Peptide-Based AIEgens: From Molecular Design, Stimuli Responsiveness to Biomedical Application

Open AccessCCS ChemistryMINI REVIEW1 Feb 2022Peptide-Based AIEgens: From Molecular Design, Stimuli Responsiveness to Biomedical Application Ben-Li Song, Xue-Hao Zhang, Zeng-Ying Qiao and Hao Wang Ben-Li Song School of Pharmaceutical Sciences, Henan Institute of Advanced Technology, Zhengzhou University, Henan 450001 CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 , Xue-Hao Zhang CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 , Zeng-Ying Qiao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 and Hao Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] School of Pharmaceutical Sciences, Henan Institute of Advanced Technology, Zhengzhou University, Henan 450001 CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 https://doi.org/10.31635/ccschem.021.202101231 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Luminogens with aggregation-induced emission (AIEgens) have a wide range of biomedical applications in bioimaging, photodynamic anticancer, antibacterial therapy, and other fields, owing to their unique photophysical properties. The precise structural design and modification of AIE molecules have aroused great interest in the past years. As peptides-AIE hybrid materials, peptide-based AIEgens generally have better solubility, biocompatibility, and lower systemic toxicity. The functional diversity, modularity, and portability of peptides provide more possibilities for the intelligent structure and functional design of AIEgens. This review summarizes the recent research progress of peptide-based AIEgens nanomaterials, from molecular design, stimuli responsiveness to biomedical application, focusing on the advantages of peptides and AIE molecules as conjugates. Finally, a summary of the challenges and opportunities of peptide-based AIEgens nanomaterials for future clinical biomedical applications is presented. Download figure Download PowerPoint Introduction Fluorescent probes have been widely used in biological imaging and sensing of cells, tissues, and animals. They provide researchers with the convenience to visualize pathological processes directly.1 In the past decades, fluorescent molecules have developed rapidly, but there are major problems involved; an example is aggregation-induced quenching (ACQ) due to the π–π interaction between molecules.2 Most traditional fluorescent probes are aggregated in aqueous solutions due to the presence of polycyclic aromatic hydrocarbons (PAHs) in their structures.3 In this case, ACQ leads to a reduction in fluorescence intensity, which greatly limits its application in a variety of physiological and pathological environments. Fortunately, Tang's group4 first proposed the concept of aggregation-induced emission (AIE) in 2001, which has become a research hotspot recently. They have made breakthrough progress in discovering the molecules with AIE structure that can emit light in concentrated solutions and the aggregate state, where the rotation of AIEgens is restricted, thereby achieving fluorescence enhancement in the aggregate state. Specifically, the mechanism governing the AIE effect is the restriction of intramolecular motion (RIM).5 Contrary to traditional ACQ fluorophores, AIEgens have inherent advantages in high signal-to-noise ratios, good light stability, large Stokes shift, low background noise, field activation ability, and other aspects.6 These excellent optical properties imply that AIE molecules have broader application prospects in the field of biological imaging compared with traditional fluorescent molecules. In recent years, owing to excellent structural design and mechanistic analysis, AIEgens have produced a wide range of applications in many fields such as optoelectronic devices, chemical sensing [reactive oxygen species (ROS) detection, volatile gas detection], and biomedicine area. AIEgens have made significant progress in imaging of specific organelles, detection of pH and enzyme activity in biological systems, binding to specific receptors on the cell membrane surface, long-term cell tracking to observe microscopic dynamics of cells, and even in vivo imaging of tumors.7 Based on the special needs of tumor physiology and pathological environment, AIEgens are required to demonstrate better water-solubility, biosafety, and lower immunogenicity, as well as minimal systemic toxicity.8 Owing to the inherent biocompatibility, biodegradability, natural biological activity, and low toxicity of the peptide, peptide-modified AIE