A liposome drug delivery system for targeting glioblastoma and its preparation method and application

Abstract

The application discloses a liposome drug delivery system for targeted treatment of glioblastoma and a preparation method and application thereof. The liposome drug delivery system comprises a polypeptide for binding to a target ANGPT2 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 target ANGPT2 protein has an amino acid sequence as shown in the following formula: GSFIHSVPRH. The polypeptide for binding to the target ANGPT2 protein is modified on the liposome, and the liposome modified with the polypeptide wraps the drug. The polypeptide modified liposome drug delivery system has strong targeting specificity, small toxic and side effects, and is applied to GBM treatment, and has good research value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a liposome delivery system for targeted treatment of glioblastoma, its preparation method, and its 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] 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 and altering the structure of liposomes and 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. Summary of the Invention

[0004] To address practical problems in the treatment of glioblastoma, this invention develops a liposomal drug delivery system for targeted therapy of glioblastoma and its preparation method. By modifying the surface of liposomes with angiopoietin 2 (ANGPT2) targeting peptides, the drug's targeting specific glioblastoma is enhanced. Adriamycin is encapsulated in liposomes capable of crossing the blood-brain barrier, increasing the efficiency of adriamycin reaching the glioma and reducing its cytotoxic effects 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.

[0005] The purpose of this invention is to provide a liposome delivery system for targeted treatment of glioblastoma and its preparation method.

[0006] The liposome delivery system for targeted treatment of glioblastoma provided by the present invention includes a polypeptide for binding to the ANGPT2 protein of glioblastoma, a drug with a killing effect on glioblastoma cells, and a liposome encapsulating the drug.

[0007] The polypeptide targeting the ANGPT2 protein has the following amino acid sequence: GSFIHSVPRH (glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His), abbreviated as GSF.

[0008] The peptide targeting the ANGPT2 protein is modified onto the liposomes, and the peptide-modified liposomes encapsulate the drug.

[0009] The drug is one that has a killing effect on glioblastoma cells, specifically doxorubicin;

[0010] The liposomes are made from lecithin and DSPE-PEG2000.

[0011] The above-mentioned liposome delivery system for targeted therapy of glioblastoma was prepared by a method including the following steps:

[0012] 1) DSPE-PEG2000-NSH was reacted with peptide GSF under alkaline conditions to obtain DSPE-PEG2000-GSF;

[0013] 2) Liposome solutions were prepared using lecithin and DSPE-PEG2000-GSF as raw materials via an ammonium sulfate gradient method;

[0014] 3) Add a drug that kills glioblastoma cells to the obtained liposome solution, incubate, and the product is obtained.

[0015] In step 1) of the above method, the molar ratio of DSPE-PEG2000-NSH to peptide GSF is 1-2:3, specifically 1.1:3;

[0016] The reaction was carried out in DMF at room temperature for 12-18 hours.

[0017] The operation of step 2) of the above method is as follows: lecithin and DSPE-PEG2000-GSF 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;

[0018] The mass ratio of lecithin to DSPE-PEG2000-GSF can be 6-9:1, specifically 9:1.

[0019] The concentration of the (NH4)2SO4 solution can be 250 mM.

[0020] The ratio of lecithin to (NH4)2SO4 solution can be 18 mg: 5 mL;

[0021] The dialysis was performed overnight in 50 mM pH 7.4 PBS.

[0022] In step 3) of the above method, the drug may specifically be doxorubicin;

[0023] The mass ratio of lecithin to the drug may be 18 mg: 1 mg;

[0024] The incubation process involves incubating at 50°C for 1-2 hours.

[0025] After incubation, dialysis is performed to remove any unencapsulated drugs.

[0026] The present invention also provides the application of the above-mentioned liposome delivery system for targeted therapy of glioblastoma in the preparation of drugs for treating human glioma or human glioma cell xenografts.

[0027] In the aforementioned application, the human glioma cells may specifically be U87-MG cells.

[0028] 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.

[0029] Compared with existing technologies, the liposome delivery system prepared by this invention has the following advantages:

[0030] 1) The liposome delivery system prepared in this invention can cross the blood-brain barrier and has a certain degree of active targeting. After binding with the targeting peptide, it enhances the targeting effect through synergistic effect and can target glioblastoma cells.

[0031] 2) The liposome drug delivery system prepared in this invention achieves active drug delivery by linking to a targeting peptide. 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.

