 μg of siRNA were mixed in phosphate buffer (12 mM phosphate, 10 mM NaCl, pH 7.4) to achieve a total volume of 50 μL at pH 7.4. The plate was incubated at 25 °C for 15 min, then the appropriate amount of dendrimer solution together with buffer solution were added to achieve a total volume of 100 μL with the final concentration of ethidium bromide at 25.4 μM, siRNA at 15.0 μM per base pair, and the desired dendrimer concentrations. This plate was further incubated at 25 °C for 30 min before exciting at 560 nm. The fluorescence emission was recorded at 590 nm (Fluostar OPTMA, BMG LABTECH, Germany). The fluorescence values

■

RESULTS AND DISCUSSION Design of siRNA Molecules with Different Overhangs. To test whether the interaction of the siRNA overhangs with dendrimeric vectors effectively contributes toward improving siRNA delivery (in addition to self-assembly of sticky siRNA), we designed siRNA molecules carrying noncomplementary sequence overhangs, complementary sequence overhangs, and conventional (dT)2 overhangs (Scheme 3). All siRNA molecules share common sequences that target the heat shock protein 27 (Hsp27) and the translationally controlled tumor protein (TCTP), respectively. Hsp27 is a molecular chaperone, which plays an important role in resistance to anticancer drugs and has been recently considered as a novel target for treating drugresistant prostate tumors and other cancer forms.38,39 TCTP, a highly conserved protein present in all eukaryotic organisms, has been lately reported to regulate cell survival in many human cancers; accordingly, it constitutes an interesting target in cancer therapy, especially in breast and prostate malignancies.40,41 While the classical siRNA bear (dT)2 overhangs, our sticky siRNA feature complementary sequence overhangs of two different lengths: (dA)5/(dT)5 and (dA)7/(dT)7. The corresponding siRNA with noncomplementary sequence overhangs were decorated with (dT)5/(dT)5, (dA)5/(dA)5, (dT)7/(dT)7, and (dA)7/(dA)7, respectively (Scheme 3). Accordingly, in what follows all of these siRNA molecules are denoted as siRNA(T2/T2), siRNA(A5/T5), siRNA(A7/T7), siRNA(T5/T5), siRNA(A5/A5), siRNA(T7/T7), and siRNA(A7/A7) for the sake of simplicity and convenience. It is to note here that we chose not to work with siRNA molecules bearing even longer overhangs for fear of such long oligonucleotides inducing undesired immune responses.42 Gene Silencing Using siRNA with Different Overhangs. The ability of our designed siRNA molecules to down-regulate Hsp27 and TCTP was assessed in human prostate cancer PC-3 and C4-2 cells, and in human breast cancer MDA-MB-231 and MCF-7 cells, respectively.43,44 The fifth generation TEA-core PAMAM dendrimer (G5) was chosen as nanovector for these siRNA molecules given its proven capacity to effectively deliver 3265



Article

Nanoscale and Stable siRNA/Dendrimer Complexes. Spurred on by these results, we next endeavored to investigate the reasons why siRNA molecules with longer overhangs of both complementary and noncomplementary sequences were more efficient in inducing gene silencing than the standard siRNA with (dT)2/(dT)2 overhangs. As the formation of stable complexes between siRNA and delivery nanovectors is one of the most important prerequisites for effective cell uptake and delivery of siRNA, we first examined the size and size distribution of various siRNA/G5 dendrimer complexes by dynamic light scattering (DLS). Also, the surface zeta potential of complexes was assessed by laser Doppler microelectrophoresis, a technique that derives the value of a surface zeta potential based on electro-osmosis measurements close to the sample surface. For the classical siRNA with (dT)2/(dT)2 overhangs, its complexes with G5 generated nanoparticles of various sizes ranging from 7 to 400 nm with large polydispersity, implying that G5 could not form stable neither uniform nanoparticles with these siRNA (Figure S2A). With siRNA harboring both complementary and noncomplementary long overhangs, however, G5 readily formed stable and uniform nanoparticles with an average size of 100 nm in diameter at N/P = 10 and a polydispersity index around 0.15 (Table 1, Figure S2). Moreover, zeta-potential measurements gave positive values for the dendrimer complexes with siRNA bearing longer overhangs (n = 5 or 7) (Table 1), implying that these siRNA/G5 nanoparticles are stable colloids. A size around 100 nm and a positive surface potential are two requisites for successful delivery of nanoparticles into cells. Hence, these data for siRNA/dendrimer complexes further corroborate our experimental observation that G5 permits effective gene silencing with siRNA bearing longer overhangs, whether complementary or not, whereas no significant gene silencing is achieved with conventional siRNA/G5 complexes. Insight into siRNA Binding to G5 by Molecular Simulations. To understand if and how different siRNA overhangs could impact dendrimer-mediated delivery, we next performed molecular dynamics (MD) simulations to assess the binding of the tested siRNA molecules to the G5 dendrimer. We commenced our in silico study by considering a situation in which monomeric siRNA form a complex with their dendrimeric nanocarrier. The relevant results are shown in Figure 2, while the corresponding numerical values are listed in Table S1. According to our calculations, binding of siRNA toward the G5 dendrimer is affected both by the nature and length of the siRNA overhangs in the following interesting manner: (i) for the two homologous siRNA series, the siRNA with (dA)n/(dA)n overhangs are characterized by the most favorable ΔGbind values, followed by the complementary overhangs (dA)n/(dT)n and, last, by the siRNA bearing (dT)n/(dT)n overhangs which show the lowest affinity for the G5 dendrimer; (ii) longer overhangs (n = 7) are more beneficial to dendrimer binding, and (iii) the lowest affinity value for the G5 dendrimer pertains to the siRNA carrying the shortest overhangs (dT)2/(dT)2. For a more rigorous and thorough understanding of the interaction between the different siRNA and G5, we then assessed the effective free energy of binding (ΔGbindeff), that is, the contribution to binding yielded by the dendrimer branches in constant and productive interaction with the nucleic acid (NA) fragment. To estimate ΔGbindeff for each siRNA/G5 complex, all G5 branches involved in nucleic acid binding (Neff) were precisely identified, and their individual contribution toward the overall binding energy estimated by a per-residue free energy decomposition technique (see SI for more details).

