Efficient, dual-stimuli responsive cytosolic gene delivery using a RGD modified disulfide-linked polyethylenimine functionalized gold nanorod.

COREL-07391; No of Pages 15 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

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Feihu Wang a, Yuanyuan Shen a, Wenjun Zhang a, Min Li a, Yun Wang a, Dejian Zhou b,⁎, Shengrong Guo a,b,⁎⁎

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Article history: Received 21 July 2014 Accepted 25 September 2014 Available online xxxx

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Keywords: Gold nanorod Disulfide-linked polyethylenimine Glutathione Near-infrared laser Photochemical effect Controlled gene delivery

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School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China School of Chemistry, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

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Controlled-release systems capable of responding to external stimuli and/or unique internal environments have received great interests in site-specific gene and/or drug delivery. In this work, a functionalized gene nanocarrier for dual-stimuli triggered cytosolic gene delivery is developed and showing high gene delivery efficacy with low cytotoxicity. The nanocarrier is prepared by conjugating gold nanorod (GNR) with multiple disulfide cross-linked short PEIs to harness the advantageous properties of GNR based near infrared (NIR) laser induced photothermal heating and intracellular stimuli-triggered degradability of disulfide cross-linked short PEIs (DSPEI). The DSPEI is further grafted with a poly(ethylene glycol) (PEG) section to afford high carrier stability in cell cultures and a terminal RGD peptide for specific targeting of cancer cells. The nanocarrier is found to effectively condense plasmid DNA to form a highly stable GNR-DSPEI-PEG-RGD/DNA complex with tumor cell-targeting ability that can be efficiently uptaken by cancer cells. Moreover, the loaded genes can be effectively released from the complex triggered by the high intracellular glutathione content and/or by photothermal effect of NIR irradiation at 808 nm. Interestingly, the GNRs-based complex can easily escape from intracellular endo-/lyso-somal compartments and release the gene load into the cytosol upon exposure to NIR irradiation, resulting in significantly improved gene transfection efficiency. Our new gene carrier exhibits high gene transfection efficiency, comparable to or even better than that of high MW PEIs, but with a much lower cytotoxicity. Additionally, neither the GNRbased carrier nor the laser treatment shows any significant evidence of cytotoxicity. This work demonstrates a promising strategy for intracellular stimuli triggered, photothermal controllable gene delivery system, which can be further applied to many other nanomedicine fields. © 2014 Published by Elsevier B.V.

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Efficient, dual-stimuli responsive cytosolic gene delivery using a RGD modified disulfide-linked polyethylenimine functionalized gold nanorod

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Gene therapy is a promising and unique approach to treat a variety of diseases, including genetic diseases, cardiovascular diseases and cancer [1–4]. Since the nucleic acids are prone to hydrolysis in biological fluids and show low cellular uptake efficiency due to their polyanionic nature, the development of gene delivery systems with high efficiency, safety and selective targeting ability is essential for gene therapy [5,6]. Although viral vectors have shown high transfection efficiency, some shortcomings such as non-specific, immunogenic and susceptibility to enzyme degradation, have limited their clinical applications. Due to the improved safety profile and ease of preparation and manipulation, nonviral gene delivery vectors are continuing to be explored and optimized [7,8]. Significant research efforts have been focused on developing controlled-release systems that are capable of responding to external stimuli and/or the unique environments of tissues.

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1. Introduction

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⁎ Corresponding author. Tel.: +44 1133436230. ⁎⁎ Correspondence to: S. Guo, School of Chemistry, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Tel.: +44 7459025683. E-mail addresses: [email protected] (D. Zhou), [email protected] (S. Guo).

The exploitation of external stimuli triggered, spatially and temporally controlled drug delivery carrier has received tremendous attention in recent years. External stimulation factors include light [9–12], temperature [13], radiofrequency [14,15], magnetic field [16] and ultrasound [17,18]. Among which, light provides a great opportunity to deliver a drug at the desired area at a specific time, which is considered a key tool to amplify drug efficacy with minimum adverse effects. In particular, near-infrared (NIR) light has been particularly attractive due to the “water window” (650–900 nm) which shows minimal absorbance by skin and tissue [19], and thus providing deep tissue penetration with high spatial precision without damaging normal biological tissues. Gold nanoparticles (GNPs) exhibit extraordinary functionality due to their unique optical and electronic properties [20]. Following excitation, the plasmons on nanoparticle surfaces can decay by either radiative damping or energetic relaxation, which create nonequilibrium “hot” electron–hole pairs [21,22]. When molecules are adsorbed on the nanoparticle surface, excited “hot” electrons can transfer to the adsorbate prior to thermalization [23,24]. Thus, photorelease strategies have sought to covalently attaching a “carrier” molecule to the nanoparticle surface through the Au\S bond. Then a “cargo” therapeutic gene

http://dx.doi.org/10.1016/j.jconrel.2014.09.026 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: F. Wang, et al., Efficient, dual-stimuli responsive cytosolic gene delivery using a RGD modified disulfide-linked polyethylenimine functionalized gold nanorod, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.09.026

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2.1. Materials

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Cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4), 5-Bromosalicylic acid (5-BrSA), silver nitrate (AgNO3), L -ascorbic acid (L-AA), polyethylenimine branched (PEI-25 KDa), L-glutathione (Reduced) (GSH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-Hydroxy succinimide (NHS), 3(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Ethidium bromide (EB), bisBenzimide H 33342 trihydrochloride (Hochest 33342), trypan blue and dimethyl sulfoxide (DMSO) were obtained from Sigma Co., Ltd. (USA). Polyethylenimine branched (PEI1.8 KDa) was purchased from Alfa Aesar (USA). 3,3′-dithiodipropionic acid, methyl thioglycolate, BOP reagent, 1-dodecanethiol (DDT) and 11-mercaptoundecanoic acid (MUDA) were purchased from Aladdin Industrial Co., Ltd. (China). YOYO-1 and Lyso Traker Red were obtained from Invitrogen Molecular Probes (USA). C(RGDyk)-NH2 (RGD) was purchased from GL Biochem (Shanghai) Ltd. (China). Maleimide polyethylene glycol N-Hydroxysuccinimide ester (MAL-PEG-NHS, MW 3500) was purchased from Jenkem Technology (China). Chloroauric acid tetrahydrate (HAuCl4·4H2O), hydrochloric acid (HCl, 36.0–38.0 wt.% in water) and nitric acid (HNO3) were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). The Dulbecco's Modified Eagle Medium (DMEM), penicillin–streptomycin, fetal bovine serum (FBS), 0.25% (w/v) trypsin–0.03% (w/v) EDTA solution and Phosphate buffer solution (PBS) were purchased from Gibco BRL (USA). Water was purified by distillation, deionization, and reverse osmosis (Milli-Q plus). All reagents were analytical grades and used without further purification. Plasmid DNA encoding for green fluorescent protein (pGFP-N1) was a kind gift from Professor Tuo Jin (School of Pharmacy, Shanghai Jiao Tong University), and was transformed in Escherichia coli DH5R and propagated in Luria-Bertani (LB) medium at 37 °C overnight, followed by isolation and purification with a commercially available plasmid purification Qiagen Plasmid Maxi Kit (Qiagen, Valencia, CA). The concentration of plasmid solution was determined by measuring the ultraviolet (UV) absorbance at 260 nm and its optical density ratio at 260 nm to 280 nm was in the range of 1.8 to 1.9.

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2.2. Cell culture

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The U-87 MG (human glioblastoma cell) cell line was kindly donated by the School of Pharmacy, Fudan University. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate (complete DMEM medium) and maintained at 37 °C in a humidified and 5% CO2 incubator. Cells grown to confluence were subcultured every other day after trypsinized with 0.25% trypsin–EDTA and diluted (1/3) in fresh growth medium.

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2.3. Synthesis of GNRs

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GNRs were synthesized using the seed-mediated growth method according to the literatures [51,52]. Briefly, a 5 mL amount of 0.5 mM HAuCl4 was mixed with 5 mL of 0.2 M CTAB solution. While the solution under vigorous stirring (1200 rpm) at 30 °C, 0.6 mL of fresh ice-cold 0.01 M NaBH4 was added. The solution color changed from yellow to brownish-yellow, and the stirring was stopped after 2 min. Afterwards, it was allowed to react for 2 h to form the CTAB-capped gold nanoparticles to be used as seeds for the synthesis of GNRs.

