A polypeptide-modified liposome for targeting treatment of glioblastoma and preparation and application thereof

Abstract

The application discloses a polypeptide-modified liposome for targeted treatment of glioblastoma and a preparation and application thereof. The liposome comprises a polypeptide for binding to a TREM2 protein of glioblastoma, a drug having a killing effect on glioblastoma cells and a liposome for wrapping the drug; the polypeptide for binding to the TREM2 protein has an amino acid sequence as shown in the following: HLRKLRKR; the polypeptide for binding to the TREM2 protein is modified on the liposome, and the polypeptide-modified liposome wraps the drug. The polypeptide-modified doxorubicin liposome prepared by the application realizes active drug delivery by connecting a targeting polypeptide, the doxorubicin having a therapeutic effect on human glioblastoma is wrapped in the liposome, the action time is prolonged, the cytotoxicity is reduced, and the stability of the whole targeted drug delivery system is improved.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a polypeptide-modified liposome for targeted therapy of glioblastoma and its preparation and application. Background Technology

[0002] Glioblastoma multiforme (GBM, WHO grade IV) is the most common and most aggressive type of malignant brain tumor. Due to its invasiveness, molecular heterogeneity, and unique location, conventional treatments (surgery, external beam radiation, and chemotherapy) have relatively limited effectiveness in improving overall survival. Currently, a major challenge in brain tumor treatment lies in the blood-brain barrier (BBB), which prevents drugs from reaching the tumor site or results in minimal drug accumulation, thus failing to achieve therapeutic effects. Therefore, improving drug targeting and delivery efficiency is an urgent problem to be solved in GBM treatment research.

[0003] In recent years, researchers have developed a drug delivery system using peptide-modified liposomes that can effectively cross the blood-brain barrier, improving drug targeting and biocompatibility. Combining the advantages of both peptides and liposomes, this technology has wide applications in drug therapy. With the development of biotechnology and molecular biology, this technology is gradually becoming a cutting-edge technique for treating malignant tumors.

[0004] Liposomes are microparticles composed of phospholipids and cholesterol, possessing excellent drug loading and metabolic capabilities. Researchers have found that encapsulating drugs in liposomes can improve drug stability and bioavailability while reducing toxic side effects on normal cells. Furthermore, peptides with the ability to specifically bind to tumor cell surface markers can enhance the binding affinity of liposomes to tumor cell surface receptors after modification. By modifying the structure of liposomes and altering the conformation of peptides, the targeting effect of liposomes can be effectively improved, enabling accurate identification and targeted therapy of tumor cells, thus improving treatment efficacy and reducing toxic side effects. Therefore, applying peptide-modified liposome drug delivery systems with strong targeting specificity and low toxicity to the treatment of GBM (genomic leukemia) has significant research value and application prospects. Summary of the Invention

[0005] To address the practical problems in glioblastoma treatment, this invention designs a peptide ligand with high affinity for the TREM2 protein, which is specifically highly expressed in glioblastoma, and provides a method for preparing a targeted drug delivery system for glioblastoma treatment. By modifying the surface of liposomes with a targeting peptide, the drug's targeting specificity to glioblastoma is enhanced. Furthermore, encapsulating doxorubicin in liposomes reduces the cytotoxic effects of doxorubicin to some extent. This drug delivery system can cross the blood-brain barrier, target U87-MG cell orthotopic xenografts, and has shown some therapeutic efficacy, demonstrating potential application prospects in glioblastoma treatment research.

[0006] The purpose of this invention is to provide a peptide-modified liposome for targeted therapy of glioblastoma.

[0007] The peptide-modified liposome for targeted therapy of glioblastoma provided by the present invention includes a peptide for binding to the TREM2 protein targeting glioblastoma, a drug with a killing effect on glioblastoma cells, and a liposome encapsulating the drug.

[0008] The polypeptide targeting TREM2 protein has the following amino acid sequence: HLRKLRKR (histidine-leucine-arginine-lysine-leucine-arginine-lysine-arginine, His-Leu-Arg-Lys-Leu-Arg-Lys-Arg), abbreviated as HLR (Sequence 1).

[0009] The peptide targeting the TREM2 protein is modified onto the liposomes, and the peptide-modified liposomes encapsulate the drug.

[0010] The drug in question is doxorubicin;

[0011] The liposomes are made from lecithin and DSPE-PEG2000.

[0012] The above-mentioned peptide-modified liposomes for targeted therapy of glioblastoma were prepared by a method comprising the following steps:

[0013] 1) DSPE-PEG2000-Mal was reacted with the peptide HLR to obtain DSPE-PEG2000-HLR;

[0014] 2) Liposome solutions were prepared using lecithin and DSPE-PEG2000-HLR as raw materials via an ammonium sulfate gradient method;

[0015] 3) Add a drug that kills glioblastoma cells to the obtained liposome solution, incubate, and the product is obtained.

[0016] In step 1) of the above method, the molar ratio of DSPE-PEG2000-Mal to peptide HLR is 1:1.5-2, specifically 1:1.5;

[0017] The reaction is carried out in water, at 4°C under inert gas protection, and for a duration of 24-48 hours, specifically 24 hours.

[0018] The operation of step 2) of the above method is as follows: lecithin and DSPE-PEG2000-HLR are dissolved in chloroform, evaporated to form a membrane, (NH4)2SO4 solution is added and hydrated; after ultrasonic dispersion, a liposome solution is prepared; uncoated (NH4)2SO4 is removed by dialysis to obtain the liposome solution;

[0019] The mass ratio of lecithin to DSPE-PEG2000-HLR can be 6-9:1, specifically 9:1.

[0020] The concentration of the (NH4)2SO4 solution can be 250 mM.

[0021] The ratio of lecithin to (NH4)2SO4 solution can be 18 mg: 5 mL;

[0022] The dialysis was performed overnight in 50 mM pH 7.4 PBS.

[0023] In step 3) of the above method, the drug that has a killing effect on glioblastoma cells can specifically be doxorubicin;

[0024] The mass ratio of lecithin to the drug may be 18 mg: 1 mg;

[0025] The incubation was carried out at 50°C for 1 hour.

[0026] After incubation, dialysis is performed to remove any unencapsulated drugs.

[0027] The present invention also provides the use of the above-mentioned peptide-modified liposomes for targeted therapy of glioblastoma in the preparation of medicaments for treating human glioma or human glioma cell xenografts.

[0028] In the aforementioned application, the human glioma cells may specifically be U87-MG cells.

[0029] According to an embodiment of the present invention, the selected human glioma cells are U87-MG cells, and the selected in vivo glioma is a BABL / c nude mouse orthotopic U87-MG cell-bearing xenograft.

