Enzyme‐Empowered “Two Birds with One Stone” Strategy for Amplifying Tumor Apoptosis and Metabolic Clearance

Abstract Nanomedicine has reshaped the landscape of cancer treatment. However, its efficacy is still hampered by innate tumor defense systems that rely on adenosine triphosphate (ATP) for fuel, including damage repair, apoptosis resistance, and immune evasion. Inspired by the naturally enzymatic reaction of glucose oxidase (GOx) with glucose, here a novel “two birds with one stone” technique for amplifying enzyme‐mediated tumor apoptosis and enzyme‐promoted metabolic clearance is proposed and achieved using GOx‐functionalized rhenium nanoclusters‐doped polypyrrole (Re@ReP‐G). Re@ReP‐G reduces ATP production while increasing H2O2 concentrations in the tumor microenvironment through GOx‐induced enzymatic oxidation, which in turn results in the downregulation of defense (HSP70 and HSP90) and anti‐apoptotic Bcl‐2 proteins, the upregulation of pro‐apoptotic Bax, and the release of cytochrome c. These processes are further facilitated by laser‐induced hyperthermia effect, ultimately leading to severe tumor apoptosis. As an enzymatic byproduct, H2O2 catalyzes the conversion of rhenium nanoclusters in Re@ReP‐G nanostructures into rhenate from the outside in, which accelerates their metabolic clearance in vivo. This Re@ReP‐G‐based “two birds with one stone” therapeutic strategy provides an effective tool for amplifying tumor apoptosis and safe metabolic mechanisms.


Synthesis of ReP and Re@ReP:
In a 100 round-bottom flask, 50 mg PVA and 19 mL deionized (Di) water were added, and the temperature was increased to 90 °C with gentle stirring for 1 h.
Then 100 mg Re2O7 pre-dissolved in 1 mL ethanol was slowly added and continuously stirred at 90 °C.After 0.5 h, 100 μL pyrrole monomer and 80 mg pyrrole-3-carboxylic acid dissolved in 1 mL Di water were added to the above mixture and stirred for 23 h.For the preparation of rhenic acid-doped polypyrrole (ReP), the mixture was cooled to room temperature and then centrifuged at 18000 rpm for 10 min to collect the precipitate, which was washed three times with Di water.For the preparation of rhenium nanoclusters-doped polypyrrole (Re@ReP), the mixture was cooled to room temperature and then 4 mL NaBH4 (4 mg mL -1 ) was added and stirred for 3 h.The final product was centrifuged at 18000 rpm for 10 min to collect the precipitate, which was washed three times with Di water.

Synthesis of Re@ReP-G:
At first, 10 mg Re@ReP was completely dispersed in 8 mL Di water, and then 2 mL of a mixed aqueous solution of EDC/NHS (EDC: 10 mg; NHS: 6.5 mg) was added and stirred for 1 h.Subsequently, 5 mg GOx (pre-dissolved in 1 mL Di water) was added dropwise to the above mixture and stirred overnight at room temperature.Finally, the mixture was purified in a dialysis bag (300 kD) for 48 h.The obtained Re@ReP-G was stored at 4 °C for further experiments.
Enzymatic activity of Re@ReP-G: 225 µg/mL of Re@ReP-G was stirred in PBS solution with or without glucose (10 mM) at various temperature (25 °C and 45 °C).At different time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 min), the absorbance of the supernatant at 560 nm was measured using a hydrogen peroxide detection kit to calculate the concentration of H2O2.The corresponding pH values were measured using a pH meter.
Photothermal performance of Re@ReP-G: Different concentrations of Re@ReP-G aqueous solutions (0, 25, 50, 100, and 200 μg mL -1 ) were exposed to an 808 nm or 1064 nm laser at various power densities (0.5, 1.0, 1.5, and 2.0 W cm -2 ) for 5 min.An IR thermal camera (Ti480U, Fluke, USA) was used to monitor the temperature change of solutions in real time.Furthermore, the photothermal stability and heating-cooling curve of Re@ReP-G aqueous solution (100 μg mL -1 ) were assessed under irradiation of an 808 nm or 1064 nm laser (1.0 W cm -2 ) for five on/off cycles (on: 6 min, off: 9 min).
In vitro degradation experiments: 100 µg mL -1 of Re@ReP-G was first dispersed in pure PBS, PBS with H2O2 (100 mM), or PBS with glucose (10 mM) and then co-cultured at 37 °C for various times.The laser irradiated group was irradiated with a 1064 nm laser with a power density of 1 W cm -2 for 5 min after 1 h of co-incubation.At defined time points (6, 12, and 24 h), certain mixtures were extracted and centrifuged for TEM observation.In the end, XPS analysis was performed to identify changes in composition.

