One-Pot Preparation of Highly Dispersed Second Near-Infrared J-Aggregate Nanoparticles Based on FD-1080 Cyanine Dye for Bioimaging and Biosensing

Open AccessCCS ChemistryCOMMUNICATION1 Feb 2022One-Pot Preparation of Highly Dispersed Second Near-Infrared J-Aggregate Nanoparticles Based on FD-1080 Cyanine Dye for Bioimaging and Biosensing Caixia Sun†, Mengyao Zhao†, Xinyan Zhu, Peng Pei and Fan Zhang Caixia Sun† Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai 200433 †C. Sun and M. Zhao contributed equally to this work.Google Scholar More articles by this author , Mengyao Zhao† Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai 200433 †C. Sun and M. Zhao contributed equally to this work.Google Scholar More articles by this author , Xinyan Zhu Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai 200433 Google Scholar More articles by this author , Peng Pei Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai 200433 Google Scholar More articles by this author and Fan Zhang *Corresponding author: E-mail Address: [email protected] Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and iChem, Fudan University, Shanghai 200433 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101332 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Bioimaging and biosensing in the second near-infrared (NIR-II) window have attracted great attention due to their unprecedented high temporal–spatial resolution, sensitivity, and penetration depth. Although some organic fluorescence dyes have been developed in this window, it is still a great challenge to synthesize hydrophilic organic contrast agents with both absorbance and emission wavelengths beyond 1300 nm. J-aggregation is a facile pathway to achieve the wavelength red-shift of organic dyes to the NIR-II window and simultaneously improve their hydrophilicity. Here, we report FD-1080 J-aggregates (FD-J) with absorbance of 1360 nm and emission of 1370 nm through heating H-aggregated FD-1080 cyanine dyes in an aqueous solution. With FD-J administration, real-time imaging of mice brain and hindlimb vasculatures can be performed beyond 1500 nm. Meanwhile, arterial and venous vessels can be clearly distinguished through dynamic imaging after injection of FD-J. In addition, reactive oxygen species-responsive NIR-II ratiometric fluorescence sensors were available based on FD-J and lanthanide nanoparticles to enable the detection of inflammation in living mice. Download figure Download PowerPoint Introduction Compared with traditional medical imaging techniques such as magnetic resonance imaging (MRI),1–3 ultrasound imaging (US),4,5 and computed tomography (CT),6 fluorescence imaging technology has attracted extensive attention and obtained rapid development, due to its advantages of nonradiation, high temporal–spatial resolution, and real-time feedback.7–9 However, traditional fluorescence imaging is mainly focused on visible light and the first near-infrared (NIR-I, 700–900 nm) window,10,11 which caused a limited tissue penetration due to the high absorbance and scattering of short-wavelength photons in biological tissues, thus hindering the application of fluorescence imaging and sensing in vivo.12,13 In recent years, the second near-infrared (NIR-II, 1000–1700 nm) window has attracted much research attention due to high temporal–spatial resolution and deep tissue penetration benefitting from low tissue absorbance and scattering and diminished autofluorescence signals.14–16 At present, NIR-II fluorescent probes are mainly divided into inorganic and organic materials. Inorganic materials, including single-walled carbon nanotubes,17,18 quantum dots,19–21 and rare earth downshifting nanoparticles (DSNPs),22–24 generally contain heavy metal elements with potential biotoxicity caused by slow excretion and long retention time in organisms, which are not conducive to clinical transformation. In contrast, organic materials, including polymers,25,26 donor–acceptor–donor (D–A–D) dyes,27,28 and cyanine dyes,29–33 are promising for clinical transformation due to their low molecular weight and flexible molecular design. So far, some organic fluorescent probes have been developed for NIR-II imaging, including the commercially available dyes indocyanine green (ICG),29 IR1061,30 S01452,31 and so on, and newly synthesized dyes FD-1080,32 LZ-dyes,33 BTC-dyes,34 CX-dyes,35 and so on. In