Nanotechnological strategies to increase the oxygen content of the tumor

Hypoxia is a negative prognostic indicator of solid tumors, which not only changes the survival state of tumors and increases their invasiveness but also remarkably reduces the sensitivity of tumors to treatments such as radiotherapy, chemotherapy and photodynamic therapy. Thus, developing therapeutic strategies to alleviate tumor hypoxia has recently been considered an extremely valuable target in oncology. In this review, nanotechnological strategies to elevate oxygen levels in tumor therapy in recent years are summarized, including (I) improving the hypoxic tumor microenvironment, (II) oxygen delivery to hypoxic tumors, and (III) oxygen generation in hypoxic tumors. Finally, the challenges and prospects of these nanotechnological strategies for alleviating tumor hypoxia are presented.


Introduction
Studies have shown that the distribution of oxygen concentration in tumors is highly heterogeneous, and the partial pressure of oxygen in many areas is less than 5 mmHg (equivalent to approximately 0.7% O 2 ), which is far lower than the partial pressure of oxygen in normal tissues (Vaupel et al., 1991;Denny, 2000). There are three main causes of hypoxia in the tumor microenvironment (TME). First, the rate of tumor apoptosis is much lower than the rate of cell growth, and the demand for oxygen and glucose is much greater than that of normal cells (Petrova et al., 2018;Matuszewska et al., 2021). Second, the tumor volume is constantly increasing. When the tumor volume increases to the oxygen diffusion limit (100-200 μm), the blood vessels cannot provide sufficient oxygen for the tissue cells (Bennewith and Dedhar, 2011;Fu et al., 2022).
Hypoxia can alter the expression of some cytokines, such as erythropoietin and metabolism-related proteins (e.g., phosphofructokinase), in the TME. These changes play an important role in the adaptation of tumor cells to hypoxia, energy storage, metastasis, proliferation, and tumor angiogenesis and eventually lead to metabolic abnormalities in tumor cells, which also exacerbates the malignant growth of tumors and drug resistance (Zhao et al., 2015). Hypoxia-inducible factor 1 (HIF-1) is involved in the regulation of cytokines and metabolism-related proteins. HIF-1 is a nuclear protein with transcriptional activity that consists of HIF-1α subunits and HIF-1β subunits. HIF-1α was degraded by OPEN ACCESS EDITED BY Meihua Yu, Shanghai University, China ubiquitination under atmospheric oxygen. HIF-1α remained stable under hypoxic conditions. HIF-1β is stably expressed in cells and plays a structural role. To exert the role of a transcription factor, the HIF-1α subunit must polymerize with the HIF-1β subunit to form a heterodimer. The gene that responds to hypoxia stress is called the hypoxia response gene (HRG), and the gene regulated by HIF-1α in HRG is the target gene of HIF-1α. The promoters or enhancers of these target genes contain different numbers of hypoxia response elements (HREs), which are the binding sites of HIF-1α. HIF-1α combines with HRE to form a transcription initiation complex, which initiates the transcription of target genes and produces various products, causing a series of reactions. For example, under hypoxic conditions, HIF-1 can bind to the HRE of the vascular endothelial growth factor (VEGF) promoter region, causing the upregulation of VEGF expression, increasing the generation of new blood vessels, and promoting the growth and metastasis of tumors (Zhang et al., 2022a;Zhang et al., 2022b).
Hypoxia also promotes proteomic and genetic changes in tumor cells, reinforces the adaptation of cancer cells to hypoxia, and enhances tumor invasion and metastasis (Rankin and Giaccia, 2016a;Al Tameemi et al., 2019;Li et al., 2020a). Moreover, hypoxia has been proven to make tumor cells resistant to conventional cancer therapies, including chemotherapy, radiotherapy, and photodynamic therapy, which is due to the need for oxygen molecules to participate in these treatments (Yuan et al., 2021a;Devarajan et al., 2021;Kopecka et al., 2021;Telarovic et al., 2021). Therefore, relieving tumor hypoxia is the key to achieving effective cancer therapy. To improve the hypoxic microenvironment, many trials have been performed. For example, hyperbaric oxygen therapy increases oxygen in the blood and tumor by delivering high concentrations of oxygen to the body (Wu et al., 2018). However, non-specific oxygen delivery and severe malformations of the tumor microvasculature have prevented further development of hypoxia palliative therapies. In addition, high concentrations of oxygen may cause serious side effects (Schwarte et al., 2019). Fortunately, the rapid development of nanotechnology and materials science has led to tremendous progress in the biological applications of nanomaterials for molecular imaging, targeted drug delivery and combination therapy. There is growing evidence that nanomedicines offer many advantages in the treatment of hypoxic tumors. For example, by virtue of the tumor-specific enhanced permeability and retention properties, nanocarriers can be enriched in tumors Yan et al., 2021;Yan et al., 2022). Then, nanomaterials with oxygen release ability can alleviate tumor hypoxia (Dai Phung et al., 2020;Zou et al., 2021;Xu et al., 2022a). In this review, we discuss the recent nanotechnologies to increase oxygen levels in tumors, including (I) improvement of the hypoxic TME, (II) oxygen-carrying nanomaterials, and (III) oxygenproduction nanomaterials ( Figure 1; Table 1). Finally, we present in detail the challenges and prospects of these tactics for alleviating tumor hypoxia.
2 Nanomaterial-mediated tumor hypoxia relief 2.1 Improving the hypoxic tumor microenvironment Most solid tumors remain in a hypoxic state throughout disease progression (Chen et al., 2022b). For their own development, tumors will plunder nutrients and oxygen through angiogenesis . Tumor blood vessels differ from normal blood vessels in various important phenotypes. The expression of hypoxia-inducible angiogenic factors, such as VEGF, which induces angiogenesis in vivo, is upregulated in hypoxic tumor sites. However, the newly formed vessels tend to be poorly organized and dysfunctional, with either variable flow directions or leakage. In addition, there are gaps between tumor-related vessel endothelial cells, which cannot complete the normal metabolic exchange of plasma and tissue fluid, resulting in leakage of blood vessels and no laminar flow, which is prone to coagulation and local tissue edema. The hypoxic tumor environment is the main cause of tumor angiogenesis, and angiogenesis is the root cause of tumor progression and metastasis. Therefore, improving the hypoxic tumor environment is a conservative tumor treatment method that can inhibit tumor progression and metastasis (Wang et al., 2020a).

