A targeting peptide-lipid linker, a targeting polypeptide liposome comprising the same and use thereof

Abstract

The application discloses a kind of targeted peptide-lipid connectors, using Kp-10 analogs to liposome active targeting to the GPR54 receptor overexpressed on the surface of breast cancer cell, and the chemotherapeutic drug doxorubicin with cytotoxicity is brought into cancer cell, so as to actively target the treatment of breast cancer, improve curative effect and reduce drug toxic side effects, especially targeted liposome 228-K3-EG8-Liposome shows excellent tumor targeting ability and enhanced cell uptake capacity.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to targeted peptide-lipid conjugates, targeted polypeptide liposomes containing the conjugates, and their applications. Background Technology

[0002] Breast cancer is the most common malignant tumor among women. Due to its ever-increasing incidence, it poses a significant threat to women's daily lives and mental and physical health. Breast cancer is classified into five subtypes based on the expression of ER, PR, and Her2 receptors in tumor tissue. Current treatment options for Her2 receptor-positive breast cancer include targeted therapies such as lapatinib (a small molecule inhibitor) and antibody drugs like trastuzumab. However, Her2 positivity only accounts for about 20% of all breast cancer patients in clinical diagnosis. For other subtypes, chemotherapy remains the mainstream treatment. Commonly used chemotherapy drugs, such as the anthracycline doxorubicin (DOX), have excellent anti-tumor effects in solid tumors. However, DOX treatment also has significant drawbacks. For example, when administered systemically via intravenous injection, its lack of targeted delivery can lead to severe cardiotoxicity and other toxic side effects. Besides the unavoidable toxic side effects, repeated administration of chemotherapy drugs can also lead to drug resistance in cancer cells, significantly affecting drug accumulation in tumor cells and weakening the drug's cytotoxic effect. Therefore, current breast cancer treatments suffer from two main problems: insufficient targeted therapies and the toxic side effects of chemotherapy drugs.

[0003] To overcome the drawbacks of chemotherapy drugs, researchers have begun using various nanocarriers to encapsulate them, thereby improving and enhancing therapeutic efficacy. Due to the enhanced permeability and retention effect (EPR) at the tumor site, nanocarriers can passively target and accumulate at the tumor site; for example, doxorubicin hydrochloride liposomes have already proven effective in breast cancer treatment. However, this targeting method is not very effective, as the therapeutic agent inevitably concentrates in reticuloendothelial organs (such as the liver and spleen) due to clearance by mononuclear phagocytes. Furthermore, due to heterogeneity and differences in vascular permeability within individual tumors, the extent of the EPR effect varies between tumors and even within tumors. Therefore, further development of nanocarriers with active targeting capabilities is needed to address the shortcomings of passive targeting and improve drug biodistribution. Summary of the Invention

[0004] Purpose of the invention: To solve the technical problems existing in the prior art, the present invention proposes a targeted liposome based on an analog of peptide Kp-10, and further applies the above-mentioned targeted liposome to drug delivery systems and the preparation of targeted drug formulations.

[0005] This invention further proposes a targeted peptide-lipid linker comprising two hydrophobic palmitic acids (PA)2, one lysine K, and a short lysine K chain. n EG (Polyethylene Glycol Chain) m An EG2 linker and an analogue of the peptide Kp-10, Kp-10-228, where n is the number of lysine residues, n = 0 or 3, EG m The polypeptide is polyethylene glycol, m = 2-45, and the sequence of the polypeptide Kp-10-228 is YNWNGFG(DW)RF (SEQ ID NO.4), where DW represents D-tryptophan; its structure is: (PA)2-K-EG m -K n -EG2-Kp-10-228.

[0006] Preferably, EG m For EG2, EG8, (EG8)2 and EG 45 Any one of them, where (EG8)2 means two EG8s linked by peptide bonds.

[0007] More preferably, in the above-mentioned targeted peptide-lipid linker, n=3, m=8, that is, its structure is: (PA) 2- K-EG8-KKK-EG2-Kp-10-228, abbreviated as 228-K3-EG8.

[0008] The present invention also provides a targeted peptide liposome comprising the above-described targeted peptide-liposome linker and liposome.

[0009] Specifically, the composition of the targeted peptide liposome is in the following molar ratio: hydrogenated soybean phosphatidylcholine: cholesterol: distearate phosphatidylethanolamine-polyethylene glycol 2000: the targeted peptide-lipid linker = (50-56): (35-40): 4-6:1, preferably, the ratio is 55:39:5:1.

[0010] The targeted liposomes were prepared by thin-film hydration.

[0011] The present invention further proposes the use of the above-mentioned targeted liposomes as a drug delivery system and in the preparation of targeted drug formulations.

[0012] As one application, this invention proposes an active drug-carrying liposome of doxorubicin hydrochloride. The liposome encapsulating doxorubicin is obtained by mixing the above-mentioned targeted liposomes with a doxorubicin solution and incubating them with shaking at 55-65°C for 1-3 hours.

[0013] Preferably, the concentration of doxorubicin is 3-5 mg / mL.

