Constructing Intramolecular Electric Fields in NIR-II-Emissive Photosensitizers to Regulate the Local Electron Density for Boosting Hypoxia-Tolerant Cancer Phototheranostics
2025; Chinese Chemical Society; Linguagem: Inglês
10.31635/ccschem.024.202405072
ISSN2096-5745
AutoresXiaoming Hu, Cheng Zhang, Jingqi Lv, Rongtian Li, Achen Qin, Caijun Zhu, Fengwei Sun, Zejing Chen, Shenghan Teng, Hongxin Lin, Zhèn Yáng, Wei Huang,
Tópico(s)Advanced Nanomaterials in Catalysis
ResumoOpen AccessCCS ChemistryRESEARCH ARTICLES17 Jan 2025Constructing Intramolecular Electric Fields in NIR-II-Emissive Photosensitizers to Regulate the Local Electron Density for Boosting Hypoxia-Tolerant Cancer Phototheranostics Xiaoming Hu†, Cheng Zhang†, Jingqi Lv, Rongtian Li, Achen Qin, Caijun Zhu, Fengwei Sun, Zejing Chen, Shenghan Teng, Hongxin Lin, Zhen Yang and Wei Huang Xiaoming Hu† College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 School of Materials Science and Engineering, East China Jiaotong University, Nanchang 330013 , Cheng Zhang† College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 , Jingqi Lv College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 , Rongtian Li Department of Clinical Pharmacy, Southern University of Science and Technology Hospital, Shenzhen 518055 , Achen Qin School of Materials Science and Engineering, East China Jiaotong University, Nanchang 330013 , Caijun Zhu School of Materials Science and Engineering, East China Jiaotong University, Nanchang 330013 , Fengwei Sun College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 , Zejing Chen School of Materials Science and Engineering, East China Jiaotong University, Nanchang 330013 , Shenghan Teng College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 , Hongxin Lin *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 , Zhen Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 and Wei Huang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] College of Photonic and Electronic Engineering, Strait Institute of Flexible Electronics (SIFE, Future Technologies), Fujian Key Laboratory of Flexible Electronics, Fujian Normal University and Strait Laboratory of Flexible Electronics (SLoFE), Fuzhou 350117 Frontiers Science Center for Flexible Electronics, Xi'an Institute of Flexible Electronics (IFE), Northwestern Polytechnical University, Xi'an 710072 https://doi.org/10.31635/ccschem.024.202405072 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookXLinked InEmail Recently emerging Type-I photodynamic therapy holds significant promise in addressing the hypoxia challenge encountered by traditional oxygen-dependent Type-II photosensitizers (PSs) in photodynamic oncotherapy. The key hurdle in engineering Type-I PSs lies in enhancing the electron transfer capabilities of these molecules, enabling them to efficiently convert H2O or oxygen-based substrates into reactive oxygen species. Herein, we propose to construct intramolecular electric fields in the second near-infrared (NIR-II) emissive organic PSs to regulate the local electron density for boosting hypoxia-tolerant cancer phototheranostics. Upon introducing the molecular cationization approach and electron programming strategy, the resultant cationic semiconducting architecture achieves electron distribution rearrangement, forms intramolecular electric fields, and facilitates electron transfer path, resulting in a 50-fold amplification of electrostatic potential difference, thereby accelerating the hydroxyl radical (•OH)-dominant Type-I photosensitization process. In vitro studies disclose that the tailor-made nanomaterial selectively targets the mitochondria and causes mitochondria-mediated cancer cell apoptosis under laser irradiation. Notably, this as-prepared nanoplatform enables NIR-II fluorescence imaging-assisted phototherapy and exhibits in vivo antitumor efficacy on 4T1-bearing mouse models. We believe that this contribution will launch the future of NIR-II emitting Type-I PSs and enlighten scientific researchers to exploit high-efficiency phototheranostic agents for cancer therapy. Download figure Download PowerPoint Introduction The emergence of phototheranostics provides new insights into malignant tumor treatment and powerful superiorities of real-time diagnosis and in situ phototherapeutic properties.1–4 Thereinto, photodynamic therapy (PDT) as a promising precision medicine technology has bred great potential for cancer therapy due to the preponderance of excellent