Synthesis of PtMn nanoparticles

Typically, Pt(acac)2 (40 mg) was mixed into the solution of 1-octadecene (ODE) and dibenzyl ether (DE) (12 mL) in a 100 mL oblique three-neck ball flask. The transparent mixture solution was stirred intensely (over 300 rpm/min) and kept at 90 °C for over 1 h under the high-purity argon (99.99%). Then quickly added Mn(acac)2 (22.5 mg), using an amount of OA and OLA of 0.7 mmol. Next, the solution was further heated to about 205 ℃ and kept for over 0.75 h. The black solution was refluxed at 300 °C for over 2 h, then naturally cooled to room temperature, and washed excess ethanol and acetone in equal amounts 3 times by high-speed centrifugation (11,000 rpm/min, 20 min). After centrifugation, the nanoparticles were dispersed in 3 mL tetrahydrofuran for further characterization. The different-sized PtMn nanoparticles (PtMn-1, PtMn-2, PtMn-3) were prepared following the same procedure but using a different volume ratio of ODE and DE (1:0, 1:1, 0:1), respectively.

Synthesis of polymers

PEG-RAFT, the pH-responsive polymer or non-pH-responsive polymer was prepared according to the previous report [53, 54]. The pH-responsive polymer was prepared using a reversible addition–fragmentation chain transfer (RAFT) polymerization strategy. PEG-RAFT (30 mg), DPA (154 mg), and AIBN (0.15 mg) were dissolved in 3 mL of dioxane and added into a flask. The flask was sealed under dry argon and was kept at 70 °C for over two days. After the reaction, the solution was dialyzed (MWCO: 3.5 KDa) using ultrapure water. Finally, the solution was lyophilized to obtain a pH-responsive polymer.

For the synthesis of non-pH-responsive polymer, MM (72 mg) which was replaced with DPA added into dioxane in the flask. The other procedures were similar to those for preparing pH-responsive polymer.

Samples for MALDI-TOF and GPC characterization

2 mg pH-responsive polymer was dispersed in 1 mL tetrahydrofuran for Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF). 20 mg pH-responsive polymer was used for Gel Permeation Chromatography (GPC). The mobile phase used in the relative molecular weight test was tetrahydrofuran.

Preparation of polymer-coated PtMn

For preparing pH-responsive polymer-coated PtMn, 50 μg of different-sized PtMn nanoparticles (PtMn-1, PtMn-2, PtMn-3) and 2 mg of pH-responsive polymer were dissolved into 1 mL of tetrahydrofuran (THF). The solution was sonicated for about 20 min and was quickly added into 4 mL of ultrapure water, followed by another 20 min of sonication. Subsequently, tetrahydrofuran was evaporated, resulting in a polymer-coated PtMn solution (R-PtMn-1, R-PtMn-2, or R-PtMn-3). The resulting solution was washed with water over 3 times using a centrifugal filter tube.

For the preparation of non-pH-responsive polymer-coated PtMn-1 nanoparticles, the main procedure was similar to that for preparing responsive polymer-coated PtMn nanoparticles, except using 2 mg of non- pH-responsive polymer.

Samples for XPS characterization

0.1 mL tetrahydrofuran of PtMn-1 (2 mg/mL) was added in HEPES buffer (10 ×, 5.4) at room temperature for 1 h. After centrifugation, the nanoparticles were dispersed in 0.1 mL tetrahydrofuran. The solution was dropped on the silicon wafer and dried at room temperature for further XPS characterization.

Measurement of metal ions released from PtMn

R-PtMn-1 or Nr-PtMn-1 (0.1 mL, Mn: 0.75 mg/mL), was incubated in 900 μL of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid buffer saline) with various buffer (2 ×, pH = 4.4, 5.4, 6.4, 7.4) at 37 °C for different time points, respectively. Then the mixture solution was filtered by a centrifugal filter tube (3 KDa) and the aliquot of the filtrate was collected to determine the concentration of Pt and Mn via inductively coupled plasma–mass spectrometry (ICP-MS).

Measurement of DLS and zeta potential

R-PtMn-1 (Mn: 5 μg/mL) was incubated in 0.1 mL of HEPES buffer with various buffers (2 ×, pH = 4.4, 5.4, 6.4, 7.4) for 12 h. R-PtMn-1, R-PtMn-2, or R-PtMn-3 (Mn: 0.1 μg/mL) dispersed in H2O, DPBS, HEPES, or DMEM for 12, 24, and 48 h, respectively. Then, the above solution was diluted (1/40) for measuring DLS. 0.1 mL of Nr-PtMn-1 or R-PtMn-1 (Mn: 5 μg/mL) was incubated in 0.1 mL of HEPES buffer with various buffers (2 ×, pH = 4.4, 5.4, 6.4, 7.4) for 6 h. Then, the above solution was diluted (1/40) for measuring zeta potential.

