Boronates as hydrogen peroxide–reactive warheads in the design of detection probes, prodrugs, and nanomedicines used in tumors and other diseases

Hydrogen peroxide (H2O2) has always been a topic of great interests attributed to its vital role in biological process. H2O2 is known as a major reactive oxygen species (ROS) which is involve in numerous physiological processes such as cell proliferation, signal transduction, differentiation, and even pathogenesis. A plenty of diseases development such as chronic disease, inflammatory disease, and organ dysfunction are found to be relevant to abnormality of H2O2 production. Thus, imminent and feasible strategies to modulate and detect H2O2 level in vitro and in vivo have gained great importance. To date, the boronate-based chemical structure probes have been widely used to address the problems from the above aspects because of the rearranged chemical bonding which can detect and quantify ROS including hydrogen peroxide (H2O2) and peroxynitrite (ONOO−). This present article discusses boronate-based probes based on the chemical structure difference as well as reactivities to H2O2 and ONOO−. In this review, we also focus on the application of boronate-based probes in the field of cell imaging, prodrugs nanoplatform, nanomedicines, and electrochemical biosensors for disease diagnosis and treatment. In a nutshell, we outline the recent application of boronate-based probes and represent the prospective potentiality in biomedical domain in the future.


Introduction
Reactive oxygen species (ROS)-which have an unpaired electron and include superoxide anions (O 2 − ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (·OH), peroxynitrite (ONOO − ), and hypochlorite (OCl − )-are generated by cells during respiration. They increase the oxidative stress level and cause oxidative damage, thereby regulating cell aging and apoptosis. Excessive ROS damage cells and cause dysfunction of tissues [1], increasing the risk of certain diseases. Hydrogen peroxide is the most critical ROS because it is more stable than others and its concentration in normal cells differs from that in abnormal cells. It is involved in several physiological and pathological processes as a signaling molecule, and the range of concentrations of physiological intracellular H 2 O 2 is approximately 10 −9 to 10 −4 M [2]. Hydrogen peroxide is a major product of oxygen metabolism that accumulates and causes oxidative stress in cells when the informatory signal increases. Hydrogen peroxide is generated in various cellular compartments by enzymatic systems such as mitochondria, the endoplasmic reticulum (ER), and peroxisomes [3,4], and this generation process involves the degradation of superoxide [5]. Superoxide is generated during electron transport in mitochondria, and antioxidant enzymes-superoxide dismutase 1 and 2 (SOD1 and SOD2) [5,6]-then convert it into hydrogen peroxide, which is a stable product. The product can be reduced to H 2 O by other antioxidant enzymes. Oxidative stress is regulated in cells by antioxidant enzymes which act as signaling molecules.
However, in some cases, the production of hydrogen peroxide can increase or become unstable. Certain factors (such as carcinogens, ultraviolet light, inflammation, and aging) can cause cells to produce excess hydrogen peroxide in the earliest tumor-initiating events that cause DNA damage and ultimately cancer. Hydrogen peroxide is thought to be involved in tumor formation [7,8]. Hydrogen peroxide is also closely related to age-associated diseases [6,9] because of its ability to regulate apoptosis; these diseases include Alzheimer disease [10], Parkinson disease [11], and diabetics related cardiovascular diseases [12]. Therefore, a sensitive method of detecting the H 2 O 2 distribution and concentration in living systems should be developed.
Traditionally, molecular imaging through optical response strategies is used to detect calcium and biometals; such strategies involve the creation of fluorescent chemosensors that detect molecular entities in biological systems by recognizing or binding to an analyte of interest [13]. However, the recognition of H 2 O 2 and other ROS is difficult because these oxygen metabolites are transient and similar in shape or size. Chemical relativity-based selective sensing that enables the accumulation of transient signals in an irreversible reaction system was developed as a secondary strategy for creating molecular probes [14]. H 2 O 2 is a highly stable ROS and plays a fundamental role in healthy and diseased living systems. The labile O-O bond of H 2 O 2 enables it to undergo chemical transformations as a two-electron electrophilic oxidant and react with arylboronic acids, aryl boronates [14], and boronate esters [15]. Scheme 1 displays the mechanism underlying the oxidation of an arylboronate (AB) with H 2 O 2 . The AB structure selectively reacts with H 2 O 2 and releases quinone methide (QM) and R groups, which can act as probes or anticancer drugs (Scheme 1) [16]. The electrophile C-B bond coordinates with H 2 O 2 to form a negatively charged tetrahedral boronate complex; the aryl bond of the nucleophile complex then transfers charge to form an intermediate boronate, which is then hydrolyzed quickly in water to produce a boric acid and the aryl structure which can go 1,6-elimination to produce QM. The QM then undergoes either hydrolysis or a reaction with the glutathione (GSH) present in the cytosol to produce a GSH-QM adduct, potentially weakening the cell's antioxidative abilities. The development of boronate-based fluorogenic probes began in 2003 Scheme 1 Mechanism of arylboronate oxidation with H 2 O 2 and releasing R group as drug or a detection probe [17,18]; these probes are based on the deboronation of aryl boronates, which results in corresponding phenolic products [19,20]. Since Lo designed and reported p-dihydroxyborylbenzyloxycarbonyl derivatives of p-nitroaniline and fluorescent 7-amino-4-methylcoumarin that react specifically with H 2 O 2 to release a chromophoric reporter [17], various boronate-based probes that detect ROS have been designed and described in the literature. In this review, we try to base on the AB model and boronate oxidation to introduce H 2 O 2 detection probes, H 2 O 2 -stimulated prodrug systems, and nanomedicines which have been developed in recent years. Target-specific drug delivery [21][22][23][24] and surface coatings for cell capture [25] have often been used in the design and construction of AB-based nanosystems that can increase the efficiency of cancer therapy. Microfluidic chips [26] and electrochemical impedance cytosensors [27] have also been employed in the design of highly sensitive biosensors for sensing cancer cells. Fluorescence spectroscopy, electroanalysis, chemiluminescence, and spectrophotometry have also been applied.

