Application of group amino acid terminal modified D-configuration peptide dendrimer in preparation of tumor drugs

Abstract

The peptide dendrimer is used for preparing the medicine for treating the breast cancer, the peptide dendrimer can promote tumor cells to start an autophagy procedure, the excellent performance in vitro and in vivo provides powerful support for wide application of the peptide dendrimer in the field of anti-tumor treatment, and the peptide dendrimer has good biocompatibility and biological safety and can be used for preparing the medicine for treating the breast cancer. Due to the unique structural design and excellent performance, a new thought is brought to the development of a nano-drug delivery system, and an important reference basis is provided for the research and development and clinical treatment of breast cancer drugs in the future.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to the use of a D-type peptide dendritic macromolecule with amino acid terminal modification in the preparation of drugs for treating breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and its treatment methods are diverse, including surgical resection, radiotherapy, and chemotherapy. Chemotherapy, in particular, is a treatment method that uses drugs to disrupt the life cycle of cancer cells, inhibiting their growth and spread. While chemotherapy can inhibit the growth or metastasis of breast cancer to some extent, its side effects and the problem of tumor drug resistance cannot be ignored.

[0003] Chemotherapy drugs not only inhibit the proliferation and growth of cancer cells but also affect their autophagy process. Studies have shown that some chemotherapeutic drugs, such as rapamycin and doxorubicin, can activate autophagy in tumor cells while killing them. However, the degree of autophagy stimulated by these anticancer drugs is often relatively mild, playing a protective role and potentially insufficient to reach the threshold for activating autophagic cell death. This low level of autophagy negatively impacts the final efficacy of chemotherapy. Furthermore, the non-specific distribution of chemotherapy drugs in the body leads to systemic toxicity, reduces drug tissue distribution and bioavailability, decreases drug efficacy, and increases side effects.

[0004] Dendritic macromolecules, as a promising biomedical polymer, possess advantages such as high branching, monodispersity, controllable molecular size, good biocompatibility, and easy clearance from the body, making them an excellent drug delivery carrier. Peptide dendritic macromolecules, with natural amino acids as the main raw material, not only inherit the advantages of traditional dendritic macromolecules but also possess characteristics such as biodegradability and low cytotoxicity. Therefore, there is an urgent need to develop a dendritic macromolecule drug delivery system that can enhance the autophagy process of tumor cells to clear abnormal substances and has lysosomal escape capabilities. Summary of the Invention

[0005] This invention provides a D-configuration peptide dendritic macromolecule with amino acid terminology modified and its use in the preparation of breast cancer drugs. This type of peptide dendritic macromolecule can regulate the autophagy process, further enhance the level of autophagy induced by chemotherapy drugs while delivering chemotherapy drugs, and has lysosomal escape function, providing a new strategy for the treatment of breast cancer.

[0006] This invention provides the use of a D-type peptide dendritic macromolecule with amino acid terminology modified in the preparation of a drug for treating breast cancer, wherein the structural formula of the peptide dendritic macromolecule is as follows:

[0007]

[0008] All R-based bases are HD-His-G3K.

[0009] Furthermore, the preparation method of the peptide dendritic macromolecules in this invention is as follows:

[0010] 1) Under N2 protection, propyneamine, Boc-D-Lys(Boc)-OH, HBTu and HOBt were dissolved in anhydrous N,N-dimethylformamide; then, N,N-diisopropylethylamine was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, and then stirred continuously at room temperature for 24 h to finally obtain the product Boc-D-G1K-Alkyne;

[0011] 2) Dissolve Boc-D-G1K-Alkyne in anhydrous dichloromethane; then add trifluoroacetic acid to the above solution and stir in an ice bath for 30 min, then continue stirring at room temperature for 6 h to obtain a white solid, which does not require further purification; under N2 protection, dissolve the white solid from the previous step, Boc-D-Lys(Boc)-OH, HBTu and HOBt in anhydrous DMF; then slowly add DIPEA to the above solution and stir in an ice bath for 0.5 h, then continue stirring at room temperature for 24 h to obtain the product Boc-D-G2K-Alkyne;

[0012] 3) Under N2 protection, Boc-D-G2K-Alkyne was dissolved in anhydrous DCM. Then, TFA was added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h to obtain a white solid, which required no further purification. Under N2 protection, the white solid from the previous step, Boc-D-Lys(Boc)-OH, HBTu, and HOBt were dissolved in anhydrous DMF. Then, DIPEA (21.1 mL, 128.0 mmol) was slowly added to the solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 36 h. Finally, the product Boc-D-G3K-Alkyne was obtained.