[0030] The advantages of the nano-targeting material prepared by this invention compared with existing technologies include:

[0031] 1) The targeted peptide designed in this invention can effectively recognize and specifically bind to human glioma U87-MG cells, and the equilibrium dissociation constant KD of its interaction with TREM2 protein is 8.17 μM, indicating a strong binding ability to TREM2 protein.

[0032] 2) The liposomes prepared in this invention can cross the blood-brain barrier and have a certain degree of active targeting. After being combined with the targeting peptide, the targeting effect of the nano-targeting material is enhanced through synergistic effect, and it can target glioblastoma cells.

[0033] 3) The polypeptide-modified doxorubicin liposomes prepared in this invention achieve active drug delivery by linking to a targeting polypeptide. Doxorubicin, which has a therapeutic effect on human glioblastoma, is encapsulated in liposomes, which prolongs the duration of action, reduces its cytotoxicity, and improves the overall stability of the targeted drug delivery system. Attached Figure Description

[0034] Figure 1 This is the structural formula of the polypeptide HLR in this invention.

[0035] Figure 2 This is a three-dimensional docking model of peptide HLR and TREM2 protein in Example 1 of the present invention.

[0036] Figure 3 This is a synthetic route diagram of the polypeptide HLR in Example 2 of the present invention.

[0037] Figure 4 The results are HPLC and MS identification of the polypeptide HLR obtained in Example 2 of this invention.

[0038] Figure 5 The results of the affinity test between HLR and TREM2 protein in Example 2 of this invention are shown.

[0039] Figure 6 The results show the effect of HLR on the survival rate of U87-MG cells in Example 3 of this invention. *p<0.05, **p<0.01.

[0040] Figure 7 This is a synthesis route diagram of FITC-HLR in Embodiment 4 of the present invention.

[0041] Figure 8 This is the structural formula of FITC-HLR in Embodiment 4 of the present invention.

[0042] Figure 9 The results are HPLC and MS identification of the FITC-HLR prepared in Example 4 of this invention.

[0043] Figure 10This is an example of immunofluorescence analysis of the binding of TREM2 protein and HLR in U87-MG cells in Example 4 of the present invention.

[0044] Figure 11 This is a flow cytometry comparison of HLR uptake in U87-MG cells in Example 4 of the present invention.

[0045] Figure 12 This is a synthesis route diagram of Cy5.5-HLR in Embodiment 5 of the present invention.

[0046] Figure 13 The structure of Cy5.5-HLR obtained in Example 5 of this invention is shown.

[0047] Figure 14 The HPLC and MS identification results are for Cy5.5-HLR prepared in Example 5 of this invention.

[0048] Figure 15 This describes the distribution and brain uptake of Cy5.5-HLR in nude mice with U87-MG cell orthotopic xenografts in Example 5 of this invention. # p<0.001, A. Fluorescence distribution at different time points after peptide uptake in nude mouse brain; B. Fluorescence intensity at different time points after peptide uptake in nude mouse brain.

[0049] Figure 16 This figure shows the uptake of Cy5.5-HLR in the organs and brain of nude mice with U87-MG cell orthotopic xenografts 24 hours after administration in Example 5 of the present invention. # p<0.001, A. Fluorescence distribution in the organs and brain of nude mice after taking up the peptide; B. Fluorescence intensity in the brain of nude mice after taking up the peptide.

[0050] Figure 17 This is the NMR spectrum of the DSPE-PEG2000-HLR in Embodiment 6 of the present invention.

[0051] Figure 18 The results are HPLC and MS identification of LRK in Example 6 of this invention.

[0052] Figure 19 This is a schematic diagram illustrating the synthesis of the liposome-encapsulated drug Dox, surface-modified peptides, and Cy5.5 in Example 6 of the present invention.

[0053] Figure 20 Performance characterization of the polypeptide-modified doxorubicin liposomes in Example 7 of this invention. A, B: Particle size; C, D: Zeta potential; E, F: Morphology analyzed by transmission electron microscopy; G, H: Encapsulation efficiency; I, J: Drug loading; K: Doxorubicin accumulation and release rate.

[0054] Figure 21The results show the effect of polypeptide-modified doxorubicin liposomes on the survival rate of U87-MG cells in Example 8 of this invention.

[0055] Figure 22 The results show the effect of peptide-modified doxorubicin liposomes on the apoptosis rate of U87-MG cells in Example 8 of this invention.

[0056] Figure 23 This shows the effect of peptide-modified doxorubicin liposomes on the invasive ability of U87-MG cells in Example 8 of this invention. **p<0.01, # p < 0.001.

[0057] Figure 24 The results show the effect of peptide-modified doxorubicin liposomes on the migration ability of U87-MG cells in Example 8 of this invention. *p<0.05, **p<0.01.

[0058] Figure 25 The results show the effect of peptide-modified doxorubicin liposomes on the survival of U87-MG cell clones in Example 8 of this invention. **p<0.01.

[0059] Figure 26 This is the result of the uptake of doxorubicin liposomes modified with peptides by U87-MG cells in Example 9 of the present invention.

[0060] Figure 27 This illustrates the uptake of peptide-modified doxorubicin liposomes across the blood-brain barrier and into the brain of nude mice with orthotopic tumors in Example 9 of this invention. A. Fluorescence distribution at different time points after uptake of peptide-modified doxorubicin liposomes into the nude mouse brain; B. Fluorescence intensity at different time points after uptake of peptide-modified doxorubicin liposomes into the nude mouse brain.

[0061] Figure 28 This image shows the uptake of peptide-modified doxorubicin liposomes in the brain 24 hours after administration in Example 9 of this invention. A. Fluorescence imaging of the organs of nude mice after uptake of peptide-modified doxorubicin liposomes; B. Fluorescence intensity of the brain of nude mice after uptake of peptide-modified doxorubicin liposomes.

[0062] Figure 29 This document describes the treatment of nude mice with orthotopic xenografts using peptide-modified doxorubicin liposomes in Example 10 of this invention. A) Comparison of fluorescence signals in orthotopic xenografts in the brains of nude mice at different time points; B) Fluorescence intensity of orthotopic xenografts in the brains of nude mice at different time points; C) Comparison of body weight of nude mice at different time points.

[0063] Figure 30 H&E staining observation of peptide-modified doxorubicin liposomes used in Example 10 of this invention for the treatment of nude mouse orthotopic xenograft tumors.

[0064] Figure 31TUNEL staining observation of peptide-modified doxorubicin liposomes used in Example 10 of this invention to treat orthotopic xenografts in nude mice.

