Biomimetic oral nano-delivery system of pterostilbene based on lactobacillus helveticus exosomes and its preparation and application

Abstract

The application discloses a bifendate biomimetic oral nanodelivery system based on lactobacillus helveticus exosomes and a preparation method and application thereof. The preparation method comprises the following steps: extracting lactobacillus helveticus exosomes from a lactobacillus helveticus culture solution by adopting a method combining continuous differential centrifugation and microporous membrane filtration; and mixing the lactobacillus helveticus exosomes with bifendate, and performing ultrasonic treatment and incubation treatment to prepare the lactobacillus helveticus exosome-bifendate biomimetic oral nanodelivery system. The application successfully extracts a lactobacillus helveticus source exosome with high yield and expandable advantages for the first time, and improves the clinical limitations of high cost and low productivity of animal source and plant source exosomes as oral delivery carriers; meanwhile, the prepared lactobacillus helveticus exosome-bifendate biomimetic oral nanodelivery system combines the natural characteristics of exosomes and the high biological safety of probiotics, and can realize the balance between the oral delivery performance and the biological safety of the nanocarrier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanodelivery technology, and particularly relates to a pterostilbene biomimetic oral nanodelivery system based on lactobacillus helveticus exosomes as well as a preparation method and application thereof. BACKGROUND

[0002] Pterostilbene, a natural phenolic compound, is mainly derived from edible plants such as grapes, blueberries and citrus fruits, and has powerful biological functions such as anti-inflammatory, antioxidant and anticancer. Pterostilbene is a dimethyl derivative of resveratrol. Due to the methylation structure, pterostilbene has stronger lipophilicity, significantly improved intestinal permeability and liver stability, and therefore has higher biological activity and better bioavailability (3 to 4 times) than resveratrol. However, free pterostilbene will face multiple biochemical and physiological barriers during oral delivery, including: (1) biochemical barriers composed of digestive enzymes, low pH and pH changes between the stomach and the intestines; (2) cell uptake barriers composed of intestinal epithelial cells; (3) and intracellular degradation effects caused by intracellular lysosomes. These biochemical and physiological barriers during oral delivery will cause a large amount of degradation and destruction of free pterostilbene, limited diffusion, and reduced effective absorption into the blood through the transcellular pathway.

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

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

[0005] The main purpose of the present application is to provide a Swiss lactobacillus exosome-based pterostilbene biomimetic oral nano delivery system and its preparation method and application, so as to overcome the shortcomings of the prior art.

[0006] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application comprises:

[0007] The present application provides a preparation method of a Swiss lactobacillus exosome-based pterostilbene biomimetic oral nano delivery system, which comprises the following steps:

[0008] The Swiss lactobacillus exosomes are extracted from the culture solution of Swiss lactobacillus by using a method combining continuous differential centrifugation and microporous membrane filtration;

[0009] And the Swiss lactobacillus exosomes are mixed with pterostilbene and subjected to ultrasonic treatment and incubation treatment to prepare Swiss lactobacillus exosome-pterostilbene biomimetic oral nano delivery system, i.e. Swiss lactobacillus exosome-based pterostilbene biomimetic oral nano delivery system.

[0010] The application also provides the Swiss lactobacillus-derived exosome-based pterostilbene biomimetic oral nanodelivery system prepared by the preparation method.

[0011] The application also provides a pterostilbene oral product, which at least comprises the Swiss lactobacillus-derived exosome-based pterostilbene biomimetic oral nanodelivery system.

[0012] The application also provides the use of the Swiss lactobacillus-derived exosome in the preparation of a pterostilbene biomimetic nanodelivery system, which comprises the Swiss lactobacillus-derived exosome and pterostilbene loaded in the cavity of the Swiss lactobacillus-derived exosome.

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

[0014] (1) The application successfully extracts a Swiss lactobacillus-derived exosome with high yield and expandable advantage for the first time, which improves the clinical limitations of high cost and low productivity of animal-derived and plant-derived exosomes as oral delivery carriers.

[0015] (2) The application realizes high loading rate of the Swiss lactobacillus-derived exosome to pterostilbene through ultrasonic technology, improves the solubility of the hydrophobic pterostilbene in water, and thus improves the bioavailability thereof.

[0016] (3) Based on the consistency (homology) between the Swiss lactobacillus-derived exosome and the membrane components of its parent cell (Swiss lactobacillus), the extracted Swiss lactobacillus-derived exosome can overcome the harsh conditions of gastrointestinal digestion, protect pterostilbene from degradation in the stomach environment, and overcome multiple barriers of oral absorption due to its high transmembrane transport efficiency and lysosome escape ability.

