Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

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Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery

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Bingwei He, Yitong Wang, Naimin Shao, Hong Chang, Yiyun Cheng ⇑

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Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, PR China

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a r t i c l e

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Article history: Received 9 February 2015 Received in revised form 17 April 2015 Accepted 24 April 2015 Available online xxxx Keywords: Dendrimer Polymer Gene transfection siRNA Fluorination

a b s t r a c t Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1–2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery. Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

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1. Introduction

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Cationic polymers are widely used as non-viral gene vectors due to advantages such as lack of immunogenicity, facile synthesis, flexibility and degradability [1–4]. Numerous polymers including polyethylenimine (PEI) [5–8], dendrimers [9,10], chitosan [11], polylysine [12] and poly(b-amino ester) [13–15] were developed as vectors for the delivery of DNA and small interfering RNA (siRNA). However, the cationic polymers such as PEI and cationic dendrimers are usually criticized by relatively low transfection efficacy and serious cytotoxicity [16]. To improve their performances in gene delivery, polymers were modified with various ligands such as lipids [17,18], amino acids [19,20], saccharides [21,22] and peptides [23,24]. These functionalized polymers show promising potential in gene delivery, but are still less than ideal [16]. Fluorination was recently developed as a new strategy to dramatically improve the transfection efficacy of cationic polymers [25–27]. Fluorinated polyamidoamine (PAMAM) and

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⇑ Corresponding author.

polypropyleneimine (PPI) dendrimers show superior efficacies to several commercial transfection reagents on commonly used cell lines [25,26]. These materials can achieve efficient gene transfection at low nitrogen to phosphorous (N/P) ratios, which ensures low cytotoxicity on the transfected cells. The improved transfection efficacy of polymers after fluorination is attributed to increased cellular uptake, serum stability, endosomal escape and easier intracellular DNA disassociation from the polymer [25]. Besides cationic dendrimers, fluorination also improves the transfection efficacy of traditional polymers such as branched PEI [28]. However, a high density of fluorous chains must be conjugated on a single polymer in these systems (Fig. 1). For example, a generation 5 (G5) PAMAM dendrimer with 128 surface amine groups should be grafted with 68 heptafluorobutyric acid moieties to achieve efficient gene transfection. Conjugation of excess fluorous chains will lead to a congested polymer surface, which prevents further modification of the polymers with other functional ligands (Fig. 1b). Here we propose a new strategy to prepare fluorinated polymers. 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT), a compound with double fluoroalkyl chains was conjugated on cationic polymers via a facile synthesis route (Fig. 1d). Due to

E-mail address: [email protected] (Y. Cheng). http://dx.doi.org/10.1016/j.actbio.2015.04.037 1742-7061/Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

Please cite this article in press as: He B et al. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.04.037

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Fig. 1. Synthesis and structures of CBT-modified polymers. (a) Cationic polymer, (b) fluorinated polymer, (c) CBT-modified polymer, (d) synthetic route of CBT-modified polymer, n denotes the average number of primary amine on the polymer, x denotes the average number of CBT moieties modified on the polymer, (e) PAMAM dendrimer, and (f) bPEI1.8K, m represents the number of repeated units in bPEI1.8K.

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the double-tailed structure of CBT and its relatively longer fluorous chains (13 fluorine atoms on each chain) compared to heptafluorobutyric acid (7 fluorine atoms on each chain), we expect to construct polymers grafted with minimal fluorous chains (average number of less than two) for efficient gene delivery. Generation 1 (G1, Fig. 1e), generation 2 (G2) and G5 PAMAM dendrimers and branched PEI with a molecular weight of 1800 Da (bPEI1.8K, Fig. 1f) were used as model polymers. Both PAMAM dendrimers and branched PEIs are synthetic polymers with branched structures and a high density of amine groups [29–31,8,32,33]. They were widely explored as non-viral gene vectors in recent years [16]. The aims of this study are to generate a new class of dendrimers or PEIs grafted with minimal fluorous chains for efficient gene delivery, and to extend the applicable scope of fluorination on improving the transfection efficacy of cationic polymers.

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2. Materials and methods

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2.1. Materials

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Ethylenediamine-cored G1 (molecular weight is 1428 Da; 8 surface primary amine groups), G2 (molecular weight is 3256 Da; 16 surface primary amine groups), G5 (molecular weight is 28826 Da; 128 surface primary amine groups) PAMAM dendrimers were purchased from Dendritech, Inc. (Midland, MI). CBT and bPEI1.8K were obtained from Sigma–Aldrich (St Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Lipofectamine 2000 (Lipo 2000) was purchased from Life Technologies (Shanghai, China). SuperFect and PolyFect were obtained from Qiagen (Germany). jetPEI was purchased from Polyplus Transfection (France). siRNA targeting firefly luciferase (siLuc, sense strand, 5’-CCCUAUUCUCCUUCUUCGCdTdT-3’) and scrambled siRNA non-specific to firefly luciferase (siNC, sense strand, 5’-UUCUCCGAACGUGUCACGUdTdT-3’) were purchased

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from GenePharma Co., Ltd (Shanghai, China). G1, G2 and G5 PAMAM dendrimers were received in aqueous solutions and the materials were lyophilized for further modification. All other chemicals were used as received without further purification.

