RESEARCH ARTICLE

Novel lysosome-targeted anticancer fluorescent agents used in zebrafish and nude mouse tumour imaging

  • Xiuli Chen 1,2 ,
  • Feng Liu 1 ,
  • Bin Chen 3 ,
  • Haiying Wu 2 ,
  • Kun Li 1 ,
  • Yongmei Xie 1 ,
  • Weihong Kuang , 4 ,
  • Zhihui Li , 1,3
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  • 1. Department of Thyroid Surgery, West China Hospital of Sichuan University, Chengdu 610041, China
  • 2. Department of Obstetrics, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou 450003, China
  • 3. Laboratory of Thyroid and Parathyroid Disease, Frontiers Science Center for Disease-related Molecular Network, Rare Diseases Center, West China Hospital of Sichuan University, Chengdu 610041, China
  • 4. Department of Psychiatry and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu 610041, China

Received date: 31 Dec 2020

Accepted date: 27 May 2021

Published date: 15 Jan 2022

Copyright

2021 Higher Education Press

Abstract

The design of three novel fatty nitrogen mustard-based anticancer agents with fluorophores incorporated into the alkene structure (CXL 118, CXL121, and CXL122) is described in this report. The results indicated that these compounds are selectively located in lysosomes and exhibit effective antitumour activity. Notably, these compounds can directly serve as both reporting and imaging agents in vitro and in vivo without the need to add other fluorescent tagging agents.

Cite this article

Xiuli Chen , Feng Liu , Bin Chen , Haiying Wu , Kun Li , Yongmei Xie , Weihong Kuang , Zhihui Li . Novel lysosome-targeted anticancer fluorescent agents used in zebrafish and nude mouse tumour imaging[J]. Frontiers of Chemical Science and Engineering, 2022 , 16(1) : 112 -120 . DOI: 10.1007/s11705-021-2075-5

