# A Biodegradable 2D Metallic MoS 2 Genesheet for Synergistic NIR-II Photothermal Immunotherapy

Lijuan Zhu, Jie Xu, Junjie Ren, Mei Yang, Changjie Yang, Xiaobo Gao, Alberto Bianco, Ding-kun Ji

► To cite this version:

Lijuan Zhu, Jie Xu, Junjie Ren, Mei Yang, Changjie Yang, et al. A Biodegradable 2D Metallic MoS 2 Genesheet for Synergistic NIR-II Photothermal Immunotherapy. Small, 2025, 21 (30), 10.1002/smll.202502577. hal-05218737

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Distributed under a Creative Commons Attribution 4.0 International License
A Biodegradable 2D Metallic MoS₂ Genesheet for Synergistic NIR-II
Photothermal Immunotherapy

Lijuan Zhu#, Jie Xu#, Junjie Ren, Mei Yang, Changjie Yang, Xiaobo Gao, Alberto Bianco*, Ding-Kun Ji*

L. Zhu, M. Yang, C. Yang, D-K. Ji

Institute of Molecular Medicine (IMM), Renji Hospital, Shanghai Jiao Tong

University School of Medicine, Shanghai, 200240, China

E-mail: dingkunji@sjtu.edu.cn

J. Xu, J. Ren

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,
Shanghai, 200240, China

X. Gao

Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China

Alberto Bianco

CNRS, Immunology Immunopathology and Therapeutic Chemistry, UPR 3572,
University of Strasbourg, ISIS, 67000 Strasbourg, France

E-mail: a.bianco@ibmc-cnrs.unistra.fr

**Keywords:** Graphene; 2D materials; drug delivery; phototherapy; cancer immunotherapy

<footnote># These authors contribute equally.</footnote>
**Abstract**

Developing promising biodegradable second near-infrared (NIR-II) immune agonists to overcome the insufficient immunogenicity of tumor cells and the poor immune response remains challenging. In this work, we explore a biodegradable “2D genesheet” for synergistic NIR-II photothermal immunotherapy. The 2D genesheet is large-scale prepared through flexible supramolecular self-assembly between metallic-MoS₂ and siRNA. The 2D genesheet not only exhibits an excellent photothermal conversion efficiency up to 56% in the NIR-II window, but also features unique H₂O₂-responsive biodegradability and high biocompatibility. Our 2D genesheet can efficiently delivery siRNA(siIDO) to tumor cells and downregulate indoleamine 2,3-dioxygenase 1 (IDO1) protein expression, exerting intrinsic immune activation and reversing IDO1-mediated immunosuppression. The spatiotemporal-controlled NIR-II phototherapy can elicit systemic immunity by evoking immunogenic cell death, promoting dendritic cell maturation, and increasing T cell infiltration. Our bioactive material is able to efficiently inhibit primary and distant tumors, achieving a 100% tumor eradication rate. This study broadens the application prospects of biodegradable NIR-II immune agonists, overcoming the limitations of current immunotherapeutic modalities.

**Keywords:** Graphene; 2D materials; RNA delivery; NIR-II phototherapy; Cancer immunotherapy
# 1. Introduction

Cancer immunotherapy, which activates the patients' immune system to eliminate primary and metastatic tumors, has achieved significant success in cancer treatment.

[1,2] Despite the promising scenarios of tumor immunotherapy, this type of therapy still faces several challenges. The systemic administration of immunotherapeutic agents, such as immune checkpoint blockers, cytokines, or adjuvants, often results in their distribution throughout normal tissues, leading to limited therapeutic benefits and potential immune-related adverse events.[3] Moreover, tumor cells can induce changes in the tumor microenvironment (TME) by overexpressing immune checkpoint proteins, which can lead to the failure of the immunotherapy. Inhibiting the TME-responsive immune checkpoints, known as immune checkpoint blockade (ICB), is considered an attractive therapeutic strategy to reduce the immunosuppressive effects in TME.[4-6] However, although ICB has shown desirable enhancement of the antitumor T-cell immunity in some cancers, its clinical responses are limited to only 10-30% of patients and certain types of tumors.[7] Therefore, alternative approaches to activate the cancer immunotherapy are highly desired.

Photothermal immunotherapy (PTI), which integrates noninvasive photothermal therapy (PTT) with immunotherapy, has gained global attention in the recent years. [8]

As previously reported, PTT not only eradicates tumor cells but also induces immunogenic cell death (ICD) in tumor tissues, thereby promoting the differentiation and infiltration of tumor-specific T cells and generating a robust anticancer immune response.[9] Second near-infrared (NIR-II) (spanning the 1000-1350 nm range) photothermal agents (PTAs) have recently become a focus of research due to their advantages in deep tissue penetration, high permission energy, excellent sensitivity and signal-to-noise ratio.[10, 11] Various types of NIR-II PTAs have been developed to meet the demands of PTI, including organic metal adjuvants,[12] semiconducting polymers,[13-18] inorganic nanoparticles,[19, 20] and small-molecule photosensitizers.

[21] However, many of these agents face practical challenges in bioapplications, such as complex preparation process, lack of degradability, and low biosafety.[12]
Therefore, the development of biodegradable NIR-II PTI nanoagents would be
beneficial in facilitating their practical applications.

2D TMDs have been proposed for various bioapplications due to their unique
physicochemical, electrical, optical, and mechanical properties.[22, 23] 2D TMDs have
been extensively utilized as PTAs owing to their biocompatibility, metabolizability,
and exceptional NIR-II photothermal performance, compared to other inorganic
nanoagents.[24-32] In particular, metallic MoS₂ (1T MoS₂) is one newly developed PTA
material, showing better biocompatibility and NIR-II photothermal performance than
commonly used 2H MoS₂.[31] Compared to graphene, M-MoS₂ is chemically and
structurally diverse and have shown better colloidal stability, better biodegradability
and higher biodegradability.[26] However, large-scale preparation of biodegradable 2D
TMDs is hardly achieved, and the integration of these materials with NIR-II light for
spatiotemporal photoregulation of immunotherapy has not yet been explored. These
limitations severely hamper their further application in practical cancer
immunotherapy. Moreover, a single photothermal effect is insufficient to suppress
tumor growth and metastasis. Cancer immunotherapy can effectively enhance the
immune response level of the tumor microenvironment through reprogramming.[33]
Small interfering RNA (siRNA) can down-regulate the immune checkpoint related
gene and protein expression and minimize off-target effect, providing a promising
pathway in treating a wide range of diseases.[33-35] However, the intrinsic difference
between 2D TMDs and siRNA pose a challenge for their precise co-delivery using a
single nanoplatform to achieve a synergistic therapeutic effect. Therefore, it is highly
desirable to develop a nano-delivery approach that can efficiently co-deliver siRNA
and 2D TMDs for a combined NIR-II PTI.

Herein we report a biodegradable 2D genesheet with efficient siRNA delivery for
synergistic NIR-II photothermal immunotherapy (Figure 1). The biodegradable 2D
M-MoS₂ nanosheet was easily large-scale prepared through a one-pot hydrothermal
treatment. These nanosheets demonstrated superior dispersibility in aqueous solutions
and efficient siRNA loading through P-S and hydrogen bonding. The resulting 2D genesheet not only exhibited superior NIR-II photothermal property with high photothermal conversion efficiency (PCE) of 56%, but also demonstrated responsive biodegradability in H₂O₂ solutions. The obtained genesheet is able to effectively deliver siRNA (siIDO) in the tumor, leading to the downregulation of IDO protein expression and exerting intrinsic immune activation. Upon NIR-II photoirradiation of the primary tumor, the 2D genesheet can induce a photothermal effect that directly ablated the tumor while promoting dendritic cell (DC) maturation and T cell priming through the induction of ICD in the cancer cells. Consequently, the 2D genesheet-induced NIR-II photothermal immunotherapy triggers a systemic immune response, efficiently inhibiting both primary and distant tumors in living mice. The superior biodegradability of the genesheet also avoids potential long-term toxicity of the nanocarrier, making it highly suitable for further clinical applications.

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Schematic illustration of biodegradable 2D genesheet-mediated NIR-II photothermal immunotherapy. (a) Preparation of 2D genesheet. (b) Schematic illustration of the mechanism of 2D genesheet for NIR-II synergetic photothermal immunotherapy.</figcaption></figure>
## 2. Results and Discussion

### 2.1. Preparation and characterization of M-MoS₂ and 2D genesheet

The biodegradable M-MoS₂ nanosheet was prepared via a one-pot hydrothermal treatment starting from MoO₃, thioacetamide and urea according to a previous work (Figure 2a).[36] After a simple filtration to remove the free small molecules and a batch ultrasonication process, black M-MoS₂ aqueous dispersion was obtained in high yield. Following the lyophilization, a black solid powder was isolated (Figure S1). It is worth noting that this method allows to easily prepare the material in gram scale. This method is simple, economic and safe to generate M-MoS₂, compared to the Li-intercalation method.[31] As-prepared M-MoS₂ is highly stable in water due to the reduced stacking of the layers, which are separated by the water molecules.[36] The immunomodulatory indoleamine 2,3-dioxygenase-1 (IDO1) siRNA sequence was then chosen as a model genetic drug, which has been proved to downregulate IDO1, relieving the immunosuppressive TME.[37] All oligonucleotide sequences are listed in Supporting Information (Table S1). M-MoS₂ was simply mixed with siRNA in an aqueous solution to construct the supramolecular MoS₂@siRNA 2D genesheet by supramolecular self-assembly.

