Lactobacillus extracellular vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a lactobacillus extracellular vesicle as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out first culture on lactobacillus to obtain a primary lactobacillus culture solution; mixing a stress agent with the primary lactobacillus culture solution to obtain a mixed culture solution; wherein the stress agent comprises vegetable oil and / or vegetable oil nanoemulsion; the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution meet the relational expression that V1: V2 is greater than or equal to 0.01: 100; performing secondary culture on the mixed culture solution to obtain a culture solution containing lactobacillus extracellular vesicles; and purifying the culture solution to obtain the lactobacillus extracellular vesicle solution. According to the preparation method, through cooperation of physical and chemical stress and biological regulation, the yield of the lactobacillus extracellular vesicles is increased, the particle size distribution of the lactobacillus extracellular vesicles is more uniform, and the lactobacillus extracellular vesicles are promoted to penetrate through brain cells more effectively so as to realize accurate drug delivery of the brain.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a lactobacillus extracellular vesicle and a preparation method and application thereof. Background Art

[0002] The blood-brain barrier has always been a challenging problem for drug delivery to the brain. As a natural barrier of the central nervous system, it strictly regulates the exchange of substances between the blood and the brain parenchyma. Although it can effectively prevent harmful substances from invading the brain, it also causes about 98% of small molecule drugs and almost all large molecule therapeutic drugs (such as peptides and proteins) to be excluded by the blood-brain barrier, resulting in limited treatment options for central nervous system diseases. Traditional methods of administration, such as oral administration and intravenous injection, are difficult for drugs to accurately reach the brain lesion area when facing the blood-brain barrier, which greatly reduces the treatment effect of brain diseases.

[0003] Extracellular vesicles are a type of endogenous nanoscale carriers secreted by all eukaryotic and prokaryotic organisms. Their structural characteristics are membrane vesicles with a lipid bilayer structure, which contain a variety of bioactive molecules such as proteins and nucleic acids. Because these nanocarriers are derived from the organism itself, they have excellent biocompatibility and low immunogenicity, and can effectively evade recognition and clearance by the host immune system. More importantly, extracellular vesicles have a unique molecular loading capacity, can carry a variety of functional molecules, and show significant advantages in crossing complex biological barriers. Through the targeted transformation of extracellular vesicles by modern bioengineering technology, it is expected to develop them into an ideal delivery system that breaks through the blood-brain barrier and provides innovative solutions for precision drug delivery and gene therapy of brain diseases. However, the current cell culture-based vesicle preparation process still faces many challenges, including complex culture process, high cost, and limited vesicle yield, which seriously restrict the clinical transformation and application of extracellular vesicles. The core of these factors is that the cell culture process is cumbersome and expensive, which makes the yield of extracellular vesicles insufficient for clinical application. Summary of the invention

[0004] The invention provides a lactobacillus extracellular vesicle and a preparation method and application thereof, so as to solve the following technical problem: how to increase the yield of the extracellular vesicle.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present application provides a method for preparing extracellular vesicles of lactobacillus, the preparation method comprising:

[0007] The lactobacillus is first cultured to obtain a primary lactobacillus culture solution;

[0008] The stress agent and the primary lactobacillus culture solution are mixed to obtain a mixed culture solution; wherein the stress agent comprises vegetable oil and / or vegetable oil nanoemulsion; the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2≥0.01:100;

[0009] Carrying out a second cultivation on the mixed culture solution to obtain a culture solution containing extracellular vesicles of lactobacillus; and

[0010] The culture solution containing the extracellular vesicles of lactobacillus is purified to obtain the extracellular vesicle solution of lactobacillus.

[0011] Optionally, when the stress agent includes vegetable oil, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.01-0.05):100; and / or

[0012] In the case where the stress agent includes plant oil nanoemulsion, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.05-2.00):100.

[0013] Optionally, when the stress agent includes plant oil nanoemulsion, the stress agent includes, by mass fraction: the plant oil: 2% to 8%, emulsifier: 10% to 30%, co-emulsifier: 10% to 30% and water: 32% to 78%.

[0014] Optionally, the emulsifier comprises at least one of polyoxyethylene castor oil, hydrogenated polyoxyethylene castor oil, Tween-80, Span-80, Tween-20 and Span-60; and / or

[0015] The auxiliary emulsifier includes at least one of the following: polyethylene glycol 400, polyethylene glycol 200, glycerol, isopropanol, propylene glycol and ethanol.

[0016] Optionally, the type of vegetable oil includes at least one of the following: pomegranate seed oil, olive oil, grape seed oil, linseed oil, linoleic acid and oleic acid.

[0017] Optionally, the temperature of the first incubation is 35° C. to 39° C., and the time of the first incubation is 6 h to 16 h; and / or

[0018] The temperature of the second incubation is 35° C. to 39° C., and the time of the second incubation is 2 h to 24 h.

[0019] Optionally, the purifying treatment of the culture fluid containing Lactobacillus extracellular vesicles to obtain a Lactobacillus extracellular vesicle solution comprises the steps of:

[0020] The culture solution containing the extracellular vesicles of lactobacillus is subjected to a first centrifugation to obtain a first centrifugation solution;

[0021] Filtering the centrifuge liquid multiple times to obtain a filtrate;

[0022] The filtrate is subjected to a second centrifugation to obtain a second centrifuge;

[0023] The second centrifuge liquid is subjected to a third centrifugation to obtain a precipitate;

[0024] Dispersing the precipitate using PBS buffer to obtain a suspension; and

[0025] The suspension is subjected to gradient centrifugation purification to obtain a lactobacillus extracellular vesicle solution;

[0026] Wherein, the speed of the first centrifugation is 3000r / min to 5000r / min, and the time of the first centrifugation is 10min to 20min; and / or

[0027] The speed of the second centrifugation is 3000 r / min to 5000 r / min, and the time of the second centrifugation is 10 min to 20 min; and / or

[0028] The third centrifugation speed is 40000r / min to 60000r / min, and the third centrifugation time is 1.5h to 2.5h; and / or

[0029] The rotation speed of the gradient centrifugation purification treatment is 10000 r / min to 12000 r / min, and the time of the gradient centrifugation purification treatment is 1.5 h to 2.0 h.

[0030] Optionally, the gradient centrifugation purification treatment includes a sucrose gradient; the sucrose gradient includes a first sucrose gradient, a second sucrose gradient and a third sucrose gradient, the first sucrose gradient is 2.4M to 3.6M, the second sucrose gradient is 1.2M to 2.0M, and the third sucrose gradient is 0.4M to 0.8M; or

[0031] The gradient centrifugation purification treatment includes being performed in the form of an iodixanol gradient; the iodixanol gradient includes a first iodixanol gradient, a second iodixanol gradient and a third iodixanol gradient, the volume V3 of the first iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V3:V4=(35-50):100, the volume V5 of the second iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V5:V4=(22-35):100, and the volume V6 of the third iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V6:V4=(5-18):100.

[0032] In a second aspect, the present application provides a lactobacillus extracellular vesicle, which is prepared by the preparation method described in the first aspect.

[0033] In a third aspect, the present application provides a drug delivery vector, which includes the lactobacillus extracellular vesicles described in the second aspect, and the drug delivery vector is used as a drug delivery vector related to central nervous system diseases.

