Dendrimer-entrapped gold nanoparticles modified with β-cyclodextrin for enhanced gene delivery applications

Jieru Qiu, Lingdan Kong, Xueyan Cao, Aijun Li, Hongru Tan and Xiangyang Shi*
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People's Republic of China. E-mail: xshi@dhu.edu.cn

Received 11th February 2016 , Accepted 2nd March 2016

First published on 3rd March 2016


Abstract

Generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers have been shown to be used as a highly efficient non-viral carrier in gene delivery. However, their high cytotoxicity and low gene transfection efficiency limit their practical applications in gene therapy. In order to improve their properties for enhanced gene delivery, the surfaces of G5 PAMAM dendrimers were grafted with β-cyclodextrin (β-CD) and were used as templates to entrap gold nanoparticles (Au NPs). The formed β-CD-modified dendrimer-entrapped Au NPs (Au DENPs-β-CD) and their ability to compact plasmid DNA (pDNA) were thoroughly characterized with different methods. The cytocompatibility of Au DENPs-β-CD was evaluated by cell viability assay. The gene delivery efficiency of the obtained Au DENPs-β-CD vector was tested by transfecting two different pDNAs encoding luciferase and enhanced green fluorescent protein into 293T cells (a human embryonic kidney cell line), respectively. Our results show that the Au DENPs-β-CD can compact the pDNA at an N/P ratio of 0.5[thin space (1/6-em)]:[thin space (1/6-em)]1 or above, possess less cytotoxicity than Au DENPs without β-CD conjugation, and enable more efficient cellular gene delivery than Au DENPs without β-CD conjugation. The developed Au DENPs-β-CD may hold great promise to be used as an efficient vector system for enhanced gene delivery applications.


Introduction

Gene therapy has been considered as a promising approach for cancer therapy due to the fact that exogenous therapeutic genes are able to be delivered to target cells to correct or compensate the genetic defect and abnormality.1–3 However, the main obstacle of gene therapy is the lack of safe and efficient carriers to deliver the genetic materials to the target cells.4,5 Due to the advantages such as low cytotoxicity, non-immunogenicity, and high genetic loading capacity, non-viral vectors have shown tremendous potential for gene delivery applications.6,7 Among the used non-viral delivery systems including cationic lipids,8,9 polymers,10,11 peptides,12,13 and nanoparticles (NPs),14,15 etc., poly(amidoamine) (PAMAM) dendrimers, a class of highly branched synthetic macromolecules,16,17 have been widely used in gene delivery18–24 owing to their unique structural characteristics such as controllability in synthesis, monodispersity, well-defined three-dimensional molecular architecture, controlled surface functionalities, and commercial availability.

It has been shown that with the number of PAMAM generation, both the cytotoxicity and gene transfection efficiency increase.25 Due to the reasonable size and abundant surface functional groups, generation 5 (G5) PAMAM dendrimers have been used as a gene delivery vector. As is known, there are three major barriers for non-viral vectors to overcome for gene delivery applications: (1) crossing the cell membrane; (2) release of DNA within the endosomes, and (3) entry of DNA into the nucleus. For improved gene delivery with reduced cytotoxicity, G5 PAMAM dendrimers have been further surface modified with functional groups.26,27 Likewise, to improve gene delivery of G5 dendrimers, Au NPs have been entrapped within the dendrimer interior.28–30 The advantages of the Au NP entrapment stem from two aspects: (1) the entrapped Au NPs are able to neutralize some of the dendrimer terminal amines due to the amine stabilization of Au NPs, decreasing the dendrimer cytotoxicity; and (2) the existence of Au NPs helps to reserve the three-dimensional spherical shape of dendrimers, thereby significantly improving the DNA compaction ability of the dendrimers.

