The current progress and futural prospects of ultrasound-targeted microbubble destruction for liver diseases: a narrative review

Introduction

Liver diseases, such as liver cancer and liver cirrhosis, are still lacking of satisfactory treatments. Alternative therapeutic options or enhanced current treatments are urgently needed. Ultrasound (US) is considered a non-invasive, low cost, and highly secure treatment that does not use hazardous ionizing radiation and is a promising method for clinical therapeutics. The wide use of US as a diagnostic medical imaging modality has allowed it to become one of the potential therapies for liver diseases (1,2). By combining with microbubble (MB), US is no longer regarded as simply a method for detecting tissue perfusion, but as a specific molecular imaging modality and targeted therapy (3,4). MBs, which commonly have diameters of 1–10 µm, are made of a shell (e.g., phospholipid, surfactant, albumin, or synthetic polymer) filled with a high molecular weight gas (e.g., sulfur hexafluoride or perfluoropropane gas) (4,5). MBs travel smoothly through the capillaries, but they cannot reach the target tissue when they encounter an endothelial gap. The US-targeted MB destruction (UTMD) technique uses low-intensity US in conjunction with MBs (6-8).

UTMD enables specific substances to penetrate barriers and reach their target tissues. Based on its potential as a non-invasive delivery mechanism, UTMD has been used in various organ systems and tumors to successfully deliver bioactive substances (e.g., genes, drugs, proteins, and gene silencing constructs) (6). Liver is an ideal target organ for UTMD because of its significant functions and acoustic accessibility. Tumor microenvironment can be remodeled by UTMD (9,10). Studies have revealed surprising uses of UTMD for treating liver cancer, hepatic gene defect diseases and hepatic fibrosis in vitro and in vivo. As a non-invasive method, UTMD has been widely discussed as a potential target treatment for human disease. In this article, we aim to narratively review the preclinical applications of UTMD to improve treatments for liver diseases. We present this article in accordance with the Narrative Review reporting checklist (available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-53/rc).

Methods

This study was performed in online databases from January 2000 to December 2023, including PubMed, ScienceDirect and Web of Science. Two independent reviewers (B.J.Y. and H.F.Z.) conducted a narrative review by conducting a systematic search. Terms sought included a combination of (“ultrasound-targeted microbubble destruction” OR “ultrasound targeted microbubble” OR “ultrasound microbubble” OR “UTMD”) AND (“liver” OR “hepatic”). Study discrepancies were resolved by another reviewer (Y.L.). The search strategy was shown in Table 1.

Results

Mechanism

MBs are usually loaded with drugs before intravenous injection for the purpose of treating the disease. The ideal ultrasonic MBs are often required for the following characters: diameter <8 µm (red blood cell diameter), sensitive targeting distribution, non-toxic, biocompatible and degradable, lasting stability, strong echo reflection, and controllable acoustic effect (11). Lipid MBs are similar to biofilm structures, with strong acoustic response, high encapsulation rate, low leakage rate, relatively simple production process and relatively low cost. At present, the preparation process has become mature, and they are the main film-forming materials. The amphiphilic properties of liposome nanobubbles can make hydrophilic drugs and RNA wrapped in the center of bubbles or coupled outside the shell, and can also contain hydrophobic drugs in the shell (12). Protein represented by albumin is another main film-forming material. Other natural polymers include polysaccharide chitosan, alginate, heparin, etc. Synthetic polymers mainly include polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone and so on. The organic polymer-assembled shell is more stable than the liposome, while the acoustic responsiveness is reduced (12-14). Nanomaterials have good histocompatibility, biostability, high reactivity and can be combined with other imaging techniques for multimodal imaging, which has become a research hotspot in recent years. Nano-MBs are easier to pass through the endothelial system with long cycle time and strong stability. However, the significant reduction of their particle size also leads to the reduction of acoustic response and the inclusion rate, and the phase-change nanodroplets are more advantageous (15). Subsequently, MBs are exposed to an US mechanical wave, causing cavitation. Cavitation is a series of US-induced biological effects on MBs, including formation, oscillation, and disruption (16,17). Oscillations are generated when MBs are sonified with US near their resonance frequency. In the presence of sufficiently high US energies, oscillation amplitudes increase and MBs collapse (6). During cavitation, the microstreaming and radiation forces generated by UTMD contribute to the rupture of barriers, such as cell membrane structure, increasing the number of agents delivered into cells and tissues (18). These features have been used to deliver a substance into a targeted region of the human body (Figure 1). Accordingly, MBs oscillate stably or violently when US is applied. Sonoporation may occur at low US intensities (0.3–3 W/cm²) when MBs oscillate in a stable motion. A higher US intensity (greater than 3 W/cm²) causes inertial cavitation, resulting in explosions and collapses of MBs. It is possible to facilitate reversible cell membrane permeation into tissues through dynamic MB motion coupled with fluid motion. In addition, energetic MB collapse (inertial cavitation) produces secondary mechanical phenomena such as liquid jetting and shockwaves, which enhance delivery. This cavitation-induced mechanical effect can produce both short-term (e.g., morphological changes) and long-term effects (e.g., temporal cell permeability changes, cell lysis, etc.), both of which can effectively increase therapeutic agent uptake and transport. The phenomenon of perforation on the cell membrane due to inertial cavitation is called sonoporation. Sonoporation can not only allow the cargo of MBs to enter the target cells, but also cause some target cells to lyse and die directly. In addition, steady-state cavitation stimulates cell endocytosis, produces reactive oxygen species to promote Ca²⁺ influx, and thermal effects increase cell membrane permeability, all of which can improve the cell’s transport capacity (19). Moreover, UTMD remodels various components of tumor microenvironment to improve treatment effects, including CD8⁺ T cells, tumour-infiltrating myeloid cells, regulatory T cells, natural killer cells and tumour vasculature (9).

