Application of nanotechnology for enhancing photodynamic therapy via ameliorating, neglecting, or exploiting tumor hypoxia

Photodynamic therapy (PDT), an oxygen‐dependent modality, has been clinically approved and is considered a promising approach for cancer treatment. However, PDT shows noticeable limits due to the hypoxic nature of most solid tumor microenvironments. In recent years, various strategies have been developed to overcome tumor hypoxia either via oxygen‐replenishing approaches or via diminishing oxygen dependence. Both of these approaches have shown promise in reversing hypoxia‐relevant PDT resistance and thus improve antitumor efficacy. However, the low oxygen level at the tumor site is also an opportunity for the development of new therapeutic modalities, such as hypoxia‐activated chemotherapy, hypoxia‐inducible drug release, and starvation therapy. Therefore, a combination of these therapeutic modalities with PDT could promote their synergetic efficacy. Herein, we present an overview of the recent trend in the modulation and utilization of tumor hypoxia via nanomedicine‐based strategies, followed by a summary of the design and mechanisms of these nanosystems to improve PDT. Finally, current challenges and future perspectives for how PDT can achieve more extensive clinical applications for cancer therapy are discussed.

modality with minimal invasiveness, low systemic toxicity, and absence of initial resistance, which has been considered a promising approach for cancer treatment. 2 The mechanism of PDT is that the tumor-localizing nontoxic photosensitizer is excited by harmless light within a specific wavelength range, followed by transference of energy, a proton or an electron to generate reactive oxygen species (ROS), usually cytotoxic singlet oxygen ( 1 O 2 ). 3 Subsequently, the generating ROS oxidizes the essential cellular macromolecules that directly lead to tumor cell apoptosis or necrosis, shuts down the tumor vasculature and further affects the immune system. 4 Generally, PDT can be classified into type I PDT and type II PDT according to two different photochemical reaction pathways. Unlike chemotherapeutic drugs that induce systemic toxicity or ionizing radiation from radiotherapy that damages neighboring healthy tissues, the three essential elements of PDT (light source, photosensitizer, and molecular oxygen) make PDT not harmful to the overall biological system. Apart from the approval required for non-cancerous disorder treatments, such as age-related macular degeneration with verteporfin (Visudyne ® ), and actinic keratosis with aminolevulinic acid (Levulan ® ), many photosensitizers appropriate for PDT have been already approved for clinical trials or applications in early and obstructive lung cancer, esophageal cancer, superficial bladder cancer, epidermal cancer, head and neck cancers, prostate cancers, and so on. 5 Despite its numerous outstanding merits and rapidly growing applications, PDT has not yet been recognized as a clinical first-line cancer therapeutic modality due to several limitations. These limitations include the lack of an ideal photosensitizer, identification of a suitable dosimetry (e.g., total light dose, light exposure time, or light delivery mode), the difficulties in monitoring the treatment outcomes, and the limited depth of tissue penetration. 6 The emergence of nanotechnology in PDT has opened up a new field to resolve some of the challenges associated with traditional photosensitizers. 2, 7 The poor performance of conventional pharmaceuticals is related to their intrinsic physicochemical properties and low tumor accumulation. 8 The unique characteristics of nano-carriers enable their cargos to be attractive candidates for drug delivery and tumor targeting. 9 However, the nanomaterial-mediated PDT is still highly oxygen-dependent since the Type 2 mechanism in PDT is more dominant, which means participation and consumption of oxygen during the PDT process. Unfortunately, insufficient oxygenation, also named hypoxia, is the unique inherent feature of most malignant tumors caused by aggressive cell proliferation and dysfunctional blood vessel formation. Hypoxia plays a crucial role in the hostile tumor microenvironment (TME) and greatly influences the treatment outcomes for therapies with oxygen involved as a critical element in killing tumors, such as chemotherapy, radiotherapy, and PDT.
Given the crucial role of hypoxia in tumor progression and its resistance to therapy, numerous efforts have been dedicated to overcoming the hypoxia-relevant limitations of PDT. Alleviating tumor hypoxia by an oxygen-replenishing strategy, which is a direct way to strengthen PDT, has been widely explored. 10 In addition, some alternative and straightforward strategies that can diminish oxygen dependence may also be a promising route for hypoxic tumor PDT. 11 Hypoxia is also an opportunity for the development of new therapeutics. In recent years, the low oxygen level and further aggravated hypoxia after PDT have also been exploited as a therapeutic target for selective cancer therapy or as a trigger to activate the cytotoxicity of bioreductive drugs and to induce the degradation of the nanostructure. 12 From this view, we herein summarize the recently reported approaches and strategies that have been focused on tumor hypoxia for enhancing PDT efficacy ( Figure 1). These approaches can be roughly classified into three categories: (a) employing oxygen-replenishing strategies to alleviate tumor hypoxia by improving the intratumoral blood flow, harnessing the hostile TME on the molecular level, generating oxygen in situ, delivering exogenous oxygen to tumors, or reducing the oxygen consumption during PDT, (b) developing an innovative new PDT to diminish oxygen dependence via fractional light irradiation and reducing the light fluence rate, oxygen-independent Type Ⅰ PDT, enhancing the 1 O 2 quantum yield, or combining one or more of these approaches with some other oxygen-independent cancer therapeutics, and (c) utilizing the inherent tumor hypoxia and magnified hypoxia after PDT, and then combining with some hypoxia-activated bioreductive therapy, hypoxia-sensitive moieties in the nanoscale vehicles, or cancer starvation therapy. After that, we discuss the above strategies in detail through design, working mechanisms, and application examples. Finally, we aim to point out the current challenges and outlook from our perspective on hypoxic tumor PDT. We seek to stimulate some new ideas and inspire ongoing effort in this field to achieve practical applications in the future.

AMELIORATING TUMOR HYPOXIA
PDT can be classified into type 1 and type 2 PDT based on the different photochemical reaction mechanisms (Figure 2). 13 For type 1 PDT, the triplet excited photosensitizer ( 3 PS * ) directly interacts with a biological substrate to form free radicals by transferring a proton or an electron. These formed radical anions or cations can then react with triplet ground state molecular oxygen ( 3 O 2 ) or water (H 2 O) to generate oxygenated products such as superoxide anion radicals (O 2 •− ), hydroxyl radicals (⋅OH), and hydrogen peroxides (H 2 O 2 ), which interfere with biological molecules, such as  Type I and Type II photodynamic therapy (PDT) lipids, proteins, and DNA, and eventually kill cancer cells. 14 Alternatively, for type 2 PDT, the 3 PS * transfers its energy directly to the surrounding 3 O 2 to form cytotoxic 1 O 2 . 15 Both type I and type II reactions can occur simultaneously, and the ratio of the two depends on several factors, for example, photosensitizer type, molecular oxygen concentration, and substrate category. In most cases, the type 2 reaction plays a dominant role in PDT, and thus PDT efficiency is determined by oxygen supply. Given this fact, directly promoting the oxygenation status within tumors is the most effective way to reverse the PDT resistance of hypoxic tumors. Such efforts will be discussed below.