It is well known that cyclodextrins (CD) possess low toxicity,31,32 non-immunogenicity and excellent hydrophobic cavity.33–35 CD has been used as an enhancer in both viral and non-viral oligonucleotide delivery due to its binding with nucleic acids, facilitating increased gene stability against nuclease.36–38 Meanwhile, positively charged CD has been explored as a new vector for gene delivery.39,40 Furthermore, various polycations covalently linked with CD have improved biocompatibility, water-solubility, and gene transfection efficiency.41 The enhanced gene delivery efficiency using CD-conjugated polymers could be due to the possible mechanisms that CD-polymer conjugates might increase the release of pDNA or the pDNA complex from endosomes following cellular uptake, although it is still unclear to what degree the membrane disruptive ability of CD-polymer conjugates contributes to the enhancing effect on gene transfer activity.42 For instance, Gonzalez et al.43,44 synthesized β-cyclodextrin (β-CD)-containing polymers for enhanced gene delivery applications, and showed that β-CD is able to be used as a biocompatibility- and solubility-enhancing moiety. It is reasonable to hypothesize that β-CD-modified G5 PAMAM dendrimers with Au NPs loaded within their interior may be developed as a highly efficient gene delivery vector.

In this study, we present an approach to using β-CD-modified dendrimer-entrapped Au NPs (Au DENPs) for enhanced gene delivery applications. G5 PAMAM dendrimers modified with β-CD were used as templates to entrap Au NPs (Scheme 1). The formed Au DENPs-β-CD vector was characterized via 1H NMR, UV-vis spectroscopy, and transmission electron microscopy (TEM). Subsequently, the number of the primary amine groups of the Au DENPs-β-CD vector was determined, and then agarose gel retardation assay was utilized to examine its pDNA binding ability. Afterwards, the Au DENPs-β-CD/pDNA polyplexes were further analyzed by dynamic light scattering (DLS) and zeta potential measurements. Moreover, thiazoyl blue tetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the vector. Finally, the gene transfection efficiency of the Au DENPs-β-CD vector was evaluated by transfecting both luciferase (Luc) reporter gene and enhanced green fluorescent protein (EGFP) gene to 293T cells in vitro. To our knowledge, this is the first report related to the use of Au DENPs-β-CD as a non-viral vector for enhanced gene delivery applications.


image file: c6ra03839e-s1.tif
Scheme 1 Schematic representation of the synthesis of Au DENPs-β-CD.

Experimental

Materials

G5 PAMAM dendrimers were purchased from Dendritech (Midland, MI). β-CD and N,N′-carbonyldiimidazole (CDI) were from J&K Scientific Ltd. (Shanghai, China). Dimethylsulfoxide (DMSO) was from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Primary amino nitrogen assay kit was from Megazyme (Wicklow, Ireland). Agarose was from Biowest (Nuaillé, France). LB broth, UNIQ-200 column plasmid medi-preps kit, and thiazoyl blue tetrazolium bromide (MTT) were from Sangon Biotech. (Shanghai, China). Luciferase assay system was from Promega Corporation (Madison, WI). Bicinchoninic acid (BCA) protein quantitation kit was from Shanghai Yeasen Biotechnology Co., Ltd. (Shanghai, China). Lightning-link® rapid Cy3 conjugation kit was purchased from Innova Biosciences (Babraham, UK). 293T cells (a human emborynic kidney cell line) were obtained from Institute of Biochemistry and Cell Biology (the Chinese Academy of Sciences, Shanghai, China). Dulbecco modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA). Penicillin and streptomycin were from Gino Biomedical Technology Co., Ltd. (Shanghai, China). Regenerated cellulose dialysis membranes with a molecular weight cutoff (MWCO) of either 8000 or 14[thin space (1/6-em)]000 were acquired from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Water used in all experiments was purified using a MilliQ-Plus 185 water purification system to have a resistivity of 18.2 MΩ cm.

Synthesis of Au DENPs-β-CD

A DMSO solution containing β-CD (63.2 mg, 55.68 μmol, 5 mL) was mixed with a DMSO solution of CDI (90.28 mg, 556.8 μmol, 5 mL) under stirring for 6 h. Then, G5·NH2 dendrimer (57.93 mg, 2.227 μmol, in 10 mL DMSO) was dropwise added to the above mixture solution under stirring at room temperature. After 3 days, the reaction mixture was purified by extensive dialysis against water using a dialysis membrane with an MWCO of 8000–14[thin space (1/6-em)]000. A further lyophilization process gave rise to a white powder of the product of G5·NH2-β-CD. The obtained G5·NH2-β-CD dendrimer was stored at −20 °C for further use.