Nanomaterials combined antiangiogenic agents
Some antiangiogenic agents normalize the vasculature by improving tumor blood flow and correspondingly delivering more oxygen to the tumor (Huang and Chen, 2010;Carmeliet and Jain, 2011). Normalizing the abnormal blood vessels in breast tumors by blocking vascular endothelial growth factor receptor 2 can facilitate the delivery of small nanoparticles (12 nm) while hindering the delivery of large nanoparticles (125 nm) (Chauhan et al., 2012). Antiangiogenic agents can increase tumor oxygen content by normalizing tumor blood supply, while normalization of vascular structure also enhances the delivery of nanodrugs, so antiangiogenic agents can synergize with nanodrugs in tumors. Li et al. (2016) used gold nanocarriers (Au NPs) to encapsulate recombinant human endostatin (rhES) to target tumors to enhance the antitumour effect. Polyethylene glycol (PEG)-modified rhES-Au-NPs (rhES-Au-NPs-PEG) can transiently normalize blood vessels and improve blood supply capacity in mouse-loaded hepatoma-22 xenografts. Then, tumor hypoxia relief, decreased vascular permeability, enhanced integrity, improved pericyte coverage, and increased blood perfusion were observed in the TME. The results of animal experiments proved that the combined treatment of rhES-Au-NPs-PEG and 5-fluorouracil was significantly more effective than the drug alone. In addition, endostatin and other antiangiogenic drugs, such as bevacizumab (anti-VEGF inhibitor), can also normalize tumor blood vessels (Isaakidou et al., 2016;Poluzzi et al., 2016).

Modulation of relevant signal pathways for tumor hypoxia based on nanomaterials
Hypoxia causes a reduction in the enzyme activity of prolyl hydroxylase and prevents HIF-1α hydroxylation, which is the master transcriptional regulator in cells under hypoxic conditions (Gilkes et al., 2014;Rankin and Giaccia, 2016b;Jing et al., 2019;Singleton et al., 2021). The stability and activity of HIF-lα depends on oxygen content and affects tumor angiogenesis, glucose metabolism, tumor stem cell proliferation, tumor cell proliferation, and multidrug resistance (Fong and Takeda, 2008;Belisario et al., 2020). In the presence of 21% oxygen, the half-life of the HIF-1α subunit was less than 5 min, but when the O 2 concentration was reduced to 1%, the half-life increased to 60 min. In the hypoxic TME, HIF-1α was no longer degraded and became stable. It binds to HIF-1β to form transcriptionally active dimers, which interact with transcriptional activation factors and finally induce the transcription and post-transcriptional regulation of downstream target genes, including erythropoiesis, glucose metabolism, angiogenesis, and drug resistance (Vaupel and Multhoff, 2018;McAleese et al., 2021). Therefore, inhibiting the HIF-1 signaling pathway is a suitable treatment strategy for hypoxic tumors. Researchers have developed several strategies to downregulate HIF-1α expression by using small interfering RNA (siRNA) Huang et al., 2022;. However, there are some obstacles to siRNA therapeutics in systemic administration, such as aggregation with serum proteins, enzymatic degradation with endogenous nucleases, and uptake by phagocytes. In addition, the distance from the hypoxic area of the solid tumor to the blood vessel and the increase in efflux transporters also increase the difficulty of siRNA delivery. Zhu et al. (2015) designed a delivery system to deliver siHIF-1α. Hybrid quantum dots with hypoxic tumor targeting properties and pH responsiveness can enhance antitumour activity and reduce toxicity to normal tissues. In addition, this siRNA delivery system based on hybrid quantum dots can realize real-time dynamic monitoring of the siRNA delivery process in vitro and in vivo. Furthermore, the delivery of drugs that downregulate HIF-1α can also reverse tumor hypoxia tolerance (Liu et al., 2019). For example, zoledronic acid (ZA) can reduce the expression of P-glycoprotein (P-gp) in multidrug-resistant (MDR) cells and inhibit P-gp transcription mediated by HIF-1α (O'Donnell et al., 2006;Riganti et al., 2013). However, when ZA is administered directly, most ZA is absorbed by bone, resulting in insufficient concentrations in the tumor. To enhance the delivery effect, Kopecka et al. (2015) coupled ZA and liposomes to form nanoparticle formulations (abbreviated as NZ). The results have shown that NZ at low concentrations increases the chemical susceptibility of otherwise multidrug-resistant cells to commonly used broad-spectrum drugs. NZ can inhibit HIF-la activity by interfering with the Ras/ERK1 pathway. In addition, NZ can also reduce the transcription of glycolytic enzymes and glucose flux. Therefore, NZ reduced the activity of adenosine triphosphatedependent adenosine triphosphate-binding transporters, thereby increasing the efficacy of chemotherapy in vitro and in vivo. To increase drug delivery efficiency, Liu et al. (2016a) utilized polyethylene glycol-poly L-lysine-poly lactic-co-glycollic acid to form nanoparticles through self-assembly, and then transferrin (Tf) was used for surface modification and loaded with daunorubicin (abbreviated as

Strategies
Nanomaterials to increase oxygen content References Improving the hypoxic TME Nanomaterials combined antiangiogenic agents Li et al. (2016) Inhibition of the HIF-1 signaling pathway based on nanomaterials DNR-Tf-NPs) to study the antitumor effect on K562 leukemia cells under hypoxia. DNR-Tf-NPs could increase the intracellular concentration of daunorubicin (DNR) and the drug sensitivity of K562 cells. At the same time, DNR-Tf-NPs could reduce the expression of HIF-la, overcome multidrug resistance induced by hypoxia and induce apoptosis. In the xenograft model, DNR-Tf-NPs remarkably inhibited the growth of tumors and significantly reduced the expression of the Ki67 proliferation marker compared with other experimental groups.

Photothermal therapy-mediated hypoxia relief
When the local temperature of the tumor tissue increases, the flow velocity increases. In recent years, a variety of nanomaterials with photothermal conversion ability have been used for tumor photothermal therapy, which could be enriched in the tumor site by passive targeting (based on the high permeability and retention effect of the tumor, i.e., the EPR effect) or active targeting (based on the binding of nanoparticle surface-coupled antibodies and tumor site-specific receptors) after being injected into mice. Under light irradiation, the local temperature of the tumor site increases locally, thereby achieving the purpose of treating and even killing tumors (Cheng et al., 2014). Based on the photothermal effect of MnS@ Bi 2 Se 3 nanomaterials, Song et al. (2015) found that a mild temperature rise (approximately 43°C) of the tumor site could be achieved under low power illumination. Interestingly, after mild photothermal treatment, the tumors were not killed, but the blood supply to the tumors could be significantly increased after a period of light exposure; thus, tumors would change from a hypoxic state to a relatively oxygen-rich state. Thus, the treated tumor could be effectively killed after oxygen-dependent X-ray radiotherapy, which was closely related to the significant increase in oxygen content inside the tumor before radiotherapy. In addition, Lu et al. (2018) synthesized CuS-modified hollow mesoporous organic silicon oxide nanoparticles (HMON@CuS) by loading oxygen-combined perfluoropentane (PFP). The heat generated by CuS after near infrared laser irradiation could convert the liquid PFP into a gaseous state and release it from the cavity of the HMON to further promote the release of oxygen and promote the diffusion of oxygen in the tumor, thereby overcoming the radiotherapy tolerance of the tumor. In addition, photothermal therapy combined with other treatment methods also proves that mild phototherapy can indeed lead to the improvement of blood supply and hypoxia in tumors, thus making tumors more sensitive to treatments (Li et al., 2020b;Li et al., 2022a;Zhang et al., 2022c).

