Streptavidin, a Protein Silver Stain Kit, and a Cell Cycle and Apoptosis Analysis Kit (PI staining) were purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Lymphoma cells used in experiments (SU-DHL-4, Daudi, CA-46, and Jeko-1) were purchased from the Kunming Cell Bank of the National Science Academy of China. (Kunming, China). A BrdU Cell Proliferation ELISA Kit was purchased from abcam-Innovating Technology. (Cambridge, UK). Artesunate (98%) was purchased from Aladdin (Shanghai, China) Primary antibodies (AKT, p-AKT, ERK, p-ERK, EGFR, β-actin, PARP, caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9, CDK1, CDK4, and CyclinB1) and secondary antibodies (rabbit and mouse) were purchased from Proteintech (Wuhan, China).

A mixture of artesunate (1 mmol), mono-propargylamine (1.2 mmol) and TEA (2 mmol) in CH₃CN (3 mL) was stirred at 0°C for 10 min. HATU (1.2 mmol) was then added in one port, and the reaction was carried out under nitrogen protection. After 3 h, the CH₃CN was removed in vacuo and the residue was purified by running on a silica gel column chromatography (X. Zhang et al., 2022). The yield of AP-1 was 84%. Nuclear magnetic resonance (NMR): ¹H NMR (400 MHz, chloroform-d) δ 6.01 (s, 1 H), 5.78 (d, J = 9.8 Hz, 1 H), 5.44 (s, 1 H), 4.23–3.75 (m, 2 H), 2.95–2.68 (m, 2 H), 2.66–2.44 (m, 3 H), 2.38 (td, J = 14.1, 3.9 Hz, 1 H), 2.23 (t, J = 2.6 Hz, 1 H), 2.03 (ddd, J = 14.6, 4.9, 3.0 Hz, 1 H), 1.89 (ddd, J = 13.4, 6.4, 3.3 Hz, 1 H), 1.75 (ddq, J = 23.3, 16.5, 3.5 Hz, 4 H), 1.63 (dt, J = 13.8, 4.5 Hz, 1 H), 1.59–1.44 (m, 1 H), 1.32 (dddd, J = 25.7, 17.4, 12.3, 5.0 Hz, 3 H), 1.13–0.99 (m, 2 H), 0.97 (d, J = 5.8 Hz, 3H), 0.85 (d, J = 7.1 Hz, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 171.7, 170.9, 104.5, 92.2, 91.5, 80.1, 71.6, 51.5, 45.2, 37.3, 36.2, 34.1, 31.8, 30.6, 29.6, 29.3, 26.0, 24.6, 22.0, 20.2, 12.1.

Cells were removed from liquid nitrogen storage and thawed in a water bath at 37°C. The cells were then resuspended in 2 mL of RPMI1640 medium and the suspension was centrifuged at 800 rpm and 4°C for 5 min. Having removed the supernatant, the cells were resuspended in 10 mL of RPMI1640 (high glucose) and placed in a 37°C, 5% CO₂, humidified incubator.

The cell cultures were centrifuged at 800 rpm and 4°C for 5 min, and having removed the supernatant, the pelleted cells were resuspended in 2 mL RPMI1640 medium. A 200-µL aliquot of this suspension was used to inoculate 10 mL of culture medium and incubated in a 5% CO₂ humidified incubator at 37°C.

Crystal structure data for HSP90 and ligand (ID: 6cyg) were downloaded from the Protein Data Bank. The intrinsic ligand and water molecules in 6cyg were removed using PYMOL2.2 software. Artesunate minimum energy optimization was performed using ChemDraw3D software, and polar hydrogens were added using Tools-1.5.6 software. Two relative positions were defined using MarvinSketch software (Chaturvedi et al., 2010), namely, the radical formed by peroxide bridge cleavage and the electronegative amino acid located in the pocket. The localization parameters were imported into MOE software, treating the intrinsic coordination cavities as binding pockets, and on the basis of this localization file and algorithm, tMOE software was used to performing docking, which was analyzed using Schrödinger.

