Rational Design and Application of an Indolium-Derived Heptamethine Cyanine with Record-Long Second Near-Infrared Emission

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE6 Jun 2022Rational Design and Application of an Indolium-Derived Heptamethine Cyanine with Record-Long Second Near-Infrared Emission Xiaoxie Ma†, Yurou Huang†, Syed Ali Abbas Abedi, Heejeong Kim, Tan Teck Boon Davin, Xiaogang Liu, Wen-Chao Yang, Yao Sun, Sheng Hua Liu, Jun Yin, Juyoung Yoon and Guang-Fu Yang Xiaoxie Ma† Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Yurou Huang† Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Syed Ali Abbas Abedi Fluorescence Research Group, Singapore University of Technology and Design, Singapore 487372 , Heejeong Kim Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750 , Tan Teck Boon Davin Fluorescence Research Group, Singapore University of Technology and Design, Singapore 487372 , Xiaogang Liu Fluorescence Research Group, Singapore University of Technology and Design, Singapore 487372 , Wen-Chao Yang Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Yao Sun Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Sheng Hua Liu Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Jun Yin *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 , Juyoung Yoon *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750 and Guang-Fu Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079 https://doi.org/10.31635/ccschem.021.202101630 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Heptamethine cyanine dyes, typified by indocyanine green, have been extensively employed as bioimaging indicators and theranostic agents. Significant efforts have been made to develop functional heptamethine cyanine dyes with outstanding bioimaging and theranostic utilities. In this work, we rationally designed and successfully developed a novel indolium-like heptamethine cyanine dye by installing indolium-derived polycyclic aromatic hydrocarbons on the terminal ends of a conjugated polyene backbone. This dye showed excellent photostability and showed bright fluorescent emission in the second near-infrared (NIR-II) window with a peak at approximately 1120 nm. Such long wavelength emission prompted a superior bioimaging resolution in vivo. In particular, this NIR-II dye had the remarkable capability of marking the blood vessels of the hindlimbs, abdomens, and brains of mice. More significantly, this dye involved a typical indolium-like heptamethine skeleton and exhibited two strong absorption bands in the 700–1300 nm NIR range, which endowed it with an intrinsic tumor-targeting capability and a high photothermal conversion efficiency (up to 68.2%), serving for the photothermal therapy of tumors under the guidance of NIR-II fluorescence imaging. This work provides an efficient design strategy for achieving indolium-like heptamethine cyanine dyes with further NIR-II emission. Download figure Download PowerPoint Introduction In recent years, heptamethine cyanine, as a type of classic NIR fluorescent dye, has been widely applied in the tracking of bioactive species, in vivo analysis, drug delivery, clinical diagnosis, phototherapy, and imaging-guided surgery.1–9 For example, indocyanine green (ICG), which fluorescence is located in the first near-infrared window (NIR-I, 700–900 nm) region and is approved by the Food and Drug Administration (FDA), has been widely applied in clinical medicine in tests of liver function and blood flow.10–20 Another popular heptamethine cyanine dye with NIR-I emission is the indolium-derived IR-780.21–36 Due to the meso-chloride moiety centrally placed on the heptamethine bridge, recent investigations showed that IR-780 with meso-chloride can form covalent or tight noncovalent adducts with albumin. And such adducts will be largely drawn to accumulate in the tumor area by its enhanced permeability and retention effect.37–39 Moreover, modifing the chloride site of IR-780 can afford the numerious derivatives of IR-780 with various biological functions.40–46 Currently, the majority of heptamethine cyanines are located in the NIR-I region.1–46 In comparison with NIR-I emission, the emission in the second near-infrared (NIR-II, 1000–2300 nm) biowindow can lead to low photon scattering, diminished autofluorescence, high penetration depth, and maximum permissible exposure to the laser.47–51 These characteristics make NIR-II fluorescent dyes promising candidates for imaging with high resolution and imaging-guided therapy for cancers.52–59 Therefore, considerable effort has been made to prepare the heptamethine-type cyanines with NIR-II emission. In genreal, two strategies