A bifunctional dendrimeric lipopeptide nanomicelle and a preparation method and application thereof

Abstract

The present application relates to a kind of bifunctional dendritic lipopeptide nanomicelles and its preparation method and application, belong to the field of medicine technology.The present application is based on the four generation lysine chiral molecule, changes its hydrophobic tail chain structure, access ROS response key, make it have low toxicity and ROS response, four generation glutamic acid dendritic macromolecule is innovatively prepared, has the effect of inducing autophagy.On this basis, a kind of ROS response and inducing autophagy bifunctional dendritic lipopeptide nanomicelles D RNP.The present application mixes two four generation dendritic macromolecules (lysine and glutamic acid) creatively, self-assembles into dendritic lipopeptide nanomicelles, changes the hydrophobic long chain structure of four generation lysine dendritic macromolecule, and adds ROS response key (TK), so that it has low toxicity and ROS response.In the activation autophagy, the expression of P63 and LC3 two proteins is verified by immunoprotein experiment, compared with existing technology, autophagy effect is better, and it also has good ROS response.

Description

Technical Field

[0001] This invention relates to a bifunctional dendritic lipopeptide nanomicelle, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Oxidative stress refers to an imbalance between oxidation and antioxidation in the body, resulting in a predominantly oxidative state. This state is caused by the excessive production of reactive oxygen species (ROS) and a decline in the body's antioxidant capacity. ROS include superoxide anions (O2·4O3 ... – Oxidizing chemicals such as hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) are normal byproducts of cellular metabolism and participate in cell signaling and biological regulation at normal levels. When ROS production exceeds the body's antioxidant system's ability to scavenge it, oxidative stress occurs, leading to damage to cells and tissues. Supplementing with antioxidants, reducing ROS production, or enhancing ROS scavenging capacity may offer new approaches to the treatment of various inflammations.

[0003] Autophagy clears accumulated lipids, damaged organelles, and protein aggregates from cells, preventing further cellular damage from the buildup of these harmful substances. This cleanup helps reduce intracellular stress and inflammation. Dysregulation of autophagy may lead to apoptosis and senescence, and promote the infiltration of inflammatory cells. In summary, autophagy, this intracellular cleanup and recycling mechanism, plays a crucial role in maintaining vascular cell homeostasis and function. Autophagy can degrade and reuse damaged or aging organelles and proteins, which is significant for preventing the accumulation of harmful substances and reducing stress and inflammation.

[0004] Against this backdrop, dendritic lipopeptides, as a novel biomolecule combining the properties of dendritic molecules and lipopeptides, offer new hope for regulating intracellular inflammatory responses and activating autophagy due to their unique structural and functional characteristics. Dendritic lipopeptides combine the highly branched structure of dendritic molecules with the bioactivity of lipopeptides, forming a stable three-dimensional structure. Their hydrophilic head and hydrophobic tail together constitute a unique amphiphilic structure, enabling dendritic lipopeptides to self-assemble into complex nanostructures such as nanofibers, nanotubes, and vesicles. These properties not only endow dendritic lipopeptides with special physicochemical properties but also demonstrate their enormous application potential in fields such as drug delivery and bioimaging.

[0005] Autophagy is considered a key process for maintaining cellular homeostasis and a response to stressors such as nutrient deficiency, which can jeopardize cellular survival. When cells encounter these stressors, autophagy, which normally occurs at low levels to balance the constant synthesis of biomolecules, is significantly upregulated. This upregulation increases cellular uptake and degradation, releasing macromolecules back into the cytoplasm to drive essential metabolic reactions and generate energy. In fact, autophagy has been found to be useful during mammalian development, and recent studies have revealed it to be an important regulator of various diseases and conditions. Rapamycin, as the most classic autophagy inducer targeting mTOR, has been widely used, but numerous clinical trials have shown that its side effects are dose-dependent and reversible. Major toxic side effects include headache, nausea, dizziness, nosebleeds, and joint pain, among others, and are considered significant.

[0006] Many existing technologies synthesize chiral dendritic macromolecular structures using amino acids such as lysine, but the preparation and purification processes are quite lengthy. Furthermore, most of the existing dendritic macromolecules composed of third-generation or higher chiral amino acids have been found to induce autophagy in different tumor cells, but not in immune cells. Moreover, most existing technologies simply synthesize chiral dendritic macromolecular structures, and the amino acids used are mostly lysine and arginine, with only one type of amino acid being verified.

[0007] Based on the above situation, this invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies. Based on the existing fourth-generation lysine chiral molecule, its hydrophobic tail chain structure is modified by incorporating a ROS-responsive bond, resulting in low toxicity and ROS responsiveness. This innovatively prepares a fourth-generation glutamic acid dendritic macromolecule with autophagy-inducing effects. Furthermore, this invention provides a dendritic lipopeptide nanomicelle with dual ROS-responsive and autophagy-inducing functions. D -RNPs. Another object of the present invention is to provide bifunctional nanomicelles. D Preparation methods of -RNPs and their in vitro applications.

[0009] Addressing the issue of lengthy preparation and purification processes in the synthesis of chiral dendritic macromolecules using existing technologies, this invention simplifies the synthesis and purification process while improving the synthesis yield. Furthermore, it modifies the hydrophobic long-chain structure by incorporating ROS-responsive bonds into the original chiral structure, resulting in a synthesized product with low toxicity and ROS responsiveness. While existing dendritic macromolecules composed of third-generation or higher chiral amino acids primarily induce autophagy in various tumor cells, this invention further explores its potential to induce autophagy in inflammatory immune cells (such as macrophages), extending its therapeutic applications beyond specific tumor cells.

[0010] Given that most existing technologies involve the simple synthesis of chiral dendritic macromolecules, primarily using lysine and arginine as amino acids, and only one specific effect is verified, this invention innovatively synthesizes a fourth-generation dendritic macromolecule prepared from glutamic acid. The synthesis and purification process is relatively simplified. Furthermore, it innovatively mixes two different dendritic macromolecules in an equimolar ratio to self-assemble into dendritic lipopeptide nanomicelles, exhibiting two effects: ROS response and autophagy induction. These two effects synergistically treat inflammation, demonstrating significant application potential.

[0011] The specific technical solution of the present invention is as follows:

[0012] In a first aspect of the present invention, a bifunctional dendritic lipopeptide nanomicelle is proposed and prepared for the first time, which is made from chiral ROS-responsive dendritic lipopeptide KRDL and glutamate dendritic lipopeptide molecule EDL.

[0013] The KRDL structure is shown in Equation I:

[0014]

[0015] in:

[0016] R1 is a dendritic polypeptide composed of lysine residues linked by amide bonds in a certain proportion. The structure of R1 is shown in Formula II:

[0017]

[0018] In Formula II, n represents D-type lysine, forming a dendritic network with amide bonds;

[0019] R2 is a carbon chain containing TK bonds. 27 H 50 O2NS2:

[0020] The structure of the EDL is shown in Equation III:

[0021]

[0022] in:

[0023] R3 is a dendritic polypeptide composed of glutamic acid residues linked by amide bonds in a certain proportion. The structure of R3 is shown in Formula IV:

[0024]

[0025] In Formula IV, n represents glutamic acid, forming a tree-like network with amide bonds;

[0026] R4 represents the carbon chain C. 18 H 33 O.

[0027] In this invention, KRDL consists of two parts: a hydrophobic tail and a hydrophilic head. The hydrophobic tail is a long hydrophobic chain composed of ROS-responsive ketithial thioglycolate and oleylamine, wherein the ROS-responsive bond refers to a bond that can break and release the drug in response to a high concentration of ROS at the plaque site, preferably a TK bond; the hydrophilic head is an amphoteric polypeptide formed by amide bonds between lysine residues in a certain proportion. EDL also consists of two parts: a hydrophobic tail and a hydrophilic head. The hydrophobic tail is a long hydrophobic chain composed of oleic acid, and the hydrophilic head is an amphoteric polypeptide formed by amide bonds between glutamic residues in a certain proportion. When KRDL and EDL are mixed in an equimolar ratio, their hydrophilic heads and hydrophobic tails can self-assemble into nanomicelles in solution. KRDL contains ROS-responsive TK bonds, which can spontaneously break in an inflammatory environment, while the D-type, compared to the L-type, can effectively prevent degradation by enzymes in vivo, thereby releasing more EDL dendritic macromolecules and D-type lysine dendritic polypeptides in an inflammatory environment, inducing macrophage autophagy.

[0028] Furthermore, the dendritic lipopeptide nanomicelles are prepared by mixing chiral ROS-responsive dendritic lipopeptide KRDL and glutamate dendritic lipopeptide molecule EDL in an equimolar ratio via a solvent injection method.

[0029] Furthermore, the preferred KRDL and EDL structures in the bifunctional dendritic lipopeptide nanomicelles of the present invention are as follows:

[0030] The structure of KRDL is shown in Equation V:

[0031]

[0032] The structure of EDL is shown in Equation VI:

[0033]

[0034] In a second aspect of the invention, a method for preparing bifunctional dendritic lipopeptide nanomicelles as described in the first aspect is provided.

