Medicine for treating brain glioma and preparation method thereof

Abstract

The present invention discloses a drug for treating brain gliomas and a method for preparing the drug, relating to the field of biomaterials. By combining the advantages of bacteria, natural exosomes, and synthetic liposomes, the present invention prepares a modular bacterium consisting of a blood-brain barrier targeting module, a photothermal module, and a ROS consumption module. The drug exhibits excellent blood-brain barrier crossing, targeting, photothermal conversion, and ROS consumption capabilities, enabling brain-targeted delivery, increasing tumor antigens, and reducing excess ROS in the tumor microenvironment, further enhancing the efficacy of immune checkpoint inhibitors against brain gliomas.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a medicine for treating brain glioma and a preparation method thereof. Background Art

[0002] Gliomas are serious tumors of the central nervous system. The current general treatment principle for gliomas is to surgically remove the tumor, supplemented by radiation or chemotherapy, and a comprehensive treatment plan for malignant gliomas that uses both radiotherapy and chemotherapy. Chemotherapy, together with surgery and postoperative radiotherapy, has become an important means of comprehensive treatment for central nervous system tumors. Due to the characteristics of gliomas such as blurred boundaries, high heterogeneity, and invasiveness, traditional treatments such as surgery and radiotherapy have limited therapeutic effects, are often harmful to normal tissues, lack tumor-selective treatment, and generally have poor prognosis, short survival, easy metastasis, and recurrence. As a new treatment method, tumor immunotherapy regulates the inherent activity of the immune system to stimulate the ability of immune cells to target malignant cells, thereby playing a role in controlling and eliminating tumor cells, and is expected to contribute to the treatment of gliomas.

[0003] Immunotherapy for glioma currently faces the following challenges:

[0004] (1) Although immune checkpoint inhibitors are the most widely used immunotherapy for glioma, their efficacy is poor. The lack of tumor antigens makes it difficult for the brain's immune cells to produce an immune response to glioma, which limits the efficacy of immunotherapy and makes it difficult to judge the treatment effect.

[0005] (2) Blood-brain barrier. It is composed of continuous brain endothelial cells, astrocytes, microglia and pericytes. It exists to protect the brain from the invasion of viruses and toxins in the blood circulation. However, this barrier also prevents cancer drugs from entering the brain.

[0006] (3) The highly immunosuppressive tumor microenvironment, especially excessive reactive oxygen species (ROS), puts survival pressure on immune cells.

[0007] Therefore, the following points are expected to regulate the immunosuppressive microenvironment of glioma and improve the sensitivity of glioma to immunotherapy:

[0008] (1) Rationally introduce synergistic therapies to increase tumor antigens and enhance the effects of immune checkpoint inhibitors on gliomas;

[0009] (2) Improve the ability to cross the blood-brain barrier and target the tumor microenvironment;

[0010] (3) Reduce excessive ROS in the tumor microenvironment.

[0011] Bacterial materials have been used in bioimaging, diagnosis, and treatment due to their wide availability, small size, and the fact that the total number of bacterial cells in the human body and on the epidermis is approximately ten times the total number of human cells. Bacteria can induce the body to produce inflammatory factors to enhance the body's innate protective immune response. By utilizing the natural toxicity of bacteria, combining with other therapies, regulating the expression of anticancer agents, and regulating the expression of tumor-specific antigens, bacteria can be modified to fight cancer. The brain environment in which gliomas reside is more complex than that of subcutaneous or other tissue tumors. How to modularly customize bacterial materials to achieve precise targeting and application of brain gliomas is currently the key to the use of bacterial materials in brain glioma immunotherapy. Summary of the Invention

[0012] Based on the deficiencies in the prior art, the purpose of the present invention is to provide a drug for treating brain glioma and a preparation method thereof; another purpose of the present invention is to provide a treatment strategy of bacteria combined with immune checkpoint inhibitors to improve the immunotherapy effect of brain glioma.

