Construction of Durvalumab/carbon nanotube/PEI/aptamer-siRNA chimera for the immunotherapy of hepatocellular carcinoma

Immunotherapy is the most promising treatment for hepatocellular carcinoma (HCC). However, the immunosuppressive microenvironment and necrosis limit its therapeutic effectiveness. Carbon nanotubes (CNTs) have good tissue permeability and can penetrate tumor necrosis area. Here we constructed a Durvalumab/CNT/PEI/aptamer-siRNA chimera (chimera/Durmab/CNT) nanoparticles for the immunotherapy of HCC. In vivo and in vitro experiments showed that aptamer-siRNA chimeras could specifically bind HCC cells and inhibit the triggering receptor expressed on myeloid cells-2 (Trem2) expression, but had no effect on Trem2 expression in normal liver and lung. Transmission electron microscope results showed that the CNT/PEI nanoparticles were 20–30 nm in diameter and 200–350 nm in length. Dense PEI attachment can be observed on CNTs. CNT/PEI nanoparticles could control the sustained release of Durvalumab for 48 h. In vitro experimental results showed that chimera/Durmab/CNT could increase the proportion of T cells and CD8 + T cells, and then promote the apoptosis of HepG2 cells, and the therapeutic effect was superior to aptamer/Durmab/CNT and Durmab/CNT. We constructed a tumor-bearing mouse model, and the results showed that chimera/Durmab/CNT significantly inhibited the growth of transplanted tumor, and the volume and proliferation was further reduced in the chimera/Durmab/CNT group compared with the aptamer/Durmab/CNT group. T cells and CD8 + T cells infiltration, and HCC cell apoptosis were significantly increased in the chimera/Durmab/CNT group. In conclusion, we constructed a Durvalumab/CNT/PEI/chimera, which can effectively treat HCC by activating anti-tumor immunity.


Introduction
Hepatocellular carcinoma (HCC) is the most common malignant tumor of the digestive system, and more than 500 000 patients are diagnosed with HCC every year worldwide. The early symptoms of HCC are not specific, and many patients have lost the opportunity for surgery by the time they are diagnosed [1]. Immunotherapy is a research hotspot in recent years. Immune checkpoint inhibitor can activate the patient's own immune function and effectively kill tumor cells [2,3]. Several checkpoint inhibitors have been approved for clinical use, including Nivolumab and Durvalumab [4,5]. Many clinical studies have shown that PD-1/PD-L1 inhibitors can effectively improve the objective remission rate and survival rate of advanced HCC patients [6,7]. Animal experiments and clinical studies have shown that there are many kinds of regulatory immune cells in HCC tissues, which may weaken the effect of immunotherapy [8]. Therefore, activating the local immunosuppressive microenvironment and recruiting more immune cells are of great significance for enhancing the effect of immunotherapy for HCC.
Triggering receptor expressed on myeloid cells-2 (Trem2), a transmembrane receptor in the immunoglobulin superfamily, is associated with the onset of Alzheimer's disease and other neurodegenerative diseases [9]. Trem2 plays an important role in the regulation of tumor immune responses and is involved in the formation of an immunosuppressive microenvironment [10]. The researchers used scRNA-Seq technology to reveal the immunosuppressive effect of Trem2 in cancer [10,11]. Co-culture experiments showed that the absence of Trem2 strongly inhibited tumor growth by enhancing T cell killing. Trem2 expression was inversely associated with overall and relapse-free survival in patients with colorectal cancer or triple-negative breast cancer [12]. Recent studies have shown that targeted Trem2 can reshape the tumor myeloid landscape and enhance the efficacy of immunotherapy [13]. Therefore, Trem2 may be a potential target to enhance the efficacy of immunotherapy for advanced HCC.
In the immunotherapy, the activated immune cells kill a large number of cancer cells, which begin to apoptosis and necrosis, and it is difficult for Nivolumab and Durvalumab to enter into the tumor. Carbon nanotubes (CNTs) have good tissue permeability and can penetrate various biological barriers [14]. Studies have found that CNTs can penetrate tumor necrosis area and tuberculous ulcer tissue to ensure that chemotherapy drugs can reach the targeted area and achieve good therapeutic effect [15]. In addition, the special nanostructure makes CNTs have huge specific surface area, so they can achieve high drug loading [16]. Here, we propose to construct nanomaterials based on CNTs for the targeted delivery of Trem2 siRNA, providing a new therapeutic approach for the clinic.

