Artemisinin derivative nano assembly with anti-tumor activity as well as preparation method and application of artemisinin derivative nano assembly

Abstract

The invention discloses an artemisinin derivative nano assembly with anti-tumor activity and a preparation method and application thereof. The nano assembly is formed by self-assembling artemisinin derivatives through intermolecular force and modifying the artemisinin derivatives with a PEG modifier; the artemisinin derivative is artesunate or dihydroartemisinin of which the side chain is binary acid or saturated fatty alcohol or unsaturated fatty alcohol; the mass ratio of the artemisinin derivative to the PEG modifier is (10: 90)-(90: 10), so that the technical effects of high drug loading capacity, good stability, low toxic and side effects and the like are realized, the urgent demand on high-efficiency and low-toxicity preparations in clinic is met, a new strategy is provided for the assembly of homotype synergistic drug nanoparticles, and the application prospect is broad. An effective nano platform is provided for developing a carrier-free nano assembly and a high-efficiency and low-toxicity nano preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations, and more specifically, to an artemisinin derivative nanoassembly with anti-tumor activity, a preparation method and application thereof. Background Art

[0002] Malignant tumors, a major global public health challenge, pose a continuing threat to human health due to their high mortality and complex pathological mechanisms. Currently, a comprehensive cancer treatment system is employed in clinical practice, encompassing multiple therapeutic approaches, including surgical intervention, chemotherapy, radiotherapy, and immunotherapy. Surgical resection demonstrates significant clinical value in the in situ and locally advanced stages of cancer, effectively achieving local control through precise excision of lesions. Technological innovations, such as molecular fluorescence-guided procedures, have significantly improved tumor clearance rates. However, for patients with advanced disease and distant metastases, surgical treatment is often limited in effectiveness and carries risks such as wound infection and postoperative complications. Chemotherapy, as a systemic treatment, utilizes cytotoxic drugs to kill tumor cells by interfering with DNA synthesis or microtubule function. However, traditional chemotherapeutic agents generally suffer from a narrow therapeutic window, and their non-selective cell-killing mechanisms can lead to adverse reactions such as myelosuppression and neurotoxicity. While radiotherapy effectively kills cancer cells, it also inevitably causes damage to normal tissues, such as radiation pneumonitis and radiation enteritis. Immunotherapy, especially immune checkpoint inhibitors and engineered T cells, can achieve lasting anti-tumor effects by relieving immunosuppression or enhancing immune recognition. However, due to tumor heterogeneity and other reasons, some patients may be insensitive to immunotherapy, resulting in certain limitations in immunotherapy. In general, current tumor treatment has formed a multimodal combined treatment paradigm, but each therapy faces common problems such as insufficient specificity, drug resistance, and toxicity accumulation. Therefore, it is urgent to develop new treatments based on new anti-tumor treatment mechanisms and innovative drug delivery technologies to supplement existing cancer therapies.

[0003] Artemisinin-based drugs are based on artemisinin (C 15 H 22O5) as the core of a class of natural medicines and their derivatives. One of the most important drug discoveries of the 20th century, this class of drugs was successfully isolated by Tu Youyou's team in the 1970s from the Asteraceae plant Artemisia annua. Their unique peroxide bridge structure (-OO-) forms the molecular basis of their pharmacological activity. After half a century of development, artemisinin-based drugs have expanded from their initial antimalarial properties to multiple therapeutic areas, including anti-tumor and immunomodulatory, demonstrating broad application prospects. Structurally, the peroxide bridge group in the artemisinin molecule is a key site of efficacy. Based on this core structure, researchers have developed a variety of derivatives with diverse physicochemical properties, including dihydroartemisinin, artesunate, and artemether. These derivatives not only retain the antimalarial activity of the parent core but also improve the drug's physicochemical properties through structural modifications. In terms of their antimalarial mechanism of action, artemisinin-based drugs cleave peroxide bridges via iron-catalyzed free radicals, which covalently bind to malarial parasite proteins, disrupting the cell membrane structure and heme metabolism, ultimately leading to parasite death. In recent years, the anti-tumor effect of artemisinin has attracted much attention. Studies have shown that in tumor tissues with high concentrations of Fe 2+ In the environment, artemisinin compounds can also exert anti-tumor effects by producing reactive oxygen species (ROS). Among them, dihydroartemisinin and artesunate have shown significant proliferation inhibition in in vitro models of various malignant tumors such as liver cancer, lung cancer, and breast cancer. However, the clinical application of artemisinin drugs still faces important challenges. On the one hand, this type of drug generally has pharmacokinetic defects such as short in vivo half-life and low oral bioavailability; on the other hand, its targeted accumulation ability in tumor tissue is limited, resulting in a narrow therapeutic window. These factors have largely limited the clinical transformation efficiency of artemisinin drugs in new indications such as anti-tumor, and have also become key scientific issues that need to be urgently addressed in current research.

[0004] With the interdisciplinary integration of molecular biology and materials science, a series of innovative drug delivery technologies have emerged, providing new technological pathways for precision cancer treatment. Innovations in drug delivery technologies have revolutionized cancer treatment strategies, demonstrating unprecedented advantages in improving drug targeting and reducing systemic toxicity. New drug delivery platforms, with their unique performance advantages, including precise control of drug release, enhanced tumor tissue penetration, improved drug biodistribution, and synergistic enhancement of therapeutic efficacy, have become the forefront of cancer treatment research. These advanced delivery systems not only optimize the in vivo behavior of therapeutic drugs but also significantly enhance their therapeutic index and clinical benefit. Currently, several anti-cancer drugs based on innovative delivery technologies have successfully achieved clinical translation, including the representative antibody-drug conjugate (ADC) drug emtansine trastuzumab (Kadcyla), polymeric micelle-delivered paclitaxel (Genexol-PM), and nucleic acid nanoparticle formulations (Onpattro). Compared to traditional chemotherapeutic drugs, these novel delivery systems not only achieve precise and targeted drug release but also significantly extend the therapeutic safety window through intelligent response mechanisms. However, existing delivery technologies primarily rely on traditional drug delivery methods such as physical encapsulation or chemical conjugation. While these technologies have achieved some success in clinical applications, they still face key challenges such as unstable drug loading efficiency, uncontrollable release kinetics, difficulty in scalable production, and potential toxicity of carrier materials. This has prompted researchers to urgently develop next-generation drug delivery platforms with greater intelligence and biocompatibility to overcome the current bottleneck in oncology drug delivery.

