Application of LHPP gene in liver cancer immunotherapy and liver cancer immunotherapy drugs

Abstract

This invention provides the application of the LHPP gene in liver cancer immunotherapy and liver cancer immunotherapy drugs. Reagents that promote LHPP gene expression can be used to prepare liver cancer immunotherapy drugs. This invention also provides an mRNA nanomedicine, which is prepared by the following method: [The invention then describes a process involving Meo-PEG-Dlink]. m A PLGA solution, an aqueous solution of LHPP gene mRNA, and a cationic liposome solution were mixed and added dropwise to water under stirring to obtain a nanosolution. The nanosolution was then transferred to an ultrafiltration membrane and centrifuged. This invention is the first to discover that LHPP can participate in the immunotherapy of liver cancer and is associated with the survival prognosis of liver cancer. LHPP enhances STAT1 nuclear translocation by affecting the expression of autophagy-regulated IFNβ, thereby promoting the expression of T cell-related chemokines secreted by liver cancer cells, and ultimately promoting CD8+ expression. + T cell infiltration. Therefore, the LHPP gene can serve as a novel target for immunotherapy of liver cancer. The mRNA nanomedicine described has advantages such as long circulation time in the blood, high accumulation in tumors, rapid mRNA release, and low cost.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically involving the application of the LHPP gene in liver cancer immunotherapy and liver cancer immunotherapy drugs. Background Technology

[0002] Chemotherapy is the cornerstone of cancer treatment; however, chemotherapy drugs lack specific targeting, causing significant damage to normal cells alongside the killing of tumor cells (liver and kidney dysfunction / bone marrow transplantation, etc.). This leads some patients to discontinue treatment due to intolerance of chemotherapy side effects. Tumor cells contain various tumor suppressor genes, which are highly expressed in normal cells and are closely related to tumor occurrence / development, invasion / metastasis. Currently, drugs targeting liver cancer-related genes mainly include small molecule kinase inhibitors, such as multi-target tyrosine kinase inhibitors (sorafenib, regorafenib, lenvatinib).

[0003] Traditional chemotherapy drugs lack specificity, are prone to side effects, and easily develop drug resistance. Other available treatment options (such as transarterial chemoembolization and percutaneous thermofrequency ablation) are all invasive procedures. Furthermore, small molecule kinase inhibitors targeting liver cancer driver genes are not highly effective, easily lead to drug resistance, and are costly to manufacture, placing a heavy financial burden on patients. Therefore, the lack of effective treatment options has become a major reason for the poor prognosis of liver cancer.

[0004] Liver cancer has been shown to be immunogenic, and immunotherapy strategies have been introduced to treat it, aiming to selectively target tumor cells by inducing or enhancing existing tumor-specific immune responses. However, the liver tends to tolerate this immune system, which may pose a challenge to immunotherapy for liver cancer, possibly due to the absence of certain important genes. Therefore, identifying key genes involved in immune tolerance in liver cancer and finding solutions based on this identification is crucial for liver cancer immunotherapy.

[0005] mRNA technology uses nucleic acids to express exogenous proteins, or surface antigens, within cells, activating the cell's immune response or enabling the production of normal functional proteins to repair damaged genes. However, exogenous RNA needs to overcome multiple physiological barriers to reach tumor cells. Therefore, it is necessary to find highly efficient delivery systems to help mRNA enter cells. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide the application of the LHPP gene in the immunotherapy of liver cancer and liver cancer immunotherapy drugs. LHPP can participate in the immunotherapy of liver cancer by affecting the expression of IFN-β regulated by autophagy, thereby enhancing STAT1 nuclear translocation, promoting the expression of T cell-related chemokines secreted by liver cancer cells, and ultimately promoting CD8. + T-cell infiltration.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] Application of reagents that promote LHPP gene expression in the preparation of immunotherapy drugs for liver cancer.

[0009] In some embodiments, the liver cancer immunotherapy drug is a liver cancer T-cell immunotherapy drug.

[0010] In some embodiments, the drug can promote CD8 + T-cell infiltration.

[0011] In some embodiments, the drug has at least one of the following effects: (1) promoting autophagy; (2) promoting the expression of IFN-β; (3) activating the JAK-STAT1 signaling pathway; (4) promoting STAT1 nuclear translocation; and (5) promoting the expression of one or more of the following chemokines: CXCL10 and CXCL11.

