CN115837007A - Oral plant exosome hydrogel for targeting regulation of intestinal tract and preparation method and application thereof - Google Patents
Oral plant exosome hydrogel for targeting regulation of intestinal tract and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an oral plant exosome hydrogel for targeting regulation of intestinal tracts, and a preparation method and application thereof, and belongs to the technical field of biology. The oral plant exosome hydrogel is obtained by sequentially embedding starch and sodium alginate in the outer layer of a mixture of plant exosomes and liposomes. The preparation method comprises the following steps: taking plant juice supernatant, adjusting the pH value to 4-6, mixing with polyethylene glycol for incubation, taking the precipitate to suspend in a PBS buffer solution, taking the supernatant, and filtering to obtain a plant exosome solution; mixing the plant exosome solution with liposome to obtain plant exosome-liposome; sequentially embedding starch layer and sodium alginate layer in the plant exosome-liposome, and dripping CaCl through needle tip 2 Solidifying in the solution to obtain the oral plant exosome hydrogel. The oral plant exosome hydrogel prepared by adopting the multistage embedding process has good stability, can realize the multistage targeted response delivery of the plant exosome, and better plays a role in intestinal regulation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to oral plant exosome hydrogel for targeting regulation of intestinal tracts, and a preparation method and application thereof.
Background
The intestinal tract is the largest digestive organ of the human body, and researches show that 80 percent of the digestion and absorption processes are finished in the intestinal tract, and the intestinal tract is not only the largest 'nutrition supply center' of the human body, but also the largest immune system of the human body. Therefore, the integrity and functionality of the intestinal barrier are the fundamental guarantee for maintaining the health of human body, unhealthy and irregular living habits can accelerate the aging of the intestinal tract, so that various diseases are easily induced, and the regulation of the health of the intestinal tract is an important means for ensuring the health of human body.
The exosome contains various active substances, such as nucleic acid (mRNA, miRNA, incRNA and circRNA), protein, lipid and other various substances, is an endogenous regulating substance, has various biological activities, can influence the dynamic balance of the microbial flora in the intestinal tract, and effectively regulates the intestinal tract. Exosomes can be divided into animal cell exosomes and plant exosomes by source. The animal cell exosome has wide sources and good clinical application prospect. However, animal cell-derived exosomes require the culture of large numbers of cells, are low in yield, and have the problem of immunogenicity of allogens. With the progress of research, scientists also find nanoparticles with physical properties and compositions similar to exosomes in the separation and purification of plants, and compared with animal exosomes, the nanoparticles have the advantages of higher yield, short extraction period, lower immunogenicity and wider application prospect due to the fact that the nanoparticles are derived from edible fruits and vegetables. However, the following problems still exist when the plant exosome liquid is directly utilized or dried for use: 1. the liquid exosome concentration is low, the requirement on storage conditions is high, and the liquid exosome needs to be refrigerated for transportation; 2. the exosome is directly freeze-dried, the structure of the exosome can be damaged by spray drying or freeze vacuum drying, and the exosome is easily affected by bile salt, gastric acid and the like after entering the body by directly using the exosome or freeze-dried exosome powder, so that the biological activity and targeting performance of the exosome are greatly reduced. Chinese patent CN114009772A provides a preparation method of a plant exosome microcapsule embedding freeze-dried powder, and the plant exosome microcapsule embedding freeze-dried powder is formed by primary coating of sodium alginate solution and secondary coating of chitosan oligosaccharide. However, the plant exosome freeze-dried powder obtained by the method still has the problems of low targeting efficiency, uncontrollable release and the like, and the effect of directly applying the plant exosome freeze-dried powder to intestinal regulation is not ideal. There is thus a need for a method of stable, targeted regulation of the intestinal tract.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an oral plant exosome hydrogel for targeting regulation of intestinal tracts, a preparation method and application thereof, and the oral plant exosome hydrogel for targeting regulation of the intestinal tracts is stable and targeting regulation of the intestinal tracts.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses an oral plant exosome hydrogel for targeting regulation of an intestinal tract, which comprises a plant exosome, porous starch and sodium alginate in a mass ratio of (0.01-0.5) - (2.5-125) - (10-500), wherein the plant exosome is embedded in an internal pore passage of the porous starch, and the porous starch is embedded in a three-dimensional network structure of the sodium alginate.
Preferably, the starch further comprises small peptide D3 and mixed phospholipid, and a mixture of the plant exosomes, the small peptide D3 and the mixed phospholipid in a mass ratio of (0.01-0.5) - (0.05-25) (1.5-125) is embedded in the inner pore canal of the porous starch; the mixed phospholipid is a mixture of phospholipid and cholesterol.
Preferably, the particle size of the mixture of plant exosomes, small peptide D3 and mixed phospholipids is in the range of 100-400 nm.
Preferably, the plant exosomes are extracted and separated under the condition of pH value of 4-6.