nanomaterials play a vital role in enhancing penetration, extending blood circulation time, improving targeting, stability, and therapeutic effects.9 The emission of the fluorescent probe is relatively low due to the water solubility of AIE, coupled with peptide-based nanomaterials. Intramolecular motion is restricted after binding to a specific target or internalization into the target cell so that their biological conjugates could form aggregates to activate AIE. Therefore, this intelligent activation of peptide-based AIEgens nanomaterial with responsive performance has a wide range of applications in bioimaging, antibacterial, and tumor diagnosis along with treatment (Figure 1).10–12 Figure 1 | An overview of the classification and theranostic modes of the peptide-based AIEgens nanomaterials. Download figure Download PowerPoint Peptide-based AIEgens have distinct advantages in improving the accuracy of imaging, clinical diagnosis, and disease treatment in vivo because the coupling of AIE and peptide improves targeting and stability, increases penetration depth, and prolongs blood circulation time. Therefore, this field is attracting the attention of researchers. Although many reviews on the mechanism, molecular design, and application of AIE have been published,1,3,7 the application of peptide-based AIEgens conjugates is still relatively limiting. This review focuses on typical examples that demonstrate the advantages of peptide-AIE conjugates and their wide application, as well as their practical progress in the field of biomedicine. Future development of peptide AIEgens conjugate is explored, and possible solutions to the current problems are proposed. Peptide-Based AIEgens Conjugates Since the discovery of the AIE phenomenon by Tang's research group in 2001,4 it has attracted much research attention due to its unique advantages. Tetraphenylethylene (TPE) is a typical representative AIEgen molecule due to the RIM mechanism, while bispyrene (BP) is quite different from it. BP can form J-type aggregates and cause aggregate luminescence. Therefore, we have divided the peptide-based AIEgens conjugate into the following parts for detailed review according to the different molecular mechanisms. TPE-peptide As one of the typical molecular structures in the AIE field, TPE with a unique propeller structure is composed of an olefin bond (stator) and four benzene rings (rotor).13 In the solution state, the benzene ring surrounding an alkene can be rotated/twisted so that the conjugation of the molecule is less restricted, and the excited state is deactivated through a nonradiation route. However, when aggregation occurs, the rotation of the benzene ring is hindered, and the highly twisted structure prevents strong π–π aggregation, thereby eliminating self-quenching. When TPE molecules are excited, they release energy through radiation transitions, so fluorescence occurs when they are in the aggregate state.3 Compared with other molecules, TPE has the advantages of its simple synthesis and easy peptide modification. Based on these advantages, the combination of TPE and peptide has a wide range of applications in biological imaging, sensor detection, and stimuli-responsive nanomaterial properties.14 The TPE molecule can be used in monitoring cancer cell apoptosis during cancer treatment. Meade and his colleagues15 designed a caspase probe 1 (CP1), a bimodal fluorescence-magnetic resonance (FL-MR) probe, which has a simultaneous FL-MR turn-on response to caspase-3/7. CP1 is composed of three parts: (1) Asp-Glu-Val-Asp (DEVD) peptide, amino acid sequence derived from the caspase-3/7 cleavage site; thus, depicts the substrate of caspase 3/7, (2) the fluorescent molecule TPE with AIE effect, and (3) the paramagnetic DOTA-Gd(III) Chelate (Figures 2a and 2b). CP1 has good solubility and dispersibility in an aqueous solution, showing a low FL/MR signal. In cancer cells, water-soluble peptide DEVD in CP1 is cleaved by caspase-3/7, and Gd(III)-AIEgen (Gad-AIE) comprise aggregates to increase FL-MR signal (Figure 2c). When the moisture content is close to 100%, Gad-AIE has a high emissivity, which is the characteristic behavior of AIEgens (Figure 2d). The critical micelle concentration (CMC) of GAD-AIE was quantified as 43 μM by fluorescence spectroscopy experiment (Figure 2e). Caspase is only activated during apoptosis and is used as a marker in the detection process. When the fluorescence intensity increases, it indicates an increase in the number of apoptotic cells. By adding inhibitors to CP1+caspase-3/7, the