Cancer Nanobombs Delivering Artoxplatin with a Polyigniter Bearing Hydrophobic Ferrocene Units Upregulate PD‐L1 Expression and Stimulate Stronger Anticancer Immunity

Abstract Poor immunogenicity seriously hampers the broader implementation of antitumor immunotherapy. Enhanced immunogenicity capable of achieving greater antitumor immunity is urgently required. Here, a novel polymer that contains hydrophobic ferrocene (Fc) units and thioketal bonds in the main chain, which further delivered a prodrug of oxaliplatin and artesunate, i.e., Artoxplatin, to cancer cells is described. This polymer with Fc units in the nanoparticle can work as a polyigniter to spark the peroxide bonds in Artoxplatin and generate abundant reactive oxygen species (ROS) to kill cancers as nanobombig for cancer therapy. Moreover, ROS can trigger the breakdown of thioketal bonds in the polymer, resulting in the biodegradation of the polymer. Importantly, nanobombig can facilitate the maturation of dendritic cells and promote the activation of antitumor immunity, through the enhanced immunogenic cell death effect by ROS generated in situ. Furthermore, metabolomics analysis reveals a decrease in glutamine in nanobombig‐treated cancer cells, resulting in the upregulation of programmed death ligand 1 (PD‐L1). Consequently, it is further demonstrated enhanced tumor inhibitory effects when using nanobombig combined with anti‐PD‐L1 therapy. Overall, the nanosystem offers a rational design of an efficient chemo‐immunotherapy regimen to promote antitumor immunity by improving tumor immunogenicity, addressing the key challenges cancer immunotherapy faced.


General instruments
Dynamic light scattering (DLS) was conducted by Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK).Inductively Coupled Plasma (ICP) analysis was conducted using Inductively Coupled Plasma Optical Emission Spectrometer (Agilent technologies 7700 series, U.S.A.).The morphology and size were measured by transmission electron microscope (TEM, Hitachi HT 7700, Japan).Flow cytometry (FCM) was conducted by Cytomics FC500 Flow Cytometry (Beckman Coulter, U.S.A.).Confocal laser scanning microscopy (CLSM) was accomplished by ZEISS LSM880. 1 H NMR spectra and 13 C NMR were measured by a 400 or 300 MHz NMR spectrometer (Bruker, USA) at room temperature.High resolution mass spectrometry (HRMS) was conducted by Agilent 1290 UPLC/6540 Q-TOF.All OD values were recorded by SpectraMax M3.X-ray photoelectron spectroscopy (XPS) was performed on the Thermo Scientific ESCALab 250Xi.Gel permeation chromatography (GPC) was conducted Agilent technologies LC-20A.

Cell lines and animals
All cells were purchased from the American Type Culture Collection (ATCC, FBS (Gibco) (v/v), respectively, and incubated at 37℃ in a 5% CO2 atmosphere.
Female BALB/c mice and KM mice (6 weeks) were obtained from Hunan SJA Laboratory (Hunan, China) and raised in SPF animal rooms.All animals were free access to standard diet and water, and kept indoors under 12 h day/12 h night.All

Synthesis of Artoxplatin
The compound Oxa(Ⅳ)-OH was synthesized according to a previously reported protocol. [1]Briefly, Oxa (3.0 g, 7.6 mmol) was added to 50-mL round-bottomed flask with 20 mL 30% H2O2.The reaction solution was centrifugated after 24 h reaction at room temperature.After washing with water (5 mL) and cold ether (5 mL) for 2 times, the solid was dried under vacuum to obtain Oxa(Ⅳ)-OH as white powder (2.8 g, yield 86%). ART (2.3 g, 6.0 mmol) was dissolved in 20 mL of anhydrous ether with the addition of dicyclohexylcarbodiimide (618 mg, 3.0 mmol).The mixture solution was stirred at room temperature overnight, and filtered to obtain clear solution.The solvent was evaporated and the residue was dissolved in 20 mL DMF with the addition of Oxa(Ⅳ)-OH (429 mg, 1.0 mmol).The mixture was heated and stirred at 50 ℃ overnight to obtain a clear yellow solution.The solvent was evaporated and purified using thin-layer chromatography to obtain Artoxplatin (382 mg, 33%).
After magnetic stirring for another 12 h at room temperature, mPEG5000-OH (1 g, 0.2 mmol) was added to the reaction mixture.After magnetic stirring for another 24 h at 50 ℃, the mixture was added into 10 mL of deionized water under sonication, followed by dialysis in a dialysis bag (MWCO: 8000-14000 Da).After 72 h, the solution was freeze-dried under reduced pressure to give P1 (800 mg) as a yellow powder.
Degradation of P1 after H2O2 treatment by GPC P1 (2 mg/mL, 2 mL) was added to a 5 mL centrifugation tube containing H2O2 (10 mM, 2 mL), then the mixture was incubated at 37 ℃ for 5 h.GPC test was carried out after the above solution was dialyzed and lyophilized.
The solution was freeze-dried under reduced pressure.Lyophilized samples were then tested by XPS.