[0065] Figure 32 This invention provides an observation of Ki67 expression in nude mice after treatment with orthotopic xenograft tumors using peptide-modified doxorubicin liposomes via immunofluorescence detection, as described in Example 10 of this invention. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] Example 1: Molecular docking of HLRKLRKR with TREM2 protein

[0069] 1) Determination of the TREM2 protein structure

[0070] The human TREM2 protein, with a total length of 230 amino acids, uses the A chain of a 5UD7 crystal structure and is scored for docking using the Cluspro algorithm.

[0071] 2) Molecular binding mode analysis

[0072] The binding mode of the highest-scoring peptide to the TREM2 protein was analyzed. The peptide sequence is histidine-leu-leucine-arginine-lysine-leucine-arginine-lysine-arginine (His-Leu-Arg-Lys-Leu-Arg-Lys-Arg, abbreviated HLRKLRKR, structural formula see [link to structural formula]). Figure 1 The results of the interaction positions of the HLRKLRKR and TREM2 docking are shown in [the table below]. Figure 2 The results showed that HLRKLRKR can effectively bind to the TREM2 protein.

[0073] Example 2: Affinity determination of HLR and TREM2 proteins

[0074] 1) Artificial synthesis of HLR

[0075] See the synthesis route diagram of HLR. Figure 3Synthesis sequence: from C-terminus to N-terminus. ① Resin swelling: Place 2-ChlorotritylChloride Resin into a reaction tube, add DCM (dichloromethane) (15 mL / g), and shake for 30 min; ② Introduce the first amino acid: Filter the solvent through a stencil, add 3 times the molar excess of Fmoc-His(Trt)-OH amino acid, add DMF (dimethylformamide) to dissolve, then add 10 times the molar excess of DIEA (diisopropylethylamine), and shake for 60 min. ③ Block with methanol; ④ Deprotection: Remove DMF, add 20% piperidine DMF solution (15 mL / g), react for 5 min, remove and add another 20% piperidine DMF solution (15 mL / g), react for 15 min; ⑤ Detection: Remove piperidine solution, take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a deep blue color indicates a positive reaction; ⑥ Washing: DMF (10 mL / g) twice, DCM (10 mL / g) twice, DMF (10 mL / g) twice; ⑦ Condensation: Protect amino acid in three-fold excess, HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) in three-fold excess, dissolve both in as little DMF as possible, add to the reaction tube, immediately add DIEA in ten-fold excess, react for 30 min; ⑧ Detection: Take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a colorless color indicates a negative reaction. ⑧ Wash twice with DMF (10 mL / g), twice with DCM (10 mL / g), twice with DMF (10 mL / g); ⑨ Repeat steps three to six, linking amino acids in the sequence from right to left; ⑩ Dry, washing steps: twice with DMF (10 mL / g), twice with methanol (10 mL / g), twice with DMF (10 mL / g), twice with DCM (10 mL / g), dry for 10 min. 11) Cut the peptide from the resin, prepare the cutting solution (10 mL / g) TFA (trifluoroacetic acid) 95%; water 1%; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120 min; blow dry and wash, blow the lysate as dry as possible with nitrogen, wash with ether six times, and then evaporate to room temperature; analyze and purify, purify the crude product by high performance liquid chromatography; freeze dry, collect the target peptide solution and concentrate it in a freeze dryer, freeze dry into powder. The HPLC and MS identification results of HLR are shown in the figure. Figure 4 .

[0076] 2) Affinity analysis of HLR and TREM2 proteins

[0077] TREM2 protein was diluted to 20 μg / mL with PBS for chip curing, and the peptide was diluted to 100 μM with PBST (pH 7.4). The TREM2 protein solution was added dropwise to the NTA chip. 200 μL of PBST (pH 7.4) was added to different wells of the cured NTA chip as a control, and 200 μL of the 100 μM peptide was added to the other wells. 250 μL of PBS buffer (pH 7.4) was injected into the sensor, and the buffer was run at the maximum flow rate (150 μL / min) until the signal baseline was reached. The flow rate was then reduced to 20 μL / min to obtain a more stable baseline. The signal of HLR binding to TREM2 protein is shown in [the figure]. Figure 5 The results showed that HLR and TREM2 protein have a strong binding interaction, and the equilibrium dissociation constant KD between them is 8.17 μM.

[0078] Example 3: Effect of HLR on U87-MG cell survival

[0079] Cell viability was determined using a CCK-8 assay kit. Human glioblastoma U87-MG cells were seeded in 96-well plates. When the cell density reached 70%, different concentrations of HLR were added, and the plates were cultured at 37°C and 5% CO2 for 24 hours. Then, 10 μL of CCK-8 reaction solution was added to each well, and the plates were cultured at 37°C and 5% CO2 for another 4 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: [A(sample well) – A(blank)] / [A(PBS) – A(blank)] × 100%. Results showed no significant difference in toxicity to U87-MG cells at concentrations of 5, 10, and 20 μg / mL. Therefore, 20 μg / mL was selected for the cell uptake experiment. Figure 6 ).

[0080] Example 4: Analysis of HLR uptake by U87-MG cells

[0081] 1) Preparation of FITC-HLR

[0082] The FITC-HLR synthesis route is as follows: Figure 7 As shown. The polypeptide synthesis is the same as steps ① to ⑧ in Example 2, with amino acids linked sequentially from right to left, followed by 5-FTIC linkage. Subsequent steps are the same as step ⑩ in Example 2. The structural formula of FITC-HLR is shown below. Figure 8 The HPLC and MS identification results are shown in the figure. Figure 9 .

[0083] 2) Qualitative analysis of HLR binding to TERM2 in U87-MG cells

[0084] U87-MG cells were seeded onto glass slides (placed in Φ35 dishes). After reaching a cell density of 60%, the cells were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde for 15 min, and allowed to air dry. Then, 1 μg / mL of FITC-HLR was added, and the cells were incubated overnight at 4°C. The nuclei were stained with 5 μg / mL of DAPI. Finally, the slides were mounted with an anti-quenching agent and observed under a fluorescence microscope. The results showed that HLR could bind to U87-MG cells (…). Figure 10 ).

