Recent advances in in situ oxygen-generating and oxygen-replenishing strategies for hypoxic-enhanced photodynamic therapy

Abstract

Cancer is a leading cause of death worldwide, accounting for an estimated 10 million deaths by 2020. Over the decades, various strategies for tumor therapy have been developed and evaluated. Photodynamic therapy (PDT) has attracted increasing attention due to its unique characteristics, including low systemic toxicity and minimally invasive nature. Despite the excellent clinical promise of PDT, hypoxia is still the Achilles’ heel associated with its oxygen-dependent nature related to increased tumor proliferation, angiogenesis, and distant metastases. Moreover, PDT-mediated oxygen consumption further exacerbates the hypoxia condition, which will eventually lead to the poor effect of drug treatment and resistance and irreversible tumor metastasis, even limiting its effective application in the treatment of hypoxic tumors. Hypoxia, with increased oxygen consumption, may occur in acute and chronic hypoxia conditions in developing tumors. Tumor cells farther away from the capillaries have much lower oxygen levels than cells in adjacent areas. However, it is difficult to change the tumor's deep hypoxia state through different ways to reduce the tumor tissue's oxygen consumption. Therefore, it will become more difficult to cure malignant tumors completely. In recent years, numerous investigations have focused on improving PDT therapy's efficacy by providing molecular oxygen directly or indirectly to tumor tissues. In this review, different molecular oxygen supplementation methods are summarized to alleviate tumor hypoxia from the innovative perspective of using supplemental oxygen. Besides, the existing problems, future prospects and potential challenges of this strategy are also discussed.

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1. Introduction

PDT is a promising modality for cancer treatment.¹ It uses a photosensitizer in the presence of oxygen to ablate cancer cells by generating reactive oxygen (ROS) at a specific wavelength of irradiation.² ROS species include singlet oxygen ¹O₂ (Type II PDT) and superoxide anion radical (O₂˙⁻), hydroxyl radical (HO˙), and hydrogen peroxide (H₂O₂) (Type I PDT).^3,4 The two photochemical pathways for the photo-inducement of photosensitizers are classified as type I and type II. In most cases, the effect is achieved through a type II mechanism involving the conversion of triplet ground molecular oxygen (³O₂) to its highly reactive singlet oxygen (¹O₂) analog.^3,5 However, the treatment method of type I PDT has also been reported in recent years.^6–9 This is the direct transfer of electrons or hydrogen atoms between photosensitizer (PS) and substrate molecules, usually resulting in the production of free radicals.^10,11 This process does not involve a large amount of O₂ consumption, and the fluorophore is a phenothiazine derivative.¹² Peng's research group has conducted a study on photodynamic therapy to improve hypoxia by using this mechanism.^11,12 The difference between the two different types of photodynamic therapy lies in whether the therapy process is oxygen-dependent, and the mechanism of the two treatment modes is shown in Fig. 1a. Most of the existing PDT systems (Type II) are highly dependent on O₂ and consume a large amount of O₂ during treatment.¹³ The critical problem affecting the result of PDT is that the tumor's hypoxia and oxygen consumption during treatment will aggravate the degree of hypoxia,¹⁴ thus making the treatment effect worse.^15,16 In solid tumors, O₂ concentration varies with location, and oxygen levels in some internal regions are very low (partial pressure of O₂ < 2.5 mmHg, severe tumor hypoxia),^17,18 contributing to the limited effectiveness of drug (doxorubicin) treatment,¹⁹ simultaneously due to the disruption of vascular growth factor balance, tumor invasiveness and resistance. To make matters worse, hypoxia in tumor tissue increases genetic instability, tumor progression, and metastasis-related problems.^20,21 while inhibiting the tumor response to cytotoxicity and targeted therapy; hypoxia is present in most cancer types,^22,23 common ones include pancreatic, breast, lung and prostate cancer. In recent years, many efforts have been made to defeat the photodynamic disturbance caused by hypoxia; among them, the method of increasing the O₂ supply of cancer tissues to treat cancer has also been proposed.^17,24,25 The core factor why anticancer drugs can hardly reach the tumor site is the separation of hypoxic tumor cells from blood.²⁶ In this case, only a few given drugs can finally get to hypoxic tumors due to the hypoxia of the microenvironment, the decrease in drug reactivity and the decrease in affinity with DNA molecules, which are the Achilles’ heel of the treatment process.^27,28 The hyperbaric oxygen (HBO) treatment method has been proposed and related studies have been carried out.²⁹ Pure oxygen is provided to patients in a pressurized chamber, breathing hyperbaric oxygen increases the pressure of oxygen, and when breathing, the oxygen content increases from 20% (50 Torr) to 100% (150–350 Torr) at 1 atm, which can advantageously reduce the hypoxia in tumor tissue and increase the sensitivity of tumor cells to drugs, increasing the curative effect in the treatment of tumor. The direct transport of oxygen molecules has been achieved using perfluorocarbon (PFC) materials approved by the FDA, such as C₅F₁₂. However, the problems of oxygen leakage and a frequent dose of tumor oxygenation strategy based on PFC have not been solved until now, which worsens the results of photodynamic therapy. Great achievements have been made in the study of anti-hypoxia-mediated tumor PDT; commonly used strategies of O₂ supplementation include using organic or inorganic catalysts to catalyze endogenous H₂O₂,^30–32 water splitting,^33,34 oxygen transportation,³⁵ PFC, and respiratory inhibitors,^36,37 and the primary purpose is to improve the therapeutic effect of PDT by relieving hypoxia.^38–41 So far, the primary methods are divided into two categories, namely: (1) improve the tumor microenvironment and relieve hypoxia; and (2) improve tumor hypoxia through various oxygen delivery carriers, for example, nanoparticles designed according to different mechanisms for tumor oxygenation and enhanced cancer therapy, including fluorocarbons, hemoglobin, and oxygen-carrying nano-platforms.

Fig. 1 Scheme illustration of the photochemical reactions for type I and type II PDT. (b) Schematic diagrams of the novel strategies to significantly promote the efficiency of PDT by relieving tumor hypoxia in different ways.

2. Improve the tumor microenvironment and relieve hypoxia

2.1 Photosynthesis produces O₂

In the emergence and evolution of life on Earth, the primitive photosynthesis of microorganisms such as cyanobacteria and Archaea has been essential for the generation of molecular oxygen.⁴² Photosynthetic cyanobacteria are a kind of prokaryote that can grow with solar energy and CO₂ as the only energy and carbon source.⁴³ Microalgae are naturally occurring single-celled microorganisms capable of photosynthesis that are used for biofuel and nutrition applications and animal feed, and as myocardial oxygenation factors for tissue-healing myocardial repair.⁴⁴ It has been reported that engineered living microalgae are transported to anoxic tumor areas to increase local oxygen levels and re-sensitize drug-resistant cancer cells to radiation and light, and heat. Through the study, we found that the oxygen produced in situ through photosynthesis mediated by microalgae significantly improves the anoxic environment in the tumor, resulting in a significant effect of radiotherapy, and many literature studies have reported that Chlorella contains a variety of immunostimulatory substances, which can be used for the immunotherapy of tumors at the same time.^45–47 Oxygen production mainly depends on the active oxygen produced by the chlorophyll of microalgae during laser irradiation, which further enhances photosensitivity and tumor cell apoptosis. It is an innovative treatment strategy for photosynthesis to produce oxygen to alleviate tumor hypoxia.

The tumor's hypoxic environment limits the cytotoxicity of many chemotherapeutic agents by blocking the ROS that are produced within the cell and leads to the inevitable recurrence and metastasis of cancer. Therefore, hypoxic tumors are rarely cured, but hypoxia in tumor tissues has been explored. Microbiology has provided some important reference value for researchers in agriculture, industry and public health research fields. As early as the late 19^th century, W. Busch and W. Coley began trying to treat tumors with microorganisms.⁴⁸ They treated sarcoma patients independently with Streptococcus pyogenes and Serratia marcescens and observed significant improvements in malignant tumors after treatment. This application is more promising because bacteria have excellent tumor targeting ability and are easy to obtain.⁴⁹ In recent years, masses of studies have used these characteristics of bacteria to target tumor tissue, activate the nano-system in different ways in situ, and finally achieve drug release or combination therapy and accurate cancer treatment^44,50

Against this background, Shi and collaborators have reported a chlorin e6 (Ce6)-integrated photosensitive cell.⁵¹ This was established by the modification of cyanobacteria (producing O₂ by photosynthesis) with the photosensitizer Ce6. With the irradiation of a 660 nm laser, photosynthesis produces O₂, which is directly used by the PDT process and increases the PDT effect (Fig. 2a). In addition to the application of Ce6 photosensitizer, the study was also extended to another negatively charged chlorin-based photosensitizer, protoporphyrin (Ppix), to construct Ppix hybrid cyanobacteria cells (Ppix-Cyan). With free Ce6, with different laser power densities (10, 20, and 30 mW cm⁻²), the change of dissolved oxygen (DO) in solution fluctuates over the range of 10 mmol⁻¹, indicating the dynamic equilibrium of oxygen dissolution–evaporation. Hybrid bacteria ceCyan can increase the DO to 24.7 μmol L⁻¹ after 10 minutes of 20 mW cm⁻² excitation power, and on increasing this to 30 mW cm⁻², DO significantly increased by 2.34 times (compared with the lower power of 20 mW cm⁻²). This indicated that cyanobacteria had high photosynthetic and oxygenation performance (Fig. 2b). When using the free radical trapping agent 2,2,6,6-tetramethylpiperidine (TEMP) to verify the free radicals’ production, cyanobacteria were added, and the free radicals increased gradually with the increase of concentration, which further indicated that O₂ supplementation would increase the production of ROS in the process of PDT. The promoting effect of oxygen mitigation on ROS was also explored, and a Singlet Oxygen Sensor Green (SOSG) probe was used to observe the production of ¹O₂ in cells. After cyanobacteria, free Ce6 photosensitizer and ceCyan cells were co-incubated with the same intensity and time of light exposure and were observed by confocal fluorescence microscope; bright fluorescence of SOSG could only be observed in ceCyan-PDT treated cells, which indicated that significant ¹O₂ was produced by cascade photosynthesis and photodynamics. The hypoxia indicator [Ru(dpp)₃]Cl₂ was also used to further verify that the hybrid bacteria irradiated by laser changed the state of hypoxia (Fig. 2c). The oxygenation effect on tumor in vivo was studied by photoacoustic (PA) imaging accompanied by B-mode ultrasound (US) imaging using oxygen saturation mode (_SO₂), showing the continuous effect of oxygen production in vivo (Fig. 2d).

Fig. 2 (a) Schematic diagram of the photodynamic process of photosynthesis enhancement. (b) After 660 nm laser irradiation for 10 minutes, the DO level changes (with or without cyanobacteria). (c) A hypoxia-sensitive probe and ROS indicator probe indicate the degree of hypoxia and ROS production of 4T1 cells after different treatments. (d) US and PA imaging and their combined images were performed on the tumor sections after intracranial injection of cyanobacteria and subsequent laser irradiation for tumor oxidation in vivo. Reproduced from ref. 51 with permission from Wiley, copyright 2019.

Gold nanorods (AuNRs) showed excellent performance in expanding the tumor vasculature; after continuously irradiating with an NIR laser, the local temperature would increase to 42 °C,⁵² and thus the results of this can be utilized for promoting the release of oxygen-dependent drugs in hypoxic tumors.⁵³ Similarly, a Chlorella AuNRs BSA-Gel was developed by Yu Seok Youn's research group;⁵⁴ this is a BSA-PEG-based hydrogel library system containing Chlorella and gold nanorods. AuNRs are an attractive material, as high temperature can cause irreversible damage to tumor cells.^55,56 Chlorella can produce oxygen in local tumor tissue through photosynthesis under 808 nm excitation, without increasing whole-body oxygen content. The tumor will be ablated, together with using the drug doxorubicin after relieving tumor hypoxia. CT imaging of the tumor tissue with Chlorella AuNRsBSA-Gel was undertaken.

In contrast to the PBS control group, the imaging ability of this system was good. The photosynthetic oxygen-production capacity of AuNRs BSA-Gel and the control group with or without 660 nm laser irradiation was evaluated. The data show its superior performance in oxygen production. The intracellular ROS production level of each group of 4T1 cells was observed by CLSM under normoxic and anoxic conditions. It was also proved that drug treatment with Dox was needed to produce ROS under the condition of oxygen to realize the ablation of tumor cells. The study on the anti-tumor effect of Chlorella AuNRs BSA-Gel on mice transplanted with 4T1 cells showed that the production of oxygen in the presence of Chlorella could promote tumor ablation.

Through Chlorella photosynthesis, the oxygen generation strategy can be combined into different nano-systems, effectively providing oxygen to hypoxic tumor cells.^57,58 Both chemotherapy drugs and photodynamic therapy have high application expectations in treating hypoxic tumors.⁵⁹ Jiang's research group designed an autotrophic light-triggered green oxygen supply machine (ALGAE),⁶⁰ which was constituted of Chlorella pyrenoidosa and alginate by calcium cross-linking. The photodynamic oxygen generation ability induced by the algal system is different. After verification, they found that the oxygen production and repetitive oxygen production of Chlorella under irradiation conditions were better than in the dark. When Ce6 is irradiated by a 635 nm laser, the energy is transferred from Ce6 to the supplementary oxygen produced by the algae system, creating more singlet oxygen. Oxygen produced by photosynthesis can alleviate hypoxia during PDT therapy, down-regulate HIF-1α and VEGF expression, and up-regulate the expression of E-cadherin. This is consistent with the previously reported hypoxia-inducible HIF-1α inducing up-regulation of VEGF expression and E-cadherin loss, even with tumor progression and metastasis.^61,62 The wound healing experiment showed the comparison group's cell fusion rate, and those of the routine PDT treatment group, and algae PDT treatment group after 24 hours, and indicated that PDT with algae treatment can better inhibit cell metastasis. Through H&E staining and TUNEL, and in vivo anticancer therapy, we can conclude that oxygen production by photosynthesis, which relied on algae implanted near the tumor tissue, can increase the proportion of apoptosis induced by PDT. The anti-cancer treatment in vivo was studied by using the model of the 4T1 tumor-bearing mouse, and the therapeutic potential of algae was proved again. The active photosynthetic bacteria Syne was also used for PDT.⁵⁰ The photosensitized encapsulated nanoparticles (HSA/ICG) were assembled by intermolecular disulfide cross-linking and linked to the surface of the Syne. Then takes shape a biomimetic system (S/HSA/ICG) with photosynthesis and PDT effects (Fig. 3a). Syne is used as the targeted photosensitizer promoting the delivery of ICG to the tumor (FL images of the mice up to 72 h) and photocatalytic oxygen production in situ. It is verified by PA imaging that Syne can achieve an anti-tumor immune response with 660 nm laser irradiation through mediation of O₂ self-sufficient PDT and immunogenic cell death (ICD). And, the temperature of S/HSA/ICG irradiated by 808 nm laser is as high as 77.8 °C. The synergistic effect of PDT/PTT can induce 84% cell death, achieve good bactericidal ability, and affect the viability and growth of bacteria to a great extent. After intravenous injection of S/HSA/ICG into mice, it accumulates effectively under laser irradiation through photosynthesis, continuously produces oxygen, significantly relieves tumor hypoxia, and enhances active oxygen production. Thus it achieved complete ablation of triple-negative breast cancer without primary tumor metastasis, and there was no tumor recurrence after 24 days of continuous treatment.

Fig. 3 (a) Schematic diagram of the photosensitizer (HSA/ICG) conjugated with Syne as an in situ photocatalytic oxygen generation system for PDT. Syne and HAS have strong tumor-targeting ability. Syne accumulates in the tumor tissue and produces oxygen under laser irradiation, enhancing the efficacy of PDT therapy and reversing the tumor immunosuppressive microenvironment. In the 4T1 MTNBC mouse model, Syne (S/HSA/ICG NP coated) can synergistically inhibit local and metastatic tumors. Reproduced from ref. 50 with permission from Wiley, copyright 2020. (b) During tumor hypoxia, laser irradiation produces oxygen through photosynthesis, and oxygen collection is used for sustainable PDT. Reproduced from ref. 63 with permission from Elsevier, copyright 2020.

