Photostable Red-Emitting Fluorescent Rhein-Magnesium(Ⅱ) Coordination Polymer Nanodot-Based Nanostructures With a Large Stokes Shift for Imaging Mitochondria in Cancer Cell

The mitochondria play a significant role in many cellular processes and are recognized as one of the most important therapeutic targets in cancer. Direct long-term imaging of the mitochondria is very crucial for treating cancer. However, the development of a red-emitting mitochondrial probe with a large Stokes shift and photostability remains highly challenging. Fluorescent metal complexes with superior physicochemical property have emerged as new fluorescent nanomaterials due to their increasing advantages in bioimaging. Herein, a luminescent pitaya-type nanostructure based on rhein-magnesium(II) (Rh-Mg) coordination polymer nanodots was used as a fluorescent nanoprobe to selectively image the mitochondria benefiting from the introduction of triphenylphosphine. The as-prepared Rh-Mg nanodot-based nanoprobe showed red emission peaking at 620 nm, a large Stokes shift (100 nm), and excellent photostability as compared with commercial mitochondrial probes. Due to these extraordinary features, this fluorescent nanoprobe was successfully used for mitochondrial targeting imaging of live cancer cell line Neuro-2a (mouse neuroblastoma) and BV2 microglial cells. Therefore, our results pave a new way for the design of fluorescent nanoprobes for imaging mitochondria in cancer cell.


INTRODUCTION
Among all cellular organelles, the mitochondria are known as the "power house" of mammalian cells and play crucial roles in biosynthesis, intracellular signal transduction, energy homeostasis, and apoptosis regulation (1)(2)(3)(4). Increasing evidence suggests that mitochondrial dysfunction is a key hallmark of many cancers, as reduced mitochondrial respiration has been observed in cancer cells, a phenomenon referred to as "Warburg effect" (5)(6)(7). Therefore, as an important therapeutic target in cancer, it is significant to track and image the mitochondria.
Deionized (DI) water (18.2 MW cm, Millipore) was used for the preparation of solutions. All chemicals were utilized as received without further purification or processing.

Instrumentation
The transmission electron microscope images were obtained using the JEM-2100 (JEOL, Japan) transmission electron microscope operating at an accelerated voltage of 200 kV. A ZEN 3600 instrument (Malvern, UK) was used to measure dynamic light scattering and zeta potential. Fourier transform infrared spectra (FT-IR) were measured on a Nicolet ™ iS ™ 10 spectrophotometer (Thermo Fisher Scientific, USA) at 4 cm −1 resolution with 32 scans. UV-vis absorbance spectra were recorded using a UH5300 spectrophotometer (Hitachi High-Technologies Corporation, Shanghai). Fluorescence spectroscopy was recorded using a PerkinElmer LS45 (PerkinElmer, USA). The images of confocal scanning laser microscopy were taken using a TCS SP8 microscope (Leica, Germany). The images of the inverted fluorescence microscope were captured by ECLIPSE-Ts2 microscope (Nikon Instruments Inc., Melville, NY, USA). In/ex vivo fluorescence images were taken by MIIS Small Animal Imaging System (Molecular Devices, USA).

Synthetic Procedures
Synthesis of Rh-Mg-PVP Rhein (0.9 mg) and MgCl 2 (0.264 mg) were dissolved in 21 ml methanol and 0.12 ml ammonia (25%-28%) and then stirred for 30 min at room temperature. PVP (2.4 mg) was added for further stirring for 30 min to obtain the Rh-Mg-PVP complex solution.