[0035] The preparation method of the bifunctional dendritic lipopeptide nanomicelles includes the following steps:

[0036] (1) Synthesis of G n K(n=2,3,4)

[0037] Synthesize chiral dendritic molecules G with D-lysine as the basic structural unit at different generations. n K(Boc) (n=2,3,4), and further remove the Boc protecting group to obtain the corresponding chiral dendritic product G. n K(n=2,3,4);

[0038] (2) Synthesis of chiral ROS-responsive dendritic lipopeptide KRDL

[0039] Based on the fourth-generation chiral lysine molecule, a demethylation reaction was first carried out in sodium hydroxide methanol solution (1 mol / L) to expose the carboxyl group. The mixture was then extracted with ethyl acetate, dried over anhydrous Na₂SO₄, concentrated under reduced pressure to remove the solvent, and dried under vacuum for 24 h to obtain a series of intermediates. These intermediates were then combined with a hydrophobic long chain composed of a ROS-responsive ketithial thioglycolate and oleylamine to synthesize a chiral ROS-responsive dendritic lipopeptide. D -KRDL;

[0040] (3) Synthesis of glutamate dendritic lipopeptide molecules EDL

[0041] Dendritic molecules of different generations with glutamic acid as the basic structural unit were synthesized. Based on the fourth-generation glutamic acid molecule, the Fmoc protecting group was further removed and then combined with a hydrophobic long chain composed of oleic acid to synthesize the glutamic acid dendritic lipopeptide molecule EDL.

[0042] (4) Preparation of nanomicelles

[0043] Will D KRDL and EDL are mixed in a 1:1 molar ratio and added to a 1:1 volume ratio solution of anhydrous ethanol and tetrahydrofuran until fully dissolved. This solution is then added dropwise to a rapidly stirred PBS solution, and stirring continues until the anhydrous ethanol and tetrahydrofuran have completely evaporated, forming nanomicelles. D -RNPs.

[0044] Furthermore, in step (1), the second-generation chiral dendritic molecule D The synthesis method of G2K is as follows:

[0045] 1) Under ice bath and nitrogen protection, the above raw materials were added to the reaction apparatus according to the molar ratio of DH-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU, and DIPEA of 1:2:3:3:6. Appropriate amounts of DMF and DIPEA were then added to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, followed by stirring at room temperature for 24 hours. After the reaction was completed, the reaction solution was extracted, washed, salted out, and dried to obtain a crude product solution. The crude reaction solution was then concentrated and purified to obtain... D -G2K(Boc)4;

[0046] 2) Take the above D In a reaction apparatus under nitrogen protection, DCM was slowly added to dissolve G2K(Boc)4, followed by slow addition of TFA. The mixture was stirred in an ice bath for 30 minutes, and then stirred again at room temperature for 3-6 hours. After the reaction was complete, the reaction solution was evaporated under reduced pressure, concentrated, and evaporated to dryness. The solution was then washed and dried under vacuum for 24 hours to obtain the desired product.D -G2K; where D The weight-to-volume ratio of -G2K(Boc)4, DCM, and TFA is 1g:3mL:3mL.

[0047] Furthermore, in step (1), the third-generation chiral dendritic molecule D The synthesis method of G3K is as follows:

[0048] 1) Under ice bath and nitrogen protection, according to D The molar ratio of -G2K, Boc-D-Lys(Boc)-OH, HOBT, HBTU, and DIPEA is 1:4:5:5:11. The above raw materials are added to the reaction apparatus, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature for 24 hours. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, recrystallized, and the crystals are filtered. After filtration to dryness, the product is vacuum dried for 24 hours to obtain... D -G3K(Boc)8;

[0049] 2) Weigh the above D In a reaction apparatus under nitrogen protection, a mixed solvent of TFA and DCM was slowly added. The mixture was stirred in an ice bath for 30 minutes under nitrogen protection, and then stirred again at room temperature for 4-6 hours. After the reaction was complete, the reaction solution was evaporated under reduced pressure, concentrated, and evaporated to dryness. The solution was then washed and vacuum dried for 24 hours to obtain the desired product. D -G3K; where D The weight-to-volume ratio of -G3K(Boc)8, TFA, and DCM is 2g:7mL:7mL.

[0050] Furthermore, in step (1), the fourth-generation chiral dendritic molecule D The synthesis method for G4K is as follows:

[0051] 1) Under ice bath and nitrogen protection, according to D The molar ratio of -G3K, Boc-D-Lys(Boc)-OH, HOBT, HBTU, and DIPEA is 1:10:12:12:30. The above raw materials are added to the reaction apparatus, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature for 36 hours. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, then recrystallized, filtered to obtain crystals, and finally dried under vacuum for 24 hours. D -G4K(Boc) 16 ;

[0052] 2) Weigh the above D -G4K(Boc) 16 In a reaction apparatus under nitrogen protection, a mixed solvent of TFA and DCM was slowly added. The mixture was stirred in an ice bath for 30 minutes under nitrogen protection, and then stirred again at room temperature for 5-7 hours. After the reaction was complete, the reaction solution was evaporated under reduced pressure, concentrated, and evaporated to dryness. The solution was then washed and vacuum dried for 24 hours to obtain the desired product. D -G4K; where D -G4K(Boc) 16 The weight-to-volume ratio of TFA to DCM is 1g:4mL:4mL.

[0053] Furthermore, in step (2), the method for synthesizing the chiral ROS-responsive dendritic lipopeptide KRDL is as follows:

[0054] (1) Take D -G4K(Boc) 16 Sodium hydroxide methanol solution was added to the reaction apparatus, and the reaction was carried out in an ice bath for 8 hours. After the reaction was completed, the pH of the reaction solution was adjusted to 3-4 with hydrochloric acid solution, and the reaction solution was extracted three times with ethyl acetate. The organic phase was collected, dried with anhydrous Na2SO4, filtered, concentrated under reduced pressure to remove the solvent, and dried under vacuum for 24 hours to obtain the final product. D -Compoud 1;

[0055] (2) Under ice bath and nitrogen protection, according to Fmoc-ethylenediamine, D The molar ratio of Compound 1, HOBT, HBTU, and DIPEA is 1:1:1.2:1.2:8. These raw materials are added to the reaction apparatus, followed by the addition of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, recrystallized, and the crystals are filtered. After filtration to dryness, the product is vacuum dried for 24 hours to obtain... D -Compoud 2;

[0056] (3) Take D -Compoud 2 was placed in a reaction apparatus, and under nitrogen protection, a mixed solvent of piperidine and DMF with a volume ratio of 1:4 was slowly added. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred again at room temperature for 6 h. After the reaction was complete, the reaction solution was transferred to another reaction apparatus, and anhydrous ethanol was added and concentrated under reduced pressure. This operation was repeated until no piperidine spots were observed on the TLC plate. After the piperidine was cleaned, the solution was concentrated under reduced pressure and evaporated to dryness. The solution was then vacuum dried for 24 h to obtain... D -Compoud 3; where, DThe weight-to-volume ratio of Compoud 2 to piperidine to DMF in the mixed solvent is 0.22 g : 5 mL;

[0057] (4) Under ice bath and nitrogen protection, according to D The molar ratio of Compound 3, TK-OAm, HOBT, HBTU, and DIPEA was 1:1.2:1.3:1.3:10. The above raw materials were added to the reaction apparatus, followed by the addition of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, then stirred at room temperature. After the reaction was complete, the reaction solution was extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation, then recrystallized. The crystals were filtered and dried under vacuum for 24 hours to obtain the final product. D -Compoud4;

[0058] (5) Take D -Compoud4 was placed in a reaction apparatus under nitrogen protection. A mixed solution of TFA and DCM at a volume ratio of 1:3 was slowly added to the apparatus. Under nitrogen protection, the mixture was stirred in an ice bath for 30 minutes, and then stirred again at room temperature for 6-8 hours. After the reaction was completed, the reaction solution was evaporated and concentrated under reduced pressure, then washed and vacuum dried for 24 hours to obtain the final product. D -KRDL; where, D The weight-to-volume ratio of the mixed solution of Compoud 4, TFA, and DCM is 0.2 g: 8 mL.

[0059] Further, in step (2), the method for synthesizing the carbon chain TK-OAm containing TK bonds is as follows: Under ice bath and nitrogen protection, the above raw materials are added to the reaction device according to the molar ratio of TK, OAM, EDCI, and NHS of 1:2.7:0.4:0.4. Then, an appropriate amount of DMF and DIPEA are added to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, and then stirred at room temperature for 4 hours. After the reaction is completed, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. Then, the crude reaction solution is concentrated and purified to obtain TK-OAm.

