An iRGD‐conjugated photothermal therapy‐responsive gold nanoparticle system carrying siCDK7 induces necroptosis and immunotherapeutic responses in lung adenocarcinoma

Abstract Although immunotherapy has improved the clinical treatment of lung adenocarcinoma (LUAD), many tumors have poor responses to immunotherapy. In this study, we confirmed that high expression of Cyclin‐Dependent Kinase 7 (CDK7) promoted an immunosuppressive macrophage phenotype and macrophage infiltration in LUAD. Thus, we have developed an internalizing‐RGD (iRGD)‐conjugated gold nanoparticle (AuNP) system which carries siCDK7 to activate the antitumor immune response. The iRGD‐conjugated AuNP/siCDK7 system exhibited good tumor targeting performance and photothermal effects. The AuNP/siCDK7 system with excellent biosafety exerted a significant photothermal antitumor effect by inducing tumor cell necroptosis. Furthermore, the AuNP/siCDK7 system ameliorated the immunosuppressive microenvironment and enhanced the efficacy of anti‐PD‐1 treatment by increasing CD8+ T cell infiltration and decreasing M2 macrophage infiltration. Hence, this iRGD‐conjugated AuNP/siCDK7 system is a potential treatment strategy for lung adenocarcinoma, which exerts its effects by triggering tumor cell necroptosis and immunotherapeutic responses.


| INTRODUCTION
Lung cancer is the leading cause of cancer death in the world according to 2020 Cancer Statistics. 1 Lung adenocarcinoma (LUAD) comprises over 50% of all lung cancer cases and its frequency is increasing. The five-year survival rate of advanced LUAD patients has been reported to be only approximately 15%. 2 Anti-programmed death 1 (PD-1) or antiprogrammed death ligand 1 (PD-L1) antibodies have revolutionized the treatment of advanced non-small-cell lung cancer (NSCLC). 3,4 However, classifying cancers based on T-cell infiltration showed that many cold tumors exhibit poor response to immunotherapy. 5 Therefore, pursuing more sensitive immunotherapy approaches for LUAD is important for curing this malignant disease.
CDK7 is a key regulator of cell cycle progression and functions as the catalytic core of the CDK-activating kinase (CAK) complex. 6,7 CAK is important for RNA polymerase II-mediated transcription. 8 CDK7 inhibition has been reported to reduce abnormal cell proliferation associated with lung cancer. 9,10 A recent study also found that CDK7 inhibition activates CD8+ T cells by triggering an intrinsic antitumor effect in small-cell lung cancer, 11 but the mechanism has not been clarified. With an increased understanding of the role of CDK7 in the progression of various tumors, great efforts have been made to inhibit CDK7 function. 12 On one hand, to date, several inhibitors targeting CDK7 have recently entered clinical trials. 13 On the other hand, the downregulation of CDK7 expression de novo may become a promising research direction.
Photothermal therapy (PTT) has become a new noninvasive method in cancer treatment that converts light energy to heat energy. 14,15 Gold nanoparticles (AuNPs) have been regarded as an important photothermal agent due to their low toxicity, high modifiability, chemical stability, and ease of synthesis and functionalization. 16,17 It has been shown that the PTT effects of AuNPs can induced ROS production which can activate the tumor cell death program. [18][19][20] AuNPs also can enhance antitumor ability and provoke antitumor immune responses in vivo when modified with inorganic molecular or bioactive substances. [21][22][23] Furthermore, as potential delivery vehicles, AuNPs can carry siRNA into the tumor site. 24 In addition, nanomedicine always exhibits cascade amplification effects for cancer therapy. 25,26 SiRNA modifications have become a very promising therapeutic strategy for anticancer research. 27,28 According to the properties of AuNPs, we designed a new AuNP/siCDK7 nanosystem that has potential for gene silencing in combination with PTT. Conjugation with polyethylenimine (PEI) and methoxy polyethylene glycol (mPEG) significantly promoted siRNA cellular uptake and increased gene knockdown efficiency. 29 A tumor-homing RGD peptide displays strongly bounding ability to cancer cells which overexpress αvβ3 and αvβ5. Internalizing-RGD (iRGD) that guided complexes binding to tumor vessels and increasing vascular and tissue permeability was utilized as a bullet, whereas the conventional RGD peptide only delivered the compounds to the blood vessels. [30][31][32] Necroptosis is one critical mechanisms of immunogenic cell death executed by the receptor interacting protein kinase 1 (RIPK1)-RIPK3-mixed lineage kinase domain-like protein (MLKL) signaling cascade. 33 Necroptosis is characterized by increased plasma membrane permeability is accompanied by the release of damage-associated molecular patterns (DAMPs) and cytokines, thereby triggering antitumor immune responses. 33,34 In this research, we studied the effect of CDK7 inhibition in triggering the immune response and developed a new iRGDconjugated AuNP@mPEG-PEI/siCDK7 system to treat LUAD. As shown in Scheme 1, the nanosystem has the following advantages: (1) tumor targeting; (2) inducing tumor cell necroptosis; and (3) inhibiting M2 macrophage polarization and induced immune response.

