Cationic Dendron-Bearing Lipids: Investigating Structure–Activity

Oct 21, 2013 - All CDLs could effectively bind small interfering RNA (siRNA) to form ... For a more comprehensive list of citations to this article, u...
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Cationic Dendron-Bearing Lipids: Investigating Structure−Activity Relationships for Small Interfering RNA Delivery Yu Zhang,†,‡ Jie Chen,† Chunsheng Xiao,† Mingqiang Li,†,‡ Huayu Tian,† and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, P. R. China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: A new family of cationic dendron-bearing lipids (CDLs) with poly(amidoamine) dendrons of first to third generation (named as A1, A2, and A3, respectively) was synthesized through a synthesis approach that permits facile variation of chemical structures. All CDLs could effectively bind small interfering RNA (siRNA) to form complexes confirmed by gel retardation analysis. In in vitro transfection experiments, A1/siRNA complexes exhibited significant gene silencing efficiency close to Lipofectamine 2000/siRNA complexes and much higher than A2/siRNA and A3/ siRNA complexes. To reveal the underlying reason, we performed a series of experimental methods. The results suggested that the CDLs with smaller dendron sizes and higher proportion of hydrophobic segments could bind siRNA to form dendriplex aggregates with more compact structures and higher surface potentials. Therefore, they could be internalized via endocytosis more easily, which was believed to be the main reason for higher gene silencing efficiency. This paper provides an efficient CDL (A1) for siRNA delivery and indicates great potential for gene therapy.

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INTRODUCTION The potent ability of siRNAs to silence the targeted genes is expected to lead to powerful new approaches for curing cancer, a potential currently explored in worldwide clinical trials. However, one of the key challenges in the development of siRNAs-based therapy is the lack of safe and efficient delivery systems enabling efficient cellular uptake and knockdown of target transcripts.1 Furthermore, the understanding of delivery systems’ mechanism of action is still incomplete. Existing gene carriers are typically categorized into two classes, viral vectors and nonviral vectors. Viral vectors are highly efficient, but their potential inflammatory, immunogenic, and mutagenic effects make them a safety risk. To avoid these adverse patient responses, increasing attention has been focused on developing nonviral vectors.2,3 Cationic polymers are one of the most commonly used nonviral vectors.4−8 Although various types of cationic polymers have exhibited efficient gene delivery capacity, they are always plagued with the flaws of undefined structures and batch to batch difference. Cationic dendrimers, a special family of cationic polymers, are promising nonviral vectors for gene delivery because of a well-defined molecular architecture, precisely controlled chemical structure, high aqueous solubility, large number of chemically versatile surface groups, and unique architecture.9,10 Poly(amidoamine) (PAMAM) dendrimers are one of the most extensively studied cationic dendrimers.11,12 The proton sponge effect generated by their tertiary amines can © XXXX American Chemical Society

help genes escape from the endosome. Many modifications have also been applied on the surface groups of PAMAM dendrimers to improve the transfection activity.9,13,14 To reduce the synthetic effort in synthesizing high-generation dendrimers and explore the advantages of the supramolecular structures formed by dendrimers, various assemblies from dendrimers have been prepared and applied in therapeutic agents delivery.15−17 The self-assembling PAMAM dendrons are further designed for gene delivery and show promise in future gene therapy.18,19 Cationic lipids are also very important nonviral vectors that are proposed as efficient carriers for the intracellular gene delivery.20−23 The positively charged headgroups can bind and complex with nucleic acids, while the hydrophobic tails can assist the assembly of the lipids into a polycationic scaffold and facilitate fusion with cell membrane, which is another strategy to help nucleic acids escape from the endosome. But up to now, few solid conclusions have been obtained on the relationships between the cationic lipids’ structures/compositions and the complexes’ transfection activities, and the empirical findings are still the main source for structure−activity relationships.24 With the advantages of both cationic dendrimers and cationic lipids, a new family of nonviral vectors has been developed. Received: August 2, 2013 Revised: October 4, 2013

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Figure 1. Chemical structures of CDLs A1, A2, and A3.

HeLa-Luc cells and Huh-7-Luc cells with or without the presence of serum, close to Lipofectamine 2000/siRNA complexes and much higher than A2/siRNA complexes and A3/siRNA complexes. To find out the underlying reason of the different transfection efficiency, we systematically investigated the capabilities of the CDLs in cellular uptake, endosome escape, and release of siRNA.

Diederich et al. prepared self-assembling vectors with CDLs of different compositions and found that the introduction of the hydrophobic part brought an unexpected strong influence on transfection effect.25 In recent years, Smith et al. have synthesized a family of CDLs with DNA binding spermine surface groups for gene delivery. They found that the hydrophobic/hydrophilic balance played a subtle role in controlling DNA binding and transfection ability and the synergistic effects were observed for the hybrid vectors with aspects of both cationic polymers and lipids.26,27 By changing the hydrophobic segments of the CDLs, they demonstrated that their self-assembly, DNA binding ability, and gene delivery potential could all be controlled in predictable ways. The study also clearly indicated the cellular delivery directly correlated with the CDLs self-assembly abilities. The self-assembled vectors significantly outperformed PEI25K in terms of delivering DNA across cell membrane. However, because of the poor DNA release, the transfection activities of the CDLs were much lower than PEI25K.28 Kono et al. have also developed a series of CDLs for DNA delivery and found both the changes of cationic dendron size and the hydrophobic moieties could affect the transfection activities.14,29,30 The CDLs with suitable dendron sizes and alkyl chains achieved high transfection activities close to Lipofectamine LTX (a commercialized agent for DNA delivery).30 Peng et al. modified different generations of PAMAM dendrons with a single alkyl chain of different chain lengths to obtain a series of amphiphilic dendrimers. They first applied the amphiphilic dendrimers in siRNA delivery and found that both the PAMAM dendrons and the hydrophobic moieties played important roles in gene silencing. However, the amphiphilic dendrimers could not effectively produce gene silencing with Luc siRNA in complete culture medium with FBS (10%).31 In this study, a new family of CDLs was synthesized by conjugating alkyne-functionalized hydrophobic units and azidemodified PAMAM dendrons. All CDLs could effectively bind siRNA to form complexes. In in vitro transfection experiments, the gene-silencing abilities of the CDLs/siRNA complexes varied significantly depending on the CDLs’ dendron sizes. A1/ siRNA complexes achieved highly efficient gene silencing in

