A method for preparing and applying biomimetic lipoprotein nanodiscs

Abstract

This invention relates to the field of biomedicine, specifically to a method for preparing and applying a biomimetic lipoprotein nanodisk to enhance the therapeutic effect on tumors. The method for preparing the biomimetic lipoprotein nanodisk provided by this invention includes the following steps: First, CCL5 peptide and DSPE-PEG2000-mal are dissolved in DMF and triethylamine, stirred at room temperature, and then the reaction solution is purified by dialysis and freeze-dried to obtain a white solid powder; then, methanol is placed in a flask, DPPC, DSPE-PEG2000-CCL5, and NG are dissolved in methanol, acetic acid is added, and the mixture is evaporated to dryness to form a thin film in the flask; then, the film is dispersed with ultrapure water, sonicated with a probe in an ice bath, and then ApoA1 peptide is added to the mixed solution, followed by thermal cycling to obtain C-LNG. This invention optimizes and modifies gemcitabine, developing a new drug NG, which significantly improves the therapeutic effect on tumors, and C-LNG has extremely high targeting and drug loading rates, further enhancing the drug's efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing and applying a biomimetic lipoprotein nanodisc. Background Technology

[0002] Since the 20th century, the global living environment has been deteriorating, and various forms of pollution have intensified. The number of carcinogenic factors surrounding humans has increased dramatically, and the incidence of tumors has risen year by year, making cancer one of the leading causes of death worldwide. Among the many types of tumors, glioblastoma multiforme (GBM) is the most common intracranial malignant tumor in adults. The median survival for GBM patients is only 12 months, with an extremely low survival rate of only 5%. High-energy ionizing radiation therapy (RT) to kill tumor cells is currently the standard clinical treatment for GBM patients after maximum safe surgical resection. However, the survival outcomes after radiotherapy are still unsatisfactory and are largely limited by low-dose radiotherapy strategies and tumor cell DNA repair responses. Therefore, finding effective treatments to overcome radiotherapy resistance in GBM is an urgent clinical need.

[0003] RAD51 plays a crucial role in the DNA dsb repair process in tumors, promoting DNA association between broken and unbroken DNA template strands and replicating homologous sequences for repair. Compared to normal brain tissue, RAD51 is highly expressed in glioblastoma tissue of GBM patients, and patients with high RAD51 expression have lower survival rates. Currently, existing technologies directly use gemcitabine (Gem) to inhibit RAD51 expression; however, gemcitabine itself has limited efficacy. Therefore, it is still necessary to develop a new drug that optimizes and modifies gemcitabine to enhance its inhibitory effect on RAD51 and further improve its efficacy.

[0004] Furthermore, some existing technologies directly use therapeutic drugs, but due to the body's metabolic processes, these drugs are often released prematurely before reaching the tumor site, resulting in low bioavailability and poor therapeutic effects. Therefore, to improve efficacy, other existing technologies encapsulate drugs in carrier materials to achieve effective controlled release. Currently, most research uses biocompatible polymers, nanoparticles, protein nanoparticles, and polymer microspheres to encapsulate drugs. While this can improve bioavailability to some extent, problems such as low drug targeting, low drug loading rate, and low bioavailability still exist.

[0005] In summary, gemcitabine currently has limited inhibitory effect on RAD51 and suffers from problems such as low drug targeting, low drug loading rate, and low bioavailability. Therefore, there is an urgent need to develop a new drug and design a new drug delivery platform to improve drug targeting and bioavailability, thereby further enhancing the therapeutic effect on tumors. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing biomimetic lipoprotein nanodiscs to further improve the therapeutic effect on tumors.

[0007] The basic solution provided by this invention is: a method for preparing biomimetic lipoprotein nanodisks, including the preparation of NG and the preparation of C-LNG, wherein the preparation of C-LNG includes the following steps:

[0008] (1) CCL5 peptide and DSPE-PEG2000-mal were dissolved in DMF and triethylamine, stirred at room temperature, and the reaction solution was purified by dialysis and freeze-dried to obtain white solid powder DSPE-PEG-CCL5; (DSPE is the abbreviation for distearylphosphatidylethanolamine, PEG is the abbreviation for polyethanol, mal is the abbreviation for maleimide, and DMF is the abbreviation for N,N-dimethylformamide)

[0009] (2) Take methanol in a flask, then dissolve DPPC, DSPE-PEG2000-CCL5 and NG in methanol, then add acetic acid, evaporate to dryness, and form a film in the flask; (DPPC mentioned in this article is the English abbreviation for dipalmitoylphosphatidylcholine)

[0010] (3) The membrane was then dispersed with ultrapure water, sonicated in an ice bath with a probe, and then ApoA1 peptide was added to the mixed solution. A thermal-cold cycle was then performed to obtain C-LNG, which is the biomimetic lipoprotein nanodisk of this invention (the biomimetic lipoprotein nanodisk is a disc-shaped nanoparticle containing the novel drug NG; the nanodisk has a ring of slits on its exterior, with ApoA1 surrounding the slits to encapsulate it). NG in this invention refers to the abbreviation of furazolidone-gemcitabine, a novel drug developed by the inventors. In the C-LNG described herein, C refers to the chemokine CCL5, and L refers to the natural high-density lipoprotein HDL. ApoA1, the main protein component of HDL, is used in the preparation of this application.

