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Gold Nanorods-Based Gene Carriers with the Capability of Photoacoustic Imaging and Photothermal Therapy Jie Chen, Hong Liang, Lin Lin, Zhaopei Guo, Pingjie Sun, Meiwan Chen, Huayu Tian, MingXiao Deng, and Xuesi Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10166 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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ACS Applied Materials & Interfaces

Gold Nanorods-Based Gene Carriers with the Capability of Photoacoustic Imaging and Photothermal Therapy Jie Chen,†,1 Hong Liang,†,‡,1 Lin Lin,† Zhaopei Guo, †,§ Pingjie Sun,† Meiwan Chen,§ Huayu Tian,*,† Mingxiao Deng,*,‡ and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, China ‡

Department of Chemistry, Northeast Normal University, Changchun 130024, China

§

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical

Sciences, University of Macau, Macao 999078, China

ABSTRACT：：Multifunctional nanoparticles with high gene transfection activity, low cytotoxicity, photoacoustic imaging ability, and photothermal therapeutic properties were prepared by conjugating low-molecular-weight polyethylenimine onto the surfaces of gold nanorods through the formation of stable S–Au bonded conjugates. Results

revealed

that

the

gene

transfection

efficiency

of

the

prepared

polyethylenimine-modified gold nanorods (GNRs-PEI1.8k) was higher and their cytotoxicity was less than those of the commercial reagent PEI25k. GNRs-PEI1.8k could also be potentially used as a photoacoustic and photothermal reagent to evaluate the

pharmacokinetics,

biodistribution,

and

antitumor

effects

of

gene/drug

nanoparticles. Therefore, GNRs-PEI1.8k can be considered a promising candidate for the clinical diagnosis and treatment of tumors.

KEYWORDS: multifunctional nanoparticles, gold nanorods, gene therapy, photoacoustic imaging, photothermal therapy

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INTRODUCTION Gene therapy is an innovative approach to treat several diseases. For instance, this technique can be applied to cure cancer via viral or non-viral carriers; such carriers introduce exogenous therapeutic genes into diseased cells to repair defective genes or inhibit oncogene expression.1-2 However, effective gene therapy is dependent on gene carrier development.3-5 Viral carriers exhibit high transfection efficiency, but safety issues severely limit their further clinical applications. By contrast, non-viral carriers are much safer, but most of them yield lower transfection efficiency than viral carriers do.6 Therefore, the transfection efficiency of non-viral carriers should be increased and their cytotoxicity should be decreased.7 Polyethylenimine (PEI) is an efficient non-viral gene carrier widely used for gene therapy because it exhibits strong electrostatic interactions with negatively charged DNA. However, the transfection efficiency of PEI is largely influenced by molecular weight. The transfection activity and cytotoxicity8 of PEI are enhanced by increasing its molecular weight. For instance, PEI25k exhibits high transfection efficiency and high cytotoxicity, whereas PEI1.8k produces less cytotoxicity and significantly low transfection efficiency.9 Gold-based nanoparticles with biomedical imaging features have been extensively investigated as gene delivery systems.10-14 Among various nanoparticles, gold nanorods (GNRs) possess interesting properties because of their unique shape effects. For example, GNRs yield a strong longitudinal surface plasmon resonance (LSPR) band that enables them to absorb near-infrared (NIR) light effectively.15 Moreover, GNRs produce a stronger and more stable photoacoustic signal than many organic dyes do.15 The bioinertness of gold nanoparticles allows them to become biocompatible and weakly cytotoxic.16 Therefore, GNRs are promising contrast agents for photoacoustic imaging, which is a noninvasive bioimaging technology providing the combined advantages of optical and acoustic imaging.17 NIR laser is usually selected as the light source for photoacoustic imaging because it can deeply penetrate the skin and underlying tissues and it is weakly absorbed by blood.18 GNRs can absorb energy and release heat after tissues are irradiated by NIR laser, which 2