general, the maximum emission wavelength of these organic fluorescent dyes can reach the NIR-II window by lengthening the conjugated chains, selecting strong electron-withdrawing and -donating terminal groups, and enhancing the rigidity of the molecular structures. However, most of the previously reported molecular dyes suffer from low quantum yield and poor hydrophilicity, which need further hydrophilic modification through conjugation with proteins or polyethylene glycol (PEG) chains or encapsulation into micelles for in vivo bioimaging.34,35 Therefore, it is urgent to develop a simple method that not only enables a red-shift of the emission wavelength of organic dyes to NIR-II window but also simultaneously obtains high hydrophilicity for bioapplications without further hydrophilic modification. J-aggregation, a highly ordered arrangement of organic dye monomer,36 is a facile way to achieve the red-shift of organic dyes to reach the NIR-II window. In J-aggregates, the transition dipole moments of the monomers are aligned almost parallel to the line joining their centers through a “head-to-tail” arrangement.37,38 This slip-stacked alignment of organic dyes causes a constructive coupling of the excited-state transition dipoles, resulting in unique optical properties including red-shifted absorbance and emission and increased extinction coefficient. Despite the significant photophysical advantages of J-aggregates, there are few reports of employing J-aggregates for in vivo imaging and sensing due to the difficulty in obtaining the necessary chromophore alignment. Many J-aggregates have been developed, including cyanine dyes,39,40 squaraine dyes,41 porphyrin derivatives,42,43 BODIPY,44,45 and perylene bisimides.46,47 Most J-aggregates are still in the visible window and the conventional NIR window, while few J-aggregates in the NIR-II window have been reported. In 2019, Sletten and co-workers48 prepared NIR-II J-aggregates with absorbance/emission wavelength at 1042/1043 nm by adjusting the solution polarity. In addition, our group reported FD-1080 J-aggregates (FD-J) with the longest absorbance/emission wavelength at 1360/1370 nm through co-assembly with 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC). Owing to the outstanding optical property and stability of NIR-II J-aggregates, in vivo bioimaging with high temporal–spatial resolution and high signal-to-noise ratio have been realized beyond 1500 nm.49 However, this co-assembly strategy resulted in a large particle size and a low encapsulation rate of the dyes, which relatively hampered the excretion of dyes in vivo. In this research, we obtain FD-J through a one-pot synthesis method by heating the FD-1080 H-aggregates (FD-H) in aqueous solution. FD-J remains stable in various normal physiological media and maintains the J-aggregated state with absorbance and emission wavelengths at 1360 and 1370 nm, respectively. Moreover, the FD-J enables high-resolution imaging of mice vessels beyond 1500 nm with a sharp vessel width of 249 μm. Aside from the superior imaging ability, FD-J also exhibits a high extinction coefficient ε = 2.7 × 104 M−1 cm−1 and a sensitive response toward the overexpressed reactive oxygen species (ROS) in a diseased microenvironment, which is further utilized to construct ROS-responsive ratiometric fluorescence nanosensors with Nd3+/Er3+-doped DSNP. The [email protected] nanosensor enables reliable detection of acute inflammation by ratiometric fluorescence imaging of the Nd channel (1300–1400 nm) and Er channel (1500–1700 nm). Care and Use of Animals All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Fudan University, in agreement with the institutional guidelines for animal handling. All of the animal experiments were authorized by the Shanghai Science and Technology Committee. Five weeks old female Balb/c nude mice were purchased from Shanghai SLRC Laboratory Animal Centre (Production License No: SCXK 2012-0002). Results and Discussion FD-1080 cyanine dyes were synthesized according to a previous report.30 The flat and hydrophobic benzo[c,d]indolium and methine chains of FD-1080 largely promoted the aggregate formation. In the presence of intermolecular forces, such as van der Waals dispersion force of the cyanine backbone and π–π stacking interactions among the dye molecules, there are mainly two aggregation states of FD-1080 