[0085] 3) Quantitative analysis of HLR uptake in U87-MG cells

[0086] Human umbilical vein endothelial cells (HUVECs) and U87-MG cells were seeded in the upper and lower chambers of a Transwell at a 1:5 ratio and cultured for 48 hours to simulate a tumor microvascular environment. The HUVEC cell culture medium in the upper chamber was aspirated and FITC-HLR (containing 10% FBS to a final concentration of 20 μg / mL) was added. The cells were incubated at 37°C and digested with 0.25% trypsin at 2 and 4 hours, washed three times with cold PBS, and then resuspended in 300 μl of cold PBS for flow cytometry analysis of cell fluorescence intensity. The results showed that the FITC fluorescence intensity of U87-MG cells at 4 hours was greater than that at 2 hours and in the PBS group, indicating that HLR could be taken up by U87-MG cells. Figure 11 ).

[0087] Example 5: HLR crossing the blood-brain barrier experiment

[0088] 1) Preparation of Cy5.5-HLR

[0089] The synthetic route of Cy5.5-HLR is as follows: Figure 12 As shown. The polypeptide synthesis follows steps ① to ⑧ of Example 2, with amino acids linked sequentially from right to left, followed by linking Cy5.5. Subsequent steps are the same as step ⑩ of Example 2. The structural formula of Cy5.5-HLR is shown below. Figure 13 The HPLC and MS identification results are shown in the figure. Figure 14 .

[0090] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0091] A mouse orthotopic tumor model of U87-MG cells was established using a stereotaxic apparatus. U87-MG-luc cells in logarithmic growth phase were harvested, digested with 0.25% trypsin, and then the cell density was adjusted to 1×10⁻⁶ cells / cells. 5 / mL, using a stereotaxic instrument, 5μL of cell suspension was aspirated and injected into the posterior ventricle of the cranium (striatum, 1.8mm to the right of the anterior fontanelle, depth 3mm) of female BABL / c nude mice. Tumor formation was observed 14 days after inoculation using in vivo imaging.

[0092] 3) Observation of intravenous administration of medication to tumor-bearing rats and HLR imaging

[0093] Tumor-bearing nude mice were injected intravenously with 100 μL (1 μg / μL) of Cy5.5-HLR saline solution as described in Example 5. Images were acquired using a small animal fluorescence in vivo imaging system at 1, 3, 6, 12, and 24 hours post-injection. Figure 15 Control group tumor-bearing mice were injected with the same volume of PBS as Cy5.5-HLR. Results showed that the brains of nude mice in the treatment group continuously displayed fluorescent signals. Twenty-four hours after injection, the mouse organs and brains were separated, and images were acquired using a small animal fluorescence in vivo imaging system. The results showed that, compared with the PBS group, the fluorescent signal in the brains of nude mice in the treatment group was significantly increased. Figure 16 This indicates that HLR can cross the blood-brain barrier and has a sustained targeted binding effect.

[0094] Example 6: Preparation of peptide-modified doxorubicin liposomes

[0095] 1) Synthesis of DSPE-PEG2000-HLR / LRK

[0096] DSPE-PEG2000-Mal and peptide HLR / LRK were dissolved in ultrapure water at a molar ratio of 1:1.5 and reacted under nitrogen protection for 24 hours to obtain the product DSPE-PEG2000-HLR / LRK. The yield was calculated by comparing the NMR spectra of the peptide and DSPE-PEG2000-HLR. Figure 17 ).

[0097] LRK is another polypeptide that targets the TREM2 protein. Its amino acid sequence is shown below: LRKLRLRL (Leucine-Arginine-Lysine-Leucine-Arginine-Leucine-Arginine-Leucine, Leu-Arg-Lys-Leu-Arg-Leu), abbreviated as LRK (Sequence 2).

[0098] The preparation method is as follows: artificial synthesis of LRK

[0099] The synthesis sequence of LRK is from the C-terminus to the N-terminus. ① After resin swelling, 2-Chlorotrityl Chloride Resin is placed in a reaction tube, DCM (dichloromethane) (15 ml / g) is added, and the mixture is shaken for 30 min. ② The first amino acid is added, the solvent is removed by vacuum filtration, 3 molar excess of Fmoc-His(Trt)-OH amino acid is added, DMF (dimethylformamide) is added to dissolve it, and then 10 molar excess of DIEA (diisopropylethylamine) is added, and the mixture is shaken for 60 min. ③ Block with methanol; ④ Deprotection: Remove DMF, add 20% piperidine DMF solution (15 mL / g), react for 5 min, remove and add another 20% piperidine DMF solution (15 mL / g), react for 15 min; ⑤ Detection: Remove piperidine solution, take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a deep blue color indicates a positive reaction; ⑥ Washing: DMF (10 mL / g) twice, DCM (10 mL / g) twice, DMF (10 mL / g) twice; ⑦ Condensation: Protect amino acid in triple excess, HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) in triple excess, dissolve both in as little DMF as possible, add to the reaction tube, immediately add DIEA in ten-fold excess, react for 30 min; ⑧ Detection: Take a dozen resin grains, wash three times with ethanol, add test reagent, heat at 105℃~110℃ for 5 min, a colorless color indicates a negative reaction. ⑧ Wash twice with DMF (10 mL / g), twice with DCM (10 mL / g), twice with DMF (10 mL / g); ⑨ Repeat steps ③ to ⑥, linking amino acids in the sequence from right to left; ⑩ Dry, washing steps: twice with DMF (10 mL / g), twice with methanol (10 mL / g), twice with DMF (10 mL / g), twice with DCM (10 mL / g), dry for 10 min. 11) Cut the peptide from the resin, prepare the cutting solution (10 mL / g) TFA (trifluoroacetic acid) 95%; water 1%; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120 min; blow dry and wash, blow the lysate as dry as possible with nitrogen, wash with ether six times, and then evaporate to dryness at room temperature; analyze and purify, purify the crude product by high performance liquid chromatography; freeze dry, collect the target peptide solution and concentrate it in a freeze dryer, freeze dry into powder.

[0100] The HPLC and MS identification results are shown in the figure. Figure 18 .

[0101] 2) Liposome-encapsulated drug Dox

[0102] Weigh 18 mg of lecithin and 2 mg of DSPE-PEG2000, dissolve them separately in 5 mL of chloroform, and evaporate them at 40 °C to form a membrane; add 5 mL of 250 mM (NH4)2SO4 solution for hydration; after ultrasonic dispersion, use a liposome extruder to obtain a liposome solution with a suitable particle size; place it in a nanodialysis device and dialyze overnight in 50 mM pH=7.4 PBS to remove uncoated (NH4)2SO4; take out the liposome solution, add 1 mg of doxorubicin, and incubate at 50 °C for 1 hour; dialyze for about 1 hour to remove uncoated doxorubicin; measure the absorbance of the solution at 480 nm, and determine the concentration according to the doxorubicin standard absorption curve.

