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Fabrication of Graphene-isolated-Au-nanocrystal Nanostructures for Multimodal Cell Imaging and Photothermal-enhanced Chemotherapy

Using nanomaterials to develop multimodal systems has generated cutting-edge biomedical functions. Herein, we develop a simple chemical-vapor-deposition method to fabricate graphene-isolated-Au-nanocrystal (GIAN) nanostructures. A thin layer of graphene is precisely deposited on the surfaces of gold nanocrystals to enable unique capabilities. First, as surface-enhanced-Raman-scattering substrates, GIANs quench background fluorescence and reduce photocarbonization or photobleaching of analytes. Second, GIANs can be used for multimodal cell imaging by both Raman scattering and near-infrared (NIR) two-photon luminescence. Third, GIANs provide a platform for loading anticancer drugs such as doxorubicin (DOX) for therapy. Finally, their NIR absorption properties give GIANs photothermal therapeutic capability in combination with chemotherapy. Controlled release of DOX molecules from GIANs is achieved through NIR heating, significantly reducing the possibility of side effects in chemotherapy. The GIANs have high surface areas and stable thin shells, as well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications.

RPMI-1640 medium, penicillin streptomycin solution and fetal bovine serum were obtained from Invitrogen. MTT was purchased from Beyotime (China). Gold nanoparticles (40 nm) were purchased from Shanghai JieYi Biotechnology. The ultrapure water used was from a Milli-Q Integral System. All other chemical reagents were analytical grade and used without further purification.
GIAN synthesis. GIAN was produced in a CVD system. First, fumed silica (1.00 g, Aladdin) was impregnated with anhydrous chloroauric acid (1%， 29.65mL) in methanol and sonicated for 2 h. Then the methanol was removed, the mixture was dried at 80 °C, and the powder was ground. Typically, 0.50 g of the powder was used for methane CVD in a tube furnace. The sample grew with a methane flow of 150 cm 3 min -1 for 10 minutes. After growth, the sample was etched with 10% HF in H 2 O (80%) and ethanol (10%) to dissolve the silica. The GIAN solid product was then washed thoroughly and collected through centrifugation. Hollow graphitic nanocapsules were obtained from a core-shell magnetic graphitic nanomaterial (MG).
MG was synthesized with the CVD system as reported previously which was similar to GIANs. The as-prepared MG was then treated with a solution of sulfuric and nitric acid to polish for 4 h, followed by solubilization in water. Excess MGs were removed by an external magnet. The HGNs were collected through centrifugation and washed thoroughly with ultrapure water.

Characterization of GIANs with transmission electron microscopy
Transmission electron microscopy was applied to characterize GIAN morphology and size distribution. Additional TEM images are shown in Fig. S1.
Several layers of shells have grown out of the Au core and isolated the Au nanocrystal.
The distance between the two shell layers was observed around 0.34 nm in the high resolution TEM (Fig. S1a), which further confirmed the graphene encapsulated Au nanocrystal structure.

Dynamic light scattering characterization of GIAN
The hydrodynamic diameters of the MGs under investigation were measured 4 using a Zetasizer Nano ZS90 DLS system equipped with a red (633 nm) laser and an Avalanche photodiode detector (APD) (quantum efficiency > 50% at 633 nm) (Malvern Instruments Ltd., Worcestershire, England). DLS measurements were performed at room temperature at a fixed scattering angle of 90°. Fig. S2 shows the size distribution of the suspended GIAN. The average size was around 70 nm, which agreed with the size measured from TEM. All size distributions reported here were based on number counting. The average particle size was obtained using a non-negative least squares (NNLS) analysis method. For each sample, two DLS measurements were conducted with a fixed 10 runs, and each run lasted 10 s.  Fig. S3 shows the ζ-potential curves of the GIAN water solution.

ζ-Potential measurement of GIAN
The GIANs, which were neutrally charged, showed good quality and very few graphene shell defects.

Two-photon luminescence characterization of GIAN
Two-photon microscopy was applied to characterize the efficient staining of GIANs on MCF-7 cells. Additionl TPL images are shown in Fig. S4. GIANs were incubated with the MCF-7 cells for 4 hours before TPL imaging. To identify the contribution of Au nanocrystal core and isolation graphene shell to TPL imaging, we incubated GIANs (Fig. S4a), Au nanoparticles (Fig. S4b) and hollow carbon capsulates ( Fig. S4c) with the MCF-7 cells and they all showed the capability for TPL imaging. Thus, both the Au nanocrystal core and graphene outer layer of the GIANs were believed to contribute to the TPL signals.

Flow cytometry characterization of the selectivity of Sgc8 aptamer
To identify the targeting capability of the Sgc8

Photostability of GIAN under NIR laser irradiation
The photostability of GIAN was investigated with longer laser irradiation time.
No obvious color change of the 0.1 mg/mL GIAN solution was observed after 1 hour of 808 nm laser (2 w/cm 2 ) irradiation, as shown in Fig. S6 inset. The UV-Vis spectra further confirm the stability of the GIANs. The absorbance of GIANs around 550 nm was found to be almost the same with or without laser irradiation (Fig. S6). The unique stability of the graphene shell of the GIAN was believed to help constrict the Au nanocrystal core inside and prevent the morphology from changing under laser irradiation. The high photostability makes GIAN a promising material for further clinical applications.