ROS‐responsive drug delivery systems

Abstract Reactive oxygen species (ROS) play an important role in signal transduction and metabolism. Over‐produced ROS in cells or tissues, however, often leads to oxidation stress that has implications in a series of diseases including cancer, aging, atherosclerosis and inflammation. Driven by the need for on‐demand drug delivery and fuelled by recent development of ROS‐responsive materials and nanomedicine, responsive drug delivery systems (DDSs) have gained increasing research interest. ROS‐responsive DDS is designed to release therapeutic agents only in targets of interest that produce excessive ROS, which may lead to both enhanced therapeutic efficiency and reduced side effects. Multiple‐stimuli responsive DDSs that are also sensitive to other stimuli can further enhance controlled drug release in sites where multiple stimuli coexist. Beyond drug delivery, multifunctional DDSs have great potential in achieving simultaneous imaging, combinatorial therapy and targeting ability by introducing multifunctional elements such as signal reporter, targeting elements and photosensitizer. This review will summarize the latest development of ROS‐responsive DDSs and discuss their design principle and biomedical applications.


| I N T R O D U C T I O N
Tremendous efforts have been devoted for the development of drug delivery systems (DDSs) that can effectively deliver therapeutic agents into disease sites. However, therapeutic efficiency is often hampered by premature drug release and rapid body clearance, which not only requires large dose of drug but also causes unwanted systemic toxicity. 1 On-demand drug delivery is thus of utmost importance in achieving sitespecific delivery with reduced side effects. In view of this, stimuliresponsive DDSs have gained increasing popularity due to their ability to release payload only in response to a specific stimulus that is associated with certain disease conditions. Typical stimuli explored by DDSs include endogenous (e.g., reactive oxygen species (ROS), redox, pH, thermal and enzyme) and exogenous (e.g., light, temperature, magnetic field and ultra sound) ones. 2 Of particular interest is the ROS-responsive DDSs because ROS overproduction has great implications on a variety of diseases and emerging ROS-responsive materials hold great promise in the development of advanced nanomedicines. An effective DDS relies on both a solid understanding of physiological conditions of the disease sites and rational design of stimuli-responsive drug carrier that can undergo sharp chemical or physical changes in response to the stimuli to allow for cargo escape. Certain pathological conditions with coexisting multiple stimuli are better targeted by multiple-stimuli responsive DDSs to enable improved drug efficacy. With the help of a photosensitizer that can produce ROS on light irradiation, controlled drug delivery can be theoretically achieved in any site of interest, which further expands the application for treatment of a wide range of diseases.
ROS are generally referred to a class of oxygen derived chemical species produced by the body. Typical ROS species include hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), superoxide (O 2 2 · ) and hydroxyl radicals (HO·), which may transform from one to another via a cascade of reactions. 3 They can be generated endogenously from mitochondrial metabolism or NADPH enzyme catalyzed reactions as well as exogenously by exposure to UV light or xenobiotic compounds. 4 While moderate level of ROS plays a vital role in physiological and pathological processes, 5  overwhelms the antioxidant defense system will lead to oxidative stress. ROS usually contain unpaired electrons or unstable bonds, which make them highly reactive. Prolonged exposure to high level ROS will cause irreversible functional alterations or complete damage to nucleic acid, proteins, lipid, and hydrocarbons. The toxic effect of excessive ROS is thus associated with an array of pathological conditions, including cancer, 6 aging, 7 diabetes, 8 cardiovascular diseases, 9 and neurodegenerative diseases. 10 There are many types of ROS-responsive materials explored in drug delivery applications, including those containing thioether, selenium/tellurium, thioketal, polysaccharide, aminoacrylate, boronic ester, peroxalate ester and polyproline. The reaction mechanism of each type of material is summarized in Scheme 1, which will be discussed in respective sections. Depending on the material or design of the carrier, the major mechanism of drug release can be attributed to solubility change induced carrier disassembly, cleavage induced carrier degradation and carrier-drug linker cleavage. The detailed synthesis and oxidation properties of most ROS-responsive materials have been covered in previous review. 11 In this review, we will discuss the progress of ROS-responsive DDS, with a focus on the design principles for various applications of DDSs with different stimuli responsiveness. Specifically, the hydrophobic poly(alkylene sulfide)s can be oxidized into more hydrophilic poly(alkylene sulfoxide) and ultimately poly(alkylene sulfone) (Scheme 1). 12 The early report of oxidation-responsive polymeric vesicles containing poly(eththelyene sulfide) in 2004 by Hubbell paved the way for development of ROS-responsive nanocarriers for DDS and biosensing applications. 12 Drug carriers containing thioethers can be easily degraded on the phase transition, leading to release of their payloads.
Inspired by Hubbell's work, Duvall's group developed ROSresponsive poly(PS-b-DMA) micelles for triggered drug release in 2012. 13 The micelle drug carrier consists of an amphiphilic diblock SCHE ME 1 Reaction scheme of ROS-responsive materials for drug release copolymer (poly(PS 74 -b-DMA 310 )) of propylene sulfide (PS)