fluorescence disappears, proving the specificity of CP1 to caspase-3/7 (Figure 2f). CP1 has a dual FL-MR turn-on response, showing sensitivity and selectivity in the presence of caspase-3/7 (Figures 2g and 2h). This feature can be used to quantify the exact concentration of the activator and accurately predict the MR signal in vitro, proving that the aggregation-driven FL-MR probe design is a unique method for quantifying the MR signal. Figure 2 | Research on the molecular design, response mechanism, and optical properties of CP1. (a) Chemical structures of CP1 and Gad-AIE, Gad-AIE is a mimic of the activated CP1. (b) The mechanism of CP1's response to caspase-3/7. (c) Fluorescence spectra of CP1 (50 μM) and Gad-AIE (50 μM) in caspase-3 buffer. (d) The relative fluorescence intensity of Gad-AIE in different H2O/dimethyl sulfoxide (DMSO) mixed solutions. (e) Fluorescence (FL) intensity of Gad-AIE at different concentrations. (f) CP1 (50 μM) and caspase-3 (0.4 μg/mL) combined time-dependent FL turn-on response. (g and h) Specificity study on the specificity and selectivity of CP1 to enzymes. Reprinted with permission from ref 15. Copyright 2019 American Chemical Society. Download figure Download PowerPoint Real-time monitoring of tumor cell apoptosis with fewer side effects is essential for the precise treatment of targeted tumors. Zhang and his collaborators16 designed a traceable self-assembled beam ([email protected]) (DOX = doxorubicin) with the DEVD peptide as the core of the caspase response, which is connected to the watery H9-PEG8 by Arg-Gly-Asp (RGD) peptide, and TPE fluorescence as a targeted drug delivery system for real-time monitoring of tumor cell apoptosis for precision cancer therapy. During drug delivery, TPE enables the micelle to be imaged at a higher quality, clearly indicating the loading and release of DOX. RGD peptides can be combined with αvβ3 integrin, which is specific to the surface expression of tumor cells, to promote cell ingestion. It shows remarkable tumor cell killing ability and opens a way for accurate tumor treatment. Wang et al.17 designed a fluorescent probe with good biocompatibility to monitor Zn2+, which is extremely important for human health. The TPE-peptide probe formed a self-assembled complex with three histidine residues in the two peptide chains in the presence of Zn2+, which led to the aggregation of the TPE part and the turn-on of fluorescence. The probe with low cytotoxicity and good stability could image Zn2+ in cells, indicating that TPE-peptide is a useful bioluminescence sensor with broad application prospects. However, the current bioapplication of TPE is mostly limited because the rotor structure of the TPE molecule causes short-wavelength absorption. Therefore, new AIE molecules with long-wavelength absorption to improve tissue penetration are worthy of further exploration and research. BP-peptide Supramolecular stacking type of organic π–π conjugated molecules affects the photophysical properties.18 The formation of J-type aggregates usually leads to fluorescence enhancement and redshift of the absorption spectrum of the fluorescent material. Our group19 developed BP as an AIE building block to monitor changes in the aggregation pattern of peptide-based nanomaterials in vivo (Figure 3a). BP is a hydrophobic molecule with π–π interaction that can self-assemble into J-type aggregates, exhibiting bright fluorescence (Figure 3b). Therefore, the self-assembly process in vivo can be embodied by fluorescence imaging technology. Through endogenous and exogenous stimulation, the in situ construction of self-assembled nanomaterials can be realized, showing some new biomedical effects. Figure 3 | Schematic illustration of in situ self-assembly and transformation based on BPs. (a) BP and its derivatives. (b) BP of self-assembled morphology and property. Reprinted with permission from ref 19. Copyright 2019 American Chemical Society. Download figure Download PowerPoint Recently, a series of achievements and progress related to peptide-based BP conjugate have been made in the imaging and antitumor area. It is well known that there are highly expressed HER2 receptors on the membrane surface of a breast cancer cell.20,21 When the HER2 receptor dimerizes, downstream signals are activated that promote rapid cancer cells' growth. Lam group22 designed and studied an AIE-based nanomaterial to inhibit cancer cell proliferation through its morphological transformation. As shown in Figure 4a, this molecule is composed of three building blocks: (1) BP, which is both a