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laser irradiation in solution and in living cells. FITC labeled GNRDSPEI-PEG-RGD was further used to investigate intracellular lighttriggered endosome escape. The transfection activity of GNR-DSPEIPEG-RGD based complexes under NIR laser treatment was also evaluated in U-87 MG cells using the pGFP as reporter genes.

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entity loads onto the “carrier” molecule via weaker, noncovalent interactions. When this type of nanocomplex is irradiated with continuouswave (CW) laser, the nanoparticle absorbs energy, which reduces the attraction between the “carrier” and “cargo” molecules either thermally or nonthermally, resulting in release of the therapeutic gene [25,26]. This “on demand” release strategy shows excellent promise for lightcontrolled delivery due to the relatively low laser power intensity and short irradiation times required to achieve the release of molecular “cargo”. Notably, research showed that low CW laser intensity irradiation of cancer cells containing endosomal GNPs leads to endosome rupture without affecting the cells' viability. This underlying photochemical mechanism facilitates the GNPs' escape from the endosome into the cytosol, which is required for the therapeutic gene to be effective [25,27,28]. Gold nanorods (GNRs), which are rod-shaped GNPs, show high promising potential as light-triggered, remotely controlled molecular release trigger in response to optical excitation. The plasmon resonance of GNRs can be tuned from the visible to NIR regions that depend on the nanorod's aspect ratio [29]. The NIR laser irradiation-triggered drug and gene release is especially attractive, because GNRs can be easily made to maximally absorb in the “water window” [30]. In addition, GNRs have the advantages of efficient large-scale synthesis and can be easily decorated with multiple molecular species to simultaneously provide biological compatibility [31–33], activated drug and gene delivery [34–36] and direct cell specific targeting [37–39]. Polyethylenimine (PEI) has been regarded as the “gold” standard for gene delivery because it shows relatively high transfection efficiency from its proton sponge effect and ability to protect DNA from degradation by enzymes [40,41]. High molecular weight PEIs such as 25-kDa PEI are highly effective in gene transfection, but also induce high cytotoxicity due to the high cationic density and lack of biodegradability [42]. Low molecular weight PEIs such as 1.8-kDa PEI have a much lower cytotoxicity, but they cannot effectively condense DNA and display very poor gene transfection activity [42,43]. In order to reduce the cytotoxicity and enhance carrier unpacking into the cytosol and/or nucleus, intracellular-cleavable disulfide-linked PEIs have been designed for gene delivery [44–46]. The disulfide linkage is stable in blood circulation [47]. Once inside cells, the disulfide bonds are cleaved under the high concentration of reductive glutathione (GSH), making it favorable to unpack the PEI shell and release the infective nucleic acid [42,48]. Therefore, reducible disulfide cross-linked short PEIs (DSPEI) was synthesized as gene carrier for controlled-release systems in response to the unique environments of cells. Integrin αvβ3, an important biomarker over-expressed on actively angiogenic endothelium and malignant glioma cell surfaces, plays a critical role in regulating tumor growth, metastasis and tumor angiogenesis [49,50]. Researches have shown that the cyclic RGD (arginine–glycine– aspartic acid) peptides (cRGD) can specifically bind with integrin αvβ3 [39,49], thus we have selected the RGD peptides as the targeting molecules for glioblastoma cell targeting and selective therapy. Here, DSPEIPEG-RGD was developed by incorporating the RGD peptide into the terminal end of hydrophilic polyethylene glycol (PEG) spacer. The tethered RGD peptide at the end of the PEG chain may behave like a free molecule in the solution due to highly flexible and well-hydrated PEG chains so that the RGD peptide should have easy access to its receptor. In this study, we developed a GNR-based gene delivery system. This nanosized gene delivery carrier consists of GNR covalently conjugated with RGD modified DSPEI via “round-trip” phase transfer ligand exchange. The success of the functionalized GNR-DSPEI-PEG-RGD was confirmed by UV–Vis–NIR spectrophotometer, 1H-NMR, transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). To investigate the general utility of this delivery system, the gene was loaded onto the carrier and the tumor cell targeting ability of the GNR-based complexes was examined by dark-field microscopy and inductively coupled plasma–mass spectrometry (ICP–MS). We evaluated YOYO-1 labeled DNA release from the complex by dual-stimuli of GSH and NIR

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2.5. Synthesis of HS-DSPEI-PEG-RGD conjugates

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The synthesis of HS-DSPEI-PEG-RGD conjugates consists of two reaction steps, as reported previously [53]. The course of reaction was shown in Fig. S1c and 1d. Briefly, in the first reaction step, 2 mmol

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2.6. HS-DSPEI-PEG-RGD functionalization of GNRs

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To synthesize DSPEI (Fig. S1a), PEI-1.8 KDa was cross-linked with 3,3′-dithiodipropionic acid (DA) at PEI primary amine:cross-link reactive group ratio of 1:1.5. Based on reports in the literature [43], we estimated the primary amine content of PEI 1800 to be 25% of the total amines. To achieve PEI primary amine:crosslink reactive group molar ratio of 1:1.5, 315 mg of 3,3′-dithiodipropionic acid was dissolved in 10 mL of anhydrous DMSO and activated by equal amounts (4 equiv/ DA) of EDC (1.15 g) and NHS (0.69 g) by stirring at room temperature for 4 h. The solution was then added drop wisely to the PEI solution containing 250 mg of PEI in 10 mL of dry DMSO, which allows the formation of an amide linkage by the reaction with primary amino groups in PEI. The resulting solution was reacted by stirring for 12 h at room temperature. Then, the reactant mixture was dialyzed against the excess amount of ddH2O for 3 days using a dialysis tube (molecular cut off 3500) to remove excess of unreacted substrates, followed by lyophilization to obtain DSPEI. The structure of the polymer was analyzed using a fourier transform infrared (FT-IR) spectrophotometer (KBr pellets, BRUKER VERTEX70, Germany). The molecular weight (Mw) and polydispersity (Mw/Mn) of DSPEI were measured by a gel permeation chromatography (GPC) system relative to PEG standards (Mp = 400–40,000 Da, Polymer Standards Service, Germany). GPC was performed on a Waters 2695 controller equipped with Ultrahydrogel columns (120 PKGD and 250 PKGD, 30 °C) and a refractive index detector (model 2414). 0.05% NaN3 aqueous solution was used as the mobile phase with a flow rate of 1 mL/min. In order to conjugate RGD to DSPEI and improve the binding ability of the DSPEI ligands to GNRs, a small number of thiol groups were introduced to the structure (Fig. S1b). Thiol-functionalized DSPEI was prepared by reacting DSPEI with methyl thioglycolate at PEI primary amine:SH ratio of 10:1. Briefly, 665 mg of BOP and 40 mg of methyl thioglycolate were dissolved in 5 mL of anhydrous DMSO. The solution was then added drop wisely to DSPEI solution containing 500 mg of DSPEI in 10 mL of dry DMSO. The mixture was reacted with stirring at 50 °C under nitrogen for 12 h to complete the formation of the partially thiol-terminated DSPEI. Then, the reactant mixture was dialyzed against water for 3 days to remove excess of unreacted substrates, followed by lyophilization to obtain DSPEI-SH. The chemical structure and degree of the thiolation of DSPEI-SH were analyzed by 1H-NMR spectra recorded on Varian Mercury Plus-400 NMR spectrometer (Varian, USA).