Figure 1 Diagram of ultrasound-targeted microbubble destruction for drug delivery and gene therapy. Treg, regulatory T cell; NK, natural killer; CTL, cytotoxic lymphocyte; DCs, dendritic cells.

In cancer cells, drug resistance is often caused by gene mutations, gene amplifications, or epigenetic changes that affect drug ingestion, metabolism, or output. Drugs have a limited ability to penetrate tumor tissues and reach lethal concentrations of tumor cells as another mechanism of anticancer drug resistance (20). Disorganized tumor vasculature and blood viscosity in tumor tissues are the main obstacles to the distribution of anticarcinogens (21). An increased drug concentration in the tissue increases the side effects. Thus, it is necessary to develop a new strategy to boost the delivery of localized drugs to tumors while minimizing the effects on the system as a whole. Fortunately, UTMD increases the micrangium permeability of drugs, enhances drug penetration through the interstitial space, and increases drug uptake in tumor cells (16,22-25).

Mutations in a single gene, including both inherited and acquired genetic disorders, result in over one hundred hepatic diseases (26). Recently, gene therapy has provided efficient approaches for treating some hepatic diseases. Both efficient delivery to the target cells and sustained expression of transgenes are required to achieve a better therapeutic effect. Similar to drug-loaded MBs, UTMD increases the delivery of the transported gene to the targeted cells or tissues following the injection of gene-loaded MBs.

In combination with other targeted delivery systems, UTMD can effectively prolong the circulation time of therapeutic agents in the body and improve the targeting efficiency, thereby improving the therapeutic effect. According to the targeted delivery mechanism, it can be divided into passive targeting delivery system (PTDS) and active targeting delivery system (ATDS). The principle of PTDS in tumor therapy mainly depends on the enhanced osmotic retention effect of solid tumors. UTMD combined with PTDS can enable tumor-related immunomodulators, chemotherapy drugs or small molecular nucleic acids to be deposited in tumor tissues under the protection of MBs. Based on the combination of UTMD and PTDS, ATDS is delivered with molecular-specific targeted modification vectors and aggregated to the target site through specific recognition and binding. The combined application strategies of UTMD and ATDS are as follows: to improve the composition of the microvesicle shell, so as to facilitate the adhesion of the targeted cells; specific ligand related to the tumor surface, such as monoclonal antibodies, glycoproteins, peptides, nucleic acids, etc. are covalent or non-covalent to the MBs to trigger specific receptor-mediated endophagocytosis; combine molecularly targeted drugs designed for the tumor growth microenvironment to control its proliferation and metastasis. According to the properties of the modified targeting groups, it can be divided into antibody-mediated and receptor-mediated active targeting preparations, mainly including monoclonal antibodies against epidermal growth factor receptor family, vascular endothelial cell growth factor receptor, insulin-like growth factor, integrin family, leukocyte differentiation antigen, etc. (15,27,28). Compared with PTDS, ATDS can attach to the target cells more closely, improving the delivery rate (29). However, there are many shortcomings: after entering the tumor vascular system, the MBs must cross different barriers to reach and enter the tumor cells; The targeted MBs will bind to the first tumor receptor near the endothelial barrier in large quantities, and the tumor penetration in other sites is less. In addition, when the targeted part of the MB vector was included, its immunogenicity and plasma protein absorption increased, and the blood circulation time and passive tumor targeting ability decreased correspondingly (15).

