Zr-based metal–organic frameworks (MOFs) using thiazolothiazole as an organic ligand exhibit excellent two-photon excited fluorescence performance, particularly in the generation of reactive oxygen species. In this study, a ZrTc nanoMOF was designed and modified with hyaluronic acid (HA) to provide good biocompatibility and cancer cell-specific targeting. In triple negative breast cancer (TNBC), the optimized composite ZrTc@HA exhibited considerable two-photon activity and excellent light-triggered O2·− generation ability at an excitation wavelength of 780 nm. ZrTc@HA can be used as a diagnostic probe for fluorescence imaging and as a therapeutic agent for photodynamic therapy (PDT) with no remarkable toxicity in vitro or in a subcutaneous tumor model in vivo. In summary, we developed a promising two-photon-activated ZrTc@HA for PDT in TNBC, with immense potential for advancements in monotherapy and synergistic phototherapy.
Triple negative breast cancer (TNBC), accounting for 15%–20% of all diagnosed breast cancer cases, is a biologically and clinically heterogeneous disease. Compared with other breast cancer types, TNBC has significantly higher rates of local and distant recurrence and mortality.1 Additionally, owing to the negative expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, it is difficult to develop specific targeted drugs; therefore, TNBC is the most refractory breast cancer subtype to treat.
Photodynamic therapy (PDT) has emerged as a promising technique for cancer therapy in recent decades owing to its minimal invasiveness, low toxicity, and high selectivity.2 PDT triggers a photochemical process in which photosensitizers (PSs) are activated by a specific wavelength of light to generate tumoricidal reactive oxygen species (ROS), which can quickly and effectively induce tumor autophagy, apoptosis, and necrosis.3
Metal–organic frameworks (MOFs), also referred to as porous coordination polymers, are hybrid crystalline materials formed by the self-assembly of metal ions or rigid clusters with multitopic organic ligands. Their unique features, including low or noninvasive nature, adjustable porosity, tunable functionality, and designable topology, make MOFs ideal for use as PSs or nanovehicles for delivering PSs in PDT for cancer treatment.4 Using organic ligands with photosensitive properties, MOFs have emerged as highly efficient multiphoton-excited fluorescence-responsive materials.5 The spatial arrangement of MOFs prevents PS aggregation and self-quenching, ensuring efficient light-to-ROS conversion; this can be further improved via metal-to-ligand charge transfer.6 Functionalized MOFs, modified via metal coordination or covalent and noncovalent interactions with ligands, typically exhibit improved physiological stability, blood circulation, biocompatibility, and specific cell- and organelle-targeting abilities, remarkably improving the selectivity of therapeutic nanoparticles (NPs).7 Furthermore, some MOFs are biodegradable and can be eliminated from the body with few side effects, making them ideal candidates for clinical applications.8
In our recent study,9 we synthesized a nanoporous ZrTc structure comprising Zr clusters (Zr6 clusters) and a thiazolothiazole-based organic ligand (4,4′-(thiazolo[5,4-d]thiazole-2,5-diyl)dibenzoic acid, Tc); this structure exhibited excellent two-photon excited fluorescence performance and improved the ability to generate superoxide anion radicals (O2·−) and singlet oxygen (1O2) in Hep-G2 cells. In the present study, to assess the potential of ZrTc to improve the photodynamic conversion efficiency in TNBC, we coated ZrTc with hyaluronic acid (HA) and evaluated its ability to generate ROS and provide PDT in preclinical models of TNBC. The potential two-photon excited fluorescence performance of ZrTc nanoMOFs makes them a promising biological tool for comprehensive fluorescence imaging and PDT of diseases under near-infrared (NIR) irradiation. HA, which introduces negative charges on the surface of ZrTc MOFs, can reduce protein adsorption, thereby resulting in improved physiological stability and biocompatibility in blood.10 CD44, a transmembrane glycoprotein involved in tumor metastasis and invasion,11 is highly expressed in many types of tumors, including TNBC.12 Therefore, this study presents a simple and effective strategy for developing a ZrTc@HA MOF for two-photon fluorescent imaging-guided tumor-specific PDT with high biosafety for treating TNBC (Scheme 1).
