A self-assembled copper-artemisinin nanoprodrug as an efficient reactive oxygen species amplified cascade system for cancer treatment

Abstract

Chemodynamic therapy (CDT) is a tumor-specific intervention methodology, which is based on the upregulation of reactive oxygen species (ROS) content by triggering the Fenton or Fenton-like reaction within the tumor microenvironment (TME). However, there are still challenges in achieving high-efficiency CDT on account of both the limited intracellular hydrogen peroxide (H₂O₂) and delivery efficiency of Fenton metal ions. Copper-based nanotherapeutic systems have attracted extensive attention and have been widely applied in the construction of nanotherapeutic systems and multimodal synergistic therapy. Herein, we propose a strategy to synergize chemotherapy drugs that upregulate intracellular ROS content with chemodynamic therapy and construct an artemisinin-copper nanoprodrug for proof-of-concept. With the proposed biomimetic self-assembly strategy, we successfully construct an injectable nanoprodrug with suitable size distribution and high drug loading content (68.1 wt%) through the self-assembly of amphiphilic artemisinin prodrug and copper ions. After reaching the TME, both Cu²⁺ ions and free AH drugs can be released from AHCu nanoprodrugs. Subsequently, the disassembled Cu²⁺ ions are converted into Cu⁺ ions by consuming the intracellular GSH. The generated Cu⁺ ions serve as a highly efficient Fenton-like reagent for robust ROS generation from both AH and tumor-over-produced H₂O₂. Results show that the nanoprodrug can realize the cascade amplification of ROS generation via artemisinin delivery and subsequent in situ Fenton-like reaction and a high tumor inhibition rate of 62.48% in vivo. This work provides a promising strategy for the design and development of an efficient nanoprodrug for tumor-specific treatment.

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Dongdong Wang

Dongdong Wang is currently a Professor at the University of Science and Technology of China. He earned his BSc in Materials Chemistry from Lanzhou University in 2013 and completed his PhD in Materials Physics and Chemistry under the guidance of Prof. Qianwang Chen at the University of Science and Technology of China in 2018. Following his PhD, he conducted postdoctoral research with Prof. Yanli Zhao at Nanyang Technological University in Singapore. Prof. Wang's research is currently centered on the development of functional nanomaterials for use in diagnostics and therapeutics.

1. Introduction

Cancer has always been a critical threat to the health of humans and seriously affects the life quality of patients all over the world.^1–3 Although traditional treatment methodologies such as surgery, chemotherapy, and radiotherapy have demonstrated varying degrees of success, inevitable physiological toxicities and unpredictable side effects usually accompany them.^4,5 The redox metabolism of the tumor microenvironment (TME) is completely different from that of healthy tissue, featuring an elevated level of reactive oxygen species (ROS) and altered redox status.⁶ Among the various intracellular ROS such as hydroxyl radical (˙OH), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), superoxide anion (˙O₂⁻), and hypochlorous acid (HClO), ˙OH is the most lethal to the biological system on account of its high reactive activity.⁷ Usually, the level of ROS within tumor cells is maintained at a certain range to regulate the normal metabolism.⁸ However, boosting generation of ROS will destroy the biomolecular structures of lipids, proteins, and DNA and induce the apoptosis of cancer cells.⁹ Very recently, Fenton or Fenton-like reaction based chemodynamic therapy (CDT) has become a new strategy for in situ treatment of tumor. Since it was proposed, CDT has attracted tremendous research attention in the biomedical field. With the over-expressed H₂O₂ in TME, chemodynamic nanoagents can efficiently induce the generation of cytotoxic ˙OH via metal ion-mediated Fenton or Fenton-like reaction, thus leading to the apoptosis or necrosis of tumor cells.^10,11 Besides, CDT is initiated without the help of external energy such as laser irradiation, thus circumventing the tissue penetration limitation of light.¹²