[0013] 4) Under N2 protection, Boc-D-G3K-Alkyne was dissolved in anhydrous DCM; then, TFA was added to the above solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h; after the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which did not require further purification; under N2 protection, the white solid from the previous step, Boc-D-His-(Boc)-OH, HBTu, and HOBt were dissolved in anhydrous DMF. Then, DIPEA was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 48 h; finally, the product Boc-D-His-G3K-Alkyne was obtained.

[0014] 5) Under N2 protection, Boc-D-His-G3K-Alkyne was dissolved in anhydrous DCM; then, TFA was added to the above solution, and the mixture was stirred in an ice bath for 30 min, and then stirred continuously at room temperature for 24 h; a white solid was obtained, which did not require further purification; under N2 protection, the white solid from the previous step, 4Arm-PEG-N3, CuSO4·5H2O and Vc·Na were dissolved in ultrapure water; then the mixture was stirred continuously at 50 °C for 48 h; finally, the peptide dendritic macromolecule as described in claim 1 was obtained.

[0015] Furthermore, the peptide dendritic macromolecules described in this invention can induce tumor cells to initiate autophagy.

[0016] Furthermore, the peptide dendritic macromolecules described in this invention possess lysosomal escape capabilities.

[0017] In this invention, specific chemical or functional groups are introduced into the structure for functional modification to enhance its stability, biocompatibility, or targeting. For example, histidine is used to modify the ends of peptide dendritic macromolecules. The imidazole group of histidine has the ability to absorb protons in an acidic environment. Through the multivalent effect, the proton sponge effect can be amplified, thereby achieving rapid osmotic expansion and membrane disruption in the endosome, promoting the escape of the nanocarrier from the endosome into the cytoplasm, protecting the drug from the effects of lysosomal enzyme degradation, and thus improving the drug's bioactivity and therapeutic effect. Introducing polyethylene glycol (PEG) groups can increase its water solubility and reduce immunogenicity, thereby improving its stability and bioavailability in vivo.

[0018] The histidine-terminated D-configuration peptide dendritic macromolecules provided by this invention possess lysosomal escape capabilities and can regulate autophagy. Their excellent performance in vitro and in vivo provides strong support for their wide application in the field of anti-tumor therapy. Furthermore, they exhibit good biocompatibility and biosafety. Their unique structural design and superior performance bring new ideas to the development of nanomedicine delivery systems and provide important reference for future breast cancer drug development and clinical treatment. Attached Figure Description

[0019] Figure 1 Synthetic route diagram for Boc-D-G1K-Alkyne

[0020] Figure 2 Synthetic route for Boc-D-G2K-Alkyne

[0021] Figure 3 Synthetic route for Boc-D-G3K-Alkyne

[0022] Figure 4 Synthetic route of Boc-D-His-G3K-Alkyne

[0023] Figure 5 The proton spectrum of Boc-D-His-G3K-Alkyne

[0024] Figure 6 Synthesis route map for 4PDHK

[0025] Figure 7 The 1H NMR (A), IR (B), and UV-Vis spectra (C) of 4PEG, 4PLHK, and 4PDHK are shown.

[0026] Figure 8 Circular dichroisms of 4PEG, 4PLHK and 4PDHK

[0027] Figure 9 (A) Graphs showing relative tumor volume changes and body weight changes during treatment for all groups (n=5). (B) Tumor weight after treatment for all groups. (C) Tumor growth inhibition rates for the three treatment groups.

