A plasmon-enhanced theranostic nanoplatform for synergistic chemo-phototherapy of hypoxic tumors in the NIR-II window

Development of simple and effective synergistic therapy by combination of different therapeutic modalities within one single nanostructure is of great importance for cancer treatment. In this study, by integrating the anticancer drug DOX and plasmonic bimetal heterostructures into zeolitic imidazolate framework-8 (ZIF-8), a stimuli-responsive multifunctional nanoplatform, DOX-Pt-tipped Au@ZIF-8, has been successfully fabricated. Pt nanocrystals with catalase-like activity were selectively grown on the ends of the Au nanorods to form Pt-tipped Au NR heterostructures. Under single 1064 nm laser irradiation, compared with Au NRs and Pt-covered Au NRs, the Pt-tipped Au nanorods exhibit outstanding photothermal and photodynamic properties owing to more efficient plasmon-induced electron–hole separation. The heat generated by laser irradiation can enhance the catalytic activity of Pt and improve the O2 level to relieve tumor hypoxia. Meanwhile, the strong absorption in the NIR-II region and high-Z elements (Au, Pt) of the DOX-Pt-tipped Au@ZIF-8 provide the possibility for photothermal (PT) and computed tomography (CT) imaging. Both in vitro and in vivo experimental results illustrated that the DOX-Pt-tipped Au@ZIF-8 exhibits remarkably synergistic plasmon-enhanced chemo-phototherapy (PTT/PDT) and successfully inhibited tumor growth. Taken together, this work contributes to designing a rational theranostic nanoplatform for PT/CT imaging-guided synergistic chemo-phototherapy under single laser activation.


Apparatus for characterization. Transmission electron microscopy (TEM) and high-resolution
transmission electron microscopy (HRTEM) measurements were carried out on a JEOL-2100 instrument operated at 200 kV. EDX High-angle annular dark field (HAADF) STEM and STEM-EDX elemental mapping were achieved on JEM-2800 (JEOL Ltd., Japan). The X-ray diffraction (XRD) patterns were tested with a Rigaku Dmax 2500 PC diffractometer equipped with Cu Kα radiation (λ = 0.15405 nm). The UV-Vis absorption spectra were recorded from SHIMADZU UV-3600 spectrophotometer (Japan). Brunauer-Emmett-Teller (BET) surface area, pore volume and pore size were measured using a Quadrasorb SI-MP instrument. Confocal fluorescence images of cells were acquired with a TCS SP5 confocal microscopy (Leica, Germany). Thermal images were taken by infrared thermal camera (Fotric 225-1, Fotric, China).
Synthesis of Au NRs. Firstly, the seed solution was prepared by injecting a freshly prepared, ice-cold NaBH 4 solution (10 mM, 600 μL) into a mixture containing HAuCl 4 (10 mM, 250 μL) and CTAB (0.1 M, 9.75 mL), followed by rapid inversion for 2 min. The resultant seed solution was kept at room temperature for 2 h prior to use. Next, the growth solution for Au NRs was prepared. covered Au NRs were prepared by as-prepared Au NRs were centrifuged (8500 rpm, 10 min) to remove 3 remaining Ag + ions from solution and were redispersed in 0.1 M CTAB, followed by reduction of platinum.

Synthesis of DOX-Pt-tipped Au@ZIF-8.
Firstly, 10 mL of the above as prepared Pt-tipped Au NRs were centrifugated at 10000 rmp for 20 min and redispersed in 10 mL of PVP (0.5g). After gently stirring for 10 h, the PVP-stabilized Au NRs were collected by centrifugation at 8,000 rpm for 30 min. The sample was redispersed in 4 mL methanol. Then, 8 mL methanol solution of 2-MIM (14.25 mg) were added and stirred for 2 min, followed by the addition of 8 mL methanol solution of Zn(NO 3 ) 2 ·6H 2 O (30.382 mg). 2 h later, the final product was washed with methanol twice at 8000 rpm for 10 min. Finally, 2 mL DOX (2 mg/mL) and 2 mL mPEG-FA (2 mg/mL) were added into the Pt-tipped Au@ZIF-8 and stirred for 48 h. The resulting DOX-Pt-tipped Au@ZIF-8 were acquired by centrifugation at 8000 rpm for 10 min twice.