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induces the thermoelastic expansion of nearby tissues and generates mechanical waves at ultrasonic frequencies.19 Ultrasonic waves can then be collected by a wide-band ultrasonic transducer placed around the detected tissue. Photoacoustic signals can be reconstructed through appropriate mathematical methods to obtain ultrasound images or X-ray computed tomography images.20 Furthermore, the NIR-radiation-mediated photothermal effects of GNRs can be used to treat tumors.18 GNRs can be utilized as photothermal agents to kill diseased cells or ablate tumor tissues. NIR light can deeply penetrate tissues without damaging normal tissues because NIR light is weakly absorbed by normal tissues.18 Different photothermal agents, such as gold nanocages, nanoshells, nanorods, graphene, and carbon nanotubes, have been widely used for in vitro and in vivo hyperthermia treatments.21-22 The strong LSPR band of GNRs effectively promotes NIR light absorption and releases large amounts of energy through thermal radiation.15 Therefore, the synergetic therapeutic effect against cancer can be achieved by combining gene therapy and photothermal therapy. Non-viral gene carriers play a critical role in tumor gene therapy, and GNRs can be potentially applied to treat tumors. GNRs are easily prepared and modified. GNRs also exhibit high photo-thermal conversion efficiency and thus can be used as contrast agents for bioimaging. In this study, low-molecular-weight PEI (PEI1.8k) was conjugated onto the surfaces of GNRs to construct a novel non-viral gene carrier with numerous properties, including photoacoustic ability and photothermal effect. The transfection efficiency and cytotoxicity of GNRs were evaluated and compared with those of commercially available PEI25k. The photoacoustic imaging ability and photothermal effects of GNRs were also examined (Scheme 1).

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Scheme 1. Schematic design of the multifunctional GNRs-PEI1.8k for photoacoustic imaging, gene and photothermal therapy

EXPERIMENTAL SECTION Materials. Tetrachloroauric acid (HAuCl4﹒4H2O) was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Silver nitrate (AgNO3, 99%) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Sodium borohydride (NaBH4, 99%) was purchased from Tianjin FuChen Chemical Reagents Factory (Tianjin, China). Ascorbic acid (AA, 99%) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Cetyltrimethylammonium bromide (CTAB, 99%) was purchased from Aladdin (Shanghai, China). N-hydroxysuccinimide (NHS,

98%)

was

purchased

from

Aladdin

(Shanghai,

China).

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC﹒HCl) was purchased from GL Biochem Ltd. (Shanghai, China). Hydrochloric acid (HCl, 36.0~38.0%) was 4


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purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Branched polyethylenimine with average molecular weights of 1800 Da (PEI1.8k, 99%) was purchased from Alfa Aesar (Tianjin, China). 3-Mercaptopropionic acid (MPA, 99%) was purchased from Alfa Aesar (Tianjin, China). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) was purchased from Amresco (Solon, Ohio, USA). Dimethylsulfoxide (DMSO) was purchased from Tianjin Yongda Chemical Reagent Company Ltd. (Tianjin, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand Island, USA). Deionized water was used throughout the experiments. Synthesis of Gold Nanorods. Seed-mediated growth method23-25 was used to prepare GNRs. Briefly, 250 µL of 0.01 M HAuCl4 was mixed with 10 mL of 0.1 M cetyltrimethylammonium bromide (CTAB) solution. Under stirring, 600 µL of 0.01 M ice-cold NaBH4 solution was added rapidly. The color of the solution immediately changed from yellow to brown. After continually stirring for 2 min, the solution was allowed to stand for 2 h at 30 °C to form CTAB-capped gold nanoparticles as seeds. Then, the growth solution is prepared. Approximately 100 mL of 0.1 M CTAB solution was added to a 250 mL conical flask and 1 mL of 0.01 M AgNO3 solution. Up to 5 mL of 0.01 M HAuCl4 solution was added in the flask. Subsequently, 1 mL of 1 M HCl was added into the solution. Then, 700 µL of 0.1 M freshly prepared ascorbic acid solution was added gently into the flask under stirring. This procedure resulted in a colorless solution. Furthermore, 100 µL of gold seed solution was added. Then, the mixture was left undisturbed overnight at 30 °C. The solution was centrifuged for 15 min at 8000 rpm and washed twice with deionized water to remove the residual reactants. Finally, the precipitate was redispersed in deionized water. PEI-Modified GNRs. PEI was bonded as a surface modifier on the surface of GNRs via sulfur–gold bond to enhance the cell transfection capacity and decrease the cytotoxicity of CTAB-capped GNRs.8-9, 26 Up to 0.1 g N-hydroxysuccinimide, 0.1 g ethylene dichloride, and 1 g PEI1.8k were added to 30 mL aqueous solution containing 0.02 g methiopropamine to prepare GNRs-PEI1.8k (1:20). The mixture was allowed to react for 24 h at 30 °C. Afterwards, the aqueous solution containing 5



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5 mg GNRs was added into the thiolated PEI solution and reacted for another 24 h at 30 °C under stirring. Then, the GNRs–PEI was separated from the reaction solution via centrifugation at 8000 rpm for 10 min. The precipitate was washed with deionized water and redispersed in deionized water again. The preparation route is illustrated in Figure 1.