[0028] Figure 10 H&E staining of major organs in mice from different treatment groups

[0029] Figure 11Transmission electron microscopy (TEM) images of 4T1 cells treated with DOX@4PDHK and DOX@4PLHK (DOX: 2 μg / mL) and their equivalent blank vectors (4PDHK and 4PLHK) for 24 hours. Scale bar: 2 μm. Autolysosomes and autophagosomes are marked with red arrows on the TEM images. (B) Quantitative analysis of autolysosome and autophagosome counts (n=3)

[0030] Figure 12 Laser confocal microscopy images of lysosomal escape after different incubation times of DOX@4PDHK.

[0031] Figure 13 Laser confocal microscopy images of lysosomal escape after different incubation times of DOX@4PLHK. Detailed Implementation

[0032] 1. Preparation of peptide dendritic macromolecules (here named 4PDHK; the preparation process for 4PLHK is the same, except that Boc-D is replaced with Boc-L in the following description)

[0033] 1.1 Synthesis of Boc-D-G1K-AIkyne

[0034] Under N2 protection, propyneamine (CAS No. 2450-71-7) (1.101 g, 20.0 mmol), Boc-D-Lys(Boc)-OH (CAS No. 204190-67-0) (8.314 g, 24.0 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTu, CAS No. 94790-37-1) (9.103 g, 24.0 mmol) and 1-hydroxybenzotriazole (HOBt, CAS No. 2592-95-2) (3.243 g, 24.0 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF, CAS No. 68-12-2) (60 mL). Subsequently, N,N-diisopropylethylamine (DIPEA, CAS No. 7087-68-5) (16.5 mL, 100.0 mmol) was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, followed by continuous stirring at room temperature for 24 h. After the reaction was complete, the reaction was monitored by thin-layer chromatography, and DMF was removed by rotary evaporation, followed by dissolution in ethyl acetate (EA). The mixed solution was washed three times each with saturated NaHCO3 solution, dilute hydrochloric acid, and saturated NaCl solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and the organic solvent was removed by rotary evaporation to obtain the crude product. Further purification was performed by silica gel column chromatography (PE:EA = 4:1) to give a white solid in 92.7% yield, named Boc-D-G1K-Alkyne.

[0035] Synthetic routes such as Figure 1 As shown

[0036] 1.2 Synthesis of Boc-D-G2K-AIkyne

[0037] Under nitrogen protection, Boc-D-G1K-Alkyne (6.136 g, 16.0 mmol) was dissolved in anhydrous dichloromethane (DCM, CAS No. 75-09-2) (23.8 mL). Trifluoroacetic acid (TFA, CAS No. 76-05-1) (23.8 mL, 320.0 mol) was then slowly added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 6 h. After the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which could be used for the next step without further purification.

[0038] Under N2 protection, the white solid from the previous step, Boc-D-Lys(Boc)-OH (13.303 g, 38.4 mmol), HBTu (18.206 g, 48.0 mmol), and HOBt (6.486 g, 48.0 mmol) were dissolved in anhydrous DMF (60 mL). Then, DIPEA (31.7 mL, 192.0 mmol) was slowly added to the solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 24 h. After the reaction was complete, the reaction was monitored by thin-layer chromatography. DMF was removed by rotary evaporation, and then dissolved in ethyl acetate. The mixture was washed three times with saturated NaHCO3 solution, dilute hydrochloric acid, and saturated NaCl solution. The organic phase was dried over anhydrous magnesium sulfate and removed by rotary evaporation to obtain the crude product. Further purification by silica gel column chromatography (PE:EA = 1:1) yielded a white solid in 73.6% yield, named Boc-D-G2K-Alkyne.

[0039] Synthetic routes such as Figure 2 As shown

[0040] 1.3 Synthesis of Boc-D-G3K-Alkyne

[0041] Under nitrogen protection, Boc-D-G2K-Alkyne (3.360 g, 4.0 mmol) was dissolved in anhydrous DCM (11.9 mL). Then, TFA (11.9 mL, 160.0 mol) was slowly added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which could be used for the next step without further purification.