Clinical data
Patients with HCC who came to our hospital from June 2019 to September 2020 were included in this study. Inclusion criteria: age >18 years old, pathological diagnosis of highly differentiated HCC. Exclusion criteria: other malignancies, autoimmune liver disease, diabetes mellitus, uremia, AIDS, cytomegalovirus infection. Patients with liver cirrhosis treated in our hospital at the same time were selected as the cirrhosis group. The general information (gender, age, weight, liver function, etc) between the two groups had no significant statistical difference (all P > 0.05). This study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of PLA Rocket Force Characteristic Medical Center (2018-11).

Immunohistochemistry
HCC tissue was fixed with 4% paraformaldehyde (Beijing ZsBio Co., Ltd) and paraffin section was made. After dewaxing, 3% hydrogen peroxide (Beijing ZsBio Co., Ltd) was added and treated at room temperature for 3 min. Washed with PBS for three times, sealed for 30 min, dripped with antiki-67 (Beijing ZsBio Co., Ltd), and spent the night in the refrigerator. After washing with PBS for three times, biotin labeled antibody (Beijing ZsBio Co., Ltd) was added and incubated at 37 • C for 30 min. After washing with PBS for three times, streptomyces ovalbumin working fluid (Beijing ZsBio Co., Ltd) was added and incubated at room temperature for 15 min. After washing with PBS for three times, the color was developed for 10 min, hematoxylin (Beijing ZsBio Co., Ltd) was re-dyed, dehydrated, transparent and sealed, and then observed under a microscope.

Construction of nanomaterials
To modify aptamer-siRNA chimera on CNTs, we firstly constructed CNT/PEI nanoparticles. The CNT/PEI nanoparticles were constructed by the methods previously reported [17]. Physical adsorption of PEI (Sigma Co., Ltd) to the CNTs (XFNANO Co., Ltd) was achieved by dispersing purified CNT (20 mg) into ethanol (25 ml) by probe sonication for 20 min. After washing with petroleum ether for three times, CNT/PEI particles were obtained by drying in a drying oven. CNT/PEI was fully dissolved into the activation buffer, EDC/NHS solution (XFNANO Co., Ltd) was added, and gently stirred for 30 min. Supernatant was discarded, coupled buffer was added, and repeated washing was performed for three times. PD-1/PD-L1 monoclonal antibody Durvalumab (1 mg) was added, stirred gently at room temperature for 12 h, centrifuged, and washed repeatedly for three times to obtain Durvalumab/CNT/PEI.

Co-culture experiments
Hepatoma cell line HepG2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (Hyclone Co., Ltd). HepG2 cells at logarithmic growth stage were divided into five groups: control group, CNT group (CNT/PEI particles were added in the medium), Durmab/CNT group (Durvalumab/CNT/PEI particles were added in the medium), Aptamer/Durmab/CNT group (Durvalumab/CNT/PEI/aptamer particles were added in the medium), Chimera/Durmab/CNT group (Durvalumab/CNT/PEI/chimera particles were added in the medium). After 24 h, the supernatant was discarded and fresh culture medium was added. The lymphocytes were separated by lymphocyte separation solution (TBD Co., Ltd) and divided into five portions in average and added into each group of medium. After co-culture for 48 h, lymphocytes were collected and added CD3 and CD8 antibodies (BD Co., Ltd). The lymphocytes were incubated at room temperature for 20 min and washed with PBS for three times. The proportion of T cells (CD3 + cell) and CD8 + T cells (CD3 + CD8 + cells) was detected by flow cytometry.

Apoptotic experiment
The experimental group was the same as before. HepG2 cells were co-cultured with lymphocytes for 48 h. Cells in each group were collected and stained with Annexin V-FITC and PI (BD Co., Ltd). After fully mixing, the cells were incubated at room temperature for 15 min. After adding binding buffer, the proportion of apoptotic cells was detected by flow cytometry.

