Method for simultaneously extracting melanin and bacterial ectovesicle compound from porphyromonas gingivalis and application of melanin and bacterial ectovesicle compound

Abstract

The invention discloses a method for simultaneously extracting melanin and a bacterial extracellular vesicle compound from porphyromonas gingivalis and application of the method. The method comprises the following steps: inoculating a solid culture medium with a porphyromonas gingivalis seed solution, and carrying out anaerobic culture overnight at 37 DEG C; picking single colonies, putting the single colonies into a liquid culture medium, performing anaerobic culture at 37 DEG C for 24 hours, and then putting the single colonies into a bacterial incubator to be continuously cultured until OD600 is equal to 1; centrifugally separating supernatant liquid, and centrifuging again to obtain clarified supernatant liquid; and filtering the clarified supernatant liquid, carrying out ultrafiltration centrifugation on the obtained filtrate, washing a filter screen with water to obtain a washing liquid, carrying out ultrasonic treatment to obtain a melanin and bacterial exovesicle compound, carrying out vacuum freeze drying, and storing the melanin and bacterial exovesicle compound in a refrigerator. The method is economical, simple and convenient, the bacterial outer membrane vesicles and the melanin can be extracted at the same time, and the melanin and bacterial outer vesicle compound can achieve the effects of an immune agonist and a photo-thermal agent at the same time under 808 nm near-infrared laser irradiation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis and an application thereof. Background Art

[0002] Cancer seriously endangers human health, often leading to a short survival time and causing great pain to patients. Although clinical treatment technology has developed in recent decades, the survival rate is still not high. The main treatments for cancer are surgery, radiotherapy and chemotherapy. However, surgery and radiotherapy often cause serious physical damage and side effects that weaken the immune system. Studies have found that most of the common chemotherapy drugs on the market are cytotoxic, which not only kills tumor cells, but also normal cells, and may induce tumor resistance. Therefore, finding and developing new and efficient tumor treatment methods to maximize the desired therapeutic effect and minimize damage and side effects has become a hot topic in current research.

[0003] The use of bacterial extracellular vesicles (OMVs) as antitumor drugs is a current research hotspot. Bacterial extracellular vesicles (OMVs) are bacterial membrane-derived spherical vesicles from Gram-positive and Gram-negative bacteria with a size range of 20-400 nm. OMVs contain features of protocells, membrane-bound and cytoplasmic proteins, enzymes, toxins, and nucleic acids as payloads, some of which, as well as surface ligands, have immunostimulatory effects.

[0004] Photothermal therapy (PTT) can utilize the high photothermal conversion efficiency of the material, inject it into the vicinity of the tumor tissue, and convert light energy into heat energy under the irradiation of an external near-infrared light source to kill cancer cells. While PTT plays a thermal ablation role and directly kills tumor cells, it can also induce immunogenic cell death (ICD) to activate systemic anti-tumor immune responses, including the redistribution and activation of immune effector cells, the expression and secretion of cytokines, and the activation of memory T lymphocytes, significantly improving the efficacy of PTT. Melanin is a biological pigment with multiple biological functions, including structural coloring, photoprotection, photosensitization, free radical scavenging, chelating metal ions, antioxidant activation, and neuroprotection. In addition, melanin can also be used as a natural drug carrier to load anticancer drugs, etc., and plays an important role in the integration of cancer diagnosis and treatment. At the same time, melanin can produce a significant photothermal effect under the continuous irradiation of near-infrared lasers, which can mediate photothermal therapy (PTT) to ablate tumor tissues and tumor blood vessels.

[0005] Porphyromonas gingivalis (Pg) is an obligate anaerobe that can stably secrete melanin in the oral cavity. At the same time, the bacteria continuously produce extracellular vesicles (OMVs) during normal growth.

[0006] At present, the methods for extracting bacterial extracellular vesicles mainly include ultracentrifugation, chromatographic column screening method and ultrafiltration concentration method. Ultracentrifugation is the most commonly used method for separating bacterial extracellular vesicles (OMV). However, ultracentrifugation requires a lot of time and specific instruments, and can only extract OMV as a substance. Chromatographic column screening method, ultrafiltration concentration method, therefore, the present invention proposes an economical and simple method, and can simultaneously extract bacterial extracellular vesicles (OMV) and melanin (Melanin), and after treatment, PgOMV@Melanin complex can be finally extracted. Summary of the invention

[0007] Technical problem to be solved: In response to the above technical problems, the present invention provides a method for simultaneously extracting melanin and bacterial outer membrane vesicle complex from Porphyromonas gingivalis and its application. The obtained complex not only has a certain immune effect, but also can utilize a near-infrared light source to mediate photothermal therapy and ablate tumor tissue and tumor blood vessels.

