Targeted liposome for treating glioblastoma and preparation method and application thereof

Abstract

The application discloses a targeted liposome for treating glioblastoma and a preparation method and application thereof. A polypeptide ligand specifically combined with myeloid cell trigger receptor 2 (TREM2) which is highly expressed in glioblastoma is designed, and finally a polypeptide sequence aiming at the TREM2 protein is obtained. A novel targeted drug delivery system is developed and prepared, the targeting property of the drug to the glioblastoma is enhanced by modifying the targeted polypeptide on the surface of the liposome, and the combination of adriamycin and the liposome reduces the toxic effect of the adriamycin on normal cells to a certain extent. The targeted drug delivery system has high targeting property, obvious treatment effect and potential clinical application value.

Description

Technical Field

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

[0002] Gliomas are characterized by rapid growth, high recurrence rate, and strong invasive growth capacity, accounting for 30-40% of brain tumors. Conventional surgical, chemotherapy, and radiotherapy treatments are not ideal. Due to the invasive growth characteristics of gliomas, surgical treatment is not only extremely difficult, but residual micro-lesions can also lead to recurrence. The presence of the blood-brain barrier (BBB) ​​and the blood-brain tumor barrier (BBTB) prevents almost all macromolecular drugs and over 98% of small molecule drugs from reaching the brain, severely impacting therapeutic efficacy. How to quickly break through traditional treatment methods, find new treatment approaches, and improve the cure rate and survival time of glioma patients is an urgent problem to be solved.

[0003] The blood-brain barrier (BBB) ​​is a structure that restricts the free exchange of substances between the blood and brain tissue, protecting normal cells from toxins and pathogens and ensuring the normal functioning of neurons. However, the BBB can hinder drugs from reaching brain tumor sites, increasing the difficulty of drug treatment. Recent studies on the BBB penetration mechanism have shown that lipid-soluble substances, amphoteric molecules, and some drugs can cross the BBB through the efflux mechanism of carriers, delivering drugs to the tumor site. Liposomes, as the most mature nanocarrier currently researched, are not only easy to prepare, inexpensive, stable, and biodegradable, but also have the advantages of long duration of action, prolonging drug action time, reducing drug toxicity, and improving drug stability, making them an ideal nanodelivery system.

[0004] Doxorubicine (Dox) is the most widely used antitumor drug in clinical practice. It has a high affinity for nucleic acids and exerts its antitumor effect by inhibiting the synthesis of RNA and DNA. However, its strong cytotoxicity limits its widespread use.

[0005] Peptides possess advantages such as high target affinity, stable efficacy, low immunogenicity, minimal toxicity, and high specificity, making them important ligand molecules for the development of targeted cancer therapies. Peptide-modified drug-encapsulated liposomes can increase drug selectivity in vivo and reduce drug toxicity. Using them as guides in targeted drug delivery systems via ligand-receptor specific binding demonstrates significant research value and application prospects. Summary of the Invention

[0006] Based on the current challenges and advancements in glioblastoma treatment, this invention designs a polypeptide ligand that specifically binds to TREM2 (Triggering Receptor Expressed on Myeloid Cells 2), a protein highly expressed in glioblastoma. This results in a polypeptide sequence targeting the TREM2 protein, leading to the development and fabrication of a novel targeted drug delivery system. By modifying the surface of liposomes with the targeting polypeptide, the drug's targeting specificity to glioblastoma is enhanced. Furthermore, the binding of doxorubicin to liposomes reduces the toxicity of doxorubicin to normal cells to some extent. This targeted drug delivery system exhibits strong targeting and significant therapeutic effects, demonstrating potential clinical application value.

[0007] One of the objectives of this invention is to provide a polypeptide that targets the TREM2 protein, the amino acid sequence of which is shown below: LRKLRLRL (Leu-Arg-Lys-Lys-Leu-Arg-Lys-Leu-Arg-Leu), abbreviated as LRK.