Further preferably, the plant exosomes are extracted and separated after being incubated with polyethylene glycol under the condition of pH value of 5.
Preferably, the plant exosomes are derived from tea leaves, mulberry bark, ginger, turmeric, onion, garlic or grapes.
The invention also discloses a preparation method of the oral plant exosome hydrogel for targeting regulation of the intestinal tract, which comprises the steps of taking plant juice supernatant, regulating the pH value to be 4-6, centrifuging, taking precipitate to suspend in PBS buffer solution, taking the supernatant, and filtering to obtain plant exosome solution; mixing the plant exosome solution with a mixture of phospholipids and cholesterol to obtain plant exosome-liposomes; mixing the plant exosome-liposome with porous starch, and removing supernatant to obtain plant exosome-liposome/porous starch granules; mixing plant exosome-liposome/porous starch granules with sodium alginate, and then utilizing CaCl 2 Solidifying the solution to obtain the oral plant exosome hydrogel.
Preferably, the supernatant of the plant juice adjusted to a pH of 4 to 6 is incubated with polyethylene glycol before centrifugation to remove the precipitate.
Preferably, the obtained plant exosome-liposome/porous starch granules are mixed with starch gelatinized into gel, and then mixed with sodium alginate.
The invention also discloses application of the oral plant exosome hydrogel for targeting regulation of the intestinal tract in preparation of medicines or health-care products for regulating the intestinal tract.
Compared with the prior art, the invention has the following beneficial effects:
according to the oral plant exosome hydrogel for targeting regulation of the intestinal tract, the porous property of the porous starch is utilized, on one hand, the porous starch is used as an adsorption carrier of the plant exosome to protect the plant exosome, and the stability of the plant exosome is improved; on the other hand, the composite gel is formed by utilizing the characteristics of the natural polymer and the sodium alginate, so that the stability of the obtained gel is further enhanced. On the one hand, sodium alginate can shrink in gastric acid and swell in the near-neutral environment of the intestinal tract, so that the gastric environment can be avoided, and the release can be triggered in the intestinal tract. On the other hand, the combination of sodium alginate and porous starch utilizes the characteristic that sodium alginate reacts to the change of pH value, starch food intake feedback adjustment and secretion of amylase has specific hydrolysis effect on the porous starch, and the obtained hydrogel has a trigger release platform of pH and amylase reaction. The preparation of the gel adopts a multi-stage embedding process, firstly, a gel shell formed by sodium alginate on the outermost layer of the plant exosome and a starch layer on the inner layer can be protected to safely and smoothly pass through the stomach to stay after being orally taken and interact with intestinal mucosa, and the gradual disintegration of the shell is realized under the double stimulation response of intestinal pH and amylase at the moment, so that the internal exosome can be slowly released. Secondly, the hierarchical structure can load more active substances to play a synergistic effect, including small molecular compounds, macromolecular proteins and the like, and finally, the raw materials in the system have good biological safety, are edible and can be used as a good carrier for oral delivery products. Therefore, the oral plant exosome hydrogel has good stability, can realize multi-stage targeted response delivery of the plant exosome, and better plays a role in intestinal regulation.
Furthermore, the released exosome is firmly anchored in the intestinal epithelial cell by virtue of the small peptide D3 peptide on the membrane surface targeting the intestinal epithelial cell, and the exosome is released out through mutual fusion with the intestinal epithelial cell membrane to play a role in regulating activity. The mixture of phospholipids and cholesterol enhances the stability of the plant exosomes on the one hand and provides a more comprehensive delivery vehicle loaded with more active ingredient on the other hand.
Furthermore, the polyethylene glycol (PEG) -based low-pH value nano vesicle separation technology can keep the integrity of the nano vesicles by capturing the nano vesicles in the reticular structure, greatly improve the yield of the plant exosomes and increase the in-vitro stability of the exosomes under the condition of not losing the integrity and key biological activity of the plant exosomes, and is simple to operate and mild in extraction process.
The preparation method of the oral plant exosome hydrogel for targeting regulation of the intestinal tract, provided by the invention, adopts a multi-stage embedding process, and sequentially embeds starch and sodium alginate in the outer layer of a mixture of the plant exosome and the liposome, so that the stable targeting delivery of active ingredients can be realized.
Further, a layer of starch film is coated on the surface of the porous starch granules, so that the stability and the controlled release of the contents can be further enhanced.