Similarly, one of the advantages of PDT therapy is that it promotes an anti-tumor immune response and sustained oxygen release. A self-enriched oxygen photodynamic therapy (Oxy-PDT) was established through loading a photosensitizer into PFC nanodroplets (Fig. 3b).⁶³ This is a novel, long-lasting PDT that is controlled by light and enables photosynthesis to produce oxygen. With the high oxygen-carrying capacity of PFC, a high oxygen content can be sustained. Therefore, although the tumor oxygen content is still limited in the PDT process, sufficient O₂ could be enriched in perfluorinated carbon nanodroplets for consumption by loaded photosensitizers. Thus, by improving the photodynamic efficiency, persistent PDT also promoted the activation of DCs and enhanced the anti-tumor immunity. After stopping the light, Chlorella further served as an adjuvant to promote (DC) activation in dendritic cells and promoted the anti-tumor immune response. Moreover, ¹O₂ exists much longer in PFC than in the cellular environment or water. Therefore, Oxy-PDT can help photosensitizers reach their full therapeutic potential. In vivo studies have revealed that tumor growth in Oxy-PDT-treated mice was inhibited after Oxy-PDT was injected into the tumor. Besides, when Oxy-PDT was injected intravenously into tumor-bearing mice, the treatment results showed that it could significantly inhibit tumor growth. At the same time, normal PDT didn't get such impressive results.

Due to the use of chlorella, the tumor tissues of mice need to be dissected before the chlorella can be implanted into the tumor tissues, and after the implantation, the chlorella needs to be sutured. Therefore, Zhang's team developed a new strategy, which relies on an injectable hydrogel (ALG-MI-S2973) to achieve the same photosynthetic function to produce oxygen, and the oxygen pump based on S.2973 is compounded with silica nanoparticles loaded with ICG.⁶⁴ After evaluating the effect of PDT, they found that PDT is feasible. This novel strategy of photo-oxygen-dynamic therapy (PODT) combined a light-driven biological oxygen pump and PDT for the rapid growth of Synechococcus elongatus UTEX 2973. This method reduces the damage to the body. Still, the biggest advantage is that the treatment process becomes fast and convenient and is expected to be used in clinic by its hypoxia relieving properties. Besides, most of the previous studies used the same excitation wavelength for photosynthesis and the PDT trigger. Zhang's team creatively separated the growth of S.2973 and the stimulus of PDT through disparate wavelengths of laser, avoiding the damaging effect of ROS on S.2973 induced by PDT in the early stages of treatment. Hence, a result of maximum oxygen production can be ensured. The excellent oxygen production of ALG-MI-S2973 can significantly reduce HIF-1α under the action of an oxygen pump, and the inhibition rate of 4T1 tumor cells is almost 100%.

Cyanobacteria are prokaryotes with a highly differentiated membrane system capable of photosynthesis. They have thylakoid membranes that perform fully functional photosynthetic and respiratory electron transport chain functions. The existence of different membrane systems gives a unique complexity to the cells of these bacteria. The efficient O₂ production of chloroplasts is attributed to their delicate Z-type structure, which is composed of two different light systems (PS-I and PS-II) that located on the chloroplast thylakoid membrane (TM).⁶⁵ Yan et al. constructed UCTM NPs by combining thylakoid membrane with upconversion nanoparticles, which was formed by modifying chloroplast thylakoid membrane with upconversion nanoparticles to release oxygen to promote the production of ROS.⁶⁶ Under the irradiation by a 980 nm laser, UCNPs can emit red light to activate PS-I and PS-II of the TM to promote the production of oxygen in water. In vivo experiments have proved that UCTM NPs have excellent biocompatibility, and 980 nm laser irradiation in PDT can effectively remove anoxic tumors in mice. Also, the publications about photosynthesis to produce oxygen were summaried in Table 1.

Table 1 Summary of publications about photosynthesis to produce oxygen

Application	O2 source	Excitation requirements (excitation wavelength, power density, time)	ROS species	PSs	Cyanobacterial cells	In vitro (cells)	In vivo (tumor-bearing mice)	DO	Ref.
Abbreviations: chlorin e6 (Ce6); Synechococcus elongatus PCC 7942 strain (S. elongatus PCC 7942); Synechococcus elongatus UTEX 2973 (S. elongatus UTEX 2973); hypoxia alleviation assessment (HAA), photosystem-I (PS-I) and photosystem-II (PS-II), thylakoid membranes (TM).
PDT	Photosynthesis	In vitro: 660 nm, 20 mW cm^−2, 15 min	^1O2	Ce6	S. elongatus PCC 7942	4T1	4T1	57.9 μmol L^−1 (30 mW cm^−2)	51
		In vivo: 660 nm, 50 mW cm^−2
PDT	Photosynthesis	635 nm, 50 mW cm^−2	^1O2	Ce6	Chlorella	4T1	4T1	∼10.0 mg L^−1 (50 mW cm^−2)	60
PDT	Photosynthesis	In vitro: 660 nm, 0.1 W cm^−2, 5 min	^1O2	ICG	S. elongatus PCC 7942	4T1	4T1 mTNBC	∼6.0 mg L^−1	50
		808 nm, 0.8 W cm^−2, 5 min
		In vivo: 660/808 (5 min), sequential radiation
PDT	Photosynthesis	In vitro: 660 nm, 1 W cm^−2	^1O2	Ce6	Chlorella	CT26	CT26	∼90 μM (170 mW cm^−2)	63
		In vivo: 660 nm, 170 mW cm^−2, 5 min (HAA); 660 nm, 1 W cm^−2, 30 s (PDT)
PDT	Photosynthesis	In vitro: 808 nm, 2 W cm^−2	^1O2	ICG	Cyanobacteria	4T1	4T1	8 mg L^−1 (0.25 W cm^−2)	64
		In vivo: 640 nm, 0.25 W cm^−2, 5 min
		808 nm, 0.25 W cm^−2, 5 min
PDT	Photosynthesis	In vitro: 980 nm, 0.5 W cm^−2, 10 min	^1O2	PS-I, PS-II	TM	3T3, 4T1	4T1	200 µg mL^−1	65
		In vivo: 980 nm, 0.5 W cm^−2, 5 min

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2.2 Improving tumor hypoxia by generating O₂in situ

2.2.1 Catalase catalysis. Hydrogen peroxide (H₂O₂) can be produced in mitochondria and has as a vital role in physiological metabolism. In both normal and tumor tissues, catalase, as an important biological enzyme, lets cells escape from the oxidative damage with its highly efficient catalytic decomposition of H₂O₂. H₂O₂ can be produced in large amounts in tumor tissue (50–100 μM), which is significantly higher than normal tissue and is closely related to the tumor's occurrence and development. A large amount of H₂O₂ will promote cell carcinogenesis and abnormal cell proliferation, angiogenesis, and DNA damage, and promote tumor cell infiltration and metastasis. Therefore, in the photodynamic therapy, the decomposition of H₂O₂ into oxygen in the tumor tissue can improve the local oxygen level and improve the therapeutic effect. This strategy is also used continuously by wrapping catalase and photosensitizer into the nano-system and applying it to the treatment of the tumor.⁶⁷

For example, the polymer vesicle named HC@P1-Vesicle was designed,⁶⁸ and H₂O₂ and polyamide dendrimer (CC-PAMAM) was assembled with ROS responsive triblock copolymers. When different excitation light was used as a trigger for oxygen production and the release of CC-PAMAM, it showed different therapeutic effects. Under irradiation (805 nm), the photothermal treatment effect was triggered, resulting in the thermal decomposition of H₂O₂. Under excitation light irradiation of 660 nm, photoactive CC-PAMAM is allowed to be released from the vesicles to achieve complete ablation of hypoxic, low-osmotic pancreatic tumors, and the therapeutic effect of this method is the best (Fig. 4a). Aiming at tumor therapy, Lei's group used nanoparticles for PDT and PTT. They designed BQ-MIL@cat-MIL nanoparticles based on the metal–organic skeleton (MOF).⁶⁹ By accurately wrapping black phosphorus quantum dots (BQ) and catalase in the inner and outer layers of the MOF, H₂O₂ was converted into O₂ in the stable outer layer of catalase in the MOF. Then O₂ was directly injected into MOF-sensitized BQ (Fig. 5a). A high singlet oxygen quantum yield was obtained, and photodynamic-thermal synergistic therapy was achieved in vitro and in vivo.

Fig. 4 (a) The treatment results of BxPC-3 tumor-bearing BALB/c nude mice with different therapies. Reproduced from ref. 68 with permission from Wiley, copyright 2017. (b) The therapeutic process and mechanism demonstration of multifunctional CMGCC nanoclusters. Reproduced from ref. 72 with permission from the American Chemical Society, copyright 2020.

Fig. 5 (a) A schematic diagram of the stepwise assembly process of BQ and catalase in homologous MOF aims to enhance therapy against hypoxic tumor cells. Reproduced from ref. 69 with permission from Wiley, copyright 2018. (b) Schematic illustration for the preparation of O₂@UiO-66@ICG@RBC and the NIR-triggered O₂ releasing and enhanced PDT mechanism. Reproduced from ref. 70 with permission from Elsevier B.V., copyright 2018. (c) The nanoparticles of PLGA-FA/IR780-H₂O₂, and illustrations of their application for PTT/PDT. Reproduced from ref. 71 with permission from the Royal Society of Chemistry, copyright 2018. (d) A schematic diagram of a nano-catalase system (Cat@PDS) that enhances chemotherapy–photodynamic therapy by decomposing and releasing oxygen through the phospholipid membrane coating. Reproduced from ref. 74 with permission from Elsevier B.V., copyright 2020.

Progress has been made in photodynamic-thermal synergistic therapy both in vivo and in vitro, and it further promotes apoptosis and activates caspase-3. Combining all the merits, it is considered a promising therapeutic platform for the anti-hypoxic tumor microenvironment. With the metal–organic skeleton (MOF), it makes use of its gas adsorption ability to prepare a bionic oxygen evolution PDT nano-platform with long cycle characteristics.⁷⁰ Simultaneously, the photothermal effect of a near-infrared thermo-sensitizer was used to catalyze oxygen production from hydrogen peroxide. UiO-66@ICG@RBC nanoparticles were designed using zirconium-based MOF (UIO-66) as an oxygen storage carrier (Fig. 5b). Afterwards this was coupled with ICG (coordination reaction) and this then continued by coating with the erythrocyte membrane. The photothermal properties of ICG can promote the sudden release of O₂ in UIO-66. Afterward, the production of O₂ was able to improve the therapeutic effect of PDT on hypoxic tumors. The same strategy used triggered photo-thermotherapy to thermally decompose H₂O₂.⁷¹ The nanoparticles (PLGA–FA/IR780–H₂O₂NPs) were constructed from core–shell poly(lactic-co-glycolic acid) nanoparticles (PLGA) and a H₂O₂/poly(vinylpyrrolidone) complex (O₂ source) covalently bound to the targeted unit of folic acid and embedded in a shell IR780 diode (Fig. 5c). In in vitro experiments, the cell survival rate was analyzed by irradiation for one minute (808 nm, 0.5 mW cm⁻²), and the cell viability was reduced by about 80%, indicating the photothermal effect and ROS production ability of this nano-system. The in vivo experiment confirmed that the photothermal effect promoted the thermal decomposition of H₂O₂ and increased the local O₂ concentration of the tumor during photothermal therapy. Simultaneously, a therapeutic experiment in HepG2 tumor-bearing mice further demonstrated the good tumor-therapeutic effect of this nano-system.

The core–shell nano-system has some special advantages; the nanoparticle RC@RFC was constructed that can combat tumor resistance to PDT, change the hypoxia state of the tumor and inhibit the expression of HIF-1α at the same time.⁷³ This nano-system, through self-assembly of hydrophobic rapamycin (RAP) and photosensitizer Ce6, forms the carrier-free double drug and then coats the surface with a layer of the metal–organic skeleton (MOF) to support catalase. The total drug loading reaches 60%, and this nano-system can be passively accumulated in the tumor tissue. For evaluation of the RC@RFC nanoparticles’ effect on tumor growth by activating the nanoparticles’ activity with the help of light irradiation, the therapeutic effect is reflected directly according to the change of tumor volume. Hyper-hypoxic tumors showed a strong effect of PDT. The in situ O₂ production of RC@TFC nanoparticles and the dissolved oxygen level under laser irradiation were analyzed, which showed that RC@TFC showed a concentration dependence and adequate continuous oxygen production capacity. Besides, the nanoparticles of Cat@PDS could target tumor tissue by wrapping catalase nanoparticles in a polyethylene glycol phospholipid membrane (Fig. 5d).⁷⁴ After the process of PDT, the tumor O₂ content was increased, and the expression of HIF-1α decreased, which finally had a remarkable inhibitory effect on tumor growth and led to 97.2% inhibition of lung metastasis. Using the catalase-catalyzed hydrogen peroxide oxygen production strategy, a tumor-targeted bioreactor LIP-IR-CAT was designed by co-loading liposomes with IR780 and catalase.⁷⁵ Based on the high fluorescence quantum yield (up to 0.127) of the IR780 photosensitizer and the specific targeting of mitochondria, the physical and chemical problems of IR780 as a single photosensitizer were solved by using this method of a NIR fluorescence probe loaded on liposomes. Intra-tumor oxygen saturation was monitored by PA imaging. The results show that the oxygen produced by LIP-IR-CAT in the tumor can supplement the O₂ consumption during PDT and provide additional O₂ to relieve tumor hypoxia. At the same time, after the application of this nano-reactor, the PDT effect in tumor-bearing mice was noteworthily stronger than that in the other control groups, and the level of ATP was significantly lower (about 68.8%). The content of glutathione (GSH) in tumor cells is at least 4 times higher than that in normal cells.^76,77 It is worth noting that the GSH in intracellular fluid is also 10–1000 times higher than in extracellular fluid.⁷⁸ Therefore, the efficiency of PDT can be improved by adding MnO₂ to nano-systems capable of targeting tumor tissue by reacting with glutathione. Based on this strategy of consuming intracellular GSH to enhance the therapeutic effect of PDT, a multi-functional nano-platform CMGCC was developed,⁷² which consists of catalase (CAT) and MnO₂ nanoparticles integrated with Ce6-modified ethylene glycol chitosan polymer micelles, in which CAT catalyzes the decomposition of hydrogen peroxide and MnO₂ can consume intracellular GSH, and produce Mn²⁺ for T₁-weighted magnetic resonance imaging (as a contrast agent). In addition, the pH-sensitive surface charge can be switched from neutral to positively charged GC polymer, and this shift can improve the accumulation of PS in the tumor area. The process of collaborative treatment is shown in Fig. 4b. After proving the good PDT performance in vitro, they continued to study the potential of the CMGCC nanoclusters as a T₁-weighted MR imaging contrast agent, and the final in vivo PDT shows that this multi-functional nano-therapy is effective at the level of the therapeutic effect on the tumor. Also, the publications about the decomposition of H₂O₂ by catalase or heat were summaried in Table 2.