II. EXPERIMENTAL SECTION
A. Development and characterization of ZrTc@HA
The synthesis process of ZrTc NPs is described in our previous study.9 In brief, 135.35 mg of Tc, 69.90 mg of ZrCl4, and 140 mg of benzoic acid were dissolved in 30 ml of dimethylformamide (DMF) solution. The solution was then transferred and sealed in a 50 ml Teflon-lined stainless-steel autoclave. The reaction was performed at a temperature of 120 °C for three days. Subsequently, the solution was centrifuged and washed three times with DMF and ethanol. The ZrTc product was freeze-dried. For HA modification, 1 mg of ZrTc and 2 mg of HA were mixed in 20 ml of deionized water. The solution was stirred at room temperature for one day. Then, ZrTc@HA was washed with deionized water three times via centrifugation and freeze-dried.
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to determine the morphology and size of the prepared NPs. The charge potentials of ZrTc and ZrTc@HA were measured based on their zeta potentials. The x-ray diffraction (XRD) patterns of ZrTc and ZrTc@HA were obtained. Dynamic light scattering (DLS) was performed to evaluate the stability of the NPs. Finally, fluorescence spectra were recorded to determine the role of the fabrication and functionalization of ZrTc@HA.
B. Two-photon excited fluorescence spectroscopy and two-photon absorption (2PA) cross section
A femtosecond laser pulse and a Ti–sapphire system (680–1080 nm, 80 MHz, and 140 fs) were used as light sources for evaluating two-photon excited fluorescence. Fluorescein (1 × 10−3 M) was used as a reference sample. The ZrTc@HA concentration was 1 mg/ml. The 2PA cross section was determined according to the method described in our previous study.13
C. Superoxide anion radical (O2·−) detection
To measure O2·− levels, we used dihydrorhodamine 123 (DHR123) as an indicator. In brief, 10 μM DHR123 and 50 μg/ml ZrTc@HA were dissolved in 2 ml of deionized water. The cuvette was then exposed to LED light (400–700 nm and 1 W/cm2) for 0–5 min. Finally, the fluorescence spectra were recorded immediately after irradiation.
D. O2·− generation in 4T1 cells under two-photon laser irradiation
The O2·− production in 4T1 cells after ZrTc@HA treatment was assessed using a confocal laser scanning microscope (CLSM). In brief, 1 × 104 4T1 cells (1 ml) were seeded into Petri dishes and incubated overnight. Then, the media was refreshed with 50 μg/ml ZrTc@HA in glucose-free RPMI-1640 media for 12 h. Thereafter, 4T1 cells were washed three times with PBS solution (pH = 7.4) and incubated with 1 μM dihydroethidium (DHE) for 0.5 h. Finally, 4T1 cells were irradiated (780 nm and 0.1 W/cm2) for 0, 1, 2, and 3 min and imaged under a CLSM.
E. One- and two-photon fluorescence imaging of ZrTc@HA in 4T1 cells
The Lecia TCS SP8 DIVE FALCON equipped with a single-wavelength laser (output wavelengths: 405, 456, 488, 514, 561, and 633 nm) and a femtosecond laser (adjustable output wavelength: 680–1080 nm, 80 MHz, and 140 fs) was used to achieve one- and two-photon fluorescence imaging. In brief, 1 × 104 4T1 cells (1 ml) were seeded into Petri dishes and incubated overnight. Then, 4T1 cells were treated with 50 μg/ml ZrTc@HA or ZrTc in glucose-free RPMI-1640 media for 12 h. After washing three times with PBS, 4T1 cells were irritated at a one-photon fluorescence emission wavelength of 405 nm (input power: 0.1 W/cm2) and a two-photon fluorescence emission wavelength of 780 nm (input power: 0.1 W/cm2). Images were captured under a CLSM. Because the cell membrane does not express the HA receptor, QSG-7701 human normal liver cells were used as a negative control.
F. In vitro therapeutic efficacy of ZrTc@HA in 4T1 cells
Flow cytometry and confocal microscopy were performed to determine the apoptotic ability of ZrTc@HA in 4T1 cells. For flow cytometry, 2 × 105 cells (2 ml) were seeded into a six-well plate and incubated overnight. Then, the medium was replaced with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 50 μg/ml ZrTc or ZrTc@HA for 12 h. The medium was washed with PBS and refreshed with DMEM. The plates were irradiated with 780 nm light for 0, 1, 3, and 5 min. Then, the cells in each group were washed, trypsinized, and collected after centrifugation at 1000 rpm for 3 min. The cells in each group were resuspended in 195 μl of binding buffer, followed by the addition of 5 μl of annexin V–FITC solution and 10 μl of propidium iodide and incubation for 20 min. The samples were then analyzed using flow cytometry.