[0008] Technical solution: In a first aspect, the present invention provides a method for simultaneously extracting melanin and bacterial outer membrane vesicle complexes from Porphyromonas gingivalis, comprising the following steps:

[0009] S1. Culture of Porphyromonas gingivalis: Inoculate the seed solution of Porphyromonas gingivalis into solid culture medium and culture it anaerobically at 37°C overnight; pick a single colony into liquid culture medium, culture it anaerobically at 37°C for 24 hours, and then continue to culture it in a bacterial incubator until the OD 600 =1;

[0010] S2. Propose the target: Absorb OD 600 = 1, centrifuge to separate the upper liquid, centrifuge again to obtain a clarified upper liquid; filter the clarified upper liquid, perform ultrafiltration centrifugation on the obtained filtrate, rinse the filter with water to obtain a rinse liquid, and obtain a melanin and bacterial extracellular vesicle complex PgOMV@Melanin after ultrasonic treatment, which is vacuum freeze-dried and stored in a refrigerator.

[0011] Preferably, the Porphyromonas gingivalis is ATCC BAA-308 Porphyromonas gingivalis.

[0012] Preferably, the solid culture medium is a brain heart infusion agar medium containing sterile defibrinated sheep blood and hemin-vitamin K.

[0013] Preferably, the liquid culture medium is a brain heart infusion broth culture medium containing sterile defibrinated sheep blood and hemin-vitamin K, which includes 3.7 g brain heart infusion broth dry powder, 50 mL distilled water, 5 mL sterile defibrinated sheep blood, 50 μL hemin and 5 μL vitamin K.

[0014] Preferably, the specific operation of filtering in step S2 is: filtering the clarified upper layer liquid through 0.45 μm and 0.22 μm filter membranes in sequence.

[0015] Preferably, the parameters of the ultrasonic treatment in step S2 are: 450w, 1min.

[0016] In a second aspect, the present invention provides the use of a melanin and bacterial extracellular vesicle complex obtained by the method described in the first aspect in the preparation of a drug for treating tumors and / or a product for diagnosing tumors.

[0017] Beneficial effects: The method of the invention is economical and simple, and can simultaneously extract bacterial extracellular vesicles (OMV) and melanin. After being treated with an ultrasonic cell crusher, the PgOMV@Melanin complex can be extracted. Under 808nm near-infrared laser irradiation, the effects of an immune agonist and a photothermal agent can be achieved simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a negatively stained TEM electron microscope image of the melanin and bacterial outer membrane vesicle complex of the present invention;

[0019] Figure 2 This is a TEM electron microscope image of the melanin and bacterial outer membrane vesicle complex of the present invention under non-negative staining;

[0020] Figure 3 This is a TEM electron microscope image of the melanin and bacterial outer membrane vesicle complex of the present invention under non-negative staining;

[0021] Figure 4 is the particle diameter diagram of the complex of melanin and bacterial outer membrane vesicles;

[0022] Figure 5 It is the SDS-PAGE electrophoresis of Porphyromonas gingivalis and the complex of melanin and bacterial outer membrane vesicles;

[0023] Figure 6 The results of the photothermal capacity test of melanin and bacterial outer membrane vesicle complexes at different concentrations are shown in Figure A, where A is a real-time thermal image of PgOMV@Melanin solutions of different concentrations irradiated with near-infrared excitation light, and Figure B is a line graph showing the changes in Figure A.