[0011] The working principle and advantages of this invention are as follows:

[0012] This invention optimizes and modifies gemcitabine, resulting in the inventors' new drug NG. No previous reports have described this compound, and this drug significantly enhances the therapeutic effect on tumors. NG, as an effective radiosensitizer, inhibits DNA chain elongation by interfering with RAD51 lesion formation, disrupting DNA damage repair pathways related to tumor survival benefits to enhance radiotherapy resistance and thus expand the efficacy of radiotherapy. Simultaneously, NO released from glutathione (GSH)-triggered NO donor conjugates can react with RT-induced oxygen free radicals (O2). − The reaction generates peroxynitrite anion (ONOO). − This is a powerful nitrifying and oxidizing agent. The resulting ONOO... − It can not only induce lipid peroxidation in tumor cells, but also inhibit the expression of RAD51. In combination with gemcitabine, it can enhance the radiotherapy of tumors and further improve the efficacy and therapeutic effect of the drug.

[0013] The C-LNG of this invention possesses dual targeting capabilities and extremely high targeting specificity. The self-organization of CCL5 enables tumor targeting, forming a chemotactic-driven drug delivery system. Simultaneously, ApoA1, a major protein in natural high-density lipoprotein, binds to SR-BI, delivering the drug to tumor tissue and further enhancing targeting. This dual targeting of CCL5 and ApoA1 enables more precise delivery of NG drugs to the lesion site, reducing drug release in non-lesion areas and preventing drug accumulation in normal tissues that could lead to toxic effects, thus significantly improving efficacy.

[0014] Furthermore, ApoA1 not only promotes targeted delivery but also serves as a sealing and binding material for the nanodisks of this invention, improving drug bioavailability. NG drugs are encapsulated in nanodisks with a ring of gaps around the outside, making leakage during transport highly likely. Therefore, ApoA1 surrounds these gaps to prevent leakage and improve bioavailability. In existing technologies, a substance typically has only one function, but the inventors of this application discovered that ApoA1 has dual functions, capable of performing multiple roles simultaneously, thus reducing costs in drug preparation.

[0015] Furthermore, existing disc-shaped nanoparticles exhibit poor encapsulation performance and extremely low drug loading rates. However, the inventors of this application discovered through extensive practice and analysis that adding acetic acid during the preparation of C-LNG makes drug encapsulation easier, resulting in better encapsulation and a higher drug loading rate. In addition, excessive use of nanodiscs can cause certain side effects on the human body. Therefore, this application uses fewer nanodiscs to encapsulate the same mass of drug, reducing side effects or toxic reactions and thus benefiting human health.

[0016] Furthermore, the mass of the CCL5 peptide in step (1) is 14-20 mg.

[0017] Furthermore, the mass of DSPE-PEG2000-mal in step (1) is 22-26 mg.

[0018] Furthermore, the stirring time in step (1) is 16-28 h.

[0019] Furthermore, in step (2), the weight ratio of DPPC, DSPE-PEG2000-CCL5 and NG is 6:3:1.

[0020] Furthermore, the acetic acid in step (2) is 0.1-0.5 ml.

[0021] Furthermore, the ultrasound time in step (3) is 1-4 minutes.

[0022] Furthermore, the temperature of the hot and cold cycle in step (3) is 4℃~50℃.

[0023] The biomimetic lipoprotein nanodiscs prepared by any of the preparation methods of the present invention are related to their application in tumor drugs.

[0024] The biomimetic lipoprotein nanodiscs prepared by any of the preparation methods of the present invention are related to the application in glioblastoma multiforme. Attached Figure Description

[0025] Figure 1 Characterization of the nanodiscs: (a) Transmission electron microscopy (TEM) images of LG, LNG, and C-LNG nanodiscs before and after incubation with GSH-containing media (PBS (pH 7.4) + 10 mM GSH) for 48 h, scale bar = 100 nm; (b) and (c) particle size distributions of LG, LNG, and CLNG nanodiscs with and without GSH-containing media; (d) average particle size of LG, LNG, and C-LNG nanodiscs with and without GSH-containing media (n = 4); (e) image of C-LNG degradation to Gem* in 10 mM GSH medium for 48 h; (f) analysis of C-LNG degradation components in response to GSH by ESI-MS; (g) NO release curves from LG, LNG, and C-LNG in PBS (pH 7.4) + 10 mM GSH (n = 4); (h) flow cytometry measurements of NO released from LG, LNG, and C-LNG nanodiscs using LG, LNG, and C-LNG respectively. NO production in LNG and C-LNG treated GL261 cells and analyzed using flow Jo; (i) GSH levels in GL261 cells incubated with LG, LNG and C-LNG nanodisks (n = 4).