Cells, artesunate, and DMSO were added to cell culture plates at concentrations of 20, 10, 5, and 2.5 μg/mL (artesunate) and 0.1% (DMSO) and incubated for 24 h. After incubation, the cells were fixed with 25 µL of 80% trichloroacetic acid for 2 h. Each well was stained with 100 µL of 4% sulforhodamine B solution for 30 min. After treatment with 100 µL of TRI lysate, the plates were shaken for 15 min and measurements were obtained using a 550-nm microplate reader.

Cell cultures were centrifuged at 200 × g for 5 min, and following removal of the supernatant, 200 µL of fixative was added to each well of plates for 30 min followed by three washes with phosphate-buffered saline (PBS). Following the addition of 100 µL of probe antibody to each well, plates were incubated for 1 h at room temperature and again washed three times with PBS. Thereafter, 100 µL of horseradish peroxidase-conjugated secondary antibody was added to each well, followed by incubation for 30 min. Having washed three times with PBS, 100 µL of TMB was added to each well to stop the reaction and measurements were performed using a 550-nm UV detector.

Following lysis, proteins were extracted from AP-1 cultured cell. To the extracted protein suspension, we added 1 µL of biotin azide (2,500 µM), 1 µL of TCEP (500 mM), 1 µL of TBTA (50,000 µM), and 500 mM CuSO₄, followed by dilution with buffer solution and mixing. The diluted solution was incubated for 3 h at 4°C, and after completion of the reaction, the mixture was centrifuged at 12,000 × g for 10 min at 4°C, and thereafter washed three times with acetone. Having allowed to dry at room temperature, 500 µL of 2.5% SDS in PBS was added to the protein preparation followed by boiling at 100°C for 5 min. The suspension was then centrifuged and the supernatant was transferred to a fresh centrifuge tube, to which 60 µL of resin was added followed by mixing for 3 h. The mixed suspension thus obtained was then centrifuged at 1,000 × g for 10 min. Having removed the supernatant, the pellet was washed with PBS, followed by the addition of loading buffer, diluted with PBS, and then boiled at 100°C for 10 min. The expression of proteins in the lymphoma cells was analyzed using western blotting.

Cells were collected in 1.5-mL Eppendorf tubes and centrifuged at 300 × g for 5 min at 4°C. The pelleted cells were washed twice with PBS, followed by the addition of 100 µL of 1× binding buffer. Following thorough mixing, 5 µL of Annexin V-FITC and 10 µL of PI staining mixture were added to the cell suspension, which was then placed in the dark for 15 min. Having added 400 µL of 1× binding buffer. PI staining was measured using Annexin V-FITC flow cytometry.

Cells were collected by centrifuging at 1,000 × g for 5 min. Having discarded the supernatant, the cells were resuspended in pre-cooled 75% ethanol and fixed overnight at 4°C. Thereafter, the cells were centrifuged at 1,000 × g for 5 min and after discarding the supernatant, the sediment was washed once with PBS. Having prepared a mixture of staining buffer, containing 10 µL of propidium iodide and 10 µL of ribonuclease A, 0.5 mL of the PI staining buffer was mixed with the cells followed by incubation for 30 min at 37°C.

Cells were collected in 15-mL centrifuge tubes, centrifuged at 1,000 × g and 4°C for 5 min, and after discarding the supernatant, the resulting cell pellets were washed twice with PBS. Having added cell lysis buffer, the suspensions were heated at 98°C for 10 min, and aliquots of the resulting lysate supernatant were subjected to gel electrophoresis. The membranes were blocked with 5% skimmed milk for 2 h, and thereafter incubated overnight with primary antibodies at 4°C. The following day, having washed five times with TBST (each for 5 min), the membranes were incubated for 2 h with secondary antibody (1:7,000), followed by five 5-min washes with TBST. Gray-scale values were obtained using Image J.

