Triphenylamine-AIEgens Photoactive Materials for Cancer Theranostics

Junjie Wang【王俊杰】^c#, Yan Wang^a,b#, Zhengdong Li^e#, Changqiang Xie^a,b, Mussmmir Khan^f, Xingzhou Peng【彭星舟】^a,b*, Fabiao Yu【于法标】^c,d*

^a State Key Laboratory of Digital Medical Engineering, School of Biomedical Engineering, Hainan University, Haikou 570228, China

^b Key Laboratory of Biomedical Engineering of Hainan Province, One Health Institute, Hainan University, Haikou 570228, China

^c Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China

^d Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

^e Precision medical center, Guangyuan City Centre hospital, Guangyuan 628099, China

^f School of Natural Sciences (SNS), National University of Science and Technology (NUST), H-12, Islamabad 44000, Pakistan

https://doi.org/10.1016/j.cclet.2023.108934Full TEXT

Graphical abstract

The photoactive agents based on triphenylamine-based aggregation-induced emission luminogens (TPA-AIEgens) for cancer theranostics were summarized, including the design principle, targeting strategies, and the latest progress in biological imaging and cancer treatment.

Abstract

Triphenylamine (TPA)-based aggregation-induced emission luminogens (TPA-AIEgens), a type of photoactive material utilizing the typical TPA moiety, has recently attracted increasing attention for the diagnostics and treatment of tumors due to their remarkable chemo-physical performance in optoelectronic research. TPA-AIEgens are distinguished from other photoactive agents by their strong fluorescence, good sensitivity, high signal-to-noise ratio, resistance to photobleaching, and lack of high concentration or aggregation-caused fluoresce quenching effects. In this review, we summarize the current advancements and the biomedical progress of TPA-AIEgens in tumor theranostics. First, the design principles of TPA-AIEgens photoactive agents as well as the advanced targeting strategies for nuclei, cell membranes, cell organelle and tumors were introduced, respectively. Next, the applications of TPA-AIEgens in tumor diagnosis and therapeutic techniques were reviewed. Last, the challenges and prospects of TPA-AIEgens for cancer therapy were performed. The given landscape of the TPA-AIEgens hereby is meaningful for the further design and utilization of the novel photoactive material, which could be beneficial for the development of clinic applications.

1.Introduction

Malignant tumors are the primary cause of human mortality, which represent a severe threat to human health and survival [1]. Nowadays, surgery, radiation, and chemotherapy are the three primary traditional treatments. Unfortunately, these traditional treatments suffer defects, such as significant systemic side effects and high recurrence rates [2,3]. Recently, phototheranostics have made significant contributions to cancer treatment due to their light-controllability, non-invasiveness, specific target, low toxicity, and good selectivity [4-6]. Normally, photodynamic therapy (PDT) and photothermal therapy (PTT) are two types of phototherapies that exogenous photosensitizing agents are involved to selectively kill tumors or inhibit tumor growth with light radiation [7,8]. In PDT, photosensitizers (PSs) are typically used to generate cytotoxic reactive oxygen species (ROS), such as singlet oxygen (¹O₂), superoxide (O₂–^–) and hydroxyl radical (–OH), which is able to eliminate tumor cells [5,9]. Photothermal agents can enhance the heating process of cells and tissues in the local area by absorbing laser energy and converting it into heat [10-12], which is similar to laser therapy [13]. The main discrepancy is that laser therapy uses endogenous chromophores with non-selectivity towards malignant cells, while PTT with exogenous agents can specifically target cancer cells. With the aid of photoactive agents, both the PDT and PTT can produce vigorous interaction with biological substances, leading to the immunogenic cell death (ICD), vascular injury, and immunological response [14-17].

Recently, the photoactive agents based on AIE luminogens (AIEgens) developed by Tang Benzhong’s research group have made breakthroughs in phototherapy research. An interesting phenomenon termed aggregation-induced emission (AIE) occurs when a series of non-emissive molecules in the dispersed state are induced to show strong emission upon aggregate formation or in solid state [18]. Due to the existence of the strong electron-vibration coupling in the dispersed state, AIE molecules demonstrate the intrinsic non-radiative transitions and fluorescence quench. In the aggregated state, the molecular environment’s restriction of intramolecular movements (RIM) might impair the electron-vibrational coupling of molecular system, resulting in depressed non-radiative transitions and enhanced fluorescence [19,20]. In general, the clinical diagnostic agents (e.g. ICG, porphyrin) suffer from certain problems such as aggregation-induced fluorescence quenching (ACQ) and photobleaching [21,22]; while AIEgens offers the following benefits compared to the traditional fluorescent dyes: 1) high-intensity fluorescence; 2) efficient light conversion; 3) strong stability; 4)luminescence regulation by flexible chemical modification; 5) high resolution in biological imaging [23,24].

