Oral spore microsphere for targeted release of intestinal tract and application of oral spore microsphere

Abstract

The invention relates to preparation of a spore-containing enteric targeted release microsphere and application of the spore-containing enteric targeted release microsphere in nicotine degradation. A probiotic bacillus subtilis 168 strain is subjected to engineering modification, so that a nicotine degrading enzyme is displayed on the surface of a spore of the probiotic bacillus subtilis 168 strain. In order to realize targeted release of intestinal tracts and reduce loss of recombinant spores in a gastric acid environment, the recombinant spores are wrapped by natural polymeric materials sodium hyaluronate and sodium alginate with pH responsiveness, and the spore microspheres with enteric targeted release are prepared. The microspheres greatly protect recombinant spores, effectively realize'interception type degradation 'of nicotine before nicotine is absorbed into blood by intestinal tracts, and continuously degrade nicotine diffused from blood by a'metabolic siphon' effect, so that the concentration of nicotine combined with a central nicotine receptor is remarkably reduced; brain dopamine reward effect induced by smoking is weakened fundamentally, positive feedback circulation of smoking-pleasant sensation is broken effectively, and visceral organs are protected from toxic and side injury of nicotine.

Description

Technical Field

[0001] This invention pertains to microspheres, specifically to an oral spore microsphere for targeted release into the intestine and its application. Background Technology

[0002] Smoking is the only preventable cause of death, yet due to the addictive and highly toxic nature of nicotine, it still directly or indirectly causes more than 6 million deaths each year. After smoking, inhaled nicotine is rapidly transported to various organs through the bloodstream. The half-life of ingested nicotine in the human body is about 2 hours. The liver, as the main metabolic organ, contains cytochrome P450 enzymes (CYP), such as CYP2A6 and CYP2B6, which are the main nicotine-metabolizing enzymes that can convert nicotine into cotinine (Pharmacogenet Genomics. 2007;17(12):1007-1015.; Arch Toxicol. 1999;73(2):65-70.). However, because nicotine can cross the blood-brain barrier and bind to nicotinic acetylcholine receptors in the brain to produce neurological effects, and because long-term smoking causes irreversible damage to the liver, the body's own metabolic system is insufficient to eliminate the effects of nicotine ingested from smoking. Traditional metabolic therapies aim to reduce nicotine metabolism and thus decrease the frequency of nicotine intake by inhibiting the activity of nicotine-metabolizing enzymes in the liver (Nicotine Tob Res.2014;16(1):18-25.; J Pharmacol Exp Ther. 2020;372(1):21-29.). However, this is ultimately a symptomatic treatment that does not address the root cause of the problem. Nicotine replacement therapy (Cochrane Database Syst Rev. 2008;(1):CD000146) follows the same treatment approach.

[0003] Effective degradation of nicotine in the blood and brain through exogenous intake of nicotine-degrading enzymes has become a hot research topic in this field in recent years. Multiple teams both domestically and internationally have been working on nicotine-degrading bacteria. Pseudomonas putidaThe drug-like properties of the nicotine-degrading enzyme NicA2 isolated from S16 were systematically studied. Although its effectiveness was verified at the animal level, its further conversion was not conducive due to the unavoidable immunogenicity of the enzyme during intravenous injection, blood half-life, cost, and ease of use. Moreover, at the animal level, the dosage of NicA2-J1 was as high as 70 mg / kg to significantly reduce the blood nicotine concentration (Biochemistry. 2005;44(50):16701-16709.; Diabetes Obes Metab. 2010;12(3):179-185.). Based on the physicochemical properties of nicotine molecules and their ability to easily travel through multiple tissues such as the brain, blood, and intestines, nicotine diffused from the blood can be continuously degraded by the "metabolic siphon" effect produced by intestinal probiotics. This significantly reduces the concentration of nicotine that binds to central nicotine receptors, fundamentally weakening the dopamine reward effect induced by smoking, effectively breaking the positive feedback loop of "smoking-pleasure," and providing a new approach for the treatment of nicotine dependence.

[0004] Previous patent applications utilized the stress resistance and unique surface protein properties of Bacillus subtilis spores to artificially imbue nicotine-degrading enzymes onto the spore surface, preparing an orally engineered live microbial preparation. This preparation, after colonization of the intestines, continuously degrades nicotine, thus reducing nicotine dependence (see patent application 202511433221.2, filed October 9, 2025). However, the oral administration of this engineered live microbial preparation was not ideal. Therefore, achieving zero leakage in the acidic gastric environment and zero-order disintegration throughout the entire intestinal tract at pH 6.0–7.5 is a pressing technical challenge in this field. Summary of the Invention

[0005] Purpose of the invention: This invention further combines recombinant spores with materials science to construct spore-containing microspheres EN@MP that are released into the intestine in a targeted manner. These spore-containing microspheres can achieve zero leakage in the gastric acid environment and zero-order disintegration and release throughout the entire intestinal segment at pH 6.0–7.5.

[0006] Technical solution: An oral spore microsphere for targeted release into the intestine, characterized in that it is prepared by the following steps: (1) A solution containing sodium alginate and sodium hyaluronate was used as the aqueous phase to encapsulate recombinant spores; (2) Using liquid paraffin as the oil phase, add emulsifier, stir and mix well, then add water phase and stir and mix well to obtain water-in-oil W / O primary emulsion. (3) Add the colostrum dropwise to the calcium chloride solution to form microspheres, then centrifuge and discard the supernatant; (4) Use one or more of chitosan, glyceryl monostearate, sodium tripolyphosphate, etc. for coating.