Synthesis of Rh-Mg-PVP@SiO 2 -TPP NPs
In order for the nanomaterial to have the targeting property, the surface of the Rh-Mg-PVP complex was firstly coated with silica shell layer; 10.15 ml methanol and 2.85 ml ammonia (25%-28%) were added to 7 ml Rh-Mg-PVP complex solution, and magnetic stirring was done at room temperature for 30 min for even dispersion. Then, 80 ml TEOS was added and the solution was stirred for 30 min after each addition of TEOS and stirred overnight after adding all to obtain Rh-Mg-PVP@SiO 2 .
The surface of Rh-Mg-PVP@SiO 2 first underwent amination with APTES. Forty milliliters of Rh-Mg-PVP@SiO 2 NP solution was added to a 100-ml round bottom flask, then about 30 ml of APTES was added. The mixture was stirred well and refluxed for 1 h in a 65°C condensation reflux device. The condensing tube was removed and then transferred to a water bath at 30°C and stirred for 12 h. TPP-COOH (1 equivalent) was dissolved in ethanol (1 ml); then, EDC (5 equivalent) and NHS (5 equivalent) were added. The active carboxylate groups are obtained by stirring for 2 h. Finally, NPs-NH 2 was conjugated through the condensation reaction between the −NH 2 group and activated −COOH group (TPP-COOH). Then, the solution was centrifuged and washed with water several times. The same operation was repeated twice. The as-prepared nanocomposites were named Rh-Mg-PVP@SiO 2 -TPP NPs.

Density Functional Theoretical Calculation
To examine the optimized structures of the compounds and its relative stability, geometry optimization with Gaussian 16 program package using standard B3LYP-6-31G (d, p) basis set was performed (27). Vibration analysis showed that the optimized structure was in accordance with the minimum points on the potential energy surface.

Cell Culture
The mouse microglia cell line BV2 and the mouse neuroblastoma cell line Neuro-2a were purchased from the ATCC. Cells were cultured at 37°C under 5% CO 2 in DMEM containing 10% (vol/ vol) FBS (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco).

Cytotoxicity Assay
The in vitro cytotoxicity of Rh-Mg-PVP@SiO 2 -TPP NPs was measured using a Cell Counting Kit-8 (CCK-8) assay in BV2 cell and Neuro-2a cell lines. Briefly, cells growing in log phase were seeded into a 96-well plate at a density of 5,000 cells per well (100 ml). The Rh-Mg-PVP@SiO 2 -TPP NPs (100 ml/well) at concentrations of 25, 50, 100, 200, and 400 mg ml −1 were added to the wells of the treatment group, and DMEM also added to the negative control group, respectively. The cells were incubated for 24 h at 37°C under 5% CO 2 . After incubation for 24 h, the medium was removed and 100 ml of CCK-8 solution was added. After 30 min, the absorbance at 450 nm was measured by a Multiskan MK3 microplate reader to determine the cell viabilities.

Cell Imaging
BV2 cells and Neuro-2a cells were plated on 15 mm small confocal laser dishes and allowed to adhere for 24 h, and the cell density was 5 × 10 4 ml −1 for 24 h, and the Rh-Mg-PVP@ SiO 2 -TPP NP solution was prepared with 200 mg ml −1 . The solution was incubated for 2, 4, and 6 h after administration. Then, the culture medium was removed and the cells were washed with PBS (1 ml) twice. One milliliter of MitoTracker Green working fluid was added and then put back into the incubator for incubation for 10-15 min. MitoTracker Green was sucked away, PBS was added along the wall, and the cells were gently washed three times by cross shaking. Finally, 1 ml of PBS was added and photographed under a confocal laser microscope. LysoTracker Green was used to investigate the distribution of nanomaterials in lysosomes, and the operation was similar to the above experiment. Luminescence imaging was performed with a PerkinElmer UltraVIEW VoX confocal fluorescence microscope and a 60 oil-immersion objective lens. Cells incubated with Rh-Mg-PVP@SiO 2 -TPP NPs were excited with a laser at 561 nm. MitoTracker Green and LysoTracker Green were excited with a laser at 488 nm.

