TAT conjugated cationic noble metal nanoparticles for gene delivery to epidermal stem cells

Biomaterials 35 (2014) 5605e5618

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TAT conjugated cationic noble metal nanoparticles for gene delivery to epidermal stem cells Li-Hua Peng a, Jie Niu a, Chen-Zhen Zhang a, Wei Yu b, Jia-He Wu a, Ying-Hui Shan a, Xia-Rong Wang a, You-Qing Shen c, Zheng-Wei Mao b, **, Wen-Quan Liang a, Jian-Qing Gao a, * a b c

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, PR China Center for Bionanoengineering and State Key Laboratory of Chemical Engineering, Zhejiang University, Hangzhou, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 March 2014 Accepted 21 March 2014 Available online 13 April 2014

Most nonviral gene delivery systems are not efficient enough to manipulate the difficult-to-transfect cell types, including non-dividing, primary, neuronal or stem cells, due to a lack of an intrinsic capacity to enter the membrane and nucleus, release its DNA payload, and activate transcription. Noble metal nanoclusters have emerged as a fascinating area of widespread interest in nanomaterials. Herein, we report the synthesis of the TAT peptide conjugated cationic noble metal nanoparticles (metal NPs@PEITAT) as highly efficient carriers for gene delivery to stem cells. The metal NPs@PEI-TAT integrate the advantages of metal NPs and peptides: the presence of metal NPs can effectively decrease the cytotoxicity of cationic molecules, making it possible to apply them in biological systems, while the cell penetrating peptides are essential for enhanced cellular and nucleus entry to achieve high transfection efficiency. Our studies provide strong evidence that the metal NPs@PEI-TAT can be engineered as gene delivery agents for stem cells and subsequently enhance their directed differentiation for biomedical application. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Metal nanoparticles TAT conjugation Gene delivery Stem cell

1. Introduction Inorganic nanoparticles (NPs) such as gold, silica, iron oxide, quantum dots, calcium phosphate, have emerged as attractive nonviral gene vectors in last decades. Among them, noble metal NPs such as AuNPs or AgNPs are of particular interest for widespread applications in biology and medicine owing to their ease of synthesis, tunable size and shape, flexible surface modification and bioconjugation, and tunable optical and electronic properties (such as absorption, fluorescence, and conductivity) [1e5]. One of the most promising biomedical applications of noble metal NPs is exploited as intracellular delivery vectors for either drugs or genes. Several attempts have been made to use AuNPs for gene delivery and transfection purposes. For example, Mirkin et al. developed

* Corresponding author. ** Corresponding author. MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, PR China. E-mail addresses: [email protected] (Z.-W. Mao), [email protected] (J.-Q. Gao). http://dx.doi.org/10.1016/j.biomaterials.2014.03.062 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

gold nanoparticles oligonucleotide nanoconjugates as intracellular gene regulation agents for the control of protein expression in cells [6]. Rotello’s group demonstrated amine-functionalized AuNPs could efficiently deliver DNA plasmids to mammalian cells [7,8]. Thomas and Klibanov have demonstrated that polyethylenimine (PEI, 2 kDa)-conjugated AuNPs deliver plasmid DNA (pDNA) to COS7 cells more efficiently as compared to PEI alone [9]. Zhong et al. prepared low molecular weight polyethylenimine (PEI 800 Da) conjugated gold nanoparticles and used them as gene carriers, which have effective gene transfection especially in serumcontaining media [10]. Tian et al. also prepared low molecular weight PEI conjugated AuNPs that exhibited 2e3 times higher transfection efficiency in cancer cells than did PEI-25K [11]. Mohan et al. examined the gene transfer efficiency and toxicity of 2 kDa PEI conjugated to gold nanoparticles in the human cornea in vitro and rabbit cornea in vivo. The results suggested that PEI capped AuNPs are safe for the cornea and can potentially be useful for corneal gene therapy in vivo [12]. Wang et al. and Gunaratne et al. demonstrated that gold nanoparticles (AuNPs) are capable of delivering microRNA and small interfering RNA into cells and efficiently down regulate target genes and modulate cell functions [13,14]. Besides, Au-PEI

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emits strong red fluorescence and can be used for bioimaging in vitro and in vivo. The development of metal nanoparticles based gene delivery system with fluorescent efficacy is facile and practical with wide application. However, until recently, although gold nanoparticles have been proved as promising candidates for gene delivery, there is no successful study relating delivery DNA by AuNPs to difficult-to-transfect cells, such as stem cells which are attractive candidates for regenerative medicine and gene therapy, has been reported. As far as we know, the engineering of sliver NPs as the gene delivery vector has not been reported else. Several factors for this situation may be that metal based nanoparticles are not efficient enough to manipulate the difficult-to-transfect cell types, due to the lack of an intrinsic capacity to enter the cells membrane and nucleus, release its DNA payload, and activate transcription [15e17], as well as the cytotoxicity to cells. Additionally, it was reported that non-viral gene complexes enter the nucleus preferentially upon the disassembly of the nuclear envelope during mitotic cell division [18]. Therefore, the nonviral vectors would be extremely inefficient when delivery DNA to the cells with slow mitosis cycle and low proliferation rate, such as epidermal stem cells (ESC). Conjugation of ligands that are able to bind specific receptors on cell membrane could largely enhance the targeting ability and the transfection efficiency of vectors [19,20]. Cell-penetrating peptides (CPPs) are short peptides, which facilitate cellular uptake of various molecular cargos (from small chemical molecules to nanosize particles), have also been used [21]. For example, the HIV-1 twinarginine translocation (TAT) peptide with the sequence of RKKRRQRRR, derived from the “transduction domain” of Tat protein, has been successful in delivering a large variety of cargos, from small particles to proteins, peptides, and nucleic acids [22e24]. In our previous study, TAT decoration was shown to affect the subcellular distribution of the particles as well, resulting in localization of the particles in the cell nucleus without causing cytotoxicity [25].