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The CTAB surfactant on the GNRs surface was replaced with 11mercaptoundecanoic acid (MUDA) by round-trip phase transfer ligand exchange as described in detail in previous report [54]. As shown in Fig. 2, round-trip ligand exchange first utilizes aqueous-to-organic phase transfer. 1 mL of concentrated GNRs (2 × 10− 8–5 × 10−8 M) with CTAB surfactants (GNR-CTAB) in water were put into contact with 1 mL of dodecanethiol (DDT). After addition of 3 mL acetone, GNRs were extracted into DDT by swirling the solution for a few seconds, upon which the aqueous phase became clear, indicating that no GNRs remained. Next, organic-to-aqueous phase transfer was performed. Excess DDT was removed by diluting the DDT coated GNRs (GNR-DDT) in 5 mL of toluene and centrifuged at 3500 rpm for 10 min. The collected GNR-DDT was resuspended in 1 mL of toluene by brief sonication, then, added to 9 mL of 0.01 M MUDA in toluene at 70 °C and vigorously stirred. Reflux and stirring continued until visible aggregation was observed (within ~ 15 min), and then the solution was allowed to settle and cool to room temperature. Aggregation indicated that GNRs were successfully coated by MUDA, which are insoluble in toluene. The aggregates were washed twice with 1 mL of toluene via decantation and then once with 1 mL of isopropanol to deprotonate the carboxylic acid. The aggregates spontaneously re-dispersed in 1 × tris-borateEDTA (TBE) buffer and were no longer soluble in toluene, suggesting that residual DDT on the NR is minimal. Once resuspended in TBE, GNR-MUDA could have their MUDA coating be further ligand-exchanged with HS-DSPEI-PEG-RGD conjugates. We incubated GNR-MUDA with 1 mM aqueous solution of HS-DSPEIPEG-RGD with stirring at room temperature under nitrogen for 12 h. Afterwards, the resulting GNRs were separated from the reaction solution via centrifugation at 10 000 rpm for 10 min, and washed with deionized water twice to remove any residual reactants. The precipitates were re-dispersed in 1 mL of ultrapure water. The HS-DSPEI or HS-DSPEI-PEG-RGD functionalized GNRs (GNRDSPEI, GNR-DSPEI-PEG-RGD) were characterized by UV–Vis–NIR spectrophotometer (Hitachi U-2910, Japan), Zetasizer NanoZS/ZEN3600 (Malvern Instruments, Herrenberg, Germany), 1H-NMR (Varian Mercury Plus-400 NMR spectrometer, Varian, USA), transmission electron microscope (TEM) (JEM-2100F, JEOL, Japan) and thermogravimetric analyzer (TGA) (Pyris 1 TGA, PerkinElmer, USA).

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RGD-NH2 peptide and 1 mmol NHS-PEG-MAL were dissolved in 5 mL anhydrous DMSO containing 60 μL of TEA. After 3 h of incubation at room temperature, cold anhydrous ether was added into the reaction solution, so that both RGD-PEG-MAL conjugate and free peptide were precipitated out as white power. After drying precipitate under vacuum, it was then dissolved in pH 9.0 sodium carbonate buffer and filtered through the 0.22 μm syringe filter in order to remove unconjugated free peptide. In the second reaction step, 2 mmol excess of RGD-PEGMAL conjugates was mixed with 1 mmol HS-DSPEI in 10 mL anhydrous DMSO and incubated at room temperature overnight. The final product, HS-DSPEI-PEG-RGD conjugates, were separated by dialysis and lyophilized. The composition of HS-DSPEI-PEG-RGD conjugates was monitored by 1H-NMR.

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To prepare the growth solution, 0.9 g of CTAB and 0.11 g 5-BrSA were dissolved in 25 mL of warm water (60 °C) in a 50 mL Erlenmeyer flask. The solution was allowed to cool to 30 °C, when 1.8 mL of 4 mM AgNO3 solution was added. The mixture was kept undisturbed at 30 °C for 15 min, after which 25 mL of 1 mM HAuCl4 solution was added. After 15 min of slow stirring (400 rpm), 200 μL of 0.064 M L-AA was added, and the solution was vigorously stirred for 60 s until it became colorless. To generate nanorods, 40 μL of the seed solution was added to the growth solution at 30 °C. The resultant mixture was stirred (1200 rpm) for 60 s and left undisturbed at 30 °C for 12 h for GNRs growth. The resulting GNRs were separated from the reaction solution via centrifugation at 11 000 rpm for 15 min, and washed with deionized water twice to remove any residual reactants. The precipitates were re-dispersed in 2 mL of ultrapure water. The morphology and size of the GNRs were measured using transmission electron microscopy (TEM) (JEM-2100F, JEOL, Japan). Absorbance measurements were carried out using a Hitachi U-2910 UV–Vis–NIR spectrophotometer.

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2.7. Preparation and characterization of DNA and GNRs-based nanocarrier 315 complexes 316 The GNR-DSPEI-PEG-RGD/DNA complexes were prepared at seven different mass ratios ranging from 1 to 15 between the GNRs nanocarriers and the pGFP. Briefly, various amounts of GNR-DSPEI-PEG-RGD in water were added into the same volume of plasmid DNA (50 μg/mL in water). The mixture was then vortexed for 30 s and incubated at room temperature for 30 min. The DNA binding ability of the GNRs nanocarriers was confirmed by agarose gel electrophoresis. The GNRs based complexes containing


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2.8.2. NIR laser triggered release of DNA The GNR-DSPEI-PEG-RGD/YOYO-1-labeled DNA complexes prepared at a mass ratio of 7.5 were added in 48-well plates. The samples were irradiated with NIR laser light (LE-LS-808-2000 T-FCA, LEO Photonics, China) at 808 nm with 5 mm diameter spot-size at 3 W/cm2 for 30 to 300 s, and the solution temperature was detected with a digital thermometer (HH508, Omega, Switzerland). After laser exposure, the amount of the released fluorescently labeled DNA was quantified by taking aliquots out of the sample. Each aliquot was centrifuged immediately to separate the GNRs from the released DNA in the supernatant and the fluorescence intensity of the supernatant was measured to determine the amount of DNA released from GNRs.

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The human glioblastoma U-87 MG cell line with overexpression of αvβ3 was selected as the test cell in this assay. U-87 MG cells were seeded onto 20 mm glass coverslips in a 12-well tissue culture plate at a density of 2 × 105 cells per well. The cells were incubated with GNRs-based complexes in the culture medium for 2 h to allow cellular uptake. For comparison, GNR-DSPEI/DNA complex nanoparticles were performed as a positive control. In the free RGD peptides competition

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2.8.3. In vitro dual-stimuli triggered DNA release The GNR-DSPEI-PEG-RGD/YOYO-1-labeled DNA complexes prepared at a mass ratio of 7.5 were irradiated with 808 nm NIR laser light at 3 W/cm2 for 120 s, and then the samples were treated with GSH at a concentration of 3 mM at 37 °C for 30 min. Afterwards, the release of the fluorescently labeled DNA was quantified as mentioned above.

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2.10. Co-localization by confocal laser scanning microscope (CLSM)

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U-87 MG cells were seeded at a density of 1 × 105 cells/well in a 12well plate over glass coverslips and allowed to attach for 24 h. Cells were incubated with FITC labeled GNR-DSPEI-PEG-RGD at a GNRs concentration of 25 μg/mL at 37 °C for 2 h to allow cellular uptake. Then, the medium was aspirated, the cells were washed twice with PBS and a fresh medium was added. Cells were immediately irradiated by the 808 nm laser at a power density of 3 W/cm2 for 120 s. Afterwards, cells were treated with Hoechst 33342 (6 μg/mL) and Lyso Tracker Red DND-99 (10 nmol/mL), and incubated at 37 °C for an additional 20 min [56]. The extracellular fluorescence was quenched with 500 μL of 0.4% trypan blue for 2 min, followed by washing with PBS the cells on coverslips were fixed with 500 μL of 4% paraformaldehyde for 30 min, then the fixed cells were mounted in anti-fluorescence quenching mounting medium and sealed with slides. Cells were visualized under a confocal laser scanning microscope (CLSM) (TCS SP5, Leica, Germany) to observe the intracellular distribution of the FITC labeled GNRs.

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U-87 MG cells were incubated with the GNR-DSPEI-PEG-RGD/DNA complexes (prepared at a mass ratio of 7.5) in an identical manner to GNRs-based complexes uptake studies. The DNA is fluorescently labeled with YOYO-1. After 4 h of incubation, the cell culture medium was aspirated, the cells were washed with PBS, trypsinized, and resuspended in cell culture medium. Half of each cell suspension was scheduled to undergo a 808 nm NIR laser treatment at 3 W/cm2 for 60 s or 120 s and the other half did not undergo laser irradiation. The cells were then plated back onto a fresh 24 well plate. After incubation at 37 °C for an additional 0.5 or 2.5 h, the medium was removed, and cells were washed three times with PBS and visualized under a fluorescence microscope (Olympus IX51, Olympus, Japan) to observe the internalization of the complexes. For quantitative assay, cells were trypsinized and resuspended in the medium. Subsequently, the cells were washed three times with PBS and fluorescence intensity was analyzed and quantified using flow cytometry (BD LSRFortessa, Becton Dickinson, USA). The data shown are the mean fluorescent signals for 10,000 cells. Samples were prepared in duplicate and experiments were performed a minimum of 3 times with results representing mean ± SD.