Photostability Investigation
Rh-Mg-PVP@SiO 2 -TPP NPs (200 mg ml −1 ) were co-incubated with BV2 cells for 2 h. After being washed with PBS, the commercially available MitoTracker Green solution was added and incubated for 15 min. The cells were placed under a confocal laser microscope and subjected to continuous laser irradiation at 561 nm for about 1 h. The fluorescence images of the red channel and the green channel within 1 h were collected and compared with the fluorescence intensity of the commercially available probe to investigate the photostability of Rh-Mg-PVP@SiO 2 -TPP NPs (19). ImageJ software was used to evaluate the fluorescence intensity of each image. For the in vivo fluorescence imaging, ICR mice were first anesthetized intraperitoneally by 10% chloral hydrate (0.1 ml/10 g animal weight). Then, mice were subcutaneously injected with 6.848 mg/kg of Rh-Mg-PVP@SiO 2 -TPP NPs in the left posterior thigh, and fluorescence imaging was performed before and after injection. For ex vivo organ imaging, mice were firstly injected with 6.848 mg/kg of Rh-Mg-PVP@SiO 2 -TPP NPs through the tail vein. Then, at different time points postinjection (1, 4, and 12 h), mice were anesthetized, sacrificed, and dissected. The dissected organs, including the heart, liver, kidney, spleen, and lung, were removed and imaged. In vivo and ex vivo fluorescence imaging was performed with MIIS small animal imaging system (Molecular Devices, in vivo fluorescent imaging: excitation wavelength = 500 nm; ex vivo organ imaging: excitation wavelength = 560 nm). The analysis of each image was conducted by ImageJ software.