[0012] In some embodiments, the reagent for promoting LHPP gene expression is selected from agonists, overexpression plasmid vectors, and nanoparticles carrying LHPP gene mRNA.

[0013] In some embodiments, the drug further comprises a PD1 antibody.

[0014] The present invention also provides an immunotherapy drug for liver cancer, the drug comprising a reagent that promotes LHPP gene expression and pharmaceutically acceptable excipients.

[0015] In some embodiments, the drug further comprises a PD1 antibody.

[0016] In some embodiments, the agent promoting LHPP gene expression is a nanoparticle, which is prepared by the following method: Meo-PEG-Dlink... m The PLGA solution, the LHPP gene mRNA aqueous solution, and the cationic liposome solution were mixed and added dropwise to water under stirring to obtain a nano solution; the nano solution was transferred to an ultrafiltration membrane and centrifuged to obtain the nanoparticles.

[0017] In some embodiments, the PEG-Dlink m The mass ratio of PLGA and LHPP gene mRNA to cationic liposomes was 250:1:25.

[0018] In some embodiments, the cationic liposomes are prepared by adding 1,2-epoxytetradecane to a type 0 PAMAM dendrimer, mixing thoroughly, and reacting at 90°C for 2 days under stirring. The reaction product is then separated by silica gel chromatography. The molar ratio of 1,2-epoxytetradecane to the type 0 PAMAM dendrimer is 7:1. The cationic liposomes are named GO-C14, and using GO-C14 to prepare nanomedicines can improve transfection efficiency.

[0019] In some embodiments, the solvent for the Meo-PEG-Dlinkm-PLGA solution is dimethylformamide.

[0020] In some embodiments, the stirring speed is 1000±200 rpm.

[0021] This invention is the first to discover that LHPP can participate in the immunotherapy of liver cancer and is associated with the survival prognosis of liver cancer. LHPP enhances STAT1 nuclear translocation by affecting the expression of autophagy-regulated IFN-β, thereby promoting the expression of T cell-related chemokines secreted by liver cancer cells, and ultimately promoting CD8+ expression. + T-cell infiltration. Therefore, the LHPP gene can serve as a novel target for liver cancer immunotherapy, and agents that promote LHPP gene expression can be used as drugs for T-cell immunotherapy of liver cancer.

[0022] Furthermore, this invention also found that the combination of reagents that promote LHPP gene expression and PD1 antibodies can produce a synergistic effect, increasing the immunotherapy efficacy against liver cancer.

[0023] This invention yielded a nanomedicine, Meo-PEG-Dlink, loaded with LHPP gene mRNA, through raw material optimization. m In PLGA polymer materials, the PEG structure can protect the loaded mRNA from attack by cationic proteins or nucleases in the blood, prolonging the blood circulation time of nanomedicines; on the other hand, the Dlink in the structure m It breaks down in the weakly acidic microenvironment within tumor cells (pH 6.5), releasing the loaded mRNA, which has a significant CD8+ characteristic. + T cells infiltrate and exert their tumor-killing effect. The nanomedicine is synthesized via a "nanoprecipitation method." During nanoparticle synthesis, raw materials can be added in proportion according to actual needs. Under different pH conditions, it can efficiently deliver mRNA into tumor cells and release mRNA under the influence of the weakly acidic microenvironment within the tumor cytoplasm, achieving the purpose of tumor treatment. It has the following advantages: (1) longer circulation time in the blood; (2) high accumulation in tumors; (3) Meo-PEG-Dlink m-PLGA accelerates the release of mRNA in a weakly acidic environment of pH 6.5; (4) It can efficiently overexpress target genes; (5) It enhances the immunotherapy of liver cancer; (6) The preparation method is simple and the cost is low.

[0024] This invention can enhance liver cancer immunotherapy and reduce the side effects of traditional treatments by overexpressing the LHPP target gene, providing a new treatment option for clinical liver cancer patients. Attached Figure Description

[0025] Figure 1 This is a size distribution diagram of nanoparticles carrying LHPP gene mRNA.

[0026] Figure 2 This is a potential diagram of nanoparticles carrying LHPP gene mRNA.