Small molecular probes
Tumors and other inflammatory sites in the body are usually under high oxidative stress. The aberrant generation of H 2 O 2 is strongly correlated with several diseases including cancer, neurodegenerative disorders, and cardiovascular diseases [28]. Cancer biology research has revealed that tumor cells exhibit higher ROS levels than do normal cells. Research has also demonstrated the major role of H 2 O 2 in cancer initiation and progression [7,8]. ROS are created by the active-energy metabolism associated with uncontrolled cell proliferation, telomere dysfunction, malfunction of mitochondrial respiration, and oncogenic stimulation [29][30][31][32][33][34][35]. The effective detection of H 2 O 2 in living cells is a crucial and complex matter. Therefore, sensitive methods of detecting H 2 O 2 distributions and concentrations in living systems are necessary. The boronate oxidation sensing method is inexpensive, convenient, and simple; hence, it is widely used in molecular sensing.

Boronate-based probes
This review divided boronate-based molecular probes into four classes on the basis of their structure and the mechanisms through which the H 2 O 2 -triggered product forms. The first class is monoboronate probes, which detect the formation of reporters through the direct and stoichiometric (1:1) oxidation of probes by oxidants (Fig. 1a). The second class is diboronate probes, which require two consecutive oxidation reactions to yield a fluorescent reporting molecule (Fig. 1b). The third class is boronobenzyl probes, the oxidation of which leads to the formation of 4-hydroxybenzyl derivatives; the primary product then releases the QM moiety to yield the final product (Fig. 2a,  b). The fourth class is p-dihydroxyborylbenzyloxycarbonyl probes, which when oxidized unmask the amine group of the final product by releasing the QM moiety and carbon dioxide (CO 2 ; Fig. 2c, d).
The first class of boronate-based probes is illustrated in Fig. 1a. Compound 1 is a water-soluble probe and is designed with a coumarin moiety that acts as the fluorescent chromophore; the end product after H 2 O 2 oxidation is a wellknown fluorophore, 7-hydroxycoumarin (umbelliferone),  [36]. Compound 2 was reported to be a series of analogous stilbene boronate ester fluorescent probes [37] with various fluorescent responses to H 2 O 2 owing to the loss or introduction of the intramolecular charge transfer's (ICT) excited state given the probes' differing functional groups. For example, when the R group was N(Me) 2 , the probe exhibited a ratiometric response, with its emission being blue shifted and its fluorescence intensity increasing upon the addition of H 2 O 2 . However, when the R group was CN, the fluorescent intensity of the probe decreased. Compounds 3 [37] and 4 [38] involve an ICT-based system. Compound 3 exhibits 3.6-fold stronger fluorescence upon H 2 O 2 addition, and compound 4 is a highly selective and sensitive naked-eye sensor for H 2 O 2 that changes from colorless to deep red. Compound 5 was developed to be a sensitive and selective fluorescent probe for the detection of acetylcholine (ACh) on the basis of enzyme-generated hydrogen peroxide (H 2 O 2 ); a strong emission peak was observed at 555 nm when compound 5 reacted with enzyme-generated H 2 O 2 , whereas the original emission peak was at 455 nm [39]. Compound 6 (4-(phenyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)amino)benzaldehyde; OTB) was developed to be a precursor molecule for chemical detection and the analysis of H 2 O 2 vapor in the context of environmental monitoring and public safety. A solid-vapor Schiff base formation reaction occurs between an OTB film and the primary amine vapor to create the probe, and the use of an arylboronate molecule with a moderate alkaline character strengthens the probe's ability to detect H 2 O 2 considerably [40]. Compounds 7 [41] and 8 [42] detect peroxynitrite in aqueous solutions and living cells. These two probes not only exhibit a turn-on response when exposed to peroxynitrite but are also highly selective of ONOO − rather than other ROS. The second class of boronate-based probes is displayed in Fig. 1b. Compounds 9 [18], 10 [43], and 11 [43] were designed, synthesized, and characterized by the laboratory of Christopher J. Chang. These compounds are fluorescein-, resorufin-, and xanthone-based fluorophores that respond to H 2 O 2 and exhibit intense green, blue, and red fluorescence, respectively. Compound 12 [44] is a dual-responsive fluorescent probe functionalized from compound 11 with an additional ROS or reactive nitrogen species trigger that can selectively detect ONOO − . Compound 12-OTBS and compound 12-OAc can be used to detect fluoride (F − ) and esterase, respectively, to yield compound 12-OH, which exhibits a considerable increase in fluorescence intensity and selectivity and sensitivity to ONOO − , which is useful in cellular imaging [44]. Compound 13 is a near-infrared fluorescent dye based on the boron-dipyrromethene (BODIPY) fluorophore and is functionalized with boronic acid groups. This dye was employed in the preparation of optodes to detect glucose in diluted whole blood; glucose is converted into hydrogen peroxide by glucose oxidase, leading to a red shift in the emission wavelength [45]. However, the main disadvantage of these diboronate probes is that they have low sensitivity because two consecutive oxidation reactions are required, unlike for monoboronate probes.

