Novel CeF3:Tm3+, Er3+ nanoparticles: NIR up-down conversion luminescence properties based on energy transfer of Tm3+ and Ce3+

Novel CeF3:Tm3+, Er3+ nanoparticles: NIR up-down conversion luminescence properties based on energy transfer of Tm3+ and Ce3+

https://doi.org/10.1016/j.ceramint.2024.05.124

Abstract

A novel CeF3:Tm3+, Er3+ NIR up-down conversion luminescent nanoparticles with highly efficient visible and near-infrared second window (NIR-II) emission were synthesized by direct precipitation and hydrothermal methods. The two different emission modes of visible-NIR II light of Er3+were realized through the energy transfer of Tm3+ and Ce3+at the excitation wavelength of 808 nm. Which successfully combined the narrow emission peaks of visible light and the deep tissue penetration advantage of NIR-II. First, the effects of preparation conditions on the structure, morphology, stability and luminescence properties of CeF3:Tm3+, Er3+ nanoparticles were analyzed by XRD, SEM, TEM and PL. The results showed that the crystallinity, morphology and luminescence intensity of CeF3:Tm3+,Er3+ nanoparticles produced by hydrothermal method (reaction time of 10 h) were optimal compared with that of direct precipitation method. CeF3: Tm3+,Er3+ nanoparticles has better thermal stability, and fluorescence lifetime of 5D0→7F2 (525 nm), 4I15/2 → 4I11/2 (1066 nm) and 4I15/2 → 4I13/2 (1386 nm) for Er3+ are 15.51 ns, 48.78 us and 57.06 us, separately. At the same time, the surface of CeF3:Tm3+, Er3+ nanoparticles was modified by PEG and Lys. And then the effects of the surface modification on the hydration size, zeta potential and up-down conversion luminescent properties (visible and NIR-II light emission) were investigated. The results show that the modification of PEG effectively slows down the agglomeration of CeF3:Tm3+, Er3+ nanoparticles and improves their stability and dispersion under the same conditions. More importantly, the modification of PEG effectively increases the up-conversion luminescence intensity of CeF3:Tm3+, Er3+ nanoparticles under 808 nm excitation. Moreover, the CeF3:Tm3+, Er3+-PEG nanoparticles with 20–40 nm showed stronger NIR up-down conversion emission intensity in aqueous solutions (pH = 5,6,7) and better thermal stability. Furthermore, the CeF3:Tm3+, Er3+-PEG nanoparticles has low cytotoxicity and good biocompatibility. This study provides a theoretical basis for the development of high-performance rare-earth NIR up-down conversion luminescence materials.

Introduction

NIR-II fluorescence imaging has longer excitation wavelengths and emission wavelengths (1000 nm–1700 nm), which can significantly reduce the light scattering rate and improve the depth of penetration in organisms [1,2]. Moreover, NIR-II fluorescence also has the advantages of low auto-fluorescence effect and high resolution [3,4]. However, only very few NIR-II fluorescence imaging materials can meet the current requirements of in vivo imaging, such as single-walled carbon nanotubes and carbon dots (low fluorescence quantum yield) [5], organic dyes (poor photo-stability) [6] and quantum dots (high toxicity) [7] etc. All of these materials are unable to be clinically applied due to their own defects. However, rare-earth NIR-II imaging materials have long fluorescence lifetime, low auto-fluorescence, high imaging signal-to-noise ratio, deep tissue penetration ability (5–20 mm), and low toxicity, which have a broad application prospect in the field of cancer diagnosis and treatment [8]. So far, the main activator rare-earth ions emitted in NIR II are Nd3+ (1060/1300 nm), Tm3+ (1470 nm), Pr3+ (1310 nm), Ho3+ (1185 nm) and Er3+ (1525 nm) [9]. For example, literature [[10], [11], [12], [13], [14]] reported that the MLnF4 (M = Na, Li; Ln = Gd, Dy, Er, Ce, Nd) luminescent nanoparticles with co-doped or mono-doped Nd3+, Tm3+, Pr3+, Ho3+ and Er3+ ions could be used as a NIR-II fluorescent probe under the excitation at 808 or 980 nm. LaF3 [15], MF2 (M = Sr, Ca) [16], GdPO4 [17], and CaS [18] luminescent nanoparticles with co-doped or mono-doped Nd3+, Tm3+, Pr3+, Ho3+, and Er3+ ions can also be used for NIR-II fluorescence imaging under 808 or 980 nm excitation in vivo, which has potential applications in tumor diagnosis and treatment. In addition, the rare-earth up-conversion luminescence requires only a low power density NIR laser (808 or 980 nm), which is low cost and more universality compared to two-photon laser excitation with high power density [19]. Among them, 808 nm is not easily absorbed by water compared to 980 nm, its absorption coefficient is only 0.023 cm−1, [19]. According to the photon transmission theory, when the light at 808 nm is irradiated on the biological tissue, the absorption of the tissue surface is very small, and the light can penetrate the deeper biological tissue. And the volume effect of light at 808 nm is large, making the biological tissue easier to solidify (the stronger the hemostatic function) [19]. Therefore, it is more suitable for the imaging field of vascular-rich deep biological tissue. More importantly, the NIR-excited rare earth nanoparticles have the tunable up- and down-conversion luminescence property, which can emit the broad-wave multiple emissions from ultraviolet/visible (UV/Vis) to NIR-II, making them ideal fluorescent probes [20]. However, the currently reported up-and down-convertion emission materials based on a single activator inhibit up-conversion luminescence efficiency and NIR-II luminescence efficiency.

Based on the above research status, a novel CeF3:Tm3+, Er3+ NIR up-down conversion luminescent materials with strong visible and NIR-II luminescence have been synthetized by direct precipitation and hydrothermal methods (Fig. 1). Firstly, the effects of different preparation methods and reaction conditions on the structure, morphology and luminescence properties of CeF3:Tm3+, Er3+ NIR up-down conversion luminescent nanoparticles were investigated. Then, the surface of CeF3:Tm3+, Er3+ nanoparticles modified by polyethylene glycol (PEG) and lysine (Lys). The effects of surface modifiers on the hydration particle size, zeta potential, luminescence properties were investigated in aqueous solutions with different pH values. Finally, the thermal stability and biocompatibility properties of CeF3:Tm3+, Er3+ NIR up-down conversion luminescent nanoparticles were investigated.

Compared with the reported research work [[30], [31], [32], [33], [34], [35]], the highlights of this research work are as follows: (1) Novel CeF3:Tm3+, Er3+ up-down conversion luminescent nanoparticles with small size and high stability were synthesized by direct precipitation and hydrothermal methods. Novel CeF3:Tm3+, Er3+ up-down conversion luminescent nanoparticles successfully combined advantage of the narrow emission peak of visible fluorescence with the deep tissue penetration advantage of NIR-II. (2) The influence of the preparation method and experiment condition of novel CeF3:Tm3+, Er3+up-down conversion luminescent nanoparticles on the luminescence performance was systematically studied, and the optimal preparation conditions were determined. (3) The influence and mechanism of surface modification on the dispersion stability and luminescence properties in different aqueous solutions (pH = 5–7) of novel CeF3:Tm3+, Er3+ up-down conversion luminescent nanoparticles were studied. (4) It reveals the influence mechanism of the surface modification and temperature change of novel CeF3:Tm3+, Er3+ up-down conversion luminescent nanoparticles on up-down conversion luminescent property.

The preparation and investigations on NIR up-down conversion luminescence properties of Novel CeF3:Tm3+, Er3+ nanoparticles with small size, high stability, biocompatibility and strong luminescence (visible and NIR-II emission) is expected to provide a practical innovative idea for the development of high-performance rare-earth NIR up-down conversion luminescence materials, therefore, it has important practical significance and research value.

Fig. 1. Schematic diagram of synthesis and luminescent property of novel CeF3:Tm3+, Er3+ NIR up-down conversion nanoparticles: (a) Negative heat quenching

mechanism, (b) Defects capture and release electrons in the crystals, (c) Multi-assisted non-radiative relaxation.

Fig. 2. XRD patterns of CeF3:Tm3+, Er3+ nanoparticles produced by hydrothermal method (a) and direct precipitation method (b).

Fig. 3. SEM images of CeF3:Tm3+,Er3+ nanoparticles obtained by hydrothermal method. (a-2 h) (b-4 h) (c-6 h) (d-8 h) (e􀀀 10 h).

Fig. 4. SEM images of CeF3:Tm3+, Er3+ nanoparticles obtained by direct precipitation method (a-S1) (b-S2) (c-S3) (d-S4) (e-S5).

Fig. 5. Up-down conversion emission spectra of CeF3:Tm3+, Er3+ nanoparticles produced by hydrothermal method.

Fig. 6. Up-down conversion emission spectra of CeF3:Tm3+, Er3+ nanoparticles obtained by direct precipitation method.

Fig. 7. (a~d) TEM images of CeF3:Tm3+, Er3+ nanoparticles (hydrothermal method, 10 h).

Fig. 8. (a,b) TEM and (c ~ g) Energy Dispersive X-ray (EDX) mapping of CeF3:Tm3+, Er3+ nanoparticles.

Fig. 9. TG plot of the CeF3:Tm3+, Er3+ nanoparticles.

Fig. 10. The temperature stability spectra of CeF3:Tm3+, Er3+ nanoparticles.

Fig. 11. Fluorescent decay curves of 5D0-7F2 (525 nm), 4I15/2 → 4I11/2 (1066 nm) and 4I15/2 → 4I13/2 (1386 nm) for Er3+.

Fig. 12. The infrared spectra of CeF3:Tm3+, Er3+, CeF3:Tm3+, Er3+-Lys and CeF3:Tm3+, Er3+-PEG nanoparticles.

Table

 

Fig. 13. Up-down conversion fluorescence spectra of CeF3:Tm3+, Er3+, CeF3: Tm3+, Er3+-PEG and CeF3:Tm3+, Er3+-Lys nanoparticles.

 

Fig. 14. Up- and down-conversion emission spectra of CeF3:Tm3+, Er3+-PEG nanoparticles in aqueous solutions (pH = 5, 6, 7).

 

Fig. 15. Cell viability of HeLa cells treated with different concentrations of CeF3:Tm3+,Er3+-PEG nanoparticles at 24 h (0 mg/ml is Control group, *P < 0.05, **P < 0.01).

Conclusion

In this paper, novel CeF3: Tm3+,Er3+ NIR up- and down-conversion luminescent nanoparticles was synthesized by direct precipitation and hydrothermal methods. Through Tm3+- Er3+-Ce3+ energy transfer process, to realize visible light and NIR-II emission modes of Er3+ at the excitation wavelength of 808 nm. And novel CeF3: Tm3+,Er3+ nanoparticles has better thermal stability, biocompatibility and fluorescence properties. Meanwhile, the effects of PEG, Lys and pH on  the hydrated particle size, stability and luminescence intensity of CeF3: Tm3+,Er3+ luminescent nanoparticles were investigated. The results show that the CeF3: Tm3+, Er3+-PEG nanoparticles still has good luminescence performance in aqueous solution, and it can be used as a good fluorescent probe because it still maintains a small hydrated particle size and good dispersion under acidic solution conditions. This work brings a new idea to combine the narrow emission peak of visible fluorescence with the deep tissue penetration advantage of NIR-II.

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Rationally designed an innovative proximity labeling near-infrared fluorogenic probe for imaging of peroxynitrite in acute lung injury

Rationally designed an innovative proximity labeling near-infrared fluorogenic probe for imaging of peroxynitrite in acute lung injury

https://doi.org/10.1016/j.cclet.2024.110082

Chinese Chemical Letters  Available online 3 June 2024, 110082

ABSTRACT

Acute lung injury (ALI) is a serious clinical condition with a high mortality rate. Oxidative stress and inflammatory responses play pivotal roles in the pathogenesis of ALI. ONOO− is a key mediator that exacerbates oxidative damage and microvascular permeability in ALI. Accurate detection of ONOO− would facilitate early diagnosis and intervention in ALI. Near-infrared fluorescence (NIRF) probes offer new solutions due to their sensitivity, depth of tissue penetration, and imaging capabilities. However, the developed ONOO− fluorescent probes face problems such as interference from other reactive oxygen species and easy intracellular diffusion. To address these issues, we introduced an innovative self-immobilizing NIRF probe, DCI2F-OTf, which was capable of monitoring ONOO− in vitro and in vivo. Importantly, leveraging the high reactivity of the methylene quinone (QM) intermediate, DCI2F-OTf was able to covalently label proteins in the presence of ONOO−, enabling in situ imaging. In mice models of ALI, DCI2F-OTf enabled real-time imaging of ONOO− levels and found that ONOO− was tightly correlated with the progression of ALI. Our findings demonstrated that DCI2F-OTf was a promising chemical tool for the detection of ONOO−, which could help to gain insight into the pathogenesis of ALI and monitor treatment efficacy.

Graphical Abstract

An innovative self-immobilizing NIRF probe DCI2F-OTf was capable of monitoring ONOO− in vitro and in vivo. DCI2F-OTf was able to covalently label proteins in the presence of ONOO−, enabling in situ imaging. In mice models of ALI, DCI2F-OTf enabled real-time imaging of ONOO− levels and found that ONOO− was tightly correlated with the progression of ALI.

