An Inter‐Cooperative Biohybrid Platform to Enable Tumor Ablation and Immune Activation

Abstract A biohybrid therapeutic system, consisting of responsive materials and living microorganisms with inter‐cooperative effects, is designed and investigated for tumor treatment. In this biohybrid system, S2O3 2−‐intercalated CoFe layered double hydroxides (LDH) are integrated at the surface of Baker's yeasts. Under the tumor microenvironment, functional interactions between yeast and LDH are effectively triggered, resulting in S2O3 2− release, H2S production, and in‐situ generation of highly catalytic agents. Meanwhile, the degradation of LDH in the tumor microenvironment induces the exposure of the surface antigen of yeast, leading to effective immune activation at the tumor site. By virtue of the inter‐cooperative phenomena, this biohybrid system exhibits significant efficacy in tumor ablation and strong inhibition of recurrence. This study has potentially offered an alternative concept by utilizing the metabolism of living microorganisms and materials in exploring effective tumor therapeutics.

was obtained from Dojindo. Naphthalene-2,3-dicarboxaldehyde (NDA) was bought from Invitrogen TM . All chemicals in this work were used directly without further purification.

Characterization
The morphology of samples was observed by a field-emission scanning electron microscope (FESEM; SU-70 Hitachi) and a transmission electron microscope (TEM, FEI Tecnai F20). High angle annular dark field images and element mapping were obtained on a Cs-corrected STEM (FEI Titan G2 80-200 C hemi STEM). The crystal structure was characterized by X-ray diffraction with Cu Kα radiation (XRD, X'pert PRO MPD). The size distributions and zeta potentials were tested by a Malvern zetasizer (Nano-ZS90, UK). All the UV-vis spectra were measured by a UV-vis spectrometer (Shimadzu, UV-2600). The fluorescence images were photographed by an inverted fluorescent microscope (Nikon, Ts2R-FL).

Synthesis of S2O3 2intercalated CoFe layered double hydroxides
Briefly, 0.6 mmol Co(NO3)2⋅6H2O and 0.2 mmol Fe(NO3)3⋅9H2O were dissolved in 20 mL of deionized water as solution A. Meanwhile, 8 mmol NaOH was dissolved in another 20 mL of deionized water to make solution B. Afterwards, under 520-rpm stirring at 65°C, solution A and B were dropwise added simultaneously into a beaker containing 0.1 mmol Na2S2O3·5H2O and 80 mL of deionized water. pH was kept around 9.5. Afterwards, the solid red-brown precipitants were collected by centrifugation, and then washed twice with the mixture of deionized water and ethanol (volume ratio: 5:1). It should be noted that all the deionized water should be boiled before use to remove dissolved carbon dioxide and the synthesis process should be accomplished within 20 min to prevent the formation of Co/Fe oxides.
Yeast culture medium preparation and yeast culture 10 g of glucose, 5 g of yeast extract and 8 g of beef extract were mixed in 1 L of deionized water to prepare liquid NYDB culture medium. For solid NYDB medium, additional 20 g of agar was added.
Baker's yeast was purchased in Yonghui supermarket in China and the brand is 'Angel'. In order to purify baker's yeast, the commercial yeast was diluted in PBS, streaked on the solid medium plate and then incubated at 37 °C overnight. Yeast colonies were then picked out and grown overnight in NYDB liquid medium in a shaking incubator (37°C, 180 rpm) before use.

Characteristics of Y@LDH
To load LDH on Yeast, 10 7 CFU yeast and different quantities of LDH were dispersed in 10 mL of deionized water. In this work, the quantity of LDH in mixture was finally decided as 0.5 mg. After stirring for 2 h, Y@LDH was collected by centrifugation and then re-suspended in ultrapure water for further use.

Culture experiment and colony model
Same yeast concentration of yeast and Y@LDH were both incubated in pH=7.4 and pH=6.5 RPMI-1640 culture medium (with 10% fetal bovine serum, pH was adjusted by HCl), respectively. Due to the influence of LDH on OD600, which was used to judge the concentration of yeast, we use Δ concentration (from ΔOD600) to estimate the influence of LDH on yeast proliferation. For colony model, same yeast concentration of yeast and Y@LDH were directly coated on NYDB solid plates and the photograph were taken after colony formation.