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2.2. Synthesis and characterization of CBT-modified polymers

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G1, G2, G5 PAMAM dendrimers and bPEI1.8K were dissolved in distilled water. CBT dissolved in ethanol was added into the polymer solutions at a molar ratio of 2:1 (4:1 for G2 dendrimer, 8:1 and 10:1 for G5 dendrimer were also synthesized to investigate the effect of conjugation ratio on transfection efficacy). The mixtures were stirred at 90 °C for 24 h and extensively dialyzed against distilled water. The products were lyophilized for further characterizations and gene transfection experiments. The number of residual amine groups on each dendrimer or PEI were estimated by a well-established ninhydrin assay [25]. Generally, 85 mg ninhydrin and 15 mg hydrindantin were dissolved in 10 mL ethylene glycol-monomethyl ether. 200 lL of the solution was added into 200 lL test samples in sodium acetate buffer (0.2 M, pH = 5.4) and the volume of the mixture was fixed to 600 lL using distilled water. The solutions were incubated in boiling water for 10 min. After cooling to room temperature, 600 lL ethanol/water (v/ v = 60/40) mixture was added to each sample. Absorbance of the samples was measured at 570 nm using a UV–Vis spectrophotometer. Unmodified dendrimers and PEI of different concentrations were tested to obtain standard curves for each polymer (G1, Abs = 1.1308C + 0.0627, R2 = 0.9899; G2, Abs = 1.3412C + 0.0321, R2 = 0.9917; bPEI1.8K, Abs = 0.3687C + 0.1032, R2 = 0.9917. Abs is the absorbance of sample at 570 nm, C is the concentration of primary amine groups, mM). CBT-modified G5 PAMAM dendrimers were characterized by fluorine element analysis (CAS Shanghai Institute of Organic Chemistry, China) because there are 128 primary amine groups on each G5 dendrimer and only a few CBT molecules were modified on each G5 dendrimer. To minimize the

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analysis errors, we used a fluorine element analysis instead of the ninhydrin assay to measure the number of CBT modified on each G5 dendrimer. The average number of CBT molecules modified on each polymer is listed in Table 1.

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2.3. Preparation and characterization of polyplexes

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Polymers and nucleic acids (EGFP plasmid or siLuc, siLuc was dissolved in diethypyrocarbonate-treated water) were mixed in distilled water at various weight ratios (w/w). The formed polyplexes were incubated at room temperature for 30 min and then diluted with DNA loading buffer. Nucleic acid binding abilities of the polymers were evaluated by an agarose gel (Biowest, Spain) retardation assay. The polyplexes were run on agarose gels at 100 V (1% w/v gel, 40 min for DNA and 1.5% w/v gel, 15 min for siRNA). The ethidium bromide stained gels were photographed using an UVIpro Gel documentation system (Tanon-2500, China). The size and zeta-potential of the prepared polyplexes in aqueous solutions were measured by dynamic light scattering and laser doppler velocimetry (LDV) in combination with phase analysis light scattering (PALS) respectively using a Zetasizer Nano ZS90 (Malvern, UK) at room temperature.

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2.4. Cell culture

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HEK293 (a human embryonic kidney cell line, ATCC) and HeLa (a human cervical carcinoma cell line, ATCC) cells were used to evaluate the DNA transfection efficacies of the fluorinated polymers. EGFP and luciferase plasmids were used as reporter genes. MDA-MB-231 (a human breast carcinoma cell line) cells and HeLa cells stably expressing firefly luciferase (MDA-MB-231-luc and HeLa-luc) were used to investigate the gene silencing efficacies of synthesized materials. HEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Inc.) and MDA-MB-231 cells were cultured in minimum essential medium (MEM, GIBCO Inc.). The mediums were supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO Inc.), 100 units/mL penicillin sulfate and 100 lg/mL streptomycin. The cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.

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2.5. Luciferase and EGFP transfection experiments

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HeLa and HEK293 cells were seeded in 24-well plates at a density around 104 cells per well. The cells were allowed to grow until 80% confluence. 0.8 lg EGFP or luciferase plasmids were mixed with polymers at various weight ratios and the mixtures were diluted with 100 lL DMEM containing 10% FBS. The polyplex solutions were incubated at room temperature for 30 min and further diluted with 150 lL medium. The cells were then incubated with the polyplex containing mediums at 37 °C for 6 h, followed by

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Table 1 Characterization of the synthesized CBT-modified polymers.