For confocal microscopy, after incubation with ZrTc or ZrTc@HA for 12 h, the cells were irradiated with a 780 nm laser light for 5 min. The cells were washed and then 195 μl of binding buffer, 5 μl of annexin V–FITC solution, and 10 μl of propidium iodide were added in turn and incubated for 20 min. Finally, the fluorescence images of each sample were obtained using an SP8 CLSM.
The methylthiazolydiphenyl-tetrazolium bromide (MTT) method was used to determine the cytotoxicity of ZrTc@HA. In brief, 1 × 104 cells (100 μl/well) were cultured in 96-well plates for 24 h with DMEM containing 10% fetal bovine serum. Then, the medium was replaced with DMEM containing ZrTc or ZrTc@HA at different concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. After incubation for 12 h, the cells were washed two times with fresh DMEM before replacing the MTT-containing medium in each well. In the photodynamic group, the cell plates were irradiated with 780 nm light for 5 min. Finally, the plates were incubated at 37 °C for 4 h. The formazan crystals were dissolved in DMSO after the MTT-containing medium was removed. Finally, the absorbance was measured at 490 nm using a microplate reader.
G. In vivo imaging of ZrTc@HA in mice bearing 4T1 cells
To establish a mouse model of TNBC, 1 × 105 4T1 cells were subcutaneously injected into four-week-old female BALB/c mice. When the tumor volume reached 80 mm3, 5 mg/kg ZrTc or ZrTc@HA in PBS was intravenously injected into the mice. At predetermined times of 0, 2, 4, 6, 8, and 12 h, the anesthetized mice were placed into a chamber for NIR imaging using an in vivo molecular imaging system equipped with a femtosecond laser pulse and Ti–sapphire system (680–1080 nm, 80 MHz, and 140 fs). For quantitative analysis, the regions of interest (ROIs) in the tumor regions were determined for comparison. After 12 h, the tumor-bearing mice were sacrificed, and the tumors and major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested for ex vivo fluorescence imaging.
H. In vivo pharmacokinetics investigation
To determine the half-life of ZrTc@HA, 5 mg/kg ZrTc@HA was injected into the tail veins of female BALB/c mice. Blood samples were collected at various time intervals: 0 min (pre-injection), 1, 3, 5, 10, 15, 30 min, 1, 3, and 8 h. Each blood sample (100 μl) was dissolved in 1 ml of 65%–68% nitric acid to dissolve the remaining ZrTc@HA. Then, 50 ml of the solution was diluted with 5 ml of deionized water. The Zr concentration was determined using inductively coupled plasma mass spectrometry.
I. In vivo therapeutic efficacy evaluation
For the tumor treatment experiment, BALB/c mice with 4T1 cells (80 mm3) were randomly divided into four groups (n = 7) and intravenously injected with PBS solution, laser, ZrTc@HA, or ZrTc@HA with laser. Laser irradiation was performed at an excitation wavelength of 780 nm for 5 min. The mice were observed for 14 days, and their tumor volumes and body weights were measured after every two days using an electronic balance and a caliper. At the end of the observation period, the mice were sacrificed, and their tumors and major organs (heart, liver, spleen, lungs, and kidneys) were collected for histological analysis. Furthermore, blood was collected for biochemical and routine blood analyses, including hematocrit (Hct), white blood cell (WBC) counts, red blood cell (RBC) counts, mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH).
J. Statistical analysis
Data are presented as mean or mean ± standard error. The Student’s t-test was used to calculate significant differences. The significance level was set at p < 0.05.
III. RESULTS AND DISCUSSION
A. Synthesis and characterization of ZrTc@HA
In a previous study, we synthesized ZrTc NPs using a solvothermal method and demonstrated their high-order multiphoton excited fluorescence performance in vitro.9 Recent studies have reported that HA is a promising target molecule in PDT for TNBC, resulting in superior tumor growth inhibition.14–16 In this study, we coated ZrTc NPs with HA to target 4T1 TNBC cells because of the high expression of CD44 in 4T1 cells. We focused on the targeted fluorescence imaging and PDT of ZrTc@HA under laser irradiation both in vitro and in vivo.