[0007] As a preferred method: The amount of sodium hyaluronate used in step (1) is 1–3% (wet / vol), and the amount of sodium alginate is 2–4% (wet / vol); the amount of spores used is the amount of spores collected from 100–1000 mL of DSM medium; mannitol or mannitol-lactose-sorbitol solution is used as a protectant for recombinant spores; and 3% (wet / vol) whey protein and 1.0% (wet / vol) calcium carbonate, sodium bicarbonate or one or more bases are added as an acid-consuming layer to neutralize the gastric acid environment; In step (2), add 0.5–1% (vol / vol) of Span-80 to the liquid paraffin, stir at 400 rpm until small bubbles appear, then add the aqueous phase dropwise to the oil phase while maintaining emulsification at 600 rpm for 10 min to obtain the W / O pre-emulsion. In step (3), the amount of calcium chloride used as a crosslinking agent is 2–4% (wet / vol); In step (4), the microspheres are resuspended in chitosan solution for repeated coating; or after chitosan coating, sodium tripolyphosphate is used as the outermost coating. The collected microspheres are resuspended in sodium tripolyphosphate and stirred at room temperature for 5 min to form a white, dense coacervate membrane instantly. After freeze-drying, spore-containing microspheres for intestinal targeted release are obtained. The sodium tripolyphosphate is first dissolved in deionized water and then the pH is adjusted to 7.5–8.0 with sodium hydroxide solution.

[0008] Alternatively, microspheres can be added to a molten mixture of glyceryl monostearate and liquid paraffin, stirred to form a paste, and then sodium chloride solution can be added. After thorough stirring, the mixture can be demulsified, centrifuged at 4°C and 3000 rpm for 5 min, the supernatant can be discarded, and the mixture can be washed with water and then freeze-dried to obtain spore-containing microspheres for targeted release into the intestine. The freeze-drying process is carried out at a temperature of -80 to -50°C for 12 to 24 hours.

[0009] The ratio of oil phase to crosslinking agent is 1:1 (vol / vol); the ratio of oil phase to water phase is 2:1 (vol / vol).

[0010] The spore microspheres are characterized by being able to achieve zero-order disintegration and release throughout the entire intestinal tract at pH 6.0–7.5, while exhibiting zero leakage in the acidic environment of the stomach.

[0011] The application of the spore microspheres in the preparation of drugs for treating nicotine addiction, assisting in smoking cessation, or reducing the toxic effects of nicotine on those exposed.

[0012] Specifically: This invention employs an emulsification-external crosslinking and double-layer coating strategy to prepare spore-containing microspheres, as detailed below: Using 2–4% (wet / vol) sodium alginate and 1–3% (wet / vol) sodium hyaluronate as the aqueous phase, and adding 3% (wet / vol) whey protein and 1% (wet / vol) calcium carbonate powder as the acid-consuming layer, the collected recombinant spores were washed with PBS and then resuspended with mannitol alone or the combined protective agent mannitol-lactose-sorbitol before being added to obtain the final aqueous phase; for the preparation of the colostrum, liquid paraffin was used as the oil phase and Span-80 was used as the emulsifier. After stirring and mixing, the aqueous phase was added with a syringe and stirred and mixed to obtain a water-in-oil (W / O) colostrum.

[0013] Gel microspheres were prepared using an external cross-linking method. The obtained colostrum was added dropwise to a 2–4% (wet / vol) calcium chloride solution using a syringe, instantly forming microspheres. The microspheres were then centrifuged, the supernatant discarded, and the microspheres were washed twice with water. They were then resuspended in a 0.6% (wet / vol) chitosan solution (pH 4.8, 1% glacial acetic acid solution), stirred at low speed for 15 min, centrifuged, and the supernatant discarded. This chitosan coating process was repeated. Preferably, 0.2% (wet / vol) sodium tripolyphosphate was used as the outermost coating, utilizing electrostatic ionic cross-linking. The -NH3 group of chitosan... + With sodium tripolyphosphate P3O 10 5- A dense ionic network is formed instantaneously through electrostatic and hydrogen bonding. The collected microspheres are resuspended in sodium tripolyphosphate (pH 7.5–8.0) and stirred at room temperature for 5 min, instantly forming a white dense coacervate membrane. After centrifugation to remove the supernatant, the microspheres are washed with water and then freeze-dried under vacuum to obtain spore-containing microspheres for targeted release into the intestine.

[0014] Preferably, 3% (wet / vol) sodium alginate and 1% (wet / vol) sodium hyaluronate are used as the aqueous phase, and 3% (wet / vol) whey protein and 1.0% (wet / vol) calcium carbonate powder are added as the acid-consuming layer. The collected recombinant spores are washed with PBS and resuspended in mannitol alone or a combined protectant mannitol-lactose-sorbitol before being added to obtain the final aqueous phase. For the preparation of the colostrum, liquid paraffin is used as the oil phase, and Span-80 is used as the emulsifier. After stirring and mixing, the aqueous phase is added using a syringe, and the mixture is stirred and mixed to obtain a water-in-oil (W / O) colostrum. Preferably, an external cross-linking method is used to prepare gel microspheres. The obtained colostrum is added dropwise to a 4% (wet / vol) calcium chloride solution using a syringe, instantly forming microspheres. The supernatant is then discarded by centrifugation, and the microspheres are washed twice with water and resuspended in a 0.6% chitosan solution (pH 4.8, 1% glacial acetic acid solution) and incubated with low-speed stirring for 15 minutes. After centrifugation for min, the supernatant was discarded, and the chitosan coating process was repeated. Preferably, 0.2% (wet / vol) sodium tripolyphosphate was used as the outermost coating, utilizing electrostatic ionic cross-linking. This allows the chitosan's -NH3... + With sodium tripolyphosphate P3O 10 5- A dense ionic network is formed instantaneously via electrostatic and hydrogen bonding. The collected microspheres are resuspended in sodium tripolyphosphate (pH 7.5–8.0) and stirred at room temperature for 5 min, instantly forming a white, dense coacervate membrane. After centrifugation to remove the supernatant, the microspheres are washed once with water and then freeze-dried under vacuum to obtain EN@MP microspheres for intestinal-targeted release.