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EXPERIMENTAL SECTION

Materials. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC·HCl, ≥98.5%) was purchased from GL Biochem (Shanghai, China) and 2,2-bis(hydroxymethyl) propionic acid (≥98%), 4-dimethylaminopyridine (DMAP), 2,2-dimethoxypropane (98%), and 3-bromopropylamine hydrobromide (98%) were purchased from Alfa Aesar company (Tianjin, China). Propargyl alcohol (98%), methyl acrylate, and ethylenediamine were purchased from Sinopharm Chemical Reagent (Beijing, China) and purified by vacuum distillation before use. Oleic acid(≥99%) and chloroquine were purchased from Sigma-Aldrich (Shanghai, China). Lissamine Rhodamine B (Rhodamine PE or Rh-PE) was supplied by Avanti Polar Lipids (Alabaster, Al). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai, China) and used as received. Poly(ethylenimine) (PEI, 25 kDa) was purchased from Sigma-Aldrich (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) and fetal bovine serum (FBS, GIBCO) were purchased from Invitrogen (Carlsbad, CA). FAM-siRNA (sense, 5′-UUC UCC GAA CGU GUC ACG U dTdT-3′; antisense, 5′-ACG UGA CAC GUU CGG AGA A dTdT-3′), Luciferase siRNA (sense, 5′-CUU ACG CUG AGU ACU UCG A dTdT-3′; antisense 5′-UCG AAG UAC UCA GCG UAA G dTdT-3′), and Rev siRNA (sense, 5′-UUC UCC GAA CGU GUC ACG U dTdT-3′; antisense, 5′-ACG UGA CAC GUU CGG AGA A dTdT-3′) used as a scramble control were purchased from Shanghai GenePharma (Shanghai, China). All siRNA products were purified by HPLC; the content of the double-stranded siRNA of full length was higher than 97%, and the average molecular weights of the siRNAs were ∼13 300 g mol−1. Clear polystyrene tissue-culture-treated 96-well plates and 6-well plates were obtained from Corning Costar (Shanghai, China). Purified deionized water was prepared by the MilliQ plus system (Millipore, Billerica, MA). All other reagents and solvents were of analytical grade. Synthesis of the CDLs. To obtain the CDLs as shown in Figure 1, we employed the copper-catalyzed cycloaddition of azides and alkynes B

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Scheme 1. Synthesis Pathway of CDLs A1, A2, and A3

(CuAAC) “click” reactions to conjugate alkyne-functionalized hydrophobic units to the focal point of the azide-modified dendrons. (See Scheme 1 for full synthetic schemes, and all the synthesis details can be seen in the Supporting Information.). The proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Bruker AV 400 NMR spectrometer. Fourier transform infrared (FT-IR) spectra were measured with the potassium bromide method on a Bio-Rad Win-IR instrument. MALDITOF was recorded by autoflex III TOF/TOF (Bruker) and MS (ESI) and HR-MS (ESI) were measured by microTOF-Q II (Bruker). Critical Aggregation Concentration Measurements of the CDLs. Pyrene was used as a fluorescence probe for critical aggregation concentration (CAC) determination. CDL dispersions at concentrations varying from 3.05 × 10−5 to 0.50 mg mL−1 were prepared, and the final pyrene concentration was 6 × 10−7 mol/L in water. The solutions were equilibrated overnight to promote the aggregation prior to fluorescence measurement. Fluorescence spectra were recorded at the emission wavelength of 390 nm on a PTI Fluorescence Master System with Felix 4.1.0 software at room temperature. The fluorescence intensity ratio of I338.5/I335.5 was analyzed as a function of CDLs’ concentration.

Preparation of CDLs/siRNA Complexes. Purified deionized water was added to rehydrate a dry thin membrane of the cationic CDLs. After sonicating for 2 min with a bath-type sonicator (JAC ULTRASONIC 4020), the aqueous dispersions of compound A1 (1 mg mL−1, 0.97 mM), A2 (1 mg mL−1, 0.67 mM), and A3 (1 mg mL−1, 0.42 mM) were obtained. Luciferase siRNA, Rev siRNA (scramble control), and FAM-siRNA were dissolved in diethypyrocarbonate (DEPC) water to get a 0.1 mg mL−1 (7.5 μM) solution, respectively. The siRNA solution was added to various volumes of the CDL dispersions and incubated for 10 min at room temperature to afford the complexes with varying N/P ratios (defined as [terminal amines in the CDLs]/[phosphates in the siRNA]), consistent with previous report of CDLs.13,29−31For the preparation of Rh-PE containing complexes, the desired amount of the CDLs was premixed with RhPE, then equilibrated at room temperature for 10 min before the addition of luciferase siRNA solution. Characterization of the Size and Morphology of the Aggregates Formed by CDLs and the CDLs/siRNA Complexes. The complexes were prepared at N/P ratio of 8. To perform the transmission electron microscope (TEM) measurement, a drop of CDLs or the complexes dispersions with the CDLs’ concentration of 0.25 mg mL−1 (Milli-Q water) was deposited onto a carbon-coated C

