Supramolecularly Engineered Conjugate of Bacteria and Cell Membrane‐Coated Magnetic Nanoparticles for Enhanced Ferroptosis and Immunotherapy of Tumors

Abstract Although various ferroptosis inducers including magnetic nanoparticles (Fe3O4) and iron‐organic frameworks have been applied in cancer treatment, the mild immunogenicity, low targeting efficiency to the tumor, and poor tissue penetration have limited the therapeutic efficacy. Herein, a supramolecularly engineered conjugate between living bacteria (facultative anaerobic Salmonella typhimurium VNP20009, VNP) and cancer cell membranes‐coated Fe3O4 nanoparticles is developed for improving targeted delivery of Fe3O4 nanoparticles into the tumor tissue and for synergistic ferroptosis and immunotherapy of tumor. The enhanced ferroptosis induced by both Fe3O4 nanoparticles and the loaded ferroptosis inducing agent (sulfasalazine (SAS)) effectively inhibits tumor growth and generates immune response via immunogenic cell death (ICD). The colonization of VNP in tumors also induces adaptive immune responses and further promotes ferroptosis. Fundamentally, the supramolecular conjugate of VNP and cell membranes‐coated Fe3O4 can potentiate the therapeutic capability of each other through mutually magnifying the ferroptosis and immunotherapy, resulting in significantly enhanced antitumor effects.

Fe 3 O 4 nanoparticles were prepared through a previously published method [2].Briefly, FeCl 3 •6H 2 O (1 g) was dissolved in 20 mL of EG to generate a uniform solution, then 3 g of NaAC and ETH (10 mL) were added in the above solution and stirred for 30 min.The mixed solution was sealed in a Teflon-lined stainless-steel autoclave and heated for 8 h at 200 °C.After the autoclave was cooled to room temperature, the dark brown products were alternately washed with ethanol and deionized water and separated by a magnet during each step.The resulting Fe 3 O 4 nanoparticles were suspended in deionized water for further use.

The loading of SAS in Fe 3 O 4 nanoparticles
To load the SAS into the pores of mesoporous Fe 3 O 4 nanoparticles, 50 mg SAS was dissolved in 10 mL DMSO to obtain a stock SAS solution with a concentration of 5 mg/mL.5 mg Fe 3 O 4 nanoparticles were mixed with 0.5 mL SAS solution (5 mg/mL) and stirred overnight.The FeA was obtained through centrifugation and washing with ethanol and deionized water.The loading amount of SAS was determined through the UV spectrum of SAS.

Preparation of CM-ADA
Briefly, 4T1 cells were first cultured with RPMI 1640 culture medium containing 10% FBS and 1% antibiotics overnight, and then 20 μM DSPE-PEG-ADA was added for incubation for 1.5 h in a 37°C incubator with 5% CO 2 .4T1 cells were harvested and washed with PBS 3 times.Subsequently, 4T1 cells were suspended with cold TM buffer (0.01M Tris and 0.001M MgCl 2 ) and placed on ice for 1-2 hours.Later, the suspension was extruded 20 times to lyse cells.1M of sucrose solution and one-third of the TM buffer volume were added to dilute the solutions and obtain the final concentration of 0.25 M.Then, the solution was centrifuged (3000 g, 4°C, 10 min) to remove the pellet mainly containing the cell nucleus.The supernatant was collected for further centrifugation with a speed of 3000 g for 30 min at 4°C.The obtained precipitate was the CM-ADA, which was then dispersed with PBS buffer and stored at -80°C for further use.

Preparation of FeAM
The suspension was mixed with CM-ADA suspension at a mass ratio of around 1:1 and sonicated for 3 min.The FeAM was obtained through centrifugation and washing with PBS.

Preparation of FeAMV
1mL FeAM (100 μg/mL) was mixed with VNP-CB[7] (10 6 CFU) in PBS buffer and incubated at 37 ℃ for 30 min.The FeAMV was gained through the host-guest interaction between ADA and CB [7].Later, the mixture was washed, centrifuged, and resuspended in PBS buffer for further use.

Nanoparticle characterization
SU-8010 (Hitachi, Japan) under the operating voltage of 3 kV was applied for SEM characterization.JEM-1011 TEM (JEOL, Japan) with an accelerating voltage of 100 kV was used for TEM imaging.The TEM species were prepared by dropping 5 μL of suspensions (0.05 mg/mL) on the copper grid and then air drying.The hydrodynamic diameters and zeta potential of samples were analyzed on the DLS (Malvern Zetasizer Nano ZS).The iron content was determined on the Thermo iCAP Qs ICP-MS.