[0005] Artemisinin-based self-assembling nanomedicines (PBSANs) represent an innovative carrier-free drug delivery strategy, offering a breakthrough solution for the efficient delivery of chemotherapeutic drugs. From a molecular design perspective, these intelligent artemisinin-based molecules cleverly achieve a "three-in-one" functional integration: acting as both a therapeutic drug and a self-assembling carrier, while also exhibiting environmentally responsive properties. This unique design concept fundamentally addresses the dual dilemmas of poor drug-carrier compatibility and low drug loading efficiency in traditional nano-drug delivery systems. At the molecular level, PBSANs employ a modular design strategy, comprising three key functional modules: 1) a drug module; 2) a response module; and 3) a modification module. The modification module, a key regulatory element in the molecular engineering of artemisinin-based derivatives, plays a crucial role in precisely controlling their self-assembly behavior. However, systematic optimization of the modification module (e.g., fatty alcohol chain length, branching degree, and degree of unsaturation) for different drug molecules and specific therapeutic needs requires the development of more comprehensive structure-activity relationship models. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide an artemisinin derivative nanoassembly with anti-tumor activity and a preparation method and application thereof.

[0007] Artemisinin drugs kill tumors by producing reactive oxygen species (ROS), but they generally have shortcomings such as short half-life, low bioavailability and poor tumor accumulation ability. Although self-assembled nanoassemblies based on prodrugs can solve these problems, selecting suitable modification modules remains a challenge. Among the numerous candidate modification groups, aliphatic compounds are widely favored due to their excellent biocompatibility and structural adjustability, among which alkane derivatives such as fatty alcohols and fatty acids have been widely used in the construction of prodrug nanoassemblies. For this reason, the inventors screened the best antitumor drugs from four angles: the presence or absence of modification modules, the length of the fatty chain, the degree of branching and the degree of unsaturation.

[0008] First, the inventors adjusted the hydrophilic / hydrophobic properties of the drug by extending the response module, so that it has both responsiveness and the ability to regulate assembly. The inventors directly connected dihydroartemisinin to 3-carboxypropyl disulfide to synthesize DHA-S, and constructed a nanoassembly DHA-S NAs by self-assembly nano-drug delivery technology. DHA-S NAs can target tumor tissue through high permeability and long retention effect (EPR effect), achieve accumulation of drugs at the tumor site, and specifically release the parent drug DHA in response to the highly reducing microenvironment of the tumor. It is worth noting that compared with conventional modular prodrugs, the drugs designed by the inventors do not rely on modified modules to drive assembly, which greatly reduces the difficulty of synthesis.

[0009] Next, the inventors systematically compared the effects of side chain length, branching degree, and unsaturation on drug performance. The inventors first linked artesunate with 1-butanol, 1-octanol, 1-dodecanol, 1-tetradecanol, and 1-octadecanol (SA) to synthesize artesunate derivatives ART-C4, ART-C8, ART-C12, ART-C14, and ART-C18. They then constructed the corresponding small molecule nanoassemblies ART-C4 NAs, ART-C8 NAs, ART-C12 NAs, ART-C14 NAs, and ART-C18 NAs using small molecule self-assembly nano-drug delivery technology and compared the effects of side chain length on drug performance.

[0010] Subsequently, the present inventors connected artesunate with 1-pentadecanol, 8-pentadecanol, and cyclopentadecanol to synthesize artesunate derivative AC 15 -L, AC 15 -B and AC 15 -C, and constructed the corresponding small molecule nanoassembly AC through small molecule self-assembly nano drug delivery technology 15-L NAs, AC 15 -B NAs and ACs 15 -C NAs, and the effect of branching degree on drugs was studied.

[0011] Finally, the inventors linked the unsaturated fatty alcohol linolenic acid with artesunate to synthesize the drug ART-LA, and compared it with the saturated compound ART-C18 (ART-SA) with the same side chain length to explore the effect of side chain saturation on drug performance.

[0012] Notably, during the exploration of side chain length, branching degree, and unsaturation, the inventors synthesized artesunate derivatives, unlike traditional modular prodrugs, that do not rely on a response module. This not only simplifies the drug synthesis process but also avoids the complex trade-off between response ability and drug balance.

[0013] To achieve the above object, the technical solution of the present invention is as follows:

[0014] Disclosed is an artemisinin derivative nanoassembly with anti-tumor activity, wherein the nanoassembly is self-assembled by artemisinin derivatives through intermolecular forces and modified with a PEG modifier; the artemisinin derivative is artesunate or dihydroartemisinin whose side chain is a dibasic acid, a saturated fatty alcohol, or an unsaturated fatty alcohol; and the mass ratio of the artemisinin derivative to the PEG modifier is 10:90 to 90:10.

[0015] Optionally, the intermolecular force includes at least one of electrostatic force, hydrogen bond force and hydrophobic force.

[0016] Optionally, the side chain comprises 3-carboxypropyl disulfide, sebacic acid, 1-butanol, 1-octanol, 1-dodecanol, 1-tetradecanol, 1-pentadecanol, 8-pentadecanol, cyclopentadecanol, 1-octadecanol or linolenic alcohol.

[0017] Optionally, the artemisinin derivative has any of the following structural formulas:

[0018]

[0019] Optionally, the PEG modifier includes at least one of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG.

[0020] Optionally, the molecular weight of the PEG modifier is 200 to 20,000.

[0021] Optionally, the PEG modifier selected for NAs prepared based on dihydroartemisinin derivatives is preferably DSPE-PEG 2K; The PEG modifier selected for NAs prepared based on artesunate derivatives is preferably DSPE-SS-PEG 2K .

[0022] The present invention also discloses a method for preparing the above-mentioned artemisinin derivative nanoassembly with anti-tumor activity, comprising the following steps:

[0023] S1. Synthesis of artemisinin derivatives;

[0024] S2. dissolving the artemisinin derivative synthesized in step S1 in an organic solvent, slowly adding the mixed solution dropwise into water under stirring to spontaneously form uniform self-assembled nanoparticles;

[0025] S3. Add an organic solvent containing a PEG modifier to the solution obtained in step S2 and mix them, then dropwise add the mixture into water under stirring to spontaneously form a uniform PEG-modified nanoassembly, and remove the organic solvent to obtain the product.

[0026] Optionally, step S1 specifically includes the following steps: when the artemisinin derivative is dihydroartemisinin with a dibasic acid side chain, including: esterifying dihydroartemisinin with 3-carboxypropyl disulfide or sebacic acid under the catalysis of DMAP and EDCI, and separating by preparative liquid chromatography under appropriate conditions; when the artemisinin derivative is artesunate with a saturated fatty alcohol or unsaturated fatty alcohol side chain, including: esterifying artesunate with side chains of different lengths, different branching degrees and different saturations under the catalysis of DMAP and EDCI, and separating by preparative liquid chromatography under appropriate conditions.