1 Introduction

Lysosomes, existing in all protozoa and multicellular animal cells, are essential organelles that degrade endocytosed poisonous molecules. Because they contain a wide spectrum of hydrolytic enzymes, lysosomes play major roles in the degradation and recycling of macromolecules [14]. Lysosome dysfunction is often involved in pathologies, including cancer [5], neurodegenerative diseases [68], atherosclerosis, and rheumatoid arthritis [9,10]. Recently, researchers have shown great interest in lysosome research and have achieved certain scientific results. Miao et al. demonstrated that the activation of lysosomes is induced by structural damage and the role of this activation pathway in extracellular matrix regeneration [11]. Fujimaki et al. revealed for the first time that lysosomal function plays an important role as a “regulatory switch” in mediating the extent eukaryotic cells quiescence [12]. These research results are of great significance for understanding important physiological processes such as tissue homeostasis, repair and regeneration and ageing of the body. In terms of fluorescent probes, Hu et al. designed a new type of fluorescent molecule that is sensitive to intracellular polarity [13]. The probe emits red and blue light in lysosomes with high polarity and lipids with low polarity, respectively. The study of Hu et al. provided a new idea for studying the mechanism of lipid droplet-lysosome interactions and related diseases. In addition, lysosomes are involved in many cell signalling pathways, e.g., endocytosis, autophagy, and apoptosis [2,14,15], and many lysosomal proteases, such as cathepsins and lysosome-associated membrane glycoprotein (Lamp) signalling pathways, are involved in apoptosis [16]. Werneburg et al. reported that a tumour necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by bcl-2 proteins [17]. These studies show that the visualization of lysosomes is critical for understanding intracellular metabolism and cell membrane recycling [18,19]. Therefore, development of lysosomal-targeted fluorescent probes is a useful endeavour.
Cell death in mammals can be characterized by two main types: apoptosis and necrosis [2022]. Autophagy, which has recently been proposed as a third distinct mode of cell death, is a homeostatic process critical for sequestering long-lived proteins and damaged organelles for subsequent degradation upon fusion with a lysosome [20,2325]. Autophagy and apoptosis are activated in parallel in cancer. In addition, autophagy plays a complex role in cancer [5,26,27].
Nitrogen mustards (N-mustards) are dichloroethylamine alkylating agents and are highly active compounds. These compounds possess an active group, bis(2-chloroethyl)amine, which can interact with amino, sulfhydryl, carboxyl and phosphoric acids in cells [2830]. The reactions eventually affect the metabolism of cells and lead to cell death. However, drawbacks to the use of N-mustards to promote cell death include poor selectivity in the intracellular milieu and reactivity with untargeted cellular components, which may result in many unwanted side effects, such as bone marrow toxicity and genotoxicity [31]. Therefore, over decades, many structural modifications have been made to the N-mustard scaffold to increase its targeting affinity/specificity. Most modifications of N-mustard compounds are directed to the carrier structure [32]. Compared with other types of N-mustards, the nitrogen atom in fatty N-mustard is more alkaline. Thus, in free fatty nitrogen mustard and fatty nitrogen mustard at physiological pH values, the nitrogen atom is more likely to cause intramolecular cyclization, resulting in the formation of ethyleneimine ions, which are highly active strong electrophilic alkylating agents [33,34]. As a consequence, fatty N-mustards have great potential for high antitumour activity in a wide range of in vitro and in vivo environments [35].
To our knowledge, most fluorescent probes, which are powerful tools used in cell biology, cannot simultaneously meet the two requirements for effective visualization and antitumour activity. Recently, our group presented several new types of mitochondria-targeting N-mustard agents with fluorophores incorporated into the main N-mustard skeleton [3639]. The molecules we synthesized exhibited highly selective activity against cancer cells. In biology and life sciences, the development of lysosome-targeting N-mustard fluorescent drugs is important for lysosome imaging and the treatment of lysosome-related diseases. In previous works, we have reported two new types of molecules, both aromatic N-mustards, with mitochondria-targeting imaging and fluorescent DNA alkylation ability [36,37]. In the present study, when we replaced aromatic N-mustard with fatty N-mustard, three lysosome-targeted agents (CXL118, CXL121, and CXL122) were formed, and then, three different fluorophores were integrated into the fatty N-mustard skeleton. As shown in Fig. 1, fluorophores 2-(2-methyl-4H-chromen-4-ylidene)malononitrile, 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium, and 3-ethyl-1,1,2-trimethyl-1H-benzoindol-3-ium were linked to fatty N-mustard skeletons by an olefin double bond. Our synthetic probes were then used for zebrafish and nude mouse tumour imaging. The experiments showed that these probes can be used for diagnostic imaging.
Fig.1 Novel functional fluorescence compounds developed in this study.

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2 Experimental

2.1 Materials and methods

All reagents are purchased with no special note. All of the solvents were dried according to the standard methods and were spectroscopic grade in the optical spectroscopic studies. Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AM-400. The 1H NMR (400 MHz) and 13C NMR (101 MHz) chemical shifts were given using CDCl3 and DMSO-d6 as the internal standard. The 1H NMR (400 MHz) chemical shifts were given in ppm relative to the internal reference trimethylchlorosilane. Electro-spray ionization mass spectral and high-resolution mass spectral data were recorded on a Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer, respectively. JUNYI Power Supply of JY300C was used for DNA agarose gel electrophoresis, data recorded in the Molecular Imager of Gel Doc TM XR+ with Image Lab TM Software of BIO-RAD. Inhibition rate is calculated by software of Graph Pad Prism 5.01 (Graph Pad Software, USA).

2.2 Synthesis and characterization of compounds CXL118, CXL121, and CXL122

The synthetic procedures of probes were depicted in Scheme 1. All the designed compounds contain two parts that are chromophoric units and N-mustards “warhead”. Initially, the intermediates of chromophoric units 4, 5, and 6 and N-mustards “warhead” including 4-(2-(bis(2-chloroethyl)amino)ethoxy)benzaldehyde (3) were synthesized. Take the case of compound synthesis process of CXL118.
Fig.2 Scheme 1 Synthetic routes of compounds CXL118, CXL121, and CXL122. (a) 1,2-dibromoethane, K2CO3, DMF, 80 °C; (b) K2CO3, acetonitrile, 80 °C, (c) SOCl2, DCM, 30 °C, and (d) piperidine, acetonitrile, 60 °C.