[0060] Furthermore, in step (3), the synthesis method of the glutamate dendritic lipopeptide molecule EDL is as follows:

[0061] 1) Under ice bath and nitrogen protection, the above raw materials were added to the reaction apparatus according to the molar ratio of Fmoc-L-Glu-OH, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU and DIPEA of 1:2.4:2.5:2.5:7.5. DMF and DIPEA were then added to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, and then stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was extracted, washed, salted out and dried to obtain a crude product solution. The crude reaction solution was then concentrated and purified to obtain Compound 6.

[0062] 2) Place Compoud 6 in a reaction apparatus. Under nitrogen protection, slowly add an appropriate amount of dichloromethane. After the solid dissolves, slowly add a 1:1 volume ratio of TFA and DCM mixed solution, and a small amount of triethylsilane. Under nitrogen protection, stir the reaction in an ice bath for 30 min, and continue stirring at room temperature for 4-6 h. Monitor the reaction progress by TLC. After the reaction is complete, evaporate and concentrate the reaction solution under reduced pressure, then wash and vacuum dry for 24 h to obtain Compoud 7. The weight-to-volume ratio of Compoud 6 to the TFA and DCM mixed solution is 2.5 g: 4 mL.

[0063] 3) The reactants were fed in the following order: the molar ratio of Compoud 7, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU and DIPEA was 1:4.8:5:5:15. The reaction solvent was DMF. The rest of the synthesis process was the same as step 1) above, to obtain Compoud 8.

[0064] 4) Prepare Compound 9 using Compound 8 as raw material, the synthesis process is the same as step 2) above;

[0065] 5) The reactants were fed in the following order: the molar ratio of Compoud 9, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU and DIPEA was 1:10:10:10:36. The reaction solvent was DMF. The rest of the synthesis process was the same as step 1) above, and Compoud 10 was obtained.

[0066] 6) Place Compoud 10 in a reaction apparatus and slowly add DMF under nitrogen protection. After the solid dissolves, slowly add a mixed solution of piperidine and DMF with a volume ratio of 1:4. Under nitrogen protection, stir the reaction in an ice bath for 30 min, and continue stirring at room temperature for 6-8 h. After the reaction is complete, transfer the reaction solution to another reaction apparatus, add a small amount of anhydrous ethanol, and concentrate by vacuum evaporation. Repeat this step to remove piperidine until no piperidine residue is detected by TLC. Dry under vacuum for 24 h to obtain Compoud 11. The weight-to-volume ratio of Compoud 10 to the mixed solution of piperidine and DMF is 1.5 g: 8 mL.

[0067] 7) The reactants were fed in a molar ratio of 1:1.8:2:2:3.7 for Compoud 11, oleic acid, HOBT, HBTU, and DIPEA, with DMF as the reaction solvent. The rest of the synthesis process was the same as in step 1) above, to obtain Compoud 12.

[0068] 8) Prepare EDL using Compound 12 as raw material. The synthesis process of EDL is the same as step 2 above.

[0069] In a third aspect of the invention, the use of bifunctional dendritic lipopeptide nanomicelles as described in the first aspect or prepared by the method described in the second aspect in the preparation of an immune cell autophagy inducer is provided.

[0070] The present invention has the following technical effects:

[0071] 1) The bifunctional dendritic lipopeptide nanomicelles prepared in this invention are prepared into nanomicelles by solvent injection, taking advantage of the amphiphilicity of dendritic lipopeptides. The structure of the dendritic lipopeptide is similar to that of a surfactant. The hydrophilic lysine sequence forms the head, which can form stable intramolecular hydrogen bonds, thereby enhancing the stability of the formed vesicles. The hydrophobic alkyl chain serves as the tail, which together with the head constitutes the unique structure of the dendritic lipopeptide. It has excellent water solubility, biocompatibility, and biodegradability, and has great application potential and prospects in drug delivery.

[0072] 2) The bifunctional dendritic lipopeptide nanomicelles of the present invention are bifunctional nanomicelles based on dendritic lipopeptide molecules. D -RNPs. In inflammatory areas, excessive ROS production leads to oxidative stress, and autophagy activity is often inhibited. Oxidative stress promotes inflammatory responses, and inhibited autophagy may lead to the continuous accumulation of intracellular damaging substances, further exacerbating cell damage, inflammatory responses, and lipid peroxidation, thus driving further inflammation. Bifunctional nanomicelles D-RNPs are ROS-responsive, able to respond to changes in ROS levels at the lesion site, clear excess ROS, reduce oxidative stress and inflammatory response, and activate autophagy by releasing D-lysine dendritic peptide knots and fourth-generation glutamate dendritic macromolecules, thereby clearing intracellular damaging substances and achieving therapeutic effects.

[0073] 3) This invention creatively mixes two fourth-generation dendritic macromolecules (lysine and glutamic acid) and self-assembles them into dendritic lipopeptide nanomicelles. Against the backdrop of existing technologies, it creatively alters the hydrophobic long-chain structure of the fourth-generation lysine dendritic macromolecule and incorporates a self-synthesized ROS-responsive bond (TK), resulting in low toxicity and ROS responsiveness. Regarding autophagy activation, the expression of two proteins, P63 and LC3, was verified through immunoprotein experiments. Compared with existing technologies, the autophagy effect is better, while also exhibiting good ROS responsiveness. Attached Figure Description

[0074] Figure 1 The nanomicelle particle size and PDI diagram are shown (Mean, n=3).

[0075] Figure 2 for L -G2K and D -G2K mass spectrum.

[0076] Figure 3 for L -G3K and D Mass spectrum of G3K.

[0077] Figure 4 for L -G4K and D -G4K mass spectrum.

[0078] Figure 5 This is the mass spectrum of TK.

[0079] Figure 6-1 This is the mass spectrum of TK-OAm.

[0080] Figure 6-2 for L -KRDL mass spectrum.

[0081] Figure 6-3 for D -KRDL mass spectrum.

[0082] Figure 7 for D Mass spectrum of KNDL.

[0083] Figure 8 This is the mass spectrum of EDL.

[0084] Figure 9chiral dendritic molecule G n CD spectrum of K.

[0085] Figure 10 This is a graph showing the protein adsorption levels after nanomicelles were incubated with FBS for 2 h and 4 h.

[0086] Figure 11 The image shows the fluorescence intensity of intracellular ROS probes after ROS removal by nanomicelles, where **** indicates p < 0.0001, ns indicates p ≥ 0.05, and n = 3.

[0087] Figure 12 The graph shows the expression levels and semi-quantitative analysis of autophagy marker proteins LC3 and p62 in foam cells after treatment with nanomicelles. In the graph, **** indicates p < 0.0001, ** indicates p < 0.01, ns indicates p ≥ 0.05, and n = 3. Detailed Implementation

[0088] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0089] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0090] Among them: L-lysine methyl ester dihydrochloride (H-Lys-OMe·2HCl) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (Boc-Lys(Boc)-OH) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; D-lysine methyl ester dihydrochloride (DH-Lys-OMe·2HCl) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; N2,N6-bis(tert-butoxycarbonyl)-D-lysine (Boc-D-Lysine) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. s(Boc)-OH), purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; L-glutamic acid di-tert-butyl hydrochloride (H-Glu(OtBu)-OtBu·HCl), purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; N-(9-fluorenylmethoxycarbonyl)-L-glutamic acid (Fmoc-L-Glu-OH), purchased from Beijing Bailingwei Technology Co., Ltd.; N-fluorenylmethoxycarbonyl-1,2-diaminoethane hydrochloride (Fmoc-ethylenediamine), purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; 3- Mercaptopropionic acid was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; triethylsilane was purchased from Shanghai Aladdin Biochemical Co., Ltd.; oleylamine (OAm) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; oleic acid (OAc) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; 1-hydroxybenzotriazole (HOBt) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; 1-ethyl... 3-Dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was purchased from Shanghai Aladdin Biochemical Co., Ltd.; N-hydroxysuccinimide (NHS) was purchased from Shanghai Aladdin Biochemical Co., Ltd.; phorbol 12-tetradecanoate 13-acetate (PMA) was purchased from Beyotime Biotechnology Co., Ltd.; lipopolysaccharide (LPS) (derived from Escherichia coli O55:B5) was purchased from Sigma-Aldrich; oxidized low-density lipoprotein (oxLDL) was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd.

[0091] Example 1: Second-generation chiral dendritic molecules L -G2K and D -G2K synthesis

[0092] L -G2K D -G2K mass spectrometry such as Figure 2 The synthetic route is as follows:

[0093]

[0094] LSynthesis of -G2K: 2.33 g (10 mmol) of H-Lys-OMe·2HCl, 7.97 g (23 mmol) of Boc-Lys(Boc)-OH, 4.05 g (30 mmol) of HOBT, 11.38 g (30 mmol) of HBTU, and 10.45 mL (60 mmol) of DIPEA were weighed. The reactants were added according to a molar ratio of 1:2:3:3:6 for H-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA. The reaction solvent was 50 mL of LDM.

[0095] Under ice bath and nitrogen protection, the above raw materials were added to a side-necked flask, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, and then stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and then washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. The crude reaction solution was then concentrated and purified by thin-layer chromatography using petroleum ether:ethyl acetate (1:1, V:V) as the mobile phase to obtain... L -G2K(Boc)4.