An emulsion was initially prepared by adding 100 mg PLGA-PEG and 50 mg artesunate powder to 1 mL of CH₂Cl₂, which was then added dropwise to 30 mL of 2% PVA solution with continuous stirring. The resulting emulsion was treated with ultrasound at 300 W for 3 min and then stirred overnight with a magnetic stirrer at 150 rpm to obtain a suspension. Following centrifugation at 12,000 rpm for 10 min, the supernatant was discarded and the precipitate was freeze-dried.

To prepare a subcutaneous xenograft model, we used 4- to 6-week-old Male nude mice (BALB/c-nu) (16–20 g), obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). All male mice were maintained at room temperature (22°C–25°C) under a 12-h light/12-h dark cycle and given free access to food and water. During experiments, all mice remained in good health. All procedures involving animal experiments followed the Guide for the Management of Laboratory Animal Affairs and the Ethical Guidelines for Animal Experimentation System of Yunnan Minzu University (2021-050). Allogeneic subcutaneous transplants were administered via intratumoral injection 4 days after transplantation. Mice were randomly divided into one of the following five groups, each containing six mice: control (saline), PLGA-PEG 400 mg/kg, artesunate-PLGA-PEG 400 mg/kg, artesunate-PLGA-PEG 200 mg/kg, and artesunate (artesunate 40 mg/kg). The mice were administered continuously with e respective preparations for 21 days, and during this period, changes in body weight were recorded. After 21 days, tumor tissues was removed from the mice subcutaneously and difference between the groups with respect to tumor weight and volume were recorded.

Dehydrated tumor tissues were embedded in paraffin wax. Slices were prepared by heating in a water bath to recover the antigen. The slices were placed in a pressure cooker, heated for 10 min together with EDTA, and after gradually cooling to room temperature, were washed three times with PBS. Having blocked with 10% goat serum (used as a blocking antibody) for 1 h and washed times with PBS, the tissue slices were incubated overnight with the primary antibody (AKT 1:100 or ERK 1:50) and the following day were washed three times with PBS. Thereafter, the slices were incubated with a secondary antibody at room temperature followed by three 20-min washes with PBS. Having initially placed the slices in DAB chromogenic substrate solution for 5–10 min, they were stained with hematoxylin for 2 min, rinsed twice with distilled water, and dehydrated for 2 min.

The quantitative data obtained in this study are presented as the means ± SE. Analyses were performed using GraphPad Prism 8 software. Student’s t-test was used to determine the statistical significance of differences between the different experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Daudi or CA-46 cells was co-incubated with AP-1. AP-1 penetrates the cell membrane, and on entering the cell cytoplasm and binds to Hsp90, thereby inhibiting cell proliferation. The binding of AP-1 to Hsp90 was assessed based immunoblotting. In the experimental group, we detected Hsp90aa1 and Hsp90ab1, whereas in the negative control group neither protein was detected, indicating that AP-1 binds to Hsp90, which has been identified as one of the targets of artesunate (Figure 1B).

We verified the binding between artesunate and Hsp90 using the AP-1 probe. On the basis of this validation, we conducted a preliminary examination of the binding pattern between artesunate and Hsp90 in silico. We hypothesized that covalent binding between artesunate and Hsp90 is induced by free radicals. With reference to the X-ray protein structure of Hsp90 in the Protein Data Bank, which was docked using the MOE method with a docking binding energy of −8.19 kcal/Mol, we used Schrödinger to analyze the covalent binding of artesunate to the ASP93 residue of Hsp90 and the binding mode of the salt bridge formed between Fe²⁺ and Ser52 (Figure 1C).

