Coordinative Amphiphiles as Tunable siRNA Transporters

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Coordinative Amphiphiles as Tunable siRNA Transporters Jin Bum Kim, Yeong Mi Lee, Jooyeon Ryu, Eunji Lee, Won Jong Kim, Gyochang Keum, and Eun-Kyoung Bang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00260 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 4, 2016

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Bioconjugate Chemistry

Coordinative Amphiphiles as Tunable siRNA Transporters Jin Bum Kim1,‡, Yeong Mi Lee2,‡, Jooyeon Ryu3, Eunji Lee3, Won Jong Kim2, Gyochang Keum1, Eun-Kyoung Bang1,* 1Center

for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea 2Center for Self-Assembly and Complexity, Institute for Basic Science (IBS), and Department of Chemistry, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang 37673, Republic of Korea 3Graduate

School of Analytical Science and Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea ‡

Kim JB and Lee YM contributed equally.

*Corresponding

author: Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea, Email: [email protected]. Tel: +82-2-958-5168.

1 Environment ACS Paragon Plus


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Abstract In this study, we developed coordinative-amphiphiles for use as novel siRNA transporters. As a modification of a conventional cationic lipid structure, we replaced the cationic head with zinc(II)-dipicolylamine complex (Zn/DPA) as a phosphate-directing group, and used various membrane-directing groups in the place of the hydrophobic tails. These simple amphiphiles are readily synthesized and easy to modify. The Zn/DPA head groups bind to the phosphate backbones of siRNAs, and to our surprise, they prevented the enzymatic degradation of siRNAs by RNase A. Interestingly, the Zn/DPA head itself exhibited moderate transfection efficiency, and its combination with a membrane-directing group—oleoyl (CA1), pyrenebutyryl (CA2), or biotin (CA3)—enhanced the delivery efficiency without imparting significant cytotoxicity. Notably, the uptake pathway was tunable depending on the nature of the membrane-directing group. CA1 delivered siRNAs mainly through caveolae-mediated endocytosis; CA2 through clathrin- and caveolin-independent endocytosis; CA3 recruited siRNAs specifically into biotin receptor–positive HepG2 cells through receptormediated endocytosis. Thus, it appears possible to develop tunable siRNA transporters simply by changing the membrane-directing parts. These are the first examples of amphiphilic siRNA transporters accompanying coordinative interactions between the amphiphiles and siRNAs. Key words. RNA interference, Delivery system, Zinc(II)-dipicolylamine complex, Amphiphiles


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Introduction RNA interference (RNAi), first reported in 1998, is currently the most efficient method for gene downregulation.1 The poor membrane permeability of small interfering RNAs (siRNAs) remains a hurdle, however, affecting their clinical applications as RNAi agents. Of the many reported delivery systems for siRNAs, 2, 3 cationic amphiphiles were the earliest to be described and remain the most popular for increasing the cellular uptake of oligonucleotides.4-8 Conventional cationic amphiphiles typically comprise a cationic head, a linker, and a hydrophobic tail. Electrostatic interactions between the heads of cationic lipids and the anionic phosphate backbones of siRNAs lead to their strong binding. In addition, the cationic character enhances cellular uptake of the siRNAs because the resulting cationic surfaces of the complexes have greater membrane affinity. Nevertheless, highly positive surface charges can induce nonspecific binding to components in blood, resulting in less efficient transport or higher toxicity. As alternatives to electrostatic attraction, other chemical interactions—e.g., sequence-specific hybridization,911

covalent bonding,12-13 and intercalation14—have also been tested as means of binding therapeutic

oligonucleotides to delivery carriers. None of these approaches has, however, been applied to deliver short siRNAs. In this study, we examined the coordinative interaction as an alternative of electrostatic interaction. When linked with various chromophores, zinc(II)-dipicolylamine complexes (Zn/DPA) has been studied widely for its ability to recognize phosphate ions.15-18 Chromophore-conjugated Zn/DPA molecules are used in the imaging of dead cells19, 20 or bacterial cells,21 and in the measuring of protein kinase activity.22, 23 The Zn/DPA group can also be used to recruit phosphopeptides,24 siRNAs25-27 into cells. For siRNA delivery, only a few examples of polymer-based delivery systems featuring Zn/DPA moieties have been described, all by the Chen group.25-27