[0017] (4) The Swiss lactobacillus-derived exosome-pterostilbene biomimetic oral nanodelivery system prepared by the application combines the natural properties of exosomes and the high biological safety of probiotics, and can balance the oral delivery performance and biological safety of the nanocarrier. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without any creative effort.

[0019] Figure 1 is a tracking analysis diagram (NTA) of the Swiss lactobacillus-derived exosome nanoparticles in Example 1 of the application;

[0020] Figure 2 is the zeta potential value graph of Lactobacillus helveticus exosome in Example 1 of the present application;

[0021] Figure 3 is the transmission electron microscopy (TEM) graph of Lactobacillus helveticus exosome in Example 1 of the present application;

[0022] Figure 4 is the scanning electron microscopy (SEM) graph of Lactobacillus helveticus exosome in Example 1 of the present application;

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

[0024] Figure 6A is the laser confocal co-localization analysis graph of Lactobacillus helveticus exosome (LHexo)-Nile-Red in Example 1 of the present application, Figure 6B is Figure 6A is the line profile analysis graph of red-green fluorescence in co-localization analysis, in which the scale is 100 nm;

[0025] Figure 7A is the ultraviolet absorption analysis graph of Lactobacillus helveticus exosome (LHexo), Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) and pterostilbene PBS solution (PTS / PBS) in Example 1 of the present application, Figure 7B is the visualized physical graph of Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) and pterostilbene PBS solution (PTS / PBS) in Example 1 of the present application;

[0026] Figure 8 is the average particle size change graph of Lactobacillus helveticus exosome (LHexo) and Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) during storage at 4℃ for 1-7 days in Example 1 of the present application;

[0027] Figure 9A is the particle size change graph of Lactobacillus helveticus exosome before (Undigested) and after (Digested) simulated gastrointestinal digestion in Example 1 of the present application, Figure 9B is the TEM and SEM analysis of Lactobacillus helveticus exosome before (Undigested) and after (Digested) simulated gastrointestinal digestion in Example 1 of the present application, in which the scale is 100 nm;

[0028] Figures 10A-10Brespectively are the uptake of Caco-2 cells and 3D heat map analysis of fluorescence intensity of Lactobacillus helveticus exosome before (Undigested) and after (Digested) simulated gastrointestinal digestion in Example 1 of the present application, the scale in the figure is 10 μm;

[0029] Figures 11A-11B respectively are the retention rate of pterostilbene in simulated gastric environment and intestinal environment in Example 1 of the present application, free pterostilbene (PTS) and Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS);

[0030] Figure 12A is the cytotoxicity analysis chart of Lactobacillus helveticus exosome (LHexo) in Example 1 of the present application, Figure 12B is the cytotoxicity analysis chart of free pterostilbene (PTS) and Lactobacillus helveticus exosome-pterostilbene (LHexo-PTS) in Example 1 of the present application;

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

[0032] Figure 14 is the laser confocal co-localization analysis and Pearson correlation coefficient (Rr) analysis chart of Lactobacillus helveticus exosome (LHexo) and organelles Golgi, endoplasmic reticulum (ER) and lysosome (Lysosome) in Example 1 of the present application, wherein Golgi, ER and Lysosome are specifically labeled as red fluorescence, and LHexo is labeled as green fluorescence, the scale in the figure is 10 μm;

[0033] Figure 15A is the pterostilbene standard curve in Examples 1-4 of the present application, Figure 15B is the loading rate and drug loading of pterostilbene in Lactobacillus helveticus exosome at different Lactobacillus helveticus exosome:pterostilbene ratios (1:2, 1:5, 1:10, 1:20) in Examples 1-4 of the present application, Figure 15C is the particle size change of Lactobacillus helveticus exosome-pterostilbene system at different Lactobacillus helveticus exosome:pterostilbene ratios (1:2, 1:5, 1:10, 1:20) in Examples 1-4 of the present application;

[0034] Figure 16 is the TEM and SEM chart of Lactobacillus helveticus exosome-pterostilbene system at different Lactobacillus helveticus exosome:pterostilbene ratios in Examples 1-4, the scale in the figure is 100 nm. DETAILED DESCRIPTION