Suk et al. [26] modified PEI/DNA complexes with Tat peptides and found that they are easily internalized by differentiated neurotypic cells and showed enhanced gene transfection efficiency up to 14fold. Therefore, cell penetrating peptides, i.e. TAT, may also be used as ligands to modify the surface of metal NPs, rendering them tremendous potential in the pharmaceutical field for intracellular drug and gene delivery. In this study, we used a facile method to prepare TAT peptide functionalized cationic gold and silver nanoparticles. Positively charged PEI molecules were used to serve as capping agents to prepare AuNPs and AgNPs. Then the nanoparticles were further functionalized via partial ligand exchange with thiol-TAT peptides (see Fig. 1 as a schematic illustration). The TAT amount can be adjusted via feeding concentration. To verify the versatility (or feasibility) of TAT-conjugated metal NPs as efficient gene carriers to stem cells, the in vitro gene transfections activity and subsequently their impact on ESC differentiation were investigated. Furthermore, the cellular uptake and intracellular distribution of NPs were studied to elucidate the delivery mechanism. 2. Experimental section 2.1. Materials HAuCl4, AgNO3, polyethelyimine (PEI, Mw 25kD), amiloride-HCl (Amil), sodium azide (Azium), chlorpromazine (CPZ), methyl-b-cyclodextrin (MBC), and 40 ,6diamidino-2-phenylindole (DAPI) were purchased from Sigma. Methylthiazoletetrazolium (MTT) was purchased from AMRESCO. Tat peptide (H-Cys-Cys-Tyr-GlyArg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH, Mw ¼ 1559) was purchased from Sangon Biotechnology Inc., Shanghai, China. MicroBCA protein assay kit was purchased from Beyotime Biotechnology Inc., Nantong, China. Ethidium bromide (EB) was purchased from Fluka. Plasmid DNAs encoding luciferase (PGL3) or EGFP were provided by the Institute of Infectious Diseases, Zhejiang University, China. Plasmid DNA encoding bFGF gene was obtained from FlyGene company (Hangzhou, China). The plasmid DNA was stored at 20  C until the transfection experiments. Threeday-old rats (18e20 g) used for ESCs extraction were supplied by Zhejiang Academy of Medical Sciences, China. Defined keratinocyte serum-free medium used for ESCs culture was purchased from Gibcol, USA. Defined keratinocyte serum-free

Fig. 1. (a) Scheme of preparation of TAT peptide-conjugated cationic noble metal nanoparticles. (b) Schematic illustration of positively charged TAT peptide-conjugated noble metal NPs binding with DNA for intercellular delivery.

L.-H. Peng et al. / Biomaterials 35 (2014) 5605e5618 medium (KSFM), penicillin, streptomycin, and trypsin were obtained from Gibco BRL (Gaithersburg, MD, USA). bFGF ELISA kit was obtained from Ming Rui Biotech Company (Shanghai, China). Rat Neuronal Nuclei (NeuN) and Glial fibrillary acidic protein (GFAP) monoclonal antibody was purchased from BD (New Jersey, U.S.A.). Goat antirabbit IgG-FITC were purchased from Santa Cruz Biotechnology (CA, USA). All other reagents were analytical grade and were used as received. Milli-Q water was used throughout the experiments. 2.2. Isolation and culture of ESCs ESCs were prepared and identified with a protocol previously reported [27]. Briefly, skin tissue biopsy was obtained from the back of adult rats was sterilized with 75% ethanol, rinsed in PBS (pH 7.4), and then minced into 2 mm wide strips treated with 0.25% Dispase II overnight. The epidermis was mechanically separated from the dermis and then incubated in trypsin-EDTA (0.05%) at 37  C for 10 min to dissociate cells. After enzyme activity was blocked with defined KSFM containing 10% FBS, the cells were suspended. The cell suspension was filtered through a stainless steel mesh to remove residual tissues. The cells were collected by centrifugation for 5 min at 1200 rpm and then plated onto 0.01% (g/g) collagen type IV coated dishes at a cell density of 1  106/mL for 10 min at room temperature. The unattached cells were removed, and the rapidly adherent epidermal cells were cultured in KSFM supplemented with 100 IU/mL penicillin at 37  C in a humidified 5% CO2 atmosphere for 3 d before replacing the medium. The medium was changed every other day. After the cells reached 80e90% confluence, they were subcultured by detaching them with trypsin-EDTA solution. 2.3. Preparation of metal NPs Firstly, gold nanoparticles (AuNPs@PEI) were synthesized by reduction of HAuCl4 (150 mg/mL) by NaBH4 (10 mg/mL) in the presence of PEI (3 mg/mL). The reaction mixture was vigorously stirred at room temperature for 15 min and then stored for at least 1 h. After that, the AuNPs@PEI were mixed with TAT solutions under constant stirring for 12 h to prepare AuNPs@PEI-TAT. PEI protected silver nanoparticles (AgNPs@PEI) were prepared by reduction of AgNO3 (170 mg/mL) by NaBH4 (4 mg/mL) in the presence of PEI (5 mg/mL). The reaction mixture was vigorously stirred at room temperature for 15 min and then stored for at least 1 h. After that, the AgNPs@PEI were mixed with TAT solutions under constant stirring for 12 h to prepare AgNPs@PEI-TAT. The free ligands were removed by dialysis. 2.4. Vector/DNA complexes formation and characterization NPs were purified by dialysis to remove excess ligand molecules. The concentration of NPs were adjusted to 20 mg/mL and verified by Inductively coupled plasma mass spectrometry (ICP-MS). Different volume of NPs solution or PEI/TAT solutions was added into a constant volume of DNA solution (100 mg/mL), vortexed for 15 s, and incubated for 30 min at 37  C prior to characterization. A series of vector/DNA complexes at various weight ratios were prepared. The vector/DNA particle sizes at different ratios were measured at room temperature by dynamic light scattering (DLS) on a Brookhaven Particle Size Analyzer (90plus). DLS measurement was carried out at a scattering angle (q) of 90 . All data were averaged from 3 to 5 parallel measurements. The zeta potential measurements were performed using an aqueous dip cell in the automatic mode (Zetasizer 3000, Malvern Instruments, Southborough, MA). The morphology of the AuNPs/DNA and AgNPs/DNA complexes at weight ratios 5 and 3 was observed using a JEOL JEM-200 transmission electron microscope (TEM), respectively. Briefly, a drop of the complexes was deposited on carbon-coated grids and dried at room temperature, and then the samples were observed.