[0096] Weigh the above L 42.0 g of G2K(Boc) was placed in a three-necked flask, a magnetic stopper was added, and the flask was sealed with a rubber stopper and placed in an ice bath. Under nitrogen protection, 6 mL of LDM was slowly added to dissolve the G2K, followed by 6 mL of LTF. The mixture was stirred in the ice bath for 30 min, and then stirred again at room temperature for 6 h. After the reaction was complete, the reaction solution was evaporated under reduced pressure and concentrated to dryness. The solution was washed with pre-cooled anhydrous diethyl ether until white, and then dried under vacuum for 24 h to obtain the desired product. L -G2K white solid 0.96 g, yield 86.9%.

[0097] DSynthesis of -G2K: 2.33 g (10 mmol) of DH-Lys-OMe·2HCl, 7.97 g (23 mmol) of Boc-D-Lys(Boc)-OH, 4.05 g (30 mmol) of HOBT, 11.38 g (30 mmol) of HBTU, and 10.45 mL (60 mmol) of DIPEA were weighed. The reactants were added in a molar ratio of DH-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA of 1:2:3:3:6. The reaction solvent was 50 mL of LDM. The remaining synthetic steps were the same as described above. L -G2K, yield rate 88.2%.

[0098] L -G2K and D The mass spectrometry results of G2K are as follows: Figure 2 As shown, MS(ESI) m / z: 417.4 [M+H] + The theoretical and calculated molecular weights of both are consistent. The 1H NMR spectroscopy results show that... L -G2K 1 H-NMR (300MHz, D2O): δ 4.39 (dd, J = 8.6, 5.6Hz, 1H), 3.99 (q, J = 6.2Hz, 1H), 3.88 (t, J = 6.7Hz, 1H), 3.71 (s, 3H), 3.18 (t, J = 7.2Hz, 2H), 2.96 (q, J = 7.0Hz, 4H), 1.98–1.03 (m, 22H). The characteristic peaks were correctly assigned. D -G2K 1 ¹H-NMR (300MHz, D₂O): δ 4.38 (dd, J = 8.6, 5.6Hz, 1H), 3.99 (t, J = 6.4Hz, 1H), 3.87 (t, J = 6.7Hz, 1H), 3.70 (s, 3H), 3.23–3.13 (m, 2H), 2.95 (q, J = 6.5Hz, 4H), 1.95–1.08 (m, 22H). The characteristic peaks were correctly assigned. Mass spectrometry and proton NMR results confirm this. L -G2K and D -G2K has been successfully synthesized.

[0099] Example 2: Third-generation chiral dendritic molecules L -G3K and D -G3K Synthesis

[0100] L -G3K D-G3K mass spectrometry Figure 3 The synthetic route is as follows:

[0101]

[0102] L -G3K synthesis: weigh L -G2K 1.22g (2.93mmol), Boc-Lys(Boc)-OH 4.67g (13.48mmol), HOBT 2.38g (17.58mmol), HBTU 6.67g (17.58mmol), DIPEA 4.54 mL (35.16mmol). According to... L The reaction mixture consisted of G2K, Boc-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA in a molar ratio of 1:4:5:5:11, and the reaction solvent was 70 mL LDM.

[0103] Under ice bath and nitrogen protection, the above raw materials were added to a side-necked flask, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, and then stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and then washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation, and then recrystallized with ethyl acetate. The crystals were filtered and washed with cold ethyl acetate, filtered to dryness, and then dried under vacuum for 24 hours to obtain the desired product. L -G3K(Boc)8 is a white solid.

[0104] Weigh the above L 82.0 g of -G3K(Boc) was placed in a three-necked flask, a magnetic stopper was added, and the flask was sealed with a rubber stopper. The flask was then placed in an ice bath. Under nitrogen protection, 14 mL of a mixed solvent of TFA and DCM (1:1, V:V) was slowly added to the flask. Under nitrogen protection, the mixture was stirred in an ice bath for 30 min, and then stirred for another 6 h at room temperature. After the reaction was complete, the reaction solution was concentrated by vacuum evaporation and then evaporated to dryness. The solution was washed with pre-cooled anhydrous diethyl ether until a white solid was obtained, and then dried under vacuum for 24 h. L -G3K white solid 0.97g, yield 90.7%.

[0105] D -G3K synthesis: weigh D-G2K 1.22g (2.93mmol), Boc-D-Lys(Boc)-OH 4.67g (13.48mmol), HOBT 2.38g (17.58mmol), HBTU 6.67g (17.58mmol), DIPEA 4.54mL (35.16mmol). According to... D The reactants were G2K, Boc-D-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA in a molar ratio of 1:4:5:5:11, with DMF as the reaction solvent. The remaining synthesis process was the same as described above. L -G3K, yield 94.0%.

[0106] L -G3K and D The mass spectrometry results of G3K are as follows: Figure 3 As shown, MS(ESI) m / z: 929.0 [M] + The theoretical and calculated molecular weights of both are consistent. The 1H NMR spectroscopy results show that... L -G3K 1 H-NMR (300MHz, D2O): δ4.33–4.11(m,3H), 4.02–3.83(m,4H), 3.70(s,3H), 3.51(q,J=7.1Hz,4H), 3.27–3.05(m,6H), 2.95(q,J=7.1Hz,8H), 1.86–1.09(m,46H), all characteristic peaks are correctly assigned. D -G3K 1 ¹H-NMR (300MHz, D₂O): δ 4.32–4.13 (m, 3H), 4.02–3.84 (m, 4H), 3.70 (s, 3H), 3.51 (q, J = 7.1Hz, 4H), 3.13 (dt, J = 21.5, 6.9Hz, 6H), 2.99–2.92 (m, 8H), 1.86–1.10 (m, 46H). The characteristic peaks were correctly assigned. Mass spectrometry and proton NMR results confirm this. L -G3K and D -G3K has been successfully synthesized.

[0107] Example 3: Fourth-generation chiral dendritic molecules L -G4K and D -G4K synthesis

[0108] L -G4K and D -G4K mass spectrometry such as Figure 4 The synthetic route is as follows:

[0109]

[0110] L -G4K synthesis: weighing L -G3K 1.5g (1.61mmol), Boc-Lys(Boc)-OH 5.82g (16.8mmol), HOBT 2.61g (19.32mmol), HBTU 7.32g (19.32mmol), DIPEA 6.24mL (48.3mmol). According to... L The reaction mixture consisted of G3K, Boc-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA in a molar ratio of 1:10:12:12:30, and the reaction solvent was 70 mL LDM.

[0111] The above-mentioned raw materials were added to a three-necked flask under ice bath and nitrogen protection, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, and then stirred at room temperature for 36 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and then washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation, and then recrystallized with ethyl acetate. The crystals were filtered and washed with cold ethyl acetate, filtered to dryness, and then dried under vacuum for 24 h to obtain the desired product. L -G4K(Boc) 16 White solid.

[0112] Weigh the above L -G4K(Boc) 16 2.0 g of the solution was placed in a three-necked flask, a magnetic stir bar was added, and the flask was sealed with a rubber stopper and placed in an ice bath. Under nitrogen protection, 16 mL of a mixed solvent of TFA and DCM (1:1, V:V) was slowly added to the flask. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred again for 7 h at room temperature. After the reaction was complete, the reaction solution was concentrated by vacuum evaporation to dryness, washed with pre-cooled anhydrous diethyl ether until a white solid was obtained, and then dried under vacuum for 24 h. L -G4K white solid 1.03g, yield 89.1%.

[0113] D -G4K synthesis: weighing D-G3K 1.5g (1.61mmol), Boc-D-Lys(Boc)-OH 5.82g (16.8mmol), HOBT 2.61g (19.32mmol), HBTU 7.32g (19.32mmol), DIPEA 6.24mL (48.3mmol). According to... D The following reactants were added in a molar ratio of 1:10:12:12:30: G3K, Boc-D-Lys(Boc)-OH, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA, with DMF as the reaction solvent. The remaining synthesis process was the same as described above. L -G4K, yield rate 91.3%.

[0114] L -G4K and D The mass spectrometry results of G4K are as follows: Figure 4 As shown, MS(ESI) m / z: 1977.79 [M+Na] + The theoretical and calculated molecular weights of both are consistent. The 1H NMR spectroscopy results show that... L -G4K 1 H-NMR (300MHz, D2O): δ 4.28–4.10 (m, 7H), 3.97 (t, J = 6.6Hz, 4H), 3.86 (t, J = 6.6Hz, 4H), 3.68 (s, 3H), 3.50 (q, J = 7.1Hz, 4H), 3.30–3.06 (m, 18H), 2.94 (q, J = 6.9Hz, 20H), 1.74–1.08 (m, 94H). The characteristic peaks were correctly assigned. D -G4K 1 1H-NMR (300MHz, D2O): δ 4.35–4.10 (m, 7H), 3.99 (t, J = 6.6Hz, 4H), 3.93–3.86 (m, 4H), 3.70 (s, 3H), 3.53 (d, J = 7.1Hz, 4H), 3.24 (d, J = 43.1Hz, 18H), 2.97 (d, J = 7.8Hz, 20H), 1.73–1.12 (m, 94H). The characteristic peaks were correctly assigned. Mass spectrometry and proton NMR results confirm this. L -G4K and D -G4K has been successfully synthesized.

