CN116251062A - Preparation method and application of bacterial membrane-liposome drug-loading system - Google Patents

Preparation method and application of bacterial membrane-liposome drug-loading system Download PDF

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CN116251062A
CN116251062A CN202310337764.9A CN202310337764A CN116251062A CN 116251062 A CN116251062 A CN 116251062A CN 202310337764 A CN202310337764 A CN 202310337764A CN 116251062 A CN116251062 A CN 116251062A
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bacterial membrane
liposome
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易玄
刘心培
顾晶钰
沈梦玲
朱敏倩
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Abstract

The invention discloses a preparation method and application of a bacterial membrane-liposome drug-loading system. According to the invention, the bacterial membrane-liposome drug-carrying system is prepared by blending the bacterial membrane and the phospholipid, so that the riding capacity of neutrophils is enhanced, and then the drug is targeted and delivered to tumor tissues, especially tumor tissues after radiotherapy, and meanwhile, the anti-tumor immune response of an organism is stimulated, so that the treatment effect is improved, and the problems of insufficient tumor targeting and insufficient autoimmune stimulation efficiency of the liposome drug-carrying system are solved.

Description

Preparation method and application of bacterial membrane-liposome drug-loading system
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of a bacterial membrane-liposome drug delivery system.
Background
Tumors have been threatening to human health, and treatments aimed at tumors mainly include surgery, radiation therapy, chemo-drug therapy, and immunotherapy. The nano medicine carrying system can improve the bioavailability of the chemotherapeutic medicine and the targeting of tumor tissues, and is widely applied to preclinical research and clinical practice. The liposome drug delivery system has a structure similar to that of a biological membrane, hydrophilic drugs can be loaded in the inner cavity of the liposome drug delivery system, and hydrophobic drugs can be loaded between the phospholipid bilayer layers. As a drug carrier, the liposome has the advantages of wide drug carrying range, slow release, easy modification, high safety, capability of reducing adverse reaction of the drug, improvement of the stability of the drug and the like. The liposome products currently on the market in China mainly comprise taxol liposome injection, amphotericin B liposome for injection and doxorubicin hydrochloride liposome injection.
Although the liposome drug delivery system plays an important role in clinical anti-tumor chemotherapy, the liposome drug delivery system can be further improved in the aspects of tumor targeting and the anti-tumor efficacy of the carrier. The liposome is used as a nano carrier of a chemotherapeutic drug, and can realize tumor-specific treatment by passively targeting to tumor tissues through the high permeability and retention (Enhanced Permeability and Retention, EPR) effect of solid tumors. However, numerous research reports and clinical practices indicate that the EPR effect of nanoparticles is not prominent, thus increasing the active targeting ability of liposomes to tumors is particularly important. The main mode adopted at present is to use saccharides and derivatives thereof, antibodies, polymers, peptides and the like to modify liposome so as to improve the active targeting of the liposome to different diseases. In the modification process, the problems of low connection efficiency, complicated separation and purification steps, uncertainty of the target performance improvement degree and the like exist. On the other hand, with the development of nano technology, a part of nano particles applied to tumor treatment can be used as a chemotherapeutic drug carrier, and can safely and effectively sensitize various tumor treatment modes (such as radiation treatment, immunotherapy and the like) so as to improve the anti-tumor curative effect. The liposome has simple components, does not have any anti-tumor efficacy when not carrying medicine, and can not excite the anti-tumor immune response of the organism.