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230-mesh copper grid and allowed to dry in air at 25 °C before being measured on a JEOL JEM-1011 transmission electron microscope with an accelerating voltage of 100 kV. For the Dynamic light scattering (DLS) measurement, the size distribution measurements of CDLs and the prepared complexes dispersions with the CDLs’ concentration of 0.25 mg mL−1 (Milli-Q water) were performed on the Wyatt QELS instrument with a vertically polarized He−Ne laser (DAWN EOS, Wyatt Technology), and the scattering angle was fixed at 90°; the data were analyzed with ASTRA and QELBatch. Characterization of the Surface Potential of the Aggregates Formed by CDLs and the CDLs/siRNA Complexes. The complexes were prepared at N/P ratio of 8. The surface potentials of the aggregates formed by CDLs (CDLs’ concentration of 0.25 mg mL−1, in Milli-Q water) and CDLs/siRNA complexes (CDLs’ concentration of 0.25 mg mL−1, in HEPES buffer solution (10 mM, pH 7.4) were measured using a Zeta Potential Analyzer (Brookhawen Instruments Corporation), we have collected the results of 10 runs, and the obtained data were analyzed with BIC PALS Zeta Potential Analyzer. Stabilities of the Dendriplex Aggregates Formed by CDLs/ siRNA. The stabilities of the dendriplex aggregates against dilution effects in 10 mM HEPES buffer (pH 7.4) and in serum-containing medium were investigated by DLS. To investigate the stabilities of the dendriplex aggregates against dilution, we prepared the CDLs/siRNA complexes dispersions with concentrations of 0.25, 0.05, and 0.013 mg mL−1 in HEPES buffer (10 mM, pH 7.4) and measured the average sizes of the dendriplex aggregates. To investigate the stabilities of the dendriplex aggregates in serum-containing medium, we further prepared the CDLs/siRNA complexes dispersions (CDLs’ concentration of 0.25 mg mL−1) in a HEPES buffer (10 mM, pH 7.4) with FBS (10%) and measured the sizes of the aggregates in the medium. Gel Retardation Analysis. The three CDLs were diluted to an appropriate concentration and stored at 4 °C. Before the analysis, the Rev siRNA was diluted with DEPC water. The solutions were mixed at various N/P ratios from 1:8 to 8:1 (Rev siRNA 0.038 nM per well) and incubated at 37 °C for 30 min. Electrophoresis was carried out on 2% agarose gel at 80 V for 25 min in TAE buffer solution (40 mM Tris−HCl, 1 v/v% acetic acid, and 1 mM EDTA). The retardation of siRNA was visualized by staining with ethidium bromide and UV light scan with a UVP EC3 bioimaging system (Upland, CA). In Vitro Gene Silencing Experiments. One day before transfection, 1 × 104 HeLa-Luc or Huh-7 cells per well were seeded in 96-well tissue culture plate in 180 μL of DMEM containing FBS (10%). Complexes of CDLs/Luc siRNA and complexes of CDLs/Rev siRNA with N/P ratio of 4, 8, and 16 were prepared with the same method as previously described. The final concentration of Rev siRNA or Luc siRNA was 75 nM. When the N/P ratio was 4, the concentration of A1, A2, and A3 was 6.13, 3.09, and 1.55 μM, respectively. When the N/P ratio was 8, the concentrations of A1, A2, and A3 were 12.25, 6.18, and 3.09 μM, respectively. When the N/P ratio was 16, the concentrations of A1, A2, and A3 were 24.5, 12.36, and 7.18 μM, respectively. For the transfection experiments in culture medium without FBS, DMEM with FBS (10%) was removed and replaced with DMEM without FBS. Then, 20 μL of the complex dispersions with different N/P ratios and other control groups was added to each well, and the plates were incubated at 37 °C for 4 h; the medium was then replaced by fresh DMEM containing FBS, in which the cells were incubated for another 48h before the luciferase assay test was carried out. For the transfection experiments in DMEM with FBS (10%), DMEM with FBS (10%) was removed and replaced with fresh DMEM with FBS (10%). Then, 20 μL of the complex dispersions with different N/P ratios and other control groups was added per well, and the plates were incubated at 37 °C for 4 h before the culture medium was changed again with DMEM with FBS (10%), then after further 48 h of incubation, the luciferase assay test was carried out. The culture medium was removed and the cells were washed with phosphatebuffered saline (PBS) three times; then, 50 μL of cell lysis buffer (2.5 × 10−3 M Tris, pH 7.8, 2.0 × 10−3 M EDTA, 2.0 × 10−3 M DTT, 10% glycerol, 1% Triton X-100) was added. After that, the 96-well plates were put in a −80 °C freezer refrigerator for 2 h. Then, 20 μL of the

lysis buffer was withdrawn for detection with a luciferase assay kit (100 μL luciferase assay buffer, Promega, Mannheim, Germany) on a luminometer for 10 s (Lumat LB9507 instrument, Berthold, Bad Wildbad, Germany). The lipoplex of Luc siRNA and Lipofectamine 2000 was used as the positive control according to the manufacture’s protocol, while naked Luc siRNA was used as the negative control. All experiments were made in triplicate, and the “inhibition of the luciferase expression” was calculated by normalizing the luminescence intensity of the groups treated with PBS as 100%. Confocal Laser Scanning Microscope Studies. For confocal laser scanning microscope (CLSM) studies, HeLa-Luc cells were seeded in six-well culture plates (a clean coverslip was put in each well) at a density of 1 × 105 cells per well and allowed to adhere for 24 h. Before transfection, the complete medium was removed and fresh complete medium with FBS or FBS-free medium was added. Then, the complexes of CDLs (Rh-PE)/FAM-siRNA reagent with N/P ratio of 8 were added. The final concentration of FAM-siRNA was adjusted to 75 nM, and the final concentration of A1, A2, and A3 was 12.25, 6.18, and 3.09 μM, respectively. After incubation at 37 °C for 4 h, the supernatant was carefully removed and the cells were washed three times with ice-cold PBS. The cells were then fixed with 1.0 mL of 4% formaldehyde in each well at 37 °C for 10 min and washed three times with ice-cold PBS again. Finally, the cell nuclei were stained with DAPI (blue). CLSM images of cells were obtained through confocal microscope (Zeiss LSM 780). Flow Cytometry Studies. For the flow cytometry (FCM) studies, ∼2 × 105 HeLa-Luc cells per well were seeded in six-well culture plates and allowed to adhere for 24 h. Before transfection, the complete medium was removed and fresh complete medium with FBS or FBSfree medium was added. Then, the complexes of CDLs(Rh-PE)/FAMsiRNA reagent with N/P ratio of 8 were added. The final concentration of FAM-siRNA was adjusted to 75 nM, and the final concentration of A1, A2, and A3 was 12.25, 6.18, and 3.09 μM, respectively. After incubation at 37 °C for 4 h, the supernatant was carefully removed and the cells were washed three times with ice-cold PBS. A total of 1 mL of 0.25% trypsin (GIBCO) solution was added thereafter, and the cells were detached from cell culture by incubation at 37 °C for 1.0 min. A single cell suspension was prepared by filtration through a 300 mesh filter. Finally, the cells suspended in 200.0 μL of PBS were subjected to FCM analysis using a Becton Dickinson FACSCalibur cytometer. Chloroquine Effect. One day before transfection, 1 × 104 HeLaLuc cells per well were seeded in 96-well tissue culture plate in 180 μL of fresh complete medium containing 10% FBS. Before transfection, complexes of CDLs/Luc siRNA and complexes of CDLs/Rev siRNA (used as control) were prepared. The desired amounts of Luc siRNA and CDLs were mixed in purified deionized water with a vortex for 10 s and left for 10 min at room temperature. 96-well tissue culture plates were added with or without chloroquine (100 μmol). After the addition of CDLs/siRNA complexes with N/P ratio of 8 (the final concentration of Rev siRNA or Luc siRNA was 75 nM, and the final concentration of A1, A2, and A3 was 12.25, 6.18, and 3.09 μM, respectively), the plates were incubated at 37 °C for 4 h, then medium with serum was removed and washed three times with ice-cold PBS. The culture plates were replaced with fresh complete medium and incubated at 37 °C for 48 h before luciferase assay test. Heparin Displacement Assay. For anion displacement assay, the complexes of CDLs/Rev siRNA at N/P ratio of 8 (Rev siRNA 0.038 nM per well) were incubated with 5 μL of heparin sodium (200 U/mg, Aladdin, Shanghai, China) in a series of weight ratios (Wheparin/WsiRNA) for 20 min at room temperature and finally analyzed on an agarose gel as previously described. Cytotoxicity Assay. Cell viabilities were assessed by the methyl thiazolyl tetrazolium (MTT) viability assays of HeLa-Luc and Huh-7Luc cells. The cells were seeded in 96-well culture plates at a density of 1 × 104 cells per well in 180 μL of DMEM containing 10% fetal bovine serum, supplemented with 50 units mL−1 penicillin and 50 units mL−1 streptomycin, and incubated at 37 °C in 5% CO2 atmosphere for 24 h. Then, the culture medium was removed and the CDLs and PEI25K (used as positive control) were added to 200 μL of complete DMEM D