[0103] 3) Liposome-encapsulated drug Dox, surface-modified with HLR / LRK

[0104] Weigh 18 mg of lecithin and 2 mg of DSPE-PEG2000-HLR / LRK, dissolve them separately in 5 mL of chloroform, and evaporate them at 40 °C to form a membrane; add 5 mL of 250 mM (NH4)2SO4 solution and hydrate; after ultrasonic dispersion, use a liposome extruder to obtain a liposome solution with a suitable particle size; place it in a nanodialysis device and dialyze overnight in 50 mM pH=7.4 PBS to remove uncoated (NH4)2SO4; take out the liposome solution, add 1 mg of doxorubicin, and incubate at 50 °C for 1 hour; dialyze for about 1 hour to remove uncoated doxorubicin; measure the absorbance of the solution at 480 nm, and determine the concentration according to the doxorubicin standard absorption curve.

[0105] 4) Liposome-encapsulated drug Dox, surface-modified peptides, and Cy5.5

[0106] Weigh 35 mg of lecithin, 1 mg of DSPE-PEG200-HLR / LRK, and 3.5 mg of DSPE-PEG2000-Cy5.5, and dissolve them separately in 5 mL of chloroform. Evaporate at 40 °C to form a membrane. Add 5 mL of 250 mM (NH4)2SO4 solution for hydration. After ultrasonic dispersion, use a liposome extruder to obtain a liposome solution of suitable particle size. Place the solution in a nanodialysis apparatus and dialyze overnight in 50 mM pH 7.4 PBS to remove uncoated (NH4)2SO4. Remove the liposome solution, add 1 mg of doxorubicin, and incubate at 50 °C for 1 hour. Dialyze for approximately 2 hours to remove the doxorubicin, yielding the target product (see schematic diagram for synthesis). Figure 19 The absorbance of the solution was measured at 480 nm, and the concentration was determined according to the standard absorption curve of doxorubicin.

[0107] Example 7: Performance characterization of peptide-modified doxorubicin liposomes

[0108] 1) Zeta potential and particle size detection

[0109] Take 10 μL of the sample to be tested, disperse it in approximately 1.5 mL of deionized water, mix thoroughly, and then place it in the sample cell of the nanoparticle size potentiometer. Select the DLS test mode and test the particle size ( Figure 20 A, B). After the test, insert the electrode into the sample cell and connect it to the particle size analyzer. Select PALS mode and test the potential ( Figure 20 C, D).

[0110] 2) Electron microscopy examination

[0111] The morphology of the sample was observed using a transmission electron microscope: 10 μL of the sample was dispersed in 2 mL of deionized water, mixed thoroughly, and then dropped onto a copper grid. After the copper grid dried, phosphotungstic acid was added again for staining, and the grid was dried further. Once the copper grid was completely dry, it was placed in a transmission electron microscope to take electron micrographs. Figure 20 E, F).

[0112] 3) Encapsulation rate

[0113] Prepare Dox solutions of known concentrations and dilute them with deionized water to different concentrations to obtain Dox solutions of different known concentrations. Measure the absorbance at 480 nm using a UV spectrophotometer to construct a Dox standard curve. Centrifuge liposome drug samples at 10000 rpm for 20 min at 4℃; the supernatant contains liposomes free of Dox. Take 10 μL of the sample to be tested, add 1% Triton X-100 to rupture the membrane, and measure the absorbance using a UV spectrophotometer. Substitute the absorbance into the standard curve to calculate the amount of drug encapsulated in the liposomes. Figure 20 (G, H). Take an equal amount of sample and directly demulsify to obtain a clear solution. The same steps can be used to obtain the sum of the encapsulated and unencapsulated drug amounts, i.e., the total drug amount. Encapsulation efficiency calculation formula: Encapsulation efficiency = Encapsulated amount / Total drug amount × 100%.

[0114] 4) Drug loading

[0115] Liposome drug samples were centrifuged at 10,000 rpm for 20 min at 4℃. The supernatant contained liposomes free of Dox. 10 μL of the sample was taken, and 1% Triton X-100 was added to perforate the membrane. The absorbance was measured using a UV spectrophotometer, and the drug loading in the liposomes was calculated by substituting the absorbance into the standard curve. Figure 20 (I, J). The total amount of Dox added during the preparation of doxorubicin liposomes is the total drug load. The formula for calculating drug loading rate is: Drug loading rate = Drug load / Total drug load × 100%.

[0116] 5) Drug release curve

[0117] The in vitro release of Dox from liposomes was detected by dialysis: A PBS buffer solution with pH 7.4 was prepared, and 2 mL of the sample solution was added to a nanodialysis device. Dialysis was performed at room temperature and a rotation speed of 1500 rpm / min. After 3, 20, 24, 44, 51, 68, and 72 hours, 50 μL of dialysate samples were collected, and 1% Triton X-100 was added to rupture the membrane. The absorbance was measured at 480 nm using a UV spectrophotometer to determine the Dox content in the liposomes. A cumulative release curve of Dox was plotted with time on the x-axis and cumulative release rate on the y-axis. Figure 20 K).

[0118] Example 8: Cellular Detection of Drug Delivery Systems

[0119] 1) U87-MG cell culture and treatment

[0120] U87-MG cells were removed from liquid nitrogen and quickly placed in a 37°C water bath. The cryovials were gently shaken to thaw the cryopreservation solution. After thawing, the cells were transferred to centrifuge tubes containing 5 mL of culture medium. The cells were collected by centrifugation at 1000 rpm for 5 min at room temperature, and the supernatant was discarded. The cells were then resuspended in MEM complete medium containing 10% fetal bovine serum, seeded into culture dishes, and gently mixed by pipetting. The cells were then cultured at 37°C under saturated humidity conditions of 5% CO2.

[0121] 2) U87-MG cell viability assay

[0122] Cell viability assays were performed using the CCK-8 assay. Four groups were set up: Dox, Lipo@Dox, HLR-modified Lipo@Dox, and LRK-modified Lipo@Dox, with effective Dox concentration gradients of 0.1, 0.5, 2, 10, and 50 μg / mL. Each group was configured with three replicates, and each well was seeded with 3000 cells in the logarithmic growth phase. Cell status was observed; when confluence reached 50-60%, the test drug was diluted to different concentrations in culture medium beforehand. After removing the medium, 100 μL of medium containing different drug concentrations was added to each well. After culturing for 48 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 4 hours. The 96-well plate was then placed in a microplate reader, and the absorbance was measured at 450 nm. Cell viability was calculated using the formula [A(sample well) – A(blank)] / [A(PBS) – A(blank)] × 100%. The results showed that after 24 hours of drug treatment, Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox all inhibited U87-MG cell viability in a dose-dependent manner, indicating that liposome-loaded Dox and peptide-modified liposome-Dox did not affect the inhibitory effect of Dox on U87-MG cells. Figure 21 ).

