A Water-Soluble Photothermal Host–Guest Complex with pH-Sensitive Superlarge Redshift Absorption

Abstract

Open AccessCCS ChemistryCOMMUNICATION1 Oct 2021A Water-Soluble Photothermal Host–Guest Complex with pH-Sensitive Superlarge Redshift Absorption Xiao-Qi Xu, Hongguang Liao, Haocheng Liu, Yanji Chu, Yonglin He and Yapei Wang Xiao-Qi Xu Department of Chemistry, Renmin University of China, Beijing 100872 , Hongguang Liao Department of Chemistry, Renmin University of China, Beijing 100872 , Haocheng Liu Department of Chemistry, Renmin University of China, Beijing 100872 , Yanji Chu Department of Chemistry, Renmin University of China, Beijing 100872 , Yonglin He Department of Chemistry, Renmin University of China, Beijing 100872 and Yapei Wang *Corresponding author: E-mail Address: [email protected] Department of Chemistry, Renmin University of China, Beijing 100872 https://doi.org/10.31635/ccschem.020.202000505 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Near infrared (NIR) absorbers for photothermal therapy are arousing great attention in tumor diagnosis and ablation. However, the inevitably wide distribution of NIR absorbers generally causes equal photothermal injuries to tumor tissues and healthy tissues. An initiative targeting strategy based on pH-sensitive redshift absorption has been proposed for NIR-induced hyperthermia only in tumor tissue. In this work, we develop a NIR absorber with superlarge redshift from the middle visible light region to NIR light region once the pH value drops. We also determined a supramolecular route to improve the water solubility as well as the thermal stability of the NIR absorber in aqueous solutions. Animal experiments reveal that the tumor microenvironment can trigger selective NIR light absorbing, which is thus responsible for the tumor-selective hyperthermia and ablation. Based on easy preparation, low cytotoxicity, and excellent tumor selectivity, this supramolecular complex is a reliable photothermal agent for intelligent tumor-specific diagnosis and treatment. Download figure Download PowerPoint Introduction Near infrared (NIR) light is showing great potential in clinical diagnosis and treatment in deeper tissues relative to other wavelengths due to less absorption by water and hemoglobin.1–5 Among the reliable methods to utilize NIR light, photothermal conversion via nonradiative emission with the highest quantum efficiency has been regarded as an attractive choice in treatment.6–10 It can generate hyperthermia in diseased tissues, particularly laying the groundwork for photothermal therapy (PTT) against cancer diseases.11–19 Nevertheless, the inevitable distribution of NIR absorbers in healthy tissues may cause undesired NIR injury, or side effects, of photothermal conversion. To surmount this obstacle, two strategies including passive and initiative targeting have been pursued to ensure photothermal conversion specifically in tumor tissues.20–22 The former aims to accumulate NIR absorbers in cancer cells based on selective cellular internalization, thus allowing photothermal conversion mainly in tumor tissues. The latter involves a chemical change of NIR absorbers based on the different environment between normal and tumor tissues. It is expected that only those NIR absorbers entering the tumor environment have the ability of photothermal conversion in the NIR window. Unlike the intense investigations of passive targeting, the study of such initiative targeting is still in its infancy because of the limited number of NIR absorbers that are capable of distinct redshift in tumors. In contrast to homeostatic condition with pH 7.4, certain tumor tissues possess lower pH values, in the range of 6.8–4.5, from the extracellular environment, to endosomes, and then to lysosomes.23 Such a distinctly acidic microenvironment in tumors relative to normal tissues renders the possibility to program the redshift of some pH-sensitive chromophores. Although a great number of pH-responsive chromophores have been exploited and investigated, few of them fulfill the requirement of a large-span redshift from visible to NIR light absorption.24,25 This requirement is crucial to practically improve the selectivity of PTT against only tumor while being innocuous to healthy tissues. In principle, the light absorption wavelength (λ) is decided by the energy gap (ΔE) of chromophores, following a formula of Δ E ∝ 1 / λ ; therefore, a large-span redshift requires a dramatic decrease in the energy gap when the chromophore is acidified in the tumor environment. The successful examples mainly relied on π-conjugated NIR absorbers, which were modified with pH-sensitive moieties.26–29 The longer light-absorption wavelength was explained by an increase in conjugation length when the chromophore was protonated at lower pH. Besides the difficulty of extending the conjugation length of chromophores within a limited