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General process: 2-(2-Methyl-4H-chromen-4-ylidene)malononitrile (4) (208 mg, 1 mmol) and 4-(2-(bis(2-chloroethyl)amino)ethoxy)benzaldehyde (3) (289 mg, 1 mmol) were dissolved in dry acetonitrile (100 mL for 14 mmol) under argon. Piperidine (91 μL, 1 mmol) was added and the solution was stirred at 60 °C for 8 h. The red solution was concentrated and the product (CXL118, (E)-2-(2-(4-(2-(bis(2-chloroethyl)amino)ethoxy)styryl)-4H-chromen-4-ylidene)malononitrile) was purified by crystallization or column chromatography. Brick red solid; Yield 26%. 1H NMR (400 MHz, CDCl3, δppm): 8.93 (t, J = 12.4 Hz, 1H, Ar–H), 7.75 (t, J = 8.2 Hz, 1H, Ar–H), 7.58–7.56 (m, 4H, Ar–H), 7.45 (t, J = 4.2 Hz, 1H, Ar–H), 6.96 (d, J = 8.4 Hz, 2H, –CH=CH–), 6.85 (s, 1H, Ar–H), 6.69 (d, J = 8.6 Hz, 1H, Ar–H), 4.10 (t, J = 6.4 Hz, 2H, –O–CH2–CH2–), 3.56 (t, J = 6.2 Hz, 4H, N(CH2)2), 3.10–3.03 (m, 4H, –CH2Cl), 2.02 (m, 2H, –O–CH2–CH2–). 13C NMR (101 MHz, DMSO, δppm): 159.08, 153.37, 152.50, 139.13, 135.81, 130.56, 126.57, 125.10, 119.49, 117.56, 116.43, 115.60, 106.54, 60.00, 56.58, 53.03. MS m/z (ESI): calcd for C26H24Cl2N3O2+: 480.12; found 480.13.
(E)-2-(4-(2-(Bis(2-chloroethyl)amino)ethoxy)styryl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium (CXL121): Yield 23%. 1H NMR (400 MHz, CDCl3, δppm): 8.30 (t, J = 8.6 Hz, 2H, Ar–H), 8.22 (d, J = 8.2 Hz, 1H, Ar–H), 7.56 (d, J = 10.4 Hz, 1H, Ar–H), 7.57–7.50 (m, 4H, Ar–H), 7.06 (t, J = 10.6 Hz, 2H, –CH=CH–), 5.05–5.00 (q, J = 14.4 Hz, 7.2 Hz, 2H, –NCH2CH3), 4.15 (t, J = 6.4 Hz, 2H, –O–CH2–CH2–), 3.52 (t, J = 8.6 Hz, 3H, –CH2Cl), 3.25–3.19 (m, 1H, –CH2Cl), 3.18–2.99 (m, 6H, N(CH2)3), 1.84 (s, 6H, –(CH3)2), 1.62 (t, J = 6.8 Hz, 3H, –CH2–CH3). 13C NMR (101 MHz, DMSO, δppm): 181.70, 162.16, 154.21, 153.84, 144.26, 140.90, 133.67, 130.10, 129.56, 128.61, 123.57, 116.22, 115.41, 110.95, 55.91, 52.58, 51.75, 28.81, 26.23, 22.53, 14.15. MS m/z (ESI): calcd for C26H33Cl2N2O+: 459.19; found 459.19.
(E)-2-(4-(2-(Bis(2-chloroethyl)amino)ethoxy)styryl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol-3-ium (CXL122): Yield 19%. 1H NMR (400 MHz, CDCl3, δppm): 8.32–8.19 (m, 4H, Ar–H), 8.12 (d, J = 8.6 Hz, 1H, Ar–H), 8.07 (t, J = 10.6 Hz, 1H, Ar–H), 7.84 (t, J = 8.4 Hz, 1H, Ar–H), 7.77–7.71 (m, 2H, Ar–H), 7.67 (t, J = 8.0 Hz, 1H, Ar–H), 7.09 (dd, J = 12.4 Hz, 8.2 Hz, 2H, –CH=CH–), 5.17 (q, J = 10.6 Hz, 8.4 Hz, 2H, –NCH2CH3), 4.16 (t, J = 8.8 Hz, 2H, –O–CH2–CH2–), 3.56 (t, J = 4.8 Hz, 3H, –CH2Cl), 3.19 (t, J = 12.0 Hz, 1H, –CH2Cl), 3.12–3.01 (m, 6H, N(CH2)3), 2.10 (s, 6H, –(CH3)2), 1.69 (t, J = 8.2 Hz , 3H, –CH2–CH3). 13C NMR (101 MHz, DMSO, δppm): 182.32, 163.12, 157.92, 153.89, 138.68, 133.71, 131.51, 131.14, 130.06, 128.82, 127.03, 123.51, 115.89, 115.46, 113.53, 109.74, 59.39, 57.46, 54.09, 53.74, 26.11, 14.34. MS m/z (ESI): calcd for C30H35Cl2N2O+: 509.21; found 509.22.