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Figure 2. HR-MS (ESI, m/z) data of (A) B1 Calcd. [M+H]+ m/z = 1229.9026; (B) A1 Calcd. [M+H]+ m/z = 1029.7977; (C) B2 Calcd. [M+2H]2+ m/z = 943.6624; (D) A2 Calcd. [M+2H]2+ m/z = 743.5575; (E) B3 Calcd. [M+2H]2+ m/z = 1600.0845; and (F) A3 Calcd. [M+2H]2+ m/z = 1199.8748. medium at various concentrations up to 32 μg mL−1, which were 31.04, 21.44, and 13.44 μM for A1, A2, and A3, respectively. The cells were subjected to MTT assay after being incubated for 48 h at 37 °C in 5% CO2 atmosphere. 490 nm light absorbance of the medium was measured by a Bio-Rad 680 microplate reader. Cell viability was calculated based on the following equation: cell viability (%) = (Asample/Acontrol) × 100, where Asample and Acontrol were denoted as absorbencies of the sample and negative control wells, respectively. Statistics. All experiments were performed at least three times and expressed as means ± standard deviation (SD). Student’s test was applied to determine the statistical significance of the obtained data. A value of P < 0.05 was considered to be statistically significant, while p < 0.01 was considered to be highly significant.

Dioleoylphosphatidylethanolamine (DOPE), which was always employed as a helper lipid in gene delivery, also had two unsaturated C18 alkyl chains (two oleic acid chains) as the hydrophobic part. Thus, when we designed our system, we conjugated the alkyne-functionalized hydrophobic part with two oleic acids chains to the focal point of the azide-modified hydrophilic PAMAM dendrons with CuAAC “click chemistry”, as shown in Figure 1. The PAMAM dendrons that had azide groups at the focal points were synthesized via a similar method reported by Lee et al.33 Then, the amines of the three obtained dendrons were protected by t-butyloxycarbonyl (Boc) groups through reaction with di-tert-butyldicarbonate. A combination of methodology previously reported by Sharpless and Smith was employed to get the first generation of alkyne-modified dendron (compound 5, Scheme 1) with two hydroxyl groups.34,35 Through ester condensation, two oleic acids were conjugated to the alkyne-modified dendron (compound 6, Scheme 1).

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RESULTS AND DISCUSSION Synthesis and Characterization of the CDLs. The CDLs faced a serious problem in siRNA delivery due to their sensitivity to serum proteins.31,32 In recent studies, the unsaturated C18 alkyl chains were found to play an important role in enhancing the stability of the CDLs/DNA dendriplex aggregates and improving their serum-resistant property.14,29 E

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Figure 3. Typical TEM micrographs of the dendron aggregates formed by the blank CDLs (A) A1, (B) A2, and (C) A3 and the dendriplex aggregates formed by the CDLs and Rev. siRNA (D) A1/siRNA complexes, (E) A2/siRNA complexes, and (F) A3/siRNA complexes at N/P ratio of 8.

and 15 ± 1.6 nm, respectively. The sizes measured by TEM were much smaller than that measured by DLS. It was because that the size measured by DLS was the hydrodynamic size, which might be affected by the water layer, and DLS was more sensitive to the interference of large particles.37 The regularity was in accordance with the results of TEM; the radii of the dendron aggregates decreased with the increase in the hydrophilic dendron size. It might be because as the sizes of hydrophilic dendron increased the steric hindrance for the CDLs assembling would increase, which would cause smaller numbers of CDLs molecules incorporated into each dendron aggregate, leading to the formation of smaller dendron aggregates.28 Besides, the TEM and DLS results of the A1 and A2 dendron aggregates indicated that the large selfassemblies might not be resulted from simple micelles. It was found that the molecules with bigger hydrophobic segments tend to form bigger aggregates in aqueous solution.38 Zeta potential measurement gave positive values of +44.1 ± 1.30, +30.6 ± 0.73, and +18.2 ± 0.59 mV for the dendron aggregates formed by A1, A2, and A3, respectively (Table S1 in the Supporting Information). For the dendron aggregates formed by A1, the higher zeta potentials might be caused by the fact that A1 with smaller hydrophilic dendron moieties and higher proportion of hydrophobic segments drove closer packing of positively charged groups on the surfaces of the dendron aggregates.27,28 For A2 and A3 self-assembling, because of the lower proportion of hydrophobic segments and bigger hydrophilic dendron moieties, after self-assembling, the loosely packed dendron aggregates with lower positive surface potential were formed. Evaluation of the SiRNA Binding Abilities of the CDLs. The abilities of the CDLs to form complexes with siRNA were examined using agarose gel electrophoresis. As can be seen in Figure 4, for all cases, the amount of free siRNA decreased with the increasing amount of the added CDLs and completely disappeared after a certain amount of CDLs was added, indicating that these CDLs had good abilities to form complexes with siRNA. In the case of A1 and A2, free siRNA disappeared above the N/P ratio of 4.0, but it still could be seen that A2 had a greater ability to form a complex with siRNA. In the case of A3, the free siRNA band was not observed above the N/P ratio of 2.0. These results indicated that the CDLs with larger sizes of the poly(amidoamine) dendrons could retard