To characterize the M-MoS₂ nanosheet and the 2D genesheet, a variety of techniques were used. The images of M-MoS₂ nanosheet obtained by high-resolution transmission electron microscopy (HRTEM) are shown in **Figure 2b**. M-MoS₂ are characterized by an ultra-thin lamellar structure and a crystal lattice pattern. The X-ray photoelectron spectroscopy (XPS) was used to detect the elemental valence of the material (Figure 2c). The bonding energies of 231.8 and 228.7 eV belong to the Mo 3d₃/₂ (Mo⁴⁺) and Mo 3d₅/₂ (Mo⁴⁺), respectively. The bonding energies of 162.7 and 161.6 eV are attributed to the S 2p₃/₂ and S 2p₃/₂, respectively, indicating that the chemical nature of product-MoS₂ is metallic 1T phase. In the UV spectrum of the materials, a broad adsorption range from 200 nm to 1380 nm was observed for M-MoS₂ (Figure 2d). In contrast, the assembly of M-MoS₂ with siRNA produced an absorbance peak at ≈ 260 nm, which is the characteristic peak of siRNA. This
suggests the complexation of the material with the siRNA. Dynamic light scattering
showed that the size of both the M-MoS₂ and the 2D genesheet was mainly in a
nanometer range of 70–250 nm (Figure 2e). The assembly of siRNA with M-MoS₂
also changed the zeta potential of the latter from -47 to -41 mV (Figure 2f). To further
confirm the successful loading of siRNA, the morphology of 2D genesheet was
observed by HRTEM (Figure 2g) and STEM (Figure 2h). As expected, the sharp
flake morphology of MoS₂ changed to be smooth to some extent after the surface
loading of siRNA. The lattice can be clearly observed in the HRTEM images. STEM
imaging further shows that the morphology of the nanoflakes has not changed
appreciably after siRNA loading (Figure 2h). The existence of siRNA along with the
Mo and S elements within the 2D genesheet was also confirmed by energy dispersive
spectroscopy (EDS) element mapping (Figure 2i-j), where all six elements display
the same distribution, indicating the uniform siRNA loading in the final 2D genesheet.
To further confirm the loading of siRNA onto M-MoS₂, we conducted a fluorescence
titration experiment (Figure S2). We found that the fluorescence of Cy5-labeled
siRNA was quenched as the amount of M-MoS₂ increased, indicating that siRNA was
efficiently adsorbed onto M-MoS₂. The stability of siRNA onto 2D genesheet was
tested both in PBS and 10% FBS buffer using polyacrylamide gel electrophoresis
(PAGE). We found that the nanostructure of the 2D genesheet increased the serum
stability of siRNA in comparison to the siRNA alone (Figure S3). This is likely due to
the protective action of M-MoS₂ against ribozyme cleavage of the nucleic acid
strand, which is consistent with our previous reported data. [38,39] These data suggest
the successful construction of the supramolecular 2D genesheet.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Preparation and characterization of M-MoS₂ and 2D genesheet. (a) Schematic illustration of the preparation of 2D genesheet. (b) HRTEM of M-MoS₂. (c) High-resolution XPS spectra of Mo 3d and S 2p for M-MoS₂. (d) UV-Vis spectra of M-MoS₂ (50 µg mL⁻¹) and 2D genesheet (M-MoS₂/siIDO=3/1, M-MoS₂=50 µg mL⁻¹) [ Insets show: (1) M-MoS₂, black line and (2) 2D genesheet dispersion, red line]. (e) DLS of M-MoS₂ (50 µg mL⁻¹) and 2D genesheet. (f) Zeta potential of M-MoS₂ (50 µg mL⁻¹) and 2D genesheet. (g) HRTEM of 2D genesheet. (h) STEM of 2D genesheet. (i) Element mapping of 2D genesheet. (j) EDS analysis of 2D genesheet.</figcaption></figure>
## 2.2. Interaction at the 2D genesheet interface driven by structure matching, P-S bonding, hydrogen bonding, and formation of water bridges

To elucidate the potential mechanisms by which M-MoS₂ interacts with siRNA, we used simulations to predict the absorption dynamics and the relevant residues of siRNA on M-MoS₂ nanosheet. We simulated the initial configurations and stable absorption configurations after 200 ns. As shown in **Figure 3a**, the contact area of siRNA with M-MoS₂ nanosheet increased at 200 ns, indicating that siRNA underwent a certain degree of conformational change to enhance the interaction with M-MoS₂. The three-dimensional structure of siRNA rearranged by bending the flexible regions or adjusting the orientation of the rigid regions, to accommodate the interactions with the M-MoS₂ nanosheet. This enhancement of the molecular contacts may be due to the interaction between specific nucleotide residues and specific sites on the surface of the MoS₂ nanosheet, including the possible formation of P-S bonds and hydrogen bonds (**Figure 3b, 3c**). Interestingly, we also found that water molecules formed bridges stabilized by hydrogen bonds with both the siRNA and the M-MoS₂ nanosheet. The P and N atoms in siRNA could form hydrogen bonding interactions with the S atoms in M-MoS₂ through the O atoms of water molecules.

In order to reveal the reasons for the changes in the contact surface, we analyzed the modifications in the number of atomic contacts between siRNA and M-MoS₂ nanosheet. The contact number changed over time and remained stable at a high contact number of 96 (**Figure 3d**). These dynamic signal fluctuations suggest siRNA adsorption and desorption events on the M-MoS₂ surface, meaning that the 2D genesheet is stabilized after 200 ns interaction. We also observed that the number of hydrogen bond contacts also greatly changed from 4 to 12, as the contact area increased during the simulation process (**Figure 3e**). By analyzing the changes in free energy between M-MoS₂ and siRNA (**Figure 3f**), we found that the free energy decreased with time and tended to be stable at -157 kcal/mol, indicating the formation of a stable state within 200 ns. Therefore, we can conclude that when M-MoS₂ nanosheet is mixed with siRNA, siRNA is adsorbed onto the M-MoS₂ surfaces. The 2D genesheet is stabilized by structure matching, P-S interactions, hydrogen bonds,
and salt-bridge interactions. The high affinity of the M-MoS₂ nanosheet for siRNA could generate a superior nanocarrier system to delivery siRNA into cells, resulting in an efficient gene therapy.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Self-assembly mechanism of 2D genesheet. (a) Molecular simulation (MD) was used to explore the adsorption of siRNA (siIDO) onto M-MoS₂ nanosheet, as well as the stable absorption residue distributions. (b) Bottom view of 2D genesheet. (c) Side view of 2D genesheet. (d) Contact numbers at the interfaces between the M-MoS₂ nanosheet and siRNA (siIDO). (e) H-bonds and free energy</figcaption></figure>
values at the interfaces between the M-MoS₂ nanosheet and siRNA.

## 2.3. NIR-II photothermal properties of 2D genesheet

With the materials in hand, we next evaluated the NIR-II photothermal conversion performance of 2D genesheet. The temperature of M-MoS₂ and 2D genesheet suspensions increased with the increase of concentration under a 1064 nm laser irradiation (Figure 4a). Interestingly, 2D genesheet displayed a higher temperature than M-MoS₂ at the same concentration of 50 µg mL⁻¹ (Figure 4b). This could be because siRNA loading hampers the stacking between the M-MoS₂ layers, increasing the photoactive area of M-MoS₂ in water. To further demonstrate the photothermal stability of the 2D genesheet, six cycles of NIR-II laser irradiation (laser on, 1 W·cm⁻², 5 min) and cooling down (laser off) of the particle suspension were subsequently recorded at a M-MoS₂ concentration of 50 µg mL⁻¹ (Figure 4c).

Apparently, there is no obvious change during the laser on-off process in both M-MoS₂ and 2D genesheet samples, demonstrating their favorable photothermal stability and promising potential as a PTT agent. PCE of M-MoS₂ nanosheet and 2D genesheet was measured in triplicate to be 47± 2% and 56± 1%, respectively (Figure 4d, 4e).