Liver cancer

UTMD mediates docetaxel (DOC)-based therapy

Chemotherapy is the main treatment method for liver cancer. With the rapid development of precision medicine, the emergence of new targeted drugs has changed the tumor treatment model and opened up the era of targeted therapy. The targeted delivery system reduces the accumulation, toxicity and drug resistance of non-target tissues due to the advantage of low dose selective concentration. Targeted therapy can not only accurately “kill the tumor”, but also reduce the risk of tumor progression, thereby extending the survival of patients. UTMD has been proposed for enhancing the effectiveness of chemotherapy by targeting drug delivery and minimizing side effects. Kang et al. explored the antitumor effect of UTMD-mediated DOC-loaded lipid MBs (DLLMs) on VX2 rabbit liver tumors (30). In this study, the DLLMs + UTMD group showed the highest inhibition rate and apoptotic index, as well as the lowest proliferating labeling index. Moreover, the DLLMs + UTMD group had the lowest extensive metastasis rate and the longest survival time. However, the UTMD strategy was not optimized, as the US frequency was much lower than the resonant frequency of the MBs. The pulse was 10 seconds on followed by 10 seconds off, which did not conform to MB dynamics. Therefore, the efficacy of UTMD in this study should be improved. Zhang et al. also explored the antitumor effects of DLLMs combined with UTMD on liver cancer (31). Compared with the other groups, DLLMs + UTMD decreased the proliferation and increased the apoptosis of MHCC-H cells. Additionally, DLLMs + UTMD resulted in a higher proportion of cells arrested in G1 phase. Moreover, a smaller tumor volume was observed in the DLLMs + UTMD group than in the control group. Besides, CD105 was successfully linked to DLLMs in this study, resulting CD105-DLLMs targeted MHCC-H cells grafted in nude mice.

UTMD mediates doxorubicin (DOX)-based therapy

Cochran et al. evaluated the biodistribution and intratumor delivery of UTMD in a rat liver cancer model compared to free DOX and polymer nanoparticles loaded with DOX (32). Drug delivery to tumors was enhanced and sustained with UTMD, which reduced plasma levels of drugs and inhibited tumor growth. In contrast to other studies, this study used a polymer MB and diagnostic US scanner. Accordingly, polymer MBs loaded with DOX show great potential as a platform for delivering chemotherapy drugs, and a common diagnostic US scanner has been used for the UTMD technique. Zhu et al. investigated the effectiveness of localized UTMD with DOX liposomes in an H22 mouse hepatocellular carcinoma (HCC) model (33). In the DOX + UTMD group, the smallest tumor volumes and weights were observed, as well as the highest tumor inhibition rate, intratumoral DOX concentration, and survival rate. Importantly, the UTMD therapy parameters were optimized in this study as follows: MB diameter, 2.30±0.25 µm; MB density, 4.0×10⁹ bubbles/mL; treatment dose, 0.2 mL per 20 g of mouse body weight; sonication frequency, 1.3 MHz; and sonication power, 2.06 W/cm². Because of the overexpression of vascular endothelial growth factor (VEGF) receptor 2 on the surface of vessels in tumors, Wu et al. used the targeted MBs conjugated with an anti-VEGF receptor 2 antibody for a mouse model bearing HepG2-RFP tumors and found UTMD can significantly enhance the antitumor effect of DOX (34). An obstacle to the clinical application of UTMD is the limited drug-loading capacity of MBs. Instead of increasing the loading of MBs, a novel MB carrying 10-hydroxycamptothecin (HCPT) developed by Li et al. requires a single low dose of injection for tumor therapy (35). HCPT-MBs require less than 20 times the dose of paclitaxel and DOX, which may overcome the limitation of the drug payload.