Cellular uptake:
To assess the cellular internalization behavior of Re@ReP-G, the nanoparticles were labeled with the near-infrared dye Cy5 and co-incubated with MCF-7 cells for different times (0, 2, 4, 8, and 12 h) at a Cy5 concentration of 5 μg mL -1 .For microscopy imaging, the nuclei were stained with DAPI and imaged by confocal laser scanning microscopy (CLSM, LSM710, Carl ZEISS, Germany).For quantitative analysis, the cells were collected and analyzed by flow cytometry (Celesta, BD, USA).
To further evaluate in vitro photothermal cytotoxicity of Re@ReP-G, MCF-7 cells were treated with PBS, Re@ReP, or Re@ReP-G (equal Re@ReP concentration: 50 or 75 μg mL -1 ) for 4 h.Afterward, cells in the laser-required groups were exposed to a 1064 nm laser with a power density of 1 W cm -2 for 5 min and continued to be incubated for 24 h.Finally, the cells were washed twice with PBS and cell viability was measured according to the standard protocol of CCK-8 assay.For cell apoptosis assays, MCF-7 cells were treated as described above and then incubated with Annexin V-FITC and propidium iodide (PI) in the dark according to the manufacturer's instructions.Finally, the cells were washed with PBS and immediately analyzed using flow cytometer (Celesta, BD, USA).
Intracellular ATP and H2O2: Cellular ATP levels were detected using the enhanced ATP assay kit.In brief, MCF-7 cells were treated with PBS, Re@ReP, or Re@ReP-G (equal Re@ReP concentration: 75 μg mL -1 ) for 24 h.Next, the cells were washed, lysed, and centrifuged to collect the suspension, which was analyzed using the ATP assay kit according to the manufacturer's instructions.As for the assessment of cellular H2O2 levels, MCF-7 cells were treated as described above and then incubated with DMEM containing BES-H2O2-Ac (10 μM) for 30 min.Finally, the cells were analyzed using flow cytometer and visualized by CLSM.
Intracellular HSP and apoptosis index: MCF-7 cells were seeded in confocal dish (1.5 × 10 5 per well) or 6-well plates (3 × 10 5 per well) and cultured for 24 h.Then, the media were replaced with fresh DMEM medium containing 75 μg mL -1 of Re@ReP or Re@ReP-G.After 4 h of incubation, cells in the groups requiring laser irradiation were exposed to a 1064 nm laser (1 W cm -2 , 5 min) and continued to be incubated for 2 h.For CLSM observation, the cells on confocal dish were co-incubated with Anti-HSP70 Monoclonal Antibody or Anti-HSP90 β Monoclonal Antibody for 2 h, and then co-incubated with Goat Anti-Rabbit IgG(H+L) Alexa Fluor 488 for 1 h.After that, the cells were washed and visualized by CLSM.As for western blotting assay, the cells on 6-well plates were collected and lysed in an ice-bath, then the total proteins were extracted with lysate buffer and quantified with different protein assay kits.In vivo biological distribution: Nude mice bearing MCF-7 tumor were intravenously injected with Cy5-labelled Re@ReP-G at Cy5 dose of 1.5 mg kg -1 .At defined time intervals (3, 6, 9, 12, and 24 h) after injection, nude mice were imaged by animal live imaging system (CRi, USA).
At the end of the experiment, all nude mice were sacrificed to collect tumors and major organs (heart, liver, spleen, lung, and kidney) for ex vivo imaging.
In vivo photoacoustic imaging: For in vivo photoacoustic (PA) imaging, nude mice bearing MCF-7 tumor were administrated with Re@ReP-G at a dose of 12.5 mg kg-1 via intravenous injection.At different time points (0, 4, 6, 12, and 24 h), PA images of the tumor site and quantitative analysis of PA signal intensities were performed and the signs were recorded.

In vivo antitumor therapy:
To investigate the therapeutic efficacy in vivo, MCF-7 tumorbearing nude mice were randomly divided into six groups (n=5): (1) PBS group (Control), (2) Laser group (L), (3) Re@ReP group, (4) Re@Re-G group, (5) Re@ReP+Laser group (Re@ReP+L), ( 6) Re@ReP-G+Laser group (Re@ReP-G+L).In groups (1) and ( 2), nude mice were injected intravenously with PBS, a 12.5 mg kg -1 dose of Re@ReP in groups (3) and ( 5), and the same dose of Re@ReP as Re@ReP was injected intravenously with Re@ReP-G in groups ( 4) and ( 6).For the groups that needed laser treatment, a 1064 nm laser (1 W cm -2 , 5 min) were irradiated to the tumor sites 12 h after injection.Furthermore, each mouse's body weight and tumor volume were assessed every two days for 16 days, and the tumor volume was determined using the formula tumor volume =1/2 × (tumor width) 2 × (tumor length).At 16 days, all tumors and organs (heart, liver, spleen, lung, and kidney) were extracted for further histological analysis.

