JP7651091B2 - Method for producing membrane vesicles containing a target substance - Google Patents

Description

The present invention relates to a method for producing membrane vesicles in which a target substance is encapsulated.

Membrane vesicles (sometimes referred to as "membrane vesicles" or "MVs" in this specification) are attracting attention as a fundamental technology for vaccine development and drug delivery due to their immune-inducing properties and their tendency to accumulate in cancer cells.

For example, in vaccine development, a technology has been developed in which membrane vesicles are prepared from bacteria and used as an immunogenic composition (Patent Documents 1 and 2). However, due to its purpose, this technology is limited to preparing membrane vesicles from bacteria, and has not yet reached the stage of encapsulating a target substance in the membrane vesicle. In addition, in the development of drug delivery technology, exosomes, which control intercellular communication, have attracted attention in recent years due to their targeting properties, and their application to pharmaceuticals is being considered (Patent Document 3). However, encapsulation of a target substance in exosomes is achieved by electroporation, freezing and thawing, ultrasonic treatment, saponification, etc., and there are naturally limitations to mass production and quality control as pharmaceuticals.

Special table 2014-520801 publication Special Publication No. 2006-503822 Special table 2019-535839 publication

The present invention aims to provide a novel method for producing membrane vesicles encapsulating a target substance.

The present inventors have been conducting intensive research focusing on the phenomenon of membrane vesicles produced by bursting bacteriolysis, and have discovered that membrane vesicles encapsulating a target substance can be produced by inducing bursting bacteriolysis in gram-negative bacteria. The present invention is based on this finding.

According to the present invention, the following inventions are provided.
[1] A method for producing membrane vesicles encapsulating a target substance, comprising: (A) producing membrane vesicles by inducing burst lysis in a gram-negative bacterium.
[2] The method for producing a gram-negative bacterium according to the above-mentioned [1], wherein burst lysis is induced in the presence of a target substance on either or both the outside and the inside of the bacterium.
[3] The method for producing the present invention described in [1] or [2] above, in which burst lysis is induced by expression of a cell wall-decomposing enzyme in a gram-negative bacterium and, optionally, an enzyme that perforates the inner membrane of the gram-negative bacterium.
[4] The method for production described in [3] above, wherein the gram-negative bacterium has a genetic modification that induces expression of a cell wall-degrading enzyme and, optionally, an enzyme that perforates the inner membrane of the gram-negative bacterium.
[5] The method for production described in [3] above, in which expression of a cell wall decomposing enzyme and, in some cases, an enzyme that perforates the inner membrane of Gram-negative bacteria is induced by applying DNA-damaging stress.
[6] The method according to the above-mentioned [5], wherein the DNA-damaging stress is induced by mitomycin, a fluoroquinolone antibiotic, active oxygen and/or nitric oxide.
[7] The method for producing a cell wall-decomposing enzyme and, in some cases, an enzyme that perforates the inner membrane of gram-negative bacteria, thereby adjusting the production amount of membrane vesicles, as described in any one of [3] to [6] above.
[8] The method according to any one of [3] to [7] above, wherein the cell wall decomposing enzyme is an endolysin.
[9] The method according to any one of the above [3] to [8], wherein the enzyme that perforates the inner membrane of gram-negative bacteria is a holin.
[10] The method for producing according to any one of [1] to [9] above, further comprising the step of (X) culturing a gram-negative bacterium prior to the induction of burst lysis.
[11] The method for producing the present invention described in [10] above, which comprises culturing a gram-negative bacterium in a medium containing the target substance.
[12] The method for producing the target substance described in [10] above, in which the target substance is produced by gram-negative bacteria.
[13] (Y) The method for producing a membrane vesicle according to any one of [10] to [12] above, further comprising a step of separating the produced membrane vesicles from the medium.
[14] The method according to [13] above, wherein a magnetic force is used in the separation step.
[15] The method according to any one of [1] to [14] above, wherein the steps of (X) culturing gram-negative bacteria, (A) producing membrane vesicles by bursting lysis, and (Y) isolating membrane vesicles are repeated one or more times.
[16] The method according to any one of the above [10] to [15], wherein the medium contains a magnesium salt at a concentration of 1 to 30 mM mass% in the medium.
[17] The method according to any one of [10] to [16] above, wherein the shaking conditions for the culture are 180 to 250 rpm.
[18] The method according to any one of [10] to [17] above, wherein the pH of the medium is 4 to 10.
[19] The method according to any one of [10] to [18] above, wherein the viscosity of the medium is 1 to 15 Pa·s.
[20] A membrane vesicle in which a target substance is encapsulated, the constituent components of the membrane vesicle being derived from cultured gram-negative bacteria.
[21] A membrane vesicle containing a target substance, produced by the production method according to any one of [1] to [19] above.
[22] A method for encapsulating a target substance in a membrane vesicle derived from a gram-negative bacterium, the method comprising a step of inducing burst lysis in the gram-negative bacterium in the presence of the target substance.