[0024] Figure 7The results of the photothermal capacity test of melanin and bacterial outer membrane vesicle complexes at different powers are shown in Figure A, where A is the real-time thermal imaging image of PgOMV@Melanin at a concentration of 1500μg / mL under near-infrared excitation light of different powers, and B is the line graph change reaction of Figure A;

[0025] Figure 8 is a schematic diagram of the photothermal reproducibility of the complex of melanin and bacterial outer membrane vesicles;

[0026] Fig. 9 This is a diagram of the inhibitory effect of Porphyromonas gingivalis, melanin and bacterial outer membrane vesicle complex on mouse squamous cell carcinoma cells;

[0027] Fig.10 It is a result diagram of the content of tumor necrosis factor TNF-α (A) and interleukin IL-6 (B) in different groups of the present invention;

[0028] Fig.11 This is the result of confocal observation of the expression of CRT, a marker protein of apoptosis, in different groups after different treatments;

[0029] Fig.12 This is the result of confocal observation of the expression of HMGB1, a marker protein of apoptosis, in different groups after different treatments;

[0030] Fig.13 This is the result of ELISA testing the expression of intracellular ATP in different groups after different treatments;

[0031] Fig.14 This is the result of inhibiting the invasion of M1 macrophages in different groups after different treatments;

[0032] Fig.15 This is the result of inhibiting the migration of M1 macrophages in different groups after different treatments;

[0033] Fig.16 This is a graph showing the live / dead status of tumor cells in different groups after different treatments observed under an inverted microscope;

[0034] Fig.17 This is the flow cytometry analysis result of macrophage transformation in different groups after different treatments;

[0035] Fig.18 This is the result of confocal observation of CD86 protein expression on the surface of M1 macrophages in different groups after different treatments. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0037] Example 1

[0038] A method for simultaneously extracting melanin and bacterial exosome complexes from Porphyromonas gingivalis, comprising the following steps:

[0039] S1,

[0040] Culture of Porphyromonas gingivalis: The culture medium is brain heart infusion broth (BHI), accurately weigh 3.7g BHI dry powder, dissolve in 50mL distilled water, sterilize at 121℃ for 15min, and cool to 37℃. Then add 5mL sterile defibrinated sheep blood and 55μL hemin-vitamin K (50μL hemin and 5μL vitamin K), shake and mix evenly. Cool to room temperature, put in a 4℃ refrigerator, keep away from light, and set aside;

[0041] The ATCC BAA-308 Porphyromonas gingivalis strain was selected, and a single Pg colony was selected using a sterile pipette and placed in a BHI blood broth medium. A disposable anaerobic gas production bag and an anaerobic gas production bag were used to culture the culture at 37°C in an anaerobic environment. After shaking in a shaker for 24 hours, the culture was continued in a bacterial incubator.

[0042] S2. Propose the target: Absorb OD 600 =1 culture medium 15mL, 3000rpm centrifugation for 15min to separate the upper liquid, further 12500rpm centrifugation for 20min, discard the bottom precipitate to obtain a clarified upper liquid; the clarified upper liquid was filtered through 0.45μm and 0.22μm filter membranes in sequence, the filtrate obtained was transferred to a Millipore ultrafiltration tube and centrifuged at 10000rpm for 20min, the filter was rinsed with distilled water, and the final product, melanin and bacterial extracellular vesicle complex PgOMV@Melanin, was obtained after ultrasonic treatment at 450w power in an ultrasonic cell crusher for 1min, and finally lyophilized in a vacuum freeze dryer and stored in a -20℃ refrigerator.

[0043] Example 2

[0044] 2.1 Electron microscopy observation:

[0045] An appropriate amount of the lyophilized product PgOMV@Melanin prepared in the example was resuspended in 5 mL of deionized water, and 5 μL of the liquid was taken with a pipette and dropped on the amorphous carbon film copper mesh carrier, and allowed to dry naturally. After drying, 4 μL of 2% uranyl acetate dye was dropped on it for negative staining for 30 seconds, and finally the copper mesh was observed by a transmission electron microscope of the Haitachi TEM system. The results are as follows: Figure 1 Shown: Clear round vesicle-like structures can be seen against the black negatively stained background.

[0046] The solution containing PgOMV@Melanin was dropped onto the amorphous carbon film copper mesh support and allowed to dry naturally. Then, the solution was observed under a transmission electron microscope using a Haitachi TEM system without negative staining. The results are as follows: Figure 2-Figure 3 As shown: A typical vesicle-like structure can be seen, and a circle of low-density uniform, cloud-like structure can be seen surrounding the vesicle.

[0047] Therefore, the PgOMV@Melanin structure is a typical structure of a Porphyromonas gingivalis vesicle surrounded by a circle of low-density melanin shadow.