This suggests that the superior photothermal conversion performance of M-MoS₂ loaded with siRNA increased the efficiency due to interlayer suppression stacking. PCE of M-MoS₂ and 2D genesheet is higher than PVP-modified 1T-MoS₂ nanodots (43.3%) and PVP-modified 2H-MoS₂ nanodots (21.3%). [31] TMDs with different crystalline phases exhibit different properties. For example, 2H-MoS₂ is a semiconductor with a relatively large bandgap of approximately 1.83 eV, while 1T-MoS₂ is metallic. [31] It has been already shown that 1T-MoS₂ has a higher absorbance in the NIR-II region compared to 2H-MoS₂. This high 1T-MoS₂ absorption arises from its inherent metallic characteristic, which is typical of the 1T crystalline phase of MoS₂. In contrast, the semiconducting nature of 2H-MoS₂ results in minimal absorbance in the NIR window.[40]
<figure><img src="image_5.png" /><figcaption>**Figure 4. NIR-II photothermal properties of 2D genesheet and its degradation in biomimetic media.**
(a) Infrared thermal imaging of M-MoS₂ at different concentration of M-MoS₂ under a 1064 nm laser irradiation (1 W·cm⁻², 10 min). (b) Temperature increasing plot of pure water and aqueous suspension of M-MoS₂ and 2D genesheet at the concentration of 50 µg mL⁻¹ under a 1064 nm laser irradiation (1 W·cm⁻², 10 min). (c) Temperature plot of the aqueous suspension of M-MoS₂ and 2D genesheet at concentrations of 50 µg mL⁻¹ during six cycles of laser on-off (laser on: 5</figcaption></figure>
min; laser off: 5 min). (d) Photothermal effect of a solution of 50 µg mL⁻¹ M-MoS₂ and 2D genesheet under irradiation with a laser for 10 min and then switched off. (e) Steady-state heating curve of M-MoS₂ (T_M) and 2D genesheet (T_MR) dispersions when irradiated with a 1064 nm laser. (f) Degradation images of 2D genesheet after 14 days. The concentrations of H₂O₂ in the samples were 0, 0.1, 1, 5, 10 mM (from left to right). TEM images of 2D genesheet in water (g) and 10 mM of H₂O₂ (h) after 1 day (Scale bar: 100 nm). (i) UV-Vis spectrum of M-MoS₂ (100 µg mL⁻¹) in H₂O₂ aqueous solution for 3 h. The concentrations of the H₂O₂ in the samples are 0, 0.1, 1, 5, 10 mM (from left to right). (j) UV-Vis spectrum of M-MoS₂, M-MoS₂ in H₂O₂, urea, MoS₃, thioacetamide.

## 2.4. Biodegradation of 2D genesheet in biomimetic conditions

As similar transition metal sulfide and selenide, M-MoS₂ holds interesting promises in the field of nanomedicine due to its intrinsic physicochemical properties.[41-43] Therefore, its excretion and biodegradation behaviors are vital in determining for this type of applications. Firstly, the colloidal stability of the M-MoS₂ and 2D genesheet dispersed in different aqueous media (H₂O, PBS, DMEM, and DMEM with 10% fetal bovine serum) was investigated through storing them at room temperature. We found that both M-MoS₂ and 2D genesheet were stable in water and cell medium with 10% fetal bovine serum, no aggregation was observed during two weeks (Figure S4). We acknowledge that the typical production H₂O₂ ranges from 0.05 to 0.1 mM into the lysosomes responsible for the degradation of xenobiotics. However, studies have shown that the degradation of MoS₂ can take few weeks to a month, as followed by in vivo studies.[44] Therefore, we set the H₂O₂ concentration in the biomimetic media to approximately one hundred times higher, at 0.1–10 mM, to facilitate a faster degradation process that can be observed within a shorter timeframe under a microscope.

The degradation of 2D genesheet in the presence of H₂O₂ was studied storing 2D genesheet at different concentration of H₂O₂ (0, 0.1, 1, 5, 10 mM) at 25 °C. With the increase in H₂O₂ concentration, the absorbance of the solution decreased and the color
of the 2D genesheet became lighter (Figure 4f and Figure S5), indicating that M-MoS₂ underwent a transformation. TEM was further used to confirm the biodegradation of M-MoS₂ (Figure 4g and 4h). Most of the M-MoS₂ nanosheet disappeared at high concentration of H₂O₂ after 1 day and the remaining materials lost the structure of M-MoS₂ nanosheet. The missing of UV characteristic peaks at 1064 nm, 245 nm and 300 nm also confirmed the structural damage of M-MoS₂ (Figure 4i and 4j). The degradation rate of M-MoS₂ was measured to be 4.8% per minute (Figure S6). This is a relatively fast process compared to other materials, such as graphene,[45] gold nanoparticles,[46] 2H MoS₂,[47] confirming the clinical suitability of this type of TDM material. The fast degradation of pristine M-MoS₂ in H₂O₂ is likely due to the oxidation of molybdenum either to MoO₃ or to soluble MoO₄²⁻ ions along the formation of SO₄²⁻ as previously reported.[44, 47-49] All these results suggest the high biodegradability of this material in the tumor microenvironment.

## 2.5. siRNA uptake and tumor microenvironment immunosuppression

IDO is overexpressed in a wide range of malignancies, including melanoma, colorectal cancer, breast cancer, endometrial cancer, and specific subtypes of lung cancer. The IDO-mediated tryptophan metabolic pathway plays a critical role in suppressing tumor immunity and facilitating immune evasion of cancer cells. Consequently, targeting IDO has emerged as a promising therapeutic strategy in the field of tumor immunotherapy. In this study, we selected the 4T1 breast cancer model due to the superficial nature of breast tumors, which renders them particularly amenable to light penetration and photothermal therapeutic interventions.

Firstly, both flow cytometry and confocal laser scanning microscopy (CLSM) were used to evaluate the cell uptake efficiency of 2D genesheet in 4T1 cells. For this purpose, siIDO was labeled with cyanine 5 (siIDO-cy5). Mouse breast cancer cells (4T1) were incubated with 1 µM siIDO-cy5 and the corresponding amount of siRNA complexed to the nanosheet M-MoS₂@siIDO-cy5 (60 µg mL⁻¹) for 4 h at 37 °C. As shown in **Figure 5a**, M-MoS₂@siIDO-cy5 exhibited higher cell uptake efficiency than free siIDO-cy5, which was ascribed to the nanosized architecture. Meanwhile,
CLSM images in **Figure 5b** corroborated the enhanced endocytosis efficiency of M-MoS₂@siIDO-cy5 in 4T1 cells compared to free siIDO-cy5. Even after 1 h of incubation, significant red fluorescence was observed (**Figure S7**). In contrast, no obvious fluorescence was detected in 4T1 cells after 8 h incubation with free siIDO-cy5, likely due to the negative charge of nucleic acid (**Figure S8**). Both CLSM and flow cytometry results indicate the improved cellular uptake efficiency of siIDO by M-MoS₂ carrier property.

It has been reported that tumor immunosuppressive microenvironment induces the overexpression of indoleamine 2, 3-dioxygenase (IDO) on tumor cell membrane.[50-51] To investigate whether our designed 2D genesheet could reverse the tumor immunosuppressive microenvironment through downregulation of IDO, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, western blot assay and immunofluorescence were conducted to evaluate the IDO expression at both mRNA and protein level using free siRNA and MoS₂@scramble (MoS₂@scr) as controls. After stimulating with 30 ng mL⁻¹ IFNγ for 12 h at 37 °C, IDO related mRNA and protein expression in 4T1 cells was significantly upregulated (**Figure 5c-e**). After treating with siIDO, M-MoS₂@scr, and 2D genesheet, the 2D genesheet caused 72% knockdown of IDO mRNA in 4T1 cells (**Figure 5c**), while only 14% IDO mRNA knockdown was observed using free siIDO, which could be ascribed to the poor cell penetration of the negatively charged siIDO. In comparison, M-MoS₂@scr did not induce a significant knockdown of IDO mRNA, implying that the anti-IDO sequence was specific. Moreover, IDO expression was detected by western blot assay and immunofluorescence. After treating with MoS₂@siIDO, the IDO expression was significantly down-regulated, while MoS₂@scr and siIDO treated cells still exhibited comparable IDO expression to IFNγ stimulated cells (**Figure 5d and e**). The results of the western blot assay and the immunofluorescence staining were consistent with that of qRT-PCR analysis, confirming that MoS₂@siIDO could effectively knockdown the target gene.

In addition, to evaluate the anticancer efficacy of the 2D genesheet in the 4T1 cell line, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and
cell apoptosis analysis were conducted using M-MoS₂@scr and free siIDO as controls. After incubating with the different conjugates at increasing concentrations, the viability of the 2D genesheet, M-MoS₂@scr and free siIDO-incubated 4T1 cells was above 80% for all samples, indicating a low cytotoxicity. However, after NIR photoirradiation at 1064 nm, the siIDO-treated 4T1 cells showed more than 90% cell viability, while M-MoS₂@scr, and 2D genesheet-incubated cells exhibited increased cytotoxicity in a concentration-dependent manner, reaching a mortality of more than 90% above 20 µg mL⁻¹ (Figure 5f). Based on the MTT assay, the calculated IC₅₀ of M-MoS₂@scr and 2D genesheet-incubated cells were 12.5 and 13.5 µg mL⁻¹, respectively. Moreover, the cytotoxic effect of 2D genesheet to 4T1 cells was further examined by apoptosis analysis using an Annexin V-FITC kit. As shown in Figure 5g, the 2D genesheet, M-MoS₂@sc, and siIDO (at 20 µg mL⁻¹ M-MoS₂ concentration) induced apoptosis level of 93.1%, 95.2% and 4.7% in 4T1 cells after NIR photoirradiation, demonstrating that the 2D genesheet, M-MoS₂@scr exhibited good photothermal effect. In contrast, only 6.7%, 10.7%, and 3.8% apoptotic cells were observed after incubating cells with the 2D genesheet, M-MoS₂@scr, and siIDO without NIR, respectively, indicating their negligible cytotoxicity.