hydrophobic core and fluorescent molecule, (2) Phe-Phe-Val-Leu-Lys (FFVLK), β-Sheet derived from amyloid (Aβ) peptide, and (3) peptide Tyr-Cys-Asp-Glu-Phe-Tyr-Ala-Cys-Tyr-Met-Asp-Val (YCDGFYACYMDV) with disulfide bond domain, which can self-assemble into nanoparticles (NPs) in aqueous solution. It is important that the self-assembled NPs display a fluorescent signal to visualize the self-assembly/disassembly process in vitro and in vivo.23 Through intravenous injection in mice, it was observed that the nanomaterial quickly accumulated on the membrane surface with high expression of HER2 receptor through receptor-ligand interaction, and the NPs were transformed into a fiber network structure in situ. The fiber inhibited the dimerization of HER2 and, at the same time, prevented downstream cell signal transduction and the expression of proliferation and survival genes in the nucleus. As shown in Figure 4b, the absorbance of the tropomyosin proteins, transformable peptide monomer 1 (TPM1), gradually decreased with increasing water content, which proved the formation of self-assembled NPs. The fluorescence intensity of BP increased significantly at 520 nm (Figure 4c), demonstrating the unique AIE fluorescence characteristic of BP in monitoring the dynamic process of TPM1. Transmission electron microscopy (TEM) observation revealed that the particle size of the peptide-based nanomaterial NPs1 is ∼ 20 nm. However, when NPs1 interacts with HER2 protein for 24 h, nanofibers (NFs) were formed with a width of ∼ 9 nm (Figure 4d). The morphological transformation of the peptide enabled it to bind more specifically to HER2 receptors and inhibited HER2 dimerization. Therefore, morphology transformation of peptide and cell surface receptor interaction to inhibit downstream pathways is a promising cancer therapy strategy, which hopefully provides new ideas for the clinical treatment of breast cancer. Figure 4 | Schematic illustration of assembly and fibrillar transformation of transformable TPM1. (a) Schematic diagram of TPM1 molecular design and morphological transformation. (b) Schematic diagram of the change of UV–vis absorption. And (c) fluorescence intensity of NPs1 in different solvent ratios, the proportion of water gradually increases (from 0 to 99.5) in DMSO (d) TEM images of NPs1 interacts with HER2 protein (MW ≈ 72 kDa) and increases with time (0.5, 6, and 24 h), and transforms into NFs (NFs1). Scale bar, 100 nm. Reprinted with permission from ref 22. Copyright 2020 Springer Nature. Download figure Download PowerPoint In addition to the antitumor applications of peptide-based BP conjugate, it has important significance in the field of bionics. It was demonstrated the use of switchable laminin (LN)-based peptide (BP-KLVFFK-GGDGR-YIGSR) to simulate artificial extracellular matrix (AECM).23 The molecule could self-assemble into NPs with strong fluorescence emission in water due to the strong π–π stacking ability and hydrophobicity of BP and the aggregation process could be monitored by fluorescence. After intravenous injection, 1-NPs accumulated at the tumor site through enhanced permeability and retention (EPR) effects.24 At the same time, it transformed into NFs, which assembled with tumor cell surface receptors to form AECM. Using the biomimetic assembly strategy,9 AECM provides a long-term barrier and effectively inhibits tumor metastasis and growth.23 Therefore, this strategy of constructing a bionic AECM in situ to inhibit tumor metastasis and invasion shows great potential for future development. BP-peptide nanomaterials can produce different biological functions by modifying different peptide sequences due to their hydrophobicity and fluorescence effect. Our group designed and prepared a self-assembling peptide-based AIE nanomaterial (BP-FFVLK-HSDVHK), which forms NPs in solution and is specific to the integrin αvβ3 on the surface of human umbilical vein endothelial cells (HUVECs). After the interaction, the morphology is transformed into a NF network, avoiding cellular endocytosis and degradation, thereby effectively inhibiting angiogenesis. In vitro experiments show that its inhibitory rate on angiogenesis is as high as 94.9%, and it has great potential as an antiangiogenic drug for adjuvant tumor treatment.25,26 One of the challenges currently faced by photodynamic therapy (PDT) is weak tissue penetration. Li's group used two-photon activation (TPA) technology to provide the possibility to solve this problem. They synthesized and designed a cationic dipeptide NP (BP-CDPNP-RB), composed of two-photon