A cholesterol succinic monoester derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, and use thereof

Abstract

The application provides a cholesterol succinic acid monoester derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition and application thereof, and relates to the technical field of medicines.The derivative or the pharmaceutically acceptable salt thereof is formed by covalently connecting cholesterol succinic acid monoester and a drug monomer (for example, phytolaccatoxin, temozolomide, (S)-10-hydroxy camptothecin, paeonol, dihydroartemisinin, etc.) with significant antitumor activity or anti-inflammatory activity through an ester bond or an amide bond.The application further provides a preparation method of the derivative and a lipid nanoparticle prepared by taking the derivative as a substrate.The derivative and the nanoparticle thereof exhibit excellent pH-sensitive release characteristics, significantly enhanced tumor cell uptake capacity, significantly reduced toxicity to normal cells and excellent in-vitro and in-vivo antitumor or anti-inflammatory activity, realize the target treatment purpose of synergistic reduction of toxicity and provide a new drug delivery technical strategy for developing high-efficiency and low-toxicity targeted antitumor drugs or anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical chemistry and drug delivery technology, and specifically relates to a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and its application. Background Technology

[0002] Inflammatory responses often accompany the development of various diseases, including neurological disorders, infections, metabolic syndrome, tumors, cardiovascular diseases, and allergic reactions. For example, studies have shown a necessary link between tumor development and chronic inflammation; various inflammatory factors produced during the inflammatory response significantly promote the proliferation, invasion, and metastasis of tumor cells. Therefore, controlling inflammation plays a crucial role in disease progression. The effective components and active ingredients of traditional Chinese medicine (TCM) possess unique advantages in anti-inflammatory and swelling-reducing effects, treating inflammation caused by injury. They can inhibit or directly block signals generated during inflammatory responses at the lesion site through multiple pathways and targets, thereby achieving anti-inflammatory, swelling-reducing, site-protecting, and repair-promoting effects. However, the low bioavailability of anti-inflammatory TCMs is due to factors such as poor water solubility and instability in most TCM monomers. For instance, while galangalol and paeonol have certain anti-inflammatory effects, their structural characteristics limit their clinical application. Studies have shown that galangin has poor solubility and thermal stability, and low bioavailability; paeonol is a small-molecule phenol with a relatively simple structure, is easily sublimated, has poor water solubility, and a rapid metabolic rate in vivo. Geraniol has a small molecular weight, is highly volatile, volatilizes at room temperature, and is unstable under high-temperature heating, easily undergoing structural changes.

[0003] In addition, malignant tumors are a major disease that seriously threatens human health, and chemotherapy remains one of the main treatment methods. Although natural products such as podophyllotoxin and (S)-10-hydroxycamptothecin, as well as chemical drugs such as temozolomide, have significant anti-tumor activity, there are still some problems: For example, podophyllotoxin has defects such as poor water solubility, high toxicity, and poor targeting, and its existing derivatives (such as etoposide) still have problems with bone marrow suppression, gastrointestinal toxicity, and drug resistance; while (S)-10-hydroxycamptothecin has problems such as poor water solubility, unstable lactone ring, and high toxicity to normal tissues, and existing camptothecin derivatives (such as irinotecan and topotecan) still have clinical defects such as bone marrow suppression, diarrhea, and drug resistance; temozolomide has poor solubility in most solutions and is easily decomposed under alkaline conditions (pH>7). Due to its short half-life, increased dosage can easily lead to a series of side effects, such as nausea, vomiting, thrombocytopenia, neutropenia, and lymphopenia, and may also cause certain damage to organs such as bone marrow, liver, and kidneys. It is evident that the clinical application of drugs such as podophyllotoxin, (S)-10-hydroxycamptothecin, and temozolomide is often limited by problems such as poor water solubility, low targeting, high systemic toxicity, and easy development of drug resistance.

[0004] In the existing technology, the structural modification or dosage form improvement of the above-mentioned drugs are mostly focused on synthesizing simple derivatives or using traditional nanocarriers for physical encapsulation. These strategies often have defects such as limited targeting efficiency, easy drug leakage, and failure to fully utilize the characteristics of the tumor microenvironment and inflammatory environment.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a series of novel cholesterol succinate monoester derivatives or their pharmaceutically acceptable salts, pharmaceutical compositions and their applications (highly efficient nano or micro drug delivery systems) with higher anti-inflammatory or anti-tumor activity, strong targeting and good stability, in order to overcome the problems of poor targeting, poor water solubility and instability of existing anti-inflammatory drugs, which lead to low bioavailability, as well as the defects of anti-tumor drugs such as poor targeting, large toxic side effects, easy metabolism and low bioavailability.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The primary objective of this invention is to provide a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof, wherein the cholesterol succinate monoester derivative is formed by covalently linking cholesterol succinate monoester with a small molecule drug via an ester bond or an amide bond; and the small molecule drug is a compound containing a hydroxyl group or an amino group that has antitumor or anti-inflammatory effects.

[0008] Furthermore, based on the above-mentioned technical solution of the present invention, the small molecule drug includes any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, alizarin, or dihydroartemisinin.

[0009] Furthermore, based on the above-described technical solution of the present invention, the cholesterol succinate monoester derivative or its pharmaceutically acceptable salt has the general structural formula shown in formula (I) or formula (II): Wherein, R1 is a small molecule drug group, selected from any one of (A1)-(A12): ; R2 is: ; In the above structural formula for R1, the asterisk " " represents a single bond connected to a group on one side of the structural formula in formula (I); In the above structural formula for R2, the asterisk " " " indicates a single bond connected to the groups on both sides of the structural formula in formula (II).

[0010] The second objective of this invention is to provide a method for preparing the above-mentioned cholesterol succinate monoester derivative or its pharmaceutically acceptable salt, specifically by esterification or amidation of the carboxyl group in cholesterol succinate monoester with the hydroxyl or amino group in a small molecule drug. Furthermore, based on the above-mentioned technical solution of the present invention, the preparation method includes the following steps: mixing cholesterol succinate monoester, small molecule drug and organic solvent, and then adding catalyst and condensing agent to carry out esterification reaction or amidation reaction to obtain cholesterol succinate monoester derivative or its pharmaceutically acceptable salt.

[0011] A third objective of this invention is to provide a pharmaceutical composition comprising a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof provided in the first objective of this invention, or a derivative or a pharmaceutically acceptable salt thereof prepared by the preparation method provided in the second objective of this invention, and pharmaceutically acceptable excipients.

[0012] The fourth objective of this invention is to provide the use of the cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof provided in the first objective of this invention, or the pharmaceutical composition provided in the third objective, in the preparation of antitumor drugs or anti-inflammatory drugs.

[0013] Compared with the prior art, the technical solution of the present invention has at least the following technical effects: (1) This invention provides a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof, wherein the cholesterol succinate monoester derivative is formed by covalently linking cholesterol succinate monoester with a small molecule drug containing hydroxyl or amino groups that has antitumor or anti-inflammatory effects via ester or amide bonds; by introducing a cholesterol succinate structure into the small molecule drug, this type of derivative can increase the targeting of the small molecule drug, reduce toxicity to normal cells, and enhance drug efficacy. Furthermore, this invention is the first to introduce cholesterol succinate monoester as a functional carrier into the design of antitumor derivatives such as podophyllotoxin, temozolomide, and (S)-10-hydroxycamptothecin, or anti-inflammatory derivatives such as paeonol, sorbitol, and dihydroartemisinin. The introduction of a functional carrier can significantly improve the solubility, stability, or pH-responsive release characteristics of the drug (or its hydrolytic capacity under lipase conditions), and effectively enhance antitumor or anti-inflammatory activity and broaden the therapeutic window. By using it as a functional modification unit and covalently binding it with active drug molecules (small molecule drugs), it is expected to comprehensively improve the physicochemical properties, pharmacokinetic behavior, and therapeutic effects of the drug.

[0014] (2) The present invention provides a pharmaceutical composition comprising the above-mentioned cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Further, the cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof can be used as a cholesterol substitute in carrier materials or drug delivery systems; carrier materials constructed from cholesterol succinate monoester derivatives or pharmaceutically acceptable salts thereof and phospholipids can achieve high encapsulation efficiency and good biocompatibility. Drug delivery systems constructed using derivatives as one of the matrix materials can significantly enhance the active enrichment and retention capacity of the target site and the tumor or inflammation suppression effect while maintaining good biosafety, thereby achieving a comprehensive improvement in drug efficacy. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is the mass spectrum of podophyllotoxin-cholesterol succinate prepared in Example 1 of the present invention; Figure 2 The image shows the nuclear magnetic resonance (NMR) spectrum of podophyllotoxin-cholesterol succinate prepared in Example 1 of this invention, wherein (a) is... 1 H-NMR spectrum, (b) is13 C-NMR spectrum; Figure 3 This is the mass spectrum of temozolomide-cholesterol succinate prepared in Example 2 of the present invention; Figure 4 The NMR spectrum of temozolomide-cholesterol succinate prepared in Example 2 of this invention is shown, wherein (a) is 1 H-NMR spectrum, (b) is 13 C-NMR spectrum; Figure 5 The mass spectrum of (S)-10-hydroxycamptothecin-cholesterol succinate prepared in Example 3 of this invention; Figure 6 The image shows the nuclear magnetic resonance spectrum of (S)-10-hydroxycamptothecin-cholesterol succinate prepared in Example 3 of this invention, wherein (a) is... 1 H-NMR spectrum, (b) is 13 C-NMR spectrum; Figure 7 This is the mass spectrum of geraniol-cholesterol succinate prepared in Example 4 of the present invention; Figure 8 The nuclear magnetic resonance spectrum of geraniol-cholesterol succinate prepared in Example 4 of this invention is shown, wherein (a) is 1 H-NMR spectrum, (b) is 13 C-NMR spectrum; Figure 9 The mass spectrum of sorbitan-cholesterol succinate prepared in Example 5 of this invention; Figure 10 The image shows the nuclear magnetic resonance spectrum of sorbitan-cholesterol succinate prepared in Example 5 of this invention, wherein (a) is... 1 H-NMR spectrum, (b) is 13 C-NMR spectrum; Figure 11 The mass spectrum of paeonol-cholesterol succinate prepared in Example 6 of this invention; Figure 12 The image shows the nuclear magnetic resonance spectrum of paeonol-cholesterol succinate prepared in Example 6 of this invention, wherein (a) is... 1 H-NMR spectrum, (b) is 13 C-NMR spectrum; Figure 13 This is the mass spectrum of dihydroartemisinin-cholesterol succinate prepared in Example 7 of the present invention; Figure 14 The nuclear magnetic resonance spectrum of dihydroartemisinin-cholesterol succinate prepared in Example 7 of this invention is shown, wherein (a) is 1 H-NMR spectrum, (b) is 13C-NMR spectrum; Figure 15 Transmission electron microscope (TEM) images of PCH-SLN, TCH-SLN, SHC-SLN, and Ge-CH-LNPs obtained in Examples 7-10 of this invention, wherein (a) is PCH-SLN, (b) is TCH-SLN, (c) is SHC-SLN, and (d) is Ge-CH-LNPs. Figure 16 The in vitro release curves of PCH-SLN prepared in Example 7 of this invention under different pH conditions (pH=5 or 7.4); Figure 17 The in vitro release curves (pH=5.5 or 7.4) of DHA-CS LNPs prepared in Example 12 and DHA LNPs prepared in Comparative Example 4 under different pH conditions are shown, where (a) is within 24 hours and (b) is within 12 hours. Figure 18 The uptake of PCH-SLN prepared in Example 7 and PPT-SLN prepared in Comparative Example 1 in human A549 tumor cells and mouse 4T1 tumor cells (size: 50 μm) are shown. (a) is human A549 tumor cells and (b) is mouse 4T1 tumor cells. Figure 19 The image shows a comparison of the fluorescence intensity of PCH-SLN prepared in Example 7 and PPT-SLN prepared in Comparative Example 1 in vitro by human A549 tumor cells and mouse 4T1 tumor cells, where (a) represents human A549 tumor cells and (b) represents mouse 4T1 tumor cells; PPT-SLN group vs. PCH-SLN group P < 0.01; Figure 20 The uptake of TCH-SLN prepared in Example 8 and TMZ-SLN prepared in Comparative Example 2 in human A549 tumor cells and mouse 4T1 tumor cells (size: 50 μm) are shown. (a) is human A549 tumor cells and (b) is mouse 4T1 tumor cells. Figure 21 The image shows a comparison of the fluorescence intensity of TCH-SLN prepared in Example 8 and TMZ-SLN prepared in Comparative Example 2 in vitro by human A549 tumor cells and mouse 4T1 tumor cells, where (a) represents human A549 tumor cells and (b) represents mouse 4T1 tumor cells; Representing the TMZ-SLN group vs. the TCH-SLN group P < 0.01; Figure 22The uptake of SHC-SLN prepared in Example 9 and SHT-SLN prepared in Comparative Example 3 in human A549 tumor cells and mouse 4T1 tumor cells (size: 50 μm) are shown. (a) is human A549 tumor cells and (b) is mouse 4T1 tumor cells. Figure 23 The image shows a comparison of the fluorescence intensity of SHC-SLN prepared in Example 9 and SHT-SLN prepared in Comparative Example 3 in vitro by human A549 tumor cells and mouse 4T1 tumor cells, where (a) represents human A549 tumor cells and (b) represents mouse 4T1 tumor cells; SHT-SLN group vs. SHC-SLN group P < 0.01, SHT-SLN group vs. SHC-SLN group P <0.05; Figure 24 The images show pseudocolor images of blood perfusion in 4T1 tumor model mice of each group (saline group, PPT-SLN group and PCH-SLN group) of this invention; Figure 25 The text compares the changes in blood perfusion in each group (saline group, PPT-SLN group, and PCH-SLN group) at different time points (a) and 30 min after drug administration (b), with a comparison between the saline group and the PCH-SLN group. P < 0.05, P <0.01; Figure 26 The images show pseudo-color images of blood perfusion in 4T1 tumor model mice of each group (saline group, TMZ-SLN group, TCH-SLN group) of this invention; Figure 27 Comparison of blood perfusion volume in each group (saline group, TMZ-SLN group, TCH-SLN group) at different time points (a) and changes in blood perfusion volume in each group 30 min after drug administration (b). Figure 28 The images show pseudocolor images of blood perfusion in 4T1 tumor model mice of each group (saline group, SHT-SLN group, SHC-SLN group) of this invention. Figure 29 Comparison of blood perfusion volume in each group (saline group, SHT-SLN group, SHC-SLN group) at different time points (a) and changes in blood perfusion volume in each group 30 min after drug administration (b); Figure 30In vivo imaging images of 4T1 tumor model mice in different groups (Blank-DIR-SLN group, PPT-DIR-SLN group, PCH-DIR-SLN group); Figure 31 In vivo imaging images of 4T1 tumor model mice in different groups (Blank-DIR-SLN group, TMZ-DIR-SLN group, TCH-DIR-SLN group); Figure 32 In vivo imaging images of 4T1 tumor model mice in different groups (Blank-DIR-SLN group, SHT-DIR-SLN group, SHC-DIR-SLN group); Figure 33 Tumor Targeting Indices (TTIs) were compared among different groups, where (a) was the PCH-DIR-SLN group, (b) was the TCH-DIR-SLN group, and (c) was the SHC-DIR-SLN group. Figure 34 The changes in body weight and tumor volume of 4T1 tumor model mice in different groups (saline group, PPT-SLN group and PCH-SLN group) at different time points are shown. (a) shows the change in body weight and (b) shows the change in tumor volume. Figure 35 Images of ex vivo tumors, comparisons of ex vivo tumor volume, and comparisons of tumor inhibition rates in 4T1 tumor model mice from different groups (saline group, PPT-SLN group, and PCH-SLN group) are shown. (a) shows images of ex vivo tumors, (b) shows comparisons of ex vivo tumor volume, and (c) shows comparisons of tumor inhibition rates. In the figures, vs. saline group... P < 0.05, P < 0.01, active pharmaceutical ingredient group vs. corresponding derivative formulation group, # P < 0.05; Figure 36 The changes in body weight and tumor volume of 4T1 tumor model mice in different groups (saline group, TMZ-SLN group, TCH-SLN group) at different time points are shown. (a) shows the change in body weight and (b) shows the change in tumor volume. Figure 37 Images of isolated tumors, comparisons of isolated tumor volume, and comparisons of tumor inhibition rates in 4T1 tumor model mice from different groups (saline group, TMZ-SLN group, and TCH-SLN group) are shown. (a) shows isolated tumor images, (b) shows comparisons of isolated tumor volume, and (c) shows comparisons of tumor inhibition rates. In the figures, vs. saline group... P < 0.05, P < 0.01, active pharmaceutical ingredient group vs. corresponding derivative formulation group, # P < 0.05; Figure 38 The changes in body weight and tumor volume of 4T1 tumor model mice in different groups (saline group, SHT-SLN group, SHC-SLN group) at different time points are shown. (a) shows the change in body weight and (b) shows the change in tumor volume. Figure 39 Images of isolated tumors, comparisons of isolated tumor volume, and comparisons of tumor inhibition rates in 4T1 tumor model mice from different groups (saline group, SHT-SLN group, and SHC-SLN group) are shown. (a) shows isolated tumor images, (b) shows comparisons of isolated tumor volume, and (c) shows comparisons of tumor inhibition rates. In the figures, vs. saline group... P < 0.05, P < 0.01, active pharmaceutical ingredient group vs. corresponding derivative formulation group, # P < 0.05; Figure 40 The figures show the MTT assay results for various cholesterol succinate derivatives and their original drug at different concentrations. (a) represents sorbitol and its derivatives, and (b) represents paeonol and its derivatives. In the figures, compared to the original drug at the same molar concentration, [the results are not shown in the original text]. ## P < 0.01, # P < 0.05; Figure 41 The effects of sorbitan and its derivatives on the expression levels of LPS-induced inflammatory cytokines TNF-α, IL-6, and IL-1β in RAW264.7 cells are shown in the figure. (a) represents TNF-α, (b) represents IL-6, and (c) represents IL-1β. Compared with the control group, [the figures are not shown in the original text]. ## P <0.01; compared with the model, P < 0.05, P < 0.01; compared with sorbitol, P < 0.05, P < 0.01; Figure 42The effects of paeonol and its derivatives on the expression levels of LPS-induced inflammatory cytokines TNF-α, IL-6, and IL-1β in RAW264.7 cells are shown in the figure. (a) represents TNF-α, (b) represents IL-6, and (c) represents IL-1β. Compared with the control group, [the figures are not shown in the original text]. ## P <0.01; compared with the model, P < 0.05, P < 0.01; compared with paeonol, P < 0.05, P < 0.01; Figure 43 The effect of different groups on NO concentration in RAW264.7 cells, where (a) is galangin and galangin combination, and (b) is paeonol and paeonol derivatives. The figure shows the effect compared with the blank. ## P < 0.01; compared with the model, P < 0.05, P < 0.01; Compared with the original drug group, P < 0.05, P < 0.01; Figure 44 The inhibitory effects of different groups (blank control group, positive control group, sorbitol group, and sorbitol derivative group) on carrageenan-induced paw swelling in mice were shown. (a) is the swelling degree graph, and (b) is the change rate of paw swelling. Figure 45 The inhibitory effects of different groups (blank control group, positive control group, paeonol group, and paeonol derivative group) on carrageenan-induced paw swelling in mice were shown. (a) is the swelling degree graph, and (b) is the change rate of paw swelling. Figure 46 The apparent effects on lung tissue in mice from different groups; Figure 47 The image shows the changes in the pathological morphology of lung tissue in mice of different groups (HE×200). Among them, A is the blank control group, B is the model group, C is the positive drug group, D1 is the sorbitol group, D2 is the sorbitol derivative group, E1 is the paeonol group, and E2 is the paeonol derivative group. Figure 48The in vitro hydrolysis release curve of paeonol cholesterol succinate; Figure 49 Figure showing the weight changes of mice in different groups at different time points; Figure 50 The images show organ indices for mice in different groups, where (a) represents the heart, (b) the liver, (c) the spleen, and (d) the kidney. Figure 51 Lung tissue sections from different groups of mice; Figure 52 The dry-to-wet ratio of the lungs of mice in different groups; in the figure, compared with the control group: p <0.001; Compared with the model group: # p <0.05, ## p <0.01, ### p <0.001; Figure 53 The figures show the serum inflammatory factor levels in mice from different groups; where (a) represents TNF-α, (b) represents IL-6, and (c) represents IL-1β. The figures are compared with the blank control group. p <0.0001; Compared with the model group: # p <0.05, ## p <0.01, ### p <0.001; Figure 54 The figures show the levels of inflammatory factors in the lung tissue of mice from different groups, where (a) represents TNF-α and (b) represents IL-6. The figures also show the comparison with the control group. p <0.0001; Compared with the model group: # p <0.05, ## p <0.01, ### p <0.001; Figure 55 The distribution of the drug in mice of different groups within 24 hours is shown in the in vivo map. Figure 56 In vitro fluorescence images of organs and tissues from different groups of mice; Figure 57 The images show fluorescence intensity of isolated mouse tissues from different groups, where (a) represents the liver and (b) represents the leg. in, Figures 49-54 In this study, control group was the blank group, model group was the model group, positive group was the positive control group, DL group was the low-dose dihydroartemisinin group (DHA-L), DH group was the high-dose dihydroartemisinin group (DHA-H), DSL group was the low-dose dihydroartemisinin cholesterol succinate group (DHA-CS-L), and DSH group was the high-dose dihydroartemisinin cholesterol succinate group (DHA-CS-H). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Process parameters not specifically specified in the following embodiments are generally performed under conventional conditions.

[0018] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0019] According to a first aspect of the invention, a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof is provided, wherein the cholesterol succinate monoester derivative or the pharmaceutically acceptable salt thereof is formed by covalently linking cholesterol succinate monoester (also referred to as cholesterol succinate monoester) with a small molecule drug via an ester bond or an amide bond; the small molecule drug is a compound containing a hydroxyl group or an amino group and having antitumor or anti-inflammatory effects.

[0020] These derivatives can increase the targeting of small molecule drugs, reduce toxicity to normal cells, and enhance drug efficacy by introducing cholesterol succinate structures into them.

[0021] As an optional embodiment of the technical solution of the present invention, the small molecule drug includes any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, alizarin, or dihydroartemisinin, preferably any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, or dihydroartemisinin.

[0022] Specifically, the cholesterol succinate monoester derivative or its pharmaceutically acceptable salt (hereinafter referred to as the derivative) of the present invention is a small molecule drug with significant antitumor activity (podophyllotoxin, temozolomide, or (S)-10-hydroxycamptothecin, etc.) or anti-inflammatory effect (geraniol, paeonol, sedge, eugenol, magnolol, alizarin, or dihydroartemisinin, etc.) of the B part, coupled by ester or amide bonds through the succinic anhydride of the C part of the linking arm. Alternatively, this invention can be understood as follows: Using small molecule drugs in part B (podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, or alizarin, etc.) as the pharmacodynamic core, a cholesterol succinate monoester group is covalently linked to the active site (hydroxyl or amino group) via an ester or amide bond that can be hydrolyzed by the tumor microenvironment or anti-inflammatory site, forming a prodrug (i.e., a cholesterol succinate monoester derivative or its pharmaceutically acceptable salt). The hydroxyl group that can serve as the active site can be an alcoholic hydroxyl group and / or a phenolic hydroxyl group, and the amino group can be an amino group from a primary or secondary amide.

[0023] Cholesterol succinate monoester, as a biocompatible lipid derivative, possesses certain cytoprotective effects and tumor proliferation-inhibiting activity, and is responsive to the weakly acidic (pH-sensitive) tumor microenvironment. Therefore, this invention, for the first time, introduces cholesterol succinate monoester as a functional carrier into the design of antitumor derivatives such as podophyllotoxin, temozolomide, and (S)-10-hydroxycamptothecin, or anti-inflammatory derivatives such as geraniol, paeonol, sorbitol, and dihydroartemisinin. Introducing this functional carrier can significantly improve the drug's solubility, stability, or pH-responsive release characteristics (or hydrolytic capacity under lipase conditions), and effectively enhance its antitumor or anti-inflammatory activity and broaden its therapeutic window. Covalently binding it as a functional modification unit with active drug molecules (small molecule drugs) is expected to comprehensively improve the drug's physicochemical properties, pharmacokinetic behavior, and therapeutic effects.