[0115] Example 4: Synthesis of ROS-responsive ketithial thioglycolate TK

[0116] TK's mass spectrometry, as shown Figure 5 The synthetic route is as follows:

[0117]

[0118] Anhydrous 3-mercaptopropionic acid (108.02 mmol) and anhydrous acetone (49.10 mmol) were placed in a three-necked flask and stirred. A catalytic amount of TFA was added, and the reaction was stirred at room temperature for 6 h. At the end of the reaction, a white solid precipitated. The white solid was filtered under vacuum and washed alternately five times each with cold n-hexane and cold deionized water to remove residual 3-mercaptopropionic acid and acetone, taking advantage of their water solubility. After vacuum drying for 24 h, 10.32 g of white solid TK was obtained, with a yield of 71.9%.

[0119] Example 5: ROS-responsive dendritic lipopeptides L -KRDL and D -KRDL Synthesis

[0120]

[0121] First, a carbon chain containing TK bonds, TK-OAm, was synthesized to serve as the hydrophobic portion of the ROS-responsive dendritic lipopeptide KRDL. Its mass spectra are shown below. Figure 6-1 The synthetic route is as follows: 1.52 g (5.39 mmol) of TK, 0.48 g (1.80 mmol) of OAm, 0.41 g (2.16 mmol) of EDCI, and 0.25 g (2.16 mmol) of NHS were weighed. The reactants were added according to a molar ratio of TK, OAm, condensing agent [carbodiimide hydrochloride (EDCI)], and NHS of 1:2.7:0.4:0.4, and the reaction solvent was 30 mL of LDM.

[0122] The above-mentioned raw materials were added to a three-necked flask under ice bath and nitrogen protection, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, and then stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate and washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. The crude reaction solution was then concentrated and purified by thin-layer chromatography using dichloromethane:methanol (45:1, V:V) as the mobile phase, yielding 384.5 mg of TK-OAm, with a yield of 42.7%.

[0123] L-type and D-type ROS-responsive dendritic lipopeptides L -KRDL, D -KRDL mass spectrometry such as Figure 6-2 6-3, the specific synthesis route is as follows:

[0124]

[0125] L -KRDL synthesis:

[0126] (1) Weighing L -G4K(Boc) 16 0.5 g (0.14 mmol) was added to a three-necked flask, a magnetic stir bar was added, and the flask was placed in an ice bath. 25 mL of 1 mol / L sodium hydroxide methanol solution was added to the flask, and the reaction was carried out in an ice bath for 8 h. After the reaction was complete, the pH of the reaction solution was adjusted to 3-4 with 1 mol / L hydrochloric acid solution, and the reaction solution was extracted three times with ethyl acetate. The organic phase was collected and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed by evaporation under reduced pressure, and the solution was dried under vacuum for 24 h. 0.385 g of a white solid, Compoud 1, was obtained, with a yield of 77.3%.

[0127] (2) Weigh 0.04 g (0.142 mmol) of Fmoc-ethylenediamine, 0.024 g (0.177 mmol) of HOBT, 0.067 g (0.177 mmol) of HBTU, and 0.2 mL (1.15 mmol) of DIPEA. Add the raw materials according to a molar ratio of 1:1:1.2:1.2:8 for Fmoc-ethylenediamine, Compoud 1, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA. Add the above raw materials to a three-necked flask under ice bath and nitrogen protection, then add 35 mL of DMF and DIPEA to dissolve the solid drug. React in an ice bath for 30 min, then stir at room temperature. After the reaction was complete, the reaction solution was extracted with ethyl acetate, and then washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation, and then recrystallized with ethyl acetate. The crystals were filtered and washed with cold ethyl acetate, filtered to dryness, and then vacuum dried for 24 hours. L -Compoud 2 white solid 0.345 g, yield 64.2%.

[0128] (3) Accurately weigh L - 20.22 g (0.231 mmol) of compound was placed in a three-necked flask, a magnetic stopper was added, and the flask was sealed with a rubber stopper. The flask was then placed in an ice bath. Under nitrogen protection, 5 mL of a mixed solvent of piperidine and DMF (1:4, V:V) was slowly added. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred for another 6 h at room temperature. After the reaction was complete, the reaction mixture was transferred to a round-bottom flask. Anhydrous ethanol was added and the mixture was concentrated under reduced pressure. This process was repeated until no piperidine spots were observed on the TLC plate. After the piperidine was washed away, the mixture was concentrated under reduced pressure and evaporated to dryness. The solution was then dried under vacuum for 24 h to obtain the desired product.L -Compoud 3 white solid 0.157 g, yield 75.7%.

[0129] (4) Weigh L -Compoud 30.208g (0.058mmol), TK-OAm 0.038g (0.0754mmol), HOBT 0.012g (0.087mmol), HBTU 0.033g (0.087mmol), DIPEA 0.1mL (0.58mmol). According to... L The molar ratios of L-Compoud, TK-OAm, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA were 1:1.2:1.3:1.3:10. Under ice bath and nitrogen protection, the above raw materials were added to a three-necked flask, followed by the addition of 30 mL of LDM and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, then stirred at room temperature. After the reaction was complete, the reaction solution was extracted with ethyl acetate and washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4 to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation and recrystallized with ethyl acetate. The crystals were filtered and washed with cold ethyl acetate, filtered to dryness, and then dried under vacuum for 24 h. 0.169 g of L-Compoud 4 white solid was obtained, with a yield of 81.6%.

[0130] (5) Weighing L -Compoud 40.2 g (0.05 mmol) was placed in a three-necked flask, a magnetic stopper was added, and the flask was sealed with a rubber stopper and placed in an ice bath. Under nitrogen protection, a mixed solution of 8 mL of LTFA and DCM (1:3, V:V) was slowly added to the flask. Under nitrogen protection, the mixture was stirred in an ice bath for 30 min, and then stirred for another 7 h at room temperature. After the reaction was complete, the reaction solution was concentrated by vacuum evaporation, evaporated to dryness, washed with pre-cooled anhydrous diethyl ether until white, and then dried under vacuum for 24 h. L -KRDL white solid 0.2 g (0.05 mmol).

[0131] D -KRDL synthesis:

[0132] (1) Weighing D -G4K(Boc) 160.5 g (0.14 mmol) was added to a three-necked flask, a magnetic stir bar was added, and the flask was placed in an ice bath. 25 mL of 1 mol / L sodium hydroxide methanol solution was added to the flask, and the reaction was carried out in an ice bath for 8 h. After the reaction was complete, the pH of the reaction solution was adjusted to 3-4 with 1 mol / L hydrochloric acid solution, and the reaction solution was extracted three times with ethyl acetate. The organic phase was collected and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed by evaporation under reduced pressure, and the solution was dried under vacuum for 24 h. D -Compoud 1, white solid, 0.399 g, yield 80.1%.

[0133] (2)-(5) are the same as steps. L -KRDL synthesis.

[0134] Comparative Example 1: ROS-free responsive dendritic lipopeptides D -KNDL Synthesis

[0135] Synthesis of ROS-free responsive dendritic lipopeptides without TK bonds D -KNDL is a control lipopeptide used in subsequent ROS response performance evaluations. D Mass spectrometry of KNDL, such as Figure 7 The synthetic route is as follows:

[0136]

[0137] Weigh out 10.5 g (0.28 mmol) of Compoud, 0.057 g (0.42 mmol) of HOBT, 0.159 g (0.42 mmol) of HBTU, and 0.2 mL (1.15 mmol) of DIPEA. Add the following materials according to a molar ratio of Compoud, oleylamine, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA of 1:2:1.5:1.5:4. Add the above materials to a three-necked flask under ice bath and nitrogen protection, then add 0.15 g (0.56 mmol) of oleylamine and dissolve the solid drug in 40 mL of DMF and DIPEA. React in an ice bath for 30 min, then stir at room temperature for 24-30 h. After the reaction was complete, the reaction solution was extracted with ethyl acetate, and then washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous Na2SO4, respectively, to obtain a crude product solution. The organic phase was concentrated by vacuum evaporation after filtration, and then recrystallized with ethyl acetate. The crystals were filtered and washed with cold ethyl acetate, filtered to dryness, and then dried under vacuum for 24 hours to obtain Compound 5 as a white solid.