[0027] Figure 3 The release curves of mRNA-loaded nanoparticles in solutions with different pH values ​​are shown.

[0028] Figure 4 CLSM images of HepG2 and Bel-7402 cells incubated with Cy5-EGFP Dm-NPs at pH 7.4 and 6.5 for 24 hours.

[0029] Figure 5 Flow cytometry profiles and mean fluorescence intensity (MFI) of EGFP Dm-NPs after incubation of HepG2 and Bel-7402 cells at pH 7.4 and 6.5 for 24 hours were obtained.

[0030] Figure 6 The pharmacokinetics of nanocarriers in response to a weakly acidic microenvironment.

[0031] Figure 7 To enable the enrichment of nanocarriers in various organs and tumor sites in response to weakly acidic microenvironments.

[0032] Figure 8 In the TCGA database, T cell-related chemokines CCL5, CXCL10, and CXCL11 are positively correlated with CD3 and CD8.

[0033] Figure 9 LHPP is significantly lowly expressed in liver cancer tumor tissues, and low LHPP expression in TCGA data predicts worse overall survival (OS) and disease-free survival (DFS).

[0034] Figure 10 The presence of CD8 and the expression of CCL5, CXCL10, and CXCL11 were increased in patients with hepatocellular carcinoma who had high LHPP expression.

[0035] Figure 11This represents the expression of LHPP in common hepatocellular carcinoma cell lines.

[0036] Figure 12 To illustrate T cell migration after LHPP overexpression.

[0037] Figure 13 The expression of chemokines in RNA and ELISA after LHPP overexpression.

[0038] Figure 14 The changes in STAT1 nuclear translocation and autophagy after LHPP overexpression.

[0039] Figure 15 The growth curves and tumor weights for each treatment group are shown.

[0040] Figure 16 The expression of CD8, GranzymB, and Perforin in tumors of each treatment group.

[0041] Figure 17 The expression of ki67 and TUNEL in tumors of each treatment group.

[0042] Figure 18 The protein expression of LHPP, p-STAT1, and LC3B in tumors of each treatment group. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0045] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0046] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0047] The following description is based on specific embodiments.

[0048] Example 1: Preparation and Performance Testing of Nanoparticles Loaded with mRNA

[0049] Nanoparticles carrying LHPP gene mRNA were prepared using a nanoprecipitation method. Dimethylformamide was selected as the solvent to prepare Meo-PEG-Dlink. m A solution of PLGA (purchased from Xi'an Ruixi Biotechnology Co., Ltd., 20 mg / ml) was then taken. m PLGA solution (125 μl), mRNA aqueous solution (10 μg, purchased from Shanghai Weihuan Biotechnology Co., Ltd., prepared to a concentration of 1 mg / ml), and cationic liposome G0-C14 solution (5 mg / ml) (50 μl) were mixed and added dropwise to deionized water under stirring (1000 rpm). The nanosolution was then transferred to an ultrafiltration membrane (EMD Millipore, MWCO 100K), the nanoparticles were separated by centrifugation, washed twice with deionized water, and the nanoparticles were collected and dispersed in 1 mL PBS buffer solution for later use. The cationic liposome G0-C14 was prepared by the following method (refer to the following literature: LoveKT, et al. (2010) Lipid-like materials for low-dose, in vivo gene silencing. ProcNatl Acad Sci USA107(5):1864–1869): 1,2-epoxytetradecane was added to the 0th generation PAMAM dendrimer at a molar ratio of 7:1. The mixture was reacted at 90°C for 2 days under vigorous stirring. The crude reaction mixture was separated by silica gel chromatography with a gradient elution solvent of CH₂Cl₂:MeO:NH₄OH = 75:22:3. The separated products were characterized by 1H NMR.

[0050] The size and potential of the nanoparticles were determined using a Nano-ZS ZEN3600 particle size analyzer. The size distribution and potential of the mRNA-loaded nanoparticles are shown in the figures below. Figure 1 and Figure 2 .