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measured on a microplate reader (Bio-Rad, model 550). The cell viability was normalized to that of non-treated cells (negative control). 2.7. Transfection assay ESCs were seeded on a 24-well plate pre-coated with 0.01% (w/w) human collagen IV and then incubated overnight to reach around 80% confluence. The culture medium was removed before transfection and the cells were rinsed with PBS. Each well received 1 mg of pDNA-EGFP, in exclusion from the vector. The vector/ pDNA-EGFP complexes were diluted in 500 mL of Opti-MEM medium. After 6 h of incubation at 37  C, the Opti-MEM medium was replaced with defined KSFM medium. After another 42 h incubation, the cells were washed with PBS twice and detached by trypsinization. Mean fluorescence intensity per cell which is positively correlated with the transfection efficiency was quantified by flowcytometry (FACS Calibur, BD). At least ten thousand cells were measured for each sample. 2.8. ESCs induced neuronal differentiation upon vector/pDNA-bFGF transfection ESCs were cultured on coverslips and transfected by vector/pDNA-bFGF complexes as mentioned before. The cells were then cultured for 21 days. The culture medium was replaced by fresh one every 3 days. In order to observe the cellular expression of GFAP, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, blocked, and permeabilized with 0.3% Triton X-100 for 30 min. Primary antibody against GFAP was applied to the cells, which were incubated for 2 h at room temperature in a humidifier chamber followed by incubation with FITC conjugated secondary antibody for 30 min according to the manufacturer’s protocol (BD Biosciences, USA). The cells were further incubated with DAPI (1%) for 15 min to stain cell nuclei. The cells were then washed with PBS, inverted (cell-side down) and mounted with a mounting medium (PBS:glycerol ¼ 1:9). The cells were then examined under a confocal laser scanning microscope (CLSM, TCS SP5, Leica). Negative isotype controls were used when imaging micrographs to ascertain that no false-positive staining had occurred. The expression levels of NeuN in the ESCs were further analyzed by Flowcytometry. Briefly, the ESC were incubated with PBS containing 5% FBS for 30 min at 4  C and then on an ice bath for another 10 min to block endogenous peroxidases. Polyoxymethylene (1 ml of a 4% solution) was added to the cells for fixation for 30 min at room temperature. The NeuN antibody was added to each 100 ml ESC and incubates in an ice bucket for 30 min followed by incubation with secondary antibody for 30 min according to the manufacturer’s protocol (BD Biosciences, USA). To wash off excess antibody following staining, cells were centrifuged and rinsed with PBS. The cells were resuspended in 1% paraformaldehyde (500 mL). Samples in which PBS was used to replace primary monoclonal antibodies were used as negative controls. The mean fluorescence intensity per cell was quantified by flowcytometery with 20,000 events being recorded for each sample. Experiments were repeated at least twice under the same conditions and settings. 2.9. Cellular uptake and intracellular distribution of vector/DNA complexes

DNA (100 mg/mL) was stained with EB at a molar ratio of 10:1 (DNA:EB). Different amount of vectors was added to this DNA/EB solution. The final volume was adjusted to 10 ml. After incubation for 15 min at room temperature, fluorescence intensity of the solution was recorded on a fluorophotometer (LS55, PerkinElmer, UK) with excitation and emission wavelengths of 510 and 605 nm, respectively. Here the vectors were added in two ways: (1) EB/DNA was formed first, then the vectors were added, and (2) vectors/DNA was formed first, then EB was added. The sequence has no significant influence on the binding assay results.