[0123] 3) U87-MG cell apoptosis experiment

[0124] The study included a control group, Dox, liposome-Dox (Lipo@Dox), HLR-modified liposome-Dox (HLR-Lipo@Dox), and LRK-modified liposome-Dox (LRK-Lipo@Dox), with effective Dox concentration gradients of 0.1, 0.5, 2, 10, and 50 μg / mL. U87-MG cells in logarithmic growth phase were washed three times with PBS, digested with 0.25% trypsin without EDTA, centrifuged at 1000 rpm for 5 min, discarded the supernatant, and washed twice with PBS to remove residual culture medium. Apoptotic cells were detected using an Annexin V / PI double staining kit. Cells were resuspended in 100 μL of 1× Binding Buffer, and 5 μL of Annexin V-FITC and 5 μL of PI were added, gently mixed, and incubated at room temperature in the dark for 10 min. 400 μL of Binding Buffer was then added for analysis. The results showed that after 24 hours of drug treatment, Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox induced apoptosis in U87-MG cells in a dose-dependent manner, indicating that liposome-loaded Dox and peptide-modified liposome-Dox did not affect Dox-induced apoptosis in U87-MG cells. Figure 22 ).

[0125] 4) U87-MG cell invasion assay

[0126] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HLR-modified liposome-Dox (HLR-Lipo@Dox), and LRK-modified liposome-Dox (LRK-Lipo@Dox), with an effective Dox concentration of 5 μg / mL. U87-MG cells in logarithmic growth phase were washed three times with PBS, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were washed twice with PBS to remove residual serum. Cells were resuspended in serum-free MEM medium, counted using a cell counting chamber, and diluted to a concentration of 4 × 10⁶ cells / mL with serum-free MEM medium. 5 Cells / mL, ready for use; 800 μL of MEM medium (containing antibiotics) with 10% FBS was added to a 24-well plate and the plate was placed in a Transwell chamber. After 1 hour, 200 μL of cell suspension from each group was inoculated into the upper chamber of the Transwell and cultured at 37°C in a 5% CO2 incubator for 24 hours; the Transwell was removed, the chamber was carefully washed once with PBS, and the cells were fixed with 70% ice-cold ethanol solution for 1 hour; the cells were stained with 0.5% crystal violet solution and placed at room temperature for 20 minutes. After washing with PBS, the unmigrated cells on one side of the upper chamber were wiped clean with a clean cotton ball; the Transwell chamber was placed under a microscope for observation and photography at 400x magnification, and 3 fields of view were taken from each group. The results showed that, after drug treatment, the invasive ability of U87-MG cells was significantly reduced in the Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups compared with the control group, and the order was: Dox group > Lipo@Dox group > LRK-Lipo@Dox group > HLR-Lipo@Dox group. Figure 23 This indicates that the targeted binding of LRK and HLR enables cells to take up more Lipo@Dox; HLR's targeted binding is superior to LRK, therefore, the HLR-Lipo@Dox group has the weakest cell invasion ability.

[0127] 5) U87-MG cell migration assay

[0128] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HLR-modified liposome-Dox (HLR-Lipo@Dox), and LRK-modified liposome-Dox (LRK-Lipo@Dox), with an effective Dox concentration of 5 μg / mL. U87-MG cells in logarithmic growth phase were washed three times with PBS, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, discarded the supernatant, and washed twice with PBS to remove residual serum. Cells were resuspended in serum-free MEM medium, counted using a cell counting chamber, and diluted to a concentration of 3 × 10⁶ cells / mL with serum-free MEM medium. 5 / mL, for later use. Thaw Matrigel at 4°C one day in advance; pre-chill the Transwell chamber, 24-well plate, and pipette tip overnight at -20°C; dilute Matrigel to a final concentration of 5 mg / mL with serum-free medium, and operate on ice; add 800 μL of pre-chilled 10% FBS at 4°C to the 24-well plate. MEM medium (containing antibiotics) was placed in a Transwell chamber. 100 μL of Matrigel (final concentration 1 mg / mL) was vertically added to the center of the bottom of the upper chamber of the Transwell chamber. The chamber was incubated at 37°C for 4–5 hours until it dried into a gel. After the Matrigel dried, 200 μL of cell suspension from each group was inoculated into the upper chamber of the Transwell chamber. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The Transwell chamber was removed, and the cells were carefully washed once with PBS. Cells were fixed with 70% ice-cold ethanol solution for 1 hour. The cells were stained with 0.5% crystal violet solution, incubated at room temperature for 20 minutes, washed once with PBS, and the unmigrated cells on one side of the upper chamber were wiped clean with a clean cotton ball. The Transwell chamber was then observed and photographed under a microscope at 400x magnification, with three fields of view for each group. The results showed that, after drug treatment, the migration ability of U87-MG cells was significantly weakened compared with the control group in the Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups. Furthermore, the order of Dox > Lipo@Dox > LRK-Lipo@Dox > HLR-Lipo@Dox group indicated that the targeted binding of LRK and HLR enabled cells to take up more Lipo@Dox. HLR's targeted binding was superior to LRK's; therefore, the HLR-Lipo@Dox group had the weakest cell migration ability. Figure 24 ).

[0129] 6) U87-MG cell cloning experiment

[0130] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HLR-modified liposome-Dox (HLR-Lipo@Dox), and LRK-modified liposome-Dox (LRK-Lipo@Dox), with an effective concentration gradient of Dox set at 5 μg / mL. U87-MG cells in logarithmic growth phase were washed three times with PBS, digested with 0.25% trypsin, and pipetted to form single cells. The cells were then suspended in culture medium for later use. The cell suspension was diluted and seeded at a density of 400 cells per well in 6-well plates. The cells were gently rotated to ensure even distribution. The culture dishes were placed in a 37°C, 5% CO2 incubator and cultured for 3 weeks. When clones appeared, the supernatant was discarded, and the cells were washed twice with PBS and fixed with 75% glacial ethanol for 15 min. The fixative was discarded, and the cells were stained with 0.5% crystal violet for 30 min. The cells were washed with PBS and counted. The colony formation rate was calculated as: colony formation rate = (number of clones / number of seeded cells) × 100%. The results showed that, after drug treatment, the cell clonal survival of U87-MG cells was significantly reduced in the Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups compared with the control group. Furthermore, the order of Dox > Lipo@Dox > LRK-Lipo@Dox > HLR-Lipo@Dox indicated that the targeted binding of LRK and HLR enabled cells to take up more Lipo@Dox. HLR's targeted binding was superior to LRK's; therefore, the HLR-Lipo@Dox group had the lowest cell survival. Figure 25 ).