| CDK7 promoted an immunosuppressive macrophage phenotype
The Cancer Genome Atlas (TCGA) dataset was used to explore the genes whose expression were correlated with CDK7 expression in LUAD tissues. Gene set enrichment analysis (GSEA) was performed.
We found that the genes, whose expression levels were negatively correlated with CDK7 expression levels, were mainly enriched in Fc gamma R-mediated phagocytosis ( Figure 1a). Furthermore, we detected the expression level of stimulating factors that promoted M1 and M2 macrophage polarization in LUAD cells. Interference with CDK7 expression significantly downregulated IL-4 and IL-13 expression in LUAD cells (Figures 1b and S1A). Next, we investigated the function of CDK7 in macrophages. We cocultured THP-1 cells with H1975 cells for 48 h, and the THP-1 cells were subsequently subjected to RNA-sequence analysis. To characterize the functions of the differentially expressed genes (DEGs) between the H1975-siNC and H1975-siCDK7 groups, KEGG pathway analyses were performed. The suggested that Akt activation may be involved in the CDK7-induced immunosuppressive macrophage phenotype. Thus, inhibiting CDK7 in cancer cells may be a potential approach to activate the macrophage immune response.

| CDK7 expression is associated with M2 macrophage infiltration in LUAD
To explore whether CDK7 affects the chemotactic migration of macrophages, we conducted a migration assay. CDK7-knockdown LUAD cells cocultured with macrophages inhibited the chemotactic migration of macrophages toward LUAD cells (Figure 2a). To further confirm this phenomenon, we measured the expression of CDK7 and a biomarker of M2 macrophages (CD206) in 43 LUAD tissues by immunohistochemistry (IHC) (Figure 2b). The results showed a positive correlation between CDK7 expression and M2 macrophage infiltration in LUAD tissues (Table 1).

| Synthesis and characterization of the AuNP@mPEG-PEI-iRGD nanosystem
The synthetic route is described in Scheme 1, and the resultant product was validated by 1 H-NMR and Fourier transform infrared (FTIR) measurements, as shown in Figure 3a

| Preparation and characterization of AuNP@mPEG-PEI-iRGD/siCDK7 complexes
We evaluated the condensation ability of AuNP@mPEG-PEI-iRGD toward siCDK7 by performing an agarose gel retardation assay.  formed a stable complex under this condition. The ζ-potential of AuNP@mPEG-PEI-iRGD/siCDK7 complexes gradually increased when the N/S ratio increased. The ζ-potential returned to basal levels when the N/S ratio was greater than 15:1 ( Figure 3h). We measured the average particle size to test the stability of AuNP/siCDK7 complex in different media. As shown in Figure S2A, the particle size of the complex remained stable in H 2 O and DMEM. The particle size in PBS decreased rapidly in the first day and remained stable in the following days. The protective effect of AuNPs on siCDK7 were also tested by UV absorption. The UV absorption spectra results showed that the absorbance of siCDK7 did not change significantly after laser irradiation ( Figure S2B), indicating that the heat produced by AuNPs did not cause much degradation of siCDK7. In addition， as shown in UV spectra, the absorption peak of AuNP@mPEG-PEI-iRGD/siCDK7 group in RNase A solution was weakened after 24 h, but still maintained a high absorption peak ( Figure S2C). The above results suggested that AuNP@mPEG-PEI-iRGD combined with siCDK7 formed positively charged and stable complexes, which facilitated the complexes of entry into cells.  laser irradiation (Figures 5f and S3E). The results suggested that interfering with CDK7 expression successfully enhanced the antitumor effects of AuNP-mPEG-PEI-iRGD-induced PTT.