[0034] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0035] The present application provides a method for preparing extracellular vesicles of lactobacillus. The method comprises adding a stressor comprising vegetable oil and / or vegetable oil nanoemulsion during the cultivation stage of lactobacillus. Based on the fatty acid components of the vegetable oil or the vegetable oil nanoemulsion, the fatty acid components can be added to provide fatty acid raw materials for the synthesis of extracellular vesicles of lactobacillus, so as to increase the yield of extracellular vesicles of lactobacillus. In addition, the volume V1 of the stressor and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2≥0.01:100, so that the primary lactobacillus culture solution has a sufficient amount of stressor. Based on the characteristic that the main component of the cell membrane is phospholipid, a sufficient amount of stressor may activate the cell membrane stress signaling pathway of lactobacillus. This stress response may upregulate the expression of genes related to the generation of extracellular vesicles of lactobacillus, so as to improve the synthesis efficiency of extracellular vesicles of lactobacillus, thereby prompting lactobacillus to enhance its adaptability to the external environment by secreting a large amount of extracellular vesicles of lactobacillus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0038] Figure 1 A schematic diagram of a method for preparing extracellular vesicles of Lactobacillus provided in an embodiment of the present application;

[0039] Figure 2 A detailed schematic diagram of a method for preparing extracellular vesicles of Lactobacillus provided in an embodiment of the present application;

[0040] Figure 3It is a bar graph of the yield of extracellular vesicles of lactobacillus isolated from pomegranate seed oil stress, olive oil stress, grape seed oil stress, linseed oil stress, linoleic acid stress and oleic acid stress in Comparative Example 1 and Examples 1-6. **, *** represent p<0.01, p<0.001, respectively, compared with the extracellular vesicles of lactobacillus without stress; #, ##, ### represent p<0.05, p<0.01, p<0.001, respectively, compared with the highest yield; In the figure, LEV: unstimulated Lactobacillus extracellular vesicles; LEV-PSO: Lactobacillus extracellular vesicles stimulated by pomegranate seed oil; LEV-OO: Lactobacillus extracellular vesicles stimulated by olive oil; LEV-GSO: Lactobacillus extracellular vesicles stimulated by grape seed oil; LEV-LO: Lactobacillus extracellular vesicles stimulated by linseed oil; LEV-LA: Lactobacillus extracellular vesicles stimulated by linoleic acid; LEV-OA: Lactobacillus extracellular vesicles stimulated by oleic acid.

[0041] Figure 4 It is a bar graph of the yield of lactobacillus extracellular vesicles isolated from pomegranate seed oil nanoemulsion stress, olive oil nanoemulsion stress, grape seed oil nanoemulsion stress and linseed oil nanoemulsion stress in Comparative Example 1 and Examples 7-10, **, *** represent p<0.01, p<0.001, respectively, compared with the yield of lactobacillus extracellular vesicles without stress; #, ### represent p<0.05, p<0.01, p<0.001, respectively, compared with the highest yield; wherein, LEV: lactobacillus extracellular vesicles without stimulation; LEV-PSO NE: lactobacillus extracellular vesicles stressed by pomegranate seed oil nanoemulsion; LEV-OO NE: lactobacillus extracellular vesicles stressed by olive oil nanoemulsion;

[0042] LEV-GSO NE: Lactobacillus extracellular vesicles stressed by grape seed oil nanoemulsion; LEV-LO NE: Lactobacillus extracellular vesicles stressed by linseed oil nanoemulsion.

[0043] Figure 5 The curves of the brain entry rate of extracellular vesicles of Lactobacillus isolated in Comparative Example 1 and Examples 1-6 under pomegranate seed oil stress, olive oil stress, grape seed oil stress, linseed oil stress, linoleic acid stress and oleic acid stress are shown over time.

[0044] Figure 6 It is a curve chart showing the change over time of the extracellular vesicles of Lactobacillus into the brain under pomegranate seed oil nanoemulsion stress, olive oil nanoemulsion stress, grape seed oil nanoemulsion stress and linseed oil nanoemulsion stress isolated in Comparative Example 1 and Examples 7-10.

[0045] Figure 7 The pharmacokinetic results of oleuropein in different tissues and the area under the drug-time curve (AUC) result diagram are shown in which: Figure 7A is a comparison of the pharmacokinetic of oleuropein in plasma under different stress-treated extracellular vesicles. Figure 7 B Comparison of the pharmacokinetics of oleuropein in the brain under different stress-treated extracellular vesicles. Figure 7 C Comparison of the pharmacokinetics of oleuropein in the heart under different stress-treated extracellular vesicles. Figure 7 D. Comparison of pharmacokinetics of oleuropein in liver under different stress-treated extracellular vesicles. Figure 7 E. Comparison of pharmacokinetics of oleuropein in spleen under different stress-treated extracellular vesicles. Figure 7 F Comparison of the pharmacokinetics of oleuropein in the lungs under different stress-treated extracellular vesicles. Figure 7 G. Comparison of the pharmacokinetics of oleuropein in the kidney under different stress-treated extracellular vesicles. Figure 7 H Oleuropein in different tissues under different stress treatment of extracellular vesicles under the area under the curve AUC comparison results; Note: *, **: free oleuropein (OL) group comparison, p < 0.05 and p < 0.001; ## :p<0.01.

[0046] Figure 8 This is the result of in vivo fluorescence imaging of mice, where: Figure 8 A is the in vivo fluorescence imaging of mice. Figure 8 B is the fluorescence dynamics curve of the living mouse brain. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0048] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0049] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions, refers to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, if the mass ratio of substance A, substance B and substance C is 1:2:3, then substance A, substance B and substance C should correspond to the proportional numbers in the proportional formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0050] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0051] It should be noted that the low yield of natural lactobacillus vesicles restricts their development and utilization.

[0052] The present application uses vegetable oil and / or vegetable oil nanoemulsion as a stimulant for lactobacillus culture, which can greatly increase the yield of lactobacillus vesicles and provide the possibility for the engineering production of lactobacillus vesicles. In addition, the use of vegetable oil and / or vegetable oil nanoemulsion as a stimulant for lactobacillus culture can enhance the ability of lactobacillus vesicles to penetrate the blood-brain barrier, so as to significantly improve the blood-brain barrier permeability and achieve efficient delivery of drugs in the brain; in addition, the lactobacillus vesicles can also reduce the distribution of drugs in other normal tissues and organs, so that the lactobacillus vesicles can improve the effectiveness and safety of the treatment of brain diseases such as Alzheimer's disease, Parkinson's disease or epilepsy.

[0053] Figure 1The schematic diagram of the process of preparing a lactobacillus extracellular vesicle provided in an embodiment of the present application is exemplarily shown;

[0054] like Figure 1 As shown, the present application embodiment provides a method for preparing extracellular vesicles of lactobacillus, the preparation method comprising:

[0055] S1. The lactobacillus is first cultured to obtain a primary lactobacillus culture solution;

[0056] S2. The stress agent and the primary lactobacillus culture solution are mixed to obtain a mixed culture solution; wherein the stress agent comprises a plant oil and / or a plant oil nanoemulsion of fat-soluble polyphenols and flavonoids; the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2≥0.01:100;

[0057] S3. subjecting the mixed culture to a second incubation to obtain a culture solution containing extracellular vesicles of Lactobacillus; and

[0058] S4. Purifying the culture solution containing the extracellular vesicles of lactobacillus to obtain an extracellular vesicle solution of lactobacillus.