Table 2 Summary of publications about the decomposition of H₂O₂ by catalase or heat

Application	Catalyst	O2 source	PSs	Excitation requirements (excitation wavelength, power density, time)	TROS species	In vitro (cell line)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: chlorin e6 (Ce6); cyanine 3 (Cy3); NIR dye (IR780 iodide); phospholipid membrane coated nanocatalase system (Cat@PDS).
PDT	Heat	H2O2	Ce6	805/660 nm, 3/5 min, 1 W cm^−2/0.1 W cm^−2	^1O2	BxPC-3	BxPC-3	68
PDT/PTT	Catalase	H2O2	Cy3	PTT: 808 nm, 1 W cm^−2, 10 min	^1O2	HeLa	HeLa	69
				PDT: 660 nm, 150 mW cm^−2, 3 min
PDT/PTT	None	UiO-66	ICG	In vitro: 808 nm, 0.06 W cm^−2, 5 min	^1O2	RAW264.7 and MCF-7	MCF-7	70
				In vivo: 808 nm, 0.06 W cm^−2, 1 min
PDT/PTT	Heat	H2O2	IR780	In vitro: 808 nm, 0.5 W cm^−2, 30 s	^1O2	HepG2 and EC	HepG2	71
				In vivo: 808 nm, 0.5 W cm^−2, 3 min
PDT	Catalase	H2O2	Ce6	In vitro: 635 nm, 0.75 W cm^−2, 2 min	^1O2	MDA-MB-231	MDA-MB-231	73
				In vivo: 635 nm, 0.75 W cm^−2, 5 min
PDT	Catalase	H2O2	Cat@PDS	In vitro: 655 nm, 1 W cm^−2, 2 min	^1O2	4T1	4T1	74
				In vivo: 655 nm, 1 W cm^−2, 5 min
PDT	Catalase	H2O2	IR780	In vitro: 808 nm, 1 W cm^−2, 20 s	^1O2	MDA-MB-231	MDA-MB-231	75
				In vivo: 808 nm, 1.5 W cm^−2, 20 s
PDT	Catalase	H2O2	Ce6	In vitro: 660 nm, 100 mW cm^−2, 5 min	^1O2	HEK 293 and HeLa	HeLa	72
				In vivo: 660 nm, 100 mW cm^−2, 10 min

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2.2.2 MnO₂ catalysis. As we all know, catalase can decompose H₂O₂ into H₂O and O₂. Other nanomaterials also have a similar effect, such as MnO₂ nanoparticles, platinum and gold nanocluster nanoparticles. They are already used in photodynamic systems.^79,80 Tumor cells use mechanisms that scavenge reactive oxygen species to resist “oxidative therapy”, such as PDT or chemotherapy. Tumor cells showed higher oxidative stress than normal cells.

To improve the abnormal oxidative state of tumor cells and reduce oxidative damage, tumor cells take measures by activating several pathways to clear ROS, including GSH, catalase (CAT), superoxide dismutase (SOD), and vitamins.^81–83 Among them, GSH is the highest antioxidant in cells,⁸⁴ which seriously hinders the efficacy of PDT. However, GSH-mediated drug resistance produces extensive challenges for various treatments,^85,86 so the consumption of GSH content helps improve the therapeutic effect.^87–89 In the tumor microenvironment, when the H₂O₂ met the MnO₂ nanosheets, the MnO₂ was reduced to Mn²⁺ together with the generation of O₂. The process is shown in the following equations:⁹⁰

pH = 5.5

MnO₂ + 2H⁺ → Mn²⁺ + H₂O + 1/2O₂ (1)

MnO₂ + H₂O₂ + 2H⁺ → Mn²⁺ + H₂O + O₂ (2)

pH = 7.4

Combined with the combination of MnO₂ and glucose depletion reaction, a tumor-targeting MnO₂ motor nano-system (MG/HA) was designed,⁹¹ which can effectively consume glucose to inhibit tumor progression and relieve hypoxia, inhibit metabolism by reducing the expression of Glut1, and further inhibit tumor invasion metastasis. In the nanocomposite, after the cancer cells with overexpression of CD44 specifically ingest MG/HA, GOD catalyzes the oxidation of glucose to gluconic acid and H₂O₂. When consuming O₂, it generates at the same time, by the reaction, H₂O₂ and acid, which seems to be a circular process (Fig. 6a). The abnormal expression of Glut1 is related to tumor proliferation, metastasis, and poor prognosis.⁹²In vitro experiments showed that the expression of Glut1 in normal COS-7 cells was lower than that in CT-26 and HeLa cells. There was a highly positive correlation between the expression level of GLT1 and the concentration of glucose. The decrease of sugar content caused by effective glucose consumption in the MG/HA group could significantly inhibit the high expression of Glut1, and the starvation toxicity of MG/HA was considerably higher than that of G/HA. In vivo observation showed that the MG/HA group showed a potent anti-tumor effect during the two-week treatment, showing an apparent O₂ cycle starvation effect. The slight weight gain in all groups indicated that each sample had good biocompatibility. In general, compared with the traditional nano-system, the overexpression of Glut1 in tumor cells can be directly suppressed by combining the MnO₂ motor with the designed MG/HA nano-system, thereby reducing tumor invasion and metastasis, thus further promoting treatment and being used to overcome the obstacle of starvation treatment. Lin's team also developed a nano-system containing MnO₂ to couple photosensitizers (Au25 nanoclusters) with platinum(IV) (Pt(IV)) prodrugs using MnO₂ nanotablets as carriers to achieve synergistic PDT and chemotherapy.⁹³ GSH can bring the content of MnO₂ nano-tablets down. Pt(IV) prodrugs in tumor tissues cooperate with MnO₂ to catalyze H₂O₂ to produce oxygen, increasing the therapeutic effect of PDT. Mn²⁺ can be used as a contrast agent in MRI to realize the integration of diagnosis and treatment.

Fig. 6 (a) The MG/HA nano-system interferes with the up-regulation of Glut1 and explains the process of ablation of tumor cells. Reproduced with permission. Reproduced from ref. 91 with permission from the American Chemical Society, copyright 2018. (b) A schematic diagram of the process of tumor treated with GO/CisPt/Ce6@MH nanoparticle CDT/PDT. (c) The kinetics of O₂ generation catalyzed by GO/CisPt/Ce6 and GO/CisPt/Ce6@MH in presence or absence of 1 mmol/L H₂O₂. Inset: photographs of each reaction tube after reaction. (d) The kinetics of ¹O₂ generation for GO/CisPt/Ce6 or GO/CisPt/Ce6@MH in the presence or absence of 1 mmol/L H₂O₂ under laser irradiation. Reproduced from ref. 97 with permission from Elsevier B.V., copyright 2020.

In recent years, two-dimensional graphene oxide (GO) has been extensively used as a drug carrier for nano-drugs because of its large surface, effective adsorption of aromatic drugs, and wealth of functional groups for drug coupling.^94–96 Therefore, Zhou's team made use of the better carrier function of GO,⁹⁷ and also used the strategy of MnO₂ catalysis to alleviate tumor hypoxia and reduce intracellular glutathione levels (Fig. 6b), to improve the sensitivity of chemo-photodynamic therapy.^94,98,99 A nanometer platform GO/CisPt/Ce6@MH was developed, CisPt was covalently bonded to the edge of the GO by ester bonds, and MnO₂ nanoparticles were further modified on a 2D surface. In in vitro experiments, dissolved oxygen was measured. This nano-platform significantly produces O₂ in the presence of hydrogen peroxide (Fig. 6c and d). The ability of this self-oxygen supply system to enhance PDT is explored. With the SOSG probe for monitoring the ¹O₂, together with H₂O₂, GO/CisPt/Ce6@MH shows a swift rate of ¹O₂ formation, which confirms the advantage of MnO₂ in enhancing PDT therapy. In in vivo tests, BALB/c mice were treated with Ce6 and CisPt (0.5 mg kg⁻¹, 1.25 mg kg⁻¹), respectively. The results showed that synergistic treatment with GO/CisPt/Ce6@MH was very useful.

A new Intelligent Nano platform that was composed of mesoporous organosilicon dioxide nanoparticles (MONS) coated with glucose oxidase (Gox) and MnO₂ nanoparticles-Ce6 was designed and named MONsGOx@MnO₂-Ce6.¹⁰⁰ Gox oxidizes glucose to gluconic acid and produces H₂O₂ at the same time. The released MnO₂ nanowires can regenerate O₂ in the presence of H₂O₂ and react with GSH (as an oxidant) to improve the photodynamic treatment effect. MONS-Gox@MnO₂-Ce6 can realize the imaging of cancer cells and reduce intracellular glucose uptake and Glut1 expression and inhibit the metabolism of cancer cells. The new Nano platform continued to be designed for relieving tumor hypoxia; through assembling a Mn ferromanganese (Mn) nanoparticle, a hollow ferromanganese (Mn) nanoparticle was designed.¹⁰¹ When exposed to a tumor hypoxic microenvironment, the vesicles disintegrate and release drugs and ferromanganese oxide nanoparticles quickly. The ferromanganese oxide can catalyze the excess H₂O₂ to produce oxygen. Combined with immunotherapy, the nanovesicles showed good efficacy in combination with chemotherapy and immunotherapy. Also, the publications about the decomposition of H₂O₂ by MnO₂ were summaried in Table 3.

Table 3 Summary of publication about the decomposition of H₂O₂ by MnO₂

Application	Catalyst	O2 source	PS	Excitation requirements (excitation wavelength, power density, time)	ROS species	In vitro (cell line)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: starving therapy (ST); glucose (GLU); chemotherapy (CT).
ST	MnO2	H2O2, GLU	NO	NO	H2O2	CT-26, COS-7 and HeLa	CT-26	91
PDT/CT	MnO2	H2O2	Au25	In vitro: 650 nm, 5 min	^1O2	HeLa	U14	93
				In vivo: 650 nm, 15 min
PDT	MnO2	H2O2	Ce6	In vitro: 635 nm, 0.1 W cm^−2, 1 min	^1O2	MDA-MB-231	MDA-MB-231	97
				In vivo: 635 nm, 0.1 W cm^−2, 5 min
PDT	MnO2	H2O2	Ce6	660 nm, 7.2 mW cm^−2, 30 min	^1O2	HeLa	NO	100

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2.2.3 Platinum (Pt) NPs. Pt NPs are the most advanced catalyst. In the current research, as a good catalyst, they can efficiently break the O–O bond of H₂O₂ and eventually contribute to the production of oxygen.^102,103

There are fatal shortcomings in the application of metal–organic framework (MOF) integrated photosensitizers in the process of PDT, but Pt-modified MOFs can be used in other treatments (chemotherapy, radiotherapy, sonodynamic therapy), although the poor efficiency of the treatment is also limited by hypoxia.^104,105 The PDT efficiency of MOFs is seriously limited by tumor hypoxia. The PCN-224-Pt nanoparticles were designed, therefore, to solve the above problems by decorating platinum nano-enzymes on the photosensitizer-integrated MOF as a new strategy.¹⁰⁶ In an in vivo experiment, in a subcutaneous tumor model, PCN-224-PT was given by intratumor injection for photodynamic therapy. Under 638 nm excitation light irradiation (1 W cm⁻²) for 10 min, the PCN-224-PT + light group's tumor ablation ability was the most significant compared with the control group after intravenous injection.

A new strategy of in situ growth was applied that decorated black phosphorus (BP) nanosheets with Pt nanoparticles (NPs), forming Pt@BP nanoparticles.¹⁰⁷ As an artificial catalase, Pt@NPs can effectively decompose the accumulated H₂O₂ and relieve tumor hypoxia in the tumor, and then improve the PDT effect of BP nanoparticles. When exposed to tumor hypoxia, BP nanosheets will not cause the decomposition of H₂O₂, while the Pt@BP nano-hydride will cause the deterioration of H₂O₂. The hypoxia environment was simulated in a cell experiment. As a hypoxia indicator probe,^108,109 the fluorescence intensity was decreased significantly when using Pt@BP nanocomposites, which indicated that Pt@BP nanohybrids had high efficiency in regulating intracellular hypoxia. In addition, after Pt@BP nanohybrid treatment, immunohistochemical staining confirmed that HIF-1α was significantly down-regulated, indicating that Pt@BP nanohybrids can also effectively reverse the hypoxia state of the tumor, and inhibit the hypoxia-related signal pathway. Zhang's research group designed and synthesized Pda-Pt@PCN-FA,¹¹⁰ which is composed of a polydopamine (Pda) nucleus, PTNP mesosphere, Zr-TCPP(PCN) porphyrin shell, and Zr6 coordination with a folic acid carboxyl group (Fig. 7a). The platinum nanoparticle intermediate layer can catalyze endogenous H₂O₂ to O₂, which plays a dual role in inhibiting the tumor cells’ growth and promoting death. Under irradiation, oxygen is converted through the PCN shell to lethal ROS. In an in vitro experiment, O₂ was measured by a dissolved oxygen meter, the liquid surface was sealed with liquid paraffin, and the O₂ production reached the maximum (about 50 mg L⁻¹) within 25 minutes after adding H₂O₂. The disodium salt of 9,10-anthracene-dipropionic acid (ADPA) was used as the probe for detecting ¹O₂,¹¹¹ and the hypoxia microenvironment in the tumor was simulated by blowing nitrogen for half an hour. The fluorescence intensity of ADPA decreased rapidly after treatment with Pda-Pt@PCN. Cell experiments were carried out again to check the oxygen-production capacity and ROS-production capacity of Pda-Pt@PCN-FA.

Fig. 7 (a) The simple preparation process of PCN-224-Pt and the schematic diagram of reactive oxygen species produced by PCN-224-Pt to kill tumor cells. Reproduced from ref. 110 with permission from John Wiley & Sons, copyright 2018. (b) Preparation of Pt@BP nano-hybrids and the scheme of tumor cell death caused by a series of reactions under NIR illumination. Reproduced from ref. 112 with permission from the American Chemical Society, copyright 2019.

They confirmed that metastasis's driving force could be caused by an anoxic microenvironment, detected cell movement by a wound healing test, and found that Pda-Pt@PCN-FA could inhibit tumor metastasis by reducing the degree of hypoxia. In in vivo experiments, the imaging and distribution of Pda-Pt@PCN-FA were studied by the small animal imaging system. Fluorescence imaging was executed accompanied by intravenous injection of the nanomaterial, and the fluorescence intensity was most substantial at 28 hours after injection, indicating a good imaging ability. Simultaneously, measurements in animals showed no significant metastatic sites in the lungs of mice injected with Pda-Pt@PCN-FA, regardless of whether radiation was or was not used. In the end, they confirmed that the inhibition of tumor metastasis is caused by the improvement of hypoxia in tumor tissue and explained the correlation between them. For overcoming the poor results of PDT, caused by premature leakage of photosensitizer and hypoxia, FA/PtBSA@MB-MSNS nanocomposites were designed and synthesized.¹¹² Platinum nanoclusters (Pt-BSA) were coated with bovine serum albumin (BSA) with oxygen and combined with mesoporous silica nanospheres to develop intelligent nano-aggregates to achieve a better therapeutic effect on anoxic tumors. Disulfide bond bridged mesoporous silica nanospheres (MSNS) contain methylene blue (MB), with BSA-encapsulated Pt nanoparticles (Pt-BSA) as a biological coating (Fig. 7b). In in vitro experiments, they evaluated the photodynamic conversion efficiency of biological tissue after nano-drug release under light irradiation. Chicken skin has a similar biological barrier effect to human tissue, so they used chicken skin with a thickness of 1 mm to stimulate the production of ¹O₂, which decreased to 72.15% after being irradiated by 635 nm laser (6 mW cm⁻²), with DPBF in FA/PtBSA@MB-MSNS aqueous solution for 120 s. This result confirmed that FA/PtBSA@MB-MSNS could produce ¹O₂ under the skin, highlighting its potential to kill cancer cells in tumor therapy. In an in vivo experiment, the ablation ability of PDT towards tumor cells in vivo was studied by using the cancer cell (HeLa) tumor model. Different treatments were used to track tumor volume change, and the therapeutic effect was by PDT treatment. Both the MB and MB-MSNS treatment groups slightly hindered tumor growth, while the PtBSA@MB-MSNS treatment group had the most significant ablation ability.