Oxygen-carrying nanocarriers
The development of efficient oxygen delivery strategies is valuable and significant for enhancing the effect of tumor therapy. At present, hyperbaric oxygen is inhaled and used clinically during tumor treatment to force more oxygen into the blood and tumors (Chu et al., 2018;Liu et al., 2021;Li et al., 2022b). However, this strategy cannot overcome the defect of insufficient oxygen supply caused by vessel structural abnormity, and it is difficult to effectively transport oxygen to the interior of the tumor. Moreover, excessive oxygen concentration in the body is prone to oxygen toxicity to the lungs and central nervous system. In recent years, artificial oxygen carriers have become increasingly widely used. Artificial oxygen carriers usually use perfluorocarbons, hemoglobin or nanomaterials to store oxygen molecules inside the carrier by dissolving or non-covalent binding. When they reach the tumor, they can release the stored oxygen into the tumor tissue, thereby increasing the oxygen content of the tumor. Next, we discuss hemoglobin/perfluorocarbon-based nanocarriers to directly deliver oxygen to hypoxic tumors.

Hemoglobin-based oxygen nanocarriers
Hemoglobin (Hb) is the core of red blood cell (RBC) binding and transporting oxygen, and oxygen forms a strong chemical covalent bond with the iron element in hemoglobin, which can reversibly bind oxygen molecules to form oxyhemoglobin (HbO 2 ). Then, oxygen is provided to various tissues and organs via the circulatory system (Gell, 2018;Olson, 2022). However, the circulation time of free Hb is short, and its stability is poor, which limits its usage and application areas. To solve this problem, researchers have used various nanocarriers to load HbO 2 by physical encapsulation to achieve the same oxygencarrying function as RBCs (Jansman and Hosta-Rigau, 2018;Kim et al., 2023). A nanobionic oxygen carrier (DHCNPs) with the functions of targeting homologous cancer cells and increasing oxygen content was prepared by using polymer-loaded doxorubicin (DOX) and Hb as the core and coating of breast cancer cell membrane on the surface (Tian et al., 2017). The DHCNPs utilize adhesion molecules of cancer cells to target and identify homologous cancer cell tumors and combine with breast cancer cells, thereby realizing targeted administration to the same tumor while delivering chemotherapy drugs and oxygen to the interior of the tumor. The results showed that the hypoxic TME was altered by targeted oxygen replenishment. Therefore, the expression of HIF-1α and p-gp was reduced. The amount of DOX pumped out of the cancer cells was reduced, thereby improving chemotherapy resistance and realizing safe and efficient chemotherapy. However, the method of encapsulating Hb in nanocarriers requires the participation of organic solvents and severe stirring or ultrasonic operation, which might affect the activity of Hb. In addition, in the process of physical loading, to minimize the mutual repulsion between protein and protein, they are fixed in the form of random orientation, which is a non-uniform state that causes partial inactivation of the protein. Moreover, this high-density loading through intermolecular forces such as ionic hydrophobic and polar interactions will cause the blocking of protein active sites in space. To avoid this problem, Hb may be coupled to the carrier by covalent binding. Wang et al. (2021a) utilized Hb and polycaprolactone self-assembled nano erythrocyte systems to deliver DOX and oxygen [V(Hb)@DOX] to reprogram the tumor immunosuppressive microenvironment to enhance chemoimmunotherapy ( Figure 2A). After administration, V(Hb) @DOX specifically targeted M2 macrophages with high CD163 expression in the tumor region. The oxygen released from V(Hb)@DOX alleviated tumor hypoxia, downregulated HIF-1α expression, and reduced the recruitment of M2-type macrophages. Since Hb is stable at neutral pH, it is partially dissociated in the acidic environment, resulting in the release of Frontiers in Pharmacology frontiersin.org 04 DOX to kill tumor cells. Ultimately, V(Hb)@DOX-mediated immune reprogramming could prevent tumor metastasis and recurrence. In addition, Hb can also be used as a sonosensitizer for cancer therapy (Yuan et al., 2021b) ( Figure 2B). To improve the cellular uptake of Hb and protect it from hydrolysis by lysosomal enzymes, zeolite imidazolium 8 (ZIF-8) (composed of Zn 2+ ions and 2-methylimidazole ligands) was used for drug encapsulation and controlled release. ZIF-8 was used as a drug carrier and could be taken up by cancer cells to achieve high Hb loading efficiency and pH-responsive O 2 release from tumor sites. Oxygen release from Hb can enhance photodynamic behavior, which then induces severe mitochondrial dysfunction and activates the mitochondrial apoptotic pathway, further effectively inhibiting tumor cell growth. Compared with RBCs, the enhanced penetration and retention effects of Hb-based nanocarriers enabled them to accumulate more in hypoxic regions of tumors and improve oxygen delivery efficiency. Therefore, nano-oxygen carriers showed better oxygen supply capacity than natural erythrocytes in the TME.

