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Being Two Is Better than Being One: A Facile Strategy to Fabricate Multicomponent Nanoparticles for Efficient Gene Delivery Fei Wang,†,‡ Lianfu Deng,‡ Jingjing Hu,† and Yiyun Cheng*,† †

Shanghai Key Laboratory of Regulatory Biology and School of Life Sciences, East China Normal University, Shanghai 200241, China Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Shanghai Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China

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S Supporting Information *

ABSTRACT: Multifunctionality is necessary in the design of efficient gene vectors due to the existence of multiple barriers during gene delivery. Traditional methods in the design of polyfunctional materials for this purpose are associated with sophisticated syntheses and high costs. Here, we proposed a facile coassembly approach to fabricate multicomponent nanoparticles for efficient gene delivery. The resulting particles contain different functional dendrimers and show favorable physicochemical characteristics. All the combinations in the fabrication of multicomponent nanoparticles show a synergistic effect in improving transfection efficacy. The prepared nanoparticles successfully address two or more barriers in the gene delivery process and show minimal toxicity on the transfected cells. The combination of high transfection efficacy and low cytotoxicity suggests that the prepared multicomponent nanoparticles as promising carriers in gene delivery.

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INTRODUCTION The success in clinical gene therapy is mainly dependent on the development of efficient and low cytotoxic gene vectors.1−4 A wide variety of polymers were developed as gene vectors during the past two decades.5−10 However, the performance of most polymers in gene therapy have been unsatisfactory because they do not possess the required transfection efficacy.5,11 The critical nanoscale design parameters (CNDPs) including size, shape, flexibility/rigidity, architecture, elemental composition, and surface chemistry of polymers should be carefully optimized to achieve efficient gene delivery.12 The “off-the-shelf” polymers such as dendrimers, polylysine. and polyethylenimine (PEI) were not specially designed for gene delivery. They lack necessary functions to breakdown the extracellular and intracellular barriers in gene delivery including DNA binding and condensation, serum stability, cellular uptake, endosomal escape, intracellular DNA unpacking, and nuclear localization.11,13 Modification of these“off-the-shelf” polymers with functional ligands may address one or two specific barriers and somewhat improve the transfection efficacy.14−17 Another approach is to precisely design polymers with multifunctions for efficient gene delivery;18,19 however, these materials are usually involved with sophisticated syntheses and the ratio of each functional ligand on the polymers is not easy to control. Scientists in this field are seeking facile strategies to fabricate polyfunctional polymers with high transfection efficacy and biocompatibility. Amino acid modification is a general method to improve the transfection efficacy of cationic polymers.20−23 The chemical diversity of amino acid residues such as guanidine, imidazole, © XXXX American Chemical Society

and phenyl groups is suitable to tailor the physicochemical characteristics of polymers.14 For example, arginine (Arg) modification improves the DNA binding capacity of polymers and the stability of resulting polyplexes. In addition, the guanidinium group in Arg has high binding affinity with phosphate groups on the cell membranes.24−26 Phenylalanine (Phe) modification modulates the balance of charge and hydrophobic contents on the polymer.27,28 The hydrophobic effect of the phenyl group in Phe can improve the cellular internalization of polyplex and thereby increase the transfection efficacy.29 Histidine (His) modification facilitates the early release of genes from the endosomal pathway into the cytosol due to the pH buffering effect of imidazole group. Besides, the His modification improves the serum-resistant property and biocompatibility of cationic polymers.30 Therefore, the three amino acids Arg, Phe, and His have beneficial effects to address the barriers in gene packaging, cell internalization, and endosomal escape, respectively. A reduction-sensitive dendronized polymer coconjugated with His and Phe or tyrosine shows high efficacy in RNA interference.31 The dual-functionalized polymers have better performance in endocytosis and endosomal escape compared to the control polymers. In a recent study, we also found that a combination of Arg, Phe, and His on the dendrimer surface generates a synergistic effect in gene delivery.32 These materials show both high transfection efficacy and minimal toxicity on the transfected cells. Though the Received: November 26, 2015 Revised: January 19, 2016

