Preparation method and application of bacterial membrane-liposome drug delivery system
The bacterial membrane-liposome drug-carrying system was prepared by blending bacterial membranes and phospholipids, and the problem of insufficient tumor targeting and immune stimulation efficacy was solved, and efficient targeting of tumor tissues after radiotherapy was achieved Delivery and immune response activation significantly improve the therapeutic effect.
Patent Information
- Application Number
- CN202310337764.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-31
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2043-03-31
AI Technical Summary
The existing liposome drug-loading system has shortcomings in tumor targeting and its own anti-tumor immune stimulation efficacy, and the extraction process of bacterial outer membrane is complex and inefficient, making it difficult to act as an effective drug carrier.
Bacterial membrane-liposome drug-loading system was prepared by blending bacterial membrane with phospholipids, using specific mass ratios of dipalmitoyl phosphatidylcholine, DSPE-PEG5000, cholesterol and bacterial membrane, combining curcumin and 1-methyl Bacterial membrane was extracted by lysozyme treatment and cell disruptors, and a bacterial membrane-liposome drug-carrying system was prepared by liposome extruder.
It improves the targeting ability of neutrophils, enhances the targeted delivery of drugs to tumor tissues after radiotherapy, and activates the body's anti-tumor immune response, significantly improving the efficacy of radiotherapy on glioblastoma.
Smart Images
Figure CN116251062B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method and application of a bacterial membrane-liposome drug-carrying system. Background Art
[0002] Tumors have always been a threat to human health. The treatment of tumors mainly includes surgery, radiotherapy, chemotherapy and immunotherapy. Nano drug delivery systems can improve the bioavailability of chemotherapeutic drugs and their targeting to tumor tissues, and have been widely used in preclinical research and clinical practice. Liposome drug delivery systems have a structure similar to that of biological membranes. Hydrophilic drugs can be loaded in their internal cavities, and hydrophobic drugs can be loaded between the layers of the phospholipid bilayer. As a drug carrier, liposomes have the advantages of a wide drug loading range, sustained release, easy modification, high safety, reduced adverse drug reactions and improved drug stability. The liposome products currently on the market in China mainly include paclitaxel liposome injection, amphotericin B liposome injection and doxorubicin hydrochloride liposome injection.
[0003] Although liposome drug delivery systems play an important role in clinical anti-tumor chemotherapy, they can be further improved in terms of tumor targeting and the anti-tumor efficacy of the carrier itself. As nanocarriers of chemotherapeutic drugs, liposomes can be passively targeted to tumor tissues through the enhanced permeability and retention (EPR) effect of solid tumors to achieve tumor-specific treatment. However, a large number of research reports and clinical practices have pointed out that the EPR effect of nanoparticles is not prominent, so it is particularly important to improve the active targeting ability of liposomes to tumors. The main method currently adopted is to modify liposomes with sugars and their derivatives, antibodies, polymers and peptides to improve their active targeting to different diseases. In these modification processes, there are problems such as low connection efficiency, cumbersome separation and purification steps, and uncertainty in the degree of improvement of targeting performance. On the other hand, with the development of nanotechnology, some nanoparticles used in tumor treatment can not only be used as chemotherapeutic drug carriers, but they can also safely and effectively sensitize a variety of tumor treatment methods (such as radiotherapy and immunotherapy) to improve the anti-tumor efficacy. Liposomes have simple ingredients and do not have any anti-tumor efficacy when not loaded with drugs, and will not stimulate the body's anti-tumor immune response.
[0004] There is inflammation inside the tumor tissue, and after surgery, radiotherapy, photothermal therapy, etc., the inflammation inside the tumor is further amplified, expressing a large number of chemokines and recruiting neutrophils in the peripheral blood. Therefore, the drug delivery system can target tumor tissues, especially tumor tissues after surgery or radiotherapy, by riding on neutrophils. In recent years, some anaerobic or facultative anaerobic bacteria have been used in the study of tumor treatment because they can specifically proliferate in the hypoxic microenvironment of tumors and induce systemic anti-tumor immune responses. Among them, BCG, as a typical representative, is a type of attenuated bovine tuberculosis bacteria that can enhance the activity of macrophages and activate T lymphocytes, thereby enhancing the body's anti-tumor cell immune function. The bacterial outer membrane collected from bacteria has a structure similar to that of liposomes, and can be targeted to tumors by riding on neutrophils and stimulate anti-tumor immunotherapy responses. However, the bacterial outer membrane has the disadvantages of complex collection process, cumbersome steps, small amount of extraction, and difficulty in loading drugs as a drug carrier. The bacterial membrane can be obtained in large quantities by dissolving bacteria, and it also has the structure of a biological membrane, which can trigger the body's anti-tumor immunity. At the same time, part of the biomembrane is used to mix with phospholipids to construct a biomembrane-doped liposome drug delivery system, which improves the various properties of the liposome. Therefore, mixing bacterial membranes with phospholipids will most likely construct a bacterial membrane-liposome drug delivery system that can ride on neutrophils, improving the tumor targeting performance and immune stimulation efficacy of liposomes. Summary of the invention
[0005] The purpose of the present invention is to prepare a bacterial membrane-liposome drug delivery system by blending bacterial membranes with phospholipids, thereby enhancing the ability to board neutrophils, thereby targeted delivery of drugs to tumor tissues, especially tumor tissues after radiotherapy, while stimulating the body's anti-tumor immune response and improving the therapeutic effect, so as to solve the problems of insufficient tumor targeting and insufficient autoimmune stimulation efficiency of the liposome drug delivery system.