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Polymer

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G1 PAMAM G2 PAMAM G5 PAMAM bPEI1.8K G1-CBT1.9 G2-CBT1.5 G5-CBT1.3 bPEI1.8K-CBT1.3

0 0 0 0 1.9 1.5 1.3 1.3

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The average number of CBT moieties conjugated on each polymer determined by ninhydrin assay or fluorine element analysis. b The molecular weights of the CBT-modified polymers are calculated according to theoretical molecular weight of the polymer and the average number of CBT moieties conjugated on each polymer.

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addition of 500 lL fresh medium into each well. After the cells were further cultured for 42 h, EGFP expressions in the cells were observed with a fluorescent microscopy (Olympus, Japan) and quantitatively analyzed by flow cytometry (BD FACSCalibur, San Jose). Bioluminescence assay of luciferase enzyme activity in the transfected cells was conducted according to the manufacturer’s protocols (Promega). The protein concentration in each well was measured using a BCA protein assay kit (Beyotime). The luciferase enzyme activity is expressed in terms of relative luciferase light units per milligram of protein (RLU/mg protein). Unmodified polymers such as G1, G2, G5 PAMAM dendrimer and bPEI1.8K, as well as commercial transfection reagents including Lipo 2000, SuperFect, PolyFect and jetPEI were tested as controls. Optimal transfection conditions for these materials were screened. Three repeats were conducted for each material.

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2.6. Gene silencing experiments

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HeLa-luc and MDA-MB-231-luc cells were seeded in 24-well plates at a density of 104 cells per well. The cells were allowed to grow until 50% confluence. 0.5 lg siRNA (siLuc or siNC) in diethypyrocarbonate-treated water (1 O.D. siRNA dissolved in 125 lL water) was mixed with polymers at different weight ratios. The formed polyplexes were diluted in 100 lL serum-free DMEM, incubated at room temperature for 30 min, and further diluted with 150 lL medium. The cells were then incubated with the polyplex solution for 6 h, followed by the addition of 500 lL medium containing 10% FBS. After 18 h incubation, the cells were washed and lysed. Then the luciferase enzyme activity was measured as described above. The relative luciferase enzyme activity of transfected cells is expressed by 100 RLUsiLuc/RLUN.C. (%), where RLUsiLuc and RLUN.C. are the luciferase activities for transfected cells and untreated cells, respectively. Unmodified polymers and Lipo 2000 were tested as a positive control. Optimal transfection conditions for these materials were screened. Three repeats were conducted for each material.

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2.7. Cytotoxicity of the fluorinated polymers

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Cytotoxicity of the fluorinated polymers on HEK293 and HeLa cells was performed by MTT assay. Generally, HEK293 and HeLa cells were plated in 96-well plates at a density around 104 cells per well and incubated for 24 h at 37 °C. The cells were then exposed to fluorinated polymers at different concentrations. After incubation for 48 h, 100 lL MTT containing medium (0.5 mg/mL) was added to each well, followed by incubation for 2 h. The medium was then replaced by 150 lL DMSO and the plate was shaken for 10 min prior to detection at 490 nm using a microplate reader (Thermo Fisher Scientific, U.S.). Viability of the cells was expressed by 100 Abstest/AbsN.C. (%), where Abstest and AbsN.C. are the absorbances for material-treated cells and untreated cells, respectively. Five repeats were conducted for each sample.

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2.8. In vivo gene silencing

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Four-week-old female BALB/c nude mice were obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The mice were housed in standard cages in a specific pathogen-free facility on a 12 h light/dark cycle at 18–22 °C with ad libitum access to food and water for two weeks. All the animal experiments were conducted according to the regulations of Association for Assessment and Accreditation of Laboratory Animal Care in Shanghai and were approved by the East China Normal University Center for Animal Research. Generally, HeLa-luc (106) cells suspended in PBS were implanted into the mice (20–25 g). When the tumor size reaches

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around 100 mm3, the mice were anesthetized using isoflurane, and intraperitoneally injected with 200 lL D-luciferin potassium salt (15 mg/mL). The mice were observed by in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton) after 5 min to measure and record the bioluminescence from implanted HeLa-luc cells. The mice were then divided into 4 groups with equivalent levels of bioluminescence (3 mice in each group) and administrated with siLuc, G1/siLuc or G1-CBT1.9/siLuc complexes (8 lg siLuc, the polymer/siLuc weight ratio is 28:1) dissolved in 100 lL 5% glucose by injection into the tumor. The mice treated with 100 lL 5% glucose were used as controls. The treatments were repeated every day and a total number of three injections were administrated for each mouse. At the fourth day, bioluminescence images of the mice were measured as described above. The relative luciferase activity was expressed by the ratio of measured luminescence after and before treatment (%).

