The recognition of HCC biomarkers in the plasma is vital for its early clinical diagnosis. Alpha-fetoprotein (AFP) is the most commonly detected tumor-associated protein and is used for the early diagnosis of HCC (Sauzay et al., 2016). However, the current diagnostic approaches have limited detection sensitivity and are time-consuming or require complex instruments, making the development of highly sensitive detection technologies an urgent requirement for detecting serum biomarkers present in very low concentrations (Ayuso et al., 2018; Singh et al., 2020). A sandwich immunoassay based on surface-enhanced resonance Raman (SERR) spectroscopy was developed that exhibited high sensitivity and specificity to human AFP (Gong et al., 2007). This system was composed of Ag/SiO₂ nanoparticles-loading rhodamine B isothiocyanate dye as the SERR-scatting (SERRS) tags and silica-coated magnetic nanoparticles as the immobilization matrix and separation tool. Polyclonal antibody and monoclonal antibody were chemically modified onto the surfaces of Ag/SiO₂ nanoparticles and silica-coated magnetic nanoparticles respectively, using glutaraldehyde as a crosslinker. The monoclonal antibody-modified silica-coated magnetic nanoparticles were first mixed with PBS buffer containing the AFP antigen and 1% bovine serum albumin and subsequently incubated with polyclonal antibody-modified Ag/SiO₂ nanoparticles. The sandwich immunocomplexes were separated from the solution under a constant magnetic field and subjected to Raman analysis. An augmented Raman signal was observed as a result of the fluorescence quenching and the Raman-enhancement ability of silver. Moreover, the detection results of the AFP antigen at diverse concentrations showed that the sandwich immunoassay had high sensitivity and specificity for AFP recognition with a detection range of 11.5 pg/ml to 0.12 μg/ml (Gong et al., 2007). Inspired by the arginine selective conductometric biosensor based on arginase and urease (Soldatkina et al., 2018), a conductometric immunoassay based on the bienzyme (arginase and urease)-functionalized nanometer-sized silica beads was developed to detect AFP (Liang et al., 2018). First, the ureases were doped into silica nanoparticles via the reverse micelle method. Then, the arginase-labeled anti-AFP secondary antibodies were coupled to the urease-doped nanosilica particles. The anti-AFP capture antibodies were coated on the microplates and the sandwich-type immunoreaction was carried out by adding the AFP-containing sample and arginase-coated nanometer-sized urease-doped silica beads. Finally, the introduced L-arginine was enzymolyzed to L-ornithine and urea by the arginase, and the produced urea was subsequently decayed into ammonia and bicarbonate under the urease enzymatic reaction, resulting in the changed conductivity of the solution that could be quantitatively monitored. The nanometer-sized silica beads increased the loading amounts of enzymes based on their high surface-to-volume ratio and quantum-size effect and further amplify the detection signal. The immunosensing system showed a highly sensitive conductometric response to AFP with a linear detection range of 0.01–100 ng/ml and a detection limit of 4.8 pg/ml. Importantly, the reproducibility and precision with a relative standard deviation of <15% and specificity with no response to other cancer biomarkers such as CA125, PSA, and hCG, etc., were satisfactory for AFP detection in HCC patients (Liang et al., 2018).

To overcome the deficiency of enzyme-based immunoassays that depend on the enzyme catalytic activity, which are determined by thermophilic scope and stability, an enzyme-free metal sequestration of a complexing agent to dominate the gold nanoparticle generation (called Scadge) strategy was carried out (Zhao et al., 2018). The detection signals originated from the in situ generated gold nanoparticles (AuNPs) that were regulated by the metal chelator ethylenediaminetetraacetic acid disodium salt (EDTA·2Na). This proposed diagnostic mode was dubbed Scadge-Diag, and silica nanoparticles (SiO₂ NPs) were utilized to further amplify the detection sensitivity via chemically grafting EDTA·2Na onto the NP surfaces (Figure 1). The Scadge-Diag system exhibited ultrasensitivity toward HBsAg and AFP in the presence of auxiliary SiO₂ NPs, with a detection limit of 2.6 × 10^–15 g/ml for HBsAg and 2.5 × 10^–19 g/ml for AFP.