The fundamental AIE motifs, such as tetraphenylene (TPE), triphenylamine (TPA), 2,3,4,5-tetraphenylsiloles (TPS), phenylvinyl anthrance, and phenyl substituted pyrrole, are integral to the creation of AIE materials [25]. In contrast to typical TPE-AIEgens, TPA-AIEgens is more flexible to be designed for in vivo applications such as high water-solubility and strong NIR II absorption. The chemical structure of TPA is composed of three benzene rings and a central nitrogen atom. Due to its unique helical structure and three phenyl rotors, TPA not only plays a role as the strong electron donor but also acts as a molecular rotor, making it easy to construct a variety of TPA-AIEgens. As an electron donor, TPA is combined with an electron acceptor to obtain a molecule with donor-acceptor (D-A) structure resulting in an adjusted intramolecular push-pull electron interaction, which benefits the red-shift of fluorescence and/or enhances intersystem crossing (ISC) process to generate more ROS [25,26]. When acting as a molecular rotor, the highly distorted conformation of TPA increases the molecular distance, reducing the intermolecular π-π packing and retaining the intramolecular rotation, which is beneficial to promote fluorescence and/or heat generation [27,28]. Therefore, TPA is a crucial functional segment in the construction of TAP-AIEgens.

Up to now, TPA-AIEgens have been developed as novel molecular materials with photosensitive and photothermal capabilities, demonstrating the huge potential in tumor theranostics [29]. Thus, the design principles, targeting strategies, and the latest progress in biological imaging and treatment based on TPA-AIEgens photoactive agents are summarized in this minireview (Scheme 1). First, the principles of design TPA-AIEgens with near-infrared absorption and good biocompatibility were summarized. Next, TPA-AIEgens targeting strategies, including targeting to nuclei, cell membranes, cell organelles and tumors, were presented. Then it introduced the application of TPA-AIEgens in biological imaging and phototherapy for the most recent years. Finally, the photoactive agents based on TPA-AIEgens for cancer treatment were prospected. We hope this review can provide valuable information for design and application of TPA-AIEgens to accelerate their clinical translation.

Scheme. 1. TPA-AIEgens for cancer theranostic, including targeting strategies, biological imaging and cancer treatment.

Fig. 1. Typical TPA-AIEgens used for cancer diagnosis and treatment. Compound 1–3 with extended π system to obtain the red-shifted and high fluorescence emission; compound 4 and 5 with rigid and strong acceptor unit to promote the fluorescence quantum yield; compound 6 and 7 with low ∆E_st values to enhance ROS generation; compound 8–10 with increased steric hindrance to red shift to NIR II emission; compound 11 and 12 with TICT state to boost photothermal conversion property.

Fig. 2. (a) Schematic illustration of MeTPAE with nuclei-targeting function for PDT. Reproduced with permission [59]. Copyright 2022, Wiley-VCH. (b) Schematic illustration of molecular design diagram of a high-performance AIE photosensitizer with cell membrane-targeting function for fluorescence imaging-guided PDT. Reproduced with permission [61]. Copyright 2022, The Royal Society of Chemistry. (c) Schematic illustration of the structure of DBP, TBP, and TBP-SO₃. Reproduced with permission [67]. Copyright 2022, The Royal Society of Chemistry. (d) Schematic illustration of the structure of TTT-1, TTT-2, TTT-3 and TTT-4. Reproduced with permission [70]. Copyright 2021, Elsevier. (e) Schematic diagram of the structure of 1,4-dihydropyridine derivatives for both LDs and ER-targeting imaging and therapy. Reproduced with permission [72]. Copyright 2023, Wiley-VCH

