Pterostilbene bionic oral nano delivery system based on lactobacillus helveticus exosome as well as preparation method and application of pterostilbene bionic oral nano delivery system

Abstract

The invention discloses a pterostilbene bionic oral nano delivery system based on a lactobacillus helveticus exosome as well as a preparation method and application of the pterostilbene bionic oral nano delivery system. The preparation method comprises the following steps: extracting the lactobacillus helveticus exosome from a lactobacillus helveticus culture solution by adopting a method of combining continuous differential centrifugation and microporous membrane filtration; and mixing the lactobacillus helveticus exosome with pterostilbene, and carrying out ultrasonic treatment and incubation treatment, so as to prepare the lactobacillus helveticus exosome-pterostilbene bionic oral nano delivery system. According to the invention, the lactobacillus helveticus source exosome with the advantages of high yield and expansibility is successfully extracted for the first time, and the clinical limitations of high cost and low productivity of animal source and plant source exosomes as oral delivery carriers are improved; meanwhile, the prepared lactobacillus helveticus exosome-pterostilbene bionic oral nano-delivery system combines the natural characteristics of the exosome and the high biological safety of the probiotics, and can realize the balance of the oral delivery performance and the biological safety of the nano-carrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-delivery, and in particular relates to a pterostilbene bionic oral nano-delivery system based on Lactobacillus helveticus exosomes, and a preparation method and application thereof. Background Art

[0002] Pterostilbene, a natural phenolic compound, is mainly derived from edible plants such as grapes, blueberries and citrus fruits. It has powerful biological functions such as anti-inflammatory, antioxidant and anti-cancer. Pterostilbene is a dimethyl derivative of resveratrol. Due to the methylated structural characteristics, pterostilbene is more lipophilic, has significantly improved intestinal permeability and liver stability, and therefore has higher bioactivity and better bioavailability (3 to 4 times) than resveratrol. However, free pterostilbene faces multiple biochemical and physiological barriers during oral delivery, including: (1) biochemical barriers formed by digestive enzymes, low pH and pH changes between the gastrointestinal tract; (2) cellular uptake barriers formed by intestinal epithelial cells; and (3) cellular internalization barriers caused by intracellular lysosomal degradation. These biochemical and physiological barriers during oral delivery will cause a large amount of degradation and destruction of free pterostilbene, restricted diffusivity, and reduced effective absorption into the blood through transcellular pathways.

[0003] In order to improve the oral bioavailability of bioactive substances, nano-delivery systems based on nanotechnology have become an emerging field for the delivery of natural active substances. Nano-delivery forms such as nanoliposomes, polymer nanoparticles / micelles, dendrimers and magnetic nanoparticles have been widely developed to improve drug stability and bioavailability. However, these synthetic nanostructures usually need to be obtained through complex chemical syntheses, and their biosafety needs to be evaluated through extensive and long-term studies. In addition, they still have certain limitations in application, such as easy disintegration under acidic conditions in the gastrointestinal environment, easy to produce complex interactions with the biological environment, difficult to cross biological barriers, and easy to cause immune clearance. Therefore, the development of a natural, biocompatible, safe and stable oral delivery system is of great significance for the effective delivery of bioactive substances.

[0004] Exosomes are nanoscale bilayer phospholipid vesicles that are naturally secreted by cells and encapsulate a variety of bioactive molecules (including nucleic acids, proteins and lipids) for intercellular communication and active molecule delivery. The particle size is usually 30-200nm. Due to the natural biological properties derived from cells, exosomes have the important characteristics of high biosafety, high tissue / cell permeability and long blood circulation half-life. Due to their good carrier properties and biosafety, the development and application of exosomes in the field of nucleic acid, protein and small molecule drug delivery have attracted much attention in recent years. The sources of exosomes are mainly divided into three categories: animal-derived (milk-derived and cell-derived), plant-derived (turmeric and lemon, etc.) and bacterial-derived (Gram-negative bacteria and Gram-positive bacteria). At present, both animal-derived and plant-derived exosomes have shown certain potential in oral delivery applications. However, the high cost and low yield of animal-derived and plant-derived exosome extraction limit their clinical availability to a certain extent. As we all know, compared with eukaryotic animals and plants, bacteria, as prokaryotes, have the characteristics of rapid proliferation in their asexual reproduction mode, which makes bacterial exosomes as delivery carriers have great cost-effectiveness and scalability advantages. Previously, research on bacterial exosomes has focused on Gram-negative pathogens (such as Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii). However, as oral delivery carriers, virulence factors (such as LPS and virulence proteins) contained in pathogenic bacteria may cause innate immune responses in organisms, causing host cell damage and inflammatory responses. Therefore, it is of great significance to develop a new type of biomimetic nano oral delivery carrier with high biocompatibility, biosafety, high yield and low cost advantages to improve the oral bioavailability of bioactive substances. Summary of the invention

[0005] The main purpose of the present invention is to provide a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes and its preparation method and application, so as to overcome the shortcomings of the prior art.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0007] The embodiment of the present invention provides a method for preparing a pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes, which comprises:

[0008] The exosomes of Lactobacillus helveticus were extracted from the culture medium of Lactobacillus helveticus by combining continuous differential centrifugation with microporous membrane filtration.

