A Combination of Guanidyl and Phenyl Groups on a Dendrimer

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A Combination of Guanidyl and Phenyl Groups on Dendrimer Enables Efficient siRNA and DNA Delivery Hong Chang, Jia Zhang, Hui Wang, Jia Lv, and Yiyun Cheng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00567 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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A Combination of Guanidyl and Phenyl Groups on Dendrimer Enables Efficient siRNA and DNA Delivery Hong Chang, Jia Zhang, Hui Wang, Jia Lv, Yiyun Cheng* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P. R. China. *Correspondence

should

be

addressed

to

Yiyun

Cheng.

E-mail:

[email protected]

KEYWORDS: dendrimer, polymer, gene delivery, guanidyl, membrane disruption.

ABSTRACT: Gene therapy has received considerable attention due to its great potential in the treatment of various diseases; however, the design of efficient and biocompatible carriers for the delivery of siRNA as well as DNA still remains a major challenge. In this study, we developed an efficient carrier for gene delivery by modification of a compound containing both guanidyl and phenyl groups on the surface of a cationic dendrimer. The guanidyl group on dendrimer facilitates nucleic acid condensation via specific guanidinium-phosphate interaction, while the phenyl group on polymer is critical for efficient endosomal escape. The combination of guanidyl and phenyl shows a synergistic effect in facilitated endocytosis. The designed material is much more efficient in siRNA and DNA delivery than control

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materials such as dendrimers engineered with guanidyl or phenyl group only, as well as intact dendrimers, and shows comparable efficacy to commercial transfection reagent Lipofectamine 2000. In addition, the material and its complex with nucleic acid show minimal toxicity on the transfected cells. This study provides a new strategy to develop multifunctional polymers for efficient siRNA and DNA delivery.

INTRODUCTION

The introduction of nucleic acid to specific cells or tissues is considered an important technique in basic biological research and clinical gene therapy.1 Though the great progress in the discovery of gene carriers has been achieved during the past decades, further efforts need to be devoted to the design of efficient and safe gene carriers.2-4 Cationic polymers are one of the most promising candidates for gene delivery due to ease of modification, available for large-scale synthesis, lack of immunogenicity and high penetration capability.5,6 Polymers such as linear or branched polyethylenimine (PEI),

dendrimers,

chitosan,

polypeptides,

and

poly(2-(dimethylaminoethyl)

methacrylate) (PDMAEMA) were proposed as gene carriers,7-13 but these materials are often criticized for either being little effective or too toxic.14

There are multiple extracellular and intracellular barriers in polymer-mediated gene delivery process.15 First, the polymer should self-assemble with nucleic acids and condense them into nanoparticles, termed polyplexes. The polyplexes are required to being stable in serum and buffer solutions, and efficiently deliver the nucleic acid into the target cells. After endocytosis, the most critical issue affecting the gene


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transfection efficacy is inefficient endosomal escape of the polyplexes.16 Finally, the nucleic acids should be released from the polymer matrix in the cytosol. For the polymers like dendrimers and PEIs, they were not specially designed for gene delivery and lack necessary function to breakdown the multiple barriers. These polymers were modified with functional ligands to address one or two barriers and somewhat improve the transfection efficacy.17 For example, modification of cationic polymers with hydrophobic ligands such as lipid and cholesterol combines the features of liposomes and polymers in gene delivery.5 The lipid-modified polymers show higher affinity to cell membranes compared to unmodified polymers. The conjugates can escape from endosomes through the fusogenic property of lipids and the proton sponge effect of cationic polymers. However, the presence of lipids or cholesterol on polymer reduces its positive charge density and nucleic acid binding capacity.18 In addition, the conjugated hydrophobic ligands on polymer may lead to irreversible cell membrane disruption and increased cytotoxicity.19,20 In an alternative strategy, multifunctional materials were synthesized by modification of various ligands on a single polymer to address the multiple extracellular and intracellular barriers in gene delivery. For example, a combination of endosomal escape moiety (i.e. histidine with an imidazole group) and hydrophobic moiety (i.e. aromatic amino acids such as phenylalanine and tyrosine) on a polymer showed high efficacy in the delivery of siRNA.21 The combination of arginine (for enhanced nucleic acid binding), phenylalanine and histidine on dendrimer at an optimal ratio also exhibited a synergistic effect in DNA delivery.17,22 Though the multifunctional polymers show


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promising efficacy, they have several disadvantages in gene delivery. For example, modification of various ligands on a polymer may generate serious spatial hindrance and the functional ligands may interfere with each other on a congested surface, and it is hard to choose the optimal ratio for each type of functional ligand on a polymer to achieve high efficacy.

Here, we proposed a facile strategy by integrating different functional ligands (i.e. phenyl and guanidyl) into a single compound (guanidinobenzoic acid, GBA) to address these issues. GBA was modified to a polyamidoamine (PAMAM) dendrimer by a condensation reaction between carboxyl group on GBA and primary amine groups on dendrimer surface. The guanidinium group on GBA is crucial for efficient nucleic acid condensation and cell membrane association through salt bridge and hydrogen bond interactions with phosphate groups on nucleic acid and phospholipids, while the phenyl groups on GBA endows the polymer with higher hydrophobicity, which is essential for cell membrane penetration and disruption. As a result, the GBA-modified dendrimer showed high efficacy in both siRNA and DNA delivery, and was dramatically more efficient than control materials such as intact dendrimers without modification, dendrimers modified with an equal number of benzoic acid (BA) or guanidinium (GA) only (Figure 1).