Figure 1. Synthesis route of GNRs-PEI. Characterizations of GNRs-PEI. Absorption spectra were taken on a UV-Vis spectrophotometer (Shimadzu 2401PC) at room temperature. To verify the stability, GNRs-PEI1.8k was performed in various conditions such as low pH (pH 6.5), blood, liver, spleen and kidney for 7 days. Then, the absorption spectra of the samples were detected. The morphology and sizes were measured by transmission electron microscopy (TEM). The TEM samples were prepared as follow: a drop of GNRs solution (0.2 mg/mL) was deposited onto a 200 mesh copper grid coated with carbon and dried at room temperature. The TEM measurements were performed on a JEOL JEM-1011 electron microscope operating at an acceleration voltage of 100 kV. The contents of gold and PEI in the product were measured by elemental analysis and thermal gravimetric analysis (TGA). Gel Retardation Assay. The binding ability of the GNRs-PEI and plasmid DNA was studied by agarose gel electrophoresis. The plasmid DNA was diluted to 0.1 mg/mL. GNRs-PEI/pDNA complexes were prepared at various weight ratios (0, 6


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0.1, 0.2, 0.3, 1.0, 1.5, 2.0 and 3.0). PEI25k and PEI1.8k were utilized as the controls. After incubation at room temperature for 30 min, 10 µL of the various solutions were analyzed by agarose gel (1%, w/v) electrophoresis at 100 V for 40 min in TAE buffer solution (40 mM Tris-HCl, 1% v/v acetic acid, and 1 mM EDTA). The retardation of the complexes were stained with ethidium bromide for 10 min and then visualized with an UV lamp a UVP EC3 imaging system (UVP Inc., Upland, CA, USA). Cell Culture and Gene Transfection. Human cervical carcinoma (HeLa) cells were cultured in the Dulbecco's modiﬁed Eagle's medium (DMEM) with high glucose content (4500 µg/mL) and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 µg/mL penicillin, and 100 µg/mL streptomycin in a 5% CO2 incubator at 37 °C under 95% humidity. Quantitative transfection efficiency was evaluated through monitoring the expression of firefly luciferase. The cells were grown in 96-well culture plates at an initial seeding density of 1.0 × 104 cells per well in 200 µL of growth medium (90% DMEM, 10% FBS) and incubated at 37 °C for 24 h before transfection to evaluate the transfection efficiency of carriers. Then, the medium was replaced with 200 µL of fresh growth medium. The complexes at various carrier/pGL3 (w/w) ratios (2.5, 5.0, 10, 20, 30, 40, and 50) were added in each well. The content of pGL3 plasmid DNA in every well was 0.2 µg. The cells were incubated for 48 h. Afterwards, the medium was removed and the cells were carefully washed with phosphate-buffered saline (PBS). Then, 50 µL of cell lysis buffer was added in each well. The culture plates were frozen at −80 °C for 30 min. After thawing out, the cell lysis was blown powerfully well by well using a pipettor for the thorough disruption of the cells. The luciferase activity was determined by detecting the light emission from 10 µL of the cell lysate mixed with 50 µL of luciferin substrate (Promega) in a luminometer (GloMax 20/20, Promega). Cytotoxicity Assay. The cytotoxicity of GNRs-PEI/pDNA complex was assessed by the MTT assay27-28 in comparison to PEI25k, PEI1.8k and CTAB capped GNRs. In brief, HeLa cells were seeded at the density of 1.0 × 104 cells per well in 96-well plates. After 24 h of incubation at 37 oC in 5% CO2 humidified atmosphere, the medium was replaced with 200 µL of fresh culture medium. Meanwhile, various 7