Catalytic Property
In this work, 3, 3′, 5, 5′-tetramethylbenzidine (TMB, 1×10 -3 M) was chosen as an indicator to detect the production of reactive oxygen species (ROS), following the relative procedures reported previously. [1] TMB can be oxidized to ox-TMB (λ=652 nm) by hydroxyl radicals (·OH). To separate the influence of some enzymes like POD outside yeast cell membranes on catalytic property, all samples were boiled at 70 °C for 10 min to inactivate the catalytic properties of yeast enzymes. DMPO was used to capture radicals. The combination of DMPO and hydroxyl radicals shows a 1:2:2:1 signal on ESR spectroscopy. To further clarify the enzyme-like activity of Y@LDH, Michaelis-Menten kinetic curve was drawn by plotting the initial ·OH producing velocities on different H2O2 concentrations, and the Michaelis-Menten constant (KM) and maximal velocity Vmax were then calculated. It should be noted that before the final test for LDH with different concentrations of Na2S, the product should be centrifuged and washed for two times.

H2S production
Different concentrations of yeast or Y@LDH with Cys/GSH were added into 20 mL glass bottle with 10 mL of antibiotic-free RPMI 1640 medium to simulate body fluid or tumor tissues with additional acid. A rubber tube was used to link this bottle with another bottle containing 2 mL of zinc acetate/sodium acetate mixture (mass ratio: 4/1) as testing bottle. All joints were sealed by Vaseline and laboratory film. The whole device (as shown in Figure S10) was then kept in a shaking incubator (37°C, 180 rpm). After specific time, N, N-dimethyl-p-phenylenediamine dihydrochloride and FeCl3 were added into the testing bottle. After incubation for another 15 min, the absorbance at 665 nm (corresponding to the production of methylene blue) of the mixture was examined, and the concentration of H2S was determined using a standard curve of Na2S.

S2O3 2release
A typical chemical titration process was used for detecting S2O3 2released. Briefly, 2.5 mg mL -1 CoFe LDH-S2O3 was dispersed in pure PBS and PBS added with acid or Na2S, respectively. At specific time points, materials were centrifuged and the supernatant containing S2O3 2was used for titration based on following reaction: Specifically, 60 mg of potassium dichromate was dissolved by 20 mL of ultrapure water in an iodine measuring bottle. Then 0.7 g of potassium iodide and 20 mL of 20% sulfuric acid were successively added and gently shaken to dissolve. After being sealed in the dark for 10 min, 150 mL of water was added and then 10 mL of diluted mixture was taken out as the indicator for titration. The supernatant was titrated with a burette. When the titration was near the end point, 2 mL of 10 mg mL -1 starch indicator solution was added. Titration process continued till the blue color disappeared and the volume of final titration solution was recorded. The colors changed during the titration can be showed in UV-vis spectra as Figure S8. The concentration of S2O3 2in supernatant can be calculated as follows: Where m is the mass of K2Cr2O7 in indicating solution (g), ΔV is the volume of supernatant used during titration (mL), and M is the molar mass of K2Cr2O7 (M=294.18 g mol -1 ).

Chemotaxis characteristic of yeast and Y@ LDH
The chemotaxis of yeast and Y@LDH was investigated via a Transwell @ migration assay (8 μm pore size, 6.5 mm diameter). To simulate the acidic tumor microenvironment, 800 μL RPMI 1640 medium with 10% fetal bovine serum (FBS) was adjusted to pH 6.0 and added to the bottom of 24-well plate.
For the control group, 800 μL RPMI 1640 medium with 10% FBS was directly added to the bottom.
At 10, 20 and 30 min, the Transwell @ inserts in specific group were removed and then let wells stand for 10 min. Afterwards, the bottom of each well was photographed and the quantities of yeast were counted.

Cytotoxicity assay
For each treatment, 4T1 cells were digested and seeded into a 24-well plate at the density of 15000 cells per well and incubated for 12 h. Except cell safety assay, all cytotoxicity experiments were carried in hypoxia condition (5% O2) to simulate the hypoxia environment in tumor tissue.
Subsequently, Transwell @ inserts (0.4 μm pore size, 6.5 mm diameter) were laid and fresh culture medium containing different therapeutic agents (LDH, Yeast and Y@LDH) was added to the inserts.
The pH and Cys concentration of medium were adjusted to meet different experimental requirements.
After incubating for another 24 h, 500 μL of 10% CCK-8 was added to each well and the absorbance at 450 nm was detected by a microplate reader. The viability of each group was calculated based on the corresponding absorbance over those of control groups.
Colony formation assay 4T1 cells were seeded into a 24-well plate at the density of 50 cells per well. After 4 h for cells adherence, PBS, LDH, Yeast and Y@LDH were added and incubated for another 12 h, respectively.
Then, the cells were cultured in fresh RPMI 1640 medium for 14 d. Afterwards, the cells were fixed by 10% formaldehyde (10 min) and stained by crystal violet (20 min).

