Antibiotic carrier based on bacterial outer membrane vesicles and preparation method thereof

Abstract

The invention provides a preparation method of an antibiotic carrier based on bacterial outer membrane vesicles, which is characterized by comprising the following steps: (1) culturing bacteria in a culture medium containing antibiotics; and (2) removing viable bacteria in the culture product, and extracting the outer membrane vesicles of the bacteria to obtain the antibiotic carrier based on the outer membrane vesicles of the bacteria, wherein the bacterium is a salmonella mutant strain QS0074, and the preservation number of the bacterium is CCTCC (China Center For Type Culture Collection) M 2023192; the antibiotic is alcamicin. The antibiotic carrier based on the bacterial outer membrane vesicles, provided by the invention, has excellent drug loading capacity on the acamicin, and has relatively good drug activity stability; the preparation method is simple and easy to popularize.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and relates to bacterial outer membrane vesicle drug carrier technology, and specifically relates to an antibiotic carrier based on bacterial outer membrane vesicles and a preparation method thereof. Background Art

[0002] Bacterial outer membrane vesicles (OMVs) are a bacterial derivative that was discovered by researchers in 1963 and gradually attracted people's attention after it was discovered again in 1999. OMVs have the characteristics of cell-to-cell interaction and can be used as a new drug delivery platform. When OMVs are used as drug carriers, they can protect the contents from the influence of the external environment and transport them to the corresponding targets, which is suitable for long-distance transportation.

[0003] In order to improve the drug loading rate and drug delivery effect of bacterial outer membrane vesicles, researchers generally prepare drug active molecules into other nanoformulations and then load them into outer membrane vesicles. However, this method is usually more complicated and not suitable for large-scale production. Therefore, it is very necessary to develop a method based on outer membrane vesicles that has a simple preparation method and good drug loading effect.

[0004] At present, in the preparation of drug carriers based on outer membrane vesicles, a relatively simple scheme is to culture bacteria in a culture medium containing drugs, so that the drugs migrate to the outer membrane vesicles during the growth of bacteria, thereby completing the drug loading. This scheme was first discovered by Kadurugamuwa and Beveridge in 1996 and has been proven to be effective since then. The advantage of this scheme is that the preparation method is simple and suitable for large-scale production, but the disadvantage is that the drug loading rate is usually low.

[0005] One of the methods to promote the production of outer membrane vesicles and improve the drug loading rate of outer membrane vesicles. Unfortunately, the mechanism of the occurrence of outer membrane vesicles has not been clearly studied (Chen Qiaoqiao, Tu Shijuan, Xia Xiuwen, etc. Research progress on the mechanism of bacterial outer membrane vesicles [J]. Journal of Taishan Medical College, 2019, 40 (12): 980-982.), so there is no clear solution to promote the production of outer membrane vesicles. Danielle M. Vermilyea et al. found that an enzyme called PPAD is important for the production of outer membrane vesicles (DOI: 10.1128 / JB.00343-20.). In addition to endogenous substances, Bauwens, A et al. found that ciprofloxacin, meropenem, fosfomycin and polymyxin B can significantly promote the production of Escherichia coli O104:H4 and Escherichia coli O157:H7 outer membrane vesicles. However, they did not further study the issue of drug loading rate (DOI10.1128 / AAC.00937-17).

[0006] In summary, it is urgently needed in this field to study a technical solution to achieve high drug loading by co-culturing bacterial outer membrane vesicles with drugs. Summary of the invention

[0007] In view of the defects of the prior art, the object of the present invention is to provide an akamycin drug carrier based on bacterial outer membrane vesicles, with high drug loading rate and simple preparation method.