cyanine dyes, H-aggregates and J-aggregates. H-aggregated molecules are formed by “head-to-head” sandwich arrangement and exhibit blue-shifted absorbance wavelength. In contrast, J-aggregated molecules are formed by “head-to-tail” or “shifted plates” arrangement and hold red-shifted absorbance wavelength (Figure 1a). Therefore, the type of aggregates in the solution could be identified by observing the position of the absorption peaks. At room temperature, FD-1080 cyanine dyes could be well dispersed in methanol as monomer with an absorption peak at 1012 nm. While in aqueous solution, FD-1080 cyan dyes existed in a variety of mixed states, predominantly in the form of H-aggregates with an absorption peak at 780 nm. However, after heating at 60 °C for 15 min, the absorption peak of FD-1080 aqueous solution exhibited a 580 nm red-shift from 780 to 1360 nm, illustrating the transformation from FD-H to FD-J (Figure 1b). The transformation was further demonstrated by circular dichroism (CD). As shown in Figure 1c, the CD signal could be detected for FD-H at 780 nm, FD-J showed a negative Cotton effect at 1360 nm, which was consistent with the maximum absorbance of FD-H and FD-J, respectively. Meanwhile, FD-J showed a high molar extinction coefficient ε = 2.7 × 104 M−1 cm−1 ( Supporting Information Figure S1). To investigate the transformation process from FD-H to FD-J caused by heating, FD-1080 aqueous solution (1 mM) was heated under various temperatures from 25 to 70 °C for 15 min. As shown in Figure 1d and Supporting Information Figure S2, the absorbance of FD-J at 1360 nm increased continuously with increasing temperature and reached a maximum at 60 °C. Meanwhile, the absorbance of FD-H at 780 nm decreased correspondingly, illustrating that the transformation from FD-H to FD-J was highly dependent on heating. The conversion efficiency from FD-H to FD-J was calculated to be 39.8%. In addition, the absorbance values of FD-J at 1360 nm increased 2.7 times, illustrating the successful transformation from FD-H to FD-J ( Supporting Information Figure S3). Furthermore, the time-dependent transformation process was also investigated. At less than 60 °C, the absorbance of FD-J at 1360 nm increased rapidly in 5 min and reached a maximum at 15 min, accompanied by the absorbance of FD-H at 780 nm gradually decreasing (Figure 1e). Similarly, the transformation process from FD-H to FD-J was observed from NIR-II images ( Supporting Information Figure S4). To explore the mechanism of the transformation from FD-H to FD-J, the energy diagram was obtained according to the absorption spectra. When FD-1080 was dispersed in aqueous solution, the monomer molecules tended to form the relatively chaotic H-aggregates. The H-exciton band corresponded to an optical transition to a state with higher energy of 12,020 cm−1. Although the J-exciton band corresponded to a transition to the state with a lower energy of 7350 cm−1, exogenous energy provided by heating was indispensable in assisting the H-aggregates to leapfrog the energy barrier and finally reach the stable J-aggregates with low energy ( Supporting Information Figure S5). Transmission electron microscopy (TEM) imaging showed the uniform and monodispersed FD-J with a size of 29 ± 3 nm, which was consistent with the hydrodynamic diameters of 30.5 nm measured by dynamic light scattering (DLS) (Figures 1f and 1g). In addition, there were two hydrophilic sulfonic groups on the FD-1080 monomer, which provided high hydrophilicity for FD-J during the assembly process. As a result, FD-J could be injected directly into the body for imaging without further modification. Figure 1 | (a) Structural illustration of FD-1080 monomer, H-aggregates, and J-aggregates and the corresponding absorbance wavelength in NIR-I and NIR-II region. (b) Normalized absorbance of FD-1080 monomer, FD-H, and FD-J. (c) CD spectra of FD-1080 monomer, FD-H, and FD-J. (d) Temperature-dependent absorbance spectra of FD-1080 in aqueous solution. Insert: absorbance at 1360 nm as a function of temperature. (e) Time-dependent absorbance spectra of FD-1080 in aqueous solution at 60 °C for different heating durations. Insert: absorbance at 1360 nm as a function of heating duration. (f) TEM images and size distribution results of FD-J. (g) DLS results of FD-J. Download figure Download PowerPoint Before using in vivo bioimaging and