Fig. 3. (a) Molecular design and synthetic route of TBP-Au. (b) Schematic illustration of TBP-Au for anticancer therapeutics through synergistic effects of photodynamic therapy and TrxR inhibition. (c) In vivo real-time fluorescence imaging in HeLa tumor-bearing nude mice after intratumor injection of TBP-Au (λex: 530 nm). Reproduced with permission [85]. Copyright 2021, American Chemical Society. (d) In vivo real-time PA images of the tumor site in the mice after tail intravenous injection with TBTO NPs. (e) In vivo real-time NIR fluorescence imaging in tumor site after intratumoral injection with TBTO NPs. Reproduced with permission [89]. Copyright 2021, Elsevier

6. Conclusion and outlook

In this review, we summarized recent advances of TPA-AIEgens for cancer theranostics. Compared to clinical photoactive agents, TPA-AIEgens shows unique and excellent advantageous such as bright emission, robust ROS generation, high photothermal conversion efficacy, resulting in improved antitumor efficacy. By rational design, TPA-AIEgens demonstrate NIR II absorption/emission, enhanced phototherapy. Owing to AIE characteristics, TPA-AIEgens is available to visualize the tumor foci, which plays a vital role in the clinic diagnosis and image-guided surgeons. Moreover, the PDT and PTT combined with immunotherapy, could be a promising approach to enhance the immunotherapy efficiency. (Table 1).

Despite impressive advancements of TPA-AIEgens in the realm of biomedicine, there are still certain problems and challenges for clinical application. First, TPA-AIEgens suffer from poor targeting capability of tumors and the accumulation of TPA-AIEgens mainly relies on the EPR effect. By modifying the size, shape, structure, surface charge, and target ligands of nanovehicles [113-115], it will be possible to create TPA-AIEgens photoactivators furnishing with unique active targeting 爌roperties for accurate and effective anti-tumor treatment in future. Second, the relationship between structure and target properties of TPA-AIEgens should be systematically studied, which will provide theoretical guidance for further development of the TPA-AIEgens to fulfill clinical requirements. Third, most of the excitation wavelengths of TPA-AIEgens are in the visible range. Compared to those dyes with an absorption in the NIR biological window, TPA-AIEgens encounter the challenges in imaging of deep tissue and high resolution requiremen [116-118]. Besides, TPA-AIEgens, as the photoactive agents, still require rigorous investigation on the therapeutic immunological mechanism and in vivo ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties. Different strategies including the structure design of the probes which have been discussed in section 3, have achieved substantial developments and will definitely promote their transition by addressing the clinical concerns. [108,119]. Last but not least, biosafety issues are inevitable for the development of novel TPA-AIEgens. Biodegradability and long-term toxicity to humans must be considered to meet the demands of clinical translation. Consequently, in order to achieve the therapeutic effect of totally curing malignancies and successfully preventing tumor metastasis and recurrence, it is imperative to conduct in-depth research on TPA-AIEgens through the aforementioned aspects. In future research, the biomedical application of TPA-AIEgens in tumor diagnosis and treatment systems can be vigorously promoted.

Table 1 The summary of TPA-AIEgens for tumor theranostics.

Near-Infrared Fluorescence Probe for Indication of the Pathological Stages of Wound Healing Process and Its Clinical Application

Xianzhu Luo【罗贤柱】,Shaowen Cheng【程少文】,Wei Zhang【张伟】,Kun Dou【窦昆】,Rui Wang【王锐】*, and Fabiao Yu【于法标】*

^a Key Laboratory of Hainan Trauma and Disaster Rescue, Department of Wound Repair, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China

^b Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Emergency and Trauma, Ministry of Education, Key

Laboratory of Hainan Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

Cite this: ACS Sens. 2024, 9, 2, 810–819Full Text

Publication Date:January 19, 2024

https://doi.org/10.1021/acssensors.3c02147

Copyright © 2024 American Chemical Society

SUBJECTS:

Fluorescence,Fluorescence imaging,Probes,Rodent models,Wound healing

Abstract

Chronic wound healing is one of the most complicated biological processes in human life, which is also a serious challenge for human health. During the healing process, multiple biological pathways are activated, and various kinds of reactive oxygen species participate in this process. Hydrogen peroxide (H2O2) involves in chronic wounds and its concentration is fluctuated in different pathological stages during the wound healing process. Therefore, H2O2 may be recognized as a powerful biomarker to indicate the wound healing process. However, the pathological roles of H2O2 cannot be fully understood yet. Herein, we proposed a near-infrared fluorescent probe DCM-H2O2 for highly sensitive and rapid detection of H2O2 in living cells and scald and incision wound mice models. DCM-H2O2 exhibited a low detection limit and high specificity with low cytotoxicity for H2O2, which had great potential for its application in vivo. The probe was successfully utilized to monitor the fluctuation of endogenous H2O2 in the proliferation process of human immortalized epidermal (HACAT) cells, which confirmed that H2O2 participated in the cells’ proliferation activity through a growth factor signaling pathway. In the scald and incision wound mice models, H2O2 concentration fluctuations at different pathological stages during the wound healing process could be obtained by in vivo fluorescence imaging. Finally, H2O2 concentrations in different stages of human diabetic foot tissues were also confirmed by the proposed probe. We expect that H2O2 could be a sensitive biomarker to indicate the wound healing process.

KEYWORDS:

near-infrared fluorescence imaging  hydrogen peroxide fluctuation  diabetes mellitus  wound healing  clinical sample test

Introduction

     Chronic wound healing remains a global challenge and one of the major common public health problems that cannot be ignored [1-3]. It is a complex physiological process that consists of hemostasis, inflammation, proliferation and remodeling [4, 5]. Unfortunately, bacterial infection or other diseases (destructive irriation, diabetes mellitus) may interfere with wound healing, leading to impaired structural and functional regeneration of entire skin tissue, which in turn may lead to severe disability and even increased mortality [6, 7]. Therefore, monitoring changes in physiological parameters during wound healing is crucial to understand the state and physiological process of wound healing, which can help identify wound infections and subsequent treatment. The current clinical evaluation of wounds healing mainly focuses on planimetry to quantitatively detect the changes of wound size and granulation tissue formation. Although this method can reflect the macroscopic changes in the wound, it cannot show the regulation at the molecular level.

Excessive infiltration of neutrophils appears to be one of the culprits of chronic wound healing [8]. Abundant ROS in neutrophils is involved in all phases of wound healing, which can cause oxidative stress and adversely affect wound healing even prevent new tissue formation [9-12]. As a critical member of ROS, hydrogen peroxide (H₂O₂) has been considered as an intracellular second messenger, which plays key roles in various physiological processes including cell growth, inflammation treatment, proliferation and differentiation, and activation of immune cells [13-15]. The previous researches indicate that H₂O₂ involves in wound healing and its concentration is fluctuated in different pathological stages during the wound healing process [16-20]. Therefore, H₂O₂ may be considered as a powerful biomarker to indicate the chronic wound healing process.

Although visual observation is the simplest and most versatile means of initial diagnosis of chronic wounds, it relies on the certain clinical experience and cannot predict the effect of treatment and the stage of healing [21]. Therefore, there is an urgent need to develop rapid and sensitive tools to detect H₂O₂ to accurately identify the various states of chronic wound healing [22]. Recently, fluorescent probes have been considered as powerful tools to monitor biological active species and processes due to their non-invasiveness, high sensitivity, and excellent spatial and temporal resolution [23-29]. Some fluorescent probes have been reported for monitoring H₂O₂ in living cells and in vivo through the careful design; however, the probes for monitoring the concentration fluctuation during the chronic wound healing process are still challenging [30-34]. Moreover, compared to the fluorescent emission in the visible light region, fluorescent probes with excitation/emission wavelength in the near-infrared (NIR) region get benefit from the low background interference, small light damage and deep penetration, which are more suitable for bioimaging [35-37]. With this in mind, we attempt to develop a NIR fluorescent probe for rapid and highly sensitive detection of H₂O₂ in living cells and in vivo to track the concentration fluctuations of H₂O₂ during the chronic wound processes.

Herein, we proposed a NIR fluorescent probe DCM-H₂O₂ with large Stokes shift for investigating the fluctuation of H₂O₂ level during chronic wound healing process in wound mice models and human diabetic foot tissues. The probe comprised of pentafluorobenzenesulfonyl ester group as recognition moiety of H₂O₂ and dicyanomethylene-benzopyran (DCM) as NIR fluorophore. In the presence of H₂O₂, DCM-H₂O₂ exhibited rapid response to H₂O₂ with large Stokes shift, emitting a brilliant fluorescent signal at 695 nm. Furthermore, DCM-H₂O₂ displayed low cytotoxicity and excellent biocompatibility. Then, the proposed probe was applied to monitor the fluctuations of H₂O₂ level during the healing process of scald and incision wound mice models and in various pathological stages of the human diabetic foot, which contributed to comprehend the role of H₂O₂ in the physiological process. The probe could act as an effective tool to help diagnose the various stages of chronic wound healing that assisted with further treatment.