[0035] In view of the defects of the prior art, the present inventors have long studied and practiced to propose the technical solutions of the present application. In the present application, the exosomes from Lactobacillus helveticus are used as the source, and the exosomes are first successfully extracted from Lactobacillus helveticus in high yield by a method combining continuous differential centrifugation and microporous membrane filtration. Then, by using ultrasonic technology, the exosome phospholipid bilayer gap is opened by ultrasonic energy, and small-molecule biological compounds pterostilbene are loaded into the cavity of the exosomes to prepare a new type of biomimetic oral nanodelivery system with high yield, high biological safety, high drug loading capacity and the ability to overcome multiple barriers for oral delivery. The prepared new type of biomimetic oral nanodelivery system (Lactobacillus helveticus-derived exosomes-pterostilbene) uses the high biological safety of probiotics and the scalability advantage brought by the rapid proliferation characteristics to solve the clinical limitations of high cost and low productivity of animal-derived and plant-derived exosomes as oral delivery carriers. By using ultrasonic technology, pterostilbene is efficiently loaded, the solubility of the hydrophobic pterostilbene in water is improved, and thus the bioavailability is improved. In addition, due to the membrane structure characteristics, the Lactobacillus helveticus exosomes can overcome the harsh conditions of gastrointestinal digestion, protect pterostilbene from degradation in the stomach environment, and overcome the multiple barriers for oral absorption by virtue of its high transmembrane transport efficiency and lysosome escape ability. The balance between the oral delivery performance and the biological safety of the nanocarrier is achieved.

[0036] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0037] Specifically, as one aspect of the technical solutions of the present application, a preparation method of a pterostilbene biomimetic oral nanodelivery system based on Lactobacillus helveticus exosomes includes:

[0038] Lactobacillus helveticus exosomes are extracted from the culture solution of Lactobacillus helveticus by a method combining continuous differential centrifugation and microporous membrane filtration;

[0039] and the Lactobacillus helveticus exosomes are mixed with pterostilbene and subjected to ultrasonic treatment and incubation to prepare a Lactobacillus helveticus exosome-pterostilbene biomimetic oral nanodelivery system, i.e., a pterostilbene biomimetic oral nanodelivery system based on Lactobacillus helveticus exosomes.

[0040] The application extracts exosomes from L helveticus culture supernatant in high yield by a method combining continuous differential centrifugation and microporous membrane filtration; the ultrasonic energy is used to open the gap between the phospholipid bilayer of the exosomes, and the pterostilbene is loaded into the cavity of the exosomes, so as to obtain a pterostilbene biomimetic oral nano delivery system based on L helveticus exosomes (L helveticus exosomes-pterostilbene).

[0041] The L helveticus in the application is a recognized safe probiotic, which belongs to gram-positive rod-shaped lactic acid bacteria and is widely present in specific food habitats and is widely used for making Italian and Swiss cheese and fermentation agents for lactic acid biological technology production. Importantly, compared with other probiotics such as bifidobacteria and lactobacilli, the L helveticus can adapt to the fermentation environment of dairy products more quickly, has excellent acid and temperature resistance, and shows adaptability to the intestinal environment, and is a high-efficiency homologous fermentation lactic acidifier. From the perspective of membrane component characteristics, the cyclopropane fatty acid (CFA) in the membrane structure of the L helveticus helps to stabilize the membrane lipid, prevent membrane degradation and turnover, and at the same time can act as a protective barrier against environmental pressure. In addition, the CFAs can reduce the permeability of protons (H+) on the outside of the membrane, constituting a permeability barrier for protons or other chemicals, which promotes the formation of excellent acid resistance of the L helveticus. The release mechanism of the exosomes leads to the consistency (homology) between the exosomes and the membrane components of the parent cells (L helveticus). Importantly, this membrane structure component with acid resistance is the key to the ability of the exosomes to resist the digestive environment of the gastrointestinal tract and effectively perform oral delivery. Therefore, the exosomes derived from the L helveticus body 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] The L helveticus is subcultured for 12-48 h to prepare a L helveticus suspension;

[0044] and the L helveticus suspension is mixed with PBS buffer and subjected to continuous low-speed centrifugation, and then the obtained supernatant is filtered by a water-based microporous filter, and then the obtained filtrate is subjected to ultrahigh-speed centrifugation to prepare L helveticus exosomes.

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

[0046] Further, the continuous low-speed centrifugation is performed by using centrifugal forces of 1000-2000xg and 8000-12000xg in sequence.

[0047] Further, the centrifugal force for centrifugation is 1000-2000xg, 8000-12000xg, and the centrifugation time is 10-45min.

[0048] Further, the pore size of the water-based microporous filter membrane includes 0.45μm and 0.22μm.