Both Au DENPs-β-CD and Au DENPs without β-CD were synthesized with the Au salt/dendrimer molar ratio of 25[thin space (1/6-em)]:[thin space (1/6-em)]1 according to a procedure described in our previous reports.45–47 In brief, G5·NH2-β-CD dendrimer (39.52 mg, 0.94 μmol) dissolved in 10 mL water was added with an HAuCl4 solution (10 mg mL−1, in 0.968 mL in water) under vigorous stirring. After 30 min, an ice cold NaBH4 solution (4.45 mg, in 5 mL water) with 5 times molar excess to the Au salt was added to the above mixture solution under stirring. The stirring process was continued for 4 h to complete the reaction. The reaction mixture was then extensively dialyzed against water (six times, 2 L) for 3 days using a dialysis membrane with an MWCO of 8000–14[thin space (1/6-em)]000 to remove the excess reactants, followed by lyophilization to obtain the Au DENPs-β-CD. Au DENPs without β-CD conjugation were also synthesized using G5·NH2 dendrimers as templates under the same experimental conditions and the characterization data can be seen in our previous reports.28,47 To be simple, G5·NH2, Au DENPs, and Au DENPs-β-CD were denoted as Q0, Q1, and Q2, respectively.

Characterization techniques

UV-vis spectroscopy was performed using a Lambda 25 UV-vis spectrophotometer (Perkin-Elmer, Waltham, MA). Samples were dissolved in water at a concentration of 0.5 mg mL−1 before measurements. 1H NMR spectrometry was carried out using a Bruker DRX 400 nuclear magnetic resonance spectrometer. Samples were dissolved in D2O before measurements. TEM was operated using a JEOL 2010F analytical electron microscope (JEOL, Tokyo, Japan) at 200 kV. TEM samples were prepared by dropping diluted sample suspension (0.1 mg mL−1) onto a carbon-coated copper grid and air-dried before measurements. The size distribution histogram was measured using ImageJ software (http://www.rsb.info.nih.gov/ij/download.html). At least 200 NPs randomly chosen from different TEM images were measured for each sample.

Determination of the number of primary amines of vectors

Q0, Q1, and Q2 were separately dissolved in water at a concentration of 2 mg mL−1. Then Megazyme's Primary Amino Nitrogen (K-PANOPA) Assay Kit was used to determine the number of the vectors' surface amines according to the manufacturer's instruction.

Preparation of vector/pDNA polyplexes

Vector/pDNA polyplexes were prepared at different N/P ratios (the molar ratio of primary amines of the vectors to phosphates in the pDNA backbone). Both vector and pDNA were dissolved in phosphate buffered saline (PBS, pH 7.4) to have a final volume of 50 μL while the concentrations of the vector changed with the N/P ratio. The vector and pDNA solutions were gently mixed for seconds, and incubated for 15–30 min at room temperature to obtain the vector/pDNA polyplexes with a final volume of 100 μL.

Agarose gel retardation assay

To evaluate the vectors' binding ability with pDNA, an agarose gel retardation assay was used. Agarose gel (1.0% w/v) containing ethidium bromide (EB, 0.5 μg mL−1) was prepared in Tris-borate-EDTA (TBE) buffer. Vector/pDNA polyplexes were prepared with 1 μg pDNA under various N/P ratios ranging from 0.25[thin space (1/6-em)]:[thin space (1/6-em)]1 to 5[thin space (1/6-em)]:[thin space (1/6-em)]1, where naked pDNA was used as a control. Gel electrophoresis was carried out for 20–30 min at 80 V. The band locations of pDNA were analyzed through a gel image analysis system (Shanghai FURI Science & Technology, Shanghai, China).

Hydrodynamic size and zeta potential measurements

The hydrodynamic size and zeta potential of the vector/pDNA polyplexes were measured by a Malvern Zetasizer Nano ZS system (Worcestershire, UK) equipped with a standard 633 nm laser. Each polyplex at the N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, or 10[thin space (1/6-em)]:[thin space (1/6-em)]1 was prepared using 5 μg of pDNA, and then diluted to a final volume of 1 mL using PBS before measurements.