[0006] In order to achieve the above object, the present invention adopts the following technical effects:
[0007] A bacterial membrane-liposome drug delivery system, comprising dipalmitoylphosphatidylcholine, DSPE-PEG 5000 , cholesterol and bacterial membrane, dipalmitoylphosphatidylcholine, DSPE-PEG 5000 , cholesterol and bacterial membrane is 3:2:1:1.
[0008] Furthermore, the bacterial membrane is made of VNP20009 strain.
[0009] Furthermore, the bacterial membrane-liposome drug delivery system contains curcumin and 1-methyl-D-tryptophan.
[0010] The preparation method of the above-mentioned bacterial membrane-liposome drug delivery system comprises the following steps:
[0011] Step 1, extraction of bacterial membrane, subculture the VNP20009 strain using LB liquid medium, centrifuge to obtain bacteria after subculture to the third generation, dissolve the bacteria in Tris HCl buffer containing lysozyme, incubate with shaking, add SDS, treat with a cell disruptor, add a mixture of chloroform, methanol and water after freeze-drying, treat with a cell disruptor, incubate, filter, and dry the filtrate to obtain a bacterial membrane;
[0012] Step 2: dipalmitoylphosphatidylcholine, DSPE-PEG 5000 , cholesterol, and bacterial membrane lyophilized powder were weighed in a mass ratio of 3:2:1:1, and the fat-soluble drug curcumin was added and dissolved in chloroform. The volatile organic reagents were removed by a rotary evaporator to form a thin film on the bottle wall. Then, 1-methyl-D-tryptophan aqueous solution was added, and the film was completely dissolved by ultrasound. The mixture was stirred and mixed, and liposomes were prepared using a liposome extruder, and the bacterial membrane-liposome drug delivery system was prepared.
[0013] Furthermore, in step 1, 2 g of bacteria were dissolved in 40 mL of 15 mg / mL lysozyme in 20 mmol / L pH 8.0 TrisHCl buffer, and the amount of SDS was 400 mg; the volume ratio of chloroform, methanol and water was 30:15:1.
[0014] The application of the above bacterial membrane-liposome drug delivery system in the preparation of tumor therapeutic drugs.
[0015] Beneficial effects:
[0016] 1. The bacterial membrane-liposome drug delivery system obtained by the present invention has a simple synthesis method, uniform size, good dispersibility, and can be loaded with hydrophilic and hydrophobic drugs at the same time.
[0017] 2. The bacterial membrane-liposome drug delivery system obtained by the present invention can be taken up by neutrophils more than liposomes, and then targeted to the tumor tissue after radiotherapy, and release drugs in an inflammatory environment.
[0018] 3. The bacterial membrane-liposome drug delivery system obtained by the present invention can activate the body's anti-tumor immune response and enhance the efficacy of radiotherapy on glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 It is a schematic diagram of the structure of the bacterial membrane-liposome drug delivery system (B-Lipo / 1-MT&Cur) in the present invention.
[0020] Figure 2 This is the transmission electron microscopy image of B-Lipo / 1-MT&Cur in Example 1.
[0021] Figure 3 This is the co-localization result of the B-Lipo / 1-MT&Cur or bacterial membrane and liposome mixture after being taken up by cells in Example 1.
[0022] Figure 4 UV-visible absorption spectra of 1-MT, Cur, liposomes loaded with two drugs (Lipo / 1-MT&Cur) and B-Lipo / 1-MT&Cur in Example 1.
[0023] Figure 5 The release of 1-MT and Cur by B-Lipo / 1-MT&Cur at different pH values in Example 1.