[0027] Optionally, in steps S2 and S3, the organic solvent comprises at least one of methanol, ethanol, and tetrahydrofuran, preferably anhydrous ethanol; the stirring speed is 200 rpm to 2000 rpm; and in step S3, the organic solvent is removed by rotary evaporation or dialysis, preferably solvent evaporation, ultrafiltration, or membrane permeation.

[0028] The present invention also discloses the use of the above-mentioned artemisinin derivative nanoassembly with anti-tumor activity or the artemisinin derivative nanoassembly with anti-tumor activity prepared by the above-mentioned preparation method in the preparation of a drug delivery system.

[0029] The present invention also discloses the use of the artemisinin derivative nanoassembly with anti-tumor activity as described above or the artemisinin derivative nanoassembly with anti-tumor activity prepared by the above preparation method in the preparation of anti-tumor drugs.

[0030] The present invention also discloses the use of the above-mentioned artemisinin derivative nanoassembly with anti-tumor activity or the artemisinin derivative nanoassembly with anti-tumor activity prepared by the above-mentioned preparation method in the preparation of an injection, oral administration or local administration system.

[0031] The implementation of the present invention will have the following beneficial effects:

[0032] 1. The present invention prepares an artemisinin derivative nanoassembly formed by self-assembly of artemisinin derivatives, which is modified with a PEG modifier to obtain an artemisinin derivative nanoassembly with anti-tumor activity. Artemisinin drugs can generate ROS at the tumor site to kill tumor cells. Furthermore, to optimize the response module and the modification module, the present invention optimizes drug performance by adjusting the length, fatty chain length, degree of branching, and degree of unsaturation of the response module, and develops drugs that are independent of the response module. This new drug can specifically release the parent drug in the tumor microenvironment, simplifying the synthesis process and having better drug performance.

[0033] 2. The artemisinin derivative nanoassembly with anti-tumor activity of the present invention achieves technical effects such as high drug loading, good stability, and low toxic side effects, meeting the urgent demand for high-efficiency and low-toxic preparations in clinical practice, providing a new strategy for the assembly of homologous synergistic drug nanoparticles, and providing an effective nanoplatform for the development of carrier-free nanoassemblies and nanoformulations with high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the synthetic route of Example 1 of the present invention.

[0035] Figure 2 The mass spectrum and NMR spectrum of DHA-S in Example 1 of the present invention are shown.

[0036] Figure 3 The mass spectrum and NMR spectrum of DHA-C in Example 1 of the present invention are shown.

[0037] Figure 4 This is the synthetic route of Example 2 of the present invention.

[0038] Figure 5 The mass spectrum and NMR spectrum of ART-C4 of Example 2 of the present invention are shown.

[0039] Figure 6 The mass spectrum and NMR spectrum of ART-C8 of Example 2 of the present invention are shown.

[0040] Figure 7 The mass spectrum and NMR spectrum of ART-C12 of Example 2 of the present invention are shown.

[0041] Figure 8The mass spectrum and NMR spectrum of ART-C14 of Example 2 of the present invention are shown.

[0042] Figure 9 The mass spectrum and NMR spectrum of ART-C18 of Example 2 of the present invention are shown.

[0043] Figure 10 This is the synthetic route for implementation 3 of the present invention.

[0044] Figure 11 AC of Example 3 of the present invention 15 -L's mass spectrum and NMR spectrum.

[0045] Figure 12 AC of Example 3 of the present invention 15 -B's mass spectrum and NMR spectrum.

[0046] Figure 13 AC of Example 3 of the present invention 15 -C mass spectrum and NMR spectrum.

[0047] Figure 14 This is the synthetic route of Example 4 of the present invention.

[0048] Figure 15 The mass spectrum and NMR spectrum of ART-LA of Example 4 of the present invention are shown.

[0049] Figure 16 This is the particle size distribution of the nanoassembly of Example 5 of the present invention.

[0050] Figure 17 This is a molecular docking diagram of the assembly process prepared in Example 6 of the present invention.

[0051] Figure 18 This is the PBS stability diagram of the nanoassembly prepared in Example 7 of the present invention.

[0052] Figure 19 This is a graph showing the stability of the nanoassembly prepared in Example 7 of the present invention in PBS containing 10% FBS.

[0053] Figure 20 These are confocal microscope photographs of the cell uptake of the C-6 (or Cy3) solution and the C-6 (or Cy3) labeled nanoassembly of Example 8 of the present invention at 0.5 hours and 2 hours.

[0054] Figure 21 This is a quantitative graph of ROS levels in 4T1 cells after drug treatment in Example 9 of the present invention.

[0055] Figure 22 This is the cytotoxicity result of the tumor cells (4T1 cells) in Example 10 of the present invention.

[0056] Figure 23 1 is a blood concentration-time curve of the DiR (or Cy7) solution and the DiR (or Cy7)-labeled nanoassembly of Example 11 of the present invention.

[0057] Figure 24 This is a graph showing the mouse tumor growth curve of the anti-tumor experiment in the 4T1 tumor model according to Example 12 of the present invention.

[0058] Figure 25 This is a statistical graph of the tumor-bearing rate of mice in the anti-tumor experiment in the 4T1 tumor model according to Example 12 of the present invention.

[0059] Figure 26 This is a graph showing changes in mouse body weight in the anti-tumor experiment in the 4T1 tumor model according to Example 12 of the present invention.

[0060] Figure 27 These are H&E staining images of the heart, liver, spleen, lung, and kidney of mice in the anti-tumor experiment in the 4T1 tumor model according to Example 12 of the present invention. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0062] Example 1: Synthesis of dihydroartemisinin derivatives

[0063] (1) Synthesis of dihydroartemisinin-3-carboxypropyl disulfide (DHA-S)

[0064] 3-Carboxypropyl disulfide (2 mmol) and EDCI (2.4 mmol) were added to a 100 mL round-bottom flask, followed by the addition of approximately 40 mL of DMF and complete sonication to dissolve the powder. Stirring was performed at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.4 mmol) and DHA (1 mmol), previously dissolved in DMF, were then added with stirring. The mixture was stirred at 25°C under nitrogen for 24 h. DMF was removed by rotary evaporation to yield a crude dihydroartemisinin derivative (DHA-S). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, mobile phase: acetonitrile / 0.1% formic acid in water = 90:10, flow rate 10 mL / min). The resulting product, DHA-S, was a pale yellow oily liquid. The product was characterized by mass spectrometry (MS) and H-NMR spectroscopy.