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amounts of carrier/pDNA complexes (2.5:1, 5:1, 10:1, 20:1, 30:1, 40:1 and 50:1) were added in each well and the cells were incubated for another 48 h. Then 20 µL of MTT solution (5 mg/mL in PBS) was added into each well. After incubation for 4 h, the MTT solution was removed from each well and then 200 µL of DMSO was added to dissolve the MTT formazan crystals. The absorbance of the degraded MTT was measured at 492 nm using an enzyme-linked immunosorbent assay microplate reader (Bio-Rad, Hercules, CA, USA). The cell viability was calculated according to the equation: Cell viability (%) = (A sample/A control) × 100 Asample is the absorbance of the cells treated with various materials and Acontrol is the absorbance of the untreated cells. Intracellular Uptake. The intracellular uptake of GNRs–PEI was analyzed by confocal laser scanning microscopy (CLSM) and cytometric analysis (FCM). For the CLSM studies, the plasmid DNA labeled with Alexa Fluor 546 was compacted with GNRs-PEI1.8k (1:20), PEI1.8k, and PEI25k. HeLa cells were seeded in 6-well plates with coverslips at a density of 1.0 × 105 cells per well and incubated for 24 h as described above. Then, the carrier/pDNA complex was added into each well. After incubation for 4 h, the cells were washed with cold PBS thrice and then fixed with 4% paraformaldehyde to be immobilized for 10 min at room temperature. Afterwards, the samples

were

washed

with

cold

PBS

thrice.

Then,

1 µL

of

4′-6-diamidino-2-phenylindole (DAPI, 1 mg/mL) was added into each well to stain the nucleus for 5 min in the dark environment. The cells were washed with cold PBS for five times after staining the nucleus with DAPI. Then, 4 µL of Alexa Flour 488 phalloidin (AF488) was added into each well to stain the cytoskeleton for 30 min at 37 °C. Finally, the coverslips were placed on slides, enclosed in glycerol, and visualized by CLSM (ZEISS LSM 780, Germany). For cytometric analysis,29 HeLa cells were seeded in 12-well plates at 1.0 × 105 cells per well for 24 h. The cells were detached and resuspended in 300 µL cold PBS after incubating with carrier/pDNA complexes for 4 h. Finally, the intracellular uptake efficiency was monitored and quantified as the percentage of cells included Alexa Fluor 546-labeled pDNA using a 8


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Guava easyCyte 6-2L Base System (Merck Millipore, USA).

In Vivo Photoacoustic Imaging. Multispectral optoacoustic tomography (MSOT) is a kind of noninvasive imaging technique. In the experiments, small animal scanner using MSOT imaging technique was utilized to study the in vivo biodistribution of GNRs–PEI. The scanner consists of a tunable optical parametric oscillator pumped by an Nd:YAG laser (Opotek Inc., Carlsbad, CA, USA) with a NIR (680–980 nm) tuning range. The laser pulse length is 8 ns and the pulse repetition frequency is 10 Hz. The optoacoustic signals were acquired via a custom-made 64-element focused transducer array (Imasonic SaS, Besancon, France) covering a solid angle of 172° around the imaged object.30-31 The nude mice were seeded under the guidance of the Animal Care and Use Committee of Northeast Normal University. Up to 200 µL of the GNRs-PEI1.8k (1:20) at a concentration of 1 mg/mL was intravenously injected into mice via tail vein. Then, the mice were fixed at a holder in supine position and submerged into water tank after being wrapped with a thin polyethylene membrane. The mice must be kept under anesthesia with anesthetic gas, which was 5% isoflourane mixture with air. The temperature of the water was kept at 34 °C throughout the experiment. The mice were scanned repeatedly at different excitation wavelengths (680, 715, 750, 785, 820, 855, 890 nm) at the liver, kidney, and spleen sites. The images were reconstructed using a linear model-based inversion after scanning. The images of tissues were finally harvested.

In Vitro and in vivo Photothermal Therapy. Human embryonic kidney cell constant expression green fluorescence protein (293T-GFP) was cultured following the above description. The cells were grown in 96-well for 24 h. Then, different amounts of GNRs-PEI1.8k (1:20; 1.6 and 5.0 µg) were added to each well. The cells without any material were utilized as the control group. NIR laser (808 nm, 1 W/cm2) was used to irradiate each well for 10 min after incubation for 4 h. Then, the fluorescent photographs were harvested using a microscope in the dark and the fluorescence intensity was quantified and analyzed with NIH ImageJ. The preliminary experiment was carried out to avoid the cell death induced by laser intensity. Briefly, different powers of laser (0.7–2.0 W/cm2) were used to irradiate the cells of the 9



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control groups. The photothermal therapy of GNRs-PEI1.8k was further verified in vivo.32-33 Briefly, HeLa cells were expanded and injected into the right anterior of the nude mice at 5.0 × 107 cells per mice to establish the subcutaneous tumors. GNRs-PEI1.8k was performed by intratumoral injection when the average diameter of the tumors reached about 0.8 cm. Then, near-infrared (NIR) laser (808 nm, 1 W/cm2) was used to irradiate the tumors for 10 min. The temperature of tumors was measured after NIR laser irradiation, and the thermographic maps were obtained using the infrared thermal camera.