2.9. Statistical analysis

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The data were presented as mean ± standard deviation. Student’s t-test was used to analyze statistically significant differences. p-values less than 0.05 were considered significant.

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3. Results and discussion

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3.1. Characterization of CBT-modified polymers and their complexes with nucleic acid

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Dendrimer or PEI was reacted with CBT by a facile reaction as shown in Fig. 1d. According to the ninhydrin assay and fluorine element analysis, the average numbers of CBT moieties conjugated on G1, G2, G5 PAMAM dendrimers and bPEI1.8K are 1.9, 1.5, 1.3 and 1.3, respectively (Table. 1). The products are termed G1-CBT1.9,

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Fig. 2. Agarose gel retardation assay used to determine the DNA binding capacity of CBT-modified polymers and unmodified polymers. (a) G1 and G1-CBT1.9, (b) G2 and G2CBT1.5, (c) G5 and G5-CBT1.3, and (d) bPEI1.8K and bPEI1.8K-CBT1.3. The weight ratio of polymer and DNA ranges from 0.5:1 to 8:1.

Fig. 3. Hydrodynamic size (a) and zeta potential (b) values of the prepared G1-CBT1.9/DNA and G2-CBT1.5/DNA polyplexes. The weight ratio of polymer and DNA ranges from 0:1 to 20:1. 0.8 lg DNA was used in each sample.


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G2-CBT1.5, G5-CBT1.3, and bPEI1.8K-CBT1.3 respectively. Structures of these materials are similar to that of a double-tailed surfactant (Fig. 1c). Due to strong fluorine–fluorine interactions, the CBTmodified polymers should be able to assemble into nanostructures in aqueous solutions (Fig. S1). All the synthesized materials show slightly lower DNA binding capacity as compared to non-modified polymers in the agarose gel retardation assay (Fig. 2), which is attributed to decreased charge densities on the synthesized polymers after CBT modification. Though decreased DNA binding capacity, the CBT-modified polymers such as G1-CBT1.9 and G2-CBT1.5 are able to condense DNA into positively charged nanoparticles (>20 mV) around 300 nm at weight ratios above 4:1 (Fig. 3). In addition, G1-CBT1.9 and G2-CBT1.5 are able to bind siRNA (siLuc) and form small nanoparticles in aqueous solutions (Fig. S2). These physicochemical properties encourage us to further investigate the DNA and siRNA delivery efficacy of CBT-modified polymers.

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3.2. In vitro DNA delivery efficacies of CBT-modified polymers

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The transfection efficacies of CBT-modified polymers on HEK293 and HeLa cells were evaluated using EGFP and luciferase plasmids as reporter genes. The optimal transfection condition for each material is screened on both cell lines (Figs. S3 and S4).

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Fig. 6. Transfection efficacies of CBT-modified polymers on HeLa cells using EGFP plasmid as the reporter gene. Unmodified polymers were used as controls. The optimal weight ratios for the polymers were selected according to the screening results in Fig. S4. Three repeats were conducted for each sample. Diamonds represent mean fluorescence intensity. ⁄⁄⁄p < 0.005 according to positive EGFP cells (%).

Fig. 4. Fluorescent microscopy images of HEK293 cells transfected with EGFP plasmids using G1-CBT1.9, G2-CBT1.5, G5-CBT1.3, and bPEI1.8K-CBT1.3 as vectors. Unmodified G1, G2, G5 PAMAM dendrimers and bPEI1.8K were tested as controls. The optimal weight ratios for the polymers were selected according to the screening results in Fig. S3.

Fig. 5. Transfection efficacies of CBT-modified polymers on HEK293 cells using EGFP (a) and luciferase (b) as reporter genes. Unmodified polymers were used as controls. The optimal weight ratios for the polymers were selected according to the screening results in Fig. S3. Three repeats were conducted for each sample. Diamonds in (a) represent mean fluorescence intensity. ⁄⁄⁄p < 0.005, ⁄⁄p < 0.01 according to positive EGFP cells (%).


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Fig. 7. Cellular uptake of YOYO-1-labeled DNA delivered by G1, G1-CBT1.9, G2 and G2-CBT1.5, respectively.