Perfluorocarbon-based oxygen nanocarriers
The solubility of O 2 in perfluorocarbon is approximately 40-50 mL O 2 per 100 mL; thus, it can be used as an oxygen carrier Wang et al., 2019;Jagers et al., 2021). In perfluorocarbons, oxygen undergoes physical dissolution, and there is a weak non-directional van der Waals force between oxygen and perfluorocarbons, which also ensures that oxygen can be dissolved or released quickly. Various perfluorocarbon (PFC)based oxygen delivery nanoplatforms, such as liposomes, hollow nanomaterials or polymers, have been successfully fabricated and utilized to alleviate tumor hypoxia and enhance oxygen-related therapeutic efficacy Gao et al., 2017;Li et al., 2018;Liang et al., 2020;Dong et al., 2022). However, the premature release of oxygen before reaching the anoxic region and the oxygen transport mainly driven by the concentration gradient are not controllable. Therefore, the development of controllable oxygen carriers is significant for the delivery of oxygen (You et al., 2020b). In view of the weak van der Waals interactions between PFC and oxygen, ultrasound and near-infrared light (NIR) are currently the main stimuli to trigger oxygen release . For example, Song et al. (2016) used human serum albumin as a stabilizer for PFC nanoemulsions and selected ultrasound to induce oxygen release to improve the hypoxic environment of tumors. The nanoemulsion was injected into tumor-bearing mice. The PFC nanoemulsion absorbed oxygen from the lungs and circulated to the hypoxic tumor site. Another common source of excitation is the NIR laser. For example, a "nano bomb" (PFH@Ag@Ch-I) was designed using indocyanine green (ICG), chitosan, and perfluorohexane (PFH) . When PFH@Ag@Ch-I was endocytosed by lysosomes, the photothermal activity of the silver nanocages made PFH reach the FIGURE 2 (A) V(Hb)@DOX enhanced cancer chemoimmunotherapy and reprogrammed the TME. Reproduced from ref. (Tian et al., 2017) with permission from Wiley, copyright 2021. (B) Nanomaterials combined with oxygen-carrying hemoglobin for sonodynamic therapy. Reproduced from ref. (Wang et al., 2021a) with permission from ACS Publications, copyright 2021.
Frontiers in Pharmacology frontiersin.org 05 boiling point and rapidly vaporize under NIR irradiation, which led to the release of oxygen. The increased internal pressure of the "nano bomb" resulted in silver nanocage explosion and enabled lysosome escape. Then, the silver nanocages were broken into small nanoparticles for a Fenton-like reaction, and the oxygen released from PFH improved the hypoxic TME and enhanced ICG-mediated photodynamic therapy (PDT), thereby achieving efficient tumor penetration and antitumour therapy. To increase the tissue penetration depth, Zhang et al. (2022d) encapsulated PFCs in functionalized polymers to prepare a photothermo-triggered "oxygen bomb." By using an NIR-II laser, the combination of photothermal therapy (PTT), PDT, and chemotherapy could be released.

Monocyte-based oxygen nanocarriers
Studies have found that several chemokines secreted by tumor cells are closely related to the recruitment of monocytes (Murdoch et al., 2004). Once monocytes reach the tumor site and further develop into tumor-associated macrophages (TAMs), they tend to be retained in hypoxic tumor tissue (Brown and Wilson, 2004). For example, bone marrow-derived monocytes were used to carry oxygen-loaded polymer bubbles, superparamagnetic iron oxide (SPOIN) and the photosensitizer chlorin e6 (Ce6) (Huang et al., 2015). The multifunctional oxygen bubble carrier (SCOPB-engulfed monocytes) was not toxic to cells without external stimulation. When irradiated with a laser (660 nm) and high-frequency magnetic field, the growth of prostate xenografts in nude mice was significantly inhibited by combined magnetothermal therapy and PDT. The histological results of the tumor sections showed that the effective therapeutic agent could relieve tumor hypoxia and then enhance the therapeutic effect of PDT.
Oxygen-carrying nanocarriers with advantages, disadvantages, and major highlights are summarized in Table 2. PFC carries more oxygen than Hb at the same concentration. As the release of oxygen in PFC is achieved by concentration gradient, while that in Hb is related to Hb oxy-deoxy conformational change, the oxygen release efficiency of PFC is higher than that of Hb, and controlled release can be realized under external stimulation.

Oxygen-production nanomaterials
The content of hydrogen peroxide (H 2 O 2 ) in tumor tissue is generally higher than that in normal tissues, which is one of the reasons for tumor invasion and metastasis. This characteristic also offers an opportunity for alleviating tumor hypoxia; for instance, catalase (CAT) can catalyze the decomposition of H 2 O 2 to generate O 2 in situ. With the development of nanotechnology, a variety of nanomaterials with catalytic properties have been discovered (Wei and Wang, 2013;Liang et al., 2019;Wang et al., 2022e). Among these nanozymes, some nanozymes with CAT-like activity have been developed. In this section, we will summarize nanomaterials combined with natural CAT enzymes and CAT-like nanozymes to enhance tumor therapy.

CAT-loaded nanoagents
CAT is a common natural enzyme in living organisms that can catalyze H 2 O 2 to produce oxygen (Goyal and Basak, 2010;Ma et al., 2019;Mu et al., 2023). To exert an excellent cascading catalysis reaction, Zhou and Li (2022) designed a three-enzyme cascade nanosystem (plasEnMOF) by embedding CAT, glucose oxidase (GOx) and horseradish peroxidase (HRP) in ZIF-8-encapsulated gold nanorods (AuNRs) ( Figure 3A). After intravenous injection of plasEnMOF in 4T1 tumor-bearing mice, the overexpressed H 2 O 2 in tumors could be converted into oxygen by CAT, thereby relieving tumor hypoxia and providing the oxygen required for GOx to react with glucose. GOx utilized oxygen to catalyze glucose to produce H 2 O 2 , which could convert into hydroxyl radicals (•OH) for CDT in the presence of HRP. In another work, to significantly increase reactive oxygen species (ROS) levels in the TME, Qin et al. (2021) prepared nanogels (FIGs-LC) by integrating lactate oxidase (LOx) and CAT into hybrid nanogels that encapsulated with Fe 3 O 4 NPs and ICG. LOx can catalyze endogenous lactic acid to produce H 2 O 2, which reacts with Fe 3 O 4 NPs to produce •OH. Meanwhile, the oxygen generated from the CAT catalytic reaction could enhance singlet oxygen production. The in vivo results showed that the tumor inhibition rate of FIG-LC reached 89.05%, and the side effects were negligible. However, the catalytic efficiency of CAT may be affected by numerous proteases in vivo, especially in tumor sites where proteases are overexpressed (Vandooren et al., 2016;Zai et al., 2021). Recently, Zai et al. (2021) developed CAT-containing Escherichia coli membrane vesicles (EMs), which showed good protease resistance for alleviating tumor hypoxia and radiotherapy enhancement ( Figure 3B). The CAT in EMs was more than 100 times more resistant to protease than free CAT and was able to maintain catalytic activity even 12 h after intratumoral injection. In a separate study, Wu et al. (2022) prepared a biodegradable nanoplatform (CSI@Ex-A) by loading CAT and ICG into silica nanoparticles and then coated it with AS1411 aptamer-modified  Frontiers in Pharmacology frontiersin.org macrophage exosomes ( Figure 3C). After endocytosis of tumor cells, the highly expressed glutathione (GSH) triggered the degradation of CSI@Ex-A to release CAT, which could catalyze H 2 O 2 to generate O 2 to alleviate tumor hypoxia. Furthermore, the therapeutic effect of sonodynamic therapy (SDT) was enhanced by GSH depletion and O 2 self-supply in vitro and in vivo.