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2.8.1. GSH-response property of GNR-DSPEI-PEG-RGD/DNA The GSH triggered reductive degradation of GNR-DSPEI-PEG-RGD based complexes was also evaluated by agarose gel electrophoresis. GNR-DSPEI-PEG-RGD/DNA complexes at different mass ratios were prepared as described above. After incubation for 30 min, GSH was added directly into the nanoparticle solution to reach a final concentration of 3 mM, which was comparable to the intracellular environment [55]. The nanoparticle solution was incubated at 37 °C for 30 min before agarose gel electrophoresis. For quantification of GSH triggered gene release from GNR-DSPEIPEG-RGD/DNA complexes, DNA was fluorescently labeled with a nucleic acid stain YOYO-1 (excitation wavelength: 491 nm, emission wavelengths: 509 nm) by mixing DNA and YOYO-1 with a ratio of 1.5 nanomole dye molecule/100 μg DNA and incubated for 30 min at room temperature in the dark. The GNR-DSPEI-PEG-RGD/YOYO-1-labeled DNA complexes were prepared as described above at a mass ratio of 7.5, and treated with GSH at a concentration of 3 mM at 37 °C for 30 min. Afterwards, the samples were immediately centrifuged at 11 000 rpm for 15 min to remove the GNRs and collect the supernatants. A fluorescence spectrophotometer (F-7000, Hitachi, Japan) was used to quantify the released DNA in supernatants. We quantified the fluorescence intensity of YOYO-1 due to YOYO-1-labeled DNA released from GNRs-based complexes.

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study, 1 μM RGD peptides was added to the incubation medium. After incubation, the cells were washed with PBS solution and fixed with 4% paraformaldehyde solution in PBS for 30 min, then the fixed cells were washed with PBS and sealed with slide. The light scattering images were recorded by using a dark-field microscope (Olympus IX71, Olympus, Japan) with an EMCCD camera (Cascade 128+, Roper Scientific, Inc.). For quantification of GNRs uptaken in cells, ICP–MS measurements were performed. After a 2 h incubation, the medium was aspirated, the cells were washed three times with cold PBS, treated with 0.2 mL of trypsin solution (containing 0.25% EDTA), and counted using flow cytometry. Then the cell pellets were sorted into a 20-mL silicon glass vial and 500 μL of aqua regia (Aqua regia is made of 3:1 of Hydrochloric acid :Nitric Acid) was added to each glass vial to completely digest the cells and dissolve the GNRs. After the aqua regia was boiled off, the content in each vial was then resuspended in 5 mL of 1% Aqua regia and filtered with 0.22 μm filters (Millipore, USA). Gold content was measured with ICP–MS (7500A, Agilent, USA), then converted to GNRs. The amount of GNRs was finally normalized to the cell number (see Materials and methods, Supporting Information).

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0.5 μg of DNA at various mass ratios were loaded on 1% agarose gel in TAE buffer (40 mM Tris–HCl, 1 v/v% acetic acid, and 1 mM EDTA) containing 0.5 μg/ mL EB and subjected to electrophoresis with a current of 100 V for 50 min. The resulting DNA bands were visualized with a UV (254 nm) illuminator and photographed with a Vilber Lourmat imaging system. The zeta-potential of GNRs-based complexes was measured with Zetasizer NanoZS/ZEN3600 (Malvern Instruments, Herrenberg, Germany). The morphology and size of complexes were observed using a transmission electron microscope (TEM) (JEM-2100F, JEOL, Japan).

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The GNR-DSPEI-PEG-RGD, GNR-DSPEI and PEI-25 kDa delivery vectors were incubated with cells in an identical manner as the in vitro GFP transfection assay. After the laser treatment, the cells were plated back onto a fresh 48-well plate and allowed to grow for 48 h, then, the MTT assay was performed. Briefly, 20 μl of 5 mg/ml MTT dissolved in PBS was added to each well. The cells were incubated for an additional 4 h at 37 °C and then the medium was discarded. Thereafter, 250 μl of DMSO was added to each well to dissolve the formazan crystals. The absorbance was read on a microplate reader (Bio-Rad 680, USA) at a test wavelength of 570 nm and reference wavelength of 630 nm. The percentage of cell growth inhibition was calculated as follows: inhibitory rate = (A570control − A570sample) / A570control × 100%.

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2.14. Statistical analysis

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The mean ± SD was determined for each treatment group. Student's t-test (two-tailed) was applied to test the significance of the difference between two groups. Differences were considered significant for p b 0.05.

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3. Results and discussion

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3.1. Synthesis and characterization of the DSPEI-PEG-RGD functionalized GNRs

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GNRs were synthesized by using the seed-mediated growth method using CTAB as soft template [51,52]. The surface plasmon absorptions of GNRs have two bands: a weak short-wavelength band appears at around 520 nm due to transverse electronic oscillations, and the other strong longitudinal plasmon band, which can be tuned from the visible to NIR region depending on the nanorod's aspect ratio (length/width) [57]. In our work, nanorods with an aspect ratio of 4.1 were chosen due to their absorption overlapping with a region of minimum extinction of the human tissues. The absorption band of the nanorods also peaks around the NIR laser wavelength at 808 nm used in our laboratory (Fig. 1A). A TEM image of the CTAB-coated GNRs showed that the average length and width were about 50 and 12 nm, respectively (Fig. 1B). The zeta potential was determined to be +34.2 mV due to the existence of the cationic surfactants (CTABs) used for capping and stabilizing the GNRs during synthesis. In order to modify the CTAB-coated GNRs with DSPEI-PEG-RGD coatings, a partially thiolated DSPEI-PEG-RGD conjugate was prepared (Fig. S1). Fig. S2 shows a comparison of the FT-IR spectra between 4000 and 500 cm−1 of the PEI and DSPEI. The absorption bands of PEI appeared at 1635 cm−1, assigned to the N–H bending vibration of the amine group. For the DSPEI two new absorption bands at 1656 and 1557 cm−1 appeared and were ascribed to the amides I and II band stretching vibrations. These results are evidence that the PEI was cross-linked with 3,3′-dithiodipropionic acid by amide linkages. The 1 H-NMR spectra of the DSPEI and thiol-terminated DSPEI (DSPEI-SH)