have been employed to construct functional indolium-derived heptamethine cyanine with NIR-II emission, as shown in Figure 1. On the one hand, some of those with emission in the NIR-I region were converted to those with NIR-II emission by various approaches, as shown in Figure 1a, mainly including protein encapsulation,17,60–63 aggregate regulation,64–66 energy transfer,67,68 equipment upgrading,15,19,69 and so on. Another strategy is to construct indolium-derived heptamethine cyanines with NIR-II emission through a chemical approach, as shown in Figure 1b. In comparison to the situation in which these physical approaches only extend the tail peaks to the NIR-II region, chemical synthesis usually affords cyanines with maximum emission in the NIR-II region. For example, benzo[cd]indolium-modified heptamethine cyanine (IR-1048) and its derivatives had typical NIR-II emission with maximum peaks at approximately 1050–1100 nm.70–74 Accordingly, the enhancement of conjugation on two terminal ends of the heptamethine backbone is a promising strategy to construct the NIR-II heptamethine cyanines. Figure 1 | The physical approach (a) and chemistry approach (b) to construct heptamethine-type cyanines with NIR-II emission. Download figure Download PowerPoint To explore the design strategy of novel indolium-derived heptamethine cyanine with farther NIR-II emission and clarify the conjugation effect of terminal groups, we further increased the conjugation of the two ends of the polyene backbon and employed a fused naphtho[3,2,1-cd]indolium to dress the conjugated polyene, affording a novel NIR-II indolium-type heptamethine cyanine in this work. This dye showed good photostability and presented a bright NIR-II emission with a peak at 1120 nm. More significantly, this NIR-II heptamethine cyanine retained integrally the speciality of excellent tumor-targeting capability as well as those meso-chlorides containing heptamethine cyanines in the NIR-I region. The findings about the manner of imaging showed that it had a remarkable resolution in marking blood vessels of the hindlimbs, abdomens, and brains of mice and could serve as a photothermal agent with a high photothermal conversion efficiency to be applied in the imaging-guided photothermal therapy (PTT) of cancer. Experimental Methods Materials All the starting chemicals were purchased from commercial suppliers, and unless stated otherwise, these materials were utilized directly without further purification. IR-1048 was purchased from Sigma-Aldrich. All reactions adopted the standard Schlenk techniques under the nitrogen atmosphere, monitored by thin-layer chromatography, and all dehydrated solvents were deaerated before use. Characterization 1H and 13C NMR spectra were collected on an Mercury Plus 400/600 spectrometer (American Varian; 400/600 MHz) with tetramethylsilane as the internal reference. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectral data were recorded on a ultrafleXtreme MALDI-TOF mass spectrometer (Bruker), with 2,5-Dihydroxybenzoic acid (DHB) as a matrix. UV–vis-NIR absorption spectra were recorded using a UV-3600 visible recording spectrophotometer (Shimadzu). The NIR-II fluorescence spectra were recorded on the FLS1000 Photoluminescence spectrometer (Edinburgh Instruments) with an excitation laser source of 980 nm. The NIR-II imaging system was obtained by Series III 900/1700 purchased from Suzhou NIR-Optics Technologies Co., Ltd. Dynamic light scattering (DLS) measurements were performed on the Zetasizer instrument ZEN3600 (Malvern, UK) with a 173 back-scattering angle and He-Ne laser (633 nm). Transmission electron microscopy (TEM) results were collected by an Tecnai G2 F20 TEM system (FEI) at 200 kV. TEM samples were prepared by dropping Cy-PA nanoparticles (NPs) onto copper grids and drying them overnight at 40 °C. Fluorescence quantum yield The fluorescence quantum yields of Cy-PA in 1,2-dichloroethane (DCE) and Cy-PA NPs were calculated according to previous literature with dye IR-26 as reference (fluorescence quantum yield has been reported as 0.05% in DCE). Briefly, a serial dilution with optical density (OD) > 0.1 of IR-26, Cy-PA in DCE and Cy-PA NPs were performed, and the fluorescent emission spectra were collected on an F1000 fluorescence spectrometer (Edinburgh Instruments) under excitation of 980 nm. All emission spectra were corrected and integrated in 1000–1400 nm region. The integrated emission intensities were plotted as a function of the OD value at maximum absorbance and fitted into a linear function. The two slopes of the linear fit between IR-26 and Cy-PA in DCE, Cy-PA NPs, respectively, were used