[0048] 2.2 Particle diameter:

[0049] NanoBrook Omni Nanoparticle Size and Zeta Potential Analyzer detects the average particle size of nanoparticles. The results are as follows: Figure 4 Shown: The average particle size is 20 nm, which is consistent with the TEM image characteristics.

[0050] 2.3 SDS-PAGE electrophoresis (staining) protein characterization:

[0051] After preparing BCA working solution and protein standard working solution, draw a protein standard curve. Dilute the Pg and PgOMV@Melanin protein samples to be tested 10 times with deionized water, then pipette 20 μL of sample into the well, then add 200 μL of BCA working solution to the well, and incubate at 37°C for 30 min. Adjust the absorbance to 562 nm, and measure the two samples with an enzyme reader. According to the measured absorbance, the protein concentration of the sample to be tested is obtained on the BCA standard curve.

[0052] SDS-PAGE electrophoresis was used to further verify the expression of PgOMV@Melanin membrane protein. The BCA membrane protein quantification method was used to quantify the membrane protein of Pg and PgOMV@Melanin. The samples were mixed with protein loading buffer at a volume ratio of 4:1, vortexed for 1 min, boiled at 100°C for 5 min, and centrifuged in a 0.5mL EP tube (2000rpm, 4°C) for 2 min. The supernatant samples were collected and 8% lower gel and 5% upper gel were prepared in the electrophoresis instrument. After gelation, Marker, Pg and PgOMV@Melanin protein samples were added to the electrophoresis channel of the upper gel in sequence, and electrophoresis was performed with 8% separation gel. When the bromophenol blue indicator reached the bottom of the lower gel, the electrophoresis was stopped, the gel block was removed, and after Coomassie brilliant blue staining for 5 min, 20 mL of decolorizing solution was prepared and washed for 10 min. After washing, the gel block was placed in deionized water for 48 h until the bands were clearly visible. The results are as follows. Figure 5As shown: The surface proteins of Porphyromonas gingivalis Pg and PgOMV@Melanin are expressed on each molecular weight marker, and the expression of surface proteins of Porphyromonas gingivalis Pg is consistent with that of Porphyromonas gingivalis Pg, indicating that PgOMV@Melanin inherits all protein information on the surface of the parent bacterium Porphyromonas gingivalis.

[0053] 2.4 Examination of photothermal effect at different concentrations:

[0054] The obtained PgOMV@Melanin solution was placed in a 2 mL EP tube and freeze-dried in a vacuum freeze dryer (-40 ° C, 12.4 Pa, 12 h) to obtain a powder. Then, different concentrations of PgOMV@Melanin solutions (1000, 1250, 1500, 1750 and 2000 μg / mL) were prepared by weighing. 1 mL of each solution was placed in an EP tube and near-infrared excitation light (808 nm, 1 W / cm 2 ) for 5 min, and use a thermal imager to record the real-time thermal image and temperature changes of the sample every 60 s. The results are as follows Figure 6 As shown in A, five different concentrations of PgOMV@Melanin solutions (1000, 1250, 1500, 1750 and 2000 μg / mL) were set up, and a control group was set up at the same time. 2 ) under the irradiation, the temperature change of the solution within 5 minutes was recorded by an infrared thermal imager. When the solution concentration was 1500μg / mL, the optimal temperature of 55℃ could be reached. Figure B is a line graph showing the temperature change of PgOMV@Melanin solutions with different concentrations in Figure A.

[0055] 2.5 Examination of photothermal effect at different powers:

[0056] The PgOMV@Melanin solution with a concentration of 1500 μg / mL was placed in a 2 mL EP tube, and the near-infrared excitation light was adjusted to different powers (0.8 w / cm 2 、1.0w / cm 2 and 1.2w / cm 2 ), using a thermal imager to record the real-time thermal image and temperature changes of the sample every 60 seconds. The results are as follows Figure 7 As shown in A: The temperature of PgOMV@Melanin solution was set to 1500 μg / mL, and the power of three different near-infrared lasers (0.8 w / cm 2 、1.0w / cm 2 and 1.2w / cm 2 ) as the parameter, the temperature change of the solution within 5 minutes was recorded by infrared thermal imager. When the near-infrared laser power was 1.0w / cm 2The optimal temperature can reach 55°C; Figure B is a line graph showing the temperature change of the PgOMV@Melanin solution at different laser powers in Figure A.