Drawings
FIG. 1 is a graph of the overall yield of PDNVs isolated by various methods of the present invention;
FIG. 2 is a graph showing the total yield of PDNVs isolated at various pH values according to the present invention;
FIG. 3 is a transmission electron microscope image of PDNVs separated by different methods of the present invention; wherein A is PDNVs obtained by a differential centrifugation method, and B is PDNVs obtained by PEG precipitation under the condition that the pH value is = 5;
FIG. 4 is a graph showing the particle size and potential of PDNVs separated at different pH values according to the present invention;
FIG. 5 is a scanning electron micrograph of a porous starch and oral plant exosome hydrogel of the present invention; wherein A is porous starch with the diameter of 20 μm; b is porous starch with the particle size of 10 mu m; c is oral plant exosome hydrogel, 20 mu m; d is oral plant exosome hydrogel with the particle size of 10 mu m;
FIG. 6 is a statistical comparison of gastrointestinal tissue fluorescence intensity of oral plant exosome hydrogels of the present invention with exosomes, plant exosomes-liposomes and plant exosomes-liposomes/porous starch granules.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides an oral plant exosome hydrogel for targeting regulation of intestinal tracts, which comprises plant exosomes, small peptide D3, mixed phospholipid, porous starch and sodium alginate in a mass ratio of (0.01-0.5) - (0.05-25) - (1.5-125) - (2.5-125) - (10-500); the mixture of the plant exosome, the small peptide D3 and the mixed phospholipid is fused to form a primary structure and is embedded in an internal pore canal of the porous starch, and the porous starch is embedded in a three-dimensional network structure of the sodium alginate to form a secondary structure.
Wherein the plant exosome is derived from any one of tea, mulberry bark, ginger, turmeric, onion, garlic and grape; in the mixture of the plant exosomes, the small peptide D3 and the mixed phospholipids, the final mass concentration of the plant exosomes is 0.02-0.1%, the final mass concentration of the small peptide D3 is 0.1-1%, and the final mass concentration of the liposome is 3-5%; the mixed phospholipid is a mixture of phospholipid and cholesterol.
Example 1
1. Plant-derived nanovesicles (PDNVs) extraction and isolation
The extraction and separation method comprises the following steps:
a) Weighing cleaned fresh rhizoma Zingiberis recens 10g, mashing, adding sterile phosphate buffer 100g, and homogenizing in high speed stirrer (750W, 3min) to completely crush;
b) Subsequently, the ginger juice was collected by filtration through a 200 μm nylon mesh, and the collected ginger juice was centrifuged for 30min (2000g, 10min;6000g, 20min);
c) The precipitate was then discarded, the pH was adjusted to pH =5 by adding 1M HCl or NaOH to the supernatant, the pH adjusted supernatant was mixed with PEG6000 and incubated overnight at 4 ℃, with a final concentration of 10% (w/v) of PEG 6000;
d) Then, the supernatant was discarded by centrifugation (8000 g, 30min), and the pellet was suspended in PBS buffer at 0.5 mg/mL;
e) Then, carrying out ultrafiltration concentration on the solution obtained in the step d) through a 100kd ultrafiltration tube, and discarding the liquid at the lower part of the ultrafiltration tube;
f) Filtering the concentrated liquid with a 0.22 μm filter membrane to obtain ginger exosome solution; by transmission electron microscopy (Tecnai G) 2 20S-TWIN, FEI, USA) morphology of exosomes was observed (specific method: the prepared sample was deposited drop-wise onto the surface of a copper grid, then stained with 1% uranyl acetate for 15 seconds, and then allowed to dry at room temperature for subsequent TEM imaging). The particle size and surface charge of exosomes were measured with a nanometer particle size analyzer (Malvern Instruments, malvern, UK) (a specific method was to dilute the exosome sample 100-fold and add it to a sample cell for measurement, which was repeated three times for each sample). And (3) determining the protein concentration of the ginger exosome solution by adopting a standard operation method of a BCA kit, adjusting the concentration to be 5.0mg/mL, and placing the ginger exosome solution as a storage solution at-80 ℃ for freezing storage to avoid repeated freezing and thawing.
Meanwhile, in order to determine the beneficial effect of low pH on exosome extraction, we further utilized different pH values to extract exosomes. I.e. the pH in step c) is adjusted to pH =6 and pH =7, respectively, the other steps being the same.
Further comparing the differences of exosomes prepared by different extraction methods, the ginger exosome is prepared by adopting a differential centrifugation method, and the specific method comprises the following steps: mashing cleaned fresh rhizoma Zingiberis recens 1g, adding sterile phosphate buffer 100g, and homogenizing in high speed stirrer (750W, 3min) to completely break; then filtering with a 200 μm nylon net to collect ginger juice, centrifuging the collected ginger juice for the first time for 10min at 1000g, taking the supernatant of the first centrifugation for the second time for 20min at 3000g, taking the supernatant of the second centrifugation for the third time for 40min at 10000g to remove impurities, finally collecting the supernatant of the third centrifugation for the fourth centrifugation for 90min at 100000g, and taking the obtained precipitate, and washing with PBS for three times to obtain the ginger exosome.
The yield of exosomes was calculated by the following formula: the yield of exosomes = the amount of collected ginger exosomes/the amount of fresh ginger input.