[0008] In order to achieve the above object, the purpose of the present invention is to provide the following technical solutions:

[0009] A method for preparing an antibiotic carrier based on bacterial outer membrane vesicles, the preparation method comprising the following steps:

[0010] (1) Cultivating bacteria in a culture medium containing antibiotics;

[0011] (2) removing live bacteria from the culture product and extracting bacterial outer membrane vesicles to obtain antibiotic carriers based on bacterial outer membrane vesicles;

[0012] Wherein, the bacteria is a mutant strain of Salmonella QS0074, and its deposit number is CCTCC M2023192;

[0013] The antibiotic is akamicin.

[0014] As can be seen from the relevant embodiments and comparative examples of the present invention, most antibiotics have a certain promoting effect on the outer membrane vesicles of the mutant strain QS0074 of Salmonella, but the promoting effect that can be produced is mostly limited. In addition, among the antibiotics that can significantly promote the outer membrane vesicles, only Acamycin can improve the drug loading rate. The possible reason is that although other antibiotics can promote the production of outer membrane vesicles, the relevant antibiotics are not loaded into the outer membrane vesicles in large quantities and are discharged.

[0015] In addition, adding a small amount of ferrous sulfate and cobalt chloride to the culture medium can further significantly increase the production and drug loading of outer membrane vesicles.

[0016] As an embodiment of the present invention, the culture medium is Brucella broth liquid culture medium.

[0017] Preferably, ferrous sulfite and cobalt chloride are also added to the culture medium.

[0018] Preferably, when the culture is performed, the culture temperature is 37° C. and the culture time is 12 hours.

[0019] Preferably, the concentration of akamicin is 250-350 μg / mL.

[0020] Preferably, when performing step (2), the culture is first centrifuged to remove the supernatant, the supernatant is filtered through a membrane, the filtrate is concentrated and then ultracentrifuged, and then the ultracentrifuged precipitate is resuspended and then filtered through a membrane.

[0021] Preferably, when membrane filtration is performed, a 0.45 μm filter membrane is used; when the culture is centrifuged, the centrifugation condition is centrifugation at 15,000×g for 10 minutes.

[0022] Preferably, a 500 kDa ultrafiltration column is used for concentration.

[0023] Preferably, the ultracentrifugation is performed under centrifugation conditions of 200,000×g and 4° C. for 2 hours.

[0024] The present invention also provides an antibiotic carrier based on bacterial outer membrane vesicles prepared by the above preparation method.

[0025] The present invention also provides the use of the above-mentioned antibiotic carrier based on bacterial outer membrane vesicles in preparing medicines for treating the indications of akamicin.

[0026] Beneficial effects of the present invention:

[0027] The antibiotic carrier based on bacterial outer membrane vesicles provided by the present invention has excellent drug loading capacity for akamycin and good drug activity stability; the preparation method of the present invention is simple and easy to promote. DETAILED DESCRIPTION

[0028] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0029] The Salmonella mutant strain QS0074 used in the examples of the present invention has a preservation number of CCTCC M2023192, is owned by the laboratory, and has been patented in another patent application. The present invention does not involve the screening and preservation of the strain.

[0030] Example 1

[0031] (1) A single clone of the Salmonella mutant strain QS0074 was cultured in 500 mL of Brucella broth at 37°C and 180 rpm overnight, wherein 300 μg / mL of acamycin was added to the Brucella broth.

[0032] (2) The culture was centrifuged at 15,000 × g for 10 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4° C. for 2 hours. The centrifuged precipitate was resuspended in a PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with akamicin.

[0033] (3) The outer membrane vesicles loaded with akamycin were treated with 0.5 M EDTA at 37°C for 3 hours, and then the empty outer membrane vesicles were removed by centrifugation. The ratio of the weight of akamycin loaded in the outer membrane vesicles to the number of outer membrane vesicles was recorded as the drug loading μg / 10 12 indivual.

[0034] (4) The outer membrane vesicles loaded with akamycin were placed in a PBS solution and stored in the dark at 4°C for 20 days to investigate the drug loading stability.

[0035] Example 2

[0036] (1) A single clone of the Salmonella mutant strain QS0074 was cultured in 500 mL of Brucella broth at 37°C and 180 rpm overnight, wherein 250 μg / mL of acamycin was added to the Brucella broth.