[0042] Under N2 protection, the white solid from the previous step, Boc-D-Lys(Boc)-OH (8.314 g, 24.0 mmol), HBTu (12.138 g, 32.0 mmol), and HOBt (4.324 g, 32.0 mmol) were dissolved in anhydrous DMF (60 mL). Then, DIPEA (21.1 mL, 128.0 mmol) was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 36 h. After the reaction was complete, the reaction was monitored by thin-layer chromatography, and DMF was removed by rotary evaporation, followed by dissolution in ethyl acetate. The mixture was washed three times with saturated NaHCO3 solution, dilute hydrochloric acid, and saturated NaCl solution. The organic phase was dried over anhydrous magnesium sulfate and removed by rotary evaporation to obtain the crude product. Further purification by silica gel column chromatography (DCM:MeOH = 20:1) yielded a white solid in 73.6% yield, named Boc-D-G3K-Alkyne.

[0043] Synthetic routes such as Figure 3 As shown

[0044] Synthesis of 1.4 Boc-D-His-G3K-Alkyne

[0045] 1.4.1 Synthesis

[0046] Under nitrogen protection, Boc-D-G3K-Alkyne (0.7 g, 0.4 mmol) was dissolved in anhydrous DCM (2.4 mL). Then, TFA (2.4 mL, 32.0 mol) was slowly added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which could be used for the next step without further purification.

[0047] Under N2 protection, the white solid from the previous step, Boc-D-His-(Boc)-OH (1.707 g, 4.8 mmol), HBTu (2.428 g, 6.4 mmol), and HOBt (0.864 g, 6.4 mmol) were dissolved in anhydrous DMF (40 mL). Then, DIPEA (4.2 mL, 25.6 mmol) was slowly added to the solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 48 h. After the reaction was complete, the reaction was monitored by thin-layer chromatography. DMF was removed by rotary evaporation, and the mixture was dissolved in ethyl acetate. The mixture was washed three times with saturated NaHCO3 solution, dilute hydrochloric acid, and saturated NaCl solution. The organic phase was dried over anhydrous magnesium sulfate and removed by rotary evaporation to obtain the crude product. Further purification by silica gel column chromatography (DCM:MeOH = 15:1) yielded a white solid in 54.3% yield, named Boc-D-His-G3K-Alkyne.

[0048] Synthetic routes such as Figure 4 As shown

[0049] 1.4.2 Product Characterization

[0050] pass 1 The product was characterized by H-NMR, such as Figure 5 The assignment of the NMR peaks shown (1H NMR (600MHz, D2O) δ 8.73–8.66 (m, 7H), 7.44–7.40 (m, 8H), 4.37–4.29 (m, 8H), 4.27–4.23 (m, 2H), 4.22–4.17 (m, 6H), 4.02–3.90 (m, 2H), 3.42–3.33 (m, 17H), 3.23–3.13 (m, 15H), 1.82–1.66 (m, 15H), 1.57–1.50 (m, 6H), 1.48–1.41 (m, 10H), 1.38–1.26 (m, 14H)) indicates the successful synthesis of this product.

[0051] 1.5 Synthesis and Verification of 4PDHK

[0052] 1.5.1 Synthesis

[0053] Under nitrogen protection, Boc-D-His-G3K-Alkyne (0.2 g, 0.055 mmol) was dissolved in anhydrous DCM (0.6 mL). Then, TFA (0.6 mL, 8.76 mol) was slowly added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which could be used for the next step without further purification.

[0054] Under nitrogen protection, the white solid from the previous step, 4Arm-PEG-N3 (9 mg, 9.12 μmol), CuSO4·5H2O (9 mg, 36.52 μmol), and Vc·Na (14 mg, 73.04 mmol) were dissolved in ultrapure water (10 mL). The mixture was then stirred continuously at 50 °C for 48 h. After the reaction was complete, the solution was dialyzed using a membrane with a MWCO of 3500 for 48 h, with water changed every 6 h. The solution was centrifuged at low speed, and the supernatant was freeze-dried to obtain a white solid with a yield of 83.2%. This solid was named 4PDHK.

