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Synergistic effect of amino acids modified on dendrimer surface in gene delivery
Biomaterials 35 (2014) 9187e9198
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Synergistic effect of amino acids modified on dendrimer surface in gene delivery Fei Wang, Yitong Wang, Hui Wang, Naimin Shao, Yuanyuan Chen, Yiyun Cheng* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2014 Accepted 19 July 2014 Available online 8 August 2014
Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triplefunctionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Dendrimer Amino acid Gene delivery Synergistic effect
1. Introduction Gene therapy remains a promising strategy in the treatment of hereditary and acquired diseases in recent years. However, the largest obstacle in gene therapy is to develop high efficient and nontoxic gene carriers [1,2]. An ideal gene carrier should possess multiple functions to overcome the barriers at different stages in gene transfection process [3]. First, the vector should condense genes into nanoparticles and the formed nanoparticles should be stable in serum. Then, the nanoparticles can be internalized into cells via specific endocytosis pathways and escaped from acidic vesicles such as endosomes and lysosomes. Finally, the vector should release the bound genes in the cytoplasm or nucleus [1]. Cationic polymers such as polyethylenimine (PEI) [4], chitosan [5], poly-L-lysine [6], diethylaminoethyl-dextran (DEAE-dextran) [7] and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) [8] are widely used as nonviral gene carriers due to their versatile structures and unique properties, but inherent cytotoxicity and relatively low transfection efficiency are associated with these polymers, which limit their applications in gene therapy [9].
* Corresponding author. E-mail address: [email protected] (Y. Cheng). http://dx.doi.org/10.1016/j.biomaterials.2014.07.027 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Dendrimers are a class of synthetic polymers with unique properties such as well-defined structure, spherical shape, low polydispersity, excellent solubility, and large number of surface functionalities and interior cavities [10e12]. Cationic dendrimers can effectively condense DNA into stable complexes due to the multivalency effect of the positive charges on dendrimer surface [13]. In addition, there is a high density of protonable tertiary amine groups in dendrimer interior, providing the “proton sponge effect” during endosomal escape process [14]. As a result, dendrimers and their conjugates were widely used as gene vectors during the past decade [15]. Dendrimer-based transfection reagents such as SuperFect and PolyFect have already entered the market. To further improve their transfection efficiency, dendrimers were modified with cyclodextrins [16], lipids, sugars, peptides, fluorous compounds [17,18], amino acids [19e24], mitochondrial targeting ligands [25] and nanoparticles [26]. Among these functionalized dendrimers, amino aciddendrimer conjugates are of great interest to the researchers [20e24,27]. Amino acids have the same fundamental structure, differing only in their residues. They can be sorted into cationic, anionic and neutral amino acids, or hydrophilic and hydrophobic amino acids. Dendrimers can be modified with amino acids via facile condensation reactions. Conjugation of cationic amino acids such as arginine (Arg) and lysine (Lys) to dendrimer directly tailors the charge density on dendrimer surface
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[19e23,28,29]. Besides, the residues of these amino acids such as guanidinium and imidazole groups play essential roles in the gene delivery processes. The guanidine group has specific affinity with cell membranes, while the imidazole group provides additional pH-buffering capacity during endosomal escape [24,30]. Arginine-rich or histidine (His)-rich peptides can be directly used as gene vectors [31]. Conjugation of hydrophobic amino acids such as phenylalanine (Phe) and leucine to dendrimer surface tailors the hydrophobicity of dendrimer surface, which is essential in the endocytosis process [32,33]. Conjugation of these amino acids can increase the transfection efficacy of dendrimers through different mechanisms, however, there are multiple barriers in gene transfection and single-functionalization cannot overcome all the barriers. A solution to this problem is multiple-functionalization of dendrimers with different amino acids such as Arg, His and Phe. Combination of these amino acids on dendrimer may simultaneously improve the membrane affinity, endocytosis and endosomal escape of the complexes. A recent study found that a combination of His and hydrophobic amino acids such as Phe and tyrosine can significantly improve the siRNA interference efficacy of a reduction-sensitive polymer [34]. Also, dual-functionalization of Arg and His on dendrimer surface allows high transfection efficacy [35]. However, these dual-functionalized polymers were synthesized in multi-steps and the synergistic effect of amino acids on these polymers still needs in-depth investigations. Here, we systematically investigate the synergistic effect of Arg, Phe and His with distinct functions on dendrimer surface in gene delivery. A one-pot approach was adopted to construct single-, dual- and triple-functionalized dendrimers (Fig. 1). Generation 5 (G5) polyamidoamine (PAMAM) dendrimer with a molecular weight of 28826 Da was used as the scaffold material. A total number of 15 dendrimer-amino acid conjugates including 5 single-, 5 dual- and 5 triple-functionalized dendrimers, respectively were synthesized. The physicochemical properties, complex formation, transfection efficacy, transfection mechanisms and cytotoxicity of these amino acid-modified dendrimers were investigated. The aims of this study are to reveal the synergistic effect of amino acids on the transfection efficacy of amino acid-modified dendrimers and to prepare multiplefunctionalized dendrimers as efficient gene vectors using a facile one-pot strategy.
2. Materials and methods 2.1. Materials G5 PAMAM dendrimer with an ethylenediamine core and surface primary amine groups was purchased from Dendritech (Midland, MI). Boc-Arg(Pbf)-OH, BocHis(Trt)-OH and Boc-Phe-OH were purchased from GL Biochem (Shanghai, China) Ltd. YOYO-1 and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, California). PolyFect was purchased from Qiagen (German). JetPEI was purchased from Polyplus-Transfection (France). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO (Gaithersburg, MD). G5 dendrimer was received in methanol solution and the solvent was distilled before use. The dendrimer was characterized by 13C NMR and polyacrylamide gel electrophoresis. All the other chemicals were used as received without further purification. 2.2. Synthesis and characterization of the single-, dual- and triple-functionalized dendrimers Amino acid-modified G5 PAMAM dendrimers were synthesized by a facile condensation reaction as described elsewhere [28]. Briefly, different amounts of Boc-Arg(pbf)-OH, Boc-Phe-OH and Boc-His(Trt)-OH were dissolved in 1.5 mL dehydrated N,N-dimethyl formamide (DMF), followed by addition of dicyclohexylcarbodiimide (DCC, 1.3 molar equivalents of carboxyl group in the protected amino acids) and N-hydroxysuccinimide (NHS, 1.2 molar equivalents of carboxyl group in the protected amino acids) to activate the carboxyl groups of amino acids for 6 h. 50 mg G5 PAMAM dendrimer was dissolved in 2 mL anhydrous dimethyl sulfoxide (DMSO) and added dropwise into the activated amino acid solution. After that, the reaction mixture was stirred at room temperature for 7 d. The molar ratios of fed amino acids to each G5 dendrimer are listed in Table 1. The reaction mixture was dialyzed against DMSO (500 mL) for two times and freeze-dried, the obtained solid was dissolved in 2 mL trifluoroacetic acid (TFA) and stirred at room temperature for 6 h to de-protect the protected groups such as Boc, Pbf and Trt. Then, TFA was removed by rotary evaporation and the crude materials were dialyzed against DMSO (500 mL, three times), PBS buffer (500 mL, three times) and distilled water (500 mL, ten times). The purified product was freeze-dried as white powders. The yielding products were characterized by 1H NMR in D2O (Varian 699.804 MHz). 2.3. Preparation of polymer/DNA complexes All the polymer/DNA complexes were freshly prepared before characterization or gene transfection experiments. Generally, amino acid-dendrimer conjugates were added into 0.8 mg plasmid DNA (EGFP or luciferase plasmid) at different N/P ratios and the sample was incubated for 30 min at room temperature. The N/P ratio was calculated according to the cationic groups (N number) on the dendrimer surface to anionic phosphate groups (P number) in plasmid DNA. The corresponding polymer/ DNA weight ratios for the complexes at different N/P ratios are also shown in Table S1. For His- and Phe-functionalized dendrimers, the N number for each conjugate was a constant of 128. For Arg-functionalized dendrimers, the N number was a sum of 128 and the number of conjugated Arg moieties since Arg has two cationic groups. Since the imidazole group in His and the tertiary amine group in dendrimer are not protonated at pH 7.4, these groups were not considered when calculating the N numbers. For the commercial transfection reagents such as Lipofectamine 2000,
Fig. 1. Synthesis of multi-functionalized dendrimers using a one-pot approach.