[0024] As an optional embodiment of the technical solution of the present invention, the general structural formula of the cholesterol succinate monoester derivative or its pharmaceutically acceptable salt is shown in formula (I) or formula (II): Wherein, R1 is a small molecule drug group, which can be an anti-tumor small molecule drug group or an anti-inflammatory small molecule drug group, specifically selected from any one of (A1)-(A12), (A1) corresponds to podophyllotoxin, (A2) corresponds to temozolomide, (A3) and (A4) correspond to (S)-10-hydroxycamptothecin, (A5) corresponds to geraniol, (A6) corresponds to galangin, (A7) corresponds to paeonol, (A8) corresponds to eugenol, (A9) and (A10) correspond to magnolol, (A11) corresponds to alizarin, and (A12) corresponds to dihydroartemisinin; ; R2 corresponds to (S)-10-hydroxycamptothecin, which is: ; In the above structural formula for R1, the asterisk " " represents a single bond connected to a group on one side of the structural formula in formula (I); In the above structural formula for R2, the asterisk " " " indicates a single bond connected to the groups on both sides of the structural formula in formula (II).

[0025] As an optional embodiment of the above technical solution of the present invention, the (A1) structure in R1 is as follows: The structure of (A12) is: ; And / or, R2 in equation (II) is: .

[0026] As an optional embodiment of the technical solution of the present invention, "pharmaceutically acceptable salt" in cholesterol succinate monoester derivative or its pharmaceutically acceptable salt refers to: (a) A base addition salt formed by the reaction of the free carboxyl group in the cholesterol succinate monoester derivative with a pharmaceutically acceptable inorganic or organic base; and / or (b) When the small molecule drug residue contains a salt-forming group, the salt formed by the salt-forming group and a pharmaceutically acceptable acid or base (inorganic or organic); Among them, inorganic bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, etc.; organic bases include, but are not limited to, aminobutanetriol, N-methylglucosamine, diethanolamine, triethanolamine, lysine, arginine, ornithine, etc. Acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

[0027] According to a second aspect of the present invention, a method for preparing a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof is provided, comprising the steps of: esterification or amidation of a carboxyl group in a cholesterol succinate monoester with a hydroxyl or amino group in a small molecule drug.

[0028] As an optional embodiment of the above-mentioned technical solution of the present invention, the small molecule drug includes any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, alizarin, or dihydroartemisinin.

[0029] By structurally modifying small molecule drugs such as podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, paeonol, sorbitol, or dihydroartemisinin, and then linking cholesterol succinate monoesters via esterification or amidation reactions, derivative drugs based on the original drugs are synthesized, namely podophyllotoxin-cholesterol succinate (referred to as podophyllotoxin derivative), temozolomide-cholesterol succinate (referred to as temozolomide derivative), (S)-10-hydroxycamptothecin-cholesterol succinate (referred to as (S)-10-hydroxycamptothecin derivative), paeonol-cholesterol succinate (referred to as paeonol derivative), sorbitol-cholesterol succinate (referred to as sorbitol derivative), and dihydroartemisinin-cholesterol succinate (referred to as dihydroartemisinin derivative).

[0030] As an optional embodiment of the above technical solution of the present invention, the preparation method of cholesterol succinate monoester derivative or its pharmaceutically acceptable salt includes the following steps: Cholesterol succinate monoester, small molecule drug and organic solvent are mixed, and then a catalyst and condensing agent are added to carry out esterification or amidation reaction to obtain cholesterol succinate monoester derivative or its pharmaceutically acceptable salt. Preferably, the condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). Preferably, the catalyst is a basic compound, specifically including at least one of 4-dimethylaminopyridine (DMAP) or 1-hydroxybenzotriazole (HOBt).

[0031] As an optional embodiment of the above-described technical solution of the present invention, the molar ratio of cholesterol succinate monoester to small molecule drug is (1-2):(1-1.5), with typical but non-limiting molar ratios being 1:1, 1:1.2, 1:1.4, 1:1.5, 1.2:1, 1.4:1, 1.5:1, 1.6:1, 1.8:1, 2.0:1, 2.0:1.2, or 2.0:1.5, etc. Generally speaking, the small molecule drug is added in excess of cholesterol succinate monoester, that is, the cholesterol succinate monoester is completely reacted to reduce purification interference.

[0032] As an optional embodiment of the above technical solution of the present invention, the molar ratio of cholesterol succinate monoester to catalyst is (0.8-1):(0.2-1.5), and typical but non-limiting molar ratios are 0.8:0.2, 0.8:0.4, 0.8:0.8, 0.8:1, 0.8:1.4, 0.8:1.5, 0.9:0.2, 0.9:0.5, 0.9:1, 0.9:1.5, 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.2 or 1:1.5, etc.

[0033] And / or, the molar ratio of cholesterol succinate monoester to condensing agent is (0.8-1):(1-1.2), with typical but non-limiting molar ratios being 0.8:1, 0.9:1, 1:1, 0.8:1.2, 0.8:1.1, 0.8:1.2, 0.9:1, or 1:1.2, etc.

[0034] By using EDCI as the condensing agent for the esterification reaction and DMAP or HOBt as the catalyst, and controlling the reaction in an appropriate ratio, small molecule drugs can be successfully linked to cholesterol succinate monoester.

[0035] According to a third aspect of the present invention, a pharmaceutical composition is also provided, comprising a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof provided in the first aspect of the present invention or a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof prepared by the preparation method provided in the second aspect of the present invention, and pharmaceutically acceptable excipients.

[0036] Different dosage forms of pharmaceutical compositions can be formed by adding different excipients. From a macroscopic product morphology perspective, the dosage form of the pharmaceutical composition can be selected from any of the following dosage forms:

[0037] (a) Oral solid dosage forms, including but not limited to capsules, tablets, granules, pills, and powders; (b) Oral liquid preparations, including but not limited to oral solutions, oral suspensions, and oral emulsions; (c) Injectable preparations, including but not limited to lyophilized powder for injection, injection solution, and injection emulsion.

[0038] From the perspective of microscopic delivery carriers, the dosage form of the drug composition can be: (1) a lipid- or lipid-like nano / submicron / micron delivery system, selected from any one of liposomes, solid lipid nanoparticles, nanoemulsions, polymer lipid hybrid nanoparticles, and polymer micelles; or, (2) Microparticle-grade sustained-release formulations, selected from any one of microspheres or microcapsules.

[0039] As a preferred embodiment of the above technical solution of the present invention, cholesterol succinate monoester derivatives or their pharmaceutically acceptable salts can be used as cholesterol substitutes in carrier materials or drug delivery systems. Preferably, the carrier material includes phospholipids and cholesterol succinate monoester derivatives or pharmaceutically acceptable salts thereof; More preferably, the dosage form of the carrier material is selected from any one of the following dosage forms: (a) Nanoscale formulations: solid lipid nanoparticles, liposomes, nanoemulsions, polymer lipid hybrid nanoparticles, and polymer micelles; (b) Submicron-sized and micron-sized formulations obtained by adjusting the particle size of the nanoscale formulation described in (a) above; (c) Microparticle formulations: microspheres, microcapsules.

[0040] As another preferred embodiment of the above-mentioned technical solution of the present invention, the dosage form of the carrier material is solid lipid nanoparticles. The carrier material of the solid lipid nanoparticles includes phospholipids, cholesterol succinate monoester derivatives or their pharmaceutically acceptable salts, and other lipids other than phospholipids and cholesterol succinate monoester derivatives (hereinafter referred to as other lipids).

[0041] This cholesterol succinate monoester derivative (a prodrug) was used as a functional lipid matrix to construct solid lipid nanoparticles with phospholipids and other lipid excipients. Its targeting and therapeutic mechanism is as follows: synergistic effect of structural modification and nanodelivery increases drug concentration at the tumor site and reduces exposure to normal tissues; environmentally responsive release occurs when the ester or amide bonds degrade in the low pH of the tumor microenvironment or in an inflammatory microenvironment, releasing the original drug.

[0042] As an optional embodiment of the technical solution of the present invention, the phospholipids include at least one of the following: natural phospholipids (such as soybean phospholipids, egg yolk lecithin, rapeseed phospholipids, sunflower seed phospholipids, etc.), modified phospholipids (such as hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, PEGylated phospholipids, etc.), preferably including natural phospholipids, more preferably including soybean phospholipids.

[0043] It should be noted that the other lipids in this invention are not limited to liquid lipids, but can also be semi-solid lipids or solid lipids, as long as they can form a stable dispersion system together with cholesterol succinate monoester derivatives.

[0044] As an optional embodiment of the technical solution of the present invention, other lipids include at least one of the following: triglycerides (e.g., medium-chain triglycerides, tricaprylic acid triglycerides, tristearate triglycerides, tripalmitoate triglycerides, trilaurate triglycerides, tricarboxylic acid triglycerides, etc.), fatty acids (e.g., oleic acid, stearic acid, lauric acid, myristic acid, etc.), fatty acid esters (caprylic acid triglycerides, isopropyl palmitate, isopropyl myristate, caprylic acid caprylic acid, etc.), vegetable oils (soybean oil, high oleic sunflower oil, olive oil, castor oil, etc.), squalene, vitamin E polyethylene glycol succinate, and octyl dodecyl alcohol.

[0045] Furthermore, other lipids may include cationic lipids, such as trimethyl-2,3-dioleoyloxypropylammonium chloride, trimethyl-2,3-dioleenoyloxypropylammonium chloride, didecyldimethylammonium bromide, dioctadecyldimethylammonium bromide, etc.

[0046] As a preferred embodiment of the above technical solution of the present invention, other lipids include medium-chain triglycerides, wherein the carbon chain of the medium-chain triglycerides is composed of 8-10 carbon atoms.

[0047] As an optional embodiment of the above-mentioned technical solution of the present invention, the mass ratio of phospholipids (e.g., soybean phospholipids), derivatives and liquid lipids (e.g., medium-chain triglycerides) is (2.8-3.2):(0.8-1.2):(1-2). Typical but non-limiting mass ratios are 2.8:0.8:1, 3:0.8:1, 3.2:0.8:1, 2.8:1:1, 3.0:1:1, 3.0:1:1.5, 3.0:1:1.6, 3.0:1:1.8, 3.0:1:2, 3.2:1:1, 3.2:1:1.2, 3.2:1:1.5, 3.2:1:1.8, 3.2:1:2, 3.2:1.2:1, 3.2:1.2:1.5 or 3.2:1.2:2, etc.

[0048] As an optional embodiment of the above technical solution of the present invention, the preparation method of solid lipid nanoparticles includes the following steps: Phospholipids (such as soybean phospholipids), cholesterol succinate monoester derivatives, organic solvents (such as anhydrous ethanol), and liquid lipids (such as medium-chain triglycerides) are mixed and the organic solvents are removed by rotary evaporation under reduced pressure at 55-60°C until a lipid film is formed. Then, an appropriate amount of deionized water is added to the lipid film, and the mixture is stirred, ultrasonically dispersed, and then ultrasonically subjected to probe-type sonication in an ice bath to obtain cholesterol succinate monoester derivative solid lipid nanoparticles.

[0049] As an optional embodiment of the above-described technical solution of the present invention, the mass ratio of phospholipids (e.g., soybean phospholipids), derivatives, liquid lipids (e.g., medium-chain triglycerides), and organic solvents (anhydrous ethanol) is (2.8-3.2):(0.8-1.2):(1-2):(2-3). Typical but non-limiting mass ratios are 2.8:0.8:1:2, 3:0.8:1:2, 3.2:0.8:1:2, 2.8:1:1:2, and 3.0:1:1:2. 3.0:1:1.5:2.6, 3.0:1:1.6:2.7, 3.0:1:1.6:3, 3.0:1:1.8:2, 3.0:1:2:2.5, 3.2:1:1:2, 3.2:1:1.2:2, 3.2:1:1.5:3, 3.2:1:1.8:2.6, 3.2:1:2:2.6, 3.2:1.2:1:3, 3.2:1.2:1.5:3 or 3.2:1.2:2:3, etc.

[0050] According to a fourth aspect of the invention, the use of the cholesterol succinate monoester derivative provided in the first aspect of the invention or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided in the third aspect of the invention, in the preparation of an antitumor drug or an anti-inflammatory drug is also provided.

[0051] As an optional embodiment of the above technical solution of the present invention, the tumor includes breast cancer, lung cancer, or liver cancer, etc.

[0052] As an optional embodiment of the above technical solution of the present invention, inflammation includes arthritis, skin inflammation, rheumatoid arthritis, gouty arthritis, kidney inflammation, gastritis, enteritis, acute lung injury or sepsis-related myocardial injury, etc.

[0053] To further illustrate the technical solution of the present invention, the following embodiments and comparative examples are provided. The raw materials used in each embodiment or experimental example are described below: Cholesterol monosuccinate (also known as cholesterol monosuccinate, cholesterol hemisuccinate, Aladdin, CAS No.: 1510-21-0), podophyllotoxin (98%, batch number: A18GS145669), geraniol (Yuanye Biotechnology, batch number M24S11X125835), galangin (98%, Shanghai Yuanye Biotechnology Co., Ltd.); paeonol (99%, Shanghai Mairui Chemical Technology Co., Ltd.) Dihydroartemisinin (DHA, Shanghai Dipo Biotechnology Co., Ltd., batch number: K913006); cholesterol (batch number L29N8F49258), dimethyl sulfoxide (DMSO) (batch number: Z28N11Y132686) and 100-200 mesh silica gel (batch number Z20A11Y121969) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 4-dimethylaminopyridine (batch number: C10220975) and DIR'[DiIC18(7)] (batch number: D864067) were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride (batch number: HW18L2719-2, Beijing Huawirui Chemical Co., Ltd.); soybean lecithin (batch number SY-SI-180501, Shanghai Avit Pharmaceutical Technology Co., Ltd.); medium-chain triglycerides (batch number: 187207, Gaffars, France, carbon chain containing 8-10 carbon atoms); succinic anhydride (batch number: 20150303) and dichloromethane (batch number 2020092011), all purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; temozolomide (98%, Shanghai Dibai Biotechnology Co., Ltd., batch number: HS25); (S)-10-hydroxycamptothecin (98%) The following reagents were used in this invention: %, Shanghai Dipo Biotechnology Co., Ltd., batch number: N26GS169094; CCK-8 cell proliferation assay kit (batch number: AC10873-500T, manufacturer: Acmec); coumarin-6 (batch number: C7810), DAPI nuclear dye solution (batch number: C0060), and 4% tissue cell fixative, all purchased from Beijing Solarbio Co., Ltd.; 96-well plates and Matrigel, both purchased from Corning Incorporated, USA; MTT (B7777743377BD, Beijing Bairddi Biotechnology Co., Ltd.); 0.25% trypsin digestion solution (HJ202802, Wuhan Sewell Biotechnology Co., Ltd.); mouse TNF-α, IL-6, IL-1β ELISA kits (all purchased from Beijing Bairddi Biotechnology Co., Ltd.); NO reagent kit (HY0821, Beijing Bairuiji Biotechnology Co., Ltd.), etc. Unless otherwise specified, all other raw materials used in this invention are commercially available products.

[0054] Instruments: Agilent Technologies 6520 Accurate-Mass Q-TOF LC / MS (Agilent Technologies, USA); AM-500 MRI scanner (Bruker, Switzerland); Ultrasonic cell disruptor (Ningbo Xinzhi Technology Co., Ltd.); Hemocytometer (Aoboxing); Carbon dioxide cell incubator (Thermo Fisher, USA); Multifunctional microplate reader (Thermo Fisher Scientific, Finland); Clean bench (Shanghai Boxun Medical Bio-Instrument Co., Ltd.); Sorall ST 8R high-speed refrigerated centrifuge (Thermo Fisher, USA); Inverted microscope (Nikon, Japan); Fluorescence inverted microscope (Nikon, Japan); Laser speckle blood flow imaging system (PeriCam, PSI-NR; Paree Medical Technology (Beijing) Co., Ltd.); Small animal in vivo optical imaging system (IVIS Lumina LT, PerkinElmer Instruments (Shanghai) Co., Ltd.), etc.

[0055] Example 1 This embodiment provides a cholesterol succinate monoester derivative (podophyllotoxin-cholesterol succinate, abbreviated PCH), the structure of which is as follows: The preparation method of podophyllotoxin-cholesterol succinate in this embodiment includes the following steps: Podophyllotoxin and cholesterol succinate monoester (intermediate) were weighed and dissolved in an appropriate amount of dichloromethane (the amount of dichloromethane was sufficient for dissolution, and it would evaporate naturally afterward). The catalyst 4-dimethylaminopyridine (DMAP) and the condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added. The molar ratio of cholesterol succinate monoester, podophyllotoxin, EDCI, and DMAP was 1:1.2:1:1.5. The mixture was stirred at 600 rpm for 7 h at room temperature on a magnetic stirrer, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by silica gel column chromatography to obtain a white amorphous powder, podophyllotoxin-cholesterol succinate. The specific synthetic route is as follows: Mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR) were used to detect the presence of ESI-MS and ESI-mass spectrometry. 1 H-NMR, 13 The structure of the derivative drug was confirmed by C-NMR, as follows: The purified podophyllotoxin-cholesterol succinate was analyzed by mass spectrometry. The mass spectrometry parameters were: ESI source (+) mode; spray voltage 3 kV; capillary temperature 350 ℃; flow rate 200 μL·min. -1 Sample injection method: The sample is injected directly through a 5 μL quantitative loop using the injection needle. ESI-MS mass spectrometry results are as follows: Figure 1 As shown, the predicted molecular weight (M) of podophyllotoxin-cholesterol succinate is 882.49, and 883.4981 in the figure represents [M+H]. + The mass spectrometry signal at 900.5237 represents [M+NH4]. + The mass spectrometry signal at 905.4800 represents [M+Na]. + The mass spectrometry signal indicates that the compound is a synthetic product of cholesterol succinate and podophyllotoxin.

[0056] The atomic numbering of the podophyllotoxin-cholesterol succinate compound structure is shown in the synthetic route diagram above. To further confirm the reaction product, [further details were provided]. 1 H-NMR and 13 C-NMR detection, 1 H-NMR and 13 The C-NMR spectrum results are shown below. Figure 2 (a) and (b). From Figure 2 (a) The 1H NMR spectrum results show that the peaks at δ 2.75-2.59 are assigned to the two methylene groups on the succinic anhydride linker at C-34 and C-35, the peak at δ 5.34 is assigned to the H group on the double bond at C6, the peak around δ 0.85 is assigned to the two methyl groups at C-18 and C-19, the peak at δ 5.90 is assigned to the methylene group on C-39, the peak at δ 5.96 is assigned to the methylene group on C-44 connected to two O groups, the peak at δ 3.75 is assigned to the H groups on the methoxy groups at C62 and C68, and the peak at δ 3.80 is assigned to the H group on the methoxy group at C65. Figure 2 (b) The carbon spectrum results show that the peaks at δ 172.91, 171.52, and 173.69 are assigned to the carbons on the three ester groups C-32, C-36, and C-52; the peaks at δ 139.40 and 122.83 are assigned to C-5 and C-6 on the carbon-carbon double bond; the peak at δ 73.81 is assigned to C-39; the peak at δ 101.58 is assigned to C-44 which is connected to two O atoms; and the peaks at δ 60.74, 56.13, and 56.11 are assigned to the C atoms on the three methoxy groups C-65, C-62, and C-68, respectively.

[0057] 1 H NMR (700 MHz, Chloroform- d) δ 6.79 (s, 1H, H-41), 6.52 (s, 1H, H-47), 6.38 (s, 2H, H-59, H-69), 5.96 (s, 2H, H-44), 5.90 (d, J = 9.7 Hz, 1H,H-39), 5.34 (t, 1H, H-6), 4.62 (dd, J = 10.9, 5.6 Hz, 1H, H-1), 4.58 (s, 1H,H-49), 4.27 (dt, J = 154.9, 9.0 Hz, 2H, H-55), 3.80 (s, 3H, H-65), 3.75 (s, 6h, H-68, H-62), 2.93 - 2.87 (m, 1H, H-51), 2.88 - 2.79 (m, 1H, H-57), 2.75 -2.59 (m, 4 h, H-34 H-35), 2.34 - 2.23 (m, 2H), 2.02 - 1.90 (m, 2H), 1.87 -1.77 (m, 3H), 1.61 - 1.39 (m, 7H), 1.38 - 1.29 (m, 3H), 1.28 - 1.20 (m, 1H),1.19 - 1.00 (m, 7H), 1.01-0.96 (m, 5H), 0.93 (dd, J = 11.7, 4.6 Hz, 3H), 0.90(d, J = 6.6 Hz, 3H, H-20), 0.85 (s, 3H, H-18), 0.85 (s, 3H, H-19), 0.66 (s,3H, H-22)。

[0058] 13C NMR (176 MHz, CDCl3) δ 173.69 (C-52), 172.91 (C-32), 171.52 (C-36), 152.61 (C-66 C-60), 148.12 (C-46), 147.58 (C-42), 139.40 (C-5), 137.11(C-58), 134.83 (C-63), 132.30 (C-48), 128.26 (C-40), 122.83 (C-6), 109.67 (C-47), 108.07 (C-41), 107.08 (C-59 C-69), 101.58 (C-44), 74.64 (C-1), 73.81 (C-39), 71.37 (C-55), 60.74 (C-65), 56.67 (C-12), 56.13 (C-62), 56.11 (C-68), 49.99, 45.53, 43.76, 42.30, 39.71, 39.51, 38.68, 38.10, 36.90, 36.56, 36.18,35.79, 31.89, 31.83, 29.38, 29.33, 28.23, 28.01, 27.74, 24.28 (C-10), 23.82(C-15), 22.83(C-18), 22.58 (C-19), 21.02 (C-24), 19.29 (C-27), 18.72 (C-20), 11.86 (C-22).

[0059] Based on the mass spectrometry results, the target product was finally identified as podophyllotoxin-cholesterol succinate.

[0060] Example 2 This embodiment provides a cholesterol succinate monoester derivative (temozolomide-cholesterol succinate, abbreviated TCH), the structure of which is as follows: The preparation method of temozolomide-cholesterol succinate in this embodiment includes the following steps: Temozolomide and cholesterol succinate monoester were weighed and dissolved in an appropriate amount of dichloromethane (DCM) (the amount of dichloromethane was sufficient for dissolution, and it would evaporate naturally afterward). 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added under ice bath conditions. The molar ratio of cholesterol succinate monoester, temozolomide, EDCI, and HOBt was 1:1.2:1:1. The reaction was carried out in the dark for 1 h, then brought to room temperature and continued for 48 h. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by silica gel column chromatography to obtain a light red solid, temozolomide-cholesterol succinate. The specific synthetic route is as follows: According to ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: The purified temozolomide-cholesterol succinate was analyzed by mass spectrometry, with the same parameters as in Example 1. The ESI-MS mass spectrometry results are as follows: Figure 3 As shown, the predicted molecular weight (M) of temozolomide-cholesterol succinate is 662.42, and 663.4490 in the figure represents [M+H]. + The mass spectrometry signal contains isotopes such as nitrogen ( 14 N and 15 The isotope ion peaks caused by N indicate that the compound is a synthetic product of temozolomide and cholesterol succinate.

