Seed-Mediated Synthesis of Tunable-Aspect-Ratio Gold Nanorods for Near-Infrared Photoacoustic Imaging
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Tunable-aspect ratio gold nanorods have been synthesized by a modified seed-mediated synthesis method. Ascorbic acid was employed as a shape controller to induce anisotropic growth, which made the aspect ratio of the synthesized gold nanorods range from 8.5 to 15.6. These nanorods possess tunable longitudinal surface plasmon resonance absorption band, covering a broad near-infrared (NIR) range, from ~ 680 to 1100 nm. When modified with thiol-polyethylene glycol (SH-PEG), the synthesized Au nanorods showed excellent biocompatibility and stability, which foreshadowed the great potential of their NIR application as photoacoustic contrast agent. Due to their adjustable absorbance in the NIR, the synthesized Au nanorods could offer stronger contrast (3.1 times to the control group without contrast agent used) and higher signal-noise ratio values (SNR; 5.6 times to the control group) in photoacoustic imaging, both in vitro and in vivo experiments. Our work presented here not only added some novel Au-based photoacoustic contrast agents but also described a possibility of contrast agent preparation covering the whole biological NIR window.
KeywordsGold nanorod Tunable-aspect ratio NIR window Photoacoustic imaging
Au typical nanorods
Cetyl trimethylammonium bromide
Methyl thiazolyl tetrazolium
Optical parametric oscillator
Selected area electron diffraction
Scanning electron microscopy
Surface plasmon resonance
Transmission electron microscopy
One-dimensional (1D) nanostructures, such as nanowires, nanorods, nanotubes, and nanobelts, are especially interesting because they are not only novel basic building blocks for nanodevices, but also possess high geometrical aspect ratio producing anisotropic features for special applications [1, 2, 3, 4, 5, 6]. Among of these 1D nanostructures, novel metal nanorods (NRs) have drawn increasing interests because of their shape-dependent surface plasmon resonance (SPR) band [7, 8], facile synthesis [9, 10, 11], favorable biocompatibility, and easy modification [12, 13, 14]. For example, Yeh et al. reported an Au nanorod (AuNR) in-shell structure smaller than 100 nm, which exhibits strong longitudinal absorbance at 600–900 nm and good applicability for the photo-induced therapies . Wang et al. successfully constructed anisotropic AuNR helical superstructures with tailored chirality, by positioning the functionalized AuNR with DNA on the origami of the designed “X” pattern of the arrangement of DNA capturing strands .
In addition, improvements in synthesis and purification of AuNRs have enabled facile tuning of the longitudinal SPR band, by adjusting the length and hence aspect ratio [15, 16, 17], for specific application, like photoacoustic imaging (PAI) and photo-induced therapies [18, 19, 20, 21, 22, 23], which need the longitudinal SPR of Au NRs to fall in the optical transparent window of biological tissue (first at 700–950 nm and second at 1000–1350 nm) [8, 18]. For instance, Huang and co-workers synthesized gold NRs with aspect ratio from 2.4 to 5.6, which displayed efficient cancer cell diagnostics and selective photothermal therapy . Jokerst et al. developed gold NRs and silica-coated gold NRs with aspect ratio of about 3.5, which showed high PAI signal for ovarian cancer detection and mesenchymal stem cell imaging [20, 21]. Yang and co-workers reported magnetic gold nanorod/PNIPAAmMA for dual magnetic resonance PAI and targeted photothermal therapy . Although many Au NR-based contrast agents have been developed, a facile, scalable synthesis of large and tunable-aspect ratio AuNRs and their absorption behavior-dependent PAI performance still remain challenges.
Herein, AuNRs with aspect ratio from 8.5 to 15.6 have been synthesized by using the modified seed-mediated growth method with the assistance of ascorbic acid. The AuNRs were demonstrated possessing with high biocompatibility and further reduced their cytotoxicity with SH-PEG modification. Benefiting from their large and tunable absorbance in the NIR region, the synthesized AuNRs could offer stronger contrast and higher signal-noise ratio (SNR) values in PAI, both in vitro and in vivo experiments. This facile method for building tunable-aspect ratio gold NRs may be utilized for fabricating contrast agent under any wavelength in the first NIR window.