For making up for the instability of most photosensitizers at present, a new nano-platform HCS@Pt-Ce6 NPs was designed,¹¹³ which has been developed to load and deliver photosensitizers. There are reported two different Pt/carbon (Pt/C) nano-enzymes that were loaded as Ce6 nano-carriers. Pt NPs as a nano-enzyme had excellent peroxidase-like and oxidase-like activity. It was verified that Pt showed higher enzyme imitation and anti-tumor activity when loaded outside the carbon shell. In an in vivo experiment, 4 hours after injection of 7.5 mg kg⁻¹ nano-particles into the tail vein, with 660 nm laser for 10 minutes, a comparison of treatment results between the experimental group and the control group showed that the tumor volumes of the HCS@Pt nanoparticle group and HCS@PtCe6 nanoparticle group were inhibited, indicating that catalytic therapy based on HCS@Pt nanoparticles has a specific effect in vivo. It also shows that HCS@Pt-Ce6 nanoparticles as photodynamic and catalytic synergistic therapy are feasible. In particular, a nano-sheet, which was constructed from a two-dimensional (2D) metal–organic skeleton Sm-tetrakis(4-carboxyphenyl) porphyrin (TCPP),¹¹⁴ was assembled with transition metal ions (Sm³⁺) and PSs (TCPP) and grew platinum enzymes mimicking catalase (CAT) on them in situ. As a new type of 2D porous nanomaterial, the 2D metal–organic framework has excellent properties for gas adsorption,^115–117 photocatalysis,^118,119 drug delivery,^120,121etc. In this nanomaterial system, the 2D MOF has a simplified one-dimensionality, which can remove the diffusion restriction on the generation of ROS, thus showing higher PDT efficiency than three-dimensional (3D) MOF. Due to the improvement of the physical and chemical properties of bulky Sm nodes and the enhancement of intersystem crossing, ¹O₂ generation capacity was enhanced. More importantly, the CAT-mimicking Pt nanoenzyme on Sm-TCPP nanoparticles can alleviate the hypoxia of the tumor microenvironment, mainly by using the decomposition of overexpressed H₂O₂ to O₂. The most important organelles that produce H₂O₂ and are very sensitive to ROS are the mitochondria. The triphenylphosphine (TPP) molecule that was introduced to Sm-TCPP-Pt achieved the function of good mitochondrion-targeting, and became an O₂ self-supply PDT system. The prodrug nanoparticles of CPT-TK-HPPH/Pt NP that were created were loaded with platinum nanoparticles (Pt NP) in order to solve the problems of targeting defects and poor efficacy of PDT in hypoxia-related solid tumors.¹²² The excellent therapeutic effects of camptothecin (CPT) and the photosensitizer – 2-(1-hexoxy ethyl)-2-decadienyl pyropheophorbide pheophorbide (HPPH) were used, respectively.^123–125 The prodrug consists of a thioketal bond connecting the CPT and HPPH, and the loaded Pt NPs are used to catalyze the release of O₂ from H₂O₂ (Fig. 8). In current studies, many photosensitizers can produce ROS and effectively decompose ROS-responsive covalent bonds by using this positive feedback regulation, such as thioketal, aryl boron ester, thioether, diselenide ether, and peroxyacetate.^126–128 By introducing drugs and photosensitizers linked with a thioketal bond as prodrugs, the ROS produced by excitation light breaks down the covalent bonds and releases the drugs. The in vitro ROS-production ability of the nano-system was verified. In order to explore the ability for intracellular ROS production, a commercial probe for detecting ROS was used. Different groups were irradiated with or without excitation, and the results showed that the CPT-TK-HPPH NP and CPT-TK-HPPH/Pt NP groups produced ROS, and the ROS contents under excitation light were 1.5 and 2.3 times higher than those without the laser, respectively. In in vivo imaging experiments, they studied whether the drug would accumulate in the tumor, directly affecting the anti-tumor effect in vivo. They evaluated the biological distribution and tumor-targeting ability of CPT-TK-HPPH/Pt NPs by NIR imaging and PA imaging. Its accumulation ability in tumor tissue was strong, and the fluorescence intensity was enhanced within 24 hours. The ablation ability of the tumor in vivo was also verified. The inhibition ability in the CPT-TK-HPPH/Pt NP + laser group was the strongest, which was due to Pt NPs in CPT-TK-HPPH/PtNP catalyzing the decomposition of hydrogen peroxide in tumor tissue to produce oxygen and improving tumor hypoxia.

Fig. 8 Schematic diagram of combined treatment of tumor cells with CPT-TK-HPPH/Pt NP under 660 nm laser irradiation. Reproduced from ref. 122 with permission from Wiley, copyright 2020.

In a recent investigation, for enhancing oxygen delivery, nanoparticle-stabilized oxygen microcapsules were prepared by interfacial polymerization.¹²⁹ The novel vehicle of polydopamine–nanoparticle-stabilized oxygen microcapsules applied for oxygen delivery is effective. In aqueous solution, it shows good biocompatibility; moreover, oxygen placed in the microcapsule can diffuse well from the core to the outside of the capsule, and thus can increase the oxygen content in the microenvironment. The feasibility of this strategy has been well proved by in vivo and in vitro experiments. In in vivo experiments, when it was injected subcutaneously, the hypoxia could be improved, and in the end suppressed the growth of the tumor. This oxygen supplementation strategy can be used in a wider range of applications, such as photodynamic therapy or other treatments with more aerobic therapy. Also, the publications about H₂O₂ decomposition by Pt NPs were summaried in Table 4

Table 4 Summary of publications about H₂O₂ decomposition by Pt NPs

Application	Catalyst	O2 source	Excitation requirements (excitation wavelength, power density, time)	PSs	ROS species	In vitro (cell line)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: tetrakis (4-carboxyphenyl) porphyrin (TCPP); black phosphorus nanosheets (BP); chlorin e6 (Ce6); Pt/carbon (Pt/C) nanozymes; catalase (CAT)-mimicking platinum nanozymes.
PDT	Pt NP	H2O2	In vitro: 638 nm, 1 W cm^−2, 10 min	TCPP	^1O2	RAW264.7, 4T1 and HeLa	H22	106
			In vivo: 638 nm, 1 W cm^−2, 8 min
PDT	Pt NP	H2O2	In vitro: 660 nm, 1 W cm^−2, 5 min	BP	^1O2	4T1	4T1	107
			In vivo: 660 nm, 1 W cm^−2, 10 min
PDT	Pt NP	H2O2	In vitro: 660 nm, 30 mW cm^−2, 2 min	H2TCPP	^1O2	CT26 and COS7	CT26	110
			In vivo: 660 nm, 220 mW cm^−2, 5 min
PDT	Pt BSA	H2O2	In vitro: 635 nm, 45 mW cm^−2, 30 min	MB	^1O2	HeLa and A549	HeLa	112
			In vivo: 635 nm, 100 mW cm^−2, 15 min
PDT	Pt/C	H2O2	In vitro (vivo): 660 nm, 0.346 W cm^−2, 10 min	Ce6	^1O2	4T1 and A549	CT26	113
PDT	Pt NP	H2O2	In vitro (vivo): 660 nm, 100 mW cm^−2, 15 min	TCPP	^1O2	MCF-7	MCF-7	114
PDT	Pt NP	H2O2	In vitro (vivo): 660 nm, 200 mW cm^−2, 5 min	CPT-TK-HPPH	^1O2	CT26	CT26	122

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2.3 CaO₂ decomposition

The ability of calcium peroxide to produce O₂ is widely used in tissue engineering, agriculture, aquaculture and relieving hypoxia in tumor tissue.^130–132 As a supplementary source of oxygen, it can release oxygen for a long time to improve the viability of organisms when they are in constant contact with water.¹³³ Calcium peroxide (CaO₂) is a promising material for regulating tumor hypoxia and improving PDT efficiency because of its good biocompatibility and high efficiency and long-lasting O₂ production. PDT uses light-activated photosensitizers to convert O₂ to ROS, while CDT uses in situ Fenton or Fenton-like reactions between H₂O₂ and the catalyst to produce ˙OH. However, a large amount of GSH in the tumor microenvironment can deplete ROS. CaO₂ is a safe solid inorganic peroxide that decomposes when it meets with water and releases O₂ and H₂O₂ at the same time. Therefore, the strategy of introducing CaO₂ into therapies involving ROS to enhance ROS production is widely used. The CaO₂ component in the nano-system is reactive to water, causing O₂ to be released in the water for a long time (eqn (4) and (5))^134–136

2CaO₂ + 2H₂O → 2Ca(OH)₂ + O₂ (4)

2CaO₂ + 4H₂O → 2Ca(OH)₂ + 2H₂O₂ → 2Ca(OH)₂ + 2H₂O + O₂ (5)

A NIR rechargeable “optical battery” for avoiding irradiating PDT was designed, which was formed by embedding three different components into biocompatible polydimethylsiloxane (namely, upconversion materials, persistent luminescence materials, photosensitizer) (Fig. 9a).¹³⁷ They used NIR irradiation (980 nm, 2 W cm⁻²) for a short charging process of 5 s, and the luminescence could be sustained for a long time (about 30 min). According to previous studies, the battery was tested using different thicknesses of pork tissue to simulate human tissue (the penetration depth is up to 1 cm). After using this way to test it, they found that when the NIR irradiation was shone at pork tissue, ¹O₂ could be produced at a depth of 4 mm. What is interesting is that the NIR rechargeable irradiation-free PDT implants can easily be made in any size and shape. In addition, the cell has good biocompatibility in the human colon adenocarcinoma cell line HT29 without light and will produce strong cytotoxicity with laser irradiation. When they encapsulated the CaO₂ in the battery, they found a higher molecular oxygen concentration. Hence, they further used oxygen production to alleviate tumor hypoxia; they implanted this device into an HT29 solid tumor, used NIR radiation for treatment (660 nm, 30 mW cm⁻²), and finally successfully solved the hypoxia condition of the tumor tissue, and a good tumor ablation effect was achieved (Fig. 9c), so it also promoted the therapeutic effect of PDT.

Fig. 9 (a) Fast charging with NIR light can achieve a long-lasting luminescence activation of a PDT “optical battery” implanted into the tumor to produce reactive oxygen species. Reproduced with permission from ref. 137. Copyright 2020, Royal Society of Chemistry (b) CaO₂/B1/NH₄HCO₃ liposome can generate O₂ for PDT under the NIR light irradiation; the bottom of the picture shows the mechanism for killing cancer cells. Reproduced from ref. 139 with permission from the American Chemical Society, copyright 2014. (c) and (d) Curve of tumor volume changes in mice during different treatments. Reproduced from ref. 137 with permission from the Royal Society of Chemistry, copyright 2020 (c). Reproduced from ref. 139 with permission from the Royal Society of Chemistry, copyright 2019 (d).

Encouraged by previous research methods, Song's research group developed a nanomaterial.¹³⁸ Through the implanted oxygen production bank, the local oxygen therapy enhanced the ablation of malignant tumors by Dox in a highly specific manner without increasing oxygen levels in tissues and organs in the whole body. The O₂ production pool's design method puts CaO₂ (contained in alginate solution) and catalase into a calcium chloride (CaCl₂) bath to form calcium cross-linked microcapsule particles. When the oxygen-producing reservoir is implanted subcutaneously near the tumor, the exposed interstitial medium will react with it, and then the O₂ is produced in situ, thus effectively reducing the anoxic area in the tumor tissue. In vitro experiments showed that the ROS level and apoptosis rate of Hep3B cells treated with Dox increased under normoxic conditions, but the ability of Dox to induce ROS was significantly decreased under hypoxic pressure, which enhanced the resistance of anoxic cells to Dox. Simultaneously, it is suggested that the oxygen-producing nanoparticles significantly enhance the cytotoxicity of Dox on ROS-induced cells. In vivo, the improvement of cytotoxicity of Dox on the tumor induced by the oxygen transport pool was further evaluated by positron emission tomography (PET). The maximum uptake of ¹⁸F-fludeoxyglucose (¹⁸F-FDG) in pellets + Dox (1.68 ± 0.06 %ID g⁻¹) was significantly lower than that in other groups, indicating the therapeutic effect of this nano-system and slowing down the metabolism and proliferation of tumor cells.

Similarly, an O₂-self-sufficient nano-platform (LipoMB/CaO₂) for PDT of anoxic tumors was designed.¹⁴⁰ In this system, a photosensitizer (MB) and CaO₂ nanoparticles are wrapped within a hydrophilic cavity. The nano-platform can amplify the production of O₂ under dual-stage light-driven PDT, regulate intracellular hypoxia and enhance the production of ¹O₂ in the process of PDT. After the first-stage laser irradiation, the release of O₂ originating from the reaction of CaO₂ and H₂O alleviated tumor hypoxia. In vivo experiments also proved that LipoMB/CaO₂ has good biocompatibility and can reduce tumor hypoxia and have an anti-tumor metastasis effect. In addition, a liposome was produced that achieved the function of being oxygen self-sufficient, named CaO₂/B1/NH₄HCO₃ liposome.¹³⁹ The liposome consists of hydrophobic aza-BODIPY dye (B1), oxygen-producing nanoparticles and hydrophilic thermally active ammonium bicarbonate (NH₄HCO₃) (Fig. 9b). The results of in vitro experiments showed that CaO₂/B1/NH₄HCO₃ had an excellent ability to produce ROS after the tumor tissue was irradiated by 730 nm and 100 mW cm⁻² for 5 minutes. At the same time, the in vivo experiments showed that this nano-platform had an excellent tumor therapeutic effects (Fig. 9d). The heat generated promotes the thermal decomposition of NH₄HCO₃ to produce oxygen, which increases the therapeutic effect of PDT and confirming the feasibility of this strategy.

In tumor therapy, the lack of substrates needed in treatment, such as hypoxia in PDT and deficiency of hydrogen peroxide in CDT, will limit the therapeutic effect of ROS. Professor Dong's team jointly designed an H₂O₂/O₂ self-sufficient nano agent (MSNs@CaO₂-ICG)@LA.¹⁴¹ This nano-system consists of manganese silicate (MSN) loaded calcium peroxide (CaO₂) and the photosensitizer indocyanine green (ICG), with further shaping of the surface, that was modified with phase-change material (LA), as an open-source and cost-saving ROS generation strategy for CDT/PDT synergistic therapy. In this nano-system, the strategy of laser irradiation to generate heat is also adopted to expose the CaO₂ in the nano-system. CaO₂ is protected by outer LA to avoid direct contact with water. Under NIR irradiation, which induces the photothermal action of photosensitizer ICG, the outer LA can melt, and the exposed CaO₂ reacts with water, resulting in the rapid formation of H₂O₂ and O₂, and further exposure of the internal MSN. The oxygen released can alleviate the hypoxia in the cells, thereby enhancing ICG-mediated PDT. The interaction between MSNs and GSH leads to the release of the Fenton-like agent Mn²⁺, which is used in H₂O₂-supplemented CDT and magnetic resonance imaging (MRI)-guided therapy; the consumption of GSH deeply destroys the intracellular redox environment, thus further improving the production efficiency of ROS. An in vitro experiment explored the synergistic therapeutic effect of MSNsCaO₂-ICG@LA-mediated CDT/PDT. The concentration of Mn²⁺ was measured at different time points, and showed the blood circulation curve of MSNs@CaO₂-ICG@LA in mice. Because the EPR effect is conducive to the accumulation of MSNs@CaO₂-ICG@LA in the tumor tissue, the brightness of the tumor site of the MSN-treated mice was higher than that of the control group, indicating that MSNs could be degraded into Mn²⁺ by GSH in vivo. It also illustrates the excellent thermal response characteristics of the nano-system, and the combined therapy of this nano-system enhances the ablation ability of the tumor. Compared with the nanometer platform designed by Zhang's research group in 2017, this topic also combined with MRI, which can realize the integration of diagnosis and treatment in the process of therapy, and provides excellent value for tumor treatment and further clinical application.

From the point of view of re-injecting oxygen into the infarcted myocardium to protect cardiomyocytes and reconstruct cardiac function in acute myocardial infarction treatment, Hao's team developed a nano-system named CMHP.¹⁴² This nano-system is formed by forming CaO₂ in a mesoporous silica nano-platform and then assembling the thermosensitive materials eicosane and polyethylene glycol. In an in vivo experiment, pathomorphological analysis showed that there was significant left ventricular wall thinning in the myocardial infarction group, while less in the CMHPv + US group. Therefore, the survival rate of cardiomyocytes under hypoxia was significantly increased, and infarcted myocardial tissue injury was reduced to a minimum.