The results are shown in fig. 1 and fig. 2, where fig. 1 shows that the exosomes produced by the low pH precipitation method are higher in yield compared to the differential centrifugation method, and fig. 2 further shows that lowering the pH to make it a weak acid environment is more beneficial to improve the exosome yield. Meanwhile, the results of fig. 3 further show that exosomes with vesicle-like structures are prepared by both the low-pH precipitation method and the differential centrifugation method, but the exosomes prepared by the low-pH precipitation method are smaller in size. The particle size and surface charge of exosomes prepared under different pH conditions in fig. 4 also further show that there is no significant difference in surface charge, but the size of exosomes increases significantly with increasing extraction pH, and the particle size of exosomes isolated under low pH conditions is smaller.
2. Preparation of plant-derived nanovesicles-liposomes (PDNVs-LP)
Weighing small peptide D3 (the synthetic sequence and method in the reference "A novel peptide protections against two-induced issue by treating the issue and modulating the gut microbiota") and adding to Pro-Lipo TM Neo (purchased from Sage Chemical (Group) co., ltd) was mixed well to obtain a phospholipid mixed solution; weighing the ginger exosome solution obtained in the step 1, slowly injecting the ginger exosome solution into the ginger exosome solution and stirring the ginger exosome solutionInjecting the phospholipid mixed solution in the state at the speed of 1-4% V/min, stirring the phospholipid mixed solution at the speed of 800-1000 rpm/min, continuously stirring for 10min after the completion, and then carrying out gradient homogenization treatment to prepare the plant exosome-liposome (PDNVs-LP) with the particle size range of 100-400 nm.
Wherein the final mass concentration of the small peptide D3 is 0.5%, the final mass concentration of the ginger exosome is 0.05%, and Pro-Lipo TM The final mass concentration of Neo is 5%.
3. Hydrogel preparation
(1) Preparation of Porous Starch (PS)
5g of corn starch (Maize starch, MS, available from Yirana food ingredients Co., ltd.) was weighed into 95g of PBS buffer to form a starch slurry, and then 12.5g of Bacillus subtilis-derived a-amylase (4000U/g, available from Sigma-Aldrich) was added to mix with the starch slurry and stirred overnight in a water bath at 37 ℃ at 200rpm/min. After the reaction is finished, 3000g of suspension is collected, centrifuged for 10min, and then absolute ethyl alcohol with the mass 2 times of that of the suspension is added to wash and precipitate for 3 times. Finally, the precipitate was dried in an oven (40 ℃ C., overnight) to obtain porous starch.
The porous starch may also be purchased directly, from a source selected from corn, potato, rice, wheat, barley or tapioca.
(2) Plant exosomes-liposomes/porous starch granules (PDNVs-LP/PS)
200g of PDNVs-LP obtained in the step 2 and 10g of PS obtained in the step 3 (1) are weighed and stirred at room temperature to be uniformly mixed (500rpm, 60min), then the mixture is kept stand for 2h, then the supernatant is gently removed, and after pre-freezing for 4h at-20 ℃, the mixture is frozen and dried overnight to obtain the PDNVs-LP/PS.
Further, in order to prevent the effective components from being reduced due to the leakage of the PDNVs-LP embedded into the inner pore channels of the PS in the subsequent operation steps, the outer layer of the PDNVs-LP/PS is coated with a starch gel film, and the specific steps are as follows:
weighing 2G of G70 high-amylose corn starch (purchased from Yiruian food ingredients Co., ltd.) and dissolving in 98G of deionized water, heating to gelatinize the starch into gel, cooling to room temperature, weighing 100G of the gel and 50G of PDNVs-LP/PS, carefully and uniformly mixing, pre-freezing at-20 ℃ overnight, and freeze-drying for 24h to obtain the corn starch coated plant exosome-liposome/porous starch granules.
(3) Plant exosomes-liposomes/porous starch/gel (PDNVs-LP/PS/SA)
4.8g of the above-mentioned corn starch-coated plant exosome-liposome/porous starch granules were weighed, and 15.2g of deionized water was added and stirred to be completely dissolved. Then 0.8g Sodium Alginate (Sodium Alginate, SA, available from Aladdin Biochemical technology Co., ltd., shanghai) was added to 19.2g deionized water to be completely dissolved, and then both were mixed at room temperature and stirred to be mixed uniformly (500 rpm/min,30 min.), and the above mixture was dropped into 100mL CaCl through a needle point (inner diameter 0.51 mm) using a peristaltic pump 2 The solution (1.5% w/v) was solidified to form hydrogel microbeads, and the microbeads were washed 2 to 3 times with a large amount of ultrapure water to obtain an oral-administration plant exosome hydrogel for targeted regulation of intestinal tracts.