in the calculation of their fluorescence quantum yields based on the following equation: QY sample = QY ref × n sample 2 n ref 2 × Slope sample Slope ref , where QYsample is the QY of Cy-PA in DCE and Cy-PA NPs, respectively; QYref is the QY of IR-26 in DCE; nsample and nref are the refractive indices of IR-26, Cy-PA in DCE and Cy-PA NPs, respectively. Computational methods We conducted density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations for IR-780, IR-820, IR-1048, and Cy-PA using the B3LYP functional accompanied by the def2SVP basis set.75 The solvent effects (water) were simulated using the solvation model based on density (SMD) model with the linear-response formalism.76 The ground state structures were optimized using Gaussian 16.77 Frequency calculations were also completed to ensure that we found the local minima on the potential energy surface. Cell culture and tumor model B16 cells were purchased from the China Infrastructure of Cell Line. B16 cells utilized in the experiments were cultured in Roswell Park Memorial Institute 1640 Medium supplemented with 10% (v/v) fetal bovine serum (Gibco) and then incubated at 37 °C under a humidified atmosphere containing 5% CO2. The female C57 mice (aged 4 weeks) were purchased from Hubei Provincial Laboratory Animal Analysis Center. B16 cells [106 in 200 μL phosphate-buffered saline (PBS)] were injected into the right hindlimbs of the mice. The B16 tumor-bearing mice were subjected to the following experiments when the tumor volume reached 150 mm3. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, and the procedures were approved by the Central China Normal University of China Animal Care and Use Committee. In vivo NIR-II fluorescence vessel imaging The female kungming (KM) mice (around 20 g weight) were purchased from Hubei Provincial Laboratory Animal Analysis Center. All KM mice were anaesthetized via intraperitoneal injection of chloral hydrate (5%, 50 μL). The tail vein-injected dose was 200 μL, 0.5 mg mL−1 Cy-PA NPs. All groups within the study contained n = 3 mice. NIR-II fluorescence imaging was conducted through a NIR-II imaging system under a 1064 nm laser (8 W cm−2, 500 ms). We used ImageJ software to conduct the fluorescence imaging analysis. Tumor-targeting imaging The C57 mice bearing tumors were anaesthetized via intraperitoneal injection of chloral hydrate (5%, 50 μL) before administration. Cy-PA NPs (0.5 mg mL−1, 200 μL) was injected to capture the tumor site and find the best PTT time. NIR-II fluorescence (12 W cm−2, 1064 nm, 1300 LP, 500 ms) images were captured at several time points (0, 1, 2, 4, 6, 8, 12, and 24 h) postinjection. The NIR-II imaging contained n = 3 mice. NIR-II fluorescence imaging was conducted through a NIR-II imaging system under 1064 nm laser irradiations. We used ImageJ software to conduct the fluorescence imaging analysis. In vivo photothermal imaging and therapy B16 tumor-bearing C57 mice were intravenously (0.5 mg mL−1, 200 μL) or intratumorally (0.5 mg mL−1, 100 μL) injected with Cy-PA NPs. Under the conditions of power density of 0.4 W cm−2 and 5 min, the infrared thermal images of tumor-bearing mice were recorded with an infrared camera Flir E4. As for PTT, the B16 tumor model was randomly divided into three groups (n = 3): (1) Cy-PA NPs + Laser; (2) only injected with Cy-PA NPs; (3) PBS + Laser; The tumors were equally irradiated for 5 min in every treatment. The infrared thermal images and the weight of these tumor-bearing mice were recorded, respectively. The tumor weight was recorded every 2 days. And the tumor lengths and widths (volume) was measured by a digital caliper every other day for 8 days for all groups to evaluate the effect of PTT. Results and Discussion Molecular design and synthesis As the only NIR organic fluorescent agent approved by the FDA, ICG has been applied in clinical medicine for over 50 years.78,79 ICG has a typical heptamethine cyanine backbone and NIR optical behavior with a maximum absorption at 780 nm and emission at 815 nm, respectively.80 In addition, another well-known NIR heptamethine cyanine dye is indolium-based IR-780, as shown in Figure 1b. From its optical behavior and molecular structure, IR-780 not only retains the photophysical properties similar to ICG but also introduces Cl reactive sites to enhance its functionalization.81,82 By the replacement of indolium moieties on two terminals of the polyene bridge into the benzo[e]indolium skeletons to form IR-820, it processes a more red-shifted absorption at 820 nm and emission at 850 nm, respectively.83 Recently, a large breakthrough came in replacing benzo[e]indolium with