Increasing the intratumoral blood flow
It is difficult for oxygen molecules to diffuse to primary avascular tumors or their metastases, and thus hypoxia occurs. However, the abnormal tumor microvasculature and chaotic blood flow always fail to rectify such an oxygen deficit. 12a Therefore, improving intratumoral blood flow has become a practical approach to increase oxygen levels in tumors. It has been reported that mild hyperthermia, about 43 • C, can enhance the intratumoral blood flow and lead to an elevation of the oxygen level in local tumors. 16 Accordingly, preor cotreatment of the tumor with mild heating is a favorable approach to enhance the hypoxic tumor PDT.
Photothermal therapy (PTT) is another phototherapeutic where the photothermal agents absorb light and dissipate the absorbed energy through non-radiative decay (heating), followed by a temperature rise in the local treatment environment resulting in irreversible cellular damage. 17 Inspired by the remarkable photothermal effect of some nanomaterials under irradiation, PTT has been introduced to increase intratumoral blood flow and thus improve tumor hypoxia for sensitizing oxygen-dependent therapeutics, including chemotherapy, 18 radiotherapy, 19 and PDT. 20 In 2013, Zheng's group reported a novel nanostructured porphyrin assembly, which converts the singlet oxygen-generating mechanism in PDT (porphyrin monomer, Photofrin) to the PTT mechanism (porphysome). Using an in vivo hypoxic tumor model, they demonstrated that the oxygen-independent PTT successfully overcomes the hypoxic conditions to achieve effective ablation of solid tumors, which could be an alternative for treating hypoxic F I G U R E 3 (A-D) Schematic illustration of the synthetic process, the working principles, and in vivo effect of GDYO@i-RBM for simultaneously overcoming diffusion-limited and perfusion-limited hypoxia. Reproduced with permission. 22 Copyright 2019, American Chemical Society. (E-G) Schematic illustration of thermal-modulated blood flow and oxygen saturation for photodynamic therapy (PDT). The blood flow velocity and oxygen saturation of tumor tissues in 4T1 tumor-bearing mice at 37 • C and 43 • C. Reprinted with permission. 23 Copyright 2016, Elsevier. Copyright 2016, Elsevier. (GDYO = graphdiyne oxide; RBM = red blood membrane) tumors. 21 Xu and Wang et al. recently developed biomimetic ultrathin graphdiyne oxide (GDYO) nanosheets coated with iRGD (CRGDKGPDC) peptide-modified red blood membrane (i-RBM), designated as GDYO@i-RBM. 22 In this nanosystem, the GYDO nanosheets efficiently catalyzed H 2 O oxidation to evolve oxygen to relieve diffusion-limited hypoxia, thereby generating abundant cytotoxic 1 O 2 under near-infrared irradiation (660 nm). Meanwhile, GDYO with its excellent light-to-heat conversion performance simultaneously enhanced tumor reoxygenation and blood perfusion to overcome perfusion-limited hypoxia for PDT ( Figure 3A-D). Consequently, the GDYO@i-RBM can relieve both diffusion-limited and perfusion-limited hypoxia and exhibit an efficient PDT for hypoxic tumors.
In addition to PTT, Cai's group fabricated a warm water bath (43 • C) to simulate a "Hot Spring" and thus investigate a thermal-modulated ROS strategy for cancer PDT using human serum albumin-chlorin e6 nanoassemblies (HSA-Ce6 NAs) ( Figure 3E). 23 Their study indicated that when the mice body temperature reaches 43 • C from 37 • C, the photosensitive reaction rate of HSA-Ce6 NAs accelerates about 20%, the blood flow velocity increases from 17.3 to 32.4 cm/s, and then the oxygen saturation in tumors increases from 52% to 79%, resulting in better tumor PDT efficacy ( Figure 3F,G). It is worth noting that many photosensitizers exhibit dualfunctionality for PDT and PTT. 24 Hence, the use of such photosensitizers with synergistic PDT/PTT effects would promote tumor blood flow and oxygenation and enhance the PDT efficacy against cancer cells.

Regulation of the tumor microenvironment on the molecular level
Several molecular abnormities in TME have been the target for regulating tumor hypoxia. For example, in contrast to the "heating" strategy discussed in the preceding section, antiangiogenic treatment is also a promising strategy to normalize the tumor microvasculature and thus increase the intratumoral blood perfusion. 25 As we know, upon PDT treatment, the tumor microvasculature rapidly shuts down partially or entirely, resulting in blood flow stasis and fast depletion of the local oxygen supply. 26 Very recently, in an investigation by Nie and his colleagues, a biomimetic PDT agent combined a tumor-targeted photosensitizer with glutathione (GSH) scavenging and anti-angiogenesis therapy was developed. 27 In this system, a porphyrinic zirconium-metal-organic framework (MOF) nanoparticle was used simultaneously as the photosensitizer and the delivery vehicle for the chemotherapeutic apatinib. Apatinib is an FDA-approved anti-angiogenic agent that shows a strong inhibition effect towards vascular endothelial growth factor receptor 2 (VEGFR2). In this design, apatinib neutralizes the PDT-induced revascularization and prevents tumor progression. After the nanoparticles selectively accumulate into tumors via the homotypic targeting effect, the combination of enhanced PDT and antiangiogenic drugs significantly improves tumor inhibition efficiency.
The transcription factor hypoxia-inducible factor 1 (HIF-1) is identified as a master regulator that adjusts cellular responses to changes in tissue oxygenation. 28 HIF-1 is a heterodimer consisting of the oxygen regulated subunit HIF-1 and the oxygen-independent subunit HIF-1 . In normoxic cells, the HIF-1 subunit undergoes proteasome degradation with inactive HIF-1. However, under hypoxic conditions, HIF-1 translocates to a nucleus where it dimerizes with HIF-1 to form HIF-1 and initiates gene transcription. In other words, under hypoxic conditions, the activated HIF-1 would upregulate a sequence of hypoxia-responsive transcription enzymes and proteins associated with tumor progression, proliferation, invasiveness, and metastasis. Therefore, various strategies that focus on targeting and downregulating the HIF-1 expression have been reported to enhance the PDT outcome, which utilizes HIF-1 inhibitors. 29 Broekgaarden et al. reported a small-molecule inhibitor of HIF-1 acriflavine-loaded cationic liposomes to inhibit the expression of HIF-1 and other hypoxia-related proteins to sensitize the PDT-mediated anticancer efficacy against human epidermoid carcinoma cells. 30 Acriflavine can effectively block HIF-1 /HIF-1 dimerization by binding HIF-1 at its Per-Arnt-Sim dimerization domain, thereby reducing the tumorigenicity of cancer cells. After A431 cells were treated with acriflavine, the HIF-1 expression was significantly suppressed. The hypoxic tumor cells are sensitized via PDT, and subsequently, the in vitro apoptosis of tumor cells is exacerbated. Other recent studies confirmed that the HIF-1 inhibition by acriflavine, 31 rapamycin, 32 and curcumin 33 could effectively sensitize PDT and radiotherapy. In another recent study by Liang and Wang et al., HIF-1 siRNA was loaded into a cationic porphyrin-grafted lipid microbubble. The microbubbles can be efficiently converted into nanoparticles in situ under ultrasound treatment, which facilitates the accumulation of photosensitizer and siRNA at the tumor site through the cavitation effect. 34 HIF-1 siRNA in this system can downregulate the hypoxia-induced HIF-1 level and enhance the PDT against triple-negative breast cancer. Also, other nano-strategies have been demonstrated as practical approaches to relieve tumor hypoxia for better PDT, such as downregulating carbonic anhydrase IX, a hypoxiarelated enzyme known to be a biomarker of poor prognosis, and decomposing hyaluronan, which was one of the primary factors contributing to the dense stroma or extracellular matrix. 35