controllability, noninvasiveness, and satisfactory anticancer efficiency.5–7 PDT utilizes a photosensitizer (PS) that, when paired with molecular oxygen and exposed to a light source, generates cytotoxic reactive oxygen species (ROS), thereby causing the death of tumor cells.8,9 As the key element of the photosensitization system, the exploration of a potent and efficient PS featured with powerful ROS generation within a neoplastic region is of great significance.10,11 However, the current antitumor system is extremely compromised because of the inherent hypoxic microenvironment of the tumor area and the deficient ROS generation of existing PSs.12–16 To overcome the intrinsic limitation of conventional photosensitization systems, efforts have been increasingly focused on the design and development of hypoxia-tolerant photosensitization nanoplatforms.17–20 Over the past few years, the primary approach for tackling the hypoxia challenge has primarily involved enhancing the oxygen content within tumor areas.21 Up to now, numerous innovative approaches have emerged, focusing on the delivery of O2-generating reagents or O2 carriers,22 encompassing metal oxide catalysts,23,24 biomimetic red blood cells,25 and fluorinated nanosystems.26 Nevertheless, these designs are constrained by numerous deficiencies, including intricate compositions and uncontrollable spatiotemporal factors.27 In order to overcome the reliance on tumor-endogenous O2, Type-I PS has emerged as an innovative photosensitive system that dramatically reduces the need for intratumoral O2 via an electron transfer pathway between activated PSs and nearby substrates,28,29 including molecular oxygen,30 water,31 RNA,32,33 proteins,34 and redox species.35 Heretofore, several Type-I PSs have been reported to be capable of transferring electrons to adjacent intracellular redox substrates and show prominent PDT efficacy.36–40 For instance, we recently proposed a feasible strategy for improving Type-I photodynamic pathway by boosting the separation of electron-hole pairs of PSs.41 Despite the progress made in the development of Type-I PSs, most of the reported photosensitization systems have been discovered by chance, and there is a notable absence of clear and feasible design directives. Therefore, there is an urgent need to develop an available design strategy to guide the engineering and preparation of novel Type-I PSs for advancing the PDT territory. The internal electric field of inorganic nanomaterials can significantly affect their optical, electrical, and magnetic properties, which can be optimized for biomedical applications.42 For example, Bu and coworkers proposed a strategy for long-lasting chemodynamic therapy of tumors by constructing a built-in electric field to drive directional electron migration in a heterojunction.43 Therefore, the internal electric field theory conduces to understanding the separation and transport mechanisms of photogenerated electrons and holes, thereby optimizing the electron transfer efficiency of materials.44–46 Inspired by inorganic nanomaterials, organic semiconducting nanomaterials possess great potential to construct intramolecular electric fields for boosting Type-I photosensitization pathways, due to the modifiable molecular structure and controllable electron distribution.47–52 Therefore, it is crucial to manage the local electron density of PSs to achieve intramolecular dipole moments to promote electron transfer for enhancing the Type-I PDT pathway. Therefore, we propose to build the intramolecular electric fields in a second near-infrared (NIR-II) emissive Type-I PS by adjusting the electron-deficient skeleton and introducing molecular cationizations with the capability of targeting the cellular mitochondria for highly efficient phototheranostics (Scheme 1). In this contribution, we legitimately design, synthesize, and compare three NIR-II-emissive organic semiconducting small molecules consisting of similar electron-deficient skeletons and electron-rich units. Firstly, an electron-deficient thiadiazoloquinoxaline (TQ) was paired with two electron-rich triphenylamine (TPA) units, affording a donor (D)-acceptor (A)-D structured organic semiconducting molecule (named as 4,4′-((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N,N-bis(4-methoxyphenyl)aniline) (OTT)) featured with photothermal therapy (PTT) effects and NIR-II fluorescence/photoacoustic (PA) signals but invalid PDT. Subsequently, the introduction