ESCs were seeded on a 24-well plate precoated with 0.01% (w/w) human collagen I and then incubated overnight to reach 30e40% confluence. After replacing the medium with Opti-MEM, the CYD-PEI/FITC-DNA complexes was added, followed by incubation for various periods. The cells were washed thrice with PBS, and the cellular uptake of the complexes by ESCs was determined with flow cytometry. In order to track DNA inside cells by CLSM, Cy3-labeled pDNA was prepared according to the protocol of Label ITR TrackerÔ intracellular nucleic acid localization kit (Mirus Bio LLC, USA). ESCs were seeded at a density of 3  104 cell cm2 and cultured for 24 h. The cells were incubated with vector/DNA complexes containing 1 mg Cy3-labeled pDNA for 6 h and then incubated with DAPI for 15 min. Cells were washed with PBS and observed under CLSM. In order to get better understanding of the intracellular distribution of metal NPs, the cells were also analyzed under TEM. After treated with vector/DNA complexes for 6 h, cells were washed twice with PBS, fixed 1 h with 3.5% (v/v) glutaraldehyde, and rinsed with PBS. Post-fixation was performed for 1.5 h in 1% (v/v) osmium tetroxide at room temperature. The samples were dehydrated in graded series of ethanol (30, 50, 70, 95, and 100 vol.%) and propylene oxide. The samples were then embedded in Durcupan (Fluka, Sigma Aldrich) (polymerization took place at 60  C for 48 h). Sections with a thickness of about 60 nm were mounted on nickel grids and stained with uranyl acetate before examination under a JEOL JEM1010 microscope.

2.6. Evaluation of cytotoxicity

2.10. Intracellular pathway of vector/DNA complexes

Cells were seeded on a 96-well culture dish (Corning) at a density of 1  104 cells/well and cultivated overnight. The medium was changed to fresh OptiMEM medium (without FBS or antibiotics), 40 ml of vector/pDNA complexes solution at various concentrations was added to each well, followed by cultivation for 6 h. Cells were further incubated in the culture medium for 48 h. The liquor/culture medium was replaced with culture medium containing 3-[4,5-dimethyl-thiazolyl2]-2,5-diphenol tetrazolium bromide (MTT) (0.5 mg/mL). After 4 h, the supernatant was aspirated, and 200 ml dimethyl sulfoxide (DMSO, Sigma, USA) was added to each well. The plate was micro-oscillated for 30 s, and then the absorbance at 570 nm was

To clarify the uptake mechanism, the energy dependence of celleparticle interaction was assessed by treatment under 4  C and with sodium azide, respectively. Different pharmacological inhibitors, including 50 mM amiloride-HCl (Amil), 10 mg/ml of chlorpromazine (CPZ), 10 mg/ml of methyl-b-cyclodextrin (MbCD), and 15 mg/ml sodium azide (Azium), were also used to treat the endothelial cells for 30 min before incubation with the vector/DNA complexes, respectively. Following medium replacement with Opti-MEM and incubation for further 30 min at 37  C, the cells were incubated with the vector/FITC-DNA complexes for 4 h and then washed with PBS for three times to remove physically adsorbed complexes. Cells were fixed

2.5. Competition binding assay

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with 4% (w/v) paraformaldehyde in PBS, permeabilized with 0.1% (v/v) Triton X-100 in PBS, and then stained with hochest 33258 (Sigma) to visualize cell nucleus. The fluorescent images were recorded on a confocal microscope (Leica TCS SP5) with a 63 oil immersion objective. The gene transfection efficiencies of vector/DNA complexes in the presence of endocytotic inhibitors were also studied. The ECSs were pretreated with various endocytotic inhibitors for 30 min and then transfected with vector/DNA complexes containing 1 mg of pDNA-EGFP in exclusion from the vector for 6 h. After further 42 h incubation, mean fluorescence intensity per cell at different time points was detected by flowcytometry, while at least ten thousand cells were measured for each sample.

3. Results and discussion 3.1. Preparation and characterization of metal NPs Metal NPs (Au@PEI or Ag@PEI NPs) were prepared via reducing HAuCl4 or AgNO3 by the classical borohydride (NaBH4) in the presence of cationic molecules PEI to stabilize the metal NPs formed. When an aliquot of NaBH4 solution was added to the mixture of metal salt and PEI, a reddish-colored solution for AuNPs and a yellowish-colored solution for AgNPs were formed within 12 h at room temperature, respectively. The NPs solutions are stable for at least one month without color change. Thermogravimetric analysis indicated a grafting density of 150 PEI molecules per AuNP and 370 PEI molecules per AgNP (Fig. 2a), indicating a quite dense coverage of PEI molecules on the NPs (0.8 molecule/nm2 for AuNPs or 0.9 molecule/nm2 for AgNPs). This observation indicates that in principle PEI is capable to stabilize the nanoparticles formed. Transmission electron microscopy (TEM) images confirm the generation of spherical nanoparticles (Fig. 2b, c). The majority of the NPs are well dispersed. However, there are some aggregates can be found. According to dynamic laser scattering (DLS) measurement, the average diameter of Au@PEI and Ag@PEI NPs are 20 nm and 35 nm, respectively (Fig. 3a). As a result, the metal NPs possess positively charge (about þ30 mV) on their surfaces (Fig. 3b). 3.2. TAT conjugation After ligand exchange with thiol-TAT, the peptide amount on NPs increased along with the elevation of its feeding concentration (Fig. 3c). There is no significant difference between AuNPs and AgNPs. In our study, the highest TAT content that can be immobilized on metal NPs is about 6% (Fig. 2a), contributing more than half of the organic capping layer. After TAT conjugation, there are no significant alternation on particle diameter (Fig. 3a), surface charge (Fig. 3b), and morphology (Fig. 3d and e).

