Pluronic F127 (Mw: 12.6 kDa) was obtained from Sigma-Aldrich (USA). Dopamine hydrochloride (DA·HCl), 1,3,5-trimethylbenzene (TMB), dimethyl sulfoxide (DMSO), cisplatin (Pt, 65%), and cyanine-5.5 (Cy5.5) were purchased from Aladdin (Shanghai, China). Ammonium hydroxide (25.0–28.0%), ethanol, potassium permanganate (KMnO4), and hydrogen peroxide (H2O2, 30% v/v) were procured from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Isopropyl-β-d-thiogalactoside (IPTG), 2-Mercaptoethanol (2-ME), 6-Carboxyfluorescence (6-FAM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), glutathione (GSH), 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA), calcein-AM, and propidium iodide (PI) were sourced from Sigma-Aldrich (USA). High-glucose Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, penicillin and streptomycin, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), and phosphate-buffered saline (PBS) were obtained from Gibco (Carlsbad, CA). Tris(hydroxymethyl)aminomethane buffer (Tris-buffer, pH 8.6), McCoy’s 5A medium supplemented with penicillin (100 U/mL) and streptomycin (100 U/mL) were acquired from Jiangsu KeyGEN BioTECH Co., Ltd (Nanjing, China). Human umbilical vein endothelial cell (HUVEC), breast cancer cell line MCF-7, and Her2-positive human ovarian cancer cell line SKOV-3 were provided by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Deionized (DI) water (>18.2 MΩ·cm) used for all experiments was purified using a Millipore system, and all chemicals were used without further purification.
Preparation of [email protected]/MnO2/PDA NPs
Mesoporous polydopamine nanoparticles (mPDA NPs) were synthesized according to a nanoemulsion assembly approach  with some slight modifications. In details, 2.0 g of Pluronic F127 and 1.0 g of DA·HCl were dissolved in 200 mL of 50% v/v ethanol, and stirred at 1000 rpm for 3 h. Then, 1 mL of TMB was added dropwise to the mixture and sonicated for 5 min to allow generation of nano-emulsion. After stirring for another 30 min at 500 rpm, 10 mL of NH4OH was added to the resultant mixture while stirring under aerobic conditions to induce the self-polymerization of dopamine. After another 3 h of continuous reaction, and then centrifugation, the mPDA NPs were collected and washed thoroughly with absolute ethanol, and then dispersed in PBS for further use. For drug loading, 200 μL of DMSO containing cisplatin (25 mg) was added to the mPDA NPs (50 mg, 50 mL in PBS), followed by sonication for 30 min, and stirred for 24 h in the dark. The cisplatin-loaded mPDA NPs ([email protected] NPs) were collected after centrifugation and washed with DI water.
The [email protected] NPs were re-dispersed into DI water (50 mL; pH 7.4), and 50 mg of KMnO4 was added while stirring at 400 rpm for 6 h. After sonication for another 6 h, the mixture was centrifuged and washed thoroughly with DI water, yielding [email protected]/MnO2 NPs. Finally, the obtained [email protected]/MnO2 NPs were re-dispersed in Tris-buffer (100 mL; pH = 8.6) and 50 mg of DA was added. The mixture was stirred (400 rpm) for 4 h, and then washed with DI water. This yielded [email protected]/MnO2/PDA NPs which was re-dispersed into DI water for further use.
Preparation and characterization of ZHer2 affibody
ZHer2 affibody was expressed and purified according to the method of our previous work  with some modifications. In details, a gene encoding the anti-Her2 affibody molecule  with adding a cysteine on the C-terminus was synthesized by GenScript (Nanjing, China) and cloned into pQE30 at the BamHI and SalI sites to construct the expression plasmid pQE30-ZHer2. The plasmid was transformed into E. coli M15 and induced overnight with 0.05 mM IPTG at 28 °C. Subsequently, the cells were collected by centrifugation, resuspended in lysis buffer (50 mM phosphate, pH 8.0, 300 mM NaCl, and 20 mM imidazole) and then sonicated on ice for 30 min to lyse the cells. The recombinant proteins in the supernatant were purified using Ni-NTA affinity chromatography according to the manual provided by the manufacturer (GenScript, Nanjing, China). The samples collected during the ZHer2 preparation process were detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using Image J software (Bethesda, MD, USA). The purified affibody was dialyzed against phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) and then quantified using the Bradford Protein Assay Kit (Beyotime, Jiangsu, China).