of four pyridine moieties towards the semiconducting skeleton 4,4′-((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N,N-bis(4-(pyridin-4-yl)phenyl)aniline) (BTT) enables amplification of the PTT effect and NIR-II fluorescence/PA signal in comparison with OTT, but exhibits weak PDT efficacy. Finally, ionization of the pyridine moieties into methylpyridinium cation affords BTT+, which achieves electron distribution rearrangement and forms intramolecular electric fields, resulting in a 50-fold amplification of electrostatic potential (ESP) difference. Such high ESP difference is beneficial to the electron separation and transfer path, thus accelerating the free radical generation for PDT.53–55 Interestingly, such cationization enhances the electron-accepting capacity of the cationic unit and promotes the NIR-II emission signal. Thus, BTT+ nanoparticles (NPs) exhibited NIR-II fluorescence imaging (FLI), photoinduced predominant hydroxyl radical (•OH) production, photothermal effects, and mitochondria targeting abilities. The all-in-one phototheranostic agent, BTT+ NPs, has been shown preeminent cancer phototherapeutic efficacy assisted with in vivo photoacoustic imaging (PAI) and NIR-II FLI. In a nutshell, the tailored BTT+ will pave the way for the long-wavelength Type-I PSs toward promoted cancer phototheranostics. Scheme 1 | Schematic representation of constructing intramolecular electric fields in NIR-II-emissive PSs to regulate the local electron density for boosting hypoxia-tolerant cancer phototheranostics. Download figure Download PowerPoint Experimental Methods Materials and reagents Amphiphilic polymer distearoyl phosphatidylethanolamine-polyethylene glycol (MW 5000) (DSPE-PEG5000), hydroxyphenyl fluorescein (HPF), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), dichlorodihydrofluorescein (DCFH), singlet oxygen sensor green (SOSG), dihydrorhodamine 123 (DHR123), aminophenyl fluorescein (APF), dihydroethidium, calcein acetoxymethyl ester (Calcein AM), propidium iodide (PI), and other chemicals were bought from Sigma-Aldrich (Saint Louis, Missouri, United States), except as otherwise mentioned, and adopted directly. Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Shanghai, China). The ultrafiltration tube (M.W. 10k) was purchased from Beyotime Biotechnology (Shanghai, China). Instrument and characterization The optical absorption spectra were collected on a UV–vis–NIR spectrophotometer (Shimadzu, Kyoto, Japan; UV-3600). NIR-II fluorescent spectra were determined by an FL spectrometer FLS980 (Edinburgh Instrument, Livingston, West Lothian, United Kingdom) with an external tunable 808 nm laser. Cyclic voltammetry assay was performed with a DH7000C electrochemical workstation (Jiangsu Donghua Analytical Instrument Co., Ltd., Jingjiang, Jiangsu, China). The in vivo and in vitro NIR-II FLI were acquired from an NIR-II in vivo imaging system (Wuhan Grand-imaging Technology Co., Ltd., Wuhan, China) equipped with a thermoelectric cooling InGaAs camera (Princeton Instruments Company, Trenton, New Jersey, United States). PAI was conducted on the Vevo LAZR-X PA imaging system (Visual-Sonics Co. Ltd, Toronto, Canada). Synthesis of BTT+ The synthetic route of BTT+ utilizing intermediates sourced from literature is depicted in Supporting Information Scheme S1. The OTT, BTT, and BTT+ were well identified by nuclear magnetic resonance (NMR) spectra and mass spectrometry ( Supporting Information Figures S1–S11). Preparation of nanoparticles OTT NPs and BTT NPs were prepared through nanoprecipitation of OTT, BTT, and amphiphilic DSPE-PEG5000 in a vigorously stirred mixture of water and tetrahydrofuran. BTT+ NPs were produced through nanoprecipitation of BTT+ and amphiphilic DSPE-PEG5000 in a mixture of water and acetonitrile (MeCN) under vigorous stirring. The aqueous solutions obtained were further refined using a centrifugal filter with a 10 kDa molecular weight cutoff to eliminate unattached DSPE-PEG5000 and excess impurities. Lastly, the NPs obtained were resuspended in phosphate buffer saline (PBS) and filtered through a 0.22 μm Millipore membrane for subsequent in vitro and intravital experiments. Detection of ROS generation In this work, a DCFH fluorescence probe was used to detect total ROS generation. The compound APF served as an indicator for detecting •OH radicals in solution. Upon the generation of •OH radicals in the system, APF undergoes oxidation and produces intense fluorescence with