Light-activated gold nanorod vesicles with NIR-II fluorescence and photoacoustic imaging performances for cancer theranostics

Fluorescence (FL) and photoacoustic (PA) imaging in the second near infrared window (NIR-II FL and NIR-II PA) hold great promise for biomedical applications because of their non-invasive nature and excellent spatial resolution properties. Methods: We develop a NIR-II PA and NIR-II FL dual-mode imaging gold nanorod vesicles (AuNR Ves) by self-assembly of amphiphilic AuNR coated with light responsive polyprodrug of Ru-complex and PEG, and NIR-II cyanine dye (IR 1061). The AuNR Ves showed strong ligh absorption property and PA imaging performance in the NIR-II windows. Moreover, the NIR-II fluorescence signal of IR 1061 loaded in the AuNR Ve is quenched. Results: The AuNR Ves can release photosensitizer Ru-complex and IR 1061 sequentially triggered by NIR light irradiation, leading to a corresponding NIR-II PA signal decrease and NIR-II FL signal recovery. Meanwhile, Ru-complex can not only serve as a chemotherapeutic drug but also generate singlet oxygen (1O2) under NIR light irradiation. The release of Ru-complex and photodynamic therapy are guided by the responsive variation of NIR-II PA and NIR-II FL signals. Conclusions: The AuNR Ve possessing not only precisely control 1O2/drug release but also the intrinsic ability to monitor therapy process offers a new strategy for the development of smart theranostic nanoplatform.

Equipments. Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AVANCE Ⅲ 500 in deuterated chloroform (CDCl 3 ) and deuterated dimethyl sulfoxide . TEM images were performed on an HT7700 transmission electron microscope (HITACHI, Japan) at 100 kV. DLS was collected through a Malvern Zetasizer Nano ZS (Malvern, U.K.). UH4150 spectrophotometer (HITACHI, Japan) have been used to record ultraviolet−visible−near-infrared light (UV−VIS−NIR) absorption spectra Fluorescence spectra were measured on Edinburgh FLS980 Spectrometer. The fluorescence images of cells were obtained on a confocal fluorescence microscope (Nikon C2). The fluorescence images in vivo were performed on IN-VIVO MASTER which provided by GRAND-IMAGING.
Synthesis of Ru(tpy)Cl 3 . 0.5 mmol RuCl 3 ·3H 2 O and 0.5 mmol 2,2′;6′,2′′-terpyridine were dissolved in 70.0 mL ethanol. Then, the mixture was heated to 80 ℃ for 3 h under vigorous stirring. After that, the solution was cooled down to room temperature. The brown powders were collected by filtration. The product was washed with ethanol and diethyl ether for three times. Finally, the product was dried for next reaction. (yield :75%)

Synthesis of [Ru(tpy)(biq)(Cl)]Cl.
[Ru(tpy)(biq)(Cl)]Cl was synthesized according to the literature. [S1] 0.2 mmol 2,2'-biquinoline and 0.2 mmol Ru(tpy)Cl 3 were dissolved in 10.0 mL mixed solvent of H 2 O amd ethanol with the volume ratio of 3:1. Ttrimethylamine was added after the mixture was bubbled with argon. After that, the mixture was refluxed for 10 h in the dark. Then, purple solution was obtained after filtration. Finally, the filtrate was evaporated under reduced pressure. The product was purified by column chromatography with silica gel (eluent: dichloromethane / methanol =8:1). (yield :30%) The solvent was evaporated and the product was obtained as violet powders.

Synthesis of AuNR Coated with PEG and Photoactivated Polymer (AuNR@PEG/PolyRu).
To prepare amphiphilic gold nanorods attached with PEG and PolyRu. AuNR@CTAB (50 nM) was mixed with 0.1 mL of 2-(2-aminoethoxy)ethanol and the mixture was stirred for 24 h. The modified AuNRs were purified by centrifugation at 9000 g for 10 min and further dispersed in 5 mL of DMSO. Amphiphilic AuNR@PEG/ PCPH was synthesized by a "grafting to" reaction. Briefly, the mixture of thiolated PEG (PEG-SH, Mn = 5000) and thiolated PCPH was slowly added into the modified AuNR dispersion, and the solution was stirred for 12 h. The AuNR@PEG/P PCPH was purified by centrifugation (10,000 g, 15 min) and dispersed in 5 mL of acetone. To synthesize photoactivated AuNR@PEG/PolyRu containing Ru complexes, AuNR@PEG/P PCPH in acetone.           17 Figure S16. Blood hemolysis using the AuNR@PEG/PolyRu Ves at different concentrations from 100 to 1600 μg mL -1 . Water was used as a positive control and PBS was used as a negative control. No visible hemolytic effect was observed when red blood cells were incubated with the nanoparticles for 12 h. Thus, the hybrid NPs were compatible with red blood cells.
18 Figure S17. Biodistribution of theVes in major organs at day 1 and day 12 post-injection.