Bionic nano-drug for improving efficacy of radiotherapy and chemotherapy, preparation method and application thereof

Abstract

The application provides a bionic nanodrug for improving the effect of radiotherapy and chemotherapy, a preparation method and application thereof. The bionic nanodrug comprises a shell, a drug carrier, a first active component and a second active component. The drug carrier is wrapped in the shell, and the drug carrier is loaded with the first active component and the second active component. The shell comprises tumor cell membranes. The first active component is an antitumor chemical drug. The second active component is a targeted interference gene. The structural formula of the drug carrier is shown as formula I, wherein n=4-8 and m=5-10. The aforementioned bionic nanodrug can be used as a tumor targeted therapy drug, realizes efficient crossing of a blood-brain barrier by a nanocarrier and precise targeting of brain glioma cells, realizes intratumoral hypoxia response / degradability of a nanosensitizer, reduces neurotoxicity caused by accumulation of the nanosensitizer in normal tissues, has good biocompatibility, realizes radiotherapy and chemotherapy sensitization effect of brain glioma and other tumors for multiple targets, and improves the treatment effect.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, specifically to a biomimetic nanomedicine for improving the efficacy of radiotherapy and chemotherapy, its preparation method, and its application. Background Technology

[0002] Malignant gliomas are highly aggressive brain tumors with a median survival of only 14.6 months. Even after surgical resection, numerous tumor cells remain infiltrating the normal brain parenchyma. Currently, radiotherapy and chemotherapy are the standard and crucial treatments after tumor surgery. However, these residual tumor cells are protected by the blood-brain barrier (BBB) ​​or blood-tumor barrier (BBTB), hindering the delivery of chemotherapeutic drugs and preventing them from reaching effective concentrations within the tumor, leading to chemoresistance. Simultaneously, the presence of normal brain tissue limits the total amount of radiation used, resulting in radioresistance due to tumor cell heterogeneity. Therefore, gliomas are highly prone to recurrence. Although the emergence of nanocarriers has enhanced the ability of drugs to cross the blood-brain barrier and target tumors, traditional nanocarriers cannot realize their full therapeutic potential due to several issues: 1. low drug concentrations within the tumor; 2. ineffective synergistic effect of radiotherapy and chemotherapy in suppressing tumor growth; and 3. strong tumor cell repair capabilities after radiotherapy and chemotherapy.

[0003] Chinese patent CN110101685A discloses a biomimetic nanomedicine, its preparation method, and its application. The invention is characterized in that the nanomedicine comprises a core and a shell covering the core; the core comprises a first component, a second component, and a carrier with sensitive side chains; the first component comprises temozolomide; the second component comprises one or more of cisplatin, lomustine, vincristine, or procarbazine; temozolomide (TMZ) and the second component (one or more of cisplatin, lomustine, vincristine, and procarbazine) are encapsulated on a carrier containing sensitive bonds. TMZ and the second component synergistically treat glioma, which is beneficial for improving therapeutic efficacy and reducing tumor cell drug resistance. The structural formula is:

[0004]

[0005] In this patent, the carrier of the sensitive bond swells rapidly in a weakly acidic environment, efficiently releasing the anticancer drug TMZ and the second component. The carrier encapsulates temozolomide and the second component as the core, with the cancer cell membrane as the outer shell, creating a nanomedicine. The cancer cell membrane possesses a certain degree of self-recognition and highly tumor-selective targeting, "homing" to homologous tumors in the body, significantly prolonging the circulation time of the nanomedicine. The cancer cell membrane, acting as the outer shell, and the carrier of the sensitive bond efficiently release the anticancer drug TMZ and the second component, ultimately killing tumor cells and achieving the goal of targeted synergistic therapy for human glioma. However, this patent simply uses the glioma cell membrane to encapsulate nanoparticles. These nanoparticles only possess the effect of multiple drugs acting simultaneously, failing to leverage the advantages of combined therapy, the tumor-suppressing characteristics of conventional radiotherapy, or address the issue of tumor cell repair after chemotherapy.

[0006] Chinese patent CN107982217B discloses a lipid-polymer with dual functions of targeting and radiosensitization, encapsulating a hydrophobic anticancer drug. The lipid-polymer comprises lipid molecules, a nitroimidazole organic polymer with radiosensitizing properties, and a hydrophobic anticancer drug. The lipid molecules include a DSPE-PEG-target, soybean lecithin, and DSPE-PEG. The nitroimidazole organic polymer with radiosensitizing properties is composed through hydrophilic-hydrophobic self-assembly. The raw materials and their mass percentages in the lipid-polymer are as follows: polynitroimidazole organic polymer, 25-90% by mass; soybean lecithin, 5-50% by mass; DSPE-PEG, 1-40% by mass; and DSPE-PEG-target targeting lipid molecules, 0.1-10% by mass. The hydrophobic anticancer drug is encapsulated in the lipid-polymer nanocarrier via hydrophobic interactions, and the amount of the hydrophobic anticancer drug accounts for 5-20% of the mass of the lipid-polymer nanocarrier. The structural formula is:

[0007]

[0008] This invention utilizes angiopep-2 as a targeting peptide to modify nanoparticles for tumor targeting, resulting in low efficiency. Furthermore, this invention uses a single polymer material, relying solely on hydrophilic-hydrophobic interactions to encapsulate doxorubicin and release it under hypoxic conditions. These nanoparticles are not innovative amphiphilic cationic / anionic polymers, therefore they cannot carry genes for synergistic therapeutic effects, and they do not address the issue of rapid tumor cell repair after radiation damage.

[0009] Therefore, there is an urgent need to provide a biomimetic drug that can increase the concentration of drugs within tumors, exert a synergistic effect with radiotherapy and chemotherapy, reduce tumor cell damage and repair after radiotherapy and chemotherapy, and has high safety. Summary of the Invention

[0010] The purpose of this application is to provide a biomimetic nanomedicine, which aims to improve the problems existing in the prior art and achieve the following effects:

[0011] 1. High drug concentration within the tumor;

[0012] 2. Fully leverage the synergistic effect of radiotherapy and chemotherapy;

[0013] 3. Effectively reduces the problem of tumor cell damage and repair after radiotherapy and chemotherapy;

[0014] 4. The material has good biocompatibility.

[0015] The objective of this invention can be achieved through the following technical solutions:

[0016] The first objective of this invention is to provide a drug carrier, the structure of which is shown in Formula I, wherein n = 4 to 8 and m = 5 to 10.

[0017]

[0018] A second objective of this invention is to provide a method for preparing the aforementioned drug carrier, the method comprising the following steps:

[0019] (1) Dissolve NE-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride (Glu-NCA) in chloroform, and add 4-aminobutyric acid.

[0020] After mixing thoroughly, stir at room temperature until the reactants become a colorless, viscous solution;

[0021] A colorless, viscous solution was poured into anhydrous diethyl ether under stirring, precipitated, filtered, and dried under vacuum to obtain a white fibrous solid. The white fibrous solid comprises compounds with the following structural formulas:

[0022] (2) The white fibrous solid obtained in (1) was completely dissolved in CF3COOH solvent. The mixture was stirred in an ice bath and 33% HBr / AcOH solution with a volume ratio of 5 to 10 was added. After the reaction, the mixture was precipitated in anhydrous diethyl ether and centrifuged to obtain a yellow crude product.

[0023] The yellow crude product was dissolved in DMSO and dialyzed.

[0024] After freeze-drying, the deprotected polymer PGlu-COOH was obtained, and the structural formula of PGlu-COOH is as follows:

[0025] (3) The polymer PGlu-COOH was dissolved in DMF, and EDCI, DMAP, and metronidazole (MIs) were added respectively; the mixture was thoroughly mixed and stirred at room temperature, and dialyzed in deionized water with a molecular weight cutoff of 5000 Da; then freeze-dried to obtain the polymer poly(MIs), the structural formula of which is:

[0026] (4) The polymer poly(MIs) was dissolved in DMF, and EDCI, DMAP and PEI were added respectively. After thorough mixing, the mixture was stirred at room temperature and dialyzed in deionized water with a molecular weight cutoff of 5000 Da. After freeze-drying, the drug carrier poly(MIs)-PEI shown in Formula I was obtained.

[0027] Preferably, during the dialysis process described in (2), (3), and (4), the dialysis fluid is changed every 8 hours.

[0028] Furthermore, in step (1), the molar ratio of NE-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride to the terminal amino group of 4-aminobutyric acid is 30-42:1.