Novel diagnostic instruments are important for effectively detecting biomarkers. A microfluidic reflectometric interference spectroscopy (RIfS) system based on a halogen light source and carboxylate groups-modified silicon nitride sensor chips was developed for the label-free AFP detection (Kurihara et al., 2012). The anti-AFP antibody was modified onto the surface of a silicon nitride chip that was immobilized on the Si wafer. This RIfS-based sensor had high reproductivity with a coefficient variation of 5.7% and no cross-reaction with other proteins such as human serum albumin, thus exhibiting the potential as an effective biomarker detection tool. The development of silica-based diagnostic approaches and instruments for the early detection of plasma biomarkers can facilitate the prevention and early diagnosis of HCC, and further improve HCC management.

MRI exhibiting a high spatiotemporal resolution and excellent soft-tissue contrast is frequently used as a noninvasive imaging modality in clinical diagnosis. Gadolinium chelate has been regularly used in T1-weighted MRI but its use has become limited because of nephrotoxicity, nonspecificity, and poor quality of images (Canavese et al., 2008; Kim et al., 2018). To develop a more sensitive, reliable, and safer MRI contrast agent for use in HCC-specific MRI, Kim et al. synthesized Mn²⁺-doped SiO₂ nanoparticles (Mn-SiO₂) (Kim et al., 2013). X-ray diffraction results of the nanospheres showed that Mn²⁺ ions were homogenously scattered in the silica structure. The inert silica structure not only improved nanoparticle safety by hindering the release of Mn²⁺ ions in the bloodstream but also detected HCC in MRI by taking advantage of the brightly enhanced MRI due to the release of Mn²⁺ under acidic conditions. The signal MRI intensities depended on Mn²⁺ concentration. A gradual but significant signal enhancement was observed in the T1-weighted signal intensity of normal tissues, which peaked at 6 h after injection. On the other hand, a different enhancement behavior was observed when HCC tissues were used and this behavior was constant for the first 6 h and enhanced brightly with the uptake of Mn²⁺ after 6 h, thus achieving a higher liver-to-HCC contrast ratio. This enhancement phase distinction between HCC and normal tissues helped in producing highcontrast enhancement HCC pictures and achieved reliable diagnosis of liver lesions over normal tissue, thus demonstrating the unique MR contrast-enhancing characteristics of Mn-SiO₂. In another study, Wang et al. synthesized arginine-rich manganese silicate nanobubbles (AMSNs) for GSH depletion-induced ferroptosis and tumor-specific MRI (Figure 2A) (Wang et al., 2018). As shown in Figure 2B, the–Mn–O– bonds in AMSNs can be cleaved and the release of free Mn²⁺ ions can be triggered by a decrease in pH or the abundance of GSH in the tumor intracellular microenvironment. Transmission electron microscope (TEM) images showed the gradual structural degradation of AMSNs as pH decreased and the GSH concentration increased. In addition, the accumulated release amount of Mn²⁺ increased along with the degradation of AMSNs under acidic and high GSH conditions. In this study, arginine (Arg) was considered a targeting moiety for HCC theranostics. HCC cells cannot produce Arg themselves due to the lack of argininosuccinate synthetase (Ensor et al., 2002). After Huh7 liver cancer cells and normal liver cells were incubated with AMSNs and DOX (Figure 2C), stronger fluorescence intensity was observed in the Huh7 liver cancer cells than in normal liver cells, indicating the selective uptake of AMSNs by HCC cells. In the tumor-bearing mice (Figure 2D), the signal intensity of T1-weighted MRI at the tumor site enhanced at 2 h after injection and reached the peak until 5 h after injection, owing to the accumulation of the released Mn²⁺ in HCC tissues. This enhancement-phase distinction between cancer cells and surrounding tissue help in producing high-contrast enhancement HCC pictures and led to the accurate diagnosis of liver lesions over normal tissues. More studies have also confirmed that Mn²⁺ doped MSN can be used as a highly sensitive MRI diagnostic agent (Chen et al., 2012; Chen et al., 2015b; Fei et al., 2020a). In general, SNFs may be effective and less toxic hepatoma-specific MRI contrast agents.