[0013] The present invention intends to use Porphyromonas gingivalis (Pg) as the main bacteria, encapsulate fusogenic liposomes, and design a modular bacteria that realizes functions in modules for the study of immunotherapy of glioma. The modular bacteria are mainly composed of a cross-blood-brain barrier targeting module, a photothermal module, and a ROS consumption module. Through the dual targeting effect of transferrin and microglial exosomes, the bacteria are more likely to be absorbed by M2 macrophages and glioma cells; through the characteristics of Pg itself containing melanin, under laser irradiation, cancer cells and tumorigenic M2 macrophages are reduced, and the lysed tumor self-antigens and Pg can be used as supplementary antigens; through the active oxygen scavenging ability of hemoglobin, ROS in the microenvironment is reduced, the pressure on the survival of immune cells is alleviated, and the surviving M1 macrophages in the glioma microenvironment participate in antigen presentation. At the same time, the use of the immune checkpoint inhibitor anti-PD-1 is combined to further enhance the anti-tumor immune response of T cells.

[0014] The technical solutions of the present invention are as follows:

[0015] A drug for treating brain glioma comprises modular bacteria, wherein the modular bacteria are Porphyromonas gingivalis, heme, transferrin and exosomes encapsulated together by lipid membrane fusion.

[0016] Preferably, the heme is a heme liposome coupled to a liposome, and the transferrin is a transferrin liposome coupled to a liposome. The heme liposome is prepared by an esterification reaction, and the transferrin liposome is prepared by a click chemistry reaction.

[0017] Preferably, the number of Porphyromonas gingivalis in the modular bacteria is 1×10 7For CFU calculation, 20 μg of heme, 20 μg of transferrin, and 20 μg of exosomes were added.

[0018] The exosomes are microglial-derived exosomes.

[0019] On the other hand, the drug for treating brain glioma also includes immune checkpoint inhibitors. When used, the modular bacteria and the immune checkpoint inhibitors are used in combination.

[0020] The present invention also provides a system for treating brain glioma, comprising the drug for treating brain glioma and a device for performing photothermal therapy.

[0021] When used for treatment, the drug for treating brain glioma is administered to the individual to be treated, and then the device for photothermal therapy is used to perform laser irradiation therapy on the brain tumor location of the individual to be treated.

[0022] The present invention also provides a method for preparing a drug for treating brain glioma, wherein the method is a method for preparing the modular bacteria, comprising the following steps:

[0023] (1) Extract and purify exosomes;

[0024] (2) Phospholipid-polyethylene glycol-hydroxyl group and heme are esterified to obtain heme liposomes;

[0025] (3) Diphenylcyclooctyne-tetraethylene glycol-active ester, transferrin, and phospholipid-polyethylene glycol-azide undergo click chemistry reaction to obtain transferrin liposomes;

[0026] (4) mixing the prepared exosomes, heme liposomes, and transferrin liposomes to react, and then co-extruding with a liposome extrusion membrane to prepare exosome-heme liposome-transferrin liposomes;

[0027] (5) Porphyromonas gingivalis is uniformly mixed with the exosome-heme liposome-transferrin liposome prepared in step (4), and the modular bacteria are prepared by co-extrusion through a liposome extrusion membrane.

[0028] The mass ratio of phospholipid-polyethylene glycol-hydroxyl group to hemoglobin is 25:4. The liposome extrusion membrane is a polycarbonate porous membrane.

[0029] The thickness of the liposome extrusion membrane in step (4) is 200 nm.

[0030] The thickness of the liposome extrusion membrane in step (5) is 1 μm.

[0031] The specific preparation method of the modular bacteria (Pg@EV-HT) involved in the present invention is as follows:

[0032] (1) The culture supernatant of mouse microglia (BV-2) was collected, possible cells and cell debris were removed, and the exosome components were precipitated and purified using a purification column, namely EV;

[0033] (2) Weigh 100 mg of DSPE-PEG-OH, 16 mg of hemin, 4.58 mg of 4-dimethylaminopyridine (DMAP), and 23 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) respectively and place them in a 100 mL round-bottom flask. Use 0.5 mL of N,N-dimethylformamide (DMF) to promote dissolution. Add 50 mL of dichloromethane (CH2Cl2) and react in anhydrous and oxygen-free room temperature in the dark for 48 h. After the reaction is completed, evaporate the mixture and wash it with CH2Cl2 three times. After dialyzing it in a 2000 MW dialysis bag, freeze-dry it for use. This is hemin liposomes (Hemsome);