[0026] Figure 2 To generate ONOO in vitro - The study included the results of DNA damage and the findings, with (a) showing CLSM images of GL261 cells under different treatments (scale bar: 25 μm); and (b) showing the results of ONOO generated by the rapid reaction of ROS and NO using probes. - (n = 4); (c) Confocal images and (d) Flow cytometry analysis of lipid peroxidation in GL261 cells after different treatments, followed by staining with the lipid peroxidation probe 11C-BODIPY (n = 4), scale bar: 50 μm; (e) Colony formation assay of GL261 cells under different treatments; (f) Relative cell viability of GL261 cells after treatment with each formulation determined by CCK-8 (n = 5); (g) DNA damage in GL261 cells under different treatments detected by CLSM after staining with anti-γ-h2ax and DAPI, scale bar: 25 μm; (h) Western blot analysis of the expression levels of γ-H2AX and RAD51 in cells under each treatment.

[0027] Figure 3 To evaluate the targeting efficiency of nanodiscs with chemokine homing effects in orthotopic GBM-bearing mice, (a) shows the distribution and signal spectrum of Lipo, HDL, and C-HDL in orthotopic GBM-bearing mice at different time points after intravenous injection, maximum: 2.5 × 10⁴, minimum: 0.1 × 10⁴; (b) shows the fluorescence signal from mouse brains in the same computational range (n = 3); (c) shows representative ex vivo fluorescence images of the brain; (d) shows the corresponding quantitative analysis of visceral organs dissected from GBM-bearing mice 12 hours after intravenous injection (n = 3); and (e) shows fluorescence images of whole brain sections from GBM-bearing mice treated with Lipo, HDL, and C-HDL, with white dashed lines representing tumor boundaries distinguished by cell density, scale bar: 250 μm.

[0028] Figure 4 (a) The average particle size of LG, LNG and C-LNG after incubation in PBS (pH 7.4) or 10% fetal bovine serum (FBS) for 48 hours; Figure 4 (b) In vivo fluorescence images of healthy mice and GBM mouse models 12 h after injection of C-HDL nanodisks. Figure 4 (c) shows the corresponding quantitative analysis (n = 3); (d) shows the H&E staining of major organs and tissues after different treatments, scale bar: 50 μm.

[0029] Figure 5The results of the in vivo antitumor efficacy of C-LNG are shown below. (a) is a schematic diagram of the antitumor efficacy study in orthotopic tumor-bearing GBM mice; (b) is the bioluminescence image of Luci+ GL261 cells in tumor-bearing mice at different time points after different treatments, with a maximum of 6.5×104 and a minimum of 1.5×103; (c) is the calculation of bioluminescence signal intensity from head images of the same region (n = 7); (d) is the t1-weighted MR image of the mouse head 30 days after different treatments; (e) is the image of tumor size observed by H&E staining on the same coronal plane; (f) is the tumor volume measured according to MRI results (n = 5), where tumor volume = maximum long axis × maximum wide axis × height × 3.14 / 6; (g) is the quantitative analysis of tumor size / brain size based on H&E staining images (n = 4); and (h) is the Kaplan-Meier survival curve of different mouse models (n = 7).

[0030] Figure 6 The tumor suppression response and effective inhibition results of orthotopic U87 glioblastoma are shown in the following images: (a) TUNEL, γ-H2AX, 4-HNE fluorescent staining images and RAD51 immunohistochemical staining images of orthotopic mouse brain tumor sections after different treatments (scale bar: 50 μm); (b) MDA content in tumor tissues after different treatments (n = 5); (c) flow cytometry and (d) corresponding quantitative results of RAD51 expression in tumor tissues of different groups (n = 5); (e) RAD51 content measured by Elias kit (n = 5); (f) MR images; and (g) corresponding quantitative tumor volume of the head of tumor-bearing mice on day 30 after different treatments (n = 5). Detailed Implementation

[0031] The following detailed explanation illustrates the specific implementation methods:

[0032] Example 1: C-LNG

[0033] The preparation method of C-LNG includes the following steps:

[0034] Step 1: Preparation of NG

[0035] (1) 5.0 g of 3,4-di(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide and 2.0 g of 7-hydroxyheptanoic acid were dissolved in 30 mL of dichloromethane, and then mixed with 4.1 g of 1,8-diazobicyclo[5,4,0]undecyl-7-ene. The mixture was stirred at 25 °C for 8 h, and the reaction solution was concentrated under reduced pressure to a yellow liquid, and then extracted with ethyl acetate and saturated brine. After concentration of the organic layer, the compound was purified by elution with ethyl acetate / petroleum ether (10:1, v / v) using a silica gel column to obtain the compound;

[0036] (2) The above 2.6 g compound, 1.9 g N,N-diisopropylethylamine and 3.2 g HATU were added to 20 mL N,N-dimethylaniline and stirred at 25 °C for 0.5 h. Then 2.0 g of the compound gemcitabine was added and stirred for 10 h. The reaction solution was concentrated under reduced pressure to obtain a yellow solid. The residue was then extracted with ethyl acetate and water. Finally, the bound organic layer was concentrated to obtain the NG product.