The manufacturing method of the present invention is advantageous in that it can easily and efficiently produce membrane vesicles in which a target substance is encapsulated. The manufacturing method of the present invention is also advantageous in that it can encapsulate a target substance in membrane vesicles at the same time as the production of the membrane vesicles without a separate step for encapsulating the target substance in the membrane vesicles, and is also advantageous in that it can avoid inactivation or disruption of the target substance during encapsulation in the membrane vesicles. The manufacturing method of the present invention is further advantageous in that it can control the production amount of membrane vesicles by external stimuli, and is also advantageous in that it can be mass-produced by scaling up the culture system or by continuous production.

Figure 1 shows the uptake of a target substance (calcein) into membrane vesicles by burst lysis. Figure 1A shows the results for calcein alone (control), and Figure 1B shows the results for burst lysis in the presence of calcein. FIG. 2 shows the amount of membrane vesicles produced per cell in each cycle of subculture. FIG. 3 shows the amount of membrane vesicles produced per cell in the absence (control) and presence of MgSO ₄ in the medium. FIG. 4 shows the amount of membrane vesicles produced per cell under each shaking condition. FIG. 5 shows the amount of membrane vesicles produced per cell in the absence (control) and presence of Ficoll in the medium. FIG. 6 shows the amount of calcein encapsulated per membrane vesicle per cell (A) and the amount of membrane vesicle produced (B) under each pH condition. FIG. 7 shows the amount of membrane vesicles recovered when membrane vesicles with iron encapsulated (Fe-MV) and without iron encapsulation (MV) (control) were recovered using a magnet. Figure 8 shows a photograph of a membrane vesicle pellet encapsulating a target substance produced by bacteria. When a signal substance (C10-HSL) was added to the medium, the membrane vesicle pellet turned purple with violacein (dark gray in the figure) (left photo), whereas when no signal substance was added (control), the membrane vesicle pellet turned purple (right photo).

Description of the Invention

The present invention is a method for producing membrane vesicles in which a target substance is encapsulated. The production method of the present invention comprises (A) a step of producing membrane vesicles by inducing explosive cell lysis in gram-negative bacteria (a step of producing membrane vesicles by explosive cell lysis). Here, "explosive cell lysis (ECL)" refers to a phenomenon in which all or a part of the bacteria in a population of gram-negative bacteria explode and die through the action of cell wall-degrading enzymes within the bacteria.

Bursting lysis can be induced by cell wall (peptidoglycan layer) degrading enzymes, for example, by expressing cell wall degrading enzymes in bacteria. Examples of cell wall degrading enzymes include endolysins derived from bacteriophages, autolysins derived from bacteria, peptidoglycan lytic enzymes derived from other bacteria such as bacteriocins, those derived from virulence factors or other antibacterial polypeptides (e.g., lysostaphin, ALE-1 lysin, mutanolysin, enterolysin), lysozyme, etc., with endolysins being preferred.

The expression of the cell wall decomposing enzyme in the gram-negative bacterium can be induced by genetic modification that results in gene expression of the cell wall decomposing enzyme. Genetic modification can be performed according to known molecular biology techniques such as recombinant gene technology. For example, as described in Turnbull L, et al., Nat Commun. 2016 Apr 14;7:11220., the cell wall decomposing enzyme can be expressed by incorporating a plasmid or expression vector encoding the cell wall decomposing enzyme into the gram-negative bacterium. The expression of the cell wall decomposing enzyme can be regulated by a control sequence such as an inducible promoter, and in the plasmid or expression vector, such a control sequence can be placed in the vicinity of the base sequence encoding the cell wall decomposing enzyme.