CAT-like nanozymes 2.3.2.1 Au-based nanozymes
Due to their excellent photothermal properties in the nearinfrared region, Au-based nanomaterials are often used as photothermal agents for PTT (Cheng et al., 2017;Ye et al., 2022). In recent years, it was found that Au-based nanomaterials have exhibited a variety of enzymatic activities, such as peroxidase, CAT, superoxide dismutase (SOD), and oxidase, among which CAT-like and SOD-like activities can be used to alleviate tumor hypoxia (Lin et al., 2014;Lou-Franco et al., 2021). pH has an important effect on the catalytic activity of AuNPs (He et al., 2013). In a neutral pH environment, AuNPs exhibit SOD-like activity that can convert superoxide into H 2 O 2 . In alkaline environments, AuNPs exhibit CAT-like activity that converts H 2 O 2 into oxygen. However, in an acidic pH environment, the SOD-like and CAT-like activities of AuNPs are significantly reduced. To improve the catalytic activity of AuNPs in acidic pH to some extent, Yang and coworkers first reported amineterminated polyamidoamine (PAMAM) dendritic moleculeencapsulated Au nanoclusters (AuNCs-NH 2 ) (Liu et al., 2017a). The 3-amines on dendritic macromolecules were easily protonated in an acidic TME, which facilitated the preadsorption of OH − on the gold nanoclusters. Furthermore, the CAT-like activity of the nanoclusters was extended to an acidic TME (pH 4.8-7.4), catalyzing the production of oxygen from overexpressed H 2 O 2 to alleviate tumor hypoxia and enhance PDT. In a similar study, Xu et al. (2019) designed a multifunctional nanocomposite (PGPAI NPs), which was polypyrrole (PPy) coated by graphene oxide (GO) flakes and then modified with PEG, Au NPs and IR820 molecules. The PGPAI NPs had good photoacoustic imaging and computed tomography imaging capabilities. Under NIR irradiation, PPy and IR820 could effectively produce heat and ROS, respectively. Au NPs not only generate oxygen by catalyzing overexpressed H 2 O 2 in the tumor, enhancing the effect of oxygen-dependent PDT but also exhibit GOx like activity and can efficiently catalyze the conversion of glucose into H 2 O 2 and gluconic acid (Li et al., 2020c;Chen et al., 2021a). Another classic example was presented by Liu et al. (2020a). An iron-based metal organic framework (GIM) doped with AuNPs was used for ROS production in hypoxic tumors via cascade catalytic reactions (Liu et al., 2020a). Due to the GOx like activity of AuNPs, GIM could catalyze glucose to H 2 O 2 and then generate •OH for CDT through the Fenton reaction. In addition, GIM could also rapidly decompose H 2 O 2 into O 2 , which not only alleviated hypoxia in the TME but also promoted the catalysis of glucose by AuNPs.

Manganese-based nanozymes
Manganese dioxide (MnO 2 ) can catalyze excessive H 2 O 2 in the TME to produce oxygen, demonstrating unprecedented advantages in the treatment of TME hypoxia due to its good degradation ability as well as high catalytic activity (Ding et al., 2020;Gao et al., 2020). A responsive cascade theranostic nanosystem (Lipo-OGzyme-AIE) was designed by encapsulating aggregation-induced emission (AIE) and OGzymes in the phospholipid bilayer ( Figure 4A) . The O 2 generated through the catalytic reaction of MnO 2 could diffuse throughout the tumor, providing O 2 for AIE to produce singlet oxygen under irradiation. In addition, bovine serum albumin (BSA) was designed to encapsulate gold nanorods (Au NRs), and MnO 2 nanoparticles were deposited at the reduction site to form Au NRs@BSA/MnO 2 (Zhou et al., 2020a). Based on the strong localized surface plasmon resonance effect of Au NRs, this nanosystem had good photothermal conversion efficiency and could be used for photothermal ablation of tumors. The MnO 2 particles from the nanosystem could decompose H 2 O 2 to produce O 2 , which in turn could be used for hypoxia improvement in the TME. However, the previously reported structures of MnO 2 are mostly nanoparticles, nanocomposites in combination with other types of nanoparticles or nanosheets, which may not be ideal for the effective loading and accurate release of drugs. Hollow nanostructures have been shown to be able to construct loading/delivery nanoplatforms for the precisely controlled release of therapeutic agents (Zhang et al., 2022f;Zhu et al., 2022). The research group of Liu developed a biodegradable hollow MnO 2 nanotherapeutic ( Figure 4B) (Yang et al., 2017). Nanotherapeutics can achieve TME-responsive imaging and specific release of drugs, improve the hypoxic environment of tumors and enhance the effect of cancer treatment. Finally, the nanotherapeutic could be rapidly decomposed in the mouse body and excreted out of the body. Based on this nanotherapeutics, the synergistic therapeutic effects of chemotherapy and PDT in vivo could be effectively improved. After nanotherapeutics are combined with checkpoint blockade, antiprogrammed death ligand 1 (anti-PD-L1) therapy can not only kill the primary tumor but also effectively inhibit the growth of distant tumors. In addition to being a nanozyme, MnO 2 can also react with H + or GSH existing in the TME and decompose to Mn 2+ , Frontiers in Pharmacology frontiersin.org which prominently enhances T1 magnetic resonance imaging contrast and can be used for tumor-specific imaging Li et al., 2022c;Pan et al., 2022).

Platinum-based nanozymes
Platinum (Pt)-based nanomaterials are widely used as nanozymes for oxygen production due to their low production cost and long-lasting catalytic properties (Chen et al., 2021b). At present, the development of nanodrug responses to the TME is a common strategy to treat tumors. A TME-activated nanodrug (PtN 4 C-SAzyme) prepared by Xu et al. (2022b) was used for the cascade catalytic reaction ( Figure 5A). SAzyme has peroxidase (POD)-like and CAT activities. The overexpressed GSH in cells was continuously consumed by Pt (IV) to generate Pt 2+ , which could reduce ROS scavenging. More importantly, PtN 4 C-SAzyme had SOD-like activity, which converted O 2 − into H 2 O 2 to supplement the consumption of H 2 O 2 , and H 2 O 2 could also further react with PtN 4 C-SAzyme to realize the cycle of •OH and O 2 − . To improve the stability of Pt-based nanomaterials in aqueous solution, Lu et al. (2020) performed PEGylation of platinum porous nanospheres (pPts) and loaded GOx ( Figure 5B). GOx could convert glucose to H 2 O 2 in the presence of oxygen; pPts subsequently decomposed the overexpressed H 2 O 2 in tumors into oxygen and water. Thus, pPts promoted the consumption of glucose in hypoxic tumors and increased cellular oxidative stress. In addition, Pt assisted by a direct current electric field and chloride ions induced the decomposition of water molecules on their surface, generating cytotoxic •OH. pPts-mediated electrokinetic therapy in synergy with starvation therapy could significantly alleviate the hypoxic microenvironment and inhibit tumor growth. Interestingly, You et al. (2020a) developed a nanomaterial (ICG-PtMGs@HGd) that could continuously replenish O 2 with low toxicity ( Figure 5C). Pt and Au successively wrapped metal-organic frameworks (MOFs) to form octahedral metal nanoshells (PtMGs), and then the surface was modified with gadolinium-chelated human serum albumin (HSA-Gd) and ICG. Oxygen production was measured with a dissolved oxygen meter. After H 2 O 2 addition, the oxygen concentration varied from 5.0 to 10.8 mgL −1 in the ICG-PtMGs@ HGd solution without significant oxygen production in the absence of ICG-MGs@HGd, and this catalytic effect was durable. In the synergistic phototherapy experiment in vivo, the results showed that the tumor volume of the ICG-PtMGs@HGd group under NIR irradiation decreased most Frontiers in Pharmacology frontiersin.org significantly, and the body weight of the tumor-bearing mice did not change significantly after treatment. In general, there is a wide range of biological applications of Pt-based nanomaterials, but the toxic side effects caused by heavy metals are still current challenges to be overcome (Duan et al., 2022;Velcheva et al., 2022).