Arylboronate-based probes
Examples of the third class of boronate probes are boronobenzyl probes ( Fig. 2a and b). Compounds 14-18 were designed using a boronobenzyl group conjugated with a fluorophore. In the presence of H 2 O 2 or ONOO − , the arylboronate group of a boronate probe rapidly eliminates p-QM and generates a final product with the quinone group; this is accompanied by a rapid turn on of fluorescence [46][47][48][49][50]. Compound 14 can be employed as a selective fluorescent turn-on probe for imaging peroxynitrite (ONOO − ) drug-damaged liver tissues; after compound 14 generates the phenolate product, the phenolic oxygen attacks the nitrile group to form the highly fluorescent coumarin derivate product through the intramolecular nucleophilic reaction [46]. Compound 19 was the first ICT-based ratiometric twophoton fluorescent probe reported to detect ONOO − [51]. The emission color red shifts from blue to green when the final product of 4-hydroxy-1,8-naphthalimide is yielded. Compounds 20-22 are excited-state intramolecular proton transfer (ESIPT)-based fluorescent probes for sensing H 2 O 2 and ONOO − [52][53][54]. After the quinone-form product is generated through oxidative hydrolysis of the arylboronate group, the ESIPT process turns on and the keto-form final product is generated and exhibits a strong fluorescent signal. Compound 23 is a phenyl boronic acid-functionalized quinone-cyanine probe that exhibits stimulus-responsive near-infrared fluorescence when detecting H 2 O 2 [55]. The arylboronate group of compound 23 is converted into the keto-form final product from a one-donor-two-acceptor system; when the product binds to DNA grooves, it exhibits turn-on near-infrared fluorescence. Compounds 24-27 consist of an exocyclic nitrogen atom masked with a positively charged arylboronate group. When these compounds react with H 2 O 2 or ONOO − , the benzyl-protecting group is cleaved to unmask the amine group, turning on the compounds' fluorescence [56][57][58][59]. The design of a positively charged amine group can increase the water solubility of the probes and enable it to act as a mitochondria-targeted carrier [59]. Figure 2c and d presents the fourth class of boronate-based probes, which contain carbamate [17,[60][61][62][63] or a carbonate group [64,65]. In the design of these probes, suitable reporters are masked with an electron-withdrawing boronatebased carbamate or carbonate-leaving group, which acts as the H 2 O 2 or ONOO − reactive trigger and is released to form the amine or phenol group and provides a turn-on or switched-fluorescent response for the ratiometric detection of H 2 O 2 and ONOO − . Compound 33 was designed to have weak absorption and emission when the π-conjugated system of the fluorophore is blocked. When the boronic ester moiety is removed through an attack by ONOO − , the π-conjugated system of the fluorophore recovers, resulting in strong fluorescence [63]. Compound

Bioapplication of small molecular probes in vitro and in vivo
The absorption and emission of small molecular probes often change in response to ROS, and small molecular probes are widely used in cellular imaging, drug discovery, and various medical applications. Common designs of these chemosensors often involve the suppression of photoinduced electron transfer, the modulation of ICT, and fluorescence resonance energy transfer (FRET) for turn-on and ratiometric sensing. Chemically transforming a probe to alter its absorption or emission properties is crucial [66]. In this section, we introduce examples of small-molecule boronatebased chemosensors and imaging agents.