ONOO–作为信号分子参与细胞信号转导和各种生理过程，包括基因表达调控和细胞凋亡。一方面，升高的ONOO–会破坏肺部抗氧化防御系统，加剧氧化应激和细胞损伤；另一方面，增加的ONOO–水平会显著增强肺微血管壁的通透性，最终损害肺功能。因此，开发用于检测ONOO–的新型工具以准确诊断ALI将有助于及时跟踪疾病进程，进行早期干预，提高患者生存率。现有的ONOO–荧光探针面临两个主要挑战：（1）易受高浓度活性氧（如过氧化氢）的干扰；（2）容易从反应位点扩散，降低成像精度和信噪比。因此，设计ONOO-激活的、邻近蛋白捕获的近红外荧光探针在细胞或活体水平上具有重要意义。

急性肺损伤（ALI）是一种严重的临床病症，具有较高的死亡率。氧化应激和炎症反应在ALI的发病机制中起着关键作用。过氧亚硝酸盐（ONOO–）是加剧ALI中氧化损伤和微血管通透性的关键介质。准确检测ONOO–有助于ALI的早期诊断和干预。目前开发的ONOO–荧光探针面临着其他活性氧物质干扰和易于细胞内扩散的问题。为了解决这些问题，我们设计了一种新型邻近标记近红外荧光探针DCI2F-OTf，该探针能够在体内外监测ONOO–。重要的是，利用亚甲基醌中间体的高反应性，DCI2F-OTf能够在ONOO–存在下共价标记蛋白质，从而实现原位成像。在ALI小鼠模型中，DCI2F-OTf实现了ONOO–的实时成像，发现ONOO–与ALI的进展密切相关。研究结果表明，DCI2F-OTf是一种有前途的化学工具，将有助于理解ALI的发病机制和监测治疗效果。

Scheme 1. (a) Schematic illustration of dual sensing and labeling NIRF probe DCI2F-OTf for ONOO–. (b) Synthesis of NIRF probe DCI2F-OTf. 

Figure 1. Fluorescence response of DCI2F-OTf (10 μM) to ONOO– excited at 490 nm. (a) Visualization of the fluorescence changes of DCI2F-OTf during titration with increasing ONOO– (0 – 35 μM) concentrations. (b) Linearity of the fluorescence intensity value of DCI2F-OTf at 652 nm with varied concentrations of ONOO– (5 – 35 μM). (c) Time-dependent fluorescence response of DCI2F-OTf before and after the addition of ONOO– (70 μM) at 37 °C recorded every 5 min. (d) The fluorescence intensities at 652 nm of DCI2F-OTf in the absence or presence of ONOO– (70 μM) over pH range from 3.0 to 7.0. (e) Values of fluorescence intensities of DCI2F-OTf treated with different competitive biological agents: 1. blank, 2. Na+ (500 μM), 3. K+ (500 μM), 4. Ser (200 μM), 5. Leu (200 μM), 6. Arg (200 μM), 7. S2O32- (100 μM), 8. HSO3– (100 μM), 9. H2S (100 μM), 10. Cys (100 μM), 11. GSH (1 mM), 12. NO2– (100 μM), 13. NO3– (100 μM), 14. NO (100 μM), 15. •OH (100 μM), 16. O21 (100 μM), 17. H2O2 (100 μM), 18. O2•– (100 μM), 19. HOCl (100 μM), 20. ONOO– (70 μM). (f) SDS-PAGE analysis of BSA (5 mg/mL) after incubation with DCI2F-OTf + ONOO–, DCI2F-OTf, DCI2F-OTf + ONOO– + UA in PBS at 37 ℃ for 30 min. Left: Coomassie brilliant blue staining; right: fluorescence imaging. λex/λem = 490/650 nm.

 

                                               

Figure 2. (a) RAW264.7 cells were pretreated with SIN-1 (200 μM, 30 min) or stimulated with LPS (1.0 μg/mL), IFN-γ (100 ng/mL) for 12 h, PMA (10 nM) for 30 min, and then incubated with DCI-OTf (10 μM, 30 min) or DCI2F-OTf (10 μM, 30 min), respectively. The red channel was collected in the range of 600-700 nm (λex = 488 nm) and the Hoechst channel was collected in the range of 430-480 nm (λex = 405 nm). (b) A549 cells were pretreated with SIN-1 (200 μM, 30 min), followed by incubation with DCI2F-OTf (10 μM, 30 min) or DCM-KA (10 μM, 30 min), before or after washout with fresh DMEM (three times, each for 6 min). (c) Normalized fluorescence intensity plots of DCI2F-OTf and DCM-KA labeled cells in (b) (n = 3). Scale bar: 50 μm.

 

                                               

Figure 3. (a) RAW264.7 cells pretreated with different concentrations of SIN-1 (0, 50, 100, 200 μM), SIN-1 (200 μM) plus ebselen (200 μM) for 30 min were incubated with DCI2F-OTf (10 μM) for 30 min. (b) The pixel intensity of DCI2F-OTf labeled cells in (a). (c) The protein levels of iNOS and IL-6 were detected by western blot analysis. (d) The relative protein expression level of iNOS and IL-6 in (c). (e) RAW264.7 cells were pretreated with SIN-1 (200 μM, 30 min), and then incubated with DCI2F-OTf (10 μM) for 0 – 30 min. (f) Flow cytometry analysis of DCI2F-OTf (10 μM) labeled SIN-1 (200 μM, 30 min) pretreated cells. Error bars represent s.d., n=3. Scale bar: 50 μm.

Figure 4. (a) RAW264.7 cells were pretreated with the stimulus LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) for different periods (0, 3, 6, 12 h), PMA (10 nM) for 30 min or LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) plus UA (200 μM) for 12 h,  PMA (10 nM) for 30 min, and then incubated with DCI2F-OTf (10 μM) for 30 min. (b) RAW264.7 cells were pretreated with LPS (0, 0.25, 0.5, 1.0 μg/mL)/IFN-γ (100 ng/mL), PMA (10 nM), or LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) plus AG (1 mM) for 12 h,  PMA (10 nM) for 30 min, and then incubated with -OTf (10 μM) for 30 min. (c) The pixel intensity of DCI2F-OTf labeled cells in (a). (d) The protein levels of iNOS, IL-6, and NF-𝜅B were detected by western blot analysis. (e) The relative protein expression level of iNOS, IL-6, and NF-𝜅B in (c). (f) The pixel intensity of DCI2F-OTf labeled cells in (b). Error bars represent s.d., n=3. Scale bar: 50 μm.

 

 

Figure 5. (a) NIRF images of control and LPS-stimulated mice after intravenous administration of DCI2F-OTf (200 μM, 45 μL). (b) NIRF imaging and photographs of dissected major organs (1: heart, 2: kidney, 3: liver, 4: spleen, 5: lung) from mice in panel (a). (c) The histogram presents the average radiant efficiency of mice in panel (a). (d) The histogram presents the average radiant efficiency of the lung in panel (b). (e) H&E staining histological images for lung tissue in control and LPS-stimulated mice. Scale bar: 50 μm. (f-h) Serum levels of inflammatory factors TNF-α, IL-6, and IL-1β in control and LPS-stimulated mice. (i) The protein levels of iNOS, COX-2, NF-κB, and IL-6 were detected by western blot analysis. (j-n) The relative protein expression level of iNOS, COX-2, NF-κB, and IL-6 in (i). (o-p) Sections of lung tissue were immunostained with an antibody that detected 3-NT. Scale bar: 50 μm. (q) NIRF images of mice fresh lung tissue sections from ALI mice in the LPS6 group. The confocal Z-axis scan images at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 µm penetration depths. λex = 488 nm; red channel: λem = 600 – 700 nm.

 

Conclusion

In conclusion, we have designed a novel NIRF probe, DCI2F-OTf, for fluorescence imaging ONOO– in live cells and mice model of ALI. Under physiological conditions, DCI2F-OTf was highly sensitive and selective for ONOO– and yielded a linear response to ONOO over a wide concentration range from 0 to 70 μM. The QM covalently labeled neighboring proteins released from the reaction of DCI2F-OTf with ONOO– could solve the problem of intracellular diffusion of the probe, enabling precise imaging of intracellular ONOO–. This strategy was superior to the vast majority of ONOO– probes that have been developed so far. The excellent sensing performance and NIRF emission characteristics enable DCI2F-OTf to image ONOO– levels in mice and tissues with ALI. In vivo imaging results indicated that SFN significantly down-regulated the oxidative stress level, thereby attenuating the severity of LPS-induced ALI, and could exert a protective role against ALI. Therefore, DCI2F-OTf was a valuable tool for studying the physiological function of ONOO– and was expected to facilitate the understanding of oxidative stress diseases such as ALI and the rapid screening of corresponding therapeutic drugs.

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Supramolecular assembly boosting the phototherapy performances of BODIPYs

Supramolecular assembly boosting the phototherapy performances of BODIPYs

Introduction

Cancer remains a major global health challenge, causing millions of deaths annually and persistently affecting human lives and wellbeing. Consequently, there is an urgent need to develop more effective anti-cancer therapies. Traditional treatments such as chemotherapy and radiotherapy, while widely applicated, are often limited by substantial systemic toxicity and adverse side effects [1], [2], [3], [4], [5]. In response to these limitations, novel clinical approaches such as photodynamic therapy (PDT) and photothermal therapy (PTT) have gained prominence. These phototherapies offer numerous advantages, including high selectivity, minimal toxic side effects, absence of drug resistance, and potent immune activation, positioning them at the cutting edge of research in chemistry, materials science, and medicine [6], [7], [8], [9], [10], [11]. According to the Jablonski diagram in Fig. 1, photosensitizers (PSs), light, and tissue oxygen are the three key components of PDT applications. PDT operates on the principle of electron or energy transfer from triplet PSs to surrounding oxygen molecules, leading to the generation of reactive oxygen species (ROS). These different ROS types, which include a variety of type I radicals like hydroxyl (•OH) and superoxide (O2−•), or singlet oxygen (1O2, type II), inflict cellular damage primarily through photochemical reactions that induce apoptosis in cancer cells [2], [3], [6], [12], [13], [14], [15]. The effectiveness of PDT hinges on the ability of PSs to generate a high yield of ROS while exhibiting low toxicity in the absence of light (dark toxicity), efficiently penetrating deep tissues, and demonstrating effective accumulation within the tumor site. These attributes are essential for targeting tumor tissues and inhibiting tumor growth when illuminated under specific light wavelengths. Thus, advancing the design and functional capabilities of PSs is crucial for enhancing the therapeutic outcomes of PDT [2], [3], [16], [6], [7], [8]. However, the limitation inherent to singular treatments of PDT is highly oxygen-dependent, especially in hypoxic tumors [17], [18]. Therefore, developing novel treatment modalities is crucial.

Similarly, PTT has become another prevalent phototherapy strategy for cancer treatment, capitalizing on the susceptibility of tumor cells to heat-induced apoptosis [9], [10], [19], [20], [21]. Unlike PDT, PTT does not rely on tissue oxygen and can effectively be carried out in hypoxic conditions. During PTT, photothermal agents (PTAs) absorb light at specific wavelengths and convert this energy into heat through non-radiative relaxation processes from the excited states to ground state, leading to tumor cell apoptosis (Fig. 1) [20], [21], [22], [23]. However, PTT tends to require high-intensity laser irradiation during treatments, since the limited transmission ability of low-intensity laser cannot penetrate deep into the organism, resulting in a lack of effective treatments for internal tumors. Thus, the single treatment of PTT is often difficult to achieve the expected effects. It can be mitigated by synergistic therapies with the other treatment modalities such as PDT or chemotherapy, combining the strengths of them [1], [11], [13], [16], [24].

Fig. 1. Schematic illustration of Jablonski diagram for the principles of PDT and PTT. Reproduced with permission [12]. Copyright 2020, the Royal Society of Chemistry.

Among various traditional PSs and PTAs characterized by planar, rigid conjugated structures, such as porphyrins [2], [10], [16], [25], [26], [27], [28], phthalocyanine [8], [29], [30], [31], [32], and perylene bisimide [5], [22], [31], [32], [33], boron dipyrromethene (BODIPY) stands out due to its distinct photochemical and photophysical properties. Since their discovery in 1968, BODIPYs, known for their intense light-absorption capabilities, high fluorescence quantum yields, long triplet excited state lifetimes, ease of functionalization, and excellent photostability, have been extensively explored for applicating in phototherapies [24], [34], [35], [36], [37], [38], [39]. Over the past few decades, the structural functionalization and applications of BODIPYs in both PDT and PTT have been rigorously investigated [3], [24], [40], [41], [42], [43], [44]. However, several inherent challenges hinder the broader application of free BODIPY molecules in PDT and PTT. For instance, their hydrophobicity, stemming from planar and rigid structures, limits their water solubility, which is crucial for biological applications. Additionally, their high fluorescence quantum yields and propensity to aggregate in water diminish their ability to generate ROS, severely impacting their effectiveness in PDT [3], [24], [34], [40], [41]. In this context, we will summarize these issues and highlight the ongoing evolution of BODIPY-based phototherapies for overcoming their limitations for enhanced cancer therapy.