[0049] Further, the ultracentrifugation is performed at a centrifugal force of 100000-150000xg.

[0050] Further, the centrifugation time for ultracentrifugation is 1-3h.

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

[0052] Dissolve pterostilbene in ethanol, and mix it with the PBS suspension of Lactobacillus helveticus exosomes to form a mixed system of Lactobacillus helveticus exosomes and pterostilbene;

[0053] Ultrasonicate the mixed system under ice bath conditions, then incubate it under ice bath for 2min, and repeat the ultrasonication and incubation.

[0054] Then, incubate the obtained product at 35-45℃ for 2h, and at 4-10℃ for 24h, and then perform ultracentrifugation to obtain the Lactobacillus helveticus exosome-pterostilbene biomimetic oral nanodelivery system.

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

[0056] Further, the ultrasonication is performed at an ultrasonic power of 100-600W.

[0057] Further, the ultrasonication is performed for 2-10min.

[0058] Further, the number of times of repeating the ultrasonication and incubation is 1-5.

[0059] Further, the ultracentrifugation is performed at a centrifugal force of 100000-150000xg and for 1-3h.

[0060] In some more specific embodiments, the preparation method of the Lactobacillus helveticus exosome-pterostilbene biomimetic oral nanodelivery system includes the following steps:

[0061] (1) Extraction of Lactobacillus helveticus exosome: Lactobacillus helveticus was subcultured in a constant temperature incubator for a predetermined time, and then the bacterial suspension was diluted with PBS buffer and subjected to continuous low-speed centrifugation. The supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm water-based microporous filter membranes, respectively, and the filtrate was collected and subjected to ultrahigh-speed centrifugation. Lactobacillus helveticus exosomes were precipitated at the bottom of the centrifuge tube, resuspended with PBS, and stored at -80°C.

[0062] (2) Preparation of Lactobacillus helveticus exosome-piceid system: Piceid was dissolved in an ethanol solution and mixed with the Lactobacillus helveticus exosome PBS suspension. An ultrasonic technique was used to treat the Lactobacillus helveticus exosome and piceid mixture under ice bath conditions using an ultrasonic probe. After 2 min of ice bath incubation, the above steps were repeated. After ultrasonic treatment, the Lactobacillus helveticus exosome-piceid obtained was incubated at 37°C for 2 h and then stored at 4°C overnight. Finally, the Lactobacillus helveticus exosome-piceid was obtained by ultrahigh-speed centrifugation and resuspension of the precipitate with PBS.

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

[0064] Preferably, in step (1), the bacterial suspension is diluted with PBS at a ratio of 2:1-1:2 (v / v).

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

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

[0067] Preferably, in step (1), the ultrahigh-speed centrifugal force is 100000 x g-150000 x g, and the centrifugation time is 1-3 h.

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

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

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

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

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

[0073] Preferably, in step (2), the ultra-high centrifugal force is 100000xg-150000xg, and the centrifugation time is 1-3h.

[0074] Another aspect of the embodiment of the present application also provides the Swiss lactobacillus exosome-based pterostilbene biomimetic oral nanodelivery system prepared by the preparation method.

[0075] Further, the loading rate of pterostilbene in the pterostilbene biomimetic oral nanodelivery system is 41-67%.

[0076] The Swiss lactobacillus exosome-based pterostilbene biomimetic oral nanodelivery system prepared by the present application takes advantage of the high biological safety and scalability of probiotics to improve the clinical limitations of animal-derived and plant-derived exosomes as oral delivery carriers, such as high cost and low productivity; the efficient loading of pterostilbene into Swiss lactobacillus exosomes by ultrasonic technology improves the solubility of hydrophobic pterostilbene in water, thereby improving its bioavailability; due to the membrane structure, Swiss lactobacillus exosomes have excellent gastrointestinal resistance, protecting pterostilbene from degradation in the gastrointestinal environment, and overcoming multiple barriers to oral absorption due to their high transmembrane transport efficiency and lysosome escape ability; the balance of high yield, biological safety, and excellent oral delivery performance of the nanocarrier is achieved.

[0077] Another aspect of the embodiment of the present application also provides a pterostilbene oral product, which at least includes the Swiss lactobacillus exosome-based pterostilbene biomimetic oral nanodelivery system.

[0078] Another aspect of the embodiment of the present application also provides the use of Swiss lactobacillus-derived exosomes in the preparation of a pterostilbene biomimetic oral nanodelivery system, which includes Swiss lactobacillus-derived exosomes and pterostilbene loaded in the cavity of the Swiss lactobacillus-derived exosomes.

