ERM is a smart material that can respond to different enzymatic environments. ERMs have been widely used as a carrier for delivering PS to tumor tissues. As Figure 1B shown, after the PS-loaded ERM enters the tumor tissues, the PS can be specially released from the ERM by responding to the unique enzymes in the tumor tissues, and then the use of light irradiation activates the PS to produce ROS to kill the tumor cells. Recently, the reported ERMs used for PS delivery mainly include hyaluronidase-responsive material, matrix metalloproteinase-responsive material, carboxylesterase-responsive material, cathepsin B-responsive material, cytochrome P450-responsive material, etc. Their detailed information of the response sites and response principles is shown in Table 1.

Hyaluronidase is an enzyme that can decompose high-molecular hyaluronic acid into small-molecule hyaluronic acid (Lv et al., 2015). Growing evidence shows that hyaluronidase is a potential cancer biomarker and therapeutic target (McAtee et al., 2014). Hyaluronic acid (HA) is a water-soluble mucopolysaccharide, which is a ligand specifically binding to CD44 overexpressed in cancer cells (Choi et al., 2011; Yu et al., 2013; Dosio et al., 2016; Liu et al., 2020). Therefore, it is a reasonable choice to use materials containing hyaluronic acid to target tumor cells and/or to release photosensitizers in response to hyaluronidase in tumor tissues. Zhou (Zhou et al., 2020) constructed a mesoporous TiO₂ doped with MnO₂ (MTM) through an improved sol-gel method to develop co-doped photosensitizer TiO₂ for enhancing PDT in the treatment of melanoma. MTM was also loaded with the antioxidant scavenger Au 25 Sv 9 and then modified by hyaluronic acid on the surface of MTM to avoid accidental release of Au 25 Sv 9. As Figure 1C shown, after the HA-modified MTM entering the tumor tissues, hyaluronidase decomposes hyaluronic acid to release Au 25 Sv 9 to remove thioredoxin reductase in tumor cells, MnO₂ catalyzes H₂O₂ to produce oxygen to improve hypoxia environment, which promoted the killing effect of photodynamic action through light-excited TiO₂. The authors found that the composite materials could improve hypoxia in tumor tissues and enhance PDT efficacy.

The use of hyaluronic acid as a sealing material of mesoporous silica can not only target CD44 receptors overexpressed in tumor cells, but also control the release of photosensitizers through enzyme response. Cheng (Cheng et al., 2021) reported a new type of nanohybrid based on GSH-responsive mesoporous organic silica nanoparticles (GMON), which could exert significant anti-tumor activity under near-infrared light irradiation. The nanosystem was composed of GSH-responsive mesoporous silica, photosensitizer IR820, hypoxia-responsive prodrug tirapazamine (TPZ) and hyaluronic acid. After administration, the nanosystem was endocytosed into tumor cells due to HA targeting CD44 receptor. The GMONs in tumor cells were degraded by the action of GSH and hyaluronidase, releasing TPZ and photosensitizer. After laser irradiation IR820 consumed oxygen to produce ROS, which further enhances the toxic effect of TPZ to tumor cells. HA has dual ability to target CD44 overexpressed in cancer cells and response to hyaluronidase overexpressed in cancer cells, which endows HA-modified materails double functions with targeted release and delivery of PS. At present, electrostatic absorption is a main method used for the modification of HA on the surface of nanomaterials. The electrostatic force is very weak and the modified nanomaterials are easily affected by the surrounding envirnoments. After entering the systemic circulation, HA on the surface of the nanomaterials is easy to drop from the materials and cause accidental release of PS. Therefore, if HA is modified on the nanomaterials through chemical bonds, the physicochemical properties and biological activity will be much improved.