The molecular probe AP-1 was obtained by covalently attaching an alkyne group to artesunate (Figure 1A). To reduce alkyne group interference, this group was located at a site other than the key bioactive site of artesunate. Subsequently, we compared the inhibition of malignant lymphoma cell proliferation by artesunate and AP-1 through experimental validation. In the BrdU assay, the survival of Daudi and CA-4 6 cells at an artesunate concentration of 20 µM was 31.91% ± 5.122% and 31.99% ± 3.88%, respectively (Figure 1D). Among the four assessed lymphoma cell lines SU-DHL-4, Daudi, CA-46, and JEKO-1, we obtained artesunate IC₅₀ values of 0.89 ± 0.12, 0.70 ± 0.29, 0.72 ± 0.05, and 1.34 ± 0.31 µM, respectively, whereas the corresponding IC₅₀ values for AP-1 were 1.23 ± 0.24, 1.89 ± 0.28, 0.59 ± 0.17, and 1.45 ± 0.28 µM (Figure 1E). These values thus indicate that the covalent linkage of alkynes to ART had no appreciable adverse effects on the inhibitory activity artesunate against cell proliferation.

The effects of artesunate-induce inhibition on the expression of Hsp90aa1, Hsp90ab1, and associated client proteins (AKT, p-AKT, ERK, p-ERK, and EGFR) were investigated in the malignant lymphoma cell lines Daudi and CA-46 using western blotting. Artesunate (20 µM) was co-incubated with tumor cells and the relative levels of client proteins were measured in the negative control and experimental groups. Although we detected no significant changes in the level of Hsp90 following the treatment with artesunate, the relative levels of AKT, p-AKT, EGFR, ERK, and p-ERK proteins in the Daudi experimental group were found to be 16% ± 12%, 33.4% ± 10.4%, 24.3% ± 10.3%, 24.8% ± 7.1%, and 18.7% ± 11.3%, respectively, compared with those in the negative control group. Similarly, in the CA-46 experimental group, the relative contents of the corresponding client proteins were 44.2% ± 9.3%, 15.5% ± 6.5%, 21.2% ± 9.8%, 11.0% ± 5%, and 14.3% ± 3.6%, respectively. These findings accordingly reveal that the expression of AKT, p-AKT, EGFR, ERK, and p-ERK in the artesunate experimental group was significantly lower than that in the control group. (Figures 1F, G).

Artesunate-induced apoptosis was confirmed using flow cytometry, which revealed rates of early apoptosis of 0.76% ± 0.21%, 2.59% ± 0.44%, 2.70% ± 0.38% and 4.90% ± 0.72% in Daudi cells treated with artesunate at doses of 0, 5, 10, and 20 μM, respectively, whereas the corresponding values obtained for late apoptosis were 1.76% ± 0.17%, 41.41% ± 2.02%, 58.77% ± 5.41%, and 83.49% ± 1.16%. Comparatively, for the SU-DHL-4 cells, the early apoptosis rates were 3.05% ± 0.84%, 8.94% ± 4.44%, 16.73% ± 1.22% and 20.95% ± 3.24%, at artesunate doses of 0, 5, 10, and 20 μM, respectively, and the corresponding late apoptosis values were 14.17% ± 1.08, 19.13% ± 5.57%, 15.33% ± 0.54%, and 21.47% ± 3.39% (Figures 2A, B).

Compared with the negative control Daudi cells, the expression levels of PARP, caspase3, cleaved caspase3, caspase9, and cleaved caspase9 proteins in the 20 µM artesunate group were 15.3% ± 7.1%, 10.3% ± 1.3%, 245.3% ± 35.4%, 27.7% ± 4.3%, and 168.7% ± 25.5%. In the SU-DHL-4 cells, compared with the negative control cells, the expression levels of PARP, caspase3, caspase3, caspase9, and caspase9 proteins in the 20 µM artesunate group were 16.4% ± 2.5%, 18.9% ± 9.3%, 540.4% ± 211.5%, 22.6% ± 22.4%, and 583.2% ± 36.1%. These findings thus indicate that whereas the expression of PARP, caspase9, and caspase3 was significantly reduced in response to treatment with artesunate, the expression of cleaved caspase9 and cleaved caspase3 was significantly enhanced. On the basis of these findings, it can be deduced that in response to treatment with artesunate, the caspase9-caspase3 signaling pathway was activated to induce apoptosis in both the assessed lymphoma cell lines (Figures 2C, D).

