Eu(NO₃)₃·6H₂O (purity: 99.99%) were purchased from the city of Shanghai Aladdin Industrial Corporation (China). Cytarabine were also purchased from the city of Shanghai Macklin biochemical technology (China). All aqueous solutions were prepared using Milli-Q 10 water (resistivity>18 MΩ cm). All chemical reagents and solvents used in this work were of analytical grade and were used without further purification.

FEI Quant 250FEG scanning electron microscopy (FEI Company, United States). ARL 3000 Desktop XRD Analyzer (Jingong Instrument (Suzhou) Co., LTD, French). ESXTAR6000 TG/DTA Thermal Difference Analyzer (Japan Seiko Electronics Nano Technology Co., LTD, Japan). Nicolet IS5 infrared spectrometer (Nicolet, United States). LEICA TCS STEDCW Ultra high-resolution microscope system (Leica Microsystems, Germany). HITACHI F-7000 Fluorescence spectrophotometer (Hitachi Scientific Instruments (Beijing) Co. LTD, Japan).

In this work, Eu-CP of cytarabine anticancer drug micro/nanospheres were prepared for the first time. In a typical synthesis procedure, 0.3 mmol of cytarabine anticancer drug was placed in a 20 ml Teflon-lined stainless-steel autoclave. Then it was dissolved in 10 ml absolute methanol under magnetic stirring. Then, 0.3 mmol Eu³⁺ was added to the above solution. Finally, we used triethylamine to adjust the PH of the above solution until the PH value was about 7. The autoclave was sealed and heated at 160°C for 6 h. The final products were collected by centrifugation and washed several times with ethanol and distilled water. The precipitate was dried at 60°C for 10 h in the end.

The tested compounds were dissolved in sterile water and diluted to the required concentration with culture. After the required concentration of solution was configured, 0.22 μm microporous membrane filter was used for filtration and sterilization. A549 cells were grown in F12 medium supplemented with 10% freshly inactivated fetal calf serum and antibiotics. L02 cells were grown in RPMI-1640 medium supplemented with 10% freshly inactivated fetal calf serum and antibiotics. The cells harvested from exponential phase (1 × 10 ⁵per ml) per well in 100 μl of medium were seeded equivalently into a 96-well plate and then incubated for 6 h at 37°C and 5% CO₂, then the tested compounds were added in a concentration gradient, and the final concentrations were, respectively, maintained at 96, 48, 24, 12, 3, 1.5, 0.5 μg/ml. The plates were kept at 37°C in a humidified atmosphere of 5% CO₂ and incubated for 48 h, the diluent of MTT of an appropriate concentration was added to each well and the plates incubated at 37°C and 5% CO₂ for 4 h. Then suck the supernatant were added 110 μl formazan solution to each well, put on a shaking table and oscillated at low speed for 10 min to fully dissolve the crystals. The measurements of absorbance of the solutions related to the number of live cells were performed on an ELISA spectrophotometer at 450 nm.

The results of the experimental data are expressed as mean ± standard error of mean. The differences between the two groups were analysed by t-test; the differences between multiple groups were compared by one-way analysis of variance using the software program R (http://www.r-project.org/). p < 0.05 was considered statistically significant.

The SEM and TEM image with sphere-shape characterization shows that complex 1 prepared by solvothermal method has the average size around 100 nm∼2.5 μm (Figures 1A–E). The SEM image of developed Eu-CP-Ara revealed uniform, smooth surface, and spherical shape (Figure 1). The amplifying TEM reveal that Eu-CP-Ara is solid spherical shape. Moreover, this product showed good dispersibility when Eu-CP-Ara was dispersed in F12 medium (Figure 1F) gand RPMI-1640 medium (Figures 1H,I). After 6 days, the dispersed solution was still very stable (Figures 1F–I).

It has been reported that different reaction time has great influence on the morphology of products (Ji et al., 2020). So the stability of the Eu-CP-Ara micro/nanosphere was tested in different reaction time and recorded by SEM observation. The SEM image show that Eu-CP-Ara micro/nanosphere was very stable (Figure 2). The morphology of products did not change shape and remained spherical.