[0015] This invention employs an emulsification-external crosslinking and double-layer coating strategy to prepare spore-containing microspheres. Preferably, 3% (wet / vol) sodium alginate and 1% (wet / vol) sodium hyaluronate are used as the aqueous phase, with 3% (wet / vol) whey protein and 1.0% (wet / vol) calcium carbonate powder added as the acid-consuming layer. The collected recombinant spores are washed with PBS and resuspended in mannitol alone or a composite protectant mannitol-lactose-sorbitol before being added to obtain the final aqueous phase. For the preparation of the colostrum, liquid paraffin is used as the oil phase, and Span-80 is used as the emulsifier. After stirring and mixing, the aqueous phase is added using a syringe, and the mixture is stirred until a water-in-oil (W / O) colostrum is obtained. Preferably, an external crosslinking method is used to prepare gel microspheres, and the obtained colostrum is added dropwise using a syringe to a concentration of 4% (wet / vol). Microspheres are instantaneously formed in a calcium chloride solution. After centrifugation and discarding the supernatant, the mixture is washed twice with a 2% calcium chloride solution and resuspended in a 0.6% chitosan solution (pH 4.8, 1% glacial acetic acid solution). After incubation with low-speed stirring for 15 min, the mixture is centrifuged and the supernatant is discarded. This chitosan coating process is repeated. Preferably, 0.2% (wet / vol) sodium tripolyphosphate is used as the outermost coating, utilizing electrostatic ionic cross-linking. The -NH3 group of chitosan... + With sodium tripolyphosphate P3O 10 5- The collected microspheres were resuspended in sodium tripolyphosphate (pH 7.5–8.0) by instantaneously forming a dense ionic network through electrostatic and hydrogen bonding. The mixture was stirred at room temperature for 5 minutes, and a white dense coacervate membrane was formed instantly. After centrifugation to remove the supernatant, the microspheres were washed with water and then freeze-dried under vacuum to obtain EN@MP microspheres for intestinal-targeted release.

[0016] Preferably, the swelling and release effects of the spore microspheres, as well as their in vitro nicotine degradation activity, were investigated using artificial gastric fluid (SGF, pH 2.0, containing pepsin) and artificial small intestinal fluid (SIF, pH 6.8, containing trypsin and bile salts).

[0017] Preferably, C57BL / 6 mice were gavaged after being labeled with the near-infrared dye DiR, and the retention and disintegration of the spore microspheres in the gastrointestinal tract were observed by in vivo imaging.

[0018] Preferably, the biocompatibility of the spore-forming microspheres was verified at the cellular level, and in C57BL / 6 mice, it was verified that they did not affect or disrupt the gut microbiota homeostasis, and could, to some extent, increase... Lactobacillus The abundance of probiotics.

[0019] Preferably, the effect of spore microspheres on nicotine withdrawal was evaluated in a mouse model of acute nicotine dependence. The effect was comparable to that of the positive control drug varenicline, and the spore microspheres did not have the safety or potential for drug abuse.

[0020] Preferably, in the storage stability study of spore microspheres, after treatment at 4, 37, and 50 ℃ for 0, 7, 14, 21, and 30 days, respectively, there were no significant differences in the number of spore germination colonies and nicotine degradation activity, indicating that the spore microspheres have extremely strong storage stability and are suitable for subsequent product shelf storage.

[0021] An oral spore microsphere for intestinal targeted release and its application, characterized in that it can be used as an adjunct to smoking cessation / nicotine dependence treatment, such as degrading nicotine in the intestine, reducing blood concentration, weakening addiction, and alleviating withdrawal symptoms.

[0022] An oral spore microsphere for intestinal targeted release and its application are characterized by their ability to prevent and treat smoking-related metabolic diseases, such as by degrading nicotine, repairing the gut microbiota damaged by nicotine, and improving fatty liver and insulin resistance.

[0023] An oral spore microsphere for targeted release into the gut and its application, characterized in that it can be used to reduce the risk of smoking-related cancers, such as reducing the carcinogenic effect of nicotine and thus reducing the risk of related cancers.

[0024] An oral spore microsphere for targeted release into the gut and its application, characterized in that it can be used to reduce the harm of secondhand smoke exposure, such as helping non-smokers degrade ingested secondhand smoke nicotine and reduce health damage.

[0025] The oral spore microspheres can also be added to various foods and dairy products to prepare new food or health products.

[0026] Beneficial effects: Oral administration offers convenience, low toxicity and side effects, and better patient compliance. Targeted release materials encapsulate engineered microorganisms, maximizing bioavailability. Sodium alginate (SA) is a natural polymer extracted from brown algae, kelp. It possesses excellent biocompatibility, non-toxicity, and biodegradability, and its structure contains a large number of -COO groups. - It exhibits polyanionic behavior in aqueous solution and can react with divalent cations (such as Ca). 2+Cross-linking forms a gel under mild conditions, effectively preventing the inactivation of active substances (Biomacromolecules. 2006;7(5):1471-1480.). Calcium alginate gel has good pH response characteristics, shrinking in the acidic environment of the stomach and swelling in the weakly alkaline intestinal environment; Sodium hyaluronate (HA) has pH response like SA (Pharm Res. 2004;21(11):2040-2047.), and HA is a natural extracellular matrix component that specifically binds to the CD44 receptor of intestinal epithelium (Wounds.2016;28(3):78-88.), prolonging the retention time of microspheres at the absorption site. Whey protein is a soluble protein found in whey, and has been used in the food industry as a protectant to improve the tolerance and survival rate of lactobacilli in the acidic environment of the stomach (J Food Sci. 2007;72(1):M031-M37.). Microspheres are formed using the above materials, but for recombinant spores, the following problems still need to be solved: (1) Zero leakage in the gastric acid environment and zero-order disintegration and release in the whole intestinal segment at pH 6.0–7.5. (2) The recombinant spores can freely colonize in the intestine.

[0027] This invention has been proven through numerous experiments: (1) The particle size of the prepared microspheres after freeze-drying is concentrated in 600–900 nm and the particle size distribution is uniform. Compared with the blank microspheres, the microspheres containing spores are yellowish-brown in color, and the TEM electron microscopy results show that the microspheres containing spores have the typical elliptical-short cylindrical shape of Bacillus subtilis spores.