Measurement of OXD activity in solution

To test catalytic activity via 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) assay, 50 μL of Nr-PtMn-1, R-PtMn-1, R-PtMn-2 or R-PtMn-3 (50 μg/mL) was incubated in 50 μL of TMB (1.5 mM) and 50 μL of various buffer (10 ×, pH = 5.4, 6.0, 6.4, 6.8, 7.4,) for 1 h. For ox-TMB of R-PtMn-1 in solution, the absorption of 100 μL of the mixture which was incubated at various time points was recorded at 650 nm with an ultraviolet–visible (UV–Vis) spectrometer to reveal the catalytic activity.

To test the dynamic process of catalytic activity, 50 μL of R-PtMn-1, R-PtMn-2, or R-PtMn-3 (50 μg/mL) was incubated in 50 μL of TMB (1.5 mM) and 50 μL of HEPES buffer (10x, pH = 6.0). After incubation for different times (0–30 min), the absorption of TMBox was recorded at 650 nm by UV–Vis spectrometer.

Measurement of GSH consumed

Various HEPES buffer (0.2 mL, 1x, pH = 7.4, 6.4, 5.4, or 4.4) containing Nr-PtMn-1 or R-PtMn-1 (50 μg/mL) and GSH (2 mM) was prepared and incubated at 37 ℃ for 12 h, respectively, followed by centrifugation. Then, 50 µL of supernatant was collected and further incubated with 50 µL of colorimetric 5,5′-dithiobis-2-(nitrobenzoicacid) (DTNB) [~ 1.2 mg/mL in dimethyl sulfoxide (DMSO)] for about 15 min. The above solution was diluted (1/6) for measurement of absorption at 412 nm (OD) to determine GSH consumed content via the following Eq. 1:

$$GSH\, comsumed\, content \left(\%\right)=\frac_-O_}_}\times 100 \%$$

(1)

Measurement of T1/T2-relaxation time

Various HEPES buffers (0.2 mL, 1 ×, pH = 7.4, 6.4, 5.4, 4.4) containing R-PtMn-1, R-PtMn-2 or R-PtMn-3 (Mn: 50 μg/mL) was incubated at 37 ℃ for 1 h. Various HEPES buffers (0.2 mL, 5 ×, pH = 7.4, 6.8, 6.4, 5.4, 4.4) containing R-PtMn-1 (Mn: 25, 50, 100, 200 μg/mL) was incubated at 37 ℃ for 6 h. Then, T1 or T2-relaxation time was tested via Bruker Minispec analyzer (60 MHz, Bruker, Germany).

For T1- or T2-MRI phantom imaging, various HEPES buffers (0.2 mL, 5 ×, pH = 7.4, 6.8, 6.4, 5.4, 4.4) containing R-PtMn-1 (Mn: 12.5, 25, 50, 100 μg/mL) was incubated at 37 ℃ for 6 h. Then, those samples were scanned by using a Bruker 7 T-MRI scanner with using T1-MRI sequence (field of view = 30 mm × 30 mm, size = 256 × 256, slice thickness = 0.7 mm, repetition time (TR) = 225.58 ms, and effective echo time (TE) = 4.5 ms) or T2-MRI sequence (field of view = 30 mm × 30 mm, size = 256 × 256, slice thickness = 0.7 mm, TR = 2500 ms, TE = 35 ms).

Cellular experiment

The mouse breast carcinoma (4T1) cells, mouse colorectal cancer (CT26) cells as cancer cells, and human embryonic kidney cell line (HEK293 cells) as normal cells were incubated on the cell culture plate in Dulbecco’s Modified Eagle Medium (DMEM) containing 1% penicillin/streptomycin and 10% of fetal bovine serum at 37 °C with 5% CO2.

Intracellular ROS evaluation

To assess the intracellular ROS production, 4T1 cells pre-seeded in optical cultured dishes were treated with Nr-PtMn-1 or R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 6 h. 4T1 cells pre-seeded in optical cultured dishes were treated with R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 2, 4, or 6 h. 4T1 cells pre-seeded in optical cultured dishes were treated R-PtMn-1 (0, 7.5, 15, 30 μg/mL) pre-seeded in optical cultured dishes for 4 h. HEK293 cells pre-seeded in optical cultured dishes were treated with R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 6 h. After being washed with DPBS over 3 times, those cells were stained with DCFH-DA (10 μM) and Hoechst (1 μg/mL) for 0.5 h, respectively. Then, the fluorescent emission of DCFH-DA (Ex = 488 nm, Em = 530 nm) was observed using a confocal laser scanning microscope (CLSM) to test intracellular ROS. The relative fluorescence intensity was measured by ImageJ software.