3.3. Competition binding assay Positively charged metal NPs show a potential to condense negatively charged DNA. To validate the binding between both, a standard ethidium bromide (EtBr)-DNA fluorescence quenching exclusion assay was carried out [28]. After combination with DNA, EB can occupy the effective binding sites on DNA and give strong fluoresce. This combination is reversible and can be replaced by other stronger interaction. When the DNA/EB was mixed with metal NPs solution, the fluorescence intensity of the solution decreased because of the squeeze out of the EB (no fluorescence emission excitated at 510 nm). Fig. 4a and b shows that the fluorescence intensity significantly decreased upon addition of metal NPs at the ratio over 1, suggesting complexation between DNA and positively charged metal NPs. The alternation of bulk composition and surface coating of the NPs has minor influence on their DNA binding ability (Fig. 4c and d). 3.4. Vector/DNA complexes formation and characterization As shown in Fig. 5a, the NPs@PEI-DNA complexes have a narrow size distribution with an average diameter of 100e200 nm. Compared to the individual metal NPs, the increase of size indicates the condensation between metal NPs and DNA. In general, there is no significant difference between the size of AuNPs/DNA and AgNPs/DNA complexes although the size of metal NPs are somehow different. All the sizes of the complexes showed similar trends as a function of vector/DNA ratio. At a very small vector/DNA ratio, both Au@PEI and Ag@PEI NPs formed relatively large complexes, which should be caused by the aggregation of the particles. When the vector/DNA ratio was increased to 1, both NPs and PEI molecules formed the complexes with smaller size. Further augment of the vector/DNA ratio caused slightly decrease and then slightly increase of the particle sizes. However, the alternation of complexes sizes is not significant. Since Au@PEI/DNA and Ag@PEI/DNA complexes have the smallest size at the vector/DNA ratio of 5 and 3 respectively, we used these ratios to study the impact of TAT content on the functions of Au@PEI-TAT/DNA and Ag@PEI-TAT/DNA complexes. Illustrated by Fig. 5b, the size of the NPs/DNA complexes increased a bit when introducing TAT onto NPs. The size kept stable along with the increase of TAT content on the NPs. But in general, the alternation of complexes sizes is small since all the NPs@PEI-TAT/DNA complexes have the size in the range of 100e 200 nm. It is worth noting that the sizes of AuNPs@PEI-TAT/DNA and AgNPs@PEI-TAT/DNA complexes are very similar although the size of AgNPs is much larger than that of AuNPs. The results

Fig. 2. (a) Weight loss of Au@PEI-TAT and Ag@PEI-TAT NPs at 400  C as a function of TAT feeding concentration which was used to modify metal@PEI NPs. TEM images of (b) Au@PEI and (c) Ag@PEI NPs.

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Fig. 3. (a) Tat contents conjugated to metal nanoparticles, (b) Particles diameter, (c) Zeta potential of Au@PEI-TAT and Ag@PEI-TAT nanoparticles as a function of TAT feeding concentration, respectively. TEM images of (d) [email protected], and (e) [email protected] nanoparticles.

Fig. 4. EtBr displacement assay of metal@PEI NPs binding DNA, showing the fluorescence intensity as a function of (a) vector/DNA ratio and (b) TAT content at a fixed vector/DNA ratio. TAT-conjugated metal NPs binding DNA, showing the fluorescence intensity as a function of vector/DNA ratio (c) and TAT content at a fixed vector/DNA ratio (d).

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Fig. 5. Particle diameter of vector/DNA complexes of as a function of (a) DNA to vector ration (w/w), and (b) TAT contents, of which Au@PEI to DNA (w/w), Ag@PEI to DNA (w/w) and PEI to DNA (w/w) ratios were 3, 5, and 1 respectively.

suggested that the sizes of metal NPs/DNA complexes are mainly controlled by the surface coating of the NPs rather than the bulk composition and size. The zeta potential of NPs@PEI/DNA complexes increased significantly when the vector/DNA ratio increased from 1 to 3, and reached a plateau afterwards: around 20 mV for AuNPs@PEI/DNA and 30 mV for AgNPs@PEI/DNA complexes, respectively (Fig. 6a). We also tested the zeta potential of NPs@PEI-TAT/DNA complexes with fixed vector/DNA ratios. As shown in Fig. 6b, the increase of TAT content of NPs did not result in significant difference of the zeta potential of NPs/DNA complexes. This might be attributed to the similar charge properties between TAT and PEI molecules and therefore the replacement of PEI by TAT molecules did not bring significant difference on the surface charge density of the NPs. The complexes possess positively surface charge, which likely facilitates the intracellular uptake and thus realize the gene transfection. 3.5. Evaluation of cytotoxicity In order to evaluate the biomedical performance of the metal NPs, rat epidermal stem cells (ESCs) were used as delegates of stem cells. As an important progenitor cells, the self-renewal ability and multilineage differentiation potential of ESCs suggest their great potential as therapeutics for regenerative medicine and gene therapy [29e32]. ESCs include at least two populations of skin mesenchymal stem cells from the basal layer of epidermis and from the hair follicles, which are the focus of dermatologic stem cell research [33]. ESCs can differentiate in vitro and in vivo into several tissue lineages such as epithelial cells, hair follicle cells, neural cells, and possibly other cell types [34], making them attractive candidates for tissue regeneration and gene therapy. Moreover, the differentiation of ESC into neural cells is thought to be a promising