molecular size, another challenge to this strategy is enabling a marked degree of redshift due to conjugation length change in the presence of an insignificant pH difference between the tumor environment and physiological conditions. This work describes the superlarge redshift of amine-capped aniline trimer (ACAT), which exhibited selective NIR light absorption in an acidic environment. Its main light absorption peak shifted from 590 to 776 nm with a wide shoulder extending to 1000 nm after acid-induced protonation (Figure 1a). Such a pH-sensitive superlarge redshift renders ACAT great promise for tumor-selective PTT. However, the hydrophobic nature of ACAT practically suppresses its solubility and pH sensitivity in aqueous medium.30–32 In addition to the discovery of the superlarge redshift of ACAT, this work also devoted effort to solve the poor solubility of ACAT in aqueous solutions via host–guest complexation. Specifically, ACAT was incorporated into a water-soluble host molecule of β-cyclodextrin (β-CD), which significantly improved the water solubility while retaining the pH sensitivity of ACAT. This supramolecular complex served as a selective NIR absorber in acidic conditions, ensuring highly selective and efficient photothermal conversion in the tumor environment. The success of tumor-selective ablation during animal experiments demonstrated the feasibility of targeted PTT based on the pH-dependent initiative targeting strategy. Figure 1 | (a) Schematic illustration of a water-soluble NIR absorber based on the host–guest complexation between ACAT and β-CD intended for pH-sensitive PTT. (b) 2D NOESY NMR spectra of ACAT-CD in D2O solution. (c) The absorbance at 550 nm as a function of the feed ratio between β-CD and ACAT. Inset is UV–vis absorption spectra of ACAT-CD in water solution at different [β-CD]∶[ACAT] ratios. Download figure Download PowerPoint Animal Studies All animal experiments were handled in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) in compliance with Chinese law for experimental animals. The experiments were performed in compliance with institutional guidelines established by the Laboratory Animal Research Center of Institute of Processing Engineering, Chinese Academy of Science, which approved our experimental protocols and procedures. Results and Discussion To prepare the supramolecular NIR absorber, ACAT and randomly methylated β-CD were dissolved in a solution of ethanol and water. Under warming at 70 °C, ACAT was encapsulated in the cavity of β-CD to form an ACAT-CD host–guest complex after the evaporation of ethanol. No Tyndall effect was observed in this supramolecular complex solution, indicating the absence of insoluble residues and the excellent solubility of the ACAT-CD host–guest complex in the aqueous medium ( Supporting Information Figure S1). A powder-like product prepared via lyophilizing the ACAT-CD solution could be redissolved in water, which confirmed the strong association between ACAT and β-CD. Detailed synthetic procedures and characterizations of ACAT and ACAT-CD are available in Supporting Information Figures S2–S4. Per the 1H NMR spectra in Supporting Information Figures S2 and S3, the chemical shift assigned to the amino group of ACAT shifts downfield upon the addition of β-CD. Further investigation by two-dimensional (2D) nuclear Overhauser enhancement spectroscopy (NOESY) identified the specific protons on ACAT and β-CD that are involved in the supramolecular interaction. It suggests that the β-CD is capped by the end benzyl ring of ACAT (Figure 1b), fully agreeing with the knowledge of CD-based supramolecular chemistry in which the host–guest interaction is facilitated by hydrophobic effect and size matching.33–36 To identify the stoichiometric ratio of ACAT-CD complex, the UV–vis spectra of ACAT in the presence of varied concentrations of β-CD are summarized in Figure 1c. Accordingly, the ACAT absorbance at 530 nm dramatically increased upon the continuous addition of β-CD, accounting for the improved solubility of ACAT. Two distinct slopes correspond to a two-step binding process: a 1∶1 complexation and 1∶2 complexation between ACAT and β-CD. With the feed ratio [β-CD]∶[ACAT] beyond 2∶1, the absorbance of ACAT approximates an equilibrium due to the complete dissolution of ACAT. Additional characterizations by Fourier transform IR (FT-IR) spectroscopy and thermogravimetric analysis (TGA) also provide substantial evidence to explain the formation of the ACAT-CD complex ( Supporting Information Figures S5 and S6). The acid-triggered redshift of ACAT was confirmed by monitoring its UV–vis–NIR absorption in a dimethylsulfoxide (DMSO) solution upon the addition of glacial acetic acid (Figure 2a). Notably, the acid addition triggered a drastic abatement of the absorption band at 590 nm along with a significant increase of a new band at 776 nm. This intriguing pH-sensitive