Scheme 3. siRNA Molecules Bearing Overhangs of Different Lengths and Sequences Studied in This Work

sticky siRNA in lieu of normal siRNA.16 As expected, no gene silencing was observed with normal siRNA delivered by G5, while gene silencing up to 90% was attained with siRNA molecules bearing complementary sequence overhangs of both lengths [(dA)5/(dT)5 and (dA)7/(dT)7, Figure 1]. This result is in agreement with our previous observations16 and can be mainly ascribed to the sticky overhangs (dA)n/(dT)n (n = 5 or 7): these complementary single-strand sequences induce siRNA self-assembly into “gene-like” longer double-stranded RNA (Scheme 1), providing significantly enhanced cooperativity and multivalency when binding toward the nanovectors. This, in turn, endows a low generation dendrimer such as G5 with greater delivery capacity. What was surprising was the considerably potent gene silencing (up to 70%) observed with siRNA bearing noncomplementary overhangs (dT)5/(dT)5, (dA)5/(dA)5, (dT)7/(dT)7, and (dA)7/(dA)7. Namely, siRNA with noncomplementary overhangs could also substantially enhance gene silencing compared to normal siRNA, although they remained less effective than their complementary sticky counterparts (Figure 1), whereas no activity was observed with any scramble siRNA molecules (Figure S1). The observed gene silencing potency depended on the length and sequence of the overhangs as well as the concentration of siRNA. Compared with conventional siRNA bearing only the two nucleotide-long overhangs (dT)2/(dT)2, siRNA molecules with longer overhangs (n > 2) were much more effective in inducing gene silencing. In addition, siRNA with (dT)n/(dT)n overhangs were slightly more efficient than those bearing (dA)n/(dA)n overhangs of equal length (n = 5 or 7). 3266



Article

Figure 1. Dendrimer G5-mediated siRNA delivery and gene silencing of heat shock protein 27 (Hsp27) and translationally controlled tumor protein (TCTP) in human prostate cancer PC-3 cells (A and E) and C4-2 cells (B and F) as well as in human breast cancer MDA-MB-231 cells (C) and MCF-7 cells (D) with siRNA carrying various overhangs at a N/P ratio of 10, respectively. The siRNA dose-dependent silencing of TCTP in PC-3 cells (G) and C4-2 cells (H) and of Hsp27 in MCF-7 cells (I). The vinculin protein was used as a reference protein. All of the transfections were done in triplicate. *, **, and *** differ from control (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively) by Student’s t test.

important finding from this study concerns the number of dendrimer branches efficiently involved in siRNA binding, Neff. As expected, Neff is the smallest (38) for the siRNA with the shortest overhangs [(dT)2/(dT)2], while a net trend is observed

The cumulative results of this analysis are reported in Figure 2 and Table S1, showing the values of Neff, the effective total free energy of binding ΔGbindeff, and its enthalpic ΔHbindeff and entropic −TΔSbindeff components for all complexes. The first 3267