[0037] Step 2: Preparation of C-LNG

[0038] (1) 20 mg of CCL5 peptide (the amount of CCL5 peptide can also be 14 mg, 16 mg, or 18 mg; this invention only shows the case where the representative amount of CCL5 peptide is 20 mg) and 24.26 mg of DSPE-PEG2000–mal (the amount of DSPE-PEG2000–mal can also be 22 mg or 26 mg; this invention only shows the case where the representative amount of DSPE-PEG2000–mal is 24.26 mg) were dissolved in 1 mL of DMF and 5 μL of triethylamine, and stirred at room temperature for 24 h (the stirring time can also be 16 h, 20 h, or 28 h; this invention only shows the case where the representative stirring time is 24 h); then the reaction solution was purified by dialysis (molecular weight cutoff of 3500 Da) for 36 h, and freeze-dried to obtain a white solid powder DSPE-PEG-CCL5;

[0039] (2) Take 10 ml of methanol into a round flask, and then weigh DPPC, DSPE-PEG2000-CCL5 and NG precisely in a weight ratio of 6:3:1 (in this example, the mass of DPPC is 6 mg, the mass of DSPE-PEG2000-CCL5 is 3 mg, and the mass of NG is 1 mg). Then dissolve DPPC, DSPE-PEG2000-CCL5 and NG in methanol, and add 0.1 ml of acetic acid (the amount of acetic acid can also be 0.3 ml or 0.5 ml; this invention only shows the case where the representative amount of acetic acid is 0.1 ml). Evaporate until dry to form a thin film in the round flask.

[0040] (3) Then the membrane is dispersed with ultrapure water and sonicated in an ice bath for 1 min with a probe (the sonication time can also be 2 min, 3 min, or 4 min; this invention only shows the case where the representative sonication time is 1 min). Then 1 mg of ApoA1 peptide is added to the mixed solution and three thermal cycles are performed between 4 °C and 50 °C to obtain C-LNG.

[0041] Comparative Example 1: LG

[0042] The LG mentioned in this invention is an abbreviation for HDL-Gem. The difference between the preparation method of LG in Comparative Example 1 and Example 1 is that the preparation steps of Step 1 and Step 2 (1) are omitted, DSPE-PEG2000-CCL5 in Step 2 (2) is replaced with DSPE-PEG2000, and NG in Step 2 (2) is replaced with 14C-Gem.

[0043] Comparative Example 2: LNG

[0044] The LNG mentioned in this invention is an abbreviation for HDL-NG. The difference between the preparation method of LNG in Comparative Example 2 and Example 1 is that the preparation step in step 2 (1) is omitted, and DSPE-PEG2000-CCL5 in step 2 (2) is replaced with DSPE-PEG2000.

[0045] Comparative Example 3: Gem

[0046] Gem drug, purchased from Chongqing Yusi Company.

[0047] I. Experimental Materials

[0048] DPPC, DSPE-PEG2000, and DSPE-PEG2000-mal were provided by Sigma-Aldrich (Shanghai, China). ApoA1-peptide mimic (sequence, PVLDLFRELLNELLEALKQKLK, PK-22), CCL5-peptide mimic (sequence, CFPYIARPLPRAHIKEYFY), and DiD were purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Shanxi, China). RAD51 antibody, SR-B1 antibody, Ki-67 antibody, HIF-1α antibody, H2AX antibody, hrp dual antibody, β-actin antibody, and DAR-1 probe were obtained from Abcam. Anti-cd31-pe, AntiCD11b-FITC, Anti-cd45-apc, and Anti-ccl5-pe were purchased from BioLegend, Inc. DAPI, glutathione, NO probe, dihydroethyl ether, Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L), Alexa Fluor 647-labeled goat anti-rabbit IgG (H+L), MDA assay kit, TUNEL assay kit, and BCA protein assay kit were purchased from Beyotime (Shanghai, China). RAD51 enzyme-linked immunosorbent assay kit was purchased from Jiangsu Meimian Industrial Co., Ltd., China. -The probes were ordered from Bestbio Ltd. The nitrotyrosine antibody was purchased from Sigma-Aldrich. The BODIPY® lipid probes were supplied by Thermo Fisher Scientific. Sodium d-luciferin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., China.

[0049] II. Experiments to demonstrate the properties of nanodisks

[0050] (I) Physicochemical properties of nanodisks

[0051] (1) Morphological characterization of nanodisks

[0052] Experiment 1

[0053] The morphology of Example 1 C-LNG, Comparative Example 1 LG, and Comparative Example 2 LNG was observed using transmission electron microscopy. Figure 1 As shown in Figure a, after staining with uranyl acetate solution, transmission electron microscopy revealed that LG, LNG, and C-LNG exhibited regular morphologies and were uniformly disc-shaped.

[0054] (2) Particle size of nanodiscs

[0055] Experiment 2

[0056] The particle size of Example 1 C-LNG, Comparative Example 1 LG, and Comparative Example 2 LNG was determined using a dynamic light scattering (DLS) laser particle size analyzer. Figure 1 As shown in b and 1d, the average diameters of LG, LNG and C-LNG are 21.48±4.49 nm, 18.96±0.87 nm and 15.93±7.11 nm, respectively.

[0057] (3) Stability of nanodisks

[0058] Experiment 3

[0059] After incubating C-LNG (Example 1), LG (Comparative Example 1), and LNG (Comparative Example 2) in PBS (pH 7.4) and 10% fetal bovine serum (FBS) for 36 hours, as shown... Figure 4 As shown in figure a, the average diameter of the nanodiscs did not change significantly, indicating that they have good stability in simulated extracellular fluid circulation and are feasible for in vivo delivery.