Small molecular probes in biosystems
Numerous fluorophores have been used in the design of boronate-based fluorescent probes. These fluorophoressuch as coumarins, naphthalimides, fluorescein and its analogs, rhodamine and its analogs, BODIPYs, and cyanineshave been widely used as signal reporters for the design of probes to detect various analytes. The first example is fluorescein derivatives used for the selective detection of H 2 O 2 in cellular imaging. Chang's laboratory first reported the design, synthesis, and characterization of several monoand di-boronate-based fluorescein-derivative probes [14]. The first-generated H 2 O 2 probe, fluorescein peroxyfluor 1, was prepared by installing boronic ester groups through 3' and 6' positions of a xanthenone scaffold; this forces the molecule into a closed, colorless, and nonfluorescent lactone form. Upon treatment with H 2 O 2 , hydrolytic deprotection of the boronates results in their structural transformation into phenols and lactone opening, which produces a colored and fluorescent fluorescein product [18]. On the basis of this reaction approach to H 2 O 2 detection, they developed xanthone-based peroxyxanthone 1 [43], resorufin-based peroxyresorufin 1 [43], Nagano's Tokyo Green-based peroxy green 1 [67], and related derivatives [14]. These probes can be used to visualize the change in H 2 O 2 levels under oxidative stress and establish boronate chemistry as a suitable approach for detecting ROS in biological samples. In addition, Chang published a tandem activity-based sensing and labeling strategy for H 2 O 2 detection that enables the capture of transcellular redox signaling within microglia-neuron communication (Fig. 3a) [68]. The probe, Peroxy Green-1 Fluoromethyl (PG1-FM), was designed to combine the boronate trigger established in the laboratory's research with a fluoromethyl group ortho to the boronate group that serves as a QM species for proximal covalent labeling upon H 2 O 2 -triggered boronate-to-phenol conversion [68]. When PG1-FM reacts with H 2 O 2 , the boronate group is converted into a phenol group, triggering fluoride elimination and generating a highly reactive QM form that can be captured by proximal proteins and leaves a fluorescence product covalently labeled with the protein. This approach minimizes background signals from extracellular H 2 O 2 reactivity and oxidized probes in cells.
Nitric oxide functions as a second messenger in cells; it is related to H 2 O 2 and plays a crucial role in signaling transduction and oxidative pathways [69].

Small boronate-molecule-targeting-probe
Some fluorescent probes are designed to be modified with a targeting group to research and understand specific organelles in living systems. The commonly used targeting Reprinted with permission from ref [68] and [70]. Copyright from 2021 National Academy of Sciences and 2012 American Chemical Society. All rights reserved groups are mitochondrial and lysosomal (Fig. 4a) [71]. Mitochondrial targeting groups are usually designed with positively charge, such as triphenylphosphonium (TPP), quaternized pyridine, indolium moiety, quaternized quinoline units, and oxonium ions, because of their negative membrane potential. Lysosome-accumulated probes are designed to have lipophilic amines, such as morpholine and tertiary amines, because lysosomes remain in the acidic range. An abnormal ROS concentration in mitochondria or lysosomes causes organelle dysfunction and is related to heart disease, neurodegenerative diseases, and aging. Therefore, the design of fluorescent targeting probes for monitoring or detecting ROS concentrations in specific cellular organelles is critical.
In 2016, Xiao et al. published articles on two organellespecific fluorescent probes, MI-H 2 O 2 and ER-H 2 O 2 , which can detect H 2 O 2 in mitochondria and the ER (Fig. 4b) [72]. MI-H 2 O 2 was synthesized using merocyanine as the fluorophore, a well-known H 2 O 2 receptor with boronic acid as a specific masking group, and lipophilic cations as a mitochondriatargetable moiety. ER-H 2 O 2 comprised a 1,8-naphthalimide fluorophore, a boronic ester recognition group, and a methyl sulphonamide group as the ER-targetable moiety. Upon reaction with H 2 O 2 , the boronic acid group of MI-H 2 O 2 is removed and converted it to an oxygen anion to form a strong push-pull conjugated system with an absorption band that shifted from 425 to 525 nm and a 13-fold fluorescence increase from a quantum yield of 0.0087 to 0.11 at 555 nm upon excitation at 525 nm. ER-H 2 O 2 is a ratiometric fluorescent probe which contains the ICT structure and exhibits the absorption from 360 to 460 nm and a 19-fold increase in emission from 458 to 558 nm upon excitation at 400 nm. The absorptive properties of MI-H 2 O 2 and ER-H 2 O 2 indicate that they can serve as naked-eye sensors for colorimetric H 2 O 2 detection. The emission properties of these probes enable multicolor fluorescence imaging of H 2 O 2 in mitochondria and the ER simultaneously; thus, endogenous H 2 O 2 levels in mitochondria and the ER during apoptosis under various stimuli can be selectively and sensitively detected.
H 2 O 2 contributes considerably to the redox process in mitochondria. It can be transformed from a free radical when energy is generated in the mitochondrial respiration chain. However, the dysregulation of oxidative stress and energy metabolism can cause neurodegenerative diseases [73,74]. Wu et al. developed a two-photon fluorescencelifetime-based probe, TFP, that can simultaneously target mitochondria and detect H 2 O 2 and adenosine triphosphate (ATP) in neurons (Fig. 4c) [75]. TFP is constructed from two independent domains, which are the H 2 O 2 and ATP reporters, and an intermediate pyridinium cation acts as a mitochondria-targeting group. The H 2 O 2 -responsive domain has naphthalimide as a fluorophore, and the naphthalimide is masked by a H 2 O 2 -cleavable benzene boronic acid pinacol ester group. The ATP-responsive domain has rhodamine as a fluorophore and a diethylenetriamine structure. When H 2 O 2 is added, the boronic group is cleaved to form the pyridine unit, and the absorption spectrum exhibits a blue shift from 401 to 380 nm, with the absorption peak decreasing in intensity; the emission intensity increases at 470 nm upon excitation at 710 nm. In the presence of ATP, the spirolactam ring in the rhodamine complex opens, and hydrogen bonds form between the amino groups and phosphate groups of ATP, which leads to a new absorption peak at 562 nm, whereas  [71,72], and [75]. Copyright from 2021 and 2016 Royal Society of Chemistry and copyright 2020 American Chemical Society. All rights reserved that at 401 nm remains unchanged. A new emission peak then forms at 590 nm upon excitation at 710 nm. However, because of the different excitation and emission spectra of naphthalimide and rhodamine, TFP can precisely detect the levels of H 2 O 2 and ATP in different signal patterns without the properties of FRET and crosstalk. TFP can also be employed to analyze the levels of H 2 O 2 and ATP in larval zebrafish because of two-photon penetration; data indicates that TFP is a suitable probe for dual imaging and the biosensing of H 2 O 2 and ATP in vivo.