RESULTS AND DISCUSSION Preparation of GNRs and GNRs–PEI. Seed-mediated growth method was used to prepare GNRs. According to previous studies,25, 34 tetrachloroauric acid (HAuCl4) was reduced by sodium borohydride (NaBH4) and stabilized by CTAB to produce gold seeds in aqueous solution. The gold seeds were mixed with growth solution and incubated overnight to produce the CTAB-capped GNRs. The absorption spectrum and geometric morphology of the CTAB-capped GNRs were obtained by UV–vis spectrophotometry and transmission electron microscopy (TEM), respectively. GNRs exhibited two absorption bands including a weak short transverse plasmon resonance band at approximately 520 nm and a strong long longitudinal plasmon resonance band at approximately 750 nm. As shown in Figure 2a, the bare GNRs and GNRs–PEI exhibit similar absorption peaks indicating that GNRs did not aggregate after they were modified by PEI1.8k. TEM imaging shows the shape and size of the bare GNRs (Figure 2b) and GNRs-PEI1.8k (Figure 2c). The average length and diameter of GNRs-PEI1.8k were approximately 60 and 20 nm, respectively. The UV–vis absorption spectrum and TEM images of the gold seed and GNRs are shown in Figure S1.

10


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Figure 2. UV-vis absorption spectrum and TEM images of the GNRs (A) UV-vis absorption spectrum of the bare CTAB-capped GNRs (black line) and GNRs-PEI1.8k (red line). (B-D) TEM images of the bare GNRs, GNRs-PEI1.8k and GNRs-PEI1.8k-DNA, respectively (scale bar=100 nm). The content of carbon, hydrogen, nitrogen, and sulfur (CHNS) in GNRs and GNRs-PEI1.8k (1:20) was detected by elemental analysis (Table 1). The results indicated that PEI was successfully conjugated on the surface of GNRs. This result was due to the content of CHNS in GNRs-PEI1.8k (1:20) exhibiting a significant enhancement compared with bare GNRs. Table 1. Elemental analysis of GNRs and GNRs-PEI1.8k (1:20) C(wt%)

H(wt%)

N(wt%)

S(wt%)

GNRs

16.50±0.002

3.37±0.010

0.92±0.014

0.065±0.008

GNRs-PEI1.8k

34.17±0.120

7.60±0.018

17.51±0.106

0.597±0.006

The content of PEI on the surface of GNRs was determined by thermal gravimetric 11



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analysis. The measurements were carried out from room temperature to 800 °C at a heating rate of 10 °C/min in N2. As shown in Figure 3, the content of CTAB in GNRs was approximately 10%. The content of PEI1.8k in GNRs-PEI1.8k (1:20) was approximately 30%. In other words, the content of gold in bare GNRs and GNRs-PEI1.8k was 90% and 70%, respectively. Furthermore, we evaluated the stability of GNRs-PEI1.8k in the conditions of different pH values (pH7.4 and pH6.5) for 7 days. Then, the UV-vis absorption spectrums of GNRs-PEI1.8k were detected. The results showed that there were no significantly differences under our experimental conditions (Figure S2A). In addition, we also tested the stability of GNRs-PEI1.8k in blood, liver, spleen and kidney for 7 days. Almost unanimous results were achieved, which could further prove the stability of GNRs-PEI1.8k (Figure S2B).

Figure 3. Thermal gravimetric analysis of GNRs and GNRs-PEI1.8k (1:20). Formation of GNRs–PEI/pDNA Complex. Necessary DNA complex and condensing ability are crucial factors for a successful non-viral gene delivery system. The DNA binding ability of the complex was characterized by gel retardation electrophoresis. The results are shown in Figure 4. The complete retardation of DNA mobility of GNRs-PEI1.8k was achieved in the range of tested weight ratios, indicating that the complexes exhibited efficient DNA binding capability. The results were similar to that of the control (PEI25k). Although PEI1.8k could also achieve the 12


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gene retarding effect, a trailing phenomenon emerged mainly due to the weakly bonding effect between PEI1.8k and pDNA. Whereas, However, GNRs-PEI1.8k required an obviously high weight ratio for complete retardation, which was possibly due to the introduction of GNRs and the low molecular weight of PEI. The zeta potentials of GNRs-PEI1.8k and GNRs-PEI1.8k/pDNA complex were +18.98 and +9.31 mV, respectively (Figure S3).