Following the bioconjugation of HA to the ZrTc NPs, we characterized its morphology using SEM and TEM [Figs. 1(a) and 1(b)]. The average diameter of ZrTc NPs was 82.50 ± 24.92 nm, whereas that of ZrTc@HA was 99.36 ± 26.42 nm. This indicates that the morphology of the ZrTc NPs did not markedly change after HA modification. The zeta potential of ZrTc NPs changed from positive to negative after HA modification, indicating successful HA wrapping [Fig. 1(c)]. The XRD patterns of ZrTc@HA NPs are shown in Fig. 1(d). The diffraction pattern of the ZrTc@HA powder was similar to that of the simulated UiO-68. To assess the stability of ZrTc@HA, we evaluated the size of the NPs every day for seven days using the DLS method. The results are shown in Fig. 1(e), which shows that ZrTc@HA remained stable in PBS and serum-containing DMEM. The absorption and fluorescence intensities of ZrTc@HA are shown in Figs. 1(f) and 1(g).
Subsequently, the nonlinear optical (NLO) properties of ZrTc@HA NPs were evaluated. An aqueous solution of ZrTc@HA exhibited strong two-photon excited fluorescence when exposed to NIR light irradiation in the range of 680–940 nm, with the highest fluorescence intensity observed at an excitation wavelength of 780 nm [Fig. 1(h)]. Furthermore, the fluorescence intensity of ZrTc@HA NPs increased as the input power increased from 1 to 1.6 W [Fig. S1(a)]. The slope of the logarithmic fitting curve for two-photon fluorescence with a laser power was calculated as 2.04, further confirming the two-photon absorption properties [Fig. S1(b)]. Herein, the red shift of the emission peak under two-photon excitation compared with single-photon excitation can be assigned to the reabsorption effect.6,9 These findings, in conjunction with the data from our previous study,9 suggest that the NLO properties of ZrTc were retained after HA coating.
To assess the photodynamic ability of ZrTc@HA, we determined the amount of ROS generated by the NPs. ROS play crucial roles in cancer cell autophagy, apoptosis, and necrosis, which are the key mechanisms of PDT in cancer treatment.17,18 First, the amount of ROS generated by ZrTc@HA must be determined to guarantee effective treatment. O2·− is a powerful member of the ROS family.19 To measure O2·− generation, we used DHR123 as a fluorescence indicator and investigated the fluorescence intensity under LED light irradiation at a wavelength of 400–700 nm and an input power of 1 W/cm2. Compared with DHR123 alone, ZrTc@HA NPs with DHR123 exhibited stronger fluorescence intensity at predetermined times of 1–5 min [Fig. S2(a)]. In addition, the fluorescence intensity increased as the irradiation time increased, reaching its maximum value after 5 min [Fig. S2(b)]. Overall, at any pretreatment time, O2·− generation was significantly higher in ZrTc@HA NPs with DHR123 than in DHR123 alone [Fig. 1(i)]. These results suggest the effective O2·−-generation ability of ZrTc@HA NPs following LED irradiation, indicating their potential for TNBC treatment.
B. Targeting and treatment ability of ZrTc@HA in 4T1 cells
The cellular uptake and water solubility of PSs are two important factors that affect the effectiveness of PDT for tumor destruction.20 Therefore, we tested the cellular uptake of ZrTc@HA by 4T1 TNBC cells.
In the cellular uptake experiment, we first verified that the HA modification of ZrTc NPs significantly enhanced their uptake by 4T1 cells. As shown in Fig. 2(a), compared with ZrTc NPs, 4T1 cells inoculated with ZrTc@HA displayed stronger green fluorescence intensity under one-photon excitation at a wavelength of 405 nm. To further confirm the active targeting ability of ZrTc@HA NPs, we performed an experiment using QSG-7701 cells, which have negative CD44 expression on their membrane, and observed almost no fluorescence signal after irradiation at 405 nm. Additionally, the NLO properties of the ZrTc@HA NPs were investigated in 4T1 cells after two-photon excitation at 780 nm, and red fluorescence was clearly observed [Fig. 2(b)]. To further compare the one-photon and two-photon efficacies of ZrTc@HA in 4T1 cells, we quantified the fluorescence intensity at the drawing bar of 6 µm; the results are demonstrated in Fig. 2(c). The fluorescence signal was significantly stronger under two-photon excitation than under one-photon excitation. These results confirm the superior cellular uptake of ZrTc@HA by 4T1 cells.