Flow cytometric analysis verified that treatment with artesunate had the effect of blocking the lymphoma cell cycle. When administered at concentrations of 20, 10, 5, and 0 μM, we observed that the percentages of Daudi cells in the S-phase of the cell cycle were 49.67% ± 2.33%, 43.67% ± 0.67%, 46.67% ± 0.88%, and 60.33% ± 0.33%, respectively. Comparatively, the corresponding percentages of CA-46 cells in the S-phase were 2.66% ± 2.66%, 8.00% ± 1%, 9.00% ± 1.15%, and 49.00% ± 0.57%, respectively (Figures 3A, B). Compared with to the control group, the percentages of cells in the G0/G1 phase were significantly increased, whereas those in the S phase were significantly reduced following treatment with artesunate (Figures 3A, B). Western blot analysis, performed to assess the effects of artesunate on the expression of CDK1, CDK4, and Cyclin B1, revealed relative levels 14.7% ± 8.6%, 30.6% ± 14.8%, and 11.7% ± 4.8%, respectively, in Daudi cells, compared with the negative control, when administered at a concentration of 20 µM. Comparatively, the corresponding values in CA-46 cells treated with 20 µM artesunate were 29.2% ± 13.7%, 5.4% ± 2.5%, and 26.9% ± 16.3%, respectively. We thus established that in both the assessed lymphoma cell lines, the expression levels of CDK1, CDK4, and Cyclin B1 were reduced following treatment with artesunate (Figures 3C, D). Collectively, our experimental results thus provided evidence to indicate that artesunate can significantly inhibit the proliferation of lymphoma cells, in which the cell cycle distribution was altered and cells were arrested in the G0/G1 phase.

Daudi cells were transplanted into the axillary tissues of nude mice, and 4 days later, the tumors were treated with drugs. Tumor size and morphology were observed, and 21 days later, tumor tissues were collected from mice and weighed. During the experiment, we detected no significant change in the body weight of the model mice (Figure 4A). On the basis of these findings and those reported previously in toxicological studies of artesunate (Wang et al., 2019; Chen et al., 2022), it can be assumed that artesunate can be safely administered to nude mice at this dose. In addition, we obtained tumor weights of 0.350 ± 0.06 g, 0.088 ± 0.03 g, 0.083 ± 0.03 g, 0.108 ± 0.03 g, and 0.285 ± 0.02 g for the control, artesunate-PLGA-PEG200 mg/kg, artesunate-PLGA-PEG400 mg/kg, artesunate, and PLGA-PEG200 mg/kg groups, respectively (Figures 4C, D). Compared with the control group, treatment with artesunate-PLGA-PEG 200 mg/kg, artesunate-PLGA-PEG 400 mg/kg, and artesunate reduced tumor volumes by 84.15%, 88.72%, and 55%, and tumor weight by 76.28%, and 69.14%, respectively. Accordingly, these findings revealed that both artesunate and artesunate-PLGA-PEG can inhibit the development of xenograft tumors in vivo (Figure 4B), and that the therapeutic effects are more pronounced when using PLGA-coated artesunate (Figures 4C, D).

We subsequently examine the expression of the heat shock protein client proteins ERK and AKT based on an immunohistochemical staining technique, with images being analyzed in gray-scale. With respect to ERK, we detected relative expression levels of 0.21 ± 0.07, 0.30 ± 0.03, 0.35 ± 0.07, and 0.91 ± 0.01 in the artesunate-PLGA-PEG400 mg/kg, artesunate-PLGA-PEG200 mg/kg, artesunate, and PLGA-PEG400 mg/kg groups respectively, which represent reductions of 79%, 70%, 65%, and 9%, respectively compared with the control group; The corresponding levels of AKT expression were 0.24 ± 0.01, 0.36 ± 0.01, 0.46 ± 0.02, and 0.91 ± 0.05, with respective reductions of 76%, 64%, 54%, and 9% compared with the control group. These findings thus indicate that artesunate can promote a significant inhibition of the expression of ERK and AKT in tumor tissues, and drugs packed within a PLGA-PEG coating showed significant tumor suppressive effects (Figures 4E, F).