Article

endowed with higher flexibility with respect to their (dA)5 counterparts, and the flexibility of the Tn-overhangs is quantified by the highest loss of entropy upon their binding with siRNA and well-qualified by the higher fluctuations (higher degree of freedom) of these overhangs in the solvent, hence leading to a less optimized siRNA/dendrimer opposite-charge contacts (Figure 3B). Lastly, the siRNA with the complementary overhangs (dA)5/(dT)5 displays a somewhat intermediate behavior (Figure 3C), both in terms of Neff and ΔGbindeff values, suggesting a possible compensatory effect between a more rigid and hence more efficient (dA)5 overhang on one side and a more flexible and less efficient (dT)5 overhang on the other. Analogous situation is found for the 27-mer siRNA/G5 complexes for which, in line with the 25-mer siRNA series, the affinity of the siRNA for the G5 dendritic vector (ΔGbindeff) increases according to the nature of the overhangs in the following order: (dT)7/(dT)7 < (dA)7/(dT)7 < (dA)7/(dA)7. Once again, the relevant simulations highlights the higher flexibility of the (dT)7 overhang and its role in dendrimer binding. Thus, in the case of the siRNA(T7/T7)/G5 complex, this property ultimately results in the smallest value of Neff (45), the least favorable enthalpic contribution (ΔHbindeff = −626.7 kcal/mol), the most unfavorable entropic term (−TΔSbindeff = +258.9 kcal/mol) and, correspondingly, the lowest value of ΔGbindeff (= −367.8 kcal/mol) (Figure 3E). On the contrary, the siRNA featuring the (dA)7/(dA)7 overhangs show the highest affinity for G5, the binding being driven by the most favorable enthalpic term (ΔHbindeff = −669.1 kcal/mol) and the lowest unfavorable entropic loss (−TΔSbindeff = +243.1 kcal/mol), in agreement with the highest value of Neff (53) and the higher rigidity (and hence, binding efficiency) of the (dA)7 overhangs (Figure 3D), respectively. Also in this case, the siRNA(A7/T7) shows an intermediate behavior between the two other members of the series (Figure 3F), confirming an overall balance between two opposing effects exerted by the two different (dA)n and (dT)n overhangs. While these results should be considered with due care given all approximations assumed in the formulations of the molecular systems involved, they do allow for the following general considerations: (i) the presence of longer overhangs on a siRNA duplex appears more advantageous for dendrimer binding when compared to shorter ones, due to the higher number of effective dendrimer charges in active and permanent contact with the nucleic acid; (ii) for siRNA with noncomplementary overhangs of a given length, the presence of less flexible [i.e., (dA)n] overhangs affords a more favorable enthalpic contribution, a less unfavorable entropic penalty, and ultimately, a higher affinity of the siRNA for binding to its dendrimeric nanocarrier; (iii) in the case of very long overhangs (e.g., n = 7), the presence of more flexible nucleotides (e.g., (dT)7/(dT)7) might be of little benefit in dendrimer complexation with respect to shorter overhangs populated by more rigid counterparts (e.g., (dA)5/(dA)5) by virtue of an enthalpy/entropy compensation effect. Concatemerization of Sticky siRNA Favors Binding with Dendrimer Vector G5. As mentioned in the Introduction, sticky siRNA can self-assemble into “gene-like” longer double-stranded RNA via hybrid bridges formed by the complementary sequence overhangs (Scheme 1). This “oligomerization” or “concatemerization” may, in turn, permit stronger cooperativity and multivalency which would assist their interaction with the vectors and, eventually, lead to better delivery efficiency. So far, the stoichiometry and structure of

Table 1. DLS Measurement of Size and Zeta Potential of the Dendrimer G5 Complexes with Various siRNA Molecules Bearing Different Overhangs siRNA(A5/T5)/G5 siRNA(T5/T5)/G5 siRNA(A5/A5)/G5 siRNA(A7/T7)/G5 siRNA(T7/T7)/G5 siRNA(A7/A7)/G5

size (nm)

polydispersity (PDI)

zeta potential (mV)

108 124 118 117 114 124

0.18 0.12 0.14 0.19 0.18 0.12

+35 +42 +43 +44 +42 +33

Figure 2. Total effective free energy (ΔGbindeff = ΔHbindeff − TΔSbindeff), enthalpic (ΔHbindeff), and entropic (−TΔSbindeff) components of binding of the siRNA molecules with different overhangs toward the G5 dendrimer as well as effective positive charges (Neff) with G5 involved in siRNA binding.

with Neff increasing from siRNA(Tn/Tn) to siRNA(An/Tn) to siRNA(An/An) for the two homologous siRNA series (Figure 2 and Table S1). Contextually, the ΔGbindeff increases (i.e., became more negative and hence more favorable) in the same order. Further examination of the main components of ΔGbindeff for all monomeric siRNA systems revealed that, although the binding process is essentially enthalpy-driven, entropy also plays a substantial role in modulating the ultimate, individual affinity of each siRNA toward the dendrimer nanovector G5. For example, the system with siRNA bearing (dA)5/(dA)5 overhangs has the most favorable effective enthalpic component (ΔHbindeff = −592.0 kcal/mol) among the three homologous assemblies with (dA)5/ (dA)5, (dA)5/(dT)5, and (dT)5/(dT)5 overhangs (Figure 2 and Table S1). This can be ascribed to the high number of favorable electrostatic interactions, characterized by the value of Neff (46), alongside other nonbonded interactions between the charged G5 residues and this specific siRNA molecule, including its overhangs. At the same time, the siRNA(A5/A5) complex with G5 presents the lowest unfavorable entropic contribution (−TΔSbindeff = +227.2 kcal/mol), by virtue of the rigid nature and aggregation propensity of the (dA)5 overhangs; this allows for more permanent contacts between the entire siRNA (including its overhangs) and the G5 peripheral positively charged terminal groups (Figure 3A). The cumulative effect of these two contributing factors results in the most favorable value of the effective free energy of binding ΔGbindeff = −364.8 kcal/mol. By contrast, the siRNA(T5/T5) in complex with G5 is characterized by the smallest Neff value (41) in the series. Accordingly, this system presents the least favorable enthalpic contribution (ΔHbindeff = −503.1 kcal/mol) and the highest entropic loss (−TΔSbindeff = +241.1 kcal/mol) which, when combined, give the lowest ΔGbindeff value (−262.0 kcal/mol). The analysis of the entire MD trajectory reveals that the (dT)5 overhangs are 3268