[0057] 2.6 Photothermal stability test:

[0058] The PgOMV@Melanin sample at a concentration of 1500 μg / mL was irradiated with a switching laser for 3 cycles, i.e., 808 nm, 1 W / cm 2 Irradiate with near-infrared laser for 5 min, turn off the laser light source, and cool to ambient temperature; then use 808 nm, 1 W / cm 2 Irradiate with near-infrared laser for 5 min, turn off the laser light source, and cool to ambient temperature; finally, use 808 nm, 1 W / cm 2 Irradiate with near-infrared laser for 5 minutes, turn off the laser light source, and cool to ambient temperature. During the photothermal stability test period, use an infrared thermal imager to monitor the temperature change of the sample in real time. The results are as follows: Figure 8 As shown in the figure: PgOMV@Melanin was heated from room temperature to 55°C within 5 minutes, and after the laser was turned off for 15 minutes, it was cooled from 55°C to room temperature, that is, 20 minutes was one cycle. A total of three cycles were performed, indicating that PgOMV@Melanin has photothermal stability.

[0059] 2.7CCK-8 cell proliferation assay:

[0060] On the first day, 1×10 5 scc-7 cells were inoculated in 96-well plates, with a total of 18 wells. 200 μL PBS was added to the adjacent wells around the cell wells to reduce the volatilization of the culture medium. The culture plate was placed in an incubator for 24 h (37°C, 5% CO2).

[0061] On the second day, after the cells attached to the wall, 100 μL of each of the five different concentrations of PgOMV@Melanin solution was added to 15 experimental wells, with three wells for each concentration. Three wells were reserved as control wells, and the experimental wells were irradiated with near-infrared laser for 5 min (808 nm, 1 w / cm 2 ), put it in the incubator and continue to culture for 2 hours; add 10 μL CCK-8 solution to each well, gently shake the well plate, and continue to incubate in the incubator for 2 hours. Use an enzyme reader to measure the absorbance value (OD value) at 450nm of each well, then:

[0062] Cell survival rate = (OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of blank well) * 100%;

[0063] The results are as follows Fig. 9As shown in the figure: With the increase of PgOMV@Melanin concentration, the survival rate of SCC-7 cells also decreased. When the concentration was 1500μg / mL, the survival rate of SCC-7 cells decreased to below 50%. This shows that PgOMV@Melanin and laser irradiation have the ability to reduce the survival of tumor cells.

[0064] 2.8 Cytokine detection:

[0065] 1×10 6 RAW264.7 cells were inoculated in a 24-well plate and cultured in a 37°C incubator overnight. PBS, PgOMV@Melanin (1500 μg / mL) and PgOMV@Melanin (1500 μg / mL) were then added and laser (808 nm, 1 w / cm 2 After irradiation for 24 hours, the contents of tumor necrosis factor TNF-a and interleukin (IL-6) in the supernatant were determined by Elisa method. Fig.10 As shown in Figure 2, the factor content increased after PgOMV@Melanin (1500 μg / mL) treatment, and PgOMV@Melanin (1500 μg / mL) and laser (808 nm, 1 w / cm 2 The contents of TNF-a (Figure A) and IL-6 (Figure B) were the highest after irradiation (5min, 10min). This indicates that RAW264.7 cells treated with PgOMV@Melanin and laser can secrete more TNF-a and IL-6 factors.

[0066] 2.9 Detection of Calreticulin:

[0067] Photothermal therapy (PTT) produces a thermal effect under the action of laser, which can induce tumor immunogenic death, directly killing tumor cells. It can also induce immunogenic cell death (ICD) to activate systemic anti-tumor immune response, causing apoptotic tumor cells to release damage-associated molecular patterns (DAMPs) to trigger the body's anti-tumor immune response. ICD is a key trigger for immune response, characterized by high expression of pro-apoptotic calreticulin (CRT) and release of high-mobility group protein 1 (HMGB1) and adenosine triphosphate (ATP).