UTMD promotes antitumor immune microenvironment

A tumor-associated antigen-specific CD8⁺ T-cell infiltration results from the immune system playing an important role in HCC disease that is an antigenic lesion expressing tumor-associated antigens and neoantigens. However, regulatory T cells, myeloid-derived suppressor cells, and M2 tumor-associated macrophages, as well as immune checkpoint regulators expressed by tumor cells, inhibit the recognition of tumor cells by CD8⁺ T cells (36,37). Thwarting the tumor immune barrier is the key to challenging HCC. To overcome the resistance to chemotherapy caused by HCC, Wischhusen et al. developed a synthetic microRNA-based molecularly targeted therapy (38). They demonstrated that microRNA-122 and anti-microRNA-21 delivered via UTMD modulated the immune microenvironment of Hepa1–6 tumors through cytokine expression. As known, the hypoxic tumor microenvironment significantly limits the efficiency of sonodynamic treatment. With the aid of UTMD, the sonosensitizer with oxygen-sufficient MBs made it more effectively enriched at the tumor site, also it directly mediated oxygen release and provide sufficient oxygen inducing HepG2 cell apoptosis (39). Using low-frequency UTMD carried the Toll-like receptor agonist and the sonosensitizer, liver cancer suppression effects were enhanced by inducing the polarization of macrophages from M2 to M1 (40). Yuan et al. observed the restriction of tumour volume in rabbit VX2 liver tumors after treatment with ganoderma applanatum polysaccharide liposome MB complexes via UTMD (41). As markers of tumour-associated macrophages, the expression levels of CD68 and CD163 were reduced in tumour tissue. A recent study showed that UTMD-mediated bone morphogenetic protein 9 delivery restored the antitumor function of natural killer cells and showed therapeutic efficacy in combination with a programmed cell death 1 ligand 1 (PD-L1) antibody in human cancer xenografts of immune-deficient mice (42).

UTMD mediates gene delivery

UTMD overcomes the limitation of the viral gene delivery system and promotes the application of this non-viral therapeutic approach for liver cancer. UTMD-mediated microRNA-206 inhibited HCC cell migration and invasion while promoting apoptosis via regulating the expressions of proteins related to apoptosis, migration, and invasion (43). The UTMD-mediated antitumor effect of the herpes simplex virus thymidine kinase (HSVTK) suicide gene system was explored on mouse xenograft liver tumors (44). Ultimately, the survival time of the tumor-bearing mice in the HSVTK + UTMD group was significantly improved. Yu et al. applied the HSVTK/ganciclovir suicide gene system and the tissue inhibitor of metalloproteinase 3 (Timp 3) gene mediated by UTMD to mouse xenograft liver tumors (45). Tumor growth was suppressed and the survival of tumor-bearing mice was significantly increased by UTMD-mediated HSVTK and Timp 3 gene delivery. UTMD also increased the number of apoptotic cells and decreased the vascular density in the tumor. A galactosylated poly-L-lysine (G-PLL)-targeted MB was used in conjunction with UTMD to deliver a c-myc antisense oligodeoxynucleotide (ASODN) in HCC tumor-bearing mice (46). The group of c-myc ASODN + G-PLL + UTMD exhibited the highest inhibition of tumor growth and cell proliferation comparing with other groups. UTMD can effectively transfer shCD133 into CD133⁺ liver cancer stem cells, and inhibited CD133 expression and the properties of liver cancer stem cells (47). Liao et al. explored a combination of hypoxia-inducible factor-1 (HIF-1) shRNA and transcatheter arterial embolization (TAE) reduces tumor growth in rats with hepatic cancer (48). There was a significant reduction in tumor size in the UTMD + TAE group. The results also indicated that UTMD-mediated HIF-1 shRNA transfection and TAE can clearly silence HIF-1 and VEGF expression, thereby inhibiting tumor growth.

Liver fibrosis

It is known that hepatocyte growth factor (HGF) can prevent the development of liver fibrosis by regulating the synthesis of extracellular matrix and the inflammatory response to inhibit the activation of hepatic stellate cells. Wang’s study (49) showed that the expression of HGF was significantly higher and the amount of fibrous septum was smaller in the HGF + UTMD group than in the other groups. Jiang et al. (50) and Zhang et al. (51) also demonstrated the feasibility and efficacy of HGF carried by UTMD for treating liver fibrosis in rats.