Catalase-based decomposition of H 2 O 2
The elevated level of H 2 O 2 , a characteristic aberrance of cancer cells, is a key signaling molecule in various physiological processes, including cell growth, cell proliferation, and tumor metastasis. 36 The endogenous enzyme catalase (CAT) protects cells from oxidative damage by specifically catalyzing H 2 O 2 decomposition to generate oxygen and H 2 O. In this regard, a CAT-based nanoplatform is considered a promising candidate for catalyzing the decomposition of intratumoral H 2 O 2 , thereby relieving tumor hypoxia. 37 In 2015, Chen et al. reported H 2 O 2 -activatable and oxygen-evolving core-shell nanoparticles (HAOP⋅NP) by co-encapsulating the photosensitizer methylene blue and CAT into a poly(lactic-co-glycolic acid) (PLGA) core and black hole quencher-3 (BHQ-3) doped into the PLGA shell. 38 After HAOP⋅NP was internalized into tumor cells, the excessive intracellular H 2 O 2 penetrated the core and generated oxygen catalyzed by CAT, resulting in PLGA shell rupture and release of the photosensitizer methylene blue. BHQ-3 in this system effectively reduced the nonselective damage to the healthy cells. This work presented a novel PDT paradigm for solid hypoxic tumors with continuous oxygenation.
In the past few years, Liu's group and their collaborators also developed a series of CAT-based nanoparticles for hypoxic tumor PDT, including (a) chemotherapeutics paclitaxel (PTX) induced co-assembly of albumin/CAT, forming smart multifunctional HSA-Ce6-CAT-PTX nanoparticles for deep intratumoral penetration, hypoxia relief, and combined PDT/chemotherapy, 39 (b) CAT modification strategy by in situ free-radical polymerization with photosensitizer meso-tetra(p-hydroxyphenyl) porphine as the cross-linker for developing multifunctional nano-theranostics with strengthened enzymatic stability and enhanced PDT efficacy, 40 and (c) bladder intravesical instillation-based PDT with self-assembled nanoparticles (CAT-Ce6/F-PEI), formed by mixing fluorinated polyethyleneimine (F-PEI) with Ce6-conjugated CAT (CAT-Ce6). 41 Recently, Phua et al. developed a novel therapeutic system based on CD44 targeting hyaluronic acid (HA), CAT, and adamantane-modified Chlorin e6 (aCe6). 42 In their design, -cyclodextrin was first functionalized onto HA, followed by conjugation with CAT to yield HA-CAT NPs. The integration of CAT with HA effectively protected the CAT from the interaction of proteinase K, and thus the physiological protein stability was much improved. Meanwhile, photosensitizer Ce6 was modified with adamantane to synthesize aCe6, which was then loaded into HA-CAT NPs, forming the named HA-CAT@aCe6 NPs. After HA-CAT@aCe6 NPs actively targeted to the CD44 receptor overexpressed MDA-MB-231 cancer cells, the loaded CAT was able to efficiently catalyze H 2 O 2 to generate additional oxygen toward the ROS production for PDT upon 660 nm light irradiation ( Figure 4A). Significant tumor regression was observed in the HA-CAT@aCe6 NP group in comparison to the control system without loading CAT ( Figure 4B). This approach could be extended to other protein-based therapeutics or enzymes.

