In the ranking of malignant tumor incidence in the world, hepatoma is the fifth. It is also one of the malignant tumors with the highest morbidity and mortality in China. Hepatoma has the characteristics of insidious onset, rapid progression, early recurrence and poor prognosis (1). Many patients with hepatoma are diagnosed at an advanced stage. With the improvement of early diagnosis rate of hepatoma, the improvement of surgery and interventional therapy and the adhibition of comprehensive therapy, the prognosis of patients with early hepatoma is satisfactory. However, the prognosis of patients with middle to advanced hepatoma is still poor.

Hepatoma is a complex, multi-step biological process involving changes in adhesion factors, matrix metalloproteinases, corresponding signal transduction matrix, and related genes. With the deepening of research on molecular signaling pathways and the tumor microenvironment of hepatoma, gene therapy for hepatoma has rapidly developed into a new treatment mode after surgery, radiotherapy, chemotherapy, and interventional therapy.

Gene therapy is a kind of treatment method which uses molecular biology technology to transplant selected genes into patients to correct abnormal gene expression. Gene therapy is mainly applied to remedy illnesses that cause grievous damage to human health, such as infectious diseases, malignant tumors, cardiovascular illnesses, and genetic illnesses (2). At present, many gene therapy methods (such as suicide genes, immune genes, RNA interference, etc.) are increasingly used in tumor therapy and have achieved exciting results. IFNγ, IL-2, and TNFα are widely used in immune gene therapy, and their curative effects are clear. The local transcription and expression of immune genes in cancer cells can increase the immunogenicity of cancer cells, thus stimulating and enhancing the body’s immune response to cancer cells and eliminating them as soon as possible. After the cancer cells are killed, the expression of immune genes also stops, and the initial triggering effect is over. IFNγ kills tumors in two ways. On the one hand, it has a direct anti-tumor effect, which is realized by inducing differentiation and accelerating tumor cell apoptosis, restraining tumor cell proliferation (3, 4). After the cancer cells are killed, the expression of immune genes also stops, and the initial triggering effect is over. IFNγ kills tumors in two ways. On the one hand, it has a direct anti-tumor effect, which is realized by inducing differentiation and accelerating tumor cell apoptosis, restraining tumor cell proliferation (5). Immunogene therapy can be combined with the body’s immune response to treat tumors, making it a worthy research direction of gene therapy for hepatoma.

Radio-gene therapy is the fusion of therapeutic genes with radiation-induced promoters. Upon radiation/radionuclide exposure, the radiation promoter is activated, and the therapeutic gene is expressed. In this process, radiotherapy and gene therapy can synergistically kill tumors, and localization of anti-tumor gene expression can be achieved by local irradiation. Moreover, the dose of radiation in the combination therapy is smaller under the premise of achieving the same anti-tumor effect (6).

However, like traditional gene therapy, this method has some shortcomings, such as issues with specificity and safety. Additionally, the general hypoxic environment of solid tumors can reduce the efficiency of the promoter induced by radiation, which affects the curative effect and application of this therapy (7, 8). Some researchers have found that inserting the hypoxia-response element (HRE) sequence upstream of the gene promoter can effectively solve the aforementioned problems. HRE is an enhancer sequence that could enhance promoter sensitivity and activity in hypoxic environments (9, 10).

Naked DNA is unstable in organism because it is easily degraded by nuclease (11). So far, there are two main gene transfer carriers: viral systems and non-viral systems, each having its own strengths and weaknesses. Viral gene vectors are currently the most efficient ones, but they pose serious biosecurity risks and have some disadvantages, such as strong self-immunogenicity, poor target specificity, and limited gene capacity, which greatly limit their clinical application. Although non-viral vectors avoid significant safety risks, the transfection efficiency of most non-viral vectors is not satisfactory, making it difficult to obtain meaningful target gene expression (12). Nowadays, the most commonly used non-viral carrier transfection methods are liposome transfection and electroporation transfection. Both methods have high transfection efficiency. However, liposomes have high cytotoxicity, which causes them to be quickly cleared by the body. Therefore, the application of liposomes must first solve this problem. Electrotransfection can only be used in vitro, and the gene is only transiently expressed in cells or tissues, so it cannot be used for transfection in vivo (13). Therefore, solving the problem of gene transfer is still the primary challenge in current gene therapy.

In recent years, nanotechnology has received much attention in various fields, including its application in the development of gene transfer vectors (14–18). Nanoparticles-based gene transfer vectors have attracted the attention of scholars (19, 20) due to their low biotoxicity and large surface area (21–25). Compared with traditional vectors, nano-vectors offer several advantages, such as slow and sustained gene release and maintenance of effective concentration for an extended period of time. Moreover, nano-vectors are highly safe and can be repeatedly injected to improve transfection efficiency. Among the various types of magnetic nanoparticles (MNPs), Fe₃O₄ MNPs are the most frequently used due to their excellent biocompatibility, low immunogenicity, superparamagnetism, and other interesting properties (26). The superparamagnetism effect allows MNPs to target tissues under an applied magnetic field, achieving efficient transfection, which enables targeted gene therapy. Additionally, MNPs can be used for hyperthermia of tumors as they can be heated through magnetic induction in an external magnetic field.