The expression of cell wall decomposition enzymes can also be induced by DNA-damaging stress. DNA-damaging stress can be imposed on the bacteria by adding a DNA replication inhibitor (e.g., mitomycin), a fluoroquinolone antibiotic (e.g., ciprofloxacin), active oxygen, nitric oxide, etc. to the medium. The amount of these substances added can be adjusted as appropriate, but can be, for example, 0.001 to 0.1% by mass in the medium.

Burst lysis can be induced more effectively by using an enzyme that perforates the inner membrane of gram-negative bacteria in addition to the cell wall decomposing enzyme. That is, by contacting the inner membrane of the bacteria with the enzyme that perforates the inner membrane of gram-negative bacteria, holes are opened in the intracellular membrane, promoting contact between the cell wall decomposing enzyme and the cell wall, and burst lysis can be induced more effectively. An example of a protein that perforates the inner membrane of gram-negative bacteria is phage-derived holin, and a preferred combination with the cell wall decomposing enzyme is holin-endolysin. The expression of the protein that perforates the inner membrane of gram-negative bacteria can be carried out by molecular biological techniques such as gene recombination techniques, in the same manner as the expression of the cell wall decomposing enzyme.

In the present invention, the bacteria that are the subject of bursting lysis are gram-negative bacteria, such as Pseudomonas aeruginosa , Escherichia coli , and acetic acid bacteria ( Acetobacter aceti , Acetobacter pasteurianus , Acetobacter liguefaciens , Acetobacter xylinum , Acetobacter polyoxygenes , Acetobacter methanolicus , Gluconobacter oxydans , Gluconobacter cerinus , Gluconobacter hansenii , and Frateuria aurantia ). Gram-negative bacteria are composed of an intracellular membrane, a peptidoglycan layer (cell wall), and an outer membrane in this order from the inside of the bacterial body, and it is considered that membrane vesicles consisting of the outer membrane (OMV; Outer Membrane Vesicle) or membrane vesicles consisting of the outer membrane and the intracellular membrane (OIMV; Outer Inner Membrane Vesicle) are formed by burst lysis. Some of the above bacteria are also used as foods or probiotic preparations (e.g., Nissle 1917 strain of Escherichia coli), and the membrane vesicles of the present invention can be derived from such bacteria from the viewpoint of safety.

The production method of the present invention may further include (X) a step of culturing gram-negative bacteria in a medium (a step of culturing gram-negative bacteria) prior to the induction of bursting bacteriolysis. The gram-negative bacteria may be cultured under normal culture conditions, but in terms of the amount of membrane vesicles produced, it is preferable to culture the bacteria under the conditions described below.

In the production method of the present invention, burst lysis is induced in a state in which a target substance is present either outside or inside or both of the gram-negative bacteria, and the target substance can be encapsulated in the membrane vesicles at the same time as the formation of the membrane vesicles. When the target substance is present outside the gram-negative bacteria, the bacteria can be cultured in a medium containing the target substance. The content of the target substance in the medium can be adjusted as appropriate, and can be, for example, 0.001 to 10% by mass (preferably 0.01 to 5% by mass) in terms of the concentration in the medium.

When the target substance is present inside the gram-negative bacteria, the target substance can be produced by the gram-negative bacteria themselves that are to be subjected to bursting lysis, and accumulated within the bacteria. The target substance can also be produced by incorporating the gene for the target substance into the bacteria using molecular biological techniques such as gene recombination technology. The target substance can be produced by the gram-negative bacteria themselves using molecular biological techniques using known methods.