[0032] This invention applies a peptide-modified liposome drug delivery system with strong targeting specificity and low toxicity to the treatment of GBM, which has good research value and application prospects. Attached Figure Description

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

[0034] Figure 2 The results are HPLC and MS identification of the polypeptide GSF obtained in Example 1 of this invention.

[0035] Figure 3 This is the structural formula of the polypeptide HSV in Example 1 of the present invention.

[0036] Figure 4 The results are HPLC and MS identification of the polypeptide HSV obtained in Example 1 of this invention.

[0037] Figure 5 The synthesis route and structural formula of DSPE-PEG2000-GSF in Example 1 of this invention are shown.

[0038] Figure 6 The synthesis route and structural formula of DSPE-PEG2000-HSV in Example 1 of this invention are shown.

[0039] Figure 7 This is the NMR spectrum of DSPE-PEG2000-GSF in Embodiment 1 of the present invention.

[0040] Figure 8 The NMR spectrum of DSPE-PEG2000-HSV in Embodiment 1 of the present invention is shown.

[0041] Figure 9 Performance characterization of the polypeptide-modified doxorubicin liposomes in Example 2 of this invention. A. Particle size; B. PDI; C. Zeta potential; D. and E. Morphology analyzed by transmission electron microscopy; F. Ultraviolet absorption wavelength.

[0042] Figure 10 The results show the effect of peptide-modified doxorubicin liposomes on cell viability in Example 3 of this invention. The groups are: ADox group; B Lipo@Dox group; C HSV-Lipo@Dox group; GSF-Lipo@Dox group.

[0043] Figure 11 The effect of peptide-modified doxorubicin liposomes on the apoptosis rate of U87-MG cells in Example 3 of this invention is shown. *p<0.05.

[0044] Figure 12 The results of flow cytometry analysis of Cy5.5 fluorescence intensity after co-culturing bEND.3 and U87-MG cells in Example 4 of this invention were used to analyze the uptake of peptide-modified doxorubicin liposomes by U87-MG cells.

[0045] Figure 13The results of flow cytometry analysis of Cy5.5 fluorescence intensity after co-culturing HUVEC and U87-MG cells in Example 4 of this invention were used to analyze the uptake of peptide-modified doxorubicin liposomes by U87-MG cells.

[0046] Figure 14 The results of flow cytometry analysis of Dox fluorescence intensity after co-culturing bEND.3 and U87-MG cells in Example 4 of this invention were used to analyze the uptake of peptide-modified doxorubicin liposomes by U87-MG cells.

[0047] Figure 15 The results of flow cytometry analysis of Dox fluorescence intensity after co-culturing HUVEC and U87-MG cells in Example 4 of this invention were used to analyze the uptake of peptide-modified doxorubicin liposomes by U87-MG cells.

[0048] Figure 16 In Example 4 of this invention, the Dox fluorescence intensity was detected by immunofluorescence after co-culturing bEND.3 and U87-MG cells to analyze the uptake of peptide-modified doxorubicin liposomes by U87-MG cells. A. Immunofluorescence detection comparison; B. Fluorescence intensity analysis; *p<0.05, # p < 0.001.

[0049] Figure 17 This invention, in Example 4, uses immunofluorescence to detect the Dox fluorescence intensity after co-culturing HUVECs and U87-MG cells, analyzing the uptake of peptide-modified doxorubicin liposomes by U87-MG cells. A. Immunofluorescence detection comparison; B. Fluorescence intensity analysis; # p < 0.001.

[0050] Figure 18 This diagram 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 4 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. *p<0.05.

[0051] Figure 19 This illustrates the uptake of peptide-modified doxorubicin liposomes in the brain 30 hours after administration in Example 4 of this invention. A. Fluorescence imaging of the organs of nude mice after uptake of peptide-modified doxorubicin liposomes; B. Fluorescence intensity analysis of the brain of nude mice after uptake of peptide-modified doxorubicin liposomes.

[0052] Figure 20 The results show the effect of peptide-modified doxorubicin liposomes on the invasive ability of U87-MG cells in Example 5 of this invention. *p<0.05, **p<0.01.

[0053] Figure 21The results show the effect of peptide-modified doxorubicin liposomes on the migration ability of U87-MG cells in Example 5 of this invention. *p<0.05, **p<0.01.