a peak at 514 nm. Compound DHR123 functioned as an indicator for detecting O2•− in solution. Upon the production of O2•− in the system, DHR123 undergoes oxidation and emits intense fluorescence with a peak wavelength of approximately 527 nm. The generation of 1O2 in solution was monitored using SOSG by observing its photoluminescence intensity. The Supporting Information provides details on the detection of ROS (containing •OH, O2•−, and 1O2) generation. Computational methods All calculations were carried out utilizing the Gaussian 09 software suite. The geometries of OTT, BTT, and BTT+ were optimized using DFT/B3LYP/6-311G(d). Based on the optimized structure, the energy calculation and electron cloud distribution were forecasted using the TDDFT/B3LYP/6-311G(d) method. The ESP distribution was obtained from the wave function computed using the B3LYP/6-311+G(2d,p) method. NIR-II fluorescence imaging and PAI assays We created an aqueous dispersion of BTT+ NPs and dispensed it into 500 μL polymerase chain reaction tubes, while saline served as the control. Subsequently, they were imaged under the NIR-II in vivo imaging system with the collected wavelength at 900–1500 nm. The intravital NIR-II FLI was performed in 4T1 tumor-bearing mice using the same procedure. In addition, in vivo PAI experiments were conducted with the assistance of a PA scanner and operational tools. The images and data were acquired using the excitation wavelength corresponding to the absorption peak. Results and Discussions Preparation and characterization In this work, we first chose the electron-rich TPA and electron-deficient TQ as the donor and acceptor, respectively, for engineering three D-A-D-type scaffolds (named OTT, BTT, and BTT+) featured with NIR absorption and NIR-II emission through the C–C coupling reaction.56 Pyridine groups and the cationization approach were introduced to further optimize the electron distribution and intersystem crossing process of the molecular framework.57 The detailed synthetic process and basic characterization of these three architectures are exhibited in the Supporting Information. The successful preparation of targeted molecules was determined by 1H NMR spectroscopy, 13C NMR spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and optical spectroscopy ( Supporting Information Figures S1–S12). These organic semiconducting molecules exhibited characteristic NIR optical absorption and NIR-II emission properties, which were attributed to the extremely conjugated frameworks with enhanced electron delocalization along the bulky scaffolds. Considering the hydrophobicity of the targeted products, an amphiphilic polymer, DSPE-PEG5000, was employed for the noncovalent functionalization of these three tailor-made molecules by a nano-coprecipitation approach, enabling aqueous dispersion and biocompatibility ( Supporting Information Figure S13). The transmission electron microscopy (TEM) imaging and dynamic light scattering (DLS) testing demonstrated that the BTT+ NPs were uniformly distributed, exhibiting an average hydrodynamic diameter of about 65 nm (Figure 1a,b). The nanoplatform's appropriate nanosize facilitated its intratumoral accumulation due to the enhanced permeation and retention (EPR) effect. Similarly, OTT NPs and BTT NPs had a uniformly distributed range of nano-sizes, with hydrodynamic diameters of approximately 55 and 60 nm, respectively ( Supporting Information Figure S14). Besides, the zeta potentials of these three OTT NPs, BTT NPs, and BTT+ NPs were determined as −13.33, −7.56, and −1.13 mV, respectively (Figure 1c), indicating that the BTT+ possessed a positive charge to neutralize the negative charge of the polyethylene glycol shell, providing the potential to target mitochondria.58 Figure 1 | (a) TEM of BTT+ NPs. (b) DLS size of BTT+ NPs. (c) Zeta potential of OTT NPs, BTT NPs, and BTT+ NPs. (d) Normalized absorption spectra of OTT NPs, BTT NPs, and BTT+ NPs in aqueous solution. (e) Normalized fluorescence spectra of OTT NPs, BTT NPs, and BTT+ NPs in aqueous solution (808 nm light excitation). (f) NIR-II fluorescence images and corresponding intensities of OTT NPs, BTT NPs, and BTT+ NPs with the same molar content. (g) Comparison of photothermal properties of OTT NPs, BTT NPs, and BTT+ NPs after photoirradiation (808 nm, 1 W cm−2) for 6 min. (h) The temperature variation curves of OTT NPs, BTT NPs, and BTT+ NPs after photoirradiation (808 nm, 1 W cm−2) for 6 min. (i) The photothermal