Photothermal performance measurement.
To measure the photothermal effect of Au NRs, Pt-tipped Au and Pt-covered Au NRs, 2 mL of an aqueous suspension containing 100 μg/mL NRs were exposed to irradiation under 1064 nm laser (1 W·cm −2 ), respectively. The real-time temperature was recorded every 60 seconds by infrared thermal camera. The photothermal stability of Pt-tipped Au@ZIF-8 aqueous solution was measured by three cycle irradiation. Immunofluorescence of tumor hypoxia. The mice were injected with PBS (control) or the DOX-Pttipped Au@ZIF-8 (200 μL, 1 mg/mL) and exposed to 1064 nm laser irradiation (1 W·cm 2 ) for 5 min after 24 h. Then, the mice were intravenously injected with pimonidazole hydrochloride (60 mg/kg). 30 min later, the mice were sacrificed and the tumors were surgically excised, formalin fixed, then embedded in paraffin.
In order to detect pimonidazole, the tissue sections were incubated with mouse FITC-conjugated antipimonidazole antibody and rabbit anti-FITC rabbit secondary antibody (dilution 1:100) according to the 6 kit's instructions. Cell nucleis were stained with DAPI (dilution 1:2000). Images were obtained by confocal fluorescence microscopy.

In vivo biodistribution analysis. For biodistribution analysis, mice bearing 4T1 tumors were injected
with DOX-Pt-tipped Au@ZIF-8 (100 μL, 20 mg/kg) intravenously. Then the mice (n = 3) were euthanized at different time points (6 h, 12 h, and 24 h). The major organs (heart, liver, spleen, lung and kidney) and Histology analysis. After 12 day treatments, all mice were euthanized for collecting the tumors and major organs including the heart, liver, spleen, lung, and kidney. The tissues were fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and then cut with a microtome. Next, the sections were stained with hematoxylin and eosin (H&E), and the images were obtained with an optical microscope. Statistical analysis. Data were given as mean ± standard deviation. The t test was adopted to execute statistical analysis with the software SPSS. Statistical significance was denoted by an asterisk (*p < 0.05, **p < 0.01).  As shown in Fig. S2, the pore size distributions of pure ZIF-8 nanocrystals and Pttipped Au@ZIF-8 core-shell nanostructures were calculated by nonlocal density functional theory. The pore size distribution of Pt-tipped Au@ZIF-8 is composed of three 8 species of micropores with diameters of 9.9, 12.7, and 15.9 Å, which were almostly consistent with that of pristine ZIF-8.  and preferentially grow on the rod tips. After tip-coating, the average length and diameter of Pt-tipped Au NRs (over 50 rods for analysis) were 58 nm and 10 nm with the average aspect ratio of 5.8. The Pt-tipped Au NRs show strong red shift to about 1060 nm in NIR-II window owing to the dumbbell-like shape and the increase of aspect ratio during deposition, in accordance with the previous report. 2 For Pt-covered Au NRs, the Au NRs were completely covered with thin Pt layer. An obvious decrease in LSPR intensity and slight red shifts was observed, which was attributed to the thin Pt layer inhibits the light absorbance. In order to further illustrate the advantages of the Pt-tipped Au NRs, we performed FDTD simulation. Taking longer Au NRs with the average length and diameter of 62 nm and 10 nm and aspect ratio of 6.2, the LSPR absorption spectrum could be centered at about 1064 nm (Fig.S5B). However, the electric field enhancement of longer Au NRs under 1064 nm laser excitation is still lower than that of the Pt-tipped Au NRs (Fig. S5C).

Calculation of Photothermal Conversion Efficiency (η):
The photothermal conversion efficiency is calculated by formula (1) 3 : where h is the heat transfer coefficient, S is the surface area of the container, T max,NP is the maximum temperature of the solution (obtained from Figure S6), T surr is the surrounding temperature (27.4 °C, obtained from Figure S6), I is the laser power density (1 W·cm −2 ), A 1064 is the absorption value (obtained from Figure 2A) of the material at 1064 nm, and Q dis is the heat generated after water and container absorbs light. To calculate hS, equations (2) and (3) were introduced: = ℎ T max,H2O is the maximum temperature of the water (34.1 °C, obtained from Figure 2B, black curve), m D is the mass of water (0.5 g), C D is the heat capacity of water (4.2 J·g -1 ·°C -1 ), and τ s is the sample system time constant, which was calculated by formula (4)