Then the “click” chemistry was carried out to couple the azides to the alkyne groups, and the compounds purification yielded the modified dendrons with hydrophobic groups connected at the focal point via triazole linkage. The compounds bearing the Boc-protected first, second, and third generations of PAMAM dendron were named as B1, B2, and B3, respectively. The Boc protecting groups of B1, B2, and B3 were finally removed from the amine surface ligands using HCl gas to obtain the final products named as A1, A2, and A3 (Figure 1), respectively. The 1H NMR (Figure S1 in the Supporting Information), 13C NMR (Figure S2 in the Supporting Information), FT-IR (Figure S3 in the Supporting Information), and MALDI-TOF and HR-MS (Figure 2) results demonstrated that the target products were successfully obtained. Self-Assembly of the CDLs. Cationic lipids are an interesting type of materials that can self-assemble into dendron aggregates in aqueous solution.19,31,34 The widely reported pyrene-probe-based fluorescence technique was used to demonstrate the formation of self-assembled aggregates. Because of the transfer of pyrene into a hydrophobic environment, a red shift of absorption band from 335.5 to 338.5 nm was observed when the copolymer concentration was increased from 3.05 × 10−5 to 0.50 mg mL−1.The CAC value was obtained from the plot of the fluorescence intensity ratio of I338.5/I335.5 versus lg c of the CDLs. The CAC values of the CDLs A1, A2, and A3 were 0.0042 (4.08 μM), 0.013 (8.75 μM), and 0.050 mg mL−1 (20.85 μM), respectively (Table S1 and Figure S4 in the Supporting Information). The dendron size had a profound effect on the CDLs’ aggregation process. With the increase in the hydrophilic dendron size and the decrease in the proportion of hydrophobic fraction, the CAC values increased from 4.08 to 20.85 μM as a consequence of less effective packing of the bigger hydrophilic headgroups and less probability of the hydrophobic part winding and interactions.36 The morphologies of the dendron aggregates were observed by TEM (Figure 3). The dendron aggregates were spherically shaped with uniform size. With the increase in the hydrophilic dendron size, the corresponding size of the dendron aggregates decreased. The radii of the dendron aggregates were also measured by DLS (Table S1 and Figure S5 in the Supporting Information), and the radii of the dendron aggregates formed by A1, A2, and A3 were 31 ± 7.2, 18 ± 4.1, F

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was irregular, and the shape of the dendriplex aggregates formed by A3/siRNA complexes seemed like aggregates of smaller particles, indicating better self-assembly behavior of A1/ siRNA complexes. The size of the dendriplex aggregates formed by CDLs/siRNA complexes became smaller as the dendron sizes increased. The radii of the dendriplex aggregates formed by A1/siRNA complexes, A2/siRNA complexes, and A3/ siRNA complexes measured by DLS were 68 ± 11.5, 51 ± 8.6, and 19 ± 6.2 nm in Milli-Q water, respectively; as shown in Figure S3 and Table S2 in the Supporting Information, the regularity was in accordance with the results of TEM, which might also be because the more hydrophobic nature of the CDLs with smaller dendron sizes and larger numbers of CDLs and siRNA molecules were incorporated into each dendriplex aggregate for the CDLs with smaller dendron sizes.30 Moreover, zeta potential measurement gave positive values of +18.5 ± 1.02, +4.2 ± 0.52, and +1.9 ± 0.53 mV for the dendriplex aggregates formed by A1/siRNA complexes, A2/ siRNA complexes, and A3/siRNA complexes at N/P ratio of 8 (Table S2 in the Supporting Information) in HEPES buffer (10 mM, pH 7.4), which was more close to the physiological conditions. After the complexation with anionic siRNA, the zeta potential values of all dendriplex aggregates formed by the CDLs/siRNA complexes decreased, but they still exhibited positive surface potentials, indicating their ability to neutralize the net anionic charge of siRNA. The surface potentials decreased as the hydrophilic dendron sizes of the CDLs increased. It might also be due to the fact that CDLs with smaller hydrophilic dendron moieties and higher proportion of hydrophobic segments drove closer packing of positively charged groups on the surfaces of the dendriplex aggregates. Thus, the higher positive surface charge densities led to higher zeta potential, as reported by Smith et al.28 Kono et al. also found that although it was with the same PAMAM headgroup, the CDL with longer alkyl chain length could form aggregates with DNA with more compact structure and higher surface potential. Evaluation of the Stabilities of the Dendriplex Aggregates Formed by CDLs/siRNA. To investigate the stabilities of the dendriplex aggregates against dilution, we prepared the CDLs/siRNA complexes dispersions with various concentrations from 0.25 to 0.013 mg mL−1 under more physiologically relevant conditions (10 mM HEPES buffer, pH 7.4) and measured the average sizes of the dendriplex aggregates with DLS. As can be seen in Table S3 in the Supporting Information, no obvious changes in size were detected, which suggested the good stabilities of the dendriplex aggregates that combined electrostatic and hydrophobic interactions in siRNA delivery. To investigate the stabilities of the dendriplex aggregates in serum-containing medium, we further prepared the CDLs/siRNA complexes dispersions in FBS (10%)-containing medium and measured the sizes of the aggregates in the medium; as can be seen in Table S4 in the Supporting Information, slight increases in the particle sizes were detected, which might be caused by the interactions of proteins in the serum. However, the sizes of the aggregates did not change much and maintained a narrow distribution, indicating the good stabilities of the dendriplex aggregates in serum-containing buffer. The results are inconsistent with previous reports by Kono et al. They found the CDLs with two C18 alkyl chains exhibited a high serum-resistant property,14 and the unsaturated chains could further improve the stabilities of the CDLs in serum-containing medium.29

Figure 4. Agarose gel electrophoretic analysis of the CDLs/Rev siRNA complexes (A) A1/siRNA complexes, (B) A2/siRNA complexes, and (C) A3/siRNA complexes at different N/P ratios.