2.4 Water splitting

For tumor tissue, hypoxia hinders the effect of PDT treatment. PDT methods to alleviate hypoxia have been constantly explored, with the use of oxygen produced by photosynthesis to alleviate tumor microenvironment hypoxia,^50,143,144 but the process of this method is tedious. The preparation of molecular hydrogen and oxygen can be accomplished by decomposing water under the action of solar energy. This method is a promising way of obtaining renewable energy storage,^145,146 so, using water in the human body as a direct resource, catalytic cracking of water to produce oxygen will be more convenient and direct. It may also be used as a lasting method of providing O₂. Inspired by this, the photolysis strategy of water in organisms has been used to generate O₂ in the body and promote ROS production or other ways to improve PDT efficiency. Among many water separation materials, carbon nitride (C₃N₄) has attracted much attention because of its adjustable band gap and position, and low-permeability to light such as ultraviolet absorption has a great effect on the body,¹⁴⁷ so C₃N₄ can be modified to drive the decomposition of water under highly permeating red light (>600 nm). This makes C₃N₄ suitable for in vivo treatment.^148,149 In 2015, Kang's research team designed a metal-free carbon nanodot–carbon nitride (C₃N₄) nanocomposite, and verified this nanocomposite had an impressive performance. After ensuring the amount of composite catalyst in water, the optimum CDots/C₃N₄ ratio is 4.8 × 10⁻³ g_CDots/g_catalyst, with CDots–C₃N₄ composites at λ = 420 ± 20 nm and λ = 580 ± 15 nm. The QEs that can decompose water into H₂ and O₂ are 16% and 6.3%, respectively. For the photocatalyst g-C₃N₄, it has the characteristics of rich resources and low cost,^150,151 and the most important thing is that the preparation is relatively simple,^152,153 so it is convenient for the application of this new catalyst. Some studies have proved that g-C₃N₄ has great potential as a photosensitizer to alleviate tumor hypoxia.^154,155 Under weak light (20 mW cm⁻²), it can produce ROS and kill tumor cells. In addition, carbon points (CdS) have the advantages of unique photoelectron transfer and luminescence properties. This study also made Yang's team think of using the combination of g-C₃N₄ and CdS to form an excellent performance PDT photosensitizer: (i) 2H₂O → H₂O₂ + H₂; (ii) 2H₂O₂ → 2H₂O + O₂.^153,156 They integrated dual-mode photodynamic therapy (Dual-PDT) into one system for multiple functions.¹⁵⁷ First, the core–shell structured upconversion nanoparticles were attached on graphitic-phase g-C₃N₄ nanosheets (one photosensitizer), and then by way of in situ growth, as-prepared nanocomposite and carbon dots were assembled in ZIF-8 metal–organic frameworks, and in the end, a dual photosensitizer mixing system for PDT was realized by stepwise water splitting. In in vivo experiments, the tumor growth rate of a UCNPS-g-C₃N₄@ZIF-8 complex culture was significantly different with or without light, indicating that the production of oxygen under light conditions alleviated tumor hypoxia and increased the therapeutic effect. This research group also reported a new multi-functional nano-platform named UCNPs@BiOCl, which is based on the integration of NaGdF₄:Yb,Tm@NaGdF₄ UCNPs and bismuth oxyhalide (BiOCl) sheets, for 980 nm near-infrared light-triggered PDT.¹⁵⁸ With the same hydrolysis strategy, the loaded UCNPs can convert near infrared light into UV/Vis region emissions, which drives the pure water pyrolysis of BiOCl sheets, and the resulting reactive oxygen species are used to kill the tumor cells.

A kind of multifunctional nanocomposite (PCCN) based on C₃N₄ was adopted to solve the problem of hypoxia restricting the efficacy of PDT.¹⁵⁹ The nanomaterial PCCN was designed and synthesized, which contains protoporphyrin photosensitizer IX (PpIX), a polyethylene glycol chain connector and an RGD sequence Arg–Gly–Asp motif, and was prepared by the accumulation of π–π bond between C₃N₄ and PpIX. PCCN uses RGD targeting and the EPR effect to reach tumor tissue, where it accumulates. PCCN produces O₂ by cracking water, which is irradiated by 630 nm laser. Photosensitizers can also redistribute the energy and transfer it to the resulting O₂ and produce cytotoxic ¹O₂. In addition, PCCN can also down-regulate the expression of hypoxia-related proteins, thus inhibiting tumor metastasis and treating tumors in two modes: ablation and inhibition of tumor metastasis. Similarly, Zhang's team (the same team as the last report) continued to develop their hydrolyzed oxygen project.¹⁶⁰ Taking advantage of the advantage of Ru(II) complex as a photosensitizer for two-photon photodynamic excitation,^161,162 a composite nanoparticle (FCRH) was designed, which was composed of iron-doped carbon nitride (Fe–C₃N₄), Ru(Bpy)₃²⁺ and hyperbranched conjugated copolymer (HOP) with a polyethylene glycol arm. By π–π bond stacking on Ru(Bpy)₃²⁺, photogenerated electrons are injected into Fe–C₃N₄ from Ru(Bpy)₃²⁺ to promote charge localization, which can more effectively separate the charge and prevent electron–hole pair recombination, thus improving the photocatalytic activity and O₂ production.^7,163 In in vitro experiments, after accumulating nanocomposites by enhancing EPR, FCRH has been proved to alleviate tumor hypoxia, thus strengthening the anti-tumor effect of PDT.^159,164,165 Under 800 nm excitation light irradiation, there was apparent oxygen production, and the EPR spectrum confirmed the production of ¹O₂. After 24 hours of intravenous injection of IR780-labeled nanocomposite material, the apparent tumor contrast IR780 fluorescence gradually increased and then decreased after 36 hours, confirming its excellent tumor-specific accumulation ability in vivo. In the treatment experiment, the tumor volume of the FCRH combined with irradiation group was suppressed entirely within 14 days, while the inhibitory effect of FCRH on 4T1 tumors was negligible under the condition of no light; this shows the same excellent therapeutic effect. In the same way as in the previous work, photocatalytic materials were developed from TiO₂, C₃N₄ and bismuth halide flakes. NIR radiation is also adopted, and the near-infrared light has the characteristics of low toxicity and vigorous penetration. The energy produced after excitation promotes the catalytic decomposition of H₂O to O₂ and improves the PDT efficiency.^157,166 The above studies have played a leading role in using the hydrolysis of water in the body to prepare O₂ to alleviate the hypoxia of tumor tissues, especially in the further improvement of the strategy in the later stages, with the use of high-penetration red light for water cleavage, which is suitable for in vivo treatment. Also, the publications about water splitting were summaried in Table 5.

Table 5 Summary of publications about water splitting

Application	Catalyst	O2 source	PSs	Excitation requirements (excitation wavelength, power density, time)	ROS species	In vitro (cell line)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: water splitting (Ws); mediating the photosensitizer (BiOCl); protoporphyrin IX (PpIX); ruthenium(II) complex (Ru(bpy)3^2+); hypoxemia after acute myocardial infarction (AMI); the phase change of heneicosane (PCH).
PDT	CaO2	Water splitting	RB	980 nm, 2 W cm^−2, 5 s	^1O2	HT29	HT29	137
PDT	CaO2	Water splitting	MB	In vitro: 660 nm, 30 mW cm^−2, 6 min	^1O2	4T1	4T1	140
				In vivo: 658 nm, 280 mW cm^−2, 10 min
PDT/PTT	CaO2	CaO2/B1/NH4HCO3	aza-BODIPY	In vitro: 730 nm, 500 mW cm^−2, 5 min	^1O2	HeLa	HeLa	139
				In vivo: 730 nm, 100 mW cm^−2, 5 min
CDT/PDT	CaO2	Water splitting	ICG	In vitro (vivo): 808 nm, 0.64 W cm^−2, 10 min	^1O2, ˙OH	MCF-7 and A549	MCF-7	141
US + CMHP	US	Water splitting	NO	1.0 MHz, 0.6 W cm^−2, 30 s	NO	H9c2	NO	142
PDT	C3N4	Ws	UCNPs	In vitro: 980 nm, 0.5 W cm^−2, 5 min	˙O2^−, ^1O2	L929	U14	157
				In vivo: 980 nm, 0.5 W cm^−2, 10 min
PDT	C3N4	Water splitting	BiOCl	In vitro (vivo): 980 nm, 0.5 W cm^−2, 10 min	OH˙, ˙O2^−	HeLa	U14	158
PDT	C3N4	PCCN	PpIX	In vitro: 630 nm He–Ne, 80 mW cm^−2, 10 min	^1O2	4T1 and MCF-7	4T1	159
				In vivo: 630 nm He–Ne, 40 mW cm^−2, 5 min
PDT	Fe–C3N4	Water splitting	Ru(bpy)3^2+	In vitro: 800 nm, 2.7 W, 3 min	^1O2	4T1	4T1	160
				In vivo: 800 nm, 2.7 W, 5 min

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3. Improve tumor hypoxia through various oxygen delivery carriers

The culprit in hypoxia is the formation of abnormal blood vessels, often forming malformed blood vessels and poor blood flow.^38,167–173 In tumor tissue this will lead to a decrease in the average level of tissue partial pressure of oxygen, which often occurs during vascular disease,^174,175 lung disease, and cancer. Normal cell metabolism or treatment (PDT, drug therapy, etc.) needs oxygen, and hypoxia will seriously affect its therapeutic effect. Therefore, increasing the concentration of O₂ by accelerating the blood flow pathway will be a new way to improve therapeutic efficacy.

3.1 Accelerate the blood flow

The former study found that changing the blood temperature (40–42 °C) can increase the blood flow speed,^176,177 and the blood will continue to carry oxygen, so the acceleration of blood flow speed will also alleviate hypoxia.^178–183 The common method of increasing blood temperature is the hyperthermia produced by PTT;^184–187 PTT absorbing light through the NIR region can accelerate the blood flow velocity of patients with a solid tumor, and thus improve the oxygenation level of the cancer and reduce the treatment resistance related to hypoxia. It converts light energy into heat energy by exciting light to irradiate photosensitizers. There are many kinds of photosensitizer, including small molecule fluorescent probes,^188–193 nanomaterials,^194,195 and so on. Therefore, the use of a mild heating method for tumor treatment is an effective way to alleviate hypoxia and promote PDT outcomes.

3.1.1 Increase the temperature through the process of PTT. Aiming to increase the temperature, the quite synergistic liquid metal nanoparticles–enzyme (LM@GOX) for combined starvation/photothermal therapy of tumors was prepared.¹⁹⁶ In this system, when irradiated with a laser (808 nm) for 15 minutes, the photothermal effect increases along with the increase of LM or light intensity. They also studied the effect of temperature on enzyme activity, and the enzyme activity was measured by monitoring the production of H₂O₂. When the temperature reached a maximum of 60 °C, the enzyme activity decreased, so the PTT process would not affect the enzyme activity. In addition, 4T1 cells were used to verify the enhanced enzyme activity of LM@GOX under in vitro irradiation. The production of H₂O₂ was confirmed by using the ROS probe 2′,7-dichlorofluorescein diacetate (DCFH-DA). The fluorescence intensity in LM@GOX-treated cells was significantly enhanced. This shows that the photothermal conversion of LM produces the heat, and under light it can improve the enzyme activity of GOX in cells. In an in vivo experiment, NIR dye Cy5 was anchored to an LM@GOX nano-system (LM@GOX-CY5) for in vivo fluorescence imaging to evaluate tumor accumulation in vivo. The results showed that LM@GOX-Cy5 could accumulate in the tumor after injection, and showed long-term retention, which was conducive to continuous light irradiation and therapy. Using the 4T1 tumor model injected subcutaneously, the anti-tumor experiment was carried out with NIR irradiation of the light group, and LM without NIR exposure could not effectively inhibit the growth of the tumor. Compared with chemotherapy or PTT alone, the synergistic effect of LM@GOX has been shown under 808 nm light, and the synergistic therapy can effectively eradicate tumor cells.

The staining experiment of HIF-1α confirmed that the tumor's hypoxia state was obviously improved.^167,197 Immunofluorescence in the LM + NIR treatment group was weak, indicating that HIF-1α was expressed at a low level in tumor tissues, and the hypoxia tolerance catalyzed by GOX may be alleviated. It was further proved that blood flow accelerated, and hypoxia was relieved during PTT treatment. Feng et al.¹⁹⁸ prepared a liposome system containing photosensitizer Ce6 and a photothermal agent (DIR). An NIR light-activated liposome Ce6 preparation was constructed, and HCe6 and DIR molecules were co-encapsulated in PEG shell liposome bilayers. Under NIR laser irradiation at 785 nm, the fluorescence of hCe6 in dir-hCe6-liposomes will be awakened. Irradiated with 660 nm LED light at 2 mW cm⁻², the function of PDT can be triggered by stimulating PS and Ce6 to produce a mild photothermal heating, which can accelerate the circulation of blood flow, lighten tumor hypoxia and promote the therapeutic effect of PDT. At the same time, it has shown great potential in skin protection and is an excellent class of activable photosensitizer.^199,200In vivo experiments show that this liposome system can significantly improve tumor blood flow and alleviate tumor hypoxia.

3.1.2 Chemotherapy drugs. In addition to accelerating the rate of blood flow in the tumor by generating heat through PTT, some chemotherapy drugs also have a similar function. By regulating the chaotic structure of tumor blood vessels, the accelerated blood flow drives more oxygen into the tumor microenvironment and improves the hypoxia. Common chemotherapy drugs include taxane, gemcitabine, cyclophosphamide, and cisplatin.

Nanoparticles containing Pt(IV) and dihydro porphyrin e6 and loaded with UCNPs were designed and developed, and can convert near-infrared light of 980 nm into emission light of 365 nm and 660 nm (Fig. 10a).²⁰¹ When UCPP decomposition is triggered by 980 nm laser irradiation, O₂ can be produced to compensate for consumption in the process of PDT, releasing active Pt(II) for synergistic photochemotherapy. The nano-system does not need to be accompanied by catalytic substances, such as MnO₂ or catalase,^57,202–204 and could solve the limitation of hypoxia PDT treatment. In an in vitro experiment, L929 cells were carefully studied by CLSM to reverse PDT-induced hypoxia. An ROS probe was used to detect ROS under hypoxia and normoxic conditions. Whether under normoxic or anoxic conditions, UCPP-treated L929 cells presented bright green fluorescence, indicating that UCPP could indeed produce O₂ inside the cells and provide enough O₂ for PDT to conquer PDT-induced hypoxia (Fig. 10b). In an in vivo experiment, two kinds of orthotopic superficial carcinoma, B16 and MDA-MB-231 tumor models, and two subcutaneous tumor models, HeLa and HCT116, were used to evaluate the anticancer activity. When B16, HCT116 and MDA-MB-231 tumor-bearing mice were treated with UCPP nanoparticles and then given 980 nm laser irradiation, all xenografts disappeared only after laser irradiation (Fig. 10c). The remarkable anti-tumor result in vivo was achieved by reasonably designed UCPP. The UCPP can generate O₂ and Pt(II) under NIR irradiation, thereby alleviating the state of hypoxia and achieving synergistic PDT-chemotherapy.

Fig. 10 (a) Schematic illustration of UCPP nanoparticles. (b) CLSM imaging of different nano-systems under normoxic and anoxic conditions. (c) The change of various cancer cells (HCT116, MDA-MB-231, and B16) in tumor-bearing mice with different treatments. Reproduced from ref. 201 with permission from Springer Nature Limited, copyright 2018.