biosensing, the stability of FD-J toward a simulated physiological environment was investigated. Absorption spectra and NIR-II emission intensity of FD-J remained stable in neutral phosphate-buffered solution (PBS), saline, and mice blood within a week (Figure 2a and Supporting Information Figure S6). Moreover, the chemical stability of FD-J was further assessed in the presence of bioactive species. No apparent absorption spectra and NIR-II emission intensity change of FD-J were observed in neutral PBS after the addition of glutathione (GSH), cysteine (CyS), and hydrogen peroxide (H2O2) at 37 °C for 6 h (Figure 2b and Supporting Information Figure S7). From the absorption spectra of FD-J at different pH values, it could be observed that FD-J remained stable in neutral and alkaline solutions (pH 7–10), while it gradually degraded in acidic solutions (pH 2–6) (Figure 2c). In addition, FD-J exhibited superior photostability in aqueous solution and mice blood under continuous irradiation for 60 min with a 1120 nm laser (Figure 2d). The relatively stable chemical and optical properties ensured the potential for the following in vivo applications. Figure 2 | (a) Absorbance values of FD-J at 1360 nm in different media (PBS, saline, and blood) within a week. (b) Absorbance values of FD-J at 1360 nm under varied concentrations of CyS, GSH, and H2O2. (c) Absorption spectra of FD-J in aqueous solution at varied pH values. Insert: absorbance values of FD-J at 1360 nm as a function of pH. (d) Photostability of FD-J in water and mice blood under irradiation of 1120 nm. Insert: the corresponding fluorescence images of FD-J in water and mice blood beyond 1500 nm. Download figure Download PowerPoint To further investigate the imaging performance of FD-J in NIR-II window, in vitro images were acquired by InGaAs camera equipped with varied long-pass (LP) emission filters (1300, 1400, and 1500 nm) under excitation of the 1120 nm laser. When capillary tubes filled with FD-J were immersed in mimicked biological tissue (1% Intralipid solution) at increased phantom depth, the sharp tube edges for 1400 and 1500 nm-LP filter groups exhibited a clearer image boundary even at 3 mm immersion depth compared with the 1300 nm-LP filter group (Figures 3a and 3b). The spatial resolution was evaluated through the full width at half-maximum (FWHM) of capillary tubes, and negligible FWHM enhancement of the 1500 nm-LP filters could be observed along with the increased penetration depth (Figure 3c). Encouraged by the outstanding in vitro NIR-II performance of FD-J, we further administrated in vivo bioimaging of mice hindlimb and cerebral vasculature with FD-J (200 μL, 1 mM) to assess the imaging performance (Figure 3d). After intravenous injection, highly resolved cerebral vasculature imaging beyond 1500 nm was obtained. The width of the vessels was measured and calculated by the Gaussian-fitted FWHM of the cross-sectional fluorescence intensity profile. The FWHM of cerebral vessels along the yellow-dashed line were measured as 307, 249, and 249 μm, respectively (Figure 3e). Similarly, the widths of the hindlimb vessels were calculated as 863, 421, 351, and 312 μm, respectively (Figure 3f), illustrating the superior bioimaging spatial resolution achieved by FD-J in the NIR-II window, especially beyond 1500 nm. Figure 3 | (a) Fluorescence images of FD-J filling capillaries at varied depths of 1% Intralipid solution in varied imaging windows. (b) Intensity decay of the fluorescence signals of FD-J as a function of penetration depth in varied imaging windows. (c) The FWHM of FD-J filling capillaries at different penetration depths in varied imaging windows. (d) Images of brain and hindlimb vessels achieved by FD-J (200 μL, 1 mM) in varied imaging windows. (e and f) The fluorescence intensity profiles (black dots) and Gaussian fitted results (red lines) in brain vessel (e) and hindlimb vessel (f) along the yellow-dashed line in (d). Download figure Download PowerPoint Benefitting from the superior in vivo imaging performance, dynamic images of the mice brain beyond 1500 nm were performed with a frame rate of 1.17 frames per second (f.p.s.) (Figures 4a–4c and Supporting Information Movie S1). Epidermal vessels were observed at 1.71 s immediately after intravenous FD-J (200 μL, 1 mM) injection, followed by NIR-II signals arising from lateral sulcus on both