Scheme 1. The molecular structure of DCM-H₂O₂ and its proposed response mechanism towards H₂O₂.

Figure 1. The spectral properties and selectivity of DCM-H₂O₂. (A) The UV-Vis absorption spectra of DCM-H₂O₂ toward H₂O₂ (0 – 50 µM) for 5 min in PBS (pH =7.4, 10 mM, PBS: DMSO=7:3, v/v). (B) The fluorescence emission spectra of DCM-H₂O₂ toward H₂O₂ (0 – 50 µM) for 5 min in PBS (pH=7.4, 10 mM, PBS: DMSO=7:3, v/v). (C) The linear relationship between the fluorescence intensity of DCM-H₂O₂ and various levels of H₂O₂. (D) Time-dependent fluorescent intensity toward H₂O₂ during 0 – 360 s, and the probe was added at 30 s. (E) The fluorescence spectra of DCM-H₂O₂ toward other analytes. (F) The fluorescent response of DCM-H₂O₂ to various reactive species at 10 min: 1. blank; 2. H₂O₂; 3. ONOO^–; 4. HNO; 5. NO; 6. ·OH; 7. ^tBuOO·; 8. O₂^·-; 9. OCl^–; 10. Cys; 11. GSH; 12. Hcy. The experiments were repeated three times and the data were shown as mean (± S.D.).

Figure 2. Fluorescence imaging of endogenous and exogenous H₂O₂ in living cells. (A) Fluorescent imaging of exogenous H₂O₂ in HeLa cells at different time points: 0 min, 5 min, 10 min, 20 min and 30 min. (B) Fluorescent imaging of exogenous H₂O₂, HClO and ONOO^– in RAW 264.7 cells. The RAW 264.7 cells were treated with H₂O₂ (10 µM, 30 µM), ClO^– and ONOO^– donor (SIN-1), and then incubated with probe (10 µM), respectively. (C) Fluorescent imaging of endogenous H₂O₂ in RAW 264.7 cells. Control group: RAW 264.7 cells was incubated with the probe DCM-H₂O₂; PMA group: The RAW 264.7 cells were treated with PMA (1 μg/mL) for 6 h and then incubated with the probe DCM-H₂O₂; PMA + NAC group, PMA+ebselen, and PMA+L-NAME group: the cells were preincubated with NAC (1 mM), ebselen (5 μM), and L-NAME (5 mM) and then tread with probe, respectively. (D-F) Relative mean fluorescent intensities in A-C. Fluorescence collection windows for red channel: λ_ex = 561 nm, λ_em = 650 – 730 nm and blue channel (DAPI): λ_ex = 405 nm, λ_em = 420 – 480 nm. The experiments were repeated five times and the data were shown as mean (± S.D.).

Figure 3. Fluorescence images and flow cytometry analysis of H₂O₂ in HACAT cells via DCM-H₂O₂. (A) The cells were treated with the probe DCM-H₂O₂ (10 μM). (B) The cells were incubated with EGF (500 ng/mL) for 30 min and then treated with probe. (C) The cells loaded with DCM-H₂O₂ were pretreated with PD153035 and then stimulated with EGF. (D) The cells loaded with DCM-H₂O₂ were pretreated with wortmannin and then stimulated with EGF. (E) The cells loaded with DCM-H₂O₂ were pretreated with apocynin before stimulated with EGF. (F) The flow cytometry analysis of the cells in A-E. (G) Relative mean fluorescent intensities in A-E. (H) Relative mean fluorescent intensities in F. The fluorescence collection windows for red channel (DCM-H₂O₂): λ_ex = 561 nm, λ_em = 650 – 730 nm and blue channel (DAPI): λ_ex = 405 nm, λ_em = 420 – 480 nm. The experiments were repeated five times and the data were shown as mean (± S.D.).