Article

Figure 3. Selected equilibrated MD snapshots of the complexes between the G5 dendrimer and the siRNA molecules with 5-nucleotide long (upper panel) and 7-nucleotide long (lower panel) overhangs. These overhangs are respectively, (A) (dA)5/(dA)5; (B) (dT)5/(dT)5; (C) (dA)5/(dT)5; (D) (dA)7/(dA)7; (E) (dT)7/(dT)7, and (F) (dA)7/(dT)7. In the panels, the dendrimer is depicted as forest green sticks, the terminal charged amine groups as light green sticks-and-balls, the siRNA as an orange ribbon, and the two overhangs (dA)n and (dT)n are highlighted in red and navy blue, respectively. Some Cl− and Na+ and counterions are shown as big light gray and small dark gray spheres, respectively. Water molecules have been omitted for clarity.

these “gene-like” siRNA are unknown. Nevertheless, Behr et al.14 clearly demonstrated that: (1) sticky siRNA capable of forming noncovalent concatemers via (dA)8/(dT)8 bridges increased siRNA/vector complex stability and hence protected them from RNase degradation; and (2) since concatemers were not detected in the absence of vectors (e.g., PEI) mainly because of the low stability of the overhang duplex, the presence of polycationic vectors may favor encounters of siRNA/nanovector intercomplexes by shielding their repulsive forces, thus favoring sticky siRNA concatemerization. To investigate these concepts, we carried out MD simulations and the appropriate ΔGbind calculations for dendrimer G5 in complex with two dimeric sticky siRNA, hereafter referred to as [siRNA(A5/T5)]2 and [siRNA(A7/T7)]2, respectively. The results obtained are shown in Figure 4 and listed in the first three columns of Table S2. When the values of the dimeric complexes are compared with those of the corresponding monomeric assemblies multiplied by two (see Figure 2 and Table S1), we see that the ΔGbind for the dimeric cases are substantially more favorable than those obtained for two monomeric systems (i.e., −975.4 kcal/mol for [siRNA(A5/T5)]2 vs 2 × (−387.4) = −774.8 kcal/mol for two siRNA(A5/T5) and −1024.5 kcal/mol for [siRNA(A7/T7)]2 vs 2 × (−422.9) = −845.8 kcal/mol for two siRNA(A7/T7), respectively). Although the models considered here likely represent a much simplified vision of the real systems, the results obtained not only support the hypothesis proposed by Behr et al.namely, preformed sticky siRNA/vector complexes favor the subsequent concatemerization of the siRNA complementary sticky endsbut also indicate a synergistic and

Figure 4. Total effective free energy (ΔGbindeff = ΔHbindeff − TΔSbindeff), enthalpic (ΔHbindeff), and entropic (−TΔSbindeff) components of binding of dimeric sticky siRNA toward the G5 dendrimer as well as effective positive charges (Neff) with G5 involved in dimeric sticky siRNA binding.

cooperative binding mechanism between sticky siRNA and dendrimer. Further supportive evidence comes from the analysis of the effective free energy of binding (ΔGbindeff) of the dimeric complexes of the sticky siRNA, [siRNA(A5/T5)]2, and [siRNA(A7/T7)]2 (Figure 4 and Table S2). Indeed, in the systems containing dimeric complexes of siRNA, the number Neff of effective G5 positive charges constantly in contact with the longer siRNA strand is more than two times that estimated for each corresponding monomeric complex {96 for the [siRNA(A5/T5)]2/G5 vs 44 3269



Article

Figure 5. Selected equilibrated MD snapshots of the complexes between dendrimer G5 and the sticky siRNA dimers [siRNA(A5/T5)]2 (A) and [siRNA(A7/T7)]2 (B). The dendrimer is depicted as forest green sticks, the terminal charged amine groups as light green sticks-and-balls, the siRNA as an orange ribbon, and the two overhangs (dA)n and (dT)n are highlighted in red and navy blue, respectively. Some Cl− and Na+ and counterions are shown as big light gray and small dark gray spheres, respectively. Water has been omitted for clarity. Binding of the dendrimer G5 toward siRNA bearing 5-nucleotide long (C) and 7-nucleotide long (D) overhangs was assessed by an ethidium bromide displacement assay.