[0055] Synthetic routes such as Figure 6 As shown

[0056] 1.5.2 Product Validation

[0057] By nuclear magnetic resonance hydrogen spectrum ( 1 The products were characterized in detail by H-NMR, with the peaks at 3.72 and 3.51 ppm originating from the protons of the alkyl group on 4-PEG-N3. The two products, 4PDHK and 4PLHK, not only exhibited peaks at these positions but also showed two distinct characteristic peaks at 8.14 and 7.18 ppm, attributed to the presence of the imidazole group in the histidine. Figure 7 A). Furthermore, a 2103 cm⁻¹ appears in the Fourier transform infrared (FT-IR) spectrum of 4-PEG-N3. -1 The stretching vibration absorption peak is observed, but the absorption peaks of the two products 4PDHK and 4PLHK at this position are significantly weakened and almost disappear. Figure 7 B). The UV-Vis spectroscopy also showed that 4PDHK and 4PLHK exhibited characteristic broad absorption of histidine in the 240–300 nm range. Figure 7 C). It is worth noting that 4PDHK and 4PLHK... 1 The H-NMR, FT-IR, and UV-vis data images are largely consistent. These results indicate that D-His-G3K-A and L-His-G3K-A were successfully covalently coupled with 4-PEG-N3 via a click reaction to synthesize 4PDHK and 4PLHK.

[0058] To test the chirality of 4PDHK and 4PLHK, circular dichroism (CD) chromatography was used. CD chromatogram ( Figure 8 The results show that the CD curves of 4PDHK exhibit a negative Cotton effect at 218 and 197 nm, while the CD curves of 4PLHK are mirror images of this. Furthermore, the CD curve of 4-PEG-N3 is a smooth curve approaching zero. Therefore, the secondary structures of 4PDHK and 4PLHK are mainly random coil conformations with opposite optical rotations, similar to those reported in the literature. This indicates that 4PDHK and 4PLHK possess completely opposite chirality, and that their chirality originates from different configurations of peptide dendritic macromolecules.

[0059] Example 2: Preparation of DOX nanomicelles for breast cancer treatment

[0060] DOX (1 mg) and 4PDHK or 4PLHK (4 mg) were dissolved in 1 mL of DMSO. The solution was then added dropwise to 9 mL of deionized water and stirred continuously overnight in the dark. The mixture was then dialyzed for 24 h using a membrane with a MWCO of 2000 to remove DMSO and unencapsulated DOX. The solution was then filtered through a 0.45 μm Millipore membrane and lyophilized to obtain DOX@4PDHK or DOX@4PLHK micelles.

[0061] Example 3 In vivo experiment

[0062] A mouse breast cancer tumor model was constructed by using 1×10 6 4T1 cells were subcutaneously implanted into the right abdomen of female BALB / C mice. Two weeks later, the tumor-bearing mice were randomly divided into four groups (n=3 per group). Every three days, mice were injected via tail vein with saline (control group), free DOX, DOX@4PLHK, and DOX@4PDHK (dose: 5 mg / kg DOX) for a total of five times. During the 16-day treatment period, mouse body weight and tumor volume were recorded every two days, with the first day's value set as 100%. After 16 days of treatment, the mice were sacrificed, the tumors were removed and weighed, and the tumor growth inhibition (TGI) value was calculated using the following formula: TGI(%) = (1 - average tumor weight in the treatment group / average tumor weight in the control group) × 100%.

