Preparation method and application of bacterial outer membrane vesicles encapsulating GLP1 analogs

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the production and use of bacterial outer membrane vesicles encapsulating glucagon-like peptide-1 analogs, which are mainly used in the treatment of type 2 diabetes (T2DM) and obesity. The bacterial outer membrane vesicles encapsulating glucagon-like peptide-1 (GLP1) analogs are loaded with a GLP1 analog with a gene coding sequence such as SEQ ID NO: 1. The bacterial outer membrane vesicles encapsulating GLP1 analogs successfully prepared by the present invention have potential clinical applicability and research value in the treatment of type 2 diabetes (T2DM) and obesity.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the production and use of bacterial outer membrane vesicles encapsulating glucagon-like peptide-1 analogs, which are mainly used in the treatment of type 2 diabetes mellitus (T2DM) and obesity. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar levels. Type 2 diabetes mellitus (T2DM) is caused by a progressive decrease in insulin secretion from pancreatic β cells in the context of insulin resistance, accounting for 90% to 95% of the diabetic population.

[0003] Glucagon-like peptide-1 (GLP1) is a peptide hormone composed of 30 amino acids, which is produced by the cleavage of proglucagon secreted from intestinal L cells, pancreatic α cells and neurons of the nucleus tractus solitarius. GLP1 acts on the gastrointestinal tract to increase the body's sensitivity to insulin. It lowers blood sugar by inhibiting glucagon and stimulating insulin secretion at the same time. It can not only correct abnormal insulin secretion, but also control blood sugar fluctuations after eating, inhibit gastric emptying, and reduce appetite. In addition, GLP1 can also increase the insulin biosynthesis rate, stimulate the growth and proliferation of β cells and inhibit their apoptosis, protect the number and function of pancreatic β cells, and promote the absorption and storage of glucose by muscle and adipose tissue. GLP1 also exhibits a protective function for nerves, suppressing appetite by acting on the feeding center, but it is difficult to locate a clear circuit in the brain. GLP1 exerts its effects through G protein-coupled receptors and is then quickly degraded by dipeptidyl peptidase-4 (DPP-4) enzyme, so the serum half-life of natural GLP1 is very short. This defect limits the direct application of GLP1 and its analogs.

[0004] Bacterial Outer Membrane Vesicles (OMVs) are vesicle structures naturally secreted by Gram-negative bacteria, mainly composed of phospholipids, proteins and lipopolysaccharides. Due to their unique nanostructure and high biocompatibility, OMVs have shown great application potential in the field of drug delivery. In recent years, studies have shown that OMVs can be used as drug carriers to deliver a variety of bioactive substances, including nucleic acids, proteins and small molecule drugs, through specific modification and engineering. In particular, OMVs can effectively enhance the stability of drugs and promote their retention and accumulation in specific tissues due to their natural immunogenicity and targeting ability.

[0005] However, despite the broad application prospects of OMVs in drug delivery, the clinical application of OMVs still faces many challenges. These mainly include how to optimize the preparation process of OMVs to ensure the efficient encapsulation rate and biological activity of GLP1 analogs and how to prevent the degradation of GLP1 analogs during the encapsulation and release process. Therefore, there is an urgent need for a new preparation method that can utilize the natural advantages of OMVs while maintaining the efficacy of GLP1-RA to achieve stable and efficient delivery of therapeutic drugs.

[0006] Based on the above background, the present invention provides a method for preparing bacterial outer membrane vesicles encapsulating GLP1 analogs and its application. Through genetic engineering modification and optimized preparation process, the problem of low OMV yield and in vitro loading rate was successfully solved, providing a more effective and convenient solution for the treatment of diabetes. This innovative method not only expands the application of OMVs in the field of peptide drug delivery, but also provides new ideas and technical support for future drug development and disease treatment. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a bacterial outer membrane vesicle encapsulating a glucagon-like peptide-1 (GLP1) analog, wherein the bacterial outer membrane vesicle carries a GLP1 analog with a gene coding sequence such as SEQ ID NO: 1.