Currently, various transition metal ions (e.g., iron, manganese, copper)-based CDT platforms have been reported and shown great promise in tumor treatment. In terms of the catalytic reactivity toward H₂O₂, copper has similar redox properties as iron.¹³ However, Fe²⁺-based Fenton reagents only work under strongly acidic conditions with a pH value of 2–4, thus compromising their CDT efficiency within a mild acidic TME. In contrast, Cu⁺ could highly catalyze the Fenton-like reaction in slightly acidic and neutral media.^14,15 More importantly, the maximum reaction rate of Cu⁺ is 160 times higher than that of Fe²⁺ for more efficient ROS generation, thus guaranteeing copper is a more suitable candidate for CDT.¹³ Although significant progress has been achieved in the area of copper-based systems for CDT, there are still many challenges that need to be solved.⁶ For instance, the high intracellular GSH level in cancer cells can quickly scavenge the generated ROS within cancer cells, thereby impairing the therapeutic effect.¹⁶ In addition, the heterolytic cleavage of intracellular H₂O₂ into H₂O and O₂ may result in the undesirable consumption of intracellular H₂O₂, further reducing the efficiency of CDT. Considering the slow generation rate and continuous consumption of endogenous H₂O₂ during CDT, the CDT efficiency will quickly decrease.¹⁷ Therefore, it is highly desirable to provide an extra supply of H₂O₂ to the tumor site for maintaining CDT efficacy.

Artemisinin and its derivatives have been recommended by the World Health Organization as affordable and safe antimalarial drugs. Early in the 1990s, researches showed the favorable antitumor activity of artemisinin in vitro. Recently, several phase I clinical trials have proven the good biosafety of oral artesunate-based formulations as chemotherapeutic agents.^18,19 Previous studies have also shown that artemisinin can generate strong cytotoxic reactive oxygen species within target cells through the Fe-mediated activation process.²⁰ Our previous works based on the co-delivery of Fe ions and artemisinin analogues have shown satisfying antitumor activity and good safety profiles both in vitro and in vivo.^21–23 Considering the structural similarity between H₂O₂ and the endoperoxide bridge of artemisinin analogues, we thus proposed the copper-mediated activation of artemisinin analogues would enhance the ROS generation ability and therapeutic efficiency. The drug self-delivery systems (DSDSs) originated from supramolecular self-assembly can largely avoid the complicated preparation steps of carriers and enhance the loading capacity of drugs, showing great potential in biomedicine.^24–27 Inspired by the coordination interactions between metal ions and organic cofactors within natural metalloproteins, researchers have developed a coordination-driven self-assembly strategy to construct self-assembled nanoprodrugs.^28,29 In addition, compared with non-covalent interactions (hydrophobic effects, van der Waals forces, hydrogen bonding, and π–π stacking) based self-assembly, the coordination interaction between metal ions and organic components is considered to be much stronger due to the nature of covalent bond.^30,31

Herein, we rationally construct a self-assembled nanoprodrug that is driven by the coordinative interactions between hydrophobic drug-amino acid conjugate (artesunate-histidine, defined as AH) and copper ions (Fig. 1). Benefiting from the strong coordination interactions between the histidine imidazole group and transition metal ion, the introduction of copper ions drives the rapid self-assemble of AH molecules in an aqueous solution into spherical nanoprodrugs (defined as AHCu). Besides, the as-prepared nanoprodrug exhibits good colloidal stability, suitable particle size distribution as well as high drug loading capacity. Within the acidic tumor microenvironment, both Cu²⁺ ions and free AH drugs can be released from AHCu nanoprodrugs. Subsequently, the disassembled Cu²⁺ ions are converted into Cu⁺ ions through consuming the intracellular GSH. The generated Cu⁺ ions serve as a highly efficient Fenton-like reagent for robust ROS generation from both AH and tumor over-produced H₂O₂. Therefore, both in vitro and in vivo results show favorable antitumor efficacy on account of the cascade amplification of ROS generation via in situ Fenton-like reaction based CDT. Our work provides a simple and effective methodology for the construction of artemisinin-based nanoprodrug through metal ion-induced supramolecular self-assemble strategy for tumor-specific treatment.

Fig. 1 The hydrophobic drug was first bonded to natural amino acid, and then, the AHCu was obtained by the coordination of metal ions, thereby obtaining tumor-specific cascade enzyme therapy in vivo.