It has been shown that bone marrow mesenchymal stem cells (BMSCs) can be used as a cytotherapy to treat liver fibrosis. However, a significant challenge is the insufficient homing of BMSCs and undefined proliferation of BMSCs, which limits the effectiveness of the program. Sun et al. found that carbon tetrachloride (CCl₄)-induced liver fibrosis in rats can be effectively and definitively alleviated by stable expression of HGF in BMSCs and UTMD technique (52). As a result of UTMD application, BMSC homing was significantly enhanced in the fibrotic liver. Biochemical markers of liver function and histopathological results improved significantly in the UTMD-mediated BMSCs-HGF group, and serum levels of biochemical markers returned to normal ranges in 12 weeks. In the UTMD-mediated BMSCs-HGF group, expression levels of liver fibrosis markers such as smooth muscle actin, collagen I, and vimentin were significantly lower. According to their results, adeno-associated virus vector-BMSCs-HGF and UTMD are potentially effective as novel therapeutic approaches for liver fibrosis.

As shown by Yang et al., UTMD-mediated delivery of an artificial microRNA targeting connective tissue growth factor (CTGF) attenuates the development of hepatic fibrosis in rats (53). It suggested UTMD is an effective method for delivering artificial miRNAs to the liver, and artificial miRNA targeting CTGF delivered by UTMD effectively silences CTGF gene expression in fibrotic liver tissue. In addition, artificial miRNA targeting CTGF significantly downregulates transforming growth factor-β1 (TGF-β1) expression, inhibits the activation of hepatic stellate cell, reduces collagen deposition, and significantly reverses liver fibrogenesis. This study suggested that artificial miRNAs delivered via UTMD for silencing the CTGF gene may be a valuable approach for the treatment of liver fibrosis.

Hepatic gene deficiency disorders

Anderson and colleagues reported a series of exciting results in the treatment of hepatic gene deficiency disorders via UTMD in mice (54,55). They proved the feasibility of delivering transposase-based vectors via UTMD in vitro and in vivo (54), and evaluated the expression and genomic integration of conventional pcDNA3 and piggyBac transposase-based pmGENIE reporter vectors. UTMD-mediated liver-specific expression of pmGENIE observed in vivo for an average of 24 days was longer than pcDNA3 observed for 4 days. The pmGENIE vector was randomly integrated into the genome in vitro but was targeted to specific areas of chromosome in vivo.

In another study, UTMD was used to deliver plasmid and transposase-based vectors encoding human factor IX (hFIX) to the livers of mice with hemophilia B (FIX^−/−) (55). These mice were divided into two groups, control groups of wild-type and mutant mice and groups treated with pZY53-hFIX (a conventional expression plasmid) or pmGENIE3-hFIX (containing a piggyBac transposon construct). At 4 to 5 days after UTMD, reductions in the clotting time and significant improvements in the percentage of FIX activity were observed in groups treated with both pZY53-hFIX and pmGENIE3-hFIX compared with untreated mutant mice. Reduced clotting times persisted for groups treated with each plasmid for 12 days after treatment. In particular, the persistent reduction in the average clotting time was evaluated in an additional set of mice treated with pmGENIE3-hFIX to assess the long-term (160 days) effects after a single treatment. UTMD was suggested to be an effective approach for treating patients with hemophilia. Anderson et al. also described that focused UTMD delivering CRISPR-Cas9 base editing vectors can introduce stop codons in mouse liver cells in vivo, which may provide a powerful platform for the development of non-viral genome editing therapies (56).

Discussion

Challenges

UTMD faces several challenges, such as inefficient transport, the short circulation time of MBs, unoptimized parameters, cavitation detection techniques, fewer animal models replicating human tumors, and a lack of clinical applications.

Although chemotherapy requires a high dose, MBs suffer from a poor drug loading capacity. Instead of increasing the drug dose, Li et al. applied a powerful drug (10-HCPT) encapsulated in MBs to mouse liver tumors; the dose was more than 20 times lower than paclitaxel or DOX. DOC exhibits very poor water solubility (35). Wei et al. resolved the water solubility problem by preparing targeted nanoparticles with a copolymer carrier (57).

While the intravenous route is sufficient for delivery, Anderson et al. utilized the intracardiac approach to deliver MBs, showing that this method worked well and was repeated safely in wild-type mice. However, this strategy has not been successfully repeated in the far more fragile FIX-deficient mice, which showed the expected results when larger or repeated doses were used (55).

