Recent Advances on Reactive Oxygen Species-Responsive Delivery

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Review Cite This: Biomacromolecules 2019, 20, 2441−2463

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Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System Huan Ye,†,⊥ Yang Zhou,†,⊥ Xun Liu,† Yongbing Chen,*,‡ Shanzhou Duan,‡ Rongying Zhu,‡ Yong Liu,*,§ and Lichen Yin*,†

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†

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, China ‡ Department of Thoracic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China § Department of Biomedical Engineering, University of Groningen and University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands S Supporting Information *

ABSTRACT: Reactive oxygen species (ROS) play crucial roles in biological metabolism and intercellular signaling. However, ROS level is dramatically elevated due to abnormal metabolism during multiple pathologies, including neurodegenerative diseases, diabetes, cancer, and premature aging. By taking advantage of the discrepancy of ROS levels between normal and diseased tissues, a variety of ROS-sensitive moieties or linkers have been developed to design ROS-responsive systems for the site-specific delivery of drugs and genes. In this review, we summarized the ROS-responsive chemical structures, mechanisms, and delivery systems, focusing on their current advances for precise drug/ gene delivery. In particular, ROS-responsive nanocarriers, prodrugs, and supramolecular hydrogels are summarized in terms of their application for drug/gene delivery, and common strategies to elevate or diminish cellular ROS concentrations, as well as the recent development of ROS-related imaging probes were also discussed.

stress and then relieve inflammation.18 In addition, ROSresponsive materials could enable the selective imaging of tumors or inflammation tissues.19−21 In this review, we describe the recent advances on ROSresponsive materials and their biomedical applications. We first provide a summary of the commonly explored ROS-responsive chemical moieties and their ROS-responsive molecular mechanisms. While the ROS-responsive systems we discuss here can often respond to various types of ROS, H2O2 as a representative type of ROS is commonly utilized to evaluate the ROS-responsive performance, because it is more stable than any other types of ROS, featuring high concentrations in cancerous or inflamed cells as a result of the dismutation of O2−.5 Subsequently, the discussion focuses on the advancement of ROS-responsive delivery systems including nanocarriers (NCs), prodrugs, and hydrogels toward the precise delivery of drugs/genes to treat cancer and diseases associated with chronic inflammation. Existing approaches to elevate or diminish the intracellular ROS levels are also reviewed. Ultimately, we briefly summarize the ROS-related imaging probes as well as sensors, and conclude this review with perspectives in the field.

1. INTRODUCTION Reactive oxygen species (ROS) are single electron reduction products of oxygen, including hydrogen peroxide (H2O2), superoxide (O2−), singlet oxygen (1O2), hydroxyl radical (· OH), peroxynitrite (ONOO−), hypochlorite (OCl−), and so on.1,2 Endogenous ROS are mainly produced in mitochondria,3,4 and they afford important physiological functions in cellular signaling and metabolism.3,5 Generally, the endogenous ROS are maintained at low levels by the intracellular reduction−oxidation balance.6 However, once the reduction− oxidation is unbalanced,7 ROS generation will be elevated, which is associated with cellular pathological states, including the initiation and progression of cancer and inflammation.3,8 The level of ROS in pathological regions, such as tumor cells and activated immune cells, can reach up to 100 × 10−6 M,9,10 2 to 3 orders of magnitude higher than in normal cells (≈20 × 10−9 M).10 Such heterogeneity of ROS concentration has motivated researchers to design ROS-responsive materials to target inflammation or cancer sites and promote the sitespecific release of encapsulated cargoes in response to high levels of ROS in pathological regions.11−14 More recently, ROS-responsive materials have been designed to increase the ROS concentration in pathological regions to inhibit cell growth or even directly induce cell death.15−17 Also, ROSresponsive materials are able to decrease the concentration of ROS to a normal value to reduce overproduction of oxidative © 2019 American Chemical Society

Received: May 7, 2019 Revised: May 21, 2019 Published: May 22, 2019 2441

DOI: 10.1021/acs.biomac.9b00628 Biomacromolecules 2019, 20, 2441−2463

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Biomacromolecules Table 1. Brief Summary of the ROS-Responsive Structures and Mechanisms

2. ROS-RESPONSIVE CHEMICAL STRUCTURES 2.1. Chemical Structures and Mechanisms. There are two major ROS-responsive mechanisms for the commonly used ROS-responsive polymers, namely ROS-induced noncleavable hydrophobic−hydrophilic transition and ROSinduced structural cleavage (Table 1). On one hand, ROS can oxidize chalcogen elements (such as S, Se, Te), changing their valence from +2 to +4 or +6. During this process, oxygen

atoms form covalent bonds with the chalcogen atoms and the polarized groups enable hydrogen bonding formation with the environmental water molecules, thus inducing the hydrophobic−hydrophilic transition of the polymer backbone without cleaving its chemical structure. On the other hand, ROS can react with chemical structures such as thioketals (TKs), phenylboronic acids/esters (PBAs/PBEs), vinyldi2442

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Biomacromolecules Scheme 1. Schematic Illustration of Different ROS-Responsive Polymers

Scheme 2. Most Commonly Used Polymerization Strategies Using RRM-Containing Monomers

1).40 Vinyldithioether can specifically respond to singlet oxygen (1O2), producing a 2 + 2 adduct that further undergoes cleavage to produce thiols.32,41 Aryl oxalates can easily react with H2O2 to form 1,2-dioxetanediones, which rapidly convert into CO2 and phenols.42 Besides, amino acids such as proline, histidine, arginine, and lysine are susceptible to ROS-mediated and metal-catalyzed oxidation.35,36 Notably, proline is the only amino acid that can form tertiary amide bonds and is known to be more susceptible to oxidation than secondary amide bonds. 2.2. Polymer Synthesis. The synthesis of ROS-responsive polymers have been reviewed elsewhere,43,44 and in this section, we only give a brief summary and highlight the most recent and important additions to previous reports. Basically, the ROS-responsive moieties (RRMs) can be (1) incorporated into polymer main chains, (2) located on the polymer side chains, or (3) used to form hyperbranched polymers (Scheme 1). In principle, the ROS-responsive polymers can be synthesized via polymerization from RRM-containing monomers (Scheme 2), or via postpolymerization modification using the RRM-containing molecules. RRMs are compatible with a variety of functional groups, and thus numerous tailored RRMcontaining monomers are designed and used for different purposes. Polymerization methods such as condensation polymerization, ring-opening polymerization (ROP), and group transfer polymerization are employed to synthesize ROS-responsive polymers.44 In addition, ROS-responsive hyperbranched polymers are often polymerized from monomers with multiple functional groups.45,46 In this section, several commonly used polymerization or postpolymerization modification strategies are summarized and discussed. 2.2.1. Thioether-Containing Structures. Step-growth polymerization,47−51 ROP,52−55 and thioacyl group transfer