The target substance to be encapsulated in the membrane vesicles is not particularly limited as long as it is a substance of a size that can be encapsulated in the membrane vesicles (for example, a diameter of 10 to 600 nm, 20 to 500 nm, or 30 to 400 nm). Examples of the target substance include low molecular weight compounds that are active ingredients of pharmaceuticals, proteins, peptides, amino acids, nucleic acids, etc. Pharmaceuticals also include biological preparations, and the active ingredients of biological preparations can be the target substance. Examples of the active ingredients of biological preparations include antibodies, antigens, interferons, interleukins, vaccines, hormones, enzymes related to blood coagulation, thrombolytic components, immunoregulatory factors, etc. Pharmaceuticals also include gene therapy preparations, and the active ingredients of gene therapy preparations can be the target substance. Examples of the active ingredients of gene therapy preparations include single-stranded or double-stranded DNA, iRNA, mRNA, siRNA, miRNA, antisense RNA, etc.

In the production method of the present invention, the production amount of membrane vesicles can be adjusted by adjusting the expression level of the cell wall decomposing enzyme and, in some cases, the expression level of the enzyme that perforates the inner membrane of the gram-negative bacteria. For example, by increasing the expression level of the enzyme, the production amount of membrane vesicles can be increased, and by decreasing the expression level of the enzyme, the production amount of membrane vesicles can be decreased.

The production method of the present invention may further include (Y) a step of separating the produced membrane vesicles from the medium (membrane vesicle separation step). The separation may be performed by a known method, for example, by removing the bacterial cells from the medium containing the bacterial cells using a centrifuge, and then filtering, purifying, or otherwise treating the supernatant as necessary to separate and recover the membrane vesicles. The supernatant may also be further purified by ultracentrifugation or density gradient centrifugation. The separation may also be performed by encapsulating the target substance and iron in the membrane vesicles during the bursting bacteriolysis, and then isolating and recovering the membrane vesicles from the medium using magnetic force. Separation using magnetic force is advantageous in that it makes it easy to separate and recover the membrane vesicles. These methods for separating and recovering membrane vesicles may be appropriately combined depending on the purpose of using the membrane vesicles.

In the production method of the present invention, one cycle is made up of (X) a gram-negative bacterium culture step, (A) a membrane vesicle production step by bursting bacteriolysis, and (Y) a membrane vesicle separation step, and this cycle can be repeated one or more times to continuously prepare membrane vesicles. In other words, once the conditions for bursting bacteriolysis and culture conditions are set, the desired membrane vesicles can be prepared in large quantities by repeating steps (X), (A), and (Y) under those conditions. There is no limit to the number of repetitions, but the upper limit can be set to, for example, 50, 40, 30, 20, or 10 times.

In the production method of the present invention, Mg ²⁺ (e.g., magnesium salts such as MgSO ₄ and MgCl ₂ ) can be added to the medium for culturing gram-negative bacteria. The amount of Mg ²⁺ added can be, for example, 1 to 30 mM (preferably 5 to 20 mM) of magnesium salt relative to the medium. The addition of Mg ²⁺ is considered to stabilize the cell membrane of gram-negative bacteria, and is advantageous in that it can increase the production amount of membrane vesicles.

In the production method of the present invention, the shaking conditions in the culture of gram-negative bacteria are preferably more than 100 rpm, and more preferably 180 to 250 rpm. The shaking conditions can be adjusted appropriately in relation to the amount of oxygen supplied to the medium, but increasing the shaking rate is advantageous in that it increases the amount of membrane vesicles produced.

In the production method of the present invention, the pH of the medium for culturing gram-negative bacteria can be adjusted, for example, in the range of pH 4 to 10 (preferably 5 to 9), but from the viewpoint of increasing the production amount of membrane vesicles, a higher pH (more alkaline), for example, pH 8 to 10 (preferably pH 8 to 9), is preferable. On the other hand, from the viewpoint of increasing the amount of the target substance encapsulated in the membrane vesicles, a lower pH (more acidic), for example, pH 4 to 6 (preferably pH 5 to 6). In the production method of the present invention, the pH of the culture medium can be appropriately set depending on the application.