[0029] Furthermore, the molar ratio of NE-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride to the terminal amino group of 4-aminobutyric acid is 40:1 or 42:1.

[0030] Furthermore, the dialysis in step (2) consists of dialysis in deionized water at 5000 Da, dialysis in ammonia water at pH 9.0, dialysis in HCl solution at pH 5.0, and finally dialysis in deionized water.

[0031] Furthermore, in step (3), the molar ratio of polymer PGlu-COOH to EDCI, DMAP and metronidazole is 1-3:50:10:30-42.

[0032] Furthermore, in step (4), the molar ratio of the polymer poly(MIs) to EDCI, DMAP and PEI is 1-5:1.2-6:0.4-2.4:1.

[0033] A third objective of this invention is to provide a biomimetic nanomedicine comprising a shell, a drug carrier, a first active component, and a second active component. The drug carrier is encapsulated within the shell and loads the first and second active components. The shell includes a tumor cell membrane. The first active component is an antitumor chemical drug. The second active component is a gene-targeting interference drug. The drug carrier has the structural formula shown in Formula I, where n = 4–8 and m = 5–10.

[0034]

[0035] The drug carrier shown in Formula I of this invention, as an innovative amphiphilic cationic polymer, can effectively encapsulate chemical drugs such as paclitaxel by exerting hydrophilic and hydrophobic effects. It can also adsorb and target interfering genes using its own positive charge. At the same time, the drug carrier will undergo responsive degradation under hypoxic microenvironment conditions, releasing the carried genes and chemotherapy drugs. It is completely fragmented in tumor tissue and avoids distribution in normal tissue. The overall nano-drug carrier has good biocompatibility.

[0036] The encapsulation of tumor cell membranes endows this biomimetic system with targeting capabilities, and an innovatively synthesized amphiphilic cationic polymer can further enhance the targeting capabilities of this biomimetic system.

[0037] Furthermore, the first active component accounts for 3.66–6.31% of the mass of the biomimetic nanomedicine, and the second active component accounts for 3.65–9.35% of the mass of the biomimetic nanomedicine.

[0038] Furthermore, the outer shell is the membrane of human glioma cells.

[0039] Furthermore, the first active component is encapsulated in a drug carrier. In some more specific instances, the first active component is a hydrophobic antitumor chemical drug; in a more specific embodiment, the first active component is one or more of paclitaxel, doxorubicin, and carmustine.

[0040] Furthermore, the second active component is loaded onto the drug carrier via charge adsorption. In some more specific examples, the second active component is selected from a targeted interference gene that inhibits DNA damage repair; in a more specific embodiment, the second component is siPGK1. The targeted interference gene of the present invention can be a gene sequence commonly used in the art, or it can be synthesized according to conventional methods in the art.

[0041] In some embodiments, the mass ratio of the drug carrier, the first active component, the second active component, and the membrane shell of the present invention is 70.3:3.66:7.35:18.69.

[0042] The fourth objective of this invention is to provide a method for preparing the aforementioned biomimetic nanomedicine, the method comprising the following steps: dissolving a first active component in an organic solvent and mixing it with a drug carrier of Formula I; dialyzing to remove the organic solvent and the free first component to obtain nanoparticles loaded with the first active component; adding a second active component to form nanoparticles simultaneously loaded with the first and second active components; mixing with a tumor cell membrane and extruding to obtain the biomimetic nanomedicine.

[0043] Furthermore, the method includes the following steps:

[0044] (1) Dissolve the first active component in an organic solvent, mix it with the drug carrier shown in Formula I, and then use an ultrasonic cleaner to sonicate at a frequency greater than 40KHz to obtain a drug carrier mixture system loaded with the first active component.

[0045] (2) The drug carrier mixture loaded with the first active component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and deionized water was used as the dialysate to remove the organic solvent and the free first active component, so as to obtain a nanoparticle solution loaded with the first component.

[0046] (3) Add the second active component to the nanoparticle solution loaded with the first component and react to form a nanocomposite loaded with both the first and second active components.

[0047] (4) Dissolve the lysed tumor cell membrane in PBS buffer solution, mix it with the nanocomposite loaded with the first and second active components, then vortex it, and extrude it sequentially and at a constant speed through an aqueous filter with reduced pore size to obtain a biomimetic nanomedicine.

[0048] Preferably, the organic solvent is DMSO, and the pore size of the aqueous phase filter is 600-800.0 nm and 200-400.0 nm, respectively.

[0049] In one particular embodiment, the method includes the following steps:

[0050] (1) Paclitaxel was dissolved in DMSO solution and mixed with the drug carrier shown in Formula I. The mixture was then ultrasonicated for 30 min at a frequency of 50 kHz using an ultrasonic cleaner to obtain a drug carrier mixture system loaded with the first active component (paclitaxel).

[0051] (2) The drug carrier mixture system loaded with the first active component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and deionized water was used as the dialysate to remove organic solvents and free paclitaxel molecules, resulting in a semi-transparent emulsion nanoparticle solution loaded with paclitaxel.

[0052] (3) Add the second active component siPGK1 to the nanoparticle solution and react for 30 min to form a nanocomposite poly(MIs) / PTX@PEI / siPGK1 simultaneously loaded with PTX and siPGK1.

[0053] (4) The lysed human glioma cell membrane (CCM) was dissolved in PBS buffer solution and mixed with the nanocomposite poly(MIs) / PTX@PEI / siPGK1 loaded with PTX and siPGK1. Then, the mixture was vortexed and extruded sequentially and at a constant speed through an aqueous filter with decreasing pore size to obtain the biomimetic nanodrug Poly(MIs) / PTX@PEI / siPGK1@CCM. The pore sizes of the aqueous filter were 800.0 nm and 400.0 nm, respectively.

[0054] A fifth objective of this invention is to provide the application of the aforementioned drug carrier or the aforementioned biomimetic nanomedicine in tumor-targeted therapy drugs.

[0055] Furthermore, the tumor is a glioma.

[0056] This invention innovatively synthesizes a drug carrier responsive to the hypoxic microenvironment of tumors, which is an amphiphilic cationic polymer, and constructs a hypoxia-responsive biomimetic nanomedicine encapsulated in tumor cell membranes (in a specific embodiment, Poly(MIs) / PTX@PEI / siPGK1@CCM), systematically revealing the relationship between its physicochemical properties and functions. Compared with the prior art, the beneficial effects of this invention are as follows:

[0057] (1) By introducing modifications to the tumor cell membrane (e.g., glioma cell membrane), the nanocarriers can efficiently cross the blood-brain barrier (BBB) ​​and precisely target tumor cells (e.g., glioma cells), thus further improving the targeting of the drug carriers of the present invention.

[0058] (2) Innovatively, a drug carrier of nitroimidazole polymer is introduced, which encapsulates the first active component, namely chemotherapy drugs (such as paclitaxel, PTX, cell apoptosis cycle regulators, and radiosensitizers) through hydrophobic interaction. The second active component (such as siPGK1, small interfering RNA targeting phosphoglycerate kinase 1, and ATP generation inhibitors) and other targeted interfering genes are adsorbed through cationic components, forming a dual-drug-loaded nanopolymer system. This system realizes the intratumoral hypoxia response / degradability of the nanosensitizer, reduces the neurotoxicity caused by its accumulation in normal tissues, and has good biocompatibility.

[0059] (3) By simultaneously loading interfering genes such as siPGK1 and chemotherapeutic drugs such as paclitaxel (PTX) onto a nanocarrier, targeting multiple points: First, increasing DNA damage and inhibiting DNA damage repair, with γH2AX expression reaching 80-90% after radiotherapy; Second, promoting cell apoptosis, with Ki67 proliferation index decreasing to 7-12% after radiotherapy; By downregulating PGK1 to 9.5-15%, a radiosensitizing effect on tumors such as gliomas is achieved, improving treatment efficacy. Once this drug delivery system is successfully developed, it will advance the process of improving the synergistic application of radiotherapy and chemotherapy with nanosensitizers, providing a new opportunity for the synergistic treatment of tumors such as gliomas! Attached Figure Description

[0060] Figure 1 The 1H NMR spectrum characterization of the amphiphilic cationic nanocarrier poly(MIs)-PEI is shown.