The inside of the tumor tissue has inflammation, and after surgery, radiotherapy, photothermal treatment, etc., the inflammation inside the tumor is further amplified, a large amount of chemokines are expressed, and neutrophils in peripheral blood are recruited. Thus, the drug delivery system can target tumor tissue, particularly tumor tissue following surgery or radiation therapy, with neutrophils. In recent years, a part of anaerobic or facultative anaerobic bacteria have been used in research on tumor treatment because they can specifically proliferate in tumor hypoxia microenvironment and induce systemic antitumor immune response in the body. The bacillus calmette-guerin is a typical representative, is an attenuated bovine type tubercle bacillus, can promote the activity of macrophages, activate T lymphocytes and strengthen the immunity of the organism against tumor cells. The bacterial outer membrane collected from the bacteria is structurally similar to liposomes and can target tumors with neutrophils and stimulate anti-tumor immunotherapeutic responses. However, the bacterial outer membrane has the defects of complex collection process, complicated steps, small extraction amount, difficult drug loading as a drug carrier and the like. The bacterial membrane can be obtained in large quantity by dissolving bacteria, also has a structure of a biological membrane, and can trigger the anti-tumor immunity of organisms. Meanwhile, a part of biological film is used for being mixed with phospholipid to construct a liposome drug carrying system doped with the biological film, so that various performances of the liposome are improved. Therefore, the bacterial membrane and the phospholipid are blended, so that a bacterial membrane-liposome drug-loading system capable of riding neutrophils is very likely to be constructed, and the tumor targeting performance and the immunostimulation efficacy of the liposome are improved.
Disclosure of Invention
The invention aims to prepare a bacterial membrane-liposome drug-carrying system by blending bacterial membranes with phospholipids, enhance the riding capacity on neutrophils, further target and deliver drugs to tumor tissues, especially tumor tissues after radiotherapy, and simultaneously excite anti-tumor immune response of organisms, so as to improve the treatment effect and solve the problems of insufficient tumor targeting and insufficient autoimmune stimulation efficiency of the liposome drug-carrying system.
In order to achieve the above purpose, the present invention adopts the following technical effects:
a bacterial membrane-liposome medicine carrying system is prepared from dipalmitoyl phosphatidylcholine and DSPE-PEG 5000 Cholesterol and bacterial membrane, dipalmitoyl phosphatidylcholine, DSPE-PEG 5000 The mass ratio of cholesterol to bacterial membrane is 3:2:1:1.
further, the bacterial membrane is made of VNP20009 strain.
Further, the bacterial membrane-liposome drug delivery system is coated with curcumin and 1-methyl-D-tryptophan.
The preparation method of the bacterial membrane-liposome drug delivery system comprises the following steps:
step 1, bacterial membrane extraction, namely, carrying out passage on a VNP20009 strain by using an LB liquid culture medium, carrying out centrifugation after passage for 3 generations to obtain bacteria, dissolving the bacteria in a Tris HCl buffer solution containing lysozyme, carrying out shaking incubation, adding SDS, treating by using a cell disruption instrument, adding a mixed solution of chloroform, methanol and water after freeze-drying, treating by using the cell disruption instrument, incubating, filtering, and drying filtrate to obtain the bacterial membrane;
step 2, dipalmitoyl phosphatidylcholine and DSPE-PEG 5000 Cholesterol and bacterial membrane freeze-dried powder according to the mass ratio of 3:2:1:1, adding fat-soluble medicine curcumin, dissolving the medicine curcumin in chloroform, removing volatile organic reagent by using a rotary evaporator to form a film on the bottle wall, then adding 1-methyl-D-tryptophan aqueous solution, completely dissolving the film by ultrasonic, stirring and mixing, and preparing liposome by using a liposome extruder, thereby obtaining the bacterial membrane-liposome medicine carrying system.
Further, 2g of the bacteria were dissolved in 40mL of 15mg/mL lysozyme in 20mmol/L Tris HCl buffer pH8.0 with SDS at 400mg in step 1; the volume ratio of the chloroform to the methanol to the water is 30:15:1.
The bacterial membrane-liposome drug-loading system is applied to the preparation of tumor therapeutic drugs.
The beneficial effects are that:
1. the bacterial membrane-liposome drug-loading system has the advantages of simple synthesis method, uniform size and good dispersibility, and can simultaneously load hydrophilic and hydrophobic drugs.