[0065] The structural formula of dihydroartemisinin-3-carboxypropyl disulfide (DHA-S) is as follows:

[0066]

[0067] (2) Synthesis of dihydroartemisinin-sebacic acid (DHA-C)

[0068] Sebacic acid (2 mmol) and EDCI (2.4 mmol) were added to a 100 mL round-bottom flask, followed by the addition of approximately 40 mL of DMF and sonication to completely dissolve the powder. Stirring was performed at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.4 mmol) and DHA (1 mmol), previously dissolved in DMF, were then added with stirring. The mixture was stirred at 25°C under nitrogen for 24 h. DMF was removed by rotary evaporation to yield a crude dihydroartemisinin derivative (DHA-C). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, mobile phase: acetonitrile / 0.1% formic acid in water = 90:10, flow rate 10 mL / min). The resulting product, DHA-C, was a pale yellow oily liquid. The product was characterized by mass spectrometry (MS) and H-NMR spectroscopy.

[0069] The structural formula of dihydroartemisinin-sebacic acid (DHA-C) is as follows:

[0070]

[0071] Synthesis route such as Figure 1 As shown, the results are Figure 2-Figure 3 As shown, the results showed that two dihydroartemisinin derivatives were successfully synthesized.

[0072] Example 2: Synthesis of artesunate derivatives with different chain lengths

[0073] (1) Synthesis of artesunate-1-butanol (ART-C4)

[0074] 1-Butanol (1.5 mmol) and EDCI (2 mmol) were added to a 100 mL round-bottom flask, followed by the addition of approximately 20 mL of dichloromethane and sonication to completely dissolve the powder. Stirring was performed at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.2 mmol) and ART (1 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C under nitrogen for 24 h. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-C4). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250 × 20 mm column, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min). ART-C4 was obtained as a colorless oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0075] The structural formula of artesunate-1-butanol (ART-C4) is as follows:

[0076]

[0077] (2) Synthesis of artesunate-1-octanol (ART-C8)

[0078] 1-Octanol (1.5 mmol) and EDCI (2 mmol) were added to a 100 mL round-bottom flask. Approximately 20 mL of dichloromethane was then added and the powder was completely dissolved by sonication. Stirring was continued at 25°C for 2 h under nitrogen to yield a colorless, transparent liquid. DMAP (0.2 mmol) and ART (1 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C for 24 h under nitrogen. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-C8). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250 × 20 mm column, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min). ART-C8 was obtained as a colorless, oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0079] The structural formula of artesunate-1-octanol (ART-C8) is as follows:

[0080]

[0081] (3) Synthesis of artesunate-1-dodecanol (ART-C12)

[0082] 1-Dodecanol (1.5 mmol) and EDCI (2 mmol) were added to a 100 mL round-bottom flask. Approximately 20 mL of dichloromethane was then added and the powder was completely dissolved by sonication. Stirring was continued at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.2 mmol) and ART (1 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C under nitrogen for 24 h. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-C12). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min). ART-C12 was obtained as a colorless oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0083] The structural formula of artesunate-1-dodecanol (ART-C12) is as follows:

[0084]

[0085] (4) Synthesis of artesunate-1-tetradecanol (ART-C14)

[0086] 1-Tetradecanol (1.5 mmol) and EDCI (2 mmol) were added to a 100 mL round-bottom flask. Approximately 20 mL of dichloromethane was then added and the powder was completely dissolved by sonication. Stirring was continued at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.2 mmol) and ART (1 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C under nitrogen for 24 h. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-C14). The crude product was then isolated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min). ART-C14 was obtained as a colorless, oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0087] The structural formula of artesunate-1-tetradecanol (ART-C14) is as follows:

[0088]

[0089] (5) Synthesis of Artesunate-1-octadecanol (ART-C18)

[0090] 1-Octadecyl alcohol (1.5 mmol) and EDCI (2 mmol) were added to a 100 mL round-bottom flask. Approximately 20 mL of dichloromethane was then added and the powder was completely dissolved by sonication. Stirring was continued at 25°C under nitrogen for 2 h to yield a colorless, transparent liquid. DMAP (0.2 mmol) and ART (1 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C under nitrogen for 24 h. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-C18). The crude product was then separated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min). ART-C18 was obtained as a colorless, oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0091] The structural formula of artesunate-1-octadecanol (ART-C18) is as follows:

[0092]

[0093] Synthesis route such as Figure 4 As shown, the results are Figure 5-Figure 9 As shown, the results showed that five artesunate derivatives of different lengths were successfully synthesized.

[0094] Example 3: Synthesis of artesunate derivatives with different branching degrees

[0095] (1) Artesunate-1-pentadecanol (AC 15 -L) synthesis

[0096] 1-Pentadecanol (0.84 mmol) and EDCI (1 mmol) were added to a 100 mL round-bottom flask, followed by the addition of about 20 mL of dichloromethane and ultrasonication to completely dissolve the powder. The mixture was stirred at 25°C for 2 h under nitrogen protection to obtain a colorless transparent liquid. DMAP (0.28 mmol) and ART (1 mmol) previously dissolved in dichloromethane were then added under stirring, and the mixture was stirred at 25°C for 24 h under nitrogen protection. The dichloromethane was removed by rotary evaporation to obtain the artesunate derivative (AC 15 The crude product was then separated and purified by preparative liquid chromatography (chromatographic column: Shim-pack PREP-ODS 250×20 mm, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min), and the obtained product AC 15 -L is a colorless oily liquid. The product was characterized by mass spectrometry (MS) and H NMR.

[0097] Artesunate-1-pentadecanol (AC 15 -L) is as follows:

[0098]

[0099] (2) Artesunate-8-pentadecanol (AC 15 -B) Synthesis

[0100] 8-pentadecanol (0.84 mmol) and EDCI (1 mmol) were added to a 100 mL round-bottom flask, and then about 20 mL of dichloromethane was added and ultrasonicated to completely dissolve the powder. The mixture was stirred at 25°C for 2 h under nitrogen protection to obtain a colorless transparent liquid. DMAP (0.28 mmol) and ART (1 mmol) which had been dissolved in dichloromethane in advance were then added under stirring, and the mixture was stirred at 25°C for 24 h under nitrogen protection. The dichloromethane was removed by rotary evaporation to obtain the artesunate derivative (AC 15 -B) crude product. The crude product was then separated and purified by preparative liquid chromatography (chromatographic column: Shim-pack PREP-ODS 250×20 mm, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min), and the obtained product AC 15 -B is a colorless oily liquid. The product was characterized by mass spectrometry (MS) and H NMR.