[0037] (2) The culture was centrifuged at 18,000 × g for 8 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4° C. for 2 hours. The centrifuged precipitate was resuspended in a PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with akamicin.

[0038] (3) The outer membrane vesicles loaded with akamycin were treated with 0.4 M EDTA at 37°C for 3 hours, and then the empty outer membrane vesicles were removed by centrifugation. The ratio of the weight of akamycin loaded in the outer membrane vesicles to the number of outer membrane vesicles was recorded as the drug loading μg / 10 12 indivual.

[0039] (4) The outer membrane vesicles loaded with akamycin were placed in a PBS solution and stored in the dark at 4°C for 20 days to investigate the drug loading stability.

[0040] Example 3

[0041] (1) A single clone of the Salmonella mutant QS0074 was cultured in 500 mL of Brucella broth at 37° C. and 180 rpm overnight, wherein 350 μg / mL of acamycin was added to the Brucella broth.

[0042] (2) The culture was centrifuged at 13,000 × g for 15 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4° C. for 2 hours. The centrifuged precipitate was resuspended in a PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with akamicin.

[0043] (3) The outer membrane vesicles loaded with akamycin were treated with 0.6 M EDTA at 37°C for 3 hours, and then the empty outer membrane vesicles were removed by centrifugation. The ratio of the weight of akamycin loaded in the outer membrane vesicles to the number of outer membrane vesicles was recorded as the drug loading μg / 10 12 indivual.

[0044] (4) The outer membrane vesicles loaded with akamycin were placed in a PBS solution and stored in the dark at 4°C for 20 days to investigate the drug loading stability.

[0045] Example 4

[0046] (1) A single clone of the Salmonella mutant strain QS0074 was cultured in 500 mL of Brucella broth at 37°C and 180 rpm overnight, wherein 350 μg / mL of acamycin, 10 μg / mL of ferrous sulfate, and 8 μg / mL of cobalt chloride were added to the Brucella broth.

[0047] (2) The culture was centrifuged at 13,000 × g for 15 minutes, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated using a 500 kDa ultrafiltration column. The concentrate was then centrifuged at 200,000 × g and 4° C. for 2 hours. The centrifuged precipitate was resuspended in a PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with akamicin.

[0048] (3) The outer membrane vesicles loaded with akamycin were treated with 0.6 M EDTA at 37°C for 3 hours, and then the empty outer membrane vesicles were removed by centrifugation. The ratio of the weight of akamycin loaded in the outer membrane vesicles to the number of outer membrane vesicles was recorded as the drug loading μg / 10 12 indivual.

[0049] (4) The outer membrane vesicles loaded with akamycin were placed in a PBS solution and stored in the dark at 4°C for 20 days to investigate the drug loading stability.

[0050] Comparative Example 1

[0051] Except that the Brucella broth liquid culture medium does not contain akamicin, the rest is consistent with the example, and the outer membrane vesicles obtained are empty outer membrane vesicles. This comparative example does not investigate the drug loading amount, but only investigates the number of outer membrane vesicles obtained.

[0052] Comparative Example 2

[0053] Except that akamicin was replaced by levofloxacin, the rest was the same as Example 1.

[0054] Comparative Example 3

[0055] Except that akamicin was replaced by ciprofloxacin, the rest was the same as Example 1.

[0056] Comparative Example 4

[0057] Except that akamycin was replaced by polymyxin B, the rest was the same as in Example 1.

[0058] Comparative Example 5

[0059] Except that akamycin was replaced by fosfomycin, the rest was the same as Example 1.

[0060] Comparative Example 6

[0061] Except that akamicin was replaced by tadalafil, the rest was the same as Example 1.

[0062] Comparative Example 7

[0063] Except that akamycin was replaced by mitomycin C, the rest was the same as Example 1.