Subsequently, to quantitatively analyze the biodistribution and tumor accumulation of the 2D genesheet, BALB/c mice bearing subcutaneous 4T1 tumors were intravenously injected with the 2D genesheet at 10 mg kg⁻¹ mice. After 1, 6, and 24 h post-injection, the tumor and the major organs were collected and the amount of Mo was measured by inductively coupled plasma mass spectroscopy (ICP-MS). Six-hours post-injection, the amount of the 2D genesheet in tumor reached the maximum (Figure 5h). To evaluate the *in vivo* photothermal property of the 2D genesheet, 4T1 tumor-bearing mice were irradiated after 6 h with a 1064 nm laser at a power density of 1.0 W·cm⁻² for 10 min. The images of the temperature changes were tracked every minute by an infrared thermal imaging camera (Figure 5i and j). The temperature of tumor tissues increased rapidly from 31.9 to 43.2 °C within 3 min laser irradiation in the mice injected with 2D genesheet, while the tumor temperature of the mice injected with PBS and M-MoS₂ only increases by 6 and 9 °C, respectively. This result
confirms that the siRNA coating could improve *in vivo* photothermal property of MoS₂ by increasing the dispersibility of M-MoS₂.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Cellular uptake behavior, *in vitro* photothermal/immunological effect, and *in vivo* biodistribution of 2D genesheet. (a) Flow cytometry of cy5-labeled 2D genesheet (M-MoS₂: siIDO = 3:1, [siIDO] = 1 µM) and siIDO ([siIDO] = 1 µM) incubated 4T1 cells at 37 °C for 4 h. (b) Confocal fluorescence images of 4T1 cells after 2 h incubation with 2D genesheet and siIDO ([siIDO] = 1 µM). Cell nucleus (blue fluorescence) was stained with Hoechst 33342, and red fluorescence represented the signals of siIDO. (c) IDO mRNA expression in 4T1 cells stimulated with IFN-γ after incubation with PBS (G1), M-MoS₂@scr (G2), siIDO (G3), 2D genesheet (G4),</figcaption></figure>
and ([siIDO] = 6.7 µg mL⁻¹, [M-MoS₂] = 20 µg mL⁻¹) for 48 h. Untreated 4T1 cells and IFN-γ stimulated 4T1 cells were used as controls. (d) Western blot image of IDO expression in 4T1 cells after incubation with PBS (G1), M-MoS₂@scr (G2), siIDO (G3), 2D genesheet (G4), ([siIDO] = 6.7 µg mL⁻¹, [M-MoS₂] = 20 µg mL⁻¹) for 48 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference. The relative intensity ratios of IDO/GAPDH normalized to IFN-γ stimulated 4T1 cells are calculated. (e) Immunofluorescence images of IDO on IFN-γ stimulated 4T1 cells after treatment with the different conjugates at 20 µg mL⁻¹ M-MoS₂ concentration for 48 h, in which untreated 4T1 cells and IFN-γ stimulated 4T1 cells were used as controls. (f) MTT data of 4T1 cells incubated with 2D genesheet, and siIDO with or without NIR photoirradiation at 1064 nm at the power intensity of 1 W·cm² for 6 min. (g) Annexin V-FITC/PI flow cytometry assay of apoptosis in 4T1 cells induced by 2D genesheet, M-MoS₂@scr, or siIDO ([siIDO] = 6.7 µg mL⁻¹, [M-MoS₂] = 20 µg mL⁻¹) with or without NIR photoirradiation at 1064 nm with the power intensity of 1 W·cm⁻² for 6 min. (h) Quantitative accumulations of 2D genesheet in major organs and tumors after 1, 6, and 24 h post-injection. (i) Thermal images of tumor-bearing mice after injection of PBS, M-MoS₂, and 2D genesheet (lines top to the bottom) and irradiated at 1064 nm with the power intensity of 1 W·cm² for different time intervals. (j) Temperature curves calculated from the thermal images.

## 2.6. *In vivo* NIR-II photothermal immunotherapy of 2D genesheet

The 2D genesheet induced *in vivo* photothermal-immunological therapy was further studied in the same tumor model. 4T1 cells were initially subcutaneously inoculated in the right flank of mice as primary tumor. After 4 days, a distant 4T1 tumor was subcutaneously inoculated in the left flank of mice (Figure 6a). The NIR photoirradiation timepoints were determined according to the results of the quantitative accumulations of 2D genesheet in tumors after different post-injection time intervals (Figure 5h). After 6 h intravenous injection, the amount of 2D genesheet in the tumor reached to maximum value and it started decreasing after 24 h. On these bases, 4T1-tumor-bearing mice were intravenously injected with M-MoS₂,
M-MoS₂@scr, or 2D genesheet every 5 days and further irradiated at 1064 nm on the primary tumors at 6 h post-injection for 8 min. Meanwhile, PBS, and the same amount of M-MoS₂-, M-MoS₂@scr- or 2D genesheet-treated mice without photoirradiation were used as controls. The tumor growth of both primary and distant tumors, body weight, and photographs of all treated mice were monitored to evaluate the *in vivo* therapeutic effect for 16 days (**Figure 6b-g**). After the treatment, the primary and the distant tumor volume curves showed that M-MoS₂, M-MoS₂@scr, or 2D genesheet under photoirradiation exhibited better primary and distant tumor growth inhibition than the non-irradiated mice (**Figure 6b and 6e**). Moreover, 2D genesheet exhibited a significant primary and distant tumor growth inhibition after photoirradiation, demonstrating that the co-delivery of M-MoS₂ and siIDO was able to improve the anticancer effect. Meanwhile, the mice treated with 2D genesheet without photoirradiation evidenced a comparable tumor inhibitory effect than those treated with M-MoS₂ and MoS₂@scr with photoirradiation, implying that the IDO inhibition could reestablish the tumor immunosuppressive microenvironment and inhibit tumor proliferation. The tumor inhibitory rate (TIR) calculated measuring the tumor weight showed that the photoirradiated 2D genesheet-treated group exhibited the highest inhibition rate on both primary and distant tumors, reaching over 90% inhibitory rate compared to PBS-treated group (**Figure 6c and 6f**). After 16-days treatment, all treated mice were sacrificed and the primary and distant tumors were photographed (**Figure 6d and 6g**). The photographs of the mice during the treatment were also recorded (**Figure S9**). Notably, no mortality was observed in any experimental group, even when tumor volumes reached over 1000 mm³ in the control group, suggesting that the subcutaneous tumor burden did not critically impact the survival within the experimental timeframe. The therapeutic effects were further confirmed by immunofluorescence staining and hematoxylin and eosin (H&E) staining. The green fluorescence signals associated to apoptotic cells were detected using TUNEL assay in both the primary and distance tumors (**Figure 6h and 6i**). From this test, upon the photoirradiation, the 2D genesheet was the most effective modality to induce tumor cell apoptosis among all the other treatments. The H&E
staining evidenced that the tumor cells of the PBS-treated group presented a large
nucleus (blue), suggesting a rapid 4T1 cell growth (Figure 6j and 6k). In contrast,
H&E staining displayed smaller nuclear fragments and nuclear shrinkage, indicating
dead cells, in both primary and distant tumors injected with the 2D genesheet and
photoirradiated, consistent with the TUNEL results. Moreover, necrotic areas were
observed. All these data suggest that the 2D genesheet could effectively inhibit the
growth of both primary and distant tumors, indicating its stronger antitumor efficacy
relative to M-MoS₂@scr and MoS₂ groups. Although external laser irradiation is
sufficient for superficial tumor ablation in murine models, clinical translation to deep-
seated tumors may require alternative light delivery methods. Minimally invasive
techniques, such as fiber-optic endoscopy, navigation bronchoscopy, or thoracoscopic-
guided laser application, can provide precise deep localized irradiation. These
strategies could facilitate the effective implementation of NIR-II photothermal
immunotherapy while minimizing invasiveness and enhancing targeting accuracy.
<figure><img src="image_7.png" /><figcaption>**Figure 6.** *In vivo anticancer effect of 2D genesheet.* (a) Schematic illustration of the tumor model implantation and the photo-immunotherapy protocol. Tumor growth curves of the primary (b) and the distant tumors (e) in 4T1-tumor-bearing mice after the different treatments. TIR of the primary (c) and the distant tumors (f) after the different treatments. Images of the primary (d) and distant tumors (g) excised from mice of each group after the treatment. TUNEL assay of the primary (h) and the distant tumors (i) after the different treatments. Blue fluorescence shows the cell nucleus stained with 4',6-diamidino-2-phenylindole (DAPI); green fluorescence</figcaption></figure>
shows the signals of apoptotic cells in tumors (Scale bar, 100 µm). H&E staining of the primary (j) and the distant tumors (k) after the different treatments (Scale bar, 100 µm). 4T1-tumor-bearing mice (n=3) were intravenously injected (7.5 mg kg⁻¹) with the different conjugates and controls: G1: PBS, G2: M-MoS₂, G3: M-MoS₂@scr, G4: 2D genesheet, M-MoS₂ (G stands for group). Statistical significance in b and e was calculated using two-tailed Student's t-test. *p* < 0.1, **p** < 0.01, ***p** < 0.001, and ****p* < 0.0001, NS: no significant difference. The mean values and SD were presented.