[0101] Artesunate-8-pentadecanol (AC15 -B) is as follows:

[0102]

[0103] (3) Artesunate-cyclopentadecanol (AC 15 -C) Synthesis

[0104] Cyclopentadecanol (0.84 mmol) and EDCI (1 mmol) were added to a 100 mL round-bottom flask, followed by the addition of about 20 mL of dichloromethane and ultrasonication to completely dissolve the powder. The mixture was stirred at 25°C for 2 h under nitrogen protection to obtain a colorless transparent liquid. DMAP (0.28 mmol) and ART (1 mmol) previously dissolved in dichloromethane were then added under stirring, and the mixture was stirred at 25°C for 24 h under nitrogen protection. The dichloromethane was removed by rotary evaporation to obtain the artesunate derivative (AC 15 The crude product was then separated and purified by preparative liquid chromatography (chromatographic column: Shim-pack PREP-ODS 250×20 mm, mobile phase: acetonitrile / water = 95:5, flow rate 10 mL / min), and the obtained product AC 15 -C was a colorless oily liquid. The product was characterized by mass spectrometry (MS) and H NMR.

[0105] Artesunate-cyclopentadecanol (AC 15 -C) is as follows:

[0106]

[0107] Synthesis route such as Figure 10 As shown, the results are Figure 11-13 As shown, the results showed that three artesunate derivatives with different branching degrees were successfully synthesized.

[0108] Example 4: Synthesis of unsaturated artesunate derivatives

[0109] (1) Synthesis of artesunate-linolenic alcohol (ART-LA)

[0110] 1-Pentadecanol (0.42 mmol) and EDCI (0.5 mmol) were added to a 100 mL round-bottom flask. Approximately 20 mL of dichloromethane was then added and the powder was completely dissolved by sonication. Stirring was continued at 25°C for 2 h under nitrogen to yield a colorless, transparent liquid. DMAP (0.14 mmol) and ART (0.5 mmol), previously dissolved in dichloromethane, were then added with stirring. The reaction was stirred at 25°C for 24 h under nitrogen. The dichloromethane was removed by rotary evaporation to yield a crude artesunate derivative (ART-LA). The crude product was then separated and purified by preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, pure acetonitrile as the mobile phase, at a flow rate of 10 mL / min). ART-LA was obtained as a colorless, oily liquid. The product was characterized by mass spectrometry (MS) and H NMR spectroscopy.

[0111] The structural formula of artesunate-linolenic acid (ART-LA) is as follows:

[0112]

[0113] Synthesis route such as Figure 14 As shown, the results are Figure 15 As shown, the results showed that the unsaturated artesunate derivatives were successfully synthesized.

[0114] Example 5: Characterization of Nanoassemblies

[0115] Using a certain amount of PEG to modify the nanoassembly can improve the stability and blood circulation time of the nanoassembly to a certain extent. Therefore, after determining the optimal conditions for preparing the non-PEGylated Artemisia annua derivative nanoassembly, we chose to modify the nanoassembly with 20% DSPE-PEG. 2K (DSPE-SS-PEG was used when screening side chain length, branching degree and saturation 2K Modification) to improve the stability of the nanoassembly and prolong its blood circulation time. The specific operation is: weigh DSPE-PEG 2K 1.25mg (or DSPE-SS-PEG 2K 1.25 mg) and 5 mg of the drug were dissolved in anhydrous ethanol to prepare a drug concentration of 5 mg / mL, DSPE-PEG 2K (or DSPE-SS-PEG 2K) mother liquor with a concentration of 1.25 mg / mL. 2 mL of deionized water was added to a 5 mL penicillin bottle, which was placed on a magnetic stirrer. 200 μL of the mother liquor was added dropwise while stirring. Stirring was continued for 3 minutes after the addition was completed. It was then transferred to a 25 mL eggplant-shaped bottle, and the organic solvent was removed by rotary evaporation at 30°C. The volume was then adjusted to 2 mL with deionized water to obtain a preparation of artemisia annua derivative nanoassemblies with a drug concentration of 0.5 mg / mL. After the artemisia annua derivative nanoassemblies were diluted 20 times with deionized water, the particle size and zeta potential were measured in parallel three times using a Malvern particle size analyzer. In addition, the prodrug nanoassemblies were stained with 2% phosphotungstic acid on the surface of a copper mesh, and the morphology of the prodrug nanoassemblies was observed using TEM.

[0116] The particle size of the two prodrug nanoassemblies was investigated using a Malvern particle size analyzer. Figure 16 Except for the ART-C4 nanoassembly with a short-chain fatty alcohol side chain, the rest of the Artemisia annua derivative nanoassemblies were able to assemble stably.

[0117] Example 6: Analysis of the Self-Assembly Mechanism of Artemisia annua Derivative Nanoassemblies

[0118] Through computer simulation, the mechanism driving prodrug assembly was explored, and molecular docking calculations were performed using the Vina program of the Yinfu cloud computing platform. The synthesized Artemisia annua derivatives were energy minimized under the MMFF94 force field to obtain a 3D structure, forming a stable nanoassembly. Semi-flexible docking was performed using the AutoDock Vina program, and the results are as follows: Figure 17 As shown in Figure 3, due to the strong hydrophobicity of the fatty alcohol side chain, there are hydrophobic forces between all Artemisia annua derivative molecules.

[0119] Example 7: Stability study of nanoassemblies

[0120] The stability of the nanoassemblies was evaluated using the particle size of the artemisia annua derivatives as an indicator. Specifically, 1 mL of an artemisia annua derivative (at a concentration of 0.5 mg / mL) was added to 9 mL of PBS (pH 7.4) and PBS containing 10% FBS (pH 7.4), respectively. The mixture was incubated at 37°C in a shaker at 100 rpm. The particle size of the nanoassemblies was measured at pre-set time points (0, 1, 2, 4, 6, 8, 10, and 12 hours).

[0121] The results are as follows Figure 18-19As shown in the figure, ART-SA is ART-C18 prepared in the above example. When the Artemisia annua derivative nanoassemblies were incubated in PBS (pH 7.4) and PBS (pH 7.4) containing 10% FBS for a certain period of time, except for the ART-C8 nanoassembly, which showed a significant change in particle size, the other Artemisia annua derivative nanoassemblies showed no significant changes. This indicates that the ART-C8 nanoassembly has poor stability, while the other Artemisia annua derivative nanoassemblies have good stability.