In the production method of the present invention, the viscosity of the medium for culturing gram-negative bacterial cells is preferably high from the viewpoint of increasing the amount of the target substance encapsulated in the membrane vesicles, and the viscosity of the culture medium can be, for example, 1 to 15 Pa·s (preferably 2 to 11 Pa·s). To increase the viscosity of the medium, for example, ficoll, alginic acid, carboxymethylcellulose, carboxyvinyl polymer, etc. can be added to the medium as a thickening agent. The content of these thickening agents in the medium can be 5 to 20% by mass for ficoll, 1 to 3.5% by mass for carboxymethylcellulose, and 0.05 to 0.3% by mass for carboxyvinyl polymer, in terms of the concentration in the medium. These thickening agents can be added alone or in any combination.

According to another aspect of the present invention, there is provided a membrane vesicle in which a target substance is encapsulated, the constituent components of the membrane vesicle being derived from gram-negative bacteria. The membrane vesicles of the present invention have a particle diameter of 10-600 nm, 20-500 nm, or 30-400 nm, and are configured in the form of either outer membrane-inner membrane vesicles (OIMV) or outer membrane vesicles (OMV). The membrane vesicles of the present invention can be produced according to the description of the production method of the present invention. Target substances encapsulated in the membrane vesicles of the present invention include the same as those described in the production method of the present invention.

According to another aspect of the present invention, the present invention provides a composition comprising the membrane vesicles of the present invention. The composition of the present invention can be provided in the form of a pharmaceutical product (e.g., a pharmaceutical composition), a food product (e.g., a food composition), a cosmetic product (e.g., a cosmetic composition), etc., depending on the nature of the substance to be encapsulated in the membrane vesicles. The composition of the present invention can be produced by containing the membrane vesicles of the present invention. Target substances to be encapsulated in the membrane vesicles contained in the composition of the present invention include the same as those described in the production method of the present invention.

According to another aspect of the present invention, the present invention further provides a method for encapsulating a target substance in a membrane vesicle derived from a gram-negative bacterium. The method for encapsulating a target substance of the present invention comprises a step of inducing burst lysis in a gram-negative bacterium in the presence of the target substance. The method for encapsulating a target substance of the present invention can be carried out according to the description of the production method of the present invention. Target substances to be encapsulated in the membrane vesicles of the present invention include the same as those described in the production method of the present invention.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1: Production of membrane vesicles by burst lysis and encapsulation of a target substance in the membrane vesicles In Example 1, burst lysis was induced in Gram-negative bacteria in the presence of a target substance, and it was demonstrated that the target substance was encapsulated in membrane vesicles.

(1) Method A: Bacterial cells The bacterial cells used were Pseudomonas aeruginosa (strain: PAO1 ΔprtN) cells transfected with a plasmid (pJN105 hol_lys, obtained from Addgene) capable of controlling the expression of holin-endolysin. The P. aeruginosa strain PAO1 ΔprtN was prepared by disrupting prtN in PAO1 (available from the RIKEN BioResource Research Center (BRC No: JCM 14847)) according to the method described in Environ Microbiol. 2014 Sep;16(9):2927-38.

B. Bacterial culture and induction of burst lysis Four ml of LB liquid medium was used, and gentamicin (Nacalai Tesque) was added at 50 μg/ml to prevent the plasmid from being removed. The bacteria were inoculated to _{an OD 600} of 0.01, and cultured at 37°C for 12 hours at 200 rpm. After 4 hours of culture, arabinose (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1% to express holin-endolysin, induce burst lysis, and produce membrane vesicles. Immediately before the addition of arabinose, 1 mM calcein (Nacalai Tesque) was added to the medium as a target substance.

C Separation, purification, and recovery of membrane vesicles The culture solution was centrifuged (4°C, 30 minutes, 7,000 x g) to pellet down the bacteria. The supernatant after centrifugation was filtered through a 0.45 μm pore size filter to remove the bacteria, and the sample was then ultracentrifuged (4°C, 1 hour, 150,000 x g). The pellet produced by ultracentrifugation was suspended in PBS. The resulting membrane vesicles were then ultracentrifuged and resuspended to wash, and this washing procedure was repeated twice. Next, the membrane vesicle pellet produced by ultracentrifugation was suspended in 45% iodixanol (AXIS-SHEILD), and 40, 35, 30, 25, 20, 15, and 10% iodixanol were layered on top, and density gradient centrifugation was performed (4°C, 3 hours, 100,000 x g, total volume 3 ml). The membrane vesicles were purified and recovered by this density gradient centrifugation.