[0028] (2) The prepared microspheres exhibit good pH responsiveness, remaining largely intact in acidic and weakly acidic environments (pH≤5.5), while showing significant swelling in neutral and weakly alkaline solutions (pH≥6.0), with a swelling ratio >10. Furthermore, in solutions with pH 6.5–7.4, the microsphere edges become noticeably blurred, and disintegration begins. Even after incubation in SGF-I solution for 2 hours, only about 10% of spores are released. However, once transferred to SIF-I solution, spores are released rapidly, and the number of spores counted after prolonged incubation in SIF-I is not significantly different from that immediately after transfer. In addition, spore counts in the upper liquid during microsphere preparation show that only <10% of spores remain unencapsulated, indicating that this spore microsphere preparation process can achieve >90% successful spore encapsulation.

[0029] (3) The microspheres prepared in this invention are biocompatible and do not cause cell damage at the cellular level, so it is expected that no safety issues will arise in vivo.

[0030] (4) The recombinant spore microspheres showed comparable activity to 3 mg of dried spores. 15 mg of recombinant spore microspheres completely degraded nicotine in the system within 20 min, with a specific enzyme activity of 27 U / mg. The number of spore germination colonies (10-10) of the recombinant spore microspheres after treatment at 4, 37, and 50 ℃ for 0, 7, 14, 21, and 30 days was also measured. 10 There was no significant difference in CFU / mL and nicotine degradation activity; both could completely degrade the added nicotine within 20 min, indicating that the spore microspheres have extremely strong storage stability and are suitable for subsequent product shelf storage.

[0031] (5) Recombinant spore microspheres indicate that the spore microspheres administered by gavage did not cause intestinal flora disorder and could increase the abundance of probiotic genus Lactobacillus to a certain extent.

[0032] Finally, it should be noted that the embodiments of the present invention are merely examples for clearly illustrating the invention and are not intended to limit the implementation of the invention. Those skilled in the art will be able to make various obvious changes, readjustments, and substitutions without departing from the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention. Attached Figure Description

[0033] Figure 1 It is a microsphere morphological observation; Figure 2 This describes the swelling of spore microspheres in solutions with different pH values; Figure 3 This describes the spore release effect of spore microspheres in simulated intestinal and gastric fluids; Figure 4 The results of the microsphere biocompatibility study include the Calcein-AM / PI experiment (A) and the CCK-8 assay (B). Figure 5 The study investigated the storage stability of spore microspheres, including spore germination count (A) and changes in activity (B). Figure 6 The images show fluorescence microscopy observation of DiR-labeled spore microspheres (A) and in vivo imaging observation (B). Figure 7 The study included the detection of fecal spores and changes in mouse body weight after gavage administration of microspheres. Figure 8 These are the 16S rDNA intestinal flora sequencing results of mouse feces after gavage administration of microspheres; Figure 9 This is a diagram showing the activity trajectory of mice in an open field experiment during a suitable oral nicotine dosage test. Figure 10These are the results of the open field test (A) and the elevated cross maze test (B) of the behavioral tests conducted on mice after their first exposure to nicotine. Figure 11 These are the results of a mechanical pain threshold assay in mice; Figure 12 This is a schematic diagram illustrating the principle of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further explained in detail below with reference to the embodiments and accompanying drawings. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available; experimental methods without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. All animal research procedures have been approved by the Animal Ethics Committee of China Pharmaceutical University.

[0035] The reagents used in the examples and their sources are as follows: liquid paraffin was purchased from Nanjing Chemical Reagent Co., Ltd.; food-grade sodium alginate was purchased from Bid Pharmaceutical; food-grade sodium hyaluronate was purchased from Shandong Freda Biochemical Co., Ltd.; pharmaceutical-grade Span-80, food-grade sodium tripolyphosphate, and chitosan were purchased from Aladdin; whey protein and glyceryl monostearate were purchased from Shanghai Yuanye Bio-Reagent Co., Ltd.

[0036] This invention relates to recombinant spores derived from patent application 202511433221.2. BS 168::ΔFrame-p43-EN-Δ、 B. subtilis 168 and BS 168::EN.

[0037] The percentage of solid substances involved in this invention is wet / vol%, i.e., weight-volume percentage. The percentage of liquid substances involved is vol / vol%, i.e., volume percentage.

[0038] Example 1, Preparation of oral spore microspheres for targeted release into the gut: To achieve targeted release into the gut, protect recombinant spores from gastric acid, and prolong their retention time in the gut, a spore-containing enteric-coated microsphere based on sodium hyaluronate and sodium alginate was designed. Specifically, based on previous studies (CN102657871B, CN111282016A), the enteric-coated microspheres were prepared using a sodium hyaluronate-sodium alginate-calcium chloride crosslinking emulsification method.

[0039] Taking the preparation of 20 mL of aqueous phase as an example, weigh out 3% (wet / vol) sodium alginate and 1% (wet / vol) sodium hyaluronate and dissolve them in 10 mL of sterile deionized water. After absorbing water and swelling, add 5 mL of whey protein solution (final concentration 3% (wet / vol), 20 mL) that has been swollen with deionized water and adjusted to pH 7.5 with sodium hydroxide solution, and 1.0% (wet / vol) calcium carbonate fine powder. Mix well, then add 5 mL of spore solution and mix well to obtain the final aqueous phase. Spores are collected from 100–1000 mL of DSM medium, equivalent to 50–500 mg of dry spore powder. After washing twice with PBS, store at 4°C and resuspend in 2% (wet / vol) mannitol or the compound protectant mannitol-lactose-sorbitol solution (6-3-3%) before the experiment. After whey protein swells, the pH needs to be adjusted to 7.5 to prevent it from subsequently complexing with sodium alginate and sodium hyaluronate. Calcium carbonate, acting as an intermediate acid-consuming layer, can raise the local pH to around 4.0.