[0122] Example 8: Cellular Uptake of Nanoparticles

[0123] Preparation of C-6 labeled nanoassemblies of Artemisia annua derivatives: Fluorescent labeled nanoassemblies of Artemisia annua derivatives were prepared using coumarin 6 (C-6) as a fluorescent marker. Specific operation: Taking the preparation of C-6 labeled DHA-S nanoassemblies as an example, 1.0 mg of C-6 was weighed, dissolved and diluted with anhydrous ethanol as a solvent to prepare a C-6 ethanol solution with a concentration of 0.8 μg / mL. 5.0 mg of DHA-S and DSPE-PEG were weighed. 2K 1.25 mg, DHA-S and DSPE-PEG were mixed with 0.5 mL of the above C-6 ethanol solution. 2K Dissolve, stir (1300rpm) C-6, DHA-S and DSPE-PEG 2K The mixed ethanol solution was slowly added dropwise to 2 mL of deionized water and stirred for 2 minutes. The organic solvent in the preparation was then removed by distillation under reduced pressure to obtain C-6-labeled DHA-S nanoassemblies. The preparation methods of other C-6-labeled Artemisia annua derivative nanoassemblies were similar to those of the C-6-labeled DHA-S nanoassemblies, except that DHA-S was replaced with other Artemisia annua derivatives.

[0124] It is worth noting that when exploring the effect of side chain unsaturation on drug properties, DSPE-SS-PEG labeled with Cy3 was used. 2K As fluorescent probes, Cy3-sp-ART-LANAs and Cy3-sp-ART-SANAs were prepared according to the preparation method described in Example 5.

[0125] Cell uptake experiment: 4T1 cells were cultured at 5×10 4Cells were seeded at a density of 100 cells / well into a 24-well plate pre-mounted with a slide and incubated for 12 hours. The old culture medium was discarded and replaced with fresh culture medium containing C-6 solution (or Cy3 solution) and C-6-labeled artemisia annua derivative nanoassemblies (or Cy3-sp-ART-LANAs and Cy3-sp-ART-SANAs), where the equivalent concentration of C-6 was 250 ng / mL (the equivalent concentration of Cy3 was 8 μg / mL), and incubated at 37°C for 0.5 or 2 hours (or 0.5 or 4 hours). Subsequently, the drug-containing culture medium was discarded, the uptake was terminated with cold PBS (pH 7.4), and the cells were washed three times. Then, the cells were fixed with 4% paraformaldehyde for 10 minutes and washed three times with cold PBS (pH 7.4). Afterwards, the cells were stained with Hoechst 33342 staining solution for 10 minutes and washed three times with cold PBS (pH 7.4). Finally, the processed slides were fixed on a glass slide with an anti-fluorescence attenuation mounting medium, and the fluorescence images of cell uptake were observed and photographed under a confocal fluorescence microscope.

[0126] like Figure 20 As shown in the figure, ART-SA is ART-C18 prepared in the above example. When investigating the effect of the extended response module on drug activity, experimental results show that compared to the blank control group, the DHA-S nanoassembly exhibited the highest fluorescence intensity, while the DHA-C nanoassembly exhibited slightly weaker fluorescence intensity. This is due to the slightly smaller particle size of the DHA-S nanoassembly, resulting in better cellular uptake. The DHA solution exhibited the weakest fluorescence intensity and the poorest cellular uptake. When investigating the effect of side chain length on drug activity, all nanoassembly groups exhibited significantly higher fluorescence intensities than the coumarin 6 solution group after the same incubation time, indicating that this nanoassembly exhibited higher cellular uptake efficiency. Notably, the fluorescence intensity of the ART-C8 nanoassembly was weaker than that of the ART-C12, ART-C14, and ART-C18 nanoassemblies. This may be due to its larger particle size and poor stability, which may hinder cellular internalization. In contrast, the ART-C18 nanoassembly, with the smallest particle size, exhibited the strongest fluorescence signal and the highest cellular uptake. The particle sizes of ART-C12 and ART-C14 nanoassemblies were similar, and the corresponding fluorescence intensities were also similar. In addition, the fluorescence intensity increased with time at 0.5 h and 2 h. Figure 20 As shown, when studying the effects of other conditions on drugs, the fluorescence intensities of 4T1 cells in the corresponding Artemisia annua derivative nanoassembly-treated groups were comparable at 0.5 and 2 h, and were much stronger than those of C-6 solution or Cy3 solution, and were time-dependent.

[0127] Example 9: Determination of intracellular ROS

[0128] DCFH-DA is used to detect intracellular ROS levels. DCFH-DA itself is non-fluorescent, but intracellular ROS can oxidize non-fluorescent DCFH-DA into fluorescent DCF. Specifically, 4T1 cells were cultured at 5×10 4 The cells were seeded into 12-well plates at a density of 100 μg / well and cultured in an incubator at 37°C containing 5% CO2 for 12 h. 4T1 cells were then treated with equal amounts of PBS, ART solution, DHA solution, and nanoassemblies of artemisinin derivatives (when exploring the effect of side chain unsaturation on the drug, 4T1 cells were treated with fresh blank culture medium, equal amounts of ART solution, LA solution, SA solution, ART-LA solution, ART-SA solution, ART-LA nanoassembly, and ART-SA nanoassembly). After incubation for 4 h, the cells were washed three times with cold PBS (pH 7.4) and then incubated with an equal amount of DCFH-DA for 30 min. The cells were then washed three times with cold PBS (pH 7.4), and the level of ROS in 4T1 cells was quantified by confocal fluorescence microscopy or flow cytometry.

[0129] like Figure 21 As shown in the figure, ART-SA is ART-C18 prepared in the above embodiment. When exploring the effect of the extended response module on the drug, according to the experimental results, it can be seen that compared with the blank control group, the DHA-S nanoassembly has the strongest fluorescence intensity, corresponding to the best ROS generation ability, and the fluorescence intensity of the DHA-C nanoassembly is second. The DHA solution and ART solution have similar and weakest fluorescence intensities.

[0130] When studying the effect of side chain length on drug activity, it was found that ART-C12 nanoassemblies exhibited the strongest fluorescence intensity compared to the blank control group. The fluorescence intensity of ART-C8 nanoassemblies and ART solution was weaker than that of ART-C12 nanoassemblies. The fluorescence intensity of ART-C14 nanoassemblies was slightly weaker than that of ART solution and ART-C8 nanoassemblies, while the fluorescence intensity of ART-C18 nanoassemblies was the weakest.