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For polymeric gene vectors, transfection and cytotoxicity are usually strongly associated. High molecular weight polymers have relatively high transfection efficacy but non-negligible cytotoxicity, while low molecular weight ones have low transfection efficacy and minimal cytotoxicity [7]. There is an urgent need to design efficient gene vectors based on low molecular weight polymers [34]. CBT modification can significantly increase the transfection efficacy of low molecular weight polymers. As shown in Figs. 4 and 5, CBT modification dramatically improves the transfection

Fig. 9. In vitro gene silencing efficacies of CBT-modified polymers on HeLa-luc cells. Unmodified polymers are tested as controls. Lipo 2000 is used as a positive control according to the product’s protocol. The optimal weight ratios for the polymers were selected according to the screening results in Fig. S8. 50 nM siLuc or siNC is used in each transfection. Three repeats were conducted for each sample. ⁄⁄⁄p < 0.005, ⁄p < 0.05 and n.s. non-statistical differences according to relative luciferase activity.

efficacy of low molecular weight polymers including G1 (1430 Da), G2 (3256 Da), and bPEI1.8K (1800 Da) on HEK293 cells. Take G1 dendrimer for example, G1-CBT1.9 transfected 73% HEK293 cells with EGFP at its optimal condition (Fig. 5a), while unmodified G1 dendrimer transfected less than 1% of the cells.

Fig. 8. Cytotoxicities of CBT-modified polymers. (a) and (b) Cytotoxicities of CBT-modified polymers and unmodified polymers at their optimal transfection conditions on HEK293 (a) and HeLa (b) cells. (c) and (d) Cytotoxicities of CBT-modified polymers, G5 PAMAM dendrimer and bPEI25K at different polymer concentrations on HEK293 (c) and HeLa (d) cells.


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Fig. 10. In vitro gene silencing efficacies of CBT-modified polymers on MDA-MB231-luc cells. Unmodified polymers are tested as controls. Lipo 2000 is used as a positive control according to the product’s protocol. The optimal weight ratios for G1-CBT1.9, G2-CBT1.5, and bPEI1.8K-CBT1.3 are 14:1, 20:1, and 4:1, respectively. 50 nM siLuc or siNC is used in each transfection. Three repeats were conducted for each sample. ⁄⁄⁄p < 0.005, ⁄⁄p < 0.01 and n.s. non-statistical differences according to relative luciferase activity.

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The efficacy of G1-CBT1.9 on luciferase expression is more than two orders of magnitude higher than that of G1 dendrimer (Fig. 5b). The efficacy of G1-CBT1.9 is superior to that of commercial transfection reagents including SuperFect and PolyFect, and approaches the efficacies of Lipo 2000 and jetPEI (Fig. S5). Similarly, bPEI1.8K-CBT1.3 transfected much more HEK293 cells than unmodified bPEI1.8K. Besides low molecular weight polymers, CBT modification in improving transfection efficacy also works on high molecular weight polymers such as G5 PAMAM dendrimer. G5-CBT1.3 shows much higher efficacy than unmodified G5 dendrimer. CBT modification also significantly improves the EGFP expression efficacy of these polymers on HeLa cells (Fig. 6). It is surprising that CBT-modified high generation dendrimers show less efficient EGFP expressions than CBT-modified low generation ones on HeLa cells. For example, G1-CBT1.9 transfected nearly 50% HeLa cells, while G5-CBT1.3 only transfected 13.22% cells. This phenomenon is distinct from that observed on HEK293 cells (Fig. 5). Probably, CBT-modified high generation dendrimers such

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as G5-CBT1.3 need a higher polymer/DNA weight ratio to transfect the HeLa cells than HEK293 cells. A higher weight ratio will cause increased cytotoxicity on the transfected cells. Exact reason behind this phenomenon needs further investigation. The transfection efficacy of a gene material depends on several parameters such as complex formation, cellular uptake, endosomal escape and intracellular DNA unpacking [16]. As shown in Fig. 7, CBT modification significantly improves the cellular uptake efficacy of dendrimer/DNA complexes at 12 h and 24 h, which is beneficial for efficient gene delivery (Fig. 7). Another possible reason to explain the higher transfection efficacy of CBT-modified polymers is their good balance in DNA packing and unpacking. Both unmodified polymers and CBT-modified polymers can condense plasmid DNA into small nanoparticles with favorable biophysical properties, however, the polyplexes with tight polymer/DNA bindings might have problems in intracellular DNA disassociation, which is a big problem for cationic polymers with a high density of positive charges [35,36]. A previous study clearly demonstrates that low generation dendrimers such as G2 PAMAM is ineffective at releasing DNA once inside the endosome due to increased protonation of the interior tertiary amine groups [37]. Upon CBT modification, the polymers with relatively weak DNA association can solve this sticky problem (Fig. 2). Therefore, the much increased transfection efficacy of CBT-modified polymers is probably attributed to increased cellular uptake and easier intracellular DNA release. Such a fluorous effect on improving transfection efficacy is in accordance with our previous findings on heptafluorobutyric acid-modified dendrimers [25]. Further increase in the number of CBT moieties on the polymers will reduce their transfection efficacies. For example, G2 dendrimer conjugated with an average number of 3.1 CBT moieties (G2-CBT3.1) is less efficient than G2-CBT1.5 (Fig. S7). G5 dendrimer conjugated with an average number of 9.5 CBT moieties (G5-CBT9.5) shows reduced transfection efficacy as compared to G5-CBT1.3 (Fig. S8). Higher CBT conjugation degree on cationic polymer further reduces its DNA binding capacity, which leads to the reduced transfection efficacy. All the CBT-modified polymers show minimal toxicity on the transfected cells at their optimal conditions (Fig. 8a and b). Especially for CBT-modified low molecular weight polymers such as G1-CBT1.9 and G2-CBT1.5, these materials show much reduced cytotoxicity on HEK293 and HeLa cells as compared to bPEI with a molecular weight of 25 kD, a widely used non-viral gene vector (Fig. 8c and d). These results together suggest that CBT modification is a facile strategy to improve the transfection efficacy of a list of cationic polymers, both low molecular weight and high molecular weight ones.