[0079] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and the accompanying drawings. The present embodiment is implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0080] In the following examples, the experimental materials used are commercially available from conventional biochemical reagent companies, unless otherwise specified.

[0081] Example 1

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

[0083] (2) Preparation of the *Lactobacillus helveticus* exosome-pterostilbene biomimetic oral nanodelivery system: Pterostilbene was dissolved in an ethanol solution and mixed with *Lactobacillus helveticus* exosome PBS suspension, wherein the ratio of *Lactobacillus helveticus* exosomes to pterostilbene was 1:10 (w / w). Using ultrasound technology, the mixture of *Lactobacillus helveticus* exosomes and pterostilbene was treated with a 200W ultrasound probe for 4 min (5 s on / 5 s off) under ice bath conditions, followed by 2 min of static incubation on ice. This process was repeated three times. After ultrasound treatment, the preliminarily obtained *Lactobacillus helveticus* exosome-pterostilbene was incubated at 37℃ for 2 h and then at 4℃ overnight. Finally, the precipitate was resuspended in PBS at 150,000 × g for 3 h to obtain the *Lactobacillus helveticus* exosome-pterostilbene biomimetic oral nanodelivery 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) revealed that the extracted Lactobacillus helveticus exosomes had a uniform particle size distribution, with the particle size concentrated at 93 nm.

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

[0087] like Figure 3 Transmission electron microscopy (TEM) results showed that the extracted Lactobacillus helveticus exosomes possessed typical exosome structural characteristics, with a teacup-shaped morphology and a particle size of approximately 100 nm, consistent with NTA analysis results. Magnified images revealed the typical phospholipid bilayer structure of the exosomes, with a relatively thick phospholipid layer, which may provide a good physical barrier to protect the bioactive components from degradation by the complex gastrointestinal environment.

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

[0089] The biomimetic oral nanodelivery system prepared in step (2) of Example 1 was characterized, gastrointestinal resistance, biological safety, cell uptake efficiency and intracellular internalization pathway analysis, specifically including:

[0090] As Figure 5 Fourier transform infrared spectroscopy (FTIR) structure analysis showed that the characteristic absorption peaks of pterostilbene were located at 1600, 1586, 1515, 1458 and 1353 cm- 1 , respectively, which were aromatic-C=C-, olefin-C-C-, aromatic ring-C=C-, -C-O- and trans-olefin-C=C- stretching vibration. In the Lactobacillus helveticus exosome-pterostilbene physical mixture group, the intensity of the characteristic peaks of pterostilbene was at the same or even higher level than that of free pterostilbene, while the intensity of each characteristic peak of pterostilbene encapsulated in Lactobacillus helveticus exosomes was significantly decreased. This indicates that pterostilbene is well encapsulated in Lactobacillus helveticus.

[0091] As Figure 6A Laser confocal microscopy (CLSM) 2D and 3D image analysis showed that the red dye Nile Red (red fluorescence signal) as a model of hydrophobic pterostilbene drug had good co-localization with Lactobacillus helveticus exosomes labeled with DID dye (green fluorescence signal), and the red fluorescence signal was completely covered in Lactobacillus helveticus exosomes. Figure 6B The red-green fluorescence signal line profile analysis further proved the co-localization of hydrophobic fluorescent substances and Lactobacillus helveticus exosomes, which proved the feasibility of loading pterostilbene into Lactobacillus helveticus exosomes by ultrasonic technology to prepare a new type of biomimetic nanometer oral delivery system. In addition, the amide I band characteristic peak of Lactobacillus helveticus exosome surface protein at 1638 cm- 1 , and the characteristic peaks of each chemical structure of pterostilbene did not shift significantly, indicating that no hydrogen bond or other non-covalent bond interaction occurred during the loading of Lactobacillus helveticus exosomes with pterostilbene. The self-assembly of Lactobacillus helveticus exosomes and pterostilbene was driven by hydrophobic interaction, and under the action of ultrasonic waves, pterostilbene was loaded into the inner phospholipid bilayer of the exosome.

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

[0093] As Figure 8 The stability analysis results of L. helveticus exosome and L. helveticus exosome-petasin show that the particle size of L. helveticus exosome and L. helveticus exosome-petasin does not change significantly within 1-7 days of storage time, which indicates that L. helveticus exosome and L. helveticus exosome-petasin system has good stability.