The aggregation form of ZHer2 was detected using SDS-PAGE with or without 2-Mercaptoethanol (2-ME) in the loading buffer. For the specific cell binding assay, ZHer2 was labelled with 6-FAM (Ruixi Biology, Xi’an, China) according to the manufacturer’s protocol: 10 μL of 6-FAM (10 mg/mL; in DMF) was added to 5 mL of ZHer2 solution (2 mg/mL; pH 8.3, in PBS). The reaction continued for 1 h at 25 °C in the dark before the mixture was dialyzed against PBS (pH 7.4) at 4 °C for 48 h to remove unreacted 6-FAM. The conjugation of 6-FAM to the ZHer2 affibody was verified by SDS-PAGE.
Conjugation of ZHer2 affibody to NPs
The [email protected]/MnO2/PDA-ZHer2 NPs were constructed by conjugating ZHer2 to [email protected]/MnO2/PDA via a Michael addition/Schiff base reaction between the amino group and the oxidized quinone form of catechol groups at weak alkaline pH conditions [49, 50]. The chemical stability of ZHer2 in reactive Tris-buffer (pH 8.6) was first evaluated before reaction. Two equal amounts of ZHer2 were diluted in PBS (pH 7.4) or Tris-buffer (pH 8.6), respectively. The samples were incubated at 37 °C while stirring. At predetermined time points, 1 mL of sample was collected and measured using a UV-Vis spectrometer.
Subsequently, [email protected]/MnO2/PDA NPs were dispersed in Tris-buffer (100 mL; 0.5 mg/mL), and 1 mL of ZHer2 solution (1 mg/mL; in PBS) was added into the dispersion followed by sonication (40 kHz; 70 W) for 30 min. Then, the mixture was stirred overnight at 25 °C. The unreacted ZHer2 was removed by centrifugation and thoroughly washed with DI water. This yielded [email protected]/MnO2/PDA-ZHer2 NPs which was re-dispersed in PBS for further use.
The morphologies of the NPs were observed by transmission electron microscopy (TEM, JEOL 2010F) at an accelerated voltage of 200 kV. The surface area and pore size of the mPDA NPs were measured using an automated surface area and porosity analyzer (Quantachrome, Autosorb-iQ). X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Fisher ESCALAB 250Xi spectrometer to determine the chemical state of MnO2. Zeta potential and particle size distribution were measured using a Malvern Zetasizer (Nano-ZS, Malvern, UK). UV-Vis absorbance spectra were recorded on a UV-2100 spectrophotometer. The concentrations of MnO2, cisplatin and ZHer2 in the NPs were analyzed by measuring Mn, Pt and S element using inductively coupled plasma-atomic emission spectrometry (ICP-AES, Prodigy, LEEMAN).
Biodegradation and in vitro drug release
[email protected]/MnO2/PDA NPs were dispersed in PBS (pH 7.4) supplemented with (1) 1 mM H2O2, (2) 1 mM H2O2, 2 mM GSH, (3) 1 mM H2O2, 5 mM GSH, and incubated at 37 °C while shaking (120 rpm). At each expected time point, 1 mL of each sample was taken for UV-Vis absorbance measurement. After 2 weeks, each sample (200 μL) was added into 24-well plates in triplicate and imaged with a digital camera. Subsequently, the residual samples were centrifugated and re-dispersed in ethanol. TEM images were collected to determine the extent of degradation.
The concentration of the drug release from the NPs was measured according to the dialysis method of our previous work  with some modifications: 3 mg of [email protected]/MnO2/PDA NPs was dispersed in PBS (2 mL; pH 7.4), and then loaded into a dialysis bag (MWCO = 7000 Da) and immersed in 18 mL of PBS containing: (1) 1 mM H2O2, pH 7.4; (2) 1 mM H2O2, 5 mM GSH, pH 7.4; (3) 1 mM H2O2, 5 mM GSH, pH 5.5. All samples were incubated at 37 °C while shaking (120 rpm) for 2 days. At predetermined time points, 1 mL of external medium was extracted and supplemented with an equal volume of fresh pre-heated medium. The concentration of the released cisplatin was determined quantitatively by ICP-AES. All experiments were evaluated in triplicate, and the data was presented as mean ± standard deviation (S.D.), n = 3.