Toxicity is one of the primary concerns and limitations for future clinical applications of the 2D genesheet. To address these points, an in vitro blood toxicity evaluation, through hemolysis analysis, was conducted. As shown in **Figure S10**, a negligible hemolysis was observed for the 2D genesheet and its degradation products, even at a concentration of 20 µg mL⁻¹, indicating the blood safety of this material. H&E staining of the major organs was then employed to evaluate the toxicity of the 2D genesheet. Staining of the tissue sections from heart, liver, spleen, lungs, and kidneys in each treated group showed intact nuclei, uniform cytoplasm, and no discernible tissue damage, cellular apoptosis, or pathological inflammation, demonstrating negligible differences between the drug-treated mice and the PBS-treated mice (**Figure S11**). Additionally, the key functionality indicators of the blood biochemistry of alkaline phosphatase (ALP), alanine transaminase (ALT), blood ureanitrogen (BUN), creatinine (Cr), and γ-glutamyltranspeptidase (γ-GT) showed no observable differences between the control and the treated groups (**Figure S12**), which indicates that liver or kidney functions were unaffected during the therapy. All these results suggest minimal adverse effects across all groups.

## 2.7. *In vivo* mechanistic study of NIR-II photothermal immunotherapy

It is well-known that PTT can activate immunogenic cell death (ICD) in tumor cells.[52, 53] To evaluate the corresponding immunological processes of ICD induced by the 2D genesheet after photoirradiation, immunofluorescence was used to observe the cell surface exposure of calreticulin (CRT) and the extracellular migration of high mobility group box-1 (HMGB-1) in tumor area after the different treatments. Only the cells treated with M-MoS₂, M-MoS₂@scr, and 2D genesheet under NIR
photoirradiation exhibited strong red fluorescence signals associated to CRT and HMGB-1, indicating the occurrence of the ICD process in tumor cells after PTT (Figure 7a and 7b). As a consequence, PTT-induced tumor-associated antigens can further maturate DCs. To assess this, we collected the primary and the distant tumor tissues to detected tumor-infiltrating lymphocytes (TILs) by flow cytometry after staining with anti-CD3, anti-CD4, and anti-CD8 antibodies (Figure 7c and 7d). The populations of TILs (i.e., CD4⁺ and CD8⁺) and cytotoxic TILs (i.e., CD8⁺) in the primary and the distant tumor tissues of the 2D genesheet-injected 4T1 tumor-bearing mice were higher than that of M-MoS₂ or M-MoS₂@scr-injected mice after NIR photoirradiation (Figure 7c). Moreover, in comparison to PBS-treated group, the frequency of cytotoxic TILs in primary and distant tumors after injecting 2D genesheet and treated without or with laser were increased to 44.0%, 41.8%, 57%, and 60.8%, corresponding to 5.3, 3.6, 6.9 and 5.2 folds higher than that of PBS groups, indicating that both siIDO and photoirradiation could enhance the population of cytotoxic TILs in both primary and distant tumor tissues. In addition, there were no significant differences in cytotoxic TILs in the primary and the distant tumors treated with M-MoS₂@scr (39.3% and 36.5%) and M-MoS₂ (38.0% and 35.3%). These TIL populations were lower than those in the 2D genesheet (57.0% and 60.8%) group after photoirradiation, demonstrating that the anti-IDO sequence was specific. DC maturation was then studied by collecting tumor-draining lymph nodes (TDLNs) and staining with anti-CD11c, anti-CD80, and anti-CD86 antibodies, using flow cytometry. Compared to PBS-treated group (with 14.1% mature DCs), 2D genesheet-treated group with laser enhanced the level of mature DCs to 41.9% (CD11c⁺/CD80⁺/CD86⁺), which were 1.7 folds higher than that of PBS-injected mice with laser (Figure 7d). In contrast, M-MoS₂ and M-MoS₂@scr-treated groups with laser exhibited much lower DC maturation frequency (26.7% and 24.5%), revealing that the 2D genesheet can significantly facilitate DC growth. All these data confirm that the 2D genesheet-mediated therapy could activate the immune system, exhibiting stronger antitumor T-cell immune responses.

Finally, immunofluorescence of IDO expression in primary and distant tumor tissues of the mice injected with M-MoS₂ and M-MoS₂@scr and 2D genesheet was measured to evaluate the down-regulation of the expression of IDO. The genesheet could significantly down-regulate the expression of IDO both in the primary and the distant
tumors, while M-MoS₂- and M-MoS₂@scr-treated group did not show changes (Figure 7e and 7f). The relative fluorescence intensity was measured and statistically analyzed to provide a more objective and robust assessment of IDO1 expression levels (Figure S13). Our analysis confirmed a significant reduction in IDO1 fluorescence intensity in the treatment group compared to the control group (p < 0.0001), which aligns with our qualitative observations. Moreover, IDO expression in the primary tumors treated with M-MoS₂, M-MoS₂@scr and 2D genesheet was further detected by western blot analysis (Figure 7g). After treating with the 2D genesheet, the IDO expression was down-regulated, consistent with that of immunofluorescence results. The serum samples of M-MoS₂-, M-MoS₂@scr- and 2D genesheet-injected mice with or without laser were collected after 3 days post-injection to detect the cytokines (e.g., TNF-α and IFN-γ) by ELISA. Both the concentration of TNF-α and IFN-γ in the sera of M-MoS₂-, M-MoS₂@scr-, and the 2D genesheet-injected mice increased after photoirradiation (Figure 7h and 7i). The ICD, DC maturation, and cytokine release profiles confirmed the enhanced inhibition of tumor proliferation by the 2D genesheet-mediated combination of NIR-II PTT and immunotherapy.
<figure><img src="image_8.png" /><figcaption>**Figure 7.** *In vivo* NIR-II photo-immunotherapy and targeted protein regulation by 2D genesheet. Immunofluorescence of (a) CRT, and (b) HMGB-1 after the different treatments. Blue fluorescence shows the cell nucleus stained with DAPI; red fluorescence represents the expression of CRT or HMGB-1 in tumors (Scale bar, 100 µm). (c) Flow cytometry analysis evidencing CD8⁺ and CD4⁺ from the primary and the distant tumors in 4T1-tumor-bearing mice after the different treatments. (d) Flow cytometry analysis of CD80⁺/CD86⁺ dual-positive DCs from the inguinal lymph nodes of 4T1-tumor-bearing mice after the different treatments. In all experiments,</figcaption></figure>
4T1-tumor-bearing mice were intravenously injected with G1: PBS, G2: M-MoS₂, G3: M-MoS₂@scr, G4: 2D genesheet (M-MoS₂: 7.5 mg kg⁻¹) (G stands for group), and the primary tumors were treated with or without NIR photoirradiation (1.0 W·cm⁻² at 1064 nm) for 8 min. Immunofluorescence showing IDO expression in (e) primary and (f) distant tumor tissues from 4T1-tumor-bearing mice injected with the different conjugated and controls, and treated with or without NIR photoirradiation (1.0 W·cm⁻² at 1064 nm) for 8 min. Blue fluorescence shows the cell nucleus stained with DAPI. Red fluorescence shows the IDO protein signals in tumors (Scale bar, 100 µm). (h) IDO protein expression in primary tumors treated with the different conjugates and controls. *In vivo* cytokine detection of (i) TNF-α and (j) IFN-γ in sera after 3 days post-injection with the different conjugates and controls with or without NIR photoirradiation (1.0 W·cm⁻² at 1064 nm) for 8 min (n = 3).