thioethers, and proline oligomers, leading to the cleavage of these structures (Table 1). In general, ethers are not oxidizable, while thioether groups can be easily converted to polar sulfoxide or sulfone in the presence of oxidative factors, resulting in a strong increase of their dipole moments (Table 1). This chemical process adjusts the aqueous solubility of the thioether-containing polymer, leading to the phase transition of the polymer from hydrophobic to hydrophilic. Thioether groups can respond to H2O2 to produce sulfoxides, while treatment of thioethers with HOCl produces the mixture of sulfoxides and sulfones.37 Although thioethers are nonresponsive to superoxide, enzymes such as superoxide dismutases (SODs) are able to convert superoxide into H2O2 and then promote the oxidation of thioethers.38 Polysulfides are commonly used at high concentration, and thus a relatively high ROS concentration is normally required (>500 μM H2O2) in order to trigger the response and drive the phase transition. Selenium and tellurium belong to the same main group as sulfur in the periodic table, while they are more sensitive to ROS than their thioether counterpart, because the electronegativity of selenium and tellurium is weaker than that of sulfur.39 The hydrophobic ferrocene (Fc) can be oxidized into hydrophilic ferricinium, and thus changes from neutral to cationic (Table 1).25,26 Normally, the TK groups are difficult to be cracked under acidic or alkali conditions, while they are readily oxidized by ROS, producing acetone and two other thiol-containing fragments (Table 1). H2O2 can specifically oxidize phenylboronic acid/ester (PBE/PBA) by inserting an oxygen atom into the C−B bond of PBE/PBA, subsequently inducing the formation of boronic ester and hydroxybenzyl alcohol (Table 2443

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Biomacromolecules Scheme 3. Synthetic Routes of Main-Chain TK-Containing Polymers and TK Cross-Linkers

polymerization,43,44,56−58 have been adopted in the synthesis of polymers containing thioether in the main chain. For instance, thioether-containing diols can react with diacids via melt polycondensation to yield poly(thioether ester)s.59,60 Monomers such as thioether-containing macrocyclic carbonate61,62 and ε-caprolactone63 can be used to synthesize polymers containing thioether in the main chain. To synthesize polymers containing thioether in the side chains, thiother-pendant monomers, such as 2-(methylthio)ethyl glycidyl ether64 and L-methionine-based N-carboxyanhydrides (NCA),65,66 have been used for polymerization.43,67 In addition, postpolymerization modification has been used to synthesize the side-chain thioether-containing polymers68 via strategies including radical thiol−ene/thiol−yne reactions,69−73 as well as nucleophilic substitutions of thiols to epoxys or halides.74,75 2.2.2. Selenium-Containing Structures. The synthesis of selenium-containing polymers has long been limited by the sensitiveness of mono- and diselenide in the presence of oxygen.76 It was not until 2010, Xu and Zhang et al. discovered that the long alkyl chain in diselenide-containing diols can increase the solubility of monomers. Step-growth polymerization was then used to obtain linear mono- and diselenidecontaining polyurethane.27,77 Recently, selenium-containing macrocyclic monomers have been developed and used to synthesize main-chain selenium-containing polymers via living ROP in the presence of lipase CA.60,78 To synthesize the sidechain selenium-containing polymers, p-methylselenostyrene and p-phenylselenostyrene were polymerized via free-radical polymerization.79 Also, postpolymerization modification of 11(benzylselanyl)undecan-1-ol onto poly(ethylene oxide-blockacrylic acid) was adopted to synthesize block copolymers containing selenium in the side chains.80 Hyperbranched selenium-containing polymers can be synthesized via direct polymerization of NaHSe with 1,3,5-tris-bromomethyl-2,4,6trimethylbenzene,45 or copolymerization of selenium-containing phosphate monomers.81 2.2.3. Tellurium-Containing Structures. The synthesis of tellurium-containing polymers is similar to that of seleniumcontaining polymers. For example, polyurethane containing tellurium in the main chain was synthesized via the condensation between di(1-hydroxyl undecyl)telluride and 2,4-toluene diisocyanate.82,83 Side-chain tellurium-containing polymers were synthesized via grafting the carboxyl group of poly(acrylic acid) (PAA) with tellurium-containing Ar−Te− OH.23,24 Hyperbranched tellurium-containing polymers were synthesized via cross-linking of 11,11′-tellurobis(undecan-1-ol) with 1,3,5-tris(bromomethyl)benzene.46 2.2.4. TK-Containing Structures. Main-chain TK-containing polymers were commonly synthesized via direct polyconden-

sation using the acetal exchange reaction between 2,2dimethoxypropane and dithiols (Scheme 3).84−86 Under optimized synthetic conditions, the formed TK-containing polymers could bear thiol groups on the main-chain terminals, which would allow subsequent modification/functionalization.84,85,87 However, the functional groups in the monomers often cannot survive under the harsh reaction conditions during polymerization, such as acidic catalysts, high temperature, and reactive thiols. Besides, the direct polycondensation often leads to TK-containing polymers with low molecular weight and wide polydispersity.84 In addition to direct polycondensation using thiols, various TK-containing crosslinkers can be synthesized and further used to synthesize mainchain TK-containing polymers,29,88−91 side-chain TK-containing polymers,28,92,93 and hyperbranched polymers.94−96 2.2.5. PBA/PBE-Containing Structures. PBEs are usually formed via the condensation of PBAs with diols (Scheme 4a),97,98 especially with pinacols. Unlike the above-mentioned structures, PBE-containing structures are seldom incorporated into the main chain of a polymer. To obtain side-chain PBEcontaining polymers, step polymerization using PBE-pendant monomers has been investigated (Scheme 4b).40,99 For example, PBE-containing diols can condense with diacids to form polyesters,40 and 2,6-bis(bromomethyl) phenylboronic acid pinacol esters were used to synthesize the PBE-pendant polysulfoniums via a two-step procedure.99 Notably, Li et al. developed a multicomponent polymerization approach to synthesize PBE-containing poly(ester-amide)s using 4-formylbenzeneboronic acid pinacol esters.100 For some PBEcontaining methacrylates, the reversible addition−fragmentation chain transfer (RAFT) polymerization is more suitable for the synthesis of block copolymers (Scheme 4c).101−104 Additionally, the postpolymerization modification strategy was extensively used to synthesize PBE-containing polymers (Scheme 4d).18,30,105,106 The hydroxyl groups on natural polymers (such as dextran30,106,107 and cyclodextrin18,108) or synthetic polymers31,105 can be modified with PBE-containing carbonochloridates or 1H-imidazole-1-carboxylates. Also, PBEcontaining halides were utilized to form quaternary ammonium on the side chains with tertiary amines.109 2.2.6. Oxalate-Containing Structures. Normally, mainchain oxalate-containing polymers were synthesized via step polymerization of oxalyl chloride with hydroxybenzyl alcohol and other aliphatic diols.34,42,110 In some cases, the phenols on therapeutic drugs, such as vanillin111 and curcumin,112 can be employed to form the oxalate-containing polymers. Alternatively, side-chain oxalate-containing polymers can be achieved via either ROP from oxalate-containing norbornene113 or RAFT polymerization from oxalate-containing methacrylate.33,114,115 2444

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Biomacromolecules Scheme 4. Synthetic Routes of PBA/PBE-Containing Structuresa

a

(a) Formation of PBEs via condensation of PBAs with diols. Step polymerization (b) and radical polymerization (c) using PBE-containing monomers. (d) Postpolymerization modification using PBE-containing moieties.