Figure 4. Gel retardation electrophoresis assay of pDNA nanocomplexes with GNRs-PEI1.8k, PEI25k and PEI1.8k under various weight ratios of carrier/pDNA complexes (0, 0.1, 0.2, 0.3, 1.0, 1.5, 2.0 and 3.0). Transfection Activity and Cytotoxicity in Vitro. The in vitro gene transfection efficiency and cytotoxicity of GNRs-PEI1.8k were detected by using HeLa cells. GNRs-PEI1.8k nanoparticles were prepared at GNRs:PEI1.8k feed ratios of 1:5, 1:10, and 1:20 (weight ratio). The palsmid DNA (pGL3-control) was used as the reporter gene and gene transfection efficiencies of carrier/pDNA complexes were evaluated at weight ratios in the range 2.5:1–50:1. Among the three nanoparticles, GNRs-PEI1.8k with a ratio of 1:20 exhibited the excellent transfection efficiency. This result was almost equivalent to that of PEI25k (Figure 5). PEI1.8k exhibited unconspicuous gene transfection effect compared with other carriers. It should be noted that GNRs-PEI1.8k showed increased transfection efficiency with the improved contents 13



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of PEI, which most likely a result of its higher binding ability with genetic materials. As shown in Figure 6, the bare GNRs exhibited high cytotoxicity because of the CTAB bilayer on their surface. GNRs-PEI1.8k with a ratio of 1:5 exhibited a certain cytotoxicity at its increased concentration. This effect was mainly due to the low quantity of PEI1.8k bonded on the surface of GNRs and the presence of large amount of CTAB. PEI1.8k and GNRs-PEI1.8k with ratios of 1:10 and 1:20 were almost non-cytotoxic within the scope of testing.

Figure 5. In vitro gene transfection efficiency of HeLa cells incubated with GNRs-PEI1.8k (including different GNRs:PEI1.8k with feed ratios: 1:5, 1:10 and 1:20 (weight ratio), respectively) for 48 h. PEI25k and PEI1.8k were used as the controls. Gene transfection efficiency under different carrier/pDNA with weight ratios: 2.5:1, 5:1, 10:1, 20:1, 30:1, 40:1 and 50:1.

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Figure 6. In vitro cytotoxicity of GNRs-PEI1.8k (including different GNRs:PEI1.8k with feed ratios: 1:5, 1:10 and 1:20 (weight ratio), respectively) for 48 h. Cytotoxicity was carried out under different weight ratios of carrier/pDNA (2.5:1, 5:1, 10:1, 20:1, 30:1, 40:1 and 50:1). Intracellular

Trafficking

of

GNRs-PEI1.8k.

Fluorescent-labeled

pDNA

(AF546-pDNA) formulations were monitored by CLSM to investigate the intracellular uptake of GNRs-PEI1.8k mediated pDNA delivery. As shown in Figure 7,

the

high

fluorescent

signals

in

HeLa

cells

treated

with

GNRs-PEI1.8k/AF546-pDNA complex were very evident and similar to that of PEI25k/AF546-pDNA complex. Whereas, PEI1.8k cannot mediate effective endocytosis. The intracellular uptake of carrier/pDNA complexes was further evaluated by FCM after incubating for 4 h with HeLa cells (Figure 8). The fluorescence peaks for GNRs-PEI1.8k/pDNA complex shifted to the right remarkably, indicating that the significant internalization efficiency was achieved and comparable with the commercial reagent PEI25k. In the control experiments, the internalization of PEI1.8k/pDNA complex was not obviously achieved. The internalization efficiencies for GNRs-PEI1.8k, PEI25k and PEI1.8k were quantified, they could achieve 96.84%, 93.26% and 25.28%, respectively (Figure S4). For the control group, PBS showed only 0.85% of internalization efficiency. The enhanced intracellular uptake capacity induced by GNRs-PEI1.8k was consistent with the former CLSM observation. These 15



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results suggest that GNRs-PEI1.8k/AF546-pDNA achieved a high level of gene transfection efficiency.