Scratching assay
Sufficient 4T1 cells were seeded into 24-well plates and incubated for 24 h. Sterile toothpick was used to scratch a line on the bottom of each plate. Subsequently, PBS was used to wash away dead cells for three times. Thereafter, PBS, LDH, Yeast and Y@LDH were added to 400 nm pore-size Transwell @ inserts on each well and incubated for 12 h, respectively. Cells were photographed by microscope every 12 h.

Fluorescence staining
In this part, different fluorochromes were used to characterize the influence of therapeutic agents on cells, respectively. Briefly, after different treatments, cells were washed with PBS for three times.

Intracellular GSH quantification
Sufficient 4T1 cells were seeded in 6-cm culture dishes and incubated for 24 h. Then PBS and Y@LDH were added, respectively. After incubation for further 8 h, the cells were washed for three times and 500 μL of RIPA lysis buffer with PMSF (10 mM) was added. To detect the GSH concentration in supernatant, 50 μL of 500 μM DTNB was mixed with 50 μL of supernatant for 5 min and the absorbance at 405 nm was detected by a microplate reader. The concentration was calculated according to the standard curve.
Macrophage polarization RAW 264.7 was used to indicate the promotion of macrophage polarization by different therapeutic agents (LDH, Yeast and Y@LDH). Nitric oxide probe and nitric oxide kits were chosen to indicate the M1 polarization. Specifically, RAW 264.7 was seeded into 24-well plate of 5000 cells per well and incubated for 12 h. Then Transwell @ inserts were laid and therapeutic agents were added. After incubation for another 12 h, the supernatant of each group was collected and tested by nitric oxide kits. 20 μM NO probe was then directly added to the remnants and incubated for 30 min. Afterwards, the cells were washed and observed under inverted fluorescent microscope.

In vivo study
Female BALB/c mice (4-6 weeks old) were purchased from Shanghai Laboratory Animal Center. All mice experiments were performed in accordance with the Guidelines for Care and Use of Laboratory

Animals of Zhejiang University and approved by the Animal Ethics Committee of Zhejiang
University. The mice were randomly divided and a 12 h light/12 h dark cycle controlled environment was set. The ethically endpoint of tumor volume was set to be 1500 cm 3 . Tumor volume was calculated as following formula: volume = (tumor length) × (tumor width) 2 × 0.52. The relative volume of tumor was calculated as the tumor volume in specific day over the tumor volume in day 0.

Median lethal dose investigation
To investigate the median lethal dose of Yeast and Y@LDH for BALB/C mice, different doses of yeast and Y@LDH (4.0, 4.6, 5.2, 5.8, 6.4 ×10 6 CFU g -1 ) were injected intravenously to mice. Each group consisted of six mice that were injected with same doses of yeast or Y@LDH. The death rate of each group was recorded and the median lethal dose of each group was calculated using the modified Karber analysis, the main formula of which is Where Xn is the logarithm of dose in the highest corresponding group; i is the difference of logarithmic doses between the two adjacent groups (high dose minus low dose); ∑m is the sum of death rate of each group.

In vivo biosafety evaluation
To evaluate the biocompatibility of therapeutic agents, after injection of LDH, Yeast, Y@LDH (10 7 CFU), the blood samples of healthy mice were collected for blood biochemistry and blood routine analysis at different time points (0, 1, 7, and 14 days, respectively). Major organs (heart, liver, spleen, lung, and kidney) of mice were collected and used for histological analysis at day 30.

Tumor ablation and immune reactivation analysis
After acclimation for 1 week, 0.1 mL (≈10 6 ) of 4T1 cells were injected into the right side back of each mouse to acquire tumor-bearing mice. When the tumor reached about 100 mm 3 , all mice were divided into six groups (6 mice for each group) and applied with various treatments three times in the

Y@LDH colonization experiment
Heart, liver, spleen, lung, kidney and tumor samples from mice were harvested and weighed after injection of Y@LDH at specific time points. All samples were then homogenized at 4°C in sterile PBS (pH = 7.2) by a grinding mill. Then the extracts of these samples were diluted with PBS for specific folds and plated on NYDB solid plates. After 24 h incubation, yeast colonies were counted.
The yeast concentration (CFU per gram of tissue) was calculated from colony counts, dilution ration and tissue weights.

Statistical analysis
All results are expressed as means ± SD as indicated. Two-tailed Student's t test was used when more than two groups were compared. Figure S1. EDS element mapping of LDH.