2. Results and discussion

2.1. Coordination-driven self-assembly of AHCu

As shown in Fig. 2a, the synthesis of artesunate-histidine (AH) was first based on a amidation reaction between artesunate and histidine using dicyclohexylcarbodiimide/N-hydroxy succinate (DCC/NHS). The detailed procedure was presented in the supplementary information. The chemical structure and molecule weight of as-prepared AH were characterized by ¹H NMR and electrospray ionization mass spectrometry (ESI-MS), respectively (Fig. S1–S3, ESI†). Subsequently, AH and Cu ions were used as starting materials for the construction of the self-assembled nanoprodrug. On account of the coordination interaction between the histidine moiety of AH and Cu ions, the as-prepared AHCu showed increased drug loading capacity and favorable colloidal stability. Within the tumor microenvironment (low pH and high GSH), both Cu²⁺ ions and AH molecules were released. The released Cu²⁺ ions will be further reduced into Cu⁺ ions via intracellular GSH-depletion. On account of the combined Cu⁺-triggered CDT from both tumor over-produced H₂O₂ and the supplied artesunate, thus endowing amplified ROS generation within the tumor site.

Fig. 2 (a) Synthesis route of artemisinin-histidine (AH) and self-assembly of AH-Cu (AHCu). (b) SEM and (c, d) TEM images of the AHCu nanoprodrug. (e) HAADF and the corresponding EDS element mapping results of AHCu nanoprodrug. (f) UV-visible absorption of AHCu nanoprodrug. (g) FTIR spectra of AH and AHCu nanoprodrug.

In the presence of both Cu²⁺ ions and AH, light blue milky turbidity was generated with the adjustment of pH value, indicating the generation of AHCu nanoprodrug. Remarkably, the metal ion-driven self-assembly process is extremely fast and can be finished within seconds. The as-prepared AHCu nanoprodrug exhibited slight blue, which should be originated from divalent copper ions. In contrast, individual AH or copper ions still maintained their initial state with the adjustment of pH. Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed a sphere morphology with a diameter of 150 nm of AHCu nanoparticles (NPs) (Fig. 2b–d). The uniform distribution of C, N, O, and Cu in AHCu NPs was confirmed by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy dispersive X-ray spectroscopy (EDS) analysis (Fig. 2e).

UV-vis spectrum of AHCu exhibited weak and broad absorption typically of colloidal nanoparticles in the visible and near-infrared regions (Fig. 2f). The self-assembly mechanism was further analyzed by Fourier transform infrared (FTIR) spectroscopy (Fig. 2g). Typically, strong C=O stretching vibration band (–CONH–) centered at 1751 cm⁻¹ and characteristic amide (–CONH–) II band (1640–1680 cm⁻¹) were observed in both AH and AHCu nanoprodrug with noticeable changes, indicating both N and O atoms from –CONH– might not be involved in coordination to Cu ions. The disappearance of the N–H stretching mode (3148 cm⁻¹) and the shift of the C=N stretching vibration (1597 cm⁻¹) provide clear evidence of the deprotonation and coordination of the imidazole group with the metal ion. These changes in the FTIR spectrum are consistent with the formation of a metal–ligand complex, where the imidazole group acts as a ligand, coordinating through its nitrogen atoms. Besides, the significant shift from 1437 cm⁻¹ to 1509 cm⁻¹ in the FTIR spectra indicates that the –COOH groups in AH are interacting strongly with Cu ions in AHCu. This shift is a clear indication of the formation of coordination bonds between the –COOH and Cu ions, highlighting the strong electrostatic interactions in the AHCu nanoprodrug. Thus, the formation of AHCu NPs initially involves the creation of coordination complexes, followed by the stacking of these complexes. This stacking process is primarily facilitated by weak molecular interactions, including hydrophobic interactions, van der Waals forces, and hydrogen bonding.³² Furthermore, the characteristic peaks of intramolecular bridging oxygen bonds (825 cm⁻¹ to the 1,2,4-trioxane ring breathing and 878 cm⁻¹ to the coupled C–O and O–O stretching modes of the O–O–C moiety) in AH were clearly shown from the FTIR spectra, indicating the good conservation of the endoperoxide bridge of AH.^33,34 The good conservation of the endoperoxide bridge in AH suggests that the mild self-assembly coordination conditions are gentle enough to maintain the structural integrity which is crucial for the functionality of the drug, as the endoperoxide bridge is responsible for the antitumor activity.