[0063] Changes in relative tumor volume during treatment, such as Figure 9 As shown in Figure A. The results showed that tumor growth was rapid in the saline group, while DOX@4PLHK slightly delayed tumor growth (p < 0.01). Notably, tumor growth was significantly inhibited in both free DOX and DOX@4PDHK (p < 0.001). Tumor weight was measured and recorded after treatment, and the tumor growth inhibition rate (TGI) was calculated. Figure 9As shown in CD, the mean tumor weight in the saline group was 1138.6±399.1 mg, the mean tumor weight in the DOX@4PDHK group was 666.8±198.0 mg (p<0.05), and the TGI was 41.4%; the mean tumor weight in the free DOX group was 443.4±162.6 mg (p<0.01), and the TGI was 61.1%; the mean tumor weight in the DOX@4PDHK group was 461.0±80.5 mg (p<0.01), and the TGI was 59.5%. Although free DOX and DOX@4PDHK showed comparable antitumor effects, mice treated with free DOX experienced a significant decrease in body weight (more than 20%) and exhibited symptoms of dyskinesia, anorexia, and lethargy. Figure 9 (B) This indicates that free DOX has severe systemic toxicity and poses significant biosafety concerns. Satisfactorily, peptide-based dendritic nanomicelles significantly improved the systemic toxicity of DOX. While DOX@4PLHK showed a faster cell uptake rate than DOX@4PDHK in cellular uptake and multicellular tumor spheroid permeation assays, DOX@4PDHK exhibited stronger antitumor effects both in vitro and in vivo. This is attributed to the stronger pH-responsive drug release characteristics of the histidine-terminated D-configuration peptide dendritic molecules, and the additional potential effects of the D-configuration peptide dendritic molecules.

[0064] After 16 days of treatment, immunohistochemical analysis was performed on the major organs of the mice. For example... Figure 10 As shown, free DOX, DOX@4PDHK, and DOX@4PLHK did not cause pathological changes in the major organs (heart, liver, spleen, and kidney) of mice. The nanomicelles did not cause hemolysis at the tested concentrations, further confirming the good biocompatibility and biosafety of the nanomicelles.

[0065] Example 4: Transmission electron microscopy observation of autophagy-related structures

[0066] Transmission electron microscopy (TEM) is widely recognized as the gold standard for visualizing autophagy structures, providing clear and conclusive evidence of autophagy induction. Therefore, intracellular autophagy-related structures are observed using TEM, such as... Figure 11As shown in Figure A, the blank 4PDHK nanoparticles exhibited a significant tendency to induce autolysomnia and autosome formation (marked with red arrows), with these autolysomnias and autophagosomes being significantly more abundant compared to cells treated with 4PLHK. This observation was further confirmed after loading the anticancer drug DOX onto these nanocarriers. When DOX@4PDHK nanoparticles were co-cultured with 4T1 cells, the presence of autolysosomes and autophagosomes was significantly increased in transmission electron microscopy images, significantly exceeding the observations in the DOX@4PLHK and blank carrier groups. These results demonstrate the superior ability of the 4PDHK structure to effectively induce autophagy.

[0067] Example 5 Lysosomal Escape Experiment

[0068] 5.1 Experimental Procedure

[0069] 4T1 cells in logarithmic growth phase were harvested, trypsinized, and counted using a cell counting chamber. 4T1 cells were placed at a density of 1 × 10⁴ cells per well in confocal culture dishes, 1 mL of culture medium was added, and the cells were cultured for 24 h. Subsequently, the cells were co-incubated with DOX@4PDHK or DOX@4PLHK micelles at a concentration of 5 μg / mL DOX for different time periods (1, 2, 8 h). After incubation, the cells were stained with Lysotracker green (50 nM) for 30 min and Hoechst 33342 for 15 min. After staining, the cells were washed twice with PBS and imaged using CLSM. Nuclei: blue, λEX = 405 nm, λEM = 430–470 nm. Lysosomes: green, λEX = 488 nm, λEM = 500–540 nm. DOX: red, λEX = 488 nm, λEM = 565–620 nm.

[0070] 5.2 Experimental Results

[0071] from Figure 12 The results show that after 1 hour of DOX@4PDHK treatment, the red fluorescence was relatively weak, mainly distributed in the cytoplasm, and co-localized with lysosomes, exhibiting yellow fluorescence. This indicates that after DOX@4PDHK micelles are endocytosed by cells, they are initially localized in lysosomes. After 2 hours, the yellow fluorescence was still present in the merged image, while red fluorescence gradually appeared, indicating that the DOX@4PDHK micelles began to escape from the lysosomes. By 8 hours, red fluorescence was abundant in the cells, while green fluorescence and yellow fluorescence decreased. Some red fluorescence overlapped with blue fluorescence, indicating that a large amount of DOX@4PDHK had escaped from the lysosomes, and the drug DOX was also released into the cytoplasm, subsequently entering the nucleus and binding to DNA to exert its effects.