H 2 O 2 -triggered drug-releasing boronate probes
Cancer is a highly common disease worldwide, and metastasis is a main cause of death. A higher level of H 2 O 2 has been observed in cancer cells than in noncancer cells because of the overproduction of ROS, which results in cellular migration and invasion [76,77]. Therefore, the properties of cancer cell environments provide advantages in the development of probes releasing cancer-targeting drugs. In 2014, Kim et al. reported an H 2 O 2 -activated theranostic agent, prodrug 36, which can deliver a drug to cancer cells and simultaneously monitor the location of the anticancer drug through strong fluorescence (Fig. 5a) [15]. Prodrug 36 has two individual domains. The first is a chemotherapeutic anticancer drug, SN-38, which is used for treatment in different carcinomas and works by inhibiting topoisomerase I [78]. The second is a fluorophore, a coumarin unit, which is conjugated with a boronate ester and exhibits turn-on fluorescence when it reacts with H 2 O 2 ; it thus enables monitoring of the release of SN-38. In the presence of H 2 O 2 , the boronate group of prodrug 36 is triggered, and the coumarin unit is released, after which the anticancer drug, SN-38, is released. The maximum absorption peak of prodrug 36 exhibits a slight red shift at λ max = 320 nm, and the emission peak also exhibits a slight red shift at 453 nm, with an approximate 8.3-fold increase in emission intensity. These properties indicate that prodrug 36 is a suitable theranostic agent for delivering SN-38 to cancer cells through a process triggered by H 2 O 2 . Furthermore, in vitro data have demonstrated that prodrug 36 specifically accumulates and releases SN-38 in cancer cells in an H 2 O 2 -activated process and can thus eliminate the side effects from direct SN-38 treatment. In vivo data have indicated the strong anticancer effect of the intratracheal injection of prodrug 36 in a metastatic lung tumor model and that this increases the survival rate.
Another example of a H 2 O 2 -triggered anticancer drug-releasing probe is Activatable Nano Pro-Drug-X (ANPD-X), which was reported in 2017 by Biswas et al. Unlike for prodrug 36, for ANPD-X, two steps are required, namely, H 2 O 2 activation and light irradiation, to release the anticancer drug. These two step reactions can more confirm and observe the H 2 O 2 detection step and the drug releasing step in real time (Fig. 5b) [79]. ANPD-X is constructed using a boronate ester as the H 2 O 2 trigger; a benzothiazole-appended p-hydroxyphenacyl (pHP) moiety as the fluorophore, based on the ESIPT phenomenon; a pHP group as the phototrigger; and a chlorambucil as the photoreleased chemotherapeutic anticancer drug. In the presence of H 2 O 2 , a boronate group is released to cause a red shift and an intense emission band at 518 nm (shifted from 448 nm), which results in a fluorescent color change from blue to green. The product is then irradiated with visible light, which triggers the release of the anticancer drug chlorambucil and results in the fluorescence returning to blue at 450 nm. This two-step fluorescent color change provides real-time information regarding the anticancer drug's release. In vitro cellular imaging has demonstrated that ANPD-X is a suitable anticancer drug delivery system that offers two-step surveillance and can cause major cytotoxicity in cancer cells.  [15] and [79]. Copyright from 2014 and 2017 American Chemical Society. All rights reserved

Electrochemistry applications in boronate systems
Hydrogen peroxide is an ROS that can affect the environment and human body. An excess of H 2 O 2 may contribute to oxidative stress and cause diseases such as cancer, neurodegenerative diseases, and diabetes. Therefore, the development of a sensor for detecting intracellular and extracellular H 2 O 2 and monitoring its changes in the body is vital.
Several types of biosensors, including fluorescent, luminescent, and spectrophotometric biosensors, have been developed for the detection of H 2 O 2 [80][81][82]. However, their applications have been limited because they require expensive equipment, have low sensitivity, and require a specific procedure for sample preparation. To resolve these problems, electrochemical sensors with simple modification processes, low costs, high sensitivity, and low detection limits have been proposed.