Figure 1. Structures of the CAs and the commonly used cationic lipid DOTAP. 3 Environment ACS Paragon Plus


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In this paper, we report the amphiphilic siRNA transporters presenting Zn/DPA as the head group. As a mimic of conventional cationic amphiphiles, we designed several simple amphipathic molecules bearing Zn/DPA moieties as potential siRNA transporters (Figure 1). We placed the cationic head group with Zn/DPA as a phosphate-directing group and used various membrane-directing groups for the hydrophobic tails. Accordingly, we call them “coordinative amphiphiles (CAs)”. Unlike the electrostatic interactions of cationic amphiphiles, the siRNA binding of our CAs occurs through coordinative bonds. As the linker, we adopted a unit described for a membrane-anchored Zn/DPA chemosensor.28-29 Employing the same Zn/DPA component as the phosphate-directing unit, we varied the membrane-directing part using oleoyl (CA1), pyrenebutyryl (CA2), and biotin (CA3) groups. The oleoyl chain, a component of phospholipids in the cellular membrane, in CA1 is among the most widely used, and efficient, hydrophobic tails.30-32 We introduced the pyrene group in CA2 with inspiration from the “pyrenebutyrate trick” for polycationic materials.33 Matile and Futaki reported that pyrenebutyrate has strong membrane affinity and increases the cellular uptake of polycations (e.g., oligoarginine).34-39 Accordingly, pyrenebutyrate has been used to deliver peptides,24, 40 drugs,41 proteins,42 and quantum dots.43-44 We expected that the pyrenebutyryl group might also have the ability to deliver siRNAs. The biotin unit provides CA3 with potential targeting ability. Biotin was recently reported as a ligand for tumor-specific vitamin receptors.45-57 We also prepared ZnBnDPA and ZnDPA, with no affinity to membranes, as simplified versions of the test compounds. Results and discussion

Scheme 1. Synthesis of PAs. a) i. Hydrazine, DCM/EtOH (1:9), reflux, 1 h, 93%. ii. Carboxylic acid, HATU, TEA, DMF, rt, 1 h; 42% for 2a; 63% for 2b; 25% for 2c. b) Zn(NO3)2, MeOH, rt, 30 min; 94% for CA1; 96% for CA2; 79% for CA3. c) NaOH, H2O/MeCN, 70 °C, 2 h. d) Zn(NO3)2, MeOH, rt, 1 h, 62%. e) Zn(NO3)2, MeOH, rt, 1 h, 70%.


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The structures of CAs are extremely simple and their syntheses are straightforward. All the CAs were readily obtained following known procedures for dipicolylamine synthesis, amide coupling, and zinc complexation (Scheme 1). Using a specific fluorescent ligand, 6,7-dihydroxy-4-sulfomethyl coumarin (7), we monitored the interactions between the Zn/DPA group and siRNAs (Figure 2A).28, 58-59 Each CA effectively quenched the fluorescence of 7 (Figure 2B) and the quenched fluorescence was recovered (Figure 2C) after adding siRNAs to the mixtures of the CAs and 7, indicating that the Zn/DPA heads bound to the siRNAs releasing the free ligand 7. Table 1 lists the dissociation constants determined from Figure 2C. All of the compounds but ZnDPA rapidly bound to the siRNAs, and CA1 forms the most stable complex with siRNAs. Interestingly, the length of the siRNA affected the binding affinity of the phosphate and Zn/DPA units. The complexes did not bind to an 8-mer double stranded RNA (dsRNA) as effectively as they did to a 21-mer siRNA, and uridine 5’-monophosphate (UMP) did not bind to Zn/DPA at the given concentration (Figures 2D, 2E, and S2).