[0059] It should be noted that the primary lactobacillus culture fluid refers to the stage in which lactobacillus produces a large amount of lactobacillus fermentation fluid after a certain period of fermentation. During this period, the lactobacillus in the primary lactobacillus culture fluid may be in the logarithmic phase, which is conducive to the stimulating effect of subsequent stress agents.

[0060] It should be noted that the present application embodiment provides a method for preparing extracellular vesicles of lactobacillus, and the preparation method improves the yield of extracellular vesicles of lactobacillus through the following mechanism, specifically:

[0061] (1) Stress agents induce bacterial stress response: Plant oil or plant oil nanoemulsion as a stress agent may activate the cell membrane stress signaling pathway of Lactobacillus, prompting the bacteria to secrete extracellular vesicles of Lactobacillus to enhance the adaptability of Lactobacillus to the external environment. This stress response can upregulate the expression of genes related to the production of extracellular vesicles of Lactobacillus and improve the synthesis efficiency of extracellular vesicles of Lactobacillus.

[0062] (2) Optimizing the culture conditions for synergistic effect: Based on the highly active bacterial flora established in the first cultivation stage, the Lactobacillus cells are promoted to be in the logarithmic growth phase. Then, in the second cultivation stage, by controlling the addition ratio of the stress agent (V1:V2 ≥ 0.01:100), the secretion of Lactobacillus extracellular vesicles can be maximized while maintaining the survival rate of Lactobacillus. In addition, the plant oil nanoemulsion can increase the permeability of the culture medium to promote the release of Lactobacillus extracellular vesicles.

[0063] (3) Multiple metabolic regulatory effects: Unsaturated fatty acids in vegetable oils have the following characteristics: 1) change the lipid composition of cell membranes and enhance membrane fluidity; 2) act as signal molecules to activate the quorum sensing system; 3) promote the transport of intracellular metabolites of lactobacilli to the extracellular space.

[0064] Therefore, an embodiment of the present application provides a method for preparing Lactobacillus extracellular vesicles. Through the synergistic effect of physicochemical stress and biological regulation, the preparation method can increase the yield of Lactobacillus extracellular vesicles compared with conventional fermentation methods, and can make the particle size distribution of Lactobacillus extracellular vesicles more uniform, so that the Lactobacillus extracellular vesicles can more effectively penetrate brain cells to achieve accurate drug delivery to the brain.

[0065] In some optional embodiments, when the stress agent includes vegetable oil, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.01-0.05):100; and / or

[0066] In the case where the stress agent includes plant oil nanoemulsion, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.05-2.00):100.

[0067] In these embodiments, when the stress agent includes vegetable oil, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.01-0.05):100, so that the primary lactobacillus culture solution has a sufficient amount of stress agent, and the sufficient amount of stress agent can provide fatty acid raw materials for the synthesis of lactobacillus extracellular vesicles and activate the cell membrane stress signal pathway of lactobacillus to improve the synthesis efficiency of lactobacillus extracellular vesicles, thereby increasing the yield of lactobacillus extracellular vesicles. In addition, when the stress agent includes a plant oil nanoemulsion, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.05-2.00):100, so that the primary lactobacillus culture solution has a sufficient amount of the stress agent. The sufficient amount of the stress agent can not only provide fatty acid raw materials for the synthesis of lactobacillus extracellular vesicles and activate the cell membrane stress signal pathway of lactobacillus, but also increase the dispersion degree of the lactobacillus extracellular vesicles through the emulsification effect of the plant oil nanoemulsion and the co-emulsifier, so as to obtain evenly dispersed and high-yield lactobacillus extracellular vesicles.

[0068] In the case where the stress agent comprises vegetable oil, the volume V1 of the stress agent may have a value of 0.01, 0.02, 0.03, 0.04 or 0.05.

[0069] When the stress agent comprises a plant oil nanoemulsion, the volume V1 of the stress agent can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.50 or 2.00.

[0070] In some optional embodiments, when the stress agent includes plant oil nanoemulsion, the stress agent includes, by mass fraction: the plant oil: 2% to 8%, emulsifier: 10% to 30%, co-emulsifier: 10% to 30% and water: 32% to 78%.

[0071] In these embodiments, where the stress agent comprises a plant oil nanoemulsion, the stress agent has the following properties:

[0072] 1. The core role of vegetable oil components:

[0073] (1) Inducing membrane structure remodeling: The effect of vegetable oil with a mass fraction of 2% to 8% is to penetrate the cell membrane of lactobacillus through the unsaturated fatty acid components of vegetable oil (such as linoleic acid and oleic acid), thereby enhancing the fluidity of the cell membrane and activating the phospholipase activity in lactobacillus, promoting the reorganization of the cell membrane structure and releasing lactobacillus extracellular vesicles.

[0074] (2) Constructing a biocompatible stress environment: This mass fraction range can not only form sufficient membrane stress stimulation, but also avoid the inhibition of lactobacillus growth caused by excessively high unsaturated fatty acid concentrations, thereby maintaining the survival rate of lactobacillus at a high level.

[0075] 2. Synergistic effect of emulsification system:

[0076] (1) Nano-scale dispersion promotes absorption: The effect of an emulsifier with a mass fraction of 10% to 30% and an emulsifier with a mass fraction of 10% to 30% can work together to form a nanoemulsion with a particle size of less than 200 nm. This fine nanoemulsion can increase the contact area between the vegetable oil and the lactic acid bacteria to a higher level.

[0077] (2) Sustained release regulation mechanism: The composite emulsifier system of emulsifier and co-emulsifier can gradually release vegetable oil during the culture process, thereby prolonging the time of stress stimulation to fully increase the production of extracellular vesicles of lactobacillus.

[0078] 3. Precise regulation of metabolic pathways:

[0079] (1) Quorum sensing activation: The polyoxyethylene groups that may exist in the emulsifier can act as signal molecule analogs and bind to the LuxS / AI-2 quorum sensing system to upregulate the expression of genes related to the secretion of extracellular vesicles of lactobacilli.

[0080] (2) Promotion of transmembrane transport: The calcium ions that may exist in the emulsifier can accelerate the efflux of extracellular vesicles of lactobacillus mediated by ABC transporters by enhancing the potential difference of the cell membrane of lactobacillus.

[0081] The mass fraction of the vegetable oil can be 2%, 3%, 4%, 5%, 6%, 7% or 8%.

[0082] The mass fraction of the emulsifier can be 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25% or 30%.

[0083] The mass fraction of the co-emulsifier can be 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25% or 30%.

[0084] In some optional embodiments, the emulsifier includes at least one of polyoxyethylene castor oil, hydrogenated polyoxyethylene castor oil, Tween-80, Span-80, Tween-20 and Span-60; and / or

[0085] The auxiliary emulsifier includes at least one of the following: polyethylene glycol 400, polyethylene glycol 200, glycerol, isopropanol, propylene glycol and ethanol.