Synthesis of Gold Nanorods
Tunable-aspect ratio AuNRs were synthesized by a modified seed-mediated synthesis method [16, 17]. In a typical procedure, a volume of 10.3 mL of 0.025 M HAuCl4 (Sinopharm Chemical Reagent Co., Ltd., ≥ 99.9%) and 3.644 g of cetyl trimethylammonium bromide (CTAB) surfactant (Tianjin Guangfu Fine Chemical Research Institute, ≥ 99.0%) were first added to a beaker. Then, deionized water (18 MΩ) was added to bring the concentration of HAuCl4 to be 2.5 × 10−3 M, and CTAB of 0.1 M. 10 mL, 4.5 mL, 4.5 mL, and 45 mL of the above-mentioned solution were separately transferred into four flasks tagged as A, B, C, and D. Then, a volume of 350 μL, 0.01 M ice-cold NaBH4 (Sinopharm Chemical Reagent Co., Ltd., ≥ 98.0%) was added into flask A and stirred for 3 min. 0.4 mL solution of flask A and 25 μL 0.1 M L(+)-ascorbic acid (AA) (Tianjin Shentai Chemical Industry Co., Ltd., ≥ 99.7%) was transferred into flask B, stirred for another 3 min. And then, 0.4 mL solution of flask B and 25 μL of 0.1 M AA was added in flask C, stirred for 3 min again. Finally, 4 mL solution of flask C and 250 μL of 0.1 M AA was added in flask D, stirred for 5 s, and then left undisturbed in a water bath at 28 °C for 12 h. The top solution was removed carefully and the precipitate was centrifuged and washed several times with distilled water to make sure the excess CTAB was fully removed. Thus, the final products were signed as Au typical nanorods (AuTR).
Repeat the above process and just change the dosage of AA, and then Au NRs with aspect ratio from 8.5 to 15.6 could be developed. The details are as follows: the dosage of AA are (35 μL, 35 μL, 350 μL) for Au rod1, (30 μL, 30 μL, 300 μL) for Au rod2, (20 μL, 20 μL, 200 μL) for Au rod3, and (15 μL, 15 μL, 150 μL) for Au rod4.
Surface Modification of AuNRs
First, 10 mg SH-PEG (Nanjing Pengsheng Biological Technology Co. Ltd) was dissolved in 1 mL deionized water and sonicated for 10 min. Then, the solution was treated with 50 mL 0.1 M NaBH4 solution under sonication for another 15 min to reduce the possible dimerized SH-PEG (PEG-S-S-PEG). Second, the cleaned-up Au NRs were dispersed into 10 mL deionized water and mixed to the above SH-PEG solution (10 ml), stirred for 5 min, and then placed undisturbedly for 5 h. Finally, the sample was centrifuged and washed with deionized water for further application.
The morphology and structure of the synthesized AuNRs were identified by scanning electron microscopy (SEM; JEOL JSM-7001F) and transmission electron microscopy (TEM; JEOL 2100F, 200 kV). UV-vis absorbances of the various AuNRs were measured by spectrophotometer (Shimadzu, 3100 UV-vis-NIR). The photoacoustic signals were recorded by the unit rotation scanning photoacoustic detection system, which contains laser device (Surelite I-20, Continuum), optical parametric oscillator (OPO) (Surelite OPO Plus), non-focused ultrasonic transducer (PMUT) (V310-SU, Olympus, 5 Hz), motor step rotating table and its motor control box (MC) (M600, Beijing Zolix Instrument Co., Ltd.), preamplifier (5077PR, Olympus), PCI4732 data acquisition (DAQ) card, and so on.
Cell Viability Experiments
All bio-experimental procedures were approved by IACUC committee at the Taiyuan University of Technology. And the experiments were carried out in accordance with the approved guidelines.
Hela cells were cultured in the standard cell medium recommended by American type culture collection (ATCC), at 37 °C under a 5% CO2 atmosphere. Cells seeded into 96-well plates were incubated with different concentrations of AuNR and AuNR-PEG for 24 h. Relative cell viabilities were determined by the standard methyl thiazolyl tetrazolium (MTT) assay and imaged under optical microscope.