Although the O2·−-generation ability of ZrTc@HA in aqueous solution after LED irradiation was confirmed, it was important to verify its ability to generate O2·− in 4T1 cells after two-photon laser irradiation. As shown in Fig. 3(a), in the presence of 1 μM DHR123 and 50 μg/ml ZrTc@HA, a weak red fluorescence was observed in 4T1 cells after 1 min of irradiation at an excitation wavelength of 780 nm and a power input of 0.1 W/cm2. Under the same excitation conditions, the red fluorescence intensity increased as the irradiation time increased from 1 to 3 min; a strong red fluorescence signal was clearly visible in 4T1 cells after 3 min of irradiation. These data provide a foundation for PDT with ZrTc@HA for treating TNBC.
Next, we evaluated the therapeutic efficacy of O2·− generated by ZrTc@HA NPs in 4T1 cells. To verify the apoptotic effect of laser irradiation on ZrTc@HA, we performed flow cytometry and confocal microscopy. The apoptotic rate of ZrTc@HA-treated 4T1 cells was significantly higher than that of ZrTc-treated cells under LED irradiation for 1, 3, and 5 min. Remarkably, the apoptotic rate of ZrTc@HA-treated 4T1 cells reached 99% under LED irradiation for 5 min [Fig. 3(b)]. To observe the in situ fluorescence of 4T1 cell apoptosis, we further imaged ZrTc- or ZrTc@HA-treated 4T1 cells under 780 nm laser irradiation for 5 min, and the signals of annexin–V FITC and PI are clearly visible in Fig. 3(c). These results suggest the remarkable treatment effect of ZrTc@HA under laser irradiation, making it a suitable candidate for use in 4T1 cell-based mouse experiments.
To confirm that the therapeutic effect was due to ROS generation, we first tested the toxicity of ZrTc and ZrTc@HA in 4T1 cells using the MTT method. The viability of 4T1 cells was almost 100% at a concentration range of 0–200 μg/ml, with no significant decrease [Fig. 3(d)]; this indicates the good biocompatibility of these NPs. Next, we examined the effect of ZrTc or ZrTc@HA with laser irradiation at 780 nm on 4T1 cells. Compared with ZrTc-treated 4T1 cells with 780 nm laser irradiation, the cell viability of ZrTc@HA-treated cells with laser irradiation for 5 min was slightly decreased at 6.25 μg/ml and remarkably decreased at concentrations of 25–200 μg/ml. These results suggest that ZrTc@HA can inhibit 4T1 cell proliferation upon laser irradiation, primarily owing to ROS generation.
C. In vivo fluorescence imaging of ZrTc@HA
Inspired by the cellular uptake of ZrTc@HA by 4T1 cells and the superior targeting ability of ZrTc@HA, we investigated its imaging and therapeutic applications in mice bearing 4T1 cells. The in vivo targeting ability of ZrTc@HA in TNBC cells was tested by injecting the NPs via the tail vein into BALB/c mice bearing 4T1 cells. As shown in Fig. 4(a), the fluorescence signal of mice treated with 5 mg/kg ZrTc@HA was higher in the 4T1 tumor regions than that of mice treated with ZrTc NPs at predetermined imaging times of 0, 2, 4, 6, 8, and 12 h. In particular, an obvious fluorescence signal was observed from 6 to 8 h after ZrTc@HA NP injection, whereas the fluorescence signal of ZrTc was consistently weak at all treated time points. Moreover, the quantified data of the ROIs in the tumor regions further confirmed the superior targeting ability of ZrTc@HA compared with ZrTc [Fig. 4(b)]. In addition, the ex vivo images of tumors and organs are shown in Fig. 4(c), which clearly demonstrates that most fluorescence signals were mainly accumulated in the tumor and liver; this indicates that ZrTc@HA has a good imaging capacity and that these NPs were mainly excreted from the liver. Taken together, these data demonstrate that the in vivo targeting specificity of ZrTc@HA in TNBC was much better than that of ZrTc, which is in accordance with the results of specific cellular uptake in vitro.