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DOI: 10.1021/acs.bioconjchem.5b00643 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. (a) Fabrication of multicomponent nanoparticles with fluorescent tags using a facile coassembly approach. G5 PAMAM dendrimers were conjugated with Arg, Phe, or His, which are beneficial for polyplex formation, cellular uptake, and endosomal escape, respectively. Each functional dendrimer was modified with a fluorescent tag to monitor the colocalization and intracellular trafficking of the prepared nanoparticles. G5-Phe71 was labeled with RBTIC (red), and G5-His65 and G5-Arg64 were labeled with FITC (green), respectively. (b) Co-localization of G5-Phe71 and G5-His65 in the prepared multicomponent nanoparticles observed by confocal microscopy. (c) DNA retardation analysis of G5-Phe71/G5-His65 at molar ratio of 3:1, 1:1, and 1:3 by an agarose gel electrophoresis assay. The molar ratios of dendrimer to DNA are 60, 150, 300, 600, and 1800, respectively. (d) Hydrodynamic radius distribution of G5-Phe71/G5-His65/DNA nanoparticles. G5-Phe71 and G5-His65 are mixed at a molar ratio of 1:1. The molar ratio of dendrimer to DNA is 1500:1.

polyfunctional polyplexes for efficient and low-cytotoxicity gene delivery.

polyfunctional materials have promising efficacy in gene delivery, they are generally synthesized with sophisticated procedures and high costs. In addition, it is hard to choose the optimal molar ratio for each amino acid on a dendrimer surface to achieve high efficacy. Furthermore, dual- or triplefunctionalization of different amino acids on a dendrimer surface may generate serious spatial hindrance and the functional amino acids may interfere with each other on a congested surface. Here, we provide a facile, rational, and practical approach to fabricate multicomponent nanoparticles with different amino acids to achieve efficient and low-toxicity gene delivery. Partially modified dendrimers with Arg, His, or Phe were coassembled with plasmid DNA to form multicomponent nanostructures (Figure 1a). Gene transfection behaviors and mechanisms of the prepared nanoparticles were investigated. The aim of this study is to develop a facile strategy to prepare

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RESULTS AND DISCUSSION Characterization of Multicomponent Nanoparticles. Generation 5 (G5) polyamidoamine (PAMAM) dendrimer was partially functionalized with Arg, His, or Phe using a facile condensation reaction. The feeding ratio of amino acid to G5 dendrimer was fixed at 83:1. The obtained functional dendrimers were characterized by 1H NMR. As shown in Figure S1, average numbers of 64 Arg, 65 His, and 71 Phe molecules were conjugated on each G5 dendrimer. The materials were termed G5-Arg64, G5-His65, and G5-Phe71, respectively. Multicomponent polyplexes were prepared using a facile coassembly strategy (Figure 1a). Two types of functional dendrimers (G5-Phe71/G5-His65, G5-Phe71/G5-Arg64, or G5B


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Figure 2. (a) Fabrication of G5-Phe71/G5-His65/DNA nanoparticles for gene delivery. (b) Fluorescent microscopy images of HeLa cells transfected by G5-Phe71/G5-His65/DNA nanoparticles at different molar ratios for 48 h. (c) EGFP expression efficacy of the nanoparticles in HeLa cells measured by flow cytometry (n = 3). (d) Luciferase activity of HeLa cells transfected with G5-Phe71/G5-His65/DNA nanoparticles (n = 3). G5Phe71, G5-His65, and G5-Phe64His40 tested at their optimal transfection conditions were used as controls. Statistically significant differences are denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 versus G5-Phe71; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus G5-His65, respectively.

of which corresponds to G5-His65 and G5-Phe71, respectively, overlap in the same locations. This fluorescence overlap indicates that G5-His65-FITC is co-localized with G5-Phe71RBITC in the prepared nanoparticles. Similarly, G5-Phe71RBITC is co-localized with G5-Arg64-FITC in related nanoparticles (Figure S3). In vitro cellular uptake experiments show that the multicomponent nanoparticles were efficiently taken up by HeLa cells after 3 h incubation. Upon uptake, the fluorescent signals from both functional dendrimers exhibit a high degree of co-localization (Figure S4). These results suggest that both functional dendrimers are included in the prepared nanoparticles and the particles are stable in serumcontaining medium and remain intact after cellular internalization. Agarose gel electrophoresis assay in Figure 1c and Figure S5 reveals that the combination of G5-Phe71, G5-His65, or G5-Arg64 mixed at different molar ratios effectively retarded the mobility of plasmid DNA above a dendrimer/DNA molar