[0008] Preferably, the GLP1 analog is also connected in series with a signal sequence and a tracer protein.

[0009] Preferably, the signal sequence is vNP, and its gene coding sequence is shown in SEQ ID NO:2.

[0010] Preferably, the tracer protein is mNerogreen, and its gene coding sequence is shown in SEQ ID NO:3.

[0011] Preferably, the bacterial outer membrane vesicles are composed of genotype Nissle 1917 ; ΔNlpI Production of engineered bacteria.

[0012] In a preferred embodiment of the present invention, the present invention provides a method for preparing the above-mentioned bacterial outer membrane vesicles, the method comprising the following steps:

[0013] (1) constructing a plasmid vector containing a signal peptide, a tag protein, and a gene encoding GLP1;

[0014] (2) Transform the plasmid vector into the host bacteria, screen and identify, and obtain the engineered strain;

[0015] (3) The engineered strain is expanded and cultured, induced with an inducer, the bacterial liquid is collected, centrifuged, the bacteria are removed and the supernatant is harvested, and the supernatant is purified by multiple ultracentrifugation steps to obtain bacterial extracellular vesicles.

[0016] Preferably, in step (2), the host bacteria is Escherichia coli Nissle 1917 ; ΔNlpI The transformation method is chemical transformation, and the identification method is Sanger sequencing.

[0017] Preferably, in step (3), the inducing agent is isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1-0.2 mM, the induction temperature is 35-40° C., and the induction time is 14-20 h.

[0018] Preferably, in step (3), the purification method comprises the following steps: the supernatant is centrifuged for the first time to remove the precipitate, and then filtered through a 0.22 μm filter membrane, the filtrate is centrifuged for the second time, the supernatant is collected, and the supernatant is removed after the third centrifugation, and resuspended with PBS to obtain bacterial outer membrane vesicles loaded with GLP1.

[0019] Preferably, the first centrifugation is performed at 6000-10000 g for 25-35 min, the second centrifugation is performed at 90000-110000 g for 55-65 min, and the third centrifugation is performed at 100000-150000 g for 55-65 min.

[0020] In another preferred embodiment of the present invention, the present invention provides an engineered strain that produces high amounts of the bacterial outer membrane vesicles, wherein the genotype of the engineered strain is Nissle 1917 ; ΔNlpI .

[0021] In another preferred embodiment of the present invention, the present invention provides use of the bacterial outer membrane vesicles or the engineered strains described in the present invention in the preparation of drugs for preventing and / or treating type 2 diabetes and obesity.

[0022] The bacterial outer membrane vesicles encapsulating the GLP1 analogues successfully prepared by the present invention have potential clinical applicability and research value in the treatment of type 2 diabetes (T2DM) and obesity.

[0023] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart for the construction of engineered bacteria and the preparation of vesicles for producing bacterial outer membrane vesicles encapsulating GLP1 analogs.

[0025] Figure 2 is the production distribution and immunoblot of GLP1 analogs, where Figure 2 The M in B stands for Marker.

[0026] Figure 3 This is the identification result of bacterial outer membrane vesicles. Figure 3 A in the figure is the transmission electron microscope result. Figure 3 B in FIG. 1 is a diagram showing the results of dynamic light scattering analysis.

[0027] Figure 4 It is a bar graph showing the changes in blood glucose, glucose tolerance, body weight and food intake in one week after a single administration.

[0028] Figure 5 It is a result chart of carcass weight, lean body mass and fat weight of each part.

[0029] Figure 6 This is the pathological result of HE staining of important organs such as heart, liver, spleen, lung, and kidney. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] The present invention connects the sequence of GLP1 analog with the signal sequence vNP and the tracer protein mNerogreen sequence in series to construct a fusion protein. In order to facilitate detection, the embodiment of the present invention adds a tag protein (6×His) on the basis of this structure, and finally designs: signal peptide (vNP)-tracer protein (mNerogreen)-GLP1 analog-tag protein (6×His), that is, vNP-mG-GLP1 analog-6×His.