D. Quantification of Membrane Vesicles The collected membrane vesicles were quantified by measuring the fluorescence intensity of FM4-64 (Invitrogen) as a membrane lipid stain using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific). The amount of membrane vesicles produced per cell was calculated as RFU (Relative Fluorescence Unit) (FM1-43 fluorescence intensity/OD ₆₀₀ ). The presence of encapsulated substances was confirmed by measuring the fluorescence intensity of calcein using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific).

(2) Results The results are shown in Figure 1. Calcein indicates the presence of the substance to be encapsulated, and FM4-64 indicates the presence of membrane vesicles. In Figure 1A, in the sample containing only calcein (control), a calcein peak was observed at around fraction number 27 to 30 (area enclosed by a dashed square). On the other hand, in Figure 1B, in the sample in which calcein was encapsulated in membrane vesicles, the FM4-64 peak and the calcein peak were observed to coincide at around fraction number 8 to 16 (area enclosed by a dashed square), confirming that calcein was encapsulated in membrane vesicles by burst lysis.

Example 2: Effect of subculture on membrane vesicle production In Example 2, the effect of subculture on membrane vesicle production by burst lysis was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis were carried out in the same manner as described in Example 1(1)B, except that calcein was not added.

C. Subculture The bacteria from the culture in which burst lysis was induced were inoculated into a new medium, and the cycle of inducing burst lysis again was repeated three times. Specifically, 12-hour culture was counted as one cycle, and the OD ₆₀₀ of the 12-hour culture was measured. The 12-hour culture used for the measurement was added to a newly prepared 4 ml LB liquid medium so that the OD ₆₀₀ was 0.01, and subcultured (second time). Subsequently, the culture after the second cycle was newly cultured in the same way, and subcultured (third time). In the second and third times, the OD ₆₀₀ of the 12-hour culture was measured in the same way as in the first time.

After each cycle of rupture lysis, membrane vesicles were separated, purified, and collected. The pelleted cells were used for the next cycle of culture. The rest of the procedure was the same as that described in Example 1(1)(c).

Quantification of Membrane Vesicles Quantification of membrane vesicles was carried out in the same manner as described in Example 1(1)E, except that FM1-43 (Invitrogen) was used as the membrane lipid staining agent.

(2) Results The results are shown in Figure 2. It was confirmed that even when subculture was performed using a medium in which burst lysis was induced, membrane vesicles could be produced by burst lysis in the second and third cycles.

Example 3: Effect of MgSO4 _on membrane vesicle production In Example 3, the effect of adding _MgSO4 to the medium on membrane vesicle production by burst lysis was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis The same procedure as described in Example 1(1)B was carried out, except that _MgSO4 was added to the LB liquid medium to a concentration of 8 mM and calcein was not added.

C. Separation, purification and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)C.

Quantification of Membrane Vesicles Quantification of membrane vesicles was carried out in the same manner as described in Example 1(1)E, using FM4-64 (Invitrogen) as a membrane lipid staining agent.

(2) Results The results are shown in Figure 3. It was confirmed that when _MgSO4 was added to the medium, the amount of membrane vesicles produced per cell was significantly increased.

Example 4: Effect of shaking conditions in bacterial culture on membrane vesicle production In Example 4, the effect of shaking conditions during culture on membrane vesicle production was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis Bacterial culture was carried out in the same manner as described in Example 1(1)(B), except that the shaking conditions during bacterial culture were 0 rpm, 100 rpm, and 190 rpm, and calcein was not added.

C. Separation, purification and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)C.

Quantification of Membrane Vesicles Quantification of membrane vesicles was carried out in the same manner as described in Example 1(1)E, except that FM1-43 (Invitrogen) was used as the membrane lipid staining agent.

(2) Results The results are shown in Figure 4. It was confirmed that when the shaking condition was 190 rpm, the amount of membrane vesicles produced per cell was significantly increased.

Example 5: Effect of medium viscosity on encapsulation of calcein in membrane vesicles In Example 5, the effect of medium viscosity on encapsulation of calcein in membrane vesicles was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis The same procedure as described in Example 1(1)B was carried out, except that 10% Ficoll (Sigma) was added to the LB liquid medium.