[0131] Example 9: Experiment on the crossing of the blood-brain barrier by peptide-modified doxorubicin liposomes

[0132] 1) U87-MG cells take up peptide-modified doxorubicin liposomes

[0133] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HLR-modified liposome-Dox (HLR-Lipo@Dox), and LRK-modified liposome-Dox (LRK-Lipo@Dox), with an effective Dox concentration of 5 μg / mL. Human umbilical vein endothelial cells (HUVECs) and U87-MG cells were seeded at a 1:5 ratio in the upper and lower chambers of a Transwell (with coverslips attached), and cultured for 48 hours to simulate a tumor microvascular environment. The HUVEC cell culture medium in the upper chamber of the Transwell was aspirated, and Dox, Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox with an effective Dox concentration of 5 μg / mL containing 10% FBS were added. The cells were incubated at 37°C, and coverslips from the lower chamber were collected after 8 hours. The cells were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde for 15 min, air-dried, and then stained with 5 μg / mL DAPI to stain the nuclei. Finally, the slides were mounted with an anti-quenching agent and scanned and analyzed using a tissue section scanner. The results showed that after co-culture and drug treatment, compared with the control and Dox groups, the Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups all showed Cy5.5 fluorescence signals, indicating that all three drugs could penetrate HUVEC cells and be taken up by U87-MG cells. Furthermore, due to the targeted binding of LRK and HLR, cells were able to take up more Lipo@Dox, with HLR showing superior targeting binding compared to LRK. Therefore, the HLR-Lipo@Dox group had the strongest fluorescence signal. Figure 26 ).

[0134] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0135] A mouse orthotopic tumor model using U87-MG-luc cells was established using a stereotaxic apparatus. U87-MG-luc cells in logarithmic growth phase were digested with 0.25% trypsin and the cell density was adjusted to 1×10⁻⁶ cells / cells. 5 / mL, using a stereotaxic instrument, 5μL of cell suspension was aspirated and injected into the posterior ventricle of the cranium (striatum, 1.8mm to the right of the anterior fontanelle, depth 3mm) of female BABL / c nude mice. Tumor formation was observed 14 days after inoculation using in vivo imaging.

[0136] 3) Experiment on cross-blood-brain barrier of peptide-modified doxorubicin liposomes

[0137] Tumor-bearing nude mice were injected via tail vein with Cy5.5-modified liposome-Dox from Example 5.

[0138] Lipo@Dox, Cy5.5 and HLR-modified liposomes-Dox (HLR-Lipo@Dox), and Cy5.5 and LRK-modified liposomes-Dox (LRK-Lipo@Dox) were administered via saline injection. The effective concentration of Dox was 2.5 mg / kg. Images were acquired using a small animal fluorescence in vivo imaging system at 2, 4, 8, and 24 hours post-injection. Figure 27 Nude mice without U87-MG cells were intravenously injected with the same volume of physiological saline as the drug. The results showed that the brains of nude mice in the Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups continuously displayed fluorescent signals, and the fluorescence signal at each time point was HLR-Lipo@Dox group > LRK-Lipo@Dox group > Lipo@Dox group, indicating that the targeted binding of LRK and HLR resulted in higher efficiency of drug uptake in the brain. Twenty-four hours after injection, the mouse organs and brains were isolated, and images were acquired using a small animal fluorescence in vivo imaging system. The results showed that the fluorescence signal intensity in the brains of tumor-bearing mice was HLR-Lipo@Dox group > LRK-Lipo@Dox group > Lipo@Dox group. Figure 28 This indicates that Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox can all cross the blood-brain barrier. However, since HLR has a stronger targeting binding effect than LRK, HLR-Lipo@Dox has a stronger targeting effect, followed by LRK-Lipo@Dox.

[0139] Example 10: Experimental use of peptide-modified doxorubicin liposomes for the treatment of glioma

[0140] 1) Drug administration and tumor observation

[0141] The experiment was divided into four groups: PBS group, Dox group, Cy5.5-modified liposome-Dox (Lipo@Dox), Cy5.5 and HLR-modified liposome-Dox (HLR-Lipo@Dox), and Cy5.5 and LRK-modified liposome-Dox (LRK-Lipo@Dox). Dox was administered via tail vein injection to nude mice at an effective concentration of 2.5 mg / kg, administered every 3 days (days 0, 3, 6, 9, and 12). Body weight changes were recorded every 3 days (days 0, 3, 6, 9, and 12), and tumor changes were observed using in vivo fluorescence imaging (days 3, 6, 9, 12, and 15). Nude mice were treated on day 15 after administration. Figure 29 A). The results showed that, compared with the PBS group and the Dox group, Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox significantly inhibited tumor growth at all time points, with HLR-Lipo@Dox showing the strongest inhibitory effect. Figure 29B). There was no significant difference in the effect of HLR-Lipo@Dox and LRK-Lipo@Dox on the body weight of nude mice. Figure 29 C) indicates that the polypeptide-modified doxorubicin liposomes have low systemic toxicity in nude mice.

[0142] 2) Paraffin slice preparation

[0143] On day 15, after treatment, the brains of nude mice were harvested and fixed with fixative for at least 24 hours. ① Dehydration: The tissue was sequentially placed in a gradient of alcohols for dehydration: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol / xylene for 5–10 minutes, xylene I for 5–10 minutes, xylene II for 5–10 minutes, wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour. ② Embedding: Melted wax was placed in an embedding frame. Before the wax solidified, the tissue was removed from the dehydration box, placed in the embedding frame according to the embedding surface requirements, and labeled accordingly. Cooling was performed at -20°C. After the wax solidified, the wax block was removed from the embedding frame and trimmed. ③ Sectioning: The trimmed wax block was sectioned using a paraffin microtome to a thickness of 3 μm. The tissue sections were floated on a 40°C warm water spreader to flatten the tissue, then spread on a glass slide and placed in a 60°C oven to bake.

[0144] 3) H&E staining

[0145] The slides were sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, and then washed with tap water. The slides were stained with hematoxylin for 3–5 min, differentiated with hydrochloric acid solution, and then blued with ammonia solution, followed by washing with water. The slides were then sequentially immersed in a gradient of 85% and 95% ethanol for dehydration, and finally stained with 1% eosin for 5 min. The slides were then sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min to achieve transparency. Finally, the slides were mounted with neutral resin, and images were acquired using a Pannoramic MIDI scanner. The results showed that, compared with the control group and the Dox group, the outlines of xenografts in the nude mouse brains of the Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox groups were all reduced, indicating that Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox could all inhibit xenograft growth. Furthermore, because HLR's targeting binding effect was superior to LRK's, the HLR-Lipo@Dox group showed the most significant inhibitory effect, followed by LRK-Lipo@Dox. Figure 30 ).