2.2. Optimization and determination of drug loading content

The self-loading behavior of amphiphilic drugs in an aqueous solution can be affected by various factors such as concentration, temperature, pH, and so on.³⁵ To optimize the preparation conditions of AHCu NPs, we fully studied the relationship between drug loading capacity and AH/Cu feeding ratio. Results showed that the particle size distribution, drug loading capacity, and encapsulation efficiency could be adjusted by changing the feeding ratio of AH/Cu (Fig. S4 and Table S1, ESI†). For the comprehensive consideration of the encapsulation efficiency and particle size, the optimal dosing ratio under our test conditions was 3.8 mM AH and 1 mM Cu²⁺. Besides, the molar ratio of AH and Cu²⁺ in AHCu nanoprodrug was calculated to be 0.82 : 1 (0.75 mM AH vs 0.91 mM Cu²⁺) through ultra-high performance liquid chromatography (UPLC) and inductively coupled plasma mass spectrometry (ICP-MS). The efficient drug loading capacity was calculated to be 68.1 wt%. It should be noted that the mass calculation of artesunate was based on its active substance, dihydroartemisinin, excluding copper and histidine.

2.3. Stability, reproducibility and responsive disassembly performance of AHCu

The stability of AHCu NPs under physiological conditions is crucial for their efficacy and directly determines the drug delivery efficiency both in vitro and in vivo.³⁶ The dynamic light scattering (DLS) curve of the AHCu nanoprodrug showed a narrow particle size distribution with an average particle size of 165.6 ± 0.7 nm and a polydispersity index (PDI) of 0.153 ± 0.012 (Fig. 3a). Zeta potential of AHCu NPs was determined to be −37.2 ± 6.5 mV (Fig. S5, ESI†). The long-term colloidal stability of the nanoprodrugs was experimentally verified. Results showed that the PDI and average particle size (Z-average) of AHCu in hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution showed no apparent fluctuations within 24 h (Fig. 3b). Besides, the UV-vis absorption at 600 nm of AHCu solution showed no obvious changes after storage at 4 °C for 24 h, further demonstrating the colloidal stability (Fig. S6, ESI†). Besides, reproducibility is also a key feature during nanoprodrug preparation. DLS measurements of three separate batches of AHCu nanoprodrugs prepared by the same experimental method showed fairly uniform particle size distribution and the same PDI, indicating a high degree of reproducibility of the experimental protocol.³⁷ (Fig. 3c and Table S2, ESI†). Overall, these results showed the favorable stability of AHCu nanoprodrugs by coordination-driven self-assembly strategy.

Fig. 3 (a) DLS profiles of AHCu at the optimal AH and copper ratio. (b) Size (Z-average, blue line) and PDI (gray line) in 24 h based on DLS measurements. (c)Reproducibility of the AHCu nanoprodrug. (d) Drug release profiles of the AHCu nanoprodrug in HEPES buffers of different pH values (5.4 and 7.4). (e) TMB degradation showing ˙OH generation after different treatments. (f) Cu 2p XPS spectrum of AHCu and AHCu + GSH.

We studied the drug release behaviors of AHCu nanoprodrugs under simulated acidic and neutral conditions. The coordination ability of the imidazole group can be tuned by changing pH. Therefore, cumulative release profiles were studied in buffers with pH 5.4 and 7.4. As shown in Fig. 3d, an enhanced drug release profile can be witnessed for pH 5.4 (73.53%) as compared with pH 7.4 (8.28%) within 24 h. The pH-responsive drug release should be attributed to the protonation of the imidazole group of AH in acidic conditions. The pH-dependent release behavior ensures specific drug release in the mild acidic tumor microenvironment, thereby minimizing chemotherapy side effects and improving antitumor efficiency.^38–40 Copper ions can drive the Fenton and Fenton-like reactions to induce the decomposition of H₂O₂ into highly cytotoxic ˙OH.^12,17 The generated ˙OH can be determined through the colorimetric method with 3,3′,5,5′-tetramethylbenzidine (TMB) as a probe.^26,41 Negligible TMB absorption change with the Cu, AHCu or H₂O₂ alone treated group could be observed, while a prominent absorption of the oxidative TMB can be witnessed in the presence of both AHCu and H₂O₂, indicating the efficient ˙OH generation through the Fenton-like reaction (Fig. 3e). In contrast, the formation of AHCu NPs is based on the synergy of coordination and other weak interactions and is sensitive to the stimulation of overexpressed GSH in cancer cells. As shown in Fig. 3f and Fig. S7 (ESI†), X-ray photoelectron spectroscopy (XPS) spectra of AHCu showed the mixed valence of Cu²⁺ (binding energies (BEs) at 933.2 and 953.2 eV) and Cu⁺ (BEs at 931.4 and 951.3 eV) assigned to the peaks of Cu 2p_3/2 and Cu 2p_1/2. The percentages of Cu²⁺ and Cu⁺ were calculated to be 81% and 19%, respectively. After GSH treatment, AHCu showed 53% percentage of Cu⁺ species, indicating the reduction of Cu²⁺. Besides, the color of AHCu solution became clear after adding 2 mM GSH, indicating the disassembly of AHCu. This phenomenon should be attributed to the stronger coordination interaction between Cu species and the –SH group of GSH.¹³ The above results indicated AHCu first reacted with GSH and subsequently triggered the generation of hydroxyl radicals from H₂O₂ through Fenton-like reaction. Moreover, the GSH depletion was verified using 5,5-dithiobis (2-nitrobenzoic acid) (Ellmans reagent, DTNB) based on its specificity to recognize sulfhydryl groups at 412 nm of absorption peak. The GSH-induced DTNB degradation notably declined with the increment of AHCu (0 to 200 μM), implying more GSH depletion, which can be attributed to GSH-promoted Cu⁺ release (Fig. S8, ESI†). Such property ensures the high efficacy of nanodrug-mediated chemodynamic therapy within tumor tissue. The depletion of GSH and the production of ROS provide an effective methodology for cancer therapy.