[0060] (4) Encapsulation efficiency and drug loading of nanodiscs

[0061] Experiment 4

[0062] The encapsulation efficiencies of Comparative Example 1 (LG), Comparative Example 2 (LNG), and Example 1 (C-LNG) were 89.77 ± 3.06%, 92.11 ± 1.93%, and 93.16 ± 0.73%, respectively. The drug loadings of LG, LNG, and C-LN were 8.98 ± 0.31%, 8.37 ± 0.18%, and 8.47 ± 0.07%, respectively, indicating the effective encapsulation of the lipophilic prodrug NG on the nanodiscs.

[0063] (II) Characterization of GSH-responsive degradation of nanodiscs

[0064] Experiment 5

[0065] After incubation with PBS (pH 7.4) + 10 mM GSH for 48 hours, the morphology of Comparative Example 2 LNG and Example 1 C-LNG was observed using transmission electron microscopy. Figure 1 As shown in Figure a, the nanoparticles exhibited significant disintegration, with the LNG in Comparative Example 2 and the C-LNG in Example 1 forming irregular shapes. DLS results further confirmed that the LNG and C-LNG increased in size after incubation in PBS (pH 7.4) + 10 mM GSH. Figure 1 As shown in c and 1d, the average sizes of LNG in Comparative Example 2 and C-LNG in Example 1 are 35.76±1.46 nm and 37.95±1.48 nm, respectively, further confirming the decomposition of nanoparticles.

[0066] like Figure 1 As shown in Figure e, the degradation of NG was determined based on the chromatograms showing a decrease or increase in peak area using high-performance liquid chromatography (HPLC), confirming the degradation of NG and the release of gemcitabine from the nanodiscs. Figure 1 As shown in f, after the nanodiscs degraded, the release of gemcitabine was analyzed by liquid chromatography-mass spectrometry (ESI-MS), indicating that gemcitabine was released responsively from NG.

[0067] The NO yield was measured using a NO fluorescent probe in Comparative Example 1 (LG), Comparative Example 2 (LNG), and Example 1 (C-LNG). Figure 1 As shown in g, in PBS (pH 7.4) + 10 mM GSH, NO fluorescence signals were easily detected in Comparative Example 2 LNG and Example 1 C-LNG at 2 h and reached a plateau before 48 h, while almost no NO fluorescence signal was detected in Comparative Example 1 LG. Meanwhile, as... Figure 1 As shown in h, the flow quantification also confirmed this result, indicating that the incorporation of the prodrug NG into the nanodisc led to the efficient generation of NO.

[0068] To further confirm the GSH responsiveness of Comparative Example 2 LNG and Example 1 C-LNG, the intracellular GSH consumption level was measured after incubation in nanodisks. Figure 1 As shown in Figure i, the GSH content in Comparative Example 2 LNG and Example 1 C-LNG cells decreased to 28.64±0.97% and 30.58±0.97%, respectively, while the GSH content in Comparative Example 1 LG cells without NG was 97.09±2.75%. These results reveal the efficient response of nanodiscs to glutathione and their role in promoting degradation.

[0069] (III) In vitro ONOO - Production and lipid peroxidation

[0070] Experiment 6

[0071] When tumor cells are exposed to radiation, the excited electrons further react with O2 to generate O2. − This means that superoxide anion free radicals are generated inside the cell. ROS reacts rapidly with NO to generate more stable and more reactive ONOO. - This is an endogenous nitrogen source. This application measures ONOO under ionizing radiation. - The generation of [something] was observed using a confocal laser scanning microscope (CLSM). For example... Figure 2 As shown in Figure a, significantly stronger fluorescence was observed in cells treated with LNG in RT+ Comparative Example 2 and C-LNG in RT+ Example 1. Furthermore, as... Figure 2 As shown in b, the fluorescent probe detected higher ONOO- fluorescence intensity in LNG of RT+ Comparative Example 2 and C-LNG of RT+ Example 1, further confirming that ONOO- - Efficient generation.

[0072] When exposed to excessive ROS, lipid peroxidation in the cell membrane can effectively disrupt the integrity of tumor cells. However, the limited lipid peroxidation is attributed to the low amount and short half-life of ROS generated by radiation. Therefore, this invention focuses on the more stable and reactive ONOO. - The induced lipid peroxidation capacity was confirmed by detecting lipid peroxidation in tumor cells using CLSM. Figure 2 As shown in Figure c, compared with LG in RT+ Comparative Example 1 and Gem in RT+ Comparative Example 3, the oxidative morphology of tumor cells in RT+ Comparative Example 2 LNG and RT+ Example 1 C-LNG is more pronounced. Furthermore, as... Figure 2 As shown in Figure d, flow cytometry analysis with C11-BODIPY probe staining further confirmed that the ONOO generated by RT+ Comparative Example 2 LNG and RT+ Example 1 C-LNG -This effectively triggered lipid peroxidation. In summary, the addition of NO enabled tumor cells to effectively produce ONOO after radiotherapy. - It can further induce lipid peroxidation, which in turn kills tumor cells.