[0064] Comparative Example 8

[0065] Except that the concentration of akamicin was adjusted to 500 μg / mL, the rest was the same as Example 1.

[0066] Comparative Example 9

[0067] Except that the concentration of akamicin was adjusted to 400 μg / mL, the rest was the same as Example 1.

[0068] Comparative Example 10

[0069] The mutant strain QS0074 of Salmonella was replaced by Escherichia coli O104:H4, and the rest was the same as in Example 1.

[0070] Experimental Example 1

[0071] The number of outer membrane vesicles formed, drug loading and stability were tested for Examples 1-3 and Comparative Examples 1-4. The drug loading was recorded as the ratio of the loaded drug weight to the number of outer membrane vesicles μg / 10 12 The drug-loaded outer membrane vesicles were placed in a PBS solution and stored at 4°C in the dark for 20 days. The drug activity was tested to investigate the drug loading stability, and free acamycin was used as the control group. The test results are shown in Table 1.

[0072] As shown in Table 1, for the mutant strain of Salmonella QS0074, the addition of akamicin is important for improving the drug loading rate. At the same time, at low concentrations of akamicin, the number of outer membrane vesicles produced can be significantly increased. However, the antibiotics used in Comparative Examples 2-7 had no significant effect on the drug loading rate and the number of outer membrane vesicles.

[0073] In addition, as shown in Example 4, adding a small amount of ferrous sulfate and cobalt chloride to the Brucella broth liquid culture medium can further significantly increase the production and drug loading of outer membrane vesicles.

[0074] Table 1

[0075]

[0076] Note 1: "-" in the table means that the corresponding experiment was not carried out. Among them, the reason why the drug activity experiment or drug loading rate detection in Comparative Examples 3-7 was not carried out was that the number of outer membrane vesicles or the drug loading rate was too low.

[0077] Note 2: The MIC of Salmonella mutant QS0074 against akamicin is 4096 μg / mL, and the MIC of Salmonella mutant QS0074 against the antibiotics used in Comparative Examples 2-7 are all over 350 μg / mL.

Claims

1. A method for preparing an antibiotic carrier based on bacterial outer membrane vesicles, characterized in that: The preparation method comprises the following steps: (1) Cultivating bacteria in a culture medium containing antibiotics; (2) removing live bacteria from the culture product and extracting bacterial outer membrane vesicles to obtain antibiotic carriers based on bacterial outer membrane vesicles; Wherein, the bacteria is a mutant strain of Salmonella QS0074, and its deposit number is CCTCC M2023192; The antibiotic is akamicin.

2. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 1, characterized in that: The culture medium is Brucella broth liquid culture medium.

3. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 2, characterized in that: The culture medium is also supplemented with ferrous sulfite and cobalt chloride.

4. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 2 or 3, characterized in that: The culture was carried out at a temperature of 37° C. for 12 hours.

5. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 1 or 2, characterized in that: The concentration of akamicin is 250-350 μg / mL.

6. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 1, characterized in that: When performing step (2), the culture is first centrifuged to remove the supernatant, the supernatant is filtered through a membrane, the filtrate is concentrated and then ultracentrifuged, and then the ultracentrifuged precipitate is resuspended and then filtered through a membrane.

7. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 6, characterized in that: When performing membrane filtration, a 0.45 μm filter membrane was used; when performing centrifugation of the culture, the centrifugation condition was centrifugation at 15,000×g for 10 minutes; and when performing concentration, a 500 kDa ultrafiltration column was used.

8. The method for preparing an antibiotic carrier based on bacterial outer membrane vesicles according to claim 7, characterized in that: Ultracentrifugation was performed at 200,000×g and 4° C. for 2 hours.

9. An antibiotic carrier based on bacterial outer membrane vesicles, characterized in that: The antibiotic carrier is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the antibiotic carrier based on bacterial outer membrane vesicles according to claim 9 in the preparation of drugs for treating the indications of akamicin.

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