Supramolecular assembly, leveraging non-covalent interactions such as hydrogen bonds, van der Waals forces, π–π interactions, and hydrophobic and hydrophilic interactions, offers an effective pathway to circumvent complex organic synthesis processes. This strategy has facilitated the construction of functional materials and has seen significant advancements over the last several decades [45], [46], [47], [48]. Notably, the rapid progress in supramolecular assembly has introduced innovative approaches to enhance the properties of BODIPYs, addressing their previous limitations. These advancements include improving water solubility and biocompatibility, shifting absorption towards the near-infrared (NIR) region, and increasing tumor accumulation and retention, collectively enhancing the performance of BODIPYs in both PDT and PTT therapies [25], [49], [50], [51], [52], [53]. By employing thoughtful design and functionalization, BODIPYs can be structured into supramolecular assemblies that exhibit red-shifted absorption suitable for deep tissue penetration, alongside enhanced biocompatibility and water solubility. This structural and functional tailoring not only facilitates more efficient tumor targeting but also improves the generation of ROS or heat during therapy, opening up vast prospects in PDT and PTT applications [1], [3], [6], [24], [37], [43], [44]. Fortunately, the facial functionalized structures of BODIPYs makes them ideal candidates as versatile building blocks for supramolecular assemblies. Recent research has made significant strides in enhancing the PDT and PTT efficacies of BODIPYs through supramolecular strategies, as demonstrated by numerous studies in recent years [3], [6], [24], [40], [42], [35], [36], [37]. Despite the breakthroughs of supramolecular assembling strategies significantly improving the performances of BODIPYs in anti-cancer phototherapies, several persisting issues and challenges must be addressed to advance their clinical applications further [24], [50]. To provide more valuable resource for researchers keen on exploring and contributing to this promising field of cancer therapy and facilitate their translation into clinical settings, systematically reviewing the latest advancements and ongoing hurdles in the formation of various BODIPY-based assemblies for single or synergistic therapies of PDT and PTT is crucial.

In this review, we aim to comprehensively summarize the strategies and existing challenges associated with designing and constructing various types of BODIPY-based assemblies for both single and synergistic treatments involving PDT and PTT. The construction of BODIPY-based assemblies has been primarily achieved through the following monomers, polymers or complexes (Table 1, Table 2, Table 3, Table 4, Table 5, Table 6): (1) amphiphilic BODIPY molecules; (2) BODIPY-based metal-coordinated macrocycles and heavy-atom-free cyclic BODIPY arrays; (3) BODIPY-encapsulated amphiphilic polymers; and (4) BODIPY-involved host–guest complexes. Each section of this review delves into the optimal design strategies and functional mechanisms of BODIPY-based assemblies, which are extensively discussed and illustrated through numerous figures (Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15, Fig. 16, Fig. 17, Fig. 18, Fig. 19, Fig. 20, Fig. 21, Fig. 22, Fig. 23, Fig. 24, Fig. 25, Fig. 26, Fig. 27, Fig. 28, Fig. 29, Fig. 30). Additionally, we provide in-depth insights into the therapeutic effects of selected BODIPY-based assemblies, demonstrated through both in vitro and in vivo experiments. These results are critically assessed to underscore their potential for advancing clinical phototherapy applications. This review is crafted to serve as a timely and essential resource for researchers and clinicians interested in the emerging field of supramolecular phototherapies involving BODIPY assemblies. We hope to foster widespread interest and encourage further innovative studies within the phototherapy domain.

Section snippets

Self-assembly of amphiphilic BODIPYs

Since BODIPY cores are composed of organic aromatic compounds, their planar rigid conjugated structures impose significant limitations on water solubility and hinder their biological applications [3], [73]. By incorporating hydrophilic groups, functionalized BODIPYs with amphiphilic structures can self-assemble into various nanostructures, including nanoparticles (NPs), nano-vesicles, and nano-micelles. These nanostructures open up novel opportunities and provide insightful approaches for

Fig. 2. (I) Schematic illustration of molecular structures of PolyFBODIPY, PSDE and PD, self-assembling NP@PolyFBODIPY, generating ROS, and decomposing to release

PSs. (II) Inhibition of tumor growth on a murine cancer model PDXMDR by NP@PolyFBODIPY. (III) Cell viability of PDCMDR cells incubated with NP@PolyBODIPY and

NP@PolyFBODIPY and exposed to irradiation under normoxic or hypoxic conditions. (IV) Tumor growth inhibition curves upon various treatments. Reproduced with

permission [60]. Copyright 2023, Wiley-VCH.

Fig. 3. (a) (I) Schematic illustration of BODIPY with the capability of enhanced ROS generation, tumor targetability, and renal clearance. (II) Relative tumor volume

of tumor-bearing mice in three groups. (III) Photographs of tumor tissues taken from the different groups of tumor-bearing mice. Reproduced with permission [61].

Copyright 2023, Wiley-VCH. (b) (I) The molecular structures and the types of aggregates formed by MBP and LMBP. (II) Mechanism of type I PDT treatment. (III)

Images of tumor tissues from different groups of tumor-bearing mice. (IV) Relative tumor volume growth profiles. Reproduced with permission [62]. Copyright 2023,

Wiley-VCH.

Fig. 4. (I) The chemical structure of Gal-OH-BDP and formation of Gal-OH-BDP NPs. (II) Galactose-targeting and (III) NIR-II fluorescence imaging-guided PTT. (IV)

Viabilities of HeLa (top) and HepG2 cells (bottom) by using Gal-OH-BDP NPs. (V) IR thermal images (left) and temperature curves of the tumors against time after

injection with PBS and Gal-OH-BDP NPs (right). (VI) Tumor volume changes of the mice in various groups with Gal-OH-BDP NPs. Reproduced with permission [64].

Copyright 2022, Elsevier.

Fig. 5. (a) (I) Self-assembled Bodiplatin-NPs for synergistic PDT/PTT. (II) Images of 4T1 cells stained by Lysotracker Green DND-26 and Hoechst 33,342 after

incubation with Bodiplatin-NPs. (III) Viability of 4T1 cells treated with Bodiplatin-NPs in the presence or absence of Vc. Reproduced with permission [65]. Copyright

2016, Wiley-VCH. (b) (I) Schematic illustration of self-assembly, tumor-targeted delivery and synergistic PDT/PTT mechanisms of 4-IBMs (Top); TEM imaging and

size distribution of 4-IBMs, self-assembly of 2-IB, 4-IB, and 8-IB (Bottom). (II) Viability of 4T1 tumor cells treated with 2-IBMs, 4-IBMs, and 8-IBMs. (III) Tumor

growth profiles of the mice treated with 2-IBMs, 4-IB, 4-IBMs, and 8-IBMs. **p < 0.01. Reproduced with permission [66]. Copyright 2021, Wiley-VCH.

Fig. 6. (I) Chemical structure of BSL. (II) Schematic representation of lactose-mediated endo-cytosis, intracellular BODIPY release triggered by GSH from NPs selfassembled

by BSL, and synergistic therapies of PDT and PTT. (III) Cell viabilities of HL7702, C2C12, HepG2, HepG2/ADR, and HeLa, in the presence of BSL NPs (top),

and Cell viabilities of HepG2, HepG2/ADR, and HeLa in the presence of BSL NPs under irradiation (bottom). (IV) Tumor volumes of BALB/C nude mice bearing

HepG2 tumors by injecting BSL NPs. *p < 0.05, **p < 0.01. Reproduced with permission [72]. Copyright 2021, Elsevier.

Fig. 7. (I) Structures of the building blocks and self-assembled triangular metallacycles. (II) Illustration of the cellular uptake of NPs prepared from triangular

metallacycles. (III) CLSM images of HeLa cells after incubation with the ligand and metallacycles (top: BODIPY ligand; middle: triangular metallacycles 1; bottom:

triangular metallacycles 2). (IV) Cytotoxicities of triangular metallacycle 2 and BODIPY ligand against HeLa cells. (V) Cytotoxicities of different formulations against

A2780cis cells. Reproduced with permission [93]. Copyright 2018, American Chemical Society.

Fig. 8. (a) (I) Structures of BODIPY ligand and the self-assembled triangular metallacycles. (II) Nanoencapsulation method of preparing NPs by using DSPE-PEGMAL.

(III) Cell viabilities of HEK293 cells (left) and U87 cells (right) after treatments. (IV) Variations of the tumor volume (left), gross appearance of the excised

tumors (middle) and body weight of mice during therapies (right). 2: metallacycle; 4: BODIPY ligand. Reproduced with permission [94]. Copyright 2022, National

Academy of Science.

Self-assembly of BODIPY-based metal-coordinated macrocycles

In recent years, a considerable number of studies on metal-coordinated BODIPY-based macrocycles for phototherapies have been reported [93], [94], [102], [103], [104], [105], [106], [107]. The use of metal-coordination driven self-assembly has proven to be an effective strategy for constructing various 2D or 3D supramolecular metal-coordination macrocycles with precisely defined sizes and shapes [31], [108], [109], [110], [111]. The integration of BODIPY units into these metal-coordinated

Fig. 9. (a) Molecular structures of RuA–RuD, and the anti-tumor mechanisms of RuD. Reproduced with permission [95]. Copyright 2023, Wiley-VCH. (b) Schematic

illustration of the synthesis of Ru1105 and the PDT in vivo. Reproduced with permission [96]. Copyright 2024, American Chemical Society.

Fig. 10. (I) The synthesis routes of M1 and Melanin-M1, cellular uptake and Pt(II) release of Melanin-M1. (II) Photothermal toxicity of cisplatin, M1 and Melanin-M1

on HeLa cells. (III) Tumor volume of mice treated with different formulations. ***p < 0.001, ****p < 0.0001. Reproduced with permission [97]. Copyright 2020,

Springer Nature.

Fig. 11. (a) The synthesis (I), anti-tumor mechanism (II), and in vivo application (III) of Ru1085. (IV) Tumor volume changes of tumor-bearing mice against time. (V)

Kaplan–Meier survival rates for various treated mice. Reproduced with permission [100]. Copyright 2022, Springer Nature. (b) (I) The preparation of Ru1100 and

NIR-II imaging guided tumor diagnosis and therapy. (II) Confocal images of A549 cells incubated with Ru1100 and JC-1 in the absence and presence of irradiation.

(III) Tumor growth inhibition after different treatments. Reproduced with permission [101]. Copyright 2022, the Royal Society of Chemistry.

Fig. 12. (I) Structure of 3d. (II) Changes in the absorption spectrum of DPBF in the presence of 3d for 1O2 characterization. (III) EPR spectra of 3d for O2􀀀

• characterization. (IV) The decay trace of triplet state of 3d. (V) Cell cytotoxicity of 3d micelles to HeLa cells demonstrated by CCK-8 assay. (VI) Images of acridine orange

(green channel, live cell marker) and propidium iodide (red channel, dead cell marker) co-stained HeLa cells incubated. Reproduced with permission [114].

Copyright 2021, the Royal Society of Chemistry.

Fig. 13. (a) (I) NIR-mediated PDT, Car-BDP-TNM construction and molecular structures of Car-BDP, PLA-PEG, and PLA-PEG-FA. Viabilities of 4T1 cells (II) and HeLa

cells (III) by Car-BDP-TNM-mediated PDT under different thickness tissue. (IV) Car-BDP-TNM-mediated PDT in deep tumor. (V) Tumor growth inhibition by Car-BDPTNM

mediated PDT in 4T1 tumors. Reproduced with permission [133]. Copyright 2016, American Chemical Society. (b) (I) The molecular structure of RET-BDP and

schematic illustration of PDT by RET-BDP-TNM. (II) Cell viabilities after treatments with RET-BDP-TNM and B2-TNM: HeLa cells (left), KP7B cells (middle), and 4T1

cells (right). (III) Photographs of the resected tumors of mice. Reproduced with permission [134]. Copyright 2017, Wiley-VCH.

Self-assembly of BODIPY-encapsulated polymers

Compared to inorganic or metallic NPs, which have raised significant concerns regarding their potential toxicity to biological tissues [122], [123], [124], encapsulating BODIPYs within polymer components has emerged as a prevalent strategy for PDT, PTT, or combined therapies of PDT and PTT. The resultant polymeric assemblies, which can be tailored in terms of composition, size, and surface properties, are generally non-toxic and degrade readily within organisms over time [7], [75], [125], [126]

Fig. 14. (I) Preparation and self-assembly of PPIAB NPs. (II) O2•􀀀 photogeneration mechanism of PPIAB NPs and its application in hypoxic cancer therapy. (III)

fluorescence images of 4T1 tumor bearing mice after PPIAB NPs injection. (IV) Tumor volume changes against time. ***p < 0.001. (V) Photograph of tumor tissues

collected from mice with different treatment. Reproduced with permission [136]. Copyright 2020, Wiley-VCH.

Fig. 15. (I) Molecular structure of BDP-5. (II) Schematic illustration of the ISC processes, reduction of ΔES1–T1 for enhancing ISC and increasing 1O2 generation. (III)

Schematic illustration of the self-assembly of BDP-5 NPs. (IV) (left) Fluorescent images of HeLa tumor-bearing mice after injection of BDP-5 NPs or normal saline;

(right) tumor growth curves in HeLa tumor-bearing mice with injection of BDP-5 NPs (blue) or normal saline (black). (V) Cell viabilities of HeLa cells after treatments

with BDP-5 NPs. Reproduced with permission [140]. Copyright 2020, Wiley-VCH.