[0138] Weigh 50.5 g (0.13 mmol) of Compoud into a three-necked flask, add a magnetic stopper, and seal the flask with a rubber stopper. Place the flask in an ice bath. Under nitrogen protection, slowly add 6 mL of a mixed solvent of LTFA and DCM (1:1, V:V). Stir the reaction mixture in an ice bath for 30 min under nitrogen protection, and continue stirring at room temperature for 8 h. After the reaction is complete, evaporate the reaction solution under reduced pressure to a dryness, wash with pre-cooled anhydrous diethyl ether until white, and dry under vacuum for 24 h. D -KRDL white solid 0.192 g, yield 66.9%.

[0139] Example 6: Synthesis of EDL, a dendritic lipopeptide with glutamic acid as its basic structural unit

[0140] A dendritic molecule with glutamic acid as the basic structural unit was synthesized through successive generations via amide condensation and OtBu removal reactions, serving as the hydrophilic head of EDL. This dendritic molecule was further deprotected by the Fmoc protecting group, exposing the amino group. Oleic acid (OAc) was used as the hydrophobic tail of the EDL, and the carboxyl group on the oleylamine was condensed with the amino group on the dendritic molecule via an amide condensation reaction to obtain the dendritic lipopeptide EDL. The mass spectra of the EDL are shown below. Figure 8 The synthetic route is as follows:

[0141]

[0142] (1) Weigh 2.58 g (7 mmol) of Fmoc-L-Glu-OH, 4.35 g (16.8 mmol) of H-Glu(OtBu)-OtBu·HCl, 2.36 g (17.5 mmol) of HOBT, 6.64 g (17.5 mmol) of HBTU, and 9.2 mL (52.5 mmol) of DIPEA. Add the reactants according to the molar ratio of Fmoc-L-Glu-OH, H-Glu(OtBu)-OtBu·HCl, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA as 1:2.4:2.5:2.5:7.5. The reaction solvent is 50 mL of LDM.

[0143] The above-mentioned raw materials were added to a three-necked flask under ice bath and nitrogen protection, followed by the addition of 50 mL of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, and then stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was extracted with ethyl acetate and washed, salted out, and dried with saturated NaHCO3, dilute hydrochloric acid, saturated NaCl, and anhydrous MgSO4, respectively, to obtain a crude product solution. The crude reaction solution was then concentrated and purified by thin-layer chromatography using petroleum ether:ethyl acetate (2:1, V:V) as the mobile phase to obtain Compound 6.

[0144] (2) Weigh 2.5 g (2.94 mmol) of Compoud 6 into a three-necked flask. Under nitrogen protection, slowly add an appropriate amount of dichloromethane (DCM). After the solid in the three-necked flask dissolves, slowly add 4 mL of a mixed solution of TFA and DCM (1:1, V:V) and a small amount of triethylsilane. Under nitrogen protection, stir the reaction in an ice bath for 30 min, and continue stirring at room temperature for 6 h. Monitor the reaction progress by TLC. After the reaction is complete, evaporate the reaction solution under reduced pressure to concentrate it to dryness, wash it with pre-cooled anhydrous diethyl ether until white, and dry it under vacuum for 24 h. 1.47 g of white solid Compoud 7 is obtained.

[0145] (3) Weigh 71.63 g (2.6 mmol) of Compoud, 3.70 g (12.5 mmol) of H-Glu(OtBu)-OtBu·HCl, 1.76 g (13.0 mmol) of HOBT, 4.94 g (13.0 mmol) of HBTU, and 6.8 mL (39.1 mmol) of DIPEA. Add the reactants according to a molar ratio of 1:4.8:5:5:15 for Compoud7, H-Glu(OtBu)-OtBu·HCl, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA. The reaction solvent is 50 mL of LDM. The remaining synthesis process is the same as in (1) above, with petroleum ether:ethyl acetate (1:1.5, V:V) as the mobile phase to obtain Compoud8.

[0146] (4) The synthesis process of Compoud 9 is the same as that in (2) above.

[0147] (5) Weigh out 1.5 g (1.31 mmol) of Compoud 9, 3.88 g (13.11 mmol) of H-Glu(OtBu)-OtBu·HCl, 1.77 g (13.1 mmol) of HOBT, 4.97 g (13.1 mmol) of HBTU, and 8.2 mL (47.16 mmol) of DIPEA. Add the reactants according to a molar ratio of 1:10:10:10:36 for Compoud 9, H-Glu(OtBu)-OtBu·HCl, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA. Use 55 mL of DMF as the reaction solvent. The rest of the synthesis process is the same as in (1) above, with dichloromethane:methanol (35:1, V:V) as the mobile phase to obtain Compoud 10.

[0148] (6) Weigh 101.5 g (0.49 mmol) of Compoud into a three-necked flask. Under nitrogen protection, slowly add 2 mL of DMF. After the solid in the three-necked flask dissolves, slowly add 2 mL of a mixed solution of piperidine and 6 mL of DMF (1:4, V:V). Under nitrogen protection, stir the reaction mixture in an ice bath for 30 min, and continue stirring at room temperature for 8 h. After the reaction is complete, transfer the reaction solution to a round-bottom flask, add a small amount of anhydrous ethanol, and concentrate by evaporation under reduced pressure. Repeat this step to remove piperidine until no piperidine residue is detected by TLC. Dry under vacuum for 24 h to obtain Compoud 11 as a white solid.

[0149] (7) Weigh out 1.0 g (0.35 mmol) of Compoud 11, 0.2 mL (0.63 mmol) of oleic acid, 0.095 g (0.7 mmol) of HOBT, 0.265 g (0.7 mmol) of HBTU, and 0.25 mL (1.31 mmol) of DIPEA. Add the reactants according to a molar ratio of 1:1.8:2:2:3.7 for Compoud 11, oleic acid, condensing agent [equimolar amounts of 1-hydroxybenzotriazole (HOBT) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU)], and DIPEA. Use 50 mL of LDM as the reaction solvent. The rest of the synthesis process is the same as described in (1). The mobile phase is dichloromethane:methanol (25:1, V:V) to obtain 0.68 g of a white solid, Compoud 12, with a yield of 62.4%.

[0150] (8) The synthesis process of EDL is the same as that in (2) above, with a yield of 76.7%.

[0151] Example 7 Dendritic G n Verification of K-chirality

[0152] The secondary structure and chirality of chiral dendritic peptides were analyzed using circular dichroism spectroscopy. L -G1K(H-LysOMe·2HCl), D -G1K(DH-Lys-OMe·2HCl), L -G2K D -G2K L -G3K D -G3K L -G4K and D -G4K was prepared into a solution with a concentration of 1.5 mg / mL and measured in the wavelength range of 190 to 250 nm. Figure 9 As shown, the results indicate that the CD spectra of D-type and L-type dendritic molecules GnK exhibit perfect mirror symmetry in the detection range of 190 nm to 250 nm, proving that the two chiral dendritic molecules are exact enantiomers.

[0153] Example 8: Preparation of nanomicelles by solvent injection method

[0154] Weigh out 5mg D -KRDL, 5mg L -KRDL, 4.44mg D -KNDL was mixed with 4.5 mg EDL (1:1, mol:mol). This mixture was dissolved in 50 μL of a mixture of anhydrous ethanol and tetrahydrofuran (1:1, V:V). The solution was then added dropwise to 1 mL of rapidly stirred PBS solution. Stirring continued until the anhydrous ethanol and tetrahydrofuran were completely evaporated, forming nanomicelles. D -RNPs, L -RNPs, D -NNPs.

[0155] PBS formulation: Dissolve 8g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.56g Na2HPO4·12H2O in 1000ml distilled water, sterilize at pH 7.2-7.4, 103kPa for 15min, and store at 4℃.

[0156] like Figure 1 Micelles prepared by solvent injection showed reduced average particle sizes of 179.5 nm, 172.0 nm, and 138.1 nm, respectively, indicating that solvent injection can produce nanomicelles with smaller particle sizes. The reduction in particle size may be attributed to the rapid dilution process in solvent injection, which reduces the aggregation tendency of amphiphilic molecules, thereby promoting the formation of smaller micelles. The polydispersity index (PDI) is a parameter measuring the uniformity of nanomicelle particle size; a smaller PDI value indicates a more uniform particle size distribution. Figure 1As shown, the corresponding PDI values ​​are 0.2070, 0.2017, and 0.2387, respectively. These data indicate that... D -RNPs, L -RNPs, D -NNPs exhibit lower PDI, indicating better particle size uniformity.

[0157] Example 9: Test of the anti-protein adsorption performance of nanomicelles

[0158] This experiment used bovine serum albumin (FBS) as a model protein and determined the anti-protein adsorption capacity of the nanogel using ultraviolet absorption spectroscopy. Different concentrations of [a specific type of nanogel] were prepared. D -RNPs, L -RNPs, D -NNPs solutions (0.1, 0.5, 1 mg / mL) were mixed with 5% FBS solution and incubated at 37°C on a shaker. Equal portions of the mixture were taken at 2 h and 4 h and centrifuged at 8000 rpm for 10 min. The supernatant was collected, and the absorbance at 275 nm was measured using a UV-Vis spectrophotometer. The amount of protein adsorbed on the sample was calculated based on the FBS standard curve. Each sample was measured in triplicate (n=3). Figure 10 As shown, the data indicates that, regardless of L -RNPs, D -NNPs or D -RNPs showed relatively low protein adsorption after incubation with bovine serum albumin (FBS) for 2 and 4 hours, indicating that all tested nanomicelles possessed a certain degree of anti-protein adsorption ability.