Other metal-based nanozymes
With the action of oxygen and GOx, glucose can be converted into gluconic acid and H 2 O 2 , thereby cutting off the nutrition source of tumor cells and inhibiting cancer cell proliferation (Huo et al., 2017;Xu et al., 2022c;Wang et al., 2022f). Considering that the catalytic activity of a single enzyme is insufficient to achieve satisfactory therapeutic effects, the development of nanozymes with multiple enzyme-mimetic functions is necessary. A multienzyme nanoreactor (IrRu-GOx@PEG NPs) for cascade catalytic reactions was prepared after the rRu alloy nanoparticles were modified with GOx and PEG ( Figure 6A) (Wei et al., 2020). In the biological catalytic stage, the glucose in the tumor was degraded to H 2 O 2 by IrRu-GOx@PEG NPs, which cut off the tumor's nutrient source and inhibited tumor growth. In the chemical catalytic stage, IrRu-GOx@PEG NPs catalyzed H 2 O 2 to generate O 2 and highly toxic singlet oxygen ( 1 O 2 ). The in vitro and in vivo results indicated that the IrRu-GOx@PEG NPs could effectively induce 4T1 cell apoptosis. In addition, a nanoplatform (DMSN@CoFe 2 O 4 /GOD-PCM) was designed for NIR II-enhanced tumor therapy by depositing ultrasmall cobalt ferrite (CoFe 2 O 4 ) and GOx into the pore size of dendritic mesoporous silica   (Figure 6B). After laser irradiation, the high temperature generated by CoFe 2 O 4 melts the phase change material (PCM) to release GOx, remodelling the TME through the glucose metabolism pathway. The resulting intensified acidic conditions and large amount of H 2 O 2 effectively initiate the cascade catalytic reaction. To achieve precise treatment, an upconversion nanoparticle (UCNP)-based smart nanosystem (UCNPs@Cu-Cys-GOx) was designed for cancer combination therapy (Wang et al., 2022a). The nanosystem remained inert (turned off) in normal tissues and was only specifically activated (turned on) in the TME through a sequence of enzymatic cascades. Moreover, the enhanced oxidative stress of the nanosystem could reverse the immunosuppressive TME. Meanwhile, the smart nanosystem combined with immunotherapy/starvation/chemokinetic synergistic therapy effectively inhibited primary tumor growth and cancer metastasis. In addition, GOx-induced starvation therapy synergized with copper death and significantly inhibited tumor growth (Fu et al., 2018;Xu et al., 2022d).

Prussian blue-based nanozymes
Prussian blue (PB) is a Food and Drug Administrationapproved antidote for heavy metal poisoning . In recent years, researchers have gradually found that PB has excellent physicochemical properties, such as photothermal conversion and catalytic activity (Liu et al., 2016b;. PB can catalyze the production of oxygen from overexpressed H 2 O 2 , which can effectively improve the malignant environment (Komkova and Karyakin, 2022). Motivated by this, Zhou et al. (2020b) designed a PB nanoplatform (SP94-PB-SF-Cy5.5 NPs) loaded with sorafenib (SF) and modified with the hepatocellular carcinoma (HCC)-specific targeting peptide SP94 and the near-infrared cyanamide dye (Cy 5.5) ( Figure 7A). In the treatment of HCC, the combination of PTT and SF could effectively reduce local tumor recurrence and the side effects caused by drugs. In addition, the POD-like activity and photothermal effect of PB could effectively reshape the hypoxic and immunosuppressive TME. When combined with anti-programmed death ligand 1 (PD-L1), immunotherapy can promote dendritic cell (DC) maturation and increase tumor infiltration of cytotoxic T lymphocytes (CTLs). More importantly, combination therapy could establish long-term immune memory and inhibit tumor metastasis and recurrence.
Excess H 2 O 2 in tumors can not only be used as a raw material for oxygen generation but also generate highly toxic ROS through the Fenton reaction, which can effectively kill tumor cells in combination with PTT. Tian et al. (2022) obtained an NIRresponsive therapeutic nanoplatform (GA-PB@MON@LA) by sequentially introducing PB and gambogic acid (GA) into the pores of mesoporous organosilicon (MONs) and coating them with the thermosensitive material lauric acid (LA) ( Figure 7B). Under NIR laser irradiation, the nanoplatform could not only induce tumor cell apoptosis by PTT but also shed LA coatings, thus facilitating the release of GA. GA inhibited the expression of HSP90 and further suppressed tumor heat resistance. In addition, the heat generated by PTT could enhance the CAT-like and Fentonlike catalytic activities of PB, promoting the production of oxygen and •OH. The in vitro and in vivo experimental results showed that GA-PB@MON@LA has good antitumour effects and can be used as a PA/MR dual-modality imaging contrast agent to provide precise guidance for cancer treatment. To minimize the effect of reticuloendothelial system clearance, nanomaterial biomineralization is an effective strategy. Cytomembranes have homology targeting and good biocompatibility, which can protect nanomaterials from immune recognition of the body to prolong the  Figure 7C). With the homing action of macrophage membranes, the nanoplatform was able to achieve enrichment in tumor tissue. The TK membrane then catalyzed the generation of oxygen from high concentrations of H 2 O 2 in the TME. During the process, the generation of oxygen promoted the rupture of the hybridized membrane, exposing Man-HMPB/HCQ. Man-HMPB/HCQ significantly enhanced macrophage internalization and induced polarization of M2 macrophages toward the M1 phenotype. In vivo results showed that the nanoplatform significantly inhibited tumor growth through a series of responses, including TAM polarization, CTL infiltration, alleviation of hypoxia and reduction in regulatory T cells.
2.3.3 Nanomaterials with self-oxygen production performance 2.3.3.1 Photocatalytic oxygen-producing nanomaterials Photocatalytic water splitting is a clean, simple and sustainable pathway for producing O 2 and hydrogen, which has led to great achievements in the fields of the environment, energy and biomedicine (Liao et al., 2020;An et al., 2021;Teng et al., 2021). Carbon nitride (C 3 N 4 ) has been applied as a nonmetallic photocatalyst for the photolysis of water in tumors to produce oxygen due to its visible light response and non-toxicity (Liu et al., 2020b;Ma et al., 2022). This endogenous oxygen production can enhance aerobic-based therapy. A cascade catalyst (PCMGH) was designed and obtained by assembling dopamine with C 3 N 4 nanosheets, coating the surface with an iron-based metal-organic framework [MIL-100(Fe)], loading GOx, and grafting hyaluronic acid ( Figure 8A) (Yu et al., 2022). A 630 nm laser could activate C 3 N 4 -mediated water splitting to generate oxygen in the tumor. Sufficient O 2 further promoted GOx to consume endogenous glucose and generate the byproduct H 2 O 2 , and finally, MIL-100(Fe) with Fenton-like activity catalyzed H 2 O 2 to generate •OH. During the cascade reaction, 808 nm NIR could elevate the reaction temperature of the tumor and enhance the catalytic performance to obtain more •OH. The efficient targeting ability of hyaluronic acid and the tumor environment response mechanism enable cascade catalysts to have both excellent biosafety and tumor efficacy. However, the disadvantages of C 3 N 4 in complex physiological environments, such as the rapid recombination of electronhole pairs and weak light absorption properties, limit its further application in biomedicine (Chen et al., 2022c). In this regard, researchers have designed ingenious modification methods to enhance the photocatalytic ability of C 3 N 4 and effectively alleviate the endogenous hypoxia of tumors. Wei et al. (2021) Zhang et al. (2022e) synthesized a heterojunction (UCNPs-C 3 N 4 -Ce6) by wrapping UCNPs with C 3 N 4 and modifying with Ce6 and COOH-FA-PEG, which prevented the recombination of C 3 N 4 electron-hole pairs, thereby enhancing the photoelectric conversion efficiency of nanomaterials ( Figure 8B). UCNPs-C 3 N 4 -Ce6 with specific recognition ability increased tumor enrichment, and ultrasound (US) and NIR simultaneously stimulated the heterojunction to generate oxygen and ROS at the C 3 N 4 interface, realizing the combined treatment of PDT, SDT, and PTT. The results of in vivo experiments showed that US + NIR-stimulated UCNPs-C 3 N 4 -Ce6 significantly upregulated the level of LC-3 protein in tumors, activated the autophagy pathway, and had good tumor elimination effects. Overall, heterostructure construction and elemental doping can improve the stability and photocatalytic activity of C 3 N 4 in vivo, which is expected to modulate and remodel the hypoxic TME and reduce tumor metastasis and recurrence Chen et al., 2021c).