| Photothermal properties and CT imaging of AuNP@mPEG-PEI-iRGD
In addition, we explored the cell death program induced by AuNP@mPEG-PEI-iRGD/siCDK7 using restoration and WB assays.

| Biological safety evaluation in vivo
Biological safety is a prerequisite for the application of materials in nanomedicine. Therefore, the toxic effects of AuNP/siCDK7 complexes on mouse main organs and blood in vivo were investigated. HE images of the main organs of mice after 14 days of different treatments are shown in Figure S4A; no obvious damage to the tissue morphology and structure of the organs were observed in any of the treatment groups. Blood chemistry parameters for evaluating liver function, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and kidney function, the blood urea nitrogen (BUN) levels, in each group were also tested ( Figure S4B). Compared with healthy mice, PBS-treated controls and mice treated with AuNP/ siCDK7 showed no significant changes in blood parameters. Therefore, we concluded that AuNP/siCDK7 complexes are safe materials and promising for use in vivo.

| DISCUSSION
Although immunotherapy has been considered as first-line treatment for NSCLC, a significant portion of patients still show no response to immunotherapy. 35 To solve this problem, immunotherapy combined with other treatments has been widely explored, thus increasing the number of patients who respond to immunotherapy. 36 Tumorassociated macrophages (TAMs) can account for up to 50% of some solid neoplasms, and M2 macrophages often play an immunosuppressive role in the tumor microenvironment (TME). 37,38 Thus, TAMs could be a promising therapeutic target in the future.
CDK7 play a critical role in mediating the transcription of key cancer dependence genes. 39 It has been demonstrated that CDK7 inhibition disrupted cell-cycle progression and induces DNA replication stress and genome instability in lung cancer while triggering immuneresponse signaling. 11 In addition, it has been reported that the high expression of CDK7 is positively correlated with poor survival rate in lung cancer. 40 In our study we first studied the association between CDK7 expression and the macrophage immune response in LUAD.
Interfering with CDK7 expression in LUAD cells decreased the expression of M2 macrophage-stimulating factors, such as IL-4 and IL-13. 41 This might explain why CDK7 promotes M2 macrophage polarization and infiltration and inhibits macrophage phagocytic abilities in LUAD. Phosphorylation of AKT and STAT are the most well-known pathways associated with macrophage M2 polarization. 42,43 We found that interfering with CDK7 expression in LUAD cells cocultured with macrophages inhibited the AKT pathway, but not the STAT pathway, in macrophages. Moreover, it has been reported that the inhibition of macrophage M2 polarization enhances the antitumor immune response and consequently induces greater proinflammatory factor secretion. 44,45 Consistently, we showed that CDK7 inhibited the expression of proinflammatory factors, such as IL-12b and CCL2, by macrophages. [46][47][48] Our work first suggested that downregulation of CDK7 expression enhanced the immune response of macrophages.
To inhibit CDK7 expression in tumors, we synthesized a tumortargeted AuNPs system to deliver siRNA. AuNPs exert multifunctional therapeutic effects in cancer treatment via photothermal effects and controllable delivery of their cargo. 49,50 PEI is a recognized standard contrast polymer for gene delivery. When the PEI/siRNA complex is endocytosed by cells, it will form connotation bodies. Then siRNA will be released into the cytoplasm to play a transfection role. 51 After the modification of PEI with mPEG, the transfection efficiency and safety of PEI will be enhanced. 29 Recently, AuNP-delivered siRNA was applied to treat glioblastoma in a first-in-human phase 0 clinical study, further indicating the clinical translational potential of AuNP-based siRNA delivery systems. 52 We demonstrated that the AuNP/siCDK7 system could be taken up by tumor cells and had satisfactory RNA interference efficiency and photothermal effects. The findings also demonstrated that the iRGD peptide had the ability to enhance the efficiency of cargo delivery by nanoparticles in vitro and in vivo, which is consistent with previous findings in other tumor models. [53][54][55] Meanwhile, noninvasive or invasive diagnosis are both key methods to assess the efficacy of treatment. 56,57 Our synthesized AuNPs had CT imaging capability in the tumor area which could guiding tumor therapy.
As an effective nanodelivery system, the nanosystem in this study could overcome the multiple obstacles to tumor treatments. 58,59 AuNPs/siCDK7 complexes were able to "kill two birds with one

| Cell culture
The LLC, H1975 and monocyte THP-1 and RAW264.7 cell lines were grown in 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cell lines were maintained in an atmosphere of 5% CO 2 at 37 C.