[0008] This invention employs molecular docking and virtual peptide library screening techniques. Considering the limitation on peptide length for crossing the blood-brain barrier, octapeptides are used as the length basis for octapeptide truncation scanning. Mechanical parameters such as the free energy, hydrogen chain, and van der Waals forces of peptide-protein binding are comprehensively evaluated, and a new target peptide with the sequence LRKLRLRL (abbreviated as LRK) is obtained through screening.

[0009] The second objective of this invention is to provide the application of the above-mentioned peptide targeting TREM2 protein in the preparation of a drug for targeted treatment of glioblastoma.

[0010] A third objective of this invention is to provide a drug for targeted treatment of glioblastoma.

[0011] The drug for targeted treatment of glioblastoma provided by the present invention includes a targeting peptide LRK for binding to glioblastoma, a drug with a killing effect on glioblastoma cells, and liposomes; wherein the targeting peptide LRK is modified on the liposomes, and the liposomes modified with peptide LRK encapsulate the drug.

[0012] The drug may specifically be doxorubicin;

[0013] The liposomes are made from lecithin and DSPE-PEG2000.

[0014] The aforementioned targeted therapy drug for glioblastoma is prepared by a method comprising the following steps:

[0015] 1) DSPE-PEG2000-Mal was reacted with peptide LRK to obtain DSPE-PEG2000-LRK;

[0016] 2) Liposome solutions were prepared using lecithin and DSPE-PEG2000-LRK as raw materials via an ammonium sulfate gradient method;

[0017] 3) Add a drug that kills glioblastoma cells to the obtained liposome solution, incubate, and the product is obtained.

[0018] In step 1) of the above method, the molar ratio of DSPE-PEG2000-Mal to peptide LRK is 1:1.5-2, specifically 1:1.5;

[0019] 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.

[0020] The operation of step 2) of the above method is as follows: lecithin and DSPE-PEG2000-LRK 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;

[0021] The mass ratio of lecithin to DSPE-PEG2000-LRK can be 6-9:1, specifically 9:1.

[0022] The concentration of the (NH4)2SO4 solution can be 250 mM.

[0023] The ratio of lecithin to (NH4)2SO4 solution can be 18 mg: 5 mL;

[0024] The dialysis was performed overnight in 50 mM pH 7.4 PBS.

[0025] In step 3) of the above method, the drug that has a killing effect on glioblastoma cells can specifically be doxorubicin;

[0026] The mass ratio of lecithin to the drug may be 18 mg: 1 mg;

[0027] The incubation was carried out at 50°C for 1 hour.

[0028] After incubation, dialysis is performed to remove any unencapsulated drugs.

[0029] The advantages of the nano-targeting material described in this invention compared to existing technologies include:

[0030] 1) The targeted peptide designed in this invention can effectively recognize and specifically bind to human glioblastoma U87-MG cells, and the equilibrium dissociation constant KD of its interaction with TREM2 protein is 4.49 μM, indicating a strong binding ability to TREM2 protein.

[0031] 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.

[0032] 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

[0033] Figure 1 This is the structural formula of the polypeptide LRK in Example 1 of the present invention.

[0034] Figure 2 This is a three-dimensional docking model of peptide LRK and TREM2 protein in Example 1 of the present invention.

[0035] Figure 3 This is a synthetic route diagram of the polypeptide LRK in Example 2 of the present invention.

[0036] Figure 4 The results are HPLC and MS identification of the polypeptide LRK obtained in Example 2 of this invention.

[0037] Figure 5 The results of the affinity test between LRK and TREM2 protein in Example 2 of this invention are shown.

[0038] Figure 6 The results show the effect of LRK on the viability of U87-MG cells in Example 3 of this invention. **p<0.01.

[0039] Figure 7 This is a synthesis route diagram of FITC-LRK in Embodiment 4 of the present invention.