MnO 2 -based and other catalase-like-based decomposition of H 2 O 2
In addition to CAT, biodegradable manganese dioxide (MnO 2 ) as an oxygen-replenishing material has attracted wide attention for hypoxic tumor treatment, thanks to its responsiveness toward excessive acid and H 2 O 2 in TME (MnO 2 + H + + H 2 O 2 → Mn 2+ + H 2 O + O 2 ). 43 For example, Prasad et al. first developed colloidal MnO 2 nanoparticles through a one-step method by reducing manganese permanganate into MnO 2 with the cationic polyelectrolyte poly(allylamine hydrochloride) as reductant and stabilizer. 44 The MnO 2 colloidal dispersions presented an average size distribution of 15 nm and zeta potential of +30 mV ( Figure 4C). After that, according to the synthesis process of colloidal MnO 2 , our group fabricated a MnO 2 -based hybrid nanosystem, CDM NPs, for oxygen-generating synergetic chemo-PDT. In this study, we hierarchically assembled doxorubicin (DOX), Ce6, and colloidal MnO 2 into an amphiphilic block copolymer poly( -caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly( -caprolactone-co-lactide) (PCLA-PEG-PCLA) by a water-in-oil-in-water (W/O/W) double emulsion solvent evaporation method. 45 After encapsulation in the nanoparticles, the positive-charged MnO 2 showed excellent colloidal stability and biocompatibility without a reduction of the TME responsiveness. This in situ oxygen generation strategy relieved tumor hypoxia and thus, PDT efficacy was significantly improved under 660 nm laser irradiation ( Figure 4D). Together with the chemotherapy by DOX, the tumor inhibition efficiency was dramatically increased in the MCF-7 tumor-bearing mice model. More importantly, taking advantage of the fluorescence and photoacoustic (PA) imaging property of Ce6 and the T 1 -weighted magnetic resonance imaging (MRI) property of Mn 2+ , 46 a degradation product of MnO 2 during the reaction with H + and H 2 O 2 , CDM NPs can achieve tri-modal real-time imaging. More recently, Yang et al. reported a similar regenerated silk fibroin (SF)@ MnO 2 nanocomplex co-loaded with indocyanine green (ICG) and DOX for an in vivo MRI/fluorescence imaging-guided tri-modal PTT/PDT/chemotherapy for 4T1 breast cancer. 47 Other MnO 2 -based nanostructures, such as nanosheets, 48 hollow nanospheres, 49 mesoporous carbon-manganese nanocomposite, 50 and other nanocomposites integrated with different types of nanostructures, 51 have also been reported by many research groups for tumor hypoxia relief and enhanced antitumor efficacy.
Also, some inorganic catalysts show CAT-like activity for catalyzing endogenous H 2 O 2 into oxygen in the TME and then promote anticancer outcomes for hypoxic tumors. Examples include novel DNA nanosponges, 52 ruthenium (Ru) nanozymes, 53 platinum (Pt) nanozymes, 54 mesoporous hollow cerium oxide nanoparticles, 55 magnetofluorescent carbon dot assemblies, 56 Pd@Pt nanosystems, 57 gold nanoparticles, 58 mesoporous copper/manganese silicate nanospheres, 59 and Prussian blue. 60 Each of these systems has been studied to decompose H 2 O 2 into oxygen within the TME. 61

Water splitting
Apart from the above strategies for decomposing intracellular H 2 O 2 into oxygen, researchers have innovatively fabricated water-splitting nanocomposites mimicking the photosynthesis process where chloroplasts in green plants absorb light energy and transfer it to H 2 O to produce large amounts of oxygen with high efficiency. Such materials include calcium peroxide (CaO 2 ) nanoparticles and carbon nitride (C 3 N 4 ) nanocomposites with specific modifications. 62 Zhang and co-workers developed a variety of nanomaterials utilizing water-splitting by the photocatalytic reaction to trigger oxygen generation for enhanced PDT. 63 In a recent study from Zhang's laboratory, a two-photon excited nanocomposite Fe-C 3 N 4 @Ru@HOP (FCRH) was constructed to alleviate tumor hypoxia and enhance PDT via generating oxygen in situ. 63c In this work, the Ru (Ⅱ) complex-loaded irondoped C 3 N 4 (Fe-C 3 N 4 ) nanoparticles were surface-modified with a hyperbranched conjugated copolymer containing poly(ethyleneglycol) arms (HOP). In this nanosystem, the HOP showed a superior two-photon action cross-section and worked as a two-photon light-harvesting agent and a donor of Förster resonance energy transfer (FRET) to generate oxygen upon two-photon light irradiation. When exposed to 808-nm two-photon irradiation, Ru (Ⅱ) was activated to generate 1 O 2 and Fe-C 3 N 4 was triggered to split H 2 O for oxygen generation. Owing to the injection of photo-induced electrons from excited Ru (Ⅱ) to Fe-C 3 N 4 , oxygen generation by Fe-C 3 N 4 was significantly accelerated. It is worth noting that the two-photon laser in this system increased penetration depth with added precision, both of which significantly enhanced PDT efficiency. Therefore, this oxygen self-supplement strategy solved the restriction of both hypoxia and tissue penetration in PDT simultaneously and showed great potential for spatiotemporally controlled tumor treatment in vivo ( Figure 4E). Most recently, Yu et al. reported that small-sized calcium peroxide (CaO 2 ) nanoparticles were employed as an oxygen-generating material via the reaction with carbon dioxide for tumor hypoxia regulation during PDT. 64 This strategy bypassed the hidden trouble that the naturally endogenous H 2 O 2 concentration in the tumor site is not enough to produce enriched oxygen. Compared with the substances that produce oxygen through other mechanisms, the water-splitting strategy features unique merits due to the abundance of H 2 O in the physiological environment for in situ oxygen generation. Hence, it has tremendous potential for clinical applications.

Hemoglobin-based oxygen carries
In the body of mammals, red blood cells (RBCs) are essential tools for oxygen and carbon dioxide transportation. Hemoglobin (Hb), an iron-containing metalloprotein inside RBCs, is responsible for carrying the inhaled oxygen from the lungs to organs and tissues and returning the metabolite carbon dioxide to the lungs for exhalation. 65 Each RBC contains 2-∼20 million Hbs, each of which can bind four oxygen molecules. 66 From this view, Tang et al. once reported an RBC-enhanced PDT approach using RBCs as the carrier of both photosensitizer ZnF 16 Pc and oxygen to improve the efficacy of oxygen-dependent PDT. 67 However, RBCs and cell-free Hbs are not perfect oxygen carriers due to their poor stability, reduced circulation half-life, and potential adverse effects. 68 With the help of nanotechnology, artificial red cells (ARCs) have been developed to serve as oxygen carriers. 69 ARCs are essentially various types of biological material with Hb as the core, including polymer/Hb assemblies, 70 PEGylated Hb spheres, 71 and other Hb-containing vesicles 72 .
In a recent attempt by Zhang's laboratory, an aggressive man-made pseudo-RBC (AmmRBC) was created to combat hypoxia-mediated PDT resistance. 72c In brief, abundant Hb (about a tenfold increase above that in natural RBCs), antioxidative enzyme-mimicking polydopamine (PDA), and polydopamine-carrying photosensitizer methylene blue were encapsulated inside the nanomedicine, which was engineered from the recombined RBC membrane (RCBM) ( Figure 5A). Together with the outer recombined RBCM shell, the introduced PDA played the role of the antioxidative enzymes existing inside RBCs, for example, superoxide dismutase (SOD) and CAT, to effectively prevent the oxygenated hemoglobin (HbO 2 ) from sustaining oxidation damage during circulation. In this way, with the absence of immunogenic clearance and long circulation half-life, this AmmRBC can significantly accumulate in tumors and then serve as an oxygen self-supply shuttle to sensitize PDT toward the extremely hypoxic tumor resulting in complete tumor elimination. Tumor hypoxia relief was confirmed with a pimonidazole-based fluorescent hypoxy-probe and evidenced by the downregulation of HIF-1 . Moreover, the oxygenation level can also be visualized by photoacoustic imaging. 43,73 However, as just mentioned, the oxygen loading efficiency for Hb is not good enough due to the limited number of oxygen-binding sites within the Hb protein. Moreover, difficulties in maintaining the conformation of the Hb active sites during the sophisticated processing and the potential Hb-induced acute nephrotoxicity and acute hypertension prevented clinical translation of the Hb-based oxygen products.