benzo[cd]indolium to form IR-1048, which exhibited typical NIR-II emission. In comparison to IR-820, IR-1048 maximum absorption shifted to 1048 nm, while the peak emission appeared at 1078 nm.84 In our previous work, we found that polycyclic aromatic hydrocarbons (PAH) had outstanding optical performance, and that the expansion of aromatic rings could greatly adjust the absorption/emission of conjugated systems.85–88 Accordingly, we proposed a hypothesis of introducing PAH to the heptamethine cyanine backbone, which was expected to possess longer NIR-II emission wavelengths. As exhibited in Figure 1b, an N-hetero naphtho[3,2,1-cd]indolium coating polyene ( Cy-PA) was designed. In comparison to the benzoindolium-based heptamethine cyanines IR-820 and IR-1048, the molecular structure of Cy-PA increased the fused aromatic ring on the benzoindolium moieties to obtain further red-shifted optical performance, which was also successfully predicted by subsequent theoretical calculations. In accordance with this rational molecular design, a highly maneuverable synthetic route for Cy-PA is outlined in Figure 2a. Crucial intermediate 1 was synthesized according to the reported literature.89 As shown, 2-bromobenzaldehyde and indolin-2-one were employed as the starting materials to prepare polycyclic aromatic intermediate 1. Subsequent N-alkylation with 1-iodopropane efficiently produced intermediate 2 in a yield of 83%, which was further treated with the Grignard reagent methylmagnesium bromide to afford 5-methyl-4-propylnaphtho[3,2,1-cd]indol-4-ium 3. Next, naphtho[3,2,1-cd]indolium 3 without further purification was directly subjected to condensation with 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde in acetic anhydride, affording the targeted heptamethine cyanine Cy-PA in 73% yield. The structures of all the unknown compounds had been confirmed by 1H and 13C spectra ( Supporting Information Figures S6–S10). Compared with the traditional heptamethine cyanines such as indolium-based IR-780, benzoindolium-based IR-820 and IR-1048, naphtho[3,2,1- cd]indolium-based Cy-PA possessed larger conjugated PAH units and was expected to have NIR-II optical performance. Figure 2 | (a) Synthetic route of Cy-PA. (b) The preparation of Cy-PA NPs. Download figure Download PowerPoint Optical properties To understand the photophysical properties of Cy-PA, we first investigated its absorption and fluorescence spectra in pure organic solvents. The absorption spectra of Cy-PA showed that it had similar absorption bands, and the intense maximum absorption was at approximately 1060 nm in organic solvents with different polarities ( Supporting Information Figure S1a). The corresponding fluorescence spectra presented strong NIR-II emission peaks at 1090–1168 nm upon excitation with a 980 nm laser ( Supporting Information Figure S1b). In PBS (pH 7.4), as shown in Figure 3a, Cy-PA exhibited an intense maximum absorption at approximately 821 nm and a relatively weak absorption at 1152 nm. Most likely, the former was ascribed to its aggregation form, while the latter was induced by its monomer. Moreover, the strong aggregation in PBS buffer solution led to a negligible NIR-II emission upon excitation with a 980 nm laser, as shown in Figure 3b. Due to their large conjugated system, cyanines easily formed the H-aggregates. To avoid strong aggregation, especially for cyanines in NIR-II windows, some disaggregation strategies were developd, including various surfactants, the complex with protein,90 encapsulation in supramolecular cavities,91 and equipment with a shielding blockage in a polymethine chain.92 For Cy-PA, we adopted an anionic surfactant sodium dodecyl sulfate (SDS) to achieve the disaggregation. When the SDS was added to the solution of Cy-PA, the original absorption at 821 nm obviously decreased while a new absorption peak at 1087 nm was observed (see Figure 3c), probably ascribed to the increase in the monomer ratio of Cy-PA. Meanwhile, its fluorescence spectrum showed a strong NIR-II emission peak at approximately 1118 nm, as shown in Figure 3d. Therefore, we can conclude that the aggregation of cyanine can be hindered by the hydrophobic pockets of SDS micelles. Positively, compared with the cyanine IR-1048 that has been currently used, both the absorbance and emission of Cy-PA presented an ∼40 nm red shift, as predicted in the molecular design. Figure 3 | Normalized absorption (a) and fluorescence spectra (b) of Cy-PA (10 μM) and Cy-PA NPs (120 μg mL−1) in PBS buffer (pH 7.4) under excitation with a 980 nm laser. Inset: NIR-II fluorescence image of Cy-PA NPs (0.5 mg mL−1) in PBS buffer (1300 nm long-pass