[0073] (iv) DNA damage and repair

[0074] Experiment 7

[0075] Ionizing radiation can effectively trigger DNA damage and can inhibit ONOO. - DNA repair and oxidative stress further exacerbate DNA damage. For example... Figure 2 As shown in g and 2h, compared with several other comparative examples, the largest DNA damage was observed in the C-LNG of Example 1 with RT+, indicating that the C-LNG nanodiscs of Example 1 can greatly increase X-ray induced DNA damage, thereby inhibiting the proliferation of cancer cells. The C-LNG of Example 1 has a better therapeutic effect on tumors than several other comparative examples.

[0076] Rapid repair of DNA damage after radiotherapy depends on the high expression of homologous recombination repair proteins, and RAD51 is a homologous recombination repair protein highly expressed in glioblastoma. Therefore, inhibiting RAD51 is an effective method to promote DNA damage and lead to tumor cell death. Western blot and immunofluorescence were used to detect the effect of RT+C-LNG from Example 1 on RAD51 expression. Figure 2 As shown in h, due to DNA damage in tumor cells, the expression of the repair protein RAD51 significantly increased after radiotherapy. Simultaneously, RAD51 expression in RT+ Comparative Example 3 Gem was significantly decreased, indicating that gemcitabine can effectively inhibit RAD51 expression, thereby achieving a radiosensitizing effect. RAD51 expression was further reduced in RT+ Comparative Example 2 LNG and RT+ Example 1 C-LNG. Therefore, the abnormal RAD51 expression in RT+ Example 1 C-LNG is attributed to ONOO. - The generation of this substance confirms that C-LNG from Example 1 can effectively inhibit the high expression of RAD51 after radiotherapy, promote DNA damage, and ultimately promote the death of tumor cells.

[0077] (v) Targeting capability of nanodisks

[0078] Experiment 8

[0079] The distribution of C-HDL in an orthotopic brain tumor model was determined using an in vivo imaging system to confirm that nanodiscs modified with ApoA1 and CCL5 mimic peptides can effectively drive nanodisc targeting of tumor tissue. Liposomes (Lipo) without ApoA1 mimic peptides and HDL nanodiscs without CCL5 mimic peptides served as negative controls. Tumor size was validated using bioluminescence immunoassay, and tumors were grouped according to size. Figure 3 As shown in Figure a, 2 hours after injection, both HDL and C-HDL (HDL nanodisks containing CCL5 mimic peptides) showed clearly observable fluorescence signals in the brain, but no signal was detected in Lipo. This indicates that the ApoA1 mimic peptide can effectively deliver HDL nanodisks into the brain. Figure 3 As shown in b, at specific time points after injection, the cumulative fluorescence intensity of C-HDL in the brain was significantly higher than that of HDL, reaching its maximum at 12 h, indicating that the homing effect mediated by CCL5 mimic peptide modification in C-HDL significantly enhances the targeting efficiency.

[0080] Major organs were collected 12 hours after injection for in vivo imaging. Figure 3 As shown in c and 3d, in vitro fluorescence images show no difference in fluorescence intensity among the heart, liver, spleen, lungs, and kidneys, while the fluorescence intensity of C-HDL in brain tissue is significantly higher than that of HDL and Lipo, further confirming the high accumulation of C-HDL in tumor tissue. The dual targeting of ApoA1 and CCL5 gives the nanodiscs extremely high targeting ability.

[0081] Furthermore, this invention uses anti-CD31 antibody to stain whole brain slices, marking tumor blood vessels, and then visualizes them under CLSM to demonstrate the accessibility of nanodisks in the tumor region. Figure 3 As shown in Figure e, abundant C-HDL fluorescence signals were observed within the entire brain tumor, while no signal was observed in normal brain tissue. Weaker fluorescence signals were observed in the tumor regions containing HDL and Lipo. These results indicate that nanodisks with chemokine homing effects can reach the interior of the tumor region, thereby improving the delivery efficiency of the nanoplatform.

[0082] This application further confirms the accessibility of C-HDL nanodisks in tumor regions and their inability to accumulate in normal brain tissue by using healthy animals and GBM models with different blood-brain barrier (BBB) ​​disruptions. Figure 4As shown in b and 4c, 12 h after administration, C-HDL fluorescence signals were visible in the brains of healthy mice, but no significant accumulation of nanoparticles was observed. These results are consistent with the previous findings, demonstrating that C-HDL can hardly cross the normal BBB and therefore cannot accumulate in normal brain tissue to exert toxic effects. Simultaneously, strong fluorescence signals were detected in the brains of GBM-bearing tumor models, further validating the accessibility of the designed nanoplatform to tumors across the blood-brain tumor barrier (BTBB).

[0083] (vi) Safety evaluation of nanodisks

[0084] Experiment 9

[0085] Blood samples were collected from gl261 orthotopic tumor-bearing mice after various treatments, and hematological and blood biochemical tests were performed. For example... Figure 4 As shown in d, no obvious abnormalities were observed in the H&E staining images of each group, indicating that the nanodiscs have very low toxicity, good biocompatibility, and can be used for tumor treatment in humans.