2.3. Design Criteria of ROS-Responsive Materials. Basically, two criteria, response efficiency and translation potential, need to be considered when responsive biomaterials are designed.116 Thus, the desired ROS-responsive materials need to respond to the local ROS with sufficient sensitivity, high selectivity, and spatiotemporal control. While different RRMs afford different ROS sensitivity, they need to be properly selected toward a specific utility. Taken drug delivery as an example, fast ROS response would contribute to instantaneous drug release, while slow ROS response could

enable sustained release. Sometimes the heterogeneity of ROS concentration may even be utilized to mediate programed and cascading drug delivery. Because RRMs involve cleavable and noncleavable transformation in response to ROS, a proper RRM needs to be properly designed toward a particular purpose. For clinical translation, the designed ROS-responsive materials should be cost-effective, amenable to scale-up, biocompatible, and stable. Libraries of ROS-responsive materials need to be developed and functionalized to fulfill the different requirements. Also, the ROS-responsive materials 2445

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Biomacromolecules Scheme 5. Schematic Illustration of Diverse ROS-Responsive Platforms Used for Drug Delivery

Figure 1. (a) Structure of PEG-PUSeSe-PEG, self-assembly of PEG-PUSeSe-PEG into micelles, and ROS- or redox-triggered disassembly of micelles. Reproduced with permission from ref 27. Copyright 2010 American Chemical Society. (b) Scheme of near-infrared light-triggered cisplatin release from NCs based on amphiphilic copolymer PEG−PUTe-PEG. Reprinted from ref 83, Copyright 2017, with permission from Elsevier.

order to prevent the preleakage of drug cargoes during blood circulation, ROS-responsive cross-linkers could be employed to construct the cross-linked, drug-loaded micelles (Scheme 5). Additionally, RRMs are also widely utilized to fabricate prodrugs and hydrogels for drug delivery purposes (Scheme 5). 3.1. Nanocarriers (NCs) for Drug Delivery. 3.1.1. Thioether-Based NCs. H2O2 responsiveness and phase transition of thiothers were first observed in 1908,118 while it was not until 2004 that the major step was made by Hubbell et al. in the application of thioether-based NCs for drug delivery.55 Among the thioether-containing polymers, poly(propylene sulfide) (PPS) has been extensively studied over the past decade owing to its unique physicochemical properties. PPS possesses a low Tg and a hydrophobic nature, which easily converts to polysulfoxide and/or polysulfone under the trigger of oxidants.43 Unilamellar NCs composed of the PEG-b-PPS-bPEG triblock polymer changed their morphologies from polymersomes to micelles in the presence of 3 vol % H2O2.55 Duvall et al. developed a PPS-based micellar carrier using block copolymer poly(propylene sulfide)-block-poly(N,N-dimethylacrylamide) (PPS-b-PDMA).119 In the presence of H2O2 (∼100 mM), less than 25% of the encapsulated cargo molecule (Nile red) was released from the micelle core, indicating the potential of these micelles for sustained drug release. Notably, the PPS-b-PDMA micelles could respond to

should be able to be constructed in different formulations and devices. In some cases, we need to combine ROSresponsiveness with other stimuli-responsiveness to achieve satisfactory efficacy.

3. ROS-RESPONSIVE DRUG DELIVERY SYSTEMS All the RRMs listed in Table 1 are hydrophobic. Therefore, theoretically, these responsive moieties can be incorporated into the main chain or side chains of hydrophobic polymer block in combination with hydrophilic block to synthesize linear or branched amphiphilic polymers (Scheme 1). These amphiphilic block polymers are then used to fabricate core− shell micelles, core-cross-linked micelles, or polymersomes (Scheme 5). The hydrophobic cores of micelles can serve to encapsulate hydrophobic therapeutics, thus improving their solubility as well as bioavailability and reducing the side effect. Polymersomes can be loaded with both hydrophilic or hydrophobic therapeutics.117 The outer shells of polymeric micelles or polymersomes are commonly composed of polymers with stealth properties, for instance, poly(ethylene glycol) (PEG), which prolongs blood circulation and enhances tumor accumulation. Once the micelles reach the diseased site (such as tumor or inflammation), the high local ROS concentration will trigger the dissociation of the micelles/ polymersomes and release the drug cargoes (Scheme 5). In 2446

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NCs to encapsulate cisplatin and indocyanine green (ICG) (Figure 1b).126 Upon tumor site-specific NIR light irradiation, ROS produced by ICG can oxidize the tellurium and trigger the dissociation of the NCs, thus rapidly releasing cisplatin due to weakened coordination interaction with tellurium. 3.1.3. TK-Based NCs. Unlike the above-mentioned NCs, the application of TK-based NCs is still in its infancy. Recently, Nam and co-workers developed biodegradable NCs based on poly(1,4-phenyleneacetone dimethylene thioketal) (PPADT),127 which can degrade in the presence of either 10 mM H2O2 or 10 mM KO2.127 Thus, the paclitaxel (PTX)loaded PPADT NCs reduced the viability of PC-3 cells down to 34.8% at 0.1 μg mL−1 of PTX. Dual-responsive thioketal nanoparticles (TKNs) were developed, which can respond to either 100 mM H2O2 or 10 mM GSH. The PTX-loaded TKNs could selectively kill PC-3 cells (cancerous) while preserve the viability of CHO cells (normal), probably resulting from the high levels of ROS and GSH in PC-3 cells that synergistically triggered the PTX release.84 In PC-3 tumor-bearing mice, the PTX-loaded TKNs can efficiently accumulate at the tumor site and suppress the tumor growth, with minimal side effect to normal tissues. Thus, the TKNs possessed great potential in enhancing the efficacy and avoiding the side effect of chemodrugs. Drug loading via hydrophobic interactions will often lead to drug preleakage during storage or systemic circulation, because hydrophobic interactions are not sufficiently strong. One solution is to introduce a strong π−π interaction between the drug and the carrier.128 For example, He and co-workers reported a novel π-conjugated thioketal, which was used to synthesize block copolymer methoxy poly(ethylene glycol)TK-poly(ε-caprolactone) (mPEG-TK-PCL). The mPEG-TKPCL NCs can not only improve the drug loading efficiency via π−π stacking and hydrophobic interaction with DOX, but also responsively released the loaded DOX under the trigger of intracellular ROS.128 Another solution to prevent drug leakage is cross-linking of the NCs. For example, we cross-linked the micelles core with a TK-containing cross-linker, which can greatly stabilize the DOX loading while enable ROS-responsive DOX release.129 3.1.4. PBA/PBE-Based NCs. PBE moieties can not only provide hydrophobic domains to construct PBE-based NCs but also allow drug loading via π−π interactions.107 The oxidation-sensitive dextran (Oxi-DEX) containing pendant PBEs, has been used to form NCs and to loaded hydrophobic therapeutics.107 The Oxi-DEX NCs were stable for more than 1 week in the absence of H2O2, while could release their cargoes with a half-life of 36 min in the presence of 1 mM H2O2. Ovalbumin (OVA)-loaded Oxi-DEX NCs induced the presentation of CD8+ T-cells with a rate 27-fold higher than that induced by OVA-loaded nonresponsive NCs, indicating their potential for immunotherapy.107 With rational design, the PBE-based NCs can respond to H2O2 at the concentration relevant to that in the activated immune cells (50−100 μM).130 The designed PBE-based NCs can release their payloads in the presence of intracellular ROS in the activated neutrophils,40 indicating their promising potentials in drug delivery to tissues undergoing oxidative stress. PBE-containing lipids were also developed and used to construct NCs. Chen and co-workers synthesized a PBE-linked PEG-lipid conjugate for ROS-responsive drug delivery.131 The PBE-based lipids formed stable NCs with a hydrodynamic diameter around 25 nm, while in the presence of 200 mM H2O2, the NCs