[0009] And, the Lactobacillus helveticus exosomes are mixed with pterostilbene and subjected to ultrasound and incubation treatment to prepare a Lactobacillus helveticus exosome-pterostilbene bionic oral nano delivery system, that is, a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes.

[0010] The embodiment of the present invention also provides a pterostilbene biomimetic oral nano-delivery system based on Lactobacillus helveticus exosomes prepared by the aforementioned preparation method, which comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of the Lactobacillus helveticus exosomes.

[0011] The embodiment of the present invention further provides a pterostilbene oral product, which at least includes the aforementioned pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes.

[0012] The embodiment of the present invention also provides the use of Lactobacillus helveticus exosomes in preparing a pterostilbene biomimetic nano oral delivery system, wherein the pterostilbene biomimetic nano oral delivery system comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of the Lactobacillus helveticus exosomes.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention successfully extracted for the first time a high-yield, scalable exosome derived from Lactobacillus helveticus, improving the clinical limitations of animal-derived and plant-derived exosomes as oral delivery carriers, which face high costs and low productivity;

[0015] (2) The present invention achieves a high loading rate of pterostilbene by Lactobacillus helveticus exosomes through ultrasonic technology, thereby increasing the solubility of hydrophobic pterostilbene in water and thus improving its bioavailability;

[0016] (3) Based on the consistency (homology) between the membrane components of Lactobacillus helveticus exosomes and their parent cells (Lactobacillus helveticus bodies), the extracted Lactobacillus helveticus exosomes can overcome the harsh conditions of gastrointestinal digestion and protect pterostilbene from degradation in the gastric environment; and overcome multiple barriers to oral absorption by virtue of their high transcellular membrane transport efficiency and lysosomal escape ability;

[0017] (4) The Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system prepared by the present invention combines the natural properties of exosomes with the high biosafety of probiotics, and can achieve a balance between the oral delivery performance and biosafety of nano-carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a graph of nanoparticle tracking analysis (NTA) of Lactobacillus helveticus exosomes in Example 1 of the present invention;

[0020] Figure 2 This is a graph of the zeta potential value of Lactobacillus helveticus exosomes in Example 1 of the present invention;

[0021] Figure 3 is a transmission electron microscopy (TEM) image of Lactobacillus helveticus exosomes in Example 1 of the present invention;

[0022] Figure 4 is a scanning electron microscope (SEM) image of Lactobacillus helveticus exosomes in Example 1 of the present invention;

[0023] Figure 5 The Fourier transform infrared (FTIR) structural analysis of Lactobacillus helveticus exosomes (LHexo), pterostilbene (PTS), Lactobacillus helveticus exosomes-pterostilbene (LHexo-PTS) and Lactobacillus helveticus exosomes + pterostilbene mixture (LHexo+PTS) in Example 1 of the present invention;

[0024] Fig. 6A This is a laser confocal co-localization analysis diagram of Lactobacillus helveticus exosomes (LHexo)-Nile Red (Nile-Red) in Example 1 of the present invention, Figure 6B yes Fig. 6A Line profile analysis of red and green fluorescence in colocalization analysis, the scale bar in the figure is 100 nm;

[0025] Fig. 7A is an ultraviolet absorption analysis diagram of Lactobacillus helveticus exosomes (LHexo), Lactobacillus helveticus exosomes-pterostilbene (LHexo-PTS) and pterostilbene PBS solution (PTS / PBS) in Example 1 of the present invention, Figure 7B It is a visualized physical picture of Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) and pterostilbene PBS solution (PTS / PBS) in Example 1 of the present invention;

[0026] Figure 8 is a graph showing the change in average particle size of Lactobacillus helveticus exosomes (LHexo) and Lactobacillus helveticus exosomes-pterostilbene (LHexo-PTS) during storage at 4° C. for 1-7 days in Example 1 of the present invention;

[0027] Fig.9A is a graph showing the particle size change of Lactobacillus helveticus exosomes before (Undigested) and after (Digested) simulated gastrointestinal digestion in Example 1 of the present invention, Fig. 9B TEM and SEM analysis of Lactobacillus helveticus exosomes before (Undigested) and after (Digested) simulated gastrointestinal digestion in Example 1 of the present invention, with the scale bar being 100 nm;

[0028] Figure 10A-10BThey are respectively the Caco-2 cell uptake of Lactobacillus helveticus exosomes before (Undigested) and after (Digested) simulated gastrointestinal digestion and 3D heat map analysis of fluorescence intensity in Example 1 of the present invention, and the scale in the figure is 10 μm;

[0029] Figure 11A-11B They are the pterostilbene retention rate diagrams of free pterostilbene (PTS) and Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) digested in simulated gastric and intestinal environments in Example 1 of the present invention, respectively;

[0030] Fig. 12A This is a graph showing the cytotoxicity of Lactobacillus helveticus exosomes (LHexo) in Example 1 of the present invention. Fig. 12B This is a diagram of cytotoxicity analysis of free pterostilbene (PTS) and Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) in Example 1 of the present invention;