enhances dendrimer binding, ultimately underlying the cooperative and multivalent nature of the dendrimeric nanovector/ nucleic acid interactions. These in silico predictions are further confirmed by experimental results aimed at assessing the binding between siRNA and the G5 dendrimer using ethidium bromide (EB) displacement fluorescence spectroscopy (Figure 5C and D). According to these tests, the siRNA carrying the complementary overhangs (dA)n/(dT)n exhibited the strongest binding toward the G5 dendrimer, the affinity of the siRNA for G5 decreasing in the order (dA)n/(dT)n > (dA)n/(dA)n > (dT)n/(dT)n (n = 5 or 7). Dissociation Process of siRNA/G5 Complexes. Importantly, once siRNA/dendrimer complexes are internalized into cells, the siRNA molecules need to be released from their corresponding supramolecular ensembles to reach the RNAi machinery to start the gene silencing process. Therefore, siRNA release from its nanovector is also a crucial step for effective siRNA delivery and gene silencing. With the purpose of investigating in more detail the process of siRNA release from its dendrimer complex, we decided to explore the siRNA/ dendrimer unbinding process using steered molecular dynamics (SMD, see SI for more details). Accordingly, during our SMD simulation, the siRNA duplex was forcedly pulled away from its vector using a pulling force given by F(t) = k[vt − (r − r0)n] where k is the force constant, v is the pulling velocity, n is the pulling direction normal, and ro and r are the position of the siRNA at the beginning and at time t of the pulling experiment, respectively. We note here that SMD simulations were not attempted on the siRNA bearing complementary overhangs

for the siRNA(A5/T5)/G5 complex, and 105 for the [siRNA(A7/T7)]2/G5 vs 47 for the siRNA(A7/T7)/G5 complex, respectively; Figure 4 and Table S2}. An inspection of the MD trajectories for these two systems reveals that the presence of the “hybridized” (dA)5/(dT)5 and (dA)7/(dT)7 double-stranded portions of these siRNA, which are more rigid and globally more charged compared to each of their corresponding single-stranded overhangs, allows for a small but significant conformational rearrangement of the dendrimer outer branches which, in turn, leads to the augmented number of favorable, stabilizing electrostatic interactions (Figure 5A and B). In addition to the total ΔGbind, evidence of the beneficial action of the “concatemerization” of two siRNA with complementary overhangs can also be found in the corresponding values of the effective quantities ΔGbindeff, ΔHbindeff, and −TΔSbindeff (Figure 4 and Table S2). In particular, it is interesting to note the concomitant synergistic enhancement in the enthalpic driving force toward binding {−1260.3 kcal/mol for [siRNA(A5/T5)]2 vs 2 × (−554.9) = −1109.8 kcal/mol for two siRNA(A5/T5) and −1441.2 kcal/mol for [siRNA(A7/T7)]2 vs 2 × (−637.3) = −1274.6 kcal/mol for two siRNA(A7/T7)} and the decrease in the entropic contribution disfavoring siRNA/G5 complexation {+372.7 kcal/mol for [siRNA(A5/T5)]2 vs 2 × (+233.4) = +446.8 kcal/mol for two siRNA(A5/T5) and +437.2 kcal/mol for [siRNA(A7/T7)]2 vs 2 × (+248.2) = +496.4 kcal/mol for two siRNA(A7/T7)}. These results demonstrate that the presence of a dimeric sticky siRNA constituted by two complementary overhangs and a central “(dA)n/(dT)n hybridized double-stranded” tract synergistically 3270



Article

(dA)n/(dT)n (n = 5 or 7) as even the simplest siRNA “dimeric concatemer” currently poses considerable technical computational problems. Figure 6A presents the time profiles of the pulling forces during the SMD simulations of the dissociation of the standard

a very strong correlation is found between these two quantities (R2 = 0.95): the larger the rupture force, the more favorable (i.e., more negative) the effective binding free energy, and hence the more reluctant the release of siRNA from their corresponding dendrimer ensembles. More interestingly, however, is the finding that, for the two siRNA with the most flexible overhangs, (dT)5/(dT)5 and (dT)7/(dT)7, the force reaches a maximum almost at the same, shortest time (∼150 ps), while the force peaks for siRNA(A7/A7) and siRNA(A5/A5) follow at ∼2.1 ns and ∼2.4 ns, respectively (Figure 6A). For the siRNA with the shortest overhangs (dT)2/ (dT)2, the rupture force maximum occurs after a considerably longer time, ∼3.2 ns (Figure 6A). Notwithstanding the fact that longer overhangs enhance the binding of the siRNA carrying noncomplementary overhangs with the dendrimer vector, they also possess increased flexibility which, in turn, facilitates their detachment from the dendrimer. Thus, the siRNA with the most flexible overhangs, (dT)7/(dT)7 and (dT)5/(dT)5, are able to unbind at earlier time points, while those featuring more rigid and/or shorter overhangs such as (dA)5/(dA)5 require longer times to dissociate from their complex. We further experimentally assessed siRNA release from their corresponding dendrimer complexes using the heparin-coupled ethidium bromide (EB) assay. In this method heparin (a highly negatively charged polysaccharide) is employed in competing with siRNA for dendrimer binding. Upon addition of heparin, siRNA is gradually displaced from the dendrimer complexes by heparin, and the released siRNA is ready to intercalate the fluorescent EB probe, which hence emits a strong fluorescence. From the intensity of the emitted EB fluorescence, the corresponding siRNA release from its nanocarrier can be estimated. As revealed in Figure 6C, in agreement with our computational predictions, the siRNA with short and flexible overhangs (dT)5/ (dT)5 are most easily displaced by heparin from their dendrimer complexes, followed in order by (dA)5/(dA)5, (dT)7/(dT)7, and (dA)7/(dA)7. Altogether, the above results show that, among the siRNA molecules with noncomplementary overhangs, those characterized by (dT)n/(dT)n overhangs appear to offer the best compromise in terms of dendrimer affinity and release. These conclusions greatly correlate with the experimental evidence, according to which siRNA(T7/T7) molecules are endowed with the best G5-mediated siRNA delivery and gene silencing among all investigated siRNA molecules carrying noncomplementary overhangs.