2.4. In vitro cytotoxicity evaluations and mechanism of action investigations

It is well-known that the hydrogen peroxide is the most abundant and stable non-radical ROS within TME with an evaluated concentration of up to 100 μM.^42,43 Such H₂O₂ over-expressed character of TME makes it quite feasible to develop copper-based nanoformulations for CDT taking advantage of the endogenous H₂O₂. In order to simulate the characteristics of over-expressed H₂O₂ in the TME, H₂O₂ with a concentration of 50 μM was used for in vitro studies. For in vitro normal cell biocompatibility and cancer cell killing studies, 4T1 and HUVEC cell lines were selected to detect the cytotoxicity of AH, AHCu, AH + H₂O₂, and AHCu + H₂O₂ (100 μM equiv.). Cell viability was determined by Cell Counting Kit 8 (CCK-8) assay. AHCu exhibited a lower cytotoxicity toward normal HUVEC cell lines (Fig. S9, ESI†). In contrast, AHCu showed obvious killing efficacy toward 4T1 cancerous cells (Fig. 4a). Besides, results showed the strongest inhibitory effect on cell viability of the AHCu + H₂O₂ group compared with other groups, indicating the favorable inhibitory effect of AHCu under an environment that imitated the tumor over-produced H₂O₂in vivo. On account of the high concentration of GSH in cancer cells, the Cu²⁺ released from AHCu would be reduced into Cu⁺ by GSH depletion, resulting in a subsequent Fenton-like reaction between Cu⁺ and H₂O₂ to generate more hydroxyl radicals.^18,44 The GSH content decreased significantly after the addition of AHCu and AHCu + H₂O₂, indicating the reaction between Cu²⁺ and GSH (Fig. 4b). In addition, GSH depletion further enhanced the killing efficacy of intracellular ROS, thus inducing the oxidation of biomolecules such as lipids, proteins, and DNA.^40,45 We also investigated the intracellular ROS generation ability of AHCu. As expected, specific doses (50 μM) of H₂O₂ alone had no significant effects on the intracellular ROS. However, ROS levels in 4T1 cells significantly increased after co-treatment with AHCu and H₂O₂ (Fig. 4c). Similar results were observed by live/dead staining, suggesting the potent anticancer activity of AHCu and H₂O₂ (Fig. 4d). In addition, flow cytometry (FCM) also indicated that the AHCu and H₂O₂ nanoprodrug showed enhanced cancer cell apoptosis (Fig. 4e and f).

Fig. 4 (a) Cytotoxicity of 4T1 cells treated with AH, AHCu, AH + H₂O₂, and AHCu + H₂O₂. (b) Changes in the content of intracellular GSH. (c) Intracellular ROS assays of differently treated 4T1 cells. (d) Live/dead cell staining assays of differently treated 4T1 cells. (e) Evaluation of apoptosis and necrosis by flow cytometry (Annexin V-FITC/PI co-staining). (f) Different cell populations obtained from (e) apoptosis and necrosis assay. *p < 0.05, **p < 0.01 versus control group. ^##p < 0.01 for the comparison between the AHCu + H₂O₂ group and AH + H₂O₂ group.