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Table 1 Characterizations of the single-, dual- and triple-functionalized dendrimers. Functionalized dendrimers
Dendrimer conjugates
Number of amino acid per G5 dendrimer Fed
Single-functionalized dendrimers
Dual-functionalized dendrimers
Triple-functionalized dendrimers
G5-Arg110 G5-Arg33 G5-Phe99 G5-Phe71 G5-His113 G5-Arg34Phe71 G5-Arg51Phe53 G5-Arg82Phe34 G5-Arg44His53 G5-Phe64His40 G5-Arg24Phe20His40 G5-Arg33Phe49His14 G5-Arg35Phe29His28 G5-Arg47Phe24His25 G5-Arg56Phe22His22
Molecular weight (Da)
Conjugated
Arg
Phe
His
Arg
Phe
His
166 0 0 0 0 83 111 124 83 0 48 85 67 83 95
0 45 166 83 0 83 55 42 0 83 24 64 33 28 24
0 0 0 0 166 0 0 0 83 83 95 43 67 55 48
110 33 0 0 0 34 51 82 44 0 24 33 35 47 56
0 0 99 71 0 71 53 34 0 64 20 49 29 24 22
0 0 0 0 113 0 0 0 53 40 40 14 28 25 22
PolyFect and JetPEI, the complexes were prepared according to the protocols at their optimal conditions. 2.4. Characterization of the polymer/DNA complexes The DNA binding capacity of amino acid-dendrimer conjugates was investigated by agarose gel (Biowest, Spain) electrophoresis. Generally, the polymer/DNA complexes at N/P ratios of 0.5:1, 1:1, 2:1 and 4:1 were prepared in deionized water and diluted with DNA loading buffer. The samples were electrophoresed on a 1% (w/v) agarose gel at 100 V for 1 h. The DNA in the gels were stained by ethidium bromide and visualized under UV illumination (Tanon-2500, China). For the DNA release assay, polymer/DNA complexes were prepared a specific molar concentration and different concentrations of heparin were added to release the bound DNA in the complexes. The resulting solutions were further analyzed by agarose gel electrophoresis as described above.
46,008 33,981 43,398 39,276 44325 44,587 44,593 46,639 42968 43733 41,005 43113 42,402 43,129 43,829
The size and zeta potential analysis of polymer/DNA complexes prepared in deionized water at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1 were carried out by dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS90 (Malvern, UK) at 25 C. 2.5. Cell culture and in vitro gene transfection experiments HeLa (ATCC, a human cervical carcinoma cell line), HEK293 (a human embryonic kidney cell line, ATCC) and NIH 3T3 (ATCC, a mouse embryonic fibroblast cell line) cells were cultured with DMEM containing 100 units/mL penicillin sulfate and streptomycin and 10% (v/v) heat-inactivated FBS at 37 C under a humidified atmosphere containing 5% CO2. Before gene transfection experiments, HeLa and NIH 3T3 cells were seeded in 24-well plates and cultured overnight to reach an appropriate cell density. The polymer/DNA complexes containing 0.8 mg plasmid DNA were diluted with 100 mL DMEM containing 10% FBS and incubated 30 min at room
Fig. 2. 1H NMR spectra of single-, dual- and triple-functionalized dendrimers in D2O.
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temperature. Then, the complexes were further diluted with 150 mL medium and incubated with the cells for 6 h. After that, 500 mL fresh medium was added to each well and the cells were further cultured for 42 h. EGFP expressions in the cells were observed by a fluorescence microscopy (Olympus, Japan) and quantitatively analyzed by flow cytometry (BD FACSCalibur, San Jose). Luciferase expressions in the cells were analyzed according to the manufacturer's protocols (Promega). The commercial gene transfection reagents including JetPEI, PolyFect and Lipofectamine 2000 were used as positive controls. 2.6. Cytotoxicity of the amino acid-dendrimer conjugates and their DNA complexes The cytotoxicity of amino acid-dendrimer conjugates and their DNA complexes were examined on NIH 3T3 cells using a well-established MTT assay. The cells were seeded in 96-well plates at a density around 104 cells per well and cultured for 12 h in 100 mL DMEM containing 10% FBS. The cells were treated with polymers or polymer/DNA complexes at different concentrations for 48 h. The chosen polymer concentrations (0.55 mM) and DNA concentration (3.2 mM) equal to those in the gene transfection experiments. 2.7. Cellular uptake of polymer/DNA complexes To analyze the cellular uptake of polymer/DNA complexes, the plasmid DNA was labeled with a green fluorescent dye YOYO-1 for 10 min according to the manufacturer's protocol (Invitrogen). The polymer/DNA complexes were then prepared as described above (the dendrimer concentration is fixed for different amino aciddendrimer conjugates in the complexes). Generally, NIH 3T3 cells was seeded in
24-well plates and cultured for 24 h to reach an appropriate cell density, and YOYO-1 (excitation at 491 nm and emission at 509 nm) labeled complexes were incubated with the cells for 1 and 2 h. After that, the cells were washed with 500 mL cold PBS for three times. Cellular uptake of the complexes was analyzed by flow cytometry. 2.8. pH-buffering capacity assay The pH buffering capacity of amino acid-dendrimer conjugates was determined as described below. Briefly, the concentration of different conjugates were fixed at a constant dendrimer concentration (22.6 nM), and pH value of the conjugate solutions was adjusted to 7.4. Then the samples were titrated with 0.12 M HCl. pH value of the conjugate solution after each titration (1 mL) was measured using a pH meter (Mettler-Toledo). The titration experiment was continued until the pH value of the solution reaches 5.0 (pH range of 7.4e5.0 mimics the endosome acidification). 2.9. Confocal microscopy NIH 3T3 cells were seeded on glass slides in 24-well plates and cultured for 24 h at 37 C. The cells were incubated with YOYO-1-labeled polymer/DNA complexes for 2 and 4 h. The medium were removed and the cells were washed with 500 mL PBS for three times. The cells were further incubated with PBST (PBS containing 0.1% Tween 20) for 5 min, and then with 0.2% BSA at room temperature for 30 min. The acidic vesicles of NIH 3T3 cells was stained with LAMP-2 (2 mg/mL) antibody conjugated with Alexa Fluor 647 (excitation at 650 nm and emission at 665 nm) for 1 h at 37 C. The cells were washed with PBS for three times and nuclei of the cells were stained by Hoechst 33342 (excitation at 346 nm and emission at 460 nm, 5 mg/mL) for
Fig. 3. DNA retardation assay of amino acid-dendrimer conjugates. The polymer/DNA complexes were prepared by mixing the synthesized polymers with EGFP plasmid at N/P ratios of 0.5:1, 1:1, 2:1 and 4:1, respectively. (a) single-functionalized dendrimers, (b) dual-functionalized dendrimers and (c) triple-functionalized dendrimers.
F. Wang et al. / Biomaterials 35 (2014) 9187e9198 10 min at room temperature. After the cells were washed with PBS for three times, co-localization of the complexes with acidic vesicles was observed by confocal microscopy (Leica SP5, Germany).
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3. Results and discussion 3.1. Synthesis and characterization of single-, dual- and triplefunctionalized dendrimers and their complexes with DNA
2.10. In vivo transfection HeLa tumor xenograft model was established as described below. 6-week-old female BALB/c nude mice at an average weight of 22.0 g were obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The animals were housed under specific-pathogen-free conditions within the animal care facility at East China Normal University. The animal experiments were carried out according to the National Institutes of Health guidelines for care and use of laboratory animals and approved by the ethics committee of East China Normal University. HeLa cells were cultured in DMEM containing 10% FBS. 100 mL cell suspensions in PBS (containing 107 cells) were subcutaneously injected into the BALB/c nude mice. When tumor size reaches 50 mm3, the mice were administrated with 25 mL G5-Arg47Phe24His25/DNA or G5-Arg110/DNA complexes in 5% glucose solution (containing 3.3 mmol polymer and 10 ug luciferase plasmid) by injection into the tumor. The animal treated with 5% glucose solution was used as a control. The treatments were repeated every day and a total number of three injections were administrated for each animal. The in vivo transfection experiments were continued for another two days. After that, 200 mL Dluciferin potassium salt (15 mg/mL) was intraperitoneally injected into the anesthetized mice. 5 min later, the mice were placed into an in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton), and the luminescence at tumor site was recorded with an exposure time of 5 min.
To investigate the synergistic effect of amino acids in gene delivery, G5 PAMAM dendrimer was functionalized with Arg, Phe and His using a facile condensation reaction. For dual- and triplefunctionalized dendrimers, mixtures of specific amino acids at different molar ratios were added to the dendrimer solution and the functionalized dendrimers were obtained by a one-pot strategy. The number of each amino acid conjugated to dendrimer was characterized by 1H NMR. As shown in Fig. 2 and Fig. S1, the four broad peaks (Ha, Hb, Hc,b0 and Hd,d0 ) in the chemical shift range of 2.0e3.5 ppm correspond to protons on dendrimer scaffold and other peaks in the spectra are assigned to protons of Arg, Phe and His, respectively [36]. The average number of amino acid conjugated to dendrimer was calculated according to the peaks areas of dendrimer protons and amino acid protons, respectively. The components of the 15 amino acid-dendrimer conjugates are listed in Table 1. For single-functionalized dendrimer, the conjugate G5-
Fig. 4. Size (a) and zeta potential (b) analysis of polymer/DNA complexes prepared at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1, respectively.