PET is a noninvasive nuclear imaging technique that can identify metabolic processes in the human body with high sensitivity. The underlying principle of PET is the detection of gamma rays emitted indirectly by radionuclides depending on the different metabolic states of tumors and normal tissues (Lu et al., 2019). Currently, chelator-based radionuclides are still widely used for synthesizing radio-labeled nanoparticles. However, considering the issues about the accessibility of specific chelation agents, undesired changes in pharmacokinetics, and long-term integrity after chelation, chelator-free labeling nanoparticles were synthesized (Chen et al., 2015a; Wall et al., 2017). Among them, Wall et al. fabricated chelator-free radiolabeling of SERR scatting (SERRS) nanoparticles called ⁶⁸Ga-labeled PET-SERRS nanoparticles (Wall et al., 2017). This nanoparticle consisted of a 60 nm diameter gold core, a Raman reporter dye layer, and a silica shell distributed with ⁶⁸Ga of 30 nm (Figure 3A). The silica shell was necessary for chelator-free radiolabeling and sulfur was added to silica surfaces to stabilize the radiolabeling of radiometal ions. Radionuclides were embedded throughout the silica shell and the SERRS nanoparticles were obtained that exhibited distinct spectral signatures and were stable regardless of photobleaching. Since the nanoparticles accumulated in the reticuloendothelial system (RES), they could be used particularly for HCC imaging. In tumor-bearing mice, the liver PET-CT image revealed clear filling defects (Figure 3B). These defects matched the tumor sizes and locations after surgically exposed (Figure 3C), intraoperative SERRS imaging (Figures 3D,E), or MRI scanning (Figures 3F–H), successfully delineating the presence of HCC. The PET-SERRS nanoparticles combined the whole-body imaging of PET and the high resolution of SERRS imaging, enabling superior visualization sensitivity of as little as 100 μm, and the possibility of a rapid preoperative roadmap and precise intraoperative surgical guidance. Thus, silica-based nanoplatforms were successful in photobleaching radionuclides in PET imaging and broadened their clinical use in HCC diagnosis.

Photoacoustic imaging (PAI) integrating optical imaging with a high contrast rate and ultrasonic imaging with a high penetration depth has garnered considerable attention in the field of medical science. At the core of this technique is a near-infrared (NIR) laser. When the energy is absorbed and converted into heat, a wind-band ultrasonic emission is produced. The resulting ultrasonic waves can be analyzed and converted into an image (Johnson et al., 2019; Liu et al., 2019a). Liao et al. constructed polydopamine-doped virus-like mesoporous nanoparticles (PVMSNs) coated with reduced graphene oxide (rGO@PVMSNs) nanocomposites (Liao et al., 2020). These nanocomposites not only had the inherent properties of the mesoporous material but also a virus-like structure. The virus-like mesoporous nanoparticles gained the extra capability of invading cells with prolonged blood circulation times than traditional MSNs (Wang et al., 2017b). Moreover, the rGO@PVMSNs exhibited excellent photothermal properties in vitro. Due to the enhanced cellular uptake by HepG₂ cells, the photoacoustic (PA) image in the tumor region was more obvious after rGO@PVMSN injection for 24 h, suggesting that rGO@PVMSNs could be a potential photoacoustic imaging contrast agent for biomedical imaging. In another study, novel hyaluronate-silica nanoparticle (HA-SiNP) conjugates were synthesized (Figure 4A) (Lee et al., 2018). The acoustic properties of SiNP, and the high biocompatibility and biodegradability of HA conferred the synthesized HA-SiNP conjugates with high liver specific-targeting efficiency, strong optical absorbance near-infrared windows, and excellent biocompatibility and biodegradability. After endocytosis specifically into HCC cells, the HA-SiNP conjugates presented an enhanced PA amplitude than that in the control group and a PA amplitude 4.4 times greater than that of SiNP (Figures 4B–H). These two SNFs made PAI useful in HCC diagnosis with high-resolution anatomical and functional information in deep tissues like the liver.