EXPERIMENTAL SECTION

Materials. Generation 5 (G5) PAMAM dendrimer with an ethanediamine core (ideal molecular weight 28826 Da, 128 primary surface amine groups) was purchased from


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Dendritech

(Midland,

MI).

GBA

hydrochloride,

BA,

and

1H-Pyrazole-1-carboxamidine hydrochloride were purchased from Sigma-Aldrich (St. Louis,

MO).

The

siRNA

targeting

firefly

luciferase

(siLuc)

(sense:

5’-CCCUAUUCUCCUUCUUCGCdTdT-3’), siRNA targeting Bcl-2 (siBcl-2) (sense: 5’-CCGGGAGAUAGUGAUGAAGdTdT-3’), siRNA targeting prolyl hydroxylase (siPHD-2) (sense: 5’-UCACGUUGAUAACCCAAAUdTdT-3’), siLuc labeled with a fluorescent dye FAM at the 5’ end (siLuc-FAM) and scrambled siRNA non-specific to any human gene (siNC) were synthesized by GenePharma Co. Ltd. (Shanghai, China). Lipofectamine 2000 (Lipo 2000) was purchased from Invitrogen (Carlsbad, CA). All the chemicals were used without further purification.

Synthesis and characterization of GBA-, BA-, and GA-modified PAMAM dendrimers. GBA (0.132 mmol) dissolved in 2 mL anhydrous N, N-dimethyl formamide was activated by dicyclohexylcarbodiimide (1.3 molar equivalent of GBA) and N-hydroxysuccinimide (1.2 molar equivalent of GBA) for 6 h at room temperature, and then added with G5 PAMAM dendrimer (1.735 µmol) dissolved in 2 mL dimethyl sulfoxide. Triethylamine (1.5 molar equivalent of GBA) was added to remove the acids and the mixture was stirred at room temperature for 7 days. After reaction, the product G5-GBA60 was purified by extensive dialysis against dimethyl sulfoxide and distilled water, and freeze-dried before further use. To investigate the effect of GBA conjugation ratio on the gene transfection efficacy of G5-GBA conjugates, the following amounts of GBA (0.097 mmol, 0.111 mmol, 0.156 mmol) were added to G5 dendrimer to synthesize polymers with different GBA conjugation


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ratios (G5-GBA31, G5-GBA40, G5-GBA76 in this study). BA-modified G5 PAMAM dendrimer was synthesized by the same procedure.18 The molar ratios of BA and G5 PAMAM dendrimer were 64:1 for G5-BA60 and 84:1 for G5-BA76. For GA-modified dendrimers, 1H-pyrazole-1-carboxamidine hydrochloride (0.114 and 0.135 mmol for G5-GA60 and G5-GA79, respectively) was added with DIEA (0.114 or 0.135 mmol) and G5 PAMAM dendrimer (1.04 µmol) in aqueous solution. The solution was stirred for 24 h at room temperature and intensively dialyzed against distilled water, and the product was lyophilized before further use. The average numbers of GBA and BA moieties modified on each G5 dendrimer were characterized by 1H NMR in deuterated water and deuterated dimethyl sulfoxide, respectively (Varian 699.804 MHz). The average number of residual primary amine groups on GA-modified dendrimer was characterized by a well-established ninhydrin assay.23

Preparation and characterization of polymer/siRNA polyplexes. GBA-, BA-, and GA-modified PAMAM dendrimers were mixed with 0.5 µg siRNA dissolved in diethylpyrocarbonate-treated water at different weight ratios (w/w, pH 7.0). The mixtures were diluted, votexed and incubated at room temperature for 30 min. The hydrodynamic size and polydispersity index of the polymer/siRNA polyplexes were measured by Zetasizer Nano ZS (Malvern Instrument). Zeta potential of the polyplex solution was measured using laser Doppler electrophoresis with the same Zetasizer. According to the obtained electrophoretic mobility, the zeta potential was calculated by the Smoluchowski equation.


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Gel electrophoresis and RNase protection assay. The polymer/siRNA polyplexes were prepared by mixing 0.25 µg siRNA with polymers at different weight ratios as described above. The samples were run on a 1.2% (w/v) agarose gel at 90 V for 15 min. For RNase protection assay, the siRNA or polymer/siRNA polyplexes (0.25 µg siRNA) at a polymer to siRNA weight ratio of 8:1 was treated with RNase (Yeasen, 10 µg/mL) for 10 min, followed by inactivation of the enzyme by an RNase inhibitor (Yeasen, 0.4 U/µL). The samples were then run on an agarose gel as described above.

