An antibiotic carrier based on bacterial outer membrane vesicles and preparation method thereof

Abstract

The present invention provides a method for preparing an antibiotic carrier based on bacterial outer membrane vesicles, characterized in that the preparation method comprises the following steps: (1) culturing bacteria in a culture medium containing antibiotics; (2) removing live bacteria from the culture medium and extracting the bacterial outer membrane vesicles, thereby obtaining an antibiotic carrier based on bacterial outer membrane vesicles; wherein the bacteria is a Salmonella mutant strain QS0074, whose deposit number is CCTCC M 2023192; and the antibiotic is acamycin. The antibiotic carrier based on bacterial outer membrane vesicles provided by the present invention has an excellent drug loading capacity for acamycin and good drug activity stability; the preparation method of the present invention is simple and easy to promote.

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), a bacterial derivative, were first discovered in 1963 and gained increasing attention after their rediscovery in 1999. OMVs possess the ability to interact with cells and serve as novel drug delivery platforms. When used as drug carriers, OMVs protect their contents from external environmental influences and transport them to their target sites, making them suitable for long-distance transport.

[0003] To improve the drug loading rate and delivery efficiency of bacterial outer membrane vesicles, researchers typically prepare active pharmaceutical molecules into other nanoparticles and then load them into outer membrane vesicles. However, this approach is often complex and unsuitable for large-scale production. Therefore, it is essential to develop a simpler preparation method based on outer membrane vesicles that can achieve high drug delivery efficiency.

[0004] Currently, a relatively simple approach to preparing drug carriers based on outer membrane vesicles is to culture bacteria in a medium containing the drug, allowing the drug to migrate into the outer membrane vesicles during bacterial growth, thus completing the drug delivery. This approach was first discovered by Kadurugamuwa and Beveridge in 1996 and has since been proven effective. While this approach offers the advantage of simple preparation and suitability for large-scale production, it also suffers from a generally low drug loading rate.

[0005] One of the methods to promote the production of outer membrane vesicles and increase the drug loading rate of outer membrane vesicles. Unfortunately, the mechanism of outer membrane vesicle formation has not yet been clearly studied (Chen Qiaoqiao, Tu Shijuan, Xia Xiuwen, et al. Research progress on the mechanism of bacterial outer membrane vesicle formation [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 could significantly promote the production of outer membrane vesicles of Escherichia coli O104:H4 and Escherichia coli O157:H7. However, they did not further study the issue of drug loading efficiency (DOI10.1128 / AAC.00937-17).

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

[0007] In view of the defects of the prior art, the object of the present invention is to provide an amikacin drug carrier based on bacterial outer membrane vesicles, which has a high drug loading rate and a 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) Cultivate the bacteria in a culture medium containing antibiotics;

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

[0012] Wherein, the bacteria is the Salmonella mutant strain QS0074, and its deposit number is CCTCC M 2023192;

[0013] The antibiotic is amikacin.

[0014] As shown in the relevant examples and comparative examples of the present invention, most antibiotics have a certain promoting effect on the outer membrane vesicles of the Salmonella mutant strain QS0074, but the promoting effect is often limited. In addition, among the antibiotics that can significantly promote the formation of outer membrane vesicles, only amikacin can increase the drug loading rate. This may be because although other antibiotics can promote the formation of outer membrane vesicles, these antibiotics are not loaded into the outer membrane vesicles in large quantities and are excreted.

[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 sulfate 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 amikacin 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 at 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 the preparation of medicines for treating amikacin indications.

[0026] Beneficial effects of the present invention:

[0027] The antibiotic carrier based on bacterial outer membrane vesicles provided by the present invention has an excellent drug loading capacity for amikacin 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 are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention 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 deposit number of CCTCC M 2023192, is owned by the laboratory, and has been patented in another patent application. The present invention does not involve the screening and preservation of this 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. 300 μg / mL of amikacin was added to the Brucella broth.

[0032] (2) The culture was centrifuged at 15,000 × g for 10 minutes, and the supernatant was collected and 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 pellet was resuspended in PBS solution and filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with amikacin.

[0033] (3) The outer membrane vesicles loaded with amikacin were treated with 0.5M EDTA at 37°C for 3 hours, and then the empty outer membrane vesicles were removed by centrifugation. The ratio of the weight of amikacin 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 amikacin were placed in 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. 250 μg / mL of amikacin was added to the Brucella broth.

[0037] (2) The culture was centrifuged at 18,000 × g for 8 minutes, and the supernatant was collected and 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 pellet was resuspended in PBS solution and filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with amikacin.

[0038] (3) The outer membrane vesicles loaded with amikacin 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 amikacin 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 amikacin were placed in 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 strain QS0074 was cultured in 500 mL of Brucella broth at 37°C and 180 rpm overnight. 350 μg / mL of amikacin was added to the Brucella broth.

[0042] (2) The culture was centrifuged at 13,000 × g for 15 minutes, and the supernatant was collected and 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 pellet was resuspended in PBS solution and filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with amikacin.

[0043] (3) The outer membrane vesicles loaded with amikacin 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 amikacin 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 amikacin were placed in 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. 350 μg / mL of amikacin, 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, and the supernatant was collected and 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 pellet was resuspended in PBS solution and filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with amikacin.

[0048] (3) The outer membrane vesicles loaded with amikacin 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 amikacin 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 amikacin were placed in 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 medium did not contain amikacin, the rest was the same as in the example, and the outer membrane vesicles obtained were empty outer membrane vesicles. This comparative example did not investigate the drug loading amount, but only investigated the number of outer membrane vesicles obtained.

[0052] Comparative Example 2

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

[0054] Comparative Example 3

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

[0056] Comparative Example 4

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

[0058] Comparative Example 5

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

[0060] Comparative Example 6

[0061] Except that amikacin was replaced by tigeracycline, the rest was the same as in Example 1.

[0062] Comparative Example 7

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

[0064] Comparative Example 8

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

[0066] Comparative Example 9

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

[0068] Comparative Example 10

[0069] The Salmonella mutant strain QS0074 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 in the dark at 4°C for 20 days. The drug activity was tested to investigate the drug loading stability. Free amikacin was used as a control group. The test results are shown in Table 1.

[0072] As shown in Table 1, the addition of amikacin is crucial for increasing drug loading efficiency in the Salmonella mutant QS0074. Furthermore, at low concentrations, amikacin significantly increased the number of outer membrane vesicles produced. In contrast, the antibiotics used in Comparative Examples 2-7 had no significant effect on drug loading efficiency or 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]

[0075] Note 1: "-" in the table indicates that the corresponding experiment was not conducted. The drug activity experiments or drug loading efficiency tests in Comparative Examples 3-7 were not conducted because the number of outer membrane vesicles or the drug loading efficiency were too low.

[0076] Note 2: The MIC of Salmonella mutant QS0074 against amikacin is 4096 μg / mL. 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) 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 amikacin, 10 μg / mL of ferrous sulfate, and 8 μg / mL of cobalt chloride were added to the Brucella broth; (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 centrifugal precipitate was resuspended in PBS solution and then filtered through a 0.45 μm filter membrane to obtain outer membrane vesicles loaded with amikacin, that is, an antibiotic carrier based on bacterial outer membrane vesicles was obtained; The bacteria is a Salmonella mutant strain QS0074, and its preservation number is CCTCC M 2023192.

2. An antibiotic carrier based on bacterial outer membrane vesicles, characterized in that: The antibiotic carrier is prepared by the preparation method according to claim 1.

3. Use of the bacterial outer membrane vesicle-based antibiotic carrier according to claim 2 in the preparation of a drug for treating amikacin-related indications.

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