Figure 4. Time-dependent fluorescence images of H₂O₂ in wound healing models. (A) Fluorescence images of H₂O₂ with DCM-H₂O₂ in healing process of scald wound mouse model at different time points (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 2 d, 3 d, 5 d, 7 d and 11 d). (B) Fluorescence images of H₂O₂ with DCM-H₂O₂ in healing process of incision wound mouse model at different time points (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 2 d, 3 d, 5 d, 7 d and 11 d). (C) Relative mean fluorescent intensities in A. (D) Relative mean fluorescent intensities in B. The fluorescence collection windows for red channel: λ_ex = 561 nm, λ_em = 650 – 730 nm. The experiments were repeated five times and the data were shown as mean (± S.D.). (E) The fluorescence detection of vitro vital organs (from left to right: heart, liver, spleen, lung, and kidney). (F) H&E and Masson staining for normal mice skin, period of inflammation of scald mice skin (8 h) and incision mice skin (48 h).

Figure 5. Fluorescent imaging in the tissues of the different physiological stages of the human diabetic foot. (A) Photographs of clinical samples. (B) H&E staining. (C) Fluorescence imaging of lesion skin tissues at lesion stages. The fluorescence collection windows for red channel: λ_ex = 561 nm, λ_em = 650 – 730 nm. (D) Three-dimensional (3D) images of (B). (E) Mapping of Z-line sequential images for dysplasia slice at a depth interval of 10 μm (diabetic foot stage V).

4. Conclusion

          In summary, we reasonably designed and synthesized a NIR probe (DCM- H₂O₂) to track the concentration fluctuations of H₂O₂ during the scald and incision wound healing process and the human diabetic foot tissues. The probe exhibited high sensitivity and specificity, and can respond quickly to H₂O₂. DCM-H₂O₂ was employed to detect exogenous and endogenous H₂O₂ and investigated the fluctuation of H₂O₂ during the proliferation of HACAT cells. Furthermore, the real-time imaging of H₂O₂ in the process of scald and incision wound mice models strongly demonstrated that the proposed probe could track the concentration fluctuations of H₂O₂ during the wound healing process. This study is helpful to better understand the role of H₂O₂ in wound healing process. Furthermore, the probe was employed to image H₂O₂ in clinical diabetic foot samples, providing a new strategy for differentiating the pathological stages of the diabetic foot.

A Fluorescent Probe with Zwitterionic ESIPT Feature for Ratiometric Monitoring of Peroxynitrite In Vitro and In Vivo

Zhenkai Wang【王振凯】 ^a,b,c,†, Miao Yan,^a,† Miaomiao Yu^a, Gang Zhang^a, Weiwei Fang【房微魏】^*,a, Fabiao Yu【于法标】^*,b,c,

• ^a Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, International Innovation Center for Forest Chemicals and Materials, College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, 210037, Nanjing, China.
• ^b Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Haikou Trauma, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China.
• ^c Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China
• †These authors contributed equally to this work

SUBJECTS:

Fluorescence,Fluorescence imaging,Mixtures,Probes, Rodent models

Abstract

Peroxynitrite (ONOO–), as a short-term reactive biological oxidant, could lead to a series of effects in various physiological and pathological processes due to its subtle concentration changes. In vivo monitoring of ONOO– and relevant physiological processes is urgently required. Herein, we describe a novel fluorescent probe termed HBT-Fl-BnB for the ratiometric detection of ONOO– in vitro and in vivo. The probe consists of an HBT core with Fl groups at the ortho and para positions responding to the zwitterionic excited-state intramolecular proton-transfer (zwitterionic ESIPT) process and a boronic acid pinacol ester with dual roles that block the zwitterionic ESIPT and recognize ONOO–. Thanks to the specificity as well as low cytotoxicity, success in imaging of endogenous and exogenous ONOO– in living cells by HBT-Fl-BnB was obtained. Additionally, the applicability of HBT-Fl-BnB to tracking the abnormal expression of ONOO– in vivo induced by inactivated Escherichia coli was also explored. This is the first report of a fluorescent probe for ONOO– sensing via a zwitterionic ESIPT mechanism.