[0131] When comparing the effects of the branching degree of the side chain on the drug, it was found that AC 15 -C nanoassembly showed the strongest fluorescence intensity, while ART solution showed weaker fluorescence intensity. 15 The fluorescence intensity of the -B nanoassembly was weaker than that of the ART solution, while the AC 15 The fluorescence intensity of the -L nanoassembly is the weakest.

[0132] When studying the effect of side chain unsaturation on drugs, flow cytometry results showed that ROS were generated in cells treated with ART solution, ART-LA solution, and ART-LA nanoassembly compared to the blank control group. However, almost no ROS were generated in cells treated with LA solution, SA solution, ART-SA solution, and ART-SA nanoassembly.

[0133] Example 10: Cytotoxicity of Nanoassemblies

[0134] The cytotoxicity of the nanoassemblies of Artemisia annua derivatives was evaluated by MTT assay. 3 (or 1×10 3 ) cells / well were seeded into 96-well plates and cultured at a constant temperature of 37°C in an incubator containing 5% CO2 for 12 hours. The cells were then treated with culture medium containing different concentrations of DHA solution, ART solution, artemisia annua derivative solution, and artemisia annua derivative nanoassembly for 48 hours (when exploring the effect of side chain unsaturation on the drug, 4T1 cells were treated with different concentrations of ART solution, LA solution, SA solution, ART-LA solution, ART-SA solution, ART-LA nanoassembly, and ART-SA nanoassembly). Subsequently, 5 mg / mL MTT (20 μL / well) was added to the 96-well plate, and the cells were incubated with MTT at 37°C for 4 hours. The culture medium was discarded, and DMSO (200 μL / well) was added to the 96-well plate to completely dissolve the generated formazan. Finally, the UV absorbance of the solution in each well at 490 nm was measured using a multifunctional microplate reader.

[0135] like Figure 22 As shown, the ART-SA in the figure is ART-C18 prepared in the above embodiment. When exploring the effect of the extended response module on the drug, its cytotoxicity results are consistent with the ROS production ability. The reason for this result is mainly due to the disulfide bond having a reduction response ability, which can consume glutathione (GSH) in the tumor, thereby reducing the consumption of ROS and enhancing the accumulation of ROS. Therefore, DHA-S has the strongest ROS generation ability and cytotoxicity, while DHA-C has no ability to consume GSH, and only the Artemisia annua structure produces ROS. Therefore, it has a cytotoxicity weaker than DHA-S. Compared with the solution, the cellular uptake ability of DHA-S nanoassembly and DHA-C nanoassembly is better than that of the solution, so the cytotoxicity is stronger than that of the solution.

[0136] When investigating the effect of side chain length on drug activity, the cytotoxicity of the artesunate derivative solution group showed a gradual decrease. The cytotoxicity of the artesunate derivative nanoassembly group initially increased and then decreased. This finding for the solution formulation is primarily due to the fact that the activity of the synthesized artesunate derivative is affected by carbon chain length, with drug activity decreasing with increasing carbon chain length. The cytotoxicity of the artesunate derivative nanoassembly group initially increased and then decreased, primarily due to the poor stability and large average particle size of the ART-C8 nanoassemblies, which resulted in poor cellular uptake. The ART-C12 nanoassemblies exhibited the best cytotoxicity due to their excellent cellular uptake and highest intracellular ROS generation. Although the cellular uptake of the ART-C14 nanoassembly was superior to that of the ART-C12 nanoassembly, its intrinsic activity was lower than that of the ART-C12 nanoassembly, and its ability to generate intracellular ROS was also weaker than that of the ART-C12 nanoassembly, resulting in its weaker cytotoxicity. The low activity of ART-C18 nanoassemblies is due to the non-toxicity of ART-C18 itself.

[0137] When comparing the effect of side chain branching on the drug, the order of solution cytotoxicity is AC 15 -C solution > ART solution > AC 15 -B solution>AC 15 -L solution. The cytotoxicity trend of the nanoassembly is similar to that of the solution, in the order of AC 15 -C nanoassembly>ART solution>AC 15 -B nanoassembly>AC 15 -L nanoassembly. This trend is due to the different binding abilities of the three synthesized artesunate derivatives to Lon protease (LONP1), AC 15 -C has the strongest binding ability to LONP1 and has the strongest ability to induce mitochondrial protein homeostasis collapse, which means that it can generate the most ROS through LONP1 and thus has the strongest cytotoxicity. The binding ability of the other groups of drugs to LONP1 is weaker than AC. 15 -C and corresponds to the trend of cytotoxicity.

[0138] When studying the effect of side chain unsaturation on the drug, the toxicity of ART-LA nanoassemblies was similar to that of ART solution. We speculate that this may be due to the fact that the polyunsaturated fatty alcohols in their structure undergo lipid peroxidation in the high ROS environment of tumor cells, resulting in chain breakage and restoration of the original ART drug activity. However, cell survival rate did not decrease significantly after treatment with ART-SA nanoassemblies, and no obvious toxicity was shown. This may be due to the fact that ART-SA does not undergo lipid peroxidation in vivo, which masks the active site.

[0139] Example 11: Pharmacokinetic Study of Nanoparticles

[0140] Sprague-Dawley rats weighing 180-220 g were randomly divided into groups and fasted for 12 hours before dosing, with free access to water. DiR solution (or Cy7 solution) and DiR-labeled nanoassemblies (or Cy7-labeled nanoassemblies) were injected into the tail vein of the SD rats to investigate their in vivo pharmacokinetic behavior (dose of 2 mg / kg DiR or Cy7). Blood was collected from the ophthalmic vein at pre-determined time points (0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after tail vein injection. The blood samples were centrifuged (8000 rpm, 3 minutes) to obtain plasma. DiR (or Cy7) was then extracted by protein precipitation, and the pharmacokinetic behavior of each formulation was determined using a microplate reader (excitation 750 nm, emission 773 nm).