[0040] Figure 8 This is the structural formula of FITC-LRK in Embodiment 4 of the present invention.

[0041] Figure 9 The results are HPLC and MS identification of the FITC-LRK prepared in Example 4 of this invention.

[0042] Figure 10 This is an example of immunofluorescence analysis of the binding of TREM2 protein and LRK in U87-MG cells in Example 4 of the present invention.

[0043] Figure 11 This is a flow cytometry comparison of LRK uptake in U87-MG cells in Example 4 of the present invention.

[0044] Figure 12 This is the NMR spectrum of DSPE-PEG2000-LRK in Embodiment 5 of the present invention.

[0045] Figure 13 This is a schematic diagram illustrating the synthesis of the liposome-encapsulated drug Dox, surface-modified peptide, and Cy5.5 in Example 5 of the present invention.

[0046] Figure 14 Performance characterization of the polypeptide-modified doxorubicin liposomes in Example 6 of this invention. A, B: Particle size; C, D: Zeta potential; E: Morphology analyzed by transmission electron microscopy; F, G: Encapsulation efficiency; H, I: Drug loading; J: Doxorubicin accumulation and release rate.

[0047] Figure 15 The results show the effect of doxorubicin liposomes modified with polypeptide LRK on the viability of U87-MG cells in Example 7 of this invention.

[0048] Figure 16 The results show the effect of doxorubicin liposomes modified with polypeptide LRK on the apoptosis rate of U87-MG cells in Example 7 of this invention.

[0049] Figure 17 The effect of LRK-modified doxorubicin liposomes on the invasive ability of U87-MG cells in Example 7 of this invention is shown. **p<0.01.

[0050] Figure 18 The results show the effect of peptide-modified doxorubicin liposomes on the migration ability of U87-MG cells in Example 7 of this invention. *p<0.05, **p<0.01.

[0051] Figure 19 The results show the effect of peptide-modified doxorubicin liposomes on the survival of U87-MG cell clones in Example 7 of this invention. **p<0.01.

[0052] Figure 20 This is the result of the uptake of doxorubicin liposomes modified with peptides by U87-MG cells in Example 8 of the present invention.

[0053] Figure 21 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 8 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.

[0054] Figure 22 This image shows the uptake of peptide-modified doxorubicin liposomes in the brain 24 hours after administration in Example 8 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.

[0055] Figure 23 This document describes the treatment of nude mice with orthotopic xenografts using peptide-modified doxorubicin liposomes in Example 9 of the present 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) Changes in body weight of nude mice at different time points.

[0056] Figure 24 H&E staining observation of peptide-modified doxorubicin liposomes used in Example 9 of this invention for the treatment of nude mouse orthotopic xenograft tumors.

[0057] Figure 25 TUNEL staining observation of peptide-modified doxorubicin liposomes used in Example 9 of this invention for the treatment of nude mouse orthotopic xenograft tumors.

[0058] Figure 26 This is an observation of Ki67 expression in nude mice after treatment with orthotopic xenograft tumors using peptide-modified doxorubicin liposomes, as described in Example 9 of this invention, using immunofluorescence detection. Detailed Implementation

[0059] 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.

[0060] 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.

[0061] Example 1: Screening of peptide ligands targeting TREM2 protein

[0062] 1) Determination of the TREM2 protein structure

[0063] 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.

[0064] 2) Establishment of virtual peptide libraries and peptide design

[0065] Considering the limitation on peptide length for crossing the blood-brain barrier, octapeptide truncation scanning was performed using octapeptides as the length basis. Several octapeptides showed energy contributions exceeding -10, accounting for over 50% of the total energy, demonstrating a relatively concentrated range of active amino acids, making truncation feasible.