[0024] Figure 6 The results are as follows: the mean fluorescence intensity of neutrophils in the blood and tumors of mice after intravenous injection of fluorescently labeled liposomes or bacterial membrane-liposomes in Example 1.
[0025] Figure 7 24 hours after intravenous administration in Example 1, the brains of mice bearing luc-stable glioblastoma in different treatment groups were collected for bioluminescence and fluorescence imaging.
[0026] Figure 8 These are confocal images of GL261 tumor cells after the neutrophils that have taken up B-Lipo / 1-MT&Cur are co-incubated with GL261 tumor cells in a normal environment or an inflammatory environment and the culture medium containing the neutrophils is removed in Example 1.
[0027] Fig. 9 The cytotoxic T cells (CD3 + CD8 + ) in CD3 + ratio in cells.
[0028] Fig.10 The regulatory T cells (CD3 + CD4 + Foxp3 + ) in CD3 + CD4 + The proportion of T cells.
[0029] Fig.11 The M1 macrophages (CD11b + F4 / 80 + CD86 + ) in CD11b + F4 / 80+ ratio in cells.
[0030] Fig.12 The M2 macrophages (CD11b + F4 / 80 + CD206 + ) in CD11b + F4 / 80 + ratio in cells.
[0031] Fig.13 This is the survival curve of mice bearing orthotopic glioblastoma in different treatment groups in Example 1. DETAILED DESCRIPTION
[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.
[0033] The VNP20009 strain used in the present invention was purchased from the American Type Culture Collection (ATCC).
[0034] Example 1
[0035] In this embodiment, a bacterial membrane-liposome drug delivery system (B-Lipo) was constructed to encapsulate the hydrophilic drug 1-methyl-tryptophan (1-Methyl-D-tryptophan, 1-MT) and the hydrophobic drug Curcumin (Cur) to obtain B-Lipo / 1-MT&Cur, which was applied to the targeted drug delivery and combined radio-immunotherapy of in situ brain glioblastoma.
[0036] The synthesis steps are as follows:
[0037] 1. Extraction of bacterial membrane:
[0038] The VNP20009 strain was subcultured using LB (Luria-Bertani) liquid medium. After the third generation, the bacteria were obtained by centrifugation at 4500rpm for 20min. 2g of bacteria were dissolved in 40mL Tris HCl buffer (20mmol / L, pH=8.0) containing lysozyme (15mg / mL), incubated at 37°C for 3h, 400mg SDS was added, treated with a cell disruptor for 10min, and lyophilized. Then chloroform, methanol and water (30:15:1, v / v / v) were added, treated with a cell disruptor for 10min, incubated at 37°C for 1h, and a 0.45μm Millipore HVLP was used. The membrane was filtered, the liquid was removed using a rotary evaporator, and the bacterial membrane was obtained after drying.
[0039] 2. Synthesis of B-Lipo / 1-MT&Cur:
[0040] Synthesis by thin film dispersion method: weigh 9 mg DPPC and 6 mg DSPE-PEG at a mass ratio of 3:2:1:1 5000 , 3mg cholesterol, 3mg bacterial membrane, add 1mg fat-soluble drug curcumin (Cur), dissolve it in 2mL chloroform, use a rotary evaporator to remove the liquid, and form a thin film on the bottle wall. Then add 1mL 1-MT aqueous solution (2mg / mL), ultrasonicate for 20min to completely dissolve it, then stir at 45℃ for 2h, and use a liposome extruder to extrude liposomes of appropriate size through 200nm and 400nm polycarbonate membranes. Use a 100KD ultrafiltration tube to wash 3 times with deionized water to remove free drugs and obtain B-Lipo / 1-MT&Cur.
[0041] The synthesis steps for preparing the liposome drug delivery system loaded with 1-MT and Cur (Lipo / 1-MT&Cur) at the same time are as follows: based on the steps for synthesizing B-Lipo / 1-MT&Cur, minus the addition of 3 mg bacterial membrane, and the rest of the steps remain the same.
[0042] The prepared drug-loaded bacterial membrane-liposome drug delivery system is as follows Figure 1 As shown, the nanoparticles were observed by transmission electron microscopy. Figure 2 As shown, it has a uniform spherical structure and a size of about 100 nm.
[0043] In order to prove that the above synthesis method can successfully fuse bacterial membrane and liposome, the bacterial membrane (BM) was stained with FITC fluorescence and the liposome was stained with DID fluorescence to prepare B-Lipo. After B-Lipo was co-incubated with cells, it was observed by confocal fluorescence microscopy. Figure 3As shown, when liposomes and bacterial membranes are simply mixed (BM+Lipo), the fluorescence of the two is basically non-overlapping, while the two fluorescences of B-Lipo merge together in the cell, proving that the bacterial membrane and liposomes have successfully fused.