Arg64/G5-His65) were mixed at a specific molar ratio, and incubated with plasmid DNA to fabricate the multicomponent nanoparticles. Co-localization of different functional dendrimers in the prepared nanoparticles was studied by confocal microscopy. G5-Arg64 and G5-His65 were tagged with a green fluorescent dye (FITC), while G5-Phe71 was modified with a red fluorescent dye (RBITC). The dye-labeled products were termed G5-Arg64-FITC, G5-His65-FITC, and G5-Phe71-RBITC, respectively. UV−vis and fluorescence spectra of these materials suggest the successful conjugation of FITC or RBITC molecules on the functional dendrimers (Figure S2). The average numbers of FITC or RBITC molecules per G5 dendrimer were 4.2 ± 0.07 for G5-His65-FITC, 2.9 ± 0.04 for G5-Arg64-FITC or 1.5 ± 0.04 for G5-Phe71-RBITC according to the calibration curve of the free dyes.33 The fluorescent dyelabeled nanoparticles were visualized by confocal microscopy. As shown in Figure 1b, FITC (green) and RBITC (red), each C


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Figure 3. (a) Cellular uptake efficacy of YOYO-1 labeled G5-Phe71/G5-His65/DNA nanoparticles. G5-Phe71 and G5-His65 are mixed at molar ratios of 3:1, 1:1, and 1:3, respectively. The experiments were conducted on HeLa cells for 3 h and 6 h, and further analyzed by flow cytometry (n = 3). Statistically significant differences are denoted by **p < 0.01 and ***p < 0.001, respectively. (b) Gene transfection efficacy of G5-Phe71/G5-His65/ DNA and G5-Phe71/DNA polyplexes in HeLa cells in the presence of nigericin (n = 3). The cells were pretreated with 0, 2.5, and 5 μM nigericin before incubation with the polyplexes. The relative luciferase activity for each material was normalized to that in the absence of nigericin. Statistically significant differences are denoted by *p < 0.05 and **p < 0.01, respectively. (c) Intracellular trafficking of the prepared G5-Phe71/G5-His65/DNA multicomponent nanoparticles visualized by confocal microscopy. G5-Phe71 was labeled with RBTIC (red), G5-His65 was labeled with FITC (green), and DNA was tagged with YOYO-1 (green), respectively. The HeLa cells were incubated with fluorescence tagged nanoparticles for 3 and 6 h, respectively.

from the results: the combination of two functional dendrimers in a polyplex structure is critical for effective gene transfection. G5-Phe71/G5-His65/EGFP nanoparticles exhibit significantly higher transfection efficacies (both percent of positive EGFP cells and mean fluorescence intensity) than single functional dendrimers (G5-Phe71 and G5-His65). G5-Phe71 and G5-His65 mixing at a molar ratio 3:1 shows the highest transfection efficacy around 40%, while single G5-Phe71 and G5-His65 only transfect 13.2% and 3.7% HeLa cells at their optimal transfection conditions (Figure 2c). Even when Phe and His were conjugated on a single G5 dendrimer, the dualfunctionalized dendrimer G5-Phe64His40 shows poor transfection efficacy. This is due to the weak DNA binding capacity of the dual-functionalized dendrimer as well as the serious spatial hindrance on G5 dendrimer surface.32 The as-prepared G5-Phe71/G5-His65 nanoparticles have high DNA binding capacity and less congested dendrimer surface, thus exhibiting a synergistic effect on improving transfection efficacy. Similar results were obtained using a luciferase reporter gene. The G5Phe71/G5-His65 multicomponent nanoparticles show much higher luciferase transfection efficacy than G5-Phe71 and G5His65, respectively (Figure 2d). We further investigated the reason the efficacy of G5-Phe71/ G5-His65 multicomponent nanoparticles is superior to G5-