Fluorescence imaging using fluorescent dyes as markers has been widely used in cytological and histological analyses. However, the disadvantages of fluorescent dyes including fluorescence bleaching and poor biocompatibility have limited their further development and clinical applications (Tan et al., 2016). To overcome these limitations, biofunctional molecules such as antibodies or aptamers were conjugated with fluorescent nanoparticles (Smith et al., 2007). Particularly, dye-doped silica nanoparticles were outstanding because they exhibited good biocompatibility and resistance to photobleaching, and their surface could be easily modified (Wang et al., 2013). Hu et al. developed carboxyl-modified fluorescein isothiocyanate (FITC)-doped silica nanoparticles (SA-FSNPs) conjugated with streptavidin (SA) (Hu et al., 2017). The functionalized silica nanostructure not only inherited the fluorescence properties of the FITC dye but was also able to emit stronger and photobleach-resistant fluorescent signals. The underlying principle of this nanosystem was based on the biotin-labeled aptamer TLS11a (Bio-TLS11a) and streptavidin-modified FSNPs. Bio-TLS11a were labeled with hepatoma cells and physically separated from FSNPs (Figure 5A). After incubation with HCC cells, the bio-TLS11a and SA-FSNP combination emitted a stronger fluorescence signal than SA-FSNPs or bio-TLS11a alone (Figure 5B). Moreover, this fluorescence remained visible for 10 min, whereas the fluorescence of FITC-TLS11a lasted only for 2 min. SA-FSNPs detected aptamer-labeled HCC with good sensitivity and excellent specificity and emitted strong, photobleach-resistant fluorescent signals without the obvious noxious effect in cells or in vivo. In another study, Yan et al. developed PEG-modified RuBpy-doped silica nanoparticles that coupled with avidin (Chen et al., 2014). The dye-doped silica nanoparticles had excellent photostability. After incubation with HepG₂ cells, these nanoparticles effectively recognized the tumor marker carcinoembryonic antigen with a magnified fluorescence signal. The SNFs exhibited enhanced sensitivity, recognition efficiency, and photostability of fluorescence imaging, and became ideal for HCC diagnosis.

Ultrasound (US) is an indispensable imaging tool of clinical diagnosis. Developing novel ultrasound contrasts is important to enhance the accuracy of clinical ultrasound diagnosis (Hunt and Romero, 2017). Nanoparticles have been widely studied as targeted imaging and therapeutic agents because of their unique size and high tissue extravasation. Solid nanoparticles such as silica and polystyrene particles were studied for their potential of enhancing the ultrasound effect (Liu et al., 2006). The in vitro results of agarose gel imaging showed that silica and polystyrene particles increased the imaging signal in a concentration-dependent manner, and the particle size had a considerable effect on the image brightness. After intravenous injection, the silica nanoparticle-mediated ultrasound signal in the liver parenchyma of mice increased over time due to the aggregation of silica nanoparticles in the lysosomes of Kupffer cells (Liu et al., 2006). Based on the potential for enhancing ultrasonography, glypican-3 protein (GPC-3) ligand peptide-modified silica nanoparticles loaded with FITC were designed for the targeted ultrasound molecular imaging of HCC cells mediated by GPC-3 overexpression on the cell surface (Di Paola et al., 2017). The in vitro results showed that the nanoparticles significantly enhanced the ultrasound signal; and at the ultrasound detection concentration they could effectively bind to the HepG₂ cells and be further uptaken. MSNs with a high specific surface area and hydrophobic surfaces could be nanobubble precursors and in situ generated microbubbles after appropriate stimuli to overcome the short lifetime of microbubbles that are used as ultrasound contrast agents (Jin et al., 2017). Perfluorodecyltriethoxysilane-modified MSNs (F-MSNs, porous and superhydrophobic) generated more durable microbubbles lasting for at least 30 min at a mechanical index of 1.0 (FDA safety guidance, MI ≤ 1.9), whereas lipid microbubbles lasted for just about 5 min. F-MSNs exhibited more pronounced and stronger ultrasound contrast signals than other nanoparticles such as MSNs (porous and hydrophilic), trimethylchlorosilane-modified MSNs (M-MSNs, porous and low hydrophobic), and perfluorodecyltriethoxysilane-modified silica nanoparticles (P-SS, superhydrophobic but nonporous) (Jin et al., 2017). The strategy of in-situ producing microbubbles from the interfacial nanobubbles (INBs) adsorbed onto the superhydrophobic surfaces of MSNs can provide potentials for developing the novel silica-based ultrasound contrast agents for HCC diagnosis. These SNFs were superior with enhanced ultrasound signals and a longer lifetime.