[0054] Figure 22 The results show the effect of peptide-modified doxorubicin liposomes on the survival of U87-MG cell clones in Example 3 of this invention. *p<0.05.

[0055] Figure 23 This document describes the treatment of nude mice with orthotopic xenografts using peptide-modified doxorubicin liposomes in Example 5 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) Changes in body weight of nude mice at different time points. *p<0.05.

[0056] Figure 24 H&E staining observation of peptide-modified doxorubicin liposomes used in Example 5 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 5 of this invention for the treatment of nude mouse orthotopic xenograft tumors.

[0058] Figure 26 This invention provides an example of observing the expression of Ki67 in nude mice after treatment with orthotopic xenograft tumors using peptide-modified doxorubicin liposomes via immunofluorescence detection, as described in Example 5 of this invention. 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: Synthesis of DSPE-PEG2000-GSF / HSV

[0062] 1) Artificial synthesis of GSF

[0063] The artificially synthesized polypeptide has the following sequence: glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His, abbreviated as GSFIHSVPRH, or GSF. The structural formula is shown below. Figure 1 ;

[0064] Fmoc-His(Boc)-Wang Resin resin with a substitution degree of 0.35 mmol / g was selected, and after swelling, the Fmoc protecting groups were removed. Coupling was performed sequentially from the C-terminus to the N-terminus according to the peptide sequence, up to Gly. Small samples were cut to verify the correctness of the peptide. The side-chain protecting groups for Ser, His, and Arg were tBu, Boc, and pbf, respectively. All amino acids had their α-amino groups protected with Fmoc. The linear peptide was reacted with the linear peptide resin using a lysis buffer (volume ratio of trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2) to obtain linear peptides with all side-chain protecting groups removed. The linear peptides were dissolved in water and purified using semi-preparative chromatography. The liquid with acceptable purity was separated, collected, and lyophilized to obtain the target peptide. The HPLC and MS identification results of GSF are shown in the figure. Figure 2 .

[0065] 2) Artificially synthesized HSV

[0066] HSV is another polypeptide that targets the ANGPT2 protein. Its amino acid sequence is shown below: HSVPRHEV (histidine-serine-valine-proline-arginine-histidine-glutamic acid-valine, His-Ser-Val-Pro-Arg-His-Glu-Val), abbreviated as HSV. The structural formula is shown below. Figure 3 ;

[0067] Fmoc-His(Boc)-Wang Resin resin with a substitution degree of 0.35 mmol / g was selected, and after swelling, the Fmoc protecting groups were removed. Coupling was performed sequentially from the C-terminus to the N-terminus according to the peptide sequence, up to His. Small samples were cut to verify the correctness of the peptide. The side-chain protecting groups for Ser, His, Arg, and Glu were tBu, Boc, pbf, and OtBu, respectively. All amino acids had their α-amino groups protected with Fmoc. The linear peptide was reacted with the linear peptide resin using a lysis buffer (volume ratio of trifluoroacetic acid: ethylenedithiol: phenol: triisopropylsilane: water = 90:4:2:2:2) to obtain linear peptides with all side-chain protecting groups removed. The linear peptides were dissolved in water and purified using semi-preparative chromatography. The liquid with acceptable purity was separated, collected, and lyophilized to obtain the target peptide. The HPLC and MS identification results of HSV are shown in the figure. Figure 4 .

[0068] 3) Synthesis of DSPE-PEG2000-GSF / HSV

[0069] The synthetic route and structural formula of DSPE-PEG2000-GSF / HSV are shown below. Figure 5 , Figure 6 100 mg of DSPE-PEG2000-NHS was dissolved in 3 mL of DMF. 129 mg of GSF / HSV PRH and 109 mg of HSV / HSV PRHEV, along with 3 molar volumes of triethylamine, were added and dissolved completely. The reaction mixture was allowed to react at room temperature for 12 hours. The reaction solution was then transferred to a dialysis bag (molecular weight cutoff 1500 Da) and dialyzed in pure water for 24 hours. The dialysate was collected and freeze-dried to obtain the product. The yield was calculated by comparing the NMR spectra of the peptide and DSPE-PEG2000-GSF / HSV. Figure 7 , 8 ).

[0070] Example 2: Preparation of peptide-modified doxorubicin liposomes

[0071] 1) Liposome-encapsulated drug Dox

[0072] 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.