Figure 2. Interaction between Zn/DPA and phosphates. (A) Simplified depiction of the interactions of Zn/DPA with 7 and a phosphate. (B) Fluorescence decay (I/I0) of 7 (10 µM) in the presence of CAs. (C) Fluorescence recovery (I/I0) after adding siRNAs to mixtures of the CAs and 7. (D, E) Binding curves for the interactions of 21-mer siRNA, 8-mer dsRNA, 21-mer ssRNA, and UMP with (D) CA1 and (E) CA2. All experiments were performed in 5 mM HEPES-NaOH buffer (pH 7.4); λex = 347 nm; λem = 480 nm. I0 represents the fluorescence intensity of 7 alone (10 μM). Table 1. Dissociation constants for the interactions of siRNAs and CAs Compound CA1 CA2 CA3 ZnBnDPA ZnDPA

Dissociation constant, Kd (M) 6.1 (± 1.1)  10–7 2.3 (± 0.5)  10–6 3.6 (± 0.5)  10–6 4.6 (± 0.5)  10–6 3.1 (± 0.8)  10–5



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The CAs appeared to bind cooperatively to the phosphate units, thereby interacting more effectively with longer siRNAs. The cooperative bindings might originate from the fact that coordination of Zn/DPAs to a phosphate backbone lessens negative charges of siRNAs. In the arginine-rich oligo/polymers, the charge repulsion between adjacent guanidinium ions is advantageous for counterion scavenging.35, 60-62 Likewise, we think that the repulsive force between two strands in the siRNA duplexes promote their attraction to Zn/DPAs, resulting in thermodynamically more stable complexes of siRNAs and Zn/DPAs. Except for CA2, CAs displayed lower affinity to a 21-mer single stranded RNA (ssRNA) than to a 21-mer siRNA, which might be due to the absence of the inter-strand charge repulsion in a single strand (Figure 2D and S2). On the contrary, CA2 displayed greatly higher affinity to ssRNAs than siRNAs (Figure 2E). We think that π-π stacking interaction of pyrenes in CA2 might be dominant over the effect of charge repulsion, and the flexible ssRNAs could aid suitable stacking of pyrene moieties than the rigid siRNA duplexes did. Figures 3A, 3C, and 3E show the band retardation of the siRNAs complexed with the CAs at various molar ratios in the agarose gel. Upon initially increasing the molar ratio of CAs to siRNAs, the mobility of the siRNAs did not change, as if complexation had failed. At higher molar ratios, however, the mobility began to decrease, indicating the formation of siRNA–CA adducts. To investigate further, we treated each complex with RNase A (Figure 3B, 3D, and 3F). Interestingly, although we observed no differences in the mobilities of the complexes, the presence of the CAs increased the stability of the siRNAs significantly. The CAs coordinated to the phosphate backbones of the siRNAs and thereby protecting siRNAs from RNase-mediated degradation. But coordination was not the only factor affecting the enzymatic stability of the siRNAs, and additional contribution from the membrane-directing parts might also be involved. The excess concentrations of the amphiphiles presumably led to self-assembled aggregates, stabilized through secondary interactions such as hydrophobic interactions (CA1 and CA2) and hydrogen bonding (CA3). These three CAs provided a band of aggregated particle with retarded mobility in the agarose gel at a molar ratio of 1000. In contrast, the compounds without membrane-directing moieties, ZnDPA and ZnBnDPA, had no effect on the mobility (Figures S4A and S4C) and no protection of siRNAs from RNase A under the same conditions (Figures S4B and S4D). In addition, CA1, which had the lowest dissociation constant (Table 1), showed the best protecting ability toward siRNAs (Figure 3B) and formed the heaviest complexes with siRNAs, which did not move at all in the gel. Since non-labelled siRNAs in the heavy complexes were not effectively stained by intercalating dyes, we additionally confirmed the complexes by using FITC-labelled siRNAs (Figure S3). We also tested the complexation between siRNAs and 2b, a compound without zinc(II) ion. In the absence of RNA staining dyes, only the compound 2b was observed in the gel due to its own fluorescence (Figure S4G). And siRNAs moved separately from the compounds 2b, shown in the gel with staining dyes (Figure S4E). They also have no protection ability on the siRNAs against to RNase A (Figure S4F). As expected, there is no interaction between siRNAs and dipicolylamine moiety via any electrostatic attraction, hydrogen bonding, or π-π stacking. Therefore, Zn/DPA is the only group which could interact to siRNAs through a coordinative bond.