4. Evaluation experiment of System
(1) Characterization of electron microscope
The surface structures of the porous starch and the composite hydrogel after freeze-drying were observed by a scanning electron microscope (SEM, hitachi, regulus8100, japan). A small amount of sample was placed on the conductive gel, gold sprayed for 2 minutes under vacuum, and SEM images were obtained at a 10kV working voltage. As can be seen from A and B in FIG. 5, the prepared starch granule has a porous structure containing a plurality of nearly circular micropores, and the porous channel structure is beneficial to embedding more contents. As can be seen from C and D in FIG. 5, the hydrogel of the oral plant exosomes was approximately spherical after lyophilization, with pores hidden from view inside, but surface encapsulation was seen after enlargement.
(2) In vitro gastric acid resistance test
And (3) incubating simulated gastric juice with the plant exosomes, the plant exosome-liposome/porous starch granules and the oral plant exosome hydrogel together to determine the concentration of free plant exosomes. The specific method comprises the following steps:
(1) simulated gastric fluid configuration: the pH of a 2mg/mL NaCl solution was adjusted to pH =2.0 with concentrated hydrochloric acid, then an amount of pepsin was added, the final concentration of pepsin was 0.3mg/mL, followed by filter sterilization with a 0.22 μm filter.
(2) Taking 1g of each of the plant exosomes, the plant exosomes-liposomes/porous starch granules and the oral plant exosome hydrogel, placing the plant exosomes, the plant exosomes-liposomes/porous starch granules and the oral plant exosome hydrogel into 9mL of simulated gastric juice preheated at 37 ℃, then placing the plant exosomes, the liposome/porous starch granules and the oral plant exosomes hydrogel into a constant temperature shaking table respectively for incubation (37 ℃,100 rpm/min), and taking 1mL of each of the four groups of mixed liquor respectively and adding NaHCO containing 0.2mol/L into the mixed liquor when the intervals are 10min, 30min, 60min, 120min and 180min 3 In sterile phosphate buffer, for 30min, and then the free plant exosome concentration was determined by BCA method. The results are shown in Table 1:
TABLE 1 gastric acid resistance test results for various groups of plant exosomes
The results in table 1 show that the plant exosomes are embedded in the hierarchy, and the gastric acid resistance stability of the plant exosomes can be enhanced.
(3) In vitro simulation of stability in the digestive tract System
And (3) incubating simulated digestive tract liquid with the plant exosomes, the plant exosomes-liposome/porous starch granules and the oral plant exosome hydrogel, and determining the concentration of free plant exosomes. The specific method comprises the following steps:
(1) a simulation model of each digestive tract simulated fluid is established, see table 2:
TABLE 2 pH and action time table of simulated fluid for each digestive tract
Wherein, the digestive tract simulation liquid in the stage 1 to the stage 6 is simulation liquid for simulating the continuous process from the esophagus to the stomach, and the preparation method is as follows: dissolving pepsin 5mg/mL in physiological saline, adjusting pH value to a specified value, and filtering for sterilization.
The digestive tract simulation solution of the stage 7 is a duodenum simulation solution, and the preparation method comprises the following steps: dissolving 10mg/mL trypsin and 3mg/mL bile salt in physiological saline respectively, adjusting pH to 5.0, and filtering for sterilization.
The digestive tract simulated fluid of the stage 8 is small intestine simulated fluid, and the preparation method is as follows: 10mg/mL trypsin and 3mg/mL bile salt are respectively dissolved in physiological saline, and 0.1mol/L NaHCO is added 3 Then the pH was adjusted to 6.5 and sterilized by filtration.
(2) Taking 1g of each of the plant exosomes, the plant exosomes-liposomes/porous starch granules and the oral plant exosome hydrogel, placing the plant exosomes-liposomes, the plant exosomes-liposomes/porous starch granules and the oral plant exosome hydrogel into 9mL of simulated digestive juice preheated at 37 ℃, then placing the plant exosomes/porous starch granules and the oral plant exosomes hydrogel into a constant temperature shaking table respectively for incubation (37 ℃ and 100 rpm/min), changing the simulated liquid of the next stage after the action time is over, centrifuging for 2min at 10000rpm/min before changing the simulated liquid each time, adding the simulated liquid of the new stage after discarding the supernatant, and vortexing for 30s to fully mix. Respectively taking 1mL of the mixed solution after the four groups of actions, and adding NaHCO with the concentration of 0.2mol/L 3 In sterile phosphate buffer, for 30min, and then the free plant exosome concentration was determined by BCA method. The results are shown in Table 3:
TABLE 3 stability of various groups of plant exosomes in simulated gut
The results in table 3 show that the plant exosomes are damaged in the digestive system to some extent, probably because the pH value of the system and the like have certain damage effects on the structure of the plant exosomes, and the damage loss can be reduced by different embedding means, and particularly, the plant exosome hydrogel system is orally taken to better protect the exosomes from being damaged by the digestive system.