[0061] Figure 2 The particle sizes and zeta potentials of poly(MIs) / PTX@PEI, poly(MIs)@PEI / siPGK1, and poly(MIs) / PTX@PEI / siPGK1@CCM are shown; among them, Figure 2 A is the particle size distribution diagram. Figure 2 B represents the Zeta potential diagram;

[0062] Figure 3 The hypoxia-responsive characterization of the poly(MIs) / PTX@PEI / siPGK1@CCM system is shown; among which, Figure 3 A represents the poly(MIs)-PEI response release mechanism. Figure 3 B is a transmission electron microscope image. Figure 3 C represents the particle size change under hypoxic conditions;

[0063] Figure 4 Serum stability of the poly(MIs) / PTX@PEI / siPGK1@CCM co-load system was demonstrated;

[0064] Figure 5 Characterization of surface membrane proteins in the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system is shown;

[0065] Figure 6 The ability of poly(MIs) / PTX@PEI / siPGK1@CCM co-load system to complex siPGK1 is shown, wherein, Figure 6 A represents the effect of the N / P ratio in the system on the loading performance of the drug siPGK1. Figure 6 B represents the effect of oxygen concentration on release performance;

[0066] Figure 7 The in vitro release capacity of PTX and siPGK1 in a simulated tumor hypoxic microenvironment was demonstrated;

[0067] Figure 8 The cytotoxicity of the poly(MIs) / PTX@PEI / siPGK1@CCM co-load system was demonstrated;

[0068] Figure 9 A quantitative analysis of the cumulative amount of poly(MIs) / PTX@PEI / siPGK1@CCM crossing the blood-brain barrier and the time is shown;

[0069] Figure 10 This demonstrates the escape of endosomal / lysosomes from the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system;

[0070] Figure 11 The release of poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system into hypoxic cells was demonstrated;

[0071] Figure 12 This demonstrates the silencing of PGK1 expression in the poly(MIs) / PTX@PEI / siPGK1@CCM co-loading system;

[0072] Figure 13 This study demonstrates the use of the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier system to sensitize PTX chemotherapy.

[0073] Figure 14 This study demonstrates the use of the poly(MIs) / PTX@PEI / siPGK1@CCM co-load system for sensitizing radiotherapy.

[0074] Figure 15 The poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier system was shown to inhibit cell colony formation.

[0075] Figure 16 This study demonstrates the synergistic effect of the poly(MIs) / PTX@PEI / siPGK1@CCM co-loading system on sensitizing radiotherapy / chemotherapy.

[0076] Figure 17 The poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier line was shown to inhibit cell proliferation.

[0077] Figure 18 This study demonstrates how the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier system enhances cell apoptosis.

[0078] Figure 19 This study illustrates the in vivo metabolic kinetics of PTX in poly(MIs) / PTX@PEI / siPGK1@CCM;

[0079] Figure 20 This study demonstrates a targeted approach to the poly(MIs) / PTX@PEI / siPGK1@CCM co-loading system, in which... Figure 20 A is a live-in imaging image of the combination of FAM-siPGK1, poly(MIs) / PTX@PEI / FAM-siPGK1@CCM and poly(MIs) / PTX@PEI / FAM-siPGK1@LipoPEG. Figure 20 B shows the organ fluorescence distribution at 8 hours;

[0080] Figure 21 This demonstrates quantitative analysis using fluorescence imaging technology on the poly(MIs) / PTX@PEI / siPGK1@CCM co-load system, where... Figure 21 A represents the in vivo metabolic kinetics of siPGK1. Figure 21 B represents the distribution of siPGK1 in the brain. Figure 21 C represents the distribution of poly(MIs) / PTX@PEI / siPGK1@CCM in the heart, liver, spleen, lungs, and kidneys;

[0081] Figure 22 This study illustrates the distribution of brain tumors using the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system.

[0082] Figure 23 The study of the tumor suppression ability of the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier system was presented, in which... Figure 23 A is a diagram showing the treatment time and plan. Figure 23 B represents the tumor volume after treatment in different groups. Figure 23 C represents a quantitative analysis of tumor volume after treatment in different groups;

[0083] Figure 24 The poly(MIs) / PTX@PEI / siPGK1@CCM and H&E slice characterization after treatment for each group are shown.

[0084] Figure 25 The following is a summary of the TUNEL staining analysis of poly(MIs) / PTX@PEI / siPGK1@CCM and each group after treatment. Figure 25 A shows TUNEL fluorescence staining of brain tumors after different treatment groups. Figure 25 B represents the quantitative analysis of the corresponding TUNEL results;

[0085] Figure 26 The analysis shows the results of γH2AX fluorescence staining after treatment for poly(MIs) / PTX@PEI / siPGK1@CCM and each group. Figure 26 A represents the immunohistochemical staining of γH2AX in brain tumors after different treatment groups. Figure 26 B is the corresponding quantitative analysis of γH2AX;

[0086] Figure 27 The following is an immunohistochemical analysis of poly(MIs) / PTX@PEI / siPGK1@CCM and PGK1 immunohistochemical analysis after treatment in each group. Figure 2 Immunohistochemistry of PGK1 in brain tumors after different treatment groups Figure 27 B is the corresponding quantitative analysis of PGK1;

[0087] Figure 28 The following is an immunohistochemical analysis of poly(MIs) / PTX@PEI / siPGK1@CCM and Ki67 after treatment in each group. Figure 28 Immunohistochemistry of Ki67 in brain tumors after different treatment groups Figure 28 Quantitative analysis of Ki67 corresponding to B

[0088] Figure 29 This study illustrates the relationship between poly(MIs) / PTX@PEI / siPGK1@CCM and post-treatment survival rates in each group.

[0089] Figure 30 This study illustrates the changes in body weight after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM in each group.

[0090] Figure 31 The study of organ toxicity after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM in each group is shown;

[0091] Figure 32 Acute toxicity studies of the poly(MIs) / PTX@PEI / siPGK1@CCM co-loaded system are shown. Detailed Implementation

[0092] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0093] In the following embodiments:

[0094] The CF3COOH refers to trifluoroacetic acid.

[0095] The HBr / AcOH solution refers to: hydrogen bromide acetic acid solution;

[0096] The DMSO refers to dimethyl sulfoxide;

[0097] The HCl mentioned refers to hydrochloric acid.

[0098] The DMF refers to N,N-dimethylamide;

[0099] The EDCI refers to: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0100] The DMAP refers to: 4-dimethylaminopyridine;

[0101] The MIs refer to: metronidazole;

[0102] The PEI refers to polyethyleneimine (source: Sigma-Aldrich, model: 408700, Mw=2000).

[0103] The siPGK1 used in the following examples can be a commonly used gene sequence in the art. For example, in the embodiments of this application, its gene sequence is: sense strand: 5'-CCA AGU CGG UAG UCC UUA UTT-3' (SEQ ID NO.1); antisense strand: 5'-AUA AGG ACU ACC GAC UUG GTT-3' (SEQ ID NO.2); however, it is not limited to the sequence used in this application. Any sequence that can interfere with PGK1 expression is within the scope of protection of this invention. Synthesis can be performed according to conventional siRNA synthesis technology.

[0104] Example 1 Synthesis and characterization of the drug carrier shown in Formula I (hereinafter also referred to as Poly(MIs)@PEI)

[0105]

[0106] In this embodiment, all n=4 and m=5. The preparation process is as follows:

[0107] (1) NE-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride (Glu-NCA, 0.2 mol) was dissolved in chloroform (80 mL), and 4-aminobutyric acid (0.005 mol) was added. After thorough mixing, the mixture was stirred at room temperature for 72 hours, and the reaction product became a colorless viscous solution. While stirring, the reaction product was poured into anhydrous diethyl ether, precipitated, filtered, and dried under vacuum to obtain a white fibrous solid. The reaction formula is as follows:

[0108]

[0109] (2) Weigh 45.0g of the above white fibrous solid product and dissolve it completely in CF3COOH solvent (30mL). Stir under ice bath and add 33% HBr / AcOH solution (150mL). After reacting for 2h, precipitate in anhydrous diethyl ether and centrifuge to obtain yellow crude product (27.5g).

[0110] The yellow crude product was dissolved in DMSO (15 mL), dialyzed in deionized water (5000 Da) for 24 h, dialyzed in ammonia water with pH = 9.0 for 24 h, dialyzed in HCl solution with pH = 5.0 for 24 h, and finally dialyzed in deionized water for 24 h (the dialysate was changed every 8 h during the above dialysis process).