3-morpholinosydnonimine (SIN-1) and peroxynitrite, two oxidants produced by activated macrophages.119 In a model of diabetic peripheral arterial disease, the curcumin-loaded NCs provide sustained, ROS dose-dependent local curcumin release and reduced ROS levels in the ischemic limb.58 Also, these curcumin-loaded NCs can significantly enhance the cell viability under exogenous oxidative stress and improve the hemoglobin oxygen saturation recovery. Taken together, this strategy provides a compelling approach for the on-demand delivery of antioxidants into localized tissues affected by chronic inflammatory diseases.58 Similarly, PEG-b-PPS NCs were used to load the antioxidant melatonin (MeI)54 and celastrol,120 which demonstrated desired efficacies toward the treatment of sepsis-induced acute liver injury54 and atherosclerosis.120 3.1.2. Selenium- and Tellurium-Based NCs. Selenium- and tellurium-based NCs have recently drawn increasing attention due to their excellent ROS sensitivity. Mono- and diselenidecontaining polymers are extremely insoluble in water and they are normally employed in the construction of amphiphilic block copolymers. Zhang and co-workers developed the diselenide-containing block copolymer PEG-PUSeSe-PEG (Figure 1a), which was formulated into core−shell micelles owing to its amphiphilic nature. Interestingly, the PEGPUSeSe-PEG micelles can respond to both glutathione (GSH) and H2O2, indicating their potential for GSH/H2O2 dualresponsive drug delivery.27 Similarly, the amphiphilic PEG-PUSe-PEG block copolymer containing monoselenide was used to form micelles and encapsulate the anticancer drug doxorubicin (DOX). Upon treatment with 0.1% H2O2, 72% of the loaded DOX could be liberated within 10 h. However, when selenium in PEG-PUSePEG was replaced by sulfur, the H2O2 sensitivity was greatly attenuated, leading to lower drug release rate.77 Subsequently, Yan et al. developed NCs from diselenide-containing amphiphilic hyperbranched polymers, achieving a high DOX loading efficiency of 61.6%. Notably, the barren NCs without encapsulating anticancer therapeutics can prevent the proliferation of cancer cells in vitro.121 This is mainly due to the internalization of diselenide-containing NCs, which can induce the apoptosis of cancer cells while selectively preserve the viability of noncancer cells.122 Synergistically, the loaded DOX can work together with NCs to eradicate the Hela cells in vitro.121 Similar results were found using monoselenide NCs, which can inhibit the proliferation of Hela cells by 50% with a NC concentration of 16 μg mL−1 over 48 h and possess ROSresponsiveness that is useful for intracellular delivery of DOX.81 More recently, diselenide-cross-linked micelles were developed by Deepagan et al.,123 and DOX was effectively loaded in the hydrophobic cores of the micelles. Upon treatment with 100 μM H2O2, the micelles dissociated and released more than 65% of the loaded DOX within 3 days. In PC3 tumor-bearing nude mice, the DOX-loaded, cross-linked micelles notably outperformed free DOX to efficiently suppress the tumor growth.123 Due to its lower electronegativity, telluride is less toxic and more sensitive to ROS than selenide and sulfide.46,124 Xu et al. fabricated the telluride-containing NCs from the triblock copolymer PEG-PUTe-PEG and used the NCs for the delivery of platinum-based anticancer drugs.125 The strong coordination interaction between platinum and tellurium atoms made the telluride-based NCs outstanding for the encapsulation of platinum-based drugs. Xu et al. further used PEG-PUTe-PEG 2447

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Figure 2. Schematic illustration of the oxidation-responsive multifunctional polymersomes that underwent ROS-triggered vesicle bilayer crosslinking, permeability switching, and enhanced intracellular drug release. Reproduced with permission from ref 101. Copyright 2016 American Chemical Society.

dissociated and the size significantly decreased.131 We recently developed PBA-pendant polypeptides which achieved ultrahigh drug loading capacity (up to 50%) and quantitative loading efficiency (nearly 100%) via the donor−acceptor coordination between PBAs and various electron-donating chemodrugs, such as DOX, epirubicin (EPI), and irinotecan (IR).31 Additionally, the encapsulated drugs can be selectively released in response to the high ROS levels (100 μM H2O2) in cancer cells.31 Apart from the morphological transformation as a result of NCs dissociation, in some cases, the NCs could maintain their morphology while change their permeability upon specific stimuli. For example, Liu et al. reported PBE-based polymersomes composed of an amphiphilic PBE-containing block copolymer (Figure 2). Once inside the cells, the intracellular ROS triggered the cleavage of PBE groups, relieving the amino groups that underwent amidation reaction. The whole process would not change the assembly structure of the polymersomes, while led to the hydrophobic-to-hydrophilic transition of the polymersome shells to enable sustained drug release inside living cells.101 Nowadays, the application of PBE-based NPs is becoming more and more disease-related. For instance, Jiang et al. reported PBE-based curcumin-loaded polymeric micelles which can reduce the oxidative stress in the inflammatory microenvironment of Alzheimer’s disease and modulate microglia to impart synergistic effect against Alzheimer’s disease.132 By targeting the abnormal microglia at an early stage, the curcumin-loaded micelles possess great potential for clinical translation. In another example, glucose oxidase (GOx)/insulin-loaded, PBE-based nanogels were used for enhanced treatment of diabetes mellitus.133 H2O2 produced by GOx and high levels of blood glucose triggered the dissociation

and swelling of the nanogels and subsequently released insulin. In a type-1 diabetes animal model, the GOx/insulin-loaded nanogels maintained blood glucose levels in a normoglcemic state for more than 16 h, with negligible inflammation effect on normal tissues.133 3.1.5. Other NCs. 3.1.5.1. Vinyldithioether-Based NCs. Vinyldithioether specifically reacts with singlet oxygen which is mostly produced via activation of tissue oxygen by a photosensitizer (PS) upon light irradiation. Since singlet oxygen possesses a lifetime of only several microseconds,134 it is often required that the vinyldithioether-based NCs encapsulate therapeutics together with a PS. 3.1.5.2. Ferrocene (Fc)-Based NCs. Fc-containing polymers are promising scaffolding materials for designing drug carriers.135 Fc units are converted from the hydrophobic, neutral state to the hydrophilic, positively charged state during oxidation, and such hydrophobic−hydrophilic transition could trigger the sol−gel transition to enable self-healing or swellingmediated drug release.25 Xu et al. developed a polymeric micelle composed of poly(N-acryloylmorpholine-block-2-acryloyloxyethyl ferrocenecarboxylate) (PACMO-b-PAEFC) for PTX encapsulation. In the presence of ROS, the micelles swelled to release the PTX.26 3.1.5.3. Proline Oligomer-Based NCs. Amino acids such as proline, histidine, arginine, and lysine are susceptible to ROSmediated and metal-catalyzed oxidation.35 The reaction of proline residues with environmental oxidants can result in the cleavage of the parent polypeptide chain at these sites. Proline oligomer has a relatively slower degradation rate under ROS than other ROS-sensitive groups, and thus it can be used for sustained release. For example, Yu et al. synthesized proline oligomers with different lengths (Pn) and exposed them to an oxidizing environment.36 Diblock copolymers of PEG and 2448