[0086] In these embodiments, the emulsifier may include at least one of the following: polyoxyethylene castor oil, hydrogenated polyoxyethylene castor oil, Tween-80, Span-80, Tween-20 and Span-60, and the co-emulsifier may include at least one of the following: polyethylene glycol 400, polyethylene glycol 200, glycerol, isopropanol, propylene glycol and ethanol, so that the composite system of the emulsifier and the co-emulsifier can prolong the time of stress stimulation and can increase the expression level of secretion-related genes of lactobacillus extracellular vesicles, thereby substantially improving the yield of lactobacillus extracellular vesicles.

[0087] In some optional embodiments, the type of vegetable oil includes at least one of the following: pomegranate seed oil, olive oil, grape seed oil, linseed oil, linoleic acid and oleic acid.

[0088] In these embodiments, the types of vegetable oils may include at least one of the following: pomegranate seed oil, olive oil, grape seed oil, linseed oil, linoleic acid and oleic acid, which may cover most types of vegetable oils, to provide fatty acid raw materials for the synthesis of extracellular vesicles of lactobacillus, activate the cell membrane stress signaling pathway of lactobacillus, and improve the synthesis efficiency of extracellular vesicles of lactobacillus, thereby increasing the production of extracellular vesicles of lactobacillus.

[0089] In some optional embodiments, the temperature of the first incubation is 35° C. to 39° C., and the time of the first incubation is 6 h to 16 h; and / or

[0090] The temperature of the second incubation is 35° C. to 39° C., and the time of the second incubation is 2 h to 24 h.

[0091] In these embodiments, the temperature of the first cultivation can be 35°C to 39°C, and the time of the first cultivation can be 6h to 16h, so that the lactic acid bacteria are fermented in large quantities and are in the logarithmic phase, thereby facilitating the stimulation effect of subsequent stress agents on these lactic acid bacteria, so as to effectively increase the production of lactic acid bacteria extracellular vesicles; in addition, the temperature of the second cultivation can be 35°C to 39°C, and the time of the second cultivation can be 2h to 24h, so that the mixed culture has sufficient temperature and sufficient time to ferment in large quantities and produce sufficient lactic acid bacteria extracellular vesicles, so as to effectively increase the production of lactic acid bacteria extracellular vesicles.

[0092] The temperature of the first incubation may be 35°C, 36°C, 37°C, 38°C or 39°C.

[0093] The first incubation time can be 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h or 16 h.

[0094] The temperature of the second incubation may be 35°C, 36°C, 37°C, 38°C or 39°C.

[0095] The second incubation time can be 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h.

[0096] Figure 2 The detailed process diagram of a method for preparing extracellular vesicles of lactobacillus provided in an embodiment of the present application is exemplarily shown;

[0097] In some optional embodiments, such as Figure 2 As shown, the culture fluid containing extracellular vesicles of lactobacillus is purified to obtain an extracellular vesicle solution of lactobacillus, comprising the steps of:

[0098] S401. performing a first centrifugation on the culture solution containing extracellular vesicles of lactobacillus to obtain a first centrifuge solution;

[0099] S402. Filtering the centrifuge several times to obtain a filtrate;

[0100] S403. The filtrate is subjected to a second centrifugation to obtain a second centrifuge;

[0101] S404. The second centrifuge solution is subjected to a third centrifugation to obtain a precipitate;

[0102] S405. Dispersing the precipitate using PBS buffer to obtain a suspension; and

[0103] S406. The suspension is subjected to gradient centrifugation purification to obtain a lactobacillus extracellular vesicle solution;

[0104] Wherein, the speed of the first centrifugation is 3000r / min to 5000r / min, and the time of the first centrifugation is 10min to 20min; and / or

[0105] The speed of the second centrifugation is 3000 r / min to 5000 r / min, and the time of the second centrifugation is 10 min to 20 min; and / or

[0106] The third centrifugation speed is 40000r / min to 60000r / min, and the third centrifugation time is 1.5h to 2.5h; and / or

[0107] The rotation speed of the gradient centrifugation purification treatment is 10000 r / min to 12000 r / min, and the time of the gradient centrifugation purification treatment is 1.5 h to 2.0 h.

[0108] In these embodiments, the small molecule impurities in the culture solution containing the lactobacillus extracellular vesicles can be removed by the first centrifugation, filtration and second centrifugation to facilitate the subsequent third centrifugation and gradient centrifugation purification process, thereby obtaining a pure lactobacillus extracellular vesicle solution. In addition, the third centrifugation and gradient centrifugation purification process can concentrate the lactobacillus extracellular vesicle solution to the greatest extent to obtain a pure lactobacillus extracellular vesicle product.

[0109] The rotation speed of the first centrifugation can be 3000r / min, 3100r / min, 3200r / min, 3300r / min, 3400r / min, 3500r / min, 3600r / min, 3700r / min, 3800r / min, 3900r / min, 4000r / min, 4500r / min or 5000r / min.

[0110] The first centrifugation time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0111] The rotation speed of the second centrifugation can be 3000r / min, 3100r / min, 3200r / min, 3300r / min, 3400r / min, 3500r / min, 3600r / min, 3700r / min, 3800r / min, 3900r / min, 4000r / min, 4500r / min or 5000r / min.

[0112] The second centrifugation time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0113] The rotation speed of the third centrifugation is 40000r / min, 41000r / min, 42000r / min, 43000r / min, 44000r / min, 45000r / min, 46000r / min, 47000r / min, 48000r / min, 49000r / min, 50000r / min, 55000r / min or 60000r / min.

[0114] The time of the third centrifugation can be 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h or 2.5 h.

[0115] In some optional embodiments, the gradient centrifugation purification process includes performing the purification process in the form of a sucrose gradient; the sucrose gradient includes a first sucrose gradient, a second sucrose gradient and a third sucrose gradient, the first sucrose gradient is 2.4M to 3.6M, the second sucrose gradient is 1.2M to 2.0M, and the third sucrose gradient is 0.4M to 0.8M; or

[0116] The gradient centrifugation purification treatment includes being performed in the form of an iodixanol gradient; the iodixanol gradient includes a first iodixanol gradient, a second iodixanol gradient and a third iodixanol gradient, the volume V3 of the first iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V3:V4=(35-50):100, the volume V5 of the second iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V5:V4=(22-35):100, and the volume V6 of the third iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V6:V4=(5-18):100.

[0117] In these embodiments, the gradient centrifugation purification process can be selectively performed in the form of a sucrose gradient or an iodixanol gradient, and a pure lactobacillus extracellular vesicle product can be obtained by the gradient centrifugation purification process.

[0118] The first sucrose gradient can be 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M or 3.6M.

[0119] The second sucrose gradient can be 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2.0M.

[0120] The third sucrose gradient can be 0.4M, 0.5M, 0.6M, 0.7M or 0.8M.

[0121] The volume V3 of the first iodixanol gradient may have a value of 35, 36, 37, 38, 39, 40, 45 or 50.

[0122] The value of the volume V5 of the second iodixanol gradient can be 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.