[0061] The atom numbering of the compound structure of temozolomide-cholesterol succinate is shown in the synthetic route diagram above. To further confirm the reaction product, the product was subjected to... 1 H-NMR and 13 C-NMR detection, specific spectral results are as follows: Figure 4 As shown in (a) and (b). From Figure 4 (a) The 1H NMR spectrum results show that the peak at δ 5.36 is assigned to the H on the double bond at C-46, the peak at δ 4.67-4.57 is assigned to the H on C-22 connected to oxygen, the peak around δ 4.39 is assigned to the H on N-14, the peak at δ 3.69 is assigned to the H on C-9 connected to two nitrogen atoms, and the peak at δ 0.67 is assigned to the H on the methyl group at C40. Figure 4(b) The carbon spectrum results show that the peak at δ 172.86 is assigned to C-19 on the ester group, the peak at δ 171.68 is assigned to C-15 and C-12 on the amide group, the peak at δ 139.59 is assigned to C-47 on the carbon-carbon double bond, the peak at δ 128.07 is assigned to C-6 on the carbon-oxygen double bond connected to two nitrogen atoms, the peaks at δ 124.77 and δ 122.72 are assigned to C-4 and C-9 on the carbon-carbon double bond connected to two nitrogen atoms, respectively, the peak at δ 120.31 is assigned to C-46 on the carbon-carbon double bond, and the peak at δ 108.57 is assigned to C-11 on the carbon-carbon double bond connected to nitrogen and carbonyl groups.

[0062] 1 H NMR (400 MHz, Chloroform- d ) δ 5.36 (t, J = 4.1 Hz, 5H), 4.67 -4.57 (m, 5H), 4.39 (s, 1H), 3.69 (s, 14H), 2.65 - 2.56 (m, 19H), 2.31 (dd, J= 9.0, 2.4 Hz, 9H), 2.04 - 1.91 (m, 10H), 1.89 - 1.76 (m, 15H), 1.64 - 0.76 (m, 172H), 0.67 (d, 14H).

[0063] 13 C NMR (101 MHz, Chloroform- d ) δ 172.86 (C-19), 171.68 (C-15, C-12), 139.59 (C-47), 128.07 (C-6), 124.77 (C-4), 122.72 (C-9), 120.31 (C-46), 108.57 (C-11), 74.38, 56.69, 56.14, 51.82, 50.01, 42.32, 39.73, 39.53, 38.05, 36.97,36.59, 36.19, 35.80, 31.91, 31.86, 29.49, 29.02, 28.24, 28.02, 27.73, 24.29, 23.84, 22.83, 22.57, 21.03, 19.32, 18.72, 11.86.

[0064] Combined with the mass spectrometry results, the target product was finally identified as temozolomide-cholesterol succinate.

[0065] Example 3 This embodiment provides a cholesterol succinate monoester derivative ((S)-10-hydroxycamptothecin-cholesterol succinate, abbreviated as SHC), the structure of which is: The preparation method of (S)-10-hydroxycamptothecin-cholesterol succinate in this embodiment includes the following steps: (S)-10-hydroxycamptothecin and cholesterol succinate monoester (intermediate) were weighed and dissolved in dichloromethane (DCM) (a suitable amount of dichloromethane was used for dissolution, which would evaporate naturally afterward). 4-Dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added. The molar ratio of cholesterol succinate monoester, (S)-10-hydroxycamptothecin, EDCI, and DMAP was 1:2.2:1:1.5. The mixture was stirred at 600 rpm for 24 h at room temperature in the dark, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by silica gel column chromatography to obtain a yellow solid (S)-10-hydroxycamptothecin-cholesterol succinate. The specific synthetic route is as follows: According to ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: The purified (S)-10-hydroxycamptothecin-cholesterol succinate was analyzed by mass spectrometry, with the same parameters as in Example 1. The ESI-MS mass spectrometry results are as follows: Figure 5 The predicted molecular weight (M) of (S)-10-hydroxycamptothecin-cholesterol succinate is 1300.63, and 1301.6319 in the figure represents [M+H]. + The mass spectrometry signal contains isotopes such as nitrogen ( 14 N and 15 The isotope ion peaks caused by (N) suggest that the compound is a synthetic product of cholesterol succinate and (S)-10-hydroxycamptothecin.

[0066] The atom numbering of the compound structure of (S)-10-hydroxycamptothecin-cholesterol succinate is shown in the above synthetic route diagram. To further confirm the reaction product, the product was subjected to... 1 H-NMR and 13 C-NMR detection, specifically as follows Figure 6 As shown in (a) and (b). From Figure 6(a) The 1H NMR spectrum results show that the peaks at δ 8.78 and 7.91 are attributed to H atoms at the ortho positions of the two double bonds on the C-9 and C-8 oxygen-bonded benzene rings. The peak at δ 8.71 is attributed to an H atom ortho to the oxygen-bonded carbon on the C-6 oxygen-bonded benzene ring. The peak at δ 8.74 is attributed to an H atom at the para position of the nitrogen atom on the nitrogen-containing six-membered ring at C-4. The peak around δ 6.08 is attributed to an H atom ortho to the oxygen atom on the lactone six-membered ring at C-19. The peak around δ 3.75 is attributed to an H atom ortho to the nitrogen atom on the nitrogen-containing five-membered ring at C-2. From... Figure 6 (b) The carbon spectroscopy results show that the peaks at δ 171.39, 171.15, 167.63, and 158.55 are assigned to the carbons on the four ester groups C-31, C-27, C-66, and C-62; the peaks at δ 157.46 and 155.11 are assigned to C-21 and C-17 on the carbon-oxygen double bond; and the peak at δ 148.81 is assigned to C-12 on the carbon-nitrogen double bond. 146.90, 145.91, 144.08, 139.47, 130.95, 130.19, 128.74, 128.55, 124.31, 122.41, 118.92, 109.01, 106.69, and 95.32 belong to C-7, C-10, C-15, C-13, C-36, C-5, C-4, C-3, C-9, C-8, C-37, C-16, C-6, and C-14, respectively.

[0067] 1 H NMR (400 MHz, Chloroform- d) δ 8.91 - 8.86 (m, 7H), 8.78 (d, J =2.0 Hz, 1H), 8.74 (s, 1H), 8.71 (s, 1H), 7.96 (s, 1H), 7.91 (d, J = 2.6 Hz,1H), 7.87 (s, 1H), 6.36 (s, 1H), 6.32 (s, 1H), 6.08 (s, 1H), 6.04 (s, 1H),5.86 - 5.83 (m, 2H), 5.78 (d, J = 5.0 Hz, 1H), 5.24 (dt, J = 11.2, 5.5 Hz,1H), 3.91 (d, J = 8.3 Hz, 1H), 3.75 (s, 23H), 3.51 (q, J = 6.9, 6.4 Hz, 3H),3.29 (td, J = 7.0, 3.7 Hz, 3H), 3.00 - 2.79 (m, 7H), 2.66 (dd, J = 12.6, 3.4Hz, 2H), 2.55 - 2.42 (m, 5H), 2.36 (dt, J = 13.1, 3.3 Hz, 2H), 2.29 - 2.14(m, 9H), 2.10 (s, 1H), 2.07 - 1.89 (m, 31H), 1.87 - 1.70 (m, 17H), 1.65 (t, J= 7.5 Hz, 13H), 1.61 - 1.39 (m, 41H), 1.32 (s, 5H)。

[0068] 13 C NMR (101 MHz, Chloroform- d) δ 171.39 (C-31), 171.15 (C-27), 167.63(C-66), 158.55 (C-62), 157.46 (C-21), 155.11 (C-17), 148.81 (C-12), 146.90(C-7), 145.91 (C-10), 144.08 (C-15), 139.47 (C-13), 130.95 (C-36), 130.19 (C-5), 128.74 (C-4), 128.55 (C-3), 124.31 (C-9), 122.41 (C-8), 118.92 (C-37), 109.01 (C-16), 106.69 (C-6), 95.32(C-14), 76.26, 74.45, 67.11, 56.59, 56.10,49.93, 49.80, 42.27, 39.66, 39.53, 39.30, 37.87, 36.77, 36.42, 36.20, 35.79,31.81, 31.78, 31.74, 30.04, 29.70, 29.22, 29.00, 28.24, 28.01, 27.60, 24.26,23.87, 22.83, 22.57, 20.93, 19.15, 18.72, 11.81, 7.60.

[0069] Combined with the mass spectrometry results, the target product was identified as (S)-10-hydroxycamptothecin-cholesterol succinate.

[0070] Example 4 This embodiment provides a cholesterol succinate monoester derivative (geraniol-cholesterol succinate), the structure of which is as follows: The preparation method of geraniol-cholesterol succinate in this embodiment includes the following steps: Take a vial, add 0.12 mmol geraniol and 0.1 mmol cholesterol succinate monoester (molar ratio 1:1.2), then add 8 mL dichloromethane. Dissolve and mix by pipetting, then add 0.075 mmol 4-dimethylaminopyridine (DMAP) and 0.15 mmol 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI). Add a stir bar and place on a magnetic stirrer at 800 r·min. -1 After reacting for 10 h, geraniol-cholesterol succinate was obtained, which was a thick, semi-fluid liquid.

[0071] The specific synthesis route is as follows: Using ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: The purified geraniol-cholesterol succinate was analyzed by mass spectrometry. The mass spectrometry parameters were: ESI source (+) mode; spray voltage 3 kV; capillary temperature 350 ℃; mobile phase: methanol:water (90:10); flow rate 200 μL•min. -1 Sample injection method: The sample is directly injected through a 5 μL quantitative loop using the injection needle. From the ESI-MS mass spectrum... Figure 7 As can be seen, the quasi-molecular ion peak m / z 645.4835 [M+Na] + This indicates that the relative molecular mass of the reaction product is 622.4835, which is similar to that of the target product. 41 H 66 The relative molecular mass of O4, 622.4859, is basically consistent, indicating that cholesterol succinate monoester and geraniol can undergo esterification under certain conditions.

[0072] To further confirm the reaction products, the products were subjected to... 1 H-NMR measurements and 13 C-NMR measurements, the specific spectral results are as follows: Figure 8 As shown in (a) and (b).

[0073] Through ESI-MS, 1 H-NMR and 13 The structure of the product was characterized and verified by C-NMR, and... 1 H-NMR spectrum and 13 The structure obtained from the C-NMR spectrum was assigned, and the results showed that geraniol-cholesterol succinate could be successfully synthesized under coupling conditions with 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) as the condensing agent and 4-dimethylaminopyridine (DMAP) as the catalyst.

[0074] Example 5 This embodiment provides a cholesterol succinate monoester derivative (izanol-cholesterol succinate), the structure of which is as follows: The preparation method of sorbitan-cholesterol succinate in this embodiment includes the following steps: An appropriate amount of sorbitan was ultrasonically dissolved in dichloromethane (DCM) (the amount of dichloromethane was sufficient for dissolution, and it would evaporate naturally afterward). Appropriate amounts of cholesterol succinate monoester, EDCI, and DMAP were added. The molar ratio of cholesterol succinate monoester, sorbitan, EDCI, and DMAP was 1:1.2:1:1.5. The mixture was stirred at room temperature for 20 h, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was washed successively with acid water, alkaline water, and saturated saline solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product (a light yellow oily substance). The product was separated by column chromatography (gradient elution: pure petroleum ether, petroleum ether: ethyl acetate = 50:1, petroleum ether: ethyl acetate = 20:1, petroleum ether: ethyl acetate = 10:1) to obtain a light yellow powder, sorbitan-cholesterol succinate.

[0075] Using ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: The purified sorbitan-cholesterol succinate was analyzed by mass spectrometry. The mass spectrometry conditions were as follows: the sample was dissolved in chromatographically pure methanol and diluted to 1–5 ppm; an electrospray ionization (ESI+) source was used; and the mass range was 50–1000. m / z The mass spectrometry results are as follows: Figure 9 As shown, the molecular weight of sorbitan-cholesterol succinate is 738. Under positive ion source conditions, 739 represents the [M+1]+ mass spectrum signal of sorbitan-cholesterol succinate. Enlarging the spectrum reveals the [M+Na]+ mass spectrum signal at 761. Figure 9 The compound is located in the upper right corner. Therefore, it can be preliminarily identified as sorbitan-cholesterol succinate.

[0076] To further confirm the reaction products, the products were subjected to... 1 H-NMR and 13 C-NMR determination, 1 H-NMR and 13 The C-NMR frequencies were 100 MHz and 400 MHz, respectively. Deuterated chloroform was used as the solvent, and tetramethylsilane was used as the internal standard. The specific spectral results are as follows: Figure 10 As shown in (a) and (b). By analyzing... 1 H-NMR spectrum and 13 The structure obtained from the C-NMR spectrum was assigned, and combined with the mass spectrometry results, the target product was finally identified as sorbitan-cholesterol succinate.

[0077] Example 6 This embodiment provides a cholesterol succinate monoester derivative (paeonol-cholesterol succinate), the structure of which is as follows: The preparation method of paeonol-cholesterol succinate in this embodiment includes the following steps: Weigh appropriate amounts of paeonol and cholesterol succinate monoester and dissolve them in an appropriate amount of dichloromethane (DCM) (the amount of dichloromethane is sufficient for dissolution and will evaporate naturally afterward). Add catalysts EDCI and DMAP. The molar ratio of cholesterol succinate monoester, paeonol, EDCI, and DMAP is 1:1.2:1:1.5. Stir the reaction for 24 h and monitor the reaction progress by TLC. After the reaction is complete, wash the reaction solution successively with acid water, alkaline water, and saturated saline solution, dry it with anhydrous sodium sulfate, and evaporate the solvent to obtain the crude product (light yellow oil). Separate it by column chromatography (petroleum ether: ethyl acetate = 100:1) to obtain the light yellow oil.

[0078] Using ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: The purified paeonol-cholesterol succinate was analyzed by mass spectrometry. The mass spectrometry conditions were as follows: the sample was dissolved in chromatographically pure methanol and diluted to 1–5 ppm; an electrospray ionization (ESI+) source was used; and the mass range was 50–1000. m / z The mass spectrometry results are as follows: Figure 11 As shown, the molecular weight of paeonol-cholesterol succinate is 634. 657 is the mass spectrometry signal of paeonol-cholesterol succinate [M+Na]+, and 369 is the signal peak of fragments generated by the fragmentation of paeonol-cholesterol succinate. For the specific structure, see [link to relevant documentation]. Figure 11 Located in the upper right corner, it can be preliminarily identified as paeonol-cholesterol succinate.

[0079] To further confirm the reaction products, the products were subjected to... 1 H-NMR and 13 C-NMR determination, 1 H-NMR and 13 The C-NMR frequencies were 100 MHz and 400 MHz, respectively. Deuterated chloroform was used as the solvent, and tetramethylsilane was used as the internal standard. The specific spectra are shown below. Figure 12 As shown in (a) and (b). By analyzing... 1 H-NMR spectrum and 13 The structure obtained from the C-NMR spectrum was assigned, and combined with mass spectrometry, the target product was finally identified as paeonol-cholesterol succinate.

[0080] Example 7 This embodiment provides a cholesterol succinate monoester derivative (dihydroartemisinin-cholesterol succinate, abbreviated as DHA-CS), the structure of which is as follows: The preparation method of dihydroartemisinin-cholesterol succinate in this embodiment includes the following steps: Synthesis of dihydroartemisinin succinate cholesterol ester: An esterification reaction system was constructed in a dichloromethane (CH2Cl2) solvent system using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) as a condensing agent and 4-dimethylaminopyridine (DMAP) as a catalyst. The reactants were precisely added in a molar ratio of cholesterol succinate:dihydroartemisinin:EDCI:DMAP = 1.0:1.0:1.5:0.2, and the reaction was continuously stirred in a magnetic stirrer at room temperature (25±2℃). The reaction progress was dynamically monitored using thin-layer chromatography (TLC) with petroleum ether-ethyl acetate (4:1, V / V) as the developing solvent. The reaction endpoint was determined to be reached when the dihydroartemisinin starting material spot completely disappeared during TLC detection.

[0081] Purification of dihydroartemisinin succinate cholesterol ester: The above reaction mixture was thoroughly ground and mixed with silica gel powder (200-300 mesh) at a volume-to-mass ratio of 1:1 (V:M, mL:g) to prepare a dry sample powder. Column chromatography was performed using a reversed-phase C18 silica gel column, with isocratic elution using three column volumes of petroleum ether-ethyl acetate (15:1, V / V) as eluent. After removing the solvent by rotary evaporation under reduced pressure for every 100 mL of eluent, dichloromethane was collected and analyzed by TLC (developing solvent as in reaction monitoring). The eluents of the target fraction with consistent and singular Rf values ​​were combined and the solvent was removed by rotary evaporation under reduced pressure (40-45℃, 0.095 MPa) to obtain a white crude solid. Column chromatography was repeated 1-2 times for further purification. The specific synthetic route is as follows: Using ESI-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR, as follows: ESI-MS structural analysis: High-purity DHA-CS samples were dissolved in trace amounts of chromatographic-grade dichloromethane to prepare a 0.1 mg / mL analyte solution. Detection was performed using a SCIEX TripleTOF® 6600+ high-resolution mass spectrometer with a positive ion electrospray ionization (ESI) source. + Full molecular weight scanning mode. Instrument parameter settings: mass scan range m / z 50~1250, high-resolution precise acquisition mode; spray voltage +5500 V, nebulizer gas temperature 550℃, auxiliary gas and sheath gas are both high-purity nitrogen; direct sample injection detection, acquisition of first-level high-resolution mass spectra, comparison of measured quasi-molecular ion peaks with theoretical molecular weight values, calculation of mass deviation, and determination of molecular formula matching degree.

[0082] Mass spectrometry results as follows Figure 13 As shown, the theoretical molecular formula of DHA-CS is C45 H 72 O7, with a theoretical molecular weight of 752.5227, and based on the sodium ion addition mode, theoretically [M+Na] + The ion peak m / z is 775.5120. Mass spectrometry observed an adduct ion peak at m / z 775.5112 [M+Na]. + 776.5144 [M+Na+ 13 C] + and 777.5169 [M+Na+2× 13 C] + Main peak [M+Na] + The measured value m / z 775.5112 deviated from the theoretical value 775.5120 by 1.0 ppm, meeting the structure determination criterion of ≤5 ppm for high-resolution mass spectrometry in organic synthesis. The isotope peak abundance distribution was consistent with the natural isotope abundances of C, H, and O. The results indicate that dihydroartemisinin and cholesterol succinate monoester undergo 1:1 esterification condensation and removal of one water molecule to generate the target coupling product, with correct molecular formula matching.

[0083] To further confirm the reaction products, the products were subjected to... 1 H-NMR and 13 The C-NMR determination was performed using deuterated chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as internal standard. 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) spectrum is shown in the figure below. Figure 14 As shown in (a) and (b).

[0084] 1H-NMR analysis: 1H NMR (400 MHz, CDCl3) δ 5.79 (d, J = 9.9 Hz, 1H), δ5.45 - 5.33 (m, 2H), 4.70 - 4.54 (m, 1H), 2.77 - 2.50 (m, 5H), 2.44 - 2.27(m, 3H), 2.02 (ddt, J = 12.2, 10.4, 3.1 Hz, 3H), 1.67 - 1.37 (m, 15H), 1.37 -1.00 (m, 19H), 1.00 - 0.92 (m, 7H), 0.92 - 0.79 (m, 12H), 0.67 (s, 3H). The doublet at δ 5.79 (J = 9.9 Hz, 1H) belongs to the C-10 alkenyl ether hydrogen of dihydroartemisinin, and the ortho-coupling constant at 9.9 Hz confirms that the double bond is in the trans (E) configuration, which is a characteristic signal of the spiroketal alkenyl ether ring in artemisinin-like compounds; the multiplet at δ 5.45-5.33 (2H) corresponds to the cholesterol A ring Δ 5 The double bond has a C-6 olefinic hydrogen (C-5 is a quaternary carbon with no proton), belonging to the steroidal Δ group. 5The characteristic resonance peaks of the alkene structure; the multiplet at δ 4.70-4.54 (1H) is the methylene hydrogen at the C-3 position of cholesterol. After the hydroxyl group at the 3-position esterifies with succinic acid, the electron-withdrawing deshielding effect of the ester group oxygen causes a significant low-field shift of this proton, directly confirming the successful construction of the intramolecular ester bond. The multiplet at δ 2.77-2.50 (5H) is composed of multiple sets of protons superimposed. The main components are the two protons of the C-2′ methylene group of the succinic acid linker arm near the ester carbonyl group at the cholesterol end and the two protons of the allyl methylene group at the C-4 position of the cholesterol core. The remaining proton comes from the C-9 hydrogen of dihydroartemisinin or the allyl hydrogen at the C-6 position of cholesterol. The multiplet at δ 2.44-2.27 (3H) belongs to the two protons of the C-3′ methylene group in the middle of the succinic acid chain, and is superimposed with a small number of methylene protons at the C-2 or C-7 positions of cholesterol. The multiple peaks at δ 2.02 (J = 12.2, 10.4, 3.1 Hz, 3H) are attributed to α-oxygen-substituted saturated methine and methylene hydrogens in the C-5, C-6, and C-9 polyoxygen spirocyclic skeleton of dihydroartemisinin, and overlap with the proton signals of the cholesterol A ring. The complex multiple coupling splitting is a typical manifestation of the crowded spirocyclic alicyclic stereochemical environment. The broad multiplet (15H) at δ 1.67–1.37 corresponds to all saturated methylene protons at C-2, C-3, C-4, C-8, and C-9 on the dihydroartemisinin fused ring skeleton. Due to the weak deshielding effect of multiple ether oxygen atoms in the ring, the chemical shift is slightly lower than that of the cholesterol alkyl saturated hydrogen. The broad envelope (19H) at δ 1.37–1.00 mainly consists of a large number of saturated methylene and methine protons on the cholesterol tetracyclic core (C-1, C-2, C-4, C-6, C-7, C-8, C-11, C-12, C-14, C-15, C-16, C-17, etc.) and the long alkyl side chain (C-20–C-27) at C17. The δ 1.00–0.92 multiplet (7H) contains the C-14 angular methyl group (3H) of dihydroartemisinin, the C-21 methyl group (3H) of cholesterol, and one methyl proton from the C-26 / C-27 isopropyl group. The δ 0.92–0.79 multiplet (12H) is an overlap signal from the C-19 angular methyl group (3H) of cholesterol, the remaining two methyl groups (6H) of the isopropyl group, and the C-15 methyl group (3H) of dihydroartemisinin. The δ 0.67 sharp singlet (3H) is the characteristic high-field signal of the C-18 quaternary carbon angular methyl group of cholesterol. Because there is no hydrogen at the adjacent carbon position, there is no spin coupling, hence the singlet without splitting. In this molecule, both hydroxyl groups of succinic acid are esterified, one end is connected to the C-10 hydroxyl group of dihydroartemisinin, and the other end is coupled to the C-3 hydroxyl group of cholesterol. There is no free carboxyl group, therefore there is no broad exchange peak corresponding to the active hydrogen of the carboxylic acid in the spectrum. The total sum of all proton integrals in the spectrum is 71H, which is basically consistent with the theoretical observable hydrogen number of 73H. The slight deviation is caused by the error in the integration interval segmentation.Based on the above-mentioned patterns of chemical shift, coupling splitting, and integral values ​​in the proton spectrum, it can be confirmed that the target product is a dihydroartemisinin-10-succinate cholesterol ester conjugate, and the connection mode of each structural segment within the molecule is completely consistent with the design expectation.