2. Compared with liposome, the bacterial membrane-liposome drug-loading system can be more ingested by neutrophils, further targets tumor tissues after radiotherapy, and releases drugs in an inflammatory environment.
3. The bacterial membrane-liposome drug-loading system can activate the anti-tumor immune response of organisms and enhance the curative effect of radiotherapy on glioblastoma.
Drawings
FIG. 1 is a schematic diagram of the bacterial membrane-liposome drug delivery system (B-Lipo/1-MT & Cur) of the present invention.
FIG. 2 is a transmission electron microscope image of B-Lipo/1-MT & Cur in example 1.
FIG. 3 shows the co-localization results of B-Lipo/1-MT & Cur or bacterial membrane and liposome mixtures taken up by cells in example 1.
FIG. 4 shows the UV-visible absorption spectra of 1-MT, cur, two drug loaded liposomes (Lipo/1-MT & Cur) and B-Lipo/1-MT & Cur of example 1.
FIG. 5 shows the release of B-Lipo/1-MT & Cur from 1-MT and Cur at different pH values in example 1.
FIG. 6 shows the mean fluorescence intensity results of neutrophils in mouse blood and tumors after intravenous injection of fluorescently labeled liposomes or bacterial membrane-liposomes as in example 1.
FIG. 7 shows the results of bioluminescence and fluorescence imaging of brains of mice bearing luc-stably transformed glioblastoma from different treatment groups collected 24 hours after intravenous administration in example 1.
FIG. 8 is a confocal image of GL261 tumor cells after removing the medium containing neutrophils after incubating the neutrophils having ingested B-Lipo/1-MT & Cur with GL261 tumor cells under normal or inflammatory conditions in example 1.
FIG. 9 is cytotoxicity of the infiltrate in glioblastoma in situ the fourth day after treatment in example 1T-cells (CD 3) + CD8 + ) In CD3 + Ratio in cells.
FIG. 10 shows regulatory T cells (CD 3) in glioblastoma in situ the fourth day after treatment in example 1 + CD4 + Foxp3 + ) In CD3 + CD4 + Ratio in T cells.
FIG. 11 is a graph showing M1 type macrophages (CD 11 b) in glioblastoma cells in situ the fourth day after treatment in example 1 + F4/80 + CD86 + ) At CD11b + F4/80 + Ratio in cells.
FIG. 12 shows M2 type macrophages (CD 11 b) in glioblastoma in situ the fourth day after treatment in example 1 + F4/80 + CD206 + ) At CD11b + F4/80 + Ratio in cells.
FIG. 13 is a plot of survival of mice bearing glioblastoma in situ in different treatment groups in example 1.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
The VNP20009 strain used in the present invention was purchased from American type culture Collection (American Type Culture Collection, ATCC).
Example 1
The embodiment constructs a bacterial membrane-liposome drug-carrying system (B-Lipo), and encapsulates a hydrophilic drug of 1-Methyl-tryptophan (1-Methyl-D-trytophan, 1-MT) and a hydrophobic drug of Curcumin (Curcumin, cur) to obtain the B-Lipo/1-MT & Cur, which is applied to targeted drug delivery and combined radiation-immunotherapy of in-situ brain glioblastoma.
The synthesis steps are as follows:
1. extraction of bacterial film:
VNP20009 strainBacteria were obtained by passaging using LB (Luria-Bertani) liquid medium, and after passage to 3 passages, centrifuging at 4500rpm for 20 min. 2g of bacteria were dissolved in 40mL Tris HCl buffer (20 mmol/L, pH=8.0) containing lysozyme (15 mg/mL), incubated at 37℃for 3h with shaking, 400mg SDS was added, treated with a cytobreaker for 10min, and lyophilized. Chloroform, methanol and water (30:15:1, v/v/v) were then added, treated with a cell disrupter for 10min, incubated at 37℃for 1h, and 0.45 μm Millipore HVLP was used
Figure BDA0004157021500000041
The membrane was filtered, the liquid was removed using a rotary evaporator and dried to obtain a bacterial membrane.