Perfluorocarbon-based oxygen carriers
Perfluorocarbons (PFCs), another type of oxygen vehicle, have been widely studied in recent years for PDT with considerable oxygen supply. PFCs are a class of materials composed of carbon and fluorine atoms with unique biocompatibility, which have been extensively explored in ultrasonography and MRI as blood substitutes, and some of them have been approved for clinical use. 74 Compared to the low oxygen loading efficiency of Hb, PFCs exhibit extraordinarily high solubility with about 40-50 mL oxygen per 100 mL PFCs, corresponding to the oxygen solubility of about 200 mL blood at 25 • C at 1 atm pressure. More importantly, the lifetime of the PDT product 1 O 2 is significantly lengthened in PFCs compared with aqueous media like the cellular environment, offering a more extended photodynamic interaction with cellular biomacromolecules. 26 In 2015, a pioneering work from Hu's group reported an oxygen self-supply PFC nanodroplet, LIP (IR780 + PFH), for enhancement of the ROS level in PDT. The near-infrared heptamethine cyanine dyes, IR-780, and perfluorohexane (PFH) were constructed into a lipid-based nanosystem (DSPE-PEG2000, lecithin, and cholesterol). 26 The abundant oxygen supply and prolonged lifetime of 1 O 2 enabled a significant improvement of PDT efficacy, either by intratumoral injection or intravenous injection of LIP (IR780 + PFH) with just one dose. Hu also constructed other types of PFC nanoemulsions utilizing human serum albumin (HSA) and liposomes for enhanced PDT and other oxygensensitive therapeutics. 75 In addition, due to their chemical inertness, PFCs can be formulated with synthetic organic and some inorganic nanostructures to facilitate oxygen delivery. 76 To further overcome the nonspecific activation of photosensitizers, in our recent work, we developed an activatable nano-photosensitizer via a redox-responsive turn-on strategy incorporated with PFH for oxygen supply to strengthen PDT. 77 In brief, photosensitizer Ce6 was conjugated to the backbone of natural polysaccharide HA via a disulfide bond to obtain amphiphilic HSC conjugates with aqueous selfassembly properties. 78 Subsequently, liquid PFH was ultrasonically emulsified with HSC conjugates and encapsulated into the core of a nanostructure, forming PFH@HSC nanoparticles. Similar formulation processing has also been utilized for enhanced fluorescence/CT/photoacoustic imaging-guided PTT. 79 Perfluorooctylbromide (PFOB), another PFC with a bromine atom, serves as the X-ray computed tomography (CT) contrast in the system. 78b,80 Fluorinated carbon chains could also be chemically anchored to the vehicles, either to confer the nanoparticles with oxygen dissolving ability or to enhance the loading efficiency and stability of PFCs in the whole system. 81 Additionally, external stimuli, including near-infrared lasers and low-power ultrasound release, have been utilized as offloading triggers for obtaining oxygen from PFCs by Liu et al., considering the low efficiency of oxygen release through passive concentration gradient diffusion. 76b,82

Reducing oxygen consumption
Apart from the above intratumoral oxygen-replenishing strategies, reducing oxygen consumption may be another practical approach to compensate for tumor hypoxiaassociated PDT resistance. Several agents, like papaverine, vandetanib (ZACTIMA TM ), and metformin (Met), have been identified to reduce oxygen consumption by tumor cells, mostly through mitochondrial respiration inhibition. 83 Recent work by Jiang's lab reported an alternative way to cut off the oxygen-consuming pathway by co-delivering the oxygen-regulator atovaquone (ATO) and photosensitizer verteporfin (VER) using sub-50-nm nanoparticles with poly(lactide-co-glycolide)-block-poly(ethyleneglycol)methylether (PLGA-PEG). 84 The antimalarial drug ATO, a homolog of the mitochondrial coenzyme Q, can interact with cytochrome bcl (mitochondrial complex III) of the respiratory chain and block the electron transfer chain, thereby blocking mitochondrial respiration and decreasing the cellular oxygen consumption rate. 85 In addition, ATO hindered the function of DHODH, downregulated pyrimidine, and thus blocked the cellular repair process. In this regard, the combination of ATO with VER promotes 1 O 2 generation in hypoxic tumors during PDT and enhances the cellular damage after phototherapy ( Figure 5B-D). Similarly, to combat the hypoxia obstacle in PDT, Feng et al. recently proposed an "oxygen-economizer" solution utilizing nitric oxide (NO) to inhibit cell respiration due to the binding competition of NO with oxygen in the mitochondrion. The NO donor sodium nitroprusside (SNP) reacts with the over-expressed thiol-bearing compounds (GSH and cysteine, RSH for short) in tumor cells, to form the S-nitrosothiol intermediate RSNO with the final product NO for efficient inhibition of cellular respiration to facilitate tetraphenylporphyrin (TPP)-mediated PDT ( Figure 5E). This strategy enables the treatment of tumors more sensitive to hypoxia-resistant treatments without an exogenous oxygen supply. 86