Figure 6. (A) Average force profile of siRNA unbinding from their G5 dendrimer complexes. Color legend: blue, siRNA(T5/T5); green, siRNA(T7/T7); yellow, siRNA(A7/A7); red, siRNA(A5/A5); black, siRNA(T2/T2). (B) Correlation between the steered molecular dynamics (SMD) peak force and the effective binding free energy ΔGbindeff for the corresponding siRNA and the G5 dendrimer. The linear fit has a correlation level of R2 = 0.95. (C) Dissociation of siRNA from the dendrimer complexes as revealed by heparin-coupled ethidium bromide assay.

■

CONCLUSIONS In our quest for safe and efficient vectors for the delivery of siRNA therapeutics, we discovered that the structurally flexible, fifth generation TEA-core PAMAM dendrimer G5 is an effective nanovector for delivering sticky siRNA.16 With the help of combined experimental and computational approaches, we have successfully identified the underlying mechanisms of interaction between the overhangs of the sticky siRNA with this dendrimeric nanovector and the contribution of siRNA overhangs to the observed improved delivery potency. siRNA molecules with complementary overhangs offer the best action in term of gene silencing. On the other hand, while siRNA bearing noncomplementary long overhangs show considerably higher gene silencing potency than normal siRNA, they are nevertheless still less effective than sticky siRNA bearing complementary overhangs. The observed gene silencing potency, however, depends on the length and nature of the overhangs. Overall, our results allowed us to formulate a sensible molecular rationale explaining the importance of the different

siRNA (siRNA(T2/T2)) and four siRNA bearing noncomplementary overhangssiRNA(T5/T5), siRNA(A5/A5), siRNA(T7/T7), and siRNA(A7/A7)from their respective G5 complexes. Clearly, an increased level of force is required to initiate movement of the siRNA double-strand away from the dendrimer, which implies that the nucleic acid encounters energy barriers to dissociation. The maximum pulling forces F are 730 pN for the siRNA(T2/T2), 753 pN for the siRNA(T5/T5), 794 pN for the siRNA(A5/A5), 824 pN for the siRNA(T7/T7), and 862 pN for the siRNA(A7/A7). After passing this peak, the force drops rapidly. In Figure 6B, these rupture forces are plotted as a function of the effective binding energies ΔGbindeff obtained from the classical MD simulations (Figure 2). As can be seen, 3271



Article

bromide assay and Ms. Virginie Baylot for her kind offer of normal TCTP siRNA.

siRNA overhangs in dendrimer-mediated siRNA delivery and gene silencing. Long and complementary overhangs on siRNA duplexes offer the best action in terms of dendrimer binding via concatemerization of the complementary overhang nucleotides. The formation of such concatemers via (dA)n/(dT)n bridges is fostered by preformed siRNA/dendrimer complexes, and the effective free energy of binding of the “gene-like” RNA/ dendrimer complex is the result of a synergistic and cooperative mechanism. Once decomplexed in the cytoplasm, the sticky siRNA concatemers fall apart to deliver siRNA molecules for effective gene silencing.14 For siRNA bearing noncomplementary overhangs, the sum of effects of overhang length, nature, and flexibility plays a major role in determining their ultimate performance in siRNA delivery and the resulting gene silencing. Thus, although higher overhang rigidity increases the siRNA binding capacity toward the G5 dendrimer, a higher overhang lengthand hence higher flexibilityis more beneficial for the subsequent nucleic acid release process. Results from computer modeling show that, among the siRNA with noncomplementary overhangs considered in the present work, those characterized by (dT)n/(dT)n overhangs appear to offer the best compromise in terms of dendrimer binding and unbinding characteristics. These conclusions greatly correlate with experimental findings according to which siRNA with (dT)n/(dT)n overhangs are endowed with the best G5-mediated siRNA delivery and gene silencing, as discussed above. Collectively the results presented here demonstrate that the adoption of this kind of multidisciplinary approach can yield fundamental insight into the importance of siRNA overhangs for dendrimer-mediated siRNA delivery. This will offer a new perspective on siRNA therapeutics and be of instrumental use in the future translational implementation of siRNA in the treatment of various diseases.