Fig. 11. In vivo luciferase gene silencing mediated by G1-CBT1.9/siLuc complexes. (a) Luminescence images of mice before and after gene silencing. The photon counts of each mouse are indicated by pseudo-color scales. (b) Relative luciferase activity of the mice before and after gene silencing.


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3.3. In vitro and in vivo siRNA delivery efficacies of CBT-modified polymers

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The siRNA delivery efficacy of CBT-modified polymers was evaluated in HeLa and MDA-MB-231 cells stably expressing luciferase (HeLa-luc and MDA-MB-231-luc). The optimal gene silencing conditions for these polymers are screened on HeLa-luc (Fig. S9). As shown in Figs. 9 and S10, unmodified G1, G2, G5 dendrimers and bPEI1.8K fail to induce luciferase suppression in HeLa-luc cells, in comparison, G1-CBT1.9, G2-CBT1.5 G5-CBT1.3 and bPEI1.8K-CBT1.3 produce significant luciferase gene silencing in the cells, confirming that CBT modification also works on improving the siRNA delivery efficacy of cationic polymers. The most efficient polymer G1-CBT1.9 silenced more than 60% luciferase in the cells. This efficacy is comparable to commercial transfection reagents such as Lipo 2000. The materials cause no significant luciferase suppression when using a non-targeting siRNA with a scrambled sequence (siNC), suggesting specific gene silencing by these CBT-modified polymers. Similarly, G1-CBT1.9, G2-CBT1.5 and bPEI1.8K-CBT1.3 efficiently inhibit the luciferase gene expression in MDA-MB-231 cells (Fig. 10). The CBT-modified polymers except bPEI1.8K-CBT1.3 cause minimal toxicity on the transfected cells at their gene silencing conditions (Fig. S11). The relative protein contents in the wells after siRNA transfection also indicate low cytotoxicity of the synthesized polymers (Figs. 9 and 10). These results encourage us to test the in vivo gene silencing efficacy of CBT-modified polymers such as G1-CBT1.9. As shown in Fig. 11, G1-CBT1.9/siLuc complex efficiently silenced luciferase gene in a HeLa-luc tumor model, while free siLuc and G1/siLuc complex failed to inhibit luciferase expression in the same model. These results indicate that CBTmodified polymers have promising potential as non-viral vectors for gene therapy.

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4. Conclusions

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A new class of fluorinated polymers including G1-CBT1.9, G2-CBT1.5, G5-CBT1.3 and bPEI1.8K-CBT1.3 were synthesized via a facile reaction. All the polymers show high efficacy in delivering EGFP and luciferase plasmids as well as siRNA into a list of commonly used cell lines. The polymers exhibit minimal toxicity on the transfected cells. The CBT modification in this study allows the conjugation of cationic polymers with minimal fluorous chains to achieve efficient gene transfection. The residual amine groups on the CBT-modified polymers can be further modified with other ligands to further improve their transfection efficacy. This study extends the applicable scope of fluorination on improving the transfection efficacy of cationic polymers. The CBT modification can be developed as a novel strategy to design efficient and low cytotoxic vectors for both DNA and siRNA delivery.

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Disclosures

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The authors declare no potential conflicts of interest with respect to authorship or publication of this article.

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Acknowledgments

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Financial supports from the National Natural Science Foundation of China (Nos. 21322405 and 21474030) and the Shanghai Municipal Science and Technology Commission (13QA1401500 and 148014518) are greatly appreciated.