| Selenium-or tellurium-containing polymers
Similar to sulfur-containing polymers, compounds containing selenium (Se) and tellurium (Te) which are also from the chalcogen group, were exploited for drug delivery applications as well. The organoselenium and organotellurium compounds can be oxidized from divalent to tetravalent states, making them attractive ROS scavengers. 15 ROS oxidation of monoselenium and monotellurium compounds may lead to phase transition from hydrophobic to hydrophilic (Scheme 1), which can be capitalized in construction of ROS-responsive drug carriers.
Early in 2010, Xu and Zhang synthesized an amphiphilic block copolymer (PEG-PUSe-PEG) with a hydrophobic monoselenidecontaining block polymer and two hydrophilic blocks of PEG. 16 The polymer self-assembles into micelles with an average diameter of 71 nm. On oxidation, the polymer micelles undergo a hydrophobic-tohydrophilic phase transition which causes disassembly of the micelles.
It was found that the PEG-PUSe-PEG polymer is more sensitive to oxidation stimuli than its sulfur analogue PEG-PUS-PEG: almost complete conversion was achieved for the former on exposure to 0.1% H 2 O 2 for 5 hr while only 30% was converted for the latter. These polymer micelles were successfully demonstrated for drug release using doxorubicin (DOX) as the model drug. The drug release profile reaches equilibrium after 10 hr with final DOX release percentage of 72%. Later, the group reported a Se-containing poly(ethylene oxide-b-acrylic acid) block copolymers with reversible self-assembly and disassembly properties on subject to repeated cycles of ROS or Vitamin C exposure. 17 Another work was also reported by the group based on Se-containing polymeric superamphiphile. 18 In 2013, Huang and Yan reported a ROSresponsive nanocarrier using a Se-containing amphiphilic hyperbranched polymer micelle. 19 The polymer consists of hydrophobic sele-nide side chains and hydrophilic dendritic backbone with phosphate segments. The micelles loaded with DOX were demonstrated for drug release in HeLa cells.
Tellurium-containing compounds are thought to have higher sensitivity due to the lower electronegativity and lower toxicity than their selenium counterparts, 20,21 making them attractive drug carriers. The higher oxidation sensitivity of telluride was verified by comparing the oxidation behaviors of telluride, selenide and sulfide dicarboxylic acids using cyclic voltammetry, indicating telluride is a better candidate as drug carrier. 22 Inspired by selenide containing materials, Xu's group further developed a number of ROS-responsive systems based on Tecontaining polymers. For example, coassembly of a hydrophobic Tecontaining polymer and phospholipids were demonstrated to have reversible redox responsiveness. 23 The amphiphilic phospholipid not only aids coassembly formation, but also provides good biocompatibility and degradability. Owing to the reversible redox nature of the Tecontaining polymer, the coassemblies can be oxidized by dilute H 2 O 2 solution and reduced by ascorbic acid. The polymer can be oxidized in 1 hr in the presence of 100 mM H 2 O 2 . The ROS oxidation, however, did not result in significant morphological changes. A Te-containing polymer micelle system was also reported to be responsive to both H 2 O 2 and 2 Gy gamma radiation which leads to NP swelling and dissociation. 22 Another example demonstrated that the Te-containing herperbranched polymer aggregates can swell under biologically relevant concentration of H 2 O 2 due to solubility switch of Te components. 21