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Figure 3. Characterization of CA/siRNA complexes. (A, C, E) Gel retardation assays and (B, D, F) enzymatic stability tests for complexes of the siRNAs with (A, B) CA1, (C, D) CA2, and (E, F) CA3. Each CA/siRNA complex was loaded in gel (A, C, E) directly or (B, D, F) after incubation in the presence (+)/absence (–) of RNase A for 30 min at 37 °C. (G) Zeta potentials and (H) sizes of the structures formed from CA1, CA2, and CA3. Corresponding charge ratios are presented in Table S1.

When the CA solutions were dispersed in siRNA solution, CA1, CA2, and CA3 formed aggregated nanosized particles (Figure 3G), from which we measured zeta potentials (Figure 3F). By increasing molar ratio, they showed smaller particle and larger zeta potentials. ZnBnDPA and ZnDPA did not give quantifiable size data, but they display negative zeta potentials (Table 2). The cryo-TEM images in Figure 4 support the presence of intermolecular hydrophobic interactions for CA1. The CA1/siRNA complexes formed multilamellar vesicle structures. In contrast, the ZnDPA/siRNA complexes formed approximately 20-nm micellar particles (Figure S5). The thickness of vesicle for CA1/siRNA complexes was ca. 5 nm, which is corresponding to the length of partially interdigitated bilayer of CA1 (Figure 4C).

Figure 4. Cryo-TEM image. (A) CA1/siRNAs complex; total concentration of CA1: 0.5 mM, molar ratio: 1000. (B) Highly magnified image of multi-lamellar vesicles observed in (A). (C) Density profile of vesicle wall thickness indicating that the vesicle wall is composed of the interdigitated bilayer of CA1/siRNAs. Table 2. Zeta potentials (mV) of structures formed from ZnBnDPA and ZnDPA. Molar ratio (Compound/siRNAs) Compound only 100

ZnBnDPA

ZnDPA

–12.5 ± 0.3 –15.0 ± 0.2

–12.5 ± 3.4 –14.5 ± 0.4

400 1000

–13.4 ± 0.5 –12.6 ± 0.6

–13.3 ± 0.9 –12.3 ± 1.8



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All compounds were less cytotoxic (Figure S7) than LipofectamineTM (Invitrogen), a commercial transfection agent. We initially screened CA1, CA2, ZnBnDPA, and ZnDPA for their transfection efficiencies (Figure 5). We mixed each compound with anti-luciferase siRNA (siLuc) and then elucidated the transfection efficiencies by measuring the residual luciferase-generated luminescence. The complexes were prepared at various molar ratios (50, 100, 200, 400, 600, 800, and 1000, at which the siRNAs were relatively stable against RNase A in Figure 3B). In Figure 5, CA1 and CA2 displayed notable transfection efficiencies. For both CA1 and CA2, the least toxic and most efficient delivery system was that with a molar ratio of 400. CA2 exhibited particularly remarkable delivery efficiency; the luminescence signal at a molar ratio of 400 was much lower than that of LipofectamineTM. Unexpectedly, ZnBnDPA and ZnDPA also displayed moderate delivery efficiencies. This might result from the coordinative interaction to Zn/DPA groups neutralizing the negative charges of the siRNAs. The additional benzyl group in ZnBnDPA provided slightly higher hydrophobicity, which increased membrane affinity, leading to uptake efficiency superior to that of ZnDPA (Figure 5, at molar ratios of 400 and 600). The uptake was enhanced after adding the specific membrane-directing groups. Our observations suggest that less-toxic Zn/DPA groups might possibly be adequate replacements for cationic head groups in lipid-based siRNA delivery. Oleoyl and pyrenebutyryl moieties are indeed suitable units for effectively recruiting siRNAs into cellular membranes.

Figure 5. Transfection efficiencies of CAs. Relative luciferase expression levels after treatment of each complex with (A) HepG2, (B) HCT116, and (C) HeLa. The expression levels of luciferase were obtained with units of RLU/mg of proteins, then normalized to the values of non-treated cells. Table S2 presents the corresponding charge ratios.