[0072] And from Figure 13The images show that DOX@4PLHK still exhibits a large amount of yellow fluorescence in the merged images at 2h and 8h, indicating that DOX@4PLHK escapes from lysosomes more slowly than DOX@4PDHK, meaning that the D conformation has a weaker drug-drug interaction force, resulting in faster drug release and stronger pH responsiveness. This demonstrates that DOX@4PDHK exhibits lysosomal escape and pH-sensitive properties, and in tumor cells, it can rapidly exert a proton sponge effect, reducing the enzymatic degradation of the drug DOX in the acidic environment of lysosomes, thereby increasing the amount and efficacy of the drug.

Claims

1. A D-terminal modified peptide dendritic macromolecule used in the preparation of a drug for treating breast cancer, characterized in that: The structural formula of the peptide dendritic macromolecule is as follows: All R-based bases are HD-His-G3K.

2. The use as described in claim 1, characterized in that: The preparation method of the peptide dendritic macromolecule is as follows: 1) Under N2 protection, propyneamine, Boc-D-Lys(Boc)-OH, HBTu and HOBt were dissolved in anhydrous N,N-dimethylformamide; then, N,N-diisopropylethylamine was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, and then stirred continuously at room temperature for 24 h to finally obtain the product Boc-D-G1K-Alkyne; 2) Dissolve Boc-D-G1K-Alkyne in anhydrous dichloromethane; then add trifluoroacetic acid to the above solution and stir in an ice bath for 30 min, then continue stirring at room temperature for 6 h to obtain a white solid, which does not require further purification; under N2 protection, dissolve the white solid from the previous step, Boc-D-Lys(Boc)-OH, HBTu and HOBt in anhydrous DMF; then slowly add DIPEA to the above solution and stir in an ice bath for 0.5 h, then continue stirring at room temperature for 24 h to obtain the product Boc-D-G2K-Alkyne; 3) Under N2 protection, Boc-D-G2K-Alkyne was dissolved in anhydrous DCM. Then, TFA was added to the solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h to obtain a white solid, which required no further purification. Under N2 protection, the white solid from the previous step, Boc-D-Lys(Boc)-OH, HBTu, and HOBt were dissolved in anhydrous DMF. Then, DIPEA was slowly added to the solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 36 h. Finally, the product Boc-D-G3K-Alkyne was obtained. 4) Under N2 protection, Boc-D-G3K-Alkyne was dissolved in anhydrous DCM; then, TFA was added to the above solution, and the mixture was stirred in an ice bath for 30 min, followed by stirring at room temperature for 12 h; after the reaction was complete, the solvent was removed by rotary evaporation as monitored by thin-layer chromatography. The resulting oily solution was then precipitated three times with anhydrous diethyl ether to obtain a white solid, which did not require further purification; under N2 protection, the white solid from the previous step, Boc-D-His-(Boc)-OH, HBTu, and HOBt were dissolved in anhydrous DMF. Then, DIPEA was slowly added to the above solution, and the mixture was stirred in an ice bath for 0.5 h, followed by stirring at room temperature for 48 h; finally, the product Boc-D-His-G3K-Alkyne was obtained. 5) Under N2 protection, Boc-D-His-G3K-Alkyne was dissolved in anhydrous DCM; then, TFA was added to the above solution and stirred in an ice bath for 30 min, and then stirred continuously at room temperature for 24 h. A white solid was obtained without further purification; under N2 protection, the white solid from the previous step, 4Arm-PEG-N3, CuSO4·5H2O and Vc·Na were dissolved in ultrapure water; then the mixture was stirred continuously at 50°C for 48 h; finally, the peptide dendritic macromolecules as described in claim 1 were obtained.

3. The use as described in claim 1, characterized in that: The aforementioned peptide dendritic macromolecules can induce tumor cells to initiate autophagy.

4. The use as described in claim 1, characterized in that: The peptide dendritic macromolecules possess lysosomal escape capabilities.

Images