5. Evaluation of Targeted modulation of intestinal Effect
(1) Intestinal targeting
(1) Selection of laboratory animals
Female C57BL/6 mice (6-8 weeks old) and Sprague-Dawley rats (4-6 weeks old) purchased from Beijing Wintolite, laboratory animal technology, inc. and were tested after being acclimatized in a pathogen-free clean animal house for one week according to standard procedures. All experiments involving mice were approved by the animal ethics committee of the experimental animal center of the university of air force military medical science.
(2) Preparation of fluorescence-labeled plant exosome hydrogel
The PDNVs were labeled with the fluorescent lipophilic dye DiR (from sigma). The specific method comprises the following steps: 0.5mg of PDNVs was weighed into 1mL of PBS buffer, 1. Mu.L of DiR dye was added thereto, and the mixture was incubated at room temperature for 30min in the absence of light. The labeled PDNVs were then passed through a 100kD ultrafiltration centrifuge tube to remove free dye to give fluorescently labeled plant exosomes (DiR-PDNVs). Then, the exosome hydrogel after fluorescent labeling is prepared according to the same method.
(3) Distribution of fluorescence in vivo
In order to confirm the intestinal targeting property, the plant exosome hydrogel prepared above was mixed with the feed uniformly, and the oral ingestion was carried out by the gavage method in mice fasted for 12 hours, the ingestion amount per mouse was 5g. Mice were sacrificed 12h after oral administration, gastric and intestinal tissues were collected, fluorescence imaging was performed using IVIS spectral series in vivo imaging system, and the fluorescence intensity of each tissue was counted.
The result is shown in fig. 6, the fluorescence intensity of the oral plant exosome gel in the stomach tissue after the hierarchical embedding is higher, which shows that the oral plant exosome gel has good intestinal targeting property and can act on the intestinal tract for a longer time.
(2) Intestinal tract regulating effect
Rats were randomly divided into two groups, with the control group fed normally 30g of feed per day, and the experimental group fed 15g of plant exosome hydrogel and 15g of normal feed per day. The feed was also fed for 14 days. The state of the rats is observed and recorded every day, the rats are weighed for 2 times every week after being fixed for the same time, and the SD rats are found to have flexible actions, smooth hair color, normal diet and drinking water and normal weight increase in the experimental period without obvious abnormal pathology and death condition.
Meanwhile, rat feces were collected 4 days before the end of the experiment, and were frozen in a refrigerator at-80 ℃ immediately after being collected every day. Then weighing 2g of rat feces, placing the rat feces in a 200mL sterilized ground triangular flask containing 0.1% peptone diluent, violently shaking the feces samples to be uniformly mixed, after uniform mixing, carrying out gradient dilution to a sterilized anaerobic tube filled with 9mL of 0.1% peptone diluent, then taking 200 mu L of the feces samples after proper dilution, respectively adding the feces samples into poured five solid plate culture media (BBL agar, bile hepta-nocturnal glucoside sodium azide agar, crystal violet neutral red bile salt agar and MRS lactobacillus agar), uniformly coating, respectively culturing bifidobacteria, enterococcus, escherichia coli, lactobacillus and the like, wherein the MRS lactobacillus agar is placed in an anaerobic jar to be cultured for 48 hours at 37 ℃, and the rest three are directly placed in an oxygen-containing incubator at 37 ℃ to be cultured for 24 hours. Colonies from each plate were then counted according to standard plate counting methods and the results are given in table 4 below:
table 4 plate counting statistical table for four kinds of bacteria in feces
Note: results are in log 10 Indicates that there was a significant difference P from the normal feed group<0.05。
Through the results, after the plant exosome hydrogel is fed, the contents of beneficial bacteria bifidobacteria and lactobacilli are increased, and the quantity of pathogenic bacteria enterococci and escherichia coli is reduced, so that the beneficial effect of regulating the intestinal tract can be exerted by influencing the intestinal tract flora and improving the distribution of the intestinal tract flora.
Meanwhile, 2g of rat feces collected continuously for 4 days are taken respectively, the rat feces are placed in a drying oven to be dried to constant weight at 105 ℃, the weight after drying is measured, and the water content is calculated as follows: stool water content (% by weight) = (wet stool weight-dry stool weight)/wet stool weight × 100% = water weight/wet stool weight × 100%. The results are given in table 5 below:
TABLE 5 stool water statistical table
Note: * Indicating a significant difference P <0.05 compared to the normal feed group.
From the results of Table 5, it was found that the water content of feces of rats fed the hydrogel group containing plant exosomes was higher. The metabolite feces is one of important indexes of body health and intestinal tract health, and the content of beneficial bacteria in the intestinal tract flora is increased, so that the feces can be softened, the water content of the feces is increased, and the feces excretion is promoted.