[0085] For products 13 C10 NMR peak assignment revealed two carbon signals in the carbonyl region at δ 172.5 and δ 167.8, which were attributed to the saturated ester carbonyl group (-COO-Chol) attached to the C-3 position of cholesterol in succinic acid and the enyl ether ester carbonyl group (-COO-DHA) attached to the C-10 position of dihydroartemisinin, respectively. Both ends of succinic acid were esterified, and the characteristic signal of the free carboxylic acid carbon at δ ~ 178 was not observed in the spectrum. This result is consistent with... 1 The observations by H NMR without carboxyl active hydrogen corroborate each other. Unsaturated sp 2 In the carbon range, δ 138.5 and 122.2 correspond to cholesterol Δ 5 The C-5 and C-6 olefinic carbons of the double bond; δ 132.8 and 126.1 belong to the C-10=C-11 olefinic ether double bond carbons of dihydroartemisinin, matching the trans-olefinic hydrogen signal at δ 5.79 in the 1H NMR spectrum. In the oxygen-saturated carbon region, δ 101.5 is the characteristic peroxyketal quaternary carbon C-12 of dihydroartemisinin, δ 93.2 is the C-10 chiral carbon linked to the ester bond, and δ 74.6 is the C-3 chiral carbon of cholesterol after esterification modification. Due to the electron-withdrawing deshielding effect of oxygen atoms, all three are in the low-field region, and the low-field shift of the cholesterol C-3 carbon is about 3 ppm compared to free cholesterol (δ 71.6), directly confirming the successful construction of two intramolecular ester bonds. The two sets of methylene carbons in the succinic acid linker arm correspond to δ 32.1 and 29.6, respectively. Within the δ 10-58 range of saturated alicyclic and alkyl carbons, δ 35-58 belong to the methine and methylene carbons on the cholesterol steroidal core and the dihydroartemisinin fused ring skeleton; δ 20-35 are the long alkyl side chain (C-20~C-27) carbon clusters at the C17 position of cholesterol; the high-field region of δ 10-20 consists entirely of various angular methyl and branched methyl carbons, with δ 11.8 corresponding to the angular methyl carbon at C-18 of cholesterol. 1 The ¹H NMR δ 0.67 singlet methyl signal corresponds one-to-one. The triplet at δ 77.26 in the spectrum is the standard solvent peak of deuterated chloroform (CDCl₃), and the weak broad peak at δ 1.60 is the signal of trace water impurities; neither of these belongs to the carbon resonance of the target product. All carbon chemical shifts, signal distribution patterns, and the three major structural units of the molecule (dihydroartemisinin polyoxyspiroacetal ring, succinate diester linker, and Δ...) are analyzed. 5 The double-bonded cholesterol sterane core and alkyl side chain are highly compatible, binding 1The H NMR proton spectrum analysis results can cross-confirm that the structure of the synthesized conjugate is completely consistent with the design target.

[0086] pass 1 The chemical shift (δ), coupling constant (J), and integration area of ​​characteristic proton signals in the H NMR spectrum, combined with... 13 The chemical shifts of the carbon skeleton signal in the CNMR spectrum confirmed the molecular structure and functional group connection mode of the compound, and all characteristic signals were a perfect match with the theoretical structure of the target compound.

[0087] Example 8: Preparation of solid lipid nanoparticles This embodiment provides a podophyllotoxin-cholesterol succinate solid lipid nanoparticle (PCH-SLN), the preparation method of which includes the following steps: Accurately weigh or measure appropriate amounts of soybean lecithin, podophyllotoxin-cholesterol succinate prepared in Example 1, anhydrous ethanol, and medium-chain triglycerides (MCT). The mass ratio of soybean lecithin, podophyllotoxin-cholesterol succinate, MCT, and anhydrous ethanol is 3:1:1.6:2.7. Place the mixture in a round-bottom flask and remove the organic solvent anhydrous ethanol by rotary evaporation under reduced pressure at 60 °C until a thin film of lipid forms at the bottom of the flask. Continue vacuuming to ensure complete removal of the organic solvent. Add an appropriate amount of deionized water (60 °C) dropwise. After the addition is complete, continue stirring for a certain period of time. After ultrasonic dispersion (600 W, 30 min), perform probe-type ultrasonication (500 W power, 5 s working time, 5 s interval) under ice bath conditions for 5 min. The resulting PCH-SLN solution is milky white, uniformly dispersed, and without stratification or precipitation.

[0088] Example 9 This embodiment provides a temozolomide-cholesterol succinate solid lipid nanoparticle (TCH-SLN) and its preparation method. Except that the podophyllotoxin-cholesterol succinate used to prepare the solid lipid nanoparticle in Example 8 is replaced with the temozolomide-cholesterol succinate prepared in Example 2, the other raw materials, amounts and preparation steps are the same as in Example 8.

[0089] Example 10 This embodiment provides (S)-10-hydroxycamptothecin-cholesterol succinate solid lipid nanoparticles (SHC-SLN) and their preparation method. Except that the podophyllotoxin-cholesterol succinate used to prepare the solid lipid nanoparticles in Example 8 is replaced with (S)-10-hydroxycamptothecin-cholesterol succinate prepared in Example 3, the other raw material types, amounts and preparation steps are the same as in Example 8.

[0090] Example 11 This embodiment provides geraniol-cholesterol succinate lipid nanoparticles (Ge-CH LNPs) and their preparation method, including the following steps: Accurately weigh 450 mg of soybean lecithin, 100.7 mg of geraniol-succinate monocholesterol ester prepared in Example 4, 500 µL of anhydrous ethanol, and 250 µL of medium-chain triglycerides (MCT). Keep warm at 60°C and place on a magnetic stirrer. While stirring, add purified water containing the stabilizing material dropwise. After the addition is complete, continue stirring for 5 min. Perform probe-type ultrasound (500 W power, 5 s working time, 5 s interval) in an ice bath for 5 min.

[0091] Example 12 This embodiment provides a dihydroartemisinin-cholesterol succinate liposome (DHA-CS LNPs), the preparation method of which includes the following steps: In a 50 mL round-bottom flask, precisely add 758 mg of soybean lecithin, 199 mg of dihydroartemisinin succinate cholesterol ester derivative, and 400 μL of medium-chain triglycerides (MCT) sequentially. Then add 800 μL of anhydrous ethanol to the flask and place it in a rotary evaporator at 45°C. Evaporate under reduced pressure for 5-10 min, maintaining a rotation speed of 20-30 rpm, until a uniform, crack-free, pale yellow lipid film forms on the inner wall of the flask. Turn off the rotary evaporator and allow the flask to stand under vacuum for 10 min to completely remove residual ethanol. Quickly add 25 mL of preheated deionized water (60°C) to the flask containing the lipid film and immediately place it in a 60°C water bath. Turn on the magnetic stirrer (600-800 rpm) and hydrate and stir for 5 min to completely detach the lipid film from the flask wall and initially disperse it to form a milky white crude liposome suspension. The crude liposome suspension was transferred to an ice bath and ultrasonically dispersed at 600W power for 15 minutes using an ultrasonic cell disruptor with a 5s ultrasonic / 5s intermittent mode. During the process, the probe was ensured to be immersed 1-2cm below the liquid surface without contacting the container wall. The result was a homogeneous and stable dihydroartemisinin-cholesterol succinate liposome suspension, which was stored in a 4°C refrigerator protected from light for later use.

[0092] Comparative Example 1 This comparative example provides podophyllotoxin solid lipid nanoparticles (PPT-SLN). The preparation method is the same as that of Example 8, except that the podophyllotoxin-cholesterol succinate is replaced with podophyllotoxin and cholesterol succinate monoester. The mass of podophyllotoxin and cholesterol succinate monoester used in this comparative example is the same as that of podophyllotoxin and cholesterol succinate monoester in Example 8. The other raw materials, amounts and preparation steps are the same as those in Example 8.

[0093] Comparative Example 2 This comparative example provides temozolomide solid lipid nanoparticles (TMZ-SLN) and their preparation method. Except that the temozolomide-cholesterol succinate in Example 9 is replaced with temozolomide and cholesterol succinate monoester, and the mass of temozolomide and cholesterol succinate monoester used in this comparative example are the same as the mass of temozolomide and cholesterol succinate monoester in temozolomide-cholesterol succinate in Example 9, the other raw material types, amounts, and preparation steps are the same as in Example 9.

[0094] Comparative Example 3 This comparative example provides (S)-10-hydroxycamptothecin solid lipid nanoparticles (SHT-SLN) and their preparation method. Except that the (S)-10-hydroxycamptothecin-cholesterol succinate in Example 10 is replaced with (S)-10-hydroxycamptothecin and cholesterol succinate monoester, and the mass of (S)-10-hydroxycamptothecin and cholesterol succinate monoester used in this comparative example are the same as those in Example 10, the mass of (S)-10-hydroxycamptothecin and cholesterol succinate monoester used in this comparative example are the same as those in Example 10, the other raw material types, amounts and preparation steps are the same.

[0095] Comparative Example 4 This comparative example provides a dihydroartemisinin liposome (DHA LNPs) and its preparation method. Except that the 199 mg dihydroartemisinin-cholesterol succinate in Example 12 is replaced with 102 mg cholesterol and 75 mg dihydroartemisinin, the other raw materials, amounts and preparation steps are the same as in Example 12.

[0096] Comparative Example 5 This comparative example provides a blank liposome (blank LNPS), the preparation method of which includes the following steps: Except for replacing 199 mg of dihydroartemisinin-cholesterol succinate in Example 12 with 102 mg of cholesterol, the other raw materials, amounts, and preparation steps are the same as in Example 12.

[0097] To compare the technical effects of the above embodiments and comparative examples, the following experimental examples are provided.

[0098] It should be noted that the data in the experimental examples were processed using statistical analysis software SAS 8.0 and GraphPad Prism 8.0. Results are expressed as mean ± standard deviation. Comparisons between groups were performed using one-way or two-way ANOVA. P < 0.05 is considered statistically significant. P < 0.01 indicates a statistically significant difference.

[0099] Experimental Example 1: Preparation and Characterization of Solid Lipid Nanoparticles (1) Transmission electron microscopy (TEM) measurement The morphology and structure of PCH-SLN (podophyllotoxin-cholesterol succinate solid lipid nanoparticles prepared in Example 8), TCH-SLN (temozolomide-cholesterol succinate solid lipid nanoparticles prepared in Example 9), SHC-SLN ((S)-10-hydroxycamptothecin-cholesterol succinate solid lipid nanoparticles prepared in Example 10), and Ge-CH-LNPs (geraniol-cholesterol succinate lipid nanoparticles prepared in Example 11) were observed using transmission electron microscopy. The results are as follows: Figure 15 As shown in (a)-(d), PCH-SLN, TCH-SLN, SHC-SLN and Ge-CH-LNPs are uniformly spherical with smooth and flat surfaces and good dispersibility, as can be seen under the microscope.

[0100] (2) Particle size and potential distribution determination a. The particle size, polydispersity index (PDI), and zeta potential of PCH-SLN, TCH-SLN, SHC-SLN, and Ge-CH-LNPs were determined using a Malvern particle size potentiometer.

[0101] The specific determination method is as follows: Take the prepared nanoparticle sample solutions (PCH-SLN, TCH-SLN, SHC-SLN, and Ge-CH-LNPs), dilute them with distilled water to 100 times, mix well, and then take 1.5 mL of the diluted solution into a particle size measuring cup. Under 25 ℃ conditions, use a Malvern laser particle size analyzer to determine the particle size and polydispersity index (PDI) of the test samples by dynamic light scattering method. Then, use a 5 mL disposable syringe to draw up the diluted solution and slowly inject it into a potential cup, carefully check, remove air bubbles, and check the potential. Each sample is measured 3 times, and the results are recorded and the average value is obtained. The specific determination results are shown in Table 1-1.

[0102] Table 1-1 Particle size, polydispersity index, and potential of PCH-SLN, TCH-SLN, SHC-SLN, and Ge-CH-LNPs The results showed that PCH-SLN, TCH-SLN, SHC-SLN and Ge-CH-LNPs did not exhibit aggregation and had relatively uniform particle size distribution.

[0103] b. The hydration size, polydispersity index (PDI), and Zeta potential of two types of lipid nanoparticles, DHA LNPs and DHA-CS LNPs, were measured using a Malvern laser particle size and Zeta potential analyzer. The specific measurement method was as follows: The prepared liposome suspension was filtered through a 0.22 μm polyethersulfone (PES) membrane and diluted 10-fold with deionized water. The detection temperature was set at 25℃, the equilibration time at 120 s, and each sample was measured in triplicate, with 10 sets of data collected per measurement. The average particle size (Z-average diameter) and polydispersity index (PDI) of the liposomes were recorded. Simultaneously, particle size distribution curves were generated using the instrument's software to visually analyze the size uniformity of the liposomes. Using the aforementioned dynamic light scattering instrument, the Zeta potential of the liposomes was measured at 25℃, again with the diluted liposome suspension as the sample.

[0104] The statistical results of particle size and potential are shown in Table 1-2.

[0105] Table 1-2 Particle size, polydispersity index, and potential of DHA LNPs and DHA-CS LNPs As shown in the table above, the PDI values ​​of both DHA LNPs and DHA-CS LNPs nanoparticles are below 0.3. According to the evaluation standards for colloidal formulations, a PDI < 0.3 indicates a narrow particle size distribution, good uniformity, no obvious aggregation or flocculation, and a stable and controllable preparation process. Potential detection results show that the Zeta potential of DHA LNPs is -52.33 ± 5.20 mV, and that of DHA-CS LNPs is -34.00 ± 0.72 mV. Both types of lipid nanoparticles carry a negative charge on their surface. The higher absolute value of the negative potential can reduce the probability of particle collision aggregation through electrostatic repulsion, further improving the long-term storage stability of the colloid.

[0106] (3) Encapsulation efficiency (EE) determination Taking PCH-SLN as an example, its encapsulation efficiency was measured.

[0107] Specific determination method: Accurately pipette 1 mL of PCH-SLN into a dialysis bag (pre-treated by boiling and activation), and use at least 10 times the volume of 20% ethanol-deionized water as the dialysis medium. Stir magnetically at 400 r / min for 96 h, changing the dialysis medium every 6 h. After 96 h, take 0.5 mL of PCH-SLN from the dialysis bag, dilute with methanol and bring the volume to 5 mL, shake well, filter through a 0.45 μm microporous membrane, and detect by liquid chromatography. Calculate the encapsulation efficiency of PCH-SLN according to the following formula.

[0108] Encapsulation rate (%) = (Total drug content in dialysis bag / Total drug content) × 100%; The specific results are shown in Table 2.

[0109] Table 2. Encapsulation efficiency (EE) measurement results of PCH-SLN The data in the table show that the average encapsulation efficiency of PCH-SLN is 81.42% and the RSD is 0.26, indicating that the preparation process can achieve a good encapsulation efficiency.

[0110] (4) Results of in vitro physical stability The in vitro physical stability of PCH-SLN, TCH-SLN, and SHC-SLN was demonstrated by measuring changes in particle size.

[0111] a. Measurement Method: PCH-SLN, TCH-SLN, and SHC-SLN were stored at 4 ℃ in the dark. Samples were taken at regular time intervals, and the polydispersity index (PDI) and particle size were measured using a Malvern laser particle size analyzer at 0, 2, 4, 7, and 15 days. The stability of the samples was assessed by the changes in particle size and PDI at different time points. Each time point was repeated three times, and the average value was calculated. The results are shown in Tables 3-5.

[0112] Table 3 Physical stability of PCH-SLN Table 4 Physical stability of TCH-SLN Table 5 Physical stability of SHC-SLN As shown in the table, after being stored at 4 ℃ in the dark and refrigerated condition for 15 days, the particle size of PCH-SLN, TCH-SLN, and SHC-SLN did not change significantly, the PDI remained below 0.3, and no stratification, precipitation, or flocculation was observed visually, with no significant color change. The experimental results indicate that PCH-SLN, TCH-SLN, and SHC-SLN maintain good physical stability even after being stored in the dark and refrigerated condition for half a month.

[0113] b. Measurement method: DHA-CS LNPs were stored in a cool, dark place for 30 consecutive days. The hydrated particle size and polydispersity index (PDI) of the system were measured periodically at 1, 3, 5, 7, 15 and 30 days. The long-term physical stability of DHA-CS LNPs was comprehensively evaluated by combining macroscopic appearance observation. The relevant test data are shown in the table below.

[0114] Table 6 Physical stability of DHA-CS LNPs As shown in Table 6, after 30 days of refrigerated and light-protected storage, the hydrated particle size of DHA-CS LNPs did not fluctuate significantly, and the PDI value remained stable around 0.2, consistently within the narrow distribution standard range. During the same period, visual observation of the formulation system revealed no colloidal instability characteristics such as stratification, precipitation, or flocculation. These results confirm that under refrigerated and light-protected storage conditions, DHA-CS LNPs can maintain good colloidal homogeneity and exhibit excellent physical stability within 30 days, meeting the requirements for subsequent in vivo animal experiments during the formulation's storage period.

[0115] (5) In vitro release assay of PCH-SLN a. Taking podophyllotoxin-cholesterol succinate solid lipid nanoparticles (PCH-SLN) as an example, their in vitro release was measured.

[0116] Assay method: Accurately measure 1.5 mL of PCH-SLN sample solution and transfer it to a dialysis bag (molecular weight cutoff 8000–14000 Da). Place the dialysis bag into a 50 mL centrifuge tube containing 30 mL of 20% ethanol-PBS release medium at pH 7.4 and 5, respectively. Dialyze at 37 °C with continuous shaking at 100 rpm. Next, samples were taken at different time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h), and 2 mL of release medium solution was taken and the same volume of fresh release medium was added. The drug concentration was determined using HPLC, and the cumulative drug release amount of PCH-SLN at a series of time points from 1 to 36 h was calculated. An in vitro drug release curve was then plotted.

[0117] The in vitro release curves of podophyllotoxin-cholesterol succinate solid lipid nanoparticles (PCH-SLN) under different pH conditions are shown in the figure. Figure 16 As shown, PCH-SLN exhibits significant release differences at pH 5 and pH 7.4. At pH 5, PCH-SLN initially shows explosive release followed by a plateau. At pH 7.4, due to partial ionization of the succinic acid groups, there is initial rapid dissolution, but the overall release rate is still lower than that at pH 5. Analysis suggests that the structural modification of podophyllotoxin, due to the presence of ester bonds, enables a pH-sensitive response; the low pH environment triggers the hydrolysis of these ester bonds, leading to rapid drug release.

[0118] b. Taking DHA LNPs and DHA-CS LNPs as examples, pH-sensitive drug release experiments were conducted on them.

[0119] Assay method: Take 1 ml of liposome solution, place it into a dialysis bag, seal it, and then place it in a beaker containing 100 mL of PBS release medium (pH=5.5 and pH=7.4) containing 20% ​​ethanol, respectively. Stir magnetically at 300 r / min. Take 5 mL samples at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and simultaneously add 5 mL of fresh release medium to maintain a constant volume. After sampling, the drug concentration in the release medium is detected by HPLC, and the cumulative release rate is calculated: Cumulative release rate = (total drug released at each time point / WT) × 100%.

[0120] Specific results are as follows Figure 17 As shown in Figures (a) and (b), DHA LNPs exhibit rapid release behavior at both pH 7.4 and pH 5.5, while DHA-CS LNPs demonstrate ideal sustained-release properties at pH 7.4 and significantly accelerated release under acidic conditions at pH 5.5. This indicates that dihydroartemisinin cholesterol succinate liposomes possess excellent sustained-release performance and pH sensitivity.

[0121] Experimental Example 2: Evaluation of in vitro antitumor activity and cytotoxicity (1) CCK-8 assay for cell proliferation inhibition Human hepatocellular carcinoma cell line (HepG2), human non-small cell lung cancer cell line (A549), and mouse breast cancer cell line (4T1) in logarithmic growth phase were cultured in 96-well plates at a concentration of 3 × 10⁻⁶ cells / well. 3Cells were cultured at a density of 100 μL / well. After 24 h in a 5% CO2 incubator, three cell types were infected with the drug. Each cell type included a positive control group (cisplatin and etoposide), a parent drug group (podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin), a derivative drug group (PCH, TCH, SHC), and an SLN group (PCH-SLN, TCH-SLN, SHC-SLN). Each drug was further prepared at five concentrations (5 μmol / L, 25 μmol / L, 50 μmol / L, 125 μmol / L, and 250 μmol / L, representing the concentration of the drug in each well), as detailed in Tables 6-8. Negative control wells (culture medium containing cells but without the test substance) and blank wells (culture medium without cells and the test substance) were also included. Each group had five replicates, and the operation was performed in triplicate. Cells were cultured at 37 °C for 24 h after drug administration in a 5% CO2 incubator. Before detection, 10 μL of cell proliferation reagent CCK-8 was added to each well, and the cells were incubated in a 37 °C, 5% CO2 incubator in the dark for 90 minutes. Finally, the OD absorbance was measured at 450 nm using a multi-mode microplate reader, and the inhibition rate of different drugs on various cell types was calculated using the following formula: Inhibition rate (%) = (OD) 阴性对照孔 - OD 实验孔 ) / (OD 阴性对照孔 - OD 空白孔 ) × 100% The IC50 values ​​of the drug against human hepatocellular carcinoma cell line (HepG2), human non-small cell lung cancer cell line (A549), and mouse breast cancer cell line (4T1) were calculated using GraphPad Prism 9.0. 50 The values ​​are shown in Table 7-9.

[0122] Table 7. Inhibition rate of A549 cancer cell proliferation (%, mean ± SD, n = 3) Note: Representative of derivative drugs vs. original drug group P < 0.05, Representative of derivative drugs vs. original drug group P < 0.01; # Representing the SLN group vs. the derivative drug group P < 0.05, ## Representing the SLN group vs. the derivative drug group P < 0.01.

[0123] Table 8 HepG2 cancer cell proliferation inhibition rate (%, mean ± SD, n = 3) Note: Representative of derivative drugs vs. original drug group P < 0.05, Representative of derivative drugs vs. original drug group P < 0.01; # Representing the SLN group vs. the derivative drug group P < 0.05, ## Representing the SLN group vs. the derivative drug group P < 0.01.

[0124] Table 9. Inhibition rate of 4T1 cancer cell proliferation (%, mean ± SD, n = 3) Note: Representative of derivative drugs vs. original drug group P < 0.05, Representative of derivative drugs vs. original drug group P < 0.01; # Representing the SLN group vs. the derivative drug group P < 0.05, ## Representing the SLN group vs. the derivative drug group P < 0.01.

[0125] As can be seen from the data in the table above, the inhibition rate of proliferation of A549 cells, HepG2 cells and 4T1 cells in all drug groups within the range of 5-250 μmol / L increased with increasing concentration, showing a dose-dependent effect.

[0126] In the inhibition of A549 cell proliferation, cisplatin, etoposide, podophyllotoxin, PCH, and PCH-SLN showed significant IC50 values. 50 The values ​​were 45.4 μmol / L, 39.24 μmol / L, 143.3 μmol / L, 76.7 μmol / L, and 47.49 μmol / L, respectively; in the inhibition of HepG2 cell proliferation, the IC50 values ​​of cisplatin, etoposide, podophyllotoxin, PCH, and PCH-SLN were [missing values]. 50The values ​​were 35.65 μmol / L, 50.75 μmol / L, 117.1 μmol / L, 63.85 μmol / L, and 39.31 μmol / L, respectively; while in the inhibition of mouse breast cancer 4T1 cell proliferation, cisplatin, etoposide, podophyllotoxin, PCH, and PCH-SLN had IC50 values ​​of 35.65 μmol / L, 50.75 μmol / L, 117.1 μmol / L, 63.85 μmol / L, and 39.31 μmol / L. 50 The values ​​were 32.26 μmol / L, 36.86 μmol / L, 90.9 μmol / L, 47.89 μmol / L, and 29.54 μmol / L, respectively. This indicates that HepG2 and 4T1 cancer cells are more sensitive to podophyllotoxin-based drugs. PCH-SLN showed better inhibitory effects on the proliferation of HepG2 cancer cells than the positive control drug etoposide, and better inhibitory effects on the proliferation of 4T1 cancer cells than the positive control drugs cisplatin and etoposide.

[0127] In the inhibition of A549 cell proliferation, etoposide, temozolomide, TCH, and TCH-SLN had different IC50 values. 50 The values ​​were 39.24 μmol / L, 161.5 μmol / L, 86.39 μmol / L, and 53.2 μmol / L, respectively; in the inhibition of HepG2 cell proliferation, the IC50 values ​​of etoposide, temozolomide, TCH, and TCH-SLN were... 50 The values ​​were 50.75 μmol / L, 145.2 μmol / L, 77.36 μmol / L, and 48.25 μmol / L, respectively; while in the inhibition of mouse breast cancer 4T1 cell proliferation, the IC50 values ​​of etoposide, temozolomide, TCH, and TCH-SLN were... 50 The values ​​were 36.86 μmol / L, 114.4 μmol / L, 59.12 μmol / L, and 35.63 μmol / L, respectively. TCH-SLN showed superior inhibitory effects on the proliferation of HepG2 and 4T1 cancer cells compared to the positive control drug etoposide.