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Arg110 represents a G5 PAMAM dendrimer conjugated with an average number of 110 Arg molecules on its surface. For dualfunctionalized dendrimer, the conjugate G5-Arg34Phe71 represents a G5 dendrimer modified with 34 Arg and 71 Phe moieties. For triple-functionalized dendrimer, the conjugate G5Arg35Phe29His28 means a conjugate with 35 Arg, 29 Phe and 28 His moieties. Gel retardation experiments were used to evaluate the DNA binding ability of the synthesized conjugates. As shown in Fig. 3, all the conjugates except G5-Phe99 successfully reduce DNA mobility above an N/P ratio of 1:1. G5-Phe99 even fails to condense DNA at an N/P ratio of 4:1. The lower DNA binding capacity of G5-Phe99 is probably due to the charge shielding effect of aromatic rings present on dendrimer surface [33]. For gene delivery, the size of polymer/DNA polyplexes is essential for efficient gene transfection. As shown in Fig. 4 and Fig. S2, the conjugates containing Arg moieties can condense plasmid DNA into nanoparticles around 200 nm (Fig. 4 and Fig. S2), while those without Arg moieties such as G5-Phe99, G5-Phe71, G5-His113 and G5-Phe64His40 fail to form small nanoparticles below 400 nm with DNA at N/P ratios of 5:1,
10:1 and 20:1. The polyplexes with sizes around 200 nm are appropriate for cellular uptake and gene delivery. This result suggests that Arg is essential in the formation of stable complexes for gene transfection [37e39]. Plasmid DNA is negatively charged at neutral conditions, and the addition of all the conjugates can turn DNA charge state from negative to positive at an N/P ratio of 5:1. The biophysical properties of the polymer/DNA complexes will be further discussed to analyze the transfection mechanisms of single-, dual- and triple-functionalized dendrimers in Section 3.3. 3.2. Synergistic effect of amino acids modified on dendrimer surface in gene delivery The transfection efficacy of single-, double- and triplefunctionalized dendrimers were tested on HeLa, HEK293 or NIH 3T3 cells using EGFP and luciferase plasmid as reporter genes. As shown in Fig. 5, a dual-functionalized dendrimer G5-Arg34Phe71 successfully transfected 75.2% HeLa cells at its optimal N/P ratio, while the single-functionalized conjugates G5-Arg33 and G5-Phe71 only transfected 21.8% and 12.1% HeLa cells at equivalent dendrimer
Fig. 5. Synergistic effect of Arg and Phe on G5-Arg34Phe71. The transfection efficacy of G5-Arg34Phe71 on HeLa cells was compared with those of G5-Arg33, G5-Phe71, G5-Arg110 and G5-Phe99, respectively. The dendrimer concentration is fixed at 0.36 mM for all the conjugates. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in HeLa cells measured by flow cytometry (n ¼ 3). Statistically significant differences are denoted by ***p < 0.001 using student's t-test.
Fig. 6. Transfection efficacies of single-, dual- and triple-functionalized dendrimers on NIH 3T3 cells for 48 h. The dendrimer concentration is fixed at 0.55 mM for all the conjugates. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3).
Fig. 7. Luciferase activities of NIH 3T3 cells transfected by single-, dual- and triple-functionalized dendrimers for 48 h. The dendrimer concentration is fixed at 0.55 mM for all the conjugates. The luciferase activity is expressed as relative luciferase light units per mg protein (RLU/mg protein, n ¼ 3).
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and EGFP plasmid molar concentrations. Also, G5-Arg34Phe71 is much more efficient (both EGFP positive cells and mean fluorescence intensity) than G5-Arg110 or G5-Phe99. Not limited to the optimal N/P ratio, G5-Arg34Phe71 shows much higher efficacy than G5-Arg110 on HeLa cells at all the N/P ratios (Fig. S3). These results suggest synergistic effect of Arg and Phe on the dual-functionalized dendrimer in gene delivery. Similar results were obtained on HEK293 cells (Fig. S4). The synergistic effect amino acids in gene delivery were further confirmed on NIH 3T3 cells. As shown in Fig. 6 and Fig. S5, all the dual- and triple-functionalized dendrimers except G5-Phe64His40 exhibit higher transfection efficacy than single-functionalized dendrimers. The triple-functionalized dendrimers G5Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient on delivering EGFP plasmid than the dual-functionalized ones. G5Phe64His40 shows poor transfection efficacy due to its weak DNA binding capacity as revealed in Fig. 4. Similar results were obtained on NIH 3T3 cells using a luciferase reporter gene (Fig. 7). The dualand triple-functionalized dendrimers show higher luciferase
activity than single-functionalized ones. The synthesized conjugates except G5-Phe99 show minimal cytotoxicity on NIH 3T3 cells at transfection concentrations (Fig. 8a). The relatively high toxicity of G5-Phe99 is due to the hydrophobic character of dendrimer surface after Phe modification. This phenomenon is also observed in a previous result [32]. Though it seems to introduce cytotoxicity after Phe modification, the dual- and triple-functionalized dendrimers containing Phe moieties show minimal cytotoxicity on NIH 3T3 cells. Even in the presence of plasmid DNA, these materials maintain high cell viability above 90% (Fig. 8b), suggesting that the dual- and triple-functionalized dendrimers can achieve high transfection efficacy with low cytotoxicity on the transfected cells. 3.3. Synergistic mechanism of amino acids modified on dendrimer surface in gene delivery The transfection efficacy of a gene material depends on several parameters such as complex formation and stability, cellular uptake, endosomal escape and intracellular DNA release [1]. As shown
Fig. 8. Cytotoxicities of single-, dual- and triple-functionalized dendrimers (a) and their DNA complexes (b) on NIH 3T3 cells for 48 h (n ¼ 5). The polymer concentrations (0.55 mM) equal to those in gene transfection experiments.
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in Figs. 5 and 6, Arg plays an essential role in gene transfection. The transfection efficacy of dual- and triple-functionalized dendrimers increases in proportion with the number of conjugated Arg moieties. Those conjugates without Arg moieties such as G5-Phe99, G5His113 and G5-Phe64His40 show extremely low transfection efficacy. First, the conjugated Arg moiety has two positive charged groups, which facilitates DNA condensation. This is evidenced by DNA retardant and DLS results in Figs. 3 and 4. Arg-containing conjugates show strong DNA binding capacity, while those without Arg moieties fail to condense plasmid DNA into nanoparticles around 200 nm at relative low N/P ratios. Second, the positive charge of the guanidinium group in Arg is delocalized on three nitrogen atoms, thus guanidinium shows better interactions with anions such as phosphates than localized cations such as ammonium [40]. Third, the guanidinium group has strong affinity to cell membranes through ionic pairing and hydrogen bonding [41]. Due to these reasons, Arg-dendrimer conjugates were widely investigated as efficient gene vectors during the past decade [19e23]. However, the dendrimer conjugated with 110 Arg moieties (G5-Arg110) shows much lower efficacy compared to the dual- and triplefunctionalized dendrimers, suggesting that His and Phe moieties also play important roles in gene delivery. This is attributed to the high charge density on G5-Arg110, which decreases the penetration of polymer/DNA complex through cell membrane via endocytosis. The high charge density on polymers may also trigger
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destabilization of self-assembled phospholipids in the cell membrane, resulting in serious cytotoxicity [9]. Increased cytotoxicity is also a limiting factor for high transfection efficacy. One solution to this problem is tailoring the balance of charge and hydrophobic contents on dendrimer surface [42]. Replacement of partial Arg moieties in G5-Arg110 with hydrophobic amino acid such as Phe can improve the complex cellular uptake. As shown in Fig. 9a, complexes of G5-Arg51Phe53, G5-Arg47Phe24His25 and G5Arg56Phe22His22 with DNA show higher cellular uptake than that of G5-Arg110. As a result, dual-functionalized dendrimer such as G5Arg51Phe53 and triple-functionalized dendrimer such as G5Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient in gene transfection than G5-Arg110. In addition, G5-Arg51Phe53/DNA complex is easier to release their bound DNA in the presence heparin than G5-Arg110/DNA complex (Fig. S6). Such a hydrophobic effect on improving gene transfection efficacy is also reported in several polymeric gene delivery systems [42e45]. Though Phe effectively tailors the hydrophobic/hydrophilic balance on dendrimer surface, the conjugation of excess Phe moieties to a G5 dendrimer also leads to increased cytotoxicity. For example, G5Phe99 is even more cytotoxic than unmodified G5 PAMAM dendrimer and it kills most of the cells at a concentration of 50 mg/mL (Fig. 9b). The incorporation of His into the conjugates can alleviate the toxicity issues of Arg and Phe. As shown in Fig. 9b, G5-Arg44His53
Fig. 9. (a) Cellar uptake of polymer/DNA complexes for 1 and 2 h by NIH 3T3 cells (n ¼ 3), statistically significant differences are denoted by *p < 0.05 and ***p < 0.001 using student's t-test. The polymer concentrations equal to those in gene transfection experiments. (b) Cytotoxicities of amino acid-dendrimer conjugates on NIH 3T3 cells at different concentrations (n ¼ 4). (c) pH-buffering capacities of G5-His113 and G5-Arg110 (n ¼ 3). (d) Confocal images of NIH 3T3 cells incubated with YOYO-1-labeled G5-Arg47Phe24His25/DNA complex (green) for 2 and 4 h. The acidic vesicles were stained with LAMP-2 conjugated with Alexa Fluor 647 (red) and the nuclei were stained with Hoechst 33342 (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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and G5-Arg47Phe24His25 are less cytotoxic than G5-Arg110 on NIH 3T3 cells. G5-His113 is non-toxic at concentrations up to 600 mg/mL. Besides the role of reducing cytotoxicity, the imidazole group (pKa ~ 6.04) in His is protonable at mild acidic conditions. As shown in Fig. 9c, G5-His113 shows better pH-buffering capacity than G5Arg110 within the pH range of 7.4 to 5.0 (endosome acidification), suggesting that the incorporation of His moieties to the conjugates can facilitate endosomal escape [24]. The triple-functionalized conjugate G5-Arg47Phe24His25 shows excellent endosomal escape ability and the complex is not co-localized with acidic vesicles within 2 h (Fig. 9d). It is worth noting that the molar ratios of Arg, Phe and His should be carefully chosen to obtain an efficient triplefunctionalized dendrimer. For example, G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are much more efficient in delivering EGFP plasmid on NIH 3T3 cells than the other three triple-functionalized dendrimers (Fig. 6). G5-Arg47Phe24His25 and G5-Arg56Phe22His22 show comparable efficacy with several commercial transfection
reagents such as JetPEI, PolyFect and Lipofectamine 2000 at their optimal conditions (Fig. 10). The triple-functionalized dendrimer G5-Arg47Phe24His25 also shows higher in vivo transfection efficacy than the single functionalized dendrimer G5-Arg110 when delivering a luciferase plasmid (Fig. 11). These results suggest that the triple-functionalized dendrimers can be developed for commercial purpose in the future. 4. Conclusions In this study, we synthesized a list of single-, dual- and triplefunctionalized dendrimers with Arg, Phe and His for gene delivery. The amino acids show synergistic effects on dual- and triplefunctionalized dendrimers. Arg in the conjugates is essential for complex formation. Phe modulates the balance of hydrophobic and hydrophilic contents on dendrimer surface, thereby facilitates the cellular uptake process. His improves pH-buffering capacity and reduces cytotoxicity of the cationic dendrimers. These amino acids
Fig. 10. Comparisons of triple-functionalized dendrimers with three commercial transfection reagents (JetPEI, PolyFect and Lipofectamine 2000) on transfection efficacy. N/P ratios for G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are 30:1 and 32:1, respectively. Gene transfection experiments for the commercial reagents were optimized according to the manufacturer's protocols. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3). Statistically significant differences are denoted by *p < 0.05 and **p < 0.01 using student's t-test.