bathochromic shift was wonderfully passed to the ACAT-CD complex, allowing acid regulation in aqueous solution. As expected, the aqueous solution consisting of the ACAT-CD complex changed from blue to cyan when the pH dropped from 7.4 to 5.0 (Figure 2b). As shown in Figure 2c, the UV–vis–NIR absorption spectra of ACAT-CD were obtained in a wide pH range while keeping the ACAT concentration constant. In alkaline or neutral conditions, the initial absorption band at 530 nm was dominant, which is ascribed to the πb–πq transition from the benzene unit (donor) to quinone unit (acceptor) in ACAT. The absorption within the NIR window, particularly at wavelengths above 780 nm were negligible. In acidic conditions, the main absorption band was shifted to 715 nm with a redshift relative to the initial absorption band as high as 160 nm. In principle, the aforesaid protonation process should be accompanied by the reconfiguration of the ACAT structure and the redistribution of molecular orbitals. The weakened absorption at 530 nm and the spontaneous appearance of a new absorption at 400 nm, both assigned to the πb–πq transition, are caused by the formation of a bipolaron (dication–diradical) structure. The acid-induced NIR absorption arising at 715 nm with a wide shoulder is attributed to the polaron transition of doped ACAT. Despite the protonated ACAT-CD in buffer solution exhibiting slight blueshift compared to the protonated ACAT in DMSO, the major light absorption of the supramolecular complex still locates in the NIR region, which ensures the use of ACAT in NIR-triggered photothermal conversion. The concentration-dependent absorption of ACAT-CD complex at pH 7.4 and 5.0 obey the Lambert–Beer law, corresponding to extinction coefficients of 0.70 L/(g·cm) at 550 nm (Figure 2d) and 1.11 L/(g·cm) at 715 nm (Figure 2e), respectively. Additionally, UV–vis–NIR spectra at different temperatures were also investigated to check the thermal stability of the acidified ACAT-CD complex (Figure 2f). The NIR absorption at 715 nm was slightly decreased along with a slightly increased absorption at 550 nm when the solution was warmed from 20 to 60 °C. This reverse change relative to the acidification process could be caused by the temperature-dependent pKa change. Immediately after the system was cooled to 30 °C again, the absorption intensity in NIR region was fully recovered. This confirms that the host–guest interaction has successfully protected ACAT from aggregation or precipitation in an aqueous environment even at high temperature. Figure 2 | (a) UV–vis–NIR spectra of ACAT in DMSO solution upon stepwise addition of glacial acetic acid. (b) Color comparison of ACAT-CD complex dissolved in water solution at pH 7.4 and 5.0. (c) UV–vis–NIR spectra of ACAT-CD complex in phosphate-buffered saline (PBS) buffer solution under different pH values. (d) Concentration-dependent UV–vis–NIR spectra of ACAT-CD complex in PBS buffer at pH 7.4. Inset is the fitting curve of the absorbance at 550 nm as a function of concentration. (e) Concentration-dependent UV–vis–NIR spectra of ACAT-CD complex in PBS buffer at pH 5.0. Inset is the fitting curve of the absorbance at 715 nm as a function of concentration. (f) Temperature-dependent UV–vis–NIR spectra of ACAT-CD complex in PBS buffer at pH 5.0. Download figure Download PowerPoint Cyclic voltammetry (CV) was used to explain the superlarge redshift of ACAT-CD complex with the pH change. According to CV investigation (Figure 3a), the electrochemical energy gap of ACAT is estimated to be 1.40 eV at neutral condition. After ACAT is protonated, its energy gap narrowed to 1.22 eV, which is consistent with the optical energy gap calculated from the absorption onset at 1000 nm. Such dramatic decrease of energy gap significantly benefits the superlarge redshift performance from neutral condition to acidic condition. In addition, the electron spin resonance (ESR) spectroscopy was investigated to understand the structural changes of ACAT-CD during protonation. As shown in Figure 3b, the appearance of an ESR signal of ACAT-CD in acidic condition implies the formation of radical cations. In analogy to polyaniline ( Supporting Information Figure S8), the transformation from quinoid to benzenoid upon proton doping generates two radical cations in the conjugated backbone of ACAT. However, the intensity of the ESR signal stayed at a relatively low level even when the ACAT-CD complex was dissolved in 1 M HCl solution, which suggests the compromised stability of the triplet state of the ACAT bipolaron model. A possible mechanism of the chemical change of ACAT led by protonation is hypothesized in Figure 3c. To be specific, the initial structure of ACAT is composed of two benzene donors and one quinone acceptor. After ACAT is protonated, the electron redistribution resulted in the transition of quinone structure to benzene structure, along with the