[0051] The controlled release curve and cellular uptake capacity of the nanoparticles were investigated using Cy5-EGFP mRNA as a template mRNA. Nanoparticles (Cy5-EGFP Dm-NPs) carrying EGFP mRNA labeled with a fluorescent dye were prepared according to the above method. After multiple washes, the nanoparticles were dispersed in 1 mL PBS. The nanoparticle solution was transferred to a dialysis tube, which was then placed at room temperature in solutions of different pH values ​​(pH 6.5 and 7.4). At specified time points, 5 μL of the nanoparticle solution was removed and added to 100 μL of dimethyl sulfoxide solution. The fluorescence intensity of the fluorescent dye was measured using a fluorescence spectrophotometer. The cumulative release rate of mRNA was calculated using the following formula: Cumulative release (%) = (M... t / M ∞ )×100; where M t The mRNA released from nanoparticles at a specified time point, M ∞ It is the total amount of mRNA loaded in the nanoparticles. Figure 3 This is the controlled release curve of mRNA from nanoparticles.

[0052] After successfully preparing Cy5-EGFP Dm-NPs and validating their pH response, we next evaluated whether the nanoparticles could utilize their pH-responsive properties to enhance cellular uptake of encapsulated mRNA. Cy5-EGFP Dm-NPs were incubated with HepG2 and Bel-7402 cells at pH 6.5 or 7.4 for 24 h. Figure 4 As shown, compared with cells incubated with Cy5-EGFP Dm-NPs at pH 7.4, cells incubated with Cy5-EGFP Dm-NPs at pH 6.5 exhibited stronger red and green fluorescence corresponding to Cy5-EGFP mRNA, indicating that more mRNA-loaded nanoparticles can be transfected at pH 6.5. Flow cytometry analysis showed that Dm-NPs significantly promoted gene transfection of encapsulated EGFP mRNA. HepG2 and Bel-7402 cells showed more than 3-fold higher uptake of EGFP Dm-NPs at pH 6.5 compared to pH 7.4. Figure 5 These results indicate that isolating PEG chains from Dm-NPs at pH 6.5 does indeed promote cellular uptake of encapsulated mRNA.

[0053] Example 2: Pharmacokinetics and tumor enrichment of mRNA-loaded nanoparticles in a weakly acidic microenvironment responsive carrier

[0054] After successfully validating the performance of the mRNA-loaded nanoparticles in a weakly acidic microenvironment and their enhanced uptake capacity after cell treatment, we further investigated their pharmacokinetics and accumulation at tumor sites in animals. Healthy mice were randomly divided into two groups (n=3). Each mouse was intravenously injected with 10 μg of mRNA: (i) free Cy5-EGFP mRNA (Naked mRNA); (ii) Cy5-EGFP mRNA-loaded nanoparticles (Dm-NPs). Orbital venous blood was collected at predetermined time intervals using a heparinized catheter. Bleeding was stopped by applying pressure to the wound for several seconds. The fluorescence intensity of Cy5-EGFP mRNA in the blood was detected using an ELISA reader. Figure 6 As shown, compared with the group of naked mRNA that was rapidly cleared from the blood, the mRNA circulation time of the Dm-NPs (cy5-EGFP-mRNA) group was significantly prolonged.

[0055] Nanoparticles loaded with Cy5-EGFP mRNA were injected into HepG2 xenograft tumor-bearing mice (10 μg per mouse) to detect the enrichment of nanoparticles in various organs and tumors. Tumor-bearing mice were randomly divided into two groups (n=3): (i) free Cy5-EGFP mRNA (Naked mRNA); (ii) nanoparticles loaded with Cy5-EGFP mRNA (Dm-NPs). Tumors and major organs were collected 24 hours after injection and imaged using the IVIS Lumina III imaging system. To quantify mRNA accumulation in tumors and organs, the fluorescence intensity of each tissue was quantified using Image-J software. Figure 7 As shown, the concentration of mRNA-loaded NPs in mouse tumors was significantly higher than that in naked mRNA.

[0056] Example 3: In a randomized controlled clinical trial, LHPP was significantly downregulated in the tissues of patients with liver cancer, and this downregulation was associated with shorter survival times in these patients.