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Fig. 11. In vivo gene transfection on a HeLa tumor xenograft model. The animals were treated with 5% glucose solution, G5-Arg47Phe24His25/DNA and G5-Arg110/DNA complexes, respectively. Luciferase plasmid was used as the reporter gene. The polyplexes were prepared in 5% glucose solution.
Fig. 12. Transfection mechanisms of single-, dual- and triple-functionalized dendrimers.
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have different functions in the gene delivery process and hence a combination of them generates synergistic effects in gene delivery (Fig. 12). The efficient triple-functionalized dendrimers achieve comparable transfection efficacy to several commercial transfection reagents on NIH 3T3 cells. In addition, the dual- and triplefunctionalized dendrimers show low cytotoxicity on the transfected cells. This study provides a new insight into the design of efficient and low cytotoxic gene vectors using a facile strategy. Acknowledgment The authors thank the grants including National Natural Science Foundation of China (No. 21322405), the Shanghai Rising Star Program (13QA1401500) and the Science and Technology of Shanghai Municipality (11DZ2260300) for financial supports. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.07.027. References [1] Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov 2005;4:581e93. [2] Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nat Mater 2013;12:967e77. [3] Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009;8:129e38. [4] Hu Y, Xu B, Ji Q, Shou D, Sun X, Xu J, et al. A mannosylated cell-penetrating peptide-graft-polyethylenimine as a gene delivery vector. Biomaterials 2014;35:4236e46. [5] Gao Y, Wang ZY, Zhang J, Zhang Y, Huo H, Wang T, et al. RVG-peptide-linked trimethylated chitosan for delivery of siRNA to the brain. Biomacromolecules 2014;15:1010e8. [6] Kadlecova Z, Rajendra Y, Matasci M, Baldi L, Hacker DL, Wurm FM, et al. DNA delivery with hyperbranched polylysine: a comparative study with linear and dendritic polylysine. J Control Release 2013;169:276e88. [7] Zarogoulidis P, Hohenforst-Schmidt W, Darwiche K, Krauss L, Sparopoulou D, Sakkas L, et al. 2-diethylaminoethyl-dextran methyl methacrylate copolymer nonviral vector: still a long way toward the safety of aerosol gene therapy. Gene Ther 2013;20:1022e8. [8] Qian X, Long L, Shi Z, Liu C, Qiu M, Sheng J, et al. Star-branched amphiphilic PLA-b-PDMAEMA copolymers for co-delivery of miR-21 inhibitor and doxorubicin to treat glioma. Biomaterials 2014;35:2322e35. [9] Mastrobattista E, Hennink WE. Polymers for gene delivery: charged for success. Nat Mater 2012;11:10e2. [10] Tomalia DA. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog Polym Sci 2005;30:294e324. [11] Svenson S, Tomalia DA. Dendrimers in biomedical applications-reflections on the field. Adv Drug Deliv Rev 2012;64:102e15. [12] Tomalia DA. Interview: an architectural journey: from trees, dendrons/dendrimers to nanomedicine. Nanomedicine 2012;7:953e6. [13] Shcharbin D, Pedziwiatr E, Bryszewska M. How to study dendriplexes I: characterization. J Control Release 2009;135:186e97. s C, Uchegbu IF, Sch€ [14] Dufe atzlein AG. Dendrimers in gene delivery. Adv Drug Deliv Rev 2005;57:2177e202. [15] 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;134:17680e7. [16] Arima H, Motoyama K, Higashi T. Sugar-appended polyamidoamine dendrimer conjugates with cyclodextrins as cell-specific non-viral vectors. Adv Drug Deliv Rev 2013;65:1204e14. [17] 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;5:4053. [18] Liu H, Wang Y, Wang M, Xiao J, Cheng Y. Fluorinated poly(propylenimine) dendrimers as gene vectors. Biomaterials 2014;35:5407e13. [19] Luo K, Li C, She W, Wang G, Gu Z. Arginine functionalized peptide dendrimers as potential gene delivery vehicles. Biomaterials 2012;33:4917e27.