absorption molecule (BP) and photosensitive drug (RB), with good biocompatibility. In NPs, co-encapsulated BP is an energy donor. After being excited by a single-photon or two-photon laser, the energy of BP was transferred to RB, which significantly promoted the production of singlet oxygen. This nanomaterial can induce cytotoxicity under both single- and two-photon irradiation, showing strong tissue penetration ability.27 BP-peptide has been proven to exhibit significant effects on in vivo imaging and treatment, which promoted the development of the field. However, BP has a low CMC in an aqueous solution and is highly hydrophobic. It can form J-type aggregates, but the monomer state is difficult to control. Compared with TPE-peptide conjugates, the regulation of the fluorescence between the monomer and aggregate states of BP is more difficult, and the structural design of nanomaterial-based on BP-peptides is relatively simple. In the future, more diverse structural and functional nanomaterials based on BP-peptides should be designed to meet the imaging and treatment needs of different diseases. Other AIE-based nanomaterials In addition to the TPE and BP, tetraphenylsilyl (TPS) and hexaphenylsilyl (HPS) as fluorescent modules are also used in the design of AIEgens, and peptide-modified TPS plays a vital role in imaging and sensing. Enzyme activity monitoring is an effective method in the process of disease diagnosis. Liu's group28 designed a dual-signal peptide-based AIE fluorescent probe for caspase cascade activation monitoring, including two AIEgens (TPS and TPE). Hydrophilic peptide (DVEDIETD) was used as the substrate for caspase-8 (IETD = Ile-Glu-Thr-Asp) and caspase-3 (DVED). The probe has no fluorescence in an aqueous solution. After H2O2-induced apoptosis in HeLa cells, caspase-8 and caspase-3 cascade was activated to break the peptide bonds, and, in turn, green and red fluorescence were turned on. This kind of excitation could produce different colors at a single wavelength, convenient for real-time monitoring of different enzymes. In addition, this real-time monitoring and imaging of cancer cells have a profound impact on the early diagnosis of cancer. Liu's group29 also designed an Ac-DEVD-TPS-cRGD (cRGD = cyclic Arg-Gly-Asp) fluorescent bioprobe. The probe has specific targeting effects toward U87 MG cells duo to a combination of cRGD and integrin receptor, monitoring and imaging cancer cell apoptosis in real-time. Compared with the near-infrared molecules developed by most researchers, HPS and TPS have been continuously improved and explored in structure due to their shorter fluorescence emission wavelengths. Therefore, the emergence of AIE molecules with long emission wavelengths is worth expecting. Stimuli-Responsive Peptide-Based AIEgens Stimuli-responsive nanomaterials can make adaptive changes in the pathological microenvironment to assist diagnosis or treatment. AIEgens have different structural change abilities by modifying targeting peptides, assembly peptides, and responsive peptides on AIE molecules. The regulation of structural changes of self-assembly peptides is considered to be a promising method in antitumor, antibacterial, and others. Peptide-based AIEgens have shown great potential in the field of personalized biomedicine by producing corresponding effects in the stimulus-responsive microenvironment (pH, ROS, enzyme). pH-responsive peptide-based AIEgens The level of pH regulation plays a vital role in biological systems. Maintaining pH at the optimal activity level of the organism helps the organism's metabolic balance.30 Abnormal pH could cause cancer, Alzheimer's disease, as many essential enzyme reactions cause pH changes by producing acidic or alkaline species. Tumor blood vessels provide a microenvironment for tumor cell growth, so current clinical strategies include embolization strategies,31 the use of vascular blockers,32 and vascular endothelial growth factor (VEGF) antibodies to block or inhibit the formation of tumor blood vessels.33,34 These approaches directly prove that the blockage of tumor blood vessels is an effective method among several for tumor treatment, which has aroused broad research interests. Wang's research team35 developed a precise and effective approach to block tumor blood vessels, regulating tumor pH and microthrombus to mimic the formation of LN fibers in situ. LN aggregates are an insoluble fiber network that could support and prevent the movement of cells as a basement membrane. The aggregation process is regulated by ligand-receptor