In vivo pharmacokinetics:
The nude mice bearing MCF-7 tumor were intravenously injected with Re@ReP-G (12.5 mg kg -1 ) (n=3).At specific time intervals (10,20,40,60,120,240,480,720, and 1440 min) after injection, the blood of nude mice was collected for ICP to measure the Re concentration.The nude mice bearing MCF-7 tumor were intravenously injected with Re@ReP-G (12.5 mg kg -1 ) (n=3).At specific time intervals (6, 12, and 24 h) after injection, the tumors and organs (heart, liver, spleen, lung, and kidney) of nude mice were collected for ICP to measure the Re concentration.

Statistical analysis:
All statistical analyses were used GraphPad Prism.The results of statistical analysis were presented as mean ± SD.Statistical significance was calculated by one-way ANOVA analysis.The statistical significance was defined as *p < 0.05; **p < 0.01; ***p < 0.001.

Animal model :
All animal experiments were approved by the Institutional Animal Care and Use Committee of Chinese Academy of Medical Sciences-Peking Union Medical College Institute of Radiation Medicine (Approval number: IRM-DWLL-2023203).Female BALB/c nude mice (17-20 g, 4-6 weeks old) were obtained from SPF Biotechnology Co. Ltd.All animal experiments were rigorously conducted in accordance with the regulations on animal use and care.5 × 10 6 MCF-7 cells suspended in PBS solution were subcutaneously injected into the right back of BALB/c nude mice.When the tumor became distinct and the tumor volume reached ~80 mm 3 , the nude mice were randomly divided into different groups.

Figure S1 .
Figure S1.(A) Hydrodynamic particle size and zeta potentials of ReP, Re@ReP, and Re@ReP-G.

Figure S2 .
Figure S2.(A) Hydrodynamic particle size and (B) corresponding TEM images of Re@ReP with different adding amounts of NaBH4.Scale bar: 200 nm.(C) Hydrodynamic particle size and (D) corresponding TEM images of Re@ReP-G with different adding amounts of GOx.Scale bar: 200 nm.(E) Hydrodynamic particle size and (F) corresponding TEM images of Re@ReP with different adding amounts of pyrrole (Py) and pyrrole-3-carboxylic acid (Py-COOH).Scale bar: 200 nm.

Figure S6 .
Figure S6.(A) Hydrodynamic diameter changes and (B) corresponding PDI changes of Re@ReP-G dispersed in different physiological media at varied time points.

Figure S7 .
Figure S7.(A) UV-vis-NIR spectra of various concentrations of Re@ReP-G.Linear relationship between concentration and absorbance of Re@ReP-G at (B) 808 and (C) 1064 nm.

Figure S8 .
Figure S8.(A) Temperature profiles of different concentrations of Re@ReP-G under laser irradiation (808 nm, 1.0 W cm -2 , 5 min) and (B) the corresponding photothermal pictures.(C) Temperature profiles of Re@ReP-G exposed to different power densities (0.5-2.0 W cm -2 ) of 808 nm laser.(D) Photothermal conversion stability of Re@ReP-G aqueous solution for five laser on/off cycles under the irradiation of 808 nm laser.(E) Photothermal heating and cooling curves of Re@ReP-G under 808 nm laser irradiation and (F) corresponding linear relationship between time and -lnθ from the cooling period.

Figure S9 .
Figure S9.(A) Temperature profiles of Re@ReP-G exposed to different power densities (0.5-2.0 W cm -2 ) of 1064 nm laser.(B) Photothermal conversion stability of Re@ReP-G aqueous solution for five laser on/off cycles under the irradiation of 1064 nm laser.(C) Photothermal heating and cooling curves of Re@ReP-G under 1064 nm laser irradiation and (D) corresponding linear relationship between time and -lnθ from the cooling period.

Figure S11 .
Figure S11.Cell viability of MCF-7 or 3T3 cells incubated with various concentrations of Re@ReP for 48 h.

Figure S13 .
Figure S13.PI and calcein AM staining to identify live/dead cells (red: dead, green: live) with different treatments, scale bar: 100 μm.

Figure S17 .
Figure S17.Detection of Re in urine and feces after injection of Re@ReP-G at various time intervals.

Figure S19 .
Figure S19.Hemolysis assay of red blood cells treated with water, PBS and Re@ReP-G dispersed in PBS at different concentrations.