[0073] 3) Liposome-encapsulated drug Dox, surface modified with GSF / HSV

[0074] Weigh 18 mg of lecithin and 2 mg of DSPE-PEG2000-GSF / HSV, dissolve them separately in 5 mL of chloroform, and 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 in a nanodialysis device 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 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. Characterization of the performance of peptide-modified doxorubicin liposomes is shown in [the table below]. Figure 9 .

[0075] 3) Liposome-encapsulated drug Dox, surface-modified peptides, and Cy5.5

[0076] Weigh 35 mg of lecithin, 1 mg of DSPE-PEG200-GSF / HSV, 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 with a 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. Measure the absorbance of the solution at 480 nm and determine the concentration based on the doxorubicin standard absorption curve.

[0077] Example 3: Cellular Detection of Drug Delivery Systems

[0078] 1) Cell Culture and Processing

[0079] bEND.3, HUVEC, and 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. bEND.3 and U87-MG cells were resuspended in DMEM complete medium containing 10% fetal bovine serum, and HUVEC cells were resuspended in DMEM complete medium containing 10% fetal bovine serum. The cells were seeded into culture dishes, gently mixed by pipetting, and cultured at 37°C under saturated humidity of 5% CO2.

[0080] 2) Cell viability experiment

[0081] Cell viability assays were performed using the CCK-8 assay. The assays included Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox (HSV-Lipo@Dox), and GSF-modified liposome-Dox (GSF-Lipo@Dox). The Dox concentrations were set at 0.1, 0.2, 1, 2, and 5 μg / mL; the Lipo@Dox concentrations were set at 0.1, 0.2, 0.5, 0.8, and 1 μg / mL; and the peptide-modified Lipo@Dox concentrations were set at 0.01, 0.02, 0.08, 0.2, and 0.5 μg / mL. Each assay was performed in 5 replicates, with 3000 cells in the logarithmic growth phase seeded in each well. Observe cell status. When confluence reaches 50-60%, dilute the test drug to different concentrations with culture medium beforehand. After removing the culture medium, add 100 μL of culture medium containing different drug concentrations to each well. After culturing for 48 hours, add 10 μL of CCK-8 solution to each well and incubate for 4 hours. Place the 96-well plate in a microplate reader and measure the absorbance at 450 nm. Cell viability is calculated using the formula [A(sample well) – A(blank)] / [A(PBS) – A(blank)] × 100%. The results show that after 24 hours of drug treatment, Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox inhibited U87-MG cell viability in a dose-dependent manner. The IC50 of the peptide-modified Lipo@Dox group was significantly higher. 50 The cytotoxicity of Lipo@Dox to glioma cells was lower than that of the Dox and Lipo@Dox groups, and the cytotoxicity of Lipo@Dox to bEND.3 and HUVEC cells was lower than that to U87-MG cells. This indicates that the peptide-modified Lipo@Dox reduced the cytotoxicity of Dox to normal cells and increased the cytotoxicity of Dox to glioma cells due to its targeting effect. Figure 10 ).

[0082] 3) U87-MG cell apoptosis experiment

[0083] The study included control, Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox group (HSV-Lipo@Dox), and GSF-modified liposome-Dox group (GSF-Lipo@Dox), with effective Dox concentration gradients of 0.1, 0.5, and 1 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, HSV-Lipo@Dox, and GSF-Lipo@Dox induced apoptosis in U87-MG cells in a dose-dependent manner. Furthermore, the apoptosis rate in the peptide-modified Lipo@Dox group was higher than that in the Lipo@Dox group, indicating that the targeting effect of peptide-modified Lipo@Dox enabled glioma cells to take up Dox more efficiently, resulting in a stronger ability to induce apoptosis in glioma cells. Figure 11 Furthermore, because GSF's targeting binding activity is superior to HSV, the GSF-Lipo@Dox group exhibited the highest apoptosis rate. Figure 11 ).