[0014] Beneficial effects: This invention screens high-affinity polypeptide ligands, uses EG linkers to connect the target peptide to the fatty acid chain, and adds lysine sequences to increase the hydrophilicity of the target peptide to prepare a target peptide exhibiting ideal target liposomes. In particular, the target liposome 228-K3-EG8-Liposome shows excellent tumor targeting ability and enhanced cellular uptake, effectively improving drug bioavailability and enhancing therapeutic effects. Attached Figure Description

[0015] Figure 1 GPR54 expression in breast cancer cell lines and other control cell lines, including: A. mRNA expression in breast cancer cell lines and other control cell lines; B. GPR54 protein expression and grayscale analysis in different types of breast cancer cell lines and control cell lines. * indicates comparison between cancer cell lines and normal breast epithelial cells MCF 10A, ***p<0.001;

[0016] Figure 2 This study examines the expression of GPR54 in different types of breast cancer cell lines and tissues. A. GPR54 mRNA expression in different subtypes of breast cancer cell lines and other control cell lines; B. GPR54 protein expression and grayscale analysis results in different subtypes of breast cancer cell lines and control cell lines; C. GPR54 mRNA expression in pathological tissues of 10 breast cancer patients and matched adjacent normal tissues; D. GPR54 protein expression and grayscale analysis results in pathological tissues of 10 breast cancer patients and matched adjacent normal tissues. N = Normal, T = Tumor; * indicates comparison between the breast cancer group and the normal group, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;

[0017] Figure 3 To analyze the cellular uptake of Kp-10 analogs by T47D and MCF-7 cell lines, the following methods were used: A. Laser confocal microscopy was used to observe the fluorescence intensity of T47D cell lines after co-incubation with FITC-labeled Kp-10 analogs for 2 hours; B. Laser confocal microscopy was used to observe the fluorescence intensity of MCF-7 cell lines after co-incubation with FITC-labeled Kp-10 analogs for 2 hours; C. Flow cytometry was used to detect the average fluorescence intensity of T47D and MCF-7 cell lines after co-incubation with FITC-labeled Kp-10 analogs for 2 hours.

[0018] Figure 4This study analyzed the cellular uptake of Kp-10 analogs by the 4T1 cell line, including: A. Laser confocal microscopy observation of the cellular uptake of Kp-10-228, 230, 231, and 240 analogs by the 4T1 cell line; scale bar: 50 μm; B. Flow cytometry detection of the cellular uptake of Kp-10-228 and 240 analogs by the 4T1 cell line; C. Laser confocal microscopy observation of the uptake of Kp-10-228 by the 4T1 cell line after blocking the GPR54 receptor with Kp-10 precursor peptide; scale bar: 50 μm.

[0019] Figure 5 Design of targeted peptide-lipid linkers;

[0020] Figure 6 A synthetic route for targeting peptide-lipid conjugates;

[0021] Figure 7 To investigate the cellular uptake of different DiD-encapsulated liposomes in T47D, MCF-7, SK-BR-3, and MDA-MB-231 cell lines: A. Flow cytometry analysis of cellular uptake of liposomes composed of ethylene glycol chains linked to targeting peptides of different lengths in four cell lines under lysine-free conditions; B. Flow cytometry analysis of cellular uptake of liposomes composed of ethylene glycol chains linked to targeting peptides of different lengths in four cell lines under lysine-modified conditions; C. Laser confocal microscopy analysis of cellular uptake of non-targeted liposomes, 228-K3-EG8-Liposome, and 228-K3-(EG8)2-Liposome in four cell lines. * indicates that compared with non-targeted liposomes, ****p < 0.0001; # indicates that compared with non-targeted liposomes, ###p < 0.001.

[0022] Figure 8The following methods were used to observe the cellular uptake of 228-K3-EG8-Liposome encapsulated with DiD after receptor blocking: (A) Laser confocal microscopy observation of cellular uptake of 228-K3-EG8-Liposome in T47D, MCF-7, MDA-MB-231, and SK-BR-3 cells after receptor blocking; (B) Flow cytometry detection of cellular uptake of 228-K3-EG8-Liposome in T47D, MCF-7, MDA-MB-231, and SK-BR-3 cells after receptor blocking; (C) Laser confocal microscopy observation of cellular uptake of non-targeted liposomes and 228-K3-EG8-Liposome in MCF 10A mammary epithelial cells; (D) Flow cytometry detection of MCF 10A cells. 10A. Cellular uptake of non-targeted liposomes and 228-K3-EG8-Liposome in mouse breast cancer cells 4T1 after receptor blocking; (E) Flow cytometry detection of cellular uptake of 228-K3-EG8-Liposome in mouse breast cancer cells 4T1 after receptor blocking; (F) Laser confocal microscopy observation of cellular uptake of 228-K3-EG8-Liposome in mouse breast cancer cells 4T1 after receptor blocking. * indicates comparison of 228-K3-EG8-Liposome with non-targeted liposomes, ***p<0.001, ****p<0.0001. # indicates comparison of 228-K3-EG8-Liposome with 228-K3-EG8-Liposome+Kp-10, ##p<0.01. ####p<0.0001;