[0146] 3) TUNEL Experiment

[0147] The TUNEL assay was used to detect apoptotic cells. The steps were as follows: ① Dewaxing of sections: Sections were sequentially immersed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min, and then rinsed with distilled water for 30 min. ② Membrane perforation: Proteinase K working solution (20 μg / mL) was added to the sections to cover the tissue, and incubated at 37°C for 25 min. The slides were then placed in PBS and washed three times on a decolorizing shaker for 5 min each time. ③ Addition of TUNEL reaction solution: An appropriate amount of reagent 1 (TdT) and reagent 2 (dUTP) from the TUNEL kit were mixed at a ratio of 1:9, and added to cover the tissue. The mixture was incubated at 37°C for 2 hours. ④ Counterstaining of cell nuclei with DAPI: Sections were washed three times with PBS for 5 min each time. After removing the PBS, DAPI staining solution (5 μg / mL) was added and incubated at room temperature in the dark for 10 min. ⑤ Mounting: The slides were mounted with anti-fluorescence quenching mounting medium. ⑥ Scanning observation and result interpretation: Images were acquired using a Pannoramic MIDI scanner. DAPI-stained cell nuclei appeared blue under UV excitation, while apoptotic cell nuclei appeared green. Results showed that Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox all induced apoptosis in U87-MG cells, with the number of apoptotic cells being HLR-Lipo@Dox > LRK-Lipo@Dox > Lipo@Dox. This indicates that the targeting binding of LRK and HLR resulted in higher efficiency in inducing apoptosis in U87-MG cells. Figure 31 Furthermore, since HLR's targeting binding effect is superior to LRK, the U87-MG cells in the HLR-Lipo@Dox group showed the most significant apoptosis.

[0148] 3) Detect Ki-67 expression

[0149] The expression of Ki67 protein was detected using immunofluorescence technology, with the following steps: ① Dewaxing of sections: Sections were sequentially immersed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, and 75% ethanol for 5 min, followed by rinsing with distilled water for 30 min. ② Antigen retrieval: Sections were placed in an antigen retrieval buffer containing EDTA and microwaved for antigen retrieval. Microwave on medium heat for 8 min, turn off for 8 min, then microwave on medium-low for 7 min. ③ Blocking: BSA was added and incubated for 30 min. ④ Primary antibody incubation: The blocking solution was gently aspirated, and Ki67 primary antibody prepared with PBS at a specific ratio was added to the sections. The sections were then incubated overnight at 4°C in a humidified chamber. ⑤ Secondary antibody conjugation: Secondary antibody of the corresponding species to the primary antibody was added to cover the tissue, and the sections were incubated at room temperature in the dark for 1 hour. ⑥ DAPI counterstaining of cell nuclei: DAPI staining solution (5 μg / mL) was added, and the sections were incubated at room temperature in the dark for 10 min. ⑦ Mounting: After slightly drying the sections, mount them with anti-fluorescence quenching mounting medium. ⑧ Scanning observation and result interpretation: Images were acquired using a Pannoracic MIDI scanner. DAPI-stained cell nuclei appear blue under UV excitation, while positive expression is indicated by red light from the corresponding fluorescein label. Results showed that Lipo@Dox, HLR-Lipo@Dox, and LRK-Lipo@Dox all inhibited Ki67 expression in U87-MG cells. The number of Ki67 cells was HLR-Lipo@Dox group < LRK-Lipo@Dox group < Lipo@Dox group (…). Figure 32 This indicates that the targeted binding of LRK and HLR is more efficient at inhibiting Ki67 expression in U87-MG cells; and because the targeted binding of HLR is superior to that of LRK, the U87-MG cells in the HLR-Lipo@Dox group showed the most significant apoptosis.

[0150] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A peptide-modified liposome for targeted therapy of glioblastoma, comprising a peptide for binding to the TREM2 protein targeting glioblastoma, a drug having a killing effect on glioblastoma cells, and a liposome encapsulating the drug. The peptide targeting the TREM2 protein is modified onto the liposomes, and the peptide-modified liposomes encapsulate the drug. in, The polypeptide targeting the TREM2 protein has the following amino acid sequence: HLRKLRKR, abbreviated as HLR.

2. The liposomes according to claim 1, characterized in that: The drug in question is doxorubicin; The liposomes are made from lecithin and DSPE-PEG2000-HLR.

3. A method for preparing the liposomes according to claim 1 or 2, comprising the following steps: 1) DSPE-PEG2000-Mal was reacted with the peptide HLR to obtain DSPE-PEG2000-HLR; 2) Liposome solutions were prepared using lecithin and DSPE-PEG2000-HLR as raw materials via an ammonium sulfate gradient method; 3) Add a drug that kills glioblastoma cells to the obtained liposome solution and incubate to obtain the final product.

4. The method according to claim 3, characterized in that: In step 1), the molar ratio of DSPE-PEG2000-Mal to peptide HLR is 1:1.5-2. The reaction is carried out in water at 4°C under inert gas protection for 24-48 hours.

5. The method according to claim 3 or 4, characterized in that: Step 2) involves dissolving lecithin and DSPE-PEG2000-HLR separately in chloroform, rotary evaporating to form a membrane, adding (NH4)2SO4 solution, and hydrating; then ultrasonically dispersing to prepare a liposome solution; and finally dialysis to remove uncoated (NH4)2SO4 to obtain the liposome solution. The mass ratio of lecithin to DSPE-PEG2000-HLR is 6-9:

1. The concentration of the (NH4)2SO4 solution was 250 mM. The ratio of lecithin to (NH4)2SO4 solution is 18 mg: 5 mL; The dialysis was performed overnight in 50 mM PBS at pH 7.

4.

6. The method according to claim 3 or 4, characterized in that: In step 3), the drug that has a killing effect on glioblastoma cells is doxorubicin; The mass ratio of lecithin to the drug is 18 mg: 1 mg; The incubation was carried out at 50°C for 1 hour. After incubation, dialysis is performed to remove any unencapsulated drugs.

7. The use of the polypeptide-modified liposome for targeted therapy of glioblastoma as described in claim 1 or 2 in the preparation of a medicament for treating human glioma or human glioma cell xenografts.

Images