ratio of 150. The sizes of these multicomponent nanoparticles are around 200 nm at a dendrimer/DNA molar ratio of 1500 (Figure 1d and Table S1). In addition, the prepared nanoparticles are positively charged (Table S1), which is responsive for efficient cellular uptake. Taken together, the method described in Figure 1a can fabricate multicomponent nanoparticles with favorable physicochemical properties for gene delivery. Gene Transfection Behaviors of G5-Phe71/G5-His65 Multicomponent Nanoparticles. The transfection efficacy of G5-Phe71/G5-His65/DNA nanoparticles was first screened on HeLa cells at different G5-Phe71/G5-His65 molar ratios (3:1 to 1:3) and dendrimer/DNA molar ratios (900−1800). The HeLa cells were incubated with the nanoparticles for 48 h in serum-containing media. After transfection, EGFP expressions in the cells were observed by fluorescence microscopy and measured by flow cytometry, and luciferase expressions in the cells were measured using a plate reader. Protein concentration in each well was measured by a BCA kit. G5-Phe71 and G5His65 were tested as controls. The screening results for the transfection efficacies of G5-Phe71/G5-His65 multicomponent nanoparticles, and G5-Phe71 and G5-His65 at their optimal polymer/DNA molar ratios are summarized in Figure S6, Figure S7, and Figure 2. An important trend can be generalized D


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Figure 4. (a) Fabrication of G5-Phe71/G5-Arg64/DNA multicomponent polyplexes for gene delivery. (b) EGFP expressions in HeLa cells transfected by the G5-Phe71/G5-Arg64/DNA nanoparticles for 48 h. (c) Transfection efficacy of the materials in (b) measured by flow cytometry (n = 3). G5-Phe71, G5-Arg64, and G5-Arg51Phe53 tested at their optimal conditions were used as controls. Statistically significant differences are denoted by **p < 0.01 versus G5-Phe71, and #p < 0.05, ##p < 0.01, and ###p < 0.001 versus G5-Arg64, respectively. (d) Cellular uptake efficacy of YOYO-1 labeled G5-Phe71/G5-Arg64/DNA nanoparticles. The dendrimers were mixed at molar ratios of 1:1 and 3:1, respectively. The experiments were conducted on HeLa cells for 3 h and 6 h, and further analyzed by flow cytometry (n = 3). Statistically significant differences are denoted by *p < 0.05 and **p < 0.01, respectively. (e) Intracellular trafficking of the prepared G5-Phe71/G5-Arg64/DNA multicomponent nanoparticles visualized by confocal microscopy. G5-Phe71 was labeled with RBTIC (red), G5-Arg64 was labeled with FITC (green), and DNA was tagged with YOYO-1 (green), respectively. The HeLa cells were incubated with fluorescence tagged nanoparticles for 3 or 6 h before confocal imaging.

Phe71 and G5-His65. As shown in Figure 3, the presence of G5Phe71 in the nanoparticles significantly improves the cellular uptake of G5-His65/DNA polyplexes due to the hydrophobic effect which is beneficial for endocytosis (Figure 3a).29,32 On the other hand, the presence of G5-His65 in the nanoparticles improves the pH buffering capacity of G5-Phe71/DNA polyplexes.30,32 Nigericin is an inhibitor of endosomal acidification which prevents the endosomal escape of polyplexes.34 As shown in Figure 3b, the gene transfection efficacy of G5Phe71/G5-His65 nanoparticles is more sensitive to nigericin as compared to G5-Phe71, suggesting the G5-His65 conjugate in the multicomponent nanoparticle is beneficial for efficient endosomal escape. The multicomponent nanoparticles can efficiently release the bound DNA after 6 h incubation with the

cells. G5-Phe71-RBITC is co-localized with G5-Arg64-FITC and YOYO-1 labeled DNA at 3 h post-incubation, while most of the functional dendrimers (red) are not overlapped with plasmid DNA (green) at 6 h (Figure 3c), suggesting efficient intracellular DNA unpacking. Taken together, the G5-Phe71/ G5-His65 multicomponent nanoparticles show high cellular internalization, improved endosomal escape, and efficient intracellular DNA release. These beneficial effects explain the high transfection efficacy of the prepared multicomponent nanoparticles. Gene Transfection Behaviors of G5-Phe71/G5-Arg64 and G5-Arg64/G5-His65 Multicomponent Nanoparticles. We further investigate whether the concept described in Figure 1a also works in the G5-Phe71/G5-Arg64 and G5-Arg64/G5E


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Figure 5. (a) Fabrication of G5-Arg64/G5-His65/DNA multicomponent polyplexes for gene delivery. (b) Fluorescent microscopy images of HeLa cells transfected by G5-Arg64/G5-His65/DNA multicomponent nanoparticles in HeLa cells. The molar ratio of G5-Arg64 to G5-His65 is fixed at 1:1. (c) EGFP expression efficacy of the nanoparticles in (b) measured by flow cytometry. G5-Arg64 and G5-His65 were tested as controls.