[0023] Figure 9 For in vivo imaging of 4T1 breast cancer mouse models at different time points: A. Fluorescence imaging at 2h, 4h, 8h, 12h, and 24h after tail vein injection of Saline, mPEG-Liposome / DiD, and 228-K3-EG8-Liposome / DiD into tumor-bearing mice, respectively. From left to right, the images in each group are Saline group, mPEG-Liposome / DiD group, and 228-K3-EG8-Liposome / DiD group; B. Semi-quantitative ROI analysis of fluorescence intensity at different time points; * indicates comparison between mPEG-Liposome group and 228-K3-EG8-Liposome, *p<0.05, **p<0.01;

[0024] Figure 10The in vivo antitumor effects of 228-K3-EG8-Liposome were as follows: A. Body weight change curves in 4T1 breast cancer model mice after intravenous injection of different DOX-encapsulated liposomes, saline, or DOX (n=8); B. Tumor volume curves in tumor-bearing mice three weeks after intravenous injection of different DOX-encapsulated liposomes, saline, or DOX (n=8); C. Kaplan-Meier survival curves in 4T1 breast cancer model mice after intravenous injection of different DOX-encapsulated liposomes, saline, or DOX (n=8); * indicates that compared with the 228-K3-EG8-Liposome group, ***p<0.001, # indicates that compared with the 228-K3-EG8-Liposome group, #p<0.05, ###<0.001. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] Kisspeptin is an endogenous ligand of the G protein-coupled receptor GPR54. The Kisspeptin / GPR54 system is mainly associated with reproductive function in vivo, and this system exhibits high expression levels in sex hormone-related tumors. This invention first observes and detects the expression of GPR54 in breast cancer cell lines and in situ tumors of breast cancer patients. Next, it screens endogenous peptide Kisspeptin-10 (Kp-10) analogs to modify the surface of PEGylated liposomes. Using the Kp-10 analogs, the liposomes are actively targeted to the GPR54 overexpressed on the surface of breast cancer cells, and the cytotoxic chemotherapeutic drug doxorubicin is delivered into the cancer cells. This actively targets and treats breast cancer, improving efficacy and reducing drug toxicity.

[0027] The meanings of the abbreviations used in the instruction manual are as follows:

[0028] Full name of English abbreviation

[0029] Fmoc-9-fluorenemethyloxycarbonyl

[0030] FITC 5(6)-fluorescein isothiocyanate

[0031] HPLC (High Performance Liquid Chromatography)

[0032] DMF N,N-dimethylformamide

[0033] DCM dichloromethane

[0034] DIEA N,N-Diisopropylethylamine

[0035] HOBt 1-hydroxybenzodiazepine

[0036] HBTU Benzotriazole-N,N,N ’ N ’ Tetramethylurea hexafluorophosphate

[0037] TFA (trifluoroacetic acid)

[0038] TIS Triisopropylsilane

[0039] Example 1: Expression of GPR54 in cancer cell lines and clinicopathological tissues.

[0040] Literature review revealed that the KISS1 / GPR54 system exhibits upregulated expression in breast cancer, colon cancer, and ovarian cancer. Therefore, the T47D and MCF-7 breast cancer cell lines with high GPR54 expression reported in the literature were selected, and normal breast cells MCF 10A, human peripheral blood neutrophils, colon cancer cells HCT 116, and colon adenocarcinoma cells RKO were used as controls to verify the feasibility of GPR54 as a targeted receptor for the treatment of breast cancer.

[0041] The expression levels of GPR54 in cultured cell lines and human peripheral blood neutrophils were detected by quantitative PCR and Western blotting. The expression levels of GPR54 mRNA in each cell line and human neutrophils are shown below. Figure 1 As shown in Figure A, the expression level of GPR54 mRNA in neutrophils and normal breast cells MCF 10A was extremely low, while the expression of GPR54 mRNA in breast cancer cell lines T47D and MCF-7 was significantly upregulated compared to neutrophils and MCF 10A (p < 0.001), with expression levels approximately 31.4-fold and 15-fold upregulated relative to MCF 10A, respectively. In the two colon cancer-related cell lines, HCT 116 showed approximately 5.6-fold upregulation of GPR54 mRNA compared to neutrophils, but the colon adenocarcinoma cell line RKO showed almost no GPR54 expression. The three ovarian cancer cell lines used in the experiment showed similar GPR54 expression results to the RKO cell line, with no detectable GPR54 mRNA in the cells.

[0042] Based on the above results, we selected four cell types—neutrophils, MCF 10A, T47D, and MCF-7—and used Western blotting to detect the GPR54 protein expression level. The GPR54 protein expression results are as follows: Figure 1As shown in Figure B, both T47D and MCF-7 cells highly express GPR54 protein. Gray-scale analysis reveals that T47D cells exhibit the highest expression level of the GPR54 receptor. In contrast, GPR54 protein expression is almost undetectable in neutrophils, and MCF 10A cells show only weak expression of GPR54.

[0043] Breast cancer is known to be classified into five subtypes based on the expression levels of three receptors: ER, PR, and Her2. According to literature reports, GPR54 expression is not correlated with the expression levels of ER, PR, and Her2. Therefore, Western blotting and Q-PCR experiments were used to compare the expression levels of T47D and MCF-7 (ER...). + PR + ), SK-BR-3 (ER) - PR - Her2 + ) and MDA-MB-231 (ER - PR - Her2 - The expression levels of the GPR54 receptor in four cell lines were analyzed, and the results are as follows: Figure 2 A and Figure 2 As shown in B, compared with the normal breast cell line MCF 10A, the expression level of GPR54 was significantly upregulated in different subtypes of breast cancer cell lines.