[0057] like Figure 8 As shown in the TCGA database, the expression of T-cell-related chemokines CCL5, CXCL10, and CXCL11 in liver cancer is positively correlated with CD3 and CD8. LHPP is significantly underexpressed in liver cancer tumor tissues, and low LHPP expression predicts shorter overall survival (OS) and progression-free survival (DFS) in liver cancer patients. Figure 9 As shown in the image. Previous studies have shown that LHPP primarily regulates tumor growth and metastasis; we are the first to discover its association with T-cell immunotherapy. We found high LHPP expression in tumor sections from 101 liver cancer patients, specifically in patients with high CD8... + T cell infiltration and increased expression of T cell-associated chemokines CCL5, CXCL10, and CXCL11, such as Figure 10 As shown.

[0058] Example 4: After treating liver cancer cells with NPs (LHPP), T cell infiltration was enhanced by promoting IFN-β expression through enhanced autophagy.

[0059] LHPP is expressed at relatively low levels in the hepatocellular carcinoma cell lines HepG2 and Bel-7402, such as Figure 11 Human hepatocellular carcinoma cells HepG2 and Bel-7402 were seeded in 6-well plates (100,000 cells per well) and incubated for 24 hours with 2 mL of medium containing 10% fetal bovine serum to allow for full adhesion. Subsequently, NPs (LHPP) were added and co-incubated with the HepG2 and Bel-7402 hepatocellular carcinoma cells at pH 6.5 and 7.4, respectively, for 24 hours, followed by incubation with normal medium for another 48 hours. Then, 500,000 activated human T cells were added to the upper chamber of a 0.5 μm transwell chamber. After 12 hours, all medium in the lower chamber was collected, centrifuged, and incubated with human CD8 flow cytometry antibody. CD8 was analyzed by flow cytometry. + T cell migration was observed. The study found that overexpression of LHPP at pH 6.5 significantly enhanced CD8 cell migration. + T cell migration ( Figure 12 ).like Figure 13 As shown, after LHPP overexpression, the T cell-related chemokines CCL5, CXCL10 and CXCL11 were significantly upregulated at the RNA and supernatant ELISA levels, and the expression of IFN-β also increased accordingly.

[0060] We treated liver cancer cells with a STAT1 inhibitor (Fludarabine) and an autophagy inhibitor (chloroquine), respectively. We found that the chemokine levels were significantly reduced after the inhibitor treatment, and the RNA and ELISA levels of IFN-β were reduced after the autophagy inhibitor treatment.

[0061] To further investigate how LHPP enhances T cell infiltration, we analyzed the transcriptome after LHPP overexpression using KEGG and GSEA. Sequencing results showed that LHPP may function through the JAK-STAT and autophagy pathways. Our further investigation revealed that LHPP overexpression enhances autophagy, promotes IFN-β elevation, activates the JAK-STAT1 pathway, promotes STAT1 nuclear translocation, and consequently increases T cell-related chemokines. Therefore, LHPP can be delivered via weakly acid-responsive nanocarriers to enhance T cell infiltration and achieve immunotherapy effects in liver cancer. Figure 14 This indicates the nuclear translocation of STAT1 and the expression of autophagy proteins after LHPP overexpression.

[0062] Example 5: Tumor-bearing mouse treatment model: Nanoparticles loaded with LHPP mRNA exert therapeutic effects on liver cancer.

[0063] We validated the role of nanoparticles carrying LHPP gene mRNA in vivo by randomly dividing 40 male NCG mice into 5 groups. Human liver cancer tumor tissue was implanted subcutaneously in the back of each mouse, and 5 to 10 million human peripheral blood mononuclear cells (PBMCs) were injected into the tail vein of each mouse. The mice were allowed to grow to a size of approximately 150 mm. 3 We administered the following treatments: 1) PBS; 2) EGPF-loaded nanomaterials NPs (EGFP); 3) LHPP-loaded nanomaterials NPs (LHPP); 4) EGPF-loaded nanomaterials NPs (EGFP) and PD1 antibody therapy; 5) LHPP-loaded nanomaterials NPs (LHPP) and PD1 antibody therapy. 100 μL (10 μg LHPP mRNA) was injected intravenously via the tail vein three times every other day. Simultaneously, PD1 antibody (150 μg / kg) was injected intraperitoneally every two days, followed by a 21-day observation period.