Shao et al. designed and synthesized a multifunctional (chromogenic, fluorescent, and electrochemical signal) probe for the detection of H 2 O 2 in living cell systems (Fig. 6a) [83]. The probe consists of a p-pinacolborylbenzyl group (H 2 O 2 reaction site) and a ferrocene-electrochemistry combined probe (FE-H 2 O 2 , visualization site). Interaction with H 2 O 2 and the boronic acid-containing group causes rearrangement and a change in color (from red to colorless) followed by the release of FE-H 2 O 2 . The hemicyanine part of FE-H 2 O 2 offers a fluorescence response, which can be applied to intracellular imaging. The redox couple provided by the ferrocene causes an electrochemical response that weakens upon the addition of H 2 O 2 . Cyclic voltammetry and differential pulse voltammetry have indicated that peak currents decrease and that the exponential fitting curve to the H 2 O 2 concentration covers 0-83 µM; the detection limit is 0.1 µM, which indicates that the multifunctional probe can be a useful tool for both intracellular and extracellular detection.
Maotian Xu et al. fabricated a microelectrode sensing platform based on ratiometric electrochemical sensing; the platform can detect H 2 O 2 in a complex environment (Fig. 6b) [84]. They deposited Au nanocones on the surface of a carbon fiber microelectrode (CFPE). They then synthesized 5-(1,2-dithiolan-3-yl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-o-lan-2-yl)phe-nyl)-pentanamide (BA), which attached to the Au/CFPE through the formation of Au-S bonds. When H 2 O 2 was present in the environment, the BA produced a phenolic hydroxyl group and caused currents. In combination with Nile Blue A as an internal reference molecule, this electrochemical platform has a low detection limit (0.02 µM), is favorable in the presence of other biological molecules or metal ions, and is applicable to detection in complex environments. In whole-blood sample detection, differential pulse voltammetry demonstrated that the electrochemical sensor has high accuracy for whole-blood, serum, and plasma samples and that it may be suitable as a platform for the early diagnosis of ROS-related diseases.
Lin et al. designed a biosensor based on an electrochemical probe 4-methoxyphenylboronic acid pinacol ester (4-MPBP) combined with a modified glassy carbon electrode (GCE), which comprised polydopamine (PDA), molybdenum sulfide (MoS 2 ), and carbon nanotubes (CNTs); the biosensor was named PDA@CNT-MoS 2 /GCE (Fig. 6c) [85]. The 4-MPBP serves as a recognition site for H 2 O 2 and produces 4-methoxy phenol (4-MP), causing a signal response. Compared with the bare GCE, which has a detection range of 0.5 to 1 mM, PDA@CNT-MoS 2 /GCE has a considerably greater limit-0.01 to 100 µM-because of its electrocatalytic properties. For the in vivo tracking analysis of a living cell system, in the presence of phorbol 12-myristate 13-acetate and spermine, which can induce the oxidative process, the current response at 0.43 V can be detected through differential pulse voltammetry in two cell lines (Caco-2 and MCF-7 cells). Adding cytochrome C peroxidase (Ccp1) does not cause peak responses, which indicates that the function of Ccp1 is a H 2 O 2 scavenger. The sensing platform combined with a redox reporter, 4-methoxyphenol, has strong analytical properties and can be applied to living cell systems. The signal amplification demonstrated by the modified electrode may become a valuable feature for future sensing devices.
In addition to H 2 O 2 , NADH is a crucial molecule and end product of several enzymatic reactions; therefore, some researchers have attempted to design a biosensor to detect both. Ho et al. used a preanodized screen-printed carbon electrode (p-SPCE) to prepare a nonenzymatic biosensor for the dual amperometric detection of NADH and H 2 O 2 (Fig. 6d) [86]. In the modification process, they used the drop-casting method to obtain 4-aminophenols, which were produced from the first interaction of 4-aminophenylboronic acid with H 2 O 2 and were adsorbed on the p-SPCE through noncovalent π-π stacking and hydrophobic interactions with oxygen functional groups. An amperometry experiment demonstrated that the biosensor has a favorable detection limit of 4.2 and 28.9 µM for NADH and H 2 O 2 , respectively; the current response was unaffected by other electroactive molecules such as ascorbic acid, uric acid, and dopamine. Most importantly, because of the level of H 2 O 2 being elevated when carcinogenesis occurs, cyclic voltammetry revealed a remarkable difference between normal cells (MC3T3E1) and cancer cells (HA22TVGH and MDA-MB-231), which indicates that this enzyme-free electrochemical probe can be employed to analyze environmental and biological samples.

Nanoparticles
The aforementioned sections fully explain the biological importance of hydrogen peroxide, which is indicative of pathogenesis, biosynthetic reactions, and cellular metabolism [87,88]. The detection of H 2 O 2 has thus been a topic of major interest. As discussed in previous sections, fluorescent probes and small molecules are widely used to detect H 2 O 2 because of their tunable fluorescence, which is also a feature of nanoparticles. Thus, this section describes several studies in which boronate-associated nanoparticles were devised and applied to biomedical domains. Gold nanoparticles (AuNPs) have been widely employed as a marker for biological molecule recognition because of their excellent physicochemical properties and controllable optical features [89][90][91][92]. One study used AuNPs into which a mannoside-boronate-sulfide (MBS) ligand had been incorporated to achieve specific H 2 O 2 detection (Fig. 7). The arylboronate structure on MBS reacts with H 2 O 2 , which Reprinted with permission from ref [93]. Copyright from 2021 Wiley-VCH GmbH triggers intramolecular electron rearrangement to create an MBSt mixture. The MBS and MBSt then coat the citratecoated AuNPs (c-AuNPs). As the mannoside on MBS@ AuNPs comes into contact with Con A, the solution's color changes quickly from red to purple because of nanoparticle aggregation. This phenomenon does not occur with MBSt@AuNPs because the ligand structure has already dissociated. For this reason, the difference in color change between the MBS@AuNPs and MBSt@AuNPs is used to create a platform for detecting H 2 O 2 through naked-eye observation [93]. Several studies have used similar strategies, including the enhancement of Raman signals, the introduction of a Au-Se interface, and the conjugation of a functional moiety onto the AuNPs, to perform H 2 O 2 tracking [94,95].