[0031] Figures 13A-13D It is a 3D heat map of the uptake of Lactobacillus helveticus exosomes of different concentrations (25 μg / mL-100 μg / mL) by Caco-2 cells at different uptake times (2h-8h) in Example 1 of the present invention, wherein the blue fluorescence is the cell nucleus labeled with DAPI, and the green fluorescence is the Lactobacillus helveticus exosomes labeled with DID, and the scale in the figure is 10 μm;

[0032] Fig.14 It is a laser confocal co-localization analysis and Pearson correlation coefficient (Rr) analysis diagram of Lactobacillus helveticus exosomes (LHexo) and cell organelles Golgi (Golgi), endoplasmic reticulum (ER) and lysosome (Lysosome) in Example 1 of the present invention, wherein Golgi, ER and Lysosome are specifically labeled with red fluorescence, LHexo is labeled with green fluorescence, and the scale in the figure is 10 μm;

[0033] Fig.15A is the pterostilbene standard curve in Examples 1-4 of the present invention, Fig. 15B is a graph of the loading rate and drug loading of pterostilbene in Lactobacillus helveticus exosomes at different Lactobacillus helveticus exosome: pterostilbene ratios (1:2, 1:5, 1:10, 1:20) in Examples 1-4 of the present invention, Fig. 15C The particle size changes of the Lactobacillus helveticus exosome-pterostilbene system at different ratios of Lactobacillus helveticus exosomes:pterostilbene (1:2, 1:5, 1:10, 1:20) in Examples 1-4 of the present invention;

[0034] Fig.16 These are TEM and SEM images of the Lactobacillus helveticus exosome-pterostilbene system at different ratios of Lactobacillus helveticus exosomes:pterostilbene in Examples 1-4, with the scale bar being 100 nm. DETAILED DESCRIPTION

[0035] In view of the defects of the prior art, the inventors of this case have proposed the technical solution of the present invention after long-term research and extensive practice. In the present invention, the probiotic Lactobacillus helveticus is used as the exosome source. By combining continuous differential centrifugation and microporous membrane filtration, exosomes are successfully extracted from Lactobacillus helveticus in high yield for the first time. Ultrasonic technology is used to open the gaps in the exosome phospholipid bilayer through ultrasonic energy, and the small molecule biological compound pterostilbene is loaded into the exosome cavity to prepare a new bionic oral nano-delivery system with high yield, high biosafety, high drug loading and the ability to overcome multiple barriers of oral delivery. The prepared new bionic oral nanodelivery system (Lactobacillus helveticus exosomes-pterostilbene) utilizes the scalability advantages brought by the high biosafety and rapid proliferation characteristics of probiotics to solve the clinical limitations of high cost and low productivity faced by animal-derived and plant-derived exosomes as oral delivery carriers; efficient loading of pterostilbene is achieved through ultrasonic technology, improving the solubility of hydrophobic pterostilbene in water, thereby increasing its bioavailability; in addition, due to the characteristics of the membrane structure, Lactobacillus helveticus exosomes can overcome the harsh conditions of gastrointestinal digestion, protect pterostilbene from degradation in the gastric environment, and overcome multiple barriers to oral absorption with their high transcellular membrane transport efficiency and lysosomal escape ability; and a balance between the oral delivery performance and biosafety of the nanocarrier is achieved.

[0036] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Specifically, as one aspect of the technical solution of the present invention, a method for preparing a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes comprises:

[0038] The exosomes of Lactobacillus helveticus were extracted from the culture medium of Lactobacillus helveticus by combining continuous differential centrifugation with microporous membrane filtration.

[0039] And, the Lactobacillus helveticus exosomes are mixed with pterostilbene and subjected to ultrasound and incubation treatment to prepare a Lactobacillus helveticus exosome-pterostilbene bionic oral nano delivery system, that is, a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes.

[0040] The present invention extracts exosomes from the culture supernatant of Lactobacillus helveticus in high yield by combining continuous differential centrifugation and microporous membrane filtration; uses ultrasonic energy to open the gaps between the exosome phospholipid bilayers, and loads pterostilbene into the exosome cavity to obtain a pterostilbene biomimetic oral nano-delivery system based on Lactobacillus helveticus exosomes (Lactobacillus helveticus exosome-pterostilbene).

[0041] Lactobacillus helveticus in the present invention is a kind of probiotic bacteria with recognized safety, belongs to Gram-positive rod-shaped lactic acid bacteria, is generally present in specific food habitats, and is widely used in making Italian and Swiss cheese and the fermentation agent produced by lactic acid biotechnology. Importantly, compared with other probiotics such as bifidobacteria and lactobacillus, Lactobacillus helveticus can adapt to the dairy fermentation environment faster, has excellent acid resistance and temperature resistance, shows adaptability to the intestinal environment, and is a kind of efficient homologous fermentation lactic acidifier). From the perspective of membrane component characteristics, cyclopropane fatty acids (CFAs) in the membrane structure of Lactobacillus helveticus help to stabilize membrane lipids, prevent membrane degradation and turnover, and can serve as a protective barrier against environmental pressure. In addition, CFAs can reduce the permeability of protons (H+) outside the membrane, constitute a permeability barrier of protons or other chemicals, which promotes the formation of excellent acid resistance of Lactobacillus helveticus. The release mechanism of exosomes causes it to have consistency (homology) with the membrane components of parent cells (Lactobacillus helveticus). Importantly, this acid-resistant membrane structure is the key to the ability of exosomes to resist the gastrointestinal digestive environment and be effectively delivered orally. Therefore, exosomes derived from Lactobacillus helveticus have important development and application potential as a new type of biomimetic nano oral delivery carrier.