[0064] Figure 15 The growth curves and tumor weights of tumors in each treatment group are shown. We found that the combination of NPs (LHPP) and PD1 drugs produced a synergistic effect, with the best therapeutic effect and significant inhibition of tumor growth; moreover, NPs (LHPP) had a significantly better therapeutic effect than NPs (EGFP).

[0065] After tumor collection, the tumors were digested into single cells and co-incubated with human antibodies against CD45, CD3, CD8, granzymB (GZMB), and perforin. The expression of CD8, granzymB, and perforin in different treatment groups was analyzed by flow cytometry. Figure 16 The infiltration and expression of CD8, GranzymB, and Perforin in tumors of each treatment group. Figure 17 The expression of ki67 and TUNEL in tumors of each treatment group. Figure 18 The protein expression of LHPP, p-STAT1, and LC3B in tumors of each treatment group was analyzed. Results showed that the NPs (LHPP) combined with PD1 group exhibited significantly reduced ki67 expression and increased TUNEL expression. Immunofluorescence (IF) analysis indicated that LHPP overexpression enhanced the expression of p-STAT1 and LC3II / I.

[0066] In summary, this invention is the first to discover that LHPP can participate in the immunotherapy of liver cancer and is associated with the survival prognosis of liver cancer. LHPP enhances STAT1 nuclear translocation by affecting the expression of autophagy-regulated IFN-β, thereby promoting the expression of T cell-related chemokines secreted by liver cancer cells, and ultimately promoting CD8+ expression.+ T cell infiltration. The nanomedicine loaded with LHPP gene mRNA provided by this invention exhibits significant CD8+ in vitro and in vivo activity. + T cells infiltrate and play a role in killing tumors, and the effect is better when used in combination with PD1 antibodies.

[0067] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of reagents that promote LHPP gene expression in the preparation of immunotherapy drugs for liver cancer, characterized in that, The reagent that promotes LHPP gene expression includes nanoparticles carrying LHPP gene mRNA. The preparation method of the nanoparticles carrying the mRNA of the LHPP gene comprises the following steps: mixing a Meo-PEG-Dlink m - mixing a PLGA solution, an mRNA aqueous solution of the LHPP gene, and a cationic liposome solution, and adding the mixture drop by drop into water under stirring to obtain a nano solution; and transferring the nano solution to an ultrafiltration membrane and centrifuging to obtain the nanoparticles. The Meo-PEG-Dlink m The mass ratio of PLGA and LHPP gene mRNA to cationic liposomes was 250:1:25; The cationic liposomes were prepared by the following method: 1,2-epoxytetradecane was added to the 0th generation PAMAM dendritic polymer, mixed well, and reacted at 90 °C for 2 days under stirring conditions. The reaction product was separated by silica gel chromatography. The molar ratio of 1,2-epoxytetradecane to the 0th generation PAMAM dendritic polymer was 7:

1. The Meo-PEG-Dlink m The solvent for the PLGA solution is dimethylformamide; The stirring speed is 1000±200 rpm.

2. The application as described in claim 1, characterized in that, The drug is also used in combination with PD1 antibodies.

3. A liver cancer immunotherapy drug, characterized in that, The drug contains a reagent that promotes LHPP gene expression and pharmaceutically acceptable excipients; The reagent that promotes LHPP gene expression is a nanoparticle, which is prepared by the following method: Meo-PEG-Dlink m The PLGA solution, the LHPP gene mRNA aqueous solution, and the cationic liposome solution were mixed and added dropwise to water under stirring to obtain a nano solution; the nano solution was then transferred to an ultrafiltration membrane and centrifuged to obtain the nanoparticles. The Meo-PEG-Dlink m The mass ratio of PLGA and LHPP gene mRNA to cationic liposomes was 250:1:25; The cationic liposomes were prepared by the following method: 1,2-epoxytetradecane was added to the 0th generation PAMAM dendritic polymer, mixed well, and reacted at 90 °C for 2 days under stirring conditions. The reaction product was separated by silica gel chromatography. The molar ratio of 1,2-epoxytetradecane to the 0th generation PAMAM dendritic polymer was 7:

1. The Meo-PEG-Dlink m The solvent for the PLGA solution is dimethylformamide; The stirring speed is 1000±200 rpm.

4. The liver cancer immunotherapy drug as described in claim 3, characterized in that, The drug is also used in combination with PD1 antibodies.

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