Gene transfection. HeLa cells (a human cervical carcinoma cell line, ATCC), HeLa cells stably expressing firefly luciferase (HeLa-luc), HEK293 cells (a human embryonic kidney cell line, ATCC), NIH3T3 cells (a mouse embryo fibroblast cell line, ATCC) and U2OS cells (a human osteosarcoma cell line, ATCC) were cultured in DMEM containing 10% FBS, 100 µg/mL streptomycin, and 100 µg/mL penicillin at 37 °C. GBA-, BA-, and GA-modified PAMAM dendrimers as well as unmodified G5 PAMAM dendrimer were complexed with siRNA or plasmid DNA at different weight ratios and the polyplex solutions were diluted with 100 µL DMEM (pH 7.4) and equilibrated for 30 min at room temperature. Then polyplex solutions were further diluted with 150 µL DMEM. The cells were incubated with the polyplex solutions for 6 h, followed by replenishment with 500 µL DMEM containing 10% FBS and further culture for 18 h or 42 h. For luciferase gene silencing experiments, the transfection was conducted for 24 h, and the firefly luciferase expression in HeLa-luc cells was analyzed according to the manufacturer’s protocols (Promega). Protein concentration in each well was determined using a BCA Protein Assay Kit (TIANGEN, China). The


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data was normalized to relative luciferase light unit per mg protein (RLU/mg protein), and relative to that of untreated cells. For Bcl-2 and Phd-2 gene silencing experiments, the transfection was conducted for 24 h, and Bcl-2 or Phd-2 gene expressions in the transfected cells were analyzed by real-time reverse transcription quantitative PCR (RT-PCR). For EGFP expression in HEK293 and HeLa cells, expressions of EGFP in the transfected cells after 48 h transfection were directly observed by a fluorescent microscopy (Olympus, Japan), and the transfection efficacy (percent of EGFP positive cells and mean fluorescence intensity of the transfected cells) was quantitatively measured using flow cytometry. Commercial transfection reagent Lipo 2000 was used as a positive control. Gene transfections by Lipo 2000 was conducted according to the product’s protocols (Invitrogen). The optimized transfection conditions for GBA-, BA-, GA-modified G5 dendrimer as well as unmodified G5 PAMAM dendrimer were screened before repeated transfection experiments. Three repeats were conducted at optimal condition for each material and the data were analyzed by Student’s t-test.

Cellular uptake and intracellular trafficking of the polyplexes. Cellular uptake of the polyplexes was quantitatively analyzed by flow cytometry (BD FACSCalibur, San Jose). Generally, HeLa cells were seeded in 24-well plate and incubated overnight. The cells were transfected with the polymer/siRNA polyplexes prepared as described above for 2 h, and then trypsinized, centrifuged and re-suspended in PBS for analysis. Three repeats were conducted for each transfection and the data were analyzed by Student’s t-test.


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Co-localization of polymer/siLuc-FAM polyplexes (1.5 µg siLuc-FAM, 12 µg G5-GBA60, and the molar ratio of G5-BA60 and G5-GA60 equals to that of G5-GBA60) with endosomes was observed by a laser scanning confocal microscope (Leica SP5, Germany) in 35 mm glass bottom cell culture dish. Generally, the cells were incubated with the prepared polyplexes for 1-4 h, washed by PBS twice, and stained with 150 nM LysoTracker Red (DND-99, Invitrogen) for 30 min and further washed by PBS twice before observation by laser scanning confocal microscope.

Cytotoxicity assay. The cytotoxicity of GBA-, GA-, BA-modified dendrimers and their polyplexes with siRNA on the transfected HeLa cells were evaluated by a well-estabilished 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HeLa cells were cultured in 96-well plate and incubated with the polymer or polyplex (polymer to siRNA weight ratio is 8:1) at various mass concentrations for 24 h, and followed by a standard MTT assay. Six repeats were conducted for each sample. RT-PCR assay. The expression of Bcl-2 gene in HeLa and U2OS cells, and the expression of Phd-2 gene in NIH3T3 cells were analyzed by RT-PCR using specific primers as follows. Bcl-2,

forward:

5’-GGACACGGACAGGATTGACA-3’;

reverse:

5’-GACATCTAAGGGCATCACAG-3’. Phd-2,

forward:

5’-CCACTGGCACTCAACTAACTCA-3’;

5’-CCGAGTTCATTTAGTGCCCGTCA-3’.


reverse:

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Total RNA was isolated from the transfected HeLa, NIH-3T3, and U2OS cells, respectively. The RNA was reverse-transcribed into cDNA using cDNA Synthesis Kit (Takara, Japan). The cDNA was subjected to RT-PCR analysis targeting Bcl-2 and Phd-2 using PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara,Japan). RESULTS AND DISCUSSION Synthesis and gene silencing efficacy of GBA-modified dendrimer. G5 PAMAM dendrimers were conjugated with GBA, BA, and GA by facile chemistries as shown in Figure 1. According to the 1H NMR and ninhydrin assay (Figure S1), the average number of GBA, BA, and GA modified on each G5 dendriemr is 60, and the products were termed G5-GBA60, G5-BA60, and G5-GA60, respectively (Table S1). The theoretical molecular weights for G5-GBA60, G5-BA60 and G5-GA60 were calculated to be 38497, 30686 and 35073 Da, respectively.