[20] Kim T, Baek J, Bai CZ, Park J. Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier. Biomaterials 2007;28:2061e7. s C. [21] Aldawsari H, Ru Edrada-Ebel A, Blatchford DR, Tate RJ, Tetley L, Dufe Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polypropylenimine polyplex. Biomaterials 2011;32:5889e99. [22] Nam HY, Nam K, Hahn HJ, Kim BH, Lim HJ, Kim HJ, et al. Biodegradable PAMAM ester for enhanced transfection efficiency with low cytotoxicity. Biomaterials 2009;30:665e73. [23] Kim T, Bai CZ, Nam K, Park J. Comparison between arginine conjugated PAMAM dendrimers with structural diversity for gene delivery systems. J Control Release 2009;136:132e9. [24] Wen Y, Zh Guo, Du Z, Fang R, Wu H, Zeng X, et al. Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives. Biomaterials 2012;33: 8111e21. [25] Wang X, Shao N, Zhang Q, Cheng Y. Mitochondrial targeting dendrimer allows efficient and safe gene delivery. J Mater Chem B 2014;2:2546e53. [26] Shan Y, Luo T, Peng C, Sheng R, Cao A, Cao X, et al. Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors. Biomaterials 2012;33:3025e35. [27] Kim ID, Lim CM, Kim JB, Nam HY, Nam K, Kim SW, et al. Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain. J Control Release 2010;142:422e30. [28] Choi JS, Nam K, Park J, Kim JB, Lee JK, Park JS. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J Control Release 2004;99:445e56. [29] Liu C, Liu X, Rocchi P, Qu F, Iovanna LJ, Peng L. Arginine-terminated generation 4 PAMAM dendrimer as an effective nanovector for functional siRNA delivery in vitro and in vivo. Bioconjugate Chem 2014;25:521e32. [30] Choi JS, Nam K, Park JS, Kim JB, Lee JK, Park JS. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with l-arginine. J Control Release 2004;99:12. €rz J, Danos O, Bechinger B. Histidine-rich amphi[31] Kichler A, Leborgne C, Ma pathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc Natl Acad Sci U S A 2002;100:1564e8. [32] Kono K, Akiyama H, Takahashi T, Takagishi T, Harada A. Transfection activity of polyamidoamine dendrimers having hydrophobic amino acid residues in the periphery. Bioconjugate Chem 2005;16:208e14. [33] Wang X, He Y, Wu J, Gao C, Xu Y. Synthesis and evaluation of phenylalaninemodified hyperbranched poly(amido amine)s as promising gene carriers. Biomacromolecules 2010;11:245e51. [34] Zeng H, Little HC, Tiambeng TN, Williams GA, Guan Z. Multifunctional dendronized peptide polymer platform for safe and effective siRNA delivery. J Am Chem Soc 2013;135:4962e5. [35] Yu GS, Bae YM, Choi H, Kong B, Choi IS, Choi JS. Synthesis of PAMAM dendrimer derivatives with enhanced buffering capacity and remarkable gene transfection efficiency. Bioconjugate Chem 2011;22:1046e55. [36] Hu J, Xu T, Cheng Y. NMR insights into dendrimer-based host-guest systems. Chem Rev 2012;112:3856e91. [37] Kawashima S. Ando Toshio. Interaction of basic oligo-l-amino acids with deoxyribonucleic acids oligo-L-arginines of various chain lengths and herring sperm DNA. J Biochem 1978;84:343e50. [38] Morris VB, Sharma CP. Enhanced in-vitro transfection and biocompatibility of L-arginine modified oligo(-alkylaminosiloxanes)-graft-polyethylenimine. Biomaterials 2010;31:8759e69. [39] Won Y-W, Kim HA, Lee M, Kim Y-H. Reducible poly(oligo-D-arginine) for enhanced gene expression in mouse lung by intratracheal injection. Mol Ther 2010;18:734e42. [40] Yang J, Liu Y, Wang H, Liu L, Wang W, Wang C, et al. The biocompatibility of fatty acid modified dextran-agmatine bioconjugate gene delivery vector. Biomaterials 2012;33:604e13. [41] Pantos A, Tsogas I, Paleos CM. Guanidinium group: a versatile moiety inducing transport and multicompartmentalization in complementary membranes. BBA-Biomembranes 2008;1778:811e23. [42] Nelson CE, Kintzing JR, Hanna A, Shannon JM, Gupta MK, Duvall CL. Balancing cationic and hydrophobic content of PEGylated siRNA polyplexes enhances endosome escape, stability, blood circulation time, and bioactivity in vivo. ACS Nano 2013;7:8870e80. [43] Wang B, He C, Tang C, Yin C. Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers. Biomaterials 2011;32:4630e8. [44] Liu Z, Zhang Z, Zhou C, Jiao Y. Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 2010;35:1144e62. [45] Yuba E, Nakajima Y, Tsukamotoa K, Iwashita S, Kojima C, Harada A, et al. Effect of unsaturated alkyl chains on transfection activity of poly(amidoamine) dendron-bearing lipids. J Control Release 2012;160:552e60.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Synergistic effect of amino acids modified on dendrimer surface in gene delivery Fei Wang, Yitong Wang, Hui Wang, Naimin Shao, Yuanyuan Chen, Yiyun Cheng* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2014 Accepted 19 July 2014 Available online 8 August 2014
Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triplefunctionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Dendrimer Amino acid Gene delivery Synergistic effect
1. Introduction Gene therapy remains a promising strategy in the treatment of hereditary and acquired diseases in recent years. However, the largest obstacle in gene therapy is to develop high efficient and nontoxic gene carriers [1,2]. An ideal gene carrier should possess multiple functions to overcome the barriers at different stages in gene transfection process [3]. First, the vector should condense genes into nanoparticles and the formed nanoparticles should be stable in serum. Then, the nanoparticles can be internalized into cells via specific endocytosis pathways and escaped from acidic vesicles such as endosomes and lysosomes. Finally, the vector should release the bound genes in the cytoplasm or nucleus [1]. Cationic polymers such as polyethylenimine (PEI) [4], chitosan [5], poly-L-lysine [6], diethylaminoethyl-dextran (DEAE-dextran) [7] and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) [8] are widely used as nonviral gene carriers due to their versatile structures and unique properties, but inherent cytotoxicity and relatively low transfection efficiency are associated with these polymers, which limit their applications in gene therapy [9].
* Corresponding author. E-mail address: [email protected] (Y. Cheng). http://dx.doi.org/10.1016/j.biomaterials.2014.07.027 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Dendrimers are a class of synthetic polymers with unique properties such as well-defined structure, spherical shape, low polydispersity, excellent solubility, and large number of surface functionalities and interior cavities [10e12]. Cationic dendrimers can effectively condense DNA into stable complexes due to the multivalency effect of the positive charges on dendrimer surface [13]. In addition, there is a high density of protonable tertiary amine groups in dendrimer interior, providing the “proton sponge effect” during endosomal escape process [14]. As a result, dendrimers and their conjugates were widely used as gene vectors during the past decade [15]. Dendrimer-based transfection reagents such as SuperFect and PolyFect have already entered the market. To further improve their transfection efficiency, dendrimers were modified with cyclodextrins [16], lipids, sugars, peptides, fluorous compounds [17,18], amino acids [19e24], mitochondrial targeting ligands [25] and nanoparticles [26]. Among these functionalized dendrimers, amino aciddendrimer conjugates are of great interest to the researchers [20e24,27]. Amino acids have the same fundamental structure, differing only in their residues. They can be sorted into cationic, anionic and neutral amino acids, or hydrophilic and hydrophobic amino acids. Dendrimers can be modified with amino acids via facile condensation reactions. Conjugation of cationic amino acids such as arginine (Arg) and lysine (Lys) to dendrimer directly tailors the charge density on dendrimer surface
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[19e23,28,29]. Besides, the residues of these amino acids such as guanidinium and imidazole groups play essential roles in the gene delivery processes. The guanidine group has specific affinity with cell membranes, while the imidazole group provides additional pH-buffering capacity during endosomal escape [24,30]. Arginine-rich or histidine (His)-rich peptides can be directly used as gene vectors [31]. Conjugation of hydrophobic amino acids such as phenylalanine (Phe) and leucine to dendrimer surface tailors the hydrophobicity of dendrimer surface, which is essential in the endocytosis process [32,33]. Conjugation of these amino acids can increase the transfection efficacy of dendrimers through different mechanisms, however, there are multiple barriers in gene transfection and single-functionalization cannot overcome all the barriers. A solution to this problem is multiple-functionalization of dendrimers with different amino acids such as Arg, His and Phe. Combination of these amino acids on dendrimer may simultaneously improve the membrane affinity, endocytosis and endosomal escape of the complexes. A recent study found that a combination of His and hydrophobic amino acids such as Phe and tyrosine can significantly improve the siRNA interference efficacy of a reduction-sensitive polymer [34]. Also, dual-functionalization of Arg and His on dendrimer surface allows high transfection efficacy [35]. However, these dual-functionalized polymers were synthesized in multi-steps and the synergistic effect of amino acids on these polymers still needs in-depth investigations. Here, we systematically investigate the synergistic effect of Arg, Phe and His with distinct functions on dendrimer surface in gene delivery. A one-pot approach was adopted to construct single-, dual- and triple-functionalized dendrimers (Fig. 1). Generation 5 (G5) polyamidoamine (PAMAM) dendrimer with a molecular weight of 28826 Da was used as the scaffold material. A total number of 15 dendrimer-amino acid conjugates including 5 single-, 5 dual- and 5 triple-functionalized dendrimers, respectively were synthesized. The physicochemical properties, complex formation, transfection efficacy, transfection mechanisms and cytotoxicity of these amino acid-modified dendrimers were investigated. The aims of this study are to reveal the synergistic effect of amino acids on the transfection efficacy of amino acid-modified dendrimers and to prepare multiplefunctionalized dendrimers as efficient gene vectors using a facile one-pot strategy.