[0032] Example 1: Preparation of bacterial outer membrane vesicles encapsulating mG-GLP1 RA

[0033] Schematic diagram of the construction of engineered bacteria and the production and characterization of bacterial outer membrane vesicles Figure 1 As shown, the specific operations are as follows:

[0034] 1. Construction and assembly of plasmid vector expressing GLP1

[0035] The gene encoding vNP-mG-GLP1-6xHis was synthesized and inserted into the MCS region using TA cloning based on the pET28 plasmid backbone.

[0036] 2. Construction of high-yield GLP1-loaded vesicle production engineering bacteria

[0037] Take a small amount of plasmid vector solution and add it to 100 μL competent medium E. coli Nissle 1917 ; ΔNlpI After gently mixing, place on ice for 30 minutes, then heat shock at 42℃ for 1.5 minutes, and finally place the mixture on ice for 2 minutes to cool to 0℃; add 900μL LB culture medium without antibiotics to the mixture, then resuscitate and culture at 37℃, 220rpm for 30 minutes, then spread the bacterial liquid on LB plate containing kanamycin, and culture at 37℃ for 12-14 hours to obtain monoclonal strains. Pick colonies with regular morphology and use universal primers T7-R;F to amplify the insertion region, and the amplified product is identified by Sanger sequencing to ensure the correct sequence.

[0038] 3. Production and Isolation of Bacterial Outer Membrane Vesicles Encapsulating GLP1

[0039] The correctly identified monoclonal strain was selected and added into 5 mL of LB culture medium containing kanamycin resistance and cultured at 37°C and 220 rpm for 12 h.

[0040] Inoculate 25 mL of TB medium containing antibiotics in a 500 mL conical flask, shake at 200 rpm (≥25 mm orbital distance) in a 37°C incubator to reach an optical density at 600 nm (OD600) of 0.8-1.0; add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 20 μg / mL, induce at 37°C for 12-14 h; collect the bacterial culture, centrifuge at 6000g for 30 min, remove the bacteria, and harvest the supernatant.

[0041] The supernatant was filtered using a 0.22 μm filter; the filtrate was centrifuged at 4°C, 100,000 g for 60 min, the liquid more than 1 cm from the bottom of the heart tube was carefully removed and centrifuged again at 4°C, 150,000 g for 60 min; the supernatant was removed, and the bottom precipitate was resuspended with PBS to obtain bacterial outer membrane vesicles encapsulating GLP1, which were named OMVs bacterial outer membrane vesicles.

[0042] Experimental Example 2: Detection and characterization of outer membrane vesicles secreted by engineered bacteria

[0043] 1. Experimental Methods

[0044] The experimental samples were the engineered bacteria constructed in Example 1 and the purified bacterial outer membrane vesicles.

[0045] Test method for the expression of the target protein of the engineered bacteria: First, inoculate the engineered bacteria in a medium containing resistance. After overnight culture, take 10 μL of the overnight bacterial solution and add it to a 5 mL shaking bacterial tube. Continue to culture at 37 ° C and 220 rpm for 4-8 hours until the OD600 of the bacterial solution reaches 0.6-0.8 by the spectrophotometer. At this time, take part of the bacterial solution as the pre-induction sample, centrifuge the sample and divide it into three parts: culture medium suspension, supernatant and bacterial resuspension. Add IPTG with a final concentration of 20 μg / mL, induce at 37 ° C for 4 hours, collect the bacterial solution as the post-induction sample, and prepare the sample according to the same steps. Prepare the sample according to the same steps after overnight culture. The sample was placed in a 12-well plate and the fluorescence was observed under a 560nm blue light.