sides of the cerebrum to the superior sagittal sinus (SSS) and the transverse sinus (TS) within 3.42 s. Then the inferior cerebral veins (ICV), SSS, superficial veins (SV), and TS were observed successively at 5.98 s. Principal component analysis (PCA) of the dynamic images was performed by time-course imaging and discriminated the arterial vessels (red, Figure 4d) from the venous vessels (blue, Figure 4e) based on hemodynamic differences (Figure 4f). Similarly, the blood flow into the femoral artery could be clearly observed by video-rate imaging upon injection FD-J (Figures 4g–4i and Supporting Information Movie S2). Aside from the main femoral artery, blood flow into many small arterial branches could also be observed with high spatial resolution. We also applied the dynamic contrast PCA approach to demonstrate the arteries and veins in the hindlimb (Figures 4j–4l). These results confirmed the capacity of NIR-II dynamic imaging to distinguish arteries from veins after FD-J administration. Figure 4 | (a–c) Time-course fluorescence images of mouse brain after FD-J (200 μL, 1 mM) administration. (d–f) PCA overlaid images of brain arterial (red) and venous (blue) vessels. (g–i) Time-course fluorescence images of mouse hindlimbs after FD-J administration. (g–l) PCA overlaid images of hindlimb arterial (red) and venous (blue) vessels. Download figure Download PowerPoint Besides on the superior imaging performance, FD-J also exhibited a high extinction coefficient at 1360 nm and a sensitive response toward the overexpressed ROS in a diseased microenvironment, which was further used to construct ROS responsive NIR-II ratiometric fluorescence nanosensor with Nd3+/Er3+-doped DSNP. In this nanosensor, NaYF4:20%[email protected]4@NaYF4:5%[email protected]4 core–shell–shell–shell DSNPs were designed and fabricated by a thermal decomposition method to provide comparable Nd3+/Er3+ fluorescence emission intensity at 1330/1550 nm under 808 nm excitation ( Supporting Information Figure S8). The emission peak of Nd3+ at 1330 nm was well overlapped with the absorbance peak of FD-J at 1360 nm, which was utilized as the NIR-II sensing signal for ROS and quenched by FD-J, while the Er3+ fluorescence emission signal at 1550 nm remained robust to provide the self-calibrated reference signal (Figures 5a–5c). The ratiometric fluorescence nanosensor (denoted as [email protected]) was prepared by coupling positively charged DSNP-NH2 with negatively charged FD-J in aqueous solution. DLS measurements showed the increased hydrodynamic diameter from 70.3 to 90.5 nm and decreased zeta potential from +20.3 to +5.6 mV, illustrating the electrostatic adherence process during the synthesis of the [email protected] nanosensor ( Supporting Information Figure S9). Upon addition of FD-J from 0 to 72 μM, NIR-II emission intensity of [email protected] at 1330 nm was gradually quenched and reached the minimum at FD-J concentration of 36 μM. On the contrary, no disturbance was observed at 1550 nm (Figure 5d). A rapid decay profile of emission ratio (Nd channel/Er channel) showed that the quenching progress changed significantly in the concentration range of 0–36 μM (Figure 5e). After FD-J conjugation, [email protected] exhibited shortened fluorescence lifetime at 1330 nm from 173 to 51 μs (Figure 5f), demonstrating energy transfer from DSNP to FD-J. Figure 5 | (a) Schematic illustration of [email protected] nanosensor. (b) Overlap of the FD-J absorbance and DSNP fluorescence emission spectra. (c) TEM image of DSNP. (d) Emission spectra of [email protected] with varied concentrations of FD-J (0–72 μM) conjugation. (e) Plot of fluorescence ratio changes as a function of FD-J concentration. (f) Fluorescence decay of DSNP and [email protected] at 1330 nm. (g) Schematic illustration of emission spectral response of [email protected] nanosensor to ClO−. (h) NIR-II fluorescence images and ratiometric fluorescence images of [email protected] under various ClO− concentration treatments. (i) The corresponding fluorescence intensity in the two channels and the emission ratio as a function of ClO− concentration. (j and k) Response selectivity of [email protected] nanosensor after various ROS treatment acquired from NIR-II fluorescence imaging (j) and emission ratio of Nd channel/Er channel (k). Download figure Download PowerPoint Similar to previously reported