[0044] The drug loading and drug releasing capabilities of the bacterial membrane-liposome drug delivery system are as follows: Figure 4 and Figure 5 As shown: The UV absorption spectrum shows that the drug delivery system successfully encapsulates the drugs 1-MT and Cur, and the encapsulation rates are: 1-MT: 35.25±0.01%; Cur: 47.23±0.01%. Then the drug delivery system's ability to release drugs was simulated in vitro. 1mL of B-Lipo / 1-MT&Cur aqueous solution was placed in a dialysis bag and stirred in a serum bottle containing 15mL of phosphate buffer with different pH (pH=5.6, 6.5, 7.4), thereby simulating the release of the material at different pH values. The contents of 1-MT and Cur were measured using a UV spectrophotometer. The results show that B-Lipo / 1-MT&Cur can achieve sustained release of Cur and 1-MT under different pH environments.
[0045] In order to verify whether bacterial membrane-liposomes can enter the tumor site, especially the tumor tissue after radiotherapy, by riding on neutrophils, first prepare liposomes or bacterial membrane-liposomes labeled with DID (fluorescent dye, maximum excitation wavelength is 644nm, maximum emission wavelength is 665nm). The specific synthesis steps are as follows: weigh 9mg DPPC, 6mg DSPE-PEG 5000 , 3 mg cholesterol (mass ratio 3:2:1), dissolved in 2 mL chloroform, added 5 μL DID, used a rotary evaporator to remove the liquid, and formed a thin film on the wall of the bottle. Add 1 mL of water, sonicate for 20 min to completely dissolve it, then stir at 45 ° C for 2 h, and use a liposome extruder to extrude liposomes of appropriate size through 200 nm and 400 nm polycarbonate membranes. Use a 100KD ultrafiltration tube to wash with deionized water 3 times to remove free substances and obtain DID-labeled liposomes (DID-Lipo). In order to obtain DID-labeled bacterial membrane-liposomes (DID-B-Lipo), 3 mg of bacterial membrane was added to the first step of the above synthesis of DID-Lipo and blended with other phospholipids, and the rest of the steps remained the same. GL261 orthotopic glioblastoma-bearing mice were divided into two groups and injected with DID-Lipo and DID-B-Lipo (DID: about 5 μg / mouse, Lipo: about 90 mg / kg, bacterial membrane: about 15 mg / kg) with the same DID fluorescence intensity through the tail vein. After 4 hours, the blood and tumors of the mice were collected, and neutrophils were extracted and separated. The fluorescence intensity of DID in neutrophils in the blood and tumors of the mice was analyzed by flow cytometry. Figure 6As shown, the results showed that liposomes containing bacterial membranes showed better affinity for neutrophils in both blood and tumors compared with ordinary liposomes.
[0046] To further verify the inflammation targeting effect, the small animal imaging system (IVIS) was used for further observation. The mice with orthotopic glioblastoma were divided into 5 groups: G1: Lipo / 1-MT&Cur; G2: X-rays+Lipo / 1-MT&Cur; G3: B-Lipo / 1-MT&Cur; G4: X-rays+B-Lipo / 1-MT&Cur+anti-Gr-1; G5: X-rays+B-Lipo / 1-MT&Cur. In each group, the intravenous injection time of the drug preparation (including Lipo / 1-MT&Cur and B-Lipo / 1-MT&Cur) was within one hour after X-ray (irradiation dose: 5Gy). The intravenous injection time of anti-Gr-1 was after X-ray irradiation and before the injection of the drug preparation, and the dose was 50μg / mouse. The content of DID, 1-MT and Cur in the drug preparation was the same, 5 μg / mouse DID, 13.19 mg / kg 1-MT and 9.06 mg / kg Cur, respectively. After 24 hours, the mouse brains were collected for bioluminescence imaging and DID fluorescence imaging and quantitative analysis. Figure 7 As shown in the figure, when the tumor site is treated with X-rays, the inflammatory effect is further enhanced. After the bacterial membrane-liposome drug delivery system is injected into the tail vein, its targeting effect is the best. After the injection of the neutrophil antibody Gr-1, it will consume the neutrophils in the body. After the drug is injected, it will be found that its targeting becomes weaker, indicating that the increase in targeting is neutrophil-dependent.