[0084] Example 4: Experiment on the crossing of the blood-brain barrier by peptide-modified doxorubicin liposomes

[0085] 1) U87-MG cells take up peptide-modified doxorubicin liposomes

[0086] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox group (HSV-Lipo@Dox), and GSF-modified liposome-Dox group (GSF-Lipo@Dox), with an effective Dox concentration of 1 μg / mL. bEND.3 or HUVEC and U87-MG cells were seeded in the upper and lower chambers of a Transwell at a ratio of 1:5 (with coverslips), and cultured for 48 hours to simulate the blood-brain barrier and blood-brain tumor barrier. The bEND.3 or HUVEC cell culture medium in the upper chamber of the Transwell was aspirated, and Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox with an effective Dox concentration of 1 μg / mL containing 10% FBS were added. The cells were incubated at 37°C, and coverslips from the lower chamber were collected at 4 and 8 hours, respectively. Samples for laser confocal microscopy 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 cell nuclei. Finally, the samples were mounted with an anti-quenching agent, photographed using a laser confocal microscope, and their fluorescence density was analyzed using ImageJ software. Samples for flow cytometry were washed three times with pre-cooled PBS, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were washed twice with PBS, and resuspended in 1 mL PBS. The fluorescence intensity of Cy5.5 and Dox was detected by flow cytometry. The results showed that, after co-culture and drug treatment, compared with the control and Dox groups, Lipo@Dox, GSF-Lipo@Dox, and HSV-Lipo@Dox showed significantly higher fluorescence intensity. and GSF-Lipo@ All DOX groups can display Cy5.5 The fluorescence signals of Lipo@Dox were higher than those of HSV-Lipo@Dox, and the fluorescence signals of GSF-Lipo@Dox were higher than those of HSV-Lipo@Dox. Figure 12 , 13 The results (14, 15) indicate that all three drug groups can penetrate bEND.3 and HUVEC cells and be taken up by U87-MG cells; in addition, due to the targeted binding of HSV and GSF, cells are able to take up more Lipo@Dox, and the targeted binding of GSF is superior to that of HSV. Therefore, the fluorescence signal of the GSF-Lipo@Dox group is the strongest. Figure 16 , 17 ).

[0087] 2) Establishment of U87-MG cell orthotopic tumor animal model

[0088] 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.

[0089] 3) Experiment on cross-blood-brain barrier of peptide-modified doxorubicin liposomes

[0090] Tumor-bearing nude mice were injected via tail vein with Cy5.5-modified liposome-Dox from Example 5.

[0091] Lipo@Dox, Cy5.5 and GSF modified liposome-Dox (GSF-Lipo@Dox), and Cy5.5 and HSV modified liposome-Dox (HSV-Lipo@Dox) were administered via saline injection. The effective concentration of Dox was 5 mg / kg. Images were acquired using a small animal fluorescence in vivo imaging system at 2, 4, 8, and 30 hours post-injection. Figure 18 Nude mice not inoculated with 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, HSV-Lipo@Dox, and GSF-Lipo@Dox groups continuously displayed fluorescent signals, and the fluorescence signal at each time point was GSF-Lipo@Dox group > HSV-Lipo@Dox group > Lipo@Dox group. Figure 18 This indicates that the targeted binding of HSV and GSF leads to higher efficiency of drug uptake in the brain. Thirty hours after injection, mouse organs and brains were isolated, and images were acquired using a small animal fluorescence in vivo imaging system. The results showed that the brains of tumor-bearing mice in the GSF-Lipo@Dox group still exhibited strong fluorescence signals. Figure 19 This indicates that GSF-Lipo@Dox has a longer retention time in the in situ tumor because its targeting binding effect is superior to that of HSV.

[0092] Example 5: Experimental study of peptide-modified doxorubicin liposomes for the treatment of glioma.

[0093] 1) U87-MG cell invasion assay

[0094] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox group (HSV-Lipo@Dox), and GSF-modified liposome-Dox group (GSF-Lipo@Dox), with the effective concentration of Dox set at 1 μg / mL. U87-MG cells in logarithmic growth phase were treated with Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox for 24 hours, washed three times with PBS, digested with 0.25% trypsin, centrifuged at 1000 rpm / min for 5 min, discarded the supernatant, washed twice with PBS to remove residual serum, resuspended in serum-free DMEM medium, and counted using a cell counting chamber. 800 μL of the solution was added to each well of a 24-well plate beforehand. Cells were cultured in DMEM medium (containing antibiotics) with 10% FBS and placed in Transwell chambers. One hour later, 200 μL of cell suspension containing 30,000 cells from each group was inoculated into the upper chamber of the Transwell. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The Transwell chambers were then removed, carefully washed once with PBS, and fixed with 70% ice-cold ethanol solution for 1 hour. Cells were stained with 0.5% crystal violet and incubated at room temperature for 20 minutes. After washing with PBS, 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 three fields of view for each group. The results showed that, after drug treatment, the invasive ability of U87-MG cells was significantly reduced in the Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox groups compared to the control group, with the order being: Dox > Lipo@Dox > HSV-Lipo@Dox > GSF-Lipo@Dox. Figure 20 This indicates that the targeted binding of HSV and GSF enables cells to take up more Lipo@Dox; GSF's targeted binding is superior to that of HSV, therefore, the GSF-Lipo@Dox group has the weakest cell invasion ability.

