Friday 12th August 2022

Structured Organic Chromophore for NIR

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Introduction

Thermal ablation of tumors is an application of hyperthermia, which will result in irreversible cell injury, the tumor apoptosis and coagulative necrosis ultimately.1,2 At present, several thermal ablation methods have been developed, including radiofrequency ablation (RFA),3,4 microwave ablation (MWA)5,6 and high-intensity focused ultrasound (HIFU).7,8 However, expensive instruments were used in these methods. Compared with methods mentioned above, non-invasive photothermal therapy (PTT) be more economical, more effective, more precise targeting at the tumor, and minimize the damage to surrounding healthy tissues. PTT can kill cancer cells and bacterial biofilm via heat energy generated by photothermal agents under NIR laser irradiation.9–15 In recent years, various inorganic nanomaterials have been actively employed for photothermal therapy of tumors, including gold nanoparticles,16 carbon nanomaterials,17 palladium nanosheets,18 and transitional metal dichalcogenide.19,20 Although superior efficiency in ablation tumors has been achieved by using inorganic photothermal agents, the potential long-term toxicity and poor biodegradation remain the major challenges. On the other hand, the NIR-absorbing (750~1700 nm) organic photothermal agents have been explored owing to their great biocompatibility and can be metabolied rapidly in biological tissue, including NIR absorbing conjugated polymers,21 cyanines22 and porphyrins.23 However, the poor photostability, severe photodegradation, and the low efficiency of photothermal conversion hamper their further applications. Dibenzamide dyes exhibit excellent molar absorption and subtly tunable optical properties, but their biological applications are mainly focused on fluorescence imaging,24 their maximum absorption and emission in the visible range are limited. Terrylenediimide-based nanomedicines have high photothermal conversion efficiency, but the preparation process with certain inaccuracies is complex and unstable.25,26 Hence, it is crucial to develop a new class of NIR absorbing organic chromophores for thermal ablation in tumor.

A-π-D structured organic chromophores have been demonstrated to be an excellent option for photothermal therapy due to their excellent photostability and strong molar absorption coefficient.27–30 Besides, chromophores with A-π-D structure have rigid planar structures and strong π-π interaction, which enhance the non-radiative decay and improve photothermal efficiency.31 To date, efforts have been made to improve the PTT efficacy of A-π-D structured organic chromophores in many researchs, the strategies proposed including increasing acceptor strength, extending conjugation length to reduce band gap, and red-shift the absorption maxima.32 To this effect, numbers of electron-deficient groups have been exploited, including diketopyrrolopyrrole,33,34 thiadiazolobenzotriazole,35,36 benzo[1,2-c:4,5c’]bis([1,2,5]thiadiazole),37,38 and indan.39,40 Among them, 2-dicyanomethylenethiazole, an electron-withdrawing core, can be combined with the electron-donating group to build an A-π-D organic chromophore for broadening the absorption spectrum, reducing the optical gap, and decreasing the HOMO level.41–45 However, 2-dicyanomethylenethiazole molecules exhibit some limitations, such as weak absorbance in the near-infrared (NIR) region and low photothermal conversion efficiency. Therefore, it is highly desirable to explore new 2-dicyanomethylenethiazole chromophores with high PTT performance.

As reported, the hyperthermia treatment can inhibit cancer cells tumorigenesis via the necroptosis pathway or DNA damage-mediation tumor apoptosis pathway.46–51 However, the underlying mechanisms are still elusive. The comprehensive understanding of photothermal agents might provide new options for efficient cancer therapy. As shown in Graphical Abstract, in this contribution, we developed an A-π-D structured organic chromophore (PTM) based on 2-dicyanomethylenethiazole for light-induced tumor thermal ablation. PTM was directly encapsulated into an amphiphilic copolymer Pluronic F-127 through the nanoprecipitation method to obtain nanoparticles, PTM NPs. These agents shown robust photostability, good biocompatibility, and high PCE (56.9%). Moreover, PTM NPs exhibited cancer cells killing capacity via DNA damage mediated apoptosis induced by heat generation in cells (Scheme 1). Both in vitro and in vivo experiments demonstrated that PTM NPs had effective anti-tumor potential. Our comprehensive studies of the mechanisms and underlying PTM NPs performance will improve the understanding of photothermal therapy and augment the future application of photothermal agents.