Nile red encapsulation and release
Nile Red dye was used as a model compound to visually examine the effect of oxidation on the dye release from the nanobomb ig .Artoxplatin (2 mg), P1 (20 mg) were co-dissolved in 1 mL DMSO was mixed with 200 μL of Nile Red solution (1 mg/mL in DMSO) and stirred for 2 h.The mixture was added to 9 mL water drop by drop.The mixture was then transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed for 24 h.After that, the solution of Nile Red loaded nanoparticle was collected and diluted to 20 mL.The effect of the H2O2-induced oxidation on the Nile Red release was studied by mixing 3 mL of Nile Red loaded nanoparticle solution with H2O2 (10 mM) and incubating at room temperature.The fluorescence spectra of the solutions were recorded at different time intervals (SpectraMax M3, λex = 540 nm) to monitor the release of Nile Red dye from the nanobomb ig .

Preparation and characterization of nanobomb ig
Artoxplatin (5 mg), P1 (50 mg) were co-dissolved in 1 mL DMSO.Then, under the condition of stirring, the solution was quickly injected into 10 mL water to selfassemble to form nanoparticles (nanobomb ig ).Free drugs and organic solvent were removed through dialysis against deionized water for 24 h.The concentration of Pt in nanobomb ig was assessed via ICP-MS (Agilent technologies 7700 series, U.S.A.).The hydrodynamic size nanobomb ig was detected via a DLS device (Malvern Zetasizer Nano, UK).The morphology and shape of nanobomb ig were visualized with a TEM device (Hitachi, Japan).

Nanoparticles uptake in the cells by CLSM and FCM
A cover slide was placed in the bottom of each well of a 24-well plate.CT26 cells (1×10 5 ) in 1 mL media were added to each well and incubated at 37 ℃ for 12 h.Then the cells were treated with nanobomb ig @Cy5.5 (2.5 μM Pt) for 1 h, 4 h, or 7 h, respectively.After being washed with cold PBS, the cells were fixed with paraformaldehyde.Cell nuclei were stained with DAPI (ThermoFisher Scientific).

The uptake and apoptosis test on 3D tumor spheroids
1% agarose gel solution (50 μL) was added to each 96-well plate.1,500 CT26 cells (200 μL) were added to each well.On the 7th day, the cell spheres were formed.For analysis of cellular uptake of nanoparticles, the 3D spheroids were treated with nanobomb ig @Cy5.5 (5 μM Pt) for 12 h and 24 h.After being washed with cold PBS, the spheroids' uptake of nanoparticles was measured via CLSM.Images were captured at intervals of 5 μm from top to bottom of the live spheroids.

Pt release kinetics of nanobomb ig
The in vitro drug release kinetics study of nanoparticles was carried out by dialysis using PBS or an aqueous solution (H2O2 = 10 mM) as the release medium.
Seal 5 mL of nanobomb ig at a Pt concentration of 100 μM in a dialysis bag (molecular retention of 3500), and then immerse them in 200 mL of release medium in a beaker covered with aluminum foil.Keep the beaker at 37°C while shaking at 100 rpm.At various time points, 1 mL of sample solution was taken from the dialysate and measured by ICP-MS.The platinum released from the micelles was expressed as the percentage of cumulative platinum in the dialysate to the total platinum in nanobomb ig .

MTT assay of various Pt containing drugs on various cancer cells
CT26, MC38, and HCT1116 cells were seeded in 96-well plates (4×10 3 cells/well) and incubated at 37 ℃ for 12 h.Subsequently, the cells were treated with PBS, Oxa, Oxa+2ART, Artoxplatin, nanobomb, and nanobomb ig at various Pt concentrations ranging from 0.005 μM to 40 μM of Pt for 48 h.The Art concentration ranged from 0.01 μM to 80 μM in Oxa+2ART group.To verify the effect of Fe 2+ , the cells were pre-treated with 10 μM of ferrous sulfate.After incubation for 10 hours, the cells were washed three times with cold PBS and then followed the above experimental procedure.Cells were then incubated with 10% MTT (5 mg/mL solution in PBS buffer) and the plates were further allowed to incubate with cells for another 4 h.Acidified SDS solution was then added (100 μL) was added in 96-well plates for each well, and the plates were kept in the dark for an additional 12 h.Measurements of absorbance were subsequently made with a Bio-Rad plate reader (SpectraMax M3) at 570 nm (peak absorbance) and subtracted at 650 nm (background absorbance).