Fractional light irradiation and reduced fluence rate
Sharply different from oxygen-replenishing strategies, directly reducing the oxygen requirement by creating a lower oxygen-dependent modality may be a choice for clinical applications. For example, fractionated illumination dividing irradiation into light-dark periods, and prolonging irradiation with a low light fluence rate have both been investigated for better tumor reoxygenation by the blood with an enhanced PDT outcome. 87 As we know, 1 O 2 generation during PDT is self-limiting, since the low partial pressure of oxygen within tumors is further diminished during the oxygen consumption process of PDT. Although the fractional PDT allows time for the replenishment of cellular oxygen, some other issues such as irradiation dependence, rapid metabolism of organic photosensitizers, and extended therapeutic cycles still need to be overcome. Hence, to further enhance the availability of fractional PDT, Akkaya et al. designed a bifunctional borondipyrromethene (BODIPY)-based fractional PDT system, in which a 2-pyridone group was grafted into the structure of BODIPY. 88 The reversible transformation between the 2-pyridone (PYR) moiety and its endoperoxide (EPO) derivative enables continuous 1 O 2 generation under both the light and dark phases of the cycle ( Figure 6A). Likewise, some PYR-containing phototherapeutic agents have also been reported by Dong's team and other labs to work continuously with fractional PDT. 89 For example, the PYR-modified aza-BODIPY nano-assembly (BDY) was synthesized for an imaging-guided PTT synergistic sustainable PDT with a high photothermal conversion efficiency of 35.7% and excellent 1 O 2 generation ability. 89b Upon light irradiation, BDY can produce 1 O 2 , along with PYR absorbing 1 O 2 on its endoperoxide form EPO to form BDY-EPO. After the light source is removed, the hyperthermia effect left upon PTT of BDY promotes 1 O 2 release via thermal-triggered cycloreversion from EPO for a sustainable PDT. This event occurs without consuming the replenished oxygen for the next round of photosensitization. Consequently, the PTT-sustainable PDT significantly inhibited tumor growth with an inhibition ratio of 93.4%. Almost no side effects occurred using this intermittent laser illumination strategy ( Figure 6B).
Generally, these techniques are considered as effective attempts to enhance the PDT by the promotion of the reperfusion of tissue oxygen and compensation of oxygen depletion. Some exceptions have been reported. 90 These approaches only affect PDT-induced oxygen depletion, whereas preexisting hypoxia cannot be reversed. Moreover, both reduction in light fluence rate and light fractionation would, in most cases, lead to a significant increase in time of irradiation required for delivery of the specific light dose.

Oxygen-independent Type I PDT
Due to their unique optical absorption properties, some nanoscale inorganic photosensitizers, such as fullerenes, titanium dioxide (TiO 2 ) nanoparticles, and zinc oxide (ZnO) nanoparticles, can be photo-induced to form electrons and hole-electron pairs which can interact with surrounding H 2 O molecules to produce various ROSs, such as⋅OH, O 2 •− , and H 2 O 2 . This strategy, a type I PDT with minimal oxygentension dependence, by changing the raw material for ROS from oxygen to H 2 O, is more suitable for hypoxic tumor therapy. Considering that most of them are excited by ultraviolet (UV) light, which is harmful and weakly penetrable in tissues, inorganic photosensitizers combined with near-infrared light via upconversion nanoparticles (UCNP), X-ray radiation, and internal self-luminescence, may achieve type 1 PDT with deep penetration and oxygen independence simultaneously. 55,91 Shi et al. reported the first example of using a nanoscintillator (SCNP) with a semiconductor for efficient radiationinduced type 1 PDT. A Ce III -doped LiYF 4 SCNP was prepared as a nano-converter that was further decorated with ultrafine ZnO semiconductor nanoparticles (SCNP@SiO 2 @ZnO). SCNP in this system has an outstanding ability to downconvert high-energy electromagnetic radiation to luminescence in the UV-Vis region and then activate ZnO to form a biotoxic ROS. 91c Very recently, a new approach introduced the radical initiator AIPH (2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride) into an albumin-based therapeutic nanoplatform (BIA NPs), in which the hyperthermia from ICG facilitates the decomposition of AIPH into oxygen-irrelevant alkyl radicals ( Figure 7A,B). The radical detection probe 2,2 ′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) verified the efficient decomposition and release of AIPH radicals at an elevated temperature (44 • C vs. 37 • C; Figure 7C). Electron spin resonance (ESR) measurements also showed alkyl radical generation under 808-nm laser irradiation in the presence of the DMPO probe ( Figure 7D). Finally, the superior synergistic photo-therapeutic efficacy of BIA NPs has been demonstrated in the A549 tumor-bearing mice model ( Figure 7E,F). 92 Apart from decomposing H 2 O, H 2 O 2 can also be utilized to generate ROS without oxygen dependence. Recently, many nanomaterials, including Fenton's reagent and catalytic nanomaterials, have been used to enhance PDT efficacy via catalyzing sufficient endogenous H 2 O 2 within tumors into oxygen and highly toxic ⋅OH. 59 in cancer cells converts downstream into highly toxic⋅OH, which may dramatically aggravate the oxidative injury and improve the anticancer efficacy. Moreover, the generated oxygen is recyclable in this cascade bio-reaction, thereby facilitating amelioration of anti-hypoxia performance ( Figure 7G). 93e

Enhancing the 1 O 2 quantum yield
In order to enhance the efficacy of PDT, efforts have also been made to design some novel photosensitizers with high 1 O 2 quantum yield. However, designing new photosensitizers is time consuming and requires sophisticated synthetic procedures. 94 Since population of the long-lived 3 PS * is a result from the transient photo-excited singlet photosensitizer ( 1 PS * ) via intersystem crossing (ISC) process, the 1 O 2 quantum yield, which governs the efficacy of PDT, is dependent on the efficiency of ISC ( 1 PS * → 3 PS * ). 95 It is known that the rate of ISC can be accelerated via the presence of heavy atoms in close proximity to the excitation as a result of enhanced spin-orbit coupling. 96 Based on this concept, Fan and his colleagues have utilized such principle to develop nano-theranostics platforms (SPN-I) containing heavy-atom iodine-enriched materials to elevate the 1 O 2 generation, thus improving the PDT efficacy. 97 The nanoparticles were composed of a NIR absorbing semiconducting polymer (PCPDTBT) serving as the photosensitizer and an iodine-grafted amphiphilic copolymer (PEG-PHEMA-I) serving as the 1 O 2 generation enhancer and nano-carrier ( Figure 8A). The high density of iodine grafted on PEG-PHEMA-I can enhance the ISC of PCPDTBT via intermolecular heavy-atom effect, thus improving the 1 O 2 quantum yield ( Figure 8B). Owing to the high X-ray attenuation coefficient of iodine in PEG-PHEMA-I, and fluorescent property of PCPDTBT, this whole nanoplatform showed efficient CT and fluorescence dual-modal imaging-guided enhanced PDT ( Figure 8C).
Although the incorporation of heavy atoms such as bromine, iodine, selenium, and certain lanthanides into the structure of photosensitizers is an effective strategy to improve spin-orbit coupling leading to facilitated ISC, this approach seems unsafe and very often leads to increased "dark toxicity." 98 An emerging molecular design approach with heavy-atom-free photosensitizers was proposed by Yoon and Park et al. to effectively generate ROS under both normoxia and hypoxia. 99 Replacing both oxygen atoms in conventional naphthalimides (RNI-O) with sulfur atoms (thionaphthalimides (RNI-S)) can remarkable enhance the ISC from 1 PS * to 3 PS * . Furthermore, the incorporation of electron-donating groups, 4-R, into the RNI-S enhanced the ROS generation ability as well as modulate between the type II and type I PDT mechanisms. The photo-toxicity experiments on HeLa cells demonstrated that their design could exert an excellent PDT efficacy even under a severely hypoxic environment (1% O 2 ).