[0141] The experimental results are as follows Figure 23 As shown in the figure, ART-SA represents ART-C18 prepared in the above examples. Due to its short half-life, the DiR (or Cy7) solution is rapidly metabolized and cleared. Compared to the solution, the circulation time of the artemisia annua derivative nanoassembly is significantly prolonged, providing a good foundation for drug accumulation in tumors in vivo. Notably, when studying the effect of side chain length on drug activity, the ART-C14 nanoassembly exhibited the highest AUC, likely due to its superior assembly capacity and optimal stability. The assembly capacity and stability of the ART-C12 nanoassembly were slightly inferior to those of the ART-C14 nanoassembly, but they also exhibited good pharmacokinetic behavior. In contrast, the ART-C18 nanoassembly had the strongest assembly capacity but poorer pharmacokinetic properties. The underlying cause of this phenomenon may be related to the presence of trace reducing substances in the blood, which induce partial dissociation of the nanoparticles. Furthermore, the strong hydrophobicity of ART-C18, combined with the complex blood environment, promotes its rapid clearance. Under complex in vivo conditions, multiple factors together lead to the poorer pharmacokinetic properties of ART-C18 nanoassemblies than those of ART-C12 and ART-C14 nanoassemblies. ART-C8 nanoassemblies have the worst pharmacokinetic properties due to their poor assembly ability and stability.

[0142] Example 12: In vivo anti-tumor experiment of nanoparticles

[0143] 4T1 cell suspension (10 7 cells / 100 μL) were inoculated on the right back of female mice. 3When the drug was administered, the mice were randomly divided into groups of 5, each group was divided into blank control group, ART solution, DHA solution, Artemisia annua derivative nanoassembly (when exploring the effect of the unsaturation of the side chain on the drug, the groups were blank control group, ART solution, ART-SA solution, ART-LA solution, ART-SA nanoassembly and ART-LA nanoassembly). The drug was administered once every other day for a total of 5 times, with an equal concentration of ART (10 mg / kg). At the same time, the tumor volume and body weight of the mice were measured and recorded every day, and the safety of the drug was preliminarily evaluated by the changes in the body weight of the mice. After the last administration, the mice were killed after an interval of 1 day, and the organs and tumors were obtained for further analysis and evaluation. The main organs (heart, liver, spleen, lungs, kidneys) were collected and fixed with 4% tissue fixative for H&E staining. The results are shown in Figures 24-25 As shown in the figure, ART-SA is ART-C18 prepared in the above example. In the group exploring the effect of the extended response module on the drug, the DHA-S nanoassembly has the best anti-tumor effect. In the group studying the effect of the length of the side chain on the drug, the ART-C14 nanoassembly has the best anti-tumor effect. In the group comparing the effect of the branching degree of the side chain on the drug, AC 15 -C nanoassembly has the best anti-tumor effect. In the group studying the effect of side chain unsaturation on drugs, ART-LA nanoassembly has the best anti-tumor effect. At the same time, a preliminary investigation was conducted on the therapeutic safety of all groups of artemisia annua derivative nanoassemblies. Figure 26 As shown in Figure 3, there was no significant difference in the body weight of tumor-bearing mice between different drug-treated groups. Figure 27 As shown, after treatment, H&E staining of the heart, liver, spleen, lungs, and kidneys showed no obvious abnormalities.

[0144] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An artemisinin derivative nanoassembly with anti-tumor activity, characterized in that: The nanoassembly is self-assembled by artemisinin derivatives through intermolecular forces and modified with a PEG modifier; The artemisinin derivative is artesunate or dihydroartemisinin with a side chain of a dibasic acid or a saturated fatty alcohol or an unsaturated fatty alcohol; The mass ratio of the artemisinin derivative to the PEG modifier is 10:90 to 90:

10.

2. The artemisinin derivative nanoassembly with anti-tumor activity according to claim 1, characterized in that: The intermolecular force includes at least one of electrostatic force, hydrogen bond force and hydrophobic force; The side chains include 3-carboxypropyl disulfide, sebacic acid, 1-butanol, 1-octanol, 1-dodecanol, 1-tetradecanol, 1-pentadecanol, 8-pentadecanol, cyclopentadecanol, 1-octadecanol, or linolenic alcohol.

3. The artemisinin derivative nanoassembly with anti-tumor activity according to claim 1, characterized in that: The PEG modifier includes at least one of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG; The molecular weight of the PEG modifier is 200 to 20,000.

4. The artemisinin derivative nanoassembly with anti-tumor activity according to claim 3, characterized in that: The PEG modifier is DSPE-PEG 2K and / or DSPE-SS-PEG 2K .

5. A method for preparing an artemisinin derivative nanoassembly with anti-tumor activity according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Synthesis of artemisinin derivatives; S2, dissolving the derivative synthesized in step S1 in an organic solvent, slowly adding the mixed solution dropwise into water under stirring to spontaneously form uniform self-assembled nanoparticles; S3. Add an organic solvent containing a PEG modifier to the solution obtained in step S2 and mix them, then dropwise add the mixture into water under stirring to spontaneously form a uniform PEG-modified nanoassembly, and remove the organic solvent to obtain the product.

6. The method for preparing the artemisinin derivative nanoassembly with anti-tumor activity according to claim 5, characterized in that: Step S1 specifically includes the following steps: When the artemisinin derivative is dihydroartemisinin with a dibasic acid side chain, the method comprises: subjecting dihydroartemisinin to an esterification reaction with 3-carboxypropyl disulfide or sebacic acid under the catalysis of DMAP and EDCI, and separating the reaction by preparing a liquid phase under appropriate conditions; When the artemisinin derivative is artesunate with a side chain of a saturated fatty alcohol or an unsaturated fatty alcohol, the method comprises: subjecting artesunate to an esterification reaction with side chains of different lengths, different degrees of branching and different degrees of saturation under the catalysis of DMAP and EDCI, and separating the reaction by preparative liquid chromatography under appropriate conditions.

7. The method for preparing the artemisinin derivative nanoassembly with anti-tumor activity according to claim 5, characterized in that: In step S2 and step S3, the organic solvent includes at least one of methanol, ethanol, and tetrahydrofuran.

8. Use of the artemisinin derivative nanoassembly with anti-tumor activity as claimed in any one of claims 1 to 4 or the artemisinin derivative nanoassembly with anti-tumor activity prepared by the preparation method as claimed in any one of claims 5 to 7 in the preparation of a drug delivery system.

9. Use of the artemisinin derivative nanoassembly with antitumor activity as claimed in any one of claims 1 to 4 or the artemisinin derivative nanoassembly with antitumor activity prepared by the preparation method as claimed in any one of claims 5 to 7 in the preparation of antitumor drugs.

10. Use of the artemisinin derivative nanoassembly with anti-tumor activity as described in any one of claims 1 to 4 or the artemisinin derivative nanoassembly with anti-tumor activity prepared by the preparation method as described in any one of claims 5 to 7 in the preparation of an injection, oral administration or local administration system.

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