[0068] CRT:

[0069] 1×10 6SCC-7 cells were inoculated in confocal microplates and incubated in a 37°C incubator overnight. PBS, PgOMV@Melanin, and PgOMV@Melanin were added to the microplates and irradiated with laser. After replacing the culture medium, the cells were cultured in a 37°C incubator for 4 h. Then:

[0070] 1) The cells in each group were permeabilized with 100 μL 0.5% cell permeabilizer for 30 min and washed with PBS three times;

[0071] 2) Each group of cells was fixed with 200 μL 4% paraformaldehyde at room temperature for 15 min and washed three times with PBS;

[0072] 3) Add 200 μL of diluted fluorescent dye Alexa Fluor 488 Anti-HMGb1 (1:100) and incubate at 4°C for 1 h;

[0073] 4) After washing with PBS three times, 300 μL DAPI was added to stain the nucleus for 8 min, and then confocal imaging was continued. The results are as follows Fig.11 As shown: The fluorescence intensity of the extranuclear CRT factor was significantly enhanced after treatment with PgOMV@Melanin or PgOMV@Melanin and laser irradiation, indicating that the CRT factor decreased inside the cell and increased outside the cell.

[0074] Conclusion: PgOMV@Melanin or PgOMV@Melanin and laser irradiation have the effect of inducing immunogenic cell death (ICD).

[0075] HMGB1:

[0076] 1×10 6 SCC-7 cells were inoculated in confocal microplates and incubated in a 37°C incubator overnight. PBS, PgOMV@Melanin, and PgOMV@Melanin were added to the microplates and irradiated with laser. After replacing the culture medium, the cells were cultured in a 37°C incubator for 4 h. Then:

[0077] 1) The cells in each group were permeabilized with 100 μL 0.5% cell permeabilizer for 30 min and washed with PBS three times;

[0078] 2) Each group of cells was fixed with 200 μL 4% paraformaldehyde at room temperature for 15 min and washed three times with PBS;

[0079] 3) Add 200 μL of diluted fluorescent dye Alexa Fluor 488 Anti-HMGb1 (1:100) and incubate at 4°C for 1 h;

[0080] 4) After washing with PBS three times, 300 μL DAPI was added to stain the nucleus for 8 min, and then confocal imaging was continued. The results are as follows Fig.12 As shown: The nuclear fluorescence intensity was significantly weakened after PgOMV@Melanin and laser irradiation treatment, indicating that the nuclear expression of HMGB1 protein was reduced.

[0081] Conclusion: PgOMV@Melanin or PgOMV@Melanin and laser irradiation have the effect of inducing immunogenic cell death (ICD).

[0082] Adenosine triphosphate (ATP) detection:

[0083] 1×10 6 SCC-7 cells were seeded in a 6-well plate and cultured overnight at 37°C and 5% CO2. Then, PBS, PgOMV@Melanin, and PgOMV@Melanin were added to the cell culture medium and irradiated with laser. After incubation for 24 hours, the culture medium was replaced with new one. After incubation for 6 hours, the cells were collected into a centrifuge tube, the supernatant was removed, and ATP extract was added (the volume of the extract was 500-1000:1. In this embodiment, 1 mL of extract was added to 5 million cells), and ultrasonically disrupted for 1 minute (200w). Centrifuge at 10,000×g and 4°C for 10 minutes; transfer the supernatant to a 2mL EP tube, add 500μL of chloroform and shake thoroughly to mix, centrifuge at 10,000×g and 4°C for 3 minutes, and take the supernatant for detection. The results are as follows: Fig.13 As shown: the intracellular ATP was higher after PBS treatment, while the intracellular ATP was significantly reduced after PgOMV@Melanin and PgOMV@Melanin and laser irradiation treatment.

[0084] Conclusion: PgOMV@Melanin or PgOMV@Melanin and laser irradiation have the effect of inducing immunogenic cell death (ICD).

[0085] 2.10 Cell invasion:

[0086] Thaw the matrix gel at 4°C overnight, dilute it with serum-free medium at a ratio of 1:10; vertically add the diluted matrix gel to the upper chamber of the transfer chamber, gently shake the chamber to evenly spread the matrix gel, and place it in an incubator (37°C, 5% CO2) for 4 hours to allow the matrix gel to polymerize into a gel film. After centrifugation to collect the cells, add serum-free medium and transfer 1×10 5RAW267.4 cells were added to the upper chamber of the transfer chamber, and 500 μL of 10% FBS culture medium was added to the lower chamber of the transfer chamber. Lipopolysaccharide, IL-6, PgOMV@Melanin (1500 μg / mL) and PgOMV@Melanin (1500 μg / mL) were added and laser irradiated. After culturing in an incubator (37°C, 5% CO2) for 24 hours, the cells were stained with 0.1% crystal violet for 20 minutes, washed three times with PBS, and the upper side of the chamber was gently wiped with a cotton swab. After proper air drying, the cells were observed under a microscope. Fig.14 As shown: the invasive ability of M1 macrophages induced by lipopolysaccharide is lower than that of M2 macrophages induced by IL-6. The invasive ability of macrophages treated with PgOMV@Melanin (1500μg / mL) and PgOMV@Melanin (1500μg / mL) and laser irradiation is reduced, and the invasive ability is further reduced after laser treatment. It shows that PgOMV@Melanin (1500μg / mL) and PgOMV@Melanin (1500μg / mL) and laser irradiation have the ability to promote the transformation of macrophages into M1 macrophages.

[0087] 2.11 Cell Migration

[0088] Thaw the cells at room temperature, add 1 mL PBS and collect the cells by centrifugation. 5 RAW267.4 cells were added to the upper chamber of the transfer chamber, and 500 μL of 10% FBS culture medium was added to the lower chamber of the transfer chamber. LPS, IL-6, PgOMV@Melanin (1500 μg / mL) and PgOMV@Melanin (1500 μg / mL) + laser treatment were added respectively. After culturing in an incubator (37°C, 5% CO2) for 24 hours, the cells were stained with 0.1% crystal violet for 20 minutes, washed three times with PBS, and the upper side of the transfer chamber was gently wiped with a cotton swab. After proper air drying, the cells were observed under a microscope. Fig.15 As shown: the migration ability of M1 macrophages induced by lipopolysaccharide is lower than that of M2 macrophages induced by IL-6. The migration ability of macrophages treated with PgOMV@Melanin (1500μg / mL) and PgOMV@Melanin (1500μg / mL) + laser is reduced, and the invasion ability is further reduced after laser treatment. This shows that PgOMV@Melanin (1500μg / mL) and PgOMV@Melanin (1500μg / mL) + laser irradiation have the ability to promote the transformation of macrophages into M1 macrophages.

[0089] 2.12 Cell live / death analysis:

[0090] 1×10 5 SCC-7 cells were seeded in a 6-well plate and cultured overnight at 37°C and 5% CO2. Then PBS, PgOMV@Melanin, and PgOMV@Melanin were added to the cell culture medium and irradiated with laser. After incubation for 24 hours, Live or Dead reagent was added for staining and imaging was performed under an inverted microscope. Fig.16 As shown: After PBS treatment, cells expressed green fluorescence, indicating that the cells were in good condition. After PgOMV@Melanin / PgOMV@Melanin+laser irradiation treatment, red fluorescence increased, indicating that some cells had died, and the red fluorescence after laser treatment was further enhanced, indicating that the number of dead cells increased further. Conclusion: PgOMV@Melanin+laser irradiation has the ability to kill tumor cells SCC-7 and can inhibit the proliferation of SCC-7 cells.

[0091] 2.13 Transformation Capability (M2→M1):

[0092] Confocal:

[0093] 1) 1×10 6 The raw264.7 cells were added to a 6-well plate, with a total of four wells. LPS and IL-6 were added to the first two wells to induce the raw264.7 cells to M1 and M2 types, respectively, and incubated in a 37°C incubator for 24 h.

[0094] 2) PgOMV@Melanin and PgOMV@Melanin were added to the last two wells and irradiated with laser for 24 h;

[0095] 3) Remove old culture medium and wash three times with PBS;

[0096] 4) The cells in each group were permeabilized with 100 μL 0.5% cell permeabilizer for 30 min and washed with PBS three times;

[0097] 5) Fix each group of cells with 200 μL 4% paraformaldehyde at room temperature for 15 min and wash with PBS three times;

[0098] 6) Add 200 μL of diluted fluorescent dye PE-CD86 (1:100) and incubate at 4°C for 12 h;

[0099] 7) After washing with PBS three times, 300 μL of DAPI was added to stain the nuclei for 8 minutes, and then confocal imaging was continued. The results are as follows Fig.18As shown: the fluorescence intensity of CD86 protein on the surface of M1 macrophages was significantly enhanced, while the intensity of CD86 protein expression on M2 macrophages was weaker. The intensity of CD86 protein on the surface of macrophages treated with PgOMV@Melanin or PgOMV@Melanin and laser irradiation was also significantly enhanced, indicating that macrophages transformed into M1 type after PgOMV@Melanin treatment.