Animal experiments
The animal model was constructed according to the methods reported in previous paper [20]. 1 × 10 6 HepG2 cells were slowly injected into the subcutaneous tissue of C57BL/six mice. After 7 d, the mice were randomly divided into five groups: control group (HepG2 cell transplantation), CNT group (injection of CNT/PEI particles around transplanted tumor), Durmab/CNT group (injection of Durvalumab/CNT/PEI particles around transplanted tumor), Aptamer/Durmab/CNT group (injection of Durvalumab/CNT/PEI/aptamer particles around transplanted tumor), Chimera/Durmab/CNT group (injection of Durvalumab/CNT/PEI/chimera particles around transplanted tumor). After 7 d, the mice were anesthetized with 0.1% pentobarbital sodium. The transplanted tumor was obtained and fixed in 4% paraformaldehyde. After paraffin sections, some tissues were stained with H&E and the other tissues were immunohistochemized to detect the expression of Ki-67 and the number of T cells, CD4 + T cells (Beijing ZsBio Co., Ltd). The apoptotic levels were detected by TUNEL staining (Roche Co., Ltd).

Statistical method
Statistical analyses were performed by using Graphpad Software 5.0. Data values were showed as mean ± SD. Multiple data comparisons were carried out via ANOVA and Bonferroni post hoc test. P < 0.05 was considered significant.

Trem2 expression was significantly elevated in HCC
H&E staining showed the formation of pseudolobules and the presence of basic liver structure in cirrhotic tissues, while the HCC tissues lost normal liver tissue structure and obvious nuclear atypia was observed. Ki-67 proliferation index and Trem2 expression of HCC were significantly higher than those of cirrhosis group. Correlation analysis showed that the proportion of Trem2+ cells was positively correlated with ki-67 proliferation index figure 1.

Characteristics of nanoparticles
Transmission electron microscope (TEM) results showed that the CNT/PEI nanoparticles were 20-30 nm in diameter and 200-350 nm in length. Dense PEI attachment can be observed on CNTs ( figure 2(A)). Sustained-release experiment showed that CNT and CNT/PEI nanoparticles could control the sustained release of Durvalumab for 48 h, there was no significant difference between the two groups ( figure 2(B)). Flow cytometry results showed that both aptamer and aptamer-siRNA chimera could specifically bind over 85% of HepG2 cells (figure 2(C)). After 48 h, the aptamer-siRNA chimera significantly inhibited Trem2 expression in HepG2 cells, while the aptamer had no significant effect ( figure 2(D)). Animal experiment results showed that aptamer-siRNA chimera could specifically bind HCC cells and inhibit their Trem2 expression, but had no effect on Trem2 expression in normal liver and lung (figure S1 available online at stacks.iop.org/BMM/17/025015/ mmedia).

Durvalumab/CNT/PEI/chimera promoted T cell proliferation
There was no statistical difference in the proportion of T cells and CD8 + T cells between the control group and CNT group. Compared with the CNT group, the proportion of T cells and CD8 + T cells in Durmab/CNT group was significantly higher. Durvalumab/CNT/PEI/chimera significantly promoted anti-tumor immune response, and the proportion of T cells and CD8 + T cells in chimera/Durmab/CNT group was significantly higher than that in the aptamer/Durmab/CNT group (figure 3).

Durvalumab/CNT/PEI/chimera promoted apoptosis of HepG2 cells
There was no statistical difference in the apoptotic ratio of HepG2 cells between the control group and CNT group. Compared with the CNT group, the apoptotic ratio of HepG2 cells in Durmab/CNT group was significantly increased. Durvalumab/CNT/PEI/chimera significantly promoted the tumor killing ability of lymphocytes, and the   apoptotic ratio of HepG2 cells in chimera/Durmab/CNT group was significantly higher than that in the aptamer/Durmab/CNT group (figure 4).

Durvalumab/CNT/PEI/chimera inhibited the growth and proliferation of transplanted tumor
There was no significant difference in the volume of transplanted tumor between the control group and CNT group. Compared with the CNT group, Durmab/CNT group had significantly lower transplanted tumor volume. Durvalumab/CNT/PEI/chimera further inhibited the growth of transplanted tumor, and the volume of transplanted tumor was further reduced in the chimera/Durmab/CNT group, compared with the aptamer/Durmab/CNT group ( figure 5(B)).
Ki-67 ratio is an important indicator of cell proliferation and has been widely used in clinicopathological detection [21]. Immunohistochemical results showed that there was no statistical difference in the proportion of Ki-67 between the control group and CNT group. Compared with the CNT group, the proportion of Ki-67 in Durmab/CNT group was significantly decreased. Durvalumab/CNT/PEI/ chimera further inhibited the proliferation of transplanted tumor, and the proportion of Ki-67 in chimera/Durmab/CNT group was further reduced compared with the aptamer/Durmab/CNT group (figures 5(A) and (C)).