[0086] III. Experiments demonstrating the effectiveness of nanodisks

[0087] (a) In vitro cytotoxicity

[0088] Experiment 10

[0089] Colony assays were conducted using GL261 cells to investigate the inhibitory effects of different components on tumor cells. For example... Figure 2 As shown in Figure e, compared with LG in RT+ Comparative Example 1 and Gem in RT+ Comparative Example 3, LNG in RT+ Comparative Example 2 and C-LNG in RT+ Example 1 were found to have greater inhibitory effects on cell proliferation. Simultaneously, cell viability assays were performed in this application, such as... Figure 2 As shown in f, Gem in RT+ Comparative Example 3 effectively killed tumor cells. In RT+ Comparative Example 1 LG, hydrophobically modified gemcitabine lipoprotein nanodiscs were more easily phagocytosed by cells, leading to more cell death. Furthermore, the cell viability of RT+ Comparative Example 2 LNG and RT+ Example 1 C-LNG decreased to 29.08±4.34% and 26.82±2.97%, respectively, due to the generation of ONOO. - It is toxic and can further inhibit tumor growth. In summary, these results indicate that the C-LNG of RT+ Example 1 can be controlled by gemcitabine and ONOO. - The strong radiosensitizing effect produced effectively inhibits the proliferation of tumor cells.

[0090] (II) Therapeutic effects of tumors

[0091] Experiment 11

[0092] like Figure 5 As shown in Figure a, gl261 tumor-bearing nude mice were randomly divided into 6 groups. The prepared nanodiscs were injected via the tail vein on days 10, 12, 14, and 16 after tumor transplantation. Twelve hours after each injection, the mice in the irradiation group were irradiated with 2 Gy, 6MV X-rays to further confirm that the biomimetic nanodiscs effectively improve radiosensitivity by inhibiting DNA repair and inducing lipid peroxidation, ultimately promoting tumor death.

[0093] The size of tumors in orthotopic Luci+gl261 tumor-bearing mice was initially monitored using bioluminescence imaging technology. Figure 5 As shown in b, the sustained increase in bioluminescence intensity during treatment with Gem in RT+ Comparative Example 3 indicates a lower inhibitory efficiency against tumor proliferation after radiotherapy. Tumor growth in RT+ Comparative Example 1 (LG) was significantly delayed, suggesting that nanodisk-mediated functionalization effectively inhibits tumor proliferation by delivering gemcitabine to the tumor site. Furthermore, the therapeutic effect of LNG in RT+ Comparative Example 2 is significantly better than that of LG in RT+ Comparative Example 1, due to its temporal and spatially distributed ONOO... - It produces toxicity in the tumor area. For example... Figure 5 As shown in c, quantitative analysis of bioluminescence intensity further revealed that C-LNG in RT+ Example 1 had the most significant inhibitory effect on tumor growth, demonstrating that the homing effect of CCL5 can effectively drive nanodisks to the tumor, thereby killing tumor cells.

[0094] Magnetic resonance imaging (MRI, T1-weighted images) was acquired 30 days after tumor implantation, such as... Figure 5 As shown in Figure d, the results indicate that RT+C-LNG from Example 1 almost completely inhibited glioblastoma derived from GL261 cells. Figure 5 As shown in f, 30 days post-surgery, the tumor volume in the PBS group (in this study, the PBS group refers to the blank experimental group treated with PBS) was (139.60±18.73) mm. 3 The tumor volume in the RT group (in this article, the RT group refers to the control group treated with high-energy ionizing radiotherapy) was (47.07±7.78) mm. 3 The tumor volume of RT+ Example 1 C-LNG was (0.52±0.29) mm. 3 In addition, such as Figure 5 As shown in e and 5g, the whole brain hematoxylin-eosin (H&E) staining images also showed that glioblastoma treated with RT+ Example 1 C-LNG was almost completely suppressed.

[0095] Compared to the LNG in RT+ Comparative Example 2, the C-LNG in RT+ Example 1 showed an enhanced effect in prolonging the survival of glioblastoma-bearing mice, thanks to the efficient delivery of the nanodiscs. Figure 5 As shown in h, compared with mice treated with inactivated PBS (32 days), RT (40 days), RT + Comparative Example 3 Gem (47 days), and RT + Comparative Example 1 LG (60 days), mice treated with RT + Comparative Example 2 LNG had a median survival of 80 days, and mice treated with RT + Example 1 C-LNG had a median survival of over 120 days, demonstrating a significant therapeutic response. In summary, these results confirm that RT + Example 1 C-LNG has a significant beneficial effect in inhibiting tumor growth and reducing tumor survival in GBM orthotopic tumor-bearing mouse models.

[0096] (III) Tumor Suppression Response

[0097] Experiment 12

[0098] Tumor cell damage response was assessed using in situ terminal transferase labeling (TUNEL) and γ-H2AX immunofluorescence staining. Figure 6 As shown in Figure a, compared with several other comparative examples, C-LNG in RT+ Example 1 induced the highest levels of apoptosis and nuclear damage in tumor cells, further demonstrating the amplified radiotherapy efficacy due to the massive DNA damage caused by the biomimetic lipoprotein-mimicking nanodiscs.

[0099] The generated ONOO - More toxic than most free radicals, lipid peroxidation damages membrane structures and promotes cell death. Therefore, this invention further investigated the levels of lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) in tumors treated with different therapies. Figure 6 As shown in Figure a, 4-HNE immunofluorescence staining revealed the most significant lipid peroxidation detected in the RT+ Example 1 C-LNG treatment. Furthermore, as... Figure 6 As shown in b, compared with several other comparative examples, the determination of MDA levels indicates that the ONOO generated by RT+ Example 1 C-LNG − It induced significant lipid peroxidation.