Figure 1. Synthesis of G5-GBA60, G5-BA60 and G5-GA60 (a) and proposed mechanism for G5-GBA60 in efficient gene delivery (b).


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Gene silencing efficacy of G5-GBA60 was tested on HeLa-luc cells stably expressing a firefly luciferase gene. siLuc specifically targeting firefly luciferase was used as the model siRNA. The siLuc concentration was 50 nM and the polymer to siRNA weight ratio (w/w) ranged from 4:1 to 20:1. G5-BA60, G5-GA60, unmodified G5 dendrimer as well as Lipo 2000 were tested as control materials. As shown in Figure 2, G5-GBA60 showed high efficacy in gene silencing on HeLa-luc cells. The luciferase gene was reduced by nearly 80% when siLuc was delivered into the HeLa-luc cells using G5-GBA60. The introduction of scramble siNC into the cells did not cause any gene knockdown, suggesting specific gene knockdown by G5-GBA60. In comparision, the control materials G5-BA60 and G5-GA60 as well as unmodified G5 PAMAM showed extremely low efficacy in silencing the luciferase gene in HeLa-luc cells (Figure 2 and Figure S2). The efficacy of G5-GBA60 was comparable to commerical transfection reagent Lipo 2000. Even at a low siLuc dose of 5 nM, G5-GBA60 efficiently down-regulated the expression of luciferase gene by 70% in HeLa-luc cells (Figure 2b and Figure S3). It is reported that ariginine modification on dendrimer can significantly improve its efficacy in gene delivery.24-26 We also compared the efficacy of G5-GBA60 with arginine-modified G5 PAMAM dendrimer with a similar grafting ratio (G5-Arg64, 64 arginine moieties modified on each G5 dendrimer). As shown in Figure S2 and Figure S4, G5-Arg64 showed weak effect on gene silencing in comparision with G5-GBA60. These results suggest that GBA is superior to BA, GA, and arginine on improving the siRNA delivery efficacy of cationic dendrimers. G5-GBA60 also efficiently down-regulated the expression of Bcl-2 gene in HeLa and U2OS cells when delivering a siRNA targeting Bcl-2 (siBcl-2, Figure 3a and 3b), and the expression of Phd-2 gene in NIH3T3 cells when delivering a siRNA targeting


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Phd-2 (siPhd-2, Figure 3c). The efficacies of G5-GBA60 are superior to Lipo 2000 in these gene silencing experiments. For example, Lipo 2000 failed to knock down the Bcl-2 gene in U2OS cells as well as the Phd-2 gene in NIH3T3 cells, while G5-GBA60 showed high efficacy in the delivery of siRNA into these cell lines, suggesting that G5-GBA60 is applicable for a much more broad scope of siRNA delivery in comparision with the golden standard transfection reagent Lipo 2000. Besides high efficacy in the delivery of siRNA, G5-GBA60 and its complex with siRNA caused minimal cell death at various concentrations. The cells treated with the polymer or polyplex showed more than 90% cell viability (Figure 3d and Figure S5), suggesting that G5-GBA60 has both high efficacy and minimal toxicity.

Figure 2. Gene silencing efficacy of polymers with 50 nM siLuc (a) and 5 nM siLuc (b) on HeLa-Luc cells. Untreated cells were used as a negative control. Lipo 2000 was tested as a positive control. The polymers with siNC were tested to confirm the specific gene silencing. The dose of G5-GBA60 was kept constant at 4 µg. The weight ratios of G5-GBA60 and siLuc in (a) and (b) are 8:1 and 80:1, respectively. The molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60. No


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significance (NS), ***p < 0.001 analyzed by student’s t-test.

Figure 3. Gene silencing efficacy of polymers with 50 nM siBcl-2 on HeLa cells (a) and U2OS cells (b), and with 50 nM siPhd-2 on NIH3T3 cells (c). Untreated cells were used as a negative control. Lipo 2000 was tested as a positive control. The dose of G5-GBA60 was 4 µg. The molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60. The cytotoxicity of G5-GBA60 and G5-GBA60/siLuc polyplex at various polymer concentrations on HeLa cells (d). The polymer to siLuc weight ratio in the polyplex is 8:1. No significance (NS) and ***p < 0.001 analyzed by student’s t-test.