[0095] 2) U87-MG cell migration assay

[0096] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox group (HSV-Lipo@Dox), and GSF-modified liposome-Dox group (GSF-Lipo@Dox), with the effective concentration of Dox set at 1 μg / mL. U87-MG cells in logarithmic growth phase were treated with Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox for 24 hours, washed three times with PBS, digested with 0.25% trypsin, centrifuged at 1000 rpm / min for 5 min, discarded the supernatant, washed twice with PBS to remove residual serum, resuspended in serum-free DMEM medium, and counted using a cell counting chamber. 800 μL of the solution was added to each well of a 24-well plate beforehand. Cells were cultured in DMEM medium (containing antibiotics) with 10% FBS and placed in Transwell chambers. One hour later, 200 μL of cell suspension containing 30,000 cells from each group was inoculated into the upper chamber of the Transwell. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The Transwell chambers were then removed, carefully washed once with PBS, and fixed with 70% ice-cold ethanol solution for 1 hour. Cells were stained with 0.5% crystal violet and incubated at room temperature for 20 minutes. After washing with PBS, 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 three fields of view for each group. The results showed that, after drug treatment, the migration ability of U87-MG cells was significantly reduced in the Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox groups compared to the control group. Furthermore, the order of Dox > Lipo@Dox > HSV-Lipo@Dox > GSF-Lipo@Dox was significantly lower. Figure 21 This indicates that the targeted binding of HSV and GSF enables cells to take up more Lipo@Dox; GSF's targeted binding is superior to that of HSV, therefore, the GSF-Lipo@Dox group has the weakest cell migration ability.

[0097] 3) U87-MG cell cloning experiment

[0098] The following groups were set up: control, Dox, liposome-Dox (Lipo@Dox), HSV-modified liposome-Dox group (HSV-Lipo@Dox), and GSF-modified liposome-Dox group (GSF-Lipo@Dox), with an effective Dox concentration of 1 μg / mL. U87-MG cells in logarithmic growth phase were treated with Dox, Lipo@Dox, HSV-Lipo@Dox, and GSF-Lipo@Dox for 24 hours, washed three times with PBS, digested with 0.25% trypsin, collected, centrifuged at 1000 rpm / min for 5 min, supernatant discarded, washed twice with PBS to remove residual serum, resuspended in serum-free DMEM, counted using a cell counting chamber, and seeded at a density of 1300 cells / mL in 35 mm diameter culture dishes. 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, the cells were washed twice with PBS, and fixed with 75% ethanol for 15 min. Discard the fixative and stain with 0.5% crystal violet for 30 min. Wash with PBS and count the cells. Calculation formula: Colony formation rate = number of clones / number of seeded cells × 100%. The results showed that after drug treatment, compared with the control group, the cell clone survival of U87-MG cells in the Dox, Lipo@Dox, HSV-Lipo@Dox and GSF-Lipo@Dox groups was significantly reduced, and the order was: Dox group > Lipo@Dox group > HSV-Lipo@Dox group > GSF-Lipo@Dox group. Figure 22 This indicates that the targeted binding of HSV and GSF enables cells to take up more Lipo@Dox; GSF's targeted binding is superior to that of HSV, therefore, the GSF-Lipo@Dox group had the least cell survival.