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Figure 3. Schematic illustration of light-strengthened protein delivery. Visible light irradiation (635 nm) of the PS at low power density (5 mW/ cm2) generated large amount of H2O2 to restore the activity of RNBC and subsequently exerted synergistic anticancer efficacy. Reproduced from ref 150 with permission of John Wiley and Sons. Copyright 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

poly(ε-caprolactone) (PCL) were carboxylated to form a terpolymer (4% PEG-86% PCL-10% cPCL), which was further cross-linked with the biaminated PEG-Pn-PEG. To verify that the proline cross-linker could accelerate the degradation of the scaffold under oxidative conditions, the material was immersed in a buffer with or without 1 mM ROS generator SIN-1 for 28 days at 37 °C. SIN-1 could simultaneously produce nitric oxide and superoxide, and further lead to the production of peroxynitrite and hydroxyl radicals in situ. Within 28 days, materials incubated under ROS conditions retained 72% of their mass, while the mass of materials incubated without ROS showed minimal change. This phenomenon was attributed to the hydrolysis of proline oligomers in the polymer network. In summary, thioether- and PBA/PBE-based based NCs have been extensively studied over the past decade, mainly due to their structural diversity and compatibility with a variety of therapeutic agents. In the future, when designing thioetherbased NC, more attention should be paid to the drug loading efficiency and ROS sensitivity. The degradation of PBA/PBEcontaining polymers generates highly active quinone methide (QM) intermediates that can react with various biomolecules, which may cause undesired adverse effects in vivo.43 This also needs to be seriously considered. Selenium/tellurium-based NCs demonstrate relatively high sensitivity to ROS, while at the meantime can respond to GSH that is widely present in cells. Therefore, improving the ROS specificity is critically demanded to ensure highly selective drug delivery to the diseased sites. For TK-based NCs, the ROS sensitivity should be improved by tailoring the composition, and more efforts

could be made to optimize the polymer preparation, selfassembly, drug loading, and drug release profile. 3.2. ROS-Responsive Prodrugs. Prodrug refers to a chemical compound that is of little pharmacological activity, while after administration, it can be converted to the active original drug via either enzymatic or chemical activation.136 Normally, compared to their parent drugs, prodrugs are designed to increase the solubility, achieve targeted delivery, and facilitate cell internalization.11 ROS-activatable prodrugs have recently been developed and explored by caging the native drug with ROS-cleavable moieties.137 ROS-responsive prodrugs can be mainly divided into small-molecular, protein, and polymer prodrugs. Theoretically, the prodrugs are composed of three functional domains, namely a ROS acceptor that can respond to ROS, an effector that is normally the native drug, and a linker between the ROS acceptor and the effector.138 In this section, we will introduce the design and application of recently developed ROS-responsive prodrugs. 3.2.1. Small-Molecular Prodrugs. Normally, small-molecular anticancer chemotherapeutics enter cancerous cells and normal cells equally. Thus, the major concern during cancer therapy is how to achieve cancer-specific drug delivery to attenuate the host toxicity. To this end, ROS-activatable smallmolecular prodrugs can respond to the high levels of ROS in most cancer cells, thus liberating the active drug to achieve selective therapeutic efficacy.136 Among all the ROS moieties as listed in Table 1, PBE/PBA moieties are most commonly used in the synthesis of prodrugs, owing to their well-defined and facile chemistry. The first 2449

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Biomacromolecules example of a PBE-based prodrug was reported in 2010,139 when Cohen et al. modified a matrix metalloproteinase inhibitor (Table S1), 1,2-HOPO-2, with PBE moieties, and the PBE-modified inhibitor could selectively inhibit the proliferation of cancer cells.139 Subsequently, various PBE/ PBA-containing small-molecular prodrugs, based on nitrogen mustards,138 camptothecin (CPT),140 5-FU,141 SAHA,142 and cinnamaldehyde,15 were designed to overcome the shortcomings of the original drugs (Table S1). The masking effect of the PBE/PBA moieties significantly diminished the cytotoxicity in normal cells, while in cancer cells with overproduced ROS, the native drugs were liberated to mediate selective anticancer efficacy. Thus, it was not surprising that these prodrugs displayed excellent cancer cell selectivity over normal cells. Notably, quinone methide (QM), a product after cleavage reaction of PBEs/PBAs, has GSH-depleting property, which can further enhance the oxidative stress in cancer cells. This unique property of PBEs/PBAs provides a possibility of conjugation with other ROS inducers such as cinnamaldehyde15 and Fc-containing moiety143 to achieve enhanced anticancer efficacy.7 Moreover, a multifunctional prodrug containing a ROS-activatable drug, a fluorescent dye, and a cancer-targeting ligand was developed, achieving cancertargeted theranostics.144 Besides the PBE/PBA moieties, very few prodrugs are fabricated from other ROS-responsive domains, mainly due to the difficulty in chemical synthesis, and some representative examples were listed in Table S1.145,146 In these cases, a PS is often necessary to produce singlet oxygen upon light irradiation, which in turn cleaves the diphenoxyethene or TK linkers to release the drugs. In addition, NCs were required to codeliver the prodrug and the PS, and they demonstrated cooperative anticancer efficacy based on the combination of chemotherapy and photodynamic therapy (PDT).147 3.2.2. Protein Prodrugs. Protein therapeutics play an important role in the medical field due to their potent pharmacological activity and specificity.148 Similar to smallmolecular drugs, proteins can also be engineered into ROSresponsive prodrugs. Modification of proteins with RRMs reversibly deactivates the protein function, while under the trigger of high ROS levels in cancer cells, the native proteins are released to recover the pharmacological activity. As such, it is normally demanded that the ROS-responsive domains should be completely removed from the proteins under ROS treatment, leaving the protein with exactly the same chemical structure. To this end, modification of proteins typically occurs on the residual amino groups of the lysin residues, onto which PBA/PBE moieties are commonly conjugated. For example, Wang et al. modified the lysine residues of RNase A with PBA (RNase-NBC), which temporarily deactivated the protein and changed the net charge of the protein from positive to negative under neutral condition.149 Thus, the RNase-NBC formed NPs with positively charged lipids via elecltrostatic interaction to facilitate cancer cell uptake. Upon H2O2 treatment, the NBC moiety was cleaved due to a self-immolation reaction, and the RNase A activity could be restored to cleave RNA and induce cytotoxic anticancer effect.149 Recently, we developed PBAmodified RNase A (RNBC) and encapsulated the prodrug into ketal-cross-linked PEI (KPEI), which were further coated with PS-modified hyaluronic acid (HA-HP), yielding the KPEI/HAHp/RNBC nanocomplexes (KHHR NCs) (Figure 3). Under long-wavelength visible light (635 nm) irradiation at low power density (5 mW/cm2), the PS-generated H2O2 could