siRNA at lower N/P ratio, which was consistent with previous reports by Jensen et al.39,40 However, in their research, the firstgeneration dendrimer lacked the capability of forming aggregates with siRNA and could not completely retard siRNA in all tested molar ratios, but in our system, the selfassembly of A1 could successfully retain the siRNA migration, indicating that the hydrophobic part played an important role for the siRNA binding, which was also noticed by Diederich et al.25 Here might be a paradox, the dendron aggregates formed by A1 had the highest surface zeta potential, but in the agarose gel electrophoresis, A1 needed the highest N/P ratio to retard all siRNA. As reported in previous studies, the reorganization of the vectors played an important role in binding siRNA.40,41 The CDLs with bigger-sized dendrons and smaller hydrophobic segments self-assembled into less compact structures, and they were of higher CAC concentrations, which might endow them the capabilities to reorganize better for siRNA binding. Besides, at the same N/P ratio, A2 and A3 possessed more tertiary amines that might help bind free siRNA.42 So, even under lower N/P ratio, A2 and A3 could still retard siRNA efficiently. The similar results were also found by Kono et al.14 In their work, they found the CDLs with longer alkyl chains could bind DNA to form more compact structure with higher surface potential, but it could not retard DNA as efficiently as the CDLs with smaller hydrophobic segment did. Thus, the fact that the CDLs with smaller-sized dendron formed dendriplex aggregates with higher surface potential is not contradictory to the fact that the CDLs with larger sized dendrons could retard siRNA at a lower N/P ratio. Evaluation of the Dendriplex Aggregates Formed by the CDLs/siRNA Complexes. Furthermore, the morphologies of the dendriplex aggregates formed by A1/siRNA complexes, A2/siRNA complexes, and A3/siRNA complexes were measured by TEM at N/P ratio of 8. As can be seen in Figure 3, the dendriplex aggregates formed by A1/siRNA complexes were spherically shaped with uniform size, the shape of the dendriplex aggregates formed by A2/siRNA complexes G

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Figure 5. Inhibition of luciferase expression of the (A,C) HeLa-Luc cells and (B,D) Huh-7-Luc cells treated with CDLs/siRNA complexes formed by Luc siRNA or Control siRNA (Rev siRNA) and CDLs of A1, A2, and A3 at different N/P ratios in (A,B) culture medium without FBS and in (C,D) complete culture medium with FBS (10%) at different N/P ratios. Lipofectamine 2000/Luc siRNA complexes and naked Luc siRNA are employed as controls. Data are presented as mean ± SD (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05; #, P > 0.05.

Scheme 2. Illustration of the siRNA Delivery by CDLs/siRNA Complexes

Gene Silencing Abilities of CDLs/siRNA Complexes. To evaluate the transfection activities of the CDLs/siRNA

complexes, we tested their abilities to deliver luciferase siRNA (Luc siRNA) and induce gene silencing on the basis of H

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Figure 6. Confocal laser scanning microscopy of HeLa-Luc cells (1 × 105 cells mL−1) after incubation with (A) naked FAM-siRNA; (B) A1(RhPE)/FAM-siRNA complexes; (C) A2(Rh-PE)/FAM-siRNA complexes; and (D) A3(Rh-PE)/FAM-siRNA complexes for 4 h at 37 °C in culture medium without FBS and complete culture medium with FBS (10%). (FAM-siRNA equivalent concentration 75 nM for all formulations and Rh-PE equivalent concentration 38.6 nM for formulations of CDLs(Rh-PE)/FAM-siRNA. The N/P ratio of CDLs(Rh-PE)/FAM-siRNA complexes is 8.) The scale bars represent 20 μm.

Figure 7. FCM histograms for counting 1 × 104 HeLa-Luc cells after incubation with (A,K) PBS; (B,L) naked FAM-siRNA; (C,M) A1(Rh-PE)/ FAM-siRNA complexes; (D,N) A2(Rh-PE)/FAM-siRNA complexes; and (E,O) A3(Rh-PE)/FAM-siRNA complexes in medium without FBS and (F,P) PBS; (G,Q) naked FAM-siRNA; (H,R) A1(Rh-PE)/FAM-siRNA complexes; (I,S) A2(Rh-PE)/siRNA complexes; and (J,T) A3(Rh-PE)/ FAM-siRNA complexes in complete medium with FBS (10%) for 4 h at 37 °C to examine the cellular uptake of FAM-siRNA and the Rh-PE marked CDLs. (FAM-siRNA equivalent concentration 75 nM for all formulations and Rh-PE equivalent concentration 38.6 nM for all formulations of CDLs(Rh-PE)/FAM-siRNA complexes. The N/P ratio of CDLs(Rh-PE)/FAM-siRNA complexes is 8.)

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could cause destabilization of endosome and promote transfer of siRNA into cytosol, was utilized to value whether the CDLs/ siRNA complexes had the capability to escape from the endosome. HeLa-Luc cells were transfected with the CDLs/ Luc siRNA complexes at N/P ratio of 8 in the presence or absence of chloroquine. As presented in Figure S7 in the Supporting Information, the presence of chloroquine had no obvious effect on the luciferase expression for the cells treated with CDLs/Luc siRNA complexes. This result suggested that all CDLs/siRNA complexes had sufficient ability to achieve efficient endosome escape, which might be caused by both the endosome buffering effect induced by the tertiary amino groups of the PAMAM dendron and the hydrophobic character of the two oleic acid chains that might enhance destabilization of endosome membranes through strong hydrophobic interaction.30,31 The results we got were consistent with previous work that demonstrated the CDLs with two C18 alkyl chains had good capabilities to escape from the endosome.30 Herein the endosome escape capability was not the limiting factor for gene-silencing abilities in this system. SiRNA Release Abilities of the CDLs/siRNA Complexes. The abilities of the CDLs/siRNA complexes to effectively release siRNA were further investigated using heparin displacement assay. Heparin was an anionic polymer, which could compete with siRNA for binding the CDLs.36 The assay was performed via gel electrophoresis using a fixed N/P ratio of 8:1 in the wells and with increasing amounts of heparin sodium being added to subsequent wells. The amount of heparin sodium required to fully displace the siRNA from the vector was a good indicator of how effectively the vector could release the siRNA from its complex intracellularly. It was interesting to note that A1/siRNA complexes showed the least effective siRNA release ability compared with its counterparts, which might be caused by the better self-assembly structure and higher surface potential of the formed dendriplex aggregates (Figure 8). The results eliminated the possibility that the siRNA release was the main factor for the poor gene silencing abilities of A2/siRNA complexes and A3/siRNA complexes. In Vitro Cytotoxicities of the CDLs. In this work, in vitro cytotoxicities of the CDLs to HeLa-Luc cells and Huh-7-Luc cells were evaluated using MTT assay with PEI25K as the positive control (Figure 9). The cells were treated with the CDLs at different concentrations and incubated for 48 h. It was observed that the cell viabilities of HeLa-Luc cells and Huh-7Luc cells treated with the CDLs were higher than 70% at all test concentrations up to 32 μg mL−1, much higher than those treated with PEI25K, indicating their low toxicities and good compatibilities. The cell viabilities of HeLa-Luc cells and Huh7-Luc cells treated with A1 were slightly lower than those treated with A2 and A3, which might be caused by higher surface potential and higher cellular uptake ratio.