[0100] Transformation capability (streaming):

[0101] 1) 1×10 6 The raw264.7 cells were added to a 6-well plate, with a total of four wells. LPS and IL-6 were added to the first two wells to induce the raw264.7 cells to M1 and M2 types, respectively, and incubated in a 37°C incubator for 24 h.

[0102] 2) PgOMV@Melanin and PgOMV@Melanin were added to the last two wells and irradiated with laser for 24 h;

[0103] 3) Remove old culture medium and wash three times with PBS;

[0104] 4) The cells in each group were permeabilized with 100 μL 0.5% cell permeabilizer for 30 min and washed with PBS three times;

[0105] 5) Fix each group of cells with 200 μL 4% paraformaldehyde at room temperature for 15 min and wash with PBS three times;

[0106] 6) Add 200 μL of diluted fluorescent dye PE-CD86 (1:100) and incubate at 4°C for 12 h;

[0107] 7) After washing with PBS three times, add 500 μL PBS to each well, transfer the cells to the flow tube after blowing gently, protect from light, and test on the flow analyzer. Fig.17 As shown: the PE fluorescence intensity of CD86 protein on the surface of M1 macrophages was significantly enhanced, while the PE fluorescence intensity of CD86 protein expressed by M2 macrophages was weaker. The PE fluorescence intensity of CD86 protein on the surface of macrophages treated with PgOMV@Melanin or PgOMV@Melanin and laser irradiation was also significantly enhanced, indicating that the macrophages treated with PgOMV@Melanin transformed into the M1 type.

[0108] In summary: the method of the present invention is economical and simple, and can simultaneously extract bacterial extracellular vesicles (OMV) and melanin. After being treated with an ultrasonic cell crusher, the PgOMV@Melanin complex can be extracted. Under 808nm near-infrared laser irradiation, the effects of immune agonist and photothermal agent can be achieved simultaneously.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously extracting melanin and bacterial outer membrane vesicle complexes from Porphyromonas gingivalis, characterized in that: The following steps are involved: S1. Culture of Porphyromonas gingivalis: Inoculate the seed solution of Porphyromonas gingivalis into solid culture medium and culture it anaerobically at 37°C overnight; pick a single colony into liquid culture medium, culture it anaerobically at 37°C for 24 hours, and then continue to culture it in a bacterial incubator until the OD 600 =1; S2. Propose the target: Absorb OD 600 = 1 of culture solution, centrifugation to separate the upper liquid, and centrifugation again to obtain a clarified upper liquid; the clarified upper liquid is filtered, the obtained filtrate is subjected to ultrafiltration centrifugation, the filter is rinsed with water to obtain a rinse liquid, and after ultrasonic treatment, a melanin and bacterial extracellular vesicle complex PgOMV@Melanin is obtained, which is vacuum freeze-dried and stored in a refrigerator.

2. A method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis according to claim 1, characterized in that: The Porphyromonas gingivalis is ATCC BAA-308 Porphyromonas gingivalis.

3. The method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis according to claim 1, characterized in that: The solid culture medium is a brain heart infusion agar culture medium containing sterile defibrinated sheep blood and hemin chloride-vitamin K.

4. The method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis according to claim 1, characterized in that: The liquid culture medium is a brain heart infusion broth culture medium containing sterile defibrinated sheep blood and hemin-vitamin K, which includes 3.7 g of brain heart infusion broth dry powder, 50 mL of distilled water, 5 mL of sterile defibrinated sheep blood, 50 μL of hemin and 5 μL of vitamin K.

5. The method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis according to claim 1, characterized in that: The specific operation of filtration in step S2 is: filtering the clarified upper layer liquid through 0.45 μm and 0.22 μm filter membranes in sequence.

6. The method for simultaneously extracting melanin and bacterial extracellular vesicle complexes from Porphyromonas gingivalis according to claim 1, characterized in that: The parameters of the ultrasonic treatment in step S2 are: 450 w, 1 min.

7. Use of the melanin and bacterial extracellular vesicle complex obtained by the method according to any one of claims 1 to 6 in the preparation of drugs for treating tumors and / or products for diagnosing tumors.

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