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are presented in Fig. S3. The peak at 7.9 ppm is from the proton of SH [58]. All of these FT-IR and 1H-NMR spectra changes indicate the formation of thiol-terminated DSPEI. The molecular weight and polydispersity of DSPEI measured by GPC were 22.5 kDa with Mw/Mn 1.38. Then DSPEI was incubated in PBS (pH 7.4) with or without the reduction agent GSH at 37 °C, to determine the profiles of the hydrolytic and reductive degradation. The result showed that incubation in physiological environment, the Mw of DSPEI decreased extremely slowly, with only 5% decrease after 5 days. On the contrary, when the DSPEI was incubated with GSH, its Mw decreased very rapidly after 15 min, which revealed that DSPEI was sensitive under the reductive circumstance, but relatively stable in physiological condition (data not shown). RGD peptide was conjugated to HS-DSPEI by using a hetrobifunctional PEG, NHS-PEG-MAL, cross-linker as described previously [53]. In the first step, the NHS ester group of heterobifunctional PEG reacted with the peptide amino terminal primary amine preactivated by the excess of TEA in anhydrous DMSO. Then, the conjugated RGDPEG-MAL was mixed with DSPEI-SH in anhydrous DMSO. As shown in Fig. S4A and B, the characteristic peaks at 6.71 ppm and 6.98 ppm were assigned to the phenyl protons of the RGD unit, which confirmed the successful synthesis of the HS-DSPEI-PEG-RGD conjugates. The conjugation ratios of HS-DSPEI-PEG-RGD conjugates, expressed as a molar ratio of RGD to DSPEI, were determined by 1H-NMR spectrum analysis. The molar ratio of RGD to DSPEI of conjugates was about 1.2 (HS-DSPEI-1.2PEG-RGD), which may exhibit as a specific and efficient gene delivery carrier [53]. GNRs are capped with CTAB surfactants, which form a bilayer structure around the gold surface [59]. The surfactants are cytotoxic and therefore must be removed before biomedical applications. In order to tune the biological properties of the GNRs, exchange must permit ligand customization. Here, we utilized an approach that via “round-trip” phase transferring to remove CTAB molecules on the GNR surface by ligand exchange with 11-mercaptoundecanoic acid (MUDA). After that, the resulting GNR-MUDA was conjugated to functionalized HSDSPEI-PEG-RGD as described in Fig. 2. The 1H-NMR spectra confirmed that DSPEI-PEG-RGD was attached on the GNR surface according to the customization (Fig. S4C). The uniform appearance and ppm values with Fig. S4A and B demonstrated that these structures were a single GNR-DSPEI-PEG-RGD. UV–vis spectra of ligand-exchanged GNRs with DSPEI-PEG-RGD (Fig. 1A) showed no significant broadening of the longitudinal surface plasmon resonance (LSPR), indicating no aggregation. Meanwhile, there was a 5 nm red shift (from 803 to 808 nm) of LSPR relative to the GNR-CTAB peak, which was due to the increased refractive index of the polymer coating [60]. Here, we purposely adjusted the LSPR wavelength of the GNR carrier to match that of the laser wavelength (808 nm) for obtaining maximal photothermal conversion efficiency. Furthermore, we found that the ligand-exchanged GNRs were stable even after 6 months of storage at high concentration (3 × 10− 8 M), and the plasmon peaks exhibited no significant changes in peak width or position, indicating a good long-term stability. We also used thermal gravimetric analysis (TGA) to calculate the content (%) of the CTABmodified GNRs and partially thiolated DSPEI-PEG-RGD-modified GNRs. The CTAB-GNRs had 6% weight loss, while GNR-DSPEI-PEG-RGD had a 22% weight loss (Fig. 1C), suggesting that the high molecule DSPEIPEG-RGD has displaced the small molecule CTAB on the GNR surface, and our ligand exchange process was successful. The DSPEI-PEG-RGD modified GNRs were imaged after negative staining with phosphotungstic acid by TEM (Fig. 1D). The images revealed that the GNRs were coated with a DSPEI-PEG-RGD conjugates layer without shape change. The averaged aspect ratio of the GNR-DSPEI-PEG-RGD was calculated to be 4.1, consistent with that of free GNR prior to DSPEI-PEG-RGD modification. Zeta potential measurements further confirmed the successful DSPEI-PEG-RGD attachment (Table 1), where both GNR-DSPEI (50.9 ± 3.2 mV) and GNR-DSPEI-PEG-RGD (48.7 ± 2.8 mV) displayed higher zeta-potentials than the parent GNR-CTAB, indicating they could have a good gene loading capacity. Therefore, we can safely conclude that

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37 °C for 4 h. After incubation the cell culture medium was aspirated, the cells were washed with PBS, trypsinized, and resupsended in cell culture medium. Half of each cell suspension was irradiated with an 808 nm laser at a power density of 3 W/cm2 for 120 s and the other half did not undergo laser irradiation. The cells were then plated back onto a fresh 24-well plate. After 24 to 48 h of incubation, the expression of green fluorescence protein (GFP) in cells was observed under a fluorescent microscope (Olympus IX51, Olympus, Japan). The transfection efficiency of the complexes GFP-positive cells was quantified by flow cytometry (BD LSRFortessa, Becton Dickinson, USA). For comparison, GNR-DSPEI/DNA and PEI-25 kDa/DNA complexes with N/P ratio of 10 were performed as a positive controls. For each formulation, the cells were transfected in triplicates.

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Fig. 1. Characterization of GNR-CTAB and GNR-DSPEI-PEG-RGD. (A) UV–Vis–NIR absorbance spectra of GNR-CTAB, GNR-DSPEI and GNR-DSPEI-PEG-RGD. (B) TEM image of GNR-CTAB. (C) TGA curves showing difference in weight loss between GNR-DSPEI-PEG-RGD and GNR-CTAB. (D)TEM image of GNR-DSPEI-PEG-RGD.

CTAB molecules on the surface of GNR have been successfully displaced by DSPEI-PEG-RGD.

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3.2. Characterization of GNR-DSPEI-PEG-RGD-based gene complexes

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GNRs-based DNA complexes were prepared by adding the GNRDSPEI-PEG-RGD solution to the DNA solution in equal volumes. The DNA concentration was kept constant. The concentration of GNRDSPEI-PEG-RGD was adjusted according to the desired GNR/DNA mass ratios from 1 to 15.

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To be effective gene vectors, cationic polymers must be able to bind and condense plasmid DNA into nanoparticles, which can then be taken up by cells. The DNA binding and condensation capability of the synthesized GNR-DSPEI-PEG-RGD were investigated by gel retardation assay and zeta potential measurements. As shown in Fig. 3A, the GNRDSPEI-PEG-RGD conjugates can bind plasmid DNA efficiently and completely retard DNA migration at a mass ratios above 7.5, indicating that the GNR-DSPEI-PEG-RGD exhibited strong ability to condense plasmid DNA. Zeta potentials of the complexes under different mass ratios were measured and shown in Fig. 3B. At mass ratios above 5, all complexes showed positive zeta potentials with positive surface charge,

Fig. 2. Scheme for GNRs ligand exchange and its DSPEI-PEG-RGD functionalization.


578 579 580 581 582 583 584 585 586 587 588


t1:3

Samples

Zeta potential (mV)

t1:4 t1:5 t1:6 t1:7

GNR-CTAB GNR-MUDA GNR-DSPEI GNR-DSPEI-PEG-RGD

34.2 −40.5 50.9 48.7

602 603

Here, we were focused on extracellular stimuli such as GSH and NIR irradiation responsive release of loaded DNA molecules. In our previous experiment, GPC traces of DSPEI in the absence and presence of GSH clearly showed that the disulfide cross-linked DSPEI were responsive to the reducing agent GSH. After incubation with GSH for 1 h at 37 °C, the average molecular weight of DSPEI decreased from 22.5 kDa to 2.1 kDa, which is very close to Mw of PEI only (1.8 kDa). To assess the release of DNA from GNR-DSPEI-PEG-RGD/DNA complexes, the complexes were incubated in PBS (pH 7.4) in the presence of 3 mM GSH, which represented the simulated intracellular redox environment. The reduction triggered release of DNA from disulfide-containing GNRbased complexes was visualized by gel electrophoresis (Fig. 4A). Compared with Fig. 3A, complexes incubated in the extracellular physiological condition, the fluorescence bands showed the presence of free DNA in GNR-DSPEI-PEG-RGD/DNA complexes after incubation with 3 mM GSH (30 min), indicating that DNA was rapidly released due to the cleavage of disulfide bonds under the reductive conditions, producing much smaller PEIs that can no longer condense the DNA. This result indicates that the GNR-DNA complexes would degrade after being internalized in the reductive intracellular environment and release their cargo efficiently intracellularly. Furthermore, the released free DNA can be quantitatively detected after YOYO-1-labeling by fluorescence spectrophotometer. With decreasing the mass ratio from 15 to 5, the fluorescence intensity was rapidly increased. Light-triggered release of DNA was investigated using an 808 nm NIR laser at 3 W/cm2 for 30 to 300 s treatment. The GNR-DSPEI-PEGRGD/DNA complexes after exposure to laser irradiation were immediately centrifuged, and released YOYO-1-labeled DNA in the supernatant was quantified by fluorescence spectroscopy. After irradiation, the supernatant fluorescence intensity at 509 nm increased, illustrating DNA release. It is obvious that the amount of DNA released was positively correlated with the increase of irradiation time (Fig. 4B blue line). Meanwhile, upon laser irradiation, the temperature of the solution was also increased due to photothermal heating afforded by the irradiated GNRs (Fig. 4B pink line). To confirm whether the DNA release via a thermally induced mechanism, the solution was slowly heated in a temperature controlled water bath (from 20 to 80 °C). After different incubation times, there was no obvious enhancement of fluorescence signal detected by fluorescence spectroscopy. Therefore, we conclude that a nonthermal mechanism but not a thermal mechanism may be responsible for light-triggered DNA release from GNR-DSPEI-PEG-RGD/DNA complexes. This nonthermal mechanism would involve a process

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3.3. In vitro glutathione and NIR laser triggered DNA release

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suggesting that they were able to condense plasmid DNA into complexes. The zeta potential of the GNR-DSPEI-PEG-RGD/DNA complexes showed a strong dependence on the mass ratios and decreased rapidly with the decreasing mass ratio. At a mass ratio = 7.5, the complex had a positive zeta potential of around + 25 mV. The size and shape of the complexes were revealed by TEM imaging after negative staining (Fig. 3C). At pH value of 7.4 and mass ratio of 7.5, the DSPEI and DNA formed compact complexes about 2 nm in thickness which were closely packed on the GNR surface due to strong electrostatic interactions between the positively charged polymer and negatively charged DNA. Nevertheless, the morphology and size of the GNR itself did not change during the complexes preparation process.