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Structure-activity relationship of G5-GBA60, G5-BA60 and G5-GA60 in siRNA delivery. To investigate the roles of guanidyl and phenyl moieties in gene delivery, we compared the behaviors of G5-GBA60, G5-BA60 and G5-GA60 on siRNA binding, cellular uptake and endosomal escape. As shown in Figure 4a, G5-GA60 efficiently bound siRNA above a polymer/siRNA weight ratio of 8:1, while G5-BA60 failed to retard the mobility of siRNA even at a weight ratio of 24:1, suggesting that the guanidinium group in G5-GA60 plays a vital role in the polyplex formation. The guanidinium can interact with the phosphate groups in siRNA by a combination of ionic and hydrogen bond interactions. The presence of phenyl groups in G5-GBA60 may cause steric hindrance in siRNA binding by the residual amine groups on dendrimer surface, as a result, G5-GBA60 showed relatively weaker siRNA binding capability in comparison with G5-GA60. It is surprising that G5-GBA60 showed the highest gene knockdown efficacy at a polymer/siRNA weight ratio of 8:1, where the polymer cannot completely bind siRNA revealed by the gel electrophoresis assay (Figure 4a). Though G5-GBA60 failed to tightly bind siRNA under an electric field, it formed stable nanoparticles around 200 nm with siRNA at all the tested weight ratios (PDI < 0.3, Figure 4b and Figure S6), and the nanoparticles are positively charged even at a weight ratio of 4:1 (Figure 4c). G5-BA60 formed large sized complexes with siRNA at a weight ratio of 8:1, which is around the isoelectric point (+6.65 mV revealed by the zeta-potential in Figure 4c). More importantly, G5-GBA60 can efficiently protect the bound siRNA from enzymatic degradation by RNase (Figure 4d). For polymer-mediated gene delivery systems, tight binding can protect the nucleic acid from enzymatic degradation, but may also lead to a problem in intracellular nucleic acid release, especially for polymers with a high density of positive charges such as dendrimers.5,27 Therefore, a balanced siRNA binding and


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release for G5-GBA60 is beneficial for its efficient siRNA delivery.

Figure 4. Agarose gel electrophoresis analysis of G5-GBA60, G5-BA60, and G5-GA60 with siRNA (a). Lane 1 represents naked siLuc. 0.25 µg siLuc was used in each sample. Hydrodynamic size (b) and zeta potential (c) of polymer/siLuc polyplexes prepared at weight ratios of 4:1, 8:1, 12:1, 16:1 and 20:1, respectively. 0.5 µg siLuc was used in each sample. RNase protection assay (d). Lane 1 represents naked siLuc. Lane 2 represents siLuc treated with RNase for 10 min, followed by enzyme inactivation with an RNase inhibitor, Lane 3-5 represent G5-GBA60/siLuc, G5-BA60/siLuc and G5-GA60/siLuc, respectively treated with RNase for 10 min, followed by RNase inactivation. 0.25 µg siLuc was used in each sample, and the polymer/siLuc weight ratio was 8:1.


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We further compared the cellular uptake of G5-GBA60/siRNA polyplex with those of G5-BA60 and G5-GA60 polyplexes. As shown in Figure 5a, G5-BA60/siRNA polyplex showed extremely weak siRNA uptake probably due to the poor siRNA complexation capability. In comparison, G5-GA60/siRNA polyplex showed moderate cellular uptake, and G5-GBA60/siRNA polyplex exhibited much stronger cell internalization than G5-GA60/siRNA. It is reported that the guanidinium group has high association affinity with the cell membrane via salt bridge and hydrogen bond interactions between guanidinium and phosphate in the phospholipids.22,28,29 The guanidyl- or arginine-modified dendrimers were expected to have high cellular uptake. By integration of a phenyl group with the guanidinium in GBA, the cellular uptake of the polyplex is significantly increased, which is attributed to the hydrophobic effect of the phenyl group. Such a phenomenon is also observed in several polymeric gene delivery systems when cationic polymers were modified with hydrophobic moieties.20,30 These results proved that the guanidyl and phenyl groups in GBA together contribute to the high cellular uptake of G5-GBA60/siRNA polyplex by the transfected cells. It is known that the endosomal escape of polyplexes is the major obstacle to achieving high efficient gene transfection.16,31 As shown in Figure 5b, G5-BA60/siRNA polyplex showed low cell internalization that is in accordance with the flow cytometry result in Figure 5a. The G5-GBA60/siLuc-FAM polyplexes were efficiently escaped from the endosomes stained by LysoTracker Red within 1 h of incubation, while most of the G5-GA60/siLuc-FAM polyplexes were co-localized with the endosome even at 4 h of incubation, suggesting the essential role of phenyl


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groups in GBA in endosomal membrane disruption. Taken together, the guanidyl group in G5-GBA60 contributed to efficient siRNA binding and cellular uptake, while the phenyl group in the polymer played essential roles in cellular uptake and endosomal escape.

Figure 5. Cellular uptake of polymer/siLuc-FAM polyplexes by HeLa-luc cells for 2 h (a). Confocal image of HeLa-luc cells treated with G5-GBA60/siLuc-FAM (b), G5-GA60/siLuc-FAM (c), and G5-BA60/siLuc-FAM (d) polyplexes for 1 h, 2 h, or 4


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h, respectively. The acidic compartments were stained with Lysotracker Red. 0.5 µg siLuc-FAM was used in each sample. The weight ratio of G5-GBA60 to siLuc-FAM was 8:1. The molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60 in the polyplexes. ***p < 0.001 analyzed by student’s t-test.