Fig. 16. (I) Molecular structures of helical-BDP and ordinary BDP (top) and the side view of the RHF optimized ground structures (bottom). (II) Cell viabilities of

CT26 cells treated with increasing concentrations of helical-BDP-NPs (left) and IRDye 700DX (right). (III) Schematic illustration of helical-BDP-NPs with anti-PD-L1

for tumor treatment. (IV) Primary (left) and artificial metastatic (right) tumor volume growth curves in tumor-bearing mice. Reproduced with permission [142].

Copyright 2020, Wiley-VCH.

Fig. 17. (a) (I) Top: self-assembly of AN-BDP and the intermolecular interaction mode in NPs. Bottom: the AN-BDP-contained NPs for PDT. (II) Cell viability of 4T1

cells treated with AN-BDP NPs. (III) Tumor volume changes of mice bearing tumors at different conditions. *p < 0.05, **p < 0.01, and ***p < 0.001. Reproduced with

permission [143]. Copyright 2021, Wiley-VCH. (b) Top: structure illustration of BDPTPA, BDPA, and ABDPTPA; Bottom: illustration of ABDPTPA NPs for 1O2

“afterglow” enhanced phototheranostics. Reproduced with permission [144]. Copyright 2021, Wiley-VCH. (c) The preparation of 1O2-nanotrap and its cytosolic

delivery for hypoxic PDT. Reproduced with permission [145]. Copyright 2022, Wiley-VCH.

Fig. 18. (I) The structure of α,β-linked BODIPYs, the process of generation of O2􀀀 • instead of 1O2, and the application for PDT in vivo. (II) The energy gaps of T1–S0 and

3O2–1O2. (III) Viabilities of HepG2 cells subjected to PS 2 with (left) or without (right) irradiation. Reproduced with permission [146]. Copyright 2021, Wiley-VCH.

Fig. 19. (I) Schematic illustrations of preparing water-stable nano-J-aggregates (J-NP). (II) The mechanism of PTT performance of J-NP. (III) Molecular structures

(BDP-H and BDP) and BDP J-dimer driven by duple Br–π interactions. (IV) UV–vis absorption spectra of BDP monomer in DMSO (blue) and J-NS NPs in water (red).

Reproduced with permission [169]. Copyright 2021, American Chemical Society.

Fig. 20. (a) (I) Constructing Nano-BFF and its PTT application. (II) 4T1 cells viabilities administrated with Nano-BFF. Without irradiation (top), under 808 (middle)

and 1064 nm (bottom) laser exposure. (III) Top: temperature variations at tumor site of the mice against time. Middle: relative tumor volumes in different treatment

groups. Bottom: survival rates of the mice bearing 4T1 tumors after different treatments. Reproduced with permission [170]. Copyright 2021, Springer Nature. (b)

Schematic illustration of Dye 2-contained J-aggregation NPs for NIR-II fluorescence imaging-guided PTT. Reproduced with permission [172]. Copyright 2022, the

Royal Society of Chemistry.

Fig. 21. (a) (I) Molecular structures of BDP1, BDP2, BisBDP1, and BisBDP2. (II) Schematic illustrations of the PA imaging–guided PTT by BisBDP2-contained Jaggregates.

(III) Temperature variations at tumor site of the mice against irradiation time. (IV) Relative fluorescence intensity in orthotopic liver tumor of the mice

with different treatments. Reproduced with permission [173]. Copyright 2022, American Association for the Advancement of Science. (b) (I) Schematic illustration of

strategic design of CT-coupled J-aggregates. (II) Cell viabilities of Hepa1-6 and HepG2 cells treated with BDP2-NPs. (III) Tumor growth curves of various groups

under different conditions. ****p < 0.0001. Reproduced with permission [178]. Copyright 2024, the Royal Society of Chemistry.

Fig. 22. (I) Protonation mechanism of NAB induced by pH changes. (II) Schematic illustration of the radiative and non-radiative transitions induced by pH changes.

(III) Tumor volume changes of different mouse groups. (**p < 0.01). (IV) Schematic illustration of pH-sensitive NAB-contained NPs for PAI and PTI guided synergistic

therapies of PDT and PTT. Reproduced with permission [180]. Copyright 2017, American Chemical Society.

Fig. 23. (a) (I) Schematic illustration of combined PDT and PTT using BODIPY-contained polymeric vesicles. Viabilities of 4T1 tumor cells treated with BODIPY

vesicles in the absence (II) or presence (III) of Vc. Reproduced with permission [186]. Copyright 2017, Wiley-VCH. (b) (I) Chemical structures of conjugated BODIPYs

and their formation of NPs. (II) Schematic illustration of photoconversion routes of CPs-based NPs. (III) Combined PDT and PTT by utilizing tri-BDP-NPs against

tumor cells. Viabilities of 4T1 tumor cells treated with di-BDP-NPs (IV) and tri-BDP-NPs (V). Reproduced with permission [187]. Copyright 2018, Wiley-VCH.

Fig. 24. (I) Schematic illustration of encapsulating BODIPY-Ir for constructing micelles. (II) Photophysical processes of generating 1O2 and photothermal conversion.

(III) Intracellular PDT/PTT treatments for cancer cells apoptosis. (IV) 1O2 quantum yields of Micelle-Ir, BODIPY-Ir and BODIPY-I by using DPBF as 1O2 scavenger. (V)

PCE of Micelle-Ir, BODIPY-Ir, and BODIPY-I. Reproduced with permission [188]. Copyright 2021, Wiley-VCH.

Fig. 25. (I) Schematic illustration of aromatic ring-fused aza-BODIPYs J-aggregates. (II) JBDP-a NPs used for combined PDT and PTT. (III) Survival rate of 4T1 cells

incubation with JBDP-a NPs or commercial IR-1061 at different concentrations. (IV) The tumor volume of various mice groups under different conditions. Reproduced

with permission [189]. Copyright 2024, Wiley-VCH.

Fig. 26. (I) Schematic illustration of the structures of PEG, BODIPY, and prodrug (PTX), the self-assembly and combination therapy processes of NPs with optimized

ratio. (II) Viabilities of cancer cells treated with Ada–BODIPY and Ada–PTX with various ratios. (III) Time-dependent tumor volumes of different mice with different

treatments. (IV) Tumor weights of mice with different treatments. (V) Body weights of the mice after different treatments. Reproduced with permission [198].

Copyright 2019, Wiley-VCH.

Fig. 27. (I) Representation of self-degradable BDP2IPh PS for PDT and chemical structure of BDP2IPh and CB[7]. (II) Decay traces of triplet states for BDP2IPh (left)

and BDP2IPh-CB[7] (right). (III) ADPA as the probe to monitor 1O2 generation abilities of BDP2IPh and BDP2IPh-CB[7]. (IV) MCF-7 cell viabilities with BDP2IPh and

BDP2IPh-CB[7] treatments in various conditions: in darkness (top left) and under light irradiation (top right); safety tests of BDP2IPh residues towards BEAS-2B cells

after PDT: in darkness (bottom left) and under irradiation (bottom right). Reproduced with permission [201]. Copyright 2020, Wiley-VCH.

Self-assembly of BODIPYs via host–guest interactions

Supramolecular host–guest chemistry has gained significant prominence since the Nobel Prize in Chemistry was awarded in 1987 to Pedersen, Cram, and Lehn [194]. Herein, we introduce three primary macrocyclic hosts: cyclodextrin (CD) [195], [196], [197], [198], cucurbituril (CB) [199], [200], [201], [202], [203], and pillar[n]arene [204], [205], [206], [207], [208], subsequently summarize how these macrocycles form supramolecular assemblies with BODIPYs to enhance their PDT and PTT performances.

Fig. 28. (I) Schematic illustration of the fabrication of host–guest complexes (HG) and generation of ROS. (II) Viabilities of HeLa cells subjected to a range of HG

concentrations without (top) and with (bottom) light-irradiation. Reproduced with permission [207]. Copyright 2022, the Royal Society of Chemistry.

Fig. 29. (I) Schematic illustrations of chemical structures, cartoon representations of P5, BDMI, MI, and CDDP, the preparation of CDDP@Suprasomes,

CDDP@Liposomes, and their anti-tumor process in vivo. (II) The concentration-dependent photothermal effect of suprasomes. (III) 4T1 cells viabilities after different

treatments. (IV) Tumor volume changes of the mice with different treatments. **p < 0.01. Reproduced with permission [209]. Copyright 2022, Wiley-VCH.

Fig. 30. (I) Schematic illustrations of the preparation and anti-tumor mechanism of LacP5⊃BSTA@DSF. (II) The images of living mice after injection of LacP5⊃BSTA. (III) Tumor sizes after treatment. (IV) Tumor volume changes during treatment. Reproduced with permission [210]. Copyright 2023, the Royal Society of Chemistry.

Conclusion and outlook

In this review, we have summarized various supramolecular assembly strategies for constructing BODIPY-based assemblies aimed at enhancing their performance in single or synergistic therapies involving PDT and PTT. The primary structures of these assemblies were constructed by amphiphilic BODIPYs, BODIPY-based metal-coordinated macrocycles, heavy-atom-free cyclic BODIPY arrays, BODIPYencapsulated polymers, and BODIPY-based host–guest complexes. Significant advancements have been made in recent years toward enhancing the PDT and PTT capabilities of BODIPYs through supramolecular assembly. This includes improved water solubility, biocompatibility, and penetration depth into tumor tissues via red-shifted NIR absorption. BODIPY-based NPs have demonstrated selective uptake by tumor cells through the EPR effect. Moreover, many NPs possess targeted therapeutic capabilities, which have amplified their therapeutic efficacy. Effective generation of ROS and heat, pivotal for inducing cancer cell death, has been achieved by modulating the intermolecular interactions within BODIPY assemblies. Additionally, several BODIPYs have been structured for synergistic therapies, addressing the limitations associated with singular PDT or PTT treatments. Consequently, BODIPYbased assemblies summarized in this review exhibited great potential for various anti-tumor phototherapies.

Despite these advancements, several challenges still pose significant hurdles for the clinical application of BODIPY-based supramolecular assemblies:

phototherapy should possess high-targeting performance, excellent water solubility and biocompatibility, robust penetration abilities, efficient tumor accumulation via EPR effects, and high ROS or heat generation capabilities. Additionally, they should be stable, not prone to leakage, and should be easily manufactured without complicated synthesis processes. Although challenges persist in the research of their supramolecular assemblies for PDT and PTT, the potential for improvement through strategic design and rational assembly is immense. As research progresses, we envision BODIPY-based assemblies becoming increasingly sophisticated and playing a vital role in antitumor treatments.

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In Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe

采用可激活的内质网靶向邻近标记远红荧光探针用于原位可视化II型糖尿病中羧酸酯酶活性检测

In Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe

Ningge Xu【胥宁格】,Dandan Tang【唐丹丹】,Heng Liu*【刘恒】,Mengyue Liu【刘梦月】,Zheng Wen【文正】,Tongmeng Jiang【蒋童蒙】*, and Fabiao Yu【于法标】*

Cite this: Anal. Chem. 2024, 96, 26, 10724–10731

Publication Date:June 19, 2024

https://doi.org/10.1021/acs.analchem.4c01721

1Department of Radiotherapy, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, 570102, China

2Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Emergency and Trauma, Ministry of Education, Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

羧酸酯酶（CE）是一种在生物体中普遍存在的酶，它参与多种关键的生理和病理过程。肝脏中CE水平的变化可能作为II型糖尿病（T2DM）的重要预兆。近期，海南医科大学于法标教授课题组报道了II型糖尿病中羧酸酯酶活性检测，相关成果以“In Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe”为题发表在国际化学权威杂志Analytical Chemistry上（DOI: 10.1021/acs.analchem.4c01721）。

研究的主要内容

我们开发出了一种创新的远红荧光探针DCI2F-Ac，它基于二氰基异佛尔酮（DCI）设计，具备内质网靶向的邻近标记功能，用于实时、准确地监测和成像CE的活性。DCI2F-Ac具有卓越的安全性，表现为极低的细胞毒性和生物毒性，同时对CE显示出极高的选择性和敏感性。与传统的CE探针相比，DCI2F-Ac的独特之处在于它能够直接共价锚定到CE上，显著减少了因扩散导致的原位荧光信号损失，提高了测量的精确性。借助“开启-关闭”荧光信号模式，DCI2F-Ac不仅能够有效区分不同的细胞系，还能精确筛选CE抑制剂。在内质网（ER）应激的研究中，我们发现毒胡萝卜素（Tg）能够显著上调CE水平，而衣霉素（Tm）则不会引发类似效应，这一发现与ER的钙稳态紧密相关。特别值得注意的是，DCI2F-Ac在T2DM患者的肝脏样本中能够有效检测到CE活性的下降，并且我们可以通过追踪CE水平的变化来评估二甲双胍、阿卡波糖以及两者联合用药的治疗效果。实验结果显示，二甲双胍与阿卡波糖的联合使用能够显著恢复CE水平至接近正常范围，展现出最佳的抗糖尿病效果。因此，DCI2F-Ac探针不仅为肝脏代谢紊乱的研究提供了新的视角，也为药物疗效评估提供了有力的工具，为探索CE在相关疾病中的潜力开辟了新途径。海南医科大学功能材料与分子影像技术研究团队硕士生胥宁格和唐丹丹为论文共同第一作者，刘恒研究员和于法标教授为共同通讯作者。本研究受到国家自然科学基金和海南省重点研发计划等项目资助。

Abstract

Abstract Image

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the “on–off” fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.