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Table 1 Zeta potentials of GNRs with different surface ligands.

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Fig. 3. Characterization of GNR-DSPEI-PEG-RGD/DNA complexes. (A) Agarose gel electrophoresis photos of GNR-DSPEI-PEG-RGD/DNA complexes at different mass ratios. (B) Zeta potentials of the complexes at various mass ratios. Error bars represent standard deviations (n = 4). (C) TEM images of GNR-DSPEI-PEG-RGD/DNA complexes at a mass ratio of 7.5.

related to the excitation of the GNRs surface plasmon. When DSPEIPEG-RGD “carrier” molecule is covalently attached to the gold surface, upon laser illumination, the excited “hot” electrons produced by plasmon decay can transfer to the adsorbate [22,34,35], which would decrease the electrostatic attraction between the DSPEI and DNA, resulting in DNA release [25,26]. Since the creation of non-equilibrium hot electrons is a direct result of plasmon excitation of the GNRs, therefore, the light-induced release begins immediately, making this method suitable for controlled delivery of therapeutic molecules [25]. It is worth


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attached to GNRs, under identical laser treatment conditions, the release of DSPEI-PEG-RGD was not detected, which demonstrated that the Au\S bond is not broken as a result of the laser treatments. For light-induced treatment, the localized heating around each GNRs results in a non-uniform thermal profile and the solution temperature does not reflect the temperature at the illuminated GNRs surface [25,26]. Perhaps, a higher temperature could be used to simulate the real conditions. However, a significant number of DNA molecules were released below 37 °C under light irradiation. Achieving DNA release at or near physiological temperature 37 °C is particularly important for avoiding hyperthermic cell death [61,62]. Therefore, we use a laser irradiation condition of 3 W/cm2 for 120 s (solution temperature 36.8 ± 1.1 °C) for the following experiment. And yet, the DNA release amount may vary over a range of experimental conditions such as laser intensity, irradiation time, GNRs concentration, DSPEI length and DNA loading capacity. Fig. 4C showed DNA release percentage from the GNR-based complexes, where a fixed concentration and mass ratio (7.5) of GNR-DSPEIPEG-RGD/DNA complexes were treated with NIR irradiation or/and 3 mM GSH. It was observed that both GSH and NIR irradiation triggered DNA release. The amounts of DNA release upon applying NIR irradiation and GSH treatment only were 15.3 ± 1.6% and 30.6 ± 2.5%, respectively, this was increased to 45.7 ± 1.8% upon application of dual-stimuli to the GNR-DSPEI-PEG-RGD/DNA complexes solution. Thus, we expected that DNA release inside cells could be induced by a combination of stimuli of GSH and NIR irradiation. The GNR-DSPEI-PEG-RGD/DNA complexes prepared at a mass ratio of 7.5 not only exhibit preferred DNA binding and condensation capability, but also favorable DNA release upon dual-stimulation. Therefore, a mass ratio of 7.5 was chosen for the following assays.

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3.4. Cell targeting of RGD modified GNRs

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Flow cytometry (FCM) analysis indicated that the glioblastoma U-87 MG cells exhibited over-expression of αvβ3 integrins. To evaluate the targeting ability of RGD-modified GNRs against αvβ3 integrins on the cells, the binding affinities of GNR-DSPEI-PEG-RGD/DNA and plain GNR-DSPEI/DNA in U-87 MG cells were investigated using dark-field microscopy. As shown in Fig. 5A, U-87 MG cells incubated with RGDmodified GNRs exhibited a strong golden color, while the cells treated with GNR-DSPEI/DNA exhibited a much weaker color, which suggests that GNR-DSPEI-PEG-RGD/DNA complex can specifically target U-87 MG cells. The role of cell surface αvβ3 integrins in GNR uptake was further evaluated by incubating U-87 MG cells with the GNR-DSPEIPEG-RGD/DNA complex in the presence of 1 mM free RGD peptides. The observations showed that the cellular uptake of RGD-modified GNRs was significantly reduced (Fig. 5A), suggesting that free RGD peptides can inhibit the binding of RGD-modified GNRs with αvβ3 integrins over-expressed tumor cells, leading to lower cellular uptake. These results indicated that enhanced cellular uptake of RGD-modified GNRs might be mainly based on the effective αvβ3 integrin-mediated endocytosis. Since the dark-field microscopy cannot definitively determine whether GNRs are endocytosed or remain outside the cell but associate with the cell membrane, inductively coupled plasma–mass spectrometry (ICP–MS) was used to confirm and quantify GNR cellular uptake. As shown in Fig. 5B, for GNR-DSPEI/DNA, GNR-DSPEI-PEG-RGD/DNA and GNR-DSPEI-PEG-RGD/DNA plus 1 mM free RGD treated cells we detected 12.1%, 48.2% and 3.1% of the GNRs were internalized by the U-87 MG cells, respectively. Furthermore, the number of GNR per cell chart shows a significant difference between the GNR-DSPEI-PEG-RGD/DNA treated cells and the other two groups (Fig. 5C). The U-87 MG cell uptake of the GNR-DSPEI-PEG-RGD/DNA complex was approximately five-fold higher than that of plain complex. This result demonstrated that the RGDs on the GNR-DSPEI-PEG-RGD/DNA complex were readily accessible to αvβ3 integrin binding, and hence the GNR-DSPEI-PEG-RGD/DNA complexes can be used as efficient, targeted intracellular gene carriers.

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Fig. 4. Stimuli-responsive release of DNA from GNR-DSPEI-PEG-RGD/DNA complexes. (A) Gel retardation analyses of GNRs-DSPEI-PEG-RGD/DNA complexes at various mass ratios in the presence of 3 mM GSH. (B) Amount of DNA released from GNR-DSPEI-PEGRGD/DNA complexes under NIR irradiation (808 nm, 3 W/cm2) for various lengths of time. (C) NIR laser and glutathione-triggered release of DNA from GNR-DSPEI-PEGRGD/DNA complexes. Error bars represent standard deviations (n = 3).

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noting that we observed no shape transformation or melting of GNRs during the laser irradiation (determined by UV–Vis–NIR spectrophotometer and TEM). Furthermore, a FITC labeled DSPEI-PEG-RGD was


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Fig. 5. Cellular uptake of GNRs with different surface modifications/DNA complexes in U-87 MG cells. (A) Bright-field and dark-field images of GNR-DSPEI/DNA, GNR-DSPEI-PEG-RGD/DNA complexes and GNR-DSPEI-PEG-RGD/DNA treated with 1 mM free RGD after incubation with U-87 MG cells for 2 h. (B) The GNRs uptake percents determined by ICP–MS. (C) Quantification of the mean GNRs number per cell. Data are presented as mean ± SD (n = 3). ***P b 0.001.