luciferase gene silencing in HeLa-Luc cells and Huh-7-Luc cells, which stably expressed the luciferase gene. Whether in culture medium with or without 10% FBS, as can be seen in Figure 5, the A1/siRNA complexes exhibited the best gene-silencing effect among all CDLs/Luc siRNA complexes and close to Lipofectamine 2000/Luc siRNA complex. As the dendron sizes increased, the silencing abilities decreased dramatically. It was accepted that for effective siRNA delivery into cells, the synthetic vector must enable the transit of the cell membrane, allow escape from the endosome after cellular uptake, and release siRNA into cytoplasm so that it could play its role in gene silencing (Scheme 2). To find out the key factors that caused the difference, a series of experiments was further designed. Cellular Uptake of the CDLs/siRNA Complexes. Cellular uptake of the CDLs/siRNA complexes by HeLa-Luc cells was studied by both the CLSM and the FCM with FAMlabeled siRNA (FAM-siRNA) and lissamine rhodamine B (RhPE) marked CDLs. As shown in Figure 6, after 4 h of incubation, the cells treated with naked FAM-siRNA exhibited the weakest FAM fluorescence. This was because the free anionic FAM-siRNA molecules could hardly approach and cross the negative cell membrane while the dendriplex aggregates with positive surface charge could be more easily internalized via endocytosis.36,43 It could also be seen that the cells treated with CDLs(Rh-PE)/FAM-siRNA complexes exhibited decreased FAM and rhodamine fluorescence intensities as the dendron size increased, which might be due to the fact that after the complexation with siRNA, CDLs with smaller dendron sizes and higher proportion of hydrophobic parts had better self-assembly abilities and formed dendriplex aggregates with more compact structures with higher surface potentials.28,36 Compared with the cells incubated in culture medium without FBS, the fluorescence intensities of both FAM and rhodamine decreased in the cells incubated in complete culture medium with FBS (10%) after the treatment with CDLs(Rh-PE)/FAM-siRNA complexes. It was because the presence of FBS could destabilize or form aggregates with CDLs(Rh-PE)/FAM-siRNA complexes, which would decrease the cellular uptake into the cells.44 The FCM was also performed to confirm the above results. The mean FAM and rhodamine fluorescence intensities in the HeLa-Luc cells were examined after incubation with different formulations for 4 h in the medium with or without FBS (10%). As shown in Figure 7, the cells treated with naked FAM-siRNA exhibited the weakest fluorescence intensity, which was in accordance with the CLSM result. (The corresponding SSC/ FSC pictures are shown in Figure S6 in the Supporting Information.) Although identical FAM-siRNA and Rh-PE concentrations were used for the CDLs(Rh-PE)/FAM-siRNA formulations, the cells exhibited decreased FAM and rhodamine fluorescence intensities as the dendron sizes of the CDLs increased, and the FAM and Rhodamine fluorescence intensities of the cells incubated in the presence of FBS were lower than those of the cells incubated without FBS, which was also in accordance with the CLSM results. Endosome Escape Abilities of the CDLs/siRNA Complexes. As generally accepted, the escaping enhancement of the introduced nucleic acids from endosome to cytoplasm was one of the most efficient strategies for the achievement of efficient transfection, and it was believed that the CDLs/siRNA complexes were taken up through the endosome pathway.28,30,31 Thus, chloroquine, an endosomotropic agent that

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CONCLUSIONS Three CDLs with hydrophilic headgroups of PAMAM dendrons and hydrophobic tails of two oleic acid chains were precisely synthesized through CuAAC “click chemistry”. The CDLs with smaller hydrophilic dendron groups and higher proportion of hydrophobic moieties exhibited lower CAC values, indicating their better self-assembly capabilities. All CDLs could efficiently bind siRNA to form complexes, and all CDLs/siRNA complexes took spherical dendriplex aggregates with uniform sizes. Zeta potential measurements showed the CDLs with smaller dendron sizes formed dendron or J

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis details, 1H NMR, 13C NMR spectra, FT-IR spectra and MALDI-TOF results of the CDLs, self-assembly data for the CDLs and the CDLs/Rev siRNA complexes, the stabilities data for the dendriplex aggregates of CDLs/Rev siRNA complexes, the SSC/FSC data of FCM, and the data of chloroquine effect in gene silencing. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (projects 21074129, 51222307, 51203153, 51021003, 51233004, 51303173, and 51273196) and Jilin province science and technology development program 20120306.

Figure 8. Gel electrophoresis images of CDLs/siRNA complexes (8:1 N/P ratio) in the presence of increasing quantities of heparin sodium (left to right): lanes 1−7 ((heparin/siRNA) 0:1, 0.31:1, 0.63:1, 1.25:1, 2.5:1, 5:1, 10:1) and lane 8 of naked siRNA.

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REFERENCES

(1) Whitehead, K. A.; Langer, R.; Anderson, D. G. Nat. Rev. Drug Discovery 2009, 8 (2), 129−138. (2) Tian, H.; Chen, J.; Chen, X. Small 2013, 9 (12), 2034−2044. (3) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109 (2), 259− 302. (4) Tian, H.; Xiong, W.; Wei, J.; Wang, Y.; Chen, X.; Jing, X.; Zhu, Q. Biomaterials 2007, 28 (18), 2899−2907. (5) Dong, X.; Tian, H.; Chen, J.; Xia, J.; Chen, X.; Wei, Y. Acta Polym. Sin. 2011, 9, 1086−1091. (6) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47 (1), 113−131. (7) Wang, Y.; Gao, S.; Ye, W.-H.; Yoon, H. S.; Yang, Y.-Y. Nat. Mater. 2006, 5 (10), 791−796. (8) Chen, L.; Tian, H.; Chen, X.; Park, T.; Maruyama, A.; Jing, X. Acta Polym. Sin. 2009, 6, 499−505. (9) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G. Adv. Drug Delivery Rev. 2005, 57 (15), 2177−2202. (10) Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem 2002, 13 (3), 510−517. (11) Esfand, R.; Tomalia, D. A. Drug Discovery Today 2001, 6 (8), 427−436. (12) Medina, S. H.; El-Sayed, M. E. H. Chem. Rev. 2009, 109 (7), 3141−3157.

dendriplex aggregates with higher surface potentials before or after the complexation with siRNA. More interestingly, the gene silencing abilities of the CDLs/siRNA complexes varied significantly depending on the CDLs’ dendron sizes, that is, the A1/siRNA complexes with smallest dendron size had the highest gene silencing ability. The low generation systems can outperform higher generation analogues in terms of their biological activity, which is one of the advantages of combining a self-assembly approach with dendron design. In detail, the CDLs with smaller hydrophilic dendron groups could bind siRNA to form dendriplex aggregates with more compact structures and higher positive surface potentials and thus could be taken up more efficiently by cells and exhibited higher gene silencing abilities. It was also noteworthy that A1/siRNA complexes achieved highly efficient siRNA silencing in the presence of serum toward HeLa-Luc and Huh-7-Luc cells, which overcame a major challenge of the CDLs in siRNA delivery and showed bright prospect for gene therapy.