Measurement of dissolved O2
The ability of MnO2-based NPs to catalyze the decomposition of H2O2 to O2 was measured using a dissolved oxygen meter (JPSJ-605, INESA). [email protected]/MnO2/PDA NPs were dispersed in PBS ([MnO2] = 2 μg/mL) and then transferred into a double-neck flask. H2O2 (30% w/v) was added to the dispersion at a final concentration of 1 mM. A blank PBS or PBS containing H2O2 was used as a control medium. The concentration of dissolved O2 was measured by the probe at predetermined time points.
GSH-triggered T1-weighted MRI
[email protected]/MnO2/PDA NPs with varied concentrations ([Mn] = 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM; in PBS) were each treated with 0 or 2 mM GSH. After 20 min, the T1-weighted relaxation times were measured using a 0.5 T NMI20 NMR Analyzing and Imaging system (Niumag, Shanghai, China) at 25 °C. The test parameters were the same as those reported in a previous work . The T1 relaxivity (r1) was acquired through a linear fitting of 1/T1 as a function of Mn concentration. In addition, T1 MRI was performed for samples at different concentrations using a clinical MR system (1.5 T, SIEMENS MAGNETOM Symphony).
In vitro cellular uptake evaluation
The expression of Her2 in the breast cancer cell line MCF-7 and human ovarian cancer cell line SKOV-3 was evaluated. MCF-7 and SKOV-3 cells were incubated in DMEM or McCoy’s 5A medium supplemented with 1% penicillin, 1% streptomycin, and 10% fetal bovine serum. The cells were cultured at 37 °C in a 5% CO2 humidified atmosphere. Afterwards, MCF-7 cells and SKOV-3 cells (2 × 105) were digested and resuspended in DMEM or McCoy’s 5A medium containing 2 or 4 μg/mL FITC-anti-Her2 antibody (Sino Biological, Beijing, China) at 37 °C for 2 h. After incubation, the cells were washed three times with PBS and responded in PBS (0.5 mL). The FITC fluorescence intensity was determined using a Becton-Dickinson FACScan analyzer (Frankin, CA, USA). Three independent experiments were conducted. The visualization of the distribution of FITC-anti-Her2 antibody in cells was further analyzed by confocal laser scanning microscopy (CLSM, Carl Zeiss LSM 700). Specifically, MCF-7 or SKOV-3 cells were seeded into 24-well plates and cultured at 37 °C. After 12 h, two types of cells were incubated with 2 or 4 μg/mL FITC-anti-Her2 antibody for another 2 h. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min at 4 °C, and then washed again three times with PBS. Finally, the cells were immediately observed using CLSM.
The specific affinity between ZHer2 and Her2-positive cancer cell lines was evaluated. Her2-negative MCF-7 cells or Her2-positive SKOV-3 cells were seeded in a confocal dish (5 × 104 cells per dish) and incubated for 12 h. After that, the medium was aspirated and replaced with 2 mL fresh medium containing 50 μg/mL FAM-ZHer2. After another 2 h of incubation, the cells were washed three times with PBS, and then fixed with 4% paraformaldehyde for 15 min at 4 °C. The cell nuclei were stained with DAPI (1 mL; 10 μg/mL) for 5 min, and then washed three times with PBS. Finally, the cells were immediately observed using CLSM. The affinity between FAM-ZHer2 and SKOV-3 cells was further evaluated by flow cytometry (FCM) assay. Approximately 2 × 105 digested cells were incubated with FAM-ZHer2 (50 μg/mL) at 37 °C for 2 h. For Her2 receptor blocking experiments, SKOV-3 cells were pre-incubated with ZHer2 (20 μg/mL) for 1 h, and then incubated with FAM-ZHer2 (50 μg/mL) for another 2 h. After that, the cells were washed three times with PBS and re-dispersed in 0.5 mL PBS for FCM assay.
In order to evaluate the affinity of monomer and dimer of the affibody to Her2 receptor, flow cytometry analysis was done. Briefly, SKOV-3 cells (2 × 105) were digested and incubated with the same molar concentration (100 nM) of monomer (in the presence of 2-ME) or dimer (in the absence of 2-ME) FAM-Her2 affibody at 37 °C for 2 h. After incubation, the cells were washed three times with PBS and redispersed in PBS (0.5 mL). The FAM fluorescence intensity was determined by flow cytometry.