generation of the bipolaron on the molecular backbone. Such a bipolaron model consists of a singlet state and a triplet state which can switch under ambient conditions. Per the density functional theory (DFT) calculations (Figure 3d), the initial energy gap of ACAT is estimated as 2.22 eV, which accounts for the absorption peak at 530 nm. By contrast, the protonated ACAT according to the bipolaron model has a narrowed energy gap of 1.63 and 1.74 eV for the singlet state and triplet state, respectively. The lower energy gap corresponds to a longer light absorbing wavelength, which explains the superlarge redshift in acid environment. Noting that the computed energy of the triplet state is only 13 kJ/mol higher than that of the singlet state, it is suggested that the singlet state is the favored form of protonated ACAT. Yet the transformation between two states is very likely to happen at room temperature, which explains the low intensity of the paramagnetic signal in the ESR spectrum. Figure 3 | (a) CV curves of ACAT-CD in DMSO solution under acidic and neutral conditions. (b) ESR spectra of ACAT-CD in 1 M HCl and pH 7.4 buffer. (c) Proposed mechanism of the chemical change of ACAT against protonation. (d) Highest occupied molecular orbital (HOMO)/lowest unoccupied molecular orbital (LUMO) of the ACAT and protonated ACAT as calculated by DFT [B3LYP/6-311g (d,p)]. Download figure Download PowerPoint The faintest fluorescence quantum yield demonstrates that nonradiative transition should be the dominant route for electron relaxation from the excited state ( Supporting Information Figure S7), which promotes the photothermal conversion of ACAT-CD after it absorbs light. To investigate the photothermal conversion performance of ACAT-CD, temperature change was recorded by a thermocouple under NIR light irradiation. As shown in Figure 4a, the temperature spiked once the ACAT-CD solution at pH 5.0 was exposed to NIR light. After irradiation for 3 min, the solution was immediately removed from the laser, and then it underwent a rapid cooling process due to the thermal dissipation. During three cycles of on/off light irradiation, the temperature change at a fixed exposure time remained almost unchanged, confirming the excellent photostability of ACAT-CD in aqueous solution. The increase in temperature reveals a positive correlation against the amplification of light power density. The temperature of ACAT-CD solution reached 48.6 °C at an irradiation power density of 1 W/cm2, which was high enough for effective tumor ablation. The increment of NIR absorption was closely related to the concentration of ACAT-CD. As shown in Figure 4b, the temperature rose from room temperature (25 °C) to 35, 42, and 50 °C as the concentration of ACAT-CD increased from 300 to 500 and 700 μg/mL, respectively. Very importantly, the control experiment revealed that ACAT-CD exhibited weaker photothermal conversion performance at pH 7.4 because of the limited NIR absorption (Figure 4c). Keeping the light power at 0.6 W/m2 and the concentration of ACAT-CD at 500 μg/mL, the temperature of the ACAT-CD solution at pH 7.4 increased by 7.9 °C, which was 9 °C lower than that achieved at pH 5.0. Such an extraordinary difference should arise from the unique advantage of superlarge redshift under different pH environments (Figure 2c), which is particularly important for tumor-selective PTT. To give a direct illustration of the photothermal conversion process, the surface temperature of pure water and ACAT-CD solution at different pH conditions were thermally imaged by IR camera. As shown in Figure 4d, ACAT-CD at pH 5.0 was confirmed to have better photothermal conversion ability than that at pH 7.4, which was fully consistent with the observation based on thermocouple detection. The photothermal conversion efficiency (ηPT) of ACAT-CD solution at pH 5.0 was calculated by eq 1: η PT = Q I (1)In which Q refers to the heat generation rate of ACAT-CD solution under light irradiation, and I refers to the light power. Taking the light power density of 0.6 W/cm2 as an example, the ηPT was calculated as high as 92.2%, indicating this supramolecular NIR absorber possesses a pronounced light use efficiency (see detailed calculation procedure in Supporting Information Figure S9). Figure 4 | The temperature change versus on/off light irradiation of ACAT-CD solution at pH 5.0: (a) with different power densities; (b) with different ACAT-CD concentrations. (c) The comparison of photothermal conversion properties of ACAT-CD at pH 7.4 and 5.0. (d) Photothermal images of water and ACAT-CD (1 mg/mL) at pH 7.4 and 5.0 under light irradiation with power density of 1.0 W/cm2. The laser with wavelength of 750 nm was chosen to match the maximum absorption band of ACAT-CD at pH 5.0. Download figure Download PowerPoint The selective photothermal conversion performance of the ACAT-CD complex was evaluated in