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3.5. Photochemical-triggered endosome escape

To explore the potential of GNR-DSPEI-PEG-RGD as a NIR laser 722 controlled gene carrier, we investigated the intracellular distribution 723 Q10 of GNRs after cellular uptake. To trace the intracellular distribution, 724 GNR-DSPEI-PEG-RGD was labeled with FITC, Hoechst 33342 and Lyso

Tracker Red DND-99 which were used for staining the nuclei and lysosomes, respectively. As shown in the CLSM images (Fig. 6), without NIR irradiation, almost all of the FITC labeled GNR-DSPEI-PEG-RGDs (green) were localized in endo/lysosomal compartments, producing yellow fluorescence spots in the merged images with Lyso tracker red fluorescence. This indicates that the cellular uptake of carrier is by


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3.6. Stimuli-responsive DNA release inside cells

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Promoting the effective DNA release from the complexes in the cytoplasm is critical for a greater gene transfection efficiency. It is well known that PEI can protect the plasmid DNA from lysosomal nuclease degradation and facilitate endosomal escape to the cytoplasm via its proton sponge effect [66,67]. The above results show that the GNRDSPEI-PEG-RGD/DNA complexes can be efficiently taken up by target cells and successfully escape from endosome following NIR laser

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induced endosomal disruption of GNRs as well as the proton sponge effect of PEI, making them highly attractive for efficient gene delivery. To study the ability of the GNR-DSPEI-PEG-RGD carrier to release DNA in vitro, fluorescence microscopy and flow cytometry were used to determine the release of YOYO-1 labeled DNA from GNR-DSPEI-PEGRGD within the U-87 MG cells. Briefly, the cells were incubated with GNR-DSPEI-PEG-RGD/DNA complexes for 4 h, after which any excess free complexes were completely removed. Then, half of the cells were used as control and the other half was irradiated with an 808 nm NIR laser at 3 W/cm2 for 60 or 120 s, followed by incubation at 37 °C for an additional 0.5 or 2.5 h. As shown in Fig. 7A, without laser treatment the green fluorescence is very weak, probably because the YOYO-1 fluorescence is quenched by the GNR [68,69]. Upon NIR laser irradiation, the light-induced transfer of “hot” electrons caused a decrease of the electrostatic adsorption between GNR-DSPEI-PEG-RGD and DNA, leading to desorption of DNA from the carrier and diffusion into the cytosol of the cells. Quantitative DNA release, reflected by the fluorescence of the whole cell, was estimated by flow cytometric analysis, which further confirmed the result observed by fluorescent microscope (Fig. 7B and C). Noticeably, the fluorescence intensity of the cells without NIR irradiation was also increased with the increasing incubation time, which may be attributed to the small number of DNAs liberated from carriers during the incubation. Following 60 or 120 s of NIR irradiation, the fluorescence signal of YOYO-1 was greatly enhanced. The mean fluorescent intensity data showed a significant difference between samples in laser off control groups and those with laser irradiation groups, indicating a substantial increase of the released DNAs into the cells after the NIR laser irradiation. Moreover, the cellular fluorescent intensity of YOYO1 was enhanced with the increasing irradiation time, indicating that a longer irradiation time caused further increase of DNA release into cells. Afterwards, we studied the time-dependent release of DNA at different incubation times (0.5 and 2.5 h). As shown in Fig. 7A and B, the intracellular fluorescence of YOYO-1 was enhanced as the samples incubation time was increased from 0.5 h to 2.5 h. As expected, cells with 2.5 h further incubation time had significantly stronger mean fluorescent intensity than those with 0.5 h. Particularly, the mean fluorescent intensity of the 120 s laser treated cells increased from 5211 to 9057 as the incubation time was increased from 0.5 h to 2.5 h. These results could attribute to the GSH triggered degradation of disulfide-containing

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endocytosis and the carriers are mostly entrapped within the endo-/ lysosomes. In contrast, after NIR irradiation (3 W/cm2 for 120 s) upon the FITC labeled GNR-DSPEI-PEG-RGD treated cells, the green fluorescence from FITC labeled GNRs was observed as more diffused spots in the cytosol, which is clearly distinguishable from the red fluorescence emanating from LysoTracker, implying successful endosomal escape of GNR-DSPEI-PEG-RGD, while only a few green fluorescent spots were found outside the endo/lysosomes over the same period in the laser off control group. The development of cooperative systems to rupture endosome via NIR laser irradiation has attracted tremendous interest recently [63–65]. Volk and co-workers have showed NIR laser-activated gold nanoparticles leading to significant endosome rupture and escape into the cytosol, and concluded that by controlling both laser power and exposure time (b 20 W/cm2 for 2 min), localized damage could be inflicted on the endosomal membrane without affecting the cell viability [28]. They have suggested one potential mechanism for the present results which may be the involvement of photochemical effects, which is, the production of free radicals by laser irradiation of gold nanoparticles, possibly as a consequence of plasmon-assisted photoemission of electrons. Some researchers also considered that the low laser intensity irradiation resulted in minimal temperature increase, suggesting the possibility of a nonthermal, photochemical mechanism that leads to endosomal membrane disruption [25–27]. Like the laser conditions used in our study (3 W/cm2 for 120 s), the photochemical effects of the GNRs could result in the GNR-DSPEI-PEG-RGD to successfully escape from the endosome before lysosomal degradation, which would induce high gene transfection efficacy by subsequent GSH-mediated DNA release in the cytosol.

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Fig. 6. Confocal laser scanning microscope images showing the intracellular distribution of GNR-DSPEI-PEG-RGD/DNA complexes in U-87 MG cells treated with or without NIR irradiation. (a) Nuclei blue-stained with DAPI; (b) green FITC labeled GNR-DSPEI-PEG-RGD; (c) Lyso-tracker red-stained lysosome; and (d) merged images of (a), (b) and (c). Yellow dots in (d) indicate the complexes trapped within acidic lysosome. (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. 7. NIR-responsive and time-dependent release of DNA from GNR-DSPEI-PEG-RGD/DNA complexes in U-87 MG cells. (A) Fluorescence microscopic images for U-87 MG cells incubated with GNR-DSPEI-PEG-RGD/DNA complexes treated without (top) or with (bottom) NIR laser irradiation, followed by 0.5 or 2.5 h of incubation. The DNA is labeled with YOYO-1 presenting as green fluorescence. (B) Flow cytometry histogram profile and (C) the mean fluorescent intensity of recovered DNA fluorescence from GNR-DSPEI-PEG-RGD/DNA complexes in U-87 MG cells upon different NIR laser irradiation times and different incubation times. Data are presented as mean ± SD (n = 5). *P b 0.05, **P b 0.01. P b 0.05 is considered significantly different. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

DSPEI and subsequent dissociation of GNR-DSPEI-PEG-RGD/DNA complexes in the reductive intracellular environment, which facilitated DNA release over time. Therefore, a longer incubation time could extend the chances of interaction between the carrier and GSH, resulting in

enhanced DNA release (Fig. 7C). Based on these observations, a schematic model of the intracellular DNA release process is illustrated in Fig. 8. After cellular uptake, the GNR-DSPEI-PEG-RGD/DNA complexes can easily escape from endosomes through photochemical induced endosomal


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3.7. In vitro transfection efficiency

of the above results, GNR-DSPEI-PEG-RGD can deliver DNA remotely and conveniently in a controllable way, and thus it could be considered as a very attractive nanotemplate for the gene delivery system. Additionally, the time-dependent GFP expression of GNR-DSPEIPEG-RGD/DNA complexes in U-87 MG cells treated with NIR laser treatment was studied. As shown in Fig. 9C, the GNR-based complexes had the maximum transfection efficiency after the cells were incubated for 40 h, while, in our study, the corresponding time for the PEI/DNA complexes was 48 h. This is mainly because the light-triggered delivery process is faster than those traditional transfection reagents such as PEI and dendrimers [26]. With a transfection reagent, DNA must slowly escape from the endosome (mainly via the proton sponge effect), dissociate from the transfection reagent, and finally diffuse in the cytosol. For the light-triggered delivery, the GNR complexes can easily escape from endosomes by photochemical induced endosomal disruption and the DNA is released during the short laser irradiation treatment (120 s). Therefore, it is unsurprising that the gene expression occurs faster due to the more efficient and rapid DNA release from the GNR-based complexes in the cytoplasm.