[0111] After freeze-drying, the deprotected polymer PGlu-COOH was obtained. The reaction formula is as follows:

[0112]

[0113] (3) Dissolve 20.0 g of polymer (0.027 mol) PGlu-COOH in DMF (100 mL), add EDCI (0.5 mol), DMAP (0.1 mol), and metronidazole (MIs, 0.42 mol), mix thoroughly, stir at room temperature for 24 hours, dialyze in deionized water (5000 Da) for 72 hours (changing the dialysate every 8 hours). Then freeze-dry to obtain polymer Poly(MIs). The reaction formula is as follows:

[0114]

[0115] (4) Dissolve 32.0 g of polymer Poly(MIs) in DMF (40 mL), add EDCI (0.024 mol), DMAP (0.008 mol), and PEI (0.02 mol), with a molar ratio of poly(MIs) to EDCI, DMAP, and PEI of 1:1.2:0.4:1. After thorough mixing, stir at room temperature for 24 hours, dialyze in deionized water (5000 Da) for 72 hours (changing the dialysate every 8 hours). After freeze-drying, obtain the drug carrier poly(MIs)-PEI as shown in Formula I. The reaction formula is as follows:

[0116]

[0117] This study characterized the structure of poly(MIs)-PEI using proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1As shown. ¹H NMR (300MHz, DMSO-d6, ppm) δ: 8.03 (s, ¹H, MN-H-4), 4.92-5.25 (m, 2H, MN-CH2-), 2.82-2.95 (m, 2H, -N-CH2-), 2.64 (s, 3H, MN-CH3).

[0118] Example 2: Preparation of tumor cell membrane

[0119] The outer shell used in this embodiment is the human glioma cell membrane, which can be extracted in a conventional manner. The preparation method of the human glioma cell membrane in this embodiment is as follows: culture a sufficient amount of human glioma cells (cell line: U87, source: ATCC), aspirate the cell culture medium, add PBS to wash away dead cells, add an appropriate amount of 2mM EDTA to digest for 3min, collect the cell fluid, centrifuge at 4℃ and 140g for 3min, discard the supernatant and keep the precipitate, add cold PBS to resuspend the cells, then centrifuge at 4℃ and 600g for 5min, aspirate the supernatant, centrifuge again at 4℃ and 600g for 1min, and carefully aspirate the remaining supernatant with a pipette. Then, membrane protein extraction solution was added to form a final PMSF concentration of 1 mM. After incubating on ice for 10-15 min, the mixture was repeatedly frozen and thawed 3 times, 30 s each time. Then, it was centrifuged at 4°C and 700g for 10 min to remove cell nuclei and unbroken cells. After centrifugation, the supernatant was carefully aspirated and centrifuged again at 4°C and 14000g for 30 min to precipitate cell membrane fragments. The solid cell membrane fragments were then freeze-dried in liquid nitrogen and stored at -80°C.

[0120] Example 3: Construction and characterization of poly(MIs) / PTX@PEI / siPGK1@CCM sensitizer

[0121] (1) The Poly(MIs)-PEI amphiphilic cationic polymer (i.e. drug carrier, 5 mg) prepared in Example 1 and paclitaxel (PTX, 0.437 mg) were mixed and slowly dripped into DMSO at pH=7.4. The mixture was ultrasonically cleaned at a frequency of 50 kHz for 30 min to finally obtain a drug carrier mixture system loaded with the first active component.

[0122] (2) The drug carrier mixture loaded with the first active component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and deionized water was used as the dialysate to remove organic solvents and free paclitaxel to obtain a Poly(MIs) / PTX@PEI solution, in which the mass percentage of successfully coated PTX was 4.94%.

[0123] (3) Subsequently, siPGK1 (0.52 mg) solution was slowly added to the above mixture, and the mixture was stirred at room temperature for 30 min to obtain the nanomedicine carrier Poly(MIs) / PTX@PEI / siPGK1 loaded with PTX and siPGK1. The hydrated particle sizes of Poly(MIs) / PTX@PEI and Poly(MIs) / PTX@PEI / siPGK1 are as follows: Figure 2 As shown in Figure A, the hydrated particle size of Poly(MIs) / PTX@PEI is 60±3.29 nm, and the hydrated particle size of Poly(MIs) / PTX@PEI / siPGK1 is 90±3.73 nm.

[0124] Example 4: Preparation of biomimetic nanomedicines (hereinafter also referred to as poly(MIs) / PTX@PEI / siPGK1@CCM)

[0125] To prepare poly(MIs) / PTX@PEI / siPGK1@CCM, the glioma cell membrane (1.32 mg) obtained by lysis in Example 2 was dissolved in 1 mL of PBS buffer solution and mixed with poly(MIs) / PTX@PEI / siPGK1 (5.79 mg) prepared in Example 3. The mixture was then vortexed and extruded sequentially and at a constant speed through an aqueous filter (800.0 nm, 400.0 nm) to obtain poly(MIs) / PTX@PEI / siPGK1@CCM.

[0126] poly(MIs) / PTX@PEI / siPGK1@CCM particle size as follows Figure 2 As shown in Figure A, the hydrated particle size is 110 ± 2.83 nm. The surface potential of poly(MIs) / PTX@PEI / siPGK1@CCM is as follows... Figure 2 As shown in B, the ζ potential is -8±1.3mV. The poly(MIs) / PTX@PEI and poly(MIs) / PTX@PEI / siPGK1 prepared in Example 3 both carry positive charges, while the nanoparticles poly(MIs) / PTX@PEI / siPGK1@CCM coated with CCM show a slight negative charge. After coating the cell membrane, it is more conducive to serum stability and prolongs blood circulation time.

[0127] Figure 3 A shows that under hypoxic conditions, the hydrophobic nitroimidazole in the poly(MIs) structure is transformed into a hydrophilic aminoimidazole, causing the nanomicelle structure to dissociate.

[0128] Figure 3 B shows that under normal oxygen conditions, poly(MIs) / PTX@PEI / siPGK1@CCM exhibits uniformly dispersed spherical nanoparticles; under hypoxic conditions, the spherical structure breaks down.

[0129] Figure 3 C shows that poly(MIs) / PTX@PEI / siPGK1@CCM swells under hypoxic conditions.

[0130] The procedure for serum stability testing is as follows:

[0131] (1) Preparation of Poly(MIs)@PEI / siPGK1: Poly(MIs)-PEI amphiphilic cationic polymer (5 mg) was slowly added dropwise to DMSO at pH 7.4. The mixture was ultrasonically cleaned at a frequency of 50 kHz for 30 min. The resulting mixture was then placed in a dialysis bag with a molecular weight cutoff of 3500 Da and deionized water was used as the dialysate to remove organic solvents and free paclitaxel, yielding Poly(MIs)@PEI. Subsequently, siPGK1 (0.52 mg) was added to the above mixture and stirred at room temperature for 30 min to obtain the siPGK1-loaded nanomedicine carrier Poly(MIs)@PEI / siPGK1.

[0132] (2) The poly(MIs)@PEI prepared in Example 1, the Poly(MIs) / PTX@PEI prepared in Example 3, and the Poly(MIs)@PEI / siPGK prepared in (1) are coated with CCM using the method of this example to obtain poly(MIs)@PEI@CCM, Poly(MIs) / PTX@PEI@CCM, and Poly(MIs)@PEI / siPGK@CCM;

[0133] (3) The poly(MIs)@PEI@CCM, Poly(MIs) / PTX@PEI@CCM, Poly(MIs)@PEI / siPGK@CCM and the poly(MIs) / PTX@PEI / siPGK1@CCM prepared in (2) were added to serum to prepare a solution with a mass concentration of 0.5 mg / ml. The solution was placed at room temperature, and the precipitation of each group was observed and recorded daily.

[0134] Serum stability test results are as follows Figure 4As shown: After standing for 7 days, poly(MIs)@PEI@CCM, Poly(MIs) / PTX@PEI@CCM, Poly(MIs)@PEI / siPGK@CCM, and poly(MIs) / PTX@PEI / siPGK1@CCM did not produce any flocculent matter or precipitation in the solution, and the particle size remained relatively stable with no significant increase in hydrated particle size. This indicates that cell membrane coating can prolong the circulation time of nanoparticles in the blood, helping them evade clearance by immune cells and enhancing their biological stability. It also shows that the cell membrane shell coating of positively charged poly(MIs)@PEI can prevent it from attracting albumin in the blood, thus preventing the formation of nanoparticle precipitation.