[0042] In some preferred embodiments, the preparation method specifically comprises:

[0043] Subculturing Lactobacillus helveticus for 12-48 hours to obtain a Lactobacillus helveticus suspension;

[0044] Furthermore, the Lactobacillus helveticus suspension is mixed with a PBS buffer and subjected to continuous low-speed centrifugation, and then the obtained supernatant is filtered using an aqueous microporous filter membrane, and then the obtained filtrate is subjected to ultrahigh-speed centrifugation to obtain Lactobacillus helveticus exosomes.

[0045] Furthermore, the volume ratio of the Lactobacillus helveticus suspension to the PBS buffer is 2:1-1:2.

[0046] Furthermore, the continuous low-speed centrifugation treatment is carried out using centrifugal forces of 1000-2000×g and 8000-12000×g in sequence.

[0047] Furthermore, the centrifugation time at a centrifugal force of 1000-2000×g and 8000-12000×g is independently 10-45 min.

[0048] Furthermore, the pore sizes used in the water system microporous filter membrane include 0.45 μm and 0.22 μm.

[0049] Furthermore, the ultra-high speed centrifugation treatment is performed using a centrifugal force of 100,000-150,000×g.

[0050] Furthermore, the centrifugation time of the ultra-high speed centrifugation treatment is 1-3 hours.

[0051] In some preferred embodiments, the preparation method specifically comprises:

[0052] Dissolving pterostilbene in ethanol and mixing the mixture with a PBS suspension of Lactobacillus helveticus exosomes to form a mixed system of Lactobacillus helveticus exosomes and pterostilbene;

[0053] First, the mixed system is subjected to ultrasonic treatment in an ice bath, and then incubated in an ice bath for 2 minutes, and then the ultrasonic treatment and incubation are repeated;

[0054] Furthermore, the obtained product is first incubated at 35-45°C for 2 hours, then allowed to stand at 4-10°C for 24 hours, and then subjected to ultra-high speed centrifugation to obtain a Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system.

[0055] Furthermore, the mass ratio of Lactobacillus helveticus exosomes to pterostilbene in the mixed system is 1:2-1:20.

[0056] Furthermore, the ultrasonic treatment uses an ultrasonic power of 100-600W.

[0057] Furthermore, the ultrasonic treatment time is 2-10 min.

[0058] Furthermore, the number of times of repeating the ultrasonic treatment and static incubation is 1-5 times.

[0059] Furthermore, the ultra-high-speed centrifugation treatment uses a centrifugal force of 100,000-150,000×g and a centrifugal time of 1-3 hours.

[0060] In some more specific embodiments, the method for preparing the Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano delivery system comprises the following steps:

[0061] (1) Extraction of Lactobacillus helveticus exosomes: Lactobacillus helveticus was placed in a constant temperature incubator for subculture until the predetermined time, the bacterial suspension was diluted with PBS buffer, and then centrifuged continuously at low speed to collect the supernatant. The supernatant was filtered through 0.45 μm and 0.22 μm water-based microporous filter membranes, and the filtrate was collected and subjected to ultra-high-speed centrifugation. The exosomes of Lactobacillus helveticus accumulated at the bottom of the centrifuge tube in the form of a precipitate, which was resuspended and collected with PBS and stored at -80°C.

[0062] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene system: Pterostilbene was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosomes. Using ultrasonic technology, the Lactobacillus helveticus exosome and pterostilbene mixed system was sonicated with an ultrasonic probe under ice bath conditions, incubated in an ice bath for 2 minutes, and the above steps were repeated. After ultrasonic treatment, the initially obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 hours and placed at 4°C overnight. Finally, the Lactobacillus helveticus exosome-pterostilbene was obtained by ultrahigh-speed centrifugation and PBS resuspended precipitation to obtain Lactobacillus helveticus exosome-pterostilbene.

[0063] Preferably, in step (1), the Lactobacillus helveticus is cultured for 12-48 hours.

[0064] Preferably, in step (1), the dilution ratio of the bacterial suspension to PBS is 2:1-1:2 (v / v).

[0065] Preferably, in step (1), the continuous low-speed centrifugal forces are 1000-2000×g and 8000-12000×g respectively.

[0066] Preferably, in step (1), the continuous low-speed centrifugation time is 10-45 min.

[0067] Preferably, in step (1), the ultra-high-speed centrifugal force is 100,000×g-150,000×g, and the centrifugation time is 1-3 h.