Cancer pharmacological treatment has revolved rapidly since their invention (Falzone et al., 2018), and chemotherapy and targeted therapy have been the major approaches for cancer treatment (Zhong et al., 2021). However, their clinical use was limited because of severe systemic adverse reactions and multidrug resistance. Generally, MSNs were used as delivery carriers of chemotherapeutic agents such as sorafenib (Zhao et al., 2017), doxorubicin (DOX) (Ye et al., 2018), cisplatin (Wang et al., 2017e), and curcumin (Kong et al., 2019b) to improve their therapeutic effects and reduce the side effects by enhancing the bioavailability of hydrophobic drugs and tumor-cell targeting. However, some disadvantages were also observed such as the sudden release or their release in circulation. Conjugating drugs into the silica skeleton could address these problems and confer other advantages such as increasing drug loading, drug stability, specific site targeting, and sustained release profile (Halimani et al., 2009; Pei et al., 2018; Wong et al., 2020).

Pei et al. immobilized cytochrome c (Cyt c) onto the surface of yolk-shell mesoporous silica nanoparticles (YMSNs) via boronic ester bonds to fabricate the tumor-targeted H₂O₂-responsive Cyt c/DOX co-delivery system (YMSN-NBC-Cyt-c-NBC-LA@DOX) (Pei et al., 2018). YMSNs, as a novel form of MSNs, were capable of a relatively high drug loading for chemotherapeutic agents. Moreover, these modifications conferred these novel MSNs with tumor-targeting features, and these MSNs could temporarily shield the bioactivity of Cyt c and further prevent DOX from premature release. In the presence of high levels of reactive oxygen species (ROS) in the TME, the boronic ester could be rapidly cleaved and the Cyt c would be removed from the surface. This process could restore the bioactivity of Cyt c and readily initiate the subsequent DOX release. The in vitro study revealed the cytotoxicity effect of H₂O₂-responsive Cyt c delivery system (YMSN-NBC-Cyt-c-NBC-LA) toward HepG₂ cells in a dose-dependent manner. Regarding the in vivo effect, the tumors of mice treated with YMSN-NBC-Cyt-c-NBC-LA@DOX were inhibited, whereas the tumors of the saline-treated group grew rapidly. In another study, Halimani et al. synthesized a series of novel berberine-capped silica nanoparticles bearing covalently linker chain lengths from C₂ to C₆ (Halimani et al., 2009). With the existence of CuSO₄ and sodium ascorbate, these nanoparticles were fabricated by the click reaction between alkyne-bearing silica nanoparticles and the azidoberberine analogs. A higher surface density of berberine in the silica-berberine combination was achieved with a hexyl linker than an ethyl linker. Multivalent or dendritic effects were observed when therapeutic molecules on the nanoparticles reacted with the interacting partners inside the cell, thereby enhancing the therapeutic effect. In accordance with these effects, the in vitro study showed that all nanoconjugates exhibited higher inhibition with the IC₅₀ values of 0.2–0.27 μm than free berberine (IC₅₀ = 1.4 μm) toward HepG₂ cells 24 h after treatment. Moreover, there was an increase in the cytotoxic effect against HCC of these nanoparticles with an increase in the linker chain length. The mechanism underlying the phenomena was the cell cycle arrest and the selective apoptotic cell death, suggesting the potential role of the spacer chain in strengthening the pharmacological effects of these nanoparticles.

In addition to covalently grafting small-molecule drugs into the framework of MSNs, metal-silica nanoparticles exhibited excellent chemotherapeutic effects. Wang et al. engineered an FA-targeted, indocyanine green (ICG)-loaded Janus silver-mesoporous silica nanoparticles (FA-JNPs@ICG) by a modified Stöber method (Wang et al., 2017c) (Figure 6A). FA-JNPs@ICG had uniform ball-stick structures consisting of silver spheres and silica rods (Figure 6B). The negatively charged ICGs were loaded on the surface and in the nanochannels since the positively charged amino groups were present in the silica body. After internalization by the cancer cells, these nanoparticles employed ICGs as the effector for photothermal therapy to activate the chemotherapeutic agent. The subsequently released silver ion response to NIR irradiation could induce apoptosis in liver cancer cells as efficiently as chemotherapy. Collectively, these nanoparticles exhibited synergistic therapeutic capabilities by integrating chemotherapy and photothermal therapy. In the in vitro study, FA-JNPs@ICG were found to be toxic toward liver cancer cells rather than normal liver cells, with an inhibition rate of ∼90% (Figure 6C). Moreover, when exposed to NIR irradiation, the designed nanosystems exhibited a promising tumor growth inhibition index of 88.9% after 16 days of treatment in a mouse model (Figure 6D). Therefore, these nanoparticles could be a promising approach for chemo/photothermal therapy with high efficiency and safety for HCC. SNF modifications have considerably improved the effects of traditional and targeted chemotherapeutic agents, and novel SNFs that exert special antitumor effects without loading antitumor drugs are worth exploring.