3.1.3 Z-Scheme heterostructures for hypoxic tumor therapy. High-Z element nanomaterials are a kind of functional nanomaterial which can simultaneously reduce tumor hypoxia and enhance tumor ionizing radiation energy deposition on a single nanometer platform,^205,206 thus improving the effectiveness of radiotherapy without the need for additional and complex O₂ delivery or O₂ generation systems; as a novel strategy, it has made great changes in improving the effect of radiotherapy for hypoxic tumors. PVP-Bi₂Se₃@Sec nanoparticles are outstanding.²⁰⁷ The X-ray attenuation and high NIR absorption performances were quite good, so these nanoparticles are equipped for use in CT/PA imaging contrast agents for imaging-guided photothermal radiation therapy.²⁰⁸ They show a noticeable enhancement of free radical production under X-ray radiation and NIR laser irradiation. The photothermal effect can increase the oxygenation within tumors by accelerating the blood flow, thus reducing the radio-resistance of cancer. PVP-Bi₂Se₃@Sec-mediated PTT/RT synergistic therapy inhibited the growth of the tumor after 5 days, and no recurrence occurred within three weeks. The final anti-tumor rate of the synergistic group was more than 98%, which further indicated that PVP-Bi₂Se₃@Sec NPs could enhance the synergistic therapeutic effect of PTT and RT on tumors. The authors speculate that the improvement in the effectiveness of synergistic therapy may be due to the hyperthermia condition that can effectively kill those cells that a not sensitive to radiotherapy. In addition, appropriate hyperthermia can increase tumor oxygenation by improving the blood flow within the tumor, which can reverse radiation resistance in tumor tissues. Cheng et al. designed novel bismuth sulfide (Bi₂S₃)@bismuth (Bi) Z-scheme nanorods (NRs), as narrow bandgap Bi₂S₃ and Bi components can generate abundant electrons and holes in NIR light (Fig. 11a).²⁰⁹ In addition, the Z-scheme heterostructure endows Bi₂S₃@Bi NRS with effective electron–hole separation ability and strong redox potential. The holes in the Bi₂S₃ valence band can react with water, providing O₂ for the electrons in the Bi conduction band, thus producing reactive oxygen species. The results show that when the oxidation potential of the valence band hole of the nanomaterial is greater than that of the water oxidation potential (1.23 V, relative to the standard hydrogen electrode), the hole will react with water to produce O₂, while when the reduction potential of the electrons in the conduction band of the nanomaterials is less than the formation potential of O₂˙⁻ (−0.13 V), the electrons can react with O₂ to form O₂˙⁻ and further form OH˙.²¹⁰In vitro, after laser irradiation, the cell injury induced by Bi₂S₃@Bi NRs was stronger than that induced by Bi₂S₃ + Bi, showing the prominent phototherapeutic effect (Fig. 11b). In an in vivo experiment, after the tumor-targeting ability of nano-particles was confirmed by CT imaging, they used 4T1 tumor-bearing mice to study the phototherapy effect of Bi₂S₃@Bi NRs. Under NIR laser irradiation, Bi₂S₃@BiNRs significantly down-regulated HIF-1α, indicating that tumor hypoxia had been relieved successfully. When the biocompatible modified Bi₂S₃@Bi NRs were injected into the tumor tissue by way of the tail vein, they showed a major inhibitory effect on tumor growth (Fig. 11b). Near-infrared irradiation could weaken the hypoxia condition of the tumor microenvironment and produce strong oxidative damage to tumor tissue, showing a good therapeutic effect of PDT on hypoxic tumors. Also, the publications about accelerating blood flow to introduce O₂ were summaried in Table 6.

Fig. 11 (a) Schematic diagram of the therapeutic process of oxygen produced by Bi₂S₃@BiNRs nanoparticles irradiated by near-infrared laser to promote ROS production. (b) After different treatments, the tumor growth curve of mice with or without laser irradiation. Reproduced from ref. 209 with permission from Wiley, copyright 2020.

Table 6 Summary of publications about accelerating blood flow to introduce O₂

Application	PS	O2 source	O2 level detection	Excitation requirements (excitation wavelength, power density, time)	ROS species	In vitro (cell lines)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: indicator ([Ru(dpp)3]Cl2); chlorin e6 (Ce6); oxygen dissolving meter (ODM); increase blood flow (IBF); pimonidazole hydrochloride (Hypoxyprobe™).
PTT	NO	IBF	NO	In vitro: 808 nm, 1.0 W cm^−2, 15 min	H2O2	4T1	4T1	196
				In vivo: 808 nm, 1.0 W cm^−2, 5 min
PDT/PTT	Chlorin e6	IBF	Hypoxyprobe™	In vitro: 785 nm, 1.0 W cm^−2, 10 min	^1O2	4T1	4T1	198
				In vivo: 785 (660) nm, 2 W cm^−2, 20 min (1 h)
PDT	Chlorin e6	UCPP	ODM	In vitro: 909 nm, 0.85 W cm^−2, 5 min	^1O2	L929	HeLa and HCT116	201
				In vivo: 909 nm, 0.85 W cm^−2, 10 min
PTT	NO	IBF	NO	In vitro: 808 nm, 1.0 W cm^−2, 10 min	Free radicals	HUVEC and MDA-MB-231	BEL-7402	207
				In vivo: 808 nm, 0.3 W cm^−2, 10 min
PDT	Bi2S3	Bi2S3 + water	[Ru(dpp)3]Cl2	In vitro: 808 nm, 0.5 W cm^−2, 10 min	O2˙^−, OH˙	4T1 and 3T3	4T1	209
				In vivo: 808 nm, 0.5 W cm^−2, 5 min

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3.2 Perfluorocarbons

PFCs are a kind of organic fluorine compound. The reason why PFCs can dissolve relatively large volumes of gas is due to the weak interactions (van der Waals force) and they are clinically used as an artificial blood substitute.^211,212 When the oxygen pressure in PFCs is lower than 760 Torr (1 atm) at room temperature (25 °C),²¹³ the amount of dissolved oxygen per 100 mL liquid (40–50 mL) is twice as much as that in normal human blood.²¹⁴ Because of its high O₂-generating capacity,^215–217 at a determined partial pressure of oxygen, it can have a higher amount of oxygen relative to the tumor matrix at a given partial oxygen pressure. From 1988, Henderson and his co-workers began using PFCs to enhance PDT by injecting porphyrin photosensitizers containing PFC nanoemulsions.²¹⁸ In addition, nano-carriers containing PFC can be used as a US imaging agent and high intensity focused ultrasound (HIFU) agent.^219,220 Gasification is a new PFC application technology that can significantly improve oxygenation and lung function in oleic acid-induced lung injury.²²¹

After applying this nanomaterials to mice, the tumor's oxygenation was enhanced, which could significantly improve the efficacy of PDT and radiotherapy. The introduction of a fluorinated chain segment increased the solubility of PS, increased the local O₂ concentration of polystyrene in the ground state (³O₂) and excited state (¹O₂), and improved the O₂ diffusivity. Additionally, PFC emulsions have been approved by the FDA.²²² Inspired by this, PFCs bring oxygen molecules into auxiliary PDT in different ways, which is a possible new method to alleviate hypoxia.

The designed nanoparticles get the most out of the O₂ and longer ¹O₂ lifetime of PFCs to significantly utilize the loaded functional photosensitizer to perfect the outcome of PDT.^212,223 In in vitro experiments, PDT was performed on LIP (IR780) and LIP (IR780 + PFH) under different hypoxia conditions (including 0.1, 1, 7, and 21 kPa O₂). The LIP (IR780 + PFH) group could significantly increase O₂ production under both hypoxia and normoxic conditions, and the PFC could successfully promote the production of ¹O₂ under hypoxic conditions. In an in vivo PDT experiment, they first measured the ¹O₂ production of LIP (IR780) and LIP (IR780 + PFH). In order to prevent the extra effect caused by the photothermal effect, which affects the reliability of the experimental results, they separated the irradiation times, and enough time was provided to restore the original temperature, so the method of a one-minute interval was adopted for two consecutive laser irradiations. In an in vivo experiment, the therapeutic effect of intratumoral injection was studied first, and the tumor volume was also tracked with or without laser irradiation. Through the study of tumor accumulation imaging and ultrasound imaging of IR780, the mice were irradiated by a 808 nm laser 24 hours after intravenous injection, and the tumor volume irradiated with light (808 nm, 10 s, 2 W cm⁻²) was about 4 times smaller than that of mice treated with IR780 alone. Tang et al.²²⁴ adopted the Oxy-PDT strategy, loading a hydrophobic NIR photosensitizer into PFC nano-droplets as an effective photodynamic nano-drug. PFH was selected as the core of the PFC. During laser irradiation, the generation of ¹O₂ in the PFC phase will be faster. In this way, when the tumor is in a state of hypoxia, the photodynamic effect is good. The model of a hypoxic CT26 subcutaneous tumor was established and confirmed in vivo. All in all, it shows that the photosensitizer loaded into perfluorinated carbon nanodroplets can successfully treat hypoxic tumors resistant to traditional PDT. Using the same method, a PCF nano-emulsion containing fluorinated porphyrin, using the PFC nanoemulsion to transport O₂ and PS simultaneously, was designed and synthesized.²²⁵ The efficacy of PDT was determined by emulsion containing photosensitizer in cellulose. The uptake of nano-emulsion by human malignant melanoma (A375) cells was first verified by a co-localization test. Under laser irradiation, cell death was indicated by NucGreen™ fluorescence and confirmed again by flow cytometry analysis.²²⁶ It was determined that the use of fluorine-containing soluble photosensitizer is the most promising method for PDT with a PFC nano-emulsion. Besides, a reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used, so plenty of amphiphilic/fluorine random copolymers with different contents of perfluorocarbon (perfluorooctyl) were prepared.²²⁷ The higher PFC content in the copolymers can produce ROS more effectively, thus improving PDT efficiency in vitro. They found that an increase in the PFC amount can carry more oxygen and therefore produce more ROS. The determination of ¹O₂ by 630 nm laser illumination (F-polymer (P₆-F59%)-HB, H-polymer (P₂-H54%)-HB) proved that the addition of PFC effectively promoted the production of ¹O₂.

Plasma gold metal nanostructures were combined with the semiconductors of Cu₂O, ASC was loaded into O₂ PFH droplets, and then LIP (ASC/PFH) nanocomposites were formed by coating liposomes (LIP).²²⁸ At the same time, due to the high O₂ capacity of PFH, under a certain oxygen partial pressure, its high oxygen content can be maintained for a certain period of time and is higher than that of the tumor matrix.²²⁹ A high quantum yield of ¹O₂ was evaluated that was nearly 0.71, which was produced by the mechanism of plasmon-induced resonance energy transfer (PIRET) that produced, from addition of Au to Cu₂O, Au@SiO₂@Cu₂O under NIR irradiation for ten minutes. In an in vitro experiment, the ¹O₂ production capacity of ASC added to Cu₂O, Au@SiO₂, and Au@Cu₂O was evaluated by a DPBF probe, and 670 nm (0.48 W cm⁻²) laser was used for irradiation for 10 minutes. In addition, the real-time oxygen-carrying capacity of PFH is as high as 168 mg L⁻¹, so it can be proved that it has good oxygen-carrying capacity. The antitumor activity of LIP (ASC/PFH) in vivo was studied, and a significant therapeutic effect in the LIP (ASC/PFH) group was shown when the laser irradiation was applied for 10 minutes every time. An H&E staining assay showed that LIP (ASC/PFH) had good biocompatibility.

Recently, a well-targeted oxygen self-sufficiency and tumor-penetration nano-platform, including IR780-loaded pH-sensitive fluorocarbon functionalized nanoparticles (SFNs) and target peptide-iRGD, has been developed (Fig. 12a).²³⁰ Through the real-time monitoring of the dissolved oxygen, a special study was carried out on the loading and release behavior of oxygen. The potential application of SFNs as oxygen storage was also studied, and the excellent O₂ loading performance and the capacity of high release were verified. An in vivo PA system and HIF-1α staining analysis were used to evaluate the hypoxia state of the tumors after the use of IR780@O₂-SFNs/iRGD, IR780@O₂-SFNs and IR780-SHN systems. The most significant change in the FL intensity of HbO₂ was in the IR780@O₂-SFNs/iRGD group (50.1%), indicating that IR780@O₂-SFNs/iRGD can transport O₂ and PS from the hydrophobic core to the deep and anoxic areas of the solid tumor, obviously improving the oxygenation of the tumor. A biodegradable hollow MoSe₂/Fe₃O₄ nano-heterostructure was used as a PDT enhancer for multimode imaging (CT/MR/IR) and synergistic anti-tumor therapy, and hence the nanoparticle O₂@PFC@MF-2@PEG/Dox was designed (Fig. 12b).²³¹ Due to effective photoinduced charge separation, MoSe₂/Fe₃O₄ can produce twice as much ROS as a single MoSe₂. On this basis, the use of a new hollow structure provides enough space for the PFC and O₂ carrier, and O₂@PFC@MF-2 can effectively overcome the anoxic microenvironment and further lead to more than 3 times the production of ROS. The anticancer drug doxorubicin (Dox) was assembled into an MF-2@PEG nanomaterial for chemotherapy so that the final O₂@PFC@MF-2@PEG/Dox realized the function of multimodal imaging and treatment. With MoSe₂/Fe₃O₄ as the PDT agent, the production ability of ROS was detected by a DCFH-DA probe, and after 808 nm laser irradiation, MoSe₂/Fe₃O₄ nanocomposites showed enhanced emission and ROS production. In order to evaluate the antitumor effect of this nano-system, the mice were classified into five groups for different treatment; the tumor volume of the O₂@PFC@MF-2@PEG + NIR and O₂@PFC@MF-2@PEG/Dox + NIR groups was significantly reduced, and the O₂@PFC@MF-2@PEG/Dox + NIR group showed the most significant tumor inhibitory effect due to the multimodal therapy (chemotherapy, PTT and PDT).

Fig. 12 (a) Structure and O₂ self-sufficient mechanism of an IR780@O₂-SFNs/iRGD nano platform. Reproduced from ref. 230 with permission from the American Chemical Society, copyright 2019. (b) The routine process of ROS production by nanoparticles under laser irradiation. Reproduced from ref. 231 with permission from the American Chemical Society, copyright 2019. (c) Schematic diagram of an O₂ delivery system for tumor imaging guidance with cooperative PDT/PTT. Reproduced from ref. 234 with permission from Dove Press Ltd, copyright 2020.

According to the advantages that IR780 can be preferentially accumulated in mitochondria and can be modified as a mitochondrial targeting agent,^232,233 Chen et al.²³⁴ designed an O₂ delivery system based on a mitochondrial liquid PFC, which was created for imaging-guided synergistic PDT/PTT of tumors (Fig. 12c). The nanoparticles can target mitochondria. Mitochondrial organelles and ROS production,²³⁵ redox state regulation and apoptosis-mediated cell death regulation play an essential role. Mitochondria are sensitive to hyperthermia and ROS, and finally induce tumor cell apoptosis by destroying the balance of mitochondrial ROS in cancer treatment; the most essential factor of applied IR780 as a PS is that it can significantly excite the triplet state of the PS and produce ¹O₂.²³⁶ So it is feasible to use a targeted mitochondrial strategy for tumor therapy. In the study of photothermal and photodynamic properties, PBS, IRP NPs and IR780 were irradiated by NIR irradiation for 5 minutes, and the temperature of the latter two groups increased to 60 °C. The temperature increased with the increase of concentration. SOSG was used to confirm the production of ¹O₂in vitro according to the fluorescence intensity. This method can verify the potential of IRP/O₂NPs as a photosensitizer. The fluorescence intensity of 5 µM SOSG containing IRP/O₂NPs (20 mg mL⁻¹) increased sharply with the extension of irradiation time (2 W cm⁻², 5 s), and increased by 4 times within 80 s. In an in vivo treatment experiment, synergistic therapy with IRP/O₂NPs (PDT/PTT), through different ways, such as the generation of ROS, damage caused by mitochondrial hyperthermic and other methods, both achieved a significant anti-tumor effect, and had good biocompatibility and minimal systemic side effects. Nanoparticles (PFH@HSC) with a PFH core, hyaluronic acid (HA) and Ce6 shell were designed.²³⁷ The photoactivation property of PFH@HSC is through destroying the structure through a “redox response” and activating it from “OFF” to “ON”; after targeting anoxic tumor tissue, laser irradiation is carried out to release Ce6 and O₂, and finally the production of more singlet oxygen boosts the efficacy of PDT. Due to the excellent imaging performance of Ce6, PFH@HSC can monitor tumor aggregation by fluorescence and PA imaging after intravenous administration into tumor-bearing mice. PFH@HSC nanoparticles showed a contrast ability for PA imaging related to the Ce6 concentration, which confirmed that PA imaging could be used to analyze the aggregation of PFH@HSC nanoparticles in tumors. They showed that PFH@HSC nanoparticles could improve the hypoxia inside the tumor by intravenous injection of PFH@HSC, using a hypoxia probe to examine both groups’ tumor sections for immunofluorescence staining. The anti-tumor effect of PFH@HSC nanoparticles on mice with MDA-MB-231 tumors was evaluated in vivo, and the redox activated and oxygen-enriched PFH@HSC nanoparticles showed the most effective tumor growth inhibition.