[0040] A W / O emulsification method was used to prepare the primary emulsion to control the final microsphere particle size. Specifically, 40 mL of liquid paraffin was measured and 0.5% (vol / vol) of Span-80 was added as an emulsifier. The mixture was stirred at 400 rpm until small bubbles appeared. Then, the aqueous phase was added dropwise to the oil phase using a 20 mL syringe while emulsifying at 600 rpm for 10 min to obtain the W / O primary emulsion.

[0041] External gelation was used to crosslink calcium chloride, forming a dense outer shell on its surface. Then, with stirring during crosslinking, ions diffused from the outside inwards, creating a gradient crosslinking structure that was dense on the outside and solid on the inside. Specifically, the proemulsion was added dropwise to 40 mL of calcium chloride solution (4%, wet / vol) using a 20 mL syringe, allowing for instantaneous crosslinking and solidification. After stirring at 600 rpm at room temperature for 1 h, and once the microspheres had mostly settled, they were transferred to a 50 mL centrifuge tube and centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was discarded, and the microspheres were washed once with 2% calcium chloride solution and once with water to obtain the crosslinked microspheres.

[0042] A double-layer coating technique was used to further process the microspheres to reduce gastric acid permeability. The cross-linked microspheres were resuspended in 15 mL of a 0.6% high-viscosity chitosan solution, stirred and incubated at 300 rpm for 15 min, then centrifuged at 4°C and 3000 rpm for 5 min. The supernatant was discarded, and the chitosan coating process was repeated. The chitosan solution was prepared with 1% glacial acetic acid, pH ≤ 5.5. Finally, the collected spore-forming microspheres were resuspended in 15 mL of a 0.2% sodium tripolyphosphate solution, stirred and incubated at room temperature at 300 rpm for 5 min, instantly forming a dense white coacervate membrane. After centrifugation and washing with water, the prepared microspheres were obtained. This process utilizes the -NH3 group of chitosan. + With sodium tripolyphosphate P3O 10 5- Sodium tripolyphosphate is first dissolved in deionized water and then the pH is adjusted to 7.5–8.0 using sodium hydroxide solution to form a dense ionic network instantaneously through electrostatic and hydrogen bonding.

[0043] Example 2, Preparation of oral spore microspheres for intestinal-targeted release (glyceryl monostearate as outer coating): Similar to Example 1, glyceryl monostearate was used instead of chitosan and sodium tripolyphosphate as the outer coating for encapsulating the spore microspheres. Specifically, glyceryl monostearate, as the outer coating, has a naturally hydrophobic surface, providing a "hard shell" for acid-sensitive substances. After entering the intestine, the lipids are emulsified by bile salts, achieving burst release, and its metabolites, glycerol and stearic acid, can serve as energy-providing substances. Specifically, 1 g of glyceryl monostearate was weighed and mixed with 10 mL of liquid paraffin, melted at 60°C, and kept at 55°C to maintain a transparent liquid state. Then, the cross-linked microspheres were added and stirred to form a paste. Two volumes of 5% (wet / vol) sodium chloride solution were added, and the mixture was stirred thoroughly to break the emulsion. The mixture was centrifuged at 4°C and 3000 rpm for 5 min, the supernatant was discarded, and the microspheres were washed with water to obtain the prepared microspheres.

[0044] The prepared microspheres were dried in a low-temperature freeze-dryer for 12–24 h until constant weight was achieved.

[0045] Example 3, Preparation of oral spore microspheres for intestinal-targeted release (with calcium chloride bilayer as outer coating): Taking the preparation of 20 mL of aqueous phase as an example, weigh out 2% (wet / vol) sodium alginate and 1% (wet / vol) sodium hyaluronate and dissolve them in 10 mL of sterile deionized water. After absorbing water and swelling, add 5 mL of whey protein solution (final concentration 3% (wet / vol), 20 mL) that has been pre-swollen with deionized water and adjusted to pH 7.5 with sodium hydroxide solution, and 1.0% (wet / vol) calcium carbonate fine powder. Mix well, then add 5 mL of spore solution and mix well to obtain the final aqueous phase. Spores are collected from 100–1000 mL of DSM medium, equivalent to 50–500 mg of dry spore powder. After washing twice with PBS, store at 4°C and resuspend in 2% (wet / vol) mannitol before the experiment. The pH of the whey protein needs to be adjusted to 7.5 after swelling to prevent subsequent complexation with sodium alginate and sodium hyaluronate. Calcium carbonate, acting as an intermediate acid-consuming layer, can raise the local pH to around 4.0.

[0046] A W / O emulsification method was used to prepare the primary emulsion to control the final microsphere particle size. Specifically, 40 mL of liquid paraffin was measured and 0.5% (vol / vol) of Span-80 was added as an emulsifier. The mixture was stirred at 400 rpm until small bubbles appeared. Then, the aqueous phase was added dropwise to the oil phase using a 20 mL syringe while emulsifying at 600 rpm for 10 min to obtain the W / O primary emulsion.

[0047] External gelation was used to crosslink calcium chloride, forming a dense outer shell on its surface. Then, with stirring during crosslinking, ions diffused from the outside inwards, creating a gradient crosslinking structure that was dense on the outside and solid on the inside. Specifically, the proemulsion was added dropwise to 40 mL of calcium chloride solution (4%, wet / vol) using a 20 mL syringe, allowing for instantaneous crosslinking and solidification. After stirring at 600 rpm at room temperature for 1 h, and once the microspheres had mostly settled, they were transferred to a 50 mL centrifuge tube and centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was discarded, and the microspheres were washed once with 2% calcium chloride solution and once with water to obtain the crosslinked microspheres.

[0048] The microspheres were further processed using a double-layer coating technique to reduce gastric acid permeability. The cross-linked microspheres were resuspended in 15 mL of a 0.6% high-viscosity chitosan solution, stirred and incubated at 300 rpm for 15 min, then centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was discarded, and the chitosan coating process was repeated. The chitosan solution was prepared with 1% glacial acetic acid, with a pH ≤ 5.5.