[0066] 3) Evaluation of docking results

[0067] The free energy, hydrogen chain, and van der Waals forces of the peptide-protein binding were calculated and comprehensively evaluated to determine the screening results. The targeted peptide was then identified. The peptide sequence is leucine-arginine-lysine-leucine-arginine-leucine-arginine-leucine (Leu-Arg-Lys-Leu-Arg-Leu-Arg-Leu, peptide sequence LRKLRLRL, abbreviated LRK, structural formula see [link to structural formula]). Figure 1 The results of its interaction positions with TREM2 are shown in [the table]. Figure 2 .

[0068] Example 2: Affinity determination of LRK and TREM2 proteins

[0069] 1) Artificial synthesis of LRK

[0070] The synthesis sequence of LRK is from the C-terminus to the N-terminus. ① After resin swelling, 2-ChlorotritylChlorideResin 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. The synthetic route diagram of peptide LRK is shown below. Figure 3 The HPLC and MS identification results of LRK are shown in [reference needed]. Figure 4 .

[0071] 2) Affinity analysis of LRK and TREM2 proteins

[0072] 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 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 LRK binding to TREM2 protein is shown in [the figure]. Figure 5 The results showed that LRK and TREM2 protein have a strong binding interaction, and the equilibrium dissociation constant KD between the two is 4.49 μM.

[0073] Example 3: Effect of LRK on U87-MG cell viability

[0074] 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%, LRK at concentrations of 5, 10, 20, 50, and 100 μg / mL was added, and the cells 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 cells were cultured at 37°C and 5% CO2 for another 4 hours. The 96-well plates were 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 no significant difference in toxicity to U87-MG cells at concentrations of 5, 10, 20, and 50 μg / mL. Figure 6 Therefore, 20 μg / mL was selected for the cell uptake experiment.

[0075] Example 4: Analysis of LRK uptake in U87-MG cells

[0076] 1) Preparation of FITC-LRK

[0077] The synthesis of LRK is the same as steps ① to ⑧ in Example 21), with amino acids linked sequentially from right to left, followed by FTIC linkage. Subsequent steps are the same as step ⑩ in Example 21). The synthetic route diagram for FITC-LRK is shown below. Figure 7 The structural formula of FITC-LRK is shown in Figure 8 The HPLC and MS identification results are shown in the figure. Figure 9 .

[0078] 2) Qualitative analysis of LRK binding to TERM2 in U87-MG cells

[0079] 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-LRK 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 LRK could bind to U87-MG cells (…). Figure 10 )

[0080] 3) Quantitative analysis of LRK uptake in U87-MG cells

[0081] Human umbilical vein endothelial cells (HUVECs) and U87-MG cells were seeded in the upper and lower chambers of a Transwell cell line 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-LRK (containing 10% FBS to a final concentration of 20 μg / mL) was added. The cells were incubated at 37°C, and after 2 and 4 hours, they were digested with 0.25% trypsin and washed three times with cold PBS. The cells were then resuspended in 300 μL of cold PBS for flow cytometry analysis to determine fluorescence intensity. The results showed that after 4 hours, the FITC fluorescence intensity of U87-MG cells was greater than that after 2 hours and in the PBS group, indicating that LRK could be taken up by U87-MG cells. Figure 11 ).

[0082] Example 5: Preparation of peptide-modified doxorubicin liposomes

[0083] 1) Synthesis of DSPE-PEG2000-LRK

[0084] DSPE-PEG2000-Mal and peptide 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-LRK. The yield was calculated by comparing the NMR spectra of the peptide and DSPE-PEG2000-LRK. Figure 12 ).

[0085] 2) Liposome-encapsulated drug Dox

[0086] 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.

[0087] 3) Liposome-encapsulated drug Dox, surface-modified with LRK

[0088] Weigh 18 mg of lecithin and 2 mg of DSPE-PEG2000-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 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.

[0089] 4) Liposome-encapsulated drug Dox, surface-modified peptides, and Cy5.5

[0090] Weigh 35 mg of lecithin, 1 mg of DSPE-PEG200-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 uncoated doxorubicin, yielding the target product (synthetic route diagram see...). Figure 13 The absorbance of the solution was measured at 480 nm, and the concentration was determined according to the standard absorption curve of doxorubicin.

