*2.1. Materials*

Poly(styrene)-b-poly((4-hydroxymethyl-[2,2-bipyri-din]-4-yl) methyl acrylate) with a molecular weight of 58,800 Da (PS-PBPyA, 42,000-b-16 800; PDI (Mw/Mn), 1.18) (P16178-SBPyA, https://www.polymersource.ca/index.php?route=product/category&path=2\_2190\_16\_105\_2205\_1069& product\_id=11572&subtract=1&serachproduct=yes&categorystart=A-1.1) and polystyrene-graftethylene-oxide functionalized with carboxy (PS-PEG-COOH, molecular weight: 36,500 Da of the PS moiety; 4600 Da of the PEG-COOH; PDI, 1.3) (P15019-SEOCOOHcomb, https://www.polymersource.ca/index.php?route=product/category&path=2\_23\_119\_2642\_1067& product\_id=2600&subtract=0&serachproduct=yes&categorystart=A-1.1) were purchased from Polymer Source, Inc. Terbium chloride hexahydrate (TbCl3 6H2O) and acetylacetone (acac) were obtained from Aladdin and SCR, Ltd. (Shanghai), respectively. 2,2-Bipyridine (bpy), tetrahydrofuran (THF) and N, N-dimethyl formamide (DMF) were provided by J&K Scientific, Ltd. (Beijing, China).

All solvents were prepared as analytical grade or chromatographically pure. All bu ffer components were of biological grade without further purification unless otherwise mentioned. Ultrapure water (≥18.2 M Ω cm) was used among all experiments. The N2 and O2 gas (>99.999%), stored in the steel cylinder, were provided by Jinghui Gas Co., Ltd. (Beijing, China). All synthetic procedures were carried out under an inert and dry nitrogen atmosphere using standard techniques.

#### *2.2. Preparation of Ru- and Tb-Metallopolymer Based NPs (Ru-Tb NPs)*

Taking advantage of the Ru-containing polymer (Ru-Poly was synthesized according to the literature [10]) and Tb-containing polymer (Tb-Poly, whose synthesis route is showed in supporting information and schematically depicted in Figure 1, along with their respective chemical structures), Ru-Tb NPs were prepared by a modified nanoprecipitation method with the schema showed in Figure 2. A mixture (200 μL, 100 ppm) of THF solution consisting of Ru-Poly, Tb-Poly, and PS-PEG-COOH at a weight ratio of 7:3:2 was quickly injected into 8 mL of distilled water in a 25 mL vial under vigorous sonication. The NPs were formed by the conglomerating of polymer as the sudden decrease in solubility and hydrophobic interaction between polymer chains. Subsequently, the solution was purged by continuous nitrogen on a 100 ◦C hot plate for 60 min to remove THF and further concentrate

the NPs suspension. The final step is filtration through a 0.2 μm micrometer syringe filter to eliminate larger aggregates. The resultant NP suspensions were stored at 4 ◦C for further use.

**Figure 1.** Schematic illustration of the synthesis of Tb-containing metallopolymer (Tb-Poly).

**Figure 2.** Schematic illustration of the preparation of Ru-Tb NPs.

#### *2.3. Oxygen Calibration of Ru-Tb NPs*

Oxygen calibrations for Ru-Tb NPs in aqueous solution were performed in a cuvette sealed with Parafilm (Chicago, IL, USA). A WITT gas mixer (KM60-2, WITT, Witten, Germany) was used to control the gas mixtures containing 0%, 5%, 10%, 15%, 20%, 25%, and 100% of O2 balanced with N2 (100%, 95%, 90%, 85%, 80%, 75%, and 0%) with an accuracy of 1% absolute, respectively. Subsequently, the O2–N2 mixtures were bubbled through the cuvette for about 10 min, at which point the luminescent intensity was recorded. The dissolved oxygen concentrations in aqueous solution were calculated by the equation

$$\text{[O}\_2\text{]}\text{/ppm} = 43 \text{ (}\% \text{O}\_2\text{/}100\text{\%)}\tag{1}$$

where % O2 is the concentration of oxygen in the bubbling gas, and 100% is the concentration of oxygen in oxygen-saturated water (43 ppm).

#### *2.4. Intracellular Localization of Ru-Tb NPs by Confocal Laser Scanning Microscopy*

HeLa cells (cancerous cervical tumor cell line) should be incubated in a 35 mm confocal culture dish at 37 ◦C for 12 h firstly. After cells adhered onto the surface of the dish and reached a density of 5 × 10<sup>4</sup> cells per dish, they could be treated with 2 mL DMEM, including Ru-Tb NPs (30 μg mL−1) for 6 h. The supernatant cell culture medium should be removed and the cells were washed by PBS three times before microscopic measurements. Unlike the adhered cells, the 3D multi-cellular tumor spheroids (MCTs) should be cultured for 7 days in a normal dish whose bottom was carpeted with agarose. Then the MCTs were treated with the same DMEM consisting Ru-Tb NPs for 12 h and transferred to the confocal dish before imaging. In all the experiments, luminescence and di fferential interference contrast (DIC) images were recorded with a Nikon A1R HD multiphoton confocal laser scanning microscope. Emission wavelengths were collected at 585–625 nm (red channel, for Ru-Poly) as the cells were excited at 488 nm, while the emissions at 525–565 nm (green channel, for Tb-Poly) were monitored under mercury lamp excitation. The same two emission channels were observed under two-photon excitation at 720 nm, while scanning Z-stack of MCTs.