 

Scheme 1. (a) Schematic illustration of dual sensing and labeling NIRF probe DCI2F-OTf for ONOO–. (b) Synthesis of NIRF probe DCI2F-OTf.

Figure 1. Fluorescence response of DCI2F-OTf (10 μM) to ONOO– excited at 490 nm. (a) Visualization of the fluorescence changes of DCI2F-OTf during titration with increasing ONOO– (0 – 35 μM) concentrations. (b) Linearity of the fluorescence intensity value of DCI2F-OTf at 652 nm with varied concentrations of ONOO– (5 – 35 μM). (c) Time-dependent fluorescence response of DCI2F-OTf before and after the addition of ONOO– (70 μM) at 37 °C recorded every 5 min. (d) The fluorescence intensities at 652 nm of DCI2F-OTf in the absence or presence of ONOO– (70 μM) over pH range from 3.0 to 7.0. (e) Values of fluorescence intensities of DCI2F-OTf treated with different competitive biological agents: 1. blank, 2. Na+ (500 μM), 3. K+ (500 μM), 4. Ser (200 μM), 5. Leu (200 μM), 6. Arg (200 μM), 7. S2O32- (100 μM), 8. HSO3– (100 μM), 9. H2S (100 μM), 10. Cys (100 μM), 11. GSH (1 mM), 12. NO2– (100 μM), 13. NO3– (100 μM), 14. NO (100 μM), 15. •OH (100 μM), 16. O21 (100 μM), 17. H2O2 (100 μM), 18. O2•– (100 μM), 19. HOCl (100 μM), 20. ONOO– (70 μM). (f) SDS-PAGE analysis of BSA (5 mg/mL) after incubation with DCI2F-OTf + ONOO–, DCI2F-OTf, DCI2F-OTf + ONOO– + UA in PBS at 37 ℃ for 30 min. Left: Coomassie brilliant blue staining; right: fluorescence imaging. λex/λem = 490/650 nm.

                                               

Figure 2. (a) RAW264.7 cells were pretreated with SIN-1 (200 μM, 30 min) or stimulated with LPS (1.0 μg/mL), IFN-γ (100 ng/mL) for 12 h, PMA (10 nM) for 30 min, and then incubated with DCI-OTf (10 μM, 30 min) or DCI2F-OTf (10 μM, 30 min), respectively. The red channel was collected in the range of 600-700 nm (λex = 488 nm) and the Hoechst channel was collected in the range of 430-480 nm (λex = 405 nm). (b) A549 cells were pretreated with SIN-1 (200 μM, 30 min), followed by incubation with DCI2F-OTf (10 μM, 30 min) or DCM-KA (10 μM, 30 min), before or after washout with fresh DMEM (three times, each for 6 min). (c) Normalized fluorescence intensity plots of DCI2F-OTf and DCM-KA labeled cells in (b) (n = 3). Scale bar: 50 μm.

                                               

Figure 3. (a) RAW264.7 cells pretreated with different concentrations of SIN-1 (0, 50, 100, 200 μM), SIN-1 (200 μM) plus ebselen (200 μM) for 30 min were incubated with DCI2F-OTf (10 μM) for 30 min. (b) The pixel intensity of DCI2F-OTf labeled cells in (a). (c) The protein levels of iNOS and IL-6 were detected by western blot analysis. (d) The relative protein expression level of iNOS and IL-6 in (c). (e) RAW264.7 cells were pretreated with SIN-1 (200 μM, 30 min), and then incubated with DCI2F-OTf (10 μM) for 0 – 30 min. (f) Flow cytometry analysis of DCI2F-OTf (10 μM) labeled SIN-1 (200 μM, 30 min) pretreated cells. Error bars represent s.d., n=3. Scale bar: 50 μm.

 

                                               

Figure 4. (a) RAW264.7 cells were pretreated with the stimulus LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) for different periods (0, 3, 6, 12 h), PMA (10 nM) for 30 min or LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) plus UA (200 μM) for 12 h,  PMA (10 nM) for 30 min, and then incubated with DCI2F-OTf (10 μM) for 30 min. (b) RAW264.7 cells were pretreated with LPS (0, 0.25, 0.5, 1.0 μg/mL)/IFN-γ (100 ng/mL), PMA (10 nM), or LPS (1.0 μg/mL)/IFN-γ (100 ng/mL) plus AG (1 mM) for 12 h,  PMA (10 nM) for 30 min, and then incubated with -OTf (10 μM) for 30 min. (c) The pixel intensity of DCI2F-OTf labeled cells in (a). (d) The protein levels of iNOS, IL-6, and NF-𝜅B were detected by western blot analysis. (e) The relative protein expression level of iNOS, IL-6, and NF-𝜅B in (c). (f) The pixel intensity of DCI2F-OTf labeled cells in (b). Error bars represent s.d., n=3. Scale bar: 50 μm.

                                               

Figure 5. (a) NIRF images of control and LPS-stimulated mice after intravenous administration of DCI2F-OTf (200 μM, 45 μL). (b) NIRF imaging and photographs of dissected major organs (1: heart, 2: kidney, 3: liver, 4: spleen, 5: lung) from mice in panel (a). (c) The histogram presents the average radiant efficiency of mice in panel (a). (d) The histogram presents the average radiant efficiency of the lung in panel (b). (e) H&E staining histological images for lung tissue in control and LPS-stimulated mice. Scale bar: 50 μm. (f-h) Serum levels of inflammatory factors TNF-α, IL-6, and IL-1β in control and LPS-stimulated mice. (i) The protein levels of iNOS, COX-2, NF-κB, and IL-6 were detected by western blot analysis. (j-n) The relative protein expression level of iNOS, COX-2, NF-κB, and IL-6 in (i). (o-p) Sections of lung tissue were immunostained with an antibody that detected 3-NT. Scale bar: 50 μm. (q) NIRF images of mice fresh lung tissue sections from ALI mice in the LPS6 group. The confocal Z-axis scan images at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 µm penetration depths. λex = 488 nm; red channel: λem = 600 – 700 nm.

Conclusion

In conclusion, we have designed a novel NIRF probe, DCI2F-OTf, for fluorescence imaging ONOO– in live cells and mice model of ALI. Under physiological conditions, DCI2F-OTf was highly sensitive and selective for ONOO– and yielded a linear response to ONOO over a wide concentration range from 0 to 70 μM. The QM covalently labeled neighboring proteins released from the reaction of DCI2F-OTf with ONOO– could solve the problem of intracellular diffusion of the probe, enabling precise imaging of intracellular ONOO–. This strategy was superior to the vast majority of ONOO– probes that have been developed so far. The excellent sensing performance and NIRF emission characteristics enable DCI2F-OTf to image ONOO– levels in mice and tissues with ALI. In vivo imaging results indicated that SFN significantly down-regulated the oxidative stress level, thereby attenuating the severity of LPS-induced ALI, and could exert a protective role against ALI. Therefore, DCI2F-OTf was a valuable tool for studying the physiological function of ONOO– and was expected to facilitate the understanding of oxidative stress diseases such as ALI and the rapid screening of corresponding therapeutic drugs.

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Rational Design of Efficient Heavy-Atom-Free Boron-Dipyrromethene Nanophotosensitizers for Two-Photon Excited Photodynamic Therapy of Cancer

Rational Design of Efficient Heavy-Atom-Free Boron-Dipyrromethene Nanophotosensitizers for Two-Photon Excited Photodynamic Therapy of Cancer

Van-Nghia Nguyen,Dong Joon Lee,Dianqi Zhang【张点奇】,Jeongsun Ha,Kunemadihalli Mathada Kotraiah Swamy,Rui Wang*【王锐】,Hwan Myung Kim*,Fabiao Yu*【于法标】, and Juyoung Yoon*

Cite this: Chem. Mater. 2024, 36, 11, 5534–5541Text Full

Publication Date:May 23, 2024

https://doi.org/10.1021/acs.chemmater.4c00482

Copyright © 2024 American Chemical Society

于法标团队及合作者发表双光子激发光动力疗法设计高效的无重原子BODIPY纳米光敏剂的癌症治疗合理方案

        5月23日，急诊创伤学院于法标教授团队在 《Chemistry of Materials》在线发表题为“Rational Design of Efficient Heavy-Atom-Free Boron-Dipyrromethene Nanophotosensitizers for Two-Photon Excited Photodynamic Therapy of Cancer”的论文。

尽管Boron-dipyrromethene（BODIPY）染料在癌症治疗领域作为光敏剂（PSs）的潜力已得到广泛认可，但在光动力疗法（PDT），特别是双光子激发光动力疗法（2PE-PDT）中开发无重原子的BODIPY光敏剂仍面临诸多挑战。本研究成功设计并合成了一种新型无重原子光敏剂（BDP-6），该光敏剂具备优化的单重态–三重态能隙和立体位阻，旨在促进系间窜越并显著提升荧光强度。为了增强BDP-6的生物相容性和肿瘤靶向能力，本研究将其封装于DSPE-PEG(2000)生物素中，制备出相应的纳米光敏剂（BDP-6 NPs）。与不含二甲基基团的对照BDP-5 NPs相比，BDP-6 NPs在水溶液中单光子激发下展现出更为明亮的深红色荧光，并显示出更高的活性氧物种（ROS）产生效率。此外，BDP-6 NPs不仅具有出色的肿瘤靶向能力和明亮的红色发射，而且在对抗癌细胞时展现出显著的光毒性，同时保持低暗毒性。尤为引人注目的是，在双光子激发条件下，BDP-6 NPs无论是在水溶液还是活细胞中均能有效生成ROS，从而在癌症细胞的2PE-PDT消融治疗中展现出卓越的性能。进一步的体内实验同样验证了BDP-6 NPs在癌症PDT治疗中的巨大潜力。本研究不仅为基于BODIPY染料的癌症2PE-PDT治疗领域提供了创新的靶向无重原子纳米光敏剂，更为未来相关治疗策略的开发和应用提供了宝贵的实践经验和策略参考。

该成果以海南医学院为共同通讯单位，急创学院功能材料与分子影像技术研究团队教师王锐研究员、于法标教授和韩国亚洲大学Hwan Myung Kim教授、梨花女子大学Juyoung Yoon教授为共同通讯作者。本研究受到国家自然科学基金何海南省重点研发项目等项目资助。

原文链接：https://doi.org/10.1021/acs.chemmater.4c00482

Abstract

Abstract Image

         Although boron-dipyrromethene (BODIPY) dyes have been widely recognized for their potential use as photosensitizers (PSs) in cancer treatment, the development of heavy-atom-free BODIPY PSs for photodynamic therapy (PDT), particularly in two-photon excited photodynamic therapy (2PE–PDT), remains challenging. Herein, a novel heavy-atom-free photosensitizer (BDP-6) with optimized singlet–triplet energy gap and steric hindrance was designed and synthesized to facilitate intersystem crossing and improve fluorescence intensity. To enhance the biocompatibility and tumor-targeting ability of BDP-6, corresponding nanophotosensitizers (BDP-6 NPs) were prepared by encapsulating BDP-6 within DSPE-PEG(2000) biotin. Compared to control BDP-5 NPs without dimethyl groups, BDP-6 NPs exhibited brighter deep red fluorescence and higher efficiency in generating reactive oxygen species (ROS) under one-photon excitation in aqueous solutions. Moreover, BDP-6 NPs displayed excellent tumor-targeting ability, bright red emission, and considerable phototoxicity with low dark toxicity toward cancer cells. Notably, under two-photon excitation, the BDP-6 NPs efficiently generated ROS both in aqueous solutions and living cells, thereby demonstrating exceptional performance in 2PE–PDT for cancer cell ablation. Furthermore, in vivo experiments revealed that BDP-6 NPs hold great promise for cancer PDT. Our work presents practical strategies for developing tumor-targeting heavy-atom-free nanophotosensitizers based on BODIPY dye for 2PE–PDT of cancer.

Figure 1. (a) Chemical structures and synthetic route of BDP dyes. (i) 4-bromo-2,6-dimethylbenzaldehyde, Pd(OAc)2, t-Bu3PHBF4 and K2CO3, toluene, reflux. (ii) DMF, POCl3. (iii) 2,4-dimethypyrrole, trifluoroacetic acid (TFA), p-chloranil, Et3N, and BF3.OEt2, CH2Cl2. (b) Schematic illustration for the preparation of BDP-6 NPs. (c) Nanoparticle size distribution of BDP PSs detected by DLS and TEM images (inset) of BDP-6 NPs. (d) UV-vis absorption and PL spectra of the BDP-5 NPs and BDP-6 NPs (10 µM, in water, lex = 490 nm). (e) ROS generation of BDP-5 NPs and BDP-6 NPs in water after irradiation by a green LED (20 mW/cm2) using DCFH-DA as ROS probe (lex = 500 nm, lem = 525 nm). (f, g) Two-photon ROS generation efficiency of BDP-6 NPs (10 µM). (f) A plot of fluorescence intensity of DHR123 (2.5 µM) with BDP-6 NPs according to the two-photon excitation wavelength. (g) A plot of fluorescence intensity for DHR123 with BDP-6 NPs against TP irradiation time at 780 nm (lex = 500 nm, lem = 525 nm).