filter) under excitation with a 1064 nm laser. (c) Absorption spectra of Cy-PA (10 μM) upon addition of SDS (0–8 mM) in PBS solution (pH 7.4). (d) Fluorescence spectra of Cy-PA (10 μM) with or without SDS (4 mM) under excitation with a 980 nm laser. (e) The DLS data of Cy-PA NPs in PBS buffer. (f) TEM image of Cy-PA NPs. Download figure Download PowerPoint To achieve biocompatibility and obtain NIR-II bioimaging with high resolution, PEGylation of the above SDS dispersive Cy-PA with surfactant distearyl phosphatidyl acetamide-methoxypolyethylene glycol-5000 (DSPE-mPEG5000) formed water-dispersible NPs, termed Cy-PA NPs, shown in Figure 2b. The solution pH of Cy-PA NPs was measured to be 7.4 on the pH meter. To assess its dispersion effect, the optical behavior of Cy-PA NPs was subsequently investigated. In comparison to the absorption at 821 nm of Cy-PA in PBS and a peak at nm of Cy-PA (120 μg mL−1, Supporting Information Figure without Cy-PA NPs showed an intense peak at 1087 nm to the monomer form of Cy-PA that was by the disaggregation of as shown in Figure In particular, the absorption of Cy-PA NPs the NIR-I and NIR-II with a high absorption × confirmed that the were to the conversion of into the intense absorption of endowed Cy-PA NPs with strong and high photothermal conversion This that Cy-PA NPs can serve as a photothermal agent with a high photothermal conversion efficiency for the PTT of cancer. of its fluorescence in Figure that it exhibited a typical NIR-II emission at approximately 1120 nm and relatively The fluorescence quantum yield of Cy-PA NPs was to be with IR-26 as reference = while Cy-PA was The of NPs through DLS confirmed that Cy-PA NPs had an of nm and a dispersion This was with the findings of TEM These findings that Cy-PA NPs has potential for NIR-II bioimaging and imaging-guided PTT. calculation To understand the molecular of spectral in cyanine we conducted DFT and calculations for IR-780, IR-820, IR-1048, and the proposed Cy-PA, as shown in Figure The calculations showed that as we from IR-780 to IR-820, the molecular molecular energy not Accordingly, in both the calculated and peak absorption and are nm). In IR-820 and IR-1048 showed a considerable in the and a nm) in their peak absorption wavelengths. The in IR-820 and IR-1048 was the of the This to have a strong on the energy that the of IR-1048 was to for IR-820, while their energy the further the energy of Cy-PA low affording further red-shifted and the of is in good with the data the of our quantum chemical calculations. Figure 4 | (a) molecular of IR-780, IR-820, IR-1048, and Cy-PA, and corresponding energy and calculated absorption and (b) in the molecular to both and the of in the The of the are to the corresponding The in IR-820 was by a the Download figure Download PowerPoint To further into in energy and the spectral of these we the to both and in the molecular we also the of the of these These for the between the cyanine derivatives in Figure from IR-780 to IR-820, the had negligible in the molecular the In by the of the rings in IR-1048 and Cy-PA, the new is involved in the molecular especially in by large these results the effects on the in molecular energy and the red from IR-780 to Cy-PA. In PTT by the strong absorption of Cy-PA NPs the NIR-I and NIR-II we their photothermal conversion in In of the that the nm laser has strong and the capability by we the nm laser as the excitation light Accordingly, we first investigated the photostability upon with a nm laser for the absorption of Cy-PA NPs compared with the while IR-780, IR-820, IR-1048, and Cy-PA obviously This that the photostability of Cy-PA NPs was greatly as shown in Figure and it was for the PTT of in vivo. The photothermal effect of Cy-PA NPs was investigated under nm laser As in Figure the of Cy-PA NPs in solution (120 μg mL−1) by °C within 5 min, while that of only by that Cy-PA NPs had an photothermal The photothermal conversion efficiency of Cy-PA NPs was calculated to be as high as ( Supporting Information Figure In addition, the photothermal also on the of Cy-PA NPs and the power density of the laser as shown in Figures and In particular, Cy-PA NPs possessed high photothermal As shown in Figure the photothermal conversion efficiency exhibited negligible of and Therefore, it was that Cy-PA NPs had a strong potential to as a photothermal agent for the of cancer. Figure 5 | (a) The photostability of IR-780 (10 IR-820 (10 IR-1048 (10 Cy-PA (10 and Cy-PA NPs (10 μg mL−1) under excitation with a nm laser W (b) conversion behavior of Cy-PA NPs of different μg mL−1) under

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