Hyperthermia is a therapeutic method used to treat cancer by destroying cancer cells and making them more sensitive to chemotherapy and radiotherapy. Therefore, it is often used in combination with gene therapy, chemotherapy, and radiotherapy to produce synergistic and supplementary effects. However, achieving uniform heating of the tumor tissue to an ideal temperature without damaging normal tissue during treatment is a challenging technical problem. Magnetic fluid hyperthermia (MFH) is a tumor hyperthermia method that combines magnetic induction heating with nanotechnology. This treatment allows for targeted localization, meaning that only tissues with magnetic nanoparticles experience an increase in temperature under an external magnetic field, while tissues without magnetic nanoparticles are unaffected by heat. As the intake of magnetic nanoparticles in tumor tissues is much higher than that in normal tissues, MFH can destroy tumor tissues while simultaneously protecting normal tissues (27–29).

In some of our previous studies, we chemically prepared Fe₂O₃ and Fe₃O₄ magnetic nanoparticles and applied them to the hyperthermia of hepatoma and lung cancer with good results. The Curie temperature of the magnetic fluid can be controlled by adjusting the iron concentration. Fe₃O₄ nanoparticles can absorb electromagnetic waves below Curie temperature and heat up in alternating magnetic fields (AMF). Once the Curie temperature is reached, Fe₃O₄ nanoparticles becomes non-magnetic, loses their capacity to take in electromagnetic waves, and the temperature begins to drop. When the temperature is below the Curie temperature, the Fe₃O₄ magnetic fluid begins to heat up again. Therefore, the temperature is cycled within the range set by Curie temperature consistently, such as 42-44 degrees Celsius, which is the effective hyperthermia temperature for cancer, and this temperature will not cause damage to normal tissues. Thus, we successfully solved the issue of temperature measurement and regulation in tumor hyperthermia, and improved the safety and stability of hyperthermia (23, 30).

Magnetic nanoparticles are coated with an albumin shell called magnetic albumin nanospheres (MANS) (31). Albumin nanospheres are often used as drug carriers, which can lower drug discharge rate and avoid drug inactivation in the course of transfer. Magnetic nanoparticles’ biocompatibility can be improved when albumin is coated with nano-materials. Placing a magnetic field outside the tumor site can make the drug-loaded magnetic albumin nanoparticles focus on tumor site. Thereby enhancing the anti-tumor effect of the antineoplastic drug and reducing the harm to normal tissues (32, 33).

Based on the above, we constructed a eukaryotic recombinant plasmid pYr-ads-8-5HRE-cfosp-IFNG and hypothesized that Fe₃O₄ nanoparticles could be used as gene carriers to target transport radiation-induced immune genes to tumor tissues in an external magnetic field. Meanwhile, Fe₃O₄ nanoparticles can also be used as magnetic media for hyperthermia, combining immunogene therapy and hyperthermia for tumor. Thus, we aim to explore an adjustable and precise combination therapy for hepatoma.

Cancer remains one of the most severe diseases facing humankind. As far as current medical knowledge goes, there is no single treatment capable of curing cancer. Recent studies have shown that multi-mode therapy is often more effective than single-mode therapy in inhibiting tumors (39–41). In our study, we investigated the combined treatment of immunogene therapy and magnetic fluid hyperthermia using Fe₃O₄ nanoparticles as the medium on cancer, which demonstrated strong synergies.

Hyperthermia is a common adjuvant treatment for cancer and is used in various cancers. Increasing the temperature of tumor tissue to 40-43 degrees Celsius can effectively induce tumor cell death (42). The hyperthermia of tumor with nano-magnetic fluid involves directly inject MNPs into malignant tumor and heating them in AMF. This method has the advantages of accuracy, targeting, and temperature control and avoids overheating the normal tissues around the tumor (43). To ensure that the liquid is dispersed in the tumor tissues and prevent overflow of the liquid, we adopted a multi-point injection strategy, injecting the magnetic nano-composite materials into the 3rd, 6th, 9th, and 12th points of each tumor. Tumors are more sensitive to heating than normal tissues because of their unique internal microenvironment, such as hypoxia, vascular disorder, acidosis, and hypoperfusion (44). Therefore, hyperthermia is a promising treatment for tumors (45, 46). In our research, the tumor inhibition rate of the MFH group was the second highest, confirming the great potential of MFH in the treatment of solid tumors.