3 Results and discussion

We performed fluorescence imaging on zebrafish and nude mouse tumours without needing to introduce other fluorescent tagging agents. Therefore, in contrast with aromatic N-mustards, fatty N-mustards have lysosome-targeting and antitumour activity. As a consequence, this study provides a reference for the development of novel lysosome-targeted anticancer drugs to inhibit cell proliferation in the field of innovative drug research.
We first investigated the absorbance (UV-visible) and fluorescence emission spectra of the three target compounds. As shown in Fig. S1 (cf. Electronic Supplementary Material, ESM) and Table S1 (cf. ESM), compounds CXL118, CXL121, and CXL122 exhibited maximum fluorescence emissions at 573, 553, and 597 nm in PBS/DMSO (7/3, v/v, pH= 7.4), respectively. The maximum UV absorbances of compounds CXL118, CXL121, and CXL122 were 450, 427, and 433 nm, respectively [40,41]. Specifically, the Stokes shift of CXL122 was greater than 160 nm, which indicated minimized excitation interference in cell-imaging experiments. Therefore, the following studies mainly focused on compound CXL122.
Because of the aforementioned fluorescent properties, these compounds can be used in the cellular environment to exert effects. To determine whether CXL118, CXL121, and/or CXL122 can specifically localize in lysosomes, HeLa cells were incubated with one of the synthetic fluorescent fatty N-mustards (2 μmol·L−1) and lysosome-tracker deep red (lysotracker deep red, a commercially available lysosome dye, 300 nmol·L−1) for 20 min [42]. Subsequently, co-localization experiments were performed using the method described. As shown in Fig. 2, after HeLa cells were incubated with these compounds, the fluorescence ascribed to CXL118, CXL121, and CXL122 clearly co-localized with lysosomes, and the Pearson correlation coefficients were 0.84, 0.74 and 0.82, respectively. Moreover, as shown in the merged images presented in Fig. 2, we confirmed that all compounds can penetrate the cell membrane, and fluorescent signals were located only in the intracellular region.
Fig.3 HeLa cells were cultured with compounds CXL118 (2 μmol·L−1), CXL121 (2 μmol·L−1), and CXL122 (2 μmol·L−1) and lysotracker deep red (300 nmol·L−1) for 20 min. The compounds CXL118, CXL121, and CXL122 possess maximum UV absorption at 400–450 nm and maximum emission wavelengths between 550 and 600 nm. For these compounds, the green channel (400–520 nm), 488 nm was the excitation wavelength; for the lysotracker deep red channel (620–750 nm), 633 nm was the excitation wavelength.