[0094] As Figures 9A-9B The particle size analysis, TEM and SEM results show that after simulated gastrointestinal digestion, the particle size, surface morphology and phospholipid bilayer structure of L. helveticus exosome are stable compared with undigested L. helveticus exosome, which is not destroyed by the gastrointestinal environment, and L. helveticus exosome has good gastric environment resistance.

[0095] As Figures 10A-10B The cell uptake and 3D heat map analysis of fluorescence intensity show that the efficiency of L. helveticus exosome uptake by intestinal epithelial Caco-2 cells is consistent before and after simulated gastrointestinal digestion, which indicates that the transcellular transport efficiency of L. helveticus exosome is not affected by gastrointestinal digestion.

[0096] As Figure 11A And Figure 11B Compared with free petasin, the retention rate of petasin encapsulated in L. helveticus exosome significantly increases after the process of simulated gastric and intestinal environment digestion, which indicates that L. helveticus exosome effectively protects petasin from degradation during gastrointestinal digestion.

[0097] Combined with the analysis results of FIGS. 9-11, it is proved that L. helveticus exosome has excellent digestion condition resistance and effective gastrointestinal degradation protection effect on petasin.

[0098] As Figure 12A The cell activity experiment results show that L. helveticus exosome shows a proliferation-promoting effect on Caco-2 cells within the concentration range of 10 ug / ml-100 ug / ml, and the proliferation-promoting effect reaches the highest (131%) at a concentration of 50 ug / ml, which indicates that L. helveticus exosome has excellent biological safety. In addition, as Figure 12B It is shown that when the treatment concentration of free petasin reaches 40 μM or 50 μM, the cell activity decreases significantly (P<0.01). However, under the same petasin concentration, the L. helveticus exosome-petasin delivery system significantly improves the cell activity (P<0.01), which indicates that L. helveticus exosome can effectively reduce the cytotoxicity of the drug petasin.

[0099] As Figures 13A-13DThe 3D heat map of the fluorescence intensity of the cells uptake and its fluorescence intensity showed that the green fluorescence distribution of the exosomes labeled with DID was larger and the fluorescence intensity was higher with the increase of the concentration of the exosomes and the co-culture time of the exosomes and Caco-2 cells, which indicated that the uptake of the L. helveticus exosomes by the intestinal epithelial cells was obviously concentration and time dependent, and had excellent uptake efficiency.

[0100] As Figure 14 The intracellular trafficking pathway analysis of the L. helveticus exosomes showed that the three organelles of Golgi, endoplasmic reticulum and lysosome were specifically labeled as red fluorescence, and the co-localization (the overlapping part was the yellow area) of these organelles and the L. helveticus exosomes (green) was observed. And the degree of co-localization of the L. helveticus exosomes and the organelles was accurately understood by calculating the overlap coefficient (r value). The results showed that the co-localization coefficient of the L. helveticus exosomes and the lysosome was the lowest (Rr==0.38), and the co-localization coefficients of the L. helveticus exosomes and the Golgi (Rr=0.83) and the endoplasmic reticulum (Rr=0.76) were higher. This indicated that the L. helveticus exosomes were directly transported to the endoplasmic reticulum or Golgi after entering the cells, without passing through the lysosome, realizing lysosome escape and avoiding intracellular hydrolytic enzyme degradation. The transport pathway of the L. helveticus exosomes through the Golgi / endoplasmic reticulum was more conducive to its intracellular transport as a nanocarrier, effectively improving the effective drug concentration and absorption utilization rate in cells.

[0101] Example 2

[0102] (1) Extraction of L. helveticus exosomes: L. helveticus was subcultured in a constant temperature incubator for 24 h, and when the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer solution (bacterial suspension: PBS = 1:1, v / v), and then subjected to continuous low-speed centrifugation at 1800xg and 10000xg centrifugal force for 15 min, and the supernatant was collected. The supernatant was filtered through 0.45μm and 0.22μm water-based microporous filter membranes in sequence, and the filtrate was collected, and subjected to ultrahigh-speed centrifugation at 150000xg centrifugal force at 4°C for 3h. The L. helveticus exosomes were accumulated in the form of precipitate at the bottom of the centrifuge tube, resuspended with PBS for collection, and stored at -80°C.