[0068] Preferably, in step (1), the ultra-high speed centrifugation time is 1-3 h.

[0069] Preferably, in step (2), in the mixed system of Lactobacillus helveticus exosome suspension and pterostilbene alcohol solution, the ratio of Lactobacillus helveticus exosome to pterostilbene is 1:2, 1:5, 1:10, 1:20 (w / w).

[0070] Preferably, in step (2), the ultrasonic power is 100-600W.

[0071] Preferably, in step (2), the ultrasonic time is 2-10 min.

[0072] Preferably, in step (2), the number of ultrasonic cycles is 1-5 times.

[0073] Preferably, in step (2), the ultra-high-speed centrifugal force is 100,000×g-150,000×g, and the centrifugation time is 1-3 h.

[0074] Another aspect of an embodiment of the present invention further provides a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes prepared by the aforementioned preparation method, wherein the pterostilbene bionic nano oral delivery system comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of Lactobacillus helveticus exosomes.

[0075] Furthermore, the loading rate of pterostilbene in the pterostilbene bionic nano oral delivery system is 41-67%.

[0076] The pterostilbene biomimetic oral nano-delivery system based on Lactobacillus helveticus exosomes prepared by the present invention utilizes the scalability advantages brought by the high biosafety and rapid proliferation characteristics of probiotics to improve the clinical limitations of high cost and low productivity faced by animal-derived and plant-derived exosomes as oral delivery carriers; the efficient loading of pterostilbene by Lactobacillus helveticus exosomes is achieved through ultrasonic technology, and the solubility of hydrophobic pterostilbene in water is improved, thereby increasing its bioavailability; due to the characteristics of the membrane structure, the Lactobacillus helveticus exosomes have excellent gastrointestinal resistance, protect pterostilbene from degradation in the gastrointestinal digestive environment, and overcome the multiple barriers to oral absorption by virtue of their high transcellular membrane transport efficiency and lysosomal escape ability; a balance between high yield, biosafety and excellent oral delivery performance of nanocarriers is achieved.

[0077] Another aspect of the embodiments of the present invention further provides a pterostilbene oral product, which at least includes the aforementioned pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes.

[0078] Another aspect of an embodiment of the present invention further provides the use of Lactobacillus helveticus exosomes in preparing a pterostilbene biomimetic nano oral delivery system, wherein the pterostilbene biomimetic nano oral delivery system comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of the Lactobacillus helveticus exosomes.

[0079] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0080] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0081] Example 1

[0082] (1) Extraction of Lactobacillus helveticus exosomes: Lactobacillus helveticus was placed in a constant temperature incubator for subculture for 24 h. When the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then centrifuged continuously at 1800×g and 10000×g for 15 min, respectively, and the supernatant was collected. The supernatant was filtered through 0.45μm and 0.22μm water-based microporous filter membranes, and the filtrate was collected and ultracentrifuged at 150000×g for 3 h at 4°C. The Lactobacillus helveticus exosomes accumulated at the bottom of the centrifuge tube in the form of a precipitate, which was resuspended and collected with PBS and stored at -80°C.

[0083] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system: Pterostilbene was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosomes, wherein the ratio of Lactobacillus helveticus exosomes to pterostilbene was 1:10 (w / w). Using ultrasound technology, the Lactobacillus helveticus exosome-pterostilbene mixed system was treated with an ultrasonic probe at 200W for 4 minutes (on 5s / off 5s) under ice bath conditions, and then incubated in an ice bath for 2 minutes. The above steps were repeated 3 times. After ultrasound treatment, the initially obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 hours and at 4°C overnight. Finally, ultracentrifugation was performed at a centrifugal force of 150,000×g and 4°C for 3 hours, and the precipitate was resuspended in PBS to obtain the Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system.

[0084] The exosomes of Lactobacillus helveticus extracted in step (1) of Example 1 were characterized and analyzed, specifically including:

[0085] like Figure 1 As shown, nanoparticle tracking analysis (NTA) showed that the particle size distribution of the extracted Lactobacillus helveticus exosomes was uniform, and the particle size was concentrated at 93 nm.

[0086] like Figure 2 The Zeta potential value analysis of Lactobacillus helveticus exosomes showed that the Zeta potential value was -10.12mV, which is within the general Zeta potential value range of exosomes. This is mainly due to the presence of lipids and proteins, and the large number of anionic sites on the surface of exosomes.

[0087] like Figure 3 The transmission electron microscopy (TEM) results showed that the extracted Lactobacillus helveticus exosomes had the typical structural characteristics of exosomes, with a teacup-shaped morphology and a particle size of about 100nm, which was consistent with the NTA analysis results. The enlarged image showed the typical phospholipid bilayer structure of exosomes, and the phospholipid layer had a high thickness, which may provide a good physical barrier for the system to protect the bioactive ingredients from degradation in the complex gastrointestinal environment.

[0088] like Figure 4 The scanning electron microscopy (SEM) results showed that the extracted Lactobacillus helveticus exosomes had a smooth spherical structure, uniform particle size distribution, and uniform morphology.