a tumor environment where the pH is lower than in normal tissues. Before the experiments on live animals, the cytotoxicity of ACAT-CD complex was first evaluated against two types of cancer cells, including MCF-7 (human breast adenocarcinoma cell line) cells and HeLa cells. As summarized in Figures 5a and 5b, ACAT-CD complex exhibited satisfactory biocompatibility to both cell lines. Fifteen nude mice were injected with MCF-7 cells to establish tumor-bearing animal models. After the tumor volume had grown to approximately 100 mm3 (ca. 3 week of incubation), the mice were divided into three groups to be subjected to the Saline-Laser treatment, ACAT-No laser treatment, and ACAT-Laser treatment. Specifically, the tumor-bearing mice in the ACAT-Laser group were intratumorally injected with ACAT-CD aqueous solution followed by the NIR light irradiation with power density of 0.6 W/cm2. For the two control groups, the Saline-Laser group received NIR light irradiation in the absence of NIR absorbers, and the ACAT-No laser group was injected with ACAT-CD solution without NIR light irradiation. The local temperature changes of the skins under NIR irradiation were recorded by IR camera at various time points (Figure 5c). The tumor injected with ACAT-CD experienced a temperature change ≤ 25.1 °C relative to body temperature, while the temperature of tumor injected with saline only increased by 9.4 °C, confirming the excellent photothermal performance of ACAT-CD in vivo. Motivated by the pH-sensitive photothermal ability of ACAT-CD, ACAT-CD injected into normal tissue provided data to support the possibility of tumor-selective ablation (ACAT-Skin). As recorded by the IR camera, the temperature changes of the ACAT-Tumor group and ACAT-Skin group maintained a difference of 5.3 °C (Figures 5d and 5e). The higher temperature resulting from better photothermal conversion performance in tumor tissue was in line with in vitro observations as stated above. Though the central temperature in ACAT-Skin group is also notable, the photothermal effect in normal tissue was much weaker than that in tumor tissue per the distinct larger thermal diffusion in the ACAT-Tumor It should be that may the photothermal difference between tumor tissue and normal including light absorption by cellular the rapid thermal by and the acidic condition in extracellular environment. Figure | (a) of MCF-7 cells with ACAT-CD at various concentrations for (b) of HeLa cells with ACAT-CD at various concentrations for (c) photothermal images of tumor or normal tissue at the ACAT-CD under NIR light irradiation. (d) The maximum temperature of tumor or normal tissue injected with ACAT-CD under the NIR laser irradiation. (e) temperature change curves of tumor or normal tissue at the ACAT-CD under NIR light irradiation. Download figure Download PowerPoint The effect of ACAT-CD was by monitoring the tumor other for 3 (Figure The tumor in mice were estimated according to the following tumor volume = Noting that the tumor is the tumor size should have compared with the tumors from the mice With the of the supramolecular NIR absorber and light irradiation, treatment in the ACAT-Laser group the tumor and the tumor In contrast, the of the control groups experienced a drastic increase by for light irradiation only and for ACAT-CD Notably, was significant body in treatment group during the therapy process, which confirms the low of ACAT-CD and light irradiation (Figure After in observations for 3 the tumor tissues were removed from the mice and were As compared in Figure the tumor of the ACAT-Laser group was much less than those of the control The optical images of tumor tissues as in Figure distinctly the successful tumor ablation via PTT. In addition, and experiments the of ACAT-Laser treatment to tumor tissues, in which as well as the of other groups (Figures and Supporting Information Figure normal including and were also from the mice in the ACAT-Laser The of per the experiments revealed that PTT based on ACAT-CD caused limited side to healthy tissues ( Supporting Information Figure A redshift as as nm was observed during the proton doping process of ACAT-CD. Such an is in the of molecular to limited conjugation The light absorption within the NIR at a protonated state was ascribed to the generation of a bipolaron transition per experiments and DFT The of acid-triggered bathochromic shift the for selective photothermal conversion under different pH conditions. in the selective photothermal conversion in the tumor which great promise for intelligent tumor diagnosis and ablation without side to normal tissues. be devoted to the of this initiative targeting strategy with other passive targeting by which supramolecular NIR absorbers could be in tumor via we to the conjugation length of ACAT to a pH-sensitive absorber which may PTT in deeper tissues. 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