Intracellular ROS assessments
CT26 cells were seeded on 24 wells plate with cell slides covered and 12 wells plate at a density of 1×10 5 and 2×10 5 respectively.Cells were treated with Oxa, Artoxplatin, nanobomb, and nanobomb ig at the same concentration of Pt (25 μM) for 7 h.Subsequently, the culture medium was replaced with a serum-free medium and then incubated with ROS indicator DCFH-DA (10 μM) for 30 mins.The samples were detected by CLSM and flow cytometry, respectively.
To verify the effect of Fe 2+ , the cells were pre-treated with 10 μM of ferrous sulfate.After incubation for 10 h, the cells were washed three times with cold PBS and then followed the above experimental procedure.

Apoptosis analysis
Cellular apoptosis was assessed with an Annexin V-FITC apoptosis detection kit (Elabscience) according to the manufacturer's instructions.In brief, CT26 cells were seeded on 12-well plates at 2×10 5 cells per well.After 12 h incubation, cells were treated with PBS, Oxa, Oxa+2ART, Artoxplatin, nanobomb, and nanobomb ig , respectively (Pt at 5 μM in Figure 1, and at 2.5 μM in Figure 3) for 48 h.The cells were then washed with PBS, and incubated with Annexin/PI reagent in the dark for 15 min at 37 ℃.Thereafter, the cells were immediately measured with FCM.
For cells requiring iron pretreatment, medium contain 10 μM ferrous sulfate was added to each well and discarded after 10 h of incubation.Then the cells were washed three times with cold PBS, and followed the drug treatment.
Live/dead stain of cancer cells CT26 cells were seeded on 6-well plates at a density of 3 × 10 5 cells per well.After 12 h incubation, cells were treated with PBS, Oxa, Artoxplatin, nanobomb, and nanobomb ig , respectively (2.5 μM Pt) for 48 h.After this time, the media was removed and the cells were then washed with cold PBS for three times.The cells were further incubated with Calcein AM/PI Cell Viability Kit (KeyGEN BioTECH) for 15 min, and the cell survival/death was assessed by CLSM.(Calcein-AM: λex = 495 nm, λem = 515 nm, PI: λex= 493 nm, λem= 617 nm)

Measurement of cell surface CRT
CRT exposure was evaluated by FCM and CLSM.For FCM analysis, CT26 cells were seeded on 12-well plates at 2×10 5 cells per well.After 12 h incubation, cells were treated with PBS, Oxa, Oxa+2ART, Artoxplatin, nanobomb, and nanobomb ig , respectively (Pt at 25 μM) for 6 h.Cells were then collected and blocked with 1% BSA (Beyotime), and then further incubated with CRT primary antibody (ab211962, Abcam) at 4℃ for 1 h.After washing with PBS for 3 times, cells were incubated with the Alexa Fluor 488-conjugated secondary antibody (ab150077, Abcam) for 30 min and then the surface fluorescence was assayed with FCM.For CLSM analysis, cover slides were placed in the bottom of each well of a 24-well plate, and CT26 cells (1×10 5 ) in 1 mL complete media were added to each well and incubated at 37 ℃ for 12 h.Then the cells were then treated with PBS, Oxa, Artoxplatin, nanobomb, and nanobomb ig at an equal Pt concentration for 6 h.Next, the cells were washed with PBS and fixed in 4% paraformaldehyde solution for 20 min, followed by incubation with 1% BSA (Beyotime) for 30 min.Then the cells were incubated with primary CRT antibody at 4 ℃ overnight, and then incubated with the Alexa Fluor 555conjugated secondary antibody (ab150078, Abcam) after three washes with PBS.

Measurement of the release of HMGB1
The passively released HMGB1 was measured via CLSM analysis.In brief, a cover slide was placed in the bottom of each well of a 24-well plate, and CT26 cells (1×10 5 ) in 1 mL media were added to each well and incubated at 37 ℃ for 12 h.Cells were then treated with different formulations like the CRT for 24 h (10 μM Pt), and washed with PBS for three times.Then, the cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 10 min.Thereafter, cells were blocked with 1% BSA for 30 min, and then incubated with HMGB1 primary antibody (https://www.metaboanalyst.ca/),and the significance of enriched pathways was calculated using Fisher's exact method.