Cytotoxicity assay

The cytotoxicity of different vectors was evaluated by MTT assay of 293T cells upon treatment with the vectors at different concentrations. Firstly, 293T cells were seeded onto a 96-well plate at a density of 8 × 103 cells per well, and then cultivated in 200 μL DMEM containing 10% FBS, 100 U mL−1 penicillin, and 100 U mL−1 streptomycin under 37 °C and 5% CO2 for 24 h. The medium was then replaced by 200 μL of fresh DMEM containing 20 μL vectors with a final concentration ranging from 50 to 2000 nM. PBS was used as control. The cells were then incubated for another 24 h under the same conditions. Thereafter, MTT (5.0 mg mL−1, 20 μL in PBS) was added into each well of the plate and the cells were continuously incubated for another 4 h. After that, the medium was replaced with 150 μL of DMSO to dissolve the formazan crystals. Each sample was measured by a Thermo Scientific Multiskan MK3 ELISA reader (Waltham, MA) at 570 nm. Mean and standard deviation of six parallel wells were reported for each sample.

Expression of the EGFP gene

293T cells were seeded at a density of 5 × 104 cell per well in a 24-well plate and cultivated at 37 °C and 5% CO2 overnight to bring the cells to a 60–70% confluence. Then, the medium was replaced by 900 μL of fresh medium without serum and 100 μL vector/pDNA polyplex with an N/P ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1 or 10[thin space (1/6-em)]:[thin space (1/6-em)]1 (1 μg EGFP pDNA for each polyplex). After transfected for 4 h, the medium was replaced with fresh medium and the cells were incubated for another 24 h. Finally, cells were observed using a Nikon Ti-S invert fluorescence microscope (Tokyo, Japan).

Flow cytometry (BD FACS Calibur, Franklin lake, NJ) was also used to detect the gene delivery efficiency of vectors through the quantification of the expression of EGFP. 293T cells were seeded in a 12-well plate at a density of 1 × 105 cells per well and cultured overnight to reach a 60–70% confluence. Subsequently, the cells were transfected with polyplexes for 4 h and medium was replaced with fresh medium. After 24 h, cells were washed once with PBS, trypsinated, centrifuged at 1000 rpm for 5 min, and resuspended into 1 mL of PBS before flow cytometry analysis. The cells were gated (Gate 1, G1) on a forward scatter/side scatter (FSC-H/SSC-H) dot plot and the fluorescence was measured at the FL1-H channel. For each vector with different N/P ratios, the experiment was repeated for 3 times and 1 × 104 cells were counted every time.

Expression of the Luc gene

Expression of the Luc gene was also carried out to confirm the gene transfection efficiency of vectors. The culture and transfection processes of the 293T cells were similar to those used for expression of the EGFP gene. Vector/pDNA polyplexes were prepared by mixing different vectors with Luc pDNA (1.0 μg per well) under N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, and 10[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively. Before the Luc activity assay, the medium was removed and the cells were washed with PBS for 3 times. According to the protocol of Promega's Luc assay, the total protein concentration of the cells was measured with a BCA protein quantitation kit. The gene delivery efficiency of each vector at different N/P ratios was characterized by relative light unit per milligram of total protein (RLU per mg, n = 3). Besides, cells without transfection or transfected with naked pDNA were used as controls.

Cellular uptake of vector/pDNA polyplexes

The gene transfection efficiency was also investigated using flow cytometry to evaluate the intracellular uptake of vector/pDNA polyplexes. The Cy3-labeled DNA was prepared according to the protocol of a Lightning-link® rapid Cy3 conjugation kit. The carrier-cargo polyplexes were prepared by mixing a vector and Cy3-labeled pDNA at different N/P ratios (1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, and 10[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively), followed by incubation at room temperature for 15–30 min.

293T cells were seeded in a 12-well plate at a density of 1 × 105 cells per well and cultured overnight to reach a 70–80% confluence. Afterwards, the cells were transfected with polyplexes for 4 h. Then the cells were washed once with PBS, trypsinated, centrifuged, and resuspended into 1 mL PBS before flow cytometry analysis. The cells were gated (G1) on a forward scatter/side scatter (FSC-H/SSC-H) dot plot and the fluorescence was measured in the FL2-H channel. For each vector with different N/P ratios, the experiment was repeated for 3 times and 1 × 104 cells were counted every time.