cyanine dyes,50 FD-J could be destroyed by ClO− and led to decreased absorbance ( Supporting Information Figure S10). As expected, the NIR-II emission intensity of [email protected] at the Nd channel (1300–1400 nm) gradually recovered after the addition of ClO− from 0 to 20 μM (Figures 5g and 5h). By using the stable emission at Er channel (1500–1700 nm) as a reference signal, the emission ratio increased with the addition of ClO− (Figure 5i). Additionally, to verify the response selectivity of [email protected] toward ClO−, the emission signal of [email protected] in the Nd channel was measured after the addition of ClO− (20 μM) and other reactive species (H2O2, OONO−, ·O2−) with an excess amount (100 μM). As shown in Figures 5j and 5k, the emission ratio of [email protected] exhibited no response in the presence of other ROS, while the ratio of ClO− treated group showed a fivefold increase, illustrating the high selectivity of [email protected] nanosensor during ClO− sensing. Encouraged by the above results, the nanosensor was applied to the in vivo ratiometric fluorescence imaging of local epidermal inflammation induced by intradermal (i.d.) injection of lipopolysaccharide (LPS). The hindlimbs of Balb/c nude mouse were i.d. injected with LPS or saline, respectively, after 4 h, and the [email protected] nanosensor was i.d. injected for ClO− detection by the NIR-II InGaAs camera (Figure 6a). Under 808 nm irradiation, the NIR-II images and signal intensity of the mouse hindlimbs were collected and measured in the two emission channels. The corresponding ratio (Nd channel/Er channel) was calculated for ClO− detection (Figures 6b–6f). Compared with the control group of a saline-treated mouse, the emission intensity of [email protected] in Nd channel was significantly enhanced in the LPS-treated mouse, and its ratio signals were about 3.5-fold higher and remained stable for at least 30 min (Figure 6f). The results demonstrated the response of the [email protected] nanosensor to distinguish an enflamed lesion from normal tissue in living mice through NIR-II ratiometric fluorescence imaging. Figure 6 | (a) Schematic illustration of NIR-II ratiometric fluorescence imaging of LPS-induced inflammation detection using a [email protected] nanosensor. (b and c) In vivo NIR-II fluorescence images and corresponding ratiometric images of saline-treated (b) and LPS-treated mouse (c) at different time points after [email protected] administration. (d and e) NIR-II signals intensity of saline (d) and LPS-treated mice (e) in the Nd channel and Er channel at different time points after [email protected] administration. (f) Ratio values obtained from the dashed yellow circle in (b) and (c). Download figure Download PowerPoint Conclusions We obtained FD-J by heating FD-H in aqueous solution at 60 °C for bioimaging and biosensing beyond 1500 nm. FD-J ensured static and dynamic imaging with high spatial resolution and penetration depth to distinguish arteries and veins of mouse brain and hindlimb. Meanwhile, the FD-J-based [email protected] nanosensor with ROS-responsiveness was hired for NIR-II ratiometric fluorescent imaging to realize inflammation detection in vivo. Overall, our study not only provides a simple pathway for the preparation of J-aggregates with NIR-II optical properties but also offers a possibility for the construction of a microenvironment responsive nanosensor for in vivo disease detection. Supporting Information Supporting Information is available and includes preparation of the FD-J, β-NaYF4:20%Er core, β-NaYF4:20%[email protected]4 core–shell, β-NaYF4:20%[email protected]4@NaYF4:5%Nd core–shell–shell, β-NaYF4:20%[email protected]4@NaYF4:5%[email protected]4 core–shell–shell–shell, and [email protected] nanosensor. Supporting Information also includes tissue phantom imaging study, responsibility test of [email protected] nanosensor in vitro, establishment of acute inflammation model, [email protected] nanosensor detection of inflammation by nanoprobes responding to microenvironment in vivo. Conflict of Interest There is no conflict of interest to report. Funding Information The work was supported by the National Key R&D Program of China (no. 2017YFA0207303), National Natural Science Foundation of China (NSFC, nos. 22088101, 21725502, 51961145403, and 22004018). 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