[0089] The biomimetic oral nano-delivery system prepared in step (2) of Example 1 was characterized, gastrointestinal resistance, biosafety, cellular uptake efficiency and cellular internalization pathway were analyzed, specifically including:

[0090] like Figure 5 The Fourier transform infrared spectroscopy (FTIR) structure analysis showed that the characteristic absorption peaks of pterostilbene are located at 1600, 1586, 1515, 1458 and 1353 cm- 1 The peaks at the left and right sides of the Lactobacillus helveticus exosomes are the stretching vibrations of aromatic -C=C-, olefin -CC-, aromatic ring -C=C-, -CO- and trans-olefin -C=C-. The intensity of the characteristic peak of pterostilbene in the physical mixture of Lactobacillus helveticus exosomes and pterostilbene is the same or even higher than that of free pterostilbene, while the intensity of the characteristic peaks of pterostilbene encapsulated in Lactobacillus helveticus exosomes is significantly decreased. This shows that pterostilbene is well encapsulated in Lactobacillus helveticus.

[0091] like Fig. 6A 2D and 3D image analysis of confocal laser scanning microscopy (CLSM) showed that the red dye Nile Red (red fluorescence signal), which is a hydrophobic pterostilbene drug model, has a good co-localization with the Lactobacillus helveticus exosomes (green fluorescence signal) labeled with DID dye, and the red fluorescence signal is completely encapsulated in the Lactobacillus helveticus exosomes. Figure 6B The red and green fluorescence signal line profile analysis further demonstrated the co-localization of hydrophobic fluorescent substances with Lactobacillus helveticus exosomes, which proved the feasibility of loading pterostilbene into Lactobacillus helveticus exosomes by ultrasound technology to prepare a new biomimetic nano oral delivery system. In addition, the surface protein of Lactobacillus helveticus exosomes is located at 1638cm- 1 The characteristic peak of the amide I band at and the characteristic peaks of the chemical structure of pterostilbene did not shift significantly, which indicates that no hydrogen bonding or other non-covalent bonding occurred during the loading of pterostilbene by Lactobacillus helveticus exosomes. The self-assembly of Lactobacillus helveticus exosomes and pterostilbene was driven by the hydrophobic interaction between the two. Under the action of ultrasound, pterostilbene was loaded into the phospholipid bilayer of the exosomes.

[0092] like Fig. 7A The UV analysis results showed that at the same concentration, the absorption peak intensity of pterostilbene in Lactobacillus helveticus exosomes-pterostilbene was significantly higher than that in pterostilbene PBS solution, which indicated that the solubility of free pterostilbene in water was significantly improved by encapsulation of Lactobacillus helveticus exosomes. Figure 7BThe visualization results also prove this point.

[0093] like Figure 8 The results of stability analysis showed that the particle size of Lactobacillus helveticus exosomes and Lactobacillus helveticus exosomes-pterostilbene did not change significantly during the storage time of 1-7 days, which indicates that Lactobacillus helveticus exosomes and Lactobacillus helveticus exosomes-pterostilbene system have good stability.

[0094] like Figure 9A-9B The particle size analysis, TEM and SEM results showed that the particle size, surface morphology and phospholipid bilayer structure of Lactobacillus helveticus exosomes after simulated gastrointestinal digestion were stable compared with undigested Lactobacillus helveticus exosomes, and were not damaged by the gastrointestinal environment. Lactobacillus helveticus exosomes have good resistance to the gastric environment.

[0095] like Figure 10A-10B The 3D heat map analysis of cellular uptake and fluorescence intensity showed that the efficiency of Lactobacillus helveticus exosomes taken up by intestinal epithelial Caco-2 cells was consistent before and after simulated gastrointestinal digestion, indicating that the transcellular transport efficiency of Lactobacillus helveticus exosomes was not affected by gastrointestinal digestion.

[0096] like Fig.11A and Fig. 11B Compared with free pterostilbene, the retention rate of pterostilbene encapsulated in Lactobacillus helveticus exosomes was significantly improved after digestion in simulated gastric and intestinal environments, indicating that Lactobacillus helveticus exosomes effectively protected pterostilbene from degradation during gastrointestinal digestion.

[0097] Combined with the analysis results of Figures 9 to 11, it is demonstrated that Lactobacillus helveticus exosomes have excellent resistance to digestive conditions and effective gastrointestinal degradation protection against pterostilbene.

[0098] like Fig. 12A The results of the cell activity experiment showed that Lactobacillus helveticus exosomes showed a proliferation-promoting effect on Caco-2 cells in the concentration range of 10ug / ml-100ug / ml, and the proliferation-promoting effect reached the highest (131%) at a concentration of 50ug / ml, indicating that Lactobacillus helveticus exosomes have excellent biosafety. Fig. 12B The results showed that when the free pterostilbene treatment concentration reached 40μM or 50μM, the cell activity decreased significantly (P<0.01). At the same pterostilbene concentration, the Lactobacillus helveticus exosome-pterostilbene delivery system significantly increased the cell activity (P<0.01), indicating that Lactobacillus helveticus exosomes can effectively reduce the cytotoxic effect of the drug pterostilbene.