[0034] (3) Dissolve transferrin (Tf) solution in PBS and react with DBCO-PEG4-NHS at a concentration 10 times that of the Tf solution. Place on a shaker in the dark and react at 300 rpm for 1 h at room temperature. Centrifuge at 7000 rpm for 30 min using a 30 kDa ultrafiltration tube to remove excess DBCO-PEG4-NHS. Mix 100 mg of DSPE-PEG-N3 with the product obtained in the previous step and react at 300 rpm for 1 h at room temperature to form transferrin liposomes (Tfsomes).

[0035] (4) Hemsomes, Tfsomes, and EVs were mixed and reacted at room temperature at a speed of 300 rpm for 1 h. EV-HT was prepared by co-extrusion using a 200 nm polycarbonate film;

[0036] (5) The solution from the culture medium containing Pg was aspirated and centrifuged, washed and resuspended in cold 1× PBS, then mixed with EV-HT and extruded multiple times through a 1 μm polycarbonate porous membrane using an Avanti liposome extruder. The excess EV-HT membrane was removed by centrifugation to obtain the modular bacterial Pg@EV-HT consisting of a trans-blood-brain barrier targeting module, a photothermal module, and a ROS consumption module.

[0037] The preparation method of modular bacteria composed of a blood-brain barrier targeting module, a photothermal module, and a ROS consumption module and its application in enhancing glioma immunotherapy have the following advantages:

[0038] The preparation of modular bacteria of the present invention uses bacteria, microglial exosomes, heme and transferrin as raw materials. Microglial exosomes are obtained by extraction, heme liposomes are obtained by esterification reaction, and transferrin liposomes are obtained by click chemistry reaction. The modular bacteria are then co-extruded with mechanical force of Pg to wrap an outer membrane on the surface of the bacteria to obtain modular bacteria.

[0039] The modular bacteria prepared by the present invention have excellent properties required for the enhancement of brain glioma immunotherapy, including crossing the blood-brain barrier, targeting cancer cells and M2 macrophages in the tumor microenvironment, photothermal and ROS consumption, and further enhancing the anti-tumor immune response of anti-PD-1-involved T cells by promoting the participation of M1 macrophages in antigen presentation.

[0040] Beneficial effects of the present invention:

[0041] The present invention combines the respective advantages of bacteria, natural exosomes and synthetic liposomes to prepare modular bacteria consisting of a blood-brain barrier targeting module, a photothermal module and a ROS consumption module. The bacteria have good blood-brain barrier crossing ability, good targeting ability, photothermal conversion ability and ROS consumption ability, and can achieve brain-targeted delivery, increase tumor antigens and reduce excessive ROS in the tumor microenvironment, further enhancing the efficacy of immune checkpoint inhibitors on brain gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the synthetic route of Hemsome;

[0043] Figure 2 The synthetic route of Tfsome;

[0044] Figure 3 are the transmission electron microscopy images of EV-HT, Pg, and Pg@EV-HT, with scales of 100 nm, 500 nm, and 500 nm, respectively;

[0045] Figure 4 is the H NMR spectrum of Hemsome;

[0046] Figure 5 is the infrared spectrum of Tfsome, and the circle represents the structure of triazole;

[0047] Figure 6 (A) Protein bands of EV, Hemsome, and Tfsome; (B) Image of EV-carrying marker proteins on the surface of Pg@EV-HT; (C) Particle sizes of Pg and Pg@EV-HT;

[0048] Figure 7 is the situation of Pg, EV-HT and Pg@EV-HT crossing the blood-brain barrier;

[0049] Figure 8 is the case for uptake of modular bacteria by macrophages of different phenotypes;

[0050] Figure 9 Infrared thermal imaging; (A) Infrared thermal imaging of Pg, EV-HT, and Pg@EV-HT solutions without or with 808 nm laser irradiation; (B) Infrared thermal imaging of mice treated with Pg, EV-HT, and Pg@EV-HT without or with 808 nm laser irradiation;