Statistical analysis

One-way ANOVA statistical analysis was performed to evaluate the experimental data. A p value of 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.

Results and discussion

Synthesis and characterization of Au DENPs-β-CD

Previous work has shown that Au DENPs are promising non-viral vectors for highly efficient gene delivery.28 Meanwhile, β-CD has been proven to be suitable biocompatible and solubility-enhancing moiety for the synthesis of biodegradable polymers. Therefore, Au DENPs-β-CD are considered to be a potential non-viral vector for enhanced gene delivery.

G5·NH2-β-CD was first synthesized by modification of G5·NH2 dendrimer with CDI-activated β-CD according to the literature.48 The structure of G5·NH2-β-CD was identified by 1H NMR spectrum (Fig. 1). Clearly, the chemical shifts between 2.25 and 3.34 ppm (HaHd′) can be assigned to methylene protons of G5 dendrimers while the proton peaks ranging from 3.5 to 4 and at about 5 ppm (H1H6) correspond to β-CD protons, confirming the success of the β-CD conjugation onto the dendrimer surface, in agreement with the results reported in the literature.48 By integrating the peak areas of β-CD and G5 dendrimer methylene protons, the number of β-CD molecules conjugated on each G5 dendrimer was calculated to be 8.4.


image file: c6ra03839e-f1.tif
Fig. 1 1H NMR spectrum of G5·NH2-β-CD (a) and the chemical structure of G5·NH2-β-CD with proton assignments (b).

The synthesized G5·NH2-β-CD dendrimers were used as templates to entrap Au NPs. Our previous work has shown that Au DENPs formed using an Au salt/dendrimer molar ratio of 25[thin space (1/6-em)]:[thin space (1/6-em)]1 are able to have the optimized gene delivery efficiency. Hence, the same Au salt/dendrimer molar ratio of 25[thin space (1/6-em)]:[thin space (1/6-em)]1 was chosen to prepare Au DENPs-β-CD in this study. The final formed Au DENPs-β-CD (Q2) vector was characterized via UV-vis spectrometry (Fig. 2), where the surface plasmon resonance (SPR) band of the particles at around 512 nm indicates the successful formation of Au NPs. The SPR band of Q2 is quite similar to Q1. In contrast, Q0 that has no Au NPs entrapped does not show the typical Au NP-related SPR band, and Q0 just shows the aliphatic dendrimer backbone-associated absorption feature.46 The size and morphology of Q2 were characterized by TEM (Fig. 3). It can be seen that the Au core particles have a close-to-spherical shape with a mean diameter of 2.9 nm and a uniform size distribution.


image file: c6ra03839e-f2.tif
Fig. 2 UV-vis spectra of Q1 and Q2 vectors.

image file: c6ra03839e-f3.tif
Fig. 3 TEM image and size distribution histogram of the Q2 vector.

The mean number of primary amines of Q0, Q1 and Q2 vectors was determined by PANOPA assay. Table 1 lists the parameters of the three vectors. Q0, Q1 and Q2 possess 94, 44 and 75 primary amines per dendrimer on their surface, respectively. Compared with Q0, the surface amine groups of Q1 and Q2 decreases, possibly due to the fact that a portion of dendrimer primary amines is contributed to stabilize the inner Au NPs. Furthermore, Q2 has more surface amine groups than Q1, possibly due to the fact that some of the hydroxyl groups of the conjugated β-CD are able to stabilize the Au NPs instead of the dendrimer primary amines.

Table 1 Physicochemical parameters of the Q0, Q1 and Q2 vectors
Vectors Q0 Q1 Q2
Calculated Mw 26[thin space (1/6-em)]010 30[thin space (1/6-em)]935 40[thin space (1/6-em)]469
Mean number of primary amines per dendrimer 94 44 75