Measurement of PD-L1 level in cancer cells
In order to determine the expression level of PD-L1, CT26 cells were cultured and treated with different formulations (2.5 μM Pt) like the HMGB1 for 48 h.Cells were stained with PE-PD-L1 (Biolegend, USA) for 1 h.Stained cells were analyzed by FCM.

Hemolytic Activity Study
For the hemolytic activity study, blood samples were obtained from mice and centrifuged at 358× g, 4°C for 5 min.The supernatant was discarded and replaced with PBS.This step was repeated three times.The following PBS addition led to a 1/5 dilution of red blood cells (RBCs).RBCs were incubated with Oxa, Artoxplatin, nanobomb, and nanobombig at a Pt concentration of 40 μM for 3 h at 37℃ (with a RBCs:drug ratio 1:1).Sterile ddH2O was used as the positive control, while PBS was used as a negative control.After incubation, the intact erythrocytes were separated by centrifugation at 2240× g, 4°C for 5 min.The supernatants were transferred into 96well plate, and the absorption at 541 nm was measured with a microplate reader.The hemolysis ratio was calculated using the following formula: Dt-experimental group, Dnc-negative control group, Dpc-positive control group

In vivo toxicological evaluation
The female KM mice (6 weeks) were randomly grouped (n=3 mice per group).Oxa, Artoxplatin, nanobomb, and nanobomb ig (3 mg/kg Pt) were injected every three days intravenously (i.v.) for a total of 4 times.The mice were monitored and weighed every three days after the first injection.Then, mice were sacrificed at 11 days after administration.Physiological and biochemical of mice blood samples were collected and examined.

Tumor models and treatment experiments
When there were ~100 mm 3 palpable tumors, mice were randomly divided into seven groups (n = 6) including PBS group, Oxa group, Artoxplatin group, nanobomb group, nanobomb ig group, PD-L1 mAb group, and nanobomb ig +PD-L1 mAb group.For groups containing Pt, mice were i.v.injection of the corresponding drugs (3 mg/kg Pt) on days 0, 3, 6, 9, respectively.And PD-L1 mAb (100 μg) was intraperitoneally (i.p.) administered to mice every 3 days for a total of 4 times.The tumor volume and mouse weight of each group was measured every three days, and the tumor volume (mm 3 ) was calculated from measurements by using the following formula: L×W 2 /2, where L represents the large diameter of the tumor, and W represents the small diameter of the tumor.The mice were sacrificed 48 h after the last treatment, tumor derived lymph nodes (TDLNs), tumor nodules and spleens were harvested for flow cytometry analysis.

Histopathological analysis
The solid tumors were harvested from tumor-bearing mice on the 11 th day of drug injection for histological observation by standard hematoxylin and eosin (H&E) staining and immunofluorescence staining.For H&E staining, the excised tumors and organs were fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with H&E.The sections were then observed under a fluorescence microscope (IX83, Olympus).Twelve-μm thick frozen tissue sections were prepared for TdT mediated-dUTP Nick-End Labeling (TUNEL) assay and immunofluorescence staining.TUNEL assay was performed according to the instrument (Solarbio).For detecting the expression of PD-L1 and infiltration of CD8 + T cells in tumor tissues, frozen tumor sections were fixed, and blocked with 1% BSA.Primary antibodies targeting PD-L1 (WLH2821, Wanleibio) and CD8 (MA1-84018, Invitrogen) were incubated overnight at 4℃ in the blocking solution and the following day for 30 min at room temperature.After extensive washing in PBS, the secondary antibody including anti-mouse Alexa Fluor 555-conjugate (ab150078, Abcam) was added to the blocking solution and incubated for 2 h.Nuclei were counterstained with DAPI (ThermoFisher Scientific).And then Images were detected and captured by CLSM.

Flow Cytometry analyses
Single-cell suspensions were prepared from TDLNs, tumors and spleens by mechanical dissociation, and followed by the treatment of red blood cell lysing buffer (Solabio) to remove red blood cells.70-μm cell strainer was used to remove debris.
animal work was done in accordance with the National Institutes of Health's Guide for the Use and Care of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University.(2018sydw0258).

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ab216986, Abcam) overnight at 4 ℃.After washing with PBS for 3 times, cells were incubated with Alexa Fluor 555-conjugated antibody for 30 min.Thereafter, cell nuclei were stained with DAPI, and imaged with a confocal microscope.(Alexa Fluor Jose, CA, USA)-ESI-Qrbitrap-MS (Orbitrap Fusion Lumos, ThermoFisher Scientific, San Jose, CA, USA).Identification and relative quantification of the data were conducted by Compound discoverer (3.1).The normalized data was imported into the SIMCA-P version 14.1 to create an OPLS-DA model.The follow-