Materials and Methods

Chemicals and Materials

All reagents and solvents were commercially provided. CCK-8 kit was provided from Dojindo Laboratories (Kumamoto, Japan). Calcein AM/PI Detection Kit and Hematoxylin and Eosin Staining Kit were purchased from NanJingKeyGen Biotech Co., Ltd. (China). 5-(10-Ethyl-phenothiazin-3-yl)thiophene-2-carbaldehyde was prepared according to reference.52

Design and Synthesis of PTM

5-(10-Ethyl-phenothiazin-3-yl)thiophene-2-carbaldehyde (3.36 g, 10 mmol) and 2-dicyanomethylenethiazole 1 (3.82 g, 10 mmol) and was added into 30 mL acetic anhydride, and stirred at 160 °C for 12 h under N2 protection. The mixture was drop added into saturated Na2CO3 aq. The dark blue solid was filtrated and purified by column chromatography using a mixture of DCM/MeOH (19: 1). Yield: 4.8 g (67%). 1H NMR (400 MHz, CDCl3), δ(ppm):7.86 (s, 1H), 7.67~7.69 (d, 2H), 7.57(s, 1H), 7.45~7.47(d, 2H), 7.40~7.42(d, 2H), 7.32~7.36(d, 2H), 7.28(s, 1H), 7.18~7.24(t, 2H), 6.94~7.05(m, 2H), 6.81(s, 1H), 3.81~3.84(d, 2H), 3.16~3.19(t, 2H), 1.59(s, 1H), 1.31~1.49(m, 8H), 0.91~0.95(m, 9H). 13C NMR (100 MHz, CDCl3), δ (ppm): 172.16, 166.78, 163.41, 148.70, 147.68, 146.71, 143.83, 141.58, 141.16, 137.38, 136.40, 134.27, 133.11, 132.03, 131.28, 130.60, 129.31, 128.16, 127.85, 126.54, 124.43, 121.11, 120.18, 119.30, 115.98, 115.12, 111.26, 95.94, 64.48, 49.56, 44.41, 31.76, 29.14, 26.55, 26.04, 15.83, 15.04, 14.33. IR (v−1, KBr): cm−1 3453, 2986, 2845,2618, 1618, 1523, 1483, 1366, 1252, 1034, 863.

PTM NPs Preparation

PTM NPs were constructed by the coprecipitation method. Pluronic F-127 (100 mg) and PTM (10 mg) were mixed in THF (1 mL). THF solution was drop added into DI water (10 mL) under ultrasonic condition. The mixture was bubbling N2 to remove THF completely. PTM was obtained after dialysis to remove the free F-127.

Animals

All the animal experiments were conducted in accordance with the institutional guidelines for the care and use of laboratory animals at Southern Medical University, Guangzhou, China, and Regulations for the Administration of Affairs Concerning Experimental Animals (1991.7, revised 2017). All the animal experiments have been approved by Southern Medical University.

Statistical Analysis

All data were expressed as means ± standard deviation (SD) or means ± standard errors. All figures shown in this article were obtained from at least three independent experiments. Analysis of variance was employed for multiple group comparisons, and results of p < 0.05 were considered statistically significant.

Result and Discussion

Synthesis, Optical and Photothermal Property of PTM NPs

NIR absorption for A-π-D structured organic chromophore (PTM) was built by using 2-dicyanomethylenethiazole as a strong acceptor, allowing for greater electron delocalization and thus lowering the band gap, phenothiazine as a donor, and planar thiophene ring as a π-conjugation unit, which provided effective conjugation and large extinction coefficient. The synthetic routine of PTM is shown in Scheme S1 and S2. The synthesis process, NMR spectrum, and IR spectrum are shown in Figure S1S5 (Supporting information).

To further enhance the biocompatibility, hydrophobic PTM and Pluronic F-127 were dissolved in THF and allowed to self-assemble into PTM NPs. First of all, the absorption property of PTM NPs was evaluated, as shown in Figure 1A, PTM NPs (200 μg/mL) have a broad UV-vis absorption arrange from 550 nm to 1000 nm with the maximum absorption located at ~680 nm, which permits deep tissue penetration and exhibits the potential to accomplish efficient photothermal therapy for deep tumor. Furthermore, the dynamic light scattering (DLS) experiment showed that PTM NPs exhibited hydrodynamic diameters around 110 nm (Figure 1B). Interestingly, the size of PTM NPs…

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