The prodrug strategy has gained widespread attention from scholars who have attempted to eliminate the side effects of chemotherapeutics. To release a drug in a controlled manner, researchers have integrated numerous linkers and moieties that respond to environmental stimuli (such as pH, radicals, and temperature) in nanomedicine systems [96][97][98][99][100]. Lu et al. conjugated camptothecin (CPT) with phenylboronic pinacol ester to form a CPT prodrug (ProCPT) [101]. The phenylboronic pinacol ester moiety is highly sensitive to ROS stimuli, providing the nanoplatform responsiveness to oxidation. The obtained polymer with poly(ethylene glycol) (PEG) and cinnamaldehyde blocks is used to form micelles in which ProCPT is encapsulated (Fig. 8a). The micelles were observed to produce free cinnamaldehyde in acidic tumor tissue, increasing levels of radicals that modulated prodrug activation. The micelles damaged normal tissues only slightly, indicating promising potential for clinical use. In another case reported by Wei et al., a dual-responsive polymersome was prepared and had a high drug loading rate and high entrapment efficiency, which are critical in drug delivery applications [102]. An amphiphilic copolymer containing N-isopropylacrylamide and boronic esters autonomously formed polymeric micelles in an aqueous form. As H 2 O 2 activated the arylboronic esters, the micelle structure collapsed, causing the release of the grafted groups; the ester bond was then cleaved by the esterase, providing the material both ROS and enzyme responsiveness. The exposed moieties, such as phenylboronic acid and a pinacol ester, play a role in the intermediate electrostatic attraction with doxorubicin, leading to high drug-loading efficiency. In this system, the boronic ester monomer functions not only as a dual stimulus-sensitive group but also a moiety that intensifies therapeutic entrapment; this is a novel strategy for specific drug delivery.
Although the prodrug strategy can result in less toxicity than do naked drugs, the administration of nanomaterials may cause undesirable immunotoxicity once the immune system detects the material [103]. Thus, whether nanoparticles containing therapeutics can escape macrophages and circulate in the body for a long period is of great concern. Much effort has been made to increase the in vivo stability of nanoparticles through PEGlyation and carbohydrate modification; the biomimetic strategy is highly promising [104,105]. In the research of Wang and his coworker, a red blood cell membrane-coated and iRGD-derived polymersome was prepared and combined with PEG and an arylboronic ester copolymer (Fig. 8b) [106]. The photosensitizer Chlorin e6 (Ce6) and hypoxia-responsive prodrug tirapazamine (denoted TPZp) were encapsulated into polymeric particles. The platform had a long period in blood circulation and the ability to deeply penetrate tumor tissues owing to the iRGD peptide enhancement. Upon light activation, radicals generated from Ce6 caused the cleavage of ROS-sensitive arylboronic esters, which synergistically released free tirapazamine. Thus, the typical features of the tumor microenvironment were utilized to develop a sensitive platform that can be used to provide synergistic therapy for malignant tumors. Another article reported a similar concept. Liu et al. synthesized a polymeric vesicle consisting of a boronic acid and boronate ester diblock copolymer and α-cyclodextrin (Fig. 8c) [107]. Upon exposure to glucose, the self-assembled vesicle swelled because of the charge interaction between glucose and phenylboronic acid. Subsequently, the large amount of H 2 O 2 in the tumor cells accelerated particle degradation, enabling the release of anticancer agents under specific conditions. H 2 O 2 and other radical species are indicative of pathological processes such as chronic inflammation and cancer cell proliferation. Therefore, theranostic nanoplatforms are promising platforms for treating tumors. Li et al. fabricated a copolymer containing a phenylboronic ester monomer and an ROS-sensitive naphthalimide-associated monomer; the copolymer disassembles with a colorimetric swift when it senses H 2 O 2 [108]. The phenyboronic ester in this framework acts not only as a moiety promoting nanoparticle degradation but also as a mediator of ratiometric fluorescence change. The difunctioned micelles can be utilized to control and monitor the drug release profile through a H 2 O 2 -mediated structure, whereas the colorimetric change approach has strong potential in malignant tumor control and diagnosis.