[0135] To verify the integrity of membrane protein function, this study analyzed the distribution characteristics of membrane proteins by SDS-PAGE gel electrophoresis. The experimental procedures were as follows: (1) The membrane proteins from the cell membrane in Example 2 and Poly(MIs) / PTX@PEI / siPGK1@CCM were added to RIPA lysis buffer, centrifuged (13000g, 5 minutes) after complete lysis, and the supernatant was collected. The total protein concentration was quantified to 1.5 mg / mL using the BCA method. (2) After preparing a 1.5% agarose gel, the sample was added to the well (20 μL) using a pipette, and electrophoretically separated (38V) for 40 minutes. (3) The separated proteins were transferred to a PVDF membrane and a protein chromogenic agent was added. (4) The gel images were acquired using the Bio-Rad Gel Doc™ XR imaging system.

[0136] Experimental results are as follows Figure 5 As shown, lane I is the total protein imprint of the cell membrane, and lane II is the total protein imprint after lysis of Poly(MIs) / PTX@PEI / siPGK1@CCM. Compared with the pure U87 cell membrane, the cell membrane proteins wrapped on the surface of poly(MIs) / PTX@PEI / siPGK1 were well preserved. The expression of the two at key molecular weights was completely consistent, indicating that no cell membrane proteins were lost during the preparation of the biomimetic nanoco-carrying system, laying a structural foundation for retaining its immune clearance evasion and homology targeting functions.

[0137] The loading capacity of the small interfering gene siPGK1 is as follows: Figure 6 As shown in Figure A, with increasing N / P ratio, the loading of siPGK1 in the biomimetic nanomedicine Poly(MIs) / PTX@PEI / siPGK1@CCM increases. The release of the small interfering gene siPGK1 under hypoxic conditions is as follows... Figure 6 As shown in Figure B, the siPGK1 release capacity gradually increases as the oxygen concentration decreases, and the siPGK1 release rate approaches 100% when the oxygen concentration is as low as 10%.

[0138] The drug release capacity of Poly(MIs) / PTX@PEI / siPGK1@CCM is as follows: Figure 7 As shown, siPGK1 and PTX are gradually released under hypoxic conditions over time; however, under normoxic conditions, the release of PTX drug and gene is minimal.

[0139] Example 5: Cell-level safety verification

[0140] Experimental Methods: U87 cells were seeded in 96-well plates and cultured overnight under normoxic conditions. Then, different concentrations of poly(MIs)@PEI / @CCM and poly(MIs) / PTX@PEI / siPGK1@CCM (100, 200, 300, 400, 500, and 800 μg / mL) were added, and the cells were incubated for another 24 h. All treated U87 cells were then incubated with CCK-8PBS solution for 4 h. Cell viability was calculated by measuring the absorbance of the cells at 450 nm using a microplate reader.

[0141] Experimental results: such as Figure 8 As shown, poly(MIs)@PEI / @CCM and poly(MIs) / PTX@PEI / siPGK1@CCM at different concentrations (100, 200, 300, 400, 500 and 800 μg / mL) exhibited good cell activity under normoxic conditions, indicating that these two nanoparticles have good biocompatibility after being coated with cell membranes.

[0142] Example 6: In vitro simulated blood-brain barrier co-culture study

[0143] Experimental methods:

[0144] (1) A layer of bEnd.3 cells was seeded onto the Transwell insert to form a dense endothelial cell monolayer in the upper chamber. Transmembrane resistance was measured using a Millicell-ERS-2 (Millipore) instrument, and the transmembrane resistance value was maintained >200 Ωcm throughout the study. 2 .

[0145] (2) Add 1 ml of FAM-siPGK1 and poly(MIs) / PTX@PEI / siPGK1@CCM solution to the upper chamber of each Transwell plug and incubate for different times (2h, 6h and 12h).

[0146] (3) The transtransformation ability of BBB at different time points was evaluated by the fluorescence intensity of siPGK1 in the lower chamber side medium of the Transwell plug.

[0147] Experimental results: such as Figure 9 As shown, the amount of siPGK1 penetrating the blood-brain barrier model was very low with prolonged observation time, proving that free siPGK1 has difficulty crossing the blood-brain barrier model. However, the fluorescence intensity of poly(MIs) / PTX@PEI / siPGK1@CCM in solution gradually increased with prolonged incubation time, indicating that poly(MIs) / PTX@PEI / siPGK1@CCM can efficiently cross the blood-brain barrier model. The main reason is that after modification of the cell membrane, the nanoparticles have excellent membrane fusion ability, making it easier for the nanoparticles to penetrate the tightly packed endothelial cells.

[0148] Example 6 Cell uptake verification

[0149] Experimental methods:

[0150] (1) Poly(MIs) / PTX@PEI / siPGK1@CCM was prepared according to the method in Example 4, wherein siPGK1 was replaced by FAM-siPGK1, and FAM fluorescently labeled poly(MIs) / PTX@PEI / siPGK1@CCM was synthesized.

[0151] (2) U87 cells were prepared at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / well into cell culture plates, culture medium was added, and the cells were cultured overnight under normoxic conditions. Poly(MIs) / PTX@PEI / siPGK1@CCM solution was added to U87 cells at a concentration of 200 μg / ml for a certain period of pretreatment.

[0152] (3) Using a confocal laser scanning microscope, cell uptake and lysosomal / endosome escape were detected by FAM fluorescence.

[0153] Experimental results: such as Figure 10 As shown, free siRNA has difficulty entering tumor cells, and only weak fluorescence was detected after 4 hours of culture. However, after co-culturing poly(MIs) / PTX@PEI / siPGK1@CCM with tumor cells for 4 hours, the fluorescence intensity inside the tumor cells gradually increased with the extension of time, indicating that poly(MIs) / PTX@PEI / siPGK1@CCM can be effectively taken up by tumor cells.

[0154] Example 7 Intracellular degradation and release verification

[0155] Experimental methods:

[0156] (1) Replace PTX in poly(MIs) / PTX@PEI / siPGK1@CCM in Example 4 with doxorubicin (DOX) and label poly(MIs) / PTX@PEI / siPGK1@CCM.

[0157] (2) U87 cells were prepared at a rate of 1×10⁻⁶. 5 Cells were seeded at a density per well into cell culture plates, cultured in culture medium, and placed in a hypoxic incubator overnight.

[0158] (3) Poly(MIs) / DOX@PEI / siPGK1@CCM was added to hypoxic and normoxic cells at a concentration of 150 μg / mL and cultured together for 4 h. The release of PTX into the cells after Poly(MIs) / DOX@PEI / siPGK1@CCM was observed by laser confocal microscopy.

[0159] Experimental results: such as Figure 11 As shown, under normoxic conditions, DOX is mainly located in the cytoplasm, indicating that poly(MIs) / DOX@PEI / siPGK1@CCM is difficult to degrade under normoxic conditions. Conversely, under hypoxic conditions, DOX molecules are transported to the cell nucleus and exhibit strong red fluorescence, indicating that the poly(MIs) / DOX@PEI / siPGK1@CCM nanomedicine is effectively decomposed under hypoxia-induced conditions.

[0160] Example 8: Verification of Intratumoral Protein Inhibition

[0161] Test method:

[0162] (1) U87 cells were prepared at a rate of 1×10 5 Cells were seeded at a low density into cell culture plates and incubated overnight in a hypoxic incubator. Poly(MIs) / PTX@PEI / siPGK1@CCM solution was added to the hypoxic U87MG cells for a pretreatment period.

[0163] (2) The expression level of siPGK1 protein in cells after treatment with different concentrations of poly(MIs) / PTX@PEI / siPGK1@CCM was determined by Western blotting.

[0164] Test results: such as Figure 12As shown, with the increase of siPGK1 concentration in poly(MIs) / PTX@PEI / siPGK1@CCM, the expression level of siPGK1 protein in tumor cells gradually decreased. The results indicate that poly(MIs) / PTX@PEI / siPGK1@CCM can effectively transfect tumor cells and interfere with PGK1 protein expression.

[0165] Example 9 Cell-level toxicity verification

[0166] Experimental methods:

[0167] (1) U87MG cells were prepared at a ratio of 1×10⁻⁶ 5 Cells were seeded at a density per well into cell culture plates and cultured overnight in both hypoxic and normoxic conditions.

[0168] (2) Poly(MIs) / PTX@PEI@CCM and poly(MIs) / PTX@PEI / siPGK1@CCM solutions were added to hypoxic U87MG cells at a concentration of 150 μg / mL for a certain period of pretreatment. Simultaneously, the same concentration of poly(MIs) / PTX@PEI / siPGK1@CCM solution was added to normoxic cells for a certain period of pretreatment.