Ferroptosis, as a new form of programmed cell death, is iron-dependent and different from apoptosis, necrosis, and autophagy. The main process of ferroptosis is the accumulation of ROS and the subsequent redox dysfunction (Yagoda et al., 2007). In the presence of Fe^2+/3+ ions, membrane polyunsaturated fatty acids (PL-PUFA-OH) are oxidated by lipoxygenases and/or ROS to produce lipid peroxides (PL-PUFA-OOH) (Cheng and Li, 2007), which can be converted into toxic lipid-free radicals (Seiler et al., 2008). Then, membrane rupture and cell death are caused by the lipid-free radicals (Cheng and Li, 2007). Since the ferroptotic process is rapid and intense without multi-level signal transduction unlike apoptosis and immune responses, it has garnered considerable attention (Fei et al., 2020b). When ferroptosis-inducing strategies and ferroptosis-based nanotherapeutics were integrated for HCC therapy, remarkable tumor suppression was achieved during the last decade (Ou et al., 2021). In all the attempts to explore ferroptotic treatment, metal elements such as iron, Mn, and copper, etc., have been found to be superior in inducing tumor cell ferroptosis, promoting ROS production, and interfering with cell communication when acting as Fenton or Fenton-like biocatalysts (Huo et al., 2019; Lin et al., 2019).

To explore new strategies for HCC therapy, our group developed Mn-doped MSNs (MnMSNs) in 2019 (Tang et al., 2019). We discovered that the manganese-oxygen chemical bonds (-Mn-O-) acted as an oxidation/reduction bond and were cleaved in malignant areas containing GSH (2–20 mM). In the acidic TME, Mn²⁺ was released after -Mn-O- cleavage, whereas intracellular GSH was consumed simultaneously. GSH consumption induced by MnMSN degradation inhibited the activity of glutathione peroxidase 4 (GPx4), thereby decreasing the accumulation of lipid-free radicals to prevent the occurrence and development of ferroptosis. Subsequently, dihydroartemisinin (DHA) was loaded onto MnMSNs (described as nanomissiles) to explore the efficiency of ferroptotic therapy that combined ROS generation and GSH depletion strategies (Figure 7A) (Fei et al., 2020a). Our in vitro results showed that the nanomissiles were degraded by GSH, leading to the release of DHA and the cleavage of -Mn-O-. The released Mn²⁺ interacted with GSH to induce a Fenton reaction. As a result, such degradation in the system led to GSH exhaustion, whereas ROS generation was driven by DHA. As shown in Figure 7B, the mean tumor volume in the nanomissile group was significantly decreased (168.83 ± 110.50 mm³) and the tumor inhibition rate was up to ∼92.56 wt%, showing better antitumor effects than those in the saline, free DHA, MnMSN, and MnMSNs@DHA groups. The ROS content was determined in the fresh tumor tissue obtained from each group to explore the ferroptotic effect of nanomissiles in vivo. As showed in Figure 7C, the nanomissile group exhibited the highest ROS generation in HCC tumors. Moreover, the nanomissiles inhibited the activity of GPx4 the most compared with that in other groups (Figure 7D). As an N-cyclohexyl compound, Fer-1 acted as a lipophilic anchor within biological membranes to prevent the accumulation of PL-PUFA-OOH, thereby inhibiting the occurrence of ferroptosis induced by FaPEG-MnMSNs@DHA (Figure 7D). These results suggested the excellent ferroptosis-inducing effect of nanomissiles in vivo. Moreover, the mice in the nanomissile group were still alive after 41 days of treatment, whereas those in the control group were dead (Figure 7E). In general, our studies demonstrated an SNF-involved, ferroptosis-based therapeutic strategy that is expected to be a candidate for the next generation of HCC therapy. Apart from the Mn ion discussed here, iron or copper ions that can act as Fenton or Fenton-like biocatalysts can be used as tumor ferroptosis inducing agents (Peng et al., 2013; Shi et al., 2016).