[0049] Example 4, Characterization of enteric-coated microspheres (microspheres obtained in Example 1): (1) Morphological observation of enteric-coated microspheres The prepared microspheres were morphologically observed using transmission electron microscopy (TEM, JEOL JEM-200EX, Japan) and scanning electron microscopy (SEM, JEOL JEM-200EX, Japan). The particle size distribution was then calculated using NanoMeasurer 1.2 software, and plotted using Origin 2024, followed by Gaussian fitting until convergence. Specifically, for SEM observation, the freeze-dried microspheres were dispersed in anhydrous ethanol, and then 5 μL was dropped onto an aluminum stage coated with conductive adhesive (carbon / copper adhesive) and allowed to air dry. Preferably, conductive coating was achieved using ion sputtering of Au powder at 5–10 nm, a current of 10 mA, and 30–60 s. The coated samples were then observed, with an accelerating voltage of 15 kV. For TEM observation, the microspheres dispersed in ethanol were dropped onto a 3 mm copper mesh (with a carbon support film) and allowed to air dry without further coating, using a voltage of 100 kV.

[0050] Result: As Figure 1 As shown, the particle size of the prepared microspheres after freeze-drying is concentrated in the range of 600–900 nm, and the particle size distribution is uniform. Compared with the blank microspheres, the microspheres containing spores have a yellowish-brown color, and the TEM electron microscopy results show that the microspheres containing spores have the typical elliptical-short cylindrical shape of Bacillus subtilis spores.

[0051] (2) pH responsiveness of enteric-coated microspheres 10 mg of dried spore-containing microspheres were weighed and added to 1 mL of solutions with pH values ​​of 1.2, 5.5, 6.0, 6.5, 7.2, and 7.4, respectively. After soaking for 30 min, the microspheres were centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the remaining solution was blotted dry with absorbent paper. The weights were then measured, and the spore swelling ratio (SR) was calculated. The results showed that the prepared microspheres had good pH responsiveness. They remained essentially unchanged in acidic and weakly acidic environments (pH ≤ 5.5), while significant swelling occurred in neutral and weakly alkaline solutions (pH ≥ 6.0), with a swelling ratio > 10. Furthermore, in solutions with pH 6.5–7.4, the edges of the microspheres became noticeably blurred, and disintegration began to occur. Figure 2 A pH response range of 6.0–7.4 covers the entire intestinal segment from the duodenum to the ileum, which is also the main area for nicotine absorption in the intestine.

[0052] (3) Encapsulation efficiency of enteric-coated microspheres and disintegration effect in simulated body fluids The spore release effect of the microspheres in simulated intestinal fluid (SIF-I, pH 6.8, enzyme-free) and simulated gastric fluid (SGF-I, pH 2.0, enzyme-free) was further investigated. Optimally, 10 mg of microspheres were weighed and added to 1 mL of SGF-I, incubated at 37℃ and 100 rpm. At 1 h and 2 h, 100 μL of the supernatant was taken, diluted to 10⁻⁵, and then 100 μL was spread onto an antibiotic resistance plate for spore counting. After 2 h of sampling, the microspheres were centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and 1 mL of SIF-I was added. Zero-order release kinetics were achieved upon addition. 100 μL of the supernatant from 2, 3, 4, and 5 h was diluted and spread onto antibiotic resistance plates for spore counting. An equal amount of spores was diluted and coated with the same dilution factor as a control group for spore counting. At the same time, the amount of spores remaining in the upper liquid during the preparation of spore microspheres was measured with the same dilution factor, and the encapsulation efficiency of the microspheres was calculated accordingly.

[0053] Result: As Figure 3 As shown, even after incubating the microspheres in SGF-I solution for 2 hours, only about 10% of the spores were released. However, once transferred to SIF-I solution, the spores were released rapidly, and the number of spores counted after prolonged incubation in SIF-I was not significantly different from that immediately after transfer. Furthermore, spore counts in the upper liquid during the microsphere preparation process showed that only <10% of the spores remained unencapsulated, indicating that this spore microsphere preparation process can achieve >90% successful spore encapsulation.

[0054] Example 5: Biosafety and activity assessment of enteric-coated microspheres: Considering that the microspheres prepared in this invention are intended for animal or human use, although most of the reagents used in this invention are food-grade and FDA-certified biosafety-safe, experimental verification at the cellular level was conducted to further provide evidence of safety and biocompatibility, and examples are provided.

[0055] (1) Biosafety study of enteric-coated microspheres Preferably, IEC-6 cells were used as the experimental subjects, and CCK8 and Calcein-AM / PI live / dead cell staining experiments were performed. The kit used for the CCK8 experiment was the SuperKine™ hypersensitive cell proliferation detection kit, and the kit used for the Calcein-AM / PI live / dead cell staining experiment was purchased from Yisheng Biotechnology. All operations were carried out in accordance with the manufacturer's instructions. Specifically, 100 μL of revived and digested cell suspension (1–5 × 10³ cells / well) was seeded into 96-well plates and cultured in a 5% CO₂ incubator for 24 h until the cells were basically adherent. Then, fresh complete culture medium was added, and 1 mg / mL microspheres were added to each well. The wells were divided into groups: PBS, blank microspheres (Blank@MP), wild-type spore microspheres (WT@MP), and recombinant spore microspheres (EN@MP). After co-incubation for another 24 h, 10 μL of CCK-8 reagent (SuperKine™ hypersensitive cell proliferation assay kit) was added to each well, and the wells were cultured for another 0.5–4 h. The absorbance at 450 nm was measured using a microplate reader (absorbance values ​​below 2.0 are practically meaningful). Each treatment had six parallel wells, and the outermost ring was blocked with PBS to avoid edge effects. After the CCK-8 assay, the solution was aspirated and the cells were washed three times with 1×PBS and the solution was discarded. 100 μL of Calcein-AM / PI live / dead cell double staining working solution was added to each well. After incubation at 37°C in the dark for 15 min, the cells were observed under an inverted fluorescence microscope IX73 (Olympus™). Live cells were observed under blue light excitation with yellow-green light, and the nuclei of dead cells were observed under yellow-green light excitation with red light.