2. Synthesis of B-Lipo/1-MT & Cur:
synthesized using a thin film dispersion method: according to the mass ratio of 3:2:1:1 weighing 9mg DPPC, 6mg DSPE-PEG 5000 3mg of cholesterol and 3mg of bacterial film, 1mg of liposoluble drug curcumin (Cur) was added, dissolved in 2mL of chloroform, and the liquid was removed by a rotary evaporator to form a film on the bottle wall. 1mL of 1-MT aqueous solution (2 mg/mL) was then added, and the solution was completely dissolved by sonication for 20min, followed by stirring at 45℃for 2h, and liposomes of appropriate size were extruded through 200nm and 400nm polycarbonate membranes using a liposome extruder. Washing with deionized water for 3 times with ultrafiltration tube of 100KD to remove free drug to obtain B-Lipo/1-MT&Cur。
The synthesis step of simultaneously preparing a liposome drug carrying system (Lipo/1-MT & Cur) loaded with 1-MT and Cur comprises the following steps: on the basis of the step of synthesizing B-Lipo/1-MT & Cur, the addition of 3mg of bacterial membrane was subtracted, the remaining steps remaining the same.
The prepared bacterial membrane-liposome drug-loading system for encapsulating the drug is shown in figure 1, and the nano particles have a uniform spherical structure and have a size of about 100nm as shown in figure 2 when observed by a transmission electron microscope.
To demonstrate that the above synthetic method allowed successful fusion of bacterial membrane and liposome, bacterial Membrane (BM) was stained with FITC fluorescence and liposome stained with DID fluorescence to prepare B-Lipo. After co-incubation of B-Lipo with cells, it was observed by confocal fluorescence microscopy. As shown in FIG. 3, the simple mixing of the liposome and bacterial membrane (BM+Lipo) resulted in substantially non-coincident fluorescence of both, while the two fluorescence of B-Lipo fused together intracellularly, demonstrating successful fusion of the bacterial membrane and liposome.
The drug carrying capacity and drug releasing capacity of the bacterial membrane-liposome drug carrying system are shown in fig. 4 and 5: the ultraviolet absorption spectrum shows that the drug carrying system successfully carries the drugs 1-MT and Cur, and the encapsulation rates are respectively as follows: 1-MT: 35.25+ -0.01%; cur:47.23 +/-0.01%. After that, the ability of the drug delivery system to release the drug was simulated in vitro, 1mL of B-Lipo/1-MT & Cur aqueous solution was placed in a dialysis bag, and placed in 15mL of serum bottles containing phosphate buffers of different pH (ph= 5.6,6.5,7.4) and stirred, thereby simulating the release of the material at different pH, and the content of 1-MT and Cur was measured using an ultraviolet spectrophotometer. The results show that under different pH environments, the slow release of Cur and 1-MT can be realized by the B-Lipo/1-MT & Cur.