Meanwhile, 0.4g of rat feces is taken and added into 4mL of sterile double distilled water, the mixture is shaken and fully mixed and placed at room temperature overnight, the mixture is shaken vigorously again the next day to be completely dispersed into homogenate, and after centrifugation is carried out for 10min at 4000rpm/min under 4 ℃, 250 mu L of supernatant is respectively sucked and placed into a 1.5mL centrifuge tube and stored in a refrigerator under 4 ℃. Taking 50 mu L of the supernatant, measuring the pH value, adding 20 mu L of 32mmol/L alpha-methyl valeric acid and 20 mu L phosphoric acid (1. The chromatographic conditions are as follows: the chromatographic column is selected to be RtxWAX667536 type, the size is 32m multiplied by 0.32mm multiplied by 0.25mm, the temperature of a chromatographic sample injector and a detector are both 250 ℃, the temperature of the column is 80 ℃, the nitrogen flow rate is 1.94mL/min, the sample injection is carried out in a split-flow mode, and the sample injection amount is 2 mu L. Meanwhile, an analytical reagent is used for accurately preparing 150mmol/L acetic acid, 50mmol/L butyric acid and 40mmol/L n-butyric acid as standard solution, and 32mmol/L alpha-methylvaleric acid as internal standard solution. The results are given in table 6 below:
TABLE 6 statistical table of changes in fecal pH and short chain fatty acids
| Feeding food | pH value | Acetic acid (mu mol/g) | Propionic acid (mu mol/g) | Butyric acid (μmol/g) |
| Normal feed | 7.02±0.06 | 32.28±1.51 | 8.86±0.81 | 8.15±0.62 |
| Feed + plant exosome hydrogel | 6.84±0.08 | 35.52±1.27* | 10.62±0.54* | 8.17±1.12 |
Note: * Indicating a significant difference P <0.05 compared to the normal feed group.
From the above results, the pH of the feces of the rats fed the plant exosome hydrogel was decreased, and the content of short-chain fatty acids was also changed, wherein butyric acid was slightly increased, and the content of acetic acid and propionic acid was significantly increased. The beneficial flora in intestinal flora such as bifidobacterium and lactobacillus are anaerobic bacteria which can ferment substrates to generate short-chain fatty acid in the presence of suitable substrates, wherein the contents of acetic acid, propionic acid and butyric acid are the highest, and the short-chain fatty acid is the main SCFA in the intestinal tract, and the SCFA can increase the excretion of cholic acid through feces, reduce the cytotoxicity of the feces, reduce the proliferation of rectal cells and promote the health of the intestinal tract.
Example 2
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising tea exosomes, small peptide D3, pro-Lipo in a mass ratio of 0.02 TM Neo, porous starch and sodium alginate; tea exosomes, small peptide D3 and Pro-Lipo TM The Neo mixture is fused to form a primary structure and embedded in the internal pore channels of the porous starch, wherein the final concentration of the tea exosomes in the primary structure is 0.02%, the final concentration of the small peptide D3 in the primary structure is 0.1%, and Pro-Lipo is added TM The final concentration of Neo is 3%; the porous starch is embedded in the three-dimensional network structure of the sodium alginate to form a secondary structure.
The above hydrogel of oral plant exosomes was prepared as in example 1.
Example 3
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising turmeric exosomes, small peptide D3, pro-Lipo in a mass ratio of 0.1 TM Neo, porous starch and sodium alginate; turmeric exosomes, small peptide D3 and Pro-Lipo TM The mixture of Neo is fused to form a primary structure and embedded in the inner pore channels of the porous starch, wherein the final concentration of the turmeric exosomes in the primary structure is 0.05%, the final concentration of the small peptide D3 in the primary structure is 0.5%, and Pro-Lipo is contained in the primary structure TM The final concentration of Neo was 3%; the porous starch is embedded in the three-dimensional network structure of the sodium alginate to form a secondary structure.
The preparation method of the oral plant exosome hydrogel is the same as that of example 1.
Example 4
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising grape exosomes, small peptide D3, pro-Lipo at a mass ratio of 0.16 TM Neo, porous starch and sodium alginate; grape exosomes, small peptides D3 and Pro-Lipo TM The mixture of Neo is fused to form a primary structure and embedded in the internal pore channels of the porous starch, wherein the final concentration of the grape exosome in the primary structure is 0.06%, the final concentration of the small peptide D3 in the primary structure is 1%, and Pro-Lipo is contained in the primary structure TM The final concentration of Neo was 4%; the porous starch is embedded in the three-dimensional network structure of the sodium alginate to form a secondary structure.
The above hydrogel of oral plant exosomes was prepared as in example 1.
Example 5
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising mulberry bark exosomes, small peptide D3, pro-Lipo at a mass ratio of 0.2 TM Neo, porous starch and sodium alginate; mulberry bark exosome, small peptide D3 and Pro-Lipo TM The mixture of Neo is fused to form a primary structure and is embedded in the inner pore canal of the porous starch, wherein the final concentration of the mulberry bark exosome in the primary structure is 0.04%, the final concentration of the small peptide D3 is 0.2%, and Pro-Lipo TM The final concentration of Neo was 3%; the porous starch is embedded in the three-dimensional network structure of the sodium alginate to form a secondary structure.