[0044] To further verify the feasibility of GPR54 as a targeted receptor for the treatment of breast cancer, the expression level of GPR54 was detected in cancerous tissue and adjacent tissue of breast cancer patients. Figure 2 C shows the GPR54 mRNA expression in the pathological tissues of 10 patients. GPR54 mRNA expression was low in the adjacent normal tissues of all 10 patients, with levels in tissues 4, 5, and 7 even being undetectable. In contrast, cancerous tissues showed high expression of GPR54 mRNA, with significantly upregulated expression compared to normal tissues.

[0045] The expression levels of GPR54 protein in the pathological tissues of 10 patients are as follows: Figure 2 As shown in Figure D, the adjacent tissues of the 10 patients showed only weak expression of GPR54 protein. Except for patient 5, the cancerous tissues of the other patients all showed strong expression of GPR54 protein, with a significantly upregulated protein expression level compared to the adjacent tissues. Based on the detection results of the pathological sections of the 10 patients (see Table 1), it can be seen that the expression level of GPR54 is not correlated with the expression of ER, PR, and Her2.

[0046] Table 1. Results of ER, PR, and Her2 assays in pathological tissues of breast cancer patients.

[0047]

[0048] In summary, these results confirm that GPR54 expression is significantly upregulated in breast cancer, while it is expressed at low levels or not at all in normal tissues. The human GPR54 receptor is most abundantly expressed in the placenta, followed by the testes. Since the placenta only appears during pregnancy, and the testes, another highly expressed tissue, are not present in the female body, GPR54 has great potential as a targeted receptor for breast cancer treatment.

[0049] Example 2 Synthesis and screening of peptide Kp-10 analogs.

[0050] (1) Synthesis of peptide Kp-10 analogues

[0051] The Kp-10 analogue was synthesized according to the polypeptide sequences in Table 2-1, where DW represents D-tryptophan, DY represents D-tyrosine, and DA represents D-alanine.

[0052] Table 2-1 Names and sequences of peptide Kp-10 analogs

[0053]

[0054] Peptides were synthesized using a solid-phase synthesis method. The steps are as follows:

[0055] ①Swelling resin: Accurately weigh 200mg of Rink Amide MBHA resin into the synthesizer, inject DCM to swell the resin for 30min, vacuum dry the DCM, and then wash with DMF for 3min each time, for a total of 3 washes.

[0056] ② Deprotection: React twice with 20% piperidine / DMF deprotection solution for 5 and 15 min to remove Fmoc. Use a vacuum pump to dry the deprotection reagent, wash the resin with DMF for 3 min, and wash a total of 4 times to remove Fmoc byproducts and residual piperidine. Take a trace amount of resin and test it with ninhydrin reagent. If the resin changes color, the protectant has been completely removed.

[0057] ③ Amino acid linkage: Calculate the required mass of amino acids, HOBt, and HBTU based on the resin substitution value, accurately weigh them, dissolve them in DMF (with DIEA added simultaneously), add them to the synthesizer, purge with argon, and shake to react for 1 hour. After the reaction is complete, dry the reaction solution, wash with DMF for 3 minutes, and repeat the washing process 3 times. Use ninhydrin reagent to detect the reaction progress; the degree of reaction is determined by whether the ninhydrin reagent changes color.

[0058] ④ Repeat steps (2) and (3) in sequence until the last amino acid of the peptide sequence.

[0059] ⑤ Ligation of the fluorescently labeled FITC molecule: Remove the protecting group from the last amino acid in the sequence, and then ligate an Acp (6-aminocaproic acid) linker molecule according to step (3). After the ligation reaction is complete, deprotection is performed, and an indole test is performed. Accurately weigh FITC, dissolve it in DMF, and add it to the synthesizer together with DIEA. Stir the reaction in the dark for 12 hours. Dry the reaction solution, wash with DMF 6 times, and perform an indole test. If the indole test reagent does not change color, the ligation is complete. Then wash the resin in the order of DCM (2 times) → methanol (1 time) → DCM (1 time) → methanol (2 times), and dry the resin under vacuum.

[0060] ⑥ Resin lysis: Add the prepared lysis buffer (TFA / ddH2O / Tis=95 / 2.5 / 2.5(v / v / v)) to the shrunken and dried resin and react for 3 hours. Collect the lysis buffer into a round-bottom flask and rotary evaporate to a volume of about 1-2 mL. Add ice-cold diethyl ether at 10 times the volume of the peptide solution to precipitate. Centrifuge at 7500 rpm for 5 minutes and wash with diethyl ether 4 times. Collect the precipitate to obtain the crude peptide.

[0061] ⑦ Peptide purification: The crude peptide product was purified by HPLC, and the pure peptide was collected by lyophilization. The purity of the peptide was analyzed by analytical HPLC, and the molecular weight was determined by mass spectrometry (ESI-TOF, maXis 4G, Bruke), as shown in Figure 2-2.