To verify whether bacterial membrane-lipids can enter tumor sites, especially tumor tissue following radiotherapy, by riding neutrophils. DID (fluorescent dye) labeled liposomes or bacterial membrane-liposomes with a maximum excitation wavelength of 644nm and a maximum emission wavelength of 665nm were first prepared. The specific synthesis steps are as follows: 9mg DPPC, 6mg DSPE-PEG are weighed 5000 3mg of cholesterol (mass ratio 3:2:1) was dissolved in 2mL of chloroform, 5. Mu.L of DID was added, and the liquid was removed by a rotary evaporator to form a film on the bottle wall. 1mL of water was added and sonicated for 20min to dissolve completely, then stirred at 45℃for 2h, and liposomes of appropriate size were extruded through 200nm and 400nm polycarbonate membranes using a liposome extruder. The free material was removed by washing 3 times with deionized water using a 100KD ultrafiltration tube to give DID-labeled liposomes (DID-Lipo). To obtain DID-labeled bacterial membrane-liposome (DID-B-Lipo), 3mg of bacterial membrane was added to blend with other phospholipids in the first step of the synthesis of DID-Lipo described above, and the remaining steps were kept consistent. GL261 in situ glioblastoma bearing mice were divided into two groups, and DID-Lipo and DID-B-Lipo (DID: about 5 μg/each mouse, lipo: about 90mg/Kg, bacterial membrane: about 15 mg/Kg) of the same DID fluorescence intensity were injected via the tail vein, respectively, and after 4 hours, the blood and tumor of the mice were taken, and neutrophils were extracted and isolated. Analysis of small using flow cytometryFluorescence intensity of DID in neutrophils in murine blood and tumor. As shown in fig. 6, the results showed that liposomes containing bacterial membranes showed better affinity to neutrophils in both blood and tumor compared to normal liposomes.
To further verify the inflammation targeting effect, further observations were made using the small animal imaging system (IVIS). Mice with glioblastoma in situ were divided into 5 groups: g1: lipo/1-MT & Cur; and G2: X-ray+lipo/1-MT & Cur; and G3: B-Lipo/1-MT & Cur; and G4: X-ray+B-Lipo/1-MT & Cur+anti-Gr-1; and G5: X-ray+B-Lipo/1-MT & Cur intravenous injection time of the pharmaceutical preparations (including Lipo/1-MT & Cur and B-Lipo/1-MT & Cur) in each group was within one hour after the irradiation of X-rays (irradiation dose: 5 Gy). The intravenous time of anti-Gr-1 was 50 μg/mouse after X-ray irradiation and before the injection of the pharmaceutical formulation. The DID,1-MT and Cur content in the pharmaceutical preparation were the same, and were 5. Mu.g/DID, 13.19mg/kg 1-MT and 9.06mg/kg Cur, respectively, per mouse. After 24 hours, the mouse brains were collected for bioluminescence imaging and DID fluorescence imaging and quantitative analysis. As shown in fig. 7, the inflammatory effect is further enhanced when the tumor site is treated with X-rays, after which the bacterial membrane-liposome drug delivery system is injected into the tail vein, and its targeting effect is the best. After the antibody Gr-1 of neutrophils was injected, it was consumed on neutrophils in vivo, and drug injection was performed again, it was found that its targeting was weakened, indicating that the increase in targeting was neutrophil dependent.
In order to examine whether neutrophils can release bacterial membrane-liposome drug-loading system under inflammatory environment and are taken up by tumor cells, neutrophils loaded with B-Lipo/1-MT & Cur were incubated with GL261 tumor cells under inflammatory environment (medium containing phorbol ester (phorbol myristate acetate, PMA: medium: 1:250)) and normal environment (normal medium), and observed by confocal microscopy, as shown in FIG. 8, the results showed that more Cur was taken up by tumor cells under inflammatory environment, indicating that better release of B-Lipo/1-MT & Cur and drug loaded by neutrophils was taken up by tumor cells under inflammatory environment. Therefore, the bacterial membrane-liposome drug-carrying system can ride neutrophils and release drugs to tumor tissues under the induction of inflammatory factors, so that a better drug delivery effect is achieved.
An immunoassay of the tumor treatment was then performed in combination with B-Lipo/1-MT & Cur. The following 4 groups were divided: g1: PBS; and G2: x-rays; and G3: X-ray+lipo/1-MT & Cur; and G4: X-ray+B-Lipo/1-MT & Cur, intravenous Lipo/1-MT & Cur and B-Lipo/1-MT & Cur,1-MT respectively within 1 hour after X-ray irradiation: 13.19mg/kg; cur:9.06mg/kg; dose of X-rays: 5Gy. Immune cells were analyzed by flow cytometry on day 4. As shown in fig. 9-12, it was found that the ratio of cytotoxic T cells in tumor was increased, the ratio of regulatory T cells was decreased, the ratio of M1 type macrophages was increased, and the ratio of M2 type macrophages was decreased after the radiotherapy was combined with the bacterial membrane-liposome drug delivery system, compared with the combined simple liposome drug delivery system, indicating that the bacterial membrane was able to activate the antitumor immune response of the body.