[0103] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nanodelivery system: Pterostilbene was dissolved in ethanol solution and mixed with Lactobacillus helveticus exosome PBS suspension, wherein the ratio of Lactobacillus helveticus exosome and pterostilbene was 1:2 (w / w). Using ultrasonic technology, under ice bath conditions, the Lactobacillus helveticus exosome and pterostilbene mixed system was treated with an ultrasonic probe at 200 W for 4 min (on 5 s / off 5 s), and incubated on ice for 2 min. The above steps were repeated 3 times. After ultrasonic treatment, the preliminarily obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 h and placed at 4°C overnight. Finally, by ultracentrifugation, under the centrifugal force of 150000 x g, the Lactobacillus helveticus exosome-pterostilbene was ultracentrifuged at 4°C for 3 hours, and the precipitate was resuspended with PBS to obtain Lactobacillus helveticus exosome-pterostilbene.

[0104] Example 3

[0105] (1) Extraction of Lactobacillus helveticus exosome: Lactobacillus helveticus was subcultured in a constant temperature incubator for 24 h, and when the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then subjected to continuous low-speed centrifugation at 1800 x g and 10000 x g for 15 min. The supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm water-based microporous filter membranes in sequence, and the filtrate was collected and ultracentrifuged at 150000 x g at 4°C for 3 h. The Lactobacillus helveticus exosome was accumulated in the form of a precipitate at the bottom of the centrifuge tube, resuspended with PBS, and stored at -80°C.

[0106] (2) Preparation of Lactobacillus helveticus exosome-pterostilbene biomimetic oral nanodelivery system: Pterostilbene was dissolved in ethanol solution and mixed with Lactobacillus helveticus exosome PBS suspension, wherein the ratio of Lactobacillus helveticus exosome and pterostilbene was 1:5 (w / w). Using ultrasonic technology, under ice bath conditions, the Lactobacillus helveticus exosome and pterostilbene mixed system was treated with an ultrasonic probe at 200 W for 4 min (on 5 s / off 5 s), and incubated on ice for 2 min. The above steps were repeated 3 times. After ultrasonic treatment, the preliminarily obtained Lactobacillus helveticus exosome-pterostilbene was incubated at 37°C for 2 h and placed at 4°C overnight. Finally, by ultracentrifugation, under the centrifugal force of 150000 x g, the Lactobacillus helveticus exosome-pterostilbene was ultracentrifuged at 4°C for 3 hours, and the precipitate was resuspended with PBS to obtain Lactobacillus helveticus exosome-pterostilbene.

[0107] Example 4

[0108] (1) Extraction of Lactobacillus helveticus exosome: Lactobacillus helveticus was subcultured in a constant temperature incubator for 24 h, and when the culture reached the predetermined time, the bacterial suspension was diluted with PBS buffer (bacterial suspension: PBS = 1:1, v / v), and then subjected to continuous low-speed centrifugation at 1800xg and 10000xg for 15 min, and the supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm water-based microporous filter membranes in sequence, and the filtrate was collected and subjected to ultrahigh-speed centrifugation at 150000xg at 4°C for 3 h. The Lactobacillus helveticus exosome was accumulated in the form of a precipitate at the bottom of the centrifuge tube, was resuspended and collected with PBS, and was stored at -80°C.

[0109] (2) Preparation of Lactobacillus helveticus exosome-piceid biomimetic oral nanodelivery system: Piceid was dissolved in an ethanol solution and mixed with a PBS suspension of Lactobacillus helveticus exosome, wherein the ratio of Lactobacillus helveticus exosome to piceid was 1:20 (w / w). Using ultrasonic technology, the Lactobacillus helveticus exosome and piceid mixture was treated with an ultrasonic probe at 200W for 4 min (on for 5 s / off for 5 s) under ice bath conditions, and was incubated on ice for 2 min. The above steps were repeated 3 times. After ultrasonic treatment, the Lactobacillus helveticus exosome-piceid obtained was incubated at 37°C for 2 h and was stored at 4°C overnight. Finally, the Lactobacillus helveticus exosome-piceid was obtained by ultrahigh-speed centrifugation at 150000xg at 4°C for 3 h, resuspension of the precipitate with PBS, and resuspension of the precipitate with PBS.

[0110] The Lactobacillus helveticus exosome-piceid prepared in Examples 1-4 was subjected to characterization analysis, specifically including:

[0111] Figure 15A is the standard curve of piceid in Examples 1-4 of the present application.

[0112] As Figure 15B the encapsulation efficiency analysis, during the ultrasonic loading of piceid, as the ratio of Lactobacillus helveticus to piceid continuously decreased (1:2-1:20), the piceid encapsulation efficiency showed a continuously decreasing trend, and the drug loading rate showed a trend of first increasing and then decreasing, with the maximum drug loading rate (55.5%) at 1:10.