[0099] like Figures 13A-13DThe 3D heat map of cellular uptake and its fluorescence intensity showed that with the increase of exosome concentration and the co-culture time of exosomes with Caco-2 cells, the green fluorescence distribution of exosomes labeled with DID became larger and the fluorescence intensity became higher, indicating that the uptake of Lactobacillus helveticus exosomes by intestinal epithelial cells showed obvious concentration and time dependence and had excellent uptake efficiency.

[0100] like Fig.14 The cellular internalization pathway of Lactobacillus helveticus exosomes was analyzed. The three organelles, Golgi apparatus, endoplasmic reticulum and lysosome, were specifically labeled with red fluorescence, and the co-localization of these organelles with Lactobacillus helveticus exosomes (green) was observed (the overlapping part is the yellow area). The overlap coefficient (r value) was calculated to accurately understand the degree of co-localization between Lactobacillus helveticus exosomes and organelles. The results show that the co-localization coefficient of Lactobacillus helveticus exosomes with lysosomes is the lowest (Rr==0.38), and the co-localization coefficient with Golgi apparatus (Rr=0.83) and endoplasmic reticulum (Rr=0.76) is higher. This shows that after Lactobacillus helveticus exosomes enter the cell, they are directly transported to the endoplasmic reticulum or Golgi apparatus without passing through the lysosome, achieving lysosomal escape and avoiding intracellular hydrolytic enzyme degradation. The transport pathway of Lactobacillus helveticus exosomes through the Golgi apparatus / endoplasmic reticulum is more conducive to its intracellular transport as a nanocarrier, effectively improving the intracellular effective drug concentration and absorption utilization rate.

[0101] Example 2

[0102] (1) Extraction of Lactobacillus helveticus exosomes: Lactobacillus helveticus was placed in a constant temperature incubator for subculture for 24 h. When the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then centrifuged continuously at 1800×g and 10000×g for 15 min, respectively, and the supernatant was collected. The supernatant was filtered through 0.45μm and 0.22μmm water-based microporous filter membranes, and the filtrate was collected and ultracentrifuged at 150000×g for 3 h at 4°C. The Lactobacillus helveticus exosomes accumulated at the bottom of the centrifuge tube in the form of a precipitate, which was resuspended and collected with PBS and stored at -80°C.

[0103] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system: Pterostilbene was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosomes, wherein the ratio of Lactobacillus helveticus exosomes to pterostilbene was 1:2 (w / w). Using ultrasound technology, the Lactobacillus helveticus exosome and pterostilbene mixed system was treated with an ultrasonic probe at 200W for 4 minutes (on 5s / off 5s) under ice bath conditions, and then incubated on ice for 2 minutes. The above steps were repeated 3 times. After ultrasound treatment, the initially obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 hours and at 4°C overnight. Finally, by ultracentrifugation at a centrifugal force of 150,000×g and 4°C for 3 hours, the precipitate was resuspended in PBS to obtain Lactobacillus helveticus exosome-pterostilbene.

[0104] Example 3

[0105] (1) Extraction of Lactobacillus helveticus exosomes: Lactobacillus helveticus was placed in a constant temperature incubator for subculture for 24 h. When the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then centrifuged continuously at 1800×g and 10000×g for 15 min, respectively, and the supernatant was collected. The supernatant was filtered through 0.45μm and 0.22μm water-based microporous filter membranes, and the filtrate was collected and ultracentrifuged at 150000×g for 3 h at 4°C. The Lactobacillus helveticus exosomes accumulated at the bottom of the centrifuge tube in the form of a precipitate, which was resuspended and collected with PBS and stored at -80°C.

[0106] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system: Pterostilbene was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosomes, wherein the ratio of Lactobacillus helveticus exosomes to pterostilbene was 1:5 (w / w). Using ultrasonic technology, the Lactobacillus helveticus exosome and pterostilbene mixed system was treated with an ultrasonic probe at 200W for 4 minutes (on 5s / off 5s) under ice bath conditions, and incubated on ice for 2 minutes. The above steps were repeated 3 times. After ultrasonic treatment, the initially obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 hours and at 4°C overnight. Finally, by ultra-high-speed centrifugation at a centrifugal force of 150,000×g and 4°C for 3 hours, the precipitate was resuspended in PBS to obtain Lactobacillus helveticus exosome-pterostilbene.

[0107] Example 4

[0108] (1) Extraction of Lactobacillus helveticus exosomes: Lactobacillus helveticus was placed in a constant temperature incubator for subculture for 24 h. When the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then centrifuged continuously at 1800×g and 10000×g for 15 min, respectively, and the supernatant was collected. The supernatant was filtered through 0.45μm and 0.22μm water-based microporous filter membranes, and the filtrate was collected and ultracentrifuged at 150000×g for 3 h at 4°C. The Lactobacillus helveticus exosomes accumulated at the bottom of the centrifuge tube in the form of a precipitate, which was resuspended and collected with PBS and stored at -80°C.