Some studies have presented nanoplatforms that can innately produce high levels of H 2 O 2 , meaning that the detection limit is low and the platforms can react with oxidation-responsive moieties [109,110]. Such systems can also be used as nanoreactors. Nie et al. constructed a nanoplatform involving glucose oxidase (GOx) encapsulation that can deprive cancer cells of glucose and thus enable cancer-starving therapy. The H 2 O 2 generated by the enzyme-catalyzed reaction accelerates particle dissociation because of the phenylboronic acid pinacol ester structure within the vesicle. In this manner, TPZp is released and prevents the growth of the tumor through synergistic chemotherapy-starving therapy [111]. In another reported research, GOX-loaded nanoreactors (denoted as theraNR) were also fabricated based on phenylboronic ester and piperidine co-polymerized polymer [109,110]. In acidic tumor tissue, piperidine segments protonated and became more hydrophilic, enhancing nanoparticle membrane permeability while allowing glucose to enter. As glucose reacted under the catalysis of glucose oxidase, abundant hydrogen peroxide produced to activate phenylboronic ester and generate quinone methide as by-products. Quinone methide is known to hinder the antioxidant ability of cancer cells owing to glutathione depletion that enables tumor starving therapy (Fig. 8d). Gu et al. established a transcutaneous patch designed specifically for insulin delivery on the basis of this concept [112]. Polymeric vesicles containing H 2 O 2 -responsive phenylboronic ester and GOx were introduced to a microneedle-array patch. Upon sensing a hyperglycemic state, the encapsulated GOx produced H 2 O 2 , accelerating vesicle decomposition and triggering insulin release (Fig. 8e). Through this platform, insulin can be released and function in the desired location with high specificity and strong control. The platform is thus a feasible option for diabetes management.

Conclusion and outlook
Arylboronate-conjugated chemical structures are constructed with an electrophilic C-B bond that can react with the labile O-O bond of ONOO − and H 2 O 2 and release QM as well as a probe or anticancer drug. This process can be easily triggered with high selectivity and sensitivity and leveraged to develop a series of boronate-based probes, prodrugs, and nanomedicines. Boronate-based probes can be divided into four classes on the basis of their chemical structure: monoboronate, diboronate, boronobenzyl, and p-dihydroxyborylbenzyloxycarbonyl probes. Several detection probes and nanomaterials have been designed with fluorescent properties that make them ideal for biological applications, including noninvasive imaging in which biological interference is minimal and the in vitro and in vivo tracing of released drugs. However, H 2 O 2 -triggered and H 2 O 2 -eliminated fluorophores must have a high fluorescence quantum yield, light stability, biocompatibility, and dispersibility in nanomaterials. Hence, fluorophores such as coumarin, naphthalimide, fluorescein, rhodamine, BODIPY, and cyanine have been widely used in this field.
We summarized the progress in the development of H 2 O 2and ONOO − -triggered boronate groups that release a probe or nanomaterial. Fluorescent probes with the turn-on property or a ratiometric fluorescent response are suitable for H 2 O 2 and ONOO − detection, whereas near-infrared and twophoton excitation are favorable for deep detection in vivo because of their strong penetration and minimal background signals. In addition, specific organelle-targeting fluorescent probes can be applied to cancer research, especially to target mitochondria, lysosomes, and the ER. Probes that release cancer-targeting drugs can also be developed because of the overproduction of H 2 O 2 in cancer cells, which causes the accumulation and release of anticancer drugs in tumor cells, ensuring high therapeutic efficiency. This prodrug strategy has also been applied in studies on nanoparticle and Fig. 8 a Particle design and formation of ProCPT@P3 micelles. b Biomimetic nanoplatform and its ROS-responsive properties. c Chemical structure of a polymer with stimulus-responsive PBA and PBEM groups. d Particle structure of theraNRs and its functions in the tumor site. e Polymer structure of mPEG-b-P(Ser-PBE) and its delivery of insulin when self-assembling into vesicles. Reprinted with permission from ref [101,106,107,109], and [112]. Copyright from 2021 and 2019 Royal Society of Chemistry and copyright from 2017 American Chemical Society. All rights reserved ◂

Nanoparticles
Releasing of anticancer prodrug Enhance the deliver efficacy of existing anticancer drug [101,106,107,109] Combination of AuNPs with physical aggregation through the protein binding Naked-eye observation [93] Electrochemistry Signal response from ferrocene and H 2 O 2 A multifunctional probe for both intracellular and extracellular detection [83] Ratiometric sensing under complex environment Increase the efficacy for bio-detection for whole blood sample [84] Signal amplification with redox reporter In vivo tracking in the living cell system [85] Dual-signal detection of NADH and H 2 O 2 Enzyme-free biosensor design [86] nanomedicine systems to increase detection sensitivity and drug efficacy. Another example of a method of increasing the H 2 O 2 detection limit is using electrochemical biosensors, which can be easily modified into multifunctional biosensors for intracellular and extracellular detection in a complex environment and provide high accuracy in whole-blood, serum, and plasma samples. A list of biological applications of our described borate-based probes is shown in Table 1. Boronate probes are a new class of redox probes that are widely used in research on H 2 O 2 biosensors, cancer diagnosis, and cancer therapy. The reaction mechanisms, spectroscopy, and molecular and cellular mechanisms of numerous boronate probes have been reported. These probes have solved numerous problems, but the progress of commercialization for the practical use such as patents, products which are under clinical trials, is limited. Despite this, boronate probes are still considered the useful tools in in vitro and in vivo biological research.