Intracellular LPO evaluation

To assess the intracellular LPO production, 4T1 cells pre-seeded in optical cultured dishes were treated with Nr-PtMn-1 or R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 2 h. 4T1 cells pre-seeded in optical cultured dishes were treated R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 0.5, 1, 1.5, or 2 h. 4T1 cells pre-seeded in optical cultured dishes were treated R-PtMn-1 (0, 7.5, 15, 30 μg/mL) pre-seeded in optical cultured dishes for 2 h. HEK293 cells pre-seeded in optical cultured dishes were treated with R-PtMn-1 (30 μg/mL) pre-seeded in optical cultured dishes for 2 h. After being washed with DPBS over 3 times, those cells were stained with liperfluo (10 μM) and Hoechst (1 μg/mL) for 0.5 h, respectively. Then, the fluorescent emission of liperfluo (Ex = 488 nm, Em = 500–550 nm) was detected using CLSM. The relative fluorescence intensity was measured by ImageJ software.

Mitochondrial membrane potential evaluation

To evaluate the change of mitochondrial membrane potential, 4T1 cells were pre-seeded in optical cultured dishes were treated with Nr-PtMn-1 or R-PtMn-1 (40 μg/mL) for 2 h. After being washed with DPBS 3 times, those cells were stained with JC-1 (10 μM) for 0.5 h. The fluorescent emission of JC-1 (Ex = 514 nm, Em = 529 nm; Ex = 585 nm, Em = 590 nm) was detected using CLSM. The relative fluorescence intensity of JC-1 was measured by ImageJ software.

Intracellular GSH evaluation

To test intracellular GSH content, 4T1 cells were pre-incubated in 6-well plates incubated with Nr-PtMn-1 or R-PtMn-1 (30 μg/mL) for 24 h. 4T1 cells pre-incubated in 6-well plates incubated with R-PtMn-1 (0, 7.5, 15, 30 μg/mL) for 24 h. 4T1 cells pre-incubated in 6-well plates incubated with R-PtMn-1 (30 μg/mL) for 6, 12, or 24 h. 4T1 cells were disrupted, and the cell lysate was frozen by liquid nitrogen and dissolved at 37 ℃ for three times. 40 μL of the supernatant was collected by centrifugation at 4 ℃ (10,000 rpm, 8 min) and was incubated with 280 μL of reagent 2 and 80 μL of reagent 3 from the GSH assay kit (BC1175, Solarbio) for 10 min. The absorption of the mixture was detected via a microplate reader at 412 nm (OD) and the concentration of GSH was calculated using the following Eq. 2:

$$GSH\, content \left(\%\right)=\frac\times 100 \%$$

(2)

Intracellular MDA evaluation

To measure intracellular malondialdehyde via malondialdehyde (MDA) assay kit, 4T1 cancer cells pre-seeded in a 6-well plate were incubated with Nr-PtMn-1 or R-PtMn-1 (30 μg/mL) for 24 h, respectively. Next, those cells were lysed and the cell lysate was collected. 50 μL of the supernatant was collected by centrifugation at 4 ℃ (12,000 rpm, 8 min) and then was mixed with 150 μL of working solution and 50 μL of reagent 3 from MDA assay kit (BC0025, Solarbio). The mixture was boiled for 1 h at over 95 ℃ and cooled to room temperature. 180 μL of supernatant was collected through centrifugation (10,000 rpm, 5 min), and the absorption of supernatant was measured via a microplate reader at 450, 532, and 600 nm (OD450, OD532, OD600). Subsequently, the concentration of MDA content was calculated according to Eqs. 3 and 4.

$$\Delta OD=OD\left(Test\right)-OD(Blank)$$

(3)

$$MDA =0.01\times (12.9\times \left(\Delta OD532-\Delta OD600\right)-2.58\times \Delta OD450)$$

(4)

Intracellular WB assays

To measure GPX4, BID, or ACSL4 expression by western blot assay, 4T1 cancer cells pre-seeded in a 6-well plate were treated with Nr-PtMn-1 or R-PtMn-1 (30 μg/mL) for about 6 h, respectively. Afterward, those cells were washed with ice-cold DPBS, harvested and the cell lysate was boiled for over 10 min. Then the proteins were further transferred to a 0.45 μm polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked in Tris-buffered saline containing Tween 20 and 5% dry skim milk (TBST), and incubated with rabbit GPX4, BID, or ACSL4 antibody (1: 1000, Absin) and β-actin antibody (1: 1000, Servicebio) for 12 h at 4 ℃. The membrane was washed with TBST and followed by the secondary antibody (1: 1000, YiShan Biotech) incubation for over 1 h. Finally, the membrane was washed with TBST and the band of each protein was captured with an enhanced chemiluminescent detection system. We have quantified the GPX4, ACSL-4, BID, and β-actin levels by image j for calculating the relative GPX4, ACSL-4, and BID levels.