[0128] In the inhibition of A549 cell proliferation, cisplatin, etoposide, (S)-10-hydroxycamptothecin, SHC, and SHC-SLN showed the highest IC50 values. 50 The values ​​were 45.4 μmol / L, 39.24 μmol / L, 127.5 μmol / L, 68.47 μmol / L, and 43.08 μmol / L, respectively; in the inhibition of HepG2 cell proliferation, cisplatin, etoposide, (S)-10-hydroxycamptothecin, SHC, and SHC-SLN had IC50 values ​​of 45.4 μmol / L, 39.24 μmol / L, 127.5 μmol / L, 68.47 μmol / L, and 43.08 μmol / L. 50The values ​​were 35.65 μmol / L, 50.75 μmol / L, 129.6 μmol / L, 71.8 μmol / L, and 44.1 μmol / L, respectively; while in the inhibition of mouse breast cancer 4T1 cell proliferation, cisplatin, etoposide, (S)-10-hydroxycamptothecin, SHC, and SHC-SLN had IC50 values ​​of 35.65 μmol / L, 50.75 μmol / L, 129.6 μmol / L, 71.8 μmol / L, and 44.1 μmol / L, respectively; 50 The values ​​were 32.26 μmol / L, 36.86 μmol / L, 100.3 μmol / L, 52.68 μmol / L, and 32.57 μmol / L, respectively. SHC-SLN showed superior inhibitory effects on the proliferation of HepG2 and 4T1 cancer cells compared to the positive control drug etoposide, and superior inhibitory effects on the proliferation of A549 cancer cells compared to the positive control drug cisplatin.

[0129] Therefore, it can be seen that modifying the original drug by introducing functional cholesterol succinate significantly improved the drug's inhibitory rate on tumor cells. P < 0.01). However, after being prepared as a solid lipid nanoparticle formulation, the derivative drug formulation showed a significantly enhanced inhibitory effect on cancer cell proliferation compared to the derivative drug group. P The value of < 0.01 demonstrates the auxiliary advantages of nano-drug delivery systems in improving the anti-tumor efficacy of drugs.

[0130] (2) CCK-8 assay for cytotoxicity Normal human hepatocytes (L02) in the logarithmic growth phase were collected at a concentration of 3 × 10⁻⁶. 3Cells were cultured at a density of 100 μL / well in 96-well plates. After culturing for 24 h in a 5% CO2 incubator, cells were infected with drugs and divided into positive control groups (cisplatin and etoposide), original drug groups (podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin), derivative drug groups (PCH, TCH, SHC), and SLN groups (PCH-SLN, TCH-SLN, SHC-SLN) according to the drugs used. Each drug was further divided into 5 concentrations (5 μmol / L, 25 μmol / L, 50 μmol / L, 125 μmol / L, and 250 μmol / L, which is the concentration of the drug in each well), as detailed in Table 10. Negative control wells (culture medium containing cells but without the test substance) and blank wells (culture medium without cells and the test substance) were also set up. Each group had 5 replicates, and the operation was performed in triplicate. After drug administration, cells were cultured in a 5% CO2 incubator at 37 °C for 24 h. Before detection, 10 μL of cell proliferation reagent CCK-8 was added to each well, and the cells were incubated in a 5% CO2 incubator at 37 °C for 90 minutes in the dark. Finally, the OD absorbance was measured at 450 nm using a multi-mode microplate reader. The inhibition rate of different drugs on normal human hepatocytes (L02) was calculated according to the above-mentioned inhibition rate formula. The results are shown in Table 10.

[0131] Table 10. Inhibition rate of normal hepatocellular toxicity (%, mean ± SD, n = 3) Note: Representative of derivative drugs vs. original drug group P < 0.05, Representative of derivative drugs vs. original drug group P < 0.01; # Representing the SLN group vs. the derivative drug group P < 0.05, ## Representing the SLN group vs. the derivative drug group P < 0.01.

[0132] As shown in Table 10, in the toxicity inhibition experiment on normal L02 hepatocytes, the average inhibition rates of cisplatin, etoposide, podophyllotoxin, PCH, and PCH-SLN on normal L02 hepatocytes at a concentration of 250 μmol / L were approximately 45.9%, 43.8%, 34.2%, 25.5%, and 13.3%, respectively. The average inhibition rates of temozolomide, TCH, and TCH-SLN on normal L02 hepatocytes at a concentration of 250 μmol / L were approximately 28.9%, 21.0%, and 10.6%, respectively. The average inhibition rates of (S)-10-hydroxycamptothecin, SHC, and SHC-SLN on normal L02 hepatocytes at a concentration of 250 μmol / L were approximately 35.9%, 28.3%, and 16.0%, respectively. Although positive control drugs cisplatin and etoposide have strong anti-tumor effects, they are highly toxic to normal cells, have low cell killing selectivity, and a narrow therapeutic window, so they should be used with caution.

[0133] The experimental results showed that, compared with the original drug group, the derivative drug group significantly improved the safety of the drug. P < 0.01), possibly because functional structural modifications enhanced the drug's protective effect on normal cells. The SLN group also showed significantly lower toxicity to normal cells (L02) compared to the derivative drug group. P (< 0.01), further enhancing efficacy and reducing toxicity, suggesting that nanodelivery systems can reduce non-specific effects. The dual-track drug delivery strategy of "structural modification-nanodelivery" can realize the construction of a highly efficient and low-toxicity anticancer drug delivery system.

[0134] Experiment Example 3: Cell Uptake Experiment Using coumarin-6 as a fluorescent probe, different formulations loaded with fluorescence were prepared. Solid lipid nanoparticles of the derivative drug loaded with 10 μL of coumarin-6 were prepared according to the preparation methods of PCH-SLN, TCH-SLN, or SHC-SLN in Examples 8-10, simply by replacing anhydrous ethanol with anhydrous ethanol containing dissolved coumarin-6. Solid lipid nanoparticles of the original drug loaded with 10 μL of coumarin-6 were prepared according to the preparation methods of PPT-SLN, TMZ-SLN, or SHT-SLN in Comparative Examples 1-3, simply by replacing anhydrous ethanol with anhydrous ethanol containing dissolved coumarin-6.

[0135] A549 cells and 4T1 cells in the logarithmic growth phase were selected, and 1 × 10⁶ cells were grown per dish. 5Standard seedings of 10 cells were placed in 35 mm culture dishes, with 3 dishes per group, and incubated overnight in a 5% CO2 incubator (37 °C) to allow cell adhesion and growth. The original culture medium was discarded, and the cells were washed three times with PBS. Then, 1 mL each of solid lipid nanoparticles loaded with fluorescent and original drugs, and solid lipid nanoparticle suspensions loaded with fluorescent and derivatized drugs were added to each dish, and the dishes were incubated for another 3 h. The old culture medium was discarded, and the cells were washed three times with PBS. Then, 1 mL of 4% paraformaldehyde solution was added to each dish to fix the cells for 15 min. The fixative was removed, and the cells were washed three times with PBS. Then, 125 μL of the nuclear dye DAPI was added to each dish, and the nuclei were stained for 15 min. After washing away the dye with PBS, 1 mL of PBS was added to each dish.

[0136] Since the original drug and the derivative drug do not exhibit autofluorescence, after co-encapsulating the fluorescent dye coumarin-6, an inverted fluorescence microscope was used to observe the uptake of PPT-SLN (i.e., the podophyllotoxin solid lipid nanoparticles prepared in Comparative Example 1), PCH-SLN (podophyllotoxin-cholesterol succinate solid lipid nanoparticles), TMZ-SLN (i.e., the temozolomide solid lipid nanoparticles prepared in Comparative Example 2), TCH-SLN (temozolomide-cholesterol succinate lipid nanoparticles), SHT-SLN (i.e., the (S)-10-hydroxycamptothecin lipid nanoparticles prepared in Comparative Example 3), and SHC-SLN ((S)-10-hydroxycamptothecin-cholesterol succinate lipid nanoparticles) in A549 cells and 4T1 cells, respectively, and quantitatively analyze their uptake.

[0137] Specifically, Figure 18 (a) and (b) show the uptake of PPT-SLN (podophyllotoxin solid lipid nanoparticles) and PCH-SLN (podophyllotoxin-cholesterol succinate solid lipid nanoparticles) in human A549 tumor cells and mouse 4T1 tumor cells, respectively. Figure 20 (a) and (b) show the uptake of TMZ-SLN (temozolomide lipid nanoparticles) and TCH-SLN (temozolomide-cholesterol succinate lipid nanoparticles) in human A549 tumor cells and mouse 4T1 tumor cells, respectively. Figure 22 (a) and (b) show the uptake of SHT-SLN ((S)-10-hydroxycamptothecin lipid nanoparticles) and SHC-SLN ((S)-10-hydroxycamptothecin-cholesterol succinate lipid nanoparticles) in human A549 tumor cells and mouse 4T1 tumor cells, respectively. Observations show that the green fluorescence signal in each group is distributed around the cell nucleus, indicating that the formulations were successfully taken up into the cytoplasm by A549 and 4T1 cells.

[0138] In the uptake of human A549 tumor cells and mouse 4T1 tumor cells, from Figure 19 , Figure 21 , Figure 23 The results of comparing the green fluorescence expression intensity of the traditional Chinese medicine formulations showed that, compared with the PPT-SLN group, the TCH-SLN group, the TMZ-SLN group, and the SHC-SLN group, the green fluorescence of the derivative drug solid lipid nanoparticles was significantly stronger than that of the original drug solid lipid nanoparticles. P < 0.01 indicates that the amount of derivative drug lipid nanoparticles taken up by A549 cells and 4T1 cells was greater than that of the original drug lipid nanoparticles. This suggests that modifying the structure of the original drug with cholesterol succinate monoester to synthesize a derivative drug can improve the ability of the drug formulation to be taken up by tumor cells. P < 0.01), thereby increasing the drug concentration and efficacy within tumor cells. This demonstrates that the derivative drug, replacing cholesterol as one of the formulation matrices, has a significant advantage in improving the uptake of the formulation by tumor cells.

[0139] Experimental Example 4: In vivo targeting and pharmacodynamics test A mouse 4T1 cell xenograft model was established. Saline, original drug lipid nanoparticles, and derivative drug lipid nanoparticles were injected via tail vein, respectively. The effects of the prepared drugs were evaluated using laser speckle imaging, in vivo imaging, and in vivo antitumor efficacy, as detailed below: (1) Establishment of 4T1 tumor model mice Frozen mouse 4T1 tumor cells were retrieved and thawed. Once the cells reached the logarithmic growth phase, they were resuspended in PBS. The cell suspension was then mixed with matrix gel at a 1:1 volume ratio to adjust the cell suspension concentration to 5 × 10⁻⁶ cells / mL. 6 0.2 mL of a mixture of 4T1 cells and matrix gel was injected subcutaneously into the right side of the back of ICR mice under aseptic conditions. Mice were allowed free movement and feeding, and tumor growth was monitored periodically until the tumor volume reached 150-200 mm. 3 Subsequent experiments can then be conducted.

[0140] (2) Laser speckle blood flow imaging detection experiment in 4T1 tumor model mice The vascular system of tumor tissue differs significantly from that of normal tissue, exhibiting structural abnormalities and functional disorders. To systematically evaluate the potential impact of the constructed drug-eluting solid lipid nanoparticles on the microcirculation of the tumor region, this invention employs laser speckle contrast imaging technology to dynamically monitor changes in local blood perfusion in the tumor area of ​​4T1 tumor-bearing mice within 30 minutes after tail vein administration.

[0141] Specifically, 4T1 tumor model mice were randomly divided into four groups: saline group, podophyllotoxin preparation group (PPT-SLN group), podophyllotoxin derivative preparation group (PCH-SLN group), temozolomide preparation group (TMZ-SLN group), temozolomide derivative preparation group (TCH-SLN group), (S)-10-hydroxycamptothecin preparation group (SHT-SLN group), and (S)-10-hydroxycamptothecin derivative preparation group (SHC-SLN group), with three mice in each group. After anesthetizing the 4T1 tumor model mice with isoflurane, the dorsal region of the mice was shaved. During imaging, the mice were fixed in a prone, static position on the platform, ensuring exposure of the back and maintaining uniform ambient light, temperature, and humidity. Before drug administration, images of the desired areas were acquired using a laser speckle blood flow imaging system. Subsequently, the saline group (200 μL) and each drug group (5 mg / kg, 200 μL) were administered via tail vein injection. The saline group received injected saline, while the PPT-SLN group received injected PPT-SLN (prepared in Comparative Example 1), the PCH-SLN group received injected PCH-SLN (prepared in Example 7), the TMZ-SLN group received injected PCH-SLN (prepared in Comparative Example 2), the TCH-SLN group received injected PCH-SLN (prepared in Example 8), the SHT-SLN group received injected PCH-SLN (prepared in Comparative Example 2), and the SHC-SLN group received injected PCH-SLN (prepared in Example 9). Mice were anesthetized 30 minutes after drug administration, and images were acquired again using a laser speckle blood flow imaging system. After acquisition, pseudo-color images were generated (respectively...). Figure 24 , Figure 26 and Figure 28 Using the bounding box tool, the tumor area and background area were selected. After removing background interference, statistical analysis of blood perfusion was performed. Specific test results are shown below. Figure 25 , Figure 27 as well as Figure 29 .

[0142] from Figures 24-29 The test results showed that, before drug administration, there was no statistically significant difference in blood perfusion values ​​among all experimental groups. P > 0.05), ensuring consistency at the experimental starting point. Thirty minutes after drug administration, no significant changes were observed in the saline group, while all drug intervention groups showed a decrease in blood perfusion. Furthermore, the derivative formulation group exhibited a greater effect on reducing blood perfusion than its corresponding original drug formulation group, showing a difference. P > 0.05, no statistically significant difference was observed. Among them, the decrease in the PPT-SLN group (Group B) and PCH-SLN group (Group C) was significantly greater than that in the saline group (Group A). P < 0.05) and highly significant differences ( P < 0.01).

[0143] Laser speckle blood flow imaging results in 4T1 tumor model mice suggest that, within a very short time after administration of drug nanoparticles, the drug can exert its therapeutic effect by actively intervening in the tumor microcirculation, rapidly reducing the blood supply to the tumor in the early stages of administration, and macroscopically creating a microenvironment unfavorable to its growth. The derivative formulation showed a stronger effect, possibly due to its superior tumor targeting and cellular uptake efficiency.

[0144] (3) In vivo imaging experiment of 4T1 tumor model mice To visually observe the distribution characteristics and tumor-targeting ability of drug-containing solid lipid nanoparticles in 4T1 tumor-bearing mice, this invention constructed various groups of drug formulations labeled with DIR fluorescence and performed dynamic monitoring from 0 to 26 hours using a small animal in vivo imaging system. To quantitatively evaluate the targeting ability of the derivative nanoparticle groups, the tumor targeting index (TTI) was further calculated. Specific steps: After hair removal, 4T1 tumor model mice were randomly divided into four groups: blank preparation (DIR fluorescence) group (abbreviated as Blank-DIR-SLN group), podophyllotoxin preparation (DIR fluorescence) group (abbreviated as PPT-DIR-SLN group), podophyllotoxin derivative preparation (DIR fluorescence) group (abbreviated as PCH-DIR-SLN group), temozolomide preparation (DIR fluorescence) group (abbreviated as TMZ-DIR-SLN group), temozolomide derivative preparation (DIR fluorescence) group (abbreviated as TCH-DIR-SLN group), (S)-10-hydroxycamptothecin preparation (DIR fluorescence) group (abbreviated as SHT-DIR-SLN group), and (S)-10-hydroxycamptothecin derivative preparation (DIR fluorescence) group (abbreviated as SHC-DIR-SLN group), with 3 mice in each group. Mice bearing tumors in each group were administered the corresponding drug via tail vein injection (5 mg / kg, 200 μL). At 1 h, 6 h, 9 h, 18 h, and 26 h post-administration, the mice were anesthetized with isoflurane and placed in a small animal in vivo imaging system. Fluorescence signals in the mice were detected and photographed at a laser wavelength / emission wavelength of 748 / 780 nm. The fluorescence signal intensity of the whole experimental animals and tumor sites was quantitatively analyzed using an IVIS optical imaging system. The ratio of fluorescence intensity in the tumor area to that in normal tissue was calculated to obtain the tumor targeting index (TTI), which was calculated using the following formula: TTI (%) = Tumor region fluorescence intensity / Whole-body fluorescence intensity × 100% In vivo imaging of 4T1 tumor model mice is shown below. Figure 30-32 As shown, the tumor targeting index is as follows: Figure 33 As shown.

[0145] from Figure 30-32It is evident that throughout the observation period, the Blank-DIR-SLN group (Group A) showed non-specific distribution only in mononuclear phagocytic organs such as the liver and spleen, with no significant fluorescence signal observed at the tumor site, indicating a lack of targeting and rapid clearance. In contrast, all DIR-loaded nanoparticle formulations achieved some enrichment in the tumor region. The fluorescence signal intensities of the PPT-DIR-SLN, TMZ-DIR-SLN, and SHT-DIR-SLN groups were weak, but the PCH-DIR-SLN, TCH-DIR-SLN, and SHC-DIR-SLN groups showed particularly outstanding performance. From 1 to 18 hours after administration, the fluorescence intensity at the tumor site of the derivative formulation (solid lipid nanoparticles) group continued to increase, significantly exceeding that of its corresponding original drug formulation group (…). P < 0.01), and as time was extended to 18-26 hours, although the whole-body fluorescence signal generally weakened, the derivative formulation group still maintained a high fluorescence intensity in the tumor area, showing excellent tumor retention ability.

[0146] from Figure 33 (a) As can be seen, the PCH-DIR-SLN group showed the best performance within 26 hours after drug administration, with TTI continuously increasing and reaching a peak at 26 hours, at which time the TTI was 17.18%. Figure 33 (b) As can be seen, the TCH-DIR-SLN group reached its peak TTI of 5.84% within 1 hour of administration. Afterward, the TTI decreased and then rapidly increased again, which may be related to its unique central nervous system affinity. However, its targeted retention effect on the tumor site was subsequently stronger, with the TTI rising again and maintaining a plateau between 9 and 26 hours. Figure 33 (c) As can be seen, the TTI in the SHC-DIR-SLN group continued to rise within 26 hours after administration, reaching a peak of 5.30% at 26 hours. The rising TTI trend and high TTI value in the derivative drug group reflect that the derivative drug has good tumor targeting and long-term retention, suggesting that derivatization modification can enhance the active targeting effect of the drug on tumors and enhance the therapeutic effect on tumors.

[0147] (3) Study on antitumor activity in tumor model mice This invention evaluates the therapeutic efficacy and biosafety of the prepared drug-containing solid lipid nanoparticles by monitoring the dynamic changes in body weight and tumor volume of 4T1 tumor-bearing mice during treatment, combined with tumor weight analysis at the treatment endpoint, as follows: The specific steps were as follows: 4T1 tumor model mice were randomly divided into seven groups: saline group (control group), podophyllotoxin preparation group (PPT-SLN group), podophyllotoxin derivative preparation group (PCH-SLN group), temozolomide preparation group (TMZ-SLN group), temozolomide derivative preparation group (TCH-SLN group), (S)-10-hydroxycamptothecin preparation group (SHT-SLN group), and (S)-10-hydroxycamptothecin derivative preparation group (SHC-SLN group). Each group of tumor-bearing mice was injected intravenously with the corresponding drug (5 mg / kg, 200 μL) every two days, for a total of three injections. Tumor volume and body weight changes were monitored daily during the treatment period. The day after the last treatment, mice were euthanized by isoflurane anesthesia and cervical dislocation, and the tumors were removed and weighed. Tumor volume and tumor inhibition rate were calculated using the following formulas: Tumor volume (mm) 3 ) = Tumor inhibition rate (%) = × 100% Specific test results are as follows: Figures 34-39 As shown.

[0148] (3.1) Body weight analysis of tumor-bearing mice in the 4T1 tumor model: The changes in body weight of 4T1 tumor-bearing mice in each drug treatment group during treatment are as follows: Figure 34 (a) Figure 36 (a) and Figure 38 As shown in (a), the body weight of tumor-bearing mice in the saline group continuously decreased during treatment, possibly due to abnormal metabolism caused by tumor growth, leading to increased consumption of nutrients and energy. In contrast, the body weight of tumor-bearing mice in each drug treatment group increased to varying degrees during treatment, with their weights later exceeding those of the saline group. This suggests that the drugs exhibit good biocompatibility in vivo and did not have significant adverse effects on tumor-bearing mice, demonstrating a relatively ideal safety profile. Among these, the tumor-bearing mice in the derivative drug groups (i.e., PCH-SLN, TCH-SLN, and SHC-SLN groups) showed larger fluctuations in body weight increase, indicating that derivatization of the original drugs improved drug tolerance in tumor-bearing mice.

[0149] (3.2) Analysis of antitumor efficacy in 4T1 tumor model mice like Figure 34 (b), 36(b) and Figure 38As shown in (b), the tumor volume in the saline group showed a rapid growth trend during treatment, indicating that the model was successfully established and the tumor progressed rapidly. All drug treatment groups showed varying degrees of tumor growth inhibition effects. The drugs had a rapid onset of action and reduced the tumor volume in 4T1 tumor model mice after both single and multiple administrations.

[0150] At the end of treatment, the dissected tumor tissue was visually displayed. Figure 35 (a) Figure 37 (a) and Figure 39 (a) The tumor volume in the derivative formulation group is smaller than that in its corresponding original drug formulation group. For example... Figure 35 (b) Figure 37 (b) and Figure 39 As shown in (b), the comparison of the ex vivo tumor volume of 4T1 tumor-bearing mice in the drug formulation group shows that the PPT-SLN group, TMZ-SLN group, and SHT-SLN group can reduce the growth of tumor volume in 4T1 tumor-bearing mice. P < 0.05), while the PCH-SLN group, TCH-SLN group, and SHC-SLN group significantly reduced the growth of tumor volume in 4T1 tumor-bearing mice ( P < 0.01).

[0151] like Figure 35 (c) Figure 37 (c) and Figure 39 As shown in (c), the comparison of tumor inhibition rates of each drug group on 4T1 tumor model mice obtained from the analysis of tumor weight data at the treatment endpoint shows that after multiple administrations, all drug groups significantly inhibited tumor growth in 4T1 tumor-bearing mice. It is noteworthy that the tumor inhibition rate of the derivative drug group was significantly higher than that of its corresponding original drug group (…). P (< 0.01), the average tumor inhibition rate of the PCH-SLN group was approximately 95.73%, the average tumor inhibition rate of the TCH-SLN group was approximately 87.84%, and the average tumor inhibition rate of the SHC-SLN group was approximately 87.12%, demonstrating outstanding tumor inhibition effects and greatly improving the therapeutic index of the drugs.

[0152] The study comprehensively confirmed that derivatizing podophyllotoxin, temozolomide, and (S)-10-hydroxycamptothecin and constructing solid lipid nanoparticles can achieve the goal of "synergistic efficacy and reduced toxicity" by improving the drug's targeting and long-term retention on tumors. The high tumor inhibition rate demonstrates the potential of this strategy in inhibiting solid tumor growth, while the stable weight gain curve supports good biocompatibility and clinical application prospects, proving the universality and robustness of the "structure modification-nanodelivery" technology.

