Rational design of non-toxic GOx-based biocatalytic nanoreactor for multimodal synergistic therapy and tumor metastasis suppression

Rationale: Glucose oxidase (GOx)-based biocatalytic nanoreactors can cut off the energy supply of tumors for starvation therapy and deoxygenation-activated chemotherapy. However, these nanoreactors, including mesoporous silica, calcium phosphate, metal-organic framework, or polymer nanocarriers, cannot completely block the reaction of GOx with glucose in the blood, inducing systemic toxicity from hydrogen peroxide (H2O2) and anoxia. The low enzyme loading capacity can reduce systemic toxicity but limits its therapeutic effect. Here, we describe a real 'ON/OFF' intelligent nanoreactor with a core-shell structure (GOx + tirazapamine (TPZ))/ZIF-8@ZIF-8 modified with the red cell membrane (GTZ@Z-RBM) for cargo delivery. Methods: GTZ@Z-RBM nanoparticles (NPs) were prepared by the co-precipitation and epitaxial growth process under mild conditions. The core-shell structure loaded with GOx and TPZ was characterized for hydrate particle size and surface charge. The GTZ@Z-RBM NPs morphology, drug, and GOx loading/releasing abilities, system toxicity, multimodal synergistic therapy, and tumor metastasis suppression were investigated. The in vitro and in vivo outcomes of GTZ@Z-RBM NPs were assessed in 4T1 breast cancer cells. Results: GTZ@Z-RBM NPs could spatially isolate the enzyme from glucose in a physiological environment, reducing systemic toxicity. The fabricated nanoreactor with high enzyme loading capacity and good biocompatibility could deliver GOx and TPZ to the tumors, thereby exhausting glucose, generating H2O2, and aggravating hypoxic microenvironment for starvation therapy, DNA damage, and deoxygenation-activated chemotherapy. Significantly, the synergistic therapy effectively suppressed the breast cancer metastasis in mice and prolonged life without systemic toxicity. The in vitro and in vivo results provided evidence that our biomimetic nanoreactor had a powerful synergistic cascade effect in treating breast cancer. Conclusion: GTZ@Z-RBM NPs can be used as an 'ON/OFF' intelligent nanoreactor to deliver GOx and TPZ for multimodal synergistic therapy and tumor metastasis suppression.


Synthesis of GOx FITC
GOx (10 mg) was treated with fluorescein isothiocyanate (FITC, 0.6 mg, 20 eq) in 50 mM borate buffer (pH 8.5, 2 mL) for 1 h at 25 °C. Excessive FITC was removed via dialysis against in PBS at pH 7.4. The conjugation efficiency of GOx FITC was quantified by the FITC/GOx molar ratio of the GOx FITC conjugate. The concentration of GOx and FITC was quantified by UV absorbance (280 nm) and fluorescence intensity (excitation at 488 nm, emission at 514 nm), respectively.

Preparation of RBM
RBC membranes (RBM) were extracted following previously published protocols with modifications. RBCs were washed with 1 × PBS in ice and then treated with a hypotonic medium. The RBCs were suspended in 0.25× PBS for 40 min at 4 °C, and then were centrifuged (9000 rpm, 5 min, 4 °C). The precipitate of pink i-RBCs was collected for further use. The RBC suspension was sonicated with a microtip using VibraCell VCX 130 (Sonic and Material, Inc., Newtown, CT, USA) (20 kHz, 130 W, 5 min). The obtained RBM suspension was further extruded through 1 μm, 400 nm, and then 200 nm polycarbonate membranes (Whatmann, Maidstone, UK) using an Avanti mini extruder device (Avanti Polar Lipids, Inc., Alabaster, AL, USA) for 11 times, respectively.

Loading Efficiency of TPZ and GOx
The payload of GOx was determined via FITC labeled by using the standard calibration curve based on the fluorescence spectra at 520 nm of the difference S3 between the supernatant collected after centrifugation and initial solution. Meanwhile, loading amount of TPZ was obtained by using the standard calibration curve based on the UV−vis absorption intensity at 470 nm of the difference between the supernatant collected after centrifugation and initial solution.
In addition, the GTZ NPs were calcinated at 250 o C for 2 h under nitrogen. The calcinated GTZ NPs were characterized by TEM.

Enzyme releasing and catalytic ability measurement
1 mg FITC labeled GTZ-RBM and GTZ@Z-RBM were dispersed into 1 mL of glucose solution (5 mM) at 7.4 and 6.0. The enzyme labeled with fluorescent molecules was released from NPs and recorded by fluorophotometer. The real time pH values and dissolved O 2 of the solutions were respectively measured with a pH meter and oxygen detector.