In our study, the tumor inhibition rate was significantly increased when MFH was combined with immunogene therapy. However, the mechanism underlying the synergistic effect of the combination therapy has not been investigated in this study. The following possibilities are speculated: hyperthermia in combination therapy of tumors was considered to influence the susceptibility to other manipulations mostly through microenvironmental factors (47). High temperature can destroy the biological integrity of the cell membrane and increase its permeability, which is conducive to the penetration and absorption of chemical drugs and gene transfer. The interior of solid tumors is often anoxic due to vascular disorder, while the anoxic cells are usually acidic and nutrient-poor, making them more vulnerable to heat damage. However, high temperature can temporarily improve the oxygen and blood supply to the tumor area, helping drugs to enter the tumor and improving the radiation susceptivity of tumor cells (48). Our next research goal is to explore the molecular mechanism of hyperthermia combined with immunogene therapy for hepatoma and to elucidate why combined therapy is superior to single therapy. This will guide the clinical selection of a more targeted and individualized treatment plan.

MNPs can not only be used as the medium of magnetic hyperthermia, but also as gene carriers in this study. Nanomaterials are used in many areas of biomedicine, including drug/gene therapy, diagnostic imaging (as contrast agents), and biological scaffolds. This is due to the advantages of nanomaterials such as high biosafety, complex surface functionalization, and easy preparation (49). MNPs have been used as gene vectors due to their good biocompatibility and large specific surface area, especially their ability to magnetically target to target tissues, which plays an important role in gene transfer (50, 51). Since DNA carries a negative charge, we connect PEI on the surface of Fe₃O₄-MNPs. PEI carries a positive charge and can bind to negatively charged DNA molecules. Thus, the plasmid can be effectively carried into HepG2 cells. Figure 6 shows specific peaks at 3,414.9 cm-1, 2,805.2cm-1 and 1,456.8 cm-1, which are consistent with the special chemical structure of PEI. This indicates that PEI has successfully modified the surface of nano Fe₃O₄. Magnetic nanoparticles are easy to agglomerate because of their small particle size. Therefore, in this study, the particle size of MNPs measured by TEM is smaller than that detected by Zetasizer Nano. The Zetasizer Nano measurement shows the hydrodynamic size of the MNPs. However, when we coated the magnetic nanoparticles with albumin, the diameter of the nanoparticles observed by TEM was not very different from that measured by DLS. MNPs modified with albumin have better dispersion. More importantly, albumin is a substance already in the human body. Wrapping magnetic nanomaterials in albumin can improve the safety of nanomaterials and effectively avoid the removal of foreign substances by the body (52, 53).

Using nano-magnetic materials as a carrier, we combined MFH and radiation-induced immunogene therapy to explore a new therapy model for hepatoma. In our research, in addition to the combined treatment group, an experimental group was also set up for each single treatment method for comparison. The consequences of our study showed that the tumor inhibition rate of combination therapy was better than that of single therapy in both cell and animal experiments. As mentioned above, we still have a lot of work to do on the molecular mechanisms. Understanding the deeper issues is helpful in guiding the formulation and selection of clinical treatment. However, this study still has high reference value and research significance in exploring the comprehensive treatment of hepatoma.

The results presented in this study demonstrate the successful synthesis and preparation of PEI-Fe₃O₄/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres for targeted delivery of IFNG to hepatoma cells. The identification of pYr-ads-8-5HRE-cfosp-IFNG confirmed the successful synthesis of the plasmid. The characterization of Fe₃O₄ nanoparticles and PEI-Fe₃O₄ nanoparticles indicated their appropriate size and surface charge for subsequent experiments. The transcript levels of IFNG in HepG2 cells were detected by qPCR, and the results showed that the IFNG transcript levels in the PEI-Fe₃O₄/pYr-ads-8-5HRE-cfosp-IFNG group were higher than those in the PEI-Fe₃O₄/pYr-ads-8-cfosp-IFNG group after hypoxia treatment, suggesting that the addition of the HRE promoter enhanced the therapeutic effect of IFNG in hypoxic conditions. The results of in vitro and in vivo experiments showed that the relative inhibition rate of cell proliferation was significantly higher in the PEI-Fe₃O₄/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres group compared to the other groups, including the negative control group and the PEI-Fe₃O₄/pDONR223-IFNG group. The results suggested that the targeted delivery of IFNG by the PEI-Fe₃O₄/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres was effective in inhibiting the proliferation of hepatoma cells. These findings suggest the potential of using targeted delivery of IFNG by nanocarriers for the treatment of hepatoma.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Ethics Committee of North Sichuan Medical College.

HZ: performed experiments, analyzed data, interpreted results of experiments, drafted manuscript, edited and revised manuscript; SL: analyzed data, approved final version of manuscript; FC: performed experiments, analyzed data; XM: approved final version of manuscript, analyzed data; ML: conceived and designed research. All authors contributed to the article and approved the submitted version.