We tested the transfection efficiency of the biotinylated CA, CA3, at a fixed molar ratio of 400. We selected HCT116 and HepG2 as biotin receptor–poor and –rich cell lines, respectively (Figure 6A).46, 48 The luciferase expression level in HepG2 cells was only 11%, whereas it was 41% in HCT116 cells, indicating that the transfection efficiency of CA3 was enhanced markedly in the presence of the biotin receptor. When these cells were incubated with biotin as a competitor, prior to the transfection of siLuc, the transfection efficiency in the HepG2 cells decreased significantly, while there was no biotin-dependency in the HCT116 cells. The confocal microscopic images for delivery of CA/FITC-labelled siRNAs complexes were illustrated in Figure S8. The green fluorescence of FITC-labelled siRNAs was superimposed to the red fluorescence of lysosome, demonstrating that all CAs effectively delivered siRNAs into cells through endocytosis.


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Figure 6. Transfection efficiencies of (A) CA3 with HCT116 and HepG2 cells in the presence / absence of biotin, and (B) the CAs with HepG2 cells at 4 °C and 37 °C, and also in the presence of genistein (300 µM), MβCD (5 mg/mL), wortmanin (200 nM), and chlorpromazine (2.5 µg/mL) at 37 °C. The expression levels of luciferase were obtained with units of RLU/mg of proteins, then normalized to the values of non-treated cells. Table S2 presents the corresponding charge ratios.

Several divalent metal complexes have been reported for their ability to hydrolysis of phosphate esters.29, 63-65 To investigate activities of compounds for hydrolysis of siRNAs, we simply incubated them with siRNAs at the physiological condition (37 ºC, pH 7.4) (Figure S6). As other divalent metal complexes, Zn/DPAs possibly hydrolysed phosphodiester linkages of siRNAs. The hydrolysis was faster with ZnBnDPA and ZnDPA than with the other CAs. We think that the self-assembled aggregates of CAs might also hinder the accessibility of water as well as RNase. However, regardless of their reactivity on hydrolysis, the reaction was slow enough to test their transfection abilities. Their cellular uptake was completed in 2 h (Figure S8) and the siRNAs were stable upto 8 h for all compounds. Thus, we could exclude the possibility of siRNA cleavages during the course of cellular uptake. We also studied the delivery mechanism in our systems by treating cells with known inhibitors for the possible endocytic pathway (Figure 6B). The possible modes of endocytosis include phagocytosis, micropinocytosis, clathrin-mediated endocytosis in non-lipid raft regions, and caveolae-mediated and clathrin-/caveolin-independent endocytosis in lipid raft domains.66 We could exclude phagocytosis because all of our CAs efficiently delivered siRNAs into non-phagotic HeLa cells (Figure 5C). Chloropromazine is an inhibitor for clathrin-mediated endocytosis. Both genistein and methyl-β-cyclodextrin (MβCD) inhibit endocytosis in lipid raft membrane domains. Genistein is a specified inhibitor for caveolae-mediated endocytosis, and wortmanin is known to block micropinocytosis. We treated cells with each inhibitor of endocytic pathway prior to the transfection of siLuc. The transfection efficiency of CA1 decreased under incubation at 4 °C and in the presence of genistein and MβCD. Thus, we suggest that the delivery of CA1/siRNA complexes was mainly related to clathrin-independent and caveolae-mediated endocytosis in lipid raft domains. CA2 displayed a different pattern of behaviour. It transported siRNAs only moderately at the low temperature, and the siRNA delivery was effectively suppressed by genistein only, not by MβCD. We conclude that CA2 delivered the siRNAs via clathrin- and caveolin-independent endocytosis. The transfection efficiency of CA3 was not affected by any of the endocytosis inhibitors, except chlorpromazine. It has been suggested that receptor-mediated endocytosis involves clathrin-coated vesicles.67 Consistently, we confirm that CA3 delivered the siRNAs effectively into cells through receptor-mediated endocytosis. As expected,