2. Materials and methods 2.1. Materials G5 PAMAM dendrimer with an ethylenediamine core and surface primary amine groups was purchased from Dendritech (Midland, MI). Boc-Arg(Pbf)-OH, BocHis(Trt)-OH and Boc-Phe-OH were purchased from GL Biochem (Shanghai, China) Ltd. YOYO-1 and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, California). PolyFect was purchased from Qiagen (German). JetPEI was purchased from Polyplus-Transfection (France). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO (Gaithersburg, MD). G5 dendrimer was received in methanol solution and the solvent was distilled before use. The dendrimer was characterized by 13C NMR and polyacrylamide gel electrophoresis. All the other chemicals were used as received without further purification. 2.2. Synthesis and characterization of the single-, dual- and triple-functionalized dendrimers Amino acid-modified G5 PAMAM dendrimers were synthesized by a facile condensation reaction as described elsewhere [28]. Briefly, different amounts of Boc-Arg(pbf)-OH, Boc-Phe-OH and Boc-His(Trt)-OH were dissolved in 1.5 mL dehydrated N,N-dimethyl formamide (DMF), followed by addition of dicyclohexylcarbodiimide (DCC, 1.3 molar equivalents of carboxyl group in the protected amino acids) and N-hydroxysuccinimide (NHS, 1.2 molar equivalents of carboxyl group in the protected amino acids) to activate the carboxyl groups of amino acids for 6 h. 50 mg G5 PAMAM dendrimer was dissolved in 2 mL anhydrous dimethyl sulfoxide (DMSO) and added dropwise into the activated amino acid solution. After that, the reaction mixture was stirred at room temperature for 7 d. The molar ratios of fed amino acids to each G5 dendrimer are listed in Table 1. The reaction mixture was dialyzed against DMSO (500 mL) for two times and freeze-dried, the obtained solid was dissolved in 2 mL trifluoroacetic acid (TFA) and stirred at room temperature for 6 h to de-protect the protected groups such as Boc, Pbf and Trt. Then, TFA was removed by rotary evaporation and the crude materials were dialyzed against DMSO (500 mL, three times), PBS buffer (500 mL, three times) and distilled water (500 mL, ten times). The purified product was freeze-dried as white powders. The yielding products were characterized by 1H NMR in D2O (Varian 699.804 MHz). 2.3. Preparation of polymer/DNA complexes All the polymer/DNA complexes were freshly prepared before characterization or gene transfection experiments. Generally, amino acid-dendrimer conjugates were added into 0.8 mg plasmid DNA (EGFP or luciferase plasmid) at different N/P ratios and the sample was incubated for 30 min at room temperature. The N/P ratio was calculated according to the cationic groups (N number) on the dendrimer surface to anionic phosphate groups (P number) in plasmid DNA. The corresponding polymer/ DNA weight ratios for the complexes at different N/P ratios are also shown in Table S1. For His- and Phe-functionalized dendrimers, the N number for each conjugate was a constant of 128. For Arg-functionalized dendrimers, the N number was a sum of 128 and the number of conjugated Arg moieties since Arg has two cationic groups. Since the imidazole group in His and the tertiary amine group in dendrimer are not protonated at pH 7.4, these groups were not considered when calculating the N numbers. For the commercial transfection reagents such as Lipofectamine 2000,
Fig. 1. Synthesis of multi-functionalized dendrimers using a one-pot approach.
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Table 1 Characterizations of the single-, dual- and triple-functionalized dendrimers. Functionalized dendrimers
Dendrimer conjugates
Number of amino acid per G5 dendrimer Fed
Single-functionalized dendrimers
Dual-functionalized dendrimers
Triple-functionalized dendrimers
G5-Arg110 G5-Arg33 G5-Phe99 G5-Phe71 G5-His113 G5-Arg34Phe71 G5-Arg51Phe53 G5-Arg82Phe34 G5-Arg44His53 G5-Phe64His40 G5-Arg24Phe20His40 G5-Arg33Phe49His14 G5-Arg35Phe29His28 G5-Arg47Phe24His25 G5-Arg56Phe22His22
Molecular weight (Da)
Conjugated
Arg
Phe
His
Arg
Phe
His
166 0 0 0 0 83 111 124 83 0 48 85 67 83 95
0 45 166 83 0 83 55 42 0 83 24 64 33 28 24
0 0 0 0 166 0 0 0 83 83 95 43 67 55 48
110 33 0 0 0 34 51 82 44 0 24 33 35 47 56
0 0 99 71 0 71 53 34 0 64 20 49 29 24 22
0 0 0 0 113 0 0 0 53 40 40 14 28 25 22
PolyFect and JetPEI, the complexes were prepared according to the protocols at their optimal conditions. 2.4. Characterization of the polymer/DNA complexes The DNA binding capacity of amino acid-dendrimer conjugates was investigated by agarose gel (Biowest, Spain) electrophoresis. Generally, the polymer/DNA complexes at N/P ratios of 0.5:1, 1:1, 2:1 and 4:1 were prepared in deionized water and diluted with DNA loading buffer. The samples were electrophoresed on a 1% (w/v) agarose gel at 100 V for 1 h. The DNA in the gels were stained by ethidium bromide and visualized under UV illumination (Tanon-2500, China). For the DNA release assay, polymer/DNA complexes were prepared a specific molar concentration and different concentrations of heparin were added to release the bound DNA in the complexes. The resulting solutions were further analyzed by agarose gel electrophoresis as described above.
46,008 33,981 43,398 39,276 44325 44,587 44,593 46,639 42968 43733 41,005 43113 42,402 43,129 43,829
The size and zeta potential analysis of polymer/DNA complexes prepared in deionized water at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1 were carried out by dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS90 (Malvern, UK) at 25 C. 2.5. Cell culture and in vitro gene transfection experiments HeLa (ATCC, a human cervical carcinoma cell line), HEK293 (a human embryonic kidney cell line, ATCC) and NIH 3T3 (ATCC, a mouse embryonic fibroblast cell line) cells were cultured with DMEM containing 100 units/mL penicillin sulfate and streptomycin and 10% (v/v) heat-inactivated FBS at 37 C under a humidified atmosphere containing 5% CO2. Before gene transfection experiments, HeLa and NIH 3T3 cells were seeded in 24-well plates and cultured overnight to reach an appropriate cell density. The polymer/DNA complexes containing 0.8 mg plasmid DNA were diluted with 100 mL DMEM containing 10% FBS and incubated 30 min at room
Fig. 2. 1H NMR spectra of single-, dual- and triple-functionalized dendrimers in D2O.
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temperature. Then, the complexes were further diluted with 150 mL medium and incubated with the cells for 6 h. After that, 500 mL fresh medium was added to each well and the cells were further cultured for 42 h. EGFP expressions in the cells were observed by a fluorescence microscopy (Olympus, Japan) and quantitatively analyzed by flow cytometry (BD FACSCalibur, San Jose). Luciferase expressions in the cells were analyzed according to the manufacturer's protocols (Promega). The commercial gene transfection reagents including JetPEI, PolyFect and Lipofectamine 2000 were used as positive controls. 2.6. Cytotoxicity of the amino acid-dendrimer conjugates and their DNA complexes The cytotoxicity of amino acid-dendrimer conjugates and their DNA complexes were examined on NIH 3T3 cells using a well-established MTT assay. The cells were seeded in 96-well plates at a density around 104 cells per well and cultured for 12 h in 100 mL DMEM containing 10% FBS. The cells were treated with polymers or polymer/DNA complexes at different concentrations for 48 h. The chosen polymer concentrations (0.55 mM) and DNA concentration (3.2 mM) equal to those in the gene transfection experiments. 2.7. Cellular uptake of polymer/DNA complexes To analyze the cellular uptake of polymer/DNA complexes, the plasmid DNA was labeled with a green fluorescent dye YOYO-1 for 10 min according to the manufacturer's protocol (Invitrogen). The polymer/DNA complexes were then prepared as described above (the dendrimer concentration is fixed for different amino aciddendrimer conjugates in the complexes). Generally, NIH 3T3 cells was seeded in
24-well plates and cultured for 24 h to reach an appropriate cell density, and YOYO-1 (excitation at 491 nm and emission at 509 nm) labeled complexes were incubated with the cells for 1 and 2 h. After that, the cells were washed with 500 mL cold PBS for three times. Cellular uptake of the complexes was analyzed by flow cytometry. 2.8. pH-buffering capacity assay The pH buffering capacity of amino acid-dendrimer conjugates was determined as described below. Briefly, the concentration of different conjugates were fixed at a constant dendrimer concentration (22.6 nM), and pH value of the conjugate solutions was adjusted to 7.4. Then the samples were titrated with 0.12 M HCl. pH value of the conjugate solution after each titration (1 mL) was measured using a pH meter (Mettler-Toledo). The titration experiment was continued until the pH value of the solution reaches 5.0 (pH range of 7.4e5.0 mimics the endosome acidification). 2.9. Confocal microscopy NIH 3T3 cells were seeded on glass slides in 24-well plates and cultured for 24 h at 37 C. The cells were incubated with YOYO-1-labeled polymer/DNA complexes for 2 and 4 h. The medium were removed and the cells were washed with 500 mL PBS for three times. The cells were further incubated with PBST (PBS containing 0.1% Tween 20) for 5 min, and then with 0.2% BSA at room temperature for 30 min. The acidic vesicles of NIH 3T3 cells was stained with LAMP-2 (2 mg/mL) antibody conjugated with Alexa Fluor 647 (excitation at 650 nm and emission at 665 nm) for 1 h at 37 C. The cells were washed with PBS for three times and nuclei of the cells were stained by Hoechst 33342 (excitation at 346 nm and emission at 460 nm, 5 mg/mL) for
Fig. 3. DNA retardation assay of amino acid-dendrimer conjugates. The polymer/DNA complexes were prepared by mixing the synthesized polymers with EGFP plasmid at N/P ratios of 0.5:1, 1:1, 2:1 and 4:1, respectively. (a) single-functionalized dendrimers, (b) dual-functionalized dendrimers and (c) triple-functionalized dendrimers.
F. Wang et al. / Biomaterials 35 (2014) 9187e9198 10 min at room temperature. After the cells were washed with PBS for three times, co-localization of the complexes with acidic vesicles was observed by confocal microscopy (Leica SP5, Germany).