Recently, PDT as an in situ vaccine can be used to increase the response rate of PD-1/PD-L1 antibodies.²³⁸ Hence, a bionic nanoemulsion expressing PD-1 membrane camouflage and applied to synergistic photodynamic immunotherapy for hypoxic breast tumors was developed. The perfluorocarbons in the nanoemulsion can provide oxygen and thus act as a PDT “energy source” against hypoxic tumors. The realization of the co-delivery function of photosensitizer and PD-1 protein by the nanoemulsion confirmed the synergistic therapeutic effect of PDT and immunotherapy, and the synergistic therapeutic completely inhibited the growth of primary and distal subcutaneous 4T1 tumors. Also, the publications about PFC as an O₂ source were summaried in Table 7.

Table 7 Summary of publications about PFC as an O₂ source

Application	PSs	O2 source	Excitation requirements (excitation wavelength, power density, time)	ROS species	In vitro (cell lines)	In vivo (tumour-bearing mice)	Ref.
Abbreviations: perfluorohexane (PFH); perfluorocarbon (PFC); chlorin e6 (Ce6); adriamycin (Dox); sinoporphyrin sodium (DVDMS).
PDT	IR780	PFH	In vitro (vivo): 808 nm, 2 W cm^−2, 20 s	^1O2	MCF-7 and CT26	CT26	229
PDT	IR780	PFH	In vitro (vivo): 808 nm, 2 W cm^−2, 10 s	^1O2	CT26	CT26	224
PDT	Fluorous porphyrin 1,2	PFH	420 nm, 8.5 mW cm^−2, 30 min	^1O2	A375, MCF7, and HEK293	NO	225
PDT	Hypocrollin B	PFC	In vitro: 630 nm, 20 mW cm^−2, 300 s	^1O2	H-1299	NO	227
PDT	Ce6	PFH	In vitro (vivo): 670 nm, 0.48 W cm^−2, 10 min	^1O2	MCF-7	MCF-7	228
PDT	IR780	Fluorinated nanoplatform	In vitro: 808 nm, 2 W cm^−2, 200 s	^1O2	4T1	4T1	230
			In vivo: 808 nm, 2 W cm^−2, 5 min
PDT/PTT	MoSe2/Fe3O4, Dox	PFC	In vitro (vivo): 808 nm, 0, 0.5, 1, 1.5 W cm^−1, 10 min	˙OH	HepG2	H22	231
PDT/PTT	IR780, ICG	PFC	In vitro: 808 nm, 2.0 W cm^−2, 5 min	^1O2	4T1	4T1	234
			In vivo: 808 nm, 1.0 W cm^−2, 5 min
PDT	Ce6	PFH	In vitro: 660 nm, 100 mW cm^−2, 5 min	^1O2 + ROS	MDA-MB-231	MDA-MB-231	237
			In vivo: 660 nm, 100 mW cm^−2, 10 min
PDT	DVDMS	Biomimetic nanoemulsion	In vitro: 635 nm, 200 mW cm^−2, 5 min	^1O2	4T1	4T1	238
			In vivo: 635 nm, 200 mW cm^−2, 30 min

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3.3 Red blood cells or hemoglobin

Red blood cells (RBC) are the staple oxygen carriers of all the vertebrates, providing enough O₂ to various tissues and organs through blood transport.²³⁹ Each red blood cell contains a lot of hemoglobin molecules (Hb),²⁴⁰ which is a metal protein that binds to oxygen and plays a role in carrying oxygen.²⁴¹ Hb is a biosafe oxygen carrier and a single Hb can bind to four oxygen molecules, so it can make good use of the ability to carry oxygen for PDT.^242,243 On account of the short cycle half-life and poor stability of Hb, exposed Hb is not suitable for oxygen transport. Because of this disadvantage, people continued to explore how to make up for the shortcomings of the original poor physical properties, so there began to be the emergence of artificial red blood cell composites.²⁴⁴ It is reported that particles camouflaged with natural erythrocyte membranes can overcome biological obstacles and extend the time of blood circulation.^245–247

In a recent study, Wang et al. prepared RBC microcarriers to selectively deliver O₂ into anoxic areas.²⁴⁸ This RBC microcarrier can surmount a series of complex biological obstacles, including transport through inflamed endothelial cells and escape from the mononuclear phagocytic system. In addition, RBC microcarriers have effective tumor accumulation capacity and can transport a mass of O₂ for PDT under NIR radiation, so achieve effective solid tumor eradication. Using this strategy, a novel site-specific hypoxia probe (HP) was combined with a PDT photosensitizer (Rose Bengal, RB) to form orthogonal excitation-emission UCNPs. The author also evaluated the formation of ROS in hypoxic U87MG cells with RBC microcarriers under alternating 980 nm and 808 nm NIR irradiation with the most significant apoptosis of RBC microcarriers, indicating a large amount of O₂ release. The ability of RBC microcarriers to overcome the biological barrier was also studied before the in vivo experiment, with mesoporous silicon microcarriers as the control group (cross-linked with HP, RB-modified UCNP, and FA), and the interaction between RBC microcarriers and a (HUVEC) fusion monolayer of human umbilical vein endothelial cells under inflammatory conditions was also studied. The results show that the use of erythrocyte microcarriers can effectively bypass tumor vessels and reduce immune clearance. Vascular oxygen saturation (sO₂) in U87MG solid tumors was evaluated by PA imaging, and caudal vein injection of RBC microcarriers increased sO₂ by 23% in half an hour. The therapeutic ability of this nano-system was evaluated in U87MG solid tumor mice. The results showed that the tumor volume decreased significantly after alternating 980 nm and 808 nm laser irradiation for two weeks. For example, Guo et al. designed an oxygen transfer biocompatible photosensitive liposome (LIH), to enhance PDT against hypoxic tumors by loading the compound with oxygen-carrier Hb and photosensitizer (Fig. 13a).²⁴⁹ Liposomes loaded with ICG and Hb (LIH) showed effective tumor accumulation. The effects of laser irradiation (808 nm, 1 W cm⁻², 1 min) and different O₂ levels on the production of ¹O₂ in the liposome solution were studied in vitro. LIH can produce a large amount of ¹O₂ under normoxic or anoxic conditions. Under the state of hypoxia, LIH can down-regulate the high expression level of HIF-1α, which proves the oxygen-carrying capacity of LIH. The PDT effect of LIH in vivo was evaluated. Mice bearing CT-26 xenografts were divided into four groups, which were treated with saline, LI + NIR irradiation, LIH or LIH + NIR irradiation, respectively. The results showed that the LIH + NIR irradiation treatment group showed the best therapeutic ability, indicating that LIH + NIR irradiation promoted cell apoptosis and inhibited proliferation.

Fig. 13 (a) Schematic diagram of a nano-system for relieving tumor hypoxia and enhancing PDT. Reproduced from ref. 249 with permission from Informa UK Limited, copyright 2018. (b) The P-Hb-B NPs by virtue of the carried O₂ achieves PDT on cancer cells. Reproduced from ref. 253 with permission from Springer Nature Switzerland AG, copyright 2018.

Although the nanoparticle drug carriers are similar. The novel nano red blood cells contain oxyhemoglobin (oxy-Hb) and the gaseous generator ammonium bicarbonate (ABC).²⁵⁰ The co-loading and controlled release of ICG and Dox resulted in synergistic therapeutic effects. After NIR laser irradiation, ICG in DIRAS exerts a PTT/PDT effect towards various breast cancer cells, and its PDT efficiency will be improved due to the oxygen participation of oxy-Hb. Due to the temperature rise caused by the PTT effect, ABC can be decomposed into NH₃ and CO₂, which further contributes to the release of Dox and plays a role in ablating tumors. In vivo experiments further evaluated the efficacy of DIRAS on PTT/PDT in 4T1 tumor-bearing mice. After irradiation, tumors treated by free ICG, ICG/RAS mixture and DIAS all showed strong green fluorescence, but the ¹O₂ produced by the DIAS group was the highest. Meanwhile, the fluorescence intensity of tumors treated with ICG/RAS mixture and DIRAS was significantly enhanced after laser irradiation, and the presence of oxy-Hb in RAS and DIRAS was conducive to the enhancement of PDT efficacy by ICG. In general, DIRAS had a strong efficacy of PTT/PDT in vivo. In 2018, Liu et al. prepared an artificial RBC nanomaterial (AmmRBC) for oxygen-self-supplied tumor PDT to overcome hypoxia-mediated tumor resistance.²⁵¹ Biomembrane recombination technology has been used to produce biovesicles composed of erythrocyte membrane (RBCM), which encapsulates a complex formed between hemoglobin and biocompatible dopamine (PDA) (Fig. 14). AmmRBCs have demonstrated great ability for tumor accumulation and a high tolerance of PDT to extreme hypoxia in the tumor, resulting in complete ablation of the tumor. In general, as a highly biocompatible component in the human body, red blood cells have great application prospects in the adjuvant treatment of cancer with photodynamic therapy. Li's group designed a multifunctional nano-complex (BP@RB-HB) through the molecule assembly method.²⁵² Because of the large overlap of emission spectra between Rose Bengal (RB) and BP, RB was chosen as the FRET receptor. Under two-photon laser irradiation, RB is indirectly excited by the intra-particle FRET mechanism, thus boosting the depth of treatment. In addition, Hb in nanocomposites can bring O₂ into the tumor through active and passive targeting mechanisms, and improve the efficiency of PDT. Liu et al. synthesized an NIR photosensitizer – brominated 4,4-difluoro-4-bora-3a,a-diaza-s-indacene (BODIPY-Br₂) with high ¹O₂ production efficiency and high fluorescence emission (Fig. 13b).²⁵³ NIR photosensitizer Hb-conjugated biodegradable peptides as O₂ carriers were synthesized through a click reaction between polypeptides and Hb-BODIPY-Br₂NPs, denoted as (p-Hb-B-NPs), and near-infrared imaging-guided PDT was achieved. The oxygen-carrying capacity and tumor tracking and treatment ability of the prepared polymer nanoparticles were verified by the study of HepG2 cancer cells. The phototoxic effect was best when the concentration of p-HB-B-NPs was 4 μM, and there was no phototoxic effect under a N₂ atmosphere. In addition, it was proved that p-Hb-B-NPs also had a strong cell killing force under hypoxia and weak light irradiation (25 mW cm⁻², 10 min).

Fig. 14 Schematic illustration of AmmRBCs for tumor therapy. Reproduced from ref. 251 with permission from Wiley, copyright 2018.

Gao et al. reported an acoustically driven and magnetically navigated analog red cell (RBCM) micromotor that serves as an O₂ and PS delivery platform that can actively transport O₂ and PS.²⁵⁴ They made use of the natural cell membrane camouflage that particles display in a long circulation of blood in living organisms and prepared a new type of RBCM micromotor by wrapping Fe₃O₄ nanoparticles and ICG with hemoglobin. By co-incubating normal hepatocytes (L02 cells) with different concentrations of RBCM, it was proved that RBCM micromotors had no obvious toxic effect on hepatocytes, indicating that RBCM micromotors had good cytocompatibility. They also used a microfluidic chip to test the ability of the RBCM micromotor to target the predetermined tumor area. With propulsion from an ultrasonic field and navigation from an external magnetic field, the RBCM micromotor quickly entered the right-side reservoir along the microfluidic channel. The O₂-carrying capacity of the RBCM micromotor was evaluated by p50. Tumor killing ability was evaluated by flow cytometry. After irradiation (808 nm, 0.35 W cm⁻², 15 min), dual-oxygen-ICG-loaded RBCM nanoparticles could kill more than 75% of the cells, and an MTT assay was carried out again to confirm this result. In conclusion, the use of an RBCM micromotor as an active transmission platform can improve the anticancer effect of photodynamic therapy.

In recent years, new fluorescent materials have developed rapidly, aiming at the many advantages of conjugated polymer nanoparticles (CPNs), such as enhanced light capture ability, low cytotoxicity and excellent photostability.^255,256 Inspired by these, Jiang et al. used conjugated polymer nanoparticles (CPNs) connected with hemoglobin (Hb) to prepare a PDT system that can luminesce and supply O₂.²⁵⁷ Hb acts as a catalyst for the luminol–H₂O₂ chemiluminescence system and provides O₂. To change the state of Hb during blood circulation, Hb-NPs were coated to form Hb-NPs@liposomes. When H₂O₂ and Hb exist at the same time, luminol emits blue light in the range of 375–550 nm, while NPs based on MEH-PPV have an absorption spectrum in the 400–550 nm range, so this situation meets the requirement of CRET as a donor–acceptor pair. After absorbing luminol radiation, Hb-NPs can sensitize oxygen molecules in Hb and produce ROS to kill cancer cells. The cell survival rate with S/deoxy-Hb-NPs@liposome at all concentrations was significantly higher than that with S/oxy-Hb-NPs@liposome, which confirmed the crucial role of oxygen in PDT. They studied the relationship between nanoparticle size and efficacy and found that smaller HB-NP groups produced more ROS.

In another effort, a hybrid protein oxygen nano-carrier was uncovered, which hybridized with Hb and HSA by an intermolecular disulfide bond (C@HPOC) to enhance the resistance of PDT to tumor growth and metastasis.²⁵⁸ C@HPOC possesses targeted delivery of photosensitizer and O₂ to the tumor, which helps to enhance the infiltration of CD8 + T cells in the tumor. C@HPOC-mediated PDT can effectively inhibit tumor cell metastasis by stimulating systemic anti-tumor immunity in the 4T1 mTNBC murine model. The killing ability of C@HPOC on the tumor was investigated in vitro, with irradiation from a 660 nm laser (0.1 W cm⁻², 2 min), indicating that C@HPOC + laser exhibited high phototoxicity against 4T1 cells compared with the control groups. To investigate the tumor cell-killing effect of C@HPOC + laser in vitro, they irradiated with a 660 nm laser (2 min, 0.1 W cm⁻²) in different groups (including the control group); the results revealed that the C@HPOC + laser group showed significant phototoxicity. To figure out whether C@HPOC-mediated PDT can trigger the emergence of ICD, they used 4T1 cells treated with C@HPOC + laser and then evaluated CRT exposure, HMGB1 release, and ATP secretion. Finally, it was shown that C@HPOC has the effect of oxygen-boosted PDT. In general, oxygen-enhanced immunogenic PDT, induced by C@HPOC, ablates the primary tumor and effectively inhibits distant tumor and lung metastasis.

A new strategy that is a kind of bionic red blood cells (BRBCs) is prepared by the layer by layer assembly method, using Fe₃O₄@CuO, an oxygen donor (oxy-Hb), an anti-cancer drug (ZnPc) and a photocrosslinking acrylate modified hyaluronic acid (HA) gel shell.²⁵⁹ The authors used Fe₃O₄@CuO as a specific recruiter for oxy-Hb, and the HA gel shell can prevent oxy-Hb from releasing O₂ in the blood, but when BRBCs enter the tumor and accumulate, the HA shell will be degraded by HAase, which triggers the release of O₂ to alleviate the hypoxia of the tumor. ZnPc consumed O₂ during PDT treatment, resulting in enhanced fluorescence of intracellular probes. The fluorescence signals of the BRBC and BRBC+ magnetic treatment groups were significantly stronger than those of other groups with HAase, indicating that BRBCs can simulate the adequate oxygen supply of erythrocytes in the PDT process in hypoxic tumors. Magnetic field-assisted tumor targeting was proved in vivo, and the accumulation of BRBCs in vivo with or without a magnetic field was studied and compared. With the same fluorescence excitation conditions, strong ZnPc fluorescence signals were detected in tumor tissues with or without a magnetic field, but the fluorescence signal intensity with BRBC + magnetism was significantly higher than that in the control group. The data show that a magnetic field can enhance tumor-targeting efficiency, and BRBCs have excellent performance in tumor targeting. Also, the publications about RBC or Hb as an O₂ vehicle were summaried in Table 8.