Figure 2. Fluorescence microscope images of (a) HeLa (cancer cells) and WI38 (normal cells) cells after treatment with BDP-6 NPs (5 µM) and (b) loaded with DCFH-DA (total ROS probe) or PI (4 µM, dead cell marker). (c) Cell viability of HeLa cells after treatment with different concentrations of BDP-6 NPs with green light irradiation.

Figure 3. Fluorescence microscopy image of HeLa cells stained with BDP-6 NPs (5 µM) and (a) DCFH-DA (20 μM, ROS probe) or (b) Hoechst 33342/PI as indicators (2 µM/ 10 µM) followed by TP scans at 5.0 mW laser power. Scale bars = 50 µm. (c) Viability of cells incubated with BDP-6 NPs and irradiated at 780 nm at individual laser powers of 1.5, 2.4, 5.0, and 6.3 mW. (d) Cell viability estimated after 780 nm TP irradiation (6.3 mW).

Figure 4. 3D image of HeLa spheroid ROS generation using ROS indicator DHR123. (a), (b) 3D images of HeLa spheroids incubated with DHR123 (15 µM) and BDP-6 NPs (100 µM) for 1 hour. (c), (d) Fluorescence images of HeLa spheroids incubated with DHR123 (15 µM) and BDP-6 NPs (100 µM) after 400 TP irradiation scans. Images were recorded in the 500-550 nm emission window after excitation at 488 nm. Scale bar = 50μm.

Figure 5. (a) In vivo imaging of BDP-6 NPs (intravenous injection, 10 μM, 100 μL in saline) in female nude BALB/c mice (λex = 490 nm, λem = 500-575 nm) at different time points of 0, 0.5, 1, 2, 4, 8, 12 h. (b) Visualization of BDP-6 NPs in the mouse breast cancer model. Photographs of untreated and PDT-treated mice models (660 nm, 200 mW/cm2, 15 min) for 1-15 days. The changes in tumor volume and weight. (c) Photographs of untreated and PDT-treated tumors within 15 days. (d) The analysis of tumor volume. (e) 2D ex vivo imaging in separated organs (tumor, heart, liver, spleen, lung, and kidney) sacrificed from mice models.

Figure 6. H&E staining of separated organs (heart, liver, spleen, lung, and kidney) sacrificed from untreated and PDT-treated mice models.

3. CONCLUSIONS

       A novel heavy-atom-free BODIPY PSs (BDP-6) comprised of an electron-donating PXZ group and an electron-withdrawing BODIPY core was designed and synthesized for tumor-targeted two-photon photodynamic therapy. The photostable and biocompatible BDP-6 NPs were produced by encapsulating BDP-6 within DSPE-PEG(2000) biotin. Compared to BDP-5 NPs without dimethyl groups, BDP-6 NPs showed brighter emission in the deep red region and more efficient ROS production efficiency under one-photon excitation in aqueous solutions. The BDP-6 NPs could selectively accumulate in cancer cells and effectively kill cancer cells by generating intracellular ROS under green light illumination (one-photon) with low dark toxicity. Interestingly, upon two-photon excitation, the BDP-6 NPs efficiently generated ROS in both aqueous solution and living cells and consequently exhibited excellent 2PE-PDT performance toward cancer cells. In vivo, results demonstrated that BDP-6 NPs showed great potential for tumor-targeted fluorescence imaging and PDT treatment. This work provides a facile, straightforward methodology for developing highly efficient nanophotosensitizers for two-photon excited photodynamic therapy based on heavy-atom-free BODIPY platforms.

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Imaging and tracking of tumor extracellular vesicles to unravel the progression of ovarian carcinoma using fluorescent membrane probes

Imaging and tracking of tumor extracellular vesicles to unravel the progression of ovarian carcinoma using fluorescent membrane probes

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Organic dye-based photosensitizers for fluorescence imaging-guided cancer phototheranostics

Organic dye-based photosensitizers for fluorescence imaging-guided cancer phototheranostics

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Highlights

  • Four types of photosensitizers with fluorescence imaging functions were summarized.
  • Design strategies for fluorescence-imaging guided phototherapy were highlighted.
  • Challenges and future opportunities of imaging-guided phototherapy were presented.

Abstract

        Precise diagnosis and treatment of tumors is the current hotspot, which has also given rise to a new subject named “theranostics”. It is an ideal precision treatment strategy if the agent can provide effective treatment while visually monitoring tumor occurrence and providing timely feedback on the efficacy. Fluorescence imaging-guided phototherapy technology is a non-invasive, simple-to-operate, highly safe and non-drug-resistant visual treatment method that can accurately monitor tumor sites, perform efficient phototherapy and feedback on tumor treatment effects. Photosensitizers with fluorescence imaging capabilities play a decisive role in the entire diagnosis and treatment process. In this review, we focus on four commonly used photosensitizers with fluorescence imaging functions, including cyanine, tetrapyrrole structures, BODIPY, and AIEgens. The design strategies and principles for improving imaging or/and therapeutic functions were highlighted based on these four organic molecular monomers or their nanoaggregates, nanocomposites, etc. The challenges and future opportunities of fluorescence imaging-guided phototherapy in the clinical translation of precision tumor treatment are also presented.

Keywords

      Fluorescence imaging-guided phototheranostics

      Cancer treatment

PhotosensitizersMonomersNanoparticals
  1. 1. Introduction
  2. 2. Design strategy for efficient phototheranostic agents
  3. 3. Cyanine-based fluorescence imaging-guided theranostics for cancer therapy
  4. 4. Tetrapyrrole structures-based fluorescence imaging-guided theranostics for cancer therapy
  5. 5. BODIPY-based fluorescence imaging-guided theranostics for cancer therapy
  6. 6. AIEgens −based fluorescence imaging-guided theranostics for cancer therapy
  7. 7. Conclusions and perspectives
  8. Declaration of competing interest
  9. Acknowledgements
  10. Data availability
  11. References

1. Introduction

        Tumors have the characteristics of high incidence rate, strong concealment in the early stage, complexity and heterogeneity of the occurrence and development process, as well as easy recurrence and metastasis, making them a killer that seriously reduces the quality of life and endangers life and health [1], [2], [3]. Traditional single or combined treatment methods such as surgery, chemotherapy, and radiotherapy are still the conventional means of clinical treatment of tumors [4], [5]. Frustratingly, the above-mentioned treatments still face shortcomings such as high invasiveness, low targeting, and high side effects [6], [7]. They are also unable to achieve real-time visual diagnosis of tumors and timely feedback on therapeutic effects. Therefore, the development of new imaging-guided, non-invasive and low-side-effect theranostic methods is of great significance for the precise treatment of tumors [8], [9], [10].

        The non-invasive, highly selective, and low-drug-resistant phototherapy has attracted widespread attention from medical workers and scientific researchers since it was used in the clinical treatment of various types of tumors more than 40 years ago [4], [11]. Photodynamic therapy (PDT) and photothermal therapy (PTT) are important components of phototherapy, which can selectively treat diseased areas and have great therapeutic effects on various types of tumors [12]. Not only that, phototherapy does not conflict with other treatments, and each treatment takes less time, making life more convenient [13], [14].

        Light, photosensitizers (PSs), oxygen (or other adjacent substrates) are indispensable and vital components of PDT [15], [16]. After the PS in the ground state (S0) absorbs excitation light of a specific wavelength, it produces toxic reactive oxygen species (ROS), damaging prominent proteins and subcellular organelles, thereby inducing apoptosis of tumor cells [17], [18]. PDT is used for anti-tumor treatment in three interrelated ways: (1) directly killing tumor cells; (2) damaging tumor blood vessels; (3) activating the immune system [19]. Different from PDT, the PS absorbs laser of a specific wavelength and reaches to the excited state from the S0, and then returns to the S0 through non-vibrational relaxation is called PTT process [20]. At the same time, it releases enough heat to damage the membrane structure of tumor cells or cause the inactivation of proteins and other substances, thereby achieving the purpose of tumor elimination [21], [22].

       Since phototherapy has inherent selectivity for disease treatment, it only needs to give light to the lesion, thereby reducing damage to normal tissue and reducing side effects [4], [23]. Based on this, the best treatment timing can be determined by real-time monitoring of the distribution of PSs in the body and its enrichment time at the tumor site, so as to achieve the lowest drug dose and the best therapeutic effect. Fluorescence imaging technology is a high-profile non-invasive imaging method with great sensitivity and resolution [24], [25], [26], [27]. By combining fluorescence imaging technology and phototherapy, the targeting effect, therapeutic efficiency and metabolic effect of PSs on tumor sites can be determined, thereby clarifying the entire dynamic process of PSs in the body and laying a theoretical foundation for clinical translation [28]. As an essential part of fluorescence imaging, fluorophores can emit fluorescence by absorbing light of specific wavelengths [29]. Fortunately, many fluorophores have been structurally modified to possess phototherapeutic properties [30]. Therefore, the development of these PSs with fluorescence imaging properties provides a feasible strategy for real-time diagnosis and precise treatment of tumors [31], [32].

        The performance of phototheranostic agents directly determines the effects of fluorescence imaging and phototherapy. Hundreds of PSs, including porphyrins, phthalocyanines, boron dipyrromethene (BODIPY), etc., have been used in clinical research on various solid tumors [33], [34], [35]. An ideal PS should have the following properties [33], [36], [37]: (1) High operability, many sites that can be modified, and simple structural modification; (2) It can possess excellent tumor targeting by itself or after modification; (3) The absorption mainly in the near-infrared region (NIR, 700–1350 nm; NIR I and NIR II), which is beneficial to reducing the interference of biological autofluorescence background and decreasing the inconvenience of life caused by the strictly avoiding light after treatment; (4) Superior quantum yield of ROS for PDT and excellent photothermal conversion efficiency (PCE) for PTT; (5) Strong safety, no dark toxicity, high biocompatibility, and easily metabolized out of the body. To meet the above requirements, researchers have designed and synthesized a series of organic molecules with fluorescent diagnostic and therapeutic functions, or constructed nanoaggregates, nanocomposites, and nanomaterials based on these functional molecules through self-assembly or co-assembly for fluorescence imaging-guided phototherapy of tumors [38].

        In this review, we focus on these fluorescent therapeutic molecules or fluorescent therapeutic aggregates, fluorescent therapeutic nanocomposites based on organic molecular structures, for phototherapy of tumors. According to the different parent structures of organic parents, they are mainly divided into cyanine, tetrapyrrole structure, BODIPY, and AIEgens, which can be used for fluorescence imaging-guided PDT, PTT, and PDT combined PTT against cancers (Fig. 1). In addition, we have in-depth summarized the impact of structural modification (molecular level) or regulated aggregation mode (nano-level) on energy dissipation, providing feasible strategies for constructing PSs that meet both imaging and therapeutic effects. Moreover, we also elaborated on the ways in which molecular structure or nanostructured phototheranostic agents can enhance the effect of phototherapy, which will provide strategies for the subsequent construction of phototheranostic agents with precise tumor imaging and treatment.

Fig. 2. Schematic of the Jablonski diagram.

Table 1. Summary of fluorescence, therapeutic modalities, advantages and disadvantages of representative PSs among cyanine-based molecular or nanomaterials.

Photosensitizer Fl. Ther. Mod. Advantages Disadvantages Ref.
heavy-atomized cyanine structures NIR I PDT (type II) broad absorption; enhanced 1O2 generation Φf is relatively reduced; O2-dependent [45], [46]
halogen-modified heptamethine cyanine NIR I PDT (type I) activatable fluorescence; anti-hypoxic PDT; ALP-overexpressed cancer targeting lower Φf (CyI) [52]
introduced TEMPO into Cy7 NIR I PDT (type II) extremely high ФΔ and
low dark cytotoxicity		 weakening of fluorescence [55]
modification of acetophenone at thiopentamethylcyanine NIR I PDT (type II) enhancement of fluorescence and ROS generation; tumor-targeted imaging O2-dependent [60]
fluorination of a squarylium indocyanine NIR I PDT (type II) enhanced ROS generation; ER-targeting; O2 supply the synthesis is relatively complex [67]
nitro imidazole modified IR-1048 dye NIR II PTT hypoxia-activated PTT; high imaging penetration depth relatively poor water solubility [73]
pyrene or TPE modified Cy7 NIR I PTT enhanced PCE; improved tumor-targeting low Φf; relatively poor water solubility [80]
hydrophilic quaternary stereo-specific cyanine and polypeptide based nanoparticles NIR II PTT increased water solubility; improved biocompatibility and PCE requires relatively high laser intensity [82]
tamoxifen modified cyanine NIR I PTT breast cancer targeted; enhanced tumor inhibition rate relatively low fluorescence penetration depth [86]
biotin modified cyanine NIR I PDT & PTT ratiometric fluorescence; tumor-targeting; pH activated phototherapy relatively low fluorescence penetration depth and photostability [102]
dimeric heptamethine cyanine with an aromatic diphenol linker NIR I PDT & PTT bright fluorescence, excellent
ROS generation capability; improved
photostability		 requires relatively high laser intensity; relatively poor water solubility [103]
nanoaggregates based on twistable TPE structure between two IR780s NIR I PDT & PTT tumor targeting; mitochondria targeted disassembly; high PCE; relatively poor water solubility [43]
crizotinib modified IR808 self-assembled with BSA NIR II PDT & PTT excellent biocompatibility and biosafety; colorectal cancer targeting requires relatively high laser intensity [116]

Fl., fluorescence; Ther. Mod., therapeutic modalities; Ref., reference; Φf, fluorescence quantum yield; TEMPO, 2,2,6,6-tetramethylpiperidinyloxy; ФΔ, singlet oxygen quantum yield; TPE, tetraphenylethene; Cy7, heptamethine cyanine; PCE, photothermal conversion efficiency.