[0123] The volume V6 of the third iodixanol gradient can have a value of 5, 6, 7, 8, 9, 10, 15, 16, 17 or 18.

[0124] Based on a general inventive concept, an embodiment of the present application provides a lactobacillus extracellular vesicle, and the lactobacillus extracellular vesicle is prepared by the preparation method.

[0125] The lactobacillus extracellular vesicles are realized based on the above-mentioned preparation method. The specific steps of the preparation method can refer to the above-mentioned embodiments. Since the lactobacillus extracellular vesicles adopt part or all of the technical solutions of the above-mentioned embodiments, they at least have all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0126] Based on a general inventive concept, an embodiment of the present application provides a drug delivery vector, which includes the lactobacillus extracellular vesicles, and is used as a drug delivery vector related to central nervous system diseases.

[0127] The drug delivery vector is realized based on the above-mentioned lactobacillus extracellular vesicles. The specific composition of the lactobacillus extracellular vesicles can refer to the above-mentioned embodiments. Since the drug delivery vector adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

[0128] Example 1

[0129] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0130] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculate it into MRS liquid culture medium, the culture conditions are temperature 37 ° C, speed 150r / min; when the culture reaches 8h, add 0.075mL of pomegranate seed oil to the culture medium, continue to culture for 16h, and terminate the culture;

[0131] S3, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 4500r / min for 15min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred to 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 3500r / min for 15min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 40000r / min for 2h, and the precipitate was suspended with the same volume of PBS;

[0132] S4. Add 2.5M, 1.6M and 0.6M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 11000r / min for 2h, collect the 1.6M layer to obtain the extracellular vesicles of lactobacillus stressed by pomegranate seed oil, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0133] Example 2

[0134] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0135] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus, inoculate it into MRS medium, start culturing, the culture conditions are 37 ° C, 150r / min; when the culture reaches 8h, add 0.03mL of olive oil to the medium, continue to culture for 16h, and terminate the culture;

[0136] S3, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 3000r / min for 20min; the supernatant was filtered using 0.45μm and 0.22μm microporous filter membranes in turn, and the filtrate was collected; the filtrate was transferred to 100kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 5000r / min for 10min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 60000r / min for 1.5h, and the precipitate was suspended with the same volume of PBS;

[0137] S4. Add 5%, 22% and 35% iodixanol solution (w / v) to a 50 mL centrifuge tube in sequence, with a volume of 10 mL for each concentration. Add 10 mL of the above PBS suspension to the top layer, centrifuge at 10000 r / min for 2 h, collect the 22% layer, obtain the extracellular vesicles of lactobacillus stressed by olive oil, merge the vesicles, and record the volume, which is the extracellular vesicle yield.

[0138] Example 3

[0139] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0140] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus, inoculate it into MRS culture medium, and culture it at a temperature of 38°C and a rotation speed of 50r / min; when the culture reaches 6h, add 0.15mL of grape seed oil to the culture medium, continue to culture for 12h, and terminate the culture;

[0141] S3, the culture solution was divided into 50mL centrifuge tubes, centrifuged at a speed of 5000r / min for 10min, and the supernatant was collected; the supernatant was filtered using 0.45μm and 0.22μm microporous filter membranes in turn, and the filtrate was collected; it was transferred to 100kDa ultrafiltration centrifuge tubes, 15mL per tube, and centrifuged at a speed of 5000r / min for 10min, and the upper concentrated liquid was collected with a test tube; the concentrated liquid was centrifuged at a speed of 40000r / min for 2.5h, and the precipitate was suspended with the same volume of PBS;

[0142] S4. Add 0.4 M, 1.2 M, and 2.4 M sucrose solutions to a 50 mL centrifuge tube in sequence, add 10 mL of the above PBS suspension to the top layer, centrifuge at 10000 r / min for 2 h, collect the 1.2 M layer to obtain the extracellular vesicles of lactobacillus stressed by grape seed oil, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0143] Example 4

[0144] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0145] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus, inoculate it into MRS medium, start culturing, the culture conditions are 37 ° C, 150r / min; when the culture reaches 8h, add 0.075mL of linoleic acid to the medium, continue to culture for 16h, and terminate the culture;

[0146] S3. The culture solution was divided into 50 mL centrifuge tubes, centrifuged at 4500 r / min for 15 min, and the supernatant was collected; the supernatant was filtered using 0.45 μm and 0.22 μm microporous filter membranes in turn, and the filtrate was collected; the filtrate was added to 50 kDa ultrafiltration centrifuge tubes, 15 mL per tube, and centrifuged at 3500 r / min for 15 min. The upper concentrated solution was collected with a test tube and combined; one volume of ultrapure water was added to the concentrated solution, and ultrafiltration was continued to remove the culture medium and collect the sample;

[0147] S4. Prepare 0.8M, 2.0M, and 3.6M sucrose solutions respectively, add 10mL of 2.5M, 10mL of 1.6M, and 10mL of 0.6M sucrose solutions in a 50mL centrifuge tube, add 10mL of sample solution to the top layer, centrifuge at 11000r / min for 2h, collect the 2.0M layer, and obtain the extracellular vesicles of Lactobacillus stressed by linoleic acid.

[0148] Example 5

[0149] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0150] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculate it into MRS culture medium, the culture conditions are temperature 35 ° C, speed 200r / min; when the culture reaches 16h, add 0.15mL of linseed oil to the culture medium, continue to culture for 14h, and terminate the culture;

[0151] S3, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 5000r / min for 10min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred into 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 5000r / min for 10min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 60000r / min for 1.5h, and the precipitate was suspended with the same volume of PBS;

[0152] S4. Add 0.4M, 1.2M and 2.4M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 12000r / min for 1.5h, collect the 1.2M layer to obtain the extracellular vesicles of lactobacillus stressed by linseed oil, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0153] Example 6

[0154] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0155] S2, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculate it into MRS culture medium, the culture conditions are temperature 35 ° C, speed 200r / min; when the culture reaches 16h, add 0.15mL of oleic acid to the culture medium, continue to culture for 14h, and terminate the culture;

[0156] S3, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 5000r / min for 10min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred into 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 5000r / min for 10min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 60000r / min for 1.5h, and the precipitate was suspended with the same volume of PBS;

[0157] S4. Add 0.4M, 1.2M and 2.4M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 12000r / min for 1.5h, collect the 1.2M layer to obtain the extracellular vesicles of Lactobacillus stressed by oleic acid, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0158] Example 7

[0159] S1. Prepare a grape seed oil nanoemulsion, which contains 8% grape seed oil, polyoxyethylene castor oil and Tween 80 (1:1) as a mixed emulsifier, with a content of 30%; PEG400 and propylene glycol (3:1) as a mixed auxiliary emulsifier, with a content of 30%; mix the grape seed oil, the mixed emulsifier and the mixed auxiliary emulsifier, then add 32% distilled water to the mixed solution, and after the distilled water is added, ultrasonicate for 5 minutes to obtain the grape seed oil nanoemulsion.