Matrix metalloproteinases (MMP) are overexpressed in a variety of tumors, and are closely related to tumor growth, invasion and metastasis (Shinagawa et al., 1994; Coussens et al., 2002; Natarajan et al., 2006; Roy et al., 2008). A large number of anti-cancer strategies related to MMP have been proposed, especially MMP sensitive or responsive substances have been widely used to deliver drugs and reduce the side effects of drugs (Winer et al., 2018). In PDT, MMP is also used as an excellent stimulus source to targetedly deliver photosensitizers. As an important subcellular organelle, mitochondria are related to cell growth, differentiation and apoptosis. The development of photosensitizers with mitochondrial targeting is an effective way to enhance photodynamic therapy. (Li et al., 2017) synthesized a cationic oligomer (p-phenylene vinylene-copolymerized benzothiazole) (OPVBT) that could accumulate in mitochondria via electrostatic interactions. OPVBT could generate a large amount of ROS upon light irradiation and exhibited significant photodynamic activity. In their studies MMP-sensitive PEG peptide (CRD-VPMSMRGG-DRC) hydrogel was used to deliver OPVBT for improving tumor selectivity and reducing side effects. Due to the high level of MMP in tumor cells, hydrogels containing MMP-cleavable (Figure 1D) substrates could gradually be degraded, locally releasing OPVBT in tumor tissues. Their results showed that the hydrogel had no toxicity to normal cells, but it could be effectively decomposed after entering tumor tissues, to release the photosensitizer, showing high selectivity and low toxicity.

MMP-2 is one of MMPs, which are overexpressed in a variety of tumor cells (Wang et al., 2019; Stephens et al., 2020). (Gao et al., 2019) designed MMP-2 responsive exfoliable prodrug vesicles (EAPV) containing pegylated photosensitizer and NLG919 prodrug (NPC). PEG was modified on the surface of the vesicle through the MMP-responsive linker—GALGLPG (A short peptide), and when the vesicles reached tumor site, the GALGLPG peptide (Figure 1D) was cleaved by MMP-2 and then removed PEG to increase the endocytosis of the vesicles. After entering tumor cells, the loaded drug NLG-919 was released in response to GSH to inactivate indoleamine 2,3-dioxygenase 1, thereby overcoming the adaptive immune resistance caused by PDT. The results demonstrated that the EAPV nanoviscles exhibited uniform size distribution, good serum stability, and dual responsiveness to MMP-2 and GSH. EAPV could also penetrate deeply into tumor tissues and be easily absorbed by tumor cells. The in-vitro and in-vivo experiments confirmed that EAPV could significantly produce ROS, induce tumor cell ICD, and ultimately enhance the immune recognition of dendritic cells to tumor cells.

Carboxylesterase (CE) is a member of α/β hydrolase folding protein, which is mainly located in the endoplasmic reticulum of various mammalian cells (Barthel et al., 2009; Löser et al., 2013). CE participates in the hydrolysis of many xenobiotics and endogenous ester or amide compounds, and plays a key role in the metabolism and biotransform of some chemotherapeutics (Ke et al., 2020). Studies have shown that CEs are highly expressed in a variety of human tumor cells, so CE is also used as a target stimulus source to deliver drugs (Sanghani et al., 2003). Cai (Cai et al., 2020) modified the tetrachloroethylene monoimine core by β-alanine methyl ester, synthesized the CE-responsive precursor P1 as the substrate of CE and the electron donor of the system, and galactose as the tumor targeting ligand and hydrophilic group. The synthesized near-infrared (NIR)-activated amphiphile P1 and folate-modified human serum albumin (HSA) could assemble into nanoclusters (FHP) with a size of about 100 nm. As Figure 1E shown, when the methyl carboxylate of P1 was selectively hydrolyzed by CE at the tumor site to remove the methyl group, FHP decreased from ∼100 to ∼10 nm, which was conducive to the penetration of FHP in the deep tissues of the tumor. At the same time, the enzyme-triggered decomposition of FHP enhanced near-infrared fluorescence (NIRF) and promoted the generation of singlet oxygen (¹O₂), thereby improving imaging-guided PDT.