Currently, a consensus on sonographic parameters for UTMD therapy for the liver has not been achieved. The process of parameter optimization is complex. There are safety problems in the application of ultrasonic MBs. In addition to microembolism and toxicity in blood circulation, the potential risk of cavitation effect cannot be ignored. In general, the US frequency is approximately 1.0 MHz, and the intensity ranges from 0.4 to 2.06 W/cm². Different studies have applied different US pulse lengths and exposure times. Therapeutic US can efficiently deliver drugs and genes to the mouse liver (58-60). MBs have been developed as an agent with both diagnostic and therapeutic capabilities (16). Diagnostic US has also been successfully applied for the same purpose (61,62), which will be convenient for clinical access. By incorporating a low pulse repetition frequency and short pulse duration, optimal hepatic expression was seen with double depth targeting at 5 and 13 mm in vivo for UTMD-mediated reporter plasmids delivery. Comparing with focused and unfocused UTMD, reporter plasmids expression was similar, but the transfection patterns were different (63). Focused UTMD resulted in patches of homogeneously transfected hepatic cells, while unfocused UTMD resulted in a diffusely scattered distribution of transfected cells throughout the liver. Although the optimization of parameters such as ultrasonic sound pressure, energy, the exposure time, and the selection of appropriate MB injection methods, nanomaterials, MB size, concentration and drug loading of MBs are expected to improve the safety and efficiency of treatment of liver diseases, they are still in the pre-clinical experimental stage, so more and more in-depth exploration is still needed in the process of clinical transformation of UTMD treatment for liver diseases.

Because of the high rates of transfection efficiency, many gene therapies depend on virus-based strategies to mediate gene transfer. However, many limitations associated with their safety and efficacy have been reported, such as low tissue specificity, strong immunogenicity and inflammatory responses, hepatotoxicity, and size limitations. Anderson et al. applied a non-viral strategy as an alternative to avoid these limitations (54). Unfortunately, the disadvantages of non-viral approaches are a low transfection efficiency and transient gene expression.

Futural perspectives

Although UTMD therapy has great qualities, many aspects must still be improved, such as sonographic parameters, drug-loading capacity, spectrum of treatable diseases and clinical applications in liver diseases. Fortunately, initial preclinical studies have reported the safety and efficacy of the technique. However, there is few clinical trial to investigate the treatments for liver disease via UTMD so far. Only two I/II phase trials about US-targeted MB delivery for hepatic metastases were found on ClinicalTrials [NCT03477019 (64) and NCT03458975 (65)]. Further clinical trials are emergent to evaluate the safety, effectiveness and long-term efficacy of UTMD in treating human liver diseases. Especially for unresectable HCC, UTMD may help to overcome drug resistance and break tumor immune barriers by undergoing immunotherapies and interventional radiology strategies.

Conclusions

UTMD has been shown in preclinical studies to enhance the therapeutic efficacy of drug delivery (Table 2) and gene therapy (Table 3) for liver cancer, liver fibrosis and hepatic gene defect diseases. Animal models in which UTMD has shown effectiveness include rabbit orthotopic liver tumor models, rat xenograft liver tumor models, rat hepatic fibrosis models, mouse xenograft liver tumor models and mouse hepatic gene deficiency disorder models. All of these studies used lipid MBs, with the exception of one study that used polymer MBs. The diameter of the MBs ranged from 1–10 µm, and the doses varied. The sonographic parameters must be optimized to improve safety and efficiency. Loaded drugs have included DOC, DOX and HCPT, but additional drugs with higher efficiency and targeting should be applied. Gene therapy, including virus-based strategies and non-viral strategies, may broaden the spectrum of liver diseases treated with UTMD.

Acknowledgments

The authors thank Pro. Hai-Bin Shi and Pro. Sheng Liu from Department of Interventional Radiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, for their advices and supports for this study. The authors thank Home for Researchers (www.home-for-researchers.com) for figure drawing by Figdraw 2.0 (ID: YSUWUbfadd).

Funding: This study was supported by the Jiangsu Provincial Innovative and Entrepreneurial Doctor Program (No. 303073543ER21), Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (No. PY2023056), and the National Natural Science Foundation of China (No. 81701802). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-53/rc

Peer Review File: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-53/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-53/coif). All authors report that this study was supported by the Jiangsu Provincial Innovative and Entrepreneurial Doctor Program (No. 303073543ER21) and Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (No. PY2023056) for H.F.Z., and the National Natural Science Foundation of China (No. 81701802) for W.Z.Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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