2.5. In vivo antitumor efficacy of AHCu

The antitumor efficacy of AHCu nanoprodrug was investigated using Balb/c mice inoculated with 4T1-Luc tumor cells (Fig. 5a). When the average volume of tumors reached 60 mm³, mice were divided randomly into 3 groups (n = 5) and intravenously administrated with PBS (control), 20 mg kg⁻¹ AH, and 20 mg kg⁻¹ AHCu every 2 days, respectively. The bioluminescence intensity of tumor was monitored every 3 days using the IVIS imaging system. After treatment for 16 days, the bioluminescence intensity of the 20 mg kg⁻¹ AHCu group was the lowest (Fig. 5b and c). As shown in Fig. 5d, after four-times administrations, the tumor volume of the AHCu group was the lowest. Compared to the PBS group, the tumor inhibition rate of 20 mg kg⁻¹ AH group was 37.83% (Fig. S10, ESI†) In contrast, the AHCu group showed an enhanced tumor inhibition rate of 62.48%, much higher than the control and AH groups. At the end of the experiment, tumors were excised and photographed (Fig. 5e). Compared with the tumors in the control group, AH and AHCu inhibited tumor cell growth in vivo, 20 mg kg⁻¹ AHCu treatment group exhibited the highest tumor inhibition effect. Besides, no significant weight losses were observed in all groups during the treatment, demonstrating the well-biological tolerance of ART-based nanoprodrugs at the tested doses (Fig. 5f). According to H&E staining results, mice treated with AHCu had the highest ratio of necrosis/late apoptosis, while a large number of viable cancer cells were found in the control group. Consistently, TdT-mediated dUTP nick end labeling (TUNEL) staining also confirmed that AHCu could efficiently induce apoptosis of cancer cells (Fig. 5g). Furthermore, histological analysis of major organs such as the heart, liver, spleen, lung, and kidney showed no detectable pathological changes, suggesting the favorable biocompatibility of the AHCu nanoprodrugs (Fig. S11, ESI†). Collectively, these results demonstrated the effectiveness of the co-delivery strategy of biocompatible artemisinin-copper self-assembly nanoprodrug for potential tumor-specific CDT treatment.

Fig. 5 (a) Illustration of drug administration and treatment process. (b) and (c) Representative IVIS spectrum images and the quantified fluorescence signal intensity of tumor sites from different groups. (d) Tumor growth curves of different groups. (e) Photo of final xenograft tumors after various treatments. (f) Average body weight profiles during the treatment. (g) H&E staining and TUNEL staining assays of tumor tissues from different groups. ***p < 0.001 versus the control group.

3. In conclusion

In summary, our study has led to the development of a self-assembled nanoprodrug, leveraging the coordinative interactions between copper ions and a hydrophobic drug-amino acid conjugate (artesunate–histidine). This innovation capitalizes on the intracellular activation of ROS-based artemisinin drugs, thereby enhancing the CDT effect of copper-based materials. Within tumor cells, Cu²⁺ ions are transformed into Cu⁺ ions through the consumption of intracellular antioxidant GSH. The resulting Cu⁺ ions serve as a highly efficient Fenton-like reagent, promoting robust ROS generation from the released artesunate-histidine and intracellular H₂O₂. The cascade amplification of ROS generation through an in situ Fenton-like reaction underpins the effectiveness of our AHCu formulation, which demonstrates remarkable therapeutic efficacy both in vitro and in vivo. This approach not only addresses the pressing challenges in tumor treatment but also offers a novel solution for the development of safe and efficient nanomedicines. Therefore, our work provides a concise and elegant scheme for the preparation of such nanomedicines. Artemisinin and its analogues, as a gift from Chinese medicine to humanity, holds significant potential value that warrants further exploration.

Data availability

The authors will supply the relevant data in response to reasonable requests.

Conflicts of interest

There is no conflict of interest for any of the authors.

Acknowledgements

X. Zhu, C. Bi and W. Cao contributed equally to this work. This investigation was supported by the National Key R&D Program of China (Grant No. 2021YFA1600202), National Natural Science Foundation of China (52373160, 22305242), Anhui Natural Science Fund Project (2208085QH238, 2308085MB34), Anhui Clinical Medical Research Transformation Special Project (202304295107020050), and the Fundamental Research Funds for the Central Universities (YD9990002021). We thank Core Facility Center for Life Sciences of the University of Science and Technology of China for imaging support.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb01237b

‡ These authors contributed equally to this work.

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