[0169] (3) All treated U87 cells were incubated with CCK-8PBS solution for 4 h. The absorbance of the cells at 450 nm was measured using an ELISA reader, and the cell viability ratio was calculated.

[0170] Experimental results: such as Figure 13 As shown, poly(MIs) / PTX@PEI / siPGK1@CCM did not exhibit significant cytotoxicity under normoxic conditions with increasing co-culture concentration. However, the cytotoxicity of poly(MIs) / PTX@PEI / siPGK1@CCM and poly(MIs) / PTX@PEI@CCM increased with increasing concentration. These results indicate that poly(MIs) / PTX@PEI@CCM possesses significant hypoxia-releasing properties and can effectively inhibit tumor cell growth.

[0171] Example 10: Verification of Cell-Level Radiosensitization

[0172] Experimental methods:

[0173] First, U87 cells were cultured overnight in 12-well plates and treated for 4 h under hypoxic and normoxic conditions with free PTX and the biomimetic nanomedicine poly(MIs) / PTX@PEI / siPGK1@CCM, respectively. Then, the incubated cells were irradiated (2 Gy) and stained with γH2AX antibody (red). Finally, the cells were analyzed and imaged using a fluorescence microscope.

[0174] Research results: such as Figure 14 As shown, the anticancer mechanism of poly(MIs) / PTX@PEI / siPGK1@CCM therapy mainly involves increasing X-ray-induced DNA damage and enhancing the fixation of these DNA damages by poly(MIs). After degradation using gene interference technology, the PGK1 protein promotes PTX-induced chemical damage and enhances X-ray-induced ionizing radiation damage; these damages can be analyzed by upregulating γH2AX. Therefore, the experimental results demonstrate that poly(MIs) / PTX@PEI / siPGK1@CCM exhibits highly efficient radiosensitization capabilities under hypoxic conditions.

[0175] Example 11 Verification of Cell Proliferation Inhibition

[0176] Experimental methods:

[0177] (1) Seed U87 cells into 6-well plates and place them in a hypoxic incubator overnight.

[0178] (2) Add PTX+siPGK1, poly(MIs) / PTX@PEI@CCM, poly(MIs)@PEI / siPGK1@CCM, and poly(MIs) / PTX@PEI / siPGK1@CCM (150 μg / mL) to the medium and treat for 4 h. Then irradiate the 6-well plate with different doses (0, 2, 4, 6, and 8 Gy). After irradiation for 1 h, discard the medium and add fresh DMEM.

[0179] (3) Alternatively, continue incubation for 2 weeks. Cell clones are fixed with 4% paraformaldehyde and stained with crystal violet. Count all colonies containing 50 or more cells.

[0180] Experimental results: such as Figure 15As shown, comparisons revealed that cells treated with poly(MIs) / PTX@PEI / siPGK1@CCM exhibited reduced colony-forming ability, indicating a significant synergistic effect with chemotherapy / radiotherapy in hypoxic tumor cells. The inhibitory effect significantly increased with increasing radiation dose (2 Gy–8 Gy), and cell colony formation was almost completely inhibited when the radiation dose exceeded 6 Gy. Furthermore, downregulation of PGK1 protein resulted in better inhibition of colony formation compared to other treatment groups, demonstrating a stronger ability to inhibit colony formation at the same radiation dose. Figure 16 As shown, the cytotoxicity results of the CCK8 assay are similar to those of the colony assay.

[0181] Example 12 Apoptosis Verification

[0182] Experimental methods:

[0183] (1) Seed U87 cells into 6-well plates and place them in a hypoxic incubator overnight.

[0184] (2) Add PTX+siPGK1, poly(MIs) / PTX@PEI@CCM, poly(MIs)@PEI / siPGK1@CCM, poly(MIs) / PTX@PEI / siPGK1@CCM (150 μg / mL) and treat for 4 h, then irradiate the 6-well plate with 6 Gy. After irradiation for 1 h, discard the culture medium and add fresh DMEM for incubation for 8 h.

[0185] (3) Cell proliferation was assessed using the EDU method: U87 cells were treated with different groups, and the EdU content was measured. The treated U87 cells were cultured in 50 μM 5-ethyl-2'-deoxyuridine (EdU) medium for 2 h, and washed multiple times with PBS. Cells were fixed with 4% formaldehyde and permeable with 0.5% Triton X-100. 643. Observe the proliferation of U87 cells. Identify the cell nucleus using DAPI reverse staining.

[0186] (4) Cell proliferation in each group was tested by flow cytometry: apoptosis was detected by Annexin V-FITC / PI apoptosis detection kit and analyzed by flow cytometry (FACSCalibur) in accordance with the manufacturer’s (Shanghai Beyotime Biotechnology Co., Ltd.) instructions.

[0187] Experimental results: such as Figure 17 As shown, EdU analysis further confirmed that poly(MIs) / PTX@PEI / siPGK1@CCM treatment had a significant inhibitory effect on tumor cell DNA replication. After Annexin V / PI double staining, flow cytometry captured cell apoptosis after different treatment groups, as shown... Figure 18 As shown, the apoptosis data are consistent with the EDU analysis results. In summary, these data indicate that chemotherapy or PGK1 gene downregulation combined with radiotherapy can effectively induce apoptosis. Furthermore, the synergistic anti-tumor effect of PGK1-mediated chemotherapy / radiotherapy is significantly superior to that of single treatment modalities.

[0188] Example 13 Targeting Validation

[0189] Experimental methods:

[0190] (1) A U87-Luci orthotopic glioma model was constructed by transplanting tumor cells into brain tissue. On day 20, the successful construction of the tumor model was verified using a small animal in vivo imaging system.

[0191] (2) Nude mice were anesthetized with 4% chloral hydrate and injected intraperitoneally with potassium fluorescein (concentration of 15 mg / ml, dose of 10 μl / g). After 10 minutes, the mice were photographed using in vivo imaging technology.

[0192] (3) After the in situ model was successfully constructed, in order to observe the targeting of nanomedicines to tumors and organ accumulation, this study used FAM-siRNA to construct experimental nanomedicines. The specific construction method was as follows: FAM-siPGK1 was obtained by labeling siPGK1 with FAM; FAM-siPGK1 was used to replace siPGK1 in Example 3, and poly(MIs) / PTX@PEI / iPGK1@CCM was constructed according to the method of Example 3; FAM-siPGK1 was used to replace siPGK1 in Example 3, and LipoPEG was used to replace CCM in Example 2. LipoPEG construction method: DSPE-PEG 2000 100 mg of chloroform and 18 mg of cholesterol were dissolved in 5 mL of chloroform. Then, the chloroform was removed by vacuum rotary evaporation to form a transparent film. 15 mL of deionized water was added, and the mixture was sonicated for 10 minutes. The resulting solution was extruded five times through an aqueous filter (200 nm) to obtain LipoPEG. Poly(MIs) / PTX@PEI / siPGK1@LipoPEG was constructed according to the method in Example 3.

[0193] (4) 200 μl of FAM-siPGK1, poly(MIs) / PTX@PEI / siPGK1@CCM, and poly(MIs) / PTX@PEI / siPGK1@LipoPEG were injected into tumor-bearing nude mice via the tail vein (siPGK1 dose: 1.0 mg / kg). The blood half-life of each experimental group was measured: First, after each group was injected via the tail vein, blood was drawn from the nude mice at different time points. Then, the blood was centrifuged, and the paclitaxel and siPGK1 contents were determined using the supernatant serum.

[0194] (5) 200 μl of FAM-siPGK1, poly(MIs) / PTX@PEI / siPGK1@CCM and poly(MIs) / PTX@PEI / siPGK1@LipoPEG were injected into tumor-bearing nude mice via the tail vein. The distribution of siPGK1 in the body was detected at different time points after injection (0h, 2h, 4h, 8h, 12h and 24h) using a small animal in vivo imaging system with an excitation wavelength of 470nm and an emission wavelength of 490nm.