Figure 7. CLSM observation the cellular uptake of GNRs-PEI1.8k, PEI25k and PEI1.8k. PEI25k and PEI1.8k are the control groups. DAPI: cell nucleus (blue); AF546: pDNA (red); AF488: cytoskeleton (green). Scale bar is 20 µm.

Figure

8.

Cellular

uptake

of

GNRs-PEI1.8k/pDNA,

PEI25k/pDNA

and

PEI1.8k/pDNA in HeLa cells. The carrier/pDNA complexes were incubated for 4 h in HeLa cells. pDNA was labeled with AF546. Photoacoustic Imaging of GNRs-PEI1.8k in Vivo. In vivo photoacoustic imaging is a noninvasive bioimaging technology that can be utilized for pharmacokinetics and biodistribution studies. Recently, the agents used for photoacoustic imaging were gold nanoparticles and NIR dyes, such as gold nanocages, nanoshells, nanorods, indocyanine green, Cy5.5, Cy7, and IRDye83035-36. GNRs have been successfully applied in photoacoustic imaging. In this study, GNRs-PEI1.8k were used as a NIR 16


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probe by this technique to observe their biodistribution in mice. On the basis of the above results, GNRs-PEI1.8k with a ratio of 1:20 was selected for further studies. The structure diagrams of small animal scanner inVision 128 (iTheraMedical, Germany) are shown in Figure S5. Up to 200 µL of GNRs-PEI1.8k 1:20 aqueous solutions (1 mg/mL) was intravenously injected into healthy nude mice through their tail vein. MSOT technology was carried out to view the entire mice. This technique depends on chromophoric molecules or particles that possess absorption spectra distributed in tissues to generate absorption greater than the background tissue absorption. Therefore, MSOT produces high resolution and high sensitivity of the photoacoustic images.20 In recent studies, Taruttis et al. exploited GNRs to achieve photoacoustic imaging through MOST technology.31 To verify this, in this study, we also investigated the photoacoustic tomography of GNRs-PEI1.8k. The clear imaging photographs of the mouse's liver (Figure 9A), kidney, and spleen (Figure 9B) were achieved by MSOT. The highlighted red regions indicated the existence of GNRs-PEI1.8k nanoparticles, which were distributed in the liver, kidney, and spleen. These results are consistent with that of previous studies.37 The intensity of the MSOT signal at different time points indicated the content variation tendency of GNRs–PEI in each organ. As shown in Figures 9C–E, the content of GNRs–PEI1.8k in the kidney improved gradually with the time increasing. Whereas, the intensity of GNRs–PEI increased during the first 70 mins for the liver and spleen, and then decreased within the scope of our testing. The data can provide a powerful guidance to the combination of gene and photothermal therapy for cancers in the future.

17



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Figure 9. In vivo photoacoustic imaging of the nude mice after intravenous injection of GNRs-PEI1.8k (1:20). The analysis of pictures taken by photoacoustic imaging. (A) visual biodistribution imaging of GNRs-PEI1.8k in the liver; (B) visual biodistribution imaging of GNRs-PEI1.8k in kidney and spleen, respectively. (C-E) the multispectral optoacoustic tomography (MSOT) signal of GNRs-PEI in the liver, kidney and spleen. The curves are normalized to their own maximum value. Photothermal Effect of GNRs-PEI1.8k in Vitro and in vivo. The in vitro photothermal effect of GNRs-PEI1.8k was evaluated by using human embryonic kidney cells with a constant expression green fluorescence protein (293T-GFP). Different powers of laser (0.7–2.0 W/cm2) were used to irradiate the cells of the control groups. This condition was applied to avoid the cell death induced by laser intensity. As shown in Figure S6, the laser would cause the cell death when the power exceeded 1 W/cm2. Therefore, we chose 1 W/cm2 as the power of laser for the whole 18


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experiments. The cells were incubated with GNRs-PEI1.8k 1:20 for 4 h and then irradiated by NIR laser (808 nm, 1 W/cm2) for 10 min. The appearance of the control groups (Figures 10A and B) without GNRs-PEI1.8k did not significantly change after irradiation as shown in Figure 10. Photothermal efficacy was exhibited when GNRs-PEI1.8k were added. The remarkable apoptosis was observed after irradiation for 10 min as illustrated in Figures 10C and D. The photothermal effect was very obvious when the content of GNRs–PEI increased from 1.6 µg/well to 5.0 µg/well (Figures 10E and F). In addition, we evaluated the quantitative fluorescence intensity based on the photothermal results (Figures 10). As shown in Figure S7, the fluorescence intensity of GNRs–PEI1.8k treated groups decreased significantly after irradiating by NIR. For the 1.6 µg and 5.0 µg of GNRs–PEI1.8k groups, the ratios of fluorescence intensity decreased about 74.3% and 84.6%, respectively. While for non-irradiation groups, the fluorescence intensity showed no obviously decreased, which could further indicate the safety of GNRs-PEI1.8k. Therefore, on the basis of gene transfection and thermal performance, we propose that GNRs-PEI1.8k can be used as a novel gene transfection regent with excellent photothermal effects.