Agarose gel retardation assay

To confirm the pDNA compaction ability of the vectors, agarose gel retardation assay was carried out. In general, naked pDNA with negative charges would migrate during the electrophoresis process, cationic polymer vectors complexed with pDNA through electrostatic interaction would neutraliuze the negative charges of pDNA, leading to its retardation upon gel electrophoresis. As shown in Fig. 4, the migration of pDNA is able to be retarded with the increase of the N/P ratio. Q1 and Q2 are able to completely retard the DNA mobility at the N/P ratio of 0.5[thin space (1/6-em)]:[thin space (1/6-em)]1 or above (Fig. 4b and c), while Q0 starts to retard the migration of pDNA at an N/P ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1 (Fig. 4a). Our results suggest that the entrapment of Au NPs within dendrimers is beneficial for enhanced pDNA compaction, in agreement with the our previous study.28 It seems that the conjugation of β-CD onto G5 dendrimers does not compromise the DNA binding capacity.
image file: c6ra03839e-f4.tif
Fig. 4 Gel retardation assay of pDNA complexed with Q0 (a), Q1 (b) and Q2 (c) at different N/P ratios. Lane 1: DNA marker; lane 2: N/P = 0.25[thin space (1/6-em)]:[thin space (1/6-em)]1; lane 3: N/P = 0.5[thin space (1/6-em)]:[thin space (1/6-em)]1; lane 4: N/P = 1[thin space (1/6-em)]:[thin space (1/6-em)]1; lane 5: N/P = 2[thin space (1/6-em)]:[thin space (1/6-em)]1; lane 6: N/P = 3[thin space (1/6-em)]:[thin space (1/6-em)]1; lane 7: N/P = 4[thin space (1/6-em)]:[thin space (1/6-em)]1; and lane 8: N/P = 5[thin space (1/6-em)]:[thin space (1/6-em)]1.

Hydrodynamic size and zeta potential measurements

The hydrodynamic size and surface potential of vectors are significant factors affecting their cytotoxicity and intracellular uptake. Therefore, dynamic light scattering (DLS) and zeta potential measurements were used to analyze the hydrodynamic size and surface potential of the vector/pDNA polyplexes, respectively (Fig. 5). The formed vectors/pDNA complexes have a size ranging from 125 to 225 nm at the N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, and 10[thin space (1/6-em)]:[thin space (1/6-em)]1 (Fig. 5a), which are suitable for gene delivery.
image file: c6ra03839e-f5.tif
Fig. 5 Mean particle size (a) and surface potential (b) of the Q0/pDNA, Q1/pDNA and Q2/pDNA polyplexes at N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1 or 10[thin space (1/6-em)]:[thin space (1/6-em)]1 (mean ± SD, n = 3).

The positive surface potential of the vector/pDNA polyplexes is essential for effective intracellular uptake through electrostatic interaction. Zeta potential measurements show that the surface potential of Q0/pDNA complexes increases with the N/P ratio, while Q1/pDNA and Q2/pDNA polyplexes have the highest surface potential at the N/P ratio of 5[thin space (1/6-em)]:[thin space (1/6-em)]1. Apparently, polyplexes formed using the vectors entrapped with Au NPs (Q1 and Q2) possess lower positive surface potentials than those formed using the vector without Au NPs (Q0). Likewise, the β-CD-modified vector (Q2)/pDNA polyplexes possess a higher surface potential than Q1 (without β-CD)/pDNA polyplexes at the same N/P ratios. The small hydrodynamic particle size with a suitable positive surface potential renders the vector/pDNA polyplexes with an ability for efficient cellular uptake for gene delivery applications.

Cytotoxicity assay

Cytotoxicity assay of the vectors was performed before their gene transfection studies. MTT viability assay of 293T cells reveals that the cell viability gradually decreases with the increase of the vector concentration (Fig. 6). At each concentration, the cytotoxicity of the vectors follows the order of Q2 < Q1 < Q0. At the highest concentration (2000 nM) tested, cells treated with Q2 still have a viability above 60%. Our results suggest that both the periphery modification of β-CD and the interior entrapment of Au NPs are beneficial to reduce the cytotoxicity of G5 dendrimers.
image file: c6ra03839e-f6.tif
Fig. 6 MTT viability assay of 293T cells treated with different vectors under different concentrations (mean ± SD, n = 6).