[0160] S2. Prepare 300 mL of MRS medium, sterilize and set aside;

[0161] S3, picking an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculated into MRS culture medium, the culture conditions are temperature 37 ° C, speed 100r / min; when the culture reaches 6h, 0.15mL of grape seed oil nanoemulsion is added to the culture medium, and the culture is continued for 12h, and the culture is terminated;

[0162] S4, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 3000r / min for 20min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred into 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 3000r / min for 20min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 40000r / min for 2.5h, and the precipitate was suspended with the same volume of PBS;

[0163] S5. Add 18%, 35% and 50% iodixanol solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 10000r / min for 2h, collect the 35% layer, obtain the extracellular vesicles of lactobacillus stressed by grape seed oil nanoemulsion, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0164] Example 8

[0165] S1. Prepare an olive oil nanoemulsion, which contains 2% olive oil, polyoxyethylene castor oil and Tween 20 (3:1) as a mixed emulsifier, with a content of 10%; PEG400 and PEG200 (1:1) as a mixed auxiliary emulsifier, with a content of 10%; mix the olive oil, the mixed emulsifier and the mixed auxiliary emulsifier, then add 78% distilled water to the mixed solution, and after the distilled water is added, ultrasonicate for 5 minutes to obtain a grape seed oil nanoemulsion.

[0166] S2. Prepare 300 mL of MRS medium, sterilize and set aside;

[0167] S3, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculate it into MRS culture medium, the culture conditions are temperature 37 ° C, speed 100r / min; when the culture reaches 16h, add 6mL of olive oil nanoemulsion to the culture medium, continue to culture for 2h, and terminate the culture;

[0168] S4, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 3000r / min for 20min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred into 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 3000r / min for 20min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 40000r / min for 2.5h, and the precipitate was suspended with the same volume of PBS;

[0169] S5. Add 0.5M, 1.4M and 3M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 10000r / min for 2h, collect the 1.4M layer to obtain the extracellular vesicles of lactobacillus stressed by olive oil nanoemulsion, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0170] Example 9

[0171] S1. Prepare a pomegranate seed oil nanoemulsion, which contains 5% pomegranate seed oil, hydrogenated polyoxyethylene castor oil as an emulsifier, with a content of 20%; isopropyl alcohol and propylene glycol (2:1) as a mixed emulsifier, with a content of 20%; the pomegranate seed oil, the emulsifier and the mixed emulsifier are mixed, and then 55% distilled water is added dropwise to the mixed solution. After the distilled water is added, ultrasonication is performed for 5 minutes to obtain a grape seed oil nanoemulsion.

[0172] S2. Prepare 300 mL of MRS medium, sterilize and set aside;

[0173] S3, picking an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculated into MRS culture medium, the culture conditions are temperature 37 ° C, speed 100r / min; when the culture reaches 14h, 1mL of pomegranate seed oil nanoemulsion is added to the culture medium, and the culture is continued for 6h, and the culture is terminated;

[0174] S4, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 4000r / min for 15min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred to 100kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 4000r / min for 15min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 50000r / min for 2h, and the precipitate was suspended with the same volume of PBS;

[0175] S5. Add 0.5M, 1.5M and 3M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 10000r / min for 1.5h, collect the 1.5M layer, obtain the extracellular vesicles of lactobacillus stressed by pomegranate seed oil nanoemulsion, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0176] Example 10

[0177] S1. Prepare a linseed oil nanoemulsion, which contains 2% linseed oil, Tween 80-Span 60 (4:1) as a mixed emulsifier, with a content of 20%; isopropanol and ethanol (3:1) as a mixed auxiliary emulsifier, with a content of 30%; mix the linseed oil, the mixed emulsifier and the mixed auxiliary emulsifier, then add 48% distilled water to the mixed solution, and after the distilled water is added, ultrasonicate for 5 minutes to obtain a linseed oil nanoemulsion.

[0178] S2. Prepare 300 mL of MRS medium, sterilize and set aside;

[0179] S3, pick an inoculation loop area (inoculation loop diameter 3mm) of Lactobacillus acidophilus (ACCC11073), inoculate it into MRS culture medium, the culture conditions are temperature 37 ° C, speed 100r / min; when the culture reaches 6h, add 1.5mL of linseed oil nanoemulsion to the culture medium, continue to culture for 12h, and terminate the culture;

[0180] S4, the culture solution was divided into 50mL centrifuge tubes, centrifuged at 4000r / min for 15min; the supernatant was taken, filtered using 0.45μm and 0.22μm microporous filter membranes in turn, the filtrate was collected, and transferred to 50kDa ultrafiltration centrifuge tubes, 15mL per tube, centrifuged at 3000r / min for 15min, and the upper concentrated solution was collected in a test tube; the concentrated solution was centrifuged at 40000r / min for 2h, and the precipitate was suspended with the same volume of PBS;

[0181] S5. Add 0.6M, 2.0M and 3.3M sucrose solutions to a 50mL centrifuge tube in sequence, with a volume of 10mL for each concentration; add 10mL of the above PBS suspension to the top layer, centrifuge at 10000r / min for 1.5h, collect the 1.5M layer, obtain the extracellular vesicles of lactobacillus stressed by linseed oil nanoemulsion, merge the vesicles and record the volume, which is the extracellular vesicle yield.

[0182] Comparative Example 1

[0183] S1. Prepare 300 mL of MRS medium, sterilize and set aside;

[0184] S2, pick a Lactobacillus acidophilus with an inoculation loop area (inoculation loop diameter 3mm), inoculate it into MRS medium, start culturing, the culture conditions are 37°C, 150r / min; terminate the culture when the culture reaches 24h;

[0185] S3. The culture solution was divided into 50 mL centrifuge tubes, centrifuged at 4500 r / min for 15 min, and the supernatant was collected; the supernatant was filtered using 0.45 μm and 0.22 μm microporous filter membranes in turn, and the filtrate was collected; the filtrate was added to 50 kDa ultrafiltration centrifuge tubes, 15 mL per tube, and centrifuged at 3500 r / min for 15 min. The upper concentrated solution was collected with a test tube and combined; one volume of ultrapure water was added to the concentrated solution, and ultrafiltration was continued to remove the culture medium and collect the sample;

[0186] S4. Prepare 2.5M, 1.6M, and 0.6M sucrose solutions respectively, add 10mL of 2.5M, 10mL of 1.6M, and 10mL of 0.6M sucrose solutions in a 50mL centrifuge tube, add 10mL of sample solution on the top layer, centrifuge at 11000r / min for 2h, collect the 1.6M layer, and obtain stress-free lactobacillus extracellular vesicles.

[0187] Experimental example

[0188] 1. Extracellular vesicle production of Lactobacillus under stimulation of no stress, different oils, and different oil nanoemulsions.

[0189] Figure 3 is the extracellular vesicle yield of lactobacillus under no stress and different oil stimulation (oil in Examples 1-6) of Comparative Example 1. Figure 3 It can be seen that the production of extracellular vesicles of lactic acid bacteria increased significantly after oil stimulation, with pomegranate seed oil as the stimulant having the highest production, followed by linoleic acid and flaxseed oil. The stimulating effects of olive oil, grape seed oil and oleic acid were relatively weak.

[0190] Figure 4The extracellular vesicle yield of lactobacillus in the absence of stress and under stimulation of different oil nanoemulsions (oil nanoemulsions of Examples 7-10) in Comparative Example 1. Figure 4 It can be seen that the production of extracellular vesicles of lactobacillus increased significantly after stimulation with oil nanoemulsion, and the production was the highest when pomegranate seed oil nanoemulsion was used as the stimulator.