Fig. 3. (a) Chemical structures of CY-C4, I-CY-C4, Br-CY-C4, and COOH-CY-C4. (b) Chemical structures of 6a and 6b.

Fig. 4. (a) Chemical structures of CyH, CyBro, CyBr, and CyI. (b) Chemical structures and reaction of CyBrP with ALP.

Fig. 5. (a) Chemical structure of dye 2. (b) Chemical structures of C2-R. (c) Fluorescence emission spectra of the various dyes in DCM. (d) Normalized DPBF absorbance decrease at 415 nm. (e) The reaction of C2-NO2 to C2-NH2 in the presence of NTR. (f) Fluorescence imaging of tumor-bearing mice after different treatments. (g) Relative tumor volume of mice over time after different treatments. Reprinted with permission from Ref. [60], copyright © 2023 The Authors. Advanced Science published by Wiley‐VCH GmbH.

Fig. 6. Chemical structures of (a) RhoSSCy and (b) HCL1-3.

Fig. 7. Chemical structures of Cy-NTR-CB and Cy-NH2 (a), FCy (b); Ru-Cyn-1, Ru-Cyn-2, Ru-Cyn-3 (c).

Fig. 8. (a) Chemical structures of Cy-830, NSCy-975, NSCy-980 and NSCy-1015 and their design stratagem; (b) The response mechanism of NSCy-1050; (c) Time-dependent whole-body NIR-II fluorescence imaging of 4T1 tumor-bearing mice after i. v. injection of NSCy-1050; (d) Ex vivo NIR-II fluorescence imaging of organs; (e) In vivo photothermal images of 4T1 tumor-bearing mice after laser irradiation various times; (f) Photographs of the tumors from different groups after therapy. Reprinted with permission from Ref. [70], copyright © 2023 Wiley‐VCH GmbH.

Fig. 9. (a) Chemical structure and response mechanism of IR1048-MZ; (b) Chemical structures of IR1-IR4 and the response mechanism of them.

Fig. 10. (a) Chemical structure of Cy-CO2Bz; (b) Chemical structures of Indocyanine and Quinoline cyanine; (c) Chemical structures of Cy7-TPE and Cy7-Pyrene; (d) Chemical structure of CY5-664; (e) Chemical structure of HQS-Cy.

Fig. 11. (a) Chemical structure of CyT, Cy and TAM; In vivo (b) and ex vivo (c) fluorescence imaging of tumor-bearing mice (b) and tumors (c) with different treatments. (d) Time-depended temperature changes at the tumor sites after laser irradiation. (e) Tumor weight of tumor-bearing mice with different treatment. Reprinted with permission from Ref. [86], copyright © 2020 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Fig. 12. (a) Chemical structure of Met-IR-782; (b) Chemical structure of response mechanism of IR-PY and its reaction in different pH; (c) Chemical structure of IR825-Cl.

Fig. 13. Chemical structure, photophysical and chemical properties of CydtPy, Mn2+-chelated CydtPy and Fe2+-chelated CydtPy. Reprinted from Ref. [92], copyright © 2023 Junfei Zhu et al. Distributed under a CC BY 4.0.

Fig. 14. Chemical structures of ICG (a), IR-52 and IR-83 (b), Cy-Bio-O (c), 26NA-NIR and 44BP-NIR (d).

Fig. 15. (a) Chemical structures of Icy-NBF and Icy-NH2. (b) Schematic illustration of O2-dependend energy dissipation. (c) Confocal imaging of calcein-AM and PI-labeled Hela cells with different treatments. (d) In vivo fluorescence imaging of tumor-bearing mice with Icy-NBF. (e) Photographs of mice in the different groups after 0, 12, and 24 days treatment. Reprinted with permission from Ref. [105] (b-e), copyright © 2020, American Chemical Society.

Fig. 16. (a) Chemical structure and its nanoaggregates of T780T; schematic illustration of the application of T780T nanoaggregates in tumor. Reprinted with permission from Ref. [43], copyright © 2021, American Chemical Society. (b) Chemical structure and its self-assembly of BTH-Cy7-TCF; schematic illustration of the application of BTH-Cy7-TCF NPs in tumor. Reprinted with permission from Ref. [110], copyright © 2023 Wiley‐VCH GmbH.

Fig. 17. Chemical structures of (a) BSS-Et and BAC808 (b). (c) The chemical structure of Crizotinib-IR808 and its self-assembly. Reprinted with permission from Ref. [116], copyright © 2023, American Chemical Society.

Table 2. Summary of fluorescence, therapeutic modalities, advantages and disadvantages of representative PSs among tetrapyrrole structures-based molecular or nanomaterials.

Photosensitizer Fl. Ther. Mod. Advantages Disadvantages Ref.
morpholine modified SiPc NIR I PDT (type I) pH activatable fluorescence; anti-hypoxic PDT; improved biocompatibility one-time treatment effect is relatively poor [121]
lipid-modified porphyrin-based nanomaterials NIR I PDT (type II) hypoxia relief; highly fluorescence; inhibit tumor growth and liver metastasis preparation of nanomaterials is relatively complicated [123]
DNBS and cRGD graft onto ZnPc NIR I PDT (type II) enhanced tumor targeting; activatable fluorescence and ROS relatively poor water solubility [131]
modifying biotin on SiPc NIR I PDT (type II) enhanced tumor targeting easy to aggregate [133]
4-sulfophenoxy mono-α-substituted ZnPc NIR I PDT (type II) turn-on fluorescence; tumor targeted imaging O2-dependent [147]
co-assembly of ZnPc and anti-cancer drug MA NIR I PDT (type II) nucleic acid-responsive fluorescence and ROS; improved anticancer effect O2-dependent [152]
self-assembly peptide modified ZnPc NIR I PDT & PTT photoactivity changes before and after transmembrane reduced fluorescence intensity [156]

Fl., fluorescence; Ther. Mod., therapeutic modalities; Ref., reference; SiPc, silicon phthalocyanine; ZnPc, zinc (II) phthalocyanine; DNBS, 2,4-dinitrobenzene sulfonic acid; cRGD, cyclic arginine-glycine-aspartic acid; MA, mitoxantrone.

Fig. 18. (a) Chemical structures of PcM and NanoPcM. Reprinted with permission from Ref. [121], copyright © 2022, American Chemical Society. (b) Chemical structures of porphyrin with O-linked cationic side chains. (c) The formation processes and biological applications of O2@PFOB@PGL NPs. Reprinted with permission from Ref. [123], copyright © 2020, American Chemical Society. (d) The formation processes of ATO/ZnPc-CA@DA. Reprinted with permission from Ref. [124], copyright © 2023 Elsevier Inc. All rights reserved.

Fig. 19. (a) Chemical structure of Ac-DEVDD-TPP and its self-assembly. Reprinted with permission from Ref. [125], copyright © 2023, American Chemical Society. (b) Chemical structure of 6. (c) Chemical structures of SiPc-biotin and compound 1.

Fig. 20. Chemical structures of P1, P2 (a) and Pt-1(b).

Fig. 21. (a) Chemical structure of PcS and its self-assembly. (b) In vivo fluorescence images of HepG2 tumor-bearing mice after post-injection of MB and NanoPcS. (c) Tumor growth of mice after various treatments. Reprinted with permission from Ref. [147], copyright © 2019, American Chemical Society. Chemical structures of PcN4 (d), ZnPcS8, ZnPcS4, ZnPcS2, ZnPcN4 and ZnPcN12 (e).

Fig. 22. Chemical structure of ZnPc-FF and its self-assembly. The spatiotemporally coupled photoactivity of PF self-assemblies is also presented. Reprinted with permission from Ref. [156], copyright © 2019 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Table 3. Summary of fluorescence, therapeutic modalities, advantages and disadvantages of representative PSs among BODIPY structures-based molecular or nanomaterials.

Photosensitizer Fl. Ther. Mod. Advantages Disadvantages Ref.
iodine disubstituted BODIPY modified erlotinib NIR I PDT (type II) high molar absorption coefficient and tumor inhibition rate; EGFR-positive tumors target O2-dependent PDT [162]
covalently connected cationic rhodamine and diiodo-substituted BODIPY NIR I PDT (type II) high tumor targeting; increased light-harvesting ability; mitochondrial anchoring ability O2-dependent PDT; the tendency for dark toxicity [164]
dibromo-substituted BODIPY grafted acetazolamide NIR I PDT (type II) relieve hypoxia; CAIX-overexpressing tumor cells targeting; inhibit tumor angiogenesis synthesis steps are relatively complex [167]
thienopyrrole-fused BODIPY NIR I PDT (type II) increased extinction coefficient and ФΔ relatively low fluorescence penetration depth [170]
multipolar triphenylamine-BODIPY two photon PDT (type I and II) higher ФΔ and better Φf; enhanced imaging depth relatively poor water solubility [173]
glycosylated Aza-BODIPY self-assembled into nanofibers NIR I PDT (type I) tumor-targeted ability; anti-hypoxia; long retention; cell membrane damage relatively low fluorescence penetration depth [51]
BODIPY modified with trifluoromethyl and CPT NIR I PTT improved PCE; enhanced biocompatibility synthesis steps are relatively complex [178]
morpholine modified aza-BODIPY based nanoparticles NIR I PDT & PTT increased water solubility; high cytotoxicity for tumor cells; rapid metabolic kinetics relatively poor tumor targeting ability [181]
rigid coplanar aza-BODIPY modified tetrastyrene NIR II PDT & PTT enhanced imaging penetration depth; improved PCE and ROS generation; unable to inhibit tumor metastasis and recurrence [186]

Fl., fluorescence; Ther. Mod., therapeutic modalities; Ref., reference; EGFR, epidermal growth factor receptor; CAIX, carbonic anhydrase IX; ФΔ, singlet oxygen quantum yield; Φf, fluorescence quantum yield; CPT, camptothecin; PCE, photothermal conversion efficiency.

Fig. 23. (a) Chemical structure of CatER. (b) In vivo whole-body fluorescence imaging of tumor-bearing mice after injection of BDP or CatER. (c) Tumor growth of mice after various treatments. Reprinted with permission from Ref. [162] (a-c), Copyright © 2022 the Author(s). Published by PNAS. Distributed under CC BY-NC-ND 4.0 DEED. Chemical structure of MBDP (d), RDM-BDP (e), pH-BODIPY (f), and AZ-BPS (g).

Fig. 24. Chemical structure of SBDPiR688 (a), Lyso-BDP (b), and (c) T-BDPn (n = 1, 2, 3). (d) Chemical structure of LMBP. (e) Chemical structure of BY-I2, BY-I8, BY-I12, BY-I16, BY-I18 and their nanoform. Reprinted with permission from Ref. [176], copyright © 2023 Wiley‐VCH GmbH.

Fig. 25. Chemical structure of BAC.

Fig. 26. Chemical structure of MAB (a), (b) TAB, TAB-I, and TAB-Br; (c) BDPN, BDPI, BDPC, and BDPJ; (d) CCNU-1060; (e) BDP 2 − 7.

Table 4. Summary of fluorescence, therapeutic modalities, advantages and disadvantages of representative PSs among AIEgens-based molecular or nanomaterials.

Photosensitizer Fl. Ther. Mod. Advantages Disadvantages Ref.
tetrastyrene derivative combined with 1O2-cleavable AA and cRGD NIR I PDT (type II) high tumor targeting; monitoring of 1O2 generation; early evaluation of the therapeutic effect synthesis steps are relatively complex; no in vivo applications [192]
alkoxyl-branched TPE modified with Br NIR I PDT (type I and II) membrane staining; efficient ROS generation relatively poor in vivo application [196]
N,N-dimethyl-substituted TPE modified with 4-vinylpyridine NIR II PDT (type I) relieve hypoxia; cancerous mitochondria-targeting; hybrid apoptosis and ferroptosis unable to inhibit tumor metastasis and recurrence [202]
TPA skeleton modified with pyridine and biotin NIR I PDT (type I and II) large stokes shift; tumor cells and mitochondrial targeting; sufficient ROS production relatively low fluorescence penetration depth [207]
TPA skeleton modified with quinolinium salt NIR I PDT (type I) time-dependent subcellular organelles target; excellent type I ROS generation relatively low fluorescence penetration depth [208]
TPA combined with thiophene and benzothiazole 2-acetonitrile NIR I PDT (type I and II) lipid droplets detection; high bioactivity and stability; enhanced ROS generation relatively poor water solubility [213]
5,5′-(6,7-diphenyl-[1,2,5]thiadiazolo[3,4–g]quinoxaline-4,9-diyl)bis(4-hexyl-N,N-bis(4-methoxyphenyl)thiophen-2-amine) NIR II PTT increased fluorescence penetration depth and control of PCE; enhanced photostability and tumor targeting the construction of nanomaterials is more complicated [222]
6, 7-diphenyl-[1,2,5]thiadiazolo[3,4g]quinoxaline as the acceptor, thiophene as the π bridge, phenothiazine serves as the acceptor, and a triphenylamine rotor donor is added to the phenothiazine NIR II PTT highest PCE (73.32 %); increased fluorescence penetration depth; noticeable therapeutic efficiency and biocompatibility relatively poor water solubility; complex synthesis steps [223]

Fl., fluorescence; Ther. Mod., therapeutic modalities; Ref., reference; AA, aminoacrylate; cRGD, cyclic arginine-glycine-aspartic acid; TPE, tetraphenylethylene; TPA, triphenylamine; PCE, photothermal conversion efficiency.