[0091] Example 6: Performance characterization of peptide-modified doxorubicin liposomes

[0092] 1) Zeta potential and particle size detection

[0093] 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 14 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 14 C, D).

[0094] 2) Electron microscopy examination

[0095] 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 14 E).

[0096] 3) Encapsulation rate

[0097] Dox solutions of known concentrations were prepared and diluted with deionized water to different concentrations to obtain Dox solutions of different known concentrations. The absorbance was then measured at 480 nm using a UV spectrophotometer to construct a Dox standard curve. 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 rupture the membrane. The absorbance was measured using a UV spectrophotometer, and the amount of drug encapsulated in the liposomes was calculated by substituting the absorbance into the standard curve. Figure 14 (F, G). 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%.

[0098] 4) Drug loading

[0099] 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 of the liposomes was calculated by substituting the absorbance into the standard curve. Figure 14 (H, I). The total amount of Dox added during the preparation of doxorubicin liposomes is the total drug load. Drug loading calculation formula: Drug loading rate = Drug load / Total drug load × 100%.

[0100] 5) Drug release curve

[0101] 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% Tritonx-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 14 J).

[0102] Example 7: Cellular Detection of Drug Delivery Systems

[0103] 1) U87-MG cell culture and treatment

[0104] 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.

[0105] 2) U87-MG cell viability assay

[0106] Cell viability assays were performed using the CCK-8 assay. Three groups were set up: Dox, liposome-Dox (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. 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 with culture medium. 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, the inhibition of U87-MG cell viability by Dox, Lipo@Dox, and LRK-Lipo@Dox was dose-dependent, indicating that liposome-loaded Dox and peptide-modified liposome-Dox did not affect the inhibitory effect of Dox on U87-MG cells. Figure 15 ).

[0107] 3) U87-MG cell apoptosis experiment

[0108] The study included a control group, Dox, liposome-Dox (Lipo@Dox), and an LRK-modified liposome-Dox group (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 LPI 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, 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 16 ).

[0109] 4) U87-MG cell invasion assay

[0110] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), and LRK-modified liposome-Dox group (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, washed twice with PBS to remove residual serum, 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. 5Cells / mL, ready for use; 800 μL of MEM medium (containing antibiotics) with 10% FBS was added to 24-well plates, and Transwell chambers were placed inside. After 1 hour, 200 μL of cell suspension from each group was inoculated into the upper chamber of the Transwell, and the cells were incubated at 37°C and 5% CO2 for 24 hours. The Transwells were removed, and the chambers 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 and incubated at room temperature for 20 min. After washing with PBS, the unmigrated cells on one side of the upper chamber were wiped clean with a clean cotton ball. The Transwell chambers were observed and photographed under a microscope at 400x magnification, with 3 fields of view for each group. The results showed that after drug treatment, the cell invasion ability of U87-MG cells was significantly reduced in the Dox, Lipo@Dox and LRK-Lipo@Dox groups compared with the control group, and the Dox group > Lipo@Dox group > LRK-Lipo@Dox group. Figure 17 This indicates that the targeted binding of LRK makes the inhibitory effect of Lipo@Dox on cell invasion more significant.