Durvalumab/CNT/PEI/chimera promoted T cell infiltration
Immunohistochemical results showed that there was no statistical difference in the number of T cells and CD8 + T cells between the control group and CNT group. Compared with the CNT group, the Durmab/CNT group significantly increased the number of T cells and CD8 + T cells. Durvalumab/CNT/PEI/chimera further promoted the infiltration of immune cells, and the number of T cells and CD8 + T cells in chimera/Durmab/CNT group was further increased compared with the aptamer/Durmab/CNT group (figures 6(A), (C) and (D)).
TUNEL staining results showed that there was no statistical difference in apoptosis ratio between the control group and CNT group. Compared with the CNT group, the apoptosis ratio of Durmab/CNT group was significantly increased. Durvalumab/CNT/PEI/chimera could further promote the apoptosis of cancer cells. Compared with the aptamer/Durmab/CNT group, the apoptosis ratio of cancer cells in the chimera/Durmab/CNT group was further increased (figures 6(B) and (E)).

Discussion
Compared with other solid organs, liver has been proved to be a relatively immunosuppressive microenvironment. Immunosuppression significantly improves the degree of malignancy of liver cancer, and the prognosis of patients is often poor [22]. Surgical resection is still the main treatment for liver cancer, but many patients have lost the opportunity of surgery. Immunotherapy can kill cancer cells by enhancing the anti-tumor immune response [23]. Therefore, targeting the immune microenvironment  in liver cancer has a good clinical application prospect. Trem2 plays an important role in the regulation of tumor immunity and is involved in the formation of an immunosuppressive microenvironment [24,25]. We collected samples from both cirrhosis and HCC and found that Trem2 expression was significantly higher on HCC cells than on hepatocytes with cirrhosis. Recent studies have shown that targeted Trem2 can effectively enhance immunotherapy [26]. We silenced the Trem2 gene in HCC cells using an aptamer-siRNA chimera. Both in vitro and animal studies showed that targeting Trem2 can significantly improve the therapeutic effect of PD-L1 inhibitors on HCC. CNTs have been widely used in tissue engineering scaffolds, drug carriers and cancer treatment [27]. CNTs have a large specific surface area because of their special nanostructures. Whether drug is adsorbed in the tube or bonded to the surface, CNTs can achieve a high drug loading. It was found that CNTs on the surface of alginate materials could significantly increase drug encapsulation efficiency and stability, and reduce drug release rate and cytotoxicity [28]. Our in vitro experiments showed that CNTs could control the sustained release of Durvalumab and achieve long-term therapeutic effect. In addition, CNTs have good penetration and can cross various biological barriers in organisms to reach various organs and tissues [29]. In the immunotherapy, HCC cells appear large necrosis, drugs are difficult to reach the target area, but easy to cause various complications. CNTs can carry Durvalumab into liver cancer, where it is more effective.
Aptamer is a nucleotide sequence that can bind proteins and cells, and has the advantages of good targeting, strong modification and easy synthesis [30].
Aptamers can carry nanomaterials to the tumor site, enhance the effect of drug treatment, reduce the occurrence of side effects [31]. Dassie et al constructed a nucleic acid chimera consisting of aptamer and siRNA. This chimera bound highly specifically to prostate cancer cells and formed siRNA with specific cleavage of the intracellular Dicer enzyme. Animal experiments showed that chimera had good tumor killing effect [18]. Here, we designed a nucleic acid chimera that targeted to bind with HCC cells and silence the Trem2 gene. In vitro experiments showed that it had good affinity and gene silencing effect. Animal experimental results showed that Durvalumab/CNT/PEI/chimera inhibited the growth and proliferation of transplanted tumor, and had a good therapeutic effect on HCC.

Conclusions
In conclusion, we constructed a Durvalumab/CNT/PEI/chimera, which can effectively treat HCC by activating anti-tumor immunity. Our study provides a new approach for the immunotherapy of HCC.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).