Efficacy of G5-GBA60 in DNA delivery. We further tested the efficacy of G5-GBA60 in DNA delivery. A plasmid encoding EGFP was used as the model DNA. The DNA dose in each transfection was 0.5 µg and the polymer dose ranged from 2 to 10 µg. The EGFP expressions in HEK293 and HeLa cells mediated by G5-GBA60 were tested and compared with G5-BA60, G5-GA60, Lipo 2000 as well as unmodified G5 dendrimer. As shown in Figure 6 and Figure S7, G5-GBA60 showed high efficacy (according to both the percent of positive EGFP cells and mean fluorescence intensity of the transfected cells) in transfecting EGFP plasmid in both HEK293 cells and HeLa cells. For example, G5-GBA60 transfected more than 80% HEK293 cells and 60% HeLa cells at its optimal condition. The efficacy is much superior to G5-BA60, G5-GA60 and unmodified G5 PAMAM dendrimer (Figure S7 and Figure S8), and is comparable to Lipo 2000. The results further confirmed the synergistic effect of guanidyl and phenyl groups in dendrimer-mediated gene delivery.


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Figure 6. EGFP transfection efficacy of polymers on HEK293 (a) and HeLa cells (b) for 48 h. Lipo 2000 was tested as a positive control. The columns in (b) and (d) represent EGFP positive cells (%) and the diamonds represent mean fluorescence intensity. 0.5 µg EGFP plasmid was used in each sample. The G5-GBA60 to DNA weight ratio is 16:1 and the molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60 in the polyplexes. No significance (NS), *p < 0.05 and ***p < 0.001 analyzed by student’s t-test.

The effect of GBA grating ratio on the gene transfection efficacy. It is known that the efficacy of surface-engineered dendrimers in gene delivery is influenced by the surface grafting ratio. To investigating the effect of GBA grating ratio on the efficacy


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of GBA-modified dendrimer, we synthesized a series of G5-GBA conjugate analogues. According to the 1H NMR analysis in Figure S9, the products were termed G5-GBA31, G5-GBA40, G5-GBA76, respectively. It is observed that the efficacy of G5-GBA conjugate increased with increasing GBA grafting ratio (Figure 7a). G5-GBA76 showed the highest efficacy in siRNA delivery. The polymer with a higher GBA grafting ratio achieved efficient gene transfection at a lower dose. For example, G5-GBA76 down-regulated the firefly luciferase gene by more than 80% even at a low polymer dose of 2 µg, while the other analogue materials only showed poor or moderate gene silencing efficacies at the same dose. High GBA grating ratios in the polymer are also beneficial for efficient EGFP plasmid expression in the cells (Figure 7b). We also synthesized dendrimers modified with average numbers of 79 GA and 76 BA, respectively. As shown in Figure S10, both G5-GA79 and G5-BA76 showed poor efficacies in the delivery of siRNA and DNA, which were dramatically lower than G5-GBA60 and G5-GBA76. These results suggested that the high efficacy of G5-GBA conjugates in siRNA and DNA delivery is not due to optimized GBA grafting ratio. It is worth noting that guanidine-engineered polymers such as arginine-modified dendrimers were reported with high efficacy in the delivery of plasmid DNA,22,24-26,32,33 however, the polymers showed only weak efficacy in the delivery of siRNA,18 and were not available for the delivery of proteins and peptides. In this study, the developed GBA-modified dendrimers such as G5-GBA60 showed high efficacy in the delivery of both plasmid DNA and siRNA, and were recently reported with dramatic efficacy in the delivery of proteins and peptides,34 suggesting that G5-GBA60 is superior to these arginine-modified dendrimers and is widely applicable for the delivery of biomacromolecules into various cell lines.


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Figure 7. The effect of GBA grafting ratio on efficacy of G5-GBA conjugate in luciferase gene silencing in HeLa-luc cells for 24 h (a), and in EGFP expression in HeLa cells for 48 h (b). 0.5 µg siLuc or EGFP plasmid was used in each sample. The columns in (b) represent EGFP positive cells (%) and the diamonds represent mean fluorescence intensity. CONCLUSIONS In summary, we synthesized a new type of polymer for efficient siRNA and DNA delivery by a rational design strategy. The dendrimer modified with GBA showed high efficacy in the delivery of both siRNA and DNA into various cells. The phenyl and guanidyl moieties in GBA modified on dendrimer surface exhibited a synergistic effect in addressing the extracellular and intracellular barriers in gene delivery. The guanidyl group in the polymer contributed to efficient nucleic acid binding and cellular uptake, and the phenyl group in the polymer was essential for efficient cellular uptake and endosomal escape. Higher GBA modification on the dendrimer yielded polymers with a higher transfection efficacy. In addition, the GBA modified dendrimer and its polyplex showed minimal toxicity on the transfected cells. This study provided a facile chemistry to design polymers with high efficacy and low toxicity. It is worth noting that the positively charged polyplexes formed between


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GBA-modified polymers and nucleic acids might have short blood circulating time when administrated to animals. Before the polymers can be used for in vivo gene therapy, we need to shield the positive charges on the polyplexes by surface modification or nanofabrication, for example, we can coat the polyplexes with a layer of anionic polymers such as hyaluronic acid for targeted gene therapy,35,36 or encapsulate the polyplexes within a degradable PLGA nanocapsule or hydrogel for local gene delivery.37,38 ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Characterization of the G5-GBA60 and its analogues, G5-BA60 and G5-GA and the in vitro gene transfection efficacies for these materials. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21474030) and the Science and Technology Commission of Shanghai Municipality (17XD1401600 and 148014518). REFERENCES (1) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Nat. Mater. 2013, 12, 967-977. (2) Lachelt, U.; Wagner, E. Chem. Rev. 2015, 115, 11043-11078.