Detection of H 2 O 2
1 mg of GTZ-RBM and GTZ@Z-RBM were respectively added into 1 mL of glucose solution (5 mM) at 7.4 and 6.0 for 30 min, then 200 μL of ammonium hydroxide and 100 μL of TiOSO 4 · xH 2 SO 4 · xH 2 O solution (5% wt) was added into the above solution to from the precipitate for 10 min. After that, the precipitate was collected and dissolved with 200 μL of H 2 SO 4 solution (2 M). Finally, homogeneous solution was recorded by the UV-Vis spectrometer.

TPZ releasing measurement
The drug release experiments were divided into two groups: (1) pH 7.4; (2) pH 6.0 phosphate buffered saline (PBS 10 mM). GTZ@Z-RBM (1 mg mL -1 5 mL) was S4 dispersed into 5 mL PBS at different pH under stirring at 37 °C in the capped vials. At predetermined time intervals, 200 µL of the buffer solution was removed for UV-vis spectrum measurement of TPZ. The cumulative amounts of TPZ released were determined according to the UV-vis absorption intensity at 470 nm, using the standard calibration curve.

Cell Cultures
According to the recommended procedure, 4T1 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin under humidified normoxic (95% air, 5% CO 2 ) conditions at 37 o C.

Cellular uptake of GTZ@Z-RBM
Cell phagocytosis was assessed via flow cytometer and CLSM. To intuitively display the cellular uptake of GTZ@Z-RBM, FITC labelled GOx was introduced into ZIF-8. 4T1 cells with 50000 cells/well in 0.5 mL of DMEM were seeded in 24-well plates and incubated for 24 h at 37 °C in 5% CO 2 atmosphere. Then the cells were further incubated with G FITC TZ@Z-RBM at 37 °C for 2, 4, and 8 h to evaluate the uptake. At last, flow cytometer and LCMS were utilized to detect the phagocytosis of NPs.

Hypoxia Measurements
4T1 cells were seeded on coverslips in a 24-well plate at a density of 5×10 4 cells per well and incubated overnight. Then the cells were incubated with fresh medium containing PBS, GTZ-RBM, and GTZ@Z-RBM at pH 7.4 or 6.5 for 4 h. The cells were incubated with fresh medium containing pimonidazole hydrochloride (60 μg/mL) S5 for another 1 h then fixed with paraformaldehyde, followed washed with PBS and stained with anti-pimonidazole mouse monoclonal antibody (FITC-Mab1, Hypoxyprobe-1 TM plus kit, Hypoxyprobe Inc.; Burlington, MA) and DAPI. The slides were observed by Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

ROS Measurements
For ROS observations on fluorescence microscope, 4T1 cells were digested and resuspended into 24-well plates with about 50000 cells/well in 0.5 mL of DMEM, and incubated for 24 h at 37 °C in 5% CO 2 atmosphere. Subsequently, 100 mM non-fluorescent 2',7'-Dichlorofluorescin diacetate (DCFH-DA) containing GTZ-RBM and GTZ@Z-RBM NPs was added into DMEM with different pH, which could form the fluorescent matter DCF (green fluorescence)

DNA Damage
4T1 cells were seeded on coverslips in a 24-well plate at a density of 5 × 10 4 per well at 37 °C overnight. Cells were further divided into the following groups: PBS, H 2 O 2 (1 mM), GTZ@Z-RBM (50 μg/mL), and GTZ@Z-RBM+CAT. All groups were then exposed to fresh medium with pH of 6.0. After incubation for 24 h, the cells were washed and fixed with 4% paraformaldehyde (PFA). Cells were incubated with anti-γ-H2AX mouse monoclonal antibody (Abcam Inc., Cambridge, MA) at a dilution

Female Balb/c mice bearing 4T1 xenografts were intravenously injected with
DiDlabeled GTZ@Z-RBM. The dose of DiD was 0.5 mg per kg mouse body weight.
At the preset times, in vivo fluorescence images were acquired on the Xenogen IVIS Lumina system (Caliper Life Sciences, Alameda, CA). Moreover, at 48 h post-injection, the organs of mice including heart, lung, liver, spleen, kidney, and tumor were collected. Then fluorescence images were also acquired on the Xenogen S8

Hematological Evaluations
The Balb/c mice (n = 5 per group) were i.v. injected with PBS, GOx, GTZ-RBM, and GTZ@Z-RBM (dose of 200 U/kg GOx, and 2 mg/kg TPZ) for 2 h. Blood from the angular vein of the eye was collected for hematology analysis, blood routine examination and blood biochemical analysis. The death of the mouse was also recorded.

Immunohistochemical and Immunofluorescence Staining Analysis
The tumor tissues were fixed in 4% formaldehyde and embedded in paraffin.
Subsequently, the tumor tissues were observed via H&E staining. Cell proliferation and apoptosis in tumor tissues were also analyzed by immunofluorescence staining of a terminal transferase dUTP nick-end labeling (TUNEL, Roche Diagnostics, Indianapolis, IN), respectively.
Statistical Analysis.