### 3. Conclusion

In summary, we have designed and prepared a biodegradable 2D genesheet using metallic MoS₂ with effective delivery of siRNA for synergistic NIR-II photothermal immunotherapy. The 2D genesheet was synthesized using a simple and efficient self-assembly strategy. It displayed a strong absorbance in the NIR-II window and a high photothermal conversion efficiency of 56%. It was able to efficiently deliver siRNA and effectively accumulate in tumor tissues of living mice following systemic administration. By effectively reverting the immunosuppressive TME through IDO1 knockdown, this material enhanced the tumor regression in a subcutaneous model. Under 1064 nm laser irradiation, the genesheet exerted an effective NIR-II phototherapy, generating heat that led to the ablation of primary tumors and the induction of immunogenic cell death. This, in turn, promoted the maturation of DCs and the activation of TILs in tumors. As a result, the 2D genesheet mediated a synergistic NIR-II photothermal immunotherapy action, leading to superior therapeutic efficacy in inhibiting the growth of both primary and distant tumors. Importantly, the 2D genesheet is biodegradable in the tumor microenvironment, which avoids long-term retention toxicity associated with the nanocarriers. This feature is
beneficial for their potential clinical applications. The flexibility of our approach
allows the complexation of other nucleic acid-based drugs into the biodegradable M-
MoS₂, creating a variety of NIR-II photoresponsive nanogene medicine to enhance
cancer immunotherapy. This research enables the simple development of
bidimensional transition metal dichalcogenide-based photothermal immunotherapy
materials to overcome the limitations of the current immunotherapeutic modalities.

Acknowledgements

We gratefully acknowledge financial support from the National Natural Science
Foundation of China (22205139, 22175116), the Natural Science Foundation of
Shanghai (22ZR1437800), the National Key Research and Development Program of
China (2023YFA1508800), the Fundamental Research Funds for the Central
Universities (2020JCPT02), the Centre National de la Recherche Scientifique
(CNRS), and the Interdisciplinary Thematic Institute SysChem, via the IdEx Unistra
(ANR-10-IDEX-0002) within the French Investments for the Future Program.

Data Availability

Data are available from the authors under reasonable request.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online.

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## Supporting Information

# A Biodegradable 2D Metallic MoS₂ Genesheet for Synergistic NIR-II
## Photothermal Immunotherapy

Lijuan Zhu#, Jie Xu#, Junjie Ren, Mei Yang, Changjie Yang, Xiaobo Gao, Alberto

Bianco*, Ding-Kun Ji*

L. Zhu, M. Yang, C. Yang, D-K. Ji

Institute of Molecular Medicine (IMM), Renji Hospital, Shanghai Jiao Tong

University School of Medicine, Shanghai 200240, China.

E-mail: dingkunji@sjtu.edu.cn

J. Xu, J. Ren

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,
Shanghai, 200240, China.

X. Gao

Department of Anatomy and Physiology, Shanghai Jiao Tong University School of
Medicine, Shanghai 200025, China.

Alberto Bianco

CNRS, Immunology Immunopathology and Therapeutic Chemistry, UPR 3572,
University of Strasbourg, ISIS, 67000 Strasbourg, France

E-mail: a.bianco@ibmc-cnrs.unistra.fr

<footnote># These authors contribute equally.</footnote>
**Experiment Section**

**Chemicals and Materials:** MoO₃, thioacetamide, and urea were purchased from Adamas-beta®. No further purification was conducted on all the chemicals. All the water used in our experiments was purified by a Milli-Q System (Millipore). The Gibco 1× PBS buffer without magnesium chloride and calcium chloride was purchased from Thermo Fisher Scientific. The McCoy's 5A cell culture media was purchased from BIOAGRIO (Shanghai, China). siIDO was purchased from Sangon Biotech. The localization probe for cell membrane, nucleus, mitochondria and lysosome were all purchased Sigma-Aldrich. IDO Rabbit mAb (#51851) and mouse interferon-γ (mIFN-γ) recombinant protein (#39127) were purchased from Cell Signaling Technology (US). Anti-HMGB1 antibody (#ab18256) and anti-Calreticulin antibody (#EPR3924) were purchased from Abcam (UK).

**Instruments:** Centrifugation were performed on an Eppendorf centrifuge 5425R. Millipore water with a resistivity of 18.2 MΩ was used in the experiments. UV-Vis spectra were recorded using a Cary 100 UV-Visible spectrophotometer from Agilent Technologies. The morphology of the samples was studied by HRTEM (TALOS F200X, Thermofisher, USA). DLS and zeta potential measurements were done with a Zetasizer Nano ZS (Malvern, U.K.). XPS was performed using a Thermo Scientific KAlpha X-ray spectrometer with a mono Xray source Al Kα excitation (1486 eV). All binding energies were calibrated to C1s peak at 284.8 eV of the surface sp3 carbon. The fine structure of the photoelectron lines was treated using Casa XPS software. A 1064 nm laser for the photothermal studies was from Changchun New Industries and a thermal imaging camera from Magnity Technologies.

**Preparation of M-MoS₂ nanosheet:** 120 mg of MoO₃, 140 mg of thioacetamide and 1.2 g of urea were dissolved in 100 ml deionized water and stirred for 2 h. The solution was poured into the autoclave and then loaded into a furnace at 200 °C for 10 h. Then, the reaction was stopped by slowly cooling the solution down to room temperature. The solution was flitted by a 200 µm pore size membrane. The filter cake was washes with DI water for 5 times. The resulting filter cake was dispersed in DI water again and sonicated for 10 min. After lyophilization, 80 mg of black solid was
obtained for further using.

**Preparation of 2D genesheet:** The 333 µg of siRNA (siIDO) in DCEP was added to 1 mL of M-MoS₂ (1 mg mL⁻¹). After stirring at room temperature for 1 h, the 2D genesheet was collected through centrifugation at the speed of 13000 rmp. Nanodrop was used to detect free siRNA in the supernatant.

To verify the sequence specificity of nucleic acids in downregulating IDO protein expression, we used a scrambled strand as a control. The preparation protocol of M-MoS₂@scr was the same of 2D genesheet.

**HRTEM imaging of 2D genesheet:** A droplet of M-MoS₂ and 2D genesheet (M-MoS₂/siIDO=10 µg mL⁻¹/3.33 µg mL⁻¹) was deposited onto carbon copper grids for HRTEM and for HAADF-STEM analyses (TALOS F200X, Thermofisher, USA).

**Hydrodynamic diameter and zeta potential measurement:** The samples of M-MoS₂ and 2D genesheet (M-MoS₂/siIDO=3/1, 10 µg mL⁻¹ of M-MoS₂) were sonicated for 5 min at room temperature. Their average hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS (Malvern, U.K.).

**Fluorescence quenching assays:** Cy5-labelled siIDO (at a concentration of 1 µM) was incubated with different amount of M-MoS₂ (0-10 µg) in DI water for 300 s, and the fluorescence was measured on a Varian Cary Eclipse fluorescence spectrophotometer using an excitation wavelength at 620 nm.

**Stability of siRNA in 2D genesheet:** 2D genesheet containing 3 µg of siIDO and 9 µg of MoS₂ was added to 20 µL of PBS or PBS with 10% FBS. The samples were sealed with sealing film and incubated at 37°C for various time points (0, 1, 2, 4, 6, 8, 24, and 48 h). After incubation, the mixtures were quenched in an ice bath, and 10 µL (0.1 µg/µL) of each sample was mixed with 6× loading buffer for electrophoresis. A 10% PAGE gel (TBE buffer) was run at 110 V for 45 min, and the bands were visualized using a chemiluminescence imager. All procedures were conducted using RNase-free consumables.

**Molecular simulation of 2D genesheet:** Two different force fields were used to adapt the various characteristics of the components within the system. The OPLS-AA force field was applied to the M-MoS₂ nanosheet due to its robust parameterization for
metallic interactions. Conversely, the AMBER force field was used for siRNA. This dual-force field approach ensured that both organic and inorganic interactions were modeled. Prior to dynamics, the system underwent energy minimization using the steepest descent method to remove any possible steric clashes and geometric inconsistencies. This was followed by an equilibration phase under NVT (constant number of particles, volume, and temperature) conditions to stabilize the temperature, followed by NPT (constant number of particles, pressure, and temperature) equilibration to stabilize both pressure and density of the system. The production molecular dynamics simulation was carried out for 200 ns.

**Photothermal properties of M-MoS₂ and 2D genesheet:** M-MoS₂ or 2D genesheet was dispersed in water with concentrations of 0, 10, 25, 50 or 75 µg mL⁻¹. Five hundred µL of the aforementioned solution in an Eppendorf tube was irradiated by a 1064 nm NIR Laser (1.0 W cm⁻²) from the top. A ThermoX infrared thermometer was used to record the temperature.

**Calculation of the photothermal conversion efficiency of M-MoS₂ and 2D genesheet:** Photothermal conversion efficiency (η) was calculated by Roper's method. To measure the photothermal conversion efficiency, 0.2 mL of an M-MoS₂ or 2D genesheet dispersion was irradiated by a 1064 nm NIR laser for 10 min. The laser was turned off to allow the sample to slowly cool down to room temperature. The real-time thermal imaging of the sample was recorded every 30 s by a ThermoX thermal camera. η was calculated via Equation (1).

$$ \eta = \frac{\text{hs}(T_{\text{max}} - T_{\text{sur}}) - Q_{\text{Dis}}}{I(1 - 10^{-A_{1064}})} \quad (1) $$

where h is the heat transfer coefficient, s is the surface area of the container, Tmax is the equilibrium temperature (°C), Tsur is the ambient temperature of the environment, I is the incident laser power (1W), QDis is the heat dissipation from the light absorbed by sample cell, and A1064 is the absorbance of M-MoS₂ at 1064 nm. The value of hs was derived according to Eq. (2):

$$ \tau = \frac{m_D c_D}{hs} \quad (2) $$
where τ is the sample system time constant and mD and cD are the mass and heat capacity of water [4.2 J g⁻¹ °C⁻¹] of the deionized water used as solvent, respectively.