In Vitro and In Vivo PAI
Two grams of agar powder (Gene Company Ltd.) was dissolved in 100 mL deionized water and mixed well by glass bar in a beaker. The turbid liquid was heated to boiling in a microwave oven (Midea Group Limited by Share Ltd.). Then, the liquid was took out and stirred in water bath for 20 min at 60 °C, until the liquid became thick. Then, the viscous materials were poured into a 4.5 cm diameter cylindric mold, cooled, and solidified. Finally, the clotted agar was used as the phantom of biological tissue, due to their approximate absorbance to NIR lasers.
A 0.9-mm diameter glass capillary was implanted to the surface of the phantom to simulate blood vessel, which would be fulfilled with fresh ox blood, or blood mixed with various concentration of AuNR-PEG in specific experiment. The phantom was placed under water, and irradiated by 680-nm or 800-nm laser, at power density of 11 mJ/cm2.
Narcotize the mouse by isoflurane transiently, then 0.04 mL/10 g 10 wt% chloral hydrate was intraperitoneally injected to make the mouse anesthesia thoroughly. The mouse head was gently shaved of hair and smoothly smeared ultrasonic coupling agent (Boline Healthcare Ltd.). The wavelength of the laser was adjusted to 800 nm, and the mouse was placed under water. Then, the cerebral blood vessels of the mouse were imaged, before and after, and the contrast agents (1 nM, 0.1 mL/10 g) were intravenously (I.V) injected into the mouse. The laser was changed to 680 nm, and repeat the experiment above. Note: When the contrast agent was changed, the I.V injection should take at least 24 h later to let the residuum completely metabolized.
Results and Discussions
The photoacoustic and optical spectra of five kinds of AuNRs and blood are shown in Fig. 3e1–e6. The multi-wavelength photoacoustic signal spectra were obtained by collecting amplitudes of photoacoustic signals at different wavelength (from 680 to 900 nm) lasers, with 1 nM aqueous solution was fulfilled in glass capillary tubes. Clearly, the graphs indicate a good agreement between the photoacoustic signal spectra and the optical spectra of the AuNRs. These results plain indicate the feasibility of applying AuNRs in PAI under suitable wavelength lasers and quantitatively give the photoacoustic effect of AuNRs at various wavelengths from 680 to 900 nm.
Comparison of contrast and SNR in PA images from b1 to b6 in Fig. 5. Contrast here is a mean value of the images for mice cerebral blood vessels; SNR in the table here denotes signal-noise ratio, which is the analysis of the whole pictures
13.2 ± 1.1
8.5 ± 0.6
By the assist of ascorbic acid, tunable-aspect ratio gold nanorods, ranging from 8.5 to 15.6, have been synthesized by a modified seed-mediated synthesis method. These gold nanorods could provide tunable absorption peaks from 680 to 1100 nm, covering the first biological NIR window. When modified with SH-PEG, the synthesized AuNRs show excellent biocompatibility and stability, which foreshadows the great potential of their near-infrared application as photoacoustic contrast agent. Both experiments in vitro and in vivo confirm that the synthesized tunable-aspect ratio AuNRs could offer stronger contrast and higher SNR values in PAI, under suitable wavelength lasers. This work provides a possible way to controllably synthesize the contrast agent under any wavelength in the first NIR window and used for visualizing diseases such as intracerebral hemorrhage and thrombus.
This work was supported by the National Natural Science Foundation of China (Grant Nos. 11602159, 51205276, and 61474079), the Special Talents in Shanxi Province (Grant No. 201605D211020), the Scientific & Technological Innovation Programs of Higher Education Institutions in Shanxi (Grant No. 2016136), and the 2018 Study Abroad Program for the University-Sponsored Young Teachers.
Availability of Data and Materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
PL conceived and designed the study and revised and rewrote the paper. YW performed most of the experiments and wrote the manuscript. DL assisted in the synthesis of Au nanorods. XS assisted in the photoacoustic experiments. CL assisted in the photoacoustic experiments. YW assisted in the bio-experiments. JH, GL, HJ, and WZ reviewed and edited the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
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