Radiotherapy (RT) is the standard clinical treatment based on radiation. While high-energy radiation eradicates the tumor cells, it also inevitably causes damage to normal tissues, resulting in serious side effects such as gastric dysfunction and bone marrow suppression. With the rapid development of nanotechnology, new radiosensitizers combining radiotherapy and nanotechnology may have high efficiency and safety for HCC treatment. Among them, Au-mesoporous silica Janus nanoparticles (GSJNs) were engineered by a modified sol-gel method (Figure 8A) (Wang et al., 2017d). Tetraethoxysilane (TEOS) was used as a silica source, cetrimonium bromide (CTAB) was used as a template, and the AuNPs were used as a substrate (Wang et al., 2015; Wang et al., 2016). The Janus NPs were formed by combining Au NPs with silica NPs together through one single unit. The modification of the framework inside MSNs was different from that in metal-dopped MSNs. While metal-dopped MSNs constitute a metal element doped into the structure of -Si-O-Si-, Janus NPs constitute metal NPs hybridized with silica NPs to form a single carrier. They are two different ways of framework modification but they are both useful to enable the synthesized SNFs with novel capacities such as high drug-loading, biodegradability, and tumor targeting. In this system, a Janus nanostructure representing an asymmetrical shape was designed to increase radiation efficiency and have sufficient surface area available for anticancer agent loading. Au can be used as a radiosensitizer to improve the radiotherapeutic effect for their high radiation absorption. Enhanced HCC targeting was achieved by conjugating folic acid (FA) onto the surface of GSJNs. After conjugation, the FA-GSJNs were endocytosed accessibly by cancer cells more than normal cells and smartly released drugs in acidic endo-lysosomes. Through the protonation and dissociation of amine groups, DOX was released from the GSJNs in a pH-responsive pattern (Figure 8B). With the assistance of X-ray irradiation, the FA-GSJNs-DOX exhibited more pronounced cell viability inhibition than the FA-GSJNs-DOX or RT alone toward HCC cells after 24 h of incubation (Figures 8C,D). Furthermore, the combination between FA-GSJNs-DOX and X-ray irradiation resulted in the highest tumor growth inhibition than in other groups after 23 days of treatment in nude mice bearing SMMC-7721 xenografts (Figures 8E–H). Notably, no obvious weight loss or cardiotoxicity was observed in mice from this group as seen with DOX treatment. In general, this novel nanosystem integrated with dual functionalities turned out to be a promising treatment for HCC therapy. In another study, tirapazamine (TPZ)-loaded Janus gold triangle-MSNs (FA-GT-MSNs@TPZ) were synthesized to improve the unsatisfactory therapeutic effects caused by the hypoxic microenvironment inside HCC (Wang et al., 2019). There was a silver triangular gold head connected to an extended silicon connector. The gold nanotriangels integrated RT and photothermal (PTT) as the dual-therapeutic agent. TPZ, as a hypoxia-activated prodrug, generated oxidizing radicals in low pH and low oxygen pressure conditions, which were usually observed in the TME. After NIR and X-ray exposure, FA-GT-MSNs@TPZ treatment killed almost all SMMC-7721 cells in hypoxia. Notably, in the in vivo study, tumor growth was completely inhibited when FA-GT-MSNs@TPZ with X-ray and NIR were used in tumor-bearing mice. In general, metal-silica Janus nanoplatforms were capable of transforming external energy to antitumor effects. Although recent studies in radiated and NIR energy are limited, more multifunctional metal-silica Janus nanoplatforms using different kinds of energies such as magnetic, electric, and sound energy are expected to be developed for HCC therapy in the future. While recent studies focused on the search for effective radiosensitizers, metal-silica hybridized nanoparticles may realize the possibility of becoming self-radiated emission agents, which exert radiotherapeutic effects without X-ray radiation, thus minimizing the side effects associated with radiation.