Microalgae-based oxygen generator
Microalgae produce oxygen by their specific photosynthesis, which has been applied for efficient oxygen production in tumors in situ to alleviate hypoxia and enhance oxygen-dependent therapies such as radiation therapy (RT) and PDT (Li et al., 2020d;Chen et al., 2021d;Zhong et al., 2021). Wang et al. (2022b) designed photosynthetic microcapsules (PMCs) by encapsulating UCNPs and cyanobacteria in an alginate microcapsule. The system achieved long-term oxygen supply by photosynthesis of cyanobacteria. PMCs could inhibit the NF-kβ pathway and downregulate the expression of HIF-1α, which created a hyperoxic microenvironment in vivo and significantly inhibited tumor growth and metastasis in hepatocellular carcinoma and mammary tumors. PMCs combined with checkpoint inhibitors (anti-PD-1) showed a powerful synergistic effect in mice with breast cancer. PMCs were intratumorally injected and had high tumor penetration. However, intravenously injected microalgae nanosystems are easily captured by the mononuclear phagocytic system (MPS)  Figure 9A) (Qiao et al., 2020). RBCM-Algae was delivered to tumors to produce oxygen in situ under red light-induced photosynthesis and improve RT. The 650 nm laser could release chlorophyll from microalgae, which further enhanced the ability to kill cancer cells by generating ROS through PDT. The rational combination of Fe 3 O 4 NPs can further improve the precise delivery of microalgae to tumor tissues. Wang et al. (2022c) obtained an intelligent robot (Volbot) by combining volvox algae with Ce6-polydopamine@Fe 3 O 4 through electrostatic interactions to achieve multimode imaging and oxygen generation ( Figure 9B). Under the control of a magnetic field, the Volbot could move on a planned route. Red light (650 nm) irradiation could enhance the motion behavior of the Volbot and boost the mixing of biological fluids, facilitate the production of oxygen and improve the effect of PDT. In addition, Volbot could absorb NIR laser to generate localized thermotherapy to treat tumors. Volbots with magnetic resonance, photoacoustic, and fluorescence multimodal imaging offer great potential for achieving precise tumor treatment. In another work, Zhong et al. (2020) prepared tumor-targeted biohybrid microswimmers (PBNs) by combining Spirulina platensis with magnetic Fe 3 O 4 NPs. The PBNs could be enriched in tumors using the guidance of magnetic fields, and oxygen production through photosynthesis could effectively alleviate hypoxia and improve the effectiveness of RT. In addition, chlorophyll can also generate ROS to enable PDT under laser irradiation. However, the application of microalgae in biomedicine is still in the preliminary stage, and some key issues remain to be solved, such as a laser selected for activating photosynthesis, microalgae size and morphology, targeting ability, biosafety, etc. Wang et al., 2021b).
Thylakoids, distributed in the chloroplast stroma and cyanobacteria cells, are small flat capsules surrounded by a single-layer membrane. The thylakoid membrane contains photosynthetic pigments and electron transport chain components, which can convert light energy into chemical energy (Zheng et al., 2018;Cheng et al., 2021). For example, Zheng et al. (2018) constructed a phototriggered nonbiological/biological thylakoid nanooxygen delivery system (PLANT) by coating the thylakoid membrane on the surface of synthetic nanoparticles, such as ZnONPs, Ag NPs, or SiO 2 NPs. The delivery system can efficiently generate oxygen in the tumor under 660 nm laser irradiation, and in vivo and in vitro results showed that PLANT could reverse tumor hypoxia, inhibit anaerobic respiration, recover the metabolism of tumor cells to normal, regulate the abnormal structure and function of tumor blood vessels, limit the migration of tumor cells and remarkably improve the curative effect of PDT or Frontiers in Pharmacology frontiersin.org antiangiogenesis treatment. In vivo results showed that, after treatment with PLANT, the levels of glycolysis-related enzymes (e.g., GLUT-1) in tumors were downregulated, and the CD31 protein and HIF-α were also significantly reduced.