To obtain τ, a dimensionless driving force temperature θ was introduced.

(3):

$$ \theta = \frac{T - T_{sur}}{T_{max} - T_{sur}} \quad (3) $$

After getting the curve of t=-τ lnθ, τ can be determined. According to Equation (3), hs was calculated. Then, according to Equation (1), η was calculated.

<table><thead><tr><th></th><th>A<sub>1064</sub></th><th>T<sub>sur</sub> (°C)</th><th>T<sub>max</sub> (°C)</th><th>hs</th><th>T (s)</th><th>η</th></tr></thead><tbody><tr><td>M-MoS<sub>2</sub></td><td>0.20</td><td>25.5</td><td>55.1 ± 2.15</td><td>0.0057</td><td>148</td><td>0.47 ± 0.02</td></tr><tr><td>Genesheet</td><td>0.21</td><td>25.5</td><td>60.0 ± 1.5</td><td>0.0060</td><td>141</td><td>0.56 ± 0.01</td></tr></tbody></table>

**Degradation of 2D genesheet:** 100 µg mL⁻¹ of M-MoS₂ with different concentration of H₂O₂ (0, 0.1, 1, 5, 10 mM) was stored at room temperature for 14 days. Their images at different points (0, 1 h, and 1, 2, 3, 7, 14 days) were taken by a digital camera.

**Calculation of the degradation rate of M-MoS₂:**

The standard curve of M-MoS₂ was measured following the UV absorption. Ten µg of M-MoS₂ were added to 200 uL of H₂O₂ (5 mM). A solution of fifty µg mL⁻¹ of M-MoS₂ with 5 mM of H₂O₂ was stored at room temperature for 60 min. The UV-Vis spectra were recorded at different time points (0, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 min). The value of degradation rate (v) was calculated according to Eq. (4):

$$ v = \frac{W_0 - W_t}{W_0 \times t} \times 100\% $$

Where W₀ is initial weight of M-MoS₂ and Wt is the residual weight of M-MoS₂ at time t.

**Cell culture and animals:** 4T1 cancer cell line was purchased from ATCC and cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin solution (100×) at 37 °C with 5% CO₂ humidified atmosphere.

**Flow cytometric analysis of cell uptake:** 4T1 cells were cultured in a 24-well culture
plate (5 × 10⁴ cells/well) overnight before treating with Cy5-labeled 2D genesheet (M-MoS₂/siIDO = 3:1) and siRNA at a Cy5 concentration of 1 µM for different time points of 4 h. Then, cells were washed, digested and tested by flow cytometry (BD FACSVerse™).

**Confocal microscopy analysis of cell uptake:** 4T1 cells were seeded on a confocal dish at a density of 5 × 10⁴ cells/well overnight before treating with Cy5-labeled 2D genesheet (M-MoS₂/siIDO = 3:1), and siRNA at a Cy5 concentration of 1 µM for 12 h. Then, cells were washed with PBS three times and stained with Hoechst 33342 for additional 5 min. Then, cells were washed with PBS three times and imaged with a confocal microscope (Leica TCS SP8).

**In vitro cell viability assay:** Cell viability assay was detected by MTT reagent. 4T1 cells were seeded in wells of a 96-well plate at the density of 7 × 10³ cells/well and incubated with complete growth medium overnight. Then, cells were treated with M-MoS₂@scr, 2D genesheet, siIDO with different concentrations for 6 h, followed by different NIR-II laser treatments (1064 nm, 1 W·cm⁻², 6 min). After culturing for another 42 h, MTT were added and incubated for another 4 h. Subsequently, absorbance at 490 nm was determined using a Microplate spectrophotometer (BioTek ELx800™). Untreated cells correspond to 100% viability. Flow cytometry was also used to analyze the treated cells in different groups.

**Quantitative real-time PCR (qRT-PCR) analysis:** To determine IDO1 mRNA expression level, 4T1 cancer cells were seeded into a 12-well culture plate at a density of 1 × 10⁵ cells per well and incubated overnight for cell attachment. Then the cells were stimulated with a mouse IFN-γ concentration of 30 ng mL⁻¹ for 12 h and incubated with M-MoS₂@siRNAscr, siIDO, and 2D genesheet ([siIDO] = 6.7 µg mL⁻¹, [M-MoS₂] = 20 µg mL⁻¹) for 48 h. After washed three times with PBS, the cells were lysed and total RNA was extracted using RNAeasy™ (Beyotime Biotechnology; Shanghai, China). Subsequently total RNA was reverse transcribed using ReverTra AceTM qPCR RT Master Mix (TOYOBO; Osaka, Japan). cDNA was then amplified using the Rotor-Gene SYBR® Green PCR Kit (Qiagen; Hilden, Germany) under the following conditions: initial activation at 95 °C for 15 min and 40 thermal cycles (10 s
denaturation at 95 °C and 45 s incubation at 52 °C). The sequences of primers were shown in Table S1.

**Western blotting analysis:** To access the IDO protein expression level, 4T1 cancer cells were seeded in a 12-well culture plate at a density of $1 \times 10^5$ cells per well and incubated overnight for attachment. The cells were then stimulated with 30 ng mL$^{-1}$ of mouse IFN-γ for 12 h and co-incubated with M-MoS$_2$@siRNAscr, siIDO, and 2D genesheet ($[\text{siIDO}] = 6.7 \ \mu\text{g mL}^{-1}$, $[\text{M-MoS}_2] = 20 \ \mu\text{g mL}^{-1}$) for 48 h. Subsequently, cells were lysed using RIPA buffer containing 1% PMSF for 10 min on ice. After quantifying the extracted proteins, equal amounts were mixed with loading buffer and heated at 95 °C for 10 min to denature the proteins. The mixture was loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, which was run at 100 V for 1.5 h before being transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 2 h, washed three times with TBST, and then subjected to immunoblotting with IDO and GAPDH antibodies at 4 °C overnight. After washing the membrane three times with TBST, it was incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody for 2 h, followed by detection using a Western-Ready ECL Substrate Kit and imaging with a ChemiDocTM imaging system (Bio-Rad, USA).

**Immunofluorescence results of IDO expression in vitro:** 4T1 cancer cells were seeded into a 24-well culture plate with a glass slide inside at a density of $5 \times 10^4$ cells per well and incubated overnight for cell attachment. Then the cells were stimulated with a mouse IFN-γ concentration of 30 ng mL$^{-1}$ for 12 h and incubated with M-MoS$_2$@scr, siIDO, and 2D genesheet ($[\text{siIDO}] = 6.7 \ \mu\text{g mL}^{-1}$, $[\text{M-MoS}_2] = 20 \ \mu\text{g mL}^{-1}$) for 48 h. After washed three times with PBS, the cells were stained with Hoechst 33342 and anti-IDO antibodies. The fluorescence images were captured using a Leica TCS SP8 confocal laser scanning microscope.

**Animals:** Female mice aged 6-8 weeks were procured from Shanghai Lingchang Experimental Animals Co., Ltd. These mice were maintained in compliance with the standards outlined in the "Guide for the Care and Use of Laboratory Animals" and the corresponding guidelines from the National Institutes of Health. The experiment
protocol was approved by the Animal Protection and Use Committee of Shanghai Jiao Tong University with approval number of A2023133-001.

**Tumor model:** $2 \times 10^6$ 4T1 cancer cells were suspended in PBS and injected subcutaneously into the right flank (primary tumors) of female BALB/c mice. Four days later, $1 \times 10^6$ 4T1 cancer cells were subcutaneously injected in the left flank (distant tumors) of the same mouse. The mice were used for *in vivo* therapy after 10 d of growth for primary tumors.

**In vivo biodistribution and tumor accumulation:** To quantitatively assess the *in vivo* accumulation of 2D genesheet in major organs and tumors, female BALB/c mice bearing 4T1 tumors were intravenously injected with 100 µL of 2D genesheet (M-MoS₂: siIDO = 3:1, M-MoS₂: 7.5 mg kg⁻¹) via the tail vein. The mice were euthanized at 1, 6 and 24 h post-injection, the animals were sacrificed and the major organs (hearts, livers, spleens, lungs, and kidneys) and tumors excised and thoroughly washed with PBS.

For inductively coupled plasma mass spectrometer (ICP-MS) analysis, analytical standards were purchased from SCP (Champlain, NY, USA) and trace metal grade nitric acid and aqua regia was purchased from Fisher Scientific (Pittsburg, PA, USA). All dilutions were done using in-house deionized water (≥18 MΩ) obtained from a Millipore water purification system.