The expression of cathepsin B is highly upregulated in malignant tumors and precancerous lesions, so cathepsin B can be used as a stimulus response target (Melancon and Li, 2011; ShiJie; Wang et al., 2020). Given that high levels of Cathepsin in tumor tissues can specifically cleave a tetra-peptide linker GFLG (Özel et al., 2015), Cheng (Cheng et al., 2020) constructed a tumor cell and mitochondrial dual-targeting nano-platform based on PpIX-GGGK (TPP)GG-GFLG-R7-RGD for the co-delivery of DOX and protoporphyrin IX (PpIX). The self-assembled nano-platform consisted of a hydrophobic PpIX-peptide conjugate (PpIX-GGGK (TPP)GG), a hydrophilic bioactive peptide with tumor-targeting RGD, cell membrane-penetrating R8 and cathepsin B. The cleaved linker GFLG is composed of self-assembled nanomicelles with a core-shell structure using hydrophilic-hydrophobic interactions, and DOX is loaded into the core through hydrophobic force and π-π interactions to form DOX@pPAC. The constructed nanosystem (DOX@pPAC) enhanced the uptake of PpIX and DOX in tumor cells through endocytosis mediated by integrin receptors. Later, the up-regulated lysosomal protease cathepsin B triggered the hydrolysis of the GFLG bond, followed by the dissociation of the DOX@ pPAC nanomicelles (Figure 1F). Then, the TPP cation of the PpIX-peptide conjugate greatly promoted the accumulation of PpIX in mitochondria, and significantly enhanced the PDT effect of PpIX on tumor cells. In this article, the researchers cleverly designed a multifunctional amphiphilic macromolecule that could self-assemble, integrating PDT, DOX, tumor targeting, mitochondrial targeting and enzyme response. The successful combination of chemotherapy and PDT amplifies the efficacy of chemotherapy and photodynamic therapy and accelerates the death of tumor cells.

Cytochrome P450 (CYP) is a multi-gene superfamily of constitutive and inducible heme-containing monooxygenases, involved in the metabolism of various xenobiotics and endogenous substrates (Blake et al., 2013; Wang et al., 2017; Dutkiewicz and Mikstacka, 2018). The CYP enzyme (P450) is located in the mitochondria and endoplasmic reticulum (Hayran et al., 2020). These enzymes use molecular oxygen and equivalent electrons transferred from the NADPH-P450 reductase in the endoplasmic reticulum or the ferredoxin and ferredoxin reductase in the mitochondria to catalyze the monooxygenase reaction (Renaud et al., 2017). Clopidogrel (CPG) is a classic antiplatelet prodrug widely used to treat thrombosis. Cytochrome P450 (CYP2C19) enzyme mediates the metabolism of CPG to form thiol-containing metabolites (Kazui et al., 2010). The formation of disulfide bonds between the cysteine residues of GSH and the thiol-containing metabolites of CPG to consume intracellular GSH is related to the antiplatelet mechanism of CPG. Inspired by the mechanism of CPG, Wang (Wang et al., 2020) used CPG to consume intracellular GSH in cancer cells expressing CYP2C19 to enhance the efficacy of PDT (Figure 1G). They firstly co-assembled CPG and photosensitizer pyropheophytin (PPa) to form nanoparticles, and then used DSPE-PEG to encapsulate the nanoparticles to form CPG/PPa NPs in order to enhance its stability. CPG and PPa formed stable and nearly spherical nanostructures through strong π-π stacking, hydrophobic interaction, hydrogen bonding and electrostatic interaction. Compared with free PPa, CPG/PPa NPs showed higher cytotoxicity to cancer cells and greater accumulation at the tumor site. After CPG/PPa NPs entering into tumor cell, CPG responded to CYP2C19 to form metabolites containing mercapto alcohols and destroy the redox homeostasis of tumor cells by consuming GSH, showed synergistic anti-tumor effect, and then enhance the efficacy of PDT in the 4T1 breast tumor xenograft model. However, the responsive materials were only modified using PEG. Although PEG modification would enhance the stability and half life of the nanoparticles in the systemic circulation, the negative charge of the PEG might make the modified nanoparticles difficult to enter into the cell. Therefore, the use of ligand molecules such as folic acid, biotin on the surface of nanoparticles might further improve the efficiency of the responsive materials in the PS delivery.