The underlying principle of sonodynamic therapy (SDT) is the combination of US irradiation and sonosensitizers to generate ROS like ¹O₂, which leads to cancer cell death depending on the redox status. SDT not only inherits a high tissue penetration depth of US but also enhances the sensitivity of tumor cells to chemotherapy. Various sonosensitizers including organic and inorganic molecules have been adopted for SDT, but some issues such as low chemical/biological stability, poor tumor targeting, and low biodegradation limit their transition to clinical application (Son et al., 2020). Thus, novel sonosensitizers with high biodegradability and chemical/biological stability need urgent exploration. With specific modifications inside the framework of MSNs, novel silica-based nanoplatforms were developed with enhanced biodegradability, biocompatibility, and higher drug-loading capacity. Li et al. developed hollow mesoporous organosilica nanoparticles (HMONs) with an organic-inorganic hybrid framework (Li et al., 2018). These HMONs were developed based on the chemical homology strategy using MSNs as the hard template and mesoporous organosilica layer were coated on the surface (Figure 11A). The organic groups referred to as disulfide bonds in the framework facilitated easy biodegradation behavior in the TME, thereby improving biocompatibility and biosafety of the nanoparticles. The inorganic mesoporous silica groups in the framework offered a larger surface area, and sufficient surface chemistry enabled the covalent anchoring of protoporphyrin (PpIX, HMONs-PpIX) into the mesopore surface and noncovalent hydrophobic-hydrophobic interaction with DOX. Furthermore, the surface conjugated RGD enabled active tumor targeting in the case of HCC and facilitated a stimuli-responsive release (Figures 11B,C). The DOX@HMONs-PpIX-RGDs were used as effective nanosonosensitizers and produced with efficient ¹O₂ when triggered by the US, leading to the apoptosis of HCC cells. As expected, when exposed to ultrasound irradiation, these nanoparticles exhibited desirable sonotoxicity and chemotoxicity toward SMMC-7721 cells. Moreover, a synergistic therapeutic effect was observed when DOX@HMONs-PpIX-RGD plus ultrasound irradiation were used to treat nude mice bearing SMMC-7721 tumor cells (Figures 11D–F). The tumor inhibition rate was 84.7%. Thus, these nanoparticles are expected to be a promising therapy for HCC. Inorganic sonosensitizers, especially the SNFs, holds immense potential for enhancing the stability, biodegradation, and therapeutic effects of HCC treatment.

Immunotherapy has also made some progress in HCC treatment. For example, in 2017, nivolumab and pembrolizumab were approved by the FDA as the second-line treatment for advanced HCC. In May 2020, the programmed cell death 1 ligand (PD-L1) antibody atelizumab combined with the target drug bevacizumab was introduced as the first-line treatment for advanced HCC, which reduced the risk of death in these patients by 42% compared with the traditional first-line treatment using sorafenib, thus bringing new hope to the long-term survival of patients with HCC (Casak et al., 2021). As one of the most widely investigated nanocarriers, the immunological effect of mesoporous silica nanoparticles in HCC treatment has been concerned and investigated. A pathogen-mimicking system based on detoxified lipopolysaccharide-modified mesoporous silica nanoparticles was designed to deliver DOX for synergistic chemo-immunotherapy of HCC (Figure 12A) (Dong et al., 2017). The pathogen-mimicking nanosystem (MSP-DOX-SP-LPS) recruited phagocytes such as neutrophils and macrophages, when injected into the tumor sites, thus simulating the pathogen infection. The recruited neutrophils generated a vast amount of extracellular ROS (Figure 12B) to trigger the loaded DOX release due to the boronic esters between MSN and the lipopolysaccharide (LPS), which could respond to the ROS, and thus, achieved the chemotherapy of HCC. The recruited macrophages could be activated by the MSN-SP-LPS to attack tumor cells directly by secreting tumor necrosis factor-α (TNF-α) or subsequently activating T cells for initiating the antitumor immune response (Figure 12C). This chemo-immunotherapy combination strategy showed an obvious synergistic therapeutic effect with inconspicuous side effects in the HCC mice model. The mice treated with MSP-DOX-SP-LPS produced a significant immune memory, showing lower recurrence of the primary tumor and lower growth rate of reinoculated tumor (Figure 12D) (Dong et al., 2017). In summary, SNF-mediated immunotherapy is effective for HCC treatment. The rapid advancement of immunotherapy based on nanoparticles holds potential for recent new approaches such as antitumor vaccines, immune checkpoint blockade, and immunogenic cell death (Liu et al., 2019b). On the other hand, the concept of realizing chemo-free treatment for cancer therapy is emerging (Zhu, 2020).