■

■

(1) Davidson, B. L.; McCray, P. B., Jr. Current prospects for RNA interference-based therapies. Nat. Rev. Genet. 2011, 12, 329−340. (2) Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discovery 2009, 8, 129−138. (3) Khurana, B.; Goyal, A. K.; Budhiraja, A.; Arora, D.; Vyas, S. P. siRNA delivery using nanocarriersan efficient tool for gene silencing. Curr. Gene Ther. 2010, 10, 139−155. (4) Schroeder, A.; Levins, C. G.; Cortez, C.; Langer, R.; Anderson, D. G. Lipid-based nanotherapeutics for siRNA delivery. J. Intern. Med. 2010, 267, 9−21. (5) Wagner, E. Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic, and precise. Acc. Chem. Res. 2012, 45, 1005−1013. (6) Liu, X.; Rocchi, P.; Peng, L. Dendrimers as non-viral vectors for siRNA delivery. New J. Chem. 2012, 36, 256−263. (7) Ravina, M.; Paolicelli, P.; Seijo, B.; Sanchez, A. Knocking down gene expression with dendritic vectors. Mini-Rev. Med. Chem. 2010, 10, 73−86. (8) Wu, J.; Zhou, J.; Qu, F.; Bao, P.; Zhang, Y.; Peng, L. Polycationic dendrimers interact with RNA molecules: polyamine dendrimers inhibit the catalytic activity of Candida ribozymes. Chem. Commun. 2005, 313− 315. (9) Zhou, J.; Wu, J.; Hafdi, N.; Behr, J. P.; Erbacher, P.; Peng, L. PAMAM dendrimers for efficient siRNA delivery and potent gene silencing. Chem. Commun. 2006, 2362−2364. (10) Liu, X. X.; Rocchi, P.; Qu, F. Q.; Zheng, S. Q.; Liang, Z. C.; Gleave, M.; Iovanna, J.; Peng, L. PAMAM dendrimers mediate siRNA delivery to target Hsp27 and produce potent antiproliferative effects on prostate cancer cells. ChemMedChem 2009, 4, 1302−1310. (11) 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.; Rossi, J. J. Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice. Mol. Ther. 2011, 19, 2228−2238. (12) Lang, M. F.; Yang, S.; Zhao, C.; Sun, G.; Murai, K.; Wu, X.; Wang, J.; Gao, H.; Brown, C. E.; Liu, X.; Zhou, J.; Peng, L.; Rossi, J. J.; Shi, Y. Genome-wide profiling identified a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. PLoS One 2012, 7, e36248. (13) Elbashir, S. M.; Martinez, J.; Patkaniowska, A.; Lendeckel, W.; Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 2001, 20, 6877− 6888. (14) Bolcato-Bellemin, A. L.; Bonnet, M. E.; Creusat, G.; Erbacher, P.; Behr, J. P. Sticky overhangs enhance siRNA-mediated gene silencing. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16050−16055. (15) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297−7301. (16) Liu, X.; Liu, C.; Laurini, E.; Posocco, P.; Pricl, S.; Qu, F.; Rocchi, P.; Peng, L. Efficient delivery of sticky siRNA and potent gene silencing in a prostate cancer model using a generation 5 triethanolamine-core PAMAM dendrimer. Mol. Pharmaceutics 2012, 9, 470−481. (17) Shen, X. C.; Zhou, J.; Liu, X.; Wu, J.; Qu, F.; Zhang, Z. L.; Pang, D. W.; Quéléver, G.; Zhang, C. C.; Peng, L. Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer complexes. Org. Biomol. Chem. 2007, 5, 3674−3681. (18) Liu, X.; Wu, J.; Yammine, M.; Zhou, J.; Posocco, P.; Viel, S.; Liu, C.; Ziarelli, F.; Fermeglia, M.; Pricl, S.; Victorero, G.; Nguyen, C.; Erbacher, P.; Behr, J. P.; Peng, L. Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus. Bioconjugate Chem. 2011, 22, 2461−2473.

ASSOCIATED CONTENT

* Supporting Information S

Additional characterization data, gene silencing data, and computational details. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Author Contributions

P.P. and X.L. contributed equally to this work Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the international ERA-Net EURONANOMED European Research project DENANORNA, Ministry of Science and Technology of China (No.2012AA022501), CNRS, INSERM, PACA Canceropôle, INCa, Association pour la Recherche sur les Tumeurs de la Prostate (XL), Association française contre les Myopathies (XL), China Scholarship Council (CC, CL), and under the auspice of European COST Action TD0802 “Dendrimers in Biomedical Applications”. The financial support from ESTECO s.r.l. (Grant DDOS) is gratefully acknowledged. Access to CINECA and CASPUR supercomputing facility was granted through the sponsored HPC Italian Supercomputing Resource Allocation (ISCRA) project MONALISA and the sponsored CASPUR HPC project NANO4HEALTH. We are grateful to Dr. Claudia Andrieu for her help with the ethidium 3272