[0051] Figure 10 Pg, EV-HT and Pg@EV-HT were treated with 808 nm laser (0.33 W cm -2 , 10 min) after irradiation of cells with ROS levels;

[0052] Figure 11 Figure 3 (A) T2W-MRI images of the brains of GL261 tumor-bearing mice in the control group (Ctrl), immune checkpoint inhibitor treatment group (I), modular bacteria treatment group (P), and immune checkpoint inhibitor combined with modular bacteria treatment group (P&I); (B) the M1 / M2 ratio of each group after treatment; (C) the number of T cells in the tumor microenvironment of each group after treatment. DETAILED DESCRIPTION

[0053] Example 1

[0054] Modular bacterial artificial liposome parts:

[0055] (1) The culture supernatant of mouse microglia (BV-2) was collected, possible cells and cell debris were removed, and the exosome components were precipitated and purified using a purification column, namely EV;

[0056] (2) Weigh 100 mg of DSPE-PEG-OH, 16 mg of hemin, 4.58 mg of 4-dimethylaminopyridine (DMAP), and 23 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) respectively and place them in a 100 mL round-bottom flask. Use 0.5 mL of N,N-dimethylformamide (DMF) to promote dissolution. Add 50 mL of dichloromethane (CH2Cl2) and react in anhydrous and oxygen-free room temperature in the dark for 48 h. After the reaction is completed, evaporate the mixture and wash it with CH2Cl2 three times. After dialyzing it with a 2000 MW dialysis bag, freeze-dry it for use. This is hemin liposomes (Hemsome). The synthesis steps are as follows: Figure 1 As shown;

[0057] (3) Dissolve transferrin (Tf) solution in PBS, react with DBCO-PEG4-NHS concentration 10 times that of Tf solution, place on a shaker in the dark, react at 300 rpm at room temperature for 1 h, centrifuge at 7000 ffmin using a 30 kDa ultrafiltration tube for 30 min to remove excess DBCO-PEG4-NHS, mix 100 mg of DSPE-PEG-N3 with the product obtained in the previous step, and react at 300 rpm at room temperature for 1 h to obtain transferrin liposomes (Tfsome); the synthesis steps are as follows: Figure 2 As shown;

[0058] (4) Hemsome, Tfsome, and EV were mixed in a mass ratio of 1:1:1, reacted at room temperature at a speed of 300 rpm for 1 h, and co-extruded using a 200 nm polycarbonate film to prepare EV-HT;

[0059] (5) From the content of 1×10 7 1 mL of Pg culture medium containing 500 CFU / mL was aspirated and centrifuged, washed, and resuspended in 1 mL of cold 1× PBS. It was then mixed with 1 mL of EV-HT at different concentrations (1, 2, 3, and 4 mg / mL) and extruded 11 times through a 1 μm polycarbonate porous membrane using an Avanti liposome extruder. The excess EV-HT membrane was removed by centrifugation to obtain the modular bacterial Pg@EV-HT consisting of a blood-brain barrier targeting module, a photothermal module, and a ROS consumption module.

[0060] The obtained modular bacteria Pg@EV-HT was characterized by transmission electron microscopy. 20 μL of sample was dropped on a copper grid. After the sample was dried naturally, the morphology was observed under a transmission electron microscope. Figure 3 As shown, it can be seen that the particle size of EV-HT is about 100 nm, and there is an obvious coating on the surface of modular bacteria.

[0061] DMSO-d6 was used as solvent. 1 H-NMR spectrum analysis of the successful preparation of hemoglobin ( Figure 4 ). Tfsome passes through the infrared region 4000 to 400 cm -1 The FT-IR spectrum was used for analysis ( Figure 5 ). The main protein bands of the modular bacterial coating were detected by SDS-PAGE ( Figure 6 A), transferrin of about 75kDa and exosome proteins can be seen. CD63 and TSG101 were used as markers to detect microglial exosomes on the surface of modular bacteria ( Figure 6 B), CD63 and TSG101 are present on the surface of modular bacteria. The particle size of Pg and Pg@EV-HT was measured using dynamic light scattering ( Figure 6 C), it can be seen that the particle size of the bacteria becomes slightly smaller after being extruded by the membrane, indicating that they are compressed.