[0153] Example 5: Evaluation of the in vitro anti-inflammatory activity and cytotoxicity of galangin-cholesterol succinate and paeonol-cholesterol succinate. (1) MTT assay for cytotoxicity This invention uses the MTT assay to investigate the toxicity of sorbitan-cholesterol succinate (sorbitan derivative) prepared in Example 5, paeonol-cholesterol succinate (paeonol derivative) prepared in Example 6, and the corresponding original drugs (sorbitan and paeonol) to mouse mononuclear macrophages (RAW264.7 cells, purchased from Peking Union Medical College Hospital Cell Biology Institute). The specific steps are as follows: RAW264.7 cells in logarithmic growth phase were treated and 1.0 × 10⁻⁶ cells were prepared. 5 A single-cell suspension of high density was added to each well of a 96-well plate at a concentration of 100 mg / L. Cells were cultured at 37 °C and 5% CO2 in an incubator for 24 h. The experiment was divided into three groups: a blank group, a zeroing group, and a drug-treated group (the group with cell-containing culture medium was the zeroing group, the group with only culture medium was the blank group, and the group with cell-containing culture medium and a series of cholesterol succinate monoester derivatives was the drug-treated group). After 24 h of cell culture, the old culture medium was discarded, and 100 μL of each medium was added to the culture medium. L contains fresh culture medium with the corresponding drug, ensuring that the final drug concentration in each dosing group is 200. M / L, 100 M / L, 50 M / L, 25 M / L and 12.5 M / L, with 6 replicates per concentration. Incubate in an incubator for 24 h. After 24 h, discard the old culture medium and add 100 M / L to each well under dark conditions. Cells were cultured in fresh medium containing 5% MTT (5%, V / V) for 4 h. The absorbance at 490 nm was measured using a microplate reader, and the cell viability was calculated using the following formula: In the formula, OD control The absorbance of the zeroing group, OD sample The absorbance of the sample group (drug administration group) is [data missing]. OD blank The absorbance is corrected using DMEM culture medium as a blank.

[0154] from Figure 40 The MTT test results in (a) show that when the concentration is less than 100... At a concentration of M / L, the cell viability of the sorbitol group reached 98%, and that of the sorbitol derivative group (sorbitol-cholesterol succinate) reached 100%. It was almost non-toxic to RAW264.7 cells. As the drug concentration increased to 200... M / L, both reduced cell viability by about 10%, the cell viability of the sorbitol group was 82%, and the cell viability of the sorbitol derivative group was as high as 94%, and it was still non-toxic to RAW264.7 cells at low concentrations (12.5 ~ 50). M / L, the cell viability of the sorbitol derivative group was significantly higher than that of the sorbitol group (M / L). P < 0.01 or P < 0.05).

[0155] Depend on Figure 40 (b) It can be seen that as the concentration of paeonol and its derivative (paeonol-cholesterol succinate) increases, cell viability decreases, especially when the concentrations of both are less than 100%. At a concentration of M / L, cell viability reached 100%. As the concentration of both increased, cell viability decreased, reaching a minimum at 200 M / L. At a concentration of M / L, cell viability was approximately 90%, and both paeonol and its derivatives exhibited low cytotoxicity against RAW264.7 cells. At a concentration of 100 M / L, the cell viability was approximately 90%, and both paeonol and its derivatives also showed low cytotoxicity against RAW264.7 cells. At M / L, the cell survival rate in the paeonol derivative group was significantly higher than that in the paeonol group.

[0156] Comprehensive analysis shows that the series of succinate cholesterol derivatives improve the survival rate of RAW264.7 cells in a concentration-dependent manner, and have better biocompatibility compared to the original drug. Taking all factors into consideration, 200... M / L represents the highest concentration used in subsequent experiments of a series of cholesterol succinate derivatives and their corresponding active ingredients.

[0157] (2) Evaluation of anti-inflammatory efficacy This invention uses LPS to stimulate RAW264.7 cells to construct an in vitro cellular inflammation model. Preliminary experiments revealed that 1 LPS at a concentration of g / mL significantly promoted the secretion of TNF-α, IL-6, IL-1β, and NO in RAW264.7 cells, successfully establishing an in vitro inflammation model. Enzyme-linked immunosorbent assay (ELISA) was then used to quantify the amounts of TNF-α, IL-6, and IL-1β released by inflammatory cells, and the Griess method was used to quantify the release of NO, thereby quantitatively studying the anti-inflammatory efficacy of succinate cholesterol derivatives. Details are as follows: Prepare 1.0 × 10⁻⁶ ppm according to the above MTT toxicity test. 5 A high density of RAW264.7 single-cell suspension, at 200 μL per well. L was inoculated into 48-well culture plates and cultured for 24 h. After culture, a series of succinate cholesterol derivatives (i.e., sorbitan-cholesterol succinate prepared in Example 5 (hereinafter referred to as sorbitan derivative) and paeonol-cholesterol succinate prepared in Example 6 (hereinafter referred to as paeonol derivative)) and their corresponding original drugs (sorbitan and paeonol) were added, and incubated for 2 h. Then, LPS solution was added (ensuring a final concentration of 1). After co-incubation for 24 h with the reagents (g / mL), the supernatant was collected. The concentrations of TNF-α, IL-6, and IL-1β in the supernatant were quantified using the following ELISA method, and the concentration of NO in the supernatant was quantified using the following Griess method, according to the kit instructions.

[0158] Steps for measuring inflammatory factors using ELISA: 1) Cell supernatant treatment: Centrifuge the cell culture supernatant at 1000 rpm for 20 min in a low-temperature refrigerated centrifuge and take the supernatant for testing.

[0159] 2) Remove the kit, stored according to the instructions, from -20℃ and -4℃ respectively, and slowly allow it to return to room temperature (18~25℃). Prepare standard samples of different concentrations, and mix 100ml each of the standard samples and cell supernatant. Add L to the plate and incubate at 37 °C for 60 min.

[0160] 3) After incubation, discard the liquid in the plate and add 100 ml of detection solution A (the finished kit, biotinylated antibody working solution) to each well. L (prepared immediately before use), incubate at 37 °C for 60 min with film covering.

[0161] 4) Discard the liquid in the well and add 350 ml of liquid to each well. Wash 5 times with L of washing solution, 1-2 minutes each time, and drain the residual liquid in the hole on the last wash.

[0162] 5) Add 100 to each hole L detection solution B (the finished kit, enzyme conjugate working solution) (prepared immediately before use), cover with a membrane, and incubate at 37 °C for 30 min.

[0163] 6) Discard the liquid in the well and add 350 ml of liquid to each well. Wash 5 times with L washing solution, 1-2 min / wash, same as operation 3, and drain all liquid from the hole on the last wash.

[0164] 7) Add 90 to each hole The mixture was developed with L 3,3',5,5'-tetramethylbenzidine (TMB) solution, coated with a film, and incubated in the dark at 37 °C, with observations made frequently (approximately 20 min).

[0165] 8) Add 50 to each hole The reaction was stopped with L stop solution, and the optical density (OD value) was measured at 450 nm using an ELISA reader.

[0166] 9) Plot a standard curve with the standard OD value as the X-axis and the concentration as the Y-axis, and calculate the concentrations of TNF-α, IL-6 and IL-1β in the cell supernatant.

[0167] Griess method for NO determination steps: 1) Cell supernatant treatment: Centrifuge the cell culture supernatant at 300 rpm for 5 min on a low-temperature refrigerated centrifuge and take the supernatant for testing.

[0168] 2) Preparation of standards at different concentrations: using 200 M NaNO2 was used as the stock solution, and 3.12 solutions were prepared sequentially using the sample diluent. M, 6.25 M, 12.5 M, 50 M and 100 M is a standard solution of NaNO2.

[0169] 3) Sample addition method: Refer to Table 11 for the sample addition method, add 50 μL of sample to each well of the 96-well plate sequentially. L standard and cell culture supernatant, then 50 Mix L Griess Reagent I and Griess Reagent II, and incubate at room temperature for 5 minutes.

[0170] 4) After the reaction is complete, measure the OD value at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Plot a standard curve based on the OD values ​​and concentrations of the standard and calculate the NO content in the cell supernatant.

[0171] Table 11 The experimental results on the effects of cholesterol succinate monoester derivatives and the original drug on the levels of inflammatory factors TNF-α, IL-6, and IL-1β are as follows: Figures 41-42 As shown, changes in the levels of TNF-α, IL-6, and IL-1β can indirectly reflect the degree of RAW264.7 cell activation, and also represent the level of inflammation in the body. Figures 41-42 In the diagram, + indicates the addition of the corresponding substance, and - indicates the absence of the corresponding substance. Compared to the blank control group (corresponding to...), ... Figure 41 LPS-, sago-, sago derivative-; corresponding Figure 42 Compared to LPS-, paeonol-, and paeonol derivative-, the model group (corresponding to LPS-, paeonol-, and paeonol derivative-) Figure 41 LPS+, sorbitan-, sorbitan derivative-; corresponding Figure 42 The levels of TNF-α, IL-6, and IL-1β in LPS+, paeonol-, and paeonol derivatives- were all significantly increased, showing highly significant differences. P The result was < 0.01, indicating that LPS successfully induced an inflammation model in RAW264.7 cells.

[0172] The effects of sorbitan and its derivatives (i.e., sorbitan cholesterol succinate) on the expression levels of inflammatory factors in RAW264.7 cells are as follows: Figure 41 As shown. Compared to the model group: low concentration 50 At M / L, sorbitan and its derivatives showed extremely weak inhibitory effects on the expression of inflammatory factors TNF-α, IL-6, and IL-1β, with no significant difference in their content. P > 0.05). With increasing concentrations of sorbitan and its derivatives, their ability to inhibit the expression of TNF-α, IL-6, and IL-1β significantly increased; at a concentration of 100... At M / L, sorbitan and its derivatives have an inhibitory effect on the expression of TNF-α, IL-6 and IL-1β. P < 0.05); concentration of 200 At M / L, sorbitan and its derivatives significantly inhibited the expression of TNF-α and IL-6. P < 0.01), which has an inhibitory effect on IL-1β expression ( P < 0.05), and compared with sorbitan derivatives, there was no significant difference in the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β at various concentrations. P > 0.05).

[0173] The effects of paeonol and its derivatives (i.e., paeonol cholesterol succinate) on the expression levels of inflammatory factors in RAW264.7 cells are as follows: Figure 42 As shown. Compared to the model group: low concentration 50 At a concentration of M / L, paeonol and its derivatives inhibited TNF-α expression in a concentration-dependent manner; the inhibitory effect became more pronounced with increasing concentration. There was a highly significant difference at M / L ( P < 0.01); for IL-6 expression, only high concentrations of 200 At M / L, paeonol and its derivatives exhibit significant inhibitory effects. P < 0.01); for IL-1β expression, low concentration 50 At M / L, paeonol has an inhibitory effect ( P < 0.05), 100 M / L and 200 At M / L, both paeonol and paeonol derivatives have inhibitory effects. P < 0.05 or P < 0.01). In addition, compared to paeonol derivatives, at a concentration of 100... M / L and 200 At M / L, the paeonol derivative showed a stronger ability to inhibit IL-1β expression than paeonol, with a significant difference. P < 0.05).

[0174] The experimental results on the effects of cholesterol succinate monoester derivatives and the original drug on NO levels are as follows: Figure 43 As shown in the figure, compared with the blank control group, the NO content in the model group was significantly increased, which was statistically significant. P < 0.01). Combined with changes in the levels of three inflammatory factors—TNF-α, IL-6, and IL-1β—it demonstrates that LPS successfully induced a reliable inflammation model in RAW264.7 cells in vitro.

[0175] The effects of sago palmitin and its derivatives (i.e., sago palmitin cholesterol succinate) on NO concentration in RAW264.7 cells are as follows: Figure 43 As shown in (a), compared to the model group, the low concentration of 50 At M / L, the release of NO in RAW264.7 cells was reduced in the sorbitol and sorbitol derivative groups, but this was not statistically significant. P > 0.05), as the concentration increases to 100~200 M / L, sorbitan and sorbitan derivatives showed strong inhibitory effects on NO release, and this effect was concentration-dependent; the higher the concentration, the stronger the inhibitory effect, and the changes in NO concentration were statistically significant. P < 0.01 or P < 0.05). In addition, when the concentration is 100... M / L and 200 At M / L, the ability of sorbitol derivatives to inhibit NO release was significantly better than that of sorbitol ( P < 0.05 indicates that under high concentration conditions, the anti-inflammatory activity of sorbitan derivative (sorbitan cholesterol succinate) is stronger than that of sorbitan, and the higher the concentration, the stronger the anti-inflammatory effect.

[0176] The effects of paeonol and its derivatives (i.e., paeonol cholesterol succinate) on NO concentration in RAW264.7 cells are as follows: Figure 43 As shown in (b). Compared with the model group, the low concentration of 50 At M / L, paeonol and its derivatives showed a certain inhibitory effect on the release of NO from RAW264.7 cells, which was statistically significant. P < 0.05), the inhibitory ability increases significantly with increasing concentration, showing a concentration-dependent relationship. When the concentration reaches 200, the inhibitory effect is enhanced. At M / L, both paeonol and its derivatives showed highly significant differences compared to the model group, which was statistically significant. P < 0.01), but there was no statistically significant difference between paeonol and paeonol derivatives ( P > 0.05).

[0177] The above in vitro experiments showed that sorbitol, paeonol, and their derivatives significantly reduced the secretion of TNF-α, IL-1β, and IL-6, as well as the concentration of NO. Furthermore, the derivatives showed superior performance compared to the original drugs in only a few specific indicators. Therefore, the series of succinate cholesterol derivatives can improve the survival rate of RAW264.7 cells in vitro in a concentration-dependent manner. Compared to the original drugs, they have better biocompatibility and can inhibit the release of pro-inflammatory factors to varying degrees, exhibiting some anti-inflammatory effects. However, their anti-inflammatory activity is not significantly altered compared to the original drugs.

[0178] Example 6: Evaluation of the in vivo anti-inflammatory activity of galangin-cholesterol succinate and paeonol-cholesterol succinate This experiment aims to conduct preliminary animal efficacy evaluations of the in vivo anti-inflammatory activity of a series of succinate cholesterol derivatives using xylene-induced ear swelling and carrageenan-induced paw swelling assays in mice. Details are as follows: (1) Xylene-induced ear swelling experiment Preparation of dexamethasone solution: Accurately weigh 20 mg of dexamethasone powder into 8 mL of physiological saline to prepare a 2.5 mg / mL dexamethasone solution for later use.

[0179] According to the random principle, 48 KM mice (male, body weight about 20 ± 5 g, purchased from Beijing Sibeifu Laboratory Animal Co., Ltd., license number: SCXK (Beijing) 2019-0010. The indoor humidity of the animal room was maintained at 65%, and the temperature was 25 ± 2 °C. After 1 week of adaptive feeding, drinking water, feed, and bedding all met the animal use standards, and the animal experiment specifications were strictly followed) were randomly divided into 8 groups, with 6 mice in each group. The groups were respectively blank control group, positive drug group (dexamethasone group), original drug group (alpinetin group, paeonol group), and derivative drug group (alpinetin cholesterol succinate group (referred to as alpinetin derivative group), paeonol cholesterol succinate group (referred to as paeonol derivative group)). The blank control group and the dexamethasone group (DEX) were intragastrically administered with normal saline and 10 mg / kg DEX every day, and the mice in the other groups were also intragastrically administered with the corresponding drug suspension solution of 40 mg / kg every day for 5 consecutive days. 30 minutes after the last administration, xylene was applied to induce inflammation. In this experiment, the self-control method of left and right ears was adopted. The specific operation was as follows: 0.02 mL of xylene was evenly applied to the front and back sides of the right ear of each mouse to induce inflammation, and the left ear was not coated with xylene as a control. After 1 hour, the mice were sacrificed by cervical dislocation, the complete left and right ears were cut off, and ear pieces were taken at the same site with a 7 mm diameter punch and weighed. The degree of swelling was expressed by the mass difference between the left and right ear pieces. The auricle swelling of mice was calculated according to the following formula, and the specific results are shown in Table 12: Inhibition rate (%) = (Average swelling degree of control group - Average swelling degree of administration group) / Average swelling degree of control group × 100% Table 12 Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; Compared with the original drug group, P < 0.05, P < 0.01 In the mouse ear swelling model, the positive drug (DEX) group, alpinetin group, alpinetin derivative group, paeonol group, and paeonol derivative group can all reduce the degree of mouse ear swelling and decrease the swelling rate to a certain extent ( P < 0.05 or P <0.01). Compared with the blank control group, the ear swelling degree of the positive drug group, alpinetin group and alpinetin derivative group was significantly improved, with statistical significance ( P < 0.05 or P< 0.01), among which sorbitan and sorbitan derivatives inhibited mouse ear swelling by 42.14% and 54.54%, respectively. Compared with sorbitan, the inhibitory effect of sorbitan derivatives was more prominent and statistically significant. P < 0.05); the inhibition rates of paeonol and paeonol derivatives on mouse ear swelling were 34.71% and 56.20%, respectively, and at the same molar concentration, the inhibitory effect of paeonol derivatives was more prominent than that of paeonol, which was statistically significant. P < 0.01).

[0180] (2) Carrageenan-induced foot edema experiment Preparation of carrageenan solution: Accurately weigh 0.1 g of carrageenan and dissolve it in 10 mL of physiological saline to prepare a 1% carrageenan solution for later use.

[0181] The grouping and administration methods were the same as in the ear swelling experiment described above. One hour after the last administration, a horizontal line was drawn on the upper part of the left hind paw of each mouse, and the thickness of the toe at that point was measured with calipers. Subsequently, 0.1 mL of 1% carrageenan was injected subcutaneously to induce inflammation, and the toe thickness was measured at the marked point at 0.5 h, 1 h, 2 h, 4 h, and 6 h after inflammation. The swelling of the mouse's toe was calculated using the following formula: Swelling thickness (mm) = Post-inflammatory toe thickness (mm) - Pre-inflammatory toe thickness (mm); Swelling rate (%) = (Post-inflammatory toe thickness - Pre-inflammatory toe thickness) / Pre-inflammatory toe thickness × 100%; Inhibition rate (%) = (average toe thickness of control group - average toe thickness of treatment group) / average toe thickness of control group × 100%, where the average toe thickness of control group and the average toe thickness of treatment group are both calculated based on 6 hours.

[0182] In a mouse paw edema model, the inhibitory effects of equimolar concentrations of the original drug and corresponding cholesterol succinate derivatives on the degree of paw edema were investigated. The effects were expressed as three indicators: edema degree, paw edema change rate, and paw edema inhibition rate. To comprehensively evaluate the change in paw edema degree over time, the area under the curve (AUC) corresponding to the paw edema change rate was used for statistical analysis. Significant differences in edema degree were not indicated. The corresponding statistical indicators and AUC are shown in Figure 44. Figure 45 As shown in Table 13.

[0183] Table 13 Note: Compared with the blank control group, # P < 0.05, ## P <0.01; Compared with the original drug group, P < 0.05, P < 0.01.

[0184] Figure 44 Experimental data showed that, compared with the blank control group, both sorbitan and its derivative (sorbitan cholesterol succinate) exhibited certain anti-inflammatory effects. P < 0.05 or P < 0.01), both sorbitol and scutellarin showed sustained inhibition of paw edema in mice starting 3 hours after inflammatory induction, with the sorbitol derivative exhibiting the most significant anti-inflammatory effect around 4 hours after inflammatory induction, and showing almost no change in anti-inflammatory activity between 5 and 6 hours. Compared with sorbitol, the sorbitol derivative showed stronger anti-inflammatory activity, which was statistically significant ( P < 0.05), the inhibition rate of sorbitol derivative was 53.79%, and the inhibition rate of sorbitol was 45.15%.

[0185] from Figure 45 As can be seen from the results, compared with the blank control group, both paeonol and paeonol cholesterol succinate have certain anti-inflammatory effects, and both can continuously inhibit paw swelling in mice starting from 2 hours after inflammation induction. Among them, paeonol cholesterol succinate has an inhibition rate as high as 57.58%, showing significant anti-inflammatory activity, and is superior to the paeonol group (37.12%), which is statistically significant. P < 0.01).

[0186] The above experimental results show that sorbitol, paeonol, and their cholesterol succinate derivatives can inhibit ear and paw swelling in mice, and have a good therapeutic effect on superficial inflammation.

[0187] (3) The therapeutic effect of cholesterol succinate monoester derivatives on deep inflammation This experiment aims to use lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as a pathological model to verify the anti-inflammatory effects of a series of cholesterol succinate monoester derivatives and to investigate their efficacy against deep inflammation, as detailed below: 1) Establishment of a lung injury model Based on the principle of randomness, 42 KM mice were randomly divided into 7 groups of 6 mice each, using a random number table. The groups were: blank group, model group, positive drug group (DEX group), sorbitol group, sorbitol derivative group, paeonol group, and paeonol derivative group.

[0188] Control group: Mice were given 0.1 mL of physiological saline by gavage for 7 consecutive days. On the 7th day, sterile physiological saline was injected intraperitoneally.

[0189] Model group: Mice were given 0.1 mL of physiological saline by gavage for 7 consecutive days. On the 7th day, 8 mg / kg of LPS was injected intraperitoneally to induce an ALI model.

[0190] Positive control group (DEX): Mice were administered 10 mg / kg of DEX solution by gavage daily for 7 consecutive days. On day 7, 8 mg / kg of LPS was injected intraperitoneally to replicate the ALI model.

[0191] Sago palmatum group: Mice were administered 40 mg / kg of sago palmatum suspension by gavage for 7 consecutive days. On day 7, mice were injected intraperitoneally with 8 mg / kg of LPS to replicate the ALI model.

[0192] Alpinia sorbitol derivative group: Mice were administered alkaloid cholesterol succinate suspension (calculated as alkaloid 40 mg / kg) by gavage for 7 consecutive days. On the 7th day, 8 mg / kg of LPS was injected intraperitoneally to replicate the ALI model.

[0193] Paeonol group: Mice were administered 40 mg / kg of paeonol suspension by gavage daily for 7 consecutive days. On day 7, mice were injected intraperitoneally with 8 mg / kg of LPS to replicate the ALI model.

[0194] Paeonol derivative group: Mice were administered paeonol cholesterol succinate suspension (calculated as paeonol 40 mg / kg) by gavage daily for 7 consecutive days. On the 7th day, 8 mg / kg of LPS was injected intraperitoneally to replicate the ALI model.

[0195] The above ALI model was constructed as follows: after continuous gavage administration for 7 days, mice in each group were injected intraperitoneally with 8 mg / kg LPS to establish the model. They were sacrificed 12 hours later, and lung tissue was separated.

[0196] 2) Lung tissue acquisition: Twelve hours after LPS modeling, mice in each group were euthanized by cervical dislocation. The mice were placed supine on the operating table, and the operating site was disinfected with 75% alcohol. The thoracic cavity was opened to fully expose the lobes of both lungs. The right lung tissue was then separated, rinsed with physiological saline to remove residual blood, and lung tissue from different locations was surgically excised and placed into pre-cooled EP tubes. The anterior lobe of the right lung was placed on weighed foil for wet-to-dry weight (W / D) measurement; the posterior lobe of the right lung was placed in a centrifuge tube containing 4% tissue fixative for H&E staining; the remaining lung tissue was rapidly placed at -80 °C for subsequent determination of inflammatory factor expression.

[0197] 3) Preparation of lung tissue homogenate: The lung tissue was obtained by means of the above method (mouse lung tissue). The obtained mouse lung tissue was washed with physiological saline to remove residual blood, the water was absorbed by filter paper, weighed and placed in a pre-labeled 2 mL EP tube. Then a certain amount of PBS solution was added to prepare a 10% tissue homogenate. The homogenate was homogenized with a multi-throughput tissue homogenizer and centrifuged at 3000 rpm for 10 min in a low-temperature refrigerated centrifuge. The supernatant was transferred to a blank EP tube and labeled for subsequent measurement of inflammatory factors.

[0198] 4) Observation indicators 4.1) General condition observation of mice: 12 h after modeling, the general condition of mice in different experimental groups was observed, including feeding and survival, mental state, responsiveness, hair luster, activity level and weight change.