[0062] Table 2-2 HPLC retention times, purity, and molecular weights of Kp-10 analogs

[0063]

[0064] (2) Cellular uptake of peptide Kp-10 analogues.

[0065] T47D and MCF-7 breast cancer cells were interacted with 10 μM FITC-Kp-10 analogue at 37°C for 2 hours, and observed and photographed under a laser confocal microscope. Figure 3 A and Figure 3 As shown in B, Kp-10-228, 230, 231 and 240 can be effectively taken up by both cell lines. Kp-10-228 is taken up most strongly by both cell lines, followed by Kp-10-230, 231 and 240. Kp-10-230 and 231 show similar uptake in the two cell lines, but Kp-10-240 shows slightly different uptake in the two cell lines.

[0066] Flow cytometry was then used to quantitatively analyze the cell uptake of the two cell lines. This was achieved by quantifying fluorescence intensity, such as... Figure 3C. Similar to the qualitative analysis results, Kp-10-228 showed the most significant enhancement in cell uptake in T47D and MCF-7 cell lines, while Kp-10-240 was weaker than 228. Kp-10-230 and Kp-10-231 showed differences in the two cell lines, with Kp-10-230 being stronger than 231 in T47D, while Kp-10-231 was slightly stronger than 230 in MCF-7.

[0067] We also examined the cellular uptake of four analogues with good targeting capabilities, namely Kp-10-228, 230, 231 and 240, in mouse 4T1 breast cancer cells. Figure 4 A shows the cellular uptake of Kp-10-228, 230, 231, and 240 in the mouse breast cancer cell line 4T1. Kp-10-228 remains the analogue with the highest cellular uptake efficiency, while Kp-10-230, 231, and 240 show similar uptake efficiencies. Kp-10-228 and 240, which showed good cellular uptake efficiency in the laser confocal microscopy assay, were then used for flow cytometry quantitative analysis. The results were consistent with the qualitative analysis results from the laser confocal microscopy assay, showing that the 4T1 cell line exhibited the highest uptake intensity of the Kp-10-228 analogue. Figure 4 B). After incubation with Kp-10 precursor peptide, cell uptake of Kp-10-228 was significantly reduced (B). Figure 4 C) indicates that the uptake of Kp-10-228 by cells is receptor-dependent. These findings confirm that Kp-10-228 acts as a targeting ligand for liposome surface modification.

[0068] Example 3 Synthesis of targeted peptide-lipid linkers and synthesis and optimization of targeted liposomes.

[0069] Figure 5 This is a schematic diagram of the design of a targeted peptide-lipid linker, which is designed according to... Figure 6 The peptides were synthesized via solid-phase peptide synthesis. The peptide-lipid conjugate sequences are shown in Table 3-1, and the purity and molecular weight are shown in Figure 3-2.

[0070] Table 3-1 Names and sequences of target peptide-lipid conjugates

[0071]

[0072] Table 3-2 Purity and molecular weight of peptide-lipid conjugates

[0073]

[0074]

[0075] Since the arginine and phenylalanine amino acids at the C-terminus of Kp-10-228 are crucial for receptor recognition and function, the C-terminal amino acids of the analogue need to be retained and in a free state. Therefore, polyethylene glycol chains of varying lengths were inserted at the N-terminus and linked to the lipids. Because the hydrophilicity or hydrophobicity of the peptide also affects the targeting efficiency of the targeted liposomes, lysine was introduced between the peptide and the polyethylene glycol chain to enhance the hydrophilicity of the targeting peptide. Finally, to minimize the interaction between the peptide and lysine, and between the peptide and polyethylene glycol (EGG),... m The interaction between the target peptide (Kp-10-228), m = 2-45, involves inserting a miniPEG (EG2) linker between the N-terminus and the lysine residue. Therefore, the conjugate consists of the following units: (1) a targeting peptide (Kp-10-228), (2) an EG2 linker, and (3) a short lysine chain (Kp-10-228). n K represents lysine, n = 0, 3), (4) polyethylene glycol chain (EG m (5) Two hydrophobic palmitic acids ((PA)2) were used to better insert the peptide-lipid linker into the lipid bilayer of the liposome. The two palmitic acid molecules were coupled to the α-amino and ε-amino groups of lysine to synthesize the targeted peptide-lipid linker. Palmitic acid was chosen instead of traditional phospholipids because it lacks phospholipid bonds, has higher chemical stability, and has increased solubility in typical solid-phase synthesis reagents (such as DMF and DCM). At the same time, one K was used to couple the two palmitic acid molecules. The connection of the two palmitic acids was to mimic the two fatty acid chains of natural phospholipids to make them more compatible.

[0076] The in vitro cellular uptake and in vivo tumor targeting capabilities of 228-K3-EG8-Liposome were investigated by incorporating DiD into all liposomes. DiD is a far-infrared fluorescent dye with a maximum excitation wavelength of 644 nm and a maximum emission wavelength of 665 nm.

[0077] Prepare various liposomes according to Table 3-2.