The preparation method of the oral plant exosome hydrogel is the same as that of example 1.
Example 6
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising onion exosomes, small peptides D3, pro-Lipo at a mass ratio of 0.5 TM Neo, porous starch and sodium alginate; onion exosomes, small peptide D3 and Pro-Lipo TM The mixture of Neo is fused to form a primary structure and embedded in the internal pore channels of the porous starch, wherein the final concentration of onion exosomes in the primary structure is 0.05%, the final concentration of small peptide D3 in the primary structure is 1%, and Pro-Lipo in the primary structure TM The final concentration of Neo was 4%; the porous starch is embedded in the three-dimensional network structure of the sodium alginate to form a secondary structure.
The preparation method of the oral plant exosome hydrogel is the same as that of example 1.
Example 7
An oral plant exosome hydrogel for targeted modulation of intestinal tracts, comprising garlic exosomes, small peptides D3, pro-Lipo in a mass ratio of 0.4 TM Neo, porous starch and sodium alginate; garlic exosomes, small peptide D3 and Pro-Lipo TM The Neo mixture is fused to form a primary structure and embedded in the inner pore canal of the porous starch, wherein the final concentration of garlic exosome in the primary structure is 0.1%, the final concentration of small peptide D3 in the primary structure is 1%, and Pro-Lipo is contained in the primary structure TM The final concentration of Neo is 5%; with porous starch embedded in sodium alginateSecondary structures are formed in the three-dimensional network structure.
The preparation method of the oral plant exosome hydrogel is the same as that of example 1.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. An oral plant exosome hydrogel for targeting regulation of intestinal tracts is characterized by comprising a plant exosome, porous starch and sodium alginate in a mass ratio of (0.01-0.5) - (2.5-125) - (10-500), wherein the plant exosome is embedded in an internal pore passage of the porous starch, and the porous starch is embedded in a three-dimensional network structure of the sodium alginate.
2. The oral plant exosome hydrogel for targeted adjustment of intestinal tract according to claim 1, further comprising small peptide D3 and mixed phospholipid, wherein a mixture of the plant exosome, the small peptide D3 and the mixed phospholipid in a mass ratio of (0.01-0.5) to (0.05-25) to (1.5-125) is embedded in the internal pore channels of the porous starch; the mixed phospholipid is a mixture of phospholipid and cholesterol.
3. The oral plant exosome hydrogel for targeted modulation of intestinal tract according to claim 2, wherein the particle size of the mixture of plant exosomes, small peptide D3 and mixed phospholipids is in the range of 100-400 nm.
4. The hydrogel for oral plant exosomes for targeted regulation of intestinal tracts according to any one of claims 1 to 3, wherein the plant exosomes are obtained by extraction and separation under the condition that the pH value is 4-6.
5. The oral plant exosome hydrogel for targeted regulation of intestinal tract according to claim 4, wherein the plant exosome is obtained by extraction and separation after being incubated with polyethylene glycol under the condition of pH value of 5.
6. The oral plant exosome hydrogel for targeted modulation of intestinal tracts according to any one of claims 1 to 3, wherein the plant exosome is derived from tea leaves, mulberry bark, ginger, turmeric, onion, garlic or grape.
7. The method for preparing the hydrogel of the oral plant exosomes for targeting and regulating the intestinal tract according to any one of claims 1 to 6, wherein the supernatant of the plant juice is taken, the pH value is regulated to be 4 to 6, the precipitate is centrifugally taken and suspended in PBS buffer solution, and the supernatant is taken and filtered to obtain the solution of the plant exosomes; mixing the plant exosome solution with a mixture of phospholipids and cholesterol to obtain plant exosome-liposomes; mixing the plant exosome-liposome with porous starch, and removing supernatant to obtain plant exosome-liposome/porous starch granules; mixing plant exosome-liposome/porous starch granules with sodium alginate, and then utilizing CaCl 2 Solidifying the solution to obtain the oral plant exosome hydrogel.
8. The method for preparing the hydrogel of oral plant exosomes for target-adjusting intestinal tract according to claim 7, wherein the plant juice supernatant with the pH value adjusted to 4-6 is mixed and incubated with polyethylene glycol before centrifugation and precipitation.
9. The oral plant exosome hydrogel for targeted regulation of intestinal tract according to claim 7, wherein the obtained plant exosome-liposome/porous starch granules are mixed with starch gelatinized into gel, and then mixed with sodium alginate.
10. Use of the hydrogel of oral plant exosomes for targeted regulation of intestinal tract according to any one of claims 1 to 6 in preparation of medicines or health products for regulating intestinal tract.
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