strategy to provide innovative therapeutical approaches for neurodegenerative diseases and nerve regeneration [35e38]. To achieve these goals, usually the ESCs are required for gene manipulation to introduce additional functions. However, the report regarding successful gene transfection of the ESCs based on non-viral vectors is scarce [39,40]. In our previous study, only two vectors, Lipofectamine 2000 and Cyclodextran modified PEI, among 15 non-viral vectors, showed slight transfection ability to the ESCs (only 1.8% and 5.4% of the cells were transfected, respectively) [41]. In view of this, much effort has been devoted to develop vectors which have enhanced transfection efficiency and reduced cytotoxicity in ESCs. Cytotoxicity of metal NPs as potential delivery vectors was firstly evaluated using a standard MTT assay. It was shown that the PEI/DNA and PEI-TAT/DNA complexes are quite toxic to the ESCs and their cytotoxicity increased along with the augment of vector/DNA weight ratio. As a result, the cell viability reached about 40% of control when the cells were exposed to PEI/ DNA or PEI-TAT/DNA complexes with the highest vector/DNA ratio. In contrast, cell viability is not significantly inhibited by the NPs@PEI/DNA (Fig. 7a), and NPs@PEI-TAT/DNA complexes at tested concentration (Fig. 7b). About 90% of the cell viability was retained when the cells were exposed to NPs@PEI/DNA complexes. This is due to the replacement of toxic PEI molecules by relatively inert metal NPs in the complexes. Additionally, cells appear to grow better with NPs@PEI-TAT/DNA complexes (the viability is larger than control), especially with the proper TAT amount. The even lower cytotoxicity of NPs@PEI-TAT vectors might be attributed to the further replacement of PEI molecules with higher molecular weight and charge density which can generate higher toxicity by TAT peptides with relatively low molecular weight and charge density. Viability studies show that metal NPs@PEI-TAT/DNA complexes are nontoxic and suitable for further biomedical application.

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Fig. 6. Zeta potential of vector/DNA complexes of as a function of (a) DNA to vector ration (w/w), and (b) TAT contents, of which Au@PEI to DNA (w/w), Ag@PEI to DNA (w/w) and PEI to DNA (w/w) ratios were 3, 5, and 1 respectively.

Fig. 7. Cell viability of epidermal stem cells (ESCs) after incubated with various vector/DNA complexes containing 1 mg DNA as a function of, (a) DNA to vector ration (w/w), and (b) Tat content, of which Au@PEI to DNA (w/w), Ag@PEI to DNA (w/w) and PEI to DNA (w/w) ratios were 3, 5, and 1 respectively.

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3.6. Tansfection assay Transfection experiments using a reporter gene, enhanced green fluorescent protein plasmid DNA (pDNA-EGFP), were firstly performed on the ESC. The mean fluorescent intensity per cell, which positively correlates to the amount of DNA which has been transferred into the cells’ nucleus was used as the indicator of transfection activity [42e44]. From the EGFP transfection results, in general, the mean fluorescent intensity of the cells treated with PEI/ DNA complexes is always weak at all vector/DNA ratios, suggesting limited transfection ability of PEI towards the ESC (Fig. 8). In contrast, NPs@PEI/DNA complexes show stronger gene transfection ability in a vector/ratio dependent manner at low vector/DNA weight rations. This is attributed to the high toxicity of NPs@PEI/ DNA complexes at higher vector/DNA rations. The AuNPs@PEI/DNA and AgNPs@PEI/DNA complexes have the best transfection ability at the vector/DNA weight ratio of 3 (Fig. 8a). The transfection ability of metal NPs@PEI-TAT/DNA complexes with an optimized vector/ DNA ratio significantly increased along with the increase of TAT content on the NPs and reached the highest values when the TAT content in the range of 3e5% of the NPs (Fig. 8b). The transfection ability of metal NPs@PEI-TAT/DNA complexes reduced with further enhanced TAT content (>6%). In contrast, PEI-TAT/DNA complexes with an optimized vector/DNA ratio did not show any improvement of the transfection ability. The results clearly demonstrated that NPs@PEI-TAT have stronger gene delivery and transfection efficacy. The in general similar gene transfection ability of Au and Ag based NPs can be attributed to their similar DNA binding ability, as well as similar size and zeta potential of resulted NPs/DNA complexes.

3.7. ESCs induced neuronal differentiation upon vector/pDNA-bFGF transfection To test the feasibility of gene transfection modulated differentiation of the ESCs, the cells were transfected with plasmid DNA encoding basic fibroblast growth factor (pDNA-bFGF) to enhance the neuronal differentiation of ESC. bFGF is one of the key growth factors that regulating the neural development and has been widely used to induce the neuronal differentiation [45,46]. Additionally, bone marrow derived mesenchymal stem cells transfected with bFGF genes to induce the neuronal cells was reported [47]. NeuN is a neuron-specific nuclear protein antigen commonly used as a biomarker for neurons [48,49]. Glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein that is expressed by numerous nueronal cell types, for example, astrocytes, is another widely used antigen for the characterization of neural cells [50]. With these two useful markers, ESCs were cultured for 21 days post vector/pDNAbFGF complexes transfection or bFGF protein incubation, followed by the identification of NeuN and GFAP expression to estimate the efficacy of the prepared vectors in delivery functional gene to ESCs for directed differentiation into neuronal cells. As shown in Fig. 9, untreated cells and cells transfected with naked DNA showed very low GFAP expression (Fig. 9a, b). Fig. 9c shows the bright GFAP expression in green (in the web version) by the positive cells treated by bFGF protein incubation for 21 days. Obvious expression of GFAP was observed in the cells transfected by metal NPs@PEI/ pDNA-bFGF (Fig. 9d, e) and Au@PEI-TAT/pDNA-bFGF and Ag@PEITAT/pDNA-bFGF complexes. However, the higher similarity of the cells morphology between the positive control (Fig. 9c) and

Fig. 8. Mean fluorescence intensity per ESC after transfected with various vector/DNA complexes containing 1 mg DNA as a function of (a) DNA to vector ration (w/w), (b) Tat content, of which Au@PEI to DNA, Ag@PEI and PEI-TAT to DNA weight ratios were 3, 5 and 1, respectively. * indicates significant difference at p < 0.05 level.