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biotin functioned as a good affinity group for receptor-mediated endocytosis, thereby simplifying the targeting formulation into CA3. The targeting group is not an additional decoration in the delivery formula, but the sole membrane-directing group in the CA system. Conclusions We have developed several coordinative amphiphiles (CAs) as effective siRNA transporters. These structures mimic conventional cationic lipids, but have a unique feature: a coordinative phosphate-directing group (Zn/DPA group) and a membrane-directing unit (oleoyl, pyrenebutyryl, biotin). These CAs are easy to synthesize and readily bound to the phosphates in the backbones of the siRNAs. The CAs aggregated in aqueous solutions and their complexes protected the siRNAs from RNase A–mediated hydrolysis. By introducing specific coordinative interaction to siRNAs, all the CAs were less cytotoxic than Lipofectamine TM. Interestingly, the Zn/DPA head itself exhibited moderate transfection efficiency, and the membrane-directing groups enhanced the uptake efficiency dramatically without adding any significant cytotoxicity. Moreover, the uptake pathways were tunable depending on the membrane-directing group. Among the molecules synthesized, CA2 and CA3 were the best in delivering siRNAs into cells. In particular, CA3, the simple biotin-tethered molecule, allowed for specific delivery into biotin receptor–rich cancer cells. Our findings open up new possibilities for developing siRNA transporters for specific purposes simply by tuning the membrane-directing parts of CAs. Acknowledgments We thank the Advanced Analysis Center in Korea Institute of Science and Technology (KIST) and Korea Basic Science Institute (KBSI) Daegu Center for HRMS analysis. This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science, and Technology (2014R1A6A3A04059719, 2013R1A2A2A04015914), by KIST through institutional project (2E26650), and by the Institute for Basic Science (IBS) [IBS-R007-D1]. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. Materials and experimental details on the syntheses of CAs, characterization of compounds, NMR spectra, binding assay of CAs, gel electrophoresis, RNA cleavage tests, cytotoxicity data, confocal microscopic images, and other Figures and Tables mentioned in the text.


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References and Notes 1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. 2. Nayerossadat, N., Maedeh, T., Ali, P. A. (2012) Viral and nonviral delivery systems for gene delivery. Adv. Biomed. Res. 1, 27–37. 3. Kanasty, R., Dorkin, J. R., Vegas, A., Anderson, D. (2013) Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977. 4. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. S., Wenz, M., Northrop, J. P., Ringold, G. M., Danielson, M. (1987) Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413–7417. 5. Zimmermann, T. S., Lee, A. C., Akinc, A., Bramlage, B., Bumcrot, D., Fedoruk, M. N., Harborth, J., Heyes, J. A., Jeffs, L. B., John, M. et al. (2006) RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114. 6. Srinivas, R., Samanta, S., Chaudhuri, A. (2009) Cationic amphiphiles: Promising carriers of genetic materials in gene therapy. Chem. Soc. Rev. 38, 3326–3338. 7. ur Rehman, Z., Zuhorn, I. S., Hoekstra, D. (2013) How cationic lipids transfer nucleic acids into cells and across cellular membranes: Recent advances. J. Control. Release 166, 46–56. 8. Zhi, D., Zhang, S., Cui, S., Zhao, Y., Wang, Y., Zhao, D. (2013) The head group evolution of cationic lipids for gene delivery. Bioconjugate Chem. 24, 487–519. 9. Branden, L. J., Mohamed, A. J., Smith, C. I. E. (1999) A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol. 17, 784–787. 10. Roulon, T., Hélène, C., Escudé, C. (2002) Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide. Bioconjugate Chem. 13, 1134–1139. 11. Srinivasan, C., Lee, J., Papadimitrakopoulos, F., Silbart, L. K., Zhao, M., Burgess, D. J. (2006) Labeling and intracellular tracking of functionally active plasmid DNA with semiconductor quantum dots. Mol. Ther. 14, 192–201. 12. Neves, C., Byk, G., Scherman, D., Wils, P. (1999) Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation. FEBS Lett. 453, 41–45. 13. Sebestyen, M. G., Ludtke, J. J., Bassik, M. C., Zhang, G., Budker, V., Lukhtanov, E. A., Hagstrom, J. E., Wolff, J. A. (1998) DNA vector chemistry: The covalent attachment of signal peptides to plasmid DNA. Nat. Biotechnol. 16, 80–85.