[0191] 2. Encapsulation efficiency of oleuropein-Lactobacillus extracellular vesicles

[0192] Take 10 mL of the lactobacillus extracellular vesicles obtained in the above embodiments and comparative examples, add 10 mg of oleuropein respectively, ultrasonicate for 1-3 min to allow oleuropein to enter the vesicles, use a 50 kd ultrafiltration tube, centrifuge at 3000 r / min for 15 min, detect the drug content outside the ultrafiltration tube by HPLC, and calculate the encapsulation efficiency according to the following formula:

[0193] Encapsulation rate (%) = (total amount of drug - drug content outside the tube) / total amount of drug * 100%.

[0194] The encapsulation efficiency of oleuropein in extracellular vesicles of Lactobacillus stimulated by different oils or nanoemulsions was between 75% and 80%.

[0195] 3. Use the PAMPA-BBB model to determine the in vitro brain penetration effect of samples.

[0196] Grind the gray matter layer of fresh pig brain, add dodecane, vortex mix, sonicate and dissolve, the sonication time should not exceed 5 minutes, and make a 20mg / mL pig brain solution; drop 16μL of pig brain solution on the Transwell membrane, place it on a shaker, and shake it at 50r / min for 1 minute to evenly spread the pig brain solution; place 1.5mL of sterile PBS solution in the lower receiving pool of Transwell, and add 0.5mL of PBS solution with a concentration of 1mg / mL oleuropein and 1mg / mL oleuropein plant oil-stressed lactobacillus extracellular vesicle solution to the upper layer. Place the Transwell in an incubator at 37°C and take it out after 0.5h, 1h, 1.5h, 2h and 4h respectively; shake it on a shaker at 50r / min for 2min, take out 0.2mL of liquid from the receiving pool, and add 0.2mL of PBS. The peak area A of oleuropein in the removed liquid was determined by HPLC 1 ; and measure the peak area A of 0.333 mg / ml oleuropein PBS solution 0 , which is taken as the peak area of ​​100% penetration. HPLC detection conditions are: C18 column (250mm×4.6mm, 5μm), mobile phase acetonitrile-water (22:78), detection wavelength 279nm. Flow rate 0.7mL / min, column temperature 35℃. The calculation formula of brain penetration rate is: Brain penetration rate (%) = A 1 / A0 *100%. Experimental results are shown in Figure 5 and Figure 6 .

[0197] Depend on Figure 5 It can be seen that the unstressed Lactobacillus extracellular vesicles and the Lactobacillus extracellular vesicles stimulated by different vegetable oils (the vegetable oils in Examples 1-6) all showed good brain penetration effects, and the brain penetration rate was significantly higher than that of the oleuropein solution; the brain penetration rate of the Lactobacillus extracellular vesicles stimulated by oil was significantly higher than that of the unstressed Lactobacillus extracellular vesicles; among the different vegetable oils, the Lactobacillus extracellular vesicles stressed by pomegranate seed oil had the best brain penetration effect, followed by grape seed oil, linoleic acid and linseed oil; the stimulation effects of oleic acid and olive oil were weaker than those of other vegetable oils.

[0198] Depend on Figure 6 It can be seen that the extracellular vesicles of Lactobacillus without stress and the extracellular vesicles of Lactobacillus stimulated by different plant oil nanoemulsions (the plant oil nanoemulsions in Examples 7-10) all showed good brain penetration effects, and the brain penetration rate was significantly higher than that of oleuropein solution; the brain penetration rate of Lactobacillus extracellular vesicles stimulated by oil nanoemulsion was significantly higher than that of the extracellular vesicles of Lactobacillus without stress; among the different plant oil nanoemulsions, the extracellular vesicles of Lactobacillus stressed by pomegranate seed oil nanoemulsion had the best brain penetration effect, and the brain penetration rate of vesicles stimulated by olive oil nanoemulsion was lower than that of other stimulated vesicles, but still significantly higher than that of unstimulated vesicles.

[0199] 4. The effect of entering the brain.

[0200] (1) In vivo pharmacokinetics and tissue distribution (e.g. Figure 7 )

[0201] The HPLC determination method of oleuropein (OL) in plasma and tissues was established; mice were intraperitoneally injected with free drug, LEV (OL) (extracellular vesicles of Lactobacillus loaded with OL), LEV-PSO (OL) (extracellular vesicles of Lactobacillus loaded with OL and stimulated with pomegranate seed oil) and LEV-PSO NE (OL) (extracellular vesicles of Lactobacillus loaded with OL and stimulated with pomegranate seed oil nanoemulsion) at a dose of 20 mg / kg OL. Four mice in each group were killed at 5 min, 10 min, 15 min, 30 min, 45 min, 60 min and 90 min after administration, and their plasma was taken to detect the blood drug concentration; the brain, heart, liver, spleen, lung and kidney of the mice were carefully removed and weighed, and a portion was homogenized with physiological saline to determine the concentration of OL in the tissue. The drug-time curve was drawn, and the AUC was calculated using DAS2.0 software. The results are shown in Figure 7 As shown. Figure 7It can be seen that intraperitoneally injected OL degrades very quickly in vivo, reaching the peak blood concentration in 10 minutes, and the prototype drug can no longer be detected after 40 minutes. LEV, LEV-PSO and LEV-PSO NE significantly reduced the distribution of OL in the liver, spleen, lungs and kidneys, and increased the concentration of OL in the brain. The peak brain concentrations of LEV (OL), LEV-PSO (OL) and LEV-PSO NE (OL) were 3, 6 and 6 times that of the free drug, respectively, and the AUC 0→90min The brain targeting abilities of the three carriers were 2.17, 7.29, and 5.96 times those of the free drug, respectively, showing the brain targeting characteristics of the three carriers, as well as the stronger brain targeting abilities of LEV-PSO and LEV-PSO NE.

[0202] (2) In vivo mouse imaging experiment Figure 8 )

[0203] Fluorescent Cy5.5 was encapsulated with LEV, LEV-PSO and LEV-PSO NE and injected intraperitoneally into mice. The mice were anesthetized at different time points and photographed using IVIS Spectrum in vivo imaging system (PerkinElmer, USA). The results are shown in Figure 2. Figure 8 As shown in the figure, compared with the free drug group, the LEV, LEV-PSO and LEV-PSO NE groups all showed obvious brain targeting effects. After 6 hours, no fluorescence was detected in the brain of the free group, while obvious residual fluorescence was still observed in the brains of the three LEV groups. At 24 hours, no fluorescence was observed in the brain of the LEV group, while a small amount of fluorescence was still observed in the LEV-PSO and LEV-PSO NE groups, indicating that LEV-PSO and LEV-PSO NE have better brain targeting ability and can prolong the drug's action time in the brain. LEV, LEV-PSO and LEV-PSO NE brain AUC 0→24h They are 2.46, 3.26 and 3.12 times of free fluorescein respectively.