[0100] Elevated expression of the homologous recombination repair protein RAD51 after radiotherapy in GBM patients is closely related to survival. Therefore, this application verifies that the functionalized nanodisc system can downregulate RAD51 expression in vivo, thereby effectively improving radiotherapy efficacy. Figure 6 As shown in Figure a, immunohistochemical analysis revealed a significant reduction in RAD51 expression in RT+ Example 1 C-LNG. Simultaneously, flow cytometry analysis was performed in this invention, as shown... Figure 6As shown in Figure c, the results indicated that the expression level of RAD51 was significantly increased in the radiotherapy group compared to the PBS group. Figure 6 As shown in Figure d, the ONOO released by RT+ Comparative Example 2 LNG and RT+ Example 1 C-LNG - It can not only directly induce lipid peroxidation, but also inhibit the expression of the DNA repair protein RAD51, thereby exacerbating DNA damage. For example... Figure 6 As shown in Figure e, ELISA data further confirmed that RT+ Example 1 C-LNG exhibited the lowest RAD51 expression, indicating that effective tumor treatment with radiosensitivity can be achieved by inhibiting DNA repair. In summary, Example 1 C-LNG derived from a NO donor-gemcitabine conjugate can enhance radiosensitivity by aggravating DNA damage and inducing lipid peroxidation.

[0101] Experiment 13

[0102] Based on the good antitumor effect of RT+C-LNG in the GL261 glioblastoma model, this application further verifies the radiosensitizing effect of C-LNG in Example 1 on U87 orthotopic human glioblastoma. The established glioblastoma model was divided into an RT group and an RT+Example 1 C-LNG group, and head MRI of mice was collected on day 30. Figure 6 As shown in f and 6g, the intracranial tumor volume in the RT group mice after radiotherapy was (75.03±15.46) mm. 3 The C-LNG from RT+ Example 1 showed a significant inhibitory effect on tumor development, with a tumor volume of (11.77±12.28) mm. 3 In summary, the C-LNG from Example 1 also exhibited good anti-tumor effects in the U87 mouse model, indicating that biomimetic lipoprotein nanodiscs can be a good strategy for improving GBM radiotherapy.

[0103] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing biomimetic lipoprotein nanodisks, comprising the preparation of NG and the preparation of C-LNG, characterized in that, The preparation of NG includes the following steps: (1) 5.0 g of 3,4-di(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide and 2.0 g of 7-hydroxyheptanoic acid were dissolved in 30 mL of dichloromethane, and then mixed with 4.1 g of 1,8-diazobicyclo[5,4,0]undecyl-7-ene; the mixture was stirred at 25 °C for 8 h, and the reaction solution was concentrated under reduced pressure to a yellow liquid, and then extracted with ethyl acetate and saturated brine; after the organic layer was concentrated, it was eluted with silica gel column and purified with ethyl acetate / petroleum ether at a volume ratio of 10:1 to obtain the compound; (2) 2.6 g of the above compound, 1.9 g of N,N-diisopropylethylamine and 3.2 g of HATU were added to 20 mL of N,N-dimethylaniline and stirred at 25 °C for 0.5 h. Then 2.0 g of gemcitabine was added and stirred for 10 h. The reaction solution was concentrated under reduced pressure to obtain a yellow solid. The residue was then extracted with ethyl acetate and water. Finally, the bound organic layer was concentrated to obtain the NG product. The preparation of C-LNG includes the following steps: (1) CCL5 peptide and DSPE-PEG2000-mal were dissolved in DMF and triethylamine, stirred at room temperature, and then the reaction solution was purified by dialysis and freeze-dried to obtain white solid powder DSPE-PEG-CCL5; (2) Take methanol in a flask, then dissolve DPPC, DSPE-PEG2000-CCL5 and NG in methanol, then add acetic acid, evaporate to dryness, and form a film in the flask; (3) The membrane was then dispersed with ultrapure water, sonicated in an ice bath with a probe, and then ApoA1 peptide was added to the mixed solution for hot and cold cycling to obtain C-LNG.

2. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 1, characterized in that: The mass of the CCL5 peptide in step (1) is 14-20 mg.

3. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 2, characterized in that: The mass of DSPE-PEG2000-mal in step (1) is 22-26 mg.

4. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 3, characterized in that: The stirring time in step (1) is 16-28 h.

5. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 4, characterized in that: In step (2), the weight ratio of DPPC, DSPE-PEG2000-CCL5 and NG is 6:3:

1.

6. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 5, characterized in that: The acetic acid in step (2) is 0.1-0.5 ml.

7. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 6, characterized in that: The ultrasound time in step (3) is 1-4 minutes.

8. The method for preparing the biomimetic lipoprotein nanodiscs as described in claim 7, characterized in that: The temperature of the hot and cold cycle in step (3) is 4℃~50℃.

9. The use of the biomimetic lipoprotein nanodiscs prepared by any one of the preparation methods according to claims 1-8 in the preparation of drugs for treating glioblastoma multiforme.

Images