Combination with other oxygen-independent therapeutics
The current trend in clinical research has gradually shifted from monotherapy to combination therapy for enhanced treatment efficacy by taking advantage of both and avoiding the disadvantages of each other. In addition to the PDT combined PTT, which exhibited less dependence on oxygen relative to only PDT, 83d,100 immunotherapy recently has become F I G U R E 9 Schematic illustration of the preparation and application of the CAGE complex for photodynamic therapy (PDT)-combined dendritic cell-based immunotherapy. Reprinted with permission. 102a Copyright 2018, American Chemical Society. a hot topic focusing on the field of cancer treatment. 101 A combination of PDT with immunotherapy may be a promising strategy to overcome the hypoxic resistance of PDT. 35b,81c,102 For example, Kim et al. recently reported a hypoxia-triggered transforming immune-modulator for immunotherapy via enhanced antigen presentation of dendritic cells (DCs) by PDT. 102a In their study, PDT was exploited for the production of immunogenic debris and the recruitment of DCs in the tumor site, followed by enhanced antigen presentation. In this case, the azobenzene linker is part of the nanoparticle skeleton, which splits under intrinsic tumor hypoxia or abrupt consumption of local oxygen after PDT, leading to the release of CpG for the delivery of DC activation (Figure 9). In another study by Liu et al., a light-triggered in situ gelation system was fabricated in order to enhance the trigger capacity of the systemic antitumor immune response. 102d A single intratumoral injection containing a hybrid hydrogel with long-term tumor retention of the photosensitizer Ce6 used for free-radical polymerization of doners and ROS generation upon irradiation, CAT for hypoxia relief, and imiquimod (R837) as the immune adjuvant, enabled a significantly enhanced immune response by multi-round stimulation. Combining the hydrogel with the anticytotoxic T-lymphocyte antigen-4 (CTLA4) checkpoint blockade, the distant tumors were inhibited by the abscopal effect, and tumor recurrence was avoided by long-term immune memory as well.

Combination with hypoxia-activated therapy
Although hypoxia is regarded as a negative prognostic indicator of anticancer efficacy, the exact reason that makes hypoxia a problem is also an opportunity for the development of new therapeutic modalities. 12a,103 Recently, some bioreductive anticancer drugs, also known as hypoxia-activated prodrugs (HAPs), have been used to treat hypoxic tumors due to their selective cytotoxicity in hypoxic conditions. Such drugs include tirapazamine (TPZ), banoxantrone (AQ4N), apaziquone (E09), TH-302, PR-104A, and mitomycin C, which have been well-studied in clinical trials and shown to have distinct hypoxia-activated cytotoxicity. 104 Therefore, co-delivery of HAPs with oxygen-consuming agents like photosensitizers through nanoparticle-based systems is appealing to achieve a high specificity along with the synergistic anticancer efficiency. 105 In this approach, PDT is used to kill tumor cells and to create a PDT-aggravated hypoxic TME by consuming available oxygen inside the solid tumors and inducing severe damage to the tumor blood vessels to delay tumor reoxygenation. Consequently, this PDT-induced hypoxia will fully activate the HAPs to destroy the remaining tumor cells, though HAPs alone cannot kill all the tumor cells as well-oxygenated tumor cells also exist.
MOFs have been tried for many biomedical applications in recent years due to their facile synthesis, biocompatibility, and porous structure. 106 Zhu, Yu, and Chen et al. synthesized an intelligent nanosystem based on UiO-66 NPs for PDT and hypoxia-triggered cascade chemotherapy, in which the co-delivery of photosensitizer and the bioreduc-tive prodrug AQ4N using MOFs (UiO-66-H/N3 NMOFs) was designed. 105f As depicted in Figure 10A, the photosensitizer photochlor (HPPH) and AQ4N were anchored onto and encapsulated into UiO-66 NPs, respectively. Moreover, by strain-promoted azide-alkyne cycloaddition, the UiO-66 NPs with azide groups were further decorated with a dense PEG layer to enhance their dispersion in the physiological environment and improve their therapeutic performance. To further strengthen the activation of AQ4N under hypoxia, Chen and Fang et al. introduced HIF-1 siRNA (siHIF-1 ) to suppress HIF-1 , thereby upregulating cytochrome P450 (CYP450, the dominant HAP-activating reductase). 105a It has been reported that oxygen and AQ4N competitively bind the heme-centered active site of CYP450, which can metabolize AQ4N into the active AQ4, a potent inhibitor of topoisomerase II. However, the adaptive elevated HIF-1 expression under hypoxic TME would downregulate CYP450, which makes HIF-1 a "hidden brake" that substantially hinders AQ4N activation under hypoxia. In light of this, the ternary system including photosensitizer, HAPs, and HIF-1 inhibitor, provides us a mechanism-based working model for PDT-combined hypoxia-activated chemotherapy.

Combination with hypoxia-responsive moieties into the vehicles
Besides HAPs, some nitroimidazole (Azomycine) analogs including 2-nitroimidazole, 4-nitrobenzyl, 4-nitrofuryl groups, and other chemical moieties such as azobenzene derivatives, quinones, aliphatic N-oxides, aromatic N-oxides, and transition metals have been reported to show hypoxia responsiveness. 103b,107 In light of this, several research groups have utilized these hypoxia-responsive chemical moieties to synthesize the vehicles for carrying photosensitizers and anticancer drugs. 108 For example, Zhu et al. fabricated a hypoxia-responsive nanomedicine named PA/HA-Ce6@TPZ NPs to incorporate the PDT agent Ce6 and bioreductive drug TPZ for synergistic anticancer therapy, in which the alkylated 2-nitroimidazole (ANI) moiety acted as the hypoxiaresponsive unit with hydrophobic-hydrophilic conversion capacity between normoxia and hypoxia for drug release ( Figure 10B). Under hypoxic conditions induced by light irradiation (660 nm, 10 mW/cm 2 ) for 30 min, the size of the nanoparticles dramatically swelled from 183 nm to ∼900 nm ( Figure 10C). It has been shown that the swelling mechanism for nanoparticles resulted from the reductive conversion of hydrophobic ANI into hydrophilic alkyl-2-aminoimidazole (AAI) under hypoxia and ROS generation ( Figure 10D,E). In vivo antitumor experiments with 4T1 tumor-bearing mice revealed a remarkable inhibition of tumor progression. The highest survival rate was noted for PA/HA-Ce6@TPZ NPs (w/L) with three intravenous administrations, which was ascribed to the cooperation between PDT and PDT-activated TPZ ( Figure 10F). The full treatment can be regarded as a self-feedback process leading to controlled cargo release and an elevated therapeutic effect. 108b Yang et al. introduced another hypoxia-responsive linker, azobenzene, to develop a size-switchable nanosystem (HCHOA) for deep tumor penetration and enhanced therapeutic efficacy of oxaliplatin combined with Ce6. 108h The diameter of the HCHOA nanosystem was 100-150 nm at the normoxia level, beneficial for long-term blood circulation and enhanced tumor accumulation through the EPR effect. However, under hypoxia, with cleavage of the azobenzene moiety in the HCHOA structure by numerous reductases, these large nanoparticles rapidly dissociate into ultrasmall ones (<10 nm), resulting in a significant increase in intratumoral permeability. More importantly, upon dissociation under hypoxia, the quenched Ce6 in the HCHOA can be activated with enhanced fluorescence and singlet oxygen production to achieve a better therapeutic outcome. From this aspect, combining PDT and hypoxia-sensitive delivery vehicles, sometimes hypoxia-activated chemotherapy can realize the temporal and spatial release of various drug cargoes, thus creating a combined anticancer efficacy.