[0062] Example 2

[0063] Analysis of modular bacteria crossing the blood-brain barrier:

[0064] The Transwell system was used to construct an in vitro blood-brain barrier (BBB) model. bEnd.3 cells were cultured at 5×10 4 The cells were seeded in the upper chamber at a density of 100 cells / cm. Blank culture medium was placed in the lower chamber. The transmembrane resistance of the bEnd.3 cells was measured daily using a transmembrane resistance meter. When the resistance reached and stabilized at 200 Ω / cm, the cells were plated. 2 The above was observed under a microscope to ensure the construction of the in vitro simulated blood-brain barrier. Cancer cells were added to the lower chamber of Transwell one day in advance, and FITC-labeled Pg, EV-HT and Pg@EV-HT were added the next day. After incubation for 3 hours, the cells in the lower chamber were collected and the proportion of cells expressing fluorescence was recorded by flow cytometry to study the penetration efficiency of the blood-brain barrier. Figure 7 It can be seen that the bottom glioma cells treated with Pg@EV-HT showed more green fluorescence compared with the Pg group and EV-HT group, which means that EV-HT modification can help Pg cross the BBB.

[0065] Example 3

[0066] Analysis of Modular Bacterial Uptake by Macrophages of Different Phenotypes:

[0067] FITC-labeled Pg, EV-HT, and Pg@EV-HT were added to macrophages of different phenotypes. After incubation for 3 hours, the cells were collected and the proportion of cells expressing fluorescence was recorded by flow cytometry to study the uptake of modular bacteria by macrophages of different phenotypes. Figure 8 As can be seen, there were no significant differences in Pg, EV-HT, and Pg@EV-HT in M0 cells. However, for M1 cell uptake, the Pg@EV-HT group showed a significant decrease compared to the Pg group. Meanwhile, M2 cell uptake of Pg@EV-HT was significantly increased compared to Pg, indicating that the bacterial coating favors M2 targets.

[0068] Example 4

[0069] Investigation of the photothermal performance of modular bacteria:

[0070] Pg, EV-HT and Pg@EV-HT solutions were placed in EP tubes respectively and illuminated with a near-infrared light source at 808 nm at 0.33 W / cm 2The power of 808 nm laser was irradiated for 10 min, and the temperature change of the solution before and after 808 nm laser irradiation was recorded by thermal imaging. Figure 9 As can be seen from Figure 5, the temperature of the Pg and Pg@EV-HT solutions can reach nearly 50 °C within 10 min.

[0071] The mice with brain glioma were given Pg, EV-HT or Pg@EV-HT solution intranasally. After 3 h, the mice were illuminated with a near-infrared light source at 808 nm at 0.33 W / cm 2 The brain tumor was irradiated with a power of 10 min, and the temperature changes of the mouse brain before and after laser irradiation were recorded by a thermal imager. Figure 9 B shows that the modular bacteria in the body can raise the temperature to 42.4°C, achieving mild photothermal therapy.

[0072] The above results indicate that modular bacteria have good photothermal properties in vitro and can be used for mild photothermal therapy in vivo.

[0073] Example 5

[0074] Investigation of ROS scavenging performance of modular bacteria:

[0075] Confocal microscopy was used to examine BV2 cells pretreated with Rosup. DCFH-DA dye was used to label cellular ROS content. Figure 10 , the fluorescence of ROS reflected by the modular bacteria-treated cell group was significantly weakened, showing good ROS scavenging performance.

[0076] Example 6

[0077] Study on the synergistic effect of modular bacteria combined with immune checkpoint inhibitors:

[0078] C57BL / 6J mice were loaded with GL261 glioma cells (2×10 5 cells / mouse). The day of tumor implantation was defined as day 0. After 7 days, the mice were given PBS, immunotherapy (anti-PD-1), photothermal therapy (Pg@EV-H-T+808nm laser), and photothermal therapy & immunotherapy (anti-PD-1+Pg@EV-H-T+808nm laser). The treatment was repeated every three days for a total of four times. On day 21, T2-weighted magnetic resonance imaging scans were performed on representative mice in each group. The results are shown in the figure. Figure 11A, The tumor size of GL261-bearing mice treated with immunotherapy or photothermal therapy was smaller than that of the control mice. Due to the resolution of T2W-MRI, it was even difficult for us to detect the tumor area in the photothermal therapy & immunotherapy group, which means that modular bacteria showed the best effect. After imaging, the brains of mice in each group were removed, weighed, and 500μL PBS was added to further lyse them into cell populations. The cells were resuspended in antibody staining buffer. APC-CD11b, FITC-CD206, Percp / Cy5.5-CD80 and PE-CD86 were used as macrophage surface markers. FITC-CD3, PE-CD4 and Percp / Cy5.5-CD8 were used as T cell surface markers. , from Figure 11 As can be seen from B, the ratio of M1 / M2 increased after treatment in the anti-PD-1+Pg@EV-H-T+808nm laser group. Figure 11 C It can be seen that modular bacteria combined with immune checkpoint inhibitors promoted the increase in the number of T cells in the tumor microenvironment.

Claims

1. A drug for treating brain glioma, characterized in that: The invention comprises modular bacteria, wherein the modular bacteria are Porphyromonas gingivalis, heme, transferrin and exosomes wrapped together by lipid membrane fusion; The method for preparing modular bacteria comprises the following steps: (1) Extraction and purification of exosomes; (2) Phospholipid-polyethylene glycol-hydroxyl and heme are esterified to obtain heme liposomes; (3) Diphenylcyclooctyne-tetraethylene glycol-active ester, transferrin, and phospholipid-polyethylene glycol-azide undergo click chemistry reaction to obtain transferrin liposomes; (4) mixing the prepared exosomes, heme liposomes, and transferrin liposomes to react, and then co-extruding them using a liposome extrusion membrane to prepare exosome-heme liposome-transferrin liposomes; (5) Porphyromonas gingivalis is mixed evenly with the exosome-heme liposome-transferrin liposome prepared in step (4), and the modular bacteria are prepared by co-extrusion through a liposome extrusion membrane.

2. The drug for treating brain glioma according to claim 1, characterized in that: The number of Porphyromonas gingivalis in modular bacteria is 1×10 7 For CFU calculation, 20 μg heme, 20 μg transferrin, and 20 μg total protein of exosomes were added.

3. The drug for treating brain glioma according to claim 1, characterized in that: The exosomes are microglial-derived exosomes.

4. The drug for treating brain glioma according to claim 1, characterized in that: It also includes immune checkpoint inhibitors. When used, the modular bacteria and the immune checkpoint inhibitors are used in combination.

5. A system for treating brain glioma, characterized in that: Comprising the drug for treating brain glioma according to any one of claims 1 to 4, and a device for performing photothermal therapy, When used for treatment, the drug for treating brain glioma is administered to the individual to be treated, and then the device for photothermal therapy is used to perform laser irradiation therapy on the brain tumor location of the individual to be treated.

6. The method for preparing the drug for treating brain glioma according to any one of claims 1 to 4, characterized in that: The method for preparing modular bacteria comprises the following steps: (1) Extraction and purification of exosomes; (2) Phospholipid-polyethylene glycol-hydroxyl and heme are esterified to obtain heme liposomes; (3) Diphenylcyclooctyne-tetraethylene glycol-active ester, transferrin, and phospholipid-polyethylene glycol-azide undergo click chemistry reaction to obtain transferrin liposomes; (4) mixing the prepared exosomes, heme liposomes, and transferrin liposomes to react, and then co-extruding them using a liposome extrusion membrane to prepare exosome-heme liposome-transferrin liposomes; (5) Porphyromonas gingivalis is mixed evenly with the exosome-heme liposome-transferrin liposome prepared in step (4), and the modular bacteria are prepared by co-extrusion through a liposome extrusion membrane.

7. The preparation method according to claim 6, characterized in that The mass ratio of phospholipid-polyethylene glycol-hydroxyl group and heme is 25:

4.

8. The preparation method according to claim 6, characterized in that The liposome extrusion membrane is a polycarbonate porous membrane, The thickness of the liposome extrusion membrane in step (4) is 200 nm. The thickness of the liposome extrusion membrane in step (5) is 1 μm.

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