[0113] As Figure 15C the particle size distribution and Figure 16 the TEM and SEM analysis, the particle size of the Lactobacillus helveticus exosome-piceid system nanoparticles gradually increased with the increase of the piceid ratio.

[0114] In addition, the present inventors also conducted tests with other raw materials, process operations, and process conditions described in the present specification with reference to the foregoing examples, and all obtained relatively ideal results.

[0115] It should be understood that the technical solutions of the present application are not limited to the above specific implementation cases, and any technical modification made according to the technical solutions of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A method for the preparation of a biosynthetic oral nanodelivery system of pterostilbene based on Lactobacillus helveticus exosomes, characterized by, The method comprises the following steps: Extracating Lactobacillus helveticus exosomes from Lactobacillus helveticus culture solution by combining continuous differential centrifugation with microporous membrane filtration; And mixing the Lactobacillus helveticus exosomes with pterostilbene and performing ultrasonic treatment and incubation to prepare Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano delivery system, i.e. pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes; wherein the mass ratio of the Lactobacillus helveticus exosomes to pterostilbene is 1:10-1:

20.

2. The production method according to claim 1, characterized by, Specifically comprising: Subculturing Lactobacillus helveticus for 12-48 h to prepare Lactobacillus helveticus suspension; And mixing the Lactobacillus helveticus suspension with PBS buffer and performing continuous low-speed centrifugation, then filtering the obtained supernatant with water-based microporous filter membrane, and then performing ultrahigh-speed centrifugation on the obtained filtrate to prepare Lactobacillus helveticus exosomes.

3. The method of claim 2, wherein: The volume ratio of the Lactobacillus helveticus suspension to PBS buffer is 2:1-1:

2.

4. The method of claim 2, wherein: The continuous low-speed centrifugation is performed with centrifugal force of 1000-2000 × g and 8000-12000 × g in sequence; and / or the continuous low-speed centrifugation is performed for 10-45 min.

5. The method of claim 2, wherein: The water-based microporous filter membrane adopts pore sizes of 0.45 μm and 0.22 μm.

6. The method of claim 2, wherein: The ultrahigh-speed centrifugation is performed with centrifugal force of 100000-150000 × g; and / or the ultrahigh-speed centrifugation is performed for 1-3 h.

7. The preparation method according to claim 1, characterized in that, Specifically comprising: Dissolving pterostilbene in ethanol and mixing the pterostilbene with PBS suspension of Lactobacillus helveticus exosomes to form a mixed system of Lactobacillus helveticus exosomes and pterostilbene; Performing ultrasonic treatment on the mixed system under ice bath condition, and then incubating the mixed system under ice bath for 2-10 min, and then repeating the ultrasonic treatment and incubation; And incubating the obtained product at 35-45℃ for 2 h, then incubating the obtained product at 4-10℃ for 24 h, and then performing ultrahigh-speed centrifugation to prepare Lactobacillus helveticus exosome-pterostilbene biomimetic oral nano delivery system.

8. The method of claim 7, wherein: The ultrasonic treatment adopts ultrasonic power of 100-200 W; and / or the ultrasonic treatment is performed for 2-4 min.

9. The method of claim 7, wherein: The repeated ultrasonic treatment and incubation are performed for 1-5 times.

10. The method of claim 7, wherein: The ultrahigh-speed centrifugation adopts centrifugal force of 100000-150000 × g and centrifugal time of 1-3 h.

11. The exosome based sirtuin mimetic bio-oral nano-delivery system based on Lactobacillus helveticus as prepared by the process of any one of claims 1-10, characterized in that: The pterostilbene biomimetic nano oral delivery system comprises Lactobacillus helveticus-derived exosomes and pterostilbene loaded in the cavities of the Lactobacillus helveticus-derived exosomes.

12. The Lactobacillus helveticus exosome based mimetic oral nano-delivery system of sptinbin according to claim 11, characterized in that: The loading rate of pterostilbene in the pterostilbene biomimetic nano oral delivery system is 41-67%.

13. An oral product of pterostilbene, characterized in that, At least comprising the pterostilbene biomimetic oral nano delivery system based on Lactobacillus helveticus exosomes in claim 11 or 12.

14. Use of Lactobacillus helveticus-derived exosomes for the preparation of a Sirtuin mimetic bionanoparticulate oral delivery system, characterized by the fact that: The pterostilbene biomimetic nano oral delivery system comprises Lactobacillus helveticus-derived exosomes and pterostilbene loaded in the cavities of the Lactobacillus helveticus-derived exosomes.

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