His65 combinations. As shown in Figure 4, Figure S8, and Figure S9, G5-Phe71/G5-Arg64 multicomponent nanoparticles show significantly higher transfection efficacy than G5-Phe71 and G5-Arg64. The G5-Phe71/G5-Arg64 nanoparticles at a molar ratio of 3:1 successfully transfect more than 50% HeLa cells, while G5-Phe71 and G5-Arg64 only transfect 18.1% and 32.9% cells at their optimal conditions (Figure 4b and c). Similar results were obtained on HeLa cells using a luciferase reporter gene (Figure S10). To investigate the reason G5-Phe71 and G5Arg64 in the prepared multicomponent nanoparticles show synergistic effect in gene delivery, we compared the cellular uptake of G5-Phe71/G5-Arg64/DNA nanoparticles by HeLa cells with those of G5-Phe71/DNA and G5-Arg64/DNA polyplexes. As shown in Figure 4d, the presence of G5-Phe71

significantly improves the cellular uptake of G5-Arg64/DNA polyplexes which is attributed to the hydrophobic effect of phenyl group in Phe. In addition, G5-Arg64 improves the cell internalization of G5-Phe71/DNA polyplexes due to the high cell membrane binding affinity of guanidinium group in Arg.32 The disassociation of G5-Phe71/G5-Arg64/DNA nanoparticles occurs in the cytosol after 6 h incubation (Figure 4e). Besides the G5-Phe71/G5-Arg64 combination, G5-Arg64/G5-His65 also show a synergistic effect in gene delivery compared to single functional dendrimers (Figure 5 and Figure S11). Efficacies of the prepared multicomponent nanoparticles are superior to several commercial transfection reagents including branched PEI, SuperFect and PolyFect and comparable to the gold standard reagent Lipofectamine 2000 (Figure S12). F


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Figure 6. Cytotoxicities of (a) G5-Phe71/G5-His65 and (b) G5-Phe71/G5-His65/DNA nanoparticles at different G5-Phe71 to G5-His65 molar ratios, and (c) G5-Phe71/G5-Arg64 and (d) G5-Phe71/G5-Arg64/DNA nanoparticles at different G5-Phe71 to G5-Arg64 molar ratios on HeLa cells for 48 h (n = 4). The polymer concentration in (a), (b), (c), and (d) equals to those in optimal transfection experiments in Figure S6 and Figure S8, respectively.

Cytotoxicity. Besides high transfection efficacy, the multicomponent nanoparticles also show good biocompatibility. The cell viability was evaluated by MTT assay. As shown in Figure 6, the G5-Phe71/G5-His65 and G5-Phe71/G5-Arg64 multicomponent nanoparticles at optimal transfection concentrations show low cytotoxicity on the HeLa cells (Figure 6a and c). Even in the presence of plasmid DNA, the toxicity of the nanoparticles on the transfected cells is minimal and all the transfected cells show more than 90% viability (Figure 6b and d). These results suggest that our strategy can fabricate multicomponent nanoparticles with both high transfection efficacy and limited cell toxicity.

following advantages. First, the current strategy is suitable for more combinations than the previous one. For example, when conjugating Phe and His on a single dendrimer, the yielding dual-functionalized dendrimer G5-Phe64His40 shows poor transfection efficacy (Figure 2c). However, the combination of G5-Phe71 and G5-His65 in multicomponent nanoparticles exhibits significantly higher transfection efficacies than single functional dendrimers (such as G5-Phe71 and G5-His65). The reason for this could be the weak DNA binding capacity of the dual-functionalized dendrimer G5-Phe64His40 as well as the serious spatial hindrance of amino acids on a G5 dendrimer surface. Second, the coassembly strategy can more easily optimize the transfection efficacy than the previous method. We only need to synthesize two or three partially modified dendrimers to start the gene transfection experiments. However, a list of multifunctional dendrimers with different amino acid molar ratios should be prepared to achieve efficient gene delivery in the previous method. This proposed combination strategy provides a new insight into the design of multicomponent nanoparticles for efficient gene delivery and this method should be applicable to many other functional polymers.