[0099] 4) Drug administration and tumor observation

[0100] The experiment was divided into four groups: PBS group, Dox group, Cy5.5 modified liposome-Dox (Lipo@Dox), Cy5.5 and GSF modified liposome-Dox (GSF-Lipo@Dox), and Cy5.5 and HSV modified liposome-Dox (HSV-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 23A). The results showed that, compared with the PBS group and the Dox group, GSF-Lipo@Dox and HSV-Lipo@Dox significantly inhibited tumor growth starting from day 6, with GSF-Lipo@Dox exhibiting the strongest inhibitory effect. Figure 23 B). GSF-Lipo@Dox and HSV-Lipo@Dox significantly increased the body weight of nude mice. Figure 23 C) indicates that the therapeutic effect of peptide-modified doxorubicin liposomes led to an increase in body weight in nude mice.

[0101] 5) Paraffin slice preparation

[0102] 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.

[0103] 6) H&E staining

[0104] 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, GSF-Lipo@Dox, and HSV-Lipo@Dox groups were all reduced, indicating that Lipo@Dox, GSF-Lipo@Dox, and HSV-Lipo@Dox could all inhibit xenograft growth. Furthermore, because GSF has a better targeting binding effect than HSV, the GSF-Lipo@Dox group showed the most significant inhibitory effect, followed by HSV-Lipo@Dox. Figure 24 ).

[0105] 7) TUNEL Experiment

[0106] 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, GSF-Lipo@Dox, and HSV-Lipo@Dox all induced apoptosis in U87-MG cells, with the number of apoptotic cells being GSF-Lipo@Dox > HSV-Lipo@Dox > Lipo@Dox. This indicates that the targeting binding of HSV and GSF resulted in higher efficiency in inducing apoptosis in U87-MG cells. Figure 25 Furthermore, since GSF has a better targeting binding effect than HSV, the U87-MG cells in the GSF-Lipo@Dox group showed the most significant apoptosis.

[0107] 8) Detect Ki-67 expression

[0108] 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 Pannoramic 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, GSF-Lipo@Dox, and HSV-Lipo@Dox all inhibited Ki67 expression in U87-MG cells, with the number of Ki67 cells being GSF-Lipo@Dox group < HSV-Lipo@Dox group < Lipo@Dox group (…). Figure 26 This indicates that the targeted binding of HSV and GSF is more efficient at inhibiting Ki67 expression in U87-MG cells; and because the targeted binding of GSF is superior to that of HSV, the apoptosis of U87-MG cells in the GSF-Lipo@Dox group is the most obvious.

[0109] 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 liposome delivery system for targeted treatment of glioblastoma, comprising a polypeptide for binding to the ANGPT2 protein of glioblastoma, a drug having a killing effect on glioblastoma cells, and a liposome encapsulating the drug; in, The polypeptide targeting the ANGPT2 protein has the following amino acid sequence: GSFIHSVPRH, glycine-serine-phenylalanine-isoleucine-histidine-serine-valine-proline-arginine-histidine, Gly-Ser-Phe-Ile-His-Ser-Val-Pro-Arg-His, abbreviated as GSF. The peptide targeting the ANGPT2 protein is modified onto the liposomes, and the peptide-modified liposomes encapsulate the drug. The drug is a drug that has a killing effect on glioblastoma cells; The drug in question is doxorubicin; The liposomes are made from lecithin and DSPE-PEG2000. The liposome delivery system for targeted treatment of glioblastoma was prepared by a method comprising the following steps: 1) DSPE-PEG2000-NSH was reacted with peptide GSF under alkaline conditions to obtain DSPE-PEG2000-GSF; 2) Liposome solutions were prepared using lecithin and DSPE-PEG2000-GSF as raw materials via an ammonium sulfate gradient method; 3) Add doxorubicin to the obtained liposome solution and incubate to obtain the final product.

2. The liposome delivery system according to claim 1, characterized in that: In step 1), the molar ratio of DSPE-PEG2000-NSH to peptide GSF is 1-2:3; The reaction was carried out in DMF at room temperature for 12-18 hours.

3. The liposome delivery system according to claim 1, characterized in that: Step 2) involves dissolving lecithin and DSPE-PEG2000-GSF 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-GSF is 6-9:

1. The concentration of the (NH4)2SO4 solution is 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.

4. The liposome delivery system according to claim 1, characterized in that: In step 3), the mass ratio of lecithin to doxorubicin 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.

5. The use of the liposome delivery system for targeted therapy of glioblastoma as described in any one of claims 1-4 in the preparation of a medicament for treating human glioma or human glioma cell xenografts.

6. The application according to claim 5, characterized in that: The human glioma cells mentioned are U87-MG cells.

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