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Appendix A. Figures with essential color discrimination

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Certain figures in this article, particularly Figs. 1, 3–11, are difficult to interpret in black and white. The full color images can be

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found in the on-line version, at: http://dx.doi.org/10.1016/j.actbio. 2015.04.037.

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Appendix B. Supplementary data

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Further data about the transfection efficacy of CBT-modified polymers are available free of charge via internet. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.04.037.

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References

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[1] Zhou J, Liu J, Cheng CJ, Patel TR, Weller CE, Piepmeier JM, et al. Biodegradable poly (amine-co-ester) terpolymers for targeted gene delivery. Nat. Mater. 2012;11:82–90. [2] Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nat. Mater. 2013;12:967–77. [3] Xu P, Li SY, Li Q, Van Kirk EA, Ren J, Murdoch WJ, et al. Virion-mimicking nanocapsules from pH-controlled hierarchical self-assembly for gene delivery. Angew. Chem. Int. Ed. Engl. 2008;47:1260–4. [4] He Y, Nie Y, Cheng G, Xie L, Shen Y, Gu Z. Viral mimicking ternary polyplexes: a reduction-controlled hierarchical unpacking vector for gene delivery. Adv. Mater. 2014;26:1534–40. [5] Bryson JM, Fichter KM, Chu W-J, Lee J-H, Li J, Madsen LA, et al. Polymer beacons for luminescence and magnetic resonance imaging of DNA delivery. Proc. Natl. Acad. Sci. U.S.A. 2009;106:16913–8. [6] Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc. Natl. Acad. Sci. U.S.A. 2005;102:5679–84. [7] Breunig M, Lungwitz U, Liebl R, Goepferich A. Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc. Natl. Acad. Sci. U.S.A. 2007;104:14454–9. [8] Yang J, Hendricks W, Liu G, McCaffery JM, Kinzler KW, Huso DL, et al. A nanoparticle formulation that selectively transfects metastatic tumors in mice. Proc. Natl. Acad. Sci. U.S.A. 2013;110:14717–22. [9] Dufes C, Uchegbu IF, Schatzlein AG. Dendrimers in gene delivery. Adv. Drug Deliv. Rev. 2005;57:2177–202. [10] Luo K, He B, Wu Y, Shen Y, Gu Z. Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors. Biotechnol. Adv. 2014;32:818–30. [11] Q-l Zhu, Zhou Y, Guan M, X-f Zhou, S-d Yang, Liu Y, et al. Low-density lipoprotein-coupled N-succinyl chitosan nanoparticles co-delivering siRNA and doxorubicin for hepatocyte-targeted therapy. Biomaterials 2014;35:5965–76. [12] Ma D, Lin Q-M, Zhang L-M, Liang Y-Y, Xue W. A star-shaped porphyrinarginine functionalized poly (l-lysine) copolymer for photo-enhanced drug and gene co-delivery. Biomaterials 2014;35:4357–67. [13] Keeney M, Ong S-G, Padilla A, Yao Z, Goodman S, Wu JC, et al. Development of poly(b-amino ester)-based biodegradable nanoparticles for nonviral delivery of minicircle DNA. ACS Nano 2013;7:7241–50. [14] Bishop CJ, Ketola T-M, Tzeng SY, Sunshine JC, Urtti A, Lemmetyinen H, et al. The effect and role of carbon atoms in poly(b-amino ester)s for DNA binding and gene delivery. J. Am. Chem. Soc. 2013;135:6951–7. [15] Deng X, Zheng N, Song Z, Yin L, Cheng J. Trigger-responsive, fast-degradable poly (b-amino ester)s for enhanced DNA unpackaging and reduced toxicity. Biomaterials 2014;35:5006–15. [16] Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat. Rev. Drug. Discov. 2005;4:581–93. [17] Liu X, Zhou J, Yu T, Chen C, Cheng Q, Sengupta K, et al. Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery systems. Angew. Chem. Int. Ed. Engl. 2014;53:11822–7. [18] Yu T, Liu X, Bolcato-Bellemin AL, Wang Y, Liu C, Erbacher P, et al. An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo. Angew. Chem. Int. Ed. Engl. 2012;51:8478–84. [19] Wang F, Wang Y, Wang H, Shao N, Chen Y, Cheng Y. Synergistic effect of amino acids modified on dendrimer surface in gene delivery. Biomaterials 2014;35:9187–98. [20] Liu C, Liu X, Rocchi P, Qu F, Iovanna JL, Peng L. Arginine-terminated generation 4 PAMAM dendrimer as an effective nanovector for functional siRNA delivery in vitro and in vivo. Bioconjug. Chem. 2014;25:521–32. [21] Arima H, Motoyama K, Higashi T. Sugar-appended polyamidoamine dendrimer conjugates with cyclodextrins as cell-specific non-viral vectors. Adv. Drug Deliv. Rev. 2013;65:1204–14. [22] Gu L, Faig A, Abdelhamid D, Uhrich K. Sugar-based amphiphilic polymers for biomedical applications: from nanocarriers to therapeutics. Acc. Chem. Res. 2014;47:2867–77. [23] Han L, Ma H, Guo Y, Kuang Y, He X, Jiang C. PH-controlled delivery of nanoparticles into tumor cells. Adv. Healthcare Mater. 2013;2:1435–9. [24] Huang S, Shao K, Kuang Y, Liu Y, Li J, An S, et al. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery. Biomaterials 2013;34:5294–302.