Figure 9. Cell viabilities of (A) HeLa-Luc cells and (B) Huh-7-Luc cells treated with A1, A2, A3, and PEI25K at different concentrations. Data are presented as mean ± SD (n = 3). *** P < 0.001; ** P < 0.01. K

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(13) Kono, K.; Akiyama, H.; Takahashi, T.; Takagishi, T.; Harada, A. Bioconjugate Chem. 2005, 16 (1), 208−214. (14) Takahashi, T.; Kojima, C.; Harada, A.; Kono, K. Bioconjugate Chem. 2007, 18 (4), 1349−1354. (15) Al-Jamal, K. T.; Ramaswamy, C.; Florence, A. T. Adv. Drug Delivery Rev. 2005, 57 (15), 2238−2270. (16) Lee, H.-i.; Lee, J. A.; Poon, Z.; Hammond, P. T. Chem. Commun. 2008, 32, 3726−3728. (17) Singh, B.; Florence, A. T. Int. J. Pharm. 2005, 298 (2), 348−353. (18) Wood, K. C.; Little, S. R.; Langer, R.; Hammond, P. T. Angew. Chem., Int. Ed. 2005, 44 (41), 6704−6708. (19) Rosen, B. M.; Wilson, C. J.; Wilson, D. A.; Peterca, M.; Imam, M. R.; Percec, V. Chem. Rev. 2009, 109 (11), 6275−6540. (20) Bhattacharya, S.; Bajaj, A. Chem. Commun. 2009, 31, 4632− 4656. (21) Yang, X. Z.; Dou, S.; Wang, Y. C.; Long, H. Y.; Xiong, M. H.; Mao, C. Q.; Yao, Y. D.; Wang, J. Acs Nano 2012, 6 (6), 4955−4965. (22) Gao, X.; Huang, L. Gene Ther. 1995, 2 (10), 710−722. (23) Foged, C. Curr. Top. Med. Chem. 2012, 12 (2), 97−107. (24) El-Aneed, A. J. Controlled Release 2004, 94 (1), 1−14. (25) Joester, D.; Losson, M.; Pugin, R.; Heinzelmann, H.; Walter, E.; Merkle, H. P.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42 (13), 1486−1490. (26) Jones, S. P.; Gabrielson, N. P.; Wong, C.-H.; Chow, H.-F.; Pack, D. W.; Posocco, P.; Fermeglia, M.; Pricl, S.; Smith, D. K. Mol. Pharm. 2011, 8 (2), 416−429. (27) Jones, S. P.; Gabrielson, N. P.; Pack, D. W.; Smith, D. K. Chem. Commun. 2008, 39, 4700−4702. (28) Barnard, A.; Posocco, P.; Pricl, S.; Calderon, M.; Haag, R.; Hwang, M. E.; Shum, V. W. T.; Pack, D. W.; Smith, D. K. J. Am. Chem. Soc. 2011, 133 (50), 20288−20300. (29) Yuba, E.; Nakajima, Y.; Tsukamoto, K.; Iwashita, S.; Kojima, C.; Harada, A.; Kono, K. J. Controlled Release 2012, 160 (3), 552−560. (30) Kono, K.; Ikeda, R.; Tsukamoto, K.; Yuba, E.; Kojima, C.; Harada, A. Bioconjugate Chem. 2012, 23 (4), 871−879. (31) Yu, T.; Liu, X.; Bolcato-Bellemin, A.-L.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.; Rocchi, P.; Behr, J.-P.; Peng, L. Angew. Chem., Int. Ed. 2012, 51 (34), 8478−8484. (32) Takahashi, T.; Harada, A.; Emi, N.; Kono, K. Bioconjugate Chem. 2005, 16 (5), 1160−1165. (33) Lee, J. W.; Kim, J. H.; Kim, B.-K.; Kim, J. H.; Shin, W. S.; Jin, S.H. Tetrahedron 2006, 62 (39), 9193−9200. (34) Barnard, A.; Smith, D. K. Angew. Chem., Int. Ed. 2012, 51 (27), 6572−6581. (35) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.; Finn, M. G.; Fokin, V. V.; Sharpless, K. B.; Hawker, C. J. Chem. Commun. 2005, 46, 5775−5777. (36) Qi, R. G.; Liu, S.; Chen, J.; Xiao, H. H.; Yan, L. S.; Huang, Y. B.; Jing, X. B. J. Controlled Release 2012, 159 (2), 251−260. (37) Li, M.; Song, W.; Tang, Z.; Lv, S.; Lin, L.; Sun, H.; Li, Q.; Yang, Y.; Hong, H.; Chen, X. ACS Appl. Mater. Interfaces 2013, 5 (5), 1781− 1792. (38) Mai, Y.; Eisenberg, A. Chem. Soc. Rev. 2012, 41 (18), 5969− 5985. (39) Jensen, L. B.; Mortensen, K.; Pavan, G. M.; Kasimova, M. R.; Jensen, D. K.; Gadzhyeva, V.; Nielsen, H. M.; Foged, C. Biomacromolecules 2010, 11 (12), 3571−3577. (40) Jensen, L. B.; Pavan, G. M.; Kasimova, M. R.; Rutherford, S.; Danani, A.; Nielsen, H. M.; Foged, C. Int. J. Pharm. 2011, 416 (2), 410−418. (41) Huebner, S.; Battersby, B. J.; Grimm, R.; Cevc, G. Biophys. J. 1999, 76 (6), 3158−3166. (42) Patil, M. L.; Zhang, M.; Taratula, O.; Garbuzenko, O. B.; He, H.; Minko, T. Biomacromolecules 2009, 10 (2), 258−266. (43) Foged, C.; Brodin, B.; Frokjaer, S.; Sundblad, A. Int. J. Pharm. 2005, 298 (2), 315−322. (44) Yang, J. P.; Huang, L. Gene Ther. 1997, 4 (9), 950−960.

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