XRD pattern of the Eu-CP-Ara is shown in Figure 3A. It obviously displays that the Eu-CP-Ara is amorphous. The peak at 3,476 cm⁻¹ and 3438 cm⁻¹ are due to the antisymmetric stretching vibration and symmetric stretching vibration of amino group (Figure 3B). The peak at 3,355 cm⁻¹ is show the absorption peak of oxyhydrogen stretching vibration (Figure 3B). By comparing the infrared image of cytarabine with its complexes, the amino peak and hydroxyl peak of cytarabine at 3,476 cm⁻¹, 3,438 cm⁻¹, and 3,355 cm⁻¹ have disappeared. But it is shown in Figure 3B that a wide peak at 3,425 cm⁻¹ is the stretching vibration of coordinate bond exists. The FT-IR results also confirms the formation of coordination polymer. TG and DTA curves of Eu-CP-Ara (Figure 3C) were also detected under atmospheric conditions at 800°C.TG analysis displays that the first mass loss in the range of 50–200°C is 12.0%, manifesting the loss of the physically absorbed water molecules. Two apparent decompositions between 200°C to 570°C and 575°C–665°C of the weight loss are ascribed to the cytarabine and Eu-CP-Ara frameworks.

Figure 4 shows elemental mapping of Eu-CP-Ara micro/nanoparticle, it can be seen that the sphere-shape micro/nanoparticle consists of Eu, C, N, and O. The distribution of nitrogen is less (Figure 5D). In addition, the uniform distributions of Eu (Figure 5B), C (Figure 5C), N (Figure 5D), and O (Figure 5E) in Eu-CP-Ara sphere-shaped were further evidenced by EDS images in Figure 5. Elementary analysis shows that the percentage of europium content is 25.90%, the carbon content is 27.53%, the nitrogen content is 10.71%, and the oxygen content is 35.86%. Based on above results, the product can be proposed to be 2Eu·3(C₉H₁₃N₃O₅)·8(H₂O).

The emission spectra of the Eu-CP-Ara excited with 394 nm were shown in Figure 6, From the emission spectrum of Eu-CP-Ara particle, these Eu-CP-Ara particles show the three peaks located in 592 nm, 619 nm, 699 nm were all the typical emission of Eu³⁺ ions, the Eu³⁺ ions exhibit dominant red emission ascribed to ⁵D₀→⁷F₁, ⁵D₀→⁷F₂, ⁵D₀→⁷F₃ transitions of Eu³⁺.

As shown in Figure 7, the A549 cells uptake situation were detected by CLSM (Zou et al., 2017; Cheng et al., 2019; Lu et al., 2020). To understand the result of cell death in this Eu-CP-Ara, we investigated the intracellular distribution of the Eu-CP-Ara under a confocal laser scanning microscopy (CLSM) (Figure 7). From the CLSM images, the fluorescence intensity in green color could be clearly seen in the cell. Eu-CP-Ara has successfully entered the A549 cell and was distributed throughout the cell. In order to prove this conclusion, the CLSM images was utilized to further confirm the distribution of Eu-CP-Ara at the light field image (Figure 7A) and the results are shown in Figure 7B. The fluorescence of Eu-CP-Ara was mostly overlapped with that of the corresponding A549 cells. The results suggested that Eu-CP-Ara tended to enter in A549 cells and had good biocompatibility (Ji et al., 2021; Zeng et al., 2019; Zhang et al., 2022).

Cytotoxicity experiments the cytotoxicity of cytarabine with Eu-CP-Ara against A549 was investigated (Figure 8). In addition, the cell viability of A549 and L02 were tested, respectively, in the presence of cytarabine, Eu-CP-Ara and europium nitrate hexahydrate (Figures 9–11). As shown in Figure 8, it is obvious to see that there is a large difference in the inhibition rate of cytarabine versus Eu-CP-Ara on A549 cell viability. We can easily draw a conclusion that both Cytarabine and Eu-CP-Ara have strong inhibition on this cell, and the cell inhibition rate was positively correlated with the drug concentration (Zeng et al., 2020). Proliferation of A549 was almost unaffected in the presence of equivalent europium nitrate hexahydrate alone. This suggests that europium ions may have a synergistic effect with cytarabine to produce a stronger toxic effect on tumor cells. And, Eu-CP Ara had lower cytotoxicity against L02 cells.