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CONCLUSIONS In summary, we proposed a facile and effective approach to fabricate multicomponent nanoparticles for nonviral gene delivery. The prepared nanoparticles contain different functional dendrimers and show favorable physicochemical characteristics. Combination of G5-Phe71/G5-His65, G5Phe71/G5-Arg64, or G5-Arg64/G5-His65 shows synergistic effects to improve the transfection efficacy. These multicomponent nanoparticles can address different barriers in the gene delivery process, e.g., G5-Arg64 is beneficial for DNA binding, G5-Phe71 for cell internalization, and G5-His65 for endosomal escape. Transfection efficacy of the multicomponent nanoparticles can be easily tailored by changing the molar ratio of functional dendrimers. In addition, the prepared nanoparticles show minimal toxicity on the transfected cells. Our major conclusion is that being two is better than being one to break down the barriers in gene delivery and improve the transfection efficacy of polymeric gene vectors. Compared with our previously reported method to fabricate multifunctional dendrimers,32 the current coassembly strategy provides the

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MATERIALS AND METHODS Materials. G5 PAMAM dendrimer with an ethylenediamine core and surface primary amine groups was purchased from Dendritech (Midland, MI). Nigericin, fluorescein-isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), and deuterium water were obtained from Sigma-Aldrich (St. Louis, MO). YOYO-1 was purchased from Invitrogen (Carlsbad, California). Boc-Arg(pbf)-OH, Boc-Phe-OH, and Boc-His(Trt)-OH were purchased from GL Biochem (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s

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G5-Phe71-RBITC/G5-Arg64-FITC/DNA polyplex nanoparticles were prepared by the same method. Cell Culture and in Vitro Gene Transfection. HeLa (a human cervical carcinoma cell line, ATCC) cells were cultured in DMEM containing 10% serum and 1% penicillin− streptomycin under humidified air containing 5% CO2 at 37 °C. The cells were plated on 24-well plates and further cultured overnight before gene transfection. The medium was replaced by 250 μL of fresh DMEM containing multicomponent nanoparticles and 10% serum. Each well contains 0.8 μg DNA and the dendrimer to DNA molar ratio ranges from 900 to 1800. After 6 h, 500 μL serum containing DMEM (10%) was added to each well and the cells were incubated for an additional 42 h. EGFP expressions in the cells was observed by fluorescence microscopy (Olympus, Japan) and quantitatively analyzed by flow cytometry (BD FACSCalibur, San Jose). Luciferase expression in the cells was analyzed according to standard protocols of luciferase assay system (Promega). The luciferase activity for each material was normalized to RLU/mg protein. Cell Uptake of Multicomponent Nanoparticles. Luciferase plasmid was labeled with YOYO-1 (Y3601, Invitrogen) for 10 min according to the manufacturer’s protocol. The YOYO-1 labeled DNA was then incubated with functional dendrimers to prepare multicomponent nanoparticles as described above. HeLa cells were treated with the YOYO-1 labeled multicomponent nanoparticles (0.8 μg DNA) for 3 h and 6 h, and washed with cold buffer for three times, followed by digestion using trypsin. Finally, the cells were resuspended with buffer and the analyzed by flow cytometry. Endosomal Escape Assay. Transfection experiments were performed with or without nigericin to estimate the endosomal escape capacity of the prepared multicomponent nanoparticles. Generally, HeLa cells were pretreated with nigericin at different concentrations in serum-free DMEM. After 30 min, the medium was removed and the multicomponent nanoparticles containing luciferase plasmid was added. The gene transfection experiments were conducted as described above. Polyplex Intracellular Trafficking. Intracellular trafficking of multicomponent nanoparticles was examined by confocal microscopy. HeLa cells were incubated with the fluorescent dye labeled nanoparticles (FITC, RBITC, or YOYO-1) for 3 or 6 h. The medium was then removed and the cells were washed with cold buffer three times, and fixed with 4% paraformaldehyde for 30 min. The nuclei of the cells were stained by Hoechst 33342 for 10 min at room temperature. Intracellular localizations of the nanoparticles were observed using confocal microscopy. Cytotoxicity of Multicomponent Nanoparticles. The cytotoxicity of multicomponent nanoparticles was evaluated on HeLa cells. The cells were seeded in 96-well plates at a density around 104 cells per well and cultured for 12 h in 100 μL serum containing DMEM. The cells were treated with multicomponent nanoparticles at their optimal gene transfection conditions for 48 h. The relative cell viability was measured using by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nontreated cells were normalized as 100% viability.