Article

(19) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B.; Wang, B. H. S.; Roitberg, A.; Seabra, G.; Kolossvai, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Liu, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M.-J.; Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M. G.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 11; University of California: San Francisco, 2010. (20) Chimera (v.1.4), Resource for Biocomputing, Visualization, and Informatics; University of California: San Francisco, CA, 2009. (21) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimeraa visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612. (22) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 2003, 24, 1999−2012. (23) Liu, Y.; Bryantsev, V. S.; Diallo, M. S.; Goddard, W. A., III. PAMAM dendrimers undergo pH responsive conformational changes without swelling. J. Am. Chem. Soc. 2009, 131, 2798−2799. (24) Posocco, P.; Laurini, E.; Dal Col, V.; Marson, D.; Karatasos, K.; Fermeglia, M.; Pricl, S. Tell me something I do not know. Multiscale molecular modeling of dendrimer/dendron organization and selfassembly in gene therapy. Curr. Med. Chem. 2012, 19, 5062−5087. (25) Karatasos, K.; Posocco, P.; Laurini, E.; Pricl, S. Poly(amidoamine)-based dendrimer/siRNA complexation studied by computer simulations: effects of pH and generation on dendrimer structure and siRNA binding. Macromol. Biosci. 2012, 12, 225−240. (26) Barnard, A.; Posocco, P.; Pricl, S.; Calderon, M.; Haag, R.; Hwang, M. E.; Shum, V. W.; Pack, D. W.; Smith, D. K. Degradable selfassembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake. J. Am. Chem. Soc. 2011, 133, 20288−20300. (27) Jones, S. P.; Gabrielson, N. P.; Wong, C. H.; Chow, H. F.; Pack, D. W.; Posocco, P.; Fermeglia, M.; Pricl, S.; Smith, D. K. Hydrophobically modified dendrons: developing structure-activity relationships for DNA binding and gene transfection. Mol. Pharmaceutics 2011, 8, 416−429. (28) Pavan, G. M.; Posocco, P.; Tagliabue, A.; Maly, M.; Malek, A.; Danani, A.; Ragg, E.; Catapano, C. V.; Pricl, S. PAMAM dendrimers for siRNA delivery: computational and experimental insights. Chem.Eur. J. 2010, 16, 7781−7795. (29) Jones, S. P.; Pavan, G. M.; Danani, A.; Pricl, S.; Smith, D. K. Quantifying the Effect of Surface Ligands on Dendron-DNA Interactions: Insights into Multivalency through a Combined Experimental and Theoretical Approach. Chem.Eur. J. 2010, 16, 4519−4532. (30) Posocco, P.; Pricl, S.; Jones, S.; Barnard, A.; Smith, D. K. Less is moremultiscale modelling of self-assembling multivalency and its impact on DNA binding and gene delivery. Chem. Sci. 2010, 1, 393−404. (31) Pavan, G. M.; Danani, A.; Pricl, S.; Smith, D. K. Modeling the multivalent recognition between dendritic molecules and DNA: understanding how ligand “sacrifice” and screening can enhance binding. J. Am. Chem. Soc. 2009, 131, 9686−9694. (32) Srinivasan, J.; Cheatham, T. E., III; Cieplak, P.; Kollman, P. A.; Case, D. A. Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate-DNA Helices. J. Am. Chem. Soc. 1998, 120, 9401−9409. (33) Gohlke, H.; Kiel, C.; Case, D. A. Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes. J. Mol. Biol. 2003, 330, 891−913. (34) Onufriev, A.; Bashford, D.; Case, D. A. Modification of the Generalized Born Model Suitable for Macromolecules. J. Phys. Chem. B 2000, 104, 3712−3720. (35) Tsui, V.; Case, D. A. Theory and applications of the generalized Born solvation model in macromolecular simulations. Biopolymers 2000, 56, 275−291.

(36) http://www.esteco.com/home/mode_frontier/mode_frontier. html (accessed June 3, 2013). (37) Cuendet, M. A.; Michielin, O. Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex. Biophys. J. 2008, 95, 3575−3590. (38) Rocchi, P.; Beraldi, E.; Ettinger, S.; Fazli, L.; Vessella, R. L.; Nelson, C.; Gleave, M. Increased Hsp27 after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis. Cancer Res. 2005, 65, 11083−11093. (39) Zoubeidi, A.; Gleave, M. Small heat shock proteins in cancer therapy and prognosis. Int. J. Biochem. Cell Biol. 2012, 44, 1646−1656. (40) Telerman, A.; Amson, R. The molecular programme of tumour reversion: the steps beyond malignant transformation. Nat. Rev. Cancer 2009, 9, 206−216. (41) Baylot, V.; Katsogiannou, M.; Andrieu, C.; Taieb, D.; Acunzo, J.; Giusiano, S.; Fazli, L.; Gleave, M.; Garrido, C.; Rocchi, P. Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer. Mol. Ther. 2012, 20, 2244−2256. (42) Bagasra, O.; Prilliman, K. R. RNA interference: the molecular immune system. J. Mol. Histol. 2004, 35, 545−553. (43) Rocchi, P.; Jugpal, P.; So, A.; Sinneman, S.; Ettinger, S.; Fazli, L.; Nelson, C.; Gleave, M. Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro. BJU Int. 2006, 98, 1082− 1089. (44) Calderwood, S. K.; Gong, J. Molecular chaperones in mammary cancer growth and breast tumor therapy. J. Cell. Biochem. 2012, 113, 1096−1103.

3273


Impact of siRNA Overhangs for Dendrimer-Mediated siRNA Delivery

Recommend Documents