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Fig. 9. (aei) CLSM images and (g) NeuN expression of the ESCs cultured for 21 days after they were treated with various vector/DNA complexes containing 1 mg pDNA-bFGF or bFGF protein. The cells were treated with (a) no DNA, (b) naked pDNA-bFGF transfection for 6 h, (c) bFGF protein (100 ng/ml) co-culture for 21 days, (d) Au@PEI/pDNA-bFGF transfection for 6 h, (e) Ag@PEI/pDNA-bFGF transfection for 6 h and (f) PEI/pDNA-bFGF transfection for 6 h, (g) Au@PEI-TAT/pDNA-bFGF for 6 h (h) Ag@PEI-TAT/pDNA-bFGF for 6 h (i) PEI-TAT/ pDNA-bFGF for 6 h. Bar represents 20 mm *indicates significant difference at p < 0.05 level, **p < 0.01 level.

NPs@PEI-TAT/pDNA-bFGF complexes treated groups (Fig. 9h, i) suggested the differentiation of the ESCs into neuronal cells in the NPs@PEI-TAT/pDNA-bFGF complexes treated group, are significantly stronger than those in NPs@PEI/pDNA-bFGF complexes treated group. It is worth noting that cells transfected by PEI-TAT/DNA complexes showed certain expression of GFAP (Fig. 9i) too. However,

the cell with positive GFAP expression was very scarce, as well as the cells viability was quite low. As a quantitative analysis, the measurement of another useful neuronal marker, NeuN expression suggested similar results: the cells transfected with metal NPs@PEITAT/pDNA-bFGF complexes showed much higher NeuN expression than that of naked DNA, NPs@PEI/pDNA-bFGF, or PEI-TAT/pDNAbFGF complexes treated cells. All the results proved that metal

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NPs@PEI-TAT are powerful vehicles for delivering functional genes into the ESCs and can even modulate their differentiation. 3.8. Cellular uptake and intracellular distribution of vector/DNA complexes In order to clarify the mechanism of excellent gene delivery ability of metal NPs@PEI-TAT/DNA complexes, their cellular uptake kinetics, nuclear targeting property, and uptake pathways were studied. To make the vector/DNA complexes detectable via flowcytometry and fluorescence microscopy, FITC labeled DNA and cy3 labeled DNA were used. As shown in Fig. 10a, b, in the cellular uptake test, the average fluorescence intensity per cell increased along with the prolongation of culture time for all types of the vector/FITC-DNA complexes and reached a highest value in the range of 4.5e6.5 h. With further prolongation of culture time, the average fluorescence intensity per cell decreased dramatically, which might be attributed to the removal of fluorescent molecules from cells by exocytosis. The ESCs took up a bit larger amount of NPs@PEI-TAT/DNA complexes than NPs@PEI/DNA complexes and PEI-TAT/DNA complexes (p < 0.05) at 6.5 h. This might be attributed to the attachment and accumulation of TAT peptides on the surface of NPs, resulted in higher TAT local concentration and subsequently enhanced cell penetrating ability. This result can be directly observed in CLSM images. As shown in Fig. 11, the cells obviously ingested more fluorescent NPs@PEI-

TAT/DNA complexes (Fig. 11f, g) than NPs@PEI/DNA complexes (Fig. 11c, d) after 6 h incubation. Besides, the overlap of signals from DNA and cell nucleus can be observed, suggesting NPs based vectors can deliver DNA not only into cells but also target to cell nucleus. In contrast, the fluorescence observed in other groups, including blank control (Fig. 11a), TAT/DNA control (Fig. 11b), PEI/ DNA complexes (Fig. 11e) and PEI-TAT/DNA complexes (Fig. 11h) treated groups are not significant. The cellular distribution and nucleus targeting of the NPs/DNA complexes was further characterized by TEM. As shown in Fig. 12, metal NPs (red arrows headed) could be found in the cytoplasm, attached to cell nuclear envelope, and inside cell nucleus (including nucleolus). All the results pointed out that NPs@PEI-TAT are able to deliver DNA into cells and cell nucleus with higher efficiency compare to PEI-TAT and NPs@PEI, resulted in excellent gene delivery ability. 3.9. Intracellular pathway of vector/DNA complexes The mechanism of TAT peptide facilitated translocation is a topic of great debate. Some studies proposed that TAT peptide can directly translocation across plasma membrane since the translocation can occur via an energy-independent process [51]. Evidence has also been presented that translocation could use several different endocytic pathways [52]. In addition, the mechanism of translocation can be dependent on whether the peptide is free or

Fig. 10. Mean fluorescence intensity of the ESCs incubated with various vector/DNA complexes containing 1 mg of FITC labeled DNA as a function of incubation time, of which Au@PEI-TAT to DNA (w/w), Ag@PEI-TAT to DNA (w/w) and PET-TAT to DNA ratios were 3, 5 and 1, respectively. TAT contents in TAT control, Au@PEI-TAT, Ag@PEI-TAT and PEI-TAT were 50 mg/ml, 4.7%, 4.3% and 4.4%, respectively.