D. Growth inhibitory effect of ZrTc@HA in 4T1 tumors
Based on the successful targeting and therapeutic effects of ZrTc@HA in vitro, its therapeutic efficacy was assessed in a 4T1 tumor-bearing mouse model. The treatment plan is shown in Fig. 5(a). Before investigating the growth inhibitory effect of ZrTc@HA on 4T1 tumors, we assessed the pharmacokinetics of ZrTc@HA by collecting the blood samples from mice at different times after ZrTc@HA injection. The half-life of ZrTc@HA in blood circulation was 1.3 h [Fig. 5(b)].
The antitumor effects of the ZrTc@HA NPs on TNBC were studied in BALB/c mice bearing 4T1 cells. The relative tumor volume data are shown in Figs. 5(c) and 5(d) and S3. Compared with control mice, mice treated with ZrTC NPs, and those treated with laser, the relative tumor volume of mice treated with ZrTc@HA under laser irradiation was significantly decreased after three days of PDT and almost disappeared 14 days after PDT. Furthermore, the pathology was evaluated using hematoxylin–eosin staining, which showed that the tumors in the mice treated with ZrTc@HA and laser irradiation had the most significant cellular damage and apoptosis, whereas the tumors in the other groups showed little or no noticeable changes [Fig. 5(e)]. These results indicate that ZrTc@HA can effectively inhibit the growth of 4T1 tumors when combined with laser irradiation at 780 nm, mainly owing to the generation of ROS.
Furthermore, the potential toxicity of the prepared NPs was evaluated by measuring mouse body weight during the treatment period, performing routine blood and biochemical tests after the observation period, and observing histological changes in the major organs. No significant difference in body weight was observed among the four groups, and no noticeable weight loss was observed in any mouse groups (Fig. S4). Additionally, after 14 days of treatment, no significant damage or inflammation was observed in the heart, liver, spleen, lungs, or kidneys of any groups (Fig. S5). Furthermore, no significant changes in Hct, WBC, RBC, MCHC, HGB, or MCV were observed in the control, laser, ZrTc@HA, and ZrTc@HA with laser groups (Fig. S6). These data indicate that ZrTc@HA is a safe nanoplatform for TNBC treatment.
In summary, ZrTc@HA NPs were synthesized using a solvothermal process and coated with negatively charged HA. Two-photon excited fluorescence spectra analyses indicated the excellent upconversion emission behavior of ZrTc@HA at 780 nm. In vitro and in vivo uptake experiments using 4T1 cells confirmed the superior targeting ability of ZrTc@HA. Upon exposure to light irradiation at 780 nm, ZrTc@HA exhibited exceptional antitumor ability with minimal toxicity. Our study provides strong evidence for the use of ZrTc@HA in NIR-mediated PDT for TNBC treatment.
Additional characterization of two-photon excited fluorescence spectra, the O2·−-generation ability of ZrTc@HA, tumor pictures, body weight of mice, histological examination, and blood biochemical and blood routine analyses treated by ZrTc@HA are given in the supplementary material.
This research was funded by the National Natural Science Foundation of China (Grant No. 22171001), the Natural Science Foundation of Anhui Province (Grant No. 2108085MB49), Anhui Medical University (Grant Nos. 2021xkj142 and 2021xkj149), and the Foundation for Advanced Talents in the First Affiliated Hospital of Anhui Medical University (Grant No. 1487).
Conflict of Interest
The authors have no conflicts to disclose.
The animal study protocol was approved by the Institutional Animal Care and Use Committee of Anhui University (2020-042).
Ningning Song: Writing – original draft (equal); Writing – review & editing (equal). Bo Li: Data curation (equal); Formal analysis (equal); Investigation (equal); Writing – original draft (equal); Writing – review & editing (equal). Dandan Li: Conceptualization (equal); Supervision (equal). Yunwen Yan: Writing – review & editing (equal).
The data that support the findings of this study are available within the article and its supplementary material and from the corresponding authors upon reasonable request.