447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515


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443 444 445

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2 May 2015 B. He et al. / Acta Biomaterialia xxx (2015) xxx–xxx 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534

[25] Wang M, Liu H, Li L, Cheng Y. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nat. Commun. 2014;5:3053. [26] Liu H, Wang Y, Wang M, Xiao J, Cheng Y. Fluorinated poly (propylenimine) dendrimers as gene vectors. Biomaterials 2014;35:5407–13. [27] Wang M, Cheng Y. The effect of fluorination on the transfection efficacy of surface-engineered dendrimers. Biomaterials 2014;35:6603–13. [28] Zhu Y, Soeriyadi AH, Parker SG, Reece PJ, Gooding JJ. Chemical patterning on preformed porous silicon photonic crystals: towards multiplex detection of protease activity at precise positions. J. Mater. Chem. B Mater. Biol. Med. 2014;2:3582–8. [29] Tomalia DA. Interview: an architectural journey: from trees, dendrons/dendrimers to nanomedicine. Nanomedicine 2012;7:953–6. [30] Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J. Chem. 2012;36:264–81. [31] Tomalia DA. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog. Polym. Sci. 2005;30:294–324.

9

[32] Svenson S, Tomalia DA. Dendrimers in biomedical applications—reflections on the field. Adv. Drug Deliv. Rev. 2012;64:102–15. [33] Kannan RM, Nance E, Kannan S, Tomalia DA. Emerging concepts in dendrimerbased nanomedicine: from design principles to clinical applications. J. Intern. Med. 2014;276:579–617. [34] Liu H, Wang H, Yang W, Cheng Y. Disulfide cross-linked low generation dendrimers with high gene transfection efficacy, low cytotoxicity, and low cost. J. Am. Chem. Soc. 2012;134:17680–7. [35] Zhang Q, Chen S, Zhuo RX, Zhang XZ, Cheng SX. Self-assembled terplexes for targeted gene delivery with improved transfection. Bioconjug. Chem. 2010;21:2086–92. [36] Maksimenko AV, Mandrouguine V, Gottikh MB, Bertrand JR, Majoral JP, Malvy C. Optimisation of dendrimer-mediated gene transfer by anionic oligomers. J. Gene Med. 2003;5:61–71. [37] Ottaviani MF, Sacchi B, Turro NJ, Chen W, Jockusch S, Tomalia DA. An EPR study of the interactions between starburst dendrimers and polynucleotides. Macromolecules 1999;32:2275–82.


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Amylose-Based Cationic Star Polymers for siRNA Delivery.

Intracellular trafficking pathways for nuclear delivery of plasmid DNA complexed with highly efficient endosome escape polymers.

siRNA Delivery Improvement by Co-formulation of Different Modified Polymers in Erythroleukemic Cell Line K562.

Fluorous microgel star polymers: selective recognition and separation of polyfluorinated surfactants and compounds in water.

Low-Molecular-Weight Polyethyleneimine Grafted Polythiophene for Efficient siRNA Delivery.

Carbon-dot-based two-photon visible nanocarriers for safe and highly efficient delivery of siRNA and DNA.

siRNA delivery with folate-low-molecular-weight polyethyleneimine-modified pullulan as non-viral carrier.

Joy and flustration with organofluorine compounds - a fluorous autobiography.

EGFP-Based Protein Nanoparticles with Cell-Penetrating Peptide for Efficient siRNA Delivery.

Rational modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking of siRNA.

A novel peptide for efficient siRNA delivery in vitro and therapeutics in vivo.

Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery.

Multivalent comb-type aptamer-siRNA conjugates for efficient and selective intracellular delivery.

Modular plasmonic nanocarriers for efficient and targeted delivery of cancer-therapeutic siRNA.

pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas.

Delivery materials for siRNA therapeutics.

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan.

Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA.

[Highly selective analysis of biogenic-related compounds utilizing fluorous chemistry].

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

Evaluation of multimeric siRNA conjugates for efficient protamine-based delivery into breast cancer cells.

Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing.

DNA nanotechnology-based development of delivery systems for bioactive compounds.