DNA Adjuvant Hydrogel‐Optimized Enzymatic Cascade Reaction for Tumor Chemodynamic‐Immunotherapy

Abstract Chemodynamic therapy (CDT) shows immense potential in cancer treatment as it not only directly kills tumor cells but also induces anti‐tumor immune responses. However, the efficacy of CDT is hampered by challenges in targeting CDT catalysts specifically to tumors using nanomaterials, along with the limitations of low H2O2 levels and short catalyst duration within the tumor microenvironment. In this study, DNA adjuvant hydrogel to arrange a glucose oxidase‐ferrocene cascade for continuously generating reactive oxygen species (ROS) from glucose in situ for tumor CDT combined with immunotherapy is employed. By precisely tuning the catalyst spacing with DNA double helix, ROS production efficiency is elevated by up to nine times compared to free catalysts, resulting in stronger immunogenetic cell death. Upon intratumoral injection, the DNA hydrogel system elicited potent anti‐tumor immune responses, thereby effectively inhibiting established tumors and rejecting re‐challenged tumors. This work offers a novel platform for integrated CDT and immunotherapy in cancer treatment.


Experimental Procedures
Materials.
The murine colon carcinoma CT26, melanoma B16F10 and breast carcinoma 4T1 cell lines were purchased from Guangzhou Xinyuan technology Co., Ltd.All cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 37 °C and 5% CO2 incubator.
Female C57BL/6 mice (6-8 weeks old) were purchased from Soochow University Laboratory Animal Center, and used under the animal experiment protocols approved by them (approval number: SYXK(Su) 2017-0043).

Fabrication and characterization of DNA hydrogel.
To construct glucose oxidase (GOx) encapsulated DNA hydrogel, the thiol-ssDNA (L3-SH) was conjugated with GOx.Briefly, L3-SH was mixed with Sulfo-SMCC at a molar ratio of 1:5 and shaken at room temperature for 0.5 h in PBS.Excess SMCC was removed by ultrafiltration.Next, L3-SH and GOx were mixed in PBS at a molar ratio of 1:5, then shaken at room temperature for 2 h.The successful coupling of DNA and GOx (L3-GOx) was identified by polyacrylamide gel electrophoresis (PAGE).
The DNA hydrogel was synthesized as previous studies.We took Gel-Fc-GOx as an example.Firstly, Yscaffold (Y1/Y2/Y3) and Linker (L1/L2-Fc20) strands were mixed in buffer (100 mM Tris-HCl, pH 6.0, 250 mM NaCl) with final concentrations of 1 mM and 1.5 mM, separately.The mixture solutions were heated to 95 °C for 5 min and then slowly cooled to 4 °C within 2 h.Then, Linker was hybridized with L3-GOx at a ratio of 1:1 for 10 min at 37 °C.Finally, Y-Scaffold and Linker were mixed with a molar ratio of 2:3 to get the A-Gel-Fc-GOx.The DNA strands were listed in Table S1, and the corresponding sequences can be changed to synthesize desired DNA hydrogels (Table S2).The prepared monomers were characterized by PAGE.

Scanning electron microscopy (SEM) characterization.
A-Gel-Fc-GOx after thoroughly freeze drying under vacuum was put onto clean Silicon plates, and their morphology were examined by SEM (HITACHI, SU8010).EDS mapping was conducted for elemental distribution analysis.

Rheological analysis.
Rheological tests were carried out on an ARES-RFS rheometer with 8 mm parallel-plate geometry.The viscosity curves were recorded under rotational runs at 25°C.Time-scan test was carried out at a fixed strain of 1% and frequency of 1 Hz at 25 °C.

Catalytic performance.
12.5 μL A-Gel-Fc-GOx was added into a series of glucose concentration (40, 80, 120, 160 mM at pH 6.0), and mixed with TMB solution (2 mg/mL).The chromogenic reaction (λ = 650 nm) was determined by the UV-vis spectra at indicated time intervals.Michaelis-Menten kinetic curve could be obtained by plotting velocity against glucose concentration.The Michaelis-Menten constant (KM) and maximal velocity Vmax were calculated via the Lineweaver-Burk plotting.
12.5 μL of different components of DNA hydrogels (A-Gel, A-Gel-Fc, A-Gel-GOx, A-Gel-Fc mix GO, A-Gel-Fc-GOx) were mixed with glucose solution (25 mM, pH 6.0) and reacted for 20 min.The production of •OH was evaluated by chromogenic reaction of TMB.