To visually track the distribution of the NPs in cells, Cy5.5, instead of Pt, loaded NPs ([email protected]/MnO2/PDA or [email protected]/MnO2/PDA-ZHer2 NPs) were prepared following the same method as described above. The SKOV-3 cellular uptake of these NPs was evaluated following a protocol similar to that described above, except that an FBS-free medium containing [email protected]/MnO2/PDA or [email protected]/MnO2/PDA-ZHer2 NPs (50 μg/mL) was added after the initial culture. To investigate Her2-dependent binding, SKOV-3 cells were pre-incubated with free ZHer2 (20 μg/mL) for 1 h prior to the incubation with [email protected]/MnO2/PDA-ZHer2 NPs (50 μg/mL). Finally, the cells were treated and probed by CLSM as described above.
FCM analysis was performed to semi-quantify the uptake of these two NPs by SKOV-3 cells. In detail, about 2 × 105 cells were incubated with [email protected]/MnO2/PDA or [email protected]/MnO2/PDA-ZHer2 NPs (50 μg/mL) after a 1 h pre-treatment with or without free ZHer2 (20 μg/mL). After 4 h incubation at 37 °C, the cells were washed three times with PBS and re-dispersed in 0.5 mL PBS for FCM assay.
The cytocompatibility of drug-free mPDA/MnO2/PDA-ZHer2 NPs to HUVECs was first studied by MTT assay. HUVECs (~1 × 104) were seeded into each well of 96-well plates and cultured overnight at 37 °C in a 5% CO2 humidified environment. The medium was replaced with fresh DMEM containing varied concentrations of mPDA/MnO2/PDA-ZHer2 NPs (1, 5, 10, 20, 50, 100, and 250 μg/mL), and the cells were incubated for another 24 h. Subsequently, the medium of each well was carefully discarded followed by the addition of MTT solution (20 μL; 10 μg/mL), and the cells were incubated for an additional 4 h. Finally, 200 μL of DMSO was added after removing the medium, and the absorbance of the wells at 570 nm was measured with a microplate reader (Multiskan FC, Thermo Scientific).
The in vitro anticancer efficacy of different cisplatin formulations was investigated using a method similar to that described above, except that media containing free cisplatin, [email protected]/MnO2/PDA NPs, or [email protected]/MnO2/PDA-ZHer2 NPs ([cisplatin] = 0.6, 3, 6, 12, 24, 48 μg/mL) was added after the initial incubation. Data are reported as mean ± S.D., with three independently performed experiments each containing three replicates. The cells treated with free ciaplatin, [email protected]/MnO2/PDA NPs or [email protected]/MnO2/PDA-ZHer2 NPs ([cisplatin] = 48 μg/mL) for 24 h were further stained with PI (staining dead cells red) and calcein-AM (staining live cells, green) in PBS solution for 30 min at 37 °C in the dark, and then imaged by inverted fluorescence microscopy (Carl Zeiss). For all experiments, PBS-treated cells were used as control.
Analysis of intracellular ROS levels
The levels of intracellular ROS were probed using DCFH-DA to evaluate the RT-sensitized effect triggered by MnO2. SKOV-3 cells were seeded onto 24-well plates at 50,000 cells/well and maintained in McCoy’s 5A medium. After 12 h incubation at 37 °C, the cells were treated with (1) PBS, (2) PBS + X-Ray, (3) MnO2-free mPDA/PDA-ZHer2 NPs (50 μg/mL) + X-Ray, (4) mPDA/MnO2/PDA-ZHer2 NPs (50 μg/mL), and (5) mPDA/MnO2/PDA-ZHer2 NPs (50 μg/mL) + X-Ray, respectively. After incubation for 4 h, the cells were irradiated with X-Ray (6 Gy). Another 1 h later, DCFH-DA solution (1 mL; 10 μM) was added to the cells for another 30 min. Subsequently, the fluorescence images were acquired by an inverted fluorescence microscopy (Carl Zeiss).
Animals and tumor model
All animal experiments were carried out with full authorization approved by the ethical committee for animal care of Shandong Cancer Hospital and Institute Shandong, First Medical University and Shandong Academy of Medical Sciences (Approval No. SDTHEC2020004083). Female BALB/C nude mice (SPF grade, 4–6 weeks old) were acquired from Beijing Huafukang Bioscience Co. Inc. (Beijing, China). Tumors were established by subcutaneous injection of SKOV-3 cells (1 × 106) dispersed in 100 μL of PBS into the right flank of each mouse. The tumor volume was monitored in real-time and calculated as length × width2/2.