Table 8 Summary of publications about RBC or Hb as an O₂ vehicle

Application	PSs	ROS species	Excitation requirements (excitation wavelength, power density, time)	ROS indicator	In vitro (cell lines)	In vivo (tumour-bearing mice)	O2 carrier	Ref.
Abbreviations: 1,3-diphenylisobenzofuran (DPBF); man-made pseudo-RBCs (mmRBCs); Rose Bengal (RB); probe for reactive oxygen species (ABDA); acoustically powered and magnetically navigated red blood cell-mimicking micromotor (RBCM); hemoglobin (Hb)-linked conjugated polymer nanoparticles (CPNs); zinc phthalocyanine (ZnPc).
PDT	Rose Bengal	^1O2	In vitro: 980 nm (0–30 min)	DPBF	U87MG	U97-MG	RBC	248
			In vivo: 980 nm (2 W cm^−2, 15 min) & 808 nm (1.5 W cm^−2, 15 min)
PDT	ICG	^1O2	In vitro: 808 nm, 1 W cm^−2, 1 min	SOSG	CT-26	S180 and CT-26	Hb	249
			In vivo: 808 nm, 1.5 W cm^−2, 3 min
PDT/PTT	ICG/Dox	^1O2	In vitro (vivo): 808 nm, 2.0 W cm^−2, 10 min	SOSG	4T1, HUVECs	4T1 and 4T1-Luc	Hb	250
PDT	MB	^1O2	In vitro (vivo): 808 nm, 2.0 W cm^−2, 10 min	DCFH-DA, COS7	4T1	4T1	mmRBC	251
PDT	RB	^1O2	800 nm, 390 mW	ABDA	MCF-7	MCF-7	Hb	252
PDT	BODIPY	^1O2	In vitro: 635 nm, 25 mW cm^−2, 10 min	DCFH-DA	HepG2	NO	Hb	253
PDT	ICG	^1O2	In vitro: 808 nm, 0.35 W cm^−2, 15 min	ABDA	HeLa and L02	NO	RBCM	254
PDT	NPs	^1O2	Chemiluminescence of luminol	DCFH	HeLa and HCT-8	—	CPNs	257
PDT	Ce6	^1O2	In vitro: 660 nm, 0.1 W cm^−2, 0.5 min	SOSG	4T1	4T1	Hb	258
			In vivo: 660 nm, 0.1 W cm^−2, 30 min
PDT	ZnPc	^1O2	In vitro: 665 nm LED, 5 min	DCFH-DA, Ru(dpp)3 Cl2	A549	A549	Oxy-Hb	259
			In vivo: 665 nm LED, 2 W, 30 min

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3.4 Hyperbaric oxygen

Hyperbaric oxygen (HBO) as a clinical adjuvant therapy has been used to cure carbon monoxide poisoning, and in wound healing, soft tissue infection and other diseases.^18,260,261 As an adjuvant therapy, it has many advantages, such as the ability to deliver oxygen to tissues without hemoglobin and the ability to reduce tumor interstitial fluid pressure (IFP). HBO can solve the problem of the slow diffusion rate and short diffusion distance of oxygen in tissue.²⁶² It can effectively oxygenate or even alleviate the anoxic area of the tumor.²⁶³ If the hypoxia environment is not changed, the therapeutic effect will be reduced. For tumor tissue hypoxia, hyperoxia to compensate for oxygen depletion is a good way to improve hypoxia. Hyperbaric oxygen can provide extra oxygen in tumor tissue by adding oxygen or substances or other molecules that can produce O₂. Another way is by modifying the structure of photosensitizers, making light-dependent photosensitizers into light-independent photosensitizers (Type II to Type I).

At present, there are few studies on the use of HBO combined with PDT in the treatment of human squamous cell carcinoma (SCC), aiming at the advantages of HBO combined with PDT. Hence, Fang et al. proposed a method using HBO combined with 5-aminolevulinic acid PDT to inhibit the proliferation of human squamous cell lines.²⁶⁴ It was found that the combination of HBO and PDT could not only inhibit the proliferation of A431 cells in vitro but also down-regulate Bcl-2 to significantly enhance PDT-induced apoptosis and autophagy. Autophagy plays a survival-promoting role in apoptosis. Therefore, the combination of HBO and PDT may be a promising treatment method for human squamous cell carcinoma. The combination of HBO and ALA-PDT cooperatively up-regulated the proteins Bax and active caspase 3, and down-regulated Bcl-2 in A431 cells. Western blot tests detected the expression of apoptosis-related proteins (Bax, Bcl-2, and caspase 3), and the results indicated that the expression of Bax and caspase in A431 cells increased cooperatively. In contrast, the expression of Bcl-2 decreased, indicating that apoptosis is an intrinsic pathway of mitochondria. The authors also reduced the production of ROS in A431 cells by dual ALA-HBO treatment, which was conducive to autophagy. In general, ALA-HBO is one of the promising methods for the treatment of human squamous cell carcinoma.

Due to the hypoxia-related environment and the difficulty of photosensitizers penetrating tumor cells away from blood vessels, the therapeutic effect is limited in deep tumors. To address this problem, Yang's group proposed a therapeutic strategy of upconverting nano-sensitizers (UNPSs) and HBO to reshape the extracellular matrix to enhance PDT (Fig. 15).²⁶⁵ UNPSs are designed to have a Nd³⁺-sensitized sandwich structure, in which the up-conversion core acts as a light transducer to transfer energy to an adjacent photosensitizer to produce ROS. The hyperbaric oxygen-assisted PDT process promotes oxygen diffusion by decomposing collagen in the tumor extracellular matrix, and promotes the penetration of UNPSs to the deep tumor. They evaluated the in vitro PDT efficacy of UNPS using MTT analysis, and cell viability was significantly reduced with increasing UNPS concentration after 808 nm laser irradiation and HBO application. HBO adjuvant PDT with UNPSs can improve the efficacy of cancer treatment. HBO-assisted PDT with UNPS was used to carry out an experiment on the mouse model of xenotransplantation. Firstly, the metabolism of UNPSs was studied; after intravenous injection, fluorescence could be detected after 6 hours and disappeared after 48 hours. They classified different treatment groups, with the group exposed to the laser being exposed for 16 minutes at 24 and 48 hours after administration; by calculating the tumor volume, the therapeutic effect could be directly reflected according to the change of tumor volume. However, the UNPSs + NIR + HBO group had the most dramatic treatment effect. In short, HBO-enhanced PDT using UNPSs represents not only a promising choice for more efficient treatment of tumors, but also a new strategy. Also, the publications about HBO as an O₂ source were summaried in Table 9.

Fig. 15 Schematic illustration of the PDT process with or without HBO; the synergistic therapy could relieve hypoxia and enhance the effect of PDT. Reproduced from ref. 265 with permission from the American Chemical Society, copyright 2018.

Table 9 Summary of publications about HBO as an O₂ source

Application	PSs	O2 source	Excitation requirements (excitation wavelength, power density, time)	ROS indicator	In vitro (cell line)	In vivo (tumor-bearing mice)	Ref.
Abbreviations: precursors of protoporphyrin IX (ALA).
PDT	ALA	HBO	630 ± 15 nm, 5 J cm^−2, 8 h	Detection kit	A431	NO	264
PDT	RB	HBO	In vivo: 808 nm, 0.75 W cm^−2, 16 min	DCFH-DA	4T1	4T1	265

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4. Summary and outlook

PDT shows great potential in clinical research and application by improving treatment efficiency with minimal invasiveness through the precise management of laser irradiation compared with traditional oncology therapies. However, hypoxia seriously limits the efficiency of PDT and leads to tumor invasion and metastasis. Therefore, the presence of adequate molecular oxygen throughout the tumor volume is critical for effective PDT, the mechanism of using photosensitizers activated by light to produce cytotoxicity (biological reactive oxygen species).

The maldistribution of blood vessels leads to highly uneven oxygen levels within the tumor, which in this case leads to the presence of severe hypoxia (deep tissue hypoxia). For drug therapy or PDT, tumor cells that lack adequate molecular oxygen are resistant to PDT in the presence of either drug therapy or photodynamic therapy. Therefore, tumor hypoxia limits the widespread clinical application of PDT to overcome the “Achilles’ heel”; so far, many strategies have been carried out to overcome hypoxia. This review paper summarizes methods for producing molecular oxygen in situ or carrying molecular oxygen into anoxic tumor areas through different carriers to improve tumor hypoxia, and this is systematically summarized and discussed. These oxygen replenishment strategies and in situ oxygen production strategies are described as follows.

• Enhance tumor oxygen delivery by utilizing hemoglobin, red blood cells, and PFCs;

• Oxygen is produced by catalyzing H₂O₂ with different catalysts;

• Light-driven water splitting increases in situ oxygen production;

• Changing blood flow in different ways increases oxygen transport;

• Oxygen is produced by photosynthesis near the tumor tissue.

These strategies significantly alleviate the hypoxia of the tumor microenvironment and improve the efficacy of PDT, and the relief of hypoxia can also enhance the therapeutic effect of some drugs. However, there are still several issues that need to be noted in terms of the disadvantages of the supplementary oxygen strategy as described below:

(1) Problems with low endogenous hydrogen peroxide content

For the in situ generation of O₂ within tumors, the amount of endogenous H₂O₂ is only 50–100 μM, so this will not achieve the goal of catalyzing endogenous hydrogen peroxide in tumor tissue with exogenous catalysts (commonly used metal ions). Therefore, this also makes the oxygen supplementation approach to PDT more difficult, which is a major drawback of the supplemental oxygen strategy. On a more optimistic note, the novel strategy of delivering exogenous H₂O₂ into tumors and using catalysts to decompose exogenous H₂O₂ into O₂ was used to solve the problem of low endogenous hydrogen peroxide content.

(2) Toxicity of metal catalysts

After solving the problem of a low content of endogenous hydrogen peroxide, another major disadvantage of the oxygen supplement strategy to improve the therapeutic effect of PDT is the toxicity of metal catalysts. MnO₂, Pt and other catalysts are often used to catalyze hydrogen peroxide for oxygen production. In addition to catalysis, these catalysts will also produce some uncontrollable cytotoxicity, such as binding with some enzymes, resulting in enzyme inactivation. Therefore, it may bring a series of problems; moreover, metal ion metabolism is also difficult, which may bring some uncontrollable side effects.

(3) The difficulty of synthesis of oxygen-delivery materials

As a complementary strategy for exogenous oxygen delivery to the tumor system, the selection of materials should be more scientific, meet better biocompatibility, and target tumor cells. Therefore, materials with better properties need to be used. These materials include: biomaterials, such as fungi, white cell membranes, cell membranes, red blood cells and hemoglobin; and chemical materials: perfluorocarbons. For photosensitive bacteria hybridized with cyanobacteria and photosensitizers with different properties for PDT therapy of photosynthesis-enhanced tumors under light stimulation, this method of in situ generation of oxygen has been recently proposed as an effective method to alleviate tumor hypoxia, but the preparation process of cyanobacteria is tedious and takes a lot of time, and surgery is needed to implant photosensitive bacteria in biological applications. If this method leads to poor wound healing there will be large scars, so in later studies, photodynamic therapy can be used to remove scars and be combined with the treatment process, so as to shield some shortcomings. For the extraction of cell membranes, the process is also more tedious, therefore, to realize the engineering of extraction process will greatly promote the application of materials. Chemical materials can be difficult to synthesize, and some materials require multi-step synthesis, which will make it more difficult to supplement oxygen therapy strategies.

(4) Excessive supplemental oxygen accelerates tumor blood vessel growth

In the human body, blood vessels can provide O₂ and nutrition for the growth of all tissues, but tumors grow much faster than normal tissues, so the demand for nutrition is higher. In this state, tumor cells begin to produce growth factors that stimulate the growth of new blood vessels, resulting in abnormal vascular morphology. Abnormal vascular morphology leads to poor blood flow and reduced O₂ supply. The insufficient supply of oxygen will cause cancer cells to metastasize and eventually form a malignant tumor. In addition, abnormal vascular morphology also makes anticancer drugs unable to reach the focus, which greatly reduces the therapeutic effect. For an oxygen supplementation strategy, oxygen is directly transported into the tumor tissue through hyperbaric oxygen. Oxygen supplements should be in an appropriate range, otherwise excess oxygen on the one hand increases oxygen pressure and brings a lot of side effects in the process of PDT. On the other hand, excess oxygen will promote the rapid growth of tumor blood vessels. Then the biggest problem will be how to balance the appropriate amount of oxygen to alleviate tumor tissue hypoxia and oxygen to promote tumor growth. More and more research data reveal that direct delivery of oxygen has certain limitations. Therefore, for the application of this method of oxygen supplementation there is a need to constantly explore how to monitor the partial pressure of oxygen and supplement the effective concentration of oxygen in the tumor tissue. Once mastered, it will become easy to balance the above problems.

Besides, other new strategies can be vigorously developed, including:

(1) Chemotherapy in combination with PDT

Nowadays, chemotherapy is still the main treatment for most cancers. However, during chemotherapy, cancer cells may develop resistance, which directly reduces the effectiveness of the treatment. Chemotherapy and PDT combined with adjuvant therapy can produce a synergistic effect, and the treatment results between them can complement each other; the combined effect can reduce the drug dose and systemic toxicity. At present, the most commonly used chemotherapy drugs are as follows: tipacamine (TPZ), Dox, AQ4N, platinum drugs, and so on.

(2) Antiangiogenic PDT

PDT also induces abnormal expression of VEGF, COX-2, and MMPs. The increased expression of HIF-1α in the tumor after PDT directly induces photodynamic drug resistance, with a high expression of VEGF. The combination of HIF-1α or VEGF inhibitors with PDT can prevent drug resistance and even improve therapeutic efficacy.

(3) PTT/PDT

PTT is targeted to tumor tissue by the photosensitizer. A low-power beam can be applied to tumor tissue to generate heat energy and use the heat to ablate the tumor. PDT and PTT both need to use irradiation and a photosensitizer, so the combined use of the photosensitizer can achieve dual functions simultaneously. The PTT process will also increase blood flow and alleviate the aggravation of hypoxia caused by the PDT process.

(4) Immunotherapy combined with PDT

Because an anti-tumor immune response can be initiated in the process of PDT, its effect on tumor ablation plays a very important role. Therefore, it has become a promising clinical model for cancer treatment. Multiple pathways have been reported for PDT to initiate anti-tumor immune responses, including:

◆ up-regulation of the expression of transcription factor nuclear factor-kB (NF-kB) and molecular chaperone heat shock protein 70 (HSP-70);

◆ promoting antigen presentation to cytotoxic T lymphocytes (CTL);

◆ stimulating the immune response of the host by promoting the secretion of cytokines.

Author contributions

Shuheng Qin: conceptualization, data curation, investigation, writing – review & editing; Yue Xu, Hua Li: supervision, visualization, formal analysis; Zhenwei Yuan, Haiyan Chen: supervision & guidance, validation, reviewing & editing.

Conflicts of interest

The author claims that they do not have competing interests for this paper.

Acknowledgements

This work was financially supported by Natural Science Foundation Committee of China (NSFC 82001891), the Post-doctoral Innovative Talent Support Program (BX20190389), China Postdoctoral science Foundation (2019M662009), and Postdoctoral Research Grant of Jiangsu Province.

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