induce uncaging of the PBA protection group to restore the anticancer function of RNase A, thus achieving a synergistic anticancer effect in vivo.150 Similarly, Pei et al. modified cytochrome c (Cyt c) with PBAs and developed a yolk−shell nanoplatform for the codelivery of the Cyt c prodrug and DOX to realize synergistic cancer therapy.151 3.2.3. Polymer Prodrugs. In general, the use of ROSresponsive polymer prodrugs possesses the following merits, including (1) the employment of hydrophilic blocks with stealth property that can prolong the blood circulation and enhance tumor accumulation, (2) a definite and high drug loading, (3) prevention of the premature drug leakage, and (4) selective drug release in the ROS-rich microenvironment. Although polymer prodrugs have been widely investigated in various fields,152 the development and application of ROSresponsive polymer prodrugs have recently been pursued (Table S2). During the synthesis of ROS-responsive polymer prodrugs, the native drug could be incorporated into the polymer via ROS-responsive linkers at three major locations, end-capping, main chain, and side chains (Scheme 5). The end-capping strategy involves the use of drugs as polymer terminals.153 For example, oligo(ethylene glycol) (OEG) was conjugated with the anticancer drug SN38 via the thioether linker.154 The amphiphilic prodrug assembled into nanocapsules which can accumulate in the tumor tissues through the enhanced permeability and retention (EPR) effect. In tumor cells, the high levels of ROS triggered the liberation of the parent SN38 to efficiently eradicate the cancer cells.154 Similarly, Pu et al. coupled DOX to the end of PEG via the TK linker, obtaining the mPEG-TK-DOX prodrug.155 Prodrugs incorporating both the drug and the RRM in the main chain are usually synthesized via step polymerization as mentioned above. Antioxidants, such as vanillin,111 vanillyl alcohol,42 and curcumin,112 were commonly used to synthesize main-chain-degradable prodrugs. In general, these antioxidantbased prodrugs can scavenge the high levels of intracellular H2O2 and the oxalates in the polymers possess CO2 bubblegenerating property in the presence of H2O2.42,112 The ROStriggered CO2 bubbles are then able to swell or destroy the structures of liposomes. For example, Shi and Farokhzad et al. developed a polymer prodrug based on anticancer drug mitoxantrone (MTO) and the TK cross-linker, which was able to self-assemble into nanoparticles (NPs).88 The polymer prodrug (polyMTO) NPs were camouflaged with PEGylated, RGD-encoded liposomes to prolong blood circulation and enhance tumor targeting. After intravenous injection of the polyMTO NPs into LNCaP tumor-bearing mice, the NPs responded to intracellular ROS to mediate a chain-breakage patterned release of the anticancer drug, leading to significant inhibition of tumor cell growth.88 ROS-responsive prodrugs containing drug molecules in the side chains are mainly synthesized via either postpolymerization modification or direct polymerization using the drug- and RRM-containing monomers. Postpolymerization modification was typically achieved via the PBA-diol chemistry. For example, naproxen, a nonsteroidal anti-inflammatory drug,98 and chlorambucil (CHL)156 can be premodified with PBA, and then the PBA-modified naproxen or CHL was conjugated to diol-containing polymers. Alternatively, naproxen-derived monomers,157 indomethacin (IND)-derived monomers,158 and CPT-derived monomers,32,33,114 were synthesized and subjected to direct polymerization. Other RRMs such as oxalate or TK are less sensitive to ROS, and thus prodrugs 2450

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hydrogel composed of methoxypoly(ethylene glycol)-blockpoly(L-methionine).166 ROS produced by activated macrophages could induce hydrogel erosion to mediate sustained drug release.166 Interestingly, Gu and Chen et al. reported a triblock copolymer consisting of a PEG block, a ROSresponsive L-methionine (Me) block, and a dextro-1-methyl tryptophan (D-1MT) block. This copolymer self-assembled to form a hydrogel at the polymer concentration higher than 8.0 wt %, into which antiprogrammed cell death-ligand 1 (aPDL1) was encapsulated.159 The loaded aPD-L1 could slowly release from the hydrogel as a result of ROS-trigger hydrogel degradation, thus stimulating CD8+ T cells to provoke anticancer immune responses.159 Besides the formation of hydrogels via physical sol-to-gel transformation, chemical cross-linking was also employed in hydrogel formation. For example, Li and Du et al. used PBEcontaining biacrylates and a 4-arm-poly(ethylene glycol) (PEG) acrylic macromonomer (4-arm-PEG-SH) to construct ROS-responsive hydrogels.167 Both insulin and GOx were encapsulated into the hydrogels, and in the presence of high levels of glucose, GOx mediated extensive production of H2O2 which in turn degraded the PBEs and triggered hydrogel dissociation to release insulin.167 To summarize this section, several factors that affect the drug loading and release profile of ROS-responsive hydrogels should be taken into account, aiming for an optimal therapeutic effect. These factors include drug-hydrogel interaction, morphologies and architectures, mechanical properties, biodegradability mechanisms and clearance time regulation of hydrogels.

based on these RRMs are commonly used in combination with other ROS amplifiers, such as glucose oxidase (GOD),115 6-Opalmitoyl-L-ascorbic acid,114 and β-lapachone,33 which can upregulate the cellular ROS production as introduced in Section 5. Protein drugs are usually fragile. In protein prodrugs, RRMs are commonly modified at the active sites of proteins to temporarily inhibit their functions, and a key requirement is that the original function of the parent proteins should be maintained after ROS treatment. Thus, the chemistry used for the synthesis of protein prodrugs should be rationally designed and more general synthetic methods need to be developed. For the synthesis of polymer prodrugs, it often requires sophisticated synthetic procedures and is not easy to scale up. Also, the chemical structures of the polymer prodrugs should be precisely characterized before they can be approved for clinical trials. To this end, new methodologies in terms of synthesis and characterization are highly demanded. 3.3. ROS-Responsive Hydrogels. In addition to various types of ROS-sensitive NCs and prodrugs, ROS-responsive hydrogels have also been developed for drug delivery and tissue engineering.159,160 The driving forces for hydrogel formation include ionic bonding, hydrogen bonding, hydrophobic interaction, or chemical cross-linking.161,162 Hydrogels have attracted considerable interest in the biomedical fields due to the high water content and the porous structure that mimic the natural extracellular matrix. Drugs or even cells can be easily mixed with the hydrogel precursor and fixed at the desired site to form hydrogels in situ after injection.162 Thereafter, the ROS-responsive hydrogels can sense the oxidative stress in the local environment, regulate cell behavior, promote on-demand drug release, and eliminate excessive ROS. Thus, ROS-responsive hydrogels provide promising utilities in the presence of host immune response and high local levels of ROS.85 To introduce ROS-responsiveness to hydrogels, one strategy is to incorporate RRMs into the backbones of the block copolymers that are used to form the hydrogels. For example, Gupta et al. synthesized a PPS-containing ABC triblock polymer, poly[(propylenesulfide)-block-(N,N-dimethylacylamide)-block-NIPAAM] (PDN), which self-assembled into micelles at room temperature. When the environmental temperature was above its lower critical solution temperature (LCST), the PNIPAAM shells of micelles changed from soluble to insoluble, leading to aggregation of the micelles and formation of hydrogels. In the presence of ROS, the PPS block underwent a gradual degradation to trigger dissociation of hydrogels.163 In a recent study, they found that the ROSresponsive PDN hydrogels were cytocompatible when cocultured with human mesenchymal cells (hMSCs). In vivo studies illustrated that the PDN hydrogels mediated sustained drug release over 12 days and allowed strong cellular infiltration.164 Similarly, Zhu et al. developed an injectable hydrogel based on four-armed star PEG-b-PPS block polymer, which was used to encapsulate and deliver hydrophobic vemurafenib.165 Under the trigger of high local ROS level induced by immune responses, the loaded vemurafenib could be released within mouse dermal wounds, promoting paradoxical mitogen-activated protein kinase (MAPK) activation in keratinocytes and wound closure.165 Another strategy to incorporate ROS-responsiveness into hydrogels is to employ polymers with ROS-responsive side chains. For example, Xu et al. synthesized a ROS-responsive