[0046] Add 5×SDS loading buffer to the supernatant, bacterial cells and bacterial liquid samples after overnight culture. Heat at 95℃ for 5min to fully denature. Separate the proteins by SDS-PAGE electrophoresis. Take the protein gel separated by SDS-PAGE electrophoresis and gently rinse the residual electrophoresis solution with pure water. Cut a 0.22μm PVDF membrane and completely immerse the PVDF membrane in methanol for about 1min. Prepare the transfer buffer and stack the filter paper, gel, protein membrane and filter paper in the transfer clip. Assemble, add ice cubes, and transfer the membrane in an ice bath. Transfer at a constant voltage of 300V and set the transfer time according to the size of the target protein. The specific ratio is: 1min for every 1KD. Rinse the membrane with TBST, then add 5% BSA blocking solution and block overnight at 4℃. Rinse the membrane with TBST, then place the membrane in His Tag primary antibody solution. After incubation overnight, remove the primary antibody incubation solution and wash the membrane with TBST. Use 10-50mL TBST each time, room temperature, 100rpm for 10min, repeat 3-5 times. Place the membrane in the corresponding secondary antibody solution and incubate for 3h at 37℃, 100rpm. Add TBST and wash the PDVF membrane. Use 10-50mL TBST each time, room temperature, 100rpm for 10min, repeat 3-5 times. Mix colorants A and B in a 1:1 ratio, completely soak the PVDF membrane, and then expose it in an exposure instrument. Determine protein expression and distribution based on the brightness and position of the bands.

[0047] 2. Experimental results

[0048] The experimental results of 560nm blue light excitation (A) and immunoblotting (B) are shown in Figure 2As shown. Before the addition of IPTG inducer, there was only weak background expression; after the addition of the inducer, the expression of the fusion protein in the bacteria increased significantly, but no fluorescence enhancement was observed in the supernatant. However, after overnight culture, the fluorescence in the culture supernatant was significantly enhanced. Immunoblotting showed that the target protein had obvious bands at specific sizes in both bacteria and OMVs. Only weak target protein expression was found in the culture supernatant, while in the purified bacterial outer membrane vesicles OMVs, the target protein was specifically expressed with high abundance. In addition, there were a variety of cleaved and degraded proteins in the culture suspension.

[0049] Experimental Example 3: Characterization of bacterial outer membrane vesicle structure and properties

[0050] 1. Experimental Methods

[0051] The experimental sample is the bacterial outer membrane vesicles prepared in Example 1.

[0052] 1.1 Detection of bacterial outer membrane vesicles by transmission electron microscopy

[0053] The prepared bacterial outer membrane vesicles were adjusted to 1×10 11 / mL; soak the carbon film-covered copper mesh in the bacterial outer membrane vesicle solution for 10 minutes, then use filter paper to absorb the copper mesh; soak the copper mesh in 4% paraformaldehyde for 5 minutes, then use filter paper to absorb the copper mesh; soak the copper mesh in deionized water for 2 minutes, then use filter paper to absorb the copper mesh, and repeat the washing 10 times; soak the copper mesh in 2% uranyl acetate for 10 minutes, then use filter paper to absorb the copper mesh; air dry overnight; use transmission electron microscopy to photograph bacterial outer membrane vesicles.

[0054] 1.2 Dynamic light scattering analysis to detect bacterial outer membrane vesicle size

[0055] Turn on the dynamic light scattering analyzer, run the self-test program, and clean the instrument with PBS; adjust the bacterial outer membrane vesicles to be tested to an appropriate concentration and add them to the cuvette and place it in the light path; turn on the temperature control switch, adjust it to 25°C, select the detection mode and parameters, wait for the temperature to stabilize, and then detect and record the abundance and distribution of the bacterial outer membrane vesicles.

[0056] 2. Experimental results

[0057] Transmission electron microscopy showed that the purified bacterial outer membrane vesicles had a characteristic saucer-like structure. Dynamic light scattering analysis showed that the vesicle particle size distribution ranged from 100 to 500 nm, with a median particle size of 154.6 nm, a Z-average of 181.1 nm, and a PDI of 0.397.