[0078] Liposomes were prepared using a thin-film hydration method. The specific steps are as follows:

[0079] (1) HPSC, CHOL, mPEG 2000 -DSPE and Peptide-K n -(EG) m -(PA)2 was restored to room temperature and then processed according to HSPC:CHOL:mPEG. 2000 -DSPE: Peptide-K n -(EG) mThe molar ratio of PA = 56-x:39:5:x, where x = 0 or 1, is used to accurately weigh the powder. When x = 0, non-targeted liposomes are obtained.

[0080] (2) Dissolve all powders thoroughly in chloroform and mix them in a round-bottom flask. Vacuum rotary evaporate at 37°C in a water bath to remove organic reagents and spread a thin lipid film on the bottom of the flask.

[0081] (3) Add PBS buffer and hydrate by shaking at 60°C.

[0082] (4) After hydration is complete, use a liposome extruder to control the particle size. Use a 0.1 μM filter membrane for extrusion filtration.

[0083] (5) Transfer the liposome solution to a centrifuge tube and store at 4°C for later use.

[0084] Preparation of DiD-encapsulated liposomes:

[0085] Liposomes were prepared using the same thin-film hydration method. DiD powder was removed and brought to room temperature. An appropriate amount was weighed and dissolved in chloroform with the other materials used for liposome preparation, then mixed in a round-bottom flask. The remaining steps were performed according to the thin-film hydration method to obtain DiD-encapsulated liposomes, which were then transferred to centrifuge tubes and stored at 4°C for later use.

[0086] Non-targeted and targeted liposomes encapsulating DiD were prepared according to the formulations in Table 3-3, and the particle size was measured. The results are also listed in Table 3-3.

[0087] Table 3-3 Liposome formulations and particle sizes

[0088]

[0089] Targeted liposome uptake by cells:

[0090] T47D, MCF-7, MDA-MB-231, and SK-BR-3 cells with non-targeted liposomes mPEG-Liposome / DiD and targeted liposomes 228-K n -EG m After co-incubation with Liposome / DiD for 4 hours, laser confocal microscopy and flow cytometry were performed. The experimental results are as follows: Figure 7 As shown.

[0091] The results showed that when lysine residues were not added between the targeting peptide and the fatty acid chain, regardless of the polyethylene glycol chain length, the targeting liposomes could not effectively enhance cellular uptake efficiency. Compared with mPEG-Liposome, 228-K0-EG m -Liposome cellular uptake efficiency was similar, with no significant difference. Figure 7 A). When three lysine residues were added after the targeting peptide, the targeting liposomes composed of polyethylene glycol chains of different lengths exhibited different cellular uptake efficiencies. Figure 7 B). 228-K3-EG8-Liposome and 228-K3-(EG8)2-Liposome showed significant increases in cellular uptake in all four cell lines, while 228-K3-EG8-Liposome showed... 45 -Liposome uptake efficiency is similar to that of non-targeted liposomes. Based on the results of the flow cytometry experiments above, 228-K3-EG8-Liposome and 228-K3-(EG8)2-Liposome, which showed relatively better performance, were selected for laser confocal imaging. Figure 7 C) The results also showed that the fluorescence intensity of 228-K3-EG8-Liposome and 228-K3-(EG8)2-Liposome in the four cell lines T47D, MCF-7, SK-BR-3 and MDA-MB-231 was significantly higher than that of non-targeted liposomes, and the uptake efficiency of 228-K3-EG8-Liposome was higher than that of 228-K3-(EG8)2-Liposome.

[0092] To verify whether the increased cellular uptake of 228-K3-EG8-Liposome is related to the GPR54 receptor, 10 μM Kp-10 was added and co-incubated with cells for 1 h before adding 228-K3-EG8-Liposome. Fluorescence intensity was detected by laser confocal microscopy and flow cytometry. Figure 8 A and Figure 8 As shown in Figure B, the cellular uptake efficiency of 228-K3-EG8-Liposome was significantly reduced after blocking the GPR54 receptor with Kp-10, indicating that the increased cellular uptake efficiency of liposome-targeted 228-K3-EG8-Liposome is dependent on the GPR54 receptor.

[0093] GPR54 was lowly expressed in MCF 10A cells. After incubation with liposomes for 4 hours, fluorescence intensity was detected by laser confocal microscopy and flow cytometry. Figure 8 C and Figure 8 As shown in D. The results indicate that although the cellular uptake of 228-K3-EG8-Liposome in MCF 10A is higher than that of non-targeted liposomes, its mean fluorescence intensity is significantly lower than that of 228-K3-EG8-Liposome in breast cancers such as T47D.

[0094] Furthermore, since a mouse model bearing tumors was subsequently established using 4T1 mouse breast cancer cells, the uptake efficiency of liposomes in 4T1 cells was detected by laser confocal microscopy and flow cytometry. Figure 8 E and Figure 8 As shown in F, 228-K3-EG8-Liposome uptake was significantly increased in 4T1 cells.

[0095] Example 4: In vitro and in vivo efficacy of peptide-targeted liposomes

[0096] (1) Determination of the breast tumor targeting ability of 228-K3-EG8-Liposome.