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Fig. 11. CLSM images of the ESCs treated with various vector/DNA complexes containing 1 mg cy3-labeled DNA (red) for 6 h: (a) Naked DNA, (b) TAT/DNA, (c) Au@PEI/DNA, (d) Ag@PEI/DNA, (e) PEI/DNA, (f) Au@PEI-TAT/DNA, (g) Ag@PEI-TAT/DNA, (h) PEI-TAT/DNA. Panel 1: black-and-white images; Panel 2: cell nucleus stained with DAPI (blue); Panel 3: cy3-labeld DNA; Panel 4: Merged images. Bar represents 5 mm. (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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Fig. 12. TEM images of (a) the pristine ESCs, and the ESCs treated with (b) Au@PEI/DNA complexes, (c) Ag@PEI/DNA complexes, (d) Au@PEI-TAT/DNA complexes, (e) Ag@PEI-TAT/ DNA complexes for 6 h, respectively. Nuclear membrane, nucleolus, and metal NPs were indicated by green, yellow and red arrows, respectively. Panel 1: whole cell and panel 2e4: amplified images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

attached to cargo. As shown in Fig. 13, the gene transfection abilities of Ag@PEI-TAT/DNA complexes were not significantly inhibited at 4  C and by all the pharmaceutical inhibitors, suggesting the complexes can enter cells via energy-independent process, i.e., direct translocation. However, the gene transfection abilities of AuNPs@PEI-TAT/DNA and PEI-TAT/DNA complexes were partially and almost completely inhibited by the pre-treatment of MbCD,

respectively. The results indicated that caveolin-mediated endocytotic pathway also contribute to the ingestion of AuNPs/PEI-TAT/ DNA and PEI-TAT/DNA complexes besides energy-independent process. In summary, the neuronal differentiation of ESCs based on genetic manipulation for the treatment of neurodegenerative disorders has caused wide attention. However, as a progenitor/primary

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Fig. 13. Mean fluorescence intensity of the ESCs transfected with various (a) metal @PEI/pDNA-EGFP complexes, and (b) metal@PEI-TAT/pDNA-EGFP complexes, containing 1 mg of pDNA-EGFP. ECSs were pretreated with various endocytosis inhibitors for 30 min before treated with vector/pDNA-EGFP complexes containing 1 mg of pDNA-EGFP for 6 h. After further 42 h of incubation, the fluorescent intensity of cells which correlated with transfection efficiency was quantified by FACS analysis.

cell, ESC is notoriously difficult to transfect with current nonviral methods due to 1) The cell membrane and nucleons are difficult to porate or pass through, especially for the progenitor/primary cells which have limited cell uptake and duplication ability. 2) Getting genes in the cell is not a natural process and is inherently toxic, especially for the non-dividing or progenitor cells that can not proliferate quickly. Results of the present study provided strong evidence for that the metal NPs@PEI-TAT integrate the advantages of PEI, metal NPs and peptides including: 1) PEI molecules render vectors positively charge and the ability to condense and protect DNA; 2) the presence of metal NPs effectively decrease the cytotoxicity of cationic molecules, making it possible to apply them in biological systems; 3) the cell penetrating peptides can obviously enhance the cellular uptake, nucleus entry and gene transfection efficiency of metal NPs. Besides, in contrast to the metal NPs@PEITAT, PEI-TAT molecules at all TAT content expressed limited cell penetrating and gene transfection ability although they have similar ability to condense DNA and generate vector/DNA complexes. The results suggested that only tethered TAT molecules on NPs can generate pores on cell membrane and facilitate cell uptake and gene transfection due to locally enhanced concentration. 4. Conclusions The conjugation of TAT peptides on noble metal NPs@PEI enhanced their ability to deliver DNA into the nucleus of stem cells with high level of cell survival. As a result, metal NPs@PEI-TAT efficiently manipulated the epidermal stem cells genetically and induced their neural differentiation. The TAT conjugated metal

nanoparticles provide a new means to engineer metal NPs for both drug and gene delivery to stem cells, which may have wide application in manipulating difficult-to-transfect cell types. Acknowledgments The study was supported by National Natural Science Foundation of China (81102393, 81273441, 51120135001), National Basic Research Program of China (No. 2014CB931901), and Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents. References [1] Agasti SS, Chompoosor A, You CC, Ghosh P, Kim CK, Rotello VM. Photoregulated release of caged anticancer drugs from gold nanoparticles. J Am Chem Soc 2009;131:5728e9. [2] Katz E, Willner I. Integrated nanoparticle-biomolecule hybrid systems: synthesis, properties, and applications. Angew Chem Int Ed Engl 2004;43:6042e 108. [3] Ghosh P, Han G, De M, Kim CK, Rotello VM. Gold nanoparticles in delivery applications. Adv Drug Deliv Rev 2008;60:1307e15. [4] Seferos DS, Prigodich AE, Giljohann DA, Patel PC, Mirkin CA. Polyvalent DNA nanoparticle conjugates stabilize nucleic acids. Nano Lett 2009;9:308e11. [5] Nakanishi J, Nakayama H, Shimizu T, Ishida H, Kikuchi Y, Yamaguchi K, et al. Light-regulated activation of cellular signaling by gold nanoparticles that capture and release amines. J Am Chem Soc 2009;131:3822e3. [6] Rosi NL, Giljohann DA, Thaxton CS, Lytton-Jean AKR, Han MS, Mirkin CA. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 2006;312:1027e30. [7] Hong R, Han G, Fernandez JM, Kim BJ, Forbes NS, Rotello VM. Glutathionemediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc 2006;128:1078e9.

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