4. ROS-RESPONSIVE GENE DELIVERY SYSTEMS Nonviral gene delivery provides tremendous potential for the treatment of human diseases.168 However, the therapeutic efficacy of nonviral gene delivery is often hurdled by the multiple physiological barriers that often pose opposing requirements for the design of delivery materials. One inconsistence is related to the gene condensation and intracellular gene release.169 For the purpose of better gene condensation, polycations with higher molecular weight and cationic charge density are usually required due to their stronger binding affinity with the anionic nucleic acids. However, it will conversely inhibit the unpackaging of nucleic acids from the polyplexes in the cytoplasm, thus compromising the transfection efficiency.170 Another inconsistence is associated with the transfection efficiency and toxicity. Polycations with higher molecular weight and cationic charge density normally afford stronger membrane activities to promote the cellular internalization, which, however, lead to higher materials toxicity at the meantime. Recently, ROSdegradable or ROS-responsive charge-reversal polycations have been designed to harmonize the above-mentioned inconsistencies to enable highly efficient gene transfection. 4.1. TK-Based Gene Delivery Systems. Groundbreaking work of using TK-based polymeric NCs to deliver gene was first reported by Merlin and Murthy et al. in 2010.86 They formed a complex using small interfering RNA (siRNA) against the pro-inflammatory cytokine tumor necrosis factoralpha (TNF-α) with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane (DOTAP), and then loaded the complex into thioketal nanoparticles (TKNs) formulated from poly(1,4-phenyleneacetone dimethylene thioketal) (PPADT). When orally administered, the TKNs could protect 2451

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Figure 4. Schematic representation of ROS-responsive charge-reversal polymer and its CRGDK-receptor targeting fusogenic lipopolyplex (RGDKFLPPs) for systemic gene delivery. (a) ROS-triggered charge reversal of B-PDEAEA to anionic poly(acrylic acid) . (b) Formulation and ROSresponsive anticancer mechanism of the fusogenic lipopolyplexes. Reproduced from ref 109 with permission of John Wiley and Sons. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

the siRNA cargo against the harsh gastrointestinal conditions and target the inflamed colon tissues with high levels of ROS that degraded the TKN to expose the positively charged DOTAP/siRNA complex, thus provoking efficient TNF-α knockdown in the inflamed colon.86 Subsequently, ROSresponsive, backbone-degradable polycations were developed to promote gene delivery. Xia et al. synthesized cationic poly(amino thioketal) (PATK) via polymerization of oligoamines with the TK-containing acrylamides. The main chain of

the polymer was easily degraded by the overproduced H2O2 in cancer cells, and thus intracellular DNA release would be accelerated to enhance the transfection efficiency.29 Similarly, we used a TK-containing cross-linker to cross-link the low molecular weight polyethylenimine (PEI), and the obtained TK-PEI with high molecular weight could efficiently condense plasmid DNA encoding p53 to form polyplexes.94 The positively charged polyplexes were coated with pheophytin a (Pha)-modified hyaluronic acid (HA-Pha) to enhance the 2452

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effectively down-regulate the TNF-α levels to protect animals from acute hepatic failure.12 TK- and PBE/PBA-based ROS-responsive gene delivery systems are most commonly studied, mostly due to the structural diversities of TK cross-linkers and PBE/PBA moieties. More efforts should be made in the future to develop gene delivery systems involving other RRMs with higher ROS sensitivity

colloidal stability and the cellular uptake of polyplexes in cancer cells. Once inside the cancer cells, nonlethal amount of ROS produced by 8 min far-red light irradiation triggered the cleavage of TK cross-linker, thus promoting intracellular DNA release to enhance the p53 transfection efficiency. At the posttransfection state, a lethal amount of ROS was produced by 30 min light irradiation, provoking PDT to synergistically kill cancer cells with p53-mediated gene therapy.94 For the treatment of wound healing disorders, such as diabetic foot ulcer, it is necessary to downregulate the overexpressed detrimental proteins, such as prolyl hydroxylase domain protein 2 (PHD2). To this end, siRNA against PHD2 was loaded into the poly(thioketal urethane) (PTK-UR) NPs,171 which could release siRNA locally within wounds with high levels of local ROS to potentiate the silencing efficiency against PHD2. In a rat model of streptozotocin (STZ)-induced type-1 diabetes, the siRNA-loaded PTK-UR NPs demonstrated effective PHD2 knockdown to promote angiogenesis, cell proliferation, and new tissue formation.171 4.2. PBE/PBA-Based Gene Delivery Systems. Besides stimuli-degradable polycations that can facilitate intracellular gene release, charge reversal polymers that can change from positive to negative upon specific triggers could also repel the gene cargoes in the cytoplasm to potentiate gene transfection.172,173 Groundbreaking work related to ROS-responsive, charge-reversal gene delivery systems was accomplished by Shen et al. in 2016.109 PBA-modified, quaternary ammonium-containing poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) was prepared and used to form spherical polyplexes with DNA (Figure 4). Furthermore, a fusogenic liposome was used to load the formed polyplexes, because the liposome can fuse with the cell membrane to facilitate direct transport into the cytosol, avoiding endolysosomal entrapment. Upon treatment with 1 mM H2O2, the PBA moieties could be oxidized to cleave the quaternary amine via self-immolation reactions and expose the carboxylic groups, thus reversing the polymer from cationic to anionic (Figure 4a). Accordingly, the loaded DNA cargoes were released from the polyplexes owing to the electrostatic repulsions. The fusogenic lipopolyplexes possessed high tumor targeting and gene expression levels in vivo, indicating their great potential for anticancer gene therapy (Figure 4b).109 Subsequently, they developed polysulfoniums with higher ROS-sensitivity as a new class of cationic, ROS-responsive gene delivery materials. The PBE/PBA fraction of polysulfoniums could be degraded into uncharged thioether after ROS treatment, which weakened the electrostatic interaction with the negatively charged DNA to promote intracellular DNA release.99 4.3. Other ROS-Responsive Gene Delivery Systems. Besides the TK- and PBE/PBA-based systems, few ROSresponsive gene delivery systems have been reported, mainly due to the lack of proper synthetic methods to incorporate the RRMs into cationic polymers. We recently developed diselenide-cross-linked PEI with increased molecular weight to improve the condensation of TNF-α siRNA (siTNF-α).12 The resulting polymer/siRNA polyplexes were subsequently coated with carboxylated mannan (Man-COOH) to allow targeting to macrophages. Once inside the cells, the polyplexes could be degraded by the intracellular ROS in inflammatory macrophages to release the loaded siRNA. After systematic administration, the siTNF-α-loaded ternary polyplexes could