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(3) Li, J.; Wang, W.; He, Y.; Li, Y.; Yan, E. Z.; Zhang, K.; Irvine, D. J.; Hammond, P. T. ACS nano 2017, 11, 2531-2544. (4) Roh, Y. H.; Deng, J. Z.; Dreaden, E. C.; Park, J. H.; Yun, D. S.; Shopsowitz, K. E.; Hammond, P. T. Angew. Chem. Int. Ed. Engl. 2016, 55, 3347-3351. (5) Wang, H.; Wang, Y.; Wang, Y.; Hu, J.; Li, T.; Liu, H.; Zhang, Q.; Cheng, Y. Angew. Chem. Int. Ed. Engl. 2015, 54, 11647-11651. (6) Wei, H.; Schellinger, J. G.; Chu, D. S.; Pun, S. H. J. Am. Chem. Soc. 2012, 134, 16554-16557. (7) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug. Discov. 2005, 4, 581-593. (8) Wang, H.; Huang, Q.; Chang, H.; Xiao, J.; Cheng, Y. Biomaterials science 2016, 4, 375-390. (9) Newland, B.; Zheng, Y.; Jin, Y.; Abu-Rub, M.; Cao, H.; Wang, W.; Pandit, A. J. Am. Chem. Soc. 2012, 134, 4782-4789. (10) Lavertu, M.; Methot, S.; Tran-Khanh, N.; Buschmann, M. D. Biomaterials 2006, 27, 4815-4824. (11) Kiang, T.; Wen, J.; Lim, H. W.; Leong, K. W. Biomaterials 2004, 25, 5293-5301. (12) Ma, P. L.; Lavertu, M.; Winnik, F. M.; Buschmann, M. D. Biomacromolecules 2009, 10, 1490-1499. (13) Engler, A. C.; Bonner, D. K.; Buss, H. G.; Cheung, E. Y.; Hammond, P. T. Soft Matter 2011, 7, 5627-5637. (14) Breunig, M.; Lungwitz, U.; Liebl, R.; Goepferich, A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14454-14459. (15) Miyata, K.; Nishiyama, N.; Kataoka, K. Chem. Soc. Rev. 2012, 41, 2562-2574. (16) Uchida, H.; Miyata, K.; Oba, M.; Ishii, T.; Suma, T.; Itaka, K.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2011, 133, 15524-15532. (17) Wang, F.; Deng, L.; Hu, J.; Cheng, Y. Bioconjug. Chem. 2016, 27, 638-646. (18) Liu, H.; Chang, H.; Lv, J.; Jiang, C.; Li, Z.; Wang, F.; Wang, H.; Wang, M.; Liu, C.; Wang, X.; Shao, N.; He, B.; Shen, W.; Zhang, Q.; Cheng, Y. Sci. Rep. 2016, 6, 25069. (19) Yang, J.; Zhang, Q.; Chang, H.; Cheng, Y. Chem. Rev. 2015, 115, 5274-5300. (20) Liu, Z.; Zhang, Z.; Zhou, C.; Jiao, Y. Prog. Polym. Sci. 2010, 35, 1144-1162. (21) Zeng, H.; Little, H. C.; Tiambeng, T. N.; Williams, G. A.; Guan, Z. J. Am. Chem. Soc. 2013, 135, 4962-4965. (22) Wang, F.; Wang, Y.; Wang, H.; Shao, N.; Chen, Y.; Cheng, Y. Biomaterials 2014, 35, 9187-9198. (23) Wang, M.; Liu, H.; Li, L.; Cheng, Y. Nat. Commun. 2014, 5, 3053. (24) Choi, J. S.; Nam, K.; Park, J. Y.; Kim, J. B.; Lee, J. K.; Park, J. S. J. Control. Release 2004, 99, 445-456. (25) Kim, T. I.; Bai, C. Z.; Nam, K.; Park, J. S. J. Control. Release 2009, 136, 132-139. (26) Nam, H. Y.; Nam, K.; Hahn, H. J.; Kim, B. H.; Lim, H. J.; Kim, H. J.; Choi, J. S.; Park, J. S. Biomaterials 2009, 30, 665-673. (27) Chang, H.; Zhang, Y.; Li, L.; Cheng, Y. J. Mater. Chem. B Mater. Biol. Med. 2015, 3, 8197-8202. (28) Pantos, A.; Tsogas, I.; Paleos, C. M. Biochim. Biophys. Acta 2008, 1778, 811-823. (29) Samanta, K.; Jana, P.; Backer, S.; Knauer, S.; Schmuck, C. Chem. Commun. (Camb.) 2016, 52, 12446-12449. (30) Nelson, C. E.; Kintzing, J. R.; Hanna, A.; Shannon, J. M.; Gupta, M. K.; Duvall, C. L. ACS nano 2013, 7, 8870-8880. (31) Nishiyama, N.; Iriyama, A.; Jang, W. D.; Miyata, K.; Itaka, K.; Inoue, Y.; Takahashi, H.; Yanagi, Y.; Tamaki, Y.; Koyama, H.; Kataoka, K. Nat. Mater. 2005, 4, 934-941. (32) Luo, K.; Li, C.; Li, L.; She, W.; Wang, G.; Gu, Z. Biomaterials 2012, 33, 4917-4927.