[0204] In summary, the present invention provides a method for preparing extracellular vesicles of lactobacillus, which has the following advantages:

[0205] 1. The present invention adds vegetable oil or nanoemulsion as a stimulant during the growth stage of lactobacillus, which can greatly increase the yield of vesicles. After being stimulated by vegetable oil or nanoemulsion, the yield of extracellular vesicles of lactobacillus is 1.2-5 times that of unstressed extracellular vesicles. Specifically, the main component of the cell membrane is phospholipids, and fatty acids are an important component of phospholipids. Vegetable oil and its nanoemulsion provide vesicles with fatty acids required for vesicle membrane synthesis, thereby greatly increasing the yield of vesicles; in addition, the emulsifier in the nanoemulsion may have a destructive effect on cell lysosomes, resulting in impaired lysosomal function and stimulating the production of vesicles; the fat-soluble polyphenols and flavonoids contained in the vegetable oil may also stimulate the growth of vesicles.

[0206] 2. Lactobacillus extracellular vesicles stimulated by vegetable oil or nanoemulsion can significantly improve the ability to enter the brain. The vesicles were encapsulated with oleuropein, and the in vitro blood-brain barrier (BBB) ​​model was used to investigate the brain entry rates of different samples. The results showed that after stimulation with vegetable oil or nanoemulsion, the production of Lactobacillus vesicles was significantly higher than that of unstimulated vesicles; at the same time, both unstressed vesicles and Lactobacillus extracellular vesicles stimulated by vegetable oil or nanoemulsion showed good brain entry effects, and the brain entry rate was significantly higher than that of oleuropein solution; the brain entry rate of Lactobacillus extracellular vesicles stimulated by oil or nanoemulsion was significantly higher than that of unstressed extracellular vesicles; at the same time, there were differences in the production and brain entry rate of vesicles stimulated by different oils or nanoemulsions. The brain entry rate of Lactobacillus extracellular vesicles increased by 90%-320% after stimulation with vegetable oil or nanoemulsion.

[0207] 3. Extracellular vesicles themselves have high biocompatibility and low immunogenicity. They are not easily engulfed by macrophages during blood circulation, which helps to concentrate in the brain. Extracellular vesicles can encapsulate both hydrophilic and lipophilic drugs. Hydrophilic drugs enter the vesicles, and lipophilic drugs are distributed in the lipid layer of the vesicles. Stimulated lactobacillus extracellular vesicles can efficiently deliver drugs to the brain, increase the concentration of drugs in the cerebral cortex, reduce the distribution of drugs in other tissues and organs, improve the effectiveness of the treatment of central nervous system diseases, and reduce the toxic side effects of drugs. They have broad application prospects in the treatment of brain diseases such as Alzheimer's disease, Parkinson's disease and brain tumors.

[0208] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by the present application.

Claims

1. A method for preparing extracellular vesicles of lactobacillus, the preparation method comprising: The lactobacillus is first cultured to obtain a primary lactobacillus culture solution; The stress agent and the primary lactobacillus culture solution are mixed to obtain a mixed culture solution; wherein the stress agent comprises vegetable oil and / or vegetable oil nanoemulsion; the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2≥0.01:100; The mixed culture solution is subjected to a second incubation to obtain a culture solution containing extracellular vesicles of lactobacillus; as well as The culture solution containing the lactobacillus extracellular vesicles is purified to obtain a lactobacillus extracellular vesicle solution.

2. The preparation method according to claim 1, wherein when the stress agent comprises vegetable oil, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.01-0.05):100; and / or In the case where the stress agent includes plant oil nanoemulsion, the volume V1 of the stress agent and the volume V2 of the primary lactobacillus culture solution satisfy the relationship: V1:V2=(0.05-2.00):

100.

3. The preparation method according to claim 1, in the case where the stress agent comprises a vegetable oil nanoemulsion, the stress agent comprises, by mass fraction: The vegetable oil is 2% to 8%, the emulsifier is 10% to 30%, the auxiliary emulsifier is 10% to 30% and the water is 32% to 78%.

4. The preparation method according to claim 3, wherein the emulsifier comprises at least one of the following: polyoxyethylene castor oil, hydrogenated polyoxyethylene castor oil, Tween-80, Span-80, Tween-20 and Span-60; and / or The auxiliary emulsifier includes at least one of the following: polyethylene glycol 400, polyethylene glycol 200, glycerol, isopropanol, propylene glycol and ethanol.

5. The preparation method according to claim 1 or 3, wherein the type of vegetable oil comprises at least one of the following: pomegranate seed oil, olive oil, grape seed oil, linseed oil, linoleic acid and oleic acid.

6. The preparation method according to claim 1, wherein the temperature of the first incubation is 35°C to 39°C, and the time of the first incubation is 6h to 16h; and / or The temperature of the second incubation is 35° C. to 39° C., and the time of the second incubation is 2 h to 24 h.

7. The preparation method according to claim 1, wherein the culture solution containing Lactobacillus extracellular vesicles is purified to obtain a Lactobacillus extracellular vesicle solution, comprising the steps of: The culture fluid containing the extracellular vesicles of lactobacillus is subjected to a first centrifugation to obtain a first centrifugation fluid; Filtering the centrifuge liquid multiple times to obtain a filtrate; The filtrate is subjected to a second centrifugation to obtain a second centrifuge; The second centrifuge liquid is subjected to a third centrifugation to obtain a precipitate; The precipitate is dispersed using PBS buffer to obtain a suspension; as well as The suspension is subjected to gradient centrifugation purification to obtain a lactobacillus extracellular vesicle solution; Wherein, the speed of the first centrifugation is 3000r / min to 5000r / min, and the time of the first centrifugation is 10min to 20min; and / or The speed of the second centrifugation is 3000 r / min to 5000 r / min, and the time of the second centrifugation is 10 min to 20 min; and / or The third centrifugation speed is 40000r / min to 60000r / min, and the third centrifugation time is 1.5h to 2.5h; and / or The rotation speed of the gradient centrifugation purification treatment is 10000 r / min to 12000 r / min, and the time of the gradient centrifugation purification treatment is 1.5 h to 2.0 h.

8. The preparation method according to claim 7, wherein the gradient centrifugation purification treatment comprises a sucrose gradient; the sucrose gradient comprises a first sucrose gradient, a second sucrose gradient and a third sucrose gradient, the first sucrose gradient is 2.4M to 3.6M, the second sucrose gradient is 1.2M to 2.0M, and the third sucrose gradient is 0.4M to 0.8M; or The gradient centrifugation purification treatment includes being performed in the form of an iodixanol gradient; the iodixanol gradient includes a first iodixanol gradient, a second iodixanol gradient and a third iodixanol gradient, the volume V3 of the first iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V3:V4=(35-50):100, the volume V5 of the second iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V5:V4=(22-35):100, and the volume V6 of the third iodixanol gradient and the volume V4 of the suspension satisfy the relationship: V6:V4=(5-18):

100.

9. A lactobacillus extracellular vesicle, wherein the lactobacillus extracellular vesicle is prepared by the preparation method according to any one of claims 1 to 8.

10. A drug delivery vector, comprising the lactobacillus extracellular vesicles according to claim 9, wherein the drug delivery vector is used as a drug delivery vector related to central nervous system diseases.

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