[0047] In order to detect whether neutrophils can release the bacterial membrane-liposome drug delivery system in an inflammatory environment and be taken up by tumor cells, neutrophils loaded with B-Lipo / 1-MT&Cur were co-incubated with GL261 tumor cells in an inflammatory environment (culture medium containing phorbolmyristate acetate (PMA) (PMA: culture medium is 1:250)) and a normal environment (normal culture medium) and observed by confocal microscopy. Figure 8 The results showed that in an inflammatory environment, tumor cells took up more Cur, indicating that in an inflammatory environment, neutrophils better released B-Lipo / 1-MT&Cur and the drugs they carried, which were then taken up by tumor cells. This shows that the bacterial membrane-liposome drug delivery system can ride on neutrophils and release drugs to tumor tissues under the induction of inflammatory factors, achieving better drug delivery effects.
[0048] Afterwards, an immune analysis of the effect of radiotherapy combined with B-Lipo / 1-MT&Cur on tumor treatment was performed. The patients were divided into the following 4 groups: G1: PBS; G2: X-rays; G3: X-rays+Lipo / 1-MT&Cur; G4: X-rays+B-Lipo / 1-MT&Cur. Lipo / 1-MT&Cur and B-Lipo / 1-MT&Cur were injected intravenously within 1 hour after X-ray irradiation. 1-MT: 13.19 mg / kg; Cur: 9.06 mg / kg; X-ray dose: 5 Gy. Immune cells were analyzed by flow cytometry on the 4th day. Figure 9-12 It was found that compared with the combination with a simple liposome drug delivery system, the proportion of cytotoxic T cells in the tumor increased, the proportion of regulatory T cells decreased, the proportion of M1 macrophages increased, and the proportion of M2 macrophages decreased after radiotherapy combined with a bacterial membrane-liposome drug delivery system, indicating that the bacterial membrane can activate the body's anti-tumor immune response.
[0049] Finally, an experiment to treat orthotopic glioblastoma was conducted to further verify the advantages of this drug delivery system. The mice were grouped into G1: PBS; G2: X-rays; G3: X-rays+Lipo / 1-MT&Cur; G4: X-rays+B-Lipo / 1-MT&Cur. Within 1 hour after X-ray irradiation, Lipo / 1-MT&Cur and B-Lipo / 1-MT&Cur were injected intravenously, 1-MT: 13.19 mg / kg; Cur: 9.06 mg / kg; X-ray dose: 5 Gy. On the day the mice were irradiated with X-rays, the weight and mortality of the mice in each group were recorded. Fig.13 As shown in the results, radiotherapy combined with bacterial membrane-liposome drug delivery system can significantly prolong the survival of mice, which is significantly better than radiotherapy combined with liposome drug delivery system. This result is mainly because compared with ordinary liposomes, bacterial membrane-liposomes can better target tumor tissues after radiotherapy and synergistically activate the body's anti-tumor immune system.
Claims
1. A bacterial membrane-liposome drug delivery system, characterized in that: Dipalmitoylphosphatidylcholine, DSPE-PEG 5000 , cholesterol, bacterial membranes, curcumin, and 1-methyl-D-tryptophan; The preparation method of the bacterial membrane-liposome drug delivery system comprises the following steps: Step 1, extraction of bacterial membrane, subculture the VNP20009 strain using LB liquid medium, centrifuge to obtain bacteria after subculture to the third generation, dissolve the bacteria in Tris HCl buffer containing lysozyme, incubate with shaking, add SDS, treat with a cell disruptor, add a mixture of chloroform, methanol and water after freeze-drying, treat with a cell disruptor, incubate, filter, and dry the filtrate to obtain a bacterial membrane; Step 2: dipalmitoylphosphatidylcholine, DSPE-PEG 5000 , cholesterol, and bacterial membrane lyophilized powder were weighed in a mass ratio of 3:2:1:1, and the fat-soluble drug curcumin was added and dissolved in chloroform. The volatile organic reagents were removed by a rotary evaporator to form a thin film on the bottle wall. Then, 1-methyl-D-tryptophan aqueous solution was added, and the film was completely dissolved by ultrasound. The mixture was stirred and mixed, and a bacterial membrane-liposome drug delivery system was prepared using a liposome extruder.
2. The bacterial membrane-liposome drug delivery system according to claim 1, characterized in that: In step 1, 2 g of bacteria were dissolved in 40 mL of 20 mmol / L pH 8.0 Tris HCl buffer containing 15 mg / mL lysozyme. The amount of SDS was 400 mg; the volume ratio of chloroform, methanol and water was 30:15:
1.
3. Use of the bacterial membrane-liposome drug delivery system according to claim 1 in the preparation of drugs for treating glioblastoma.