cyclic curve of BTT+ NPs (808 nm, 1 W cm−2). Download figure Download PowerPoint Subsequently, the optical properties of these tailoring nanoplatforms were further investigated. The dimethylformamide (DMF) solutions of OTT, BTT, and BTT+ absorb a maximum signal at 791, 755, 731 nm and emit in the NIR-II region with fluorescence peaks at 1116, 1082, and 1056 nm, respectively ( Supporting Information Figure S12). These three tailor-made organic chromophores possessed excellent NIR absorption in aqueous solution, with molar absorption coefficients, ε, of 1.67 × 104, 2.29 × 104, and 2.12 × 104 M−1 cm−1, respectively, at their NIR absorption peaks, and 5.02 × 104, 9.66 × 104, and 12.73 × 104 M−1 cm−1 at their maximum absorption points, respectively ( Supporting Information Figure S15). The remarkable light utilization ability within the NIR region of these molecules confers significant superiority for their deep-tissue biological practices. It is worth noting that the BTT and BTT+ showed absorption/emission blue-shift effects compared with the counterpart OTT with the introduction of pyridine groups and cationization effect. The electrophilic methylpyridinium groups could cause the electron redistribution of the delocalized structure and shift the D-A-D-type structure to A'-D-A-D-A' skeleton, thus reducing the intramolecular charge transfer (ICT) effect of the whole conjugated skeleton and generating the blue-shift phenomenon. Intriguingly, the BTT+ NPs in the aqueous solution possessed a longer wavelength absorption than BTT NPs (Figure 1d), which was opposite to the optical absorption observed in DMF solvents. The bathochromic-shift absorption of BTT+ NPs in the aqueous solution was mainly attributed to the formation of strong intermolecular electrostatic interactions in an aggregate state. Besides, the broader fluorescent emission of BTT+ NPs further indicated the generation of the aggregate state (Figure 1e). Then, the NIR-II FLI assays were recorded under a 1000 nm long-pass (LP) filter with laser radiation of 808 nm. Results indicated that the fluorescence intensity of OTT, BTT, and BTT+ gradually increased, which was opposite to the shorter emissive wavelength (Figure 1f). Taking advantage of IR26 as a reference (QY = 0.5% at dichloroethane, Supporting Information Figure S16), the quantum yields (QY) of the DMF solution were investigated and calculated to be 0.038%, 0.34%, and 0.51%, respectively.59 Based on these results, we deem that the excited state of longer-wavelength emissive fluorophores is much more likely to be invalidated through vibration or rotation paths, thus causing the lower QY.60 Furthermore, to deeply explore the aggregation behavior of BTT+ NPs, we collected changes in the absorption spectra of BTT+ in water/MeCN mixtures at different water fractions (fw). Results demonstrated that with the increase of fw, the absorption spectra of BTT+ exhibited a red-shift trend ( Supporting Information Figure S17a). Compared to the BTT MeCN solution, BTT+ NPs in aqueous solution exhibited an absorption red shift of 47 nm ( Supporting Information Figure S17b), which indicated the J-aggregation potential of BTT+ NPs. Besides, BTT+ NPs in aqueous solution showed a brighter NIR-II fluorescent signal than in MeCN solution, and the fluorescent brightness of BTT+ nearly exhibited a decreasing trend with the increase of fw ( Supporting Information Figure S18), which manifested the typical NIR-II fluorescent behavior of D-A-D-type chromophores. In a nutshell, the tailoring BTT+ NPs could act as a highly potent NIR-II fluorescence contrast agent for accurate deciphering of biological imaging. Given the terrific NIR absorption properties of these phototheranostics, we also recorded their photothermal behaviors in the aqueous phase. Results indicated that the temperature of these nano-system solutions increased rapidly from 25.0 to 65.3 °C, and 80.5 °C and 85.7 °C for OTT, BTT, and BTT+ NPs (100 μM), respectively, after 808 nm laser irradiation (Figure 1g,h). Additionally, harnessing higher-power laser exposure was conducive to the conversion of light to heat, which was in accord with the routine reports ( Supporting Information Figure S19a). Subsequently, we recorded the temperature variation of BTT+ NPs with different contents. Results displayed that the temperature increment was positively related to the concentration of the nano agent ( Supporting Information Figure S19b,c). Thereafter, by collecting the