摘要

过氧亚硝酸盐（ONOO−）作为一种短期反应性生物氧化剂，由于其浓度的微妙变化，可对多种生理和病理过程产生一系列影响。迫切需要对 ONOO− 和相关生理过程进行体内监测。

在此，作者描述了一种称为 HBT-Fl-BnB 的新型荧光探针，用于体外和体内 ONOO− 的比例检测。该探针由一个在邻位和对位具有 Fl 基团的 HBT 核心组成，该核心响应两性离子激发态分子内质子转移（两性离子 ESIPT）过程，以及具有双重作用的硼酸频哪醇酯，可阻断两性离子 ESIPT 并识别 ONOO− 。由于其特异性和低细胞毒性，HBT-Fl-BnB 成功对活细胞中的内源性和外源性 ONOO− 进行成像。此外，还探讨了 HBT-Fl-BnB 在追踪灭活大肠杆菌诱导的体内 ONOO− 异常表达方面的适用性。这是通过两性离子 ESIPT 机制进行 ONOO− 传感的荧光探针的第一份报告。

图文解析

方案1.设计HBT-Fl-BnB及其相关合成路线。

图 1. (a) 含有过量 ONOO− 的 HBT-Fl-BnB、HBT-Fl 和 HBT-Fl-BnB 的荧光光谱（10 μM 和 10 mM PBS 缓冲液，含有 5% THF、0.2% Tween，pH = 7.4 ，λex = 340 nm）和（b）HBT-Fl（10 μM）在 584 nm 处的荧光强度与 PBS 缓冲液-THF 混合物中的水分数（%，fw）的关系图，λex = 360 nm。 (c, d) HBT-Fl-BnB Me导数的分子前沿轨道和能级的DFT计算。

Figure 1. (a) Fluorescence spectra of HBT-Fl-BnB, HBT-Fl and HBT-Fl-BnB with excess ONOO⁻ (10 μM, and 10 mM PBS buffer with 5% THF, 0.2% Tween, pH = 7.4, λ_ex = 340 nm), and (b) the plot of fluorescent intensity of HBT-Fl (10 μM) at 584 nm vs. water fractions (%, f_w) in the PBS buffer-THF mixture, λ_ex = 360 nm; and (c,d) DFT calculations of the molecular frontier orbitals and energy levels of the Me derivative of HBT-Fl-BnB.

图 2. (a) 添加 ONOO− (0−100 μM) 后 HBT-Fl-BnB 的荧光光谱。插图：FI583/FI396 与 ONOO− (0.0−100 μM) 的比率。 (b) FI583/FI396 比率与 ONOO− 浓度 (0.0−25 μM) 的线性关系。 (c) 添加 ONOO- 或其他干扰分析物以及 (d) 在竞争性分析物存在下添加 ONOO- 后 HBT-F1-BnB 的荧光强度比 (FI583/FI396)。测试条件：10 μM HBT-Fl-BnB、10 mM PBS 缓冲液（含 5% THF、0.2% Tween）、pH = 7.4 和 λex = 340 nm。其他干扰分析物：ONOO− , 1 O2, ·OH, HOCl, H2O2, NO, TBHP, GSH, L-Cys, Sec, Cys-SSH, Fe3+ , Zn2+ , F− , I − , SO4 2− , NO2 , HCO3 、CO3 2− 和 S2O3 2− 。

Figure 2. (a) Fluorescence spectra of HBT-Fl-BnB upon the addition of ONOO⁻ (0‒100 μM), inset: the ratio of FI₅₈₃/FI₃₉₆ vs. ONOO⁻ (0.0-100 μM); (b) Linearity for the ratio of FI₅₈₃/FI₃₉₆ vs. ONOO⁻ concentrations (0.0-25 μM); Fluorescent intensity ratio (FI₅₈₃/FI₃₉₆) of HBT-Fl-BnB upon (c) adding ONOO⁻ or other interfering analytes respectively, and (d) adding ONOO⁻ in the presence of competitive analytes. Test conditions: 10 μM HBT-Fl-BnB, and 10 mM PBS buffer with 5% THF, 0.2% Tween, pH = 7.4, λ_ex = 340 nm). Other interfering analytes: ONOO^‒, ¹O₂, ·OH, HOCl, H₂O₂, NO, TBHP, GSH, L-Cys, Sec, Cys-SSH, Fe³⁺, Zn²⁺, F^‒, I^‒, SO₄^2‒, NO₂^‒, HCO₃^‒, CO₃^2‒ and S₂O₃^2‒.

方案2. HBT-Fl-BnB检测ONOO的拟议反应机制。

Scheme 2. Proposed reaction mechanism of HBT-Fl-BnB towards detecting ONOO⁻.