[0097] like Figure 9 From 2 hours to 24 hours, compared with the tumor-bearing mice injected with mPEG-Liposome / DiD, 228-K3-EG8-Liposome / DiD showed a significant accumulation behavior at the tumor site over time, demonstrating excellent breast tumor targeting ability.

[0098] (2) Preparation of liposomes encapsulating doxorubicin.

[0099] Liposomes were prepared using a thin-film hydration method. The specific steps are as follows:

[0100] ①Please wait for HPSC, CHOL, mPEG 2000 -DSPE, Peptide-K n -(EG) m - PA powder returned to room temperature, according to HSPC:CHOL:mPEG 2000 -DSPE: Peptide-K n -(EG) m The molar ratio of -PA = 56-x:39:5:x

[0101] Accurately weigh the powder mentioned above (X = 0 or 1).

[0102] ② Dissolve all powders thoroughly in chloroform and mix them in a round-bottom flask. Evaporate under vacuum at 37°C in a water bath to remove organic reagents, and spread a thin lipid film on the bottom of the flask.

[0103] ③ Add 10 mL of (NH4)2SO4 buffer solution and hydrate by shaking at 60°C.

[0104] ④ After sufficient hydration, control the particle size using a liposome extruder. Filter using a 0.1 μM filter membrane.

[0105] ⑤ Dialyze the liposome solution using a dialysis bag with a molecular weight cutoff of 10000 Da. The dialysate is HBS buffer (NaCl 150mM, HEPES 20mM, pH 7.5). Dialyze twice, 1 hour each time, to ensure complete removal of (NH4)2SO4 from the aqueous phase of the liposome.

[0106] ⑥ Dissolve doxorubicin hydrochloride powder in HBS buffer to a concentration of 5 mg / mL.

[0107] ⑦ Mix the liposome solution and doxorubicin solution in a round-bottom flask, and incubate with shaking at 60°C for 1 hour to obtain liposomes encapsulated with doxorubicin.

[0108] ⑧ Dialyze the liposome solution again, using HBS buffer as the dialysate, maintaining the same molecular weight cutoff. Change the dialysate multiple times to thoroughly remove any unencapsulated DOX.

[0109] ⑨ Transfer the liposome solution to a centrifuge tube and store at 4°C for later use.

[0110] (3) Encapsulation efficiency determination

[0111] Determination of the standard curve:

[0112] Determination of the DOX standard curve: DOX powder was dissolved in HBS buffer to prepare standard solutions with concentrations of 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL. The blank control group consisted of HBS buffer without DOX. The absorbance values ​​of each concentration were measured at 480 nm using a UV spectrophotometer. The standard curve was plotted with absorbance values ​​on the ordinate and DOX concentration on the abscissa.

[0113] Determination of encapsulation efficiency:

[0114] 50 μL of undialyzed DOX liposomes and 50 μL of fully dialyzed DOX liposomes were incubated in 950 μL of 10% Triton X-100 HBS solution at 60°C for 10 min to induce membrane permeation. A negative control group was prepared using HBS buffer (containing Triton X-100). The absorbance of the samples before and after dialysis was measured at 480 nm using a UV spectrophotometer. The DOX concentration was calculated based on the standard curve, and the encapsulation rate was determined as the OD of the dialyzed sample. 480 OD of predialysis sample 480 ×100%

[0115] Targeted and non-targeted liposomes encapsulated with doxorubicin were prepared according to the formulations in Table 4, and their particle size and other parameters are given in the table. The DOX concentration was calculated based on the standard curve, and the encapsulation efficiency was determined by the OD500 of the dialysis sample. 480 OD of predialysis sample 480×100%.

[0116] Table 4. Formulation and particle size parameters of liposomes encapsulating doxorubicin.

[0117]

[0118] (4) Antitumor effect of peptides encapsulating doxorubicin targeting liposomes.

[0119] To evaluate the therapeutic effect of targeted liposomes on tumors, the mouse 4T1 breast cancer model was used again. Tumor-bearing mice were randomly divided into four groups: Saline group, DOX group, mPEG-Liposome / DOX group, and 228-K3-EG8-Liposome / DOX group. The drugs were administered via tail vein on days 1, 3, 5, 7, 9, and 11, for a total of six administrations.

[0120] To assess systemic toxicity in vivo, mouse body weight was measured throughout the study, and the results are as follows: Figure 10 As shown in Figure A, the DOX group exhibited significant toxicity during the study and was euthanized on day 9 due to rapid weight loss (>15%). No significant weight changes were observed in the Saline and 228-K3-EG8-Liposome / DOX groups, while the mPEG-Liposome / DOX group showed lower systemic toxicity. These results indicate that targeting liposomes with 228-K3-EG8-Liposome / DOX can significantly reduce the systemic toxicity of doxorubicin.

[0121] In vivo efficacy results showed ( Figure 10 (B) Although mPEG-Liposome / DOX treatment reduced the tumor burden in mice, the tumor burden increased over time, indicating that it could reduce but not completely inhibit tumor growth. Compared with the Saline control group and the mPEG-Liposome / DOX group, 228-K3-EG8-Liposome / DOX significantly inhibited tumor growth and had similar efficacy to free DOX.