[0199] The experimental results showed that 12 hours after modeling, the mice in the blank control group who received intraperitoneal injection of saline showed normal general condition, including mental state, food intake, responsiveness, hair growth, and weight. Their breathing was stable and rhythmic, with no significant difference from before modeling. In the model group induced by intraperitoneal injection of LPS solution, most mice showed significant weight loss, poor mental state, sluggishness, darkened and partially erect fur, and reduced movement. Palpation of the chest cavity revealed obvious symptoms of respiratory distress and increased respiratory rate. The mice in the drug-treated groups showed varying conditions; although their mental state was slightly worse, their responsiveness and activity levels were still acceptable, and the increased respiratory rate was reduced. The mice in the succinate cholesterol derivative group showed the best activity level.

[0200] 4.2) Pathological observation of lung tissue: The fixed right lung tissue was removed and dehydrated, embedded, and sectioned in sequence. Then, the sections were dewaxed, cleared, and baked. After staining with hematoxylin and eosin, the tissue was dehydrated, cleared, and mounted. Finally, the lung tissue was observed under a CX21 fluorescence inverted microscope, and images were acquired and analyzed.

[0201] The apparent effects of different drugs on mouse lung tissue, such as Figure 46 As shown in the figure. The results showed that the lung tissue of the normal group mice was pink, and compared with the model group, the lung tissue volume was smaller and there was no edema. Both the model group and the drug-treated group showed varying degrees of edema. The specific results were analyzed in conjunction with HE-stained sections.

[0202] Pathological morphological changes in mouse lung tissue as follows Figure 47As shown in the figure. The results showed that the lung tissue structure of the mice in the blank control group was clear and intact, with no obvious necrotic cells, uniform alveolar size distribution, thin alveolar walls, and basically normal alveolar septa. There was no obvious hemorrhage or edema in the lung tissue, and the overall pathological changes were not obvious. Compared with the blank control group, 12 hours after intraperitoneal injection of LPS, the lung tissue morphology and alveolar structural integrity of the model group were significantly disrupted. The mice's lungs showed symptoms such as alveolar atrophy, focal thickening of alveolar walls, and widening of alveolar septa. Congestion and inflammatory cell infiltration were visible in the lung tissue, showing diffuse pulmonary edema. This indicates that intraperitoneal injection of LPS successfully caused inflammatory damage to the lungs. Compared with the model group, the positive control group (DEX) significantly improved the lung tissue damage in mice. The alveolar collapse, alveolar wall thickening, alveolar septal widening, lung congestion, and degree of inflammatory cell infiltration were all reduced, and the pathological changes were close to those of the blank control group mice.

[0203] Mice treated with galangin and its derivatives showed significantly improved lung tissue damage compared to the model group. Alveolar collapse, alveolar wall thickening, and lung congestion were reduced, along with a marked decrease in inflammatory cell infiltration. The galangin derivative group showed a more significant reduction in lung tissue damage, primarily manifested in a marked reduction in alveolar wall thickening. Mice treated with paeonol and its derivatives showed similar recovery to the galangin and derivative groups, with a substantial reduction in lung pathology and a degree of improvement. Furthermore, the derivative group exhibited the same pattern as the original drug group, showing greater improvement than the original drug group.

[0204] Therefore, the above results indicate that sorbitol, paeonol and their derivatives can significantly reduce the degree of LPS-induced lung injury to a certain extent, maintain the normal morphology and integrity of lung tissue, and the degree of reduction in the derivative treatment group is better than that in the original drug treatment group.

[0205] 4.3) Measurement of lung wet / dry weight (W / D) The wet-to-dry weight ratio (W / D) of lung tissue is mainly used to measure the degree of fluid exudation and edema in the lungs, and it can indirectly reflect the degree of pathological damage to the lungs. The specific measurement method is as follows: the anterior lobe of the right lung, placed on tin foil, is weighed on a precision balance, and the wet weight of the lung tissue is recorded. W The lung tissue, along with the aluminum foil, was then placed in a 60°C incubator for 72 hours to allow for complete dehydration. It was then weighed again using a precision balance, and the dry weight of the lung tissue was recorded. D ), calculate the net wet weight of lung tissue ( W ) / Net dry weight ( D The ratio of ) is the lung tissue water content, and the specific results are shown in Table 14.

[0206] Table 14 Note: Compared with the blank control group, ## P < 0.01; compared with the model group, P < 0.05, P <0.01; Compared with the original drug group, P < 0.05, P < 0.01 The data in the table above show that, compared with the blank control group, the W / D ratio in the model group was significantly higher 12 hours after modeling, which was statistically significant. P The value < 0.01 indicates that intraperitoneal injection of LPS can induce lung inflammation, and the model was successfully established. Compared with the model group, the lung tissue W / D ratio of mice treated with the positive drug (DEX) was significantly reduced, showing a highly significant difference. P < 0.01), the W / D value of the sorbitol group decreased accordingly, showing a significant difference ( P < 0.05), the sorbitol derivative group showed a highly significant difference ( P < 0.01), while there was no significant difference between the sorbitan group and the sorbitan derivative group ( P >0.05), but the W / D value of the sorbitol derivative group was lower than that of sorbitol.

[0207] Similarly, for the paeonol group and the paeonol derivative group, compared with the model group, the W / D ratio of the lung tissue of mice treated with paeonol derivatives was significantly reduced, showing a highly significant difference. P < 0.01), the paeonol group showed a corresponding decrease, indicating a significant difference ( P < 0.05), and the paeonol group showed a significant difference compared to the paeonol derivative group ( P <0.05) Overall analysis shows that the original drug group (ziziphus julibrissin group, paeonol group) and the derivative group (ziziphus julibrissin derivative group, paeonol derivative group) can reduce the dry-wet weight ratio of lung tissue induced by LPS to a certain extent. Among them, the derivatives have the most obvious anti-inflammatory effect compared with the original drug.

[0208] 4.4) Detection of inflammatory factors TNF-α, IL-6, and IL-1β using NO content. Take the supernatant of the lung tissue homogenate prepared in section 3) above, and use an ELISA kit to detect the concentrations of TNF-α, IL-6, and IL-1β. Determine the NO content using the Griess method. The procedure must be strictly followed according to the instructions. Specific steps are detailed in the aforementioned ELISA procedure for determining inflammatory factors and the Griess method for determining NO. See Table 15 for the specific results. Table 15 Note: Compared with the blank control group, ## P < 0.01; Compared with the model group, P < 0.05, P <0.01; Compared with the original drug group, P < 0.05, P < 0.01.

[0209] The results showed that, compared with the model group, the expression levels of TNF-α, IL-6, and IL-1β in the lung tissue homogenate of mice treated with sorbitol, sorbitol derivatives, paeonol, and paeonol derivatives were significantly reduced, and the differences were statistically significant. P < 0.05 or P < 0.01). The expression levels of IL-1β differed significantly between sorbitan and its derivatives. P < 0.05, meaning that the sorbitol derivative has a better ability to inhibit IL-1β expression than sorbitol, while there is no significant difference in the expression levels of TNF-α and IL-6. P > 0.05). Compared with paeonol, paeonol derivatives significantly reduced the expression levels of IL-6 and IL-1β in mouse lung tissue homogenate, which was statistically significant. P < 0.05), TNF-α expression level was not statistically significant ( P >0.05). Paeonol and its derivatives exhibit the same pattern as sorbitol and its derivatives: the succinate cholesterol derivatives obtained by modifying the original drug show a strong ability to inhibit the expression of TNF-α, IL-6 and IL-1β.

[0210] The NO content in the lung tissue homogenates of each group of mice is shown in Table 16 below: Table 16 Note: Compared with the blank control group, ## P< 0.01; Compared with the model group, P < 0.05, P <0.01; Compared with the original drug group, P < 0.05, P < 0.01 The NO content in the lung tissue homogenate of the sorbitol and sorbitol derivative group and the paeonol and paeonol derivative group was significantly different from that of the model group. P < 0.05), but there was no statistically significant difference between sorbitan and sorbitan derivatives. P > 0.05). Compared with the paeonol group, the paeonol derivative groups were all statistically significant ( P The value of paeonol derivatives (< 0.05) indicates that the ability of paeonol derivatives to inhibit NO release is superior to that of the original paeonol, and that they have a strong preventive effect against acute lung injury in mice.

[0211] Experimental Example 7: Stability Evaluation of Paeonol Cholesterol Succinate This invention uses paeonol cholesterol succinate as an example to demonstrate its stability in artificial gastrointestinal fluid and in the presence of lipase (inflammatory microenvironment) from the aspects of gastrointestinal stability and in vitro release. It verifies whether paeonol cholesterol succinate can remain stable in gastrointestinal fluid and can continuously accumulate and release paeonol in an inflammatory microenvironment. Specifically: (1) Stability in artificial gastrointestinal fluid Preparation of artificial gastrointestinal fluid: According to the method in Part IV of the 2020 edition of the Chinese Pharmacopoeia, and based on the actual experimental dosage, a certain scale was reduced to prepare 100 mL of artificial gastric and intestinal fluid. The operation method was scaled down 10 times from the pharmacopoeia method, while the other operations were the same. The fluid should be prepared and used immediately.

[0212] Stability test in artificial gastric fluid: A certain amount of paeonol cholesterol succinate (prepared in Example 6) was accurately weighed and dissolved in 6 mL of artificial gastric fluid. The sample was placed in a 37 ℃ constant temperature water bath shaker for horizontal shaking incubation, with three replicates per group. Samples were taken at 0 h, 1 h, 2 h, and 3 h, and continuously for 3 h. Each 150 μL sample was added with an appropriate amount of methanol to inactivate pepsin, and then diluted with 0.22 mL of methanol. Liquid phase samples were prepared using a microporous membrane, and the contents of paeonol cholesterol succinate and paeonol were determined by UPLC: when determining paeonol cholesterol succinate, the content of paeonol cholesterol succinate at 0 h was set as 100%; when determining paeonol, the content of paeonol contained in paeonol cholesterol succinate at 0 h was set as 100%, and the content of paeonol in artificial gastric fluid at 0 h was set as 0.

[0213] Stability test in artificial intestinal fluid: A certain amount of paeonol cholesterol succinate (prepared in Example 6) was accurately weighed and dissolved in 6 mL of artificial intestinal fluid. The sample was placed in a 37 ℃ constant temperature water bath shaker for horizontal shaking incubation, with 3 replicates per group. Samples were taken at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, for a continuous 6 h. Each time, 150 μL of sample was added to an appropriate amount of methanol to inactivate pepsin, and then diluted with 0.22 mL of methanol. Liquid phase samples were prepared using microporous membranes, and the contents of paeonol cholesterol succinate and paeonol were determined by UPLC: When determining paeonol cholesterol succinate, the content of paeonol cholesterol succinate at 0 h was set as 100%; when determining paeonol, the content of paeonol contained in paeonol cholesterol succinate at 0 h was set as 100%, and the content of paeonol in artificial intestinal fluid at 0 h was set as 0.

[0214] The average residual amounts of paeonol cholesterol succinate in artificial gastric and intestinal fluids are shown in Tables 17 and 18.

[0215] Table 17 Average residual amount of paeonol cholesterol succinate in simulated gastric fluid and average amount of paeonol produced ( x± s , n =3) Table 18 Average residual amount of paeonol cholesterol succinate in artificial intestinal fluid and average amount of paeonol produced ( x± s , n =3) Normally, after entering the body, drugs remain in the stomach for 2-3 hours and in the small intestine for 4-6 hours. As can be seen from the data in Tables 17 and 18 above, in simulated gastric fluid, the average remaining amount of paeonol cholesterol succinate after 3 hours of incubation was approximately 86.19%, and the average amount of paeonol generated after 3 hours of incubation was approximately 9.77%. In simulated intestinal fluid, the average remaining amount of paeonol cholesterol succinate after 6 hours of incubation was approximately 89.22%, and the average amount of paeonol generated was approximately 11.66%.

[0216] In summary, paeonol cholesterol succinate exhibits good stability in artificial gastric and intestinal fluids, with better stability in artificial intestinal fluids than in artificial gastric fluids. Gastrointestinal administration can be considered as a possible route of administration when investigating tissue distribution in later stages.

[0217] (2) Hydrolysis in body fluids within the artificial inflammatory microenvironment: In this experiment, 200 U / mL lipase was added to artificial normal body fluid to create an artificial inflammatory microenvironment, namely PBS buffer containing (200 U / mL lipase + 0.5% SDS).

[0218] The ability of paeonol cholesterol succinate to release paeonol via hydrolysis was evaluated in an artificial inflammatory microenvironment fluid. First, 2 mg of paeonol cholesterol succinate was dissolved in both artificial normal body fluid and artificial inflammatory microenvironment fluid, and the ends were tied tightly. Dialysis bags were then placed in 45 mL of each fluid, and the mixture was shaken in a constant temperature water bath at 70 rpm and 37 °C. Samples (1 mL) were taken at 0.15 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h of shaking, and an equal volume of the corresponding release medium was added. The solutions were then diluted with 0.22 mL of the solution. Liquid phase samples were prepared using a filter membrane, and the concentration of paeonol after hydrolysis was determined by UPLC.

[0219] With the amount of paeonol contained in paeonol cholesterol succinate set as 100%, and the content of paeonol in 0-hour artificial normal body fluid and artificial inflammatory microenvironment body fluid set as 0%, in vitro drug release curves of paeonol cholesterol succinate in two different release media were plotted with the cumulative release percentage at each time point as the ordinate and the sampling time as the abscissa. The results are as follows: Figure 48 As shown.

[0220] In an artificial inflammatory microenvironment where lipase is present, paeonol cholesterol succinate can hydrolyze and release paeonol. The cumulative release after 24 hours is approximately 96.33%, and after 48 hours, approximately 98.00%. Following this trend, paeonol cholesterol succinate can be completely hydrolyzed over time. However, in artificial normal body fluids, due to the absence of lipase, the hydrolysis rate of paeonol cholesterol succinate is extremely low, with only 19.15% of paeonol released after 48 hours. This indicates that paeonol cholesterol succinate has weak hydrolytic ability and low paeonol release in artificial normal body fluids.

[0221] In summary, paeonol cholesterol succinate can be hydrolyzed to release paeonol under artificial inflammatory microenvironment conditions in the presence of lipase, and can be completely hydrolyzed over time to release paeonol.

[0222] Experimental Example 8 In Vivo Efficacy Verification of Dihydroartemisinin-Cholesteryl Succinate Liposomes To systematically compare and evaluate the protective effects of DHA and DHA-CS on acute lung injury, a mouse acute lung injury model induced by intraperitoneal injection of LPS was used as the research system. Dexamethasone, a first-line clinical anti-inflammatory drug, was simultaneously set as the positive control group (positive drug group). From multiple dimensions including dynamic monitoring of the animals' growth status for 7 consecutive days, toxicity assessment of key organs including heart, liver and kidney, regulation of splenic immune homeostasis, semi-quantitative analysis of lung histopathology, quantification of the degree of pulmonary edema by lung wet-to-dry weight ratio, and the expression levels of core pro-inflammatory factors in peripheral plasma and local lung tissue, the in vivo protective effects of different doses of DHA and DHA-CS on mice with acute lung injury were systematically compared and investigated, the dose-effect dependency relationship of the two drugs and the structure-activity improvement advantage after cholesteryl succinate modification were clarified, and the anti-inflammatory activity of the derivative was determined on the basis of fully verifying the systemic administration safety of the test substances.

[0223] Experimental animals: Kunming (KM) mice, male, weighing about 25 ± 5 g, were purchased from Beijing Sibeifu Experimental Animal Co., Ltd., with the license number: SCXK (Beijing) 2019-0010. The indoor humidity of the animal room was maintained at 65%, and the temperature was 25 ± 2 ℃. The animals were adaptively reared for 1 week, the rearing environment had a 12 h / 12 h circadian rhythm, and the animals had free access to food and water. The feed and bedding materials all met the SPF-grade animal use standards. The whole experiment strictly followed the "Guidelines for the Ethics and Use of Experimental Animals", and all animal operation procedures were reviewed and approved by the Experimental Animal Ethics Committee of our university. During the experiment, animal pain and injury were minimized to ensure the standardization and ethical compliance of the animal experiment.

[0224] Main experimental reagents: Lipopolysaccharide (LPS, Shanghai Jizhi Biochemical Technology Co., Ltd.; Article No.: AL8880); Dexamethasone (DEX, 99%, Shanghai Yuanye Bio-Technology Co., Ltd.); 4% paraformaldehyde tissue fixative solution (Beijing Bairuiji Biotechnology Co., Ltd., Batch No. 20251030); Mouse interleukin-1β ELISA kit (Mouse IL-1 beta Uncoated ELISA, Batch No.: 462134-001, Thermo Fisher Scientific); Mouse tumor necrosis factor-α ELISA kit (Mouse TNF-a ELISA Kit, Batch No.: RRDC2604015, Servicebio); Mouse interleukin-6 ELISA kit (Mouse IL-6 ELISA Kit, Batch No.: RRDC2510010, Servicebio); Conventional reagents such as sterile normal saline, PBS buffer, hematoxylin-eosin staining solution, and 75% medical disinfectant alcohol are all of analytical grade.

[0225] Preparation of main reagent (LPS solution): Accurately weigh 20 mg of LPS powder and dissolve it in 10 mL of sterile physiological saline. Vortex until the powder is completely dissolved and the solution is clear and free of precipitate to prepare an LPS modeling solution with a final concentration of 2 mg / mL. All LPS solutions were prepared on the day of the experiment and stored briefly at low temperature and protected from light.

[0226] 1) Establishment of a lung injury model Based on the random number table method, 42 healthy male KM mice were randomly divided into 7 groups of 6 mice each. The groups were: blank group, model group, positive drug group, low-dose DHA group, high-dose DHA group, low-dose DHA-CS group, and high-dose DHA-CS group.

[0227] Control group: Sterile saline was injected intraperitoneally daily for 7 consecutive days. On the 7th day, an equal volume of sterile saline was injected intraperitoneally simultaneously. No model was established. Model group: Sterile saline was injected intraperitoneally daily for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to induce an acute lung injury (ALI) model in mice.

[0228] Positive control group: 2.5 mg / kg dexamethasone solution was administered intraperitoneally at regular intervals for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to induce an acute lung injury model of ALI in mice.

[0229] The low-dose DHA group (DHA-L group, abbreviated as DL) was administered 50 mg / kg DHA nanoliposomes via intraperitoneal injection daily at regular intervals, with an administration volume of 0.2 mL, for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to replicate the ALI model.

[0230] The high-dose DHA group (DHA-H group, abbreviated as DH) was administered 100 mg / kg DHA nanoliposomes via intraperitoneal injection daily at regular intervals, with an administration volume of 0.2 mL, for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to replicate the ALI model.

[0231] The low-dose DHA-CS group (DHA-CS-L group, abbreviated as DSL) was administered 50 mg / kg (based on DHA equivalent) of DHA-CS nanoliposomes via intraperitoneal injection daily for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to replicate the ALI model.

[0232] The high-dose DHA-CS group (DHA-CS-H group, abbreviated as DSH) was given 100 mg / kg (based on DHA equivalent) of DHA-CS nanoliposomes by intraperitoneal injection daily for 7 consecutive days. On the 7th day, 8 mg / kg LPS was injected intraperitoneally to replicate the ALI model.

[0233] The dosage volume for each group was uniformly 0.2 mL, administered intraperitoneally at a fixed time each day, and the mice had free access to food and water during the intervention period.

[0234] Construction of the ALI model: Mice in each group were treated with the drug continuously for 7 days. One hour after the last administration, except for the control group, mice in the other groups were injected intraperitoneally with 8 mg / kg LPS to establish an acute lung injury model; the control group was injected intraperitoneally with an equal volume of sterile saline. After modeling, mice were fed normally for 12 hours, and samples were collected to detect various indicators.

[0235] 2) Acquisition and packaging of lung tissue Twelve hours after LPS modeling, mice in each group were euthanized using the cervical dislocation method. Mice were fixed supine on a sterile operating table. The chest wall skin was disinfected with 75% alcohol, and the thoracic cavity was cut layer by layer to fully expose both lung lobes. The intact right lung tissue was separated, and residual blood on the lung tissue surface was rinsed with pre-cooled sterile saline. Surface moisture was absorbed with sterile filter paper, and tissues were collected and packaged separately: the anterior lobe of the right lung was placed on pre-weighed sterile foil for determining the wet-to-dry weight ratio of the lung; the middle lobe of the right lung was immersed in 4% paraformaldehyde tissue fixative and fixed at room temperature for 24 hours for subsequent pathological staining; the posterior lobe of the right lung was quickly placed in sterile cryovials and stored at -80 ℃ for detecting lung tissue inflammatory factors.

[0236] 3) Preparation of lung tissue homogenate Mouse lung tissue stored at -80 ℃ was collected, rinsed with sterile saline to remove residual blood, blotted dry with filter paper, and accurately weighed. Pre-cooled PBS buffer was added according to the mass-to-volume ratio to prepare a 10% lung tissue homogenate system. The homogenate was then thoroughly homogenized in a cryogenic homogenizer until no visible tissue fragments remained. Homogenization parameters: 60 Hz, 90 s, 30 s interval, 2 cycles. After homogenization, the homogenate was centrifuged at 3000 rpm at 4 ℃ for 10 min. The supernatant of the clear homogenate was aliquoted, labeled, and stored at -80 ℃ for the detection of inflammatory factors and NO content.

[0237] 4) Observation indicators 4.1) General observation of mice: Within 12 hours of LPS modeling, the survival status, food and water intake, spontaneous activity, fur fluff and luster, sensitivity to external stimuli, and nasal secretions of each group of mice were observed and recorded at regular intervals. The weight of the mice was measured before collection, and the mortality and lethargy of each group of mice were recorded.

[0238] The body weight changes of mice in each group were monitored dynamically for 7 consecutive days, such as... Figure 49 As shown in the figure. The results showed that the body weight of mice in each group showed a steady growth trend, with a consistent growth trajectory, and there were no statistically significant differences in body weight among the groups at different time points. Body weight is a direct indicator for evaluating the acute toxicity and systemic adverse reactions of the test substance. The above results suggest that, under the dosing regimen of this experiment, the test drug did not significantly interfere with the normal growth and development of mice, and had no significant systemic toxic side effects.

[0239] The experimental endpoint was the determination of organ indices in the mouse heart, liver, kidney, and spleen. Specific results are as follows: Figure 50 As shown. Statistical results showed no significant differences in cardiac, liver, and kidney indices among the groups of mice; compared with the blank control group, the spleen index of the treatment group was significantly reduced ( p <0.05). As an important peripheral immune organ, the spleen is a key site for the settlement, activation, and proliferation of T and B lymphocytes. A decrease in organ index usually indicates changes in the number and proliferation level of immune cells in the spleen, suggesting that the tested drug may have a certain regulatory effect on spleen growth and immune homeostasis in mice.

[0240] 4.2) Pathological observation of lung tissue Lung tissue was fixed in 4% paraformaldehyde, then dehydrated using an automated dehydrator, embedded in paraffin, and sectioned using a rotary microtome (5 μm thickness). Sections were dewaxed to water, graded hydration, stained with hematoxylin and eosin, cleared, and mounted with neutral resin. Pathological images of the lung tissue were acquired using a fluorescence inverted microscope, and analyzed using Image-Pro Plus 6.0 pathological section analysis software with standardized parameters for imaging and archiving. Differences in alveolar structural integrity, inflammatory cell infiltration, and alveolar congestion and edema pathological morphology were observed among the groups.

[0241] Lung tissue pathological microscopic examination results as follows Figure 51 The results showed that the alveolar structures of the control group mice were regular in morphology and had clear boundaries, with uniform alveolar cavities and no swelling in the lung tissue. No obvious inflammatory cell infiltration was observed in the alveolar interstitium, and the overall morphology of the lung tissue was normal. The lung tissue of the model group mice showed significant pathological damage, with impaired alveolar wall structure and disruption of integrity. A large number of alveoli merged to form bullae-like changes, and the alveolar walls were diffusely thickened. The alveolar interstitium was congested and edematous, accompanied by the recruitment and infiltration of a large number of neutrophils. The lung tissue was swollen and lesions were prominent, presenting typical pulmonary inflammatory pathological features.