RESULTS AND DISCUSSION
The synthesis procedure of Rh-Mg-PVP@SiO 2 -TPP NPs is illustrated in Scheme 1. Firstly, rhein-magnesium (Rh-Mg) coordination polymer nanodots with an average particle size of ∼3.3 nm were first synthesized in methanol phase (Figures 1A, B). Second, the coordination polymer nanodots were protected with PVP(Rh-Mg-PVP) to avoid aggregation and to increase stability (28). Using a modified Stöber method (29), a SiO 2 shell was fabricated, and multiple Rh-Mg-PVP nanodot core-silica shell nanoparticles (multi-Rh-Mg-PVP@SiO 2 NPs) were obtained. The TEM images showed that the size of Rh-Mg-PVP@SiO 2 was ∼30 nm in diameter with a narrow size distribution In addition, small spherical particles were observed in the TEM images dispersed in the SiO 2 nanomaterials. The size of these spherical particles was ∼2.4 nm according to the ImageJ analysis, which was similar to the size of Rh-Mg-PVP. Thus, after coating the silica shell layer, each silica nanoparticle encapsulated several Rh-Mg-PVP nanodots, forming a similar "pitaya-type" structure. It was preliminarily inferred from the FT-IR spectra that the SiO 2 shell was successfully coated. As can be seen from Supplementary Figure  S2, 1,092 cm −1 is the absorption peak of Si-O-Si antisymmetric stretching vibration, and 799 cm −1 is the absorption peak of Si-O-Si symmetric stretching vibration. Accordingly, it can be concluded that silica particles with a large amount of hydroxyl groups on the surface were prepared by the classical Stöber method. To enable nanodots to target the mitochondria, the Rh-Mg-PVP@SiO 2 first underwent amination with APTES. Then, the mitochondrial targeting group (4-carboxybutyl) triphenylphosphine bromide was activated using EDC and NHS (30,31). Finally, Rh-Mg-PVP@SiO 2 -NH 2 was conjugated through the condensation reaction between the −NH 2 group and activated −COOH group, and the as-prepared nanocomposites were named Rh-Mg-PVP@SiO 2 -TPP NPs. TEM images ( Figures 1C, D) showed that the prepared Rh-Mg-PVP@ SiO 2 -TPP NPs were spherical with a particle size of 25.4 ± 3.5 nm. The average hydrodynamic particle size of Rh-Mg-PVP@SiO 2 -TPP NPs is about 102.7 nm, and the PDI value is 0.143, which indicated that the Rh-Mg-PVP@SiO 2 -TPP NPs have narrow particle size distributions ( Figure 1E). In the FT-IR spectra of Rh-Mg-PVP@ SiO 2 -TPP NPs ( Figure 1F), the characteristic peak at 1,113 cm −1 was the stretching vibration peak of the P-C bond. The absorption peaks located at 509 and 692 cm −1 were attributed to the bending vibration of the P-C bond. Thus, the TPP-COOH was successfully anchored to the Rh-Mg-PVP@SiO 2 . The surface modification process was further monitored with z potential evaluations.  The corresponding absorption and emission spectra of Rh-Mg-PVP@SiO 2 -TPP NPs were measured in water. The UV-vis absorption spectrum shows that the maximum absorption wavelength of Rh-Mg-PVP@SiO 2 -TPP NPs is 520 nm, similar to the spectrum of the Rh-Mg coordination polymer nanodots (Figure 2A). Compared with the maximum absorption wavelength of rhein at 430 nm, the spectrum of Rh-Mg-PVP@SiO 2 -TPP NPs has a red shift. To investigate the influence of magnesium on the UV-vis spectra of rhein, theoretical studies of electronic structures were carried out using the density functional theory (DFT) method (32). On the basis of optimizing the structure of ground state, the excited states of Rh-Mg and rhein in methanol were studied by the B3LYP/TD-DFT method. As shown in Figure 3, the energy gaps (E gap ) of HOMO-1 and LUMO of rhein and Rh-Mg are 3.498 and 2.870 eV, respectively. Thus, forming a metal complex made a dramatic change in the energy levels of excited states, leading to the red shift in absorption spectra of Rh-Mg. When excited at 520 nm, Rh-Mg-PVP@SiO 2 -TPP NPs displayed strong emissions at 620 nm ( Figure 2B). The results demonstrate that Rh-Mg-PVP@SiO 2 -TPP NPs exhibit large Stokes shifts, approximately 100 nm, which results in an efficient separation of the absorbance and emission maxima ( Figure 2C). Next, we recorded the fluorescence quantum yields of Rh-Mg-PVP@SiO 2 -TPP NPs using the integrating sphere setting and got the value of 0.49 in water, which is a considerable value among many reported fluorescence probes (19,26).  Figure 4. The red and green color fluorescent images were obtained after excited by 561 and 488 nm, respectively. The red fluorescence represented the distribution of Rh-Mg-PVP@SiO 2 -TPP NPs, and green represented MTG. As can be seen from the merged images, the red image and the green image were well merged after co-incubation of Rh-Mg-PVP@SiO 2 -TPP NPs and cell lines for 4 h. The mean co-localization coefficients of Rh-Mg-PVP@SiO 2 -TPP NPs and MTG from Neuro-2a cells and from BV2 cells were 0.59 and 0.61, respectively (Figures 4, 5). These results demonstrated that Rh-Mg-PVP@SiO 2 -TPP NPs could stain mitochondria in live cells with high selectivity. In addition, we found that a portion of nanoprobes was also distributed in lysosomes (Supplementary Figure S5).
Photostability is one of the most important parameters for fluorescent bioprobes. Here, to verify the work ability of Rh-Mg-PVP@SiO 2 -TPP NPs, the fluorescence imaging under long-time laser irradiation was investigated. After 1 h of laser irradiation, the fluorescence intensities of Rh-Mg-PVP@SiO 2 -TPP NPs in Neuro-2a cells remained at 97.1% ( Figure 6A), whereas the fluorescence intensity of MitoTracker Green remained at 52.3% ( Figure 6B and Video S1). The repeated experiment was performed in BV2 cells and similar results were obtained (Supplementary Figure S6). Thus, Rh-Mg-PVP@SiO 2 -TPP NPs could be believed as photostable, because it maintained more than 50% fluorescence intensities after 1 h of laser irradiation ( Figure 6A). Therefore, the Rh-Mg-PVP@SiO 2 -TPP NPs show great potential for long-term live cell imaging.
In addition to cellular imaging, Rh-Mg-PVP@SiO 2 -TPP can also be used for in vivo imaging. Here, we selected the ICR mice as biological models, and the in vivo fluorescence imaging capability of Rh-Mg-PVP@SiO 2 -TPP NPs was investigated by MIIS small animal imaging system. After subcutaneous injection, compared with before injection, an obvious fluorescence signal was observed at the injection site (Supplementary Figure S7 Figure S8 in Supporting Information). The overlays of fluorescence images and brightfield images of organs showed that the signal originated predominantly from the liver, kidney, and lung. These results

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by The Animal Care and Ethics Committee of Beijing University of Chinese Medicine.