[0122] By plotting the median survival time of tumor-bearing mice, the antitumor effects of two DOX-loaded liposomes can be evaluated. Figure 10As shown in Figure C, no mouse deaths occurred in the 228-K3-EG8-Liposome / DOX group during the study, while mouse deaths occurred in both the Saline and mPEG-Liposome / DOX groups. The median survival times for the two groups were 20 days and 24 days, respectively. Mice in the DOX group, due to the severe systemic toxicity of DOX, died quickly, with a median survival of only 9 days. This demonstrates that 228-K3-EG8-Liposome / DOX can significantly prolong the survival time of mice.

[0123] In summary, targeting liposome 228-K3-EG8-Liposome / DOX can significantly improve drug efficacy and reduce systemic toxicity in breast cancer treatment.

[0124] This invention first detected the expression of GPR54 in human breast cancer cell lines (T47D, MCF-7, SK-BR-3, and MDA-MB-231), mammary epithelial cells (MCF 10A), and human blood neutrophils using quantitative PCR and Western blotting experiments. The results showed that GPR54 expression was significantly upregulated in breast cancer cell lines and was not correlated with the expression levels of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). In contrast, GPR54 was only expressed at low levels in MCF 10A cells and was not expressed in human blood neutrophils. Simultaneously, quantitative PCR and Western blotting data showed that GPR54 expression was also significantly upregulated in in situ tumor tissues of breast cancer patients, and significantly higher than in adjacent normal tissues.

[0125] Next, the experiments examined the uptake of a low-activity antagonist analog of the GPR54 receptor's endogenous ligand Kp-10 in breast cancer cells. Using FITC-labeled analogs, flow cytometry and laser confocal microscopy experiments determined that Kp-10-228 was the analog with the highest cellular uptake efficiency.

[0126] Then, the length of the polyethylene glycol chain connecting the targeting peptide and lipid, as well as the hydrophilicity of the targeting peptide, were systematically optimized and screened. Targeted liposomes were prepared using a thin-film hydration method, and their in vitro cellular uptake efficiency was studied. Experimental results showed that the targeted liposome 228-K3-EG8-Liposome modified with peptide Kp-10-228, compared with the non-targeted liposome mPEG-Liposome, had significantly higher cellular uptake efficiency in breast cancer cell lines, and the increase in cellular uptake efficiency depended on the GPR54 receptor.

[0127] Finally, small animal imaging and a mouse 4T1 breast cancer model were used to study the in vivo targeted distribution and anti-tumor effects of 228-K3-EG8-Liposome. The results showed that compared with tumor-bearing mice injected with mPEG-Liposome, 228-K3-EG8-Liposome accumulated significantly more at the tumor site, demonstrating a stronger targeting ability for breast tumors. In the efficacy study of the two liposomes, the results showed that mice injected with 228-K3-EG8-Liposome had significantly prolonged survival, slowed tumor growth, reduced doxorubicin toxicity, improved bioavailability, and enhanced therapeutic effect.

[0128] In summary, this study validated the significant upregulation of GPR54 expression in breast cancer. Utilizing its modified high-affinity peptide ligand Kp-10-228, a targeting peptide was linked to a fatty acid chain via an EG8 linker. A lysine residue was added to increase the hydrophilicity of the targeting peptide, resulting in a targeted liposome exhibiting ideal targeting properties. The 228-K3-EG8-Liposome demonstrated excellent tumor targeting ability and enhanced cellular uptake, effectively improving drug bioavailability and enhancing therapeutic efficacy. Therefore, liposome drug delivery systems based on the Kisspeptin / GPR54 system hold promise as a novel strategy for treating breast cancer.

Claims

1. A targeted peptide-lipid linker, characterized in that, Its structural formula is (PA). 2- K-EG8-KKK-EG2-Kp-10-228 comprises two hydrophobic palmitic acids (PA)2, one lysine K, a short lysine chain KKK, a polyethylene glycol chain EG8, an EG2 linker, and a polypeptide Kp-10-228; the sequence of the polypeptide Kp-10-228 is YNWNGFG(DW)RF, where DW represents D-type tryptophan.

2. A targeted polypeptide liposome, characterized in that, Includes the targeted peptide-liposome linker and liposome as described in claim 1.

3. The targeted peptide liposome according to claim 2, wherein the composition of the targeted peptide liposome is in the following molar ratio: hydrogenated soybean phosphatidylcholine: cholesterol: distearate phosphatidylethanolamine-polyethylene glycol 2000: the targeted peptide-lipid linker according to claim 1 = (50-56): (35-40): (4-6):

1.

4. The targeted polypeptide liposome according to claim 2, characterized in that, The targeted polypeptide liposomes were prepared by thin-film hydration.

5. Use of the targeted polypeptide liposomes according to any one of claims 2-4 in the preparation of anti-breast cancer targeted drug formulations.

6. A doxorubicin hydrochloride active drug-carrying liposome, characterized in that, By mixing the targeted polypeptide liposomes according to any one of claims 2-4 with a doxorubicin solution and incubating them with shaking at 55-65°C for 1-3 hours, liposomes encapsulating doxorubicin are obtained.

7. The doxorubicin hydrochloride active drug-carrying liposome according to claim 6, characterized in that, The concentration of doxorubicin is 3-5 mg / mL.

Images