图 3. Raw264.7 细胞的共焦荧光成像。

Figure 3. Confocal fluorescence imaging of Raw264.7 cells. (a) Control group: cells were only incubated with HBT-Fl-BnB. Cells were stimulated with exogenous ROS/RNS: (b) H₂O₂; (c) HOCl; (d) NO; (e) O₂·⁻; (f) ONOO⁻ (10 μM in PBS buffer), then with HBT-Fl-BnB. 430ch: λ_em= 415-515 nm, 583ch: λ_em=550-650 nm. Ratio images generated from 583ch/430ch. λ_ex=405 nm, scale bar: 20 μm. (g) Normalized average fluorescence intensity of Fl₅₈₃/Fl₄₃₀. *** P<0.001. The experiments were repeated three times and the data were shown as mean (±S.D.).

图 4. Raw264.7 细胞的共焦荧光成像。

Figure 4. Confocal fluorescence imaging of Raw264.7 cells. Cells were stimulated with different concentrations of ONOO⁻, (a) 0; (b) 2); (c) 4); (d) 6; (e) 8 and (f) 10 μM, then with HBT-Fl-BnB. 430ch: λ_em= 415-515 nm, 583ch: λ_em=550-650 nm. Ratio images generated from 583ch/430ch. λ_ex=405 nm, scale bar: 20 μm. (g) Normalized average fluorescence intensity of Fl₅₈₃/Fl₄₃₀. * P<0.05, ** P<0.01 *** P<0.001. The experiments were repeated three times and the data were shown as mean (±S.D.).

图 5. Raw264.7 细胞的共焦荧光成像。

Figure 5. Confocal fluorescence imaging of Raw264.7 cells. (a) Control group: cells were only incubated with HBT-Fl-BnB. Cells were stimulated with different stimuli: (b) LPS (1 μg/mL); (c) IFN-γ (50 ng/mL); (d) PMA (10 nmol/L); (e) LPS (1 μg/mL) + IFN-γ (50 ng/mL); (f) LPS (1 μg/mL) + IFN-γ (50 ng/mL), then with PMA (10 nmol/L); and (g) AG (1 mmol/L) + LPS ( 1 μg/mL) + IFN-γ (50 ng/mL), then with PMA (10 nmol/L); then were further incubated with HBT-Fl-BnB. 430ch: λ_em= 415-515 nm, 583ch: λ_em=550-650 nm. Ratio images generated from 583ch/430ch. λ_ex=405 nm, scale bar: 20 μm. (h) Normalized average fluorescence intensity of Fl₅₈₃/Fl₄₃₀. ** P<0.01 *** P<0.001. The experiments were repeated three times and the data were shown as mean (±S.D.).

图 6. 在大肠杆菌引起急性腹膜炎期间用小鼠荧光成像监测 ONOO−。

Figure 6. Fluorescence imaging of mice to monitor ONOO⁻ during the Escherichia coli caused acute peritonitis. Mice were intraperitoneally injected with inactivated Escherichia coli (2 × 10⁷ CFU), and after (a) 0, (b) 6, (c)12, (d) 24, (e) 36, (f) 72 h, HBT-Fl-BnB (50 μM, 200 μL) was injected through the tail vein. (g) Normalized average fluorescence intensity of Fl₅₈₃/Fl₄₃₀. 430ch: λ_em= 415-515 nm, 583ch: λ_em=550-650 nm. λ_ex=405 nm. (h) Changes of WBC during acute peritonitis in mice. * P<0.05, ** P<0.01 *** P<0.001. The experiments were repeated three times and the data were shown as mean (±S.D.).

Conclusion

        In summary, a novel ratiometrically fluorescent probe HBT-Fl-BnB featuring a zwitterionic ESIPT was firstly synthesized to detect ONOO⁻ in vitro and in vivo. An HBT core with two Fl groups at the ortho and para positions of OH responding to the zwitterionic ESIPT, and a boronic acid pinacol ester possessing dual roles that blocking the zwitterionic ESIPT as well as sensing ONOO⁻ are both presented in HBT-Fl-BnB. This designed probe enabled a quantitative and ratiometric detection of ONOO⁻ with high selectivity and sensitivity. Considering the low cytotoxicity and high selectivity of HBT-Fl-BnB towards ONOO⁻, the success in monitoring and imaging of endogenous and exogenous ONOO⁻ in living cells and mice was obtained. Importantly, the abnormal expression of ONOO⁻ in vivo induced by inactivated Escherichia coli was also confirmed by HBT-Fl-BnB, which provided a potential strategy to understand relevant pathogenic mechanism. We envision that this strategy will inspire future probe design with infrared emission.

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