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A Combination of Guanidyl and Phenyl Groups on Dendrimer Enables Efficient siRNA and DNA Delivery Hong Chang, Jia Zhang, Hui Wang, Jia Lv, Yiyun Cheng*

For Table of Contents Use Only


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TOC 86x34mm (300 x 300 DPI)


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Figure 1. Synthesis of G5-GBA60, G5-BA60 and G5-GA60 (a) and proposed mechanism for G5-GBA60 in efficient gene delivery (b). 177x70mm (300 x 300 DPI)


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Figure 2. Gene silencing efficacy of polymers with 50 nM siLuc (a) and 5 nM siLuc (b) on HeLa-Luc cells. Untreated cells were used as a negative control. Lipo 2000 was tested as a positive control. The polymers with siNC were tested to confirm the specific gene silencing. The dose of G5-GBA60 was 4 µg. The molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60. No significance (NS), ***p < 0.001 analyzed by student’s t-test. 177x58mm (300 x 300 DPI)


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Figure 3. Gene silencing efficacy of polymers with 50 nM siBcl-2 on HeLa cells (a) and U2OS cells (b), and with 50 nM siPhd-2 on NIH3T3 cells (c). Untreated cells were used as a negative control. Lipo 2000 was tested as a positive control. The dose of G5-GBA60 was 4 µg. The molar concentration of G5-BA60 and G5GA60 equals to that of G5-GBA60. The cytotoxicity of G5-GBA60 and G5-GBA60/siLuc polyplex at various polymer concentrations on HeLa cells (d). The polymer to siLuc weight ratio in the polyplex is 8:1. No significance (NS) and ***p < 0.001 analyzed by student’s t-test. 177x126mm (300 x 300 DPI)


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Figure 4. Agarose gel electrophoresis analysis of G5-GBA60, G5-BA60, and G5-GA60 with siRNA (a). Lane 1 represents naked siLuc. 0.25 µg siLuc was used in each sample. Hydrodynamic size (b) and zeta potential (c) of polymer/siLuc polyplexes prepared at weight ratios of 4:1, 8:1, 12:1, 16:1 and 20:1, respectively. 0.5 µg siLuc was used in each sample. RNase protection assay (d). Lane 1 represents naked siLuc. Lane 2 represents siLuc treated with RNase for 10 min, followed by enzyme inactivation with an RNase inhibitor, Lane 3-5 represent G5-GBA60/siLuc, G5-BA60/siLuc and G5-GA60/siLuc, respectively treated with RNase for 10 min, followed by RNase inactivation. 0.25 µg siLuc was used in each sample, and the polymer/siLuc weight ratio was 8:1. 203x145mm (300 x 300 DPI)


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Figure 5. Cellular uptake of polymer/siLuc-FAM polyplexes by HeLa-luc cells for 2 h (a). Confocal image of HeLa-luc cells treated with G5-GBA60/siLuc-FAM (b), G5-GA60/siLuc-FAM (c), and G5-BA60/siLuc-FAM (d) polyplexes for 1 h, 2 h, or 4 h, respectively. The acidic compartments were stained with Lysotracker Red. 0.5 µg siLuc-FAM was used in each sample. The weight ratio of G5-GBA60 to siLuc-FAM was 8:1. The molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60 in the polyplexes. ***p < 0.001 analyzed by student’s t-test. 203x193mm (300 x 300 DPI)


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Figure 6. EGFP transfection efficacy of polymers on HEK293 (a) and HeLa cells (b) for 48 h. Lipo 2000 was tested as a positive control. The columns in (b) and (d) represent EGFP positive cells (%) and the diamonds represent mean fluorescence intensity. 0.5 µg EGFP plasmid was used in each sample. The G5-GBA60 to DNA weight ratio is 16:1 and the molar concentration of G5-BA60 and G5-GA60 equals to that of G5-GBA60 in the polyplexes. No significance (NS), *p < 0.05 and ***p < 0.001 analyzed by student’s t-test. 177x134mm (300 x 300 DPI)


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Figure 7. The effect of GBA grafting ratio on efficacy of G5-GBA conjugate in luciferase gene silencing in HeLa-luc cells for 24 h (a), and in EGFP expression in HeLa cells for 48 h (b). 0.5 µg siLuc or EGFP plasmid was used in each sample. The columns in (b) represent EGFP positive cells (%) and the diamonds represent mean fluorescence intensity. 80x27mm (300 x 300 DPI)


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A Combination of Guanidyl and Phenyl Groups on a Dendrimer

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