interaction and lowering pH. The AIE-peptide-based nanomaterial, LN mimic peptide (LMMP) BP-FFVLK(OEG-CREKA)-His6 is composed of hydrophobic and fluorescence imaging unit BP, pH-responsive unit His6, a self-assembly unit (FFVLK), and the target sequence Cys-Arg-Glu-Lys-Ala (CREKA) (Figure 5a). By accurately and effectively simulating the formation of LN fibers in tumor blood vessels, the network of LN-like fibers is constructed, with 3-day timeliness in tumor blood vessels. These fibers can capture red blood cells, causing blockage of tumor blood vessels and inhibiting tumor growth. The UV–vis spectrum experiment shows that a different water content caused a redshift of the material, indicating the aggregation of BP units in LMMP material (Figure 5b). LMMP exhibits a strong excimer emission at 520 nm in an aqueous solution (Figure 4c). Therefore, the process of self-assembly in vivo can be visualized by fluorescence imaging, which proves the formation of J-type aggregates, and at the same time, enhances the stability of the peptide (Figure 5c). By lowering the pH, the hydrophilicity of the material could be increased, resulting in the conversion from NPs to NFs (Figure 5d). In summary, LMMP could transform from NPs to NFs in an acidic environment. Tumor vascular obstruction could cause a decrease in oxygenated hemoglobin (HbO2). This approach of changing the shape of AIE peptide-based nanomaterials through pH response to achieve tumor suppression provides new ideas for tumor treatment. Figure 5 | Schematic diagram of the pH response from the transformation of LMMP. (a) The molecular structure of LMMP. (b) UV–vis spectra of LMMP with different water content. (c) Fluorescence spectra of LMMP-NP solution at different time points when the pH is 6.5. (d) TEM schematic diagram of the morphological changes of LMMP at different time points and pH. Reprinted with permission from ref 35. Copyright 2020 American Chemical Society. Download figure Download PowerPoint The development of AIEgens peptide-based nanomaterials with morphological transformation in a physiological environment is essential for tumor diagnosis, imaging, and treatment. Hu's research group36 designed and synthesized a pH-responsive AIEgens peptide-based nanomaterial, consisting of four parts, including STP (sequence: Ser-Lys-Asp-Glu-Glu-Trp-His-Lys-Asn-Asn-Phe-Pro-Leu-Ser-Pro,SKDEEWHKNNFPLSP) peptide containing α-helix structural fragments, hydrophobic TPE, anticancer drug DOX, and P18. The self-assembled NPs are transformed into NFs in the environment of tumor blood vessels and low pH, targeting the VEGFR2 receptor overexpressed on the surface of tumor cell membranes to release drugs more efficiently and achieve the therapeutic effects. The successful design of responsive materials has important implications for targeted imaging diagnosis, detections, and specific cancer treatments. By using the characteristics of the tumor microenvironment, Wang and his colleagues37 developed a nano-carrier drug delivery system, STD Nano-Micelle (STD-NM), responsive to pH and specific markers, which is invisible in the normal environment but activated in the acidic environment of the tumor. It binds specifically to the receptors on the membrane surface of cancer cells; owing to the AIE characteristics of STD-NM, the fluorescence could be turned on when the cells are apoptotic. Therefore, this design has great application potential in tumor imaging and cancer treatment. Gao and his colleagues38 used DOX-encapsulated polyethylene glycol block peptides Phe-Phe-Lys-Tyr (FFKY) combined with TPE (PEG-PEP-TPE/DOX) to form NPs, in which 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO) can induce a fluorescence resonance energy transfer (FRET)-pair with DOX, dynamically detecting drug release. ROS-responsive peptide-based AIEgens ROS, including superoxide anions,39 hydrogen peroxide,40 hydroxyl radicals,41 and so on, is a normal product of oxygen metabolism, produced mainly in the mitochondria. Normal cells are in a state of balanced oxidation-reduction; the disruption of this balance leads to a series of diseases such as cancer, Alzheimer's disease,42 and epilepsy. Therefore, monitoring and real-time imaging of ROS are of great significance in the early detection of biological abnormalities.43 Xia and his colleagues44 designed and synthesized a TPE derivative [Tyr-containing TPE (TT)] that could recognize H2O2 through charge interaction used in confocal laser scanning microscopy (CLSM) measurements in cancer and normal cells. The specific molecular design