| Immunohistochemistry
The protein expression in tumor tissues was measured using an immunoperoxidase method. Slides were incubated with primary antibodies, followed by incubation with secondary antibodies and being stained with a DAB kit. The IHC intensity of each tissue slide was independently evaluated by two experienced clinical pathologists who were blinded to the patients' clinical data.

| Characterization
The absorbance of mPEG-PEI-iRGD and AuNPs was determined by UV spectrophotometry in the range of 200-1000 nm. Fourier transform infrared (FTIR) spectra were recorded by an FTIR spectrophotometer in the range of 4000-399 cm À1 . The morphology of AuNPs was characterized by transmission electron microscopy (TEM). The chemical structures were characterized by 1H NMR spectroscopy using deuterium oxide (D2O) as the solvent and analyzed by MestReNova software. Thermogravimetric (TG) analysis was carried out to determine the compositions of the AuNPs using thermogravimetry. The TG settings were as follows: temperature range of 25-800 C and heating rate of 10 C/min. The ζ-potential and hydrodynamic diameter were recorded by a Zetasizer Nano ZS (Malvern) apparatus with size and ζ-potential analyzer software.
AuNP@iRGD dispersions with a series of Au concentrations (5 $ 80 mM) were placed in 0.3 ml Eppendorf tubes, and all the tubes were loaded into the CT imaging system and scanned sequentially. In vivo CT imaging ability was performed using LLC tumor-bearing mice. The mice were injected with 100 μg/g AuNP@mPEG-PEI-iRGD via the tail vein. CT images were captured, and Hounsfield units (HU) were measured.

| Agarose gel retardation assay
The siCDK7 complexation ability of AuNP@mPEG-PEI-iRGD was evaluated by performing an agarose gel retardation assay. AuNP@m-PEG-PEI-iRGD/siCDK7 complexes with different N/S ratios were separated by 1% agarose gel at 140 V for 30 min. A fluorescence detector was used to capture images.

| Photothermal performance
AuNP@mPEG-PEI-iRGD dispersions with a series of Au concentrations were placed in cuvettes. All cuvettes were irradiated with 808 nm near-infrared light at different powers for 5 min. In vivo PTT was performed using LLC tumor-bearing mice. The mice were injected with 100 μl of saline or 100 μg/g AuNP@mPEG-PEI-iRGD via the tail vein. Twenty-four hours after injection, the tumor region was irradiated with an 808 nm laser at a power density of 1 w cm À2 for 5 min.
The temperature was measured by a thermocouple thermometer and captured by a thermal imaging camera.  Table S1.

| Phagocytosis assay
RAW264.7 cells were stained with DiO, and LLC cells were stained with Dil for 15 min. RAW264.7 cells were incubated with LLC cells exposed to different treatments for 6 h. Finally, the samples were analyzed using flow cytometry (FCM).

| Transfection assay
The cellular uptake of AuNP complexes with and without iRGD was analyzed to confirm the targeting ability of iRGD. Cy5-labeled, lipo-siCDK7, AuNP@mPEG-PEI/siCDK7 or AuNP@mPEG-PEI-iRGD/ siCDK7 complexes were used to treat the cells. The injected amounts of siCDK7 and AuNPs were 0.5 and 10 μg/ml, respectively. After incubation for 4 h, the cells in each group were washed with PBS and cultured in medium supplemented with 10% FBS for 48 h.

| Apoptosis assay
The cell apoptosis rate was determined by an Annexin V 633 Apoptosis Detection Kit. The cells were collected and washed twice with cold PBS, followed by incubation with Annexin V/PI for 30 min on ice.

| Transwell assay
Transwell chambers were used to investigate cell migration. After

| Safety evaluation
After treatment for 2 weeks, the main organs (heart, liver, spleen, lung, and kidney) and serum of the mice were collected for further investigation. Major organs were sectioned for HE staining analysis.
The serum biochemistry (ALT, AST, BUN) was analyzed using an Automatic Biochemical Analyzer.