Metal peroxide-based oxygen generator
Metal peroxides can decompose to produce H 2 O 2 and metal ions under the acidic TME . H 2 O 2 has a wide range of applications in tumor therapy, such as increasing oxidative stress, providing oxygen, and providing catalytic substrate (Fu et al., 2018;Fu et al., 2021). Motivated by this, a combined nanotherapeutic (DCI) was synthesized by coloading CuO 2 and ICG with dendritic mesoporous organosilica (DMOS) as a carrier and modifying with hyaluronic acid ( Figure 10A) (Wang et al., 2022d). DCI can produce oxygen and H 2 O 2 under acidic conditions to alleviate hypoxia. The 1 O 2 produced by NIR photoexcitation of ICG and the •OH produced by Cu 2+mediated Fenton-like reaction lead to the death of tumor cells. However, the limited efficiency of the Fenton-like reaction greatly affects metal peroxides for chemodynamic therapy (Wang et al., 2020b). Therefore, developing multicomponent catalytic metal composites through synergistic catalytic effects can achieve more satisfying catalytic performance . Koo et al. (2022) reported a nanomaterial composite (CFp NPs) based on Cu-Fe perovskite nanoparticles ( Figure 10B). The results showed that the CFp NPs synthesized with a Cu/Fe ratio of 7: 3 had the best catalytic performance and could successfully ablate tumors at a low dose of 3.7 mg/kg. At the same time, CFp NPs alleviated tumor hypoxia by TME-responsive oxygen generation ability. The responsive release of ferric ions could also enhance T1weighted MRI, enabling in vivo monitoring of tumors. Furthermore, metal ions dissociated by metal peroxides are also able to enhance the efficacy of tumor therapy. For example, Ca 2+ released from CaO 2 could induce calcium overload and cause mitochondrial damage ; Ba 2+ produced by BaO 2 could act as a potassium ion channel inhibitor and inhibit tumor cell proliferation . In addition, Chen et al. (2022a) designed a multifunctional nanoscavenger (ECMT NSs) by self-assembly of CAT, the digestive enzymes chymotrypsin (CHY), calcium peroxide nanoparticles (CaP), and the photosensitizer Ce6 and modified it with fibronectin-targeting CLT1 peptide ( Figure 10C). Upon reaching solid tumors, the synergistic effect of CHY and ROS could effectively destroy the tumor matrix and facilitate the penetration of ECMT NSs. CaP could generate large amounts of Ca 2+ and H 2 O 2 in the acidic TME, which facilitated calcium ion therapy. Meanwhile, the generated H 2 O 2 could be converted to oxygen in the presence of CAT, thus favoring the remodelling of the tumor hypoxic environment. The cooperation of calcium ion therapy and PDT could promote apoptosis and immunogenic cell death of tumor cells, which induce the activation of CTLs, thus reversing the immunosuppressive environment.

Conclusion and future perspectives
Hypoxia is one of the characteristics of the TME, and there is no linear relationship between tumor size and the degree of hypoxia, which may be present even if the tumor diameter is less than 1 mm (Li et al., 2007). Hypoxia leads to high invasion and metastasis of cancer cells, further making matters worse. In this paper, we review the advances in nanomaterial-based approaches to enhance the efficacy of various oxygen-related antitumour therapies by improving the TME by oxygen delivery and generation. Nanomaterials have achieved desirable results in enhancing tumor therapy. In clinical trials of hypoxic tumor treatment, a variety of personalized drugs have been developed (Table 3). NVX-108, the oxygen carrier of the second generation PFC,  (2016) Frontiers in Pharmacology frontiersin.org exhibits significantly higher oxygen delivery capacity, lower recommended dose, and fewer side effects than Flusol-DA with respect to direct oxygen delivery. At the same time, polyhedral hemoglobin has been further explored and practiced in clinical practice. Currently, a variety of drugs targeting hypoxia-related signaling pathways have been approved, but the results of various clinical trials are far from being as expected. Therefore, many critical issues must be carefully elaborated. First, nanomaterials themselves lack active targeting and only take advantage of the EPR of tumors. However, EPR is a controversial topic in nanomedicine, and its reliability has been doubted by many researchers (Nichols and Bae, 2014). However, the accumulation of nanotherapeutics in the tumor site after intravenous injection is still limited even by the EPR effect, and most nanotherapeutics are mainly distributed in various organs Dai et al., 2022). In addition, due to multiple factors, such as the high interstitial fluid pressure of the tumor and dense intercellular substances, most nanotherapeutics reaching the tumor site are blocked around the tumor and cannot penetrate into the tumor, especially vascular insufficiency at the hypoxia site . Therefore, nanotherapeutics should be carefully designed to have highly efficient tumor enrichment and deep penetration abilities.
Second, the biocompatibility of nanotherapeutics for alleviating tumor hypoxia in animals needs to be studied in depth. Metal-based nanoplatforms such as manganese, cerium, copper, and iron are used for oxygen delivery and generation, and non-specific accumulation in the body may be potentially toxic to normal tissues and organs (Lin et al., 2021). Although short-term (≤30 days) toxicity assessments of these nanoplatforms have been reported in most literature; however, long-term toxicity (≥6 months) still needs to be evaluated for future clinical transformation. In addition, due to the complex and variable physiological environment, nanotherapeutics may release oxygen prematurely in the circulation, which may lead to cytotoxicity to other organs. Therefore, it is important to develop nanomaterials with controlled release and monitor their biosafety. Therefore, it is of great significance to develop nanoplatforms with good biological safety and controllable oxygen release.
Third, the design of nanoscale oxygen-carrying platforms is considered suitable for clinical transformation. At present, oxygen delivery is mainly used as the source of ROS production in various oxygen-related treatments. How much oxygen can be carried by the nanoplatform, how much oxygen can be supplied for the tumor, and how long its oxygen supply can last all need to be considered. Therefore, when designing nanomaterials, it is necessary to consider the oxygen supply level, oxygen retention time and oxygen consumption required for treatment in the tumor to obtain an oxygen production/delivery nanoplatform. In addition, the design of facile synthetic nanotherapeutics is conducive to industrial production.
Fourth, nanomaterials should have imaging capabilities that can reflect hypoxia information during tumor treatment. The current polarographic Clark electrodes for measuring the partial pressure of oxygen have technical limitations, are invasive, and cannot be used for the clinical evaluation of tumor hypoxia. Immunostaining of hypoxia markers in tumor tissue only shows the hypoxic area of the tumor and does not provide accurate quantitative information in real time. The combination of imaging technologies such as CT, MRI, and positron emission computed tomography (PET) can fill the gap in tumor hypoxia assessment (Liu et al., 2017b). For example, hypoxia-sensitive fluorescent nanoprobes can be used for hypoxia monitoring. Furthermore, the combined use of these imaging modalities may provide additional valuable information about tumor physiology. Therefore, the development of nanoplatforms with hypoxia relief and imaging abilities is a future trend.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.