[0195] (6) At the peak of organ fluorescence accumulation (8h), three nude mice were randomly selected from each group, euthanized, and dissected to obtain major organs such as the heart, liver, spleen, lungs, kidneys, and brain. Under light-protected conditions, the distribution of siPGK1 was detected using a small animal in vivo imaging system. Subsequently, the organ tissues were homogenized using a grinder, 700 μl of chromatographic grade acetonitrile was added and incubated overnight, centrifuged at 12,000 rpm, and the supernatant was collected. After evaporation and drying, 200 μl of chromatographic grade acetonitrile was added, centrifuged at 12,000 rpm, and the supernatant was collected. Finally, the siPGK1 content was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0196] Experimental results: such as Figure 19 As shown, after encapsulation in liposome form, the blood circulation time of PTX is significantly prolonged. On the other hand, as... Figure 20 As shown, where Figure 20 A is a live imaging image of the FAM-siPGK1, poly(MIs) / PTX@PEI / siPGK1@CCM, and poly(MIs) / PTX@PEI / siPGK1@LipoPEG groups. Figure 20B shows the organ fluorescence distribution at 8 hours. Two hours after tail vein injection, free siPGK1 was mainly located in the kidneys and was almost completely excreted from the body by 24 hours, indicating that free siPGK1 is eliminated rapidly and has poor targeting. Compared with free siPGK1, poly(MIs) / PTX@PEI / siPGK1@LipoPEG liposomes showed more significant tumor accumulation 8 hours after tail vein injection, with cumulatively enhanced tumor fluorescence intensity, which continued until 12 hours after injection. Subsequently, FAM-siPGK1 fluorescence began to weaken, indicating that poly(MIs) / PTX@PEI / siPGK1@LipoPEG liposomes can passively accumulate at the tumor site through enhanced permeability retention (EPR effect). However, due to the lack of active targeting, the amount accumulated is small and cannot be effectively internalized by tumor cells, making it easily metabolized by the body. Compared with the other two groups, the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system showed a strong tumor accumulation effect 4 hours after tail vein injection, reaching its peak 8 hours after injection. At 24 hours, the FAM-siPGK1 fluorescence in the tumor tissue was still strong, indicating that the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrying system not only has good targeting, but is also easily phagocytosed by tumor cells and not easily metabolized and cleared by the body.

[0197] To further compare the differences in drug distribution among organs, three nude mice were randomly selected from each group at six time points (0h, 2h, 4h, 8h, 14h, and 24h) after tail vein injection. The mice were dissected in the dark, and major organs, including the heart, liver, spleen, lungs, kidneys, and brain, were removed. In vivo fluorescence imaging technology was used to quantify the fluorescence values ​​and plot bar charts for analysis. Figure 21 The results showed that FAM-siPGK1 was rapidly cleared from the blood, with a half-life of only 15 minutes. The blood half-lives of poly(MIs) / PTX@PEI / siPGK1@LipoPEG and poly(MIs) / PTX@PEI / siPGK1@CCM were 3.5 h and 1.8 h, respectively. This comparison demonstrates that poly(MIs) / PTX@PEI / siPGK1@CCM exhibits good biocompatibility and a long in vivo circulation time. After in vivo circulation, poly(MIs) / PTX@PEI / siPGK1@CCM gradually accumulates within the brain tumor, reaching its maximum accumulation at 8 h post-injection, and then gradually clears. Compared to other organs (heart, liver, spleen, lung, kidney), poly(MIs) / PTX@PEI / siPGK1@CCM demonstrates specific targeting ability at the brain tumor site.

[0198] To further observe the distribution of poly(MIs) / PTX@PEI / siPGK1@CCM in brain tumor tissue, brain tissues treated with different groups were dissected, frozen sections were prepared, and the distribution of siPGK1 in different groups was directly observed under a fluorescence microscope. The results are as follows: Figure 22 As shown, poly(MIs) / PTX@PEI / siPGK1@CCM exhibits better brain tumor targeting performance compared to the other two groups (poly(MIs) / PTX@PEI / siPGK1@Lipo and PTX+siPGK1).

[0199] The above experimental results all suggest that the poly(MIs) / PTX@PEI / siPGK1@CCM co-carrier system has good targeting and long-circulating properties, laying a pharmacokinetic basis for in vivo inhibition of tumor proliferation.

[0200] Example 14 Verification of In Situ Brain Tumor Suppression

[0201] Experimental methods:

[0202] (1) Using the in-situ model constructed in Example 13, the nude mice were weighed and randomly divided into 7 groups (n=10):

[0203] The treatment for Saline (group 1) was: injection of normal saline;

[0204] The treatment for Saline+RT (group 2) was: radiotherapy followed by saline injection;

[0205] The treatment for Free PTX+naked siPGK1+RT (group 3) was: paclitaxel injection followed by radiotherapy;

[0206] The treatment method for poly(MIs) / PTX@PEI / siPGK1@CCM (without RT, group 4) is: poly(MIs) / PTX@PEI / siPGK1@CCM injection;

[0207] The treatment for poly(MIs)@PEI / siPGK1@CCM+RT (group 5) was: poly(MIs)@PEI / siPGK1@CC injection followed by radiotherapy;

[0208] The treatment for poly(MIs) / PTX@PEI@CCM+RT (group 6) is: poly(MIs) / PTX@PEI@CCM injection followed by radiotherapy;

[0209] The treatment for poly(MIs) / PTX@PEI / siPGK1@CCM+RT (group 7) is: poly(MIs) / PTX@PEI / siPGK1@CCM injection followed by radiotherapy;

[0210] (2) The mice were administered different treatments via tail vein injection (once every 2 days). The dosages of the chemotherapy drugs PTX and siPGK1 in each group were 1.8 mg PTX and 2.3 mg siPGK1 per kilogram of mouse body weight.

[0211] (3) Whole brain radiotherapy using X-ray equipment (2Gy, 0.2Gy / min).

[0212] (4) The tumor suppression effect of each group was evaluated by capturing in vivo bioluminescence images at different time points using the Maestro animal imaging system. The formula for calculating the relative tumor size is as follows: Relative tumor size (Vt / V0) = Bioluminescence intensity on day t (Vt) / Bioluminescence intensity on day 10 (V0). Throughout the study, mice were weighed every 2 days, and survival curves for each group of mice were plotted.

[0213] (5) At the end of the experiment, the pathological sections and immunohistochemistry of each group were analyzed.

[0214] Experimental results:

[0215] like Figure 23 As shown in A, to evaluate the antitumor effects of each group in vivo, an orthotopic model of U87 glioblastoma was established using Balb / c mice, and treatment was administered on days 12, 14, and 16, with tumor size evaluated on days 20 and 30.

[0216] like Figure 23As shown in Figure B, in vivo bioluminescence imaging using fluorescein injection easily monitors the effects of different treatment strategies on the growth of glioblastoma cells in situ. Clearly, the Saline treatment group (Group 1) had no effect on the proliferation of glioblastoma cells in situ, and the tumor volume on day 30 was significantly larger than on day 10. Overall, the experimental results showed significant differences in tumor suppression among the various treatment strategies after continuous treatment. Notably, poly(MIs) / PTX@PEI / siPGK1@CCM+RT (Group 7) exhibited the best anti-tumor effect, with lower bioluminescence intensity than other treatment groups. More importantly, mice treated with either siPGK1 alone (Group 5) or PTX alone (Group 6) showed varying degrees of tumor suppression. These results indicate that PGK1 or PTX alone can suppress tumor growth. However, combining both, through PGK1-mediated chemotherapy and radiotherapy, has a synergistic sensitizing effect. On the other hand, CCM plays a crucial role in tumor recognition and deep penetration, which is essential for subsequent PGK1 downregulation, PTX-induced chemotherapy, and radiotherapy. For example... Figure 23 As shown in Figure C, tumor growth almost stopped after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT. Figure 24 As shown, H&E staining of the brain further verified the tumor-suppressing ability of poly(MIs) / PTX@PEI / siPGK1@CCM+RT, and it can be seen that the volume of the brain tumor is very small.

[0217] like Figure 25 As shown in Figure A, immunofluorescence comparison revealed that TUNNEL expression was highest in tumor tissue after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, indicating significant apoptosis. Figure 25 As shown in B, quantitative analysis revealed that approximately 90% of cells underwent apoptosis after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, which was significantly higher than that of radiotherapy alone (45%) and the Saline control group (2.5%).

[0218] like Figure 26 As shown in Figure A, the immunohistochemical results of γH2AX indicate that the expression level of γH2AX in tumor tissue was highest after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT. Figure 26As shown in Figure B, approximately 40% of tumor cells exhibit DNA breaks after radiotherapy. Following treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, γH2AX expression in tumor cells reached as high as 90%, demonstrating its significant radiosensitizing ability.

[0219] like Figure 27 As shown in Figure A, the immunohistochemical results of PGK1 indicate that PGK1 expression was lowest in tumor tissue after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT. Figure 27 As shown in Figure B, after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, the expression level of PGK1 protein in tumor cells decreased to 10.5%, indicating that it can effectively inhibit the expression of PGK1 protein.