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3.8. Cell viability assay

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In our previous study, the MTT assay against U-87 MG cells was carried out to determine the cytotoxicity of disulfide-containing DSPEI, and PEI-1.8 kDa and PEI-25 kDa were used as controls. The results shown in Fig. S5A indicated that the cell viability decreased with increasing polymer molecular weight and dosage. Under the same conditions, DSPEI showed significantly lower cytotoxicity than that of PEI-25 kDa. The low cytocompatibility of PEI-25 kDa is probably due to its high binding and aggregation on the cell surface and internalization, which results in significant necrosis [70]. Since DSPEI is made of disulfide cross-linked short PEI-1.8 kDa, the disulfide bonds will be cleaved inside cells due to the high concentration of intracellular GSH. As a result, the high molecular weight DSPEI will be converted into low molecular weight PEIs and hence low cytotoxicity. Therefore, the DSPEI polycation with low cytotoxicity could be a much safer alternative gene carrier over the PEI-25 kDa. As shown in Fig. S5B, the cell viability remained above 80%, when the concentration of GNR-DSPEI-PEG-RGD was as high as 25 μg/mL, revealing that the GNR-DSPEI-PEG-RGD did not have significant cytotoxic under such relatively high doses. The cell viabilities of the U-87 MG cells after incubation with the GNR-DSPEI-PEG-RGD vectors with and without subsequent laser treatment were also investigated by MTT assay (Fig. 10). The GNRDSPEI and GNR-DSPEI-PEG-RGD are proven to be less toxic to U-87 MG cells than PEI-25 kDa under the “carrier” concentrations used in

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The transfection activity of GNR-DSPEI-PEG-RGD based complexes 820 was evaluated in U-87 MG cells using the pGFP (expressing green fluo821 rescence protein) as the reporter gene. Branched PEI-25 kDa and GNR822 DSPEI were used as controls. For the transfection experiment, cells 823 were incubated with complexes for 4 h and images were taken after fur824 ther 40 h culture for the GNR-based complexes and 48 h for PEI/DNA 825 complexes. 826 Fig. 9A depicts green fluorescence of U-87 MG cells after gene trans827 fection derived from pGFP qualitatively by fluorescent microscopy 828 images. It could be seen that the cells incubated with PEI/DNA and 829 GNRs-based complexes showed bright intracellular green fluorescence. 830 The strongest fluorescent signal was observed for cells incubated with 831 the GNR-DSPEI-PEG-RGD/DNA complexes and executed with NIR laser 832 treatment. To determine the percentage of cells that actually expressed 833 GFP, we counted the number of GFP-positive cells using flow cytometry 834 (Fig. 9B). For cells without laser treatment, the GNR-DSPEI/DNA com835 plex exhibited significant GFP expression (66.1 ± 3.6% of the cells 836 were GFP positive) due to the buffering effect of DSPEI at endosomal 837 compartments and the disulfide bonds breakage promoted DNA release. 838 Moreover, grafting of αvβ3 integrin-binding RGD peptide into DSPEI 839 via PEG spacer further enhanced the transfection efficiency with 840 82.3 ± 4.1% cells being GFP positive, which was benefited from the 841 more efficient uptake of the complexes by αvβ3 integrin-mediated en842 docytosis. When cells were further treated with NIR laser irradiation, 843 the GFP expression cells for the GNR-DSPEI-PEG-RGD/DNA complexes 844 reached the maximum with 97.1 ± 3.4% of cells being GFP positive. 845 Moreover, the GFP positive cells in GNR-DSPE/DNA complexes group 846 also increased to 84.6 ± 4.5%. Therefore, the laser-treated cells exhibit847 ed an additional 15% and 18% GFP expression, respectively, compared to 848 those without laser treatment. However, there was no significant differ849 ence in GFP positive cell percentiles between the PEI/DNA groups when 850 the cells treated with NIR laser or not. This difference in gene expression 851 indicates that the laser treatment triggered further DNA release from 852 Q11 the GNR-DSPEI-PEG-RGD carrier inside cells since the laser irradiation 853 takes place within the “water window”, such that the cellular environ854 ment should have a minimal effect on the GNRs response to laser irradi855 Q12 ation [30]. Thus, the release of DNA is due to the generation of “hot 856 electrons” on the gold surface, which should not be affected by the 857 Q13 cellular environment significantly [25]. Therefore, with consideration

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disruption and the proton sponge effect of PEI, which was followed by NIR laser irradiation and GSH-triggered release of the DNAs into the cytosol.

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Fig. 8. Illustration of the mechanism of cytosolic DNA release triggered by NIR laser and GSH after NIR laser induced endosomal disruption and the proton sponge effect.


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Fig. 9. In vitro transfection efficiencies of GNR-DSPEI-PEG-RGD/DNA, GNR-DSPEI/DNA and PEI/DNA complexes for U-87 MG cells. (A) Fluorescent photomicrographs of transfection and (B) transfection efficiencies of GNRs-based complexes for U-87 MG cells treated with or without NIR laser irradiation. PEI-25 kDa/DNA complexes with N/P ratio of 10 was set as positive controls. (C) time-dependent GFP expression of GNR-DSPEI-PEG-RGD/DNA complexes in U-87 MG cells treated with NIR laser irradiation. Data displayed as mean ± SD (n = 3). *P b 0.05, **P b 0.01. P b 0.05 is considered significantly different.

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the GFP transfection assay. A small decrease in cell survival in the GNR conjugates samples could possibly be due to the surface DSPEI. The GNR-DSPEI-PEG-RGD exhibited a slightly higher cytotoxicity than the GNR-DSPEI, this may be contribute to the higher cell uptake of GNRDSPEI-PEG-RGD. It is worth noting that, cells treated with the NIR laser had no significant difference in the percentage of cell survival than those without NIR laser treatment, indicating that the NIR laserinduced photothermal effect of GNRs did not affect cell viability significantly. This may come as a surprise considering that GNRs are wellknown for their use in photothermal cancer therapy. However, the GNR concentration, laser power and exposure time used in this study

were significantly lower than those used for photothermal induction of cell death [71–73]. Therefore, we can conclude that under certain circumstances, the GNR-DSPEI-PEG-RGD developed herein could be a safe and efficient gene vector.

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4. Conclusion

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In summary, we have developed an efficient NIR laser-triggered gene delivery system based on intracellular reductive environment cleavable DSPEI functionalized integrin-targeting GNRs. This GNRDSPEI-PEG-RGD can effectively condense gene into stable complex

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Acknowledgment

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This work is supported by the National Natural Science Foundation of China (NSFC, Grant No.81171439), the National Basic Research Program of China (973 Program, Grant 2010CB529902), the National Key Technology R & D Program of the Ministry of Science and Technology (2012BAI18B01) and the European Research Council via a Marie Curie International Incoming Fellowship to S.G. (Grant No: PIIF-GA-2012331281).

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2014.09.026.

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nanoparticles with positive surface charges and exhibit excellent tumor cell-targeting ability. The GNR-based complexes exhibited controlled gene release by dual-stimuli of NIR laser irradiation and intracellular high GSH content in living cells. Importantly, the GNR-DSPEI-PEGRGD/DNA complexes were able to escape from endosomes by photoinduced endosomal disruption of GNRs and the proton sponge effect of DSPEI, resulting in significantly enhanced gene transfection efficiency than those without irradiation. Therefore, the GNR-DSPEI-PEG-RGD can efficiently deliver gene intra-cellularly in a remotely NIR laser controllable way, and thus it is considered to be a very attractive nanotemplate for site-specific gene delivery. This proof of concept of “on demand” release from GNR is potentially a powerful method for improving drug delivery efficiencies, which can be further applied to many other nanomedicine fields.

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Fig. 10. MTT assay for assessing the cell viability of U-87 MG cells incubated with PEI 25 kDa, GNR-DSPEI and GNR-DSPEI-PEG-RGD under the “carrier” concentration used in the GFP transfection assay with or without NIR laser treatment. Data displayed as mean ± SD (n = 5). **P b 0.01.

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Bioreducible guanidinylated polyethylenimine for efficient gene delivery.

Construction of a tumor cell-targeting non-viral gene delivery vector with polyethylenimine modified with RGD sequence-containing peptide.

EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation.

Increased tumor targeted delivery using a multistage liposome system functionalized with RGD, TAT and cleavable PEG.

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Efficient gene delivery to articular cartilage using electroporation.

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Cyclodextrin and polyethylenimine functionalized mesoporous silica nanoparticles for delivery of siRNA cancer therapeutics.

liquid crystalline elastomer composites prepared by sequential thiol-click chemistry.