Finally, experiments for treating in-situ glioblastoma are carried out, and the advantages of the drug carrying system are further verified. Mice were grouped as G1: PBS; and G2: x-rays; and G3: X-ray+lipo/1-MT & Cur; and G4: X-ray+B-Lipo/1-MT & Cur. Intravenous injection of Lipo/1-MT & Cur and B-Lipo/1-MT & Cur,1-MT, respectively, within 1 hour after X-ray irradiation: 13.19mg/kg; cur:9.06mg/kg; dose of X-rays: 5Gy. Mice on the day of X-ray exposure, mice in each group began to record body weight and death. As shown in fig. 13, the survival time of mice can be prolonged significantly by combining radiotherapy with a bacterial membrane-liposome drug-loading system, which is obviously better than that of the radiotherapy with a liposome drug-loading system. This result is mainly because bacterial membrane-liposomes can better target tumor tissue after radiotherapy and synergistically activate the body's anti-tumor immune system compared to normal liposomes.

Claims (6)

1. A bacterial membrane-liposome drug delivery system, characterized by: from dipalmitoyl phosphatidylcholine, DSPE-PEG 5000 Cholesterol and bacterial membrane, dipalmitoyl phosphatidylcholine, DSPE-PEG 5000 The mass ratio of cholesterol to bacterial membrane is 3:2:1:1.
2. the bacterial membrane-liposome drug delivery system of claim 1, wherein: the bacterial membrane is made of VNP20009 strain.
3. The bacterial membrane-liposome drug delivery system of claim 1, wherein: the bacterial membrane-liposome drug delivery system comprises curcumin and 1-methyl-D-tryptophan.
4. The method for preparing the bacterial membrane-liposome drug delivery system of claim 1, wherein the method comprises the steps of: the method comprises the following steps:
step 1, bacterial membrane extraction, namely, carrying out passage on a VNP20009 strain by using an LB liquid culture medium, carrying out centrifugation after passage for 3 generations to obtain bacteria, dissolving the bacteria in a Tris HCl buffer solution containing lysozyme, carrying out shaking incubation, adding SDS, treating by using a cell disruption instrument, adding a mixed solution of chloroform, methanol and water after freeze-drying, treating by using the cell disruption instrument, incubating, filtering, and drying filtrate to obtain the bacterial membrane;
step 2, dipalmitoyl phosphatidylcholine and DSPE-PEG 5000 Cholesterol and bacterial membrane freeze-dried powder according to the mass ratio of 3:2:1:1, adding fat-soluble medicine curcumin, dissolving the medicine curcumin in chloroform, removing volatile organic reagent by using a rotary evaporator to form a film on the bottle wall, then adding 1-methyl-D-tryptophan aqueous solution, completely dissolving the film by ultrasonic, stirring and mixing, and preparing liposome by using a liposome extruder, thereby obtaining the bacterial membrane-liposome medicine carrying system.
5. The method of manufacturing according to claim 4, wherein: in step 1, 2g of bacteria were dissolved in 40mL of 15mg/mL lysozyme in 20mmol/L Tris HCl buffer pH8.0, with SDS being used in an amount of 400mg; the volume ratio of the chloroform to the methanol to the water is 30:15:1.
6. Use of the bacterial membrane-liposome drug delivery system of claim 1 for the preparation of a medicament for the treatment of tumors.
CN202310337764.9A 2023-03-31 2023-03-31 Preparation method and application of bacterial membrane-liposome drug-loading system Pending CN116251062A (en)

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