**Thermal efficacy evaluation of NIR-II PTT:** Mice with 4T1 tumors were intravenously injected with PBS, M-MoS₂, 2D genesheet at the dose of (M-MoS₂: siIDO = 3:1, M-MoS₂: 7.5 mg kg⁻¹), respectively, and the tumors were then exposed to 1064 nm laser (3 min). The value of the laser spot size was 1 cm². Thermal camera was applied to capture mouse thermal images, and the temperatures of tumors were recorded.

**In vivo anticancer effects:** 4T1-tumor-bearing BALB/c mice were randomly divided into eight groups (three mice per group). When the primary tumor volumes reached approximately 150 mm³, the mice were intravenously injected with 200 µL PBS containing M-MoS₂, M-MoS₂@scr, or 2D genesheet ([M-MoS₂] = 7.5 mg kg⁻¹) on day 0, 5, 10. At 6 h of each post-injection timepoint, primary tumor of each mouse
was irradiated with or without NIR laser (1W·cm⁻² at 1064 nm) for 8 min. Then the sizes of primary and distant tumors and the body weights of mice were measured every 2 d for 15 d. The tumor volume was calculated as follows: volume = (tumor length) × (tumor width)²/2. After 15 d treatments, the mice in each group were euthanized, and the tumor tissues were dissected, washed, wiped, weighed, and photographed. collected tumor inhibition rate (TIR) was calculated based on the tumor weight using the following formula: TIR (%) = (W₀-Wt)/W₀ × 100%. Where W₀ is the average tumor mass in the control group, and Wt is the average tumor mass in the treated group. Then the tumor was used for subsequent H&E and immunofluorescence staining (TUNEL, IDO, CRT, and HMGB-1).

**Hemolysis assay:** Red blood cells (RBCs) were collected from BALB/c mice through orbital blood and separated from the serum by centrifuging at 3000 rpm for 5 min. After washing with PBS three times, RBCs were diluted with PBS (4%, v/v), and then 250 µL of this RBC suspension were mixed with 250 µL of deionized water (positive control), PBS (negative control), or Genesheet at different concentrations (10, 20, 40 µg mL⁻¹) with or without 5 mM H₂O₂ treatment. After incubation at 37 °C for 6 h, the mixtures were centrifuged at 3000 rpm for 5 min. The obtained supernatant (100 µL) was added to wells of a 96-well plate. Finally, the absorbance at 570 nm was determined with a microplate spectrophotometer, and the percentage hemolysis of RBCs was calculated. Hemolysis ratio (%) = (A (sample, 570 nm) – A (negative, 570 nm)) / (mean value of A (positive, 570 nm) – A (negative, 570 nm)) × 100%.

**In vivo evaluation of T cell and myeloid-derived suppressor cell population:** After 15 d under the different treatments, the mice in each group were euthanized, and the primary and distant tumors were collected and digested with 1 mg mL⁻¹ type I collagenase, 100 µg mL⁻¹ type IV collagenase, and 100 µg mL⁻¹ DNase I for 2 h at 37°C, then the tissues were filtered through a 70 µm nylon cell strainer. Then the red blood cells of the obtained tumor single-cell suspension were removed using ACK lysis, and the cells were washed with cell staining buffer. After that, the cells were incubated with anti-CD16 antibody for 10 min. For the cell surface antigen staining, the cells were stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-Gr-1, and
anti-CD11b antibodies for another 30 min. After washed with cell staining buffer, the cells were analyzed by Fortessa X20 (BD Biosciences).

**In vivo evaluation of DC maturation and cytokine release:** After 5 d under the different treatments, the mice in each group were euthanized, and the tumor-draining lymph nodes were collected and directly ground. Then the obtained single cells were incubated with anti CD16 antibody for 10min, and then stained with anti-CD11c, anti-CD80, and anti CD86 antibodies for another 30 min. After washed three times, the cells were analyzed by Fortessa X20 (BD Biosciences). After 15 d under the different treatments, the mice in each group were euthanized, and the blood samples were collected for the detection of TNF-α and IFN-γ by ELISA.

**Statistical analysis:** The experimental data are expressed as the mean ± standard deviation (mean ± SD). Significant differences were calculated using one-way ANOVA with the Turkey-Kramer formula, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.
<table><caption>Table S1. Detailed information of siRNA sequences</caption><thead><tr><td>Name</td><td>Sequence from 5' to 3'</td></tr></thead><tbody><tr><td>IDO1 siRNA</td><td>GCAAUAUUGCUGUUCCCUATT</td></tr><tr><td>IDO1 siRNA scramble</td><td>UUCUCCGAACGUGUCACGUTT</td></tr><tr><td>IDO1 primer forward sequence</td><td>GTGTGAATGGTCTGGTCTCTGTG</td></tr><tr><td>IDO1 primer reverse sequence</td><td>TGAGGGCTCTTCCGACTTGTC</td></tr><tr><td>GAPDH primer forward sequence</td><td>CGTGGAGTCTACTGGTGTCTTC</td></tr><tr><td>GAPDH primer reverse sequence</td><td>CGGAGATGATGACCCTTTTGGC</td></tr></tbody></table>
Additional Figure S1-S13

a

<figure><img src="image_9.png" /></figure>

b

<figure><img src="image_10.png" /><figcaption>**Figure S1.** Digital images of M-MoS₂ (1 mg mL⁻¹) aqueous dispersion and solid powder.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure S2.** Fluorescence quenching of fluorescently-labelled siIDO (1 µM) by M-MoS₂ at increasing concentrations (0, 1, 2, 3, 4, 5, 6, 8, 10 µg).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Figure S3.** Stability test of free siIDO and siIDO in 2D genesheet during 48 h. (a) PAGE gel image of free siIDO and 2D genesheet in PBS or 10% FBS buffer at 37°C for various time points (0, 1, 2, 4, 6, 8, 24, and 48 h). (b) Quantitative analysis of the gel band intensities.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Figure S4.** Colloidal stability of M-MoS₂ (100 µg mL⁻¹) and 2D genesheet (M-MoS₂/siIDO = 3:1, M-MoS₂=100 µg mL⁻¹).</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure S5.** Degradation images of 2D genesheet (M-MoS₂/siIDO = 3:1, M-MoS₂=100 µg mL⁻¹) in 14 days. The concentrations of the H₂O₂ in the samples are 0, 0.1, 1, 5, 10 mM (from left to right).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure S6.** The calculation of degradation rate of M-MoS₂. a) UV-VIS spectra of M-MoS₂ in different concentration (0, 10, 20, 30, 40, 50 µg/mL). b) The standard curve of M-MoS₂ according the UV absorption of M-MoS₂ at 307 nm. c) The UV-VIS spectra of M-MoS₂ (50 µg/mL) in 5 nM of H₂O₂ during 60 min. d) The Abs intensity of M-MoS₂ (50 µg/mL) in 5 nM of H₂O₂ at 307 nm.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure S7.** Confocal fluorescence images of 4T1 cells after incubation with 2D genesheet ([siIDO] = 1 µM) at different time intervals (1, 4, 6 and 8 h). Cell nucleus (blue fluorescence) was stained with Hoechst 33342, and red fluorescence represented the signals of siIDO (Scale bar: 75 µm).</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Figure S8.** Confocal fluorescence images of 4T1 cells after incubation with siIDO ([siIDO] = 1 µM) at different time intervals (1, 4, 6 and 8 h). Cell nucleus (blue fluorescence) was stained with Hoechst 33342, and red fluorescence represented the signals of siIDO (Scale bar: 75 µm).</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Figure S9.** Images of 4T1 tumor implanted BALB/c mice treated with different conjugates and controls. G1: PBS, G2: M-MoS₂, G3: M-MoS₂@scr, G4: 2D genesheet (corresponding to M-MoS₂: 7.5 mg kg⁻¹) (G strands for group).</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure S10.** Hemolysis assay. (a) Photograph of hemolytic analysis and (b) relative hemolysis ratio of deionized water (positive control), PBS (negative control), or Genesheet at different concentrations (5, 10, 20 µg mL⁻¹) with or without 5 mM H₂O₂ treatment after 6 h.</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S11.** H&E staining for major organs of mice treated with different onjugates and controls. G1: PBS, G2: M-MoS₂, G3: M-MoS₂@scr, G4: 2D genesheet (corresponding to M-MoS₂: 7.5 mg kg⁻¹) (G strands for group) (Scale bar: 100 µm).</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Figure S12.** Blood biochemistry data after administration of the different conjugates and controls by tail vein injection. Level of alkaline phosphatase (ALP), alanine transaminase (ALT), blood ureanitrogen (BUN), creatinine (Cr), and γ-glutamyltranspeptidase (γ-GT) were measured.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure S13.** Quantification of IDO expression in primary tumors and distant tumors of 4T1-tumor-bearing mice by immune fluorescence staining after the different treatments (n = 3). p**** < 0.0001.</figcaption></figure>