[0056] Result: As Figure 4 As shown, similar to PBS treatment, cells in each microsphere treatment group grew normally, and the cells were mostly green under the microscope. There were fewer dead cells with nuclei stained red, indicating that the microspheres prepared in this invention are biocompatible and do not cause cell damage at the cellular level. Therefore, it is not expected that there will be no safety issues in vivo.

[0057] (2) Enteric-coated microsphere activity Preferably, based on the counting of spore-bearing microspheres and the calculation of the specific enzyme activity of recombinant spores, 15 mg of spore-bearing microspheres is used. Specifically, 15 mg of recombinant spore-bearing microspheres (dry weight) are resuspended in 500 μL of 1×PBS (pH 7.4), and 10 μL of 400 μM nicotine is added. After mixing, the mixture is incubated at 37°C. 100 μL samples are taken at 0, 10, 20, 30, 40, 50, and 60 min after incubation. The reaction is terminated by adding twice the volume of methanol, and 5 μL of internal standard d3-nicotine (500 μM, m / z=166.01, Shanghai Zhenzhun Biotechnology) is added. 2 μL of the sample is injected into LC-MS / MS, eluted with 95% methanol for 2 min, and the values ​​of 163.15 (NIC), 161.10 (NMM), and 166.01 are recorded using MRM positive ion mode. The signal abundances were 179.01 (PON), 163.15>130.15, 161.10>119.10, 179.01>106.15, and 166.01>130.15. A standard curve was plotted using the peak area of ​​the analyte / internal standard in the second-order response versus the concentration of the analyte / internal standard, and the enzyme activity of the recombinant spores was calculated based on the standard curve. Spore enzyme activity was defined as the amount of spore microspheres required to catalyze the degradation of 1 nM nicotine per minute at 37°C and a 1×PBS system. MS parameters in MRM mode are shown in Table 1 below.

[0058] Table 1. MS parameters for multiple reaction monitoring (MRM) in positive ion mode for analytes and internal standards. .

[0059] Results: The activity was comparable to that of 3 mg of dry spores. 15 mg of recombinant spore microspheres could completely degrade nicotine in the system within 20 min, and its specific enzyme activity was calculated to be 27 U / mg.

[0060] (3) Storage stability of enteric-coated microspheres To determine the storage conditions and shelf life of the enteric-coated microspheres prepared in this invention, in order to meet the relevant regulations and quality agreements of CDE and FDA, and to provide data support for industrialization, the differences in the activity and spore germination rate of the microspheres after treatment at different temperatures for different durations were investigated. Three 15 mg portions of spore-containing microspheres were weighed and treated at 4, 37, and 50°C for 0, 7, 14, 21, and 30 days, respectively. After resuspending in 500 μL of 1×PBS (pH 7.4), 10 μL of 400 μM nicotine was added and reacted for 20 min. 100 μL of the reaction solution was then used to terminate the reaction with an equal volume of methanol before LC-MS / MS analysis. Simultaneously, the reaction solution was serially diluted to 10⁻⁷ and plated onto antibiotic resistance plates to determine the spore germination rate after treatment at different temperatures for different durations. Microspheres stored at 4°C were used as a control.

[0061] Result: As Figure 5 As shown, there was no significant difference in the number of spore germination colonies (10¹⁰ CFU / mL) and nicotine degradation activity. Both could completely degrade the added nicotine in the system within 20 min, indicating that the spore microspheres have extremely strong storage stability and are suitable for subsequent product shelf storage.

[0062] Example 6: Effects of in vivo imaging and spore-forming microspheres on mouse gut microbiota: (1) In vivo imaging observation of recombinant spore microspheres DiR is a lipophilic fluorescent dye with strong near-infrared light penetration into cells or tissues, and exhibits low autofluorescence levels in the near-infrared region. These properties make it particularly suitable for in vivo imaging or tracing experiments. To determine the appropriate time interval between spore microspheres and nicotine gavage, and to observe the retention and distribution of spore microspheres in vivo, it is preferable to label the spore microspheres with 10 μM DiR dye (Yisheng Biotechnology) for in vivo imaging observation. Specifically, 15 mg of spore microspheres are weighed, resuspended in 500 μL of deionized water, and then DiR dye is added to a final concentration of 10 μM. After incubation at 37°C in the dark with shaking for 1 h, the microspheres are centrifuged at 5000 rpm for 5 min to remove unlabeled dye. The microspheres are then washed three times with deionized water and placed in a 6-well plate for observation under an inverted fluorescence microscope. Figure 6As shown in Figure A, the microspheres were essentially labeled with red fluorescence (DiR+). C57BL / 6 mice were then gavaged with 15 mg of the labeled microspheres, and DiR fluorescence (Em=748 nm, Ex=780 nm) was monitored at 1, 3, 6, 12, and 24 h post-gavage using the IVIS Lumina® III in vivo imaging system (Living Image® 4.8.2, Revvity). Mice were placed in a matching small animal gas anesthesia chamber, anesthetized with isoflurane, and then placed in a dark chamber. Mice gavaged with unlabeled microspheres (DiR-) served as controls, with three mice in each group (repeated). To facilitate abdominal imaging and avoid the influence of hair and temporary hair removal cream on the imaging results, the mice's abdomens were shaved with hair removal cream and an electric razor one day prior to the gavage. After gavage with the microspheres, the mice were allowed normal water intake. After in vivo imaging observation, the mice were euthanized by cervical dislocation, and the heart (H), liver (L), spleen (S), lung (P), kidney (K), brain (B), large intestine (LI), and small intestine (SI) were dissected and separated to further observe the distribution of fluorescence throughout the mouse body.