5. UP-REGULATION OF ROS IN MALIGNANT CELLS/TISSUES ROS is a double-edged sword for cancer cells. Chronic and continuous ROS production may induce cancer, and the transformed cells could generate more ROS during their exuberant metabolic activities.10 In order to survive from the high levels of intracellular ROS, ROS-scavenging enzymes such as SOD, glutathione peroxidase, and peroxiredoxin are often up-regulated in malignant cells.7 The increase in thioredoxin activity along with elevated oxidant stress may contribute to facilitated transcription and stimulated cell proliferation.7 However, when the balance between ROS generation and elimination is interrupted via either up-regulation of ROS generation or down-regulation of ROS elimination, the augmented ROS, particularly induced by therapeutics, can push these stressed malignant cells beyond their limit, thus damaging the cell membranes, proteins, lipids, and nucleic acids, or even inducing cell apoptosis (Figure 5).6 The overall

Figure 5. Killing cancer cells via ROS-mediated mechanisms. Adapted by permission from Springer Nature, ref 6. Copyright 2009 Macmillan Publishers Limited, https://www.nature.com/nrd/.

cellular ROS levels are determined by the rates of ROS generation and elimination. Thus, exogenous agents that increase ROS generation or inhibit ROS elimination can induce significant accumulation of ROS in cancer cells, leading to oxidative damage and cell death.6 Besides the ROSmediated direct cell killing, the augmented ROS generation could also serve to strengthen the ROS-responsive performance, because the innate ROS level in tissues/cells is often insufficient to trigger the efficient and complete transformation of ROS-responsive drug/gene delivery systems. In this section, we will introduce some recent studies on enhancing ROS generation and GSH consumption toward cancer therapies. 2453

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covalent conjugation with RRMs in prodrugs15,174 or simple encapsulation using ROS-responsive NCs.28,33,114 Although there are innumerous other therapeutics that can be used to induce the intracellular ROS and cell apoptosis,7 their mechanisms still need to be unraveled and their applications together with ROS-responsive systems still need to be explored. Due to the redox adaptation mechanism of cancer cells, the use of ROS generators alone may not be sufficient to kill cancer cells with up-regulated antioxidant capacity. Combinations of ROS generators with compounds capable of eliminating cellular antioxidant systems may impart synergistic effects.6 Big steps have been made toward the up-regulation of ROS levels to sensitize ROS-responsive behaviors. However, the tissue/cell selectivity needs to be precisely controlled, thereby avoiding undesired ROS generation in normal tissues/cells that would cause side toxicity. For instance, nonspecific GOx release during circulation or in normal cells will dramatically elevate H2O2 concentrations and reduce the environmental pH, leading to overwhelming toxicity. Visible light is often used to activate PSs to generate ROS. However, its poor tissue penetration depth greatly hurdles the clinical application. Thus, development of PSs that can absorb near-infrared light are of great demand.181,182

5.1. Enhanced ROS Generation. 5.1.1. Fenton Reaction. The Fenton reaction refers to a catalytic process that converts hydrogen peroxide into a highly toxic hydroxyl free radical, thereby increasing the oxidizing capability of ROS. Ferrocene undergoes oxidation in the presence of ROS to produce Fe2+, which subsequently acts as the catalyst for Fenton reaction. For example, Kwon et al. synthesized a Fenton reaction-performing polymer (PolyCAFe) which possessed a hydrophobic backbone (composed of benzoyloxycinnamaldehyde (BCA) and ferrocene) and a hydrophilic PEG domain.174 BCA was released in the acidic environment and produced H2O2, which was rapidly converted to hydroxyl radicals by ferrocene to induce apoptotic cell death. Besides polymeric systems, some small-molecular prodrugs were also investigated.143 For example, Daum et al. designed an N-alkylaminoferrocenebased prodrug which could be converted to a ROS amplifier (electron rich ferrocene).175 The lysosome-specific prodrug could target lysosomes with the help of an alkylated piperidine fragment. The iron ions in the prodrugs, which was loosely bound onto lysosomes, could catalyze the conversion of H2O2 into highly toxic ·OH.175 In addition, iron oxide NPs33 and Fe3+ ion delivery systems176,177 were also investigated for enhanced ROS generation. 5.1.2. Enzymatic Reaction. Certain enzymes such as superoxide dismutase and GOx have been used to generate ROS in the presence of specific substrates. Among them, GOx is a well-known example, which generates H2O2 when exposed to glucose and oxygen. To protect the enzymatic activity, GOx could be loaded in the inner cavity of ROS-responsive polymersomes.178 The generated H2O2 can trigger the degradation of ROS-responsive linkers, such as PBEs/PBAs and oxalates, thus changing the permeability of the polymersomes and triggering release of the encapsulated cargoes.102,105,115 Besides, the ROS-sensitive hydrogel is also a powerful platform that is widely used to encapsulate GOx.167 During the enzymatic process mediated by GOx, cellular glucose will be converted to gluconic acid, thus generating H2O2 to degrade the hydrogels and release the drug cargoes. 5.1.3. Photosensitizers (PSs). PSs are commonly used to achieve high local levels of ROS upon light irradiation, thus mediating cancer cell eradication or “on-demand” drug release. PSs absorb energy from light irradiation and transfer a proportion of the energy to activate the tissue oxygen and generate singlet oxygen. In PDT-mediated cancer treatment, the highly reactive singlet oxygen leads to the breakdown of lipids, DNA, and proteins, thereby inducing cancer cell apoptosis.179 Also, singlet oxygen has been used to trigger the degradation of ROS-responsive linkers such as TK and oxalates, thereby enhancing the ROS responsiveness of the NCs. PSs, either porphyrin-based pheophorbide a, 180 5,10,15,20-tetraphenylchlorin (TPC),146 and tetramethylhematoporhyrin (TMHP), 1 1 0 or nonporphyrin-based Ce6,32,93,95,96 ICG,181,182 and rhodamine 6G (Rho-6G),145 have been investigated for combination therapy. Such a lightmediated strategy features spatiotemporal precision in terms of ROS production, and normally requires light irradiation with low optical power density (