[0109] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system: Pterostilbene was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosomes, wherein the ratio of Lactobacillus helveticus exosomes to pterostilbene was 1:20 (w / w). Using ultrasound technology, the Lactobacillus helveticus exosome and pterostilbene mixed system was treated with an ultrasonic probe at 200W for 4 minutes (on 5s / off 5s) under ice bath conditions, and incubated on ice for 2 minutes. The above steps were repeated 3 times. After ultrasound treatment, the initially obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 hours and at 4°C overnight. Finally, by ultra-high-speed centrifugation at a centrifugal force of 150,000×g and 4°C for 3 hours, the precipitate was resuspended in PBS to obtain Lactobacillus helveticus exosome-pterostilbene.

[0110] The characterization analysis of Lactobacillus helveticus exosomes-pterostilbene prepared in Examples 1-4 specifically includes:

[0111] Fig.15A It is the pterostilbene standard curve in Examples 1-4 of the present invention.

[0112] like Fig. 15B The encapsulation rate analysis showed that during the ultrasonic loading of pterostilbene, as the ratio of Lactobacillus helveticus to pterostilbene continued to decrease (1:2-1:20), the pterostilbene encapsulation rate showed a downward trend, and the drug loading rate showed a trend of first increasing and then decreasing. The drug loading rate was the highest (55.5%) at 1:10.

[0113] like Fig. 15C The particle size distribution and Fig.16 TEM and SEM analysis showed that the nanoparticle size of Lactobacillus helveticus exosome-pterostilbene system gradually increased with the increase of pterostilbene ratio.

[0114] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0115] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes, characterized in that: include: The exosomes of Lactobacillus helveticus were extracted from the culture medium of Lactobacillus helveticus by combining continuous differential centrifugation with microporous membrane filtration. And, the Lactobacillus helveticus exosomes are mixed with pterostilbene and subjected to ultrasound and incubation treatment to prepare a Lactobacillus helveticus exosome-pterostilbene bionic oral nano delivery system, that is, a pterostilbene bionic oral nano delivery system based on Lactobacillus helveticus exosomes.

2. The preparation method according to claim 1, characterized in that: Specifically include: Subculturing Lactobacillus helveticus for 12-48 hours to obtain a Lactobacillus helveticus suspension; Furthermore, the Lactobacillus helveticus suspension is mixed with a PBS buffer and subjected to continuous low-speed centrifugation, and then the obtained supernatant is filtered using an aqueous microporous filter membrane, and then the obtained filtrate is subjected to ultrahigh-speed centrifugation to obtain Lactobacillus helveticus exosomes.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the Lactobacillus helveticus suspension to the PBS buffer solution is 2:1-1:

2.

4. The preparation method according to claim 2, characterized in that: The continuous low-speed centrifugation treatment is performed using centrifugal forces of 1000-2000×g and 8000-12000×g in sequence; and / or the continuous low-speed centrifugation treatment lasts for 10-45 minutes; And / or, the pore sizes of the water system microporous filter membrane include 0.45 μm and 0.22 μm; And / or, the ultra-high speed centrifugation treatment is performed using a centrifugal force of 100,000-150,000×g; and / or, the ultra-high speed centrifugation treatment is performed for a centrifugal time of 1-3 hours.

5. The preparation method according to claim 1, characterized in that: Specifically include: Dissolving pterostilbene in ethanol and mixing the mixture with a PBS suspension of Lactobacillus helveticus exosomes to form a mixed system of Lactobacillus helveticus exosomes and pterostilbene; First, the mixed system is subjected to ultrasonic treatment in an ice bath, and then incubated in an ice bath for 2-10 minutes, and then the ultrasonic treatment and incubation are repeated; Furthermore, the obtained product is first incubated at 35-45° C. for 2 hours, then allowed to stand at 4-10° C. for 24 hours, and then subjected to ultra-high-speed centrifugation to obtain a Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano-delivery system.

6. The preparation method according to claim 5, characterized in that: The mass ratio of Lactobacillus helveticus exosomes to pterostilbene in the mixed system is 1:2-1:

20.

7. The preparation method according to claim 5, characterized in that: The ultrasonic power used in the ultrasonic treatment is 100-600W; and / or, the ultrasonic treatment time is 2-10min; And / or, the number of times of repeating the ultrasonic treatment and static incubation is 1-5 times; And / or, the ultra-high speed centrifugation treatment uses a centrifugal force of 100,000-150,000×g and a centrifugal time of 1-3 h.

8. The pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The pterostilbene bionic nano oral delivery system comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of the Lactobacillus helveticus exosomes; preferably, the loading rate of pterostilbene in the pterostilbene bionic nano oral delivery system is 41-67%.

9. An oral product of pterostilbene, characterized in that: At least comprising the pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes as described in claim 8.

10. Use of Lactobacillus helveticus-derived exosomes in the preparation of a pterostilbene biomimetic nano oral delivery system, characterized in that: The pterostilbene bionic nano oral delivery system comprises Lactobacillus helveticus exosomes and pterostilbene loaded in the cavity of the Lactobacillus helveticus exosomes.

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