[0111] 5) U87-MG cell migration assay

[0112] The following groups were selected: control, Dox, liposome-Dox (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. Melt 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 using serum-free medium and operate on ice. Add 800 μL of pre-chilled 10% FBSMEM medium (containing antibiotics) at 4°C to the 24-well plate, place it in the Transwell chamber, and vertically add 100 μL of Matrigel to the center of the bottom of the upper chamber of the Transwell chamber. Incubate at 37°C for 4–5 hours until dry. After the Matrigel dried to a gel-like consistency, 200 μL of cell suspension from each group was inoculated into the upper chamber of a Transwell and incubated at 37°C with 5% CO2 for 24 hours. The Transwell was then removed, and the chambers 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, incubated at room temperature for 20 minutes, washed once with PBS, and any unmigrated cells on one side of the upper chamber were wiped clean with a clean cotton ball. The Transwell chambers were then observed and photographed under a microscope at 400x magnification, with three fields of view for each group. The results showed that, compared with the control group, the migration ability of U87-MG cells treated with the drug was significantly reduced in the Dox, Lipo@Dox, and LRK-Lipo@Dox groups, and the Dox group > Lipo@Dox group > LRK-Lipo@Dox group. Figure 18 This indicates that the targeted binding of LRK makes the inhibitory effect of Lipo@Dox on cell migration more significant.

[0113] 6) U87-MG cell cloning experiment

[0114] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), and LRK-modified liposome-Dox group (LRK-Lipo@Dox), with an effective Dox concentration gradient of 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 alcohol 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 number of surviving cell clones in U87-MG cells was significantly reduced in the Dox, Lipo@Dox, and LRK-Lipo@Dox groups compared to the control group, and the Dox group > Lipo@Dox group > LRK-Lipo@Dox group. Figure 19 This indicates that the targeted binding of LRK makes the inhibitory effect of Lipo@Dox on cell clone survival more significant.

[0115] Example 8: Experiment on cross-blood-brain barrier of peptide-modified doxorubicin liposomes

[0116] 1) U87-MG cells take up peptide-modified doxorubicin liposomes

[0117] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), and LRK-modified liposome-Dox group (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, and LRK-Lipo@Dox (with an effective Dox concentration of 5 μg / mL and 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 cell 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, both the Lipo@Dox and LRK-Lipo@Dox groups exhibited Cy5.5 fluorescence signals compared to the control and Dox groups, indicating that both drugs could penetrate HUVEC cells and be taken up by U87-MG cells. Furthermore, due to the targeted binding of LRK, cells were able to take up more Lipo@Dox. Figure 20 ).

[0118] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0119] 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.

[0120] 3) Experiment on cross-blood-brain barrier of peptide-modified doxorubicin liposomes

[0121] Tumor-bearing nude mice were injected via tail vein with Cy5.5-modified liposome-Dox from Example 5.

[0122] Lipo@Dox and Cy5.5 and LRK-modified liposome-Dox (LRK-Lipo@Dox), with an effective Dox concentration of 2.5 mg / kg, were administered. Images were acquired using a small animal fluorescence in vivo imaging system at 2, 4, 8, and 24 hours post-injection. Nude mice without U87-MG cells were intravenously injected with the same volume of PBS as the drug. Results showed that the brains of nude mice in both the Lipo@Dox and LRK-Lipo@Dox groups continuously displayed fluorescent signals. Both Lipo@Dox and LRK-Lipo@Dox crossed the blood-brain barrier, and the fluorescence signal at each time point was significantly higher in the LRK-Lipo@Dox group than in the Lipo@Dox group, indicating that the targeted binding of LRK resulted in higher efficiency of drug uptake in the brain. Figure 21 Twenty-four hours after injection, the organs and brains of mice 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 greater in the LRK-Lipo@Dox group than in the Lipo@Dox group. Figure 22 This indicates that the targeting effect of LRK makes the retention of Lipo@Dox in the brain more significant.

[0123] Example 9: Experimental study of peptide-modified doxorubicin liposomes for the treatment of glioblastoma.

[0124] 1) Drug administration and tumor observation

[0125] The experiment was divided into three groups: PBS group, Dox group, Cy5.5-modified liposome-Dox (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). The results of nude mice treated on day 15 showed that, compared with the PBS and Dox groups, Lipo@Dox and LRK-Lipo@Dox significantly inhibited tumor growth at all time points. Figure 23 (A, B) and LRK-Lipo@Dox showed a more significant inhibitory effect on tumor growth, indicating that the targeted binding of LRK leads to higher efficiency in drug uptake by the tumor. After 15 days of treatment, the nude mice showed minimal weight change, indicating that the peptide-modified doxorubicin liposomes had low systemic toxicity in nude mice. Figure 23 C).