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As shown in Fig. 3, an 3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide (MTT) assay was performed to test the antiproliferative activity of the fluorescent agents (CXL118, CXL121, and CXL122) at gradient concentrations (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 μmol·L−1) in different cell lines (A549, DU145, HeLa and BCPAP cells). The results are shown in Table 1. Compounds CXL118, CXL121, and CXL122 showed different antiproliferative activities. Overall, CXL118, CXL121, and CXL122 have a certain inhibitory effect on A549 cells, DU145 cells, HeLa cells and BCPAP cells, with particularly potent inhibition of A549 cells.
Fig.4 MTT assay of different cell lines treated with the compounds. The inhibition rate of CXL122 in different cells was assayed at different drug concentrations (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 μmol·L−1). The antiproliferative activity of all compounds was evaluated by MTT assay in different cell types, including A549 cells (a lung cancer cell line), DU145 cells (a human prostate cancer cell line), and HeLa cells (a cervical cancer cell line). The values represent mean±S.D. (n = 3).

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Tab.1 IC50 values for the synthesized compounds
Cell line IC50/(μmol·L−1) a)
A549 DU145 HeLa BCPAP
CXL118 5.78±1.5 7.18±1.1 8.64±1.3 >20
CXL121 2.06±1.1 10.28±1.3 17.35±1.2 >20
CXL122 1.55±1.3 7.87±1.1 6.72±1.4 >40
Bis(2-chloroethyl)amine 2.78±1.2 8.94±1.4 11.63±1.3 >30

a) Cytotoxicity was determined by MTT assay after 72 h of treatment. The data are representative of three independent experiments.

Next, Western blot assays were performed to examine the effect of CXL122 on the LC3-I, LC3-II, P62, and Bcl-2 protein expression in intact cells; LC3-I, LC3-II and P62 are the typical substrates evaluated in autophagy research. As shown in Fig. 4(A), the autophagy rate increased in a CXL122 concentration-dependent manner. Furthermore, the level of P62 was lower in the CXL122-treated groups than in the control group. In addition, we also investigated the effects of CXL122 on Bcl-2 protein expression in the apoptotic pathway. The results showed that CXL122 decreased the levels of Bcl-2 in the A549 cell line in a dose-dependent manner. In addition, bis(2-chloroethyl)amine was used as a positive control to study cell death. The Western blot assays indicated a reduction in Bcl-2 protein expression with an increase in the concentration of bis(2-chloroethyl)amine, which suggested that apoptosis was the main cause of cell death. All these results suggest that CXL122-induced autophagy in lung cancer cells may occur simultaneously with cancer cell apoptosis.
Finally, fluorescence-assisted flow cytometry was used to study the ability of the fluorescent fatty-N-mustards to be taken up by A549 cells (Fig. 4(B)) [43,44]. In this experiment, no compound was added to the control group, only the commercially available stains Annexin V and PI were added (Fig. 4(B-a); (b) shows the result of adding only CXL122 at a concentration of 10 μmol·L−1 without additional stain). Notably, the performance of the experimental group without any additional dye was similar to that of the experimental group in which additional dyes were added in the physiological range (Fig. S2, cf. ESM). The flow cytometry experiments illustrate that the fluorescent fatty nitrogen mustard molecules eliminated background interference and the misleading data associated with unreacted labelling dyes, which makes the analysis more efficient and economical.
Fig.5 (A) Western blot analysis of LC3-I, LC3-II, P62, and Bcl-2 protein expression in A549 cells treated with the indicated doses of CXL122 and bis(2-chloroethyl)amine for 72 h; (B) Flow cytometry to evaluate the effect of CXL122. (a) Control with Annexin V and PI added; (b) without additional stain, i.e., only CXL122 (10 μmol·L−1) was added to cells.