temperature elevation and natural cooling curve, we calculated the photothermal conversion efficiency (PCE) of BTT+ NPs with 33.68%, which was considerably higher than OTT NPs (22%) and nearly equal to BTT NPs (33.02%) ( Supporting Information Figure S20).61,62 Furthermore, the photothermal stability was further explored by collecting the five "heat-cool" cyclic curves (Figure 1i). This stability assessment indicated that the temperature reduction and increment in all cycles exhibited a negligible variation, demonstrating the splendid photothermal stability of the prepared BTT+ NPs. Combined with the aforementioned NIR-II fluorescence results, the simultaneous increment of PCE and QY of BTT+ NPs in comparison with the counterpart OTT NPs is very exciting since it is considered difficult to balance molecular energy to achieve a synergistic increase. Finally, the optical stability of BTT+ NPs was further investigated, and the commercial indocyanine green (ICG) and zinc phthalocyanine (ZnPc) acted as the experimental control group. After persistent light exposure at 808 nm for over 25 min, BTT+ NPs and ZnPc NPs showed an almost unchanged optical absorption and solution color; nevertheless, the control ICG exhibited a dramatic absorption reduction and appeared as a nearly colorless aqueous solution ( Supporting Information Figure S21). These assays displayed that the tailored organic semiconducting D-A-D-type BTT+ NPs possessed good optical stability.63 Evaluation of photosensitization effect Given the brilliant optical behavior due to the large π-conjugated delocalization of these prepared nanoplatforms, the photodynamic effect was further explored under 808 nm laser irradiation (Figure 2a). The ROS generation capabilities of OTT, BTT, and BTT+ NPs were evaluated by a ROS-sensitive probe nonemissive DCFH, which could convert into the fluorescent 2′,7′-dichlorofluorescein (DCF) after treatment of ROS. The ROS release was determined by measuring the fluorescence change of DCF. Results showed the DCFH alone and DCFH + OTT NPs treated with laser exposure could not induce fluorescence increment of DCF, indicating the negligible ROS generation capacity of OTT NPs (Figure 2b,c). Besides, BTT NPs exhibited a weak ROS release after 808 nm irradiation (Figure 2d). Intriguingly, after cationization treatment of BTT, the resultant BTT+ NPs could dramatically induce the strong fluorescent emission of DCF, manifesting a powerful ROS generation ability of the cationized BTT+ NPs (Figure 2e). Hence, BTT+ NPs enabled fast production of ROS under irradiation, while OTT NPs and BTT NPs showed no or weak ROS generation potency (Figure 2f). Figure 2 | (a) Schematic description of the constructing intramolecular electric fields in NIR-II-emissive BTT+ NPs for ·OH-based Type-I PDT. Fluorescence spectra of ROS-sensitive DCFH treated with (b) PBS, (c) OTT NPs, (d) BTT NPs, and (e) BTT+ NPs under different photoirradiation times. (f) Comparison of the fluorescent intensity (F/F0) of DCFH for ROS detection. Fluorescence spectra of ·OH-sensitive APF treated with (g) PBS and (h) BTT+ NPs under different photoirradiation times. Download figure Download PowerPoint Subsequently, the generation of •OH, O2•−, and 1O2 of BTT+ NPs were systematically investigated by the specific fluorescence probes, APF, DHR123, and SOSG, respectively. We observed that BTT+ NPs with irradiation enabled the amplification of APF's fluorescence emission, indicating the excellent •OH production capacity, while APF alone group showed no response (Figure 2g,h). In addition, further results showed that BTT+ NPs do not respond to DHR123 and SOSG, manifesting the inappreciable generation performance of O2•− and 1O2 ( Supporting Information Figures S22 and S23). All in all, the as-prepared BTT+ NPs can serve as a Type-I PS with predominant •OH production for further tumor hypoxia-tolerant PDT treatment. Exploration of the potential photosensitization mechanism for boosting Type-I photodynamic process Further theoretical exploration was motivated by the evident distinction of these three nano-systems in NIR-II fluorescence, photothermal, and photosensitization properties (Figure 3a). The theoretical calculations based on time-dependent density functional theory (TD-DFT) at the B3LYP/6-311G(d) level were performed. First, the optimized geometries and frontier molecular orbitals of OTT, BTT, and BTT+ are recorded (Figure 3b)
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