19



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Figure 10. In vitro photothermal effect of 293T-GFP cells incubated with different amounts of GNRs-PEI1.8k (1:20) for 4 h, non-irradiated and irradiated for 10 min by NIR laser (808 nm, 1 W/cm2). (A, B) Non-irradiated and irradiated control groups without GNRs-PEI1.8k, respectively. (C, D) Non-irradiated and irradiated cells incubated with 1.6 µg of GNRs-PEI1.8k/well. (E, F) Non-irradiated and irradiated cells incubated with 5.0 µg of GNRs-PEI1.8k/well. The photothermal effects were then confirmed in the HeLa xenograft tumor bearing nude mice.33 After NIR laser irradiation, the elevation of temperature could be seen directly, indicating the photothermal activity of GNRs-PEI1.8k was kept in vivo. For tumor-bearing mice injected with GNRs-PEI1.8k and PBS, the local tumor temperature reached 60.2 oC and 39.6 oC in the first 100 s, respectively. This could indicate the effective in vivo photothermal effect of GNRs-PEI1.8k (Figure 11A and 11B) Furthermore, the temperature change in tumor site was obtained using the infrared thermal camera during irradiation (Figure 11C). The increasement of temperature could be seen obviously compared with PBS group, which further indicated GNRs-PEI1.8k possessed effective photothermal activity in vivo. A recent study by Shen et al. also demonstrated cationic carrier-modified GNRs with transfection performance and photothermal effect.38 The risk from the high molecular weight of PEI25k mainly restricted its further application. In this study, we firstly showed three functions of GNRs-PEI1.8k in one work including high gene transfection efficiency, photoacoustic imaging ability and photothermal therapy property. Combined with the former data, GNRs-PEI1.8k with its excellent multifunctions could further validate its potential application in diagnosis and treatment of tumors.

20


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Figure 11. In vivo photothermal effect of GNRs-PEI1.8k. The HeLa tumor-bearing mice were intratumorally injected with saline and GNRs-PEI1.8k. Then, the thermographic images and temperature change of tumors were measured after NIR irradiation (1.0 W/cm2, 10 min). The near-infrared thermographic images captured after 100 s of tumor-bearing mice after photothermal therapy. A) PBS; B) GNRs-PEI1.8k. C) The thermographic monitoring in the tumors of PBS and GNRs-PEI1.8k-injected nude mice.

CONCLUSIONS GNRs-PEI1.8k yielded high gene transfection efficiency and low cytotoxicity. Its pharmacokinetics

and

biodistribution

properties

were

investigated

through

photoacoustic imaging. Our results demonstrated that GNRs-PEI1.8k exhibited excellent photothermal effect and thus could be applied as an anticancer agent through photothermal therapy. Therefore, GNRs-PEI1.8k can be used as a promising multi-functional gene carrier for future cancer therapy. However, the metabolism, organismal toxicity, synergetic antitumor and side effects of the proposed gene carrier should be further investigated.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Tel: +86 431 85262539 *E-mail: [email protected]. Tel: +86 431 85099667 21



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*E-mail: [email protected]. Tel: +86 431 85262112 Author Contributions 1

These authors contributed equally to this work.

The paper was written through contributions of all authors. All authors have given approval to the ﬁnal version of the paper. Notes The authors declare no competing financial interest.

ACKNOWLEGEMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 51303173, 51233004, 51390484, 21474104, 51403205, 51503200 and 51520105004), "Ten thousand talent program" outstanding young scholars, Jilin province science and technology development program (20160204032GX) and the Fundamental Research Funds for the Central Universities (2412016KJ042).

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Graphic for manuscript

Caption: Schematic design of the multifunctional GNRs-PEI1.8k for photoacoustic imaging, gene and photothermal therapy

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Gold-Nanorods-Based Gene Carriers with the ... - ACS Publications

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