In vivo MRI and biodistribution
Aiming to demonstrate the TME-triggered MRI ability of the MnO2 layer, 50 μL of PBS containing [email protected]/MnO2/PDA NPs ([Mn] = 50 mM) was indirectly injected into the tumor sites or the muscle on the opposite side. At pre-expected time points (0 min, 5 min, and 120 min), the SKOV-3 tumor-bearing mice were scanned using an MR analysis and imaging system (1.5 T, SIEMENS MAGNETOM Symphony).
When the tumor volume reached approximately 200 mm3, [email protected]/MnO2/PDA NPs or [email protected]/MnO2/PDA-ZHer2 NPs ([Mn] = 3 mM, 100 μL in PBS per mouse) were intravenously injected into the tumor-bearing mice, and T1-weighted MR images were obtained at different time points after injection. The following MR scanning parameters were set: TE = 16.9 ms, TR = 760 ms, FOV = 10 cm × 10 cm, slice thickness = 3 mm, and point resolution = 512 mm × 512 mm.
The mice were executed at 12 h, and the heart, liver, spleen, lung, kidney, brain, muscle, and tumors were extracted and weighed, and then dissolved in aqua regia solution (2 mL; 65 °C) for 24 h. Finally, the Pt content present in different organs and tumors was quantified by ICP-AES.
Immunofluorescence and bio-TEM assays
The SKOV-3 tumor-bearing mice were intravenously injected with 100 μL of PBS containing [email protected]/MnO2/PDA NPs or [email protected]/MnO2/PDA-ZHer2 NPs (1 mg/mL). For the immunofluorescence assay, tumors were collected after 12 h post-injection and fixed with OCT (Sakura) for further sections under frozen conditions. The tumor sections were incubated overnight with rabbit anti-mouse Her2 primary antibody (dilution 1:200, Abcam) against Her2 at 4 °C, and then for 60 min with goat anti-rabbit secondary antibody (dilution 1:200, Abcam) at 37 °C. The cell nuclei were stained with DAPI. Finally, the obtained slices were scanned using an imaging system (Nikon DS-U3).
For bio-TEM observation, tumors were treated with 1% OsO4 for 2 h at 25 °C, and embedded in resin after the cells were dehydrated. Then, ultrathin sections (70–90 nm) of tumor tissues were cut and further studied by bio-TEM (Hitachi HT7700, Tokyo, Japan).
In vivo antitumor efficacy and safety evaluation
When the volume of tumors reached ~50 mm3, the tumor-bearing mice were randomly divided into four groups (n = 5) and intravenously injected with (1) PBS; (2) free cisplatin; (3) [email protected]/MnO2/PDA NPs; and (4) [email protected]/MnO2/PDA-ZHer2 NPs (doze of cisplatin = 2 mg/kg) every two days. Tumor volume and body weights were monitored and recorded every day since the first injection. On the 14th day, 0.5 mL of blood from each group of mice (n = 3) was withdrawn for biochemistry, after which the experiment was halted. The tumors were collected and stained with terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) for the apoptosis assay, while the major organs (heart, liver, spleen, lung, and kidney) were extracted and stained with haematoxylin and eosin (H&E) for histological analysis.
For chemo-radiation combined therapy, when the volume of tumors reached ~50 mm3, four groups of tumor-bearing mice (n = 5) were treated with (5) PBS, (6) PBS + X-Ray, (7) MnO2-free [email protected]/PDA-ZHer2 NPs + X-Ray; and (8) [email protected]/MnO2/PDA-ZHer2 NPs + X-Ray. All groups of mice except for group (5) received cisplatin and MnO2 doses of 2 mg/kg and 3.4 mg/kg, respectively, every two days. For radiotherapy, mice received an X-Ray radiation at a dose of 6 Gy for 24 h post-injection every four days. Tumor sizes and body weights were recorded every day. After 14 days, all the mice were sacrificed, and tumors were collected for volume measurement and weighting. Finally, to evaluate tumor hypoxia levels, the tumor tissues from groups (1)–(4) and group (7) were HIF-1α stained according to the procedure provided by the manufacturer.
All results are presented as mean ± standard deviation (S.D.), and the significance of the data was determined using one-way ANOVA. A p value < 0.05 indicates statistical significance, and data are represented as (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001. NS was considered to be no statistically significant.