Fig. 27. Chemical structure of TPECM-1TPP, TPECM-2TPP (a), (b) TPE-TThPy, TPE-TPys; (c) TPTB; (d) TPE-PTB.

Fig. 28. (a) Chemical structure of TPEQM-DMA. (b) 1O2 detection by using SOSG as probe. (c) OH• detection by using HPF as probe. (d) The live/dead cell staining assays under normoxic and hypoxic conditions. (e) In vivo NIR-I and NIR-II fluorescence imaging of tumor-bearing mice after injection of TPEQM-DMA. (f) Tumor growth of mice after various treatments. Reprinted with permission from Ref. [202], copyright © 2023, American Chemical Society.

Fig. 29. Chemical structure of (a) TPATrzPy-3+; (b) DCMT; (c) TBTCP; (d) TSBPy-OH; (e) TSPy-B; (f) CTQ-S; (g) MTOTPy; (h) ADB; (i) TTVP; (j) MeTTSN; (k) DPP-BPYS.

Fig. 30. Chemical structure of (a) DCMa, DCls, DCPy and DCFu; (b) DSABBT; (c) DPpy, DMPpy and DMPSI.

Fig. 31. (a) Chemical structure of NHTDP and nanostructure of NHTDP@M. (b) Photothermal effect and photostability of DHTDP NP@M (c) In vivo NIR-II fluorescence imaging of tumor-bearing mice after injection of DHTDP NP and DHTDP NP@M. (d) Time-depended photothermal imaging in vivo. (e) Tumor growth of mice after various treatments. Reprinted with permission from Ref. [222], copyright © 2023 Wiley‐VCH GmbH.

Fig. 32. (a) Chemical structure of TPTQ and nanostructure of TPTQ NPs. (b) Photothermal effect TPTQ NPs (c) In vivo NIR-II fluorescence imaging of tumor-bearing mice after injection of TPTQ NPs. (d) Time-depended photothermal imaging in vivo. (e) Tumor growth of mice after various treatments. Reprinted with permission from Ref. [223], copyright © 2023 Elsevier B.V. All rights reserved.

7. Conclusions and perspectives

       In this review, we summarized the organic molecules inculding cyanines, porphyrins, phthalocyanines, BODIPY, and AIEgens or nanoaggregates and nanocomposite based on these organic molecules for fluorescence imaging-guided phototherapy. Special attention is paid to the strategy of visual elimination of tumors through molecular structure modification, controlled aggregation, and assembly of functionalized nanomaterials. Obviously, this integrated approach to diagnosis and treatment demonstrates the huge potential for clinical transformation of precision tumor treatment. However, fluorescence imaging-guided phototherapy for tumor elimination still faces great challenges: (1) There is currently no fully mature method that can accurately control the fluorescence, photodynamic or photothermal intensity of all kinds of PSs. The strategies used by different PSs molecules are not completely consistent, and the design methods need to be adjusted according to the structure. (2) Maintaining photostability of fluorescence after continuous high-intensity output is a huge challenge. Meanwhile, fluorescence imaging will also face interference from background. To solve the above problems, some afterglow luminescent structures have been studied. Such structures store the energy of the illuminated light and then slowly emit photons. Song et al. have achieved tumor glycolysis and chemotherapy resistance by constructing afterglow structure; [224] in addition, this kind of materials can also quantify and image targets such as pH, superoxide anion, and aminopeptidase [225]. (3) Quantitatively evaluating the accuracy of detecting early tumor lesions through fluorescence intensity and the effectiveness of phototherapy is still limited. (4) The problem of penetration depth. There is an urgent need to further expand the fluorescence emission wavelength so that it can be used for the visual treatment of deep-seated solid tumors. Currently, photoacoustic imaging is favored due to its deeper penetration depth [226]. In addition to tumors, diseases such as atherosclerosis can also be diagnosed early through photoacoustic imaging [227], [228]. (5) Phototherapy has the disadvantage of locality, and metastatic tumors are difficult to detect or treat through this method. In this case, phototherapy needs to be used in conjunction with systemic treatments such as chemotherapy and immunotherapy to enhance the therapeutic effect of metastatic tumors.

Collectively, the use of fluorescence-imaging guided phototherapy for anti-tumor is a promising strategy. This review summarizes the progress made in this field in recent years as well as the opportunities and challenges faced. We believe that with the continuous efforts of scientific researchers and clinicians, fluorescence imaging-guided phototherapy will become a prominent feature in the field of precision tumor treatment in the future.

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Triphenylamine-AIEgens Photoactive Materials for Cancer Theranostics

Triphenylamine-AIEgens Photoactive Materials for Cancer Theranostics

 

Junjie Wang【王俊杰】c#, Yan Wanga,b#, Zhengdong Lie#, Changqiang Xiea,b, Mussmmir Khanf, 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 ∆Est 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.

Name Absorption/

Emission

 Target Diagnosis Therapeutics Tumor model Ref
β-TPA-PIO 405 nm/543nm Endoplasmic reticulum FLI PDT SKOV3 subcutaneous tumor model [6]
tri(3-pyridylphenyl)amine 405 nm/

480-580 nm

 Nucleus FLI PDT HeLa xenograft tumor model [58]
MeTPAE 405 nm/

600?0 nm

 Nucleus FLI PDT 4T1 subcutaneous tumor model [59]
TBMPEI 465 nm/780 nm Cell membrane FLI PDT 4T1 subcutaneous tumor model [61]
TPANPF6 405 nm/

520-650 nm

 Mitochondrion FLI PDT / [63]
TPATrzPy-3+ 405 nm/

570-650 nm

 Mitochondrion FLI PDT Zebrafish model of liver tumor [64]
T-BDP 363, 631 nm/

724 nm

 Lysosome FLI PDT+PTT / [69]
DTPAP-P 456 nm/543 nm Nucleus FLI PDT / [71]
TPA-DHPy-Py 488 nm/

600-700nm

 Endoplasmic reticulum and lipid droplets FLI PDT MDA-MB-231 subcutaneous model [72]
BDPTPA 808 nm/- Tumor PAI PDT+PTT 143B xenograft tumor model [74]
TPA-T-TQ 780 nm/- Tumor PAI PTT 4T1 subcutaneous tumor model [75]
Ce6@HA-Cys-TTDTT 635 nm/

700-750 nm

 Tumor NIR FLI PDT+PTT HeLa subcutaneous tumor model [76]
TPA-DPPy 405 nm/- Saturated fatty acids and mitochondrion FLI PDT / [82]
TBP-Au 488, 870 nm/

575-630nm

 Lysosome and lipid droplets 2P- FLI PDT HeLa subcutaneous tumor model [85]
TPA-2PI 960 nm/

600-800 nm

 DNA and?mitochondrion 2P- FLI PDT 4T1 subcutaneous tumor model [86]
CyQN-BTT 671, 800 nm/

900-1100 nm

 Tumor NIR-II FLI+PAI PDT+PTT 4T1 subcutaneous tumor model [91]
MeO-TPA-TPP 455 nm/

550-620 nm

 Mitochondrion FLI PDT 4T1 breast tumor model [97]
TPA-DHPy 488 nm/

500-650 nm

 Endoplasmic reticulum and lipid droplets FLI PDT HeLa subcutaneous tumor model [98]
PQ-TPAOC1 NPs 660 nm/- Tumor / PDT+PTT 4T1 tumor model [105]
TPA-TDPP 638 nm/743 nm Tumor FLI PDT+PTT HeLa tumor model [106]
tTDCR 488 nm/627 nm Tumor FLI PDT+immuno-

therapy

 4T1 subcutaneous tumor model [109]
TPA-DCR 460 nm/

620?5 nm

 / FLI PDT+immuno-

therapy

 4T1 subcutaneous tumor model [110]
α-Th-TPA-PIO 465 nm/660 nm Endoplasmic reticulum FLI PDT+immune-

therapy

 B16-F10 subcutaneous tumor model [111]
Platinum(II) Triphenylamine 405 nm/

620 ?20 nm

 Mitochondrion FLI PDT+immune-

therapy

 U14 subcutaneous tumor model [112]
DCP-PTPA 680 nm/- Tumor PAI / 4T1 xenograft tumor model [120]
BOPHY-2TPA 561 nm/

630-690 nm

 HeLa cell FLI PDT / [121]

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

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.

Abstract Image

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: λex = 561 nm, λem = 650 – 730 nm and blue channel (DAPI): λex = 405 nm, λem = 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): λex = 561 nm, λem = 650 – 730 nm and blue channel (DAPI): λex = 405 nm, λem = 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: λex = 561 nm, λem = 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: λex = 561 nm, λem = 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.

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

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 Yua, Gang Zhanga, 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
Cite this: Anal. Chem. 2024, 96, 8, 3600–3608Full Text

Publication Date:February 19, 2024

https://doi.org/10.1021/acs.analchem.3c05718

Copyright © 2024 American Chemical Society

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.

Abstract Image

摘要

图片图片图片

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

图片图片图片

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, λex = 340 nm), and (b) the plot of fluorescent intensity of HBT-Fl (10 μM) at 584 nm vs. water fractions (%, fw) in the PBS buffer-THF mixture, λex = 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, λex = 340 nm). Other interfering analytes: ONOO‒, 1O2, ·OH, HOCl, H2O2, NO, TBHP, GSH, L-Cys, Sec, Cys-SSH, Fe3+, Zn2+, F‒, I‒, SO42‒, NO2‒, HCO3‒, CO32‒ and S2O32‒.

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: λem= 415-515 nm, 583ch: λem=550-650 nm. Ratio images generated from 583ch/430ch. λex=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: λem= 415-515 nm, 583ch: λem=550-650 nm. Ratio images generated from 583ch/430ch. λex=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: λem= 415-515 nm, 583ch: λem=550-650 nm. Ratio images generated from 583ch/430ch. λex=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: λem= 415-515 nm, 583ch: λem=550-650 nm. λex=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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GSK343 modulates macrophage M2 polarization through the EZH2MST1YAP1 signaling axis to mitigate neurological damage induced by hypercalcemia in CKD mice

GSK343 modulates macrophage M2 polarization through the EZH2MST1YAP1 signaling axis to mitigate neurological damage induced by hypercalcemia in CKD mice

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Associated Detection of Superoxide Anion and Mercury(II) under Chronic Mercury Exposure in Cells and Mice Models via a Three-Channel Fluorescent Probe

Analytical Chemistry, 2018, 90, 16, 9769-9778Full text

Wang, Yue;Gao, Min ; Chen, Qingguo ;Yu, Fabiao*; Jiang, Guibin ; Chen, Lingxin*

https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b01442

 

Abstract

As a cytotoxic heavy metal ion, mercury(II) ion (Hg2+) induces severe oxidative stress and further results in physiological dysfunction. Although mercury poisoning can be treated with many drugs, such as sodium selenite, the therapeutic effect is relatively poor, and it seems that the damage to human health continues. However, the interpretation for the pathogenesis has not been clarified yet. We supposed that the reason is attributed to Hg2+-caused intracellular oxidative stress. To confirm our hypothesis, we strived to design a three-channel ratio fluorescent probe, HCy–SeH, for superoxide anion (O2•–) and Hg2+combined detection. O2•– is a vital precursor for other reactive oxygen species (ROS), which is involved in many physiological and pathological processes. However, until now there is no efficient chemical tool for O2•– and Hg2+ combined detection in cells and in vivo. The fluorescence response of our probe is initiated by a hydrogen abstraction reaction from the hydrocyanine fluorophore moiety. Once oxidized by O2•–, HCy–SeH recovers its π-conjugated system back to a heptamethine cyanine derivative, Cy–SeH. Cy–SeH coexists with its conjugate base, Cy═Se. One emits red fluorescence, and the other one emits green fluorescence. The response unit, −SeH, can trap Hg2+ via a Se–Hg antagonism reaction to afford an orange-emitting final product, Keto–Cy. The probe offers high selectivity and sensitivity toward O2•– and Hg2+. When applied for O2•– and Hg2+detection in HEK 293 cells, the imaging results indicate that our probe can provide a combined response for O2•– and Hg2+ in real time and in situ. Flow cytometry analysis is well-consistent with the results from fluorescence imaging. When applied to image O2•– and Hg2+ in mice models, we find that Hg2+dominantly accumulates in the kidney and induces a burst of O2•–. We confirm that chronic mercurialism can cause severe oxidative damage and renal fibrosis. HCy–SeH further provides a new information that, even when intracellular Hg2+ has been antagonized, the outbreak of O2•– caused by mercury poisoning still lasts.

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