Team:Tongji Shanghai/Materials Synthesis

Tongji_Shanghai-2016.igem.org Tongji Shanghai

Project

Our project is our story.

Overview

    We have synthesized the AuNRs (gold nanorods) and detoxified them for photothermal therapy, also known as optical hyperthermia or photothermal ablation, which is an emerging strategy for treating solid tumors. Gold nanoparticles are capable of confining resonant photons, further inducing coherent surface plasmon oscillation of their conduction band electrons. For non-invasive therapy, near infrared (NIR) radiation is chosen because it penetrates tissue more deeply. And we find that AuNRs with a strong SPR(surface plasmon resonance) in the NIR region can show intense absorption of light in the NIR region, also biocompatibility. Even more, it can accumulate in tumor tissue via passive targeting phenomena. PEG can be used to increase biocompatibility, suppress immunogenic responses and decrease adsorption.

Experiment:

Part 1. Synthesis of AuNRs

    GNRs are synthesized through the seedless growth route. The growth solution is made from a stepwise mixing CTAB (cetyl trimethyl ammonium bromide) aqueous solution and HAuCl4 aqueous solution (1mM,5ml). Then add AgNO3 aqueous solution slowly with stirring, followed by AA aqueous solution, and stop stirring when the solution is transparent. Finally the growth of AuNRs is induced by injecting freshly-prepared ice-cold NaBH4 aqueous solution. The resultant solution needs to be kept undisturbed at room temperature for 6 hours.[2]

The concentration and material volume are listed as following:

CTAB(cetyl trimethyl ammonium bromide): 0.2M 5ml

HAuCl4(chloroauric acid):1mM5ml

NaBH4(sodium borohydrate):0.01M 15μl

AA(ascorbic acid):78.8mM 70μl

AgNO3(silver nitrate)：4mM 300μl


Part 2. Au concentration measurements

    The as-synthesized AuNRs is purified by three cycles of centrifugation to remove excessive Au ions. Afterwards, the precipitates are dissolved with aqua regia and diluted by deionized water. An ICP-OES (inductively coupled plasma optical emission spectrometer) is applied for measuring the ratio of reacted HAuCl4.

Part 3. Photothermal application of AuNRs

    First, we test UV–vis-NIR extinction spectra of the AuNRs.
    Second, we test the photothermal effect of the AuNRs. We prepare a 200μl aqueous suspension of AuNRs with different concentration in anadiabatic box. Then irradiate with laser for 10 minutes. The laser we use is an 808nm laser generator. We test the AuNRs at different power density.
    Third, we test the photothermal stability of the AuNRs. We irradiate the GNRs solution from an ambient temperature to the top temperature for consecutive four cycles, testing whether the top temperature will change or not.
    We record the temperature every 1 minute.

Part 4. Surface modification

    In our experiment, we use poly ethylene glycol (PEG) to minimized dose-related side effects of single AuNRs.
    The treatment is mixing prepared AuNRs with PEG and leaving undisturbed at room temperature for 24h. The final concentration of PEG is 0.8mg/ml.[3]

Result:

    We get AuNRs with an aspect ratio of~3.9(41.9 × 10.6 nm), the transmission electron microscopy(TEM) image is shown as figure1(a),the TEM image clearly indicates that the products are single crystals and the shape is quite even.
    As to the AuNRs with the aspect ratio of 3.9, two LSPR peaks at 546 and 770 nm are observed (figure 1(b)).The former arises from the transverse resonant oscillation, while the latter results from the resonant oscillation[1], the LSPR range of the AuNRs is wide in near infrared region.
    The temperature variation of the GNRs is revealed by irradiating a 200μl aqueous solution using an 808 nm laser. The temperature increases obviously(figure 1(c)) as the increase of the GNRs concentration. In addition, as the laser power density increases, the temperature increment is higher, because more energy is absorbed by the solution (figure 1(d)).Figure 1(e) shows that the top temperature is almost unchanged after four cycles, which indicates the photothermal stability of GNRs. These results indicate that the GNRs synthesized by us possess good photothermal property and stability.

Biosafety:

    The photothermal stability test result indicates that the AuNRs exhibits excellent photothermal stability. So it can be irradiated by laser many times with its properties unchanged. The cyto-toxicity of CTAB-GNRs is independent of their aspect ratio[4]. And the cell toxicity experiment shows that the toxicity of the AuNRs is dose-related.When the AuNRs concentration is low, it doesn’t show obvious cell toxicity, but it exhibits cell toxicity with the concentration increase. The cell toxicity is the result of the AuNRs caninduce cell apoptosis and autophagy by damaging mitochondria and activating intracellular reactive oxygen species (ROS).[4]Surface modifications have been extensively used to improve the biocompatibility and stability of gold nanorods. Our results show that with the surface modification of PEG, the toxicity of the AuNRs decreased dramatically. In a word, all of the results testify that our material have well-controlled biosafety both in material stability and its cell toxicity.

Refference:

    [1]Chen H, Shao L, Li Q, et al. Gold nanorods and their plasmonic properties[J]. Chemical Society Reviews, 2013, 42(7): 2679-2724.
    [2]Wang W, Li J, Lan S, et al. Seedless synthesis of gold nanorods using resveratrol as a reductant[J]. Nanotechnology, 2016, 27(16): 165601.
    [3]李媛, 王小慧, 庄远, 等. 不同尺寸, 形貌金纳米粒子小鼠体内急性毒性研究[J]. 军事医学 ISTIC, 2013, 37(6).
    [4]Wan J, Wang J H, Liu T, et al. Surface chemistry but not aspect ratio mediates the biological toxicity of gold nanorods in vitro and in vivo[J]. Scientific reports, 2015, 5.

Figure1a


Figure1b


Figure1c


Figure1d


Figure1e


Figure1.(a)The TEM image of the AuNRs. (b)The UV–vis-IR absorption spectrum.(c)The temperature incrementwith different concentration of AuNRs.The power density of the 808 nm laser is fixed at 2.80W/cm2.(d)The temperature increment with three different laser power density. The concentration of the AuNRs is 100 μg/ml. (e)The temperature variation of 19.2 μg/ml AuNRs suspension as irradiated by the 1.80 W/cm2 808 nm laser for four cycles.