Membrane protein characterization
The membrane protein of FeM was characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Briefly, extracted CM-ADA and FeM nanoparticles were lysed with RIPA lysate, then the total protein concentration was decided by BCA Protein Assay Kit (Beyotime, China).All samples were mixed with SDS-PAGE sample loading buffer (Beyotime, China), and heated at 100 ℃ for 10 min.Then, 40 g/well of each sample was run on a 10% SDS-polyacrylamide gel at 80 V, lasting for 30 min first, followed by 120 V for 1 h.Later, the gel was dyed with Coomassie Blue for 20 min, washed with deionized water, and eventually imaged.

Drug release behavior
To analyze the drug release behavior of SAS, 2 mg of FeAM were suspended in 2 mL of PBS buffer solution at different pH of 7.4, 6.5, and 5.5, respectively, to yield 1 mg/mL suspension, and the suspensions were transferred into dialysis bags (M D =1000 Da).The dialysis bags were soaked in 20 mL of release medium (buffer with different pH values), respectively, and stirred gently at room temperature.At different time points, including 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, 1 mL of release solution was collected for absorption measurement, and 1 mL fresh buffer solution at different pH values was supplemented to keep release medium volume constant.The drug release behavior of bare FeA in the pH 7.4 PBS buffer (0.02 M) was used as a control.SAS has a characteristic absorption peak at 359 nm, and the absorbance of SAS at 359 nm was linear with SAS concentration.The release amount of SAS in the dialysis bag at each time point was measured by DR600 UV-Vis Spectrophotometer.Each experiment was performed in triplicate.The encapsulation efficiency of SAS in the nanoparticles were measured by a DR600 UV-Vis spectrophotometer.

In vitro biocompatibility evaluation
MTT assay was used to assess the biocompatibility of nanoparticles against LO2 cells.First, LO2 cells were inoculated in a 96-well plate with a density of 10 4 cells/well and cultured overnight.Then, Fe 3 O 4 , FeM, and FeMV with different concentrations of Fe 3 O 4 (0, 12.5, 25, 50, and 100 μg/mL) were added and treated for 24 h.Later, the culture medium was discarded, and 100 μL culture medium involving MTT (5 mg/mL) was applied to each well, subsequently incubated for another 4 h.Then the medium was discarded, followed by adding 100 μL DMSO to dissolve formazan.Eventually, the cytotoxicity of nanoparticles to LO2 cells was assessed through a Microplate reader.

In vitro cytotoxicity assay
Similarly, an MTT assay was applied to investigate the cytotoxicity of different formulations against 4T1 cells.First, 4T1 cells were inoculated in a 96-well plate with a density of 10 4 cells/well and incubated overnight.When cells reached around 70% confluence, cells were incubated with PBS, VNP, SAS, Fe 3 O 4 , FeA, FeAM, FeMV, and FeAMV containing the same concentration of SAS (20 μg/mL), Fe 3 O 4 (100 μg/mL) and VNP (10 6 CFU).After treated for 24 h, the culture medium was removed, and the FBS-free medium involving MTT (5 mg/mL) was added and incubated for another 4 h.Then the medium was discarded, followed by adding 100 μL DMSO to each well to dissolve formazan.Eventually, the cytotoxicity of different groups against 4T1 cells was assessed by a Microplate reader at 490 nm.

Intracellular GSH level measurement
The intracellular GSH level was measured in FeAMV-treated 4T1 cells by using the GSH assay kit (Beyotime, China).4T1 cells were inoculated with a density of 2×10 5 each well in the 6-well plate.Then PBS, SAS, Fe 3 O 4 , VNP, FeA, FeMV, and FeAMV with an equivalent concentration of SAS (20 μg/mL), Fe 3 O 4 (100 μg/mL), or VNP (10 6 CFU) were added to the 4T1 cells culture.After incubation for 12 h, the cells were treated with trypsin to collect cell suspension; then, the suspension was washed with PBS through centrifugation.The supernatant was discarded and treated with a 3-fold cell volume of protein removal reagent S solution.The sample was rapidly freeze-thawed twice using liquid nitrogen and a 37°C water bath.The sample was then placed in the ice bath for 5 min and centrifuged (10,000 g) at 4°C for 10 min.Last, the supernatant was used to determine total GSH by the GSH assay kit.