[0242] Compared with the model group, the positive control group showed significant reversal of lung tissue pathological damage, basic restoration of alveolar structural integrity, near disappearance of alveolar fusion and bullous changes, mild alveolar wall thickening, and significant reduction in interstitial congestion and edema. Only a small number of scattered inflammatory cell infiltrations were observed in the alveolar interstitium, and the lung pathological morphology was close to that of the normal control group, demonstrating the best anti-inflammatory and repair effects. The DHA-L group showed limited improvement in lung tissue pathological damage, with alveolar fusion, interstitial swelling, and neutrophil infiltration remaining relatively prominent. In the DHA-H group, alveolar wall thickening was alleviated, alveolar walls thinned, lung tissue swelling decreased, and the number of neutrophils infiltrating the alveolar interstitium significantly decreased. The improvement level of lung tissue pathology in the DHA-CS-L group was roughly equivalent to that in the DHA-H group; the DHA-CS-H group showed significant restoration of lung tissue pathological morphology, approaching the normal control group, with improved alveolar wall thickness, significant reduction in lung tissue swelling, no obvious neutrophil infiltration in the alveolar interstitium, and effective relief of lung inflammation.

[0243] 4.3) Determination of lung wet / dry weight (W / D) ratio Remove the aluminum foil wrapping from the mouse's right anterior lung lobe, accurately weigh it using an electronic analytical balance, and record the wet weight (W) of the lung tissue. Place the aluminum foil along with the lung tissue into a 60 ℃ constant temperature drying oven for 72 h until the lung tissue mass is constant. After cooling to room temperature, weigh the dry weight (D), subtract the blank mass of the aluminum foil, and calculate the ratio of net wet weight to net dry weight of the lung (W / D). The higher the ratio, the more severe the pulmonary edema. The calculation formula is as follows: Lung tissue W / D ratio = net wet weight of lung tissue (W) / net dry weight of lung tissue (D).

[0244] The wet-to-dry weight (W / D) ratio of lung tissue is a core objective indicator for quantitatively evaluating the degree of pulmonary edema. A higher ratio indicates increased vascular permeability in the lung tissue and more severe inflammatory exudation and tissue edema. Statistical results are as follows: Figure 52 As shown, compared with the blank control group, the W / D ratio of lung tissue in the LPS-induced model group mice was significantly increased ( P <0.01), indicating that LPS stimulation caused disordered pulmonary vascular permeability in mice, with a large amount of fluid accumulating in the alveoli and pulmonary interstitium; combined with HE pathological sections, the model group showed typical pulmonary edema pathological features such as extensive alveolar congestion, severe interstitial swelling, and increased alveolar effusion. The stable and reliable construction of the acute pulmonary edema model was confirmed from both quantitative indicators and microscopic morphology.

[0245] Compared to the model group, the lung tissue W / D ratio in the low-dose DHA group (DL) did not decrease significantly. P<0.05 indicates that low-dose DHA has no significant effect on improving LPS-mediated pulmonary vascular leakage and edema; the lung W / D ratio of mice in the high-dose DHA group (DH), low-dose DHA-CS group (DSL), and high-dose DHA-CS group (DSH) were all significantly reduced. P <0.05), and the W / D levels of the above three groups were not significantly different from those of the positive drug group.

[0246] The above results suggest that DHA and its esterified derivative DHA-CS can exert a significant protective effect against pulmonary edema by inhibiting increased pulmonary vascular permeability and reducing interstitial and alveolar inflammatory exudation. This protective effect exhibits a clear dose-dependent characteristic. At the same dose equivalent, DHA-CS modified with cholesterol succinate structure is more effective than parent DHA. High-dose DHA-CS has the most prominent effect on improving pulmonary edema, and its efficacy is comparable to that of clinically positive anti-inflammatory drugs.

[0247] 4.4) Detection of inflammatory factor levels a. Reagent preparation ①TNF-α and IL-6: Lyophilized standards were reconstituted with their respective diluents to obtain stock solutions of 600 pg / mL and 500 pg / mL, respectively. These stock solutions were then mixed with an equal volume of diluent A to obtain the highest concentration standard solutions (TNF-α 300 pg / mL, IL-6 250 pg / mL). Six gradients were prepared by 2-fold serial dilution. Diluent A served as a blank control. Enzyme-labeled antibodies were diluted 1:100 with diluent B before use. ②IL-1β: 10× coating buffer and 5× detection diluent were diluted to prepare 1× working solution. Capture antibody and detection antibody were diluted 1:250, and HRP streptavidin was diluted 1:100. All solutions were prepared 30 min before use. Lyophilized standards were reconstituted to obtain a 10 ng / mL stock solution, which was then diluted to 1000 pg / mL and prepared by 2-fold serial dilution to obtain seven gradients.

[0248] b. Specific testing procedures TNF-α and IL-6 detection procedure (Saiwell reagent kit) ①TNF-α, IL-6 (pre-coated plate): Set up blank wells, standard wells, and sample wells. Add 100 μL of the corresponding standard solution, sample, or dilution solution A to each well. After sealing the plate, incubate at room temperature with shaking at 500 rpm for 1.5 h. Wash the plate 7 times (300 μL / well). Add 100 μL of enzyme-labeled antibody working solution and incubate with shaking for 0.5 h. Wash the plate 5 times. Add 90 μL of TMB chromogenic solution and develop the color in the dark for 10-30 min. Add 50 μL of stop solution and read the values ​​at dual wavelengths of 450 nm and 630 nm. ②IL-1β (uncoated plate): Add 100 μL of capture antibody working solution to each well and incubate overnight at 4 ℃; wash the plate twice, add 200 μL of detection diluent and block at room temperature for 1 h; after washing the plate, add 100 μL each of standard solution and sample, and incubate with shaking at 400 rpm for 2 h; wash the plate 5 times, add 100 μL of detection antibody working solution and incubate for 1 h; after washing the plate, add 100 μL of HRP enzyme working solution and incubate for 30 min; after washing the plate, add 100 μL of TMB substrate solution and develop color in the dark for 15~30 min, then add stop solution to terminate the reaction, and read the values ​​at dual wavelengths of 450 nm / 620 nm.

[0249] c. Results Calculation and Quality Control Standards: Both standards and samples were tested in triplicate, and the average OD value was used after subtracting the blank. A standard curve was fitted with standard concentration as the x-axis and OD value as the y-axis (R²). 2 (≥0.99), calculate sample concentration based on curve, and calculate dilution factor for diluted samples. The limit of detection for TNF-α was 2.56 pg / mL, for IL-6 it was 0.72 pg / mL, and the linear range for IL-1β was 15.6–1000 pg / mL; intra-assay CV <10%, inter-assay CV <15%, and recovery rate was 75%–105%.

[0250] d. Statistical analysis: All experimental measurement data are expressed as follows: Mean ± s indicates statistical significance. SPSS 26.0 statistical software was used for analysis. One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0251] e. Results Analysis Plasma inflammatory factor test results as follows Figure 53 As shown in the figure. The results showed that compared with the blank control group, the levels of three pro-inflammatory factors, TNF-α, IL-6, and IL-1β, in the plasma of mice in the model group were significantly increased ( p <0.001 indicates that the mice exhibited a significant systemic inflammatory response after modeling, confirming the successful establishment of the inflammatory model. Compared to the model group, the plasma TNF-α and IL-1β levels in mice of all treatment groups were significantly downregulated ( p<0.05); while IL-6 levels showed a significant decreasing trend only in the high-dose DHA-CS-H group, with no significant statistical differences in the other treatment groups. Further comparative analysis with the positive control group revealed that, in terms of plasma TNF-α and IL-6, only the DHA-CS-H group showed no significant difference from the positive control group; regarding plasma IL-1β, except for the low-dose DHA-L group which showed a significant statistical difference, all other treatment groups showed no significant difference from the positive control group.

[0252] Results of local inflammatory factor detection in lung tissue, such as Figure 54 As shown, the trend is basically consistent with that of plasma: compared with the blank control group, the levels of TNF-α and IL-6 in the lung tissue of mice in the model group were significantly upregulated ( p The result was <0.001, further validating the effectiveness and stability of the lung inflammation model. Compared to the model group, all treatment groups effectively reduced the levels of inflammatory factors TNF-α and IL-6 in lung tissue, and the differences were statistically significant. p <0.05). Intergroup comparison of the positive drug group showed that there was no significant difference in TNF-α content in lung tissue between the drug administration groups and the positive drug group; only the low-dose DHA-L group showed a significant statistical difference in IL-6 content in lung tissue, while the levels in the other drug administration groups were similar to those in the positive drug group.

[0253] The results of plasma and lung tissue inflammatory factor detection showed that DHA-CS has excellent in vivo anti-inflammatory activity and can effectively inhibit the inflammatory response in the lungs and the whole body. Its effect in regulating the expression of pro-inflammatory factors is similar to that of clinical positive drugs, and the high-dose DHA-CS group has the best overall anti-inflammatory effect.

[0254] Based on comprehensive experimental results including general mouse condition, organ indices, lung tissue pathological morphology, and plasma and lung tissue inflammatory factors, this invention confirms that the self-made DHA derivative DHA-CS has significant in vivo anti-inflammatory and acute lung injury protective effects. Regarding drug safety, neither DHA nor DHA-CS affects normal growth and development in mice, and there is no significant toxic damage to vital organs such as the heart, liver, and kidneys, indicating safe and reliable use. In terms of efficacy, DHA-CS effectively improves LPS-induced alveolar structural damage and lung edema, inhibits neutrophil inflammatory infiltration, significantly reduces the expression of systemic and local pro-inflammatory factors in the lungs, and alleviates excessive inflammatory immune responses, with overall efficacy showing a good dose-dependent effect. Compared to the parent DHA, DHA-CS, after structural modification, exhibits significantly enhanced efficacy, overcoming the shortcomings of the parent drug's narrow anti-inflammatory spectrum and weak efficacy at low doses. High-dose DHA-CS showed the best intervention effect, with overall anti-inflammatory and lung-protective efficacy comparable to clinically proven positive anti-inflammatory drugs, demonstrating good potential for future development and application.

[0255] Experiment Example 9: Validation of Inflammation Targeting by Dihydroartemisinin-Cholesterol Succinate Liposomes In the present invention, blank LNPs (i.e., Comparative Example 5), DHA LNPs (i.e., Comparative Example 4), and DHA-CS LNPs (i.e., Example 12) are labeled with DIR (the added amount of DIR fluorescent dye is 5 μL), respectively, to form DIR-loaded LNPs (abbreviated as DIR LNPs), DIR-loaded DHA LNPs (abbreviated as DIR DHA LNPs), and DIR-loaded DHA-CS LNPs (abbreviated as DIR DHA-CS LNPs). A mouse local granulomatous inflammation model is established, and through continuous in vivo fluorescence imaging from 2 h to 24 h combined with end-point ex vivo organ fluorescence quantification, the whole-body metabolism patterns of the three nanoparticles and the accumulation characteristics in reticuloendothelial organs such as the liver and spleen are dynamically compared. The enrichment rate and sustained retention capacity of the nanoparticles before and after modification at the inflammatory lesion are investigated with emphasis, so as to clarify the improvement effect of cholesterol succinate structure modification on the in vivo pharmacokinetic behavior and inflammation targeting performance of the nanoparticles, and provide delivery mechanism support at the in vivo distribution level for the previous in vivo anti-inflammatory efficacy results.

[0256] Experimental animals: 4-week-old SPF-grade male Kunming mice with a body weight range of 20-25 g, purchased from Speifu (Beijing) Biotechnology Co., Ltd., with the animal production license number: SCXK (Beijing) 2019-0010. All mice are housed in a standard SPF-grade animal feeding environment, with feeding conditions set at a temperature of 22±2 °C, a relative humidity of 50%±10%, a 12 h / 12 h day-night light-dark cycle, free access to food and water, and the formal experiment is carried out after 3 days of adaptive feeding. All animal operation procedures in this experiment strictly follow the "Guidelines for Ethical Review of Laboratory Animal Welfare", and have been reviewed and approved by the Institutional Laboratory Animal Ethics Committee, which complies with the ethical norms for animal experiments.

[0257] Main reagents used in the experiment: Turpentine oil (Shanghai Macklin Biochemical Technology Co., Ltd., batch number: C11405635, analytical grade); near-infrared fluorescent dye DiIC18(7) (DIR, Shanghai Macklin Biochemical Technology Co., Ltd., batch number: D864067); isoflurane (RWD Life Technology Co., Ltd., batch number: 21020401, special for animal anesthesia); normal saline (Sichuan Kelun Pharmaceutical Co., Ltd., sterile grade); special depilatory cream for mice, sterile PBS buffer.

[0258] Experimental instruments: Tanon ABL X5 small animal in vivo imaging system (Shanghai Tanon Technology Co., Ltd.); MATRX small animal dedicated inhalation anesthesia machine (MATRX Corporation, USA); electronic analytical balance (Mettler-Toledo Instruments Co., Ltd.); pipette (Eppendorf, Germany) Statistical analysis: All experimental measurement data are expressed as mean ± standard deviation (x±s). SPSS 26.0 statistical software was used for analysis. One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons. P<0.05 was considered statistically significant.

[0259] 1) Construction of a mouse hind leg granulomatous inflammation model Healthy Kunming mice weighing 20-25 g were selected and, after acclimatization, an inflammation model was established. This experiment used a local intramuscular injection of turpentine oil to establish a mouse hind leg granulomatous inflammation model. The specific procedure was as follows: 50 μL of sterile turpentine oil was precisely injected into the gastrocnemius muscle of the mouse's right hind leg at a fixed time each day for 3 consecutive days. The injection site and depth were consistent throughout the process to avoid damage to blood vessels and tendons. After model establishment, the mice were routinely fed and observed for 3 days, with continuous monitoring of swelling, congestion, and limb movement in the right hind leg. The local granulomatous inflammation model was considered successfully established when the mouse's right hind leg exhibited significant redness, swelling, tissue thickening, and granulomatous proliferation, and was ready for subsequent fluorescence imaging experiments.

[0260] 2) Mouse in vivo near-infrared fluorescence imaging experiment Twelve Kunming mice with successful modeling and uniform weight were selected and randomly divided into three groups of four mice each using a random number table: DIR-loaded LNPs group (DIR LNPs), DIR-loaded DHA LNPs group (DIR DHA LNPs), and DIR-loaded DHA-CS LNPs group (DIR DHA-CS LNPs). All mice were distinguished by ear tagging. Each group received a single tail vein administration at a uniform dose of 0.2 mg / kg (based on DIR) and a volume of 0.2 mL. Free DIR and both nanoparticle formulations were dispersed in sterile saline solution. All administration procedures were performed in a sterile laminar flow hood to ensure aseptic technique and operational consistency.

[0261] Before drug administration, mice underwent standardized pretreatment: After isoflurane inhalation anesthesia, hair was thoroughly removed from the trunk, limbs, and inflamed hind legs using mouse-specific depilatory cream. Residual depilatory cream and dirt were wiped away with physiological saline, and the mice were dried before use to eliminate interference from hair and impurities on fluorescence imaging signals. In vivo fluorescence imaging was performed at 2 h, 4 h, 8 h, 12 h, and 24 h after drug administration. Mice were anesthetized with isoflurane inhalation for 3–5 min at each time point to ensure stable positioning and physiological state. Anesthetized mice were stably placed in the dark chamber of the Tanon ABL X5 small animal in vivo imaging system. Imaging parameters were fixed: excitation wavelength 750 nm, emission wavelength 780 nm. Exposure time, focal length, and signal gain were maintained uniformly throughout the imaging process. Fluorescence imaging of each group of mice was completed and images were saved for subsequent in vivo fluorescence distribution and signal intensity analysis.

[0262] Fluorescence distribution images were continuously acquired at different time points over 24 hours using an in vivo imaging system to dynamically track the in vivo transport behavior of nanoparticles. The imaging results are shown in Figure 55. The 24-hour continuous in vivo fluorescence imaging results show that the fluorescence accumulation trends in the DIR LNPs group and the DIR DHA LNPs group are similar. The fluorescence signals of both groups peaked 4–8 hours after administration, mainly accumulating in reticuloendothelial metabolic organs such as the liver, spleen, and kidneys. Over time, the fluorescence signals gradually decreased, and by 12–24 hours, the organ fluorescence had essentially faded, with no significant residual fluorescence signal. In stark contrast, a clearly discernible fluorescence signal was still detectable in the DIR DHA-CS LNPs group at the 24-hour observation endpoint, indicating that the nanoparticles modified with cholesterol succinate had a longer in vivo circulation retention time, effectively prolonging the drug's in vivo action period.

[0263] The comparison of targeted fluorescence signals at the inflammatory site of the leg further confirms the advantages of derivative modification: DIRDHA LNPs showed significant fluorescence enrichment in the inflammatory area 8 hours after administration; while DIR DHA-CS LNPs accumulated rapidly at the inflammatory site 2 hours after administration, and the fluorescence signal was maintained throughout the 24-hour observation period. These differences in spatiotemporal distribution demonstrate that DHA-CS lipid nanoparticles possess excellent inflammation-targeting properties, not only accumulating more quickly at the inflammatory lesion but also remaining at the injury site for a longer period to continuously deliver the drug; effectively prolonging the duration of drug action at the lesion from a physical perspective of drug delivery, increasing local drug exposure concentration, and thus improving the efficiency of drug utilization in vivo.

[0264] 3) Fluorescence imaging experiments of isolated mouse organs and tissues After completing the 24-hour endpoint in vivo imaging detection, mice were painlessly euthanized using the cervical dislocation method. Normal muscles of the left hind leg, inflamed granulomatous muscles of the right hind leg, and major organ tissues (heart, liver, spleen, lung, and kidney) were rapidly dissected and separated. All excised tissues were immediately rinsed three times with sterile saline to thoroughly remove residual blood and tissue fluid, avoiding interference from autofluorescence in the detection results. Excess moisture was then gently blotted with sterile filter paper to ensure a smooth and dry surface, ready for testing.

[0265] All pre-processed tissues and organs were placed on the in vivo imaging system platform. Images were acquired and analyzed using the same fluorescence imaging parameters as in "2) Mouse In vivo Near-Infrared Fluorescence Imaging Experiment." The fluorescence distribution characteristics and fluorescence signal intensity of different tissues and organs in each group of mice were recorded. The targeting enrichment performance and in vivo tissue distribution patterns of free DIR, DIR-DHA LNPs, and DIR-DHA-CSLNPs in inflamed tissues were compared and evaluated. All imaging experiments were performed in triplicate to ensure the reliability and repeatability of the experimental data.

[0266] Fluorescence images of isolated organs were acquired using a live in vivo imaging system. The fluorescence distribution results of isolated tissues are as follows: Figure 56 As shown. The in vitro fluorescence imaging pattern highly matches the 24-hour in vivo whole-body fluorescence distribution trend: both the blank DIR LNPs and DIR DHA LNPs groups showed moderate fluorescence enrichment signals in the liver and spleen; DIR DHA-CS LNPs also showed significant accumulation in the liver and spleen, the two major reticuloendothelial organs, but compared to the other two nanoparticles, the fluorescence signal intensity at the inflammatory lesions in the legs was significantly higher. The fluorescence intensity of the in vitro tissues was compared to... Figure 57 As shown in the figure. Combined with the results of previous organ index experiments, this confirms that nanoparticles can participate in the regulation of inflammation in the body through the spleen's immune-related pathway.

[0267] Semi-quantitative grayscale analysis showed that the liver was the primary organ for accumulation of the three nanoparticles, with no significant difference in fluorescence intensity over 24 hours. Notably, DIR DHA-CS LNPs exhibited a significant advantage in targeted accumulation at inflammatory lesions, in addition to their distribution in normal organs: at the same time point, the fluorescence grayscale intensity of inflamed tissues was much higher than that of DIR LNPs and DIR DHA LNPs. Furthermore, high-intensity fluorescence could still be detected in ex vivo samples after 24 hours, indicating that the DHA-CS modified carrier can prolong the retention time at the inflammatory site, increase the drug concentration in the target tissue, and optimize the in vivo drug utilization efficiency.

[0268] The above in vivo fluorescence tracing results fully demonstrate that cholesterol succinate modification can significantly improve the in vivo circulation dynamics and inflammation-targeting performance of DHA lipid nanoparticles: prolonging in vivo circulation time, accelerating the enrichment of inflammatory lesions, and persistently remaining in damaged tissues, increasing local drug exposure. This explains the superior in vivo anti-inflammatory and lung injury protective effects of DHA-CS compared to parent DHA from the perspective of drug delivery, providing key experimental evidence for the in vivo distribution of DHA-CS nanodelivery systems for inflammation-related injuries.

[0269] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art, all within the spirit and principles of the invention.

Claims

1. A cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof, characterized in that, The cholesterol succinate monoester derivative is formed by covalently linking cholesterol succinate monoester with a small molecule drug via an ester bond or an amide bond; the small molecule drug is a compound containing a hydroxyl group or an amino group that has antitumor or anti-inflammatory effects.

2. The cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The small molecule drug includes any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, alizarin, or dihydroartemisinin.

3. The cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Its general structural formula is shown in formula (I) or formula (II): Wherein, R1 is a small molecule drug group, selected from any one of (A1)-(A11): ； R2 is: ; In the above structural formula for R1, the asterisk " " represents a single bond connected to a group on one side of the structural formula in formula (I); In the above structural formula for R2, the asterisk " " represents a single bond connected to the groups on both sides of the structural formula in formula (II); Preferably, the (A1) structure in R1 is: The structure of (A12) is: ; Alternatively, R2 in equation (II) is: .

4. A method for preparing the cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The cholesterol succinate monoester derivative is prepared by esterification or amidation of the carboxyl group in cholesterol succinate monoester with the hydroxyl or amino group in a small molecule drug.

5. The preparation method according to claim 4, characterized in that, The preparation method includes the following steps: mixing cholesterol succinate monoester, a small molecule drug and an organic solvent, and then adding a catalyst and a condensing agent to carry out an esterification reaction or an amidation reaction to obtain a cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof.

6. The preparation method according to claim 5, characterized in that, The small molecule drug includes any one of podophyllotoxin, temozolomide, (S)-10-hydroxycamptothecin, geraniol, paeonol, galangin, eugenol, magnolol, alizarin, or dihydroartemisinin. And / or, the condensing agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; And / or, the catalyst comprises 4-dimethylaminopyridine or 1-hydroxybenzotriazole; And / or, the organic solvent includes dichloromethane.

7. A pharmaceutical composition, characterized in that, It comprises a cholesterol succinate monoester derivative as described in any one of claims 1-3 or a pharmaceutically acceptable salt thereof, or a derivative or a pharmaceutically acceptable salt thereof prepared by the preparation method described in any one of claims 4-6, and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The cholesterol succinate monoester derivative or its pharmaceutically acceptable salt can be used as a cholesterol substitute in carrier materials or drug delivery systems. Preferably, the carrier material comprises phospholipids and the cholesterol succinate monoester derivative or a pharmaceutically acceptable salt thereof; Preferably, the dosage form of the carrier material is selected from any one of the following dosage forms: (a) Nanoscale formulations: solid lipid nanoparticles, liposomes, nanoemulsions, polymer lipid hybrid nanoparticles, and polymer micelles; (b) Submicron-sized and micron-sized formulations obtained by adjusting the particle size of the nanoscale formulation described in (a) above; (c) Microparticle formulations: microspheres, microcapsules.

9. The use of the cholesterol succinate monoester derivative of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 7 or 8, in the preparation of an antitumor drug or an anti-inflammatory drug.

10. The application according to claim 9, characterized in that, The tumors include breast cancer, glioma, lung cancer, or liver cancer; And / or, the inflammation includes arthritis, skin inflammation, rheumatoid arthritis, gouty arthritis, kidney inflammation, gastritis, enteritis, acute lung injury, or sepsis-related myocardial injury.

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