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3. Results and discussion 3.1. Synthesis and characterization of single-, dual- and triplefunctionalized dendrimers and their complexes with DNA
2.10. In vivo transfection HeLa tumor xenograft model was established as described below. 6-week-old female BALB/c nude mice at an average weight of 22.0 g were obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The animals were housed under specific-pathogen-free conditions within the animal care facility at East China Normal University. The animal experiments were carried out according to the National Institutes of Health guidelines for care and use of laboratory animals and approved by the ethics committee of East China Normal University. HeLa cells were cultured in DMEM containing 10% FBS. 100 mL cell suspensions in PBS (containing 107 cells) were subcutaneously injected into the BALB/c nude mice. When tumor size reaches 50 mm3, the mice were administrated with 25 mL G5-Arg47Phe24His25/DNA or G5-Arg110/DNA complexes in 5% glucose solution (containing 3.3 mmol polymer and 10 ug luciferase plasmid) by injection into the tumor. The animal treated with 5% glucose solution was used as a control. The treatments were repeated every day and a total number of three injections were administrated for each animal. The in vivo transfection experiments were continued for another two days. After that, 200 mL Dluciferin potassium salt (15 mg/mL) was intraperitoneally injected into the anesthetized mice. 5 min later, the mice were placed into an in vivo imaging system (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton), and the luminescence at tumor site was recorded with an exposure time of 5 min.
To investigate the synergistic effect of amino acids in gene delivery, G5 PAMAM dendrimer was functionalized with Arg, Phe and His using a facile condensation reaction. For dual- and triplefunctionalized dendrimers, mixtures of specific amino acids at different molar ratios were added to the dendrimer solution and the functionalized dendrimers were obtained by a one-pot strategy. The number of each amino acid conjugated to dendrimer was characterized by 1H NMR. As shown in Fig. 2 and Fig. S1, the four broad peaks (Ha, Hb, Hc,b0 and Hd,d0 ) in the chemical shift range of 2.0e3.5 ppm correspond to protons on dendrimer scaffold and other peaks in the spectra are assigned to protons of Arg, Phe and His, respectively [36]. The average number of amino acid conjugated to dendrimer was calculated according to the peaks areas of dendrimer protons and amino acid protons, respectively. The components of the 15 amino acid-dendrimer conjugates are listed in Table 1. For single-functionalized dendrimer, the conjugate G5-
Fig. 4. Size (a) and zeta potential (b) analysis of polymer/DNA complexes prepared at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1, respectively.
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Arg110 represents a G5 PAMAM dendrimer conjugated with an average number of 110 Arg molecules on its surface. For dualfunctionalized dendrimer, the conjugate G5-Arg34Phe71 represents a G5 dendrimer modified with 34 Arg and 71 Phe moieties. For triple-functionalized dendrimer, the conjugate G5Arg35Phe29His28 means a conjugate with 35 Arg, 29 Phe and 28 His moieties. Gel retardation experiments were used to evaluate the DNA binding ability of the synthesized conjugates. As shown in Fig. 3, all the conjugates except G5-Phe99 successfully reduce DNA mobility above an N/P ratio of 1:1. G5-Phe99 even fails to condense DNA at an N/P ratio of 4:1. The lower DNA binding capacity of G5-Phe99 is probably due to the charge shielding effect of aromatic rings present on dendrimer surface [33]. For gene delivery, the size of polymer/DNA polyplexes is essential for efficient gene transfection. As shown in Fig. 4 and Fig. S2, the conjugates containing Arg moieties can condense plasmid DNA into nanoparticles around 200 nm (Fig. 4 and Fig. S2), while those without Arg moieties such as G5-Phe99, G5-Phe71, G5-His113 and G5-Phe64His40 fail to form small nanoparticles below 400 nm with DNA at N/P ratios of 5:1,
10:1 and 20:1. The polyplexes with sizes around 200 nm are appropriate for cellular uptake and gene delivery. This result suggests that Arg is essential in the formation of stable complexes for gene transfection [37e39]. Plasmid DNA is negatively charged at neutral conditions, and the addition of all the conjugates can turn DNA charge state from negative to positive at an N/P ratio of 5:1. The biophysical properties of the polymer/DNA complexes will be further discussed to analyze the transfection mechanisms of single-, dual- and triple-functionalized dendrimers in Section 3.3. 3.2. Synergistic effect of amino acids modified on dendrimer surface in gene delivery The transfection efficacy of single-, double- and triplefunctionalized dendrimers were tested on HeLa, HEK293 or NIH 3T3 cells using EGFP and luciferase plasmid as reporter genes. As shown in Fig. 5, a dual-functionalized dendrimer G5-Arg34Phe71 successfully transfected 75.2% HeLa cells at its optimal N/P ratio, while the single-functionalized conjugates G5-Arg33 and G5-Phe71 only transfected 21.8% and 12.1% HeLa cells at equivalent dendrimer
Fig. 5. Synergistic effect of Arg and Phe on G5-Arg34Phe71. The transfection efficacy of G5-Arg34Phe71 on HeLa cells was compared with those of G5-Arg33, G5-Phe71, G5-Arg110 and G5-Phe99, respectively. The dendrimer concentration is fixed at 0.36 mM for all the conjugates. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in HeLa cells measured by flow cytometry (n ¼ 3). Statistically significant differences are denoted by ***p < 0.001 using student's t-test.
Fig. 6. Transfection efficacies of single-, dual- and triple-functionalized dendrimers on NIH 3T3 cells for 48 h. The dendrimer concentration is fixed at 0.55 mM for all the conjugates. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3).
Fig. 7. Luciferase activities of NIH 3T3 cells transfected by single-, dual- and triple-functionalized dendrimers for 48 h. The dendrimer concentration is fixed at 0.55 mM for all the conjugates. The luciferase activity is expressed as relative luciferase light units per mg protein (RLU/mg protein, n ¼ 3).
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and EGFP plasmid molar concentrations. Also, G5-Arg34Phe71 is much more efficient (both EGFP positive cells and mean fluorescence intensity) than G5-Arg110 or G5-Phe99. Not limited to the optimal N/P ratio, G5-Arg34Phe71 shows much higher efficacy than G5-Arg110 on HeLa cells at all the N/P ratios (Fig. S3). These results suggest synergistic effect of Arg and Phe on the dual-functionalized dendrimer in gene delivery. Similar results were obtained on HEK293 cells (Fig. S4). The synergistic effect amino acids in gene delivery were further confirmed on NIH 3T3 cells. As shown in Fig. 6 and Fig. S5, all the dual- and triple-functionalized dendrimers except G5-Phe64His40 exhibit higher transfection efficacy than single-functionalized dendrimers. The triple-functionalized dendrimers G5Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient on delivering EGFP plasmid than the dual-functionalized ones. G5Phe64His40 shows poor transfection efficacy due to its weak DNA binding capacity as revealed in Fig. 4. Similar results were obtained on NIH 3T3 cells using a luciferase reporter gene (Fig. 7). The dualand triple-functionalized dendrimers show higher luciferase
activity than single-functionalized ones. The synthesized conjugates except G5-Phe99 show minimal cytotoxicity on NIH 3T3 cells at transfection concentrations (Fig. 8a). The relatively high toxicity of G5-Phe99 is due to the hydrophobic character of dendrimer surface after Phe modification. This phenomenon is also observed in a previous result [32]. Though it seems to introduce cytotoxicity after Phe modification, the dual- and triple-functionalized dendrimers containing Phe moieties show minimal cytotoxicity on NIH 3T3 cells. Even in the presence of plasmid DNA, these materials maintain high cell viability above 90% (Fig. 8b), suggesting that the dual- and triple-functionalized dendrimers can achieve high transfection efficacy with low cytotoxicity on the transfected cells. 3.3. Synergistic mechanism of amino acids modified on dendrimer surface in gene delivery The transfection efficacy of a gene material depends on several parameters such as complex formation and stability, cellular uptake, endosomal escape and intracellular DNA release [1]. As shown
Fig. 8. Cytotoxicities of single-, dual- and triple-functionalized dendrimers (a) and their DNA complexes (b) on NIH 3T3 cells for 48 h (n ¼ 5). The polymer concentrations (0.55 mM) equal to those in gene transfection experiments.