[0058] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0059] Experimental Example 4: Effect of bacterial outer membrane vesicles on obesity treatment in vivo

[0060] 1. Experimental Methods

[0061] The experimental sample was the bacterial outer membrane vesicle prepared according to Example 1. Eighteen 12-week-old C57 / BL6 male mice were selected and a high-fat diet (XHF60 from Synergistic Biology) was used to establish an induced obese mouse model. The mice were randomly divided into three groups: a. Control group, n=6: high-fat diet, subcutaneous injection of 100 μL PBS per week; b. Semaglutide treatment group, n=6: high-fat diet, subcutaneous injection of 100 μL Semaglutide (10 μg / mL) per week; c. OMVs treatment group, n=6: high-fat diet, subcutaneous injection of 100 μL OMVs (10 μg / mL) per week.

[0062] During the experiment, the body weight, food intake and blood sugar changes of the mice were measured and recorded regularly. After 5 weeks of treatment, the mice were killed, and their organs and adipose tissue were removed for HE staining and fat weight weighing to evaluate the effect and safety of the treatment.

[0063] 2. Experimental results

[0064] The experimental results of blood glucose changes, glucose tolerance, body weight and food intake after a single dose for one week are as follows: Figure 4 As shown. After a single subcutaneous injection, OMVs showed a blood sugar control effect comparable to Semaglutide. Glucose tolerance test results showed that after 5 weeks of treatment, OMVs had comparable blood sugar maintenance capabilities to the positive control drug Semaglutide. Body weight change results showed that during the treatment, the body weight of mice in the OMVs group decreased significantly. Daily average food intake results showed that food intake was significantly reduced within the first three days after administration.

[0065] Figure 5 The results of fat weight in various parts showed that OMVs significantly reduced the accumulation of fat in various parts. Figure 6 In the pathological results, HE staining showed that 5 weeks after subcutaneous injection of Semaglutide, the villi of the small intestine were significantly damaged, while no obvious damage was observed in mice injected with OMVs. OMVs did not show obvious pathological changes in important organs such as the heart, liver, spleen, lungs, and kidneys.

[0066] In summary, the bacterial outer membrane vesicles encapsulating GLP1 involved in the present invention have significant effects in blood sugar control and obesity treatment, and do not show obvious safety issues.

[0067] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A bacterial outer membrane vesicle encapsulating a glucagon-like peptide-1 (GLP1) analog, wherein the bacterial outer membrane vesicle carries a GLP1 analog having a gene coding sequence such as SEQ ID NO: 1; The GLP1 analog is also connected in series with a signal sequence and a tracer protein, the signal sequence is vNP, and its gene coding sequence is shown in SEQ ID NO: 2, and the tracer protein is mNerogreen, and its gene coding sequence is shown in SEQ ID NO: 3; The bacterial outer membrane vesicles are prepared by an engineered strain with the genotype of Nissle 1917; ΔNlpI.

2. The method for preparing the bacterial outer membrane vesicles according to claim 1, comprising the following steps: (1) constructing a plasmid vector containing a signal peptide, a tag protein, and a gene encoding GLP1; (2) Transform the plasmid vector into the host bacteria, screen and identify, and obtain the engineered strain; (3) The engineered strain is expanded and cultured, induced with an inducer, the bacterial liquid is collected, centrifuged, the bacteria are removed and the supernatant is harvested, and the supernatant is purified by multiple ultracentrifugation steps to obtain bacterial extracellular vesicles.

3. The preparation method according to claim 2, characterized in that: In step (2), the host bacteria is Escherichia coli Nissle 1917; ΔNlpI, the transformation method is chemical transformation, and the identification method is Sanger sequencing.

4. The preparation method according to claim 2, characterized in that: In step (3), the induction agent is isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1-0.2 mM, the induction temperature is 35-40° C., and the induction time is 14-20 h.

5. The preparation method according to claim 2, characterized in that: In step (3), the purification method includes the following steps: the supernatant is centrifuged for the first time to remove the precipitate, and then filtered through a 0.22 μm filter membrane, the filtrate is centrifuged for the second time, the supernatant is collected, and the supernatant is removed after the third centrifugation, and resuspended with PBS to obtain bacterial outer membrane vesicles loaded with GLP1.

6. Use of the bacterial outer membrane vesicles according to claim 1 in the preparation of drugs for preventing and / or treating type 2 diabetes and obesity.

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