In vitro DCs maturation experiment
Bone marrow-derived iDCs were harvested from BALB/c mice aged 6 to 8 weeks following the instruction reported before [3].The 24-well transwell system with a 0.4 μm polycarbonate porous membrane was used to study in vitro DCs maturation experiment.First, 2×10 4 4T1 cells per well were inoculated in the upper well.Subsequently, cells were incubated with PBS, SAS, Fe 3 O 4 , VNP, FeA, FeAM, and FeAMV with an equivalent concentration of SAS (20 μg/mL), Fe 3 O 4 (100 μg/mL), or VNP (10 6 CFU) for 24 h.After that, the upper well was transferred and co-incubated with DCs previously inoculated in the bottom well.After co-culture for 24 h, DCs in the bottom were harvested and stained with anti-CD11c antibody and anti-CD86 antibody for 1 h.Later, DCs were resuspended by PBS and assessed by flow cytometry after washing by centrifugation.

In vitro immune analysis
The TNF-α and IL-6 levels were evaluated using ELISA kits (Lai Er Bio-Tech, China) in a 24-well transwell system with a 0.4 μm polycarbonate porous membrane.First, 4T1 cells were inoculated in the upper well.When the cells reached 70-80% confluence, PBS, SAS, Fe 3 O 4 , VNP, FeA, FeAM, and FeAMV with an equivalent concentration of SAS (20 μg/mL), Fe 3 O 4 (100 μg/mL), or VNP (10 6 CFU) were added to the cells and incubated for 24 h.Afterward, the upper wells were transferred and co-incubated with Raw 264.7 previously inoculated in the bottom well.After the co-culture, the supernatant was obtianed for cytokine TNF-α and IL-6 analysis by ELISA assay kits.

In vivo bio-distribution study
Female BALB/c mice (6 to 8 weeks) were obtained from the Animal Center of the University of Macau. 10 6 4T1 cells were injected to the hind legs of mice and observed for 12 days.Then, the mice were separated into 5 groups at random (n=3), and the free Cy5.5, Fe 3 O 4 , VNP, FeAM, and FeAMV were intravenously injected into the mice through the tail vein (2 mg/kg Cy5.5).At predetermined time points after injection (0, 6, 12, 24, 36, and 48 h), mice were anesthetized, and an in vivo imaging system (IVIS) was applied to capture fluorescence signals.All mice were sacrificed 48 hours later to collect the organs and tumor for fluorescence imaging.
Later, 0.02 mg tissue was cut from the organs or tumors for grinding to observe the distribution of Fe in different organs.The ground mixture was then soaked in aqua regia overnight, diluted with a 2% nitric acid solution, and detected through the Thermo iCAP Qs ICP-MS to measure Fe content.
Similarly, 0.02 mg tissue was cut from the organs or tumors for grinding to observe the distribution of VNP in different organs.Single-cell suspension was obtained using a Falcon® 40 µm cell strainer.After centrifuging (300 g, 5 min), the supernatant was diluted at different times for plating and colony counting.Later, 100 μL bacteria solution was added to the agar plate, spread uniformly, and incubated at 37℃ overnight for colony counting.

In vivo anti-tumor study
Female BALB/c mice (6 to 8 weeks) were obtained from the Animal Center of the University of Macau. 10 6 4T1 cells were injected to the hind legs to get 4T1 tumor-bearing mice.Once the tumor volume reached about 80 mm 3 , the mice were separated into 7 groups at random and intravenously injected with PBS, SAS, VNP, FeA, FeAM, FeMV and FeAMV via the tail vein with an equivalent SAS concentration of 5 mg/kg on day 1, 3, 5, 7, 9.The tumor volume and mice weight were recorded every 3 days until the test ended.The tumor volume was calculated as the following formula: V = (L × W 2 )/2, where L and W denoted the maximum and minimum diameters.When the 14-day treatment ended, the mice were sacrificed to harvest their major organs, tumors, and blood for H&E and TUNEL staining, Fe and VNP distribution evaluation, the study of M1/M2 macrophage ratio in tumor section, and IL-6 and TNF-α expression level in the tumor site.

Figure S3 .
Figure S3.Size distribution of A) Fe 3 O 4 nanoparticles and B) FeM on Day 0 and Day 7, respectively.

Figure S4 .
Figure S4.The mean hydrodynamic diameters of Fe 3 O 4 and FeM after incubation in 10% FBS for different durations determined by DLS.

Figure S6 .
Figure S6.The proliferation profiles of VNP and FeMV for up to 12 h.

Figure
Figure S7.A) N 2 absorption-desorption isotherms and B) pore size distribution of Fe 3 O 4 nanoparticles.

Figure S8 .
Figure S8.In vitro cytotoxicity of Fe 3 O 4 nanoparticles, FeM, and FeMV with different concentrations of Fe 3 O 4 against LO2 cells after 24 h incubation.