[0159] Example 10: Investigation of ROS scavenging ability of nano-micelles

[0160] Intracellular ROS levels were determined using the DCFH-DA fluorescent probe method. RAW 264.7 cells in logarithmic growth phase were collected by centrifugation, and the supernatant was discarded. 5 mL of fresh complete culture medium was added, and the cells were gently resuspended by pipetting. The homogenized cell suspension was loaded onto a hemocytometer for counting, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells / mL. 1 mL of cell suspension was seeded into each well of a 12-well plate and incubated for 24 h to allow complete cell adhesion. A blank control group, a drug-treated group, and a positive control group were set up. The blank group (N-CTLs) was treated with basal medium; the drug-treated groups were treated with 1 mL of medium containing 200 μM H₂O₂. D -RNPs, D -NNPs, L- RNPs solution (final concentration of nanomicelles: 1 mg / mL); positive control group P-CTL was added to 1 mL of basal medium containing 200 μM H2O2. After culturing for 30 min, the culture medium was aspirated and each well was washed 3 times with PBS buffer. 100 μL of DCFH-DA probe was added to each well, and after culturing for 20 min, each well was washed 3 times with PBS buffer. 400 μL of PBS solution was added to each well, and the cells were gently pipetted until they detached. The cell suspension was collected in a centrifuge tube, and the cell suspension was gently pipetted again to disperse the cells evenly. The fluorescence intensity of DCFH-DA was measured by flow cytometry (EX: 488 nm, EM: 525 nm). Two replicates were set for each sample (n = 3). Figure 11 The results showed L -RNPs, D Compared with the P-CTL group, the fluorescence intensity of the -RNPs group was significantly reduced, showing a significant difference, but there was no significant difference compared with the blank control group N-CTL without added H2O2. D The fluorescence intensity of the -NNPs group was similar to that of the P-CTL group, indicating that there was no ROS scavenging effect. This demonstrates the presence of ROS-responsive TK nanomicelles. L -RNPs, D -RNPs have a strong ROS scavenging ability, while TK nanomicelles lack ROS-responsive bonds. D -NNPs do not have the ability to remove ROS.

[0161] Example 11: Investigation of the effect of nanomicelles on the expression of autophagy marker proteins

[0162] The expression changes of autophagy-related proteins p62 and LC3-II in cells were investigated using Western blot. The differences in protein expression levels were quantitatively analyzed using grayscale measurements to determine the abundance of autophagosomes in the cells. THP-1 cells in logarithmic growth phase were collected by centrifugation. After discarding the supernatant, 5 mL of fresh complete culture medium was added, and the cells were gently resuspended by pipetting. The homogenized cell suspension was loaded onto a hemocytometer for counting, and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 5Cells were cultured with PMA (10 mg / mL) and LPS (1 mg / mL) to a final PMA concentration of 1 μg / mL and a final LPS concentration of 500 ng / mL. The cell suspension containing PMA and LPS was seeded into 60 mm cell culture dishes (5 mL cell suspension per dish). The culture dishes were then incubated at 37°C with 5% CO2 for 12 h until cell adhesion was achieved. The experiment included a blank control group (N-CTL), a drug-treated group, a Rapa control group, and a model control group (M-CTL). The culture medium was aspirated and washed three times with RPMI-1640 medium. Fresh medium was added to the blank group (N-CTL), while 5 mL of serum-free medium containing 50 μg / mL oxLDL was added to the other groups. After 36 h of incubation, 5 mL of fresh medium was added to the N-CTL and P-CTL groups; 5 mL of medium containing 1 mg / mL oxLDL was added to the drug-treated groups. D -RNPs, D -NNPs, L -RNPs; Rapa control group: 1 mL of culture medium containing 100 nM Rapa solution was added. After administration, the samples were incubated at 37°C and 5% CO2 for 24 h. Proteins were extracted by lysis with RIPA lysis buffer, and protein concentration was determined by the BCA method. The content of each sample was calculated based on the measured absorbance. The proteins in each group were diluted to the same concentration with RIPA lysis buffer, and 5×SDS protein loading buffer was added at a ratio of 1:4. The samples were then heated to denature. Gel preparation, loading, electrophoresis, transfer to a membrane, blocking, incubation with primary and secondary antibodies, and development were performed. Figure 12 The results showed that LC3-II protein expression... D The LC3-II expression level in the RNPs group was significantly higher than that in the Rapa group. D The LC3-II expression level in the -NNPs group was slightly higher than that in the Rapa group, while L - The RNPs group had lower LC3-II expression levels. This demonstrates... D -RNPs may have a stronger autophagy activation effect. Regarding p62 protein expression, D - The p62 expression level in the RNPs group was close to that in the Rapa group, while D p62 expression in the -NNPs group was significantly higher than that in Rapa, indicating that D -RNPs may have an autophagy activation effect similar to rapamycin, indicating that... D -NNPs may involve blocked autophagy pathways or incomplete autophagy. Considering the changes in both LC3-II and p62, compared to the Rapa group, D -NNPs may have similar autophagy activation effects, while D The -RNPs group showed a significantly stronger autophagy activation effect. Conversely, L-RNPs do not have the ability to activate autophagy.

[0163] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A bifunctional dendritic lipopeptide nanomicelle, characterized in that, The bifunctional dendritic lipopeptide nanomicelles are composed of chiral ROS-responsive dendritic lipopeptides. D- It is made from KRDL and EDL (glutamate dendritic lipopeptide) molecules: The aforementioned D- The KRDL structure is shown in Equation V: Formula V; The structure of the EDL is as shown in Equation VI: Formula VI; The dendritic lipopeptide nanomicelles are composed of chiral ROS-responsive dendritic lipopeptides. D- KRDL and glutamate dendritic lipopeptide molecules EDL were mixed in an equimolar ratio and prepared by solvent injection.

2. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of G n K, n = 2, 3 and 4 Synthesize chiral dendritic molecules G with D-lysine as the basic structural unit at different generations. n K(Boc), n = 2, 3 and 4, and further removing the Boc protecting group to obtain the corresponding chiral dendritic molecular product G. n K, n = 2, 3 and 4; (2) Synthesis of chiral ROS-responsive dendritic lipopeptides D- KRDL Based on the fourth-generation chiral lysine molecule, a demethylation reaction was first carried out with 1 mol / L sodium hydroxide methanol solution to expose the carboxyl group. The mixture was then extracted with ethyl acetate, dried over anhydrous Na₂SO₄, concentrated under reduced pressure to remove the solvent, and dried under vacuum for 24 h to obtain a series of intermediates. These intermediates were then combined with a hydrophobic long chain composed of a ROS-responsive ketithiolide and oleylamine to synthesize a chiral ROS-responsive dendritic lipopeptide. D -KRDL; (3) Synthesis of glutamate dendritic lipopeptide molecules EDL Dendritic molecules of different generations with glutamic acid as the basic structural unit were synthesized. Based on the fourth-generation glutamic acid molecule, the Fmoc protecting group was further removed and then combined with a hydrophobic long chain composed of oleic acid to synthesize the glutamic acid dendritic lipopeptide molecule EDL. (4) Preparation of nanomicelles Will D KRDL and EDL are mixed in a 1:1 molar ratio and added to a 1:1 volume ratio solution of anhydrous ethanol and tetrahydrofuran until fully dissolved. This solution is then added dropwise to a rapidly stirred PBS solution, and stirring continues until the anhydrous ethanol and tetrahydrofuran have completely evaporated, forming nanomicelles. D -RNPs.

3. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 2, characterized in that, In step (1), the second-generation chiral dendritic molecule D The synthesis method of -G2K is as follows: 1) Under ice bath and nitrogen protection, the above raw materials were added to the reaction apparatus according to the molar ratio of DH-Lys-OMe·2HCl, Boc-Lys(Boc)-OH, HOBT, HBTU, and DIPEA of 1:2:3:3:

6. Appropriate amounts of DMF and DIPEA were then added to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, followed by stirring at room temperature for 24 hours. After the reaction was completed, the reaction solution was extracted, washed, salted out, and dried to obtain a crude product solution. The crude reaction solution was then concentrated and purified to obtain... D -G2K(Boc)4; 2) Take the above D In a reaction apparatus under nitrogen protection, DCM was slowly added to dissolve G2K(Boc)4, followed by slow addition of TFA. The mixture was stirred in an ice bath for 30 min, and then stirred again at room temperature for 3-6 h. After the reaction was complete, the reaction solution was evaporated under reduced pressure, concentrated, and evaporated to dryness. The solution was then washed and dried under vacuum for 24 h to obtain the desired product. D -G2K; where D The weight-to-volume ratio of -G2K(Boc)4, DCM, and TFA is 1g:3mL:3mL.

4. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 2, characterized in that, In step (1), the third-generation chiral dendritic molecule D The synthesis method of -G3K is as follows: 1) Under ice bath and nitrogen protection, according to D The molar ratio of -G2K, Boc-D-Lys(Boc)-OH, HOBT, HBTU, and DIPEA is 1:4:5:5:

11. The above raw materials are added to the reaction apparatus, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature for 24 hours. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, recrystallized, and the crystals are filtered. After filtration to dryness, the product is vacuum dried for 24 hours to obtain... D -G3K(Boc)8 White solid; 2) Weigh the above D In a reaction apparatus under nitrogen protection, a mixed solvent of TFA and DCM was slowly added. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred again at room temperature for 4-6 h. After the reaction was complete, the reaction solution was evaporated under reduced pressure, concentrated, and evaporated to dryness. The solution was then washed and vacuum dried for 24 h to obtain the desired product. D -G3K; where D The weight-to-volume ratio of -G3K(Boc)8, TFA, and DCM is 2g:7mL:7mL.

5. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 2, characterized in that, In step (1) described above, fourth-generation chiral dendritic molecules D The synthesis method for -G4K is as follows: 1) Under ice bath and nitrogen protection, according to D The molar ratio of -G3K, Boc-D-Lys(Boc)-OH, HOBT, HBTU, and DIPEA is 1:10:12:12:

30. The above raw materials are added to the reaction apparatus, followed by the addition of appropriate amounts of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature for 36 hours. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, then recrystallized, filtered to obtain crystals, and finally dried under vacuum for 24 hours. D -G4K(Boc) 16 ; 2) Weigh the above D -G4K(Boc) 16 In a reaction apparatus under nitrogen protection, a mixed solvent of TFA and DCM was slowly added. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred again at room temperature for 5-7 h. After the reaction was complete, the reaction solution was concentrated by vacuum evaporation, washed, and vacuum dried for 24 h to obtain the desired product. D -G4K; where D -G4K(Boc) 16 The weight-to-volume ratio of TFA to DCM is 1g:4mL:4mL.

6. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 2, characterized in that, In step (2), chiral ROS-responsive dendritic lipopeptides are synthesized. D- The synthesis method of KRDL is as follows: (1) Take D -G4K(Boc) 16 In the reaction apparatus, sodium hydroxide methanol solution was added, and the reaction was carried out under ice bath conditions for 8 h. After the reaction was completed, the pH of the reaction solution was adjusted to 3-4 with hydrochloric acid solution, and the reaction solution was extracted three times with ethyl acetate. The organic phase was collected, dried with anhydrous Na2SO4, filtered, concentrated under reduced pressure to remove the solvent, and dried under vacuum for 24 h to obtain the final product. D -Compoud1; (2) Under ice bath and nitrogen protection, according to Fmoc-ethylenediamine, D The molar ratio of Compound 1, HOBT, HBTU, and DIPEA is 1:1:1.2:1.2:

8. These raw materials are added to the reaction apparatus, followed by the addition of DMF and DIPEA to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, then stirred at room temperature. After the reaction is complete, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase is concentrated by vacuum evaporation, then recrystallized. The crystals are filtered and dried under vacuum for 24 hours to obtain the final product. D -Compoud 2; (3) Take D -Compoud 2 was placed in a reaction apparatus, and under nitrogen protection, a mixed solvent of piperidine and DMF with a volume ratio of 1:4 was slowly added. The mixture was stirred in an ice bath for 30 min under nitrogen protection, and then stirred again at room temperature for 6 h. After the reaction was complete, the reaction solution was transferred to another reaction apparatus, and anhydrous ethanol was added and concentrated under reduced pressure. This operation was repeated until no piperidine spots were observed on the TLC plate. After the piperidine was cleaned, the solution was concentrated under reduced pressure and evaporated to dryness. The solution was then dried under vacuum for 24 h to obtain... D -Compoud 3; where, D The weight-to-volume ratio of Compoud 2 to piperidine to DMF is 0.22 g : 5 mL. (4) Under ice bath and nitrogen protection, according to D The molar ratio of Compound 3, TK-OAm, HOBT, HBTU, and DIPEA was 1:1.2:1.3:1.3:

10. The above raw materials were added to the reaction apparatus, followed by the addition of DMF and DIPEA to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 minutes, then stirred at room temperature. After the reaction was complete, the reaction solution was extracted, washed, salted out, and dried to obtain a crude product solution. After filtration, the organic phase was concentrated by vacuum evaporation, then recrystallized. The crystals were filtered and dried under vacuum for 24 hours to obtain the final product. D -Compoud 4; (5) Take D -Compoud 4 In a reaction apparatus under nitrogen protection, a mixed solution of TFA and DCM with a volume ratio of 1:3 was slowly added to the above reaction apparatus. Under nitrogen protection, the mixture was stirred in an ice bath for 30 min, and then stirred for another 6-8 h at room temperature. After the reaction was completed, the reaction solution was evaporated and concentrated under reduced pressure, then washed and vacuum dried for 24 h to obtain the desired product. D -KRDL; where, D The weight-to-volume ratio of the mixed solution of Compoud 4, TFA, and DCM is 0.2 g : 8 mL.

7. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 6, characterized in that, In step (2), the method for synthesizing carbon chain TK-OAm containing TK bonds is as follows: Under ice bath and nitrogen protection, the above raw materials are added to the reaction device according to the molar ratio of TK, OAm, EDCI, and NHS of 1:2.7:0.4:0.

4. Then, an appropriate amount of DMF and DIPEA are added to dissolve the solid drug. The reaction is first carried out in an ice bath for 30 minutes, and then stirred at room temperature for 4 hours. After the reaction is completed, the reaction solution is extracted, washed, salted out, and dried to obtain a crude product solution. Then, the crude reaction solution is concentrated and purified to obtain TK-OAm.

8. The method for preparing bifunctional dendritic lipopeptide nanomicelles according to claim 2, characterized in that, In step (3), the synthesis method of glutamate dendritic lipopeptide molecule EDL is as follows: 1) Under ice bath and nitrogen protection, the above raw materials were added to the reaction apparatus according to the molar ratio of Fmoc-L-Glu-OH, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU and DIPEA of 1:2.4:2.5:2.5:7.

5. DMF and DIPEA were then added to dissolve the solid drug. The reaction was first carried out in an ice bath for 30 min, and then stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was extracted, washed, salted out and dried to obtain a crude product solution. The crude reaction solution was then concentrated and purified to obtain Compound6. 2) Place Compoud 6 in a reaction apparatus. Under nitrogen protection, slowly add an appropriate amount of dichloromethane. After the solid dissolves, slowly add a 1:1 volume ratio of TFA and DCM mixed solution, and a small amount of triethylsilane. Under nitrogen protection, stir the reaction in an ice bath for 30 min, and continue stirring at room temperature for 4-6 h. Monitor the reaction progress by TLC. After the reaction is complete, evaporate and concentrate the reaction solution under reduced pressure, then wash and vacuum dry for 24 h to obtain Compoud 7. The weight-to-volume ratio of Compoud 6 to the TFA and DCM mixed solution is 2.5 g: 4 mL. 3) The reactants were fed in the following proportions: Compoud 7, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU, and DIPEA in a molar ratio of 1:4.8:5:5:

15. The reaction solvent was DMF. The rest of the synthesis process was the same as in step 1) above, to obtain Compoud 8. 4) Prepare Compound 9 using Compound 8 as raw material, the synthesis process is the same as step 2 above). 5) The reactants were fed in the following order: the molar ratio of Compoud 9, H-Glu(OtBu)-OtBu·HCl, HOBT, HBTU and DIPEA was 1:10:10:10:

36. The reaction solvent was DMF. The rest of the synthesis process was the same as in step 1) above, and Compoud 10 was obtained. 6) Place Compoud 10 in a reaction apparatus and slowly add DMF under nitrogen protection. After the solid dissolves, slowly add a mixed solution of piperidine and DMF with a volume ratio of 1:

4. Under nitrogen protection, stir the reaction in an ice bath for 30 min, and continue stirring at room temperature for 6-8 h. After the reaction is complete, transfer the reaction solution to another reaction apparatus, add a small amount of anhydrous ethanol, and concentrate by vacuum evaporation. Repeat this step to remove piperidine until no piperidine residue is detected by TLC. Dry under vacuum for 24 h to obtain Compoud 11. The weight-to-volume ratio of Compoud 10 to the mixed solution of piperidine and DMF is 1.5 g: 8 mL. 7) The reactants were fed in a molar ratio of 1:1.8:2:2:3.7 for Compoud 11, oleic acid, HOBT, HBTU, and DIPEA, with DMF as the reaction solvent. The rest of the synthesis process was the same as in step 1) above, to obtain Compoud 12. 8) Prepare EDL using Compoud 12 as raw material. The synthesis process of EDL is the same as step 2 above.

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