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Zebrafish, which have been widely used in biological and biochemical research, are important model organisms with rapid embryonic development and short generation times [45,46]. Zebrafish embryos and larvae have frequently been used for in vivo imaging [47]. Here, we described the results of fluorescence imaging of zebrafish using CXL122 as the probe, which was introduced into zebrafish embryos and larvae by soaking. Green in vivo fluorescence imaging with CXL122 used as a probe was observed, as shown in Fig. 5, which presents bright field and fluorescence images of embryos and larvae after soaking with CXL122. As shown in Fig. 5(A), the fluorescence images of embryos 3 h post-fertilization (hpf) become brighter with increasing concentrations of CXL122, whereas the bright field images did not differ from each other. Next, fluorescence imaging of zebrafish embryos after soaking with CXL122 (at 2.5 μmol·L−1) enabled the visualization of embryo development at different periods, namely, from the single cell stage to larval stage (Fig. 5(B)). We found that CXL122 did not disturb embryo development, demonstrating the low toxicity and high biocompatibility of CXL122. Finally, zebrafish larvae were used as models to validate the results of the imaging application of CXL122in vivo. Fig. 5(C) shows whole bodies of zebrafish larvae 78 hpf after soaking in CXL122 solution at different concentrations for 6 h. The zebrafish larvae became increasingly brighter with increasing concentration, showing a concentration-dependent reaction, illustrating that CXL122 was successfully introduced into the larvae. These results validate the practicability of using CXL122 as an imaging probe.
Fig.6 Bright field and fluorescence images of zebrafish embryos and zebrafish. A: Bright field (upper) and fluorescence (lower) images of zebrafish embryos 3 hpf after soaking for 3 h in CXL122 solution of different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 μmol·L−1). The Images acquired under bright light show the yolk sac (ys) and the inner mass of the embryos (ime). A 4× objective lens was used. Scale bars, 200 μm. (B) Bright field (upper) and fluorescence (lower) images taken at different time points of zebrafish embryos after soaking in 2.5 μmol·L−1CXL122 solution for 3 h: (a) 3, (b) 6, (c) 12, (d) 24, and (e) 48 hpf. A 4× objective lens was used. Scale bars, 200 μm. (C) Bright field (upper) and fluorescence (lower) images of the whole bodies of zebrafish larvae taken 78 hpf after soaking for 6 h in CXL122 solution of different concentrations. A 4× objective lens was used. Scale bars, 200 μm.

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Among various diagnostic imaging techniques, fluorescence imaging agents provide accurate outcomes for cancer diagnosis through non-invasive, real-time, and high-resolution imaging [4850]. In vivo imaging of various fluorescent chemical compounds has been reported [51,52]. Therefore, to provide further evidence on the fluorescence imaging capacity of CXL122in vivo, dye (20 mmol·L−1, 25 μL) was injected into the tumour site of nude mice bearing BxPC-3 cells and imaged at various times (0, 5, 10, 15, 20, 25 and 30 min). As depicted in Fig. 6, the fluorescence intensity within the tumour was markedly high and increased over time after the intratumour injection of CXL122; however, the control did not show any significant fluorescence in vivo. Thirty minutes after the injection, the fluorescence imaging of CXL122 demonstrated a stronger fluorescence intensity at tumour sites than at healthy sites, attesting to the outstanding tumour imaging capacity and long-term preservation time of the probe at tumour sites.
Fig.7 Representative images of BxPC-3 tumour-bearing mice acquired at different times after intratumour injection with CXL122in vivo.

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4 Conclusions

In conclusion, we developed three novel related multifunctional fatty N-mustard compounds (CXL118, CXL121, and CXL122) that were selectively located in the lysosomes of living cells. Because of the excellent fluorescent properties of these compounds, using flow cytometry, we directly analysed the results without the need for incorporating additional dyes or stains. These fatty N-mustard compounds have potent antitumour with inhibitory cell proliferation activity. Zebrafish embryos and larvae were used as models to validate the practicability of using CXL122 as an in vivo imaging probe. In vivo tumour imaging in a BxPC-3 tumour-bearing mouse experiment showed that CXL122 had outstanding tumour imaging capacity and long-term preservation time at tumour sites. We anticipate that this design concept will enable the development of additional fluorescent molecules containing fatty N-mustard and inspire the production of more efficient fluorescent probes for use in vivo.

Acknowledgements

This work was financially supported by Science and Technology Department of Sichuan Province (Nos. 2020YJ0237, 2018SZ0030, 2019YFH0119), National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University (Z20191006) and the 1ˑ3ˑ5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (Nos. ZYJC18025 and ZYJC18003).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11705-021-2075-5 and is accessible for authorized users.

Compliance with Ethics Guidelines

Xiuli Chen, Feng Liu, Bin Chen, Haiying Wu, Kun Li, Yongmei Xie, Weihong Kuang and Zhihui Li declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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