Expression of the EGFP gene

To investigate the enhanced gene transfection efficiency of Q2, EGFP expression assay was performed. Fluorescence microscopic images (Fig. 7) show that for the vectors of Q0 and Q1, cells transfected with EGFP gene display the strongest green fluorescence signals at an N/P ratio of 5[thin space (1/6-em)]:[thin space (1/6-em)]1. Q2 seems to be the best vector in terms of EGFP gene transfection, and cells transfected with Q2 display much stronger green fluorescence signal intensity than Q1 and Q0 at all studied N/P ratios.
image file: c6ra03839e-f7.tif
Fig. 7 Fluorescence microscopic images (100×) of EGFP gene expression in 293T cells using Q0 (a), Q1 (b), and Q2 (c) vectors at the N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, 10[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively. Images were taken 24 h after transfection under similar instrumental conditions.

To quantify the gene transfection efficacy, cells expressed EGFP were assayed by flow cytometry (Fig. 8). Cells without transfection were used as control. For all vectors, the relative mean fluorescence (RMF) of cells transfected with EGFP increases with the N/P ratio. Clearly, through the use of Q2 vector, the RMF of EGFP-transfected cells is much higher than that transfected with other vectors at the same N/P ratios.


image file: c6ra03839e-f8.tif
Fig. 8 Flow cytometry analysis of 293T cells transfected using Q0, Q1, and Q2 vectors at the N/P ratios of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, 10[thin space (1/6-em)]:[thin space (1/6-em)]1, respectively. Cells without treatment were used as controls. Data are shown with mean ± SD (n = 3).

Expression of the Luc gene

To further quantify the gene delivery efficiency, firefly Luc gene expression assay was carried out (Fig. 9). It can be seen that all vectors are able to effectively transfect Luc gene to cells, compared to cells without transfection or cells transfected with naked pDNA. At the N/P ratios of 5[thin space (1/6-em)]:[thin space (1/6-em)]1 and 10[thin space (1/6-em)]:[thin space (1/6-em)]1, the Luc expression using Q2 vector reaches 6.2 × 106 and 5.9 × 106 RLU per mg, respectively, significantly higher than that using Q0 and Q1 vectors (p < 0.01). It can be safely conclude that Q2 exhibits the most effective gene delivery efficiency. The Luc gene transfection results are consistent with the EGFP gene expression assay.
image file: c6ra03839e-f9.tif
Fig. 9 Luc gene transfection efficiency in 293T cells using Q0, Q1 and Q2 as vectors. Cells without treatment (none) and cells treated with vector-free pDNA (pDNA) were used as controls (mean ± SD, n = 3).

Cellular uptake of vector/pDNA polyplexes

To further investigate the underlying mechanism associated with the enhanced gene delivery efficiency, the cellular uptake of the Q2/pDNA polyplexes was assessed by flow cytometry. Cy3-labeled pDNA was complexed with different vectors at different N/P ratios, while non-transfected cells were used as control. As shown in Fig. 10, the RMF of cells increases with the N/P ratio, similar to that of cells transfected with EGFP gene. Cells treated with the Q1/pDNA polyplex display the largest RMF (about 50%) at an N/P ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1. Importantly, cells treated with the Q2/pDNA polyplex display much higher RMF than those treated with the Q0/pDNA and Q1/pDNA polyplexes at the same N/P ratios (p < 0.001). Overall, our results imply that the surface conjugation of β-CD and the interior entrapment of Au NPs render the G5 dendrimers with enhanced cellular uptake, thereby significantly improving their gene delivery efficiency.
image file: c6ra03839e-f10.tif
Fig. 10 Flow cytometry analysis of the intracellular uptake of the vector/Cy3-labeled pDNA polyplexes in 293T cells at three different N/P ratios. Cells without treatment were used as control. Data are shown as mean ± SD (n = 3).

Conclusion

In summary, we synthesized a novel non-viral vector of Au DENPs-β-CD for enhanced gene delivery applications. G5 dendrimers modified with β-CD are able to be used as templates to synthesize Au NPs. The formed Au DENPs-β-CD with an Au core size of 2.9 nm display reduced cytotoxicity and improved DNA compaction ability, and are able to transfect both EGFP and Luc genes to cells with improved gene delivery efficiency. The developed Au DENPs-β-CD may be employed as a new non-viral vector for various gene delivery applications.

Acknowledgements

This research is financially supported by the National Natural Science Foundation of China (21273032, 31400816), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, and the Fundamental Research Funds for the Central Universities (for X. Cao).

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