In vitro cytotoxicity.
B16F10 cells were seeded at a density of 1×10 4 cells per well in a 96-well plate and cultured overnight.
After that the medium was changed with fresh DMEM with pH 6.0, the followed by the addition of 6.25 μL different DNA hydrogels (A-Gel, A-Gel-Fc, A-Gel-GOx, A-Gel-Fc mix GOx, A-Gel-Fc-GOx) to make a total volume of 100 μL.Each group was conducted in triplicate.After 12 h incubation, the medium was removed, and fresh medium containing 10% CCK8 was added to each well.After incubation for another 30 min, the 96well plate was measured by a microplate reader at the absorbance of 450 nm.
A-Gel-Fc-GOx with a series concentration (depending on GOx) was dispersed in DMEM medium with pH adjusted to 6.0 or 7.4.The cell cytotoxicity was evaluated by CCK8 assay as above.

ATP assay.
B16F10 cells were cultured in a 6-well plate.In Fig. 2g, the intracellular ATP content was assessed by collecting supernatants after various treatments, followed by processing with an ATP assay kit.In Fig. 3c, the secreted ATP content was evaluated by collecting cell medium.The ATP levels were quantified using enhanced chemiluminescence measured with a microplate reader.

Intracellular ROS detection and imaging.
B16F10 cells were inoculated at 5×10 4 per well in 24-well plates and cultured overnight.Then the medium was changed to fresh DMEM with pH 6.0, following by addition of 12.5 μL different DNA hydrogels (A-Gel, A-Gel-Fc, A-Gel-GOx, A-Gel-Fc mix GO, A-Gel-Fc-GOx) to produce ROS, and PBS as control.After incubation for 12 h, the medium was removed and DCFH-DA diluted in serum-free DMEM was added to each well.Flow cytometry was used to quantify the intracellular ROS.
Using confocal microscope dishes to incubate cells and treated in the same way, CLSM (Nikon Eclipse Ti) was used to detect the production of intracellular ROS.

In vivo anti-tumor efficacy
CT26 cells (1×10 6 /each) were inoculated subcutaneously in 5-week-old C57BL/6 mice to construct a colon cancer model.The mice were divided into 6 groups equally, one group was the control group using saline, and the remaining 5 groups were treated with Gel no CpG, A-Gel, GOx mix Fc, Gel-Fc-GOx, and A-Gel-Fc-GOx.At day 7, 100 μL of each sample was injected to the tumor in situ.The tumor volumes and mice body weights were monitored every other day.The survival time of the remaining mice were monitored until day 60 post the first injection.Tumor volume was calculated as the following formula: Tumor volume = 0.5×L×W 2 (L: the longest dimension, W: the shortest dimension).
The mice subcutaneous melanoma and orthotopic breast cancer models were conducted as above.

In vivo immune responses
On day 16 after treatment of CT26 tumor-bearing mice with different fractions, some mice were taken from each group.The lymph nodes were harvested and filtrated through a 200-mesh filter to get single cells.
To verify the intratumor infiltration of T lymphocytes, on day 16 after treatment of CT26 tumor-bearing mice with different fractions, some mice were taken from each group.The tumor tissues were collected and making into frozen slices, using IHC easy CD8 Ready-To-Use IHC (Proteintech) for immunohistochemical analysis.
To analyze the mouse cytokine and interferon responses, mice were euthanized and serum was collected after 16 days.Tumor necrosis factor α (TNF-α) and Interferon γ (IFN-γ) were measured using ELISA kits (Solarbio) according to the manufacturer's instructions.

Anti-tumor metastatic ability assay
Mouse 4T1 breast cancer lung metastasis models were constructed.The tumor-bearing mice were equally divided into 4 groups, one of which was the control, and the remaining 3 groups were treated with using straight GOx mix Fc, A-Gel, and A-Gel-Fc-GOx.At day 7, 100 μL of each was injected in situ in the tumor.
The tumor volumes and mice body weights were monitored every other day.The survival time of the remaining mice for each group were monitored until day 24 post the first injection.After 24 days some mice were taken from each group.The lungs were collected and making into frozen evaluated with Hematoxylin/eosin (H&E).

Tumor rechallenge studies
After complete cure of the mice with A-Gel-Fc-GOx, the mice were allowed to recover for 57 days and then inoculated again with CT26 cells (3×10 6 /each) on the other side of the mice, as well as untreated mice.
The tumor volume size of the mice was measured every other day, while the survival of each group of mice was observed until day 60 after reinoculation to test the presence of immune memory.The spleen was harvested and filtrated through a 200-mesh filter to get single cells.The effector memory T cells (CD44 + CD62L -in CD3 + CD8 + cells) were measured by flow cytometry.

Statistical analysis.
All statistical analyses were conducted on Origin software.Data from the experiments were performed for three times and the results are presented as the mean ± standard deviation (SD).One-way ANOVA with Tukey's multiple comparisons test was performed for statistical analysis of the difference between the two groups.P value < 0.05 was considered statistically significant between the data sets, where all significant values were indicated as follows: *p< 0.05, **p< 0.01, ***p< 0.001.Table S1.Gel-GOx Y1, Y2, Y3, L1, L2, L3-GOx Gel-Fc Y1, Y2, Y3, L1, L2-Fc20