C. Separation, purification and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)C.

D. Quantification of membrane vesicles and calcein Quantification of membrane vesicles was performed in the same manner as described in Example 1(1)C, using FM4-64 as a membrane lipid stain. The fluorescence intensities of FM4-64 and calcein were measured using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific). The amount of calcein encapsulated per membrane vesicle was calculated as the fluorescence intensity of "calcein/FM4-64".

(2) Results The results are shown in Figure 5. It was confirmed that the amount of calcein encapsulated in membrane vesicles increased with increasing viscosity of the medium.

Example 6: Effect of medium pH on membrane vesicle production and inclusion of calcein in membrane vesicles In Example 6, the effect of medium pH on membrane vesicle production and inclusion of calcein in membrane vesicles was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis The same procedure as described in Example 1(1)B was carried out except that the pH of the LB liquid medium was adjusted to pH 5, pH 7, or pH 9.

C. Separation, purification and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)C.

Quantitation of membrane vesicles and calcein The procedure was carried out in the same manner as described in Example 5(1)(E).

(2) Results The results are shown in Figure 6. It was confirmed that the amount of calcein encapsulated in membrane vesicles decreased with increasing pH (Figure 6A), whereas the amount of membrane vesicles produced per cell increased with increasing pH (Figure 6B).

Example 7: Recovery of membrane vesicles using a magnet In Example 7, recovery of membrane vesicles using a magnet was examined.

(1) Method A: Bacterial Cells The bacterial cells described in Example 1(1)A were used.

Bacterial culture and induction of burst lysis The same procedure as described in Example 1(1)B was carried out, except that 1 mM iron oxide was added to the medium instead of calcein immediately before the addition of arabinose.

C. Recovery and quantification of membrane vesicles 900 μl each of membrane vesicle solution (MV) and membrane vesicle solution encapsulating iron (Fe-MV) was prepared so that the fluorescence intensity of FM1-43 was 3. Each solution was placed on a magnetic stand (Toyobo) and left to stand for 1 minute under stationary conditions, after which only the supernatant was removed using an aspirator. Next, 900 μl of PBS was added and stirred using a vortex. The membrane vesicles in this solution were then quantified by measuring the fluorescence intensity of FM1-43.

(2) Results The results are shown in Figure 7. It was confirmed that iron-encapsulating membrane vesicles could be collected using a porcelain stand.

Example 8: Encapsulation of bacterially produced substances in membrane vesicles In Example 8, the encapsulation of bacterially produced substances in membrane vesicles was examined.

(1) Method A: Bacterial Cells Chromobacterium violaceum , an AHL synthesis gene-deficient strain (strain: VIR24, prepared from Chromobacterium violaceum Bergonzini (ATCC12472) according to the description in Microbes Environ. 2009;24(4):305-14.), was used as the bacterial cell.

Bacterial culture: Bacteria were inoculated into LB medium to give an OD ₆₀₀ of 0.01, and cultured at 30° C. for 12 hours at 100 rpm.

C. Induction of membrane vesicle production Mitomycin C (Nacalai tesque) was used to induce the production of membrane vesicles derived from Chromobacterium violaceum. Mitomycin C was added when the OD ₆₀₀ was in the range of 0.4 to 0.6, and was added to a concentration of 200 ng/ml.

Addition of signal substances
The VIR24 strain cannot produce N-acyl-L-homoserine lactone (AHL) by itself, so purple pigment production is not induced (even when this strain is cultured, the culture medium does not turn purple). Therefore, C10-HSL (Cayman Chemical), a type of AHL, was added to this strain to induce the production of the purple pigment (violacein) by the VIR24 strain. The concentration of C10-HSL added was 100 nM at the final concentration, added 1 hour after the start of culture.

Isolation of membrane vesicles and confirmation of inclusion of bacterially produced substances in membrane vesicles The culture solution was centrifuged (4°C, 30 minutes, 7000 x g) to pellet down the bacterial cells. The supernatant after centrifugation was filtered through a 0.45 μm pore size filter to remove the bacterial cells, and then 7.0 ml of the sample was ultracentrifuged (4°C, 1 hour, 150,000 x g) to separate the membrane vesicles. Since the substance (violacein) produced by the bacterial cells is purple, the presence or absence of inclusion in the membrane vesicles was visually confirmed.