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in Figs. 5 and 6, Arg plays an essential role in gene transfection. The transfection efficacy of dual- and triple-functionalized dendrimers increases in proportion with the number of conjugated Arg moieties. Those conjugates without Arg moieties such as G5-Phe99, G5His113 and G5-Phe64His40 show extremely low transfection efficacy. First, the conjugated Arg moiety has two positive charged groups, which facilitates DNA condensation. This is evidenced by DNA retardant and DLS results in Figs. 3 and 4. Arg-containing conjugates show strong DNA binding capacity, while those without Arg moieties fail to condense plasmid DNA into nanoparticles around 200 nm at relative low N/P ratios. Second, the positive charge of the guanidinium group in Arg is delocalized on three nitrogen atoms, thus guanidinium shows better interactions with anions such as phosphates than localized cations such as ammonium [40]. Third, the guanidinium group has strong affinity to cell membranes through ionic pairing and hydrogen bonding [41]. Due to these reasons, Arg-dendrimer conjugates were widely investigated as efficient gene vectors during the past decade [19e23]. However, the dendrimer conjugated with 110 Arg moieties (G5-Arg110) shows much lower efficacy compared to the dual- and triplefunctionalized dendrimers, suggesting that His and Phe moieties also play important roles in gene delivery. This is attributed to the high charge density on G5-Arg110, which decreases the penetration of polymer/DNA complex through cell membrane via endocytosis. The high charge density on polymers may also trigger
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destabilization of self-assembled phospholipids in the cell membrane, resulting in serious cytotoxicity [9]. Increased cytotoxicity is also a limiting factor for high transfection efficacy. One solution to this problem is tailoring the balance of charge and hydrophobic contents on dendrimer surface [42]. Replacement of partial Arg moieties in G5-Arg110 with hydrophobic amino acid such as Phe can improve the complex cellular uptake. As shown in Fig. 9a, complexes of G5-Arg51Phe53, G5-Arg47Phe24His25 and G5Arg56Phe22His22 with DNA show higher cellular uptake than that of G5-Arg110. As a result, dual-functionalized dendrimer such as G5Arg51Phe53 and triple-functionalized dendrimer such as G5Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient in gene transfection than G5-Arg110. In addition, G5-Arg51Phe53/DNA complex is easier to release their bound DNA in the presence heparin than G5-Arg110/DNA complex (Fig. S6). Such a hydrophobic effect on improving gene transfection efficacy is also reported in several polymeric gene delivery systems [42e45]. Though Phe effectively tailors the hydrophobic/hydrophilic balance on dendrimer surface, the conjugation of excess Phe moieties to a G5 dendrimer also leads to increased cytotoxicity. For example, G5Phe99 is even more cytotoxic than unmodified G5 PAMAM dendrimer and it kills most of the cells at a concentration of 50 mg/mL (Fig. 9b). The incorporation of His into the conjugates can alleviate the toxicity issues of Arg and Phe. As shown in Fig. 9b, G5-Arg44His53
Fig. 9. (a) Cellar uptake of polymer/DNA complexes for 1 and 2 h by NIH 3T3 cells (n ¼ 3), statistically significant differences are denoted by *p < 0.05 and ***p < 0.001 using student's t-test. The polymer concentrations equal to those in gene transfection experiments. (b) Cytotoxicities of amino acid-dendrimer conjugates on NIH 3T3 cells at different concentrations (n ¼ 4). (c) pH-buffering capacities of G5-His113 and G5-Arg110 (n ¼ 3). (d) Confocal images of NIH 3T3 cells incubated with YOYO-1-labeled G5-Arg47Phe24His25/DNA complex (green) for 2 and 4 h. The acidic vesicles were stained with LAMP-2 conjugated with Alexa Fluor 647 (red) and the nuclei were stained with Hoechst 33342 (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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and G5-Arg47Phe24His25 are less cytotoxic than G5-Arg110 on NIH 3T3 cells. G5-His113 is non-toxic at concentrations up to 600 mg/mL. Besides the role of reducing cytotoxicity, the imidazole group (pKa ~ 6.04) in His is protonable at mild acidic conditions. As shown in Fig. 9c, G5-His113 shows better pH-buffering capacity than G5Arg110 within the pH range of 7.4 to 5.0 (endosome acidification), suggesting that the incorporation of His moieties to the conjugates can facilitate endosomal escape [24]. The triple-functionalized conjugate G5-Arg47Phe24His25 shows excellent endosomal escape ability and the complex is not co-localized with acidic vesicles within 2 h (Fig. 9d). It is worth noting that the molar ratios of Arg, Phe and His should be carefully chosen to obtain an efficient triplefunctionalized dendrimer. For example, G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are much more efficient in delivering EGFP plasmid on NIH 3T3 cells than the other three triple-functionalized dendrimers (Fig. 6). G5-Arg47Phe24His25 and G5-Arg56Phe22His22 show comparable efficacy with several commercial transfection
reagents such as JetPEI, PolyFect and Lipofectamine 2000 at their optimal conditions (Fig. 10). The triple-functionalized dendrimer G5-Arg47Phe24His25 also shows higher in vivo transfection efficacy than the single functionalized dendrimer G5-Arg110 when delivering a luciferase plasmid (Fig. 11). These results suggest that the triple-functionalized dendrimers can be developed for commercial purpose in the future. 4. Conclusions In this study, we synthesized a list of single-, dual- and triplefunctionalized dendrimers with Arg, Phe and His for gene delivery. The amino acids show synergistic effects on dual- and triplefunctionalized dendrimers. Arg in the conjugates is essential for complex formation. Phe modulates the balance of hydrophobic and hydrophilic contents on dendrimer surface, thereby facilitates the cellular uptake process. His improves pH-buffering capacity and reduces cytotoxicity of the cationic dendrimers. These amino acids
Fig. 10. Comparisons of triple-functionalized dendrimers with three commercial transfection reagents (JetPEI, PolyFect and Lipofectamine 2000) on transfection efficacy. N/P ratios for G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are 30:1 and 32:1, respectively. Gene transfection experiments for the commercial reagents were optimized according to the manufacturer's protocols. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3). Statistically significant differences are denoted by *p < 0.05 and **p < 0.01 using student's t-test.
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Fig. 11. In vivo gene transfection on a HeLa tumor xenograft model. The animals were treated with 5% glucose solution, G5-Arg47Phe24His25/DNA and G5-Arg110/DNA complexes, respectively. Luciferase plasmid was used as the reporter gene. The polyplexes were prepared in 5% glucose solution.
Fig. 12. Transfection mechanisms of single-, dual- and triple-functionalized dendrimers.
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have different functions in the gene delivery process and hence a combination of them generates synergistic effects in gene delivery (Fig. 12). The efficient triple-functionalized dendrimers achieve comparable transfection efficacy to several commercial transfection reagents on NIH 3T3 cells. In addition, the dual- and triplefunctionalized dendrimers show low cytotoxicity on the transfected cells. This study provides a new insight into the design of efficient and low cytotoxic gene vectors using a facile strategy. Acknowledgment The authors thank the grants including National Natural Science Foundation of China (No. 21322405), the Shanghai Rising Star Program (13QA1401500) and the Science and Technology of Shanghai Municipality (11DZ2260300) for financial supports. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.07.027. References [1] Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nat Rev Drug Discov 2005;4:581e93. [2] Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nat Mater 2013;12:967e77. [3] Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009;8:129e38. [4] Hu Y, Xu B, Ji Q, Shou D, Sun X, Xu J, et al. A mannosylated cell-penetrating peptide-graft-polyethylenimine as a gene delivery vector. Biomaterials 2014;35:4236e46. [5] Gao Y, Wang ZY, Zhang J, Zhang Y, Huo H, Wang T, et al. RVG-peptide-linked trimethylated chitosan for delivery of siRNA to the brain. Biomacromolecules 2014;15:1010e8. [6] Kadlecova Z, Rajendra Y, Matasci M, Baldi L, Hacker DL, Wurm FM, et al. DNA delivery with hyperbranched polylysine: a comparative study with linear and dendritic polylysine. J Control Release 2013;169:276e88. [7] Zarogoulidis P, Hohenforst-Schmidt W, Darwiche K, Krauss L, Sparopoulou D, Sakkas L, et al. 2-diethylaminoethyl-dextran methyl methacrylate copolymer nonviral vector: still a long way toward the safety of aerosol gene therapy. Gene Ther 2013;20:1022e8. [8] Qian X, Long L, Shi Z, Liu C, Qiu M, Sheng J, et al. Star-branched amphiphilic PLA-b-PDMAEMA copolymers for co-delivery of miR-21 inhibitor and doxorubicin to treat glioma. Biomaterials 2014;35:2322e35. [9] Mastrobattista E, Hennink WE. Polymers for gene delivery: charged for success. Nat Mater 2012;11:10e2. [10] Tomalia DA. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog Polym Sci 2005;30:294e324. [11] Svenson S, Tomalia DA. Dendrimers in biomedical applications-reflections on the field. Adv Drug Deliv Rev 2012;64:102e15. [12] Tomalia DA. Interview: an architectural journey: from trees, dendrons/dendrimers to nanomedicine. Nanomedicine 2012;7:953e6. [13] Shcharbin D, Pedziwiatr E, Bryszewska M. How to study dendriplexes I: characterization. J Control Release 2009;135:186e97. s C, Uchegbu IF, Sch€ [14] Dufe atzlein AG. Dendrimers in gene delivery. Adv Drug Deliv Rev 2005;57:2177e202. [15] 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;134:17680e7. [16] Arima H, Motoyama K, Higashi T. Sugar-appended polyamidoamine dendrimer conjugates with cyclodextrins as cell-specific non-viral vectors. Adv Drug Deliv Rev 2013;65:1204e14. [17] 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;5:4053. [18] Liu H, Wang Y, Wang M, Xiao J, Cheng Y. Fluorinated poly(propylenimine) dendrimers as gene vectors. Biomaterials 2014;35:5407e13. [19] Luo K, Li C, She W, Wang G, Gu Z. Arginine functionalized peptide dendrimers as potential gene delivery vehicles. Biomaterials 2012;33:4917e27.
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