Combination with cancer starvation therapy
In the current world, strategies that block nutrients and oxygen supply to cancer cells starve tumors, leading to subsequent cellular necrosis and apoptosis. 10b Therefore, the combination of PDT with certain cancer starvation therapeutic agents can collaboratively consume the food tumors need for their survival and growth, and ultimately starve or suffocate cancer. 109 A proof-of-concept study by Bu and Shi et al. reported magnesium silicide (Mg 2 Si) nanoparticles as an efficient oxygen-consuming agent. 110 The Mg 2 Si nanoparticles degrade in response to the mildly acidic TME, thereby generating the highly reactive intermediate silane (SiH 4 ), which is an oxygen scavenger (reaction: Mg 2 Si + 4H + → 2Mg 2+ + SiH 4 ; SiH 4 + 2O 2 → 2H 2 O +SiO 2 ). The by-product SiO 2 can aggregate in situ to block tumor capillaries and shut down the oxygen and nutrient supplies of the tumors. The collaborative starving and suffocating effects would result in excellent antitumor efficacy. Zhang and other researchers have also demonstrated this enhanced cancer therapy via the combination of cancer starvation therapy with chemotherapy, immunotherapy, and PDT. 111 Recently, an intriguing "mutual promotion cycle" hybrid nanozyme (rMGB) was designed by Sun and Qian et al. for enhanced starvation therapy and PDT against hypoxic tumors, which was composed of MnO 2 NPs, Ce6-albumin, and glucose oxidase (GOx) ( Figure 11A). 112 With oxygen involvement, the intratumoral glucose is catalyzed by GOx to degrade into H 2 O 2 . In their system, MnO 2 was introduced to react with endogenous H 2 O 2 and H + to produce oxygen, which promotes the catalytic efficiency of GOx for starvation therapy. Remarkably, this process not only blocked the nutrient supply for starvation therapy but also provided H 2 O 2 (a by-product of glucose oxygenolysis) to enhance PDT synergistically. Moreover, GOx also provides abundant H + (via gluconic acid), which causes MnO 2 to accelerate further oxygen generation for alleviating tumor hypoxia and enhancing PDT efficacy ( Figure 11B). The biochemical reaction cycle, consisting of MnO 2 , O 2 , GOx, and H + , was triggered by the TME and promoted mutually to achieve self-supplied H + and accelerate oxygen generation. Hence, starvation therapy is enhanced, alleviating tumor hypoxia, and accelerating the ROS generation in PDT. A similar synergetic mechanism is utilized by Li and Tang et al. to achieve starvation therapy combined with PDT against tumors and tumor metastasis ( Figure 11C). 113

SUMMARY AND PERSPECTIVES
In summary, PDT is a promising antitumor strategy due to its minimal invasiveness, feasibility, and high efficiency. Although several PDT agents have been approved for clinical trial and application, the full potential of PDT as a first-line treatment option has yet to be realized. Recent progress in nanotechnology has created a new horizon combating the tumor hypoxia challenge by providing diverse solutions. Considering that tumor hypoxia is one of the critical factors affecting PDT efficacy, we herein provide this comprehensive review of different nanomedicine-based strategies to overcome the limits of hypoxia for the advancement of PDT-mediated cancer therapy: (a) harnessing tumor hypoxia via diverse oxygen-replenishing methods; (b) neglecting hypoxia with oxygen-independent treatment modalities; (c) utilizing tumor hypoxia to exaggerate the antitumor outcomes through some unique mechanisms.
The recent studies listed in this review indicate that nano-strategies offer tremendous potential to modulate tumor hypoxia and thus reinforce PDT. We believe the research focus on tumor hypoxia will continue to be under the spotlight and may soon become one of the primary concerns together with other therapies for cancer therapy. However, most of these nanosystems are in a very early phase, and a more critical evaluation of such therapeutic systems is needed. For example, the level of hypoxia and other parameters like H 2 O 2 concentration vary among tumor types, which can change as the tumor progresses, even in the same patient and with the same tumor type. Therefore, the precise correlation between tumor oxygenation level and PDT efficacy still needs to be carefully determined, which would assist the further rational design of hypoxia modulated PDT carriers. Although hypoxia is undoubtedly one of the most significant concerns, PDT still faces a variety of challenges that must be overcome before its extensive use in the clinical environment. These challenges include complicated factors within the TME or the inherent limits of PDT, such as the inadequate penetration depth of the light source, the undesired hydrophobicity and tumor non-selectivity of most photosensitizers, and lack of specific accumulations in deep tumor tissues or sub-cellular organelles for enhancing toxicity. More importantly, the simplicity, biocompatibility, immunogenicity, and pharmacokinetics of the PDT vehicles should be systemically evaluated in preclinical studies. As most of the highly efficient catalysis nanosystems for oxygenation are based on inorganic materials, the in vivo degradation profiles of such substances and the toxicity of the degradation products should also be carefully evaluated. The interdisciplinary nature of PDT will inspire specialists and technicians in medicine, physics, chemistry, biology, and engineering. With all the challenges addressed in this field tackled step-by-step, it is expected that hypoxia-overcoming, hypoxia-ignoring, and hypoxia-utilizing systems will open up a new avenue for PDT and other oxygen-dependent therapeutics to boost their potential in clinical antitumor treatment.