Intracellular cytotoxicity evaluation

To investigate the inhibition of cellular viability, CT26 cells and 4T1 cells received the following treatment:

CT26 cells pre-seeded into 96-well plates were treated with R-PtMn-1, R-PtMn-2, or R-PtMn-3with various concentrations (0, 30, 60, 120, 240 μg/mL) for 24 h, respectively. 4T1 cells pre-seeded in a 96-well plate were treated with R-PtMn-1 (240 μg/mL) for different incubation times (0, 4, 10, 18, 30 h), respectively. 4T1 cells pre-seeded in a 96-well plate were treated with Nr-PtMn-1, R-PtMn-1, R-PtMn-2 or R-PtMn-3 at different concentrations (0, 30, 60, 120, 240 μg/mL) for 24 h, respectively.

HEK cells pre-seeded into 96-well plates were treated with R-PtMn-1 with various concentrations (0, 30, 60, 120, 240 μg/mL) for 24 h, respectively.

After being washed with DPBS 3 times, those cells were incubated with 200 μL of DMEM containing MTT (0.5 mg/mL) for 3 h. After that, the solution was removed and each well was added with 180 μL of DMSO. After incubation at 37 ℃ for over 0.5 h, the absorption at 490 nm was detected via a microplate reader, and the relative cell viability was calculated according to the standard MTT method.

Cancer imaging in vivo

All animal experiments were approved by the Institutional Animal Care and Use Committee of Hunan University (SYXK 2018-0006).

For preparing the tumor model, female BALB/c mice were subcutaneously injected using about 50 μL DPBS solution with 4T1 or CT26 tumor cells (~ 1 × 106).

For in vivo T1 or T2 MRI imaging, 4T1 tumor-bearing mice were i.t. injected with Nr-PtMn-1 or R-PtMn-1 (25 μL, Mn: 20 μg/mL) or i.v. injected with R-PtMn-1 (200 μL, Mn: 700 μg/mL), respectively. Then, those mice were immediately anesthetized with isoflurane in oxygen and scanned by 7 T-MRI scanners (PharmaScan 70/16 US, Burker), using T1-MRI sequence (size = 384 × 384, FOV = 30 mm × 30 mm, slice thickness = 0.7 mm, TR = 230.5 ms, and TE = 4.5 ms) or T2-MRI sequence (size = 256 × 256, FOV = 30 mm × 30 mm, slice thickness = 0.7 mm, TR = 2500 ms, and TE = 35 ms).

Catalytic cancer therapy in vivo

For cancer therapy in vivo, both i.t. and i.v. the administration was performed. CT26 tumor-bearing female mice were randomly divided into 4 groups (n = 5) and followed the following administration: (1) None treatment as the control group; (2) Nr-PtMn-1 (25 µL, Mn: 0.7 mg/mL), (3) R-PtMn-1 (25 µL, Mn: 0.7 mg/mL, i.t.), (4) R-PtMn-1 (200 µL, Mn: 0.7 mg/mL, i.v.). 4T1 tumor bearing female mice were randomly divided into 2 groups (n = 5) and received the following administration: (1) None treatment as the control group; (2) R-PtMn-1 (200 µL, Mn: 0.7 mg/mL, i.v.). The body weights and tumor volumes of mice from each group were recorded every other day during the 14 days of study. The volume of the tumor was calculated as Length × Width2/2. All groups of mice were sacrificed on the 14th day, and then the tumor weight of each group was recorded. Representative tumors were taken out on the second day, and the main five organs of representative mice from each group were collected after the 14th-day post-injection for H&E and TUNEL staining, via the standard protocol, and examined using a Pannoramic MIDI microscope (3DHIESTECH, Hungary).

For DCFH-DA or liperfluo staining, those tissues were collected from mice for cryo-sections. Then, tumors’ slices were stained with DCFH-DA (10 µM, Ex = 488 nm, Em = 530 nm) and DPAI (1 μg/mL, Ex = 358 nm, Em = 461 nm) for about 2 h, respectively. Finally, the fluorescent confocal images of those tissues were collected by CLSM. The relative fluorescence intensity was measured by ImageJ software.

Statistical analysis

Statistics analysis was shown as mean ± standard deviation (SD). All experiments were carried out at least three times. A significant difference (*p < 0.05, **p < 0.01, ***p < 0.001) was done by Student’s t-test.

Data availability

All relevant data are available from the authors.