(2) Results The results are shown in Figure 8. The photograph shows the pellet of membrane vesicles after ultracentrifugation. The photograph on the left shows the membrane vesicle pellet derived from the VIR24 strain with the addition of C10-HSL, and the photograph on the right shows the membrane vesicle pellet derived from the VIR24 strain without the addition of C10-HSL. It was confirmed that the production of violacein (purple) in the VIR24 strain was induced by the addition of a signal (C10-HSL), and the produced violacein was encapsulated in the membrane vesicles.

Claims (20)

A method for producing membrane vesicles in which a target substance is encapsulated, comprising the steps of (A) producing membrane vesicles by inducing bursting bacteriolysis in gram-negative bacteria, and (X) culturing the gram-negative bacteria before inducing bursting bacteriolysis, in which the gram-negative bacteria are cultured in a medium containing the target substance, the shaking conditions for the culture are 180 to 250 rpm, and the viscosity of the medium is 1 to 15 Pa·s. The method according to claim 1, in which burst lysis is induced in the presence of the target substance on either or both the outside and the inside of the gram-negative bacteria. The method according to claim 1 or 2, in which burst lysis is induced by a cell wall decomposing enzyme in a gram-negative bacterium. The method according to claim 1 or 2, in which burst lysis is induced by expression of a cell wall decomposing enzyme in a gram-negative bacterium and an enzyme that perforates the inner membrane of the gram-negative bacterium. The method according to claim 3 or 4, wherein the gram-negative bacterium has a genetic modification that induces the expression of a cell wall decomposing enzyme. The method according to claim 3 or 4, wherein the gram-negative bacteria have genetic modifications that induce the expression of a cell wall decomposing enzyme and an enzyme that perforates the inner membrane of the gram-negative bacteria. The method according to claim 3 or 4, in which expression of the cell wall decomposition enzyme is induced by DNA-damaging stress. The method according to claim 3 or 4, in which expression of a cell wall decomposing enzyme and an enzyme that perforates the inner membrane of a gram-negative bacterium is induced by DNA-damaging stress. The method according to claim 8, wherein the DNA-damaging stress is induced by mitomycin, a fluoroquinolone antibiotic, active oxygen, and/or nitric oxide. The method according to any one of claims 3 to 9, in which the production amount of membrane vesicles is adjusted by adjusting the expression level of the cell wall decomposing enzyme. The method according to any one of claims 3 to 9, in which the production amount of membrane vesicles is adjusted by adjusting the expression level of a cell wall decomposing enzyme and an enzyme that perforates the inner membrane of gram-negative bacteria. The method according to any one of claims 3 to 11, wherein the cell wall decomposing enzyme is an endolysin. The method according to any one of claims 3 to 12, wherein the enzyme that perforates the inner membrane of a gram-negative bacterium is a holin. The method according to any one of claims 1 to 13, in which the target substance is produced by gram-negative bacteria. (Y) The method for producing a membrane vesicle according to any one of claims 1 to 14, comprising a step of separating the produced membrane vesicles from the medium. The method of claim 15, wherein magnetic force is used in the separation step. The method according to any one of claims 1 to 16, in which the steps of (X) culturing gram-negative bacteria, (A) producing membrane vesicles by bursting lysis, and (Y) isolating the membrane vesicles are repeated one or more times. The method according to any one of claims 1 to 17, wherein the medium contains a magnesium salt at a concentration of 1 to 30 mM. The method according to any one of claims 1 to 18, wherein the pH of the medium is 4 to 10. A method for encapsulating a target substance in membrane vesicles derived from gram-negative bacteria, comprising the steps of inducing burst lysis in the gram-negative bacteria in the presence of the target substance, and (X) culturing the gram-negative bacteria before inducing burst lysis, in which the gram-negative bacteria are cultured in a medium containing the target substance, the shaking conditions for the culture are 180 to 250 rpm, and the viscosity of the medium is 1 to 15 Pa·s.

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