[0063] Result: As Figure 6 As shown in Figure B, the fluorescence signal was relatively strong within 3 hours after gavage, with strong fluorescence signals observed in the large and small intestines, while no fluorescence was detected in other organs. After 6 hours of gavage, the fluorescence signal significantly weakened, and no fluorescence signal was detected at 24 hours, indicating that the outer layer of the microspheres was essentially digested and excreted. The fact that fluorescence signals were generally detectable within 12 hours also suggests that the spores released by the microspheres were detectable in the intestines for at least 12 hours. The in vivo imaging results further provide evidence for the disintegration and release of the microspheres in vivo.

[0064] (2) Effects of spore-forming microspheres on the gut microbiota of mice In Example 5, the biocompatibility of the cellular-level spore microspheres was investigated. The results showed that the spore microspheres prepared in this invention are biosafe and do not cause cell damage. Furthermore, the encapsulated spores possess both nicotine degradation activity and catalase activity, degrading nicotine without causing oxidative stress damage to cells. Considering that the final site of action of the spore microspheres is the intestine, although Bacillus subtilis is a probiotic and the reagents used in the microsphere preparation process are mostly food-grade, the effects of the spore microspheres on the intestinal flora of mice were investigated for safety reasons. C57BL / 6 mice were preferably used as experimental subjects. After one week of acclimatization feeding, the experiment began. Six C57BL / 6 mice were administered blank microspheres (Blank@MP) or spore microspheres (EN@MP) by gavage. Mice not administered microspheres by gavage served as controls. The spore microspheres were resuspended in 0.5% sodium carboxymethyl cellulose to prevent microsphere aggregation. After administering 15 mg microspheres via gavage for 3 consecutive days, gavage was stopped and the mice were washed until day 7. Fresh feces were collected periodically using sterile containers and forceps, and the weight changes of the mice were recorded. Feces were collected at 0 d (A1), Blank@MP-1d, Blank@MP-3d (A2), Blank@MP-5d (A3), Blank@MP-7d (A4), EN@MP-1d, EN@MP-3d (C1), EN@MP-5d (C2), and EN@MP-7d (C3), and were plated and sent to Lianchuan Biotechnology for 16S rDNA sequencing analysis.

[0065] Result: As Figure 7As shown, no Bacillus subtilis growth was observed in the feces of mice that were not gavaged with spore-forming microspheres and those gavaged with Blank@MP on the resistance plate surface. However, the feces of mice gavaged with EN@MP for 1 day and 3 days contained spore counts of 4.1 × 10⁵ CFU / g and 4.7 × 10⁵ CFU / g, respectively. During the cleansing period after cessation of gavage, the spore count detected in the EN@MP group mice decreased to 1.2 × 10⁵ CFU / g on day 5 and further decreased to 6.3 × 10⁴ CFU / g on day 7. This indicates that the gavage-administered spores are excreted with the feces and gradually decrease, making it unlikely that excessive colonization would affect the intestinal flora. Alpha diversity analysis refers to the diversity within a specific habitat (or sample) to assess the richness and diversity of species in an environmental community. Chao1, observed_species, and ace indices primarily reflect community richness, used to estimate the number of species in the community. Shannon and Simpson indices primarily reflect community diversity; higher values ​​indicate greater richness and species homogeneity. 16S rDNA sequencing results also showed no significant difference in species diversity between groups C1, C2, and C3 and groups A1, A2, A3, and A4, except that group A2 had slightly lower species richness, possibly due to sampling error. Figure 8 Beta diversity analysis was used to analyze species differences between different environmental communities (or samples). The figure shows the results of principal component analysis (PCA), principal coordinate analysis (PCoA), and non-metric multidimensional scaling (NMDS). In different groups, the closer the samples are, the more similar the microbial composition and the smaller the differences, indicating that different treatments have little impact on the distribution of the bacterial community. Anosim similarity analysis results for each group also support this conclusion. Further species composition analysis revealed that although there was no significant impact on the bacterial community in terms of Alpha and Beta diversity, the EN@MP group clearly increased the abundance of the gut probiotic genus *Lactobacillus*, while *Bacillus subtilis* was only found in the top 200 abundance at the Genus level. Figure 8 The above results all indicate that the oral administration of spore-forming microspheres did not cause intestinal flora dysbiosis and could, to some extent, increase the abundance of the probiotic genus Lactobacillus.

[0066] Example 7, Pharmacodynamics of enteric-coated microspheres in animals: (1) Exploring the oral dosage of nicotine Mice are ideal model animals because they share more than 90% homology with human functional genes and are similar in genome sequence, development, biochemical metabolism and physiological characteristics. The metabolism of nicotine in the human body is mainly carried out by the cytochrome P450 enzyme CYP2A6 in the liver (Drug Metab Dispos. 2005;33(8):1166-1173.; Psychopharmacology (Berl). 2006;184(3-4):401-408.). The CYP2A5 in mice shares 84% ​​amino acid sequence similarity with it (J Pharmacol Exp Ther. 1997;282(3):1608-1614.; Biochem Pharmacol. 2001;62(8):1025-1036.), while in rats, nicotine is metabolized by enzymes of the CYP2B family (Biochem Pharmacol. 1993;45(12):2554-2556.). To simulate the scenario of nicotine intake from smoking, nicotine was administered to mice by gavage in this embodiment. After oral administration of nicotine to mice, it is usually absorbed into the brain within 5–15 minutes. Although the oral bioavailability of nicotine is only 20–30%, and it is strongly metabolized by the liver, according to previous reports (Mol Neurobiol. 2017;54(2):904-921.), subcutaneous injection of 0.2 mg / kg of nicotine tartrate can significantly increase the amount of spontaneous activity in mice during passive swimming. Therefore, the effects of oral administration of nicotine at low, medium, and high concentrations of 0.5, 1.0, and 1.5 mg / kg on the activity of mice were first investigated to observe the excitatory dose for subsequent experimental investigations. Three