[0126] 2) Paraffin slice preparation

[0127] 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.

[0128] 3) H&E staining

[0129] 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, they were mounted with neutral resin, and images were acquired using Pannoramic MIDI scanning. The results showed that, compared with the control group and the Dox group, the outlines of the xenografts in the nude mouse brains of both the Lipo@Dox and LRK-Lipo@Dox groups were reduced, indicating that both Lipo@Dox and LRK-Lipo@Dox could inhibit xenograft growth. Figure 24 ).

[0130] 4) TUNEL Experiment

[0131] 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 both Lipo@Dox and LRK-Lipo@Dox induced apoptosis in U87-MG cells, with the number of apoptotic cells exceeding that in the LRK-Lipo@Dox group. This indicates that LRK's targeted binding effect resulted in a higher efficiency in inducing apoptosis in U87-MG cells. Figure 25 ).

[0132] 5) Detect Ki-67 expression

[0133] 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 PannoramicMIDI scanning slides. 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 both Lipo@Dox and LRK-Lipo@Dox inhibited Ki67 expression in U87-MG cells. The number of Ki67 cells was lower in the LRK-Lipo@Dox group than in the Lipo@Dox group, indicating that LRK targeting and binding resulted in a higher efficiency in inhibiting Ki67 expression in U87-MG cells (see...). Figure 26 ).

[0134] 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 polypeptide targeting the TREM2 protein, the amino acid sequence of which is shown below: LRKLRLRL, leucine-arginine-lysine-leucine-arginine-leucine-arginine-leucine, Leu-Arg-Lys-Leu-Arg-Leu-Arg-Leu, abbreviated as LRK.

2. The use of the peptide targeting TREM2 protein as described in claim 1 in the preparation of a medicament for targeted treatment of glioblastoma.

3. A drug for targeted therapy of glioblastoma, comprising a glioblastoma-targeting peptide LRK, a drug with killing effects on glioblastoma cells, and liposomes; wherein, The targeting peptide LRK is modified onto the liposomes, and the liposomes modified with peptide LRK encapsulate the drug. The amino acid sequence of the targeting peptide LRK is as follows: LRKLRLRL, leucine-arginine-lysine-leucine-arginine-leucine-arginine-leucine.

4. The drug for targeted treatment of glioblastoma according to claim 3, characterized in that: The drug that has a killing effect on glioblastoma cells is doxorubicin; The liposomes are made from lecithin and DSPE-PEG2000.

5. A method for preparing the drug for targeted treatment of glioblastoma as described in claim 3 or 4, comprising the following steps: 1) reacting DSPE-PEG2000-Mal with polypeptide LRK to obtain DSPE-PEG2000-LRK; 2) preparing a liposome solution using lecithin and DSPE-PEG2000-LRK as raw materials via an ammonium sulfate gradient method; 3) adding a drug with killing effect on glioblastoma cells to the obtained liposome solution and incubating to obtain the drug.

6. The method according to claim 5, characterized in that: In step 1), the molar ratio of DSPE-PEG2000-Mal to peptide LRK is 1:1.5-2; The reaction is carried out in water under inert gas protection for 24-48 hours.

7. The method according to claim 5, characterized in that: Step 2) involves dissolving lecithin and DSPE-PEG2000-LRK 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-LRK 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 pH 7.4 PBS.

8. The method according to claim 5, 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.

9. A liposome modified with peptide LRK, wherein the amino acid sequence of peptide LRK is as follows: LRKLRLRL, leucine-arginine-lysine-leucine-arginine-leucine-arginine-leucine, Leu-Arg-Lys-Leu-Arg-Leu-Arg-Leu.

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