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65. Fife, T. H., Pujari, M. P. (1990) Divalent metal ion catalyzed reactions of acyl phosphates. J. Am. Chem. Soc. 112, 5551–5557. 66. El-Sayed, A., Harashima, H. (2013) Endocytosis of gene delivery vectors: From clathrin-dependent to lipid raft-mediated endocytosis. Mol. Ther. 21, 1118–1130. 67. Doherty, G. J., McMahon, H. T. (2009) Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902.


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Table of Contents Graphics



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Synthesis of PAs. a) i. Hydrazine, DCM/EtOH (1:9), reflux, 1 h, 93%. ii. Carboxylic acid, HATU, TEA, DMF, rt, 1 h; 42% for 2a; 63% for 2b; 25% for 2c. b) Zn(NO3)2, MeOH, rt, 30 min; 94% for CA1; 96% for CA2; 79% for CA3. c) NaOH, H2O/MeCN, 70 °C, 2 h. d) Zn(NO3)2, MeOH, rt, 1 h, 62%. e) Zn(NO3)2, MeOH, rt, 1 h, 70%. 84x84mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Structures of the CAs and the commonly used cationic lipid DOTAP. 105x130mm (300 x 300 DPI)



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Interaction between Zn/DPA and phosphates. (A) Simplified depiction of the interactions of Zn/DPA with 7 and a phosphate. (B) Fluorescence decay (I/I0) of 7 (10 µM) in the presence of CAs. (C) Fluorescence recovery (I/I0) after adding siRNAs to mixtures of the CAs and 7. (D, E) Binding curves for the interactions of 21-mer siRNA, 8-mer dsRNA, 21-mer ssRNA, and UMP with (D) CA1 and (E) CA2. All experiments were performed in 5 mM HEPES-NaOH buffer (pH 7.4); λex = 347 nm; λem = 480 nm. I0 represents the fluorescence intensity of 7 alone (10 µM). 101x122mm (300 x 300 DPI)


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Characterization of CA/siRNA complexes. (A, C, E) Gel retardation assays and (B, D, F) enzymatic stability tests for complexes of the siRNAs with (A, B) CA1, (C, D) CA2, and (E, F) CA3. Each CA/siRNA complex was loaded in gel (A, C, E) directly or (B, D, F) after incubation in the presence (+)/absence (–) of RNase A for 30 min at 37 °C. (G) Zeta potentials and (H) sizes of the structures formed from CA1, CA2, and CA3. Corresponding charge ratios are presented in Table S1. 52x32mm (300 x 300 DPI)



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Cryo-TEM image. (A) CA1/siRNAs complex; total concentration of CA1: 0.5 mM, molar ratio: 1000. (B) Highly magnified image of multi-lamellar vesicles observed in (A). (C) Density profile of vesicle wall thickness indicating that the vesicle wall is composed of the interdigitated bilayer of CA1/siRNAs. 24x7mm (300 x 300 DPI)


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Figure 5. Transfection efficiencies of CAs. Relative luciferase expression levels after treatment of each complex with (A) HepG2, (B) HCT116, and (C) HeLa. The expression levels of luciferase were obtained with units of RLU/mg of proteins, then normalized to the values of non-treated cells. Table S2 presents the corresponding charge ratios. 39x8mm (300 x 300 DPI)



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Figure 6. Transfection efficiencies of (A) CA3 with HCT116 and HepG2 cells in the presence / absence of biotin, and (B) the CAs with HepG2 cells at 4 °C and 37 °C, and also in the presence of genistein (300 µM), MβCD (5 mg/mL), wortmanin (200 nM), and chlorpromazine (2.5 µg/mL) at 37 °C. The expression levels of luciferase were obtained with units of RLU/mg of proteins, then normalized to the values of non-treated cells. Table S2 presents the corresponding charge ratios. 34x14mm (300 x 300 DPI)


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Table of Contents Graphics 39x19mm (300 x 300 DPI)


Coordinative Amphiphiles as Tunable siRNA Transporters

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