[0220] like Figure 28 As shown in Figure A, Ki67 immunohistochemical images revealed the lowest Ki67 expression level in tumor tissue after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT. Figure 27 As shown in Figure B, after treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, the expression level of Ki67 protein in tumor cells decreased to 8.76%, indicating that it can effectively inhibit the proliferation of tumor cells.

[0221] To further evaluate the antitumor efficacy of each group, we recorded and monitored the median survival time of mice after treatment in each group. The results are as follows: Figure 29 As shown, mice treated with poly(MIs) / PTX@PEI / siPGK1@CCM+RT had a median survival of up to 82 days. In contrast, mice in groups 4 (n=4) without RT treatment had a relatively shorter median survival of 50 days. This indicates that radiotherapy has a significant impact on tumor suppression, primarily because the hypoxic tumor environment always occurs in deep tissues or the central region of solid tumors, which limits the in vivo degradation of poly(MIs) / PTX@PEI / siPGK1@CCM. However, the local hypoxia caused by radiotherapy effectively addresses this issue.

[0222] To further assess in vivo safety, we recorded body weight during treatment, and the results are as follows: Figure 30As shown, the results indicate that the poly(MIs) / PTX@PEI / siPGK1@CCM nanomedicine exhibits good biocompatibility after intravenous injection into mice with orthotopic brain tumors. After treatment with poly(MIs) / PTX@PEI / siPGK1@CCM+RT, the mice did not experience a significant decrease in body weight, demonstrating good in vivo safety and superior tumor-inhibiting activity compared to the PBS group, although the PBS group showed a faster decrease in body weight.

[0223] like Figure 31 As shown, we then further analyzed the pathological sections of the heart, liver, spleen, lungs, and kidneys of mice treated with different strategies. Mice treated with poly(MIs) / PTX@PEI / siPGK1@CCM did not show significant damage in these major organs, indicating that poly(MIs) / PTX@PEI / siPGK1@CCM has long-term biocompatibility.

[0224] like Figure 32 As shown, in mice treated intravenously with poly(MIs) / PTX@PEI / siPGK1@CCM, the effects of blood biochemistry and hematological results on liver and kidney function or hematological parameters were negligible. In conclusion, these results further demonstrate the promising clinical application of poly(MIs) / PTX@PEI / siPGK1@CCM biomimetic nanomedicines in the treatment of gliomas.

[0225] In summary, the biomimetic nanomedicine prepared by this invention can serve as a targeted tumor therapy drug, achieving efficient crossing of the blood-brain barrier (BBB) ​​and precise targeting of glioma cells by the nanocarrier; realizing the intratumoral hypoxia response / degradability of the nanosensitizer, reducing the neurotoxicity caused by its accumulation in normal tissues, and exhibiting good biocompatibility; and achieving radiotherapy and chemotherapy sensitization effects on tumors such as gliomas by targeting multiple sites (increasing DNA damage, inhibiting DNA damage repair, and promoting cell apoptosis), thereby improving the therapeutic effect.

[0226] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A drug carrier, characterized in that, The structural formula of the drug carrier is shown in Formula I, where n = 4~8, m = 5~10. Formula I.

2. A method for preparing the drug carrier according to claim 1, characterized in that, The method includes the following steps: (1) Dissolve N-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride in chloroform and add 4-aminobutyric acid; After mixing thoroughly, stir at room temperature until the reactants become a colorless, viscous solution; With stirring, the colorless viscous solution was poured into anhydrous diethyl ether, precipitated, filtered, and dried under vacuum to obtain a white fibrous solid; (2) The white fibrous solid obtained in (1) was completely dissolved in CF3COOH solvent, stirred in an ice bath and 33% HBr / AcOH solution with a volume ratio of 5 to 10 was added. After the reaction, the product was precipitated in anhydrous diethyl ether and centrifuged to obtain a yellow crude product. The yellow crude product was dissolved in DMSO and dialyzed. After freeze-drying, the deprotected polymer PGlu-COOH was obtained; (3) Dissolve polymer PGlu-COOH in DMF, add EDCI, DMAP and metronidazole; mix thoroughly, stir at room temperature, dialyze in 5000 Da of deionized water; then freeze dry to obtain polymer poly(MIs); (4) Dissolve the polymer poly(MIs) in DMF, add EDCI, DMAP and PEI; after thorough mixing, stir at room temperature, dialyze in 5000 Da of deionized water; after freeze drying, obtain the block polymer poly(MIs)-PEI shown in Formula I.

3. The method for preparing a drug carrier according to claim 2, characterized in that, During the dialysis process described in steps (2), (3), and (4), the external dialysis fluid is changed every 8 hours.

4. The method for preparing a drug carrier according to claim 2, characterized in that, In step (1), the molar ratio of N-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride to the terminal amino group of 4-aminobutyric acid is 30~42:

1.

5. The method for preparing a drug carrier according to claim 4, characterized in that, The molar ratio of the N-benzyloxycarbonyl-L-glutamic acid-N-carboxylic anhydride to the terminal amino group of 4-aminobutyric acid is 40:1 or 42:

1.

6. The method for preparing a drug carrier according to claim 2, characterized in that, The dialysis in step (2) consists of dialysis in deionized water at 5000 Da, dialysis in ammonia water at pH 9.0, dialysis in HCl solution at pH 5.0, and finally dialysis in deionized water.

7. The method for preparing a drug carrier according to claim 2, characterized in that, In step (3), the molar ratio of polymer PGlu-COOH to EDCI, DMAP and metronidazole is 1~3: 50: 10: 30~42.

8. The method for preparing a drug carrier according to claim 2, characterized in that, The molar ratio of the polymer poly(MIs) in step (4) to EDCI, DMAP and PEI is 1~5: 1.2~6: 0.4~2.4:

1.

9. A biomimetic nanomedicine, characterized in that, The biomimetic nanomedicine comprises a shell, a drug carrier, a first active component, and a second active component. The drug carrier is encapsulated within the shell and loads the first and second active components. The shell includes a tumor cell membrane. The first active component is an antitumor compound. The second active component is a gene-targeting interference compound. The structural formula of the drug carrier is shown in Formula I, where n = 4~8 and m = 5~10. Equation I; The first active component is a hydrophobic antitumor compound.

10. The biomimetic nanomedicine according to claim 9, characterized in that, The first active component accounts for 3.66-6.31% of the mass of the biomimetic nanomedicine, and the second active component accounts for 3.65-9.35% of the mass of the biomimetic nanomedicine.

11. The biomimetic nanomedicine according to claim 9, characterized in that, The outer shell is the membrane of human glioma cells.

12. The biomimetic nanomedicine according to claim 9, characterized in that, The first active ingredient is one or a combination of paclitaxel, doxorubicin, and carmustine.

13. The biomimetic nanomedicine according to claim 9, characterized in that, The second active component is selected from targeted interference genes that inhibit DNA damage repair.

14. The biomimetic nanomedicine according to claim 13, characterized in that, The second active component is siPGK1.

15. A method for preparing the biomimetic nanomedicine according to claim 9, characterized in that, The method includes the following steps: dissolving a first active component in an organic solvent and mixing it with a drug carrier of Formula I; dialyzing to remove the organic solvent and the free first component to obtain nanoparticles loaded with the first active component; adding a second active component to form nanoparticles simultaneously loaded with the first and second active components; mixing with a tumor cell membrane and extruding to obtain the biomimetic nanomedicine.

16. The preparation method according to claim 15, characterized in that, Includes the following steps: (1) Dissolve the first active component in an organic solvent, mix it with the drug carrier shown in Formula I, and then use an ultrasonic cleaner to sonicate it at a frequency greater than 40 KHz to obtain a drug carrier mixture system loaded with the first active component. (2) The drug carrier mixture system loaded with the first active component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and deionized water was used as the dialysate to remove organic solvents and free molecules of the first active component, so as to obtain a nanoparticle solution loaded with the first component. (3) Add the second active component to the nanoparticle solution loaded with the first component and react to form a nanocomposite loaded with both the first and second active components. (4) The lysed tumor cell membrane was dissolved in PBS buffer solution, mixed with the nanocomposite loaded with the first and second active components, vortexed, and extruded sequentially through an aqueous filter with reduced pore size to obtain a biomimetic nanomedicine.

17. The preparation method according to claim 16, characterized in that, The organic solvent is DMSO, and the pore size of the aqueous filter is 600~800 nm and 200~400 nm, respectively.

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