modified Eagle’s medium (DMEM) were obtained from GIBCO (Gaithersburg, MD). G5 PAMAM dendrimer was received in aqueous and the solvent was lyophilized before use. Quality of the dendrimer was characterized by 13C NMR and polyacrylamide gel electrophoresis. All the other chemicals were used as received without further purification. Synthesis and Characterization of Partially Modified Dendrimers with Arg, Phe, and His. The amino acids were conjugated to the G5 dendrimer surface by a facile condensation reaction as reported elsewhere.24 Briefly, BocArg(pbf)-OH, Boc-Lys-OH, or Boc-His(Trt)-OH was dissolved in 1 mL anhydrous N,N-dimethylformamide, followed by addition of dicyclohexylcarbodiimide (1.3 mol equiv of amino acids), and N-hydroxysuccinimide (1.2 mol equiv of amino acids) to activate the carboxyl groups for 6 h. Then, 30 mg G5 PAMAM dendrimer dissolved in 1 mL anhydrous dimethyl sulfoxide was added dropwise into the activated amino acid solution and stirred at room temperature for 7 d. The feed molar ratio of amino acid to G5 dendrimer is fixed at 83:1. After the reaction, the mixture solution was dialyzed against 500 mL dimethyl sulfoxide twice and freeze−dried. The obtained crude material was then dissolved into 2 mL trifluoroacetic acid/dichloromethane solution (4:1, v/v) and stirred at room temperature for 6 h to remove the protected groups such as Boc, pbf, and Trt. The remaining trifluoroacetic acid and dichloromethane in the product were removed by rotary evaporation and the synthesized materials were intensively dialyzed against dimethyl sulfoxide, PBS buffer, and distilled water. The purified polymers were obtained by freeze−drying and characterized by 1H NMR in deuterium water (Varian 699.804 MHz). According to the NMR results, average numbers of 64 Arg, 65 His, and 71 Phe molecules were conjugated on each G5 dendrimer. The products were termed G5-Arg64, G5-His65, and G5-Phe71, respectively. Synthesis and Characterization of Fluorescent Dye Labeled Dendrimers Engineered with Amino Acids. In order to confirm the colocalization of different functional dendrimers in a polyplex, G5-Arg64 and G5-His65 conjugates were labeled with a green fluorescent dye FITC, while G5Phe71 conjugate was labeled with a red fluorescent dye RBITC. Generally, dendrimer−amino acid conjugates and FITC or RBRITC were mixed in aqueous solutions at a dendrimer/dye molar ratio of 1:4. The mixture was stirred for 24 h at room temperature and dialyzed against distilled water under dark environment to remove the unreacted dyes. The purified products were obtained by freeze−drying and characterized by UV−vis and fluorescence spectrometer. Fabrication and Characterization of Multicomponent Nanoparticles. Two types of functional dendrimers were mixed at specific molar ratios (1:3 to 3:1). The dendrimer mixtures were further incubated with 0.8 μg plasmid DNA at room temperature for 30 min. The molar ratio of dendrimer to DNA ranges from 60 to 1800. The formed nanoparticles were characterized by agarose gel electrophoresis and dynamic light scattering (Malvern Zetasizer Nano ZS 90, UK). To confirm the co-localization of different functional dendrimers in a polyplex nanoparticle, G5-Phe71-RBITC and G5-His65-FITC at a molar ratio of 1:2 were mixed together and incubated with plasmid DNA for 30 min. The dendrimer to DNA molar ratio is fixed at 1500 (optimal molar ratio in gene transfection experiments). The polyplex solution was then naturally air-dried on a glass slide and the nanoparticles were characterized by confocal microscopy (Leica SP5, Germany).

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00643. H


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Further data on characterization of the synthesized materials, polyplexes, intracellular trafficking behavior and in vitro gene transfection results (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank financial support from the National Natural Science Foundation of China (No. 21322405 and No. 21474030) and the Shanghai Municipal Science and Technology Commission (148014518) on this work.

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REFERENCES

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