| Boronic ester-containing polymers
Boronic ester, particularly arylboronic acid pinacol ester, has been frequently employed in drug delivery applications due to their ability to be oxidized by H 2 O 2 at physiological pH and temperature to produce phenol and pinacol borate (Scheme 1). 24 Two examples of polysaccharide modified at their hydroxyls with arylboronic ester groups have been demonstrated for drug delivery based on a solubility switch strategy. Once modified with boronic ester, the water soluble polysaccharide becomes organic soluble, which facilitates payload encapsulation.
Upon exposure to ROS species and oxidation of boronic esters, the polysaccharides are converted back to the water soluble parent form, which concurrently release the payloads. In one example, an oxidation sensitive dextran-boronic ester conjugate was synthesized for vaccination application. 25 The modified dextran was designed to encapsulate ovalbumin (OVA), a widely used antigen for immunization. Upon uptake by phagosomes of antigen-presenting cells (APCs), the OVA loaded nanoparticles (NPs) are degraded by the ROS heavily produced in APC, leading to OVA release. Results showed that OVA loaded NPs increased antigen presentation to CD8 1 T-cells by 27-fold as compared to the non-responsive NPs.
In another example, b-cyclodextrin conjugated with boronic ester (Ox-bCD) was demonstrated for drug delivery in both in vitro and in vivo model. 26 As shown in Figure 1A, core shell NPs were formed between Ox-bCD and poly(ethylene glycol)-distearoylphosphatidylethanolamine (DSPE-PEG) or PEG-adamantyl (Ada) via assembly/nanoprecipitation method taking advantage of hydrophobic interaction In comparison to solubility switch approach, aryl boronic ester can also be incorporated into polymers which backbone can be degraded in response to ROS. In one example, boronic esters are introduced to each motif of a polymer that forms NP. 27 In the presence of H 2 O 2 , bor-onic ester is cleaved, followed by quinone methide rearrangement to degrade the polymer into smaller pieces. Such a design has an enhanced ROS sensitivity as each boronic ester hydrolysis leads to degradation of polymer backbone. The system has been demonstrated for drug release using Nile red as indicator. In another work, the authors have developed a general strategy to fabricate on-demand drug delivery based on a chain shattering approach. 28 The polymer is constructed with alternating trigger-responsive domains (TRDs) and drugs both on the backbone. The protecting group in the TRD can either be a UV-

| DDS with thioketal linkers
Thioketal linkers have been frequently used in recent years as they can be readily cleaved by ROS oxidants, producing ketones and thiols (Scheme 1). 30 Thioketal linkers are employed in a number of examples to deliver therapeutic agents to inflammation sites or cancer cells that are rich in ROS species. For example, a direct complexation between a cationic poly(amino thioketal) (PATK) and negatively charged DNA was used to deliver gene to prostate cancer cells. 31  Other examples which use thioketal as a linker between the carrier and drug will be covered in later sections.

| ROS-and light-responsive DDSs
Light is one of the most commonly used external stimuli to trigger drug release or therapy activation. In contrast to internal stimuli which may introduce additional complexity and instability, light triggered drug release provides a more reliable spatiotemporal control of release with ease of operation. When integrated with ROS-responsive DDS, a photosensitizer (PS) is usually used as a light-sensitive element to generate ROS, mainly singlet oxygen (SO), which in turn activates the ROStriggered drug delivery. As the SO generated in excess has cytotoxicity to cells or tissues, the integrated system can be potentially applied for photodynamic therapy (PDT) to result in enhanced therapeutic efficiency. Use of light source in higher wavelengths, especially in the near infrared (NIR) range, is beneficial in achieving non-invasive therapy with improved tissue penetration.
Different from monoselenides which undergo solubility switch upon ROS oxidation, diselenides can be rapidly cleaved by ROS (Scheme 1), which allows disruption of diselenide-containing drug carriers. ROS-and light-responsive DDSs have been explored using diselenides polymers with the help of PS as a source of ROS. In one example, triblock copolymer micelles (PEG-PUSeSe-PEG) with different PEG lengths were tested for ROS-responsiveness with addition of a typical PS, a porphyrin derivative 9,10-anthracenedipropionic acid (ADPA). 33 It was found that SO released by ADPA upon red light illumination can effectively cleave Se-Se bond, releasing the DOX encapsulated by the polymer micelles. The drug release efficiency was higher for micelles with shorter PEG chains due to better penetration of SO to react with Se-Se bond. In another example, the group demonstrated visible light responsive diselenide-containing layer-by-layer films for potential application of combinational chemotherapy and PDT. 34 As shown in Figure 2 into the immersing media, the release percentage was found to be as high as 80% after 5 hr visible light irradiation. Importantly, part of the ROS generated may also be used for killing of cells or bacteria, making such a system useful for dual modal therapy applications.
Polysaccharides are reported to be depolymerized by ROS species (Scheme 1), which make them promising ROS-activatable carriers for therapeutic agents. For example, hyaluronic acid (HA)-Chlorin e6 (Ce6) conjugate has been used for ROS-triggered PDT and fluorescence imaging. 35 The fluorescence of the PS Ce6 is quenched when forming NPs. Excess ROS can depolymerize HA to recover fluorescence and concurrently cause phototoxicity to cells. PS is believed to undergo two types of reactions-via electron transfer to generate type I (e.g.,  Figure 3A, the polymer consists of an AIE PS (TPECM) conjugated with DNA-binding low molecular weight oligoethylenimine (OEI) via an AA linker with hydrophilic PEG side chains. OEI was used as it is believed to facilitate endo/lysosomal escapes via "proton sponge effect" and it has lower toxicity as compared to its high molecular weight counterpart. The polymer then forms complex with negatively charged DNA to form highly emissive water soluble NPs. Upon cell uptake of the NPs through endocytosis and subsequent light irradiation, ROS is generated by PS to disrupt endo/lysosomal membranes to allow for vector escape. Concurrently, the generated ROS can break the AA linker to degrade polymer into smaller components, leading to can be generalized to deliver other types of therapeutic drugs. A similar work was reported recently using a Ce6 conjugated PPS for DOX deliver and endo/lysosomal escape. 45 Taking advantage of the light-up characteristics of AIE probe, another work was reported for tracking the activation of PDT via ROS-triggered fluorescence turn on. 46 DDS that combines diagnostic, therapeutic and targeting functions in a single platform are highly desirable for biomedicine. In this regard, Liu's group has developed an integrated system based on thioketalcontaining conjugated polyelectrolytes (CPEs) for combined PDT and controlled drug delivery with targeting ability. 47

| ROS and Enzyme dual/multiple stimuli responsive DDSs
Dual responsiveness to both ROS and enzymes are beneficial for DDS in achieving enhanced therapeutic effects as both stimuli are commonly coexistent in pathological conditions such as tumor and inflammation.
In such a dual-responsive system, an enzyme cleavable substrate is usually incorporated to modulate drug release. For example, an amphiphilic block copolymer was designed to enable drug release in response to both ROS and matrixmetalloproteinase-2 (MMP-2). 48  In addition to dual-responsive system, DDS with multiresponsiveness to light, ROS and enzymes were also reported based on inorganic NPs. In one example, gold NPs (AuNPs) were used as carrier and fluorescence quencher for FRET based tumor imaging and light manipulated on-demand drug release. 49 As shown in Figure 4A, PEG Another example is based on a multifunctional ZnO cocktail for combinatorial therapy. 50 Multiple elements were carried to achieve and ROS. In vivo study of the system showed that significant apoptosis was induced by the cocktail (71.2 6 8.2%) as compared to free DOX (12.9 6 5.2%).

| ROS-and pH-responsive DDSs
Acidic pH is one of the key characteristics of pathological conditions including tumor, inflammation and organelles like endo/lysosome. In a typical ROS and pH dual-responsive system, the pH responsive elements generally improve drug release by inducing a morphology change of the drug carrier upon protonation or deprotonation. Based on this strategy, a ROS and pH dual-responsive system was reported for drug delivery to inflammatory areas with oxidative stress and reduced pH conditions. 51 The DDS involves a Cy3-labelled pH-responsive N-palmitoyl chitosan (NPCS) which forms NP with a polythioketal and therapeutic agent curcumin ( Figure 5A). On one hand, the encapsulated hydrophobic polythioketal is degraded to hydrophilic fragments, which causes NP disintegration. On the other hand, the low pH causes protonation of amine group in NPCS and subsequent morphology change of NPCS that favors NP dissociation. The mechanism of anti-inflammatory effect is attributed to both extracellular radical scavenging and intracellular inhibition which downregulates the proinflammatory cascades. Cy3 and curcumin serve as a FRET pair for monitoring of curcumin release behaviors and oxidant inhibitory effect.
It was found that 50% of encapsulated curcumin can be released in Results show that the curcumin-NPs can be better retained than free drugs and the increasing intensity for curcumin-NP in inflamed ankles indicates effective drug release triggered by oxidative stress and lowered pH in the inflamed condition. A luminescent probe L-012 was intravenously administered to detect the ROS level in vivo. The much lower chemiluminescence at the inflamed ankle with curcumin-NP treatment than that with saline treatment indicates much reduced ROS level and good inhibitory effect for the former ( Figure 5C).
Oligoproline was also found to be cleaved by ROS species (Scheme 1). Early in 2011, polymeric scaffolds cross-linked with proline oligomers were tested by Sung's group for oxidation responsiveness as a potential drug delivery carrier. 52 The same group later reported a polyproline-containing pH liable block terpolymer for gene delivery. 53 The terpolymer contains an equimolar ratio of positively charged block and negatively charged block at physiological pH. The polymer was further complexed with plasmid DNA for pathological vascular targeted gene delivery. At reduced pH in endosomes, protonation of the negatively charged block caused destabilization of the polymer core and membrane disruption, leading to endomal escape of the nanocarrier.  55 Under inflammation conditions, the excessive ROS will react with peroxalate ester bond while acidic pH will cleave the acetal linkages to release vanillin. As a result, the therapeutic effect is attributed to both the ROS scavenging ability As discussed so far, the major delivery mechanism is based on chemical bond cleavage or solubility switch that degrades drug carrier.
A recent work reports a novel DDS that releases drug upon shell disruption by gas bubbles generated in response to both ROS exposure and in situ created acidic miliew. 58 The DDS consists of a poly lactic-

| ROS-and thermal-responsive DDSs
Temperature is another common stimuli that has been widely investigated in oncology. In view of the slightly higher temperature of tumor microenvironment than that of normal tissues, thermal-responsive materials are designed to collapse in response to elevated temperature in tumor or upon externally induced local hyperthermia to release its payload. The temperature difference between ambient and physiological conditions may also require thermal-responsive materials for drug administration or loading purposes. A thermoresponsive hydrogel based on PPS containing triblock polymer was reported for temperature modulated ROS-triggered drug release. 59 As shown in Figure 6A 60 Finally, the Nile red loaded hydrogel was injected subcutaneously into male BALB/c mice to monitor local retention of the drug released. As shown in Figure 6C, the drug loaded triblock hydrogel provides a sustained local release over two weeks, whereas the control with diblock (withought NIPAAM) polymer shows rapid drug diffusion and poor retention.
Another example of ROS and thermal dual-responsive DDS was reported by Chen's group. 61 The triblock polymer consists of alternating polyethylene glycol (PEG) as the shell and a thermal and oxidation dual-responsive thioether containing polymer as the core. The hydrophobic drugs such as Nile red are encapsulated into the collapsed carrier at elevated temperature and released upon ROS exposure.

| Dual redox-responsive DDSs
The intracellular environment is known to have high reduction level due to presence of reducing agents such as glutathione (GSH) (0.5-10 mM). The GSH level in tumor cells is several fold higher than the normal ones, making it a useful stimuli for targeting tumor cells and triggering drug delivery. 62 Two types of materials have been reported for redox-responsive DDS.
The Se-Se bond can either be oxidized to seleninic acid by ROS or be reduced to selenol by reducing agents. A triblock copolymer micelle system (PEG-PUSeSe-PEG) with a polyurethane (PU) block containing diselenide and two blocks of PEG were demonstrated for dual response to both H 2 O 2 and GSH. 63 The amphiphilic block copolymer self-assembled in aqueous solution to form NPs of 76 nm size. Either oxidation or reduction will lead to polymer chain dissociation and NP disassembly. The cargo release behavior was studied using Rhodamine B (RB) loaded NPs. It took 1.1 hr and 2 hr to release 80% RB in the presence of 0.1% H 2 O 2 (equivalent to 30 mM) or 0.1 mg/mL GSH, respectively. Later in 2014, the group further developed a general strategy using a phospholipid and diselenide-containing block copolymer that form coassemblies by electrostatic interaction for dual redox response. 64 A thioether containing DDS responsive to both glutathione (GSH) and ROS was also reported for tumor therapy. 65 To construct such a tumor heterogeneity-responsive system, both a GSH-responsive phenol ester linker and ROS-responsive thioether linker were used. As shown in Figure 7A, the prodrug consists of a camptothecin-based topoisomerase I inhibitor 7-ethyl-10-hydroxyl-camptothecin (SN38) conjugated with the PEG via the phenol ester and thioether linkers, which then self-assembles to form nanocapsules (OEG-2S-SN38) with 100 nm diameter. When internalized by tumors that are rich in both GSH and ROS, the nanocapsules can either undergo thiolysis or ROStriggered hydrolysis of phenol ester to release SN38. Results revealed that 80% SN38 could be released within 15 min in the presence of 10 mM, which is much faster than the diselenides mentioned earlier.
Two hour was needed to release 80% SN38 in the presence of 10 mM GSH at pH 7.4. The nanocapsules were tested with Bcap37 (BC) cells, showing enhanced in vitro cytotoxicity, which was proved to result from triggered SN38 release. In vivo antitumor activities were also studied using Bcap37 breast tumor xenograft model for OEG-2S-SN38a and a clinically used SN38 prodrug irinotecan. Figure 7B shows the tumor volume of mice treated with PBS and two prodrugs, indicating a much higher inhibition rate achieved by OEG-2S-SN38a prodrug, which is attributed to the combination of enhanced permeation and retention (EPR) effect and therapeutic effect of released SN38 drug. Depending on the nature of payloads, suitable drug carriers can be selected to load drugs via hydrophobic interaction, electrostatic interaction and covalent bonding, which allow drugs to be released via different mechanisms. DDSs with polymers containing thioether and monoselenium or monotellurium generally release encapsulated drug based on a phase change induced carrier disassembly and they have increased ROS sensitivity with increasing electronegativity. Polymers containing diselenides and oxylate are usually degraded into small pieces to release the cargo. Other materials based on boronic ester, thioketal and aminoacrylate are frequently used as ROS-cleavable linker which directly liberate the drugs linked. While no direct relationship has been established between drug release efficiency and the type of ROS-responsive material, it is generally believed that the carrier material with high sensitivity to ROS and reaction with easy access of ROS that leads to direct drug release (such as DDS with ROS-linker conjugated drug) will result in higher release efficiency.
Multiple-stimuli responsive DDSs that are also sensitive to other internal stimuli such as pH, temperature, enzymatic activity and reducing agents hold the promise to offer better targeting ability to diseased sites. In addition to target sites that overproduce ROS, the ROSresponsive DDS may also be applied to other sites of interest by introduction of a PS that can generate ROS in situ upon light irradiation.
The PS not only allows for light triggered drug release, but also kills cells or bacteria though PDT, enabling combinatorial therapy with improved therapeutic efficiency. Multifunctional DDS that combines therapy with imaging capability is beneficial for revealing the spatial and temporal location of the drug, facilitating the pharmacokinetic study and early diagnosis of disease. It is believed that the applications of the DDS can be greatly expanded by recruitment of multifunctional elements such as targeting ligand, imaging contrast agents, for theranostic platform, multi-modal therapy and multiple-stimuli sensitive drug delivery applications. It is hoped that this review will inspire the development more advanced DDS and clinically relevant applications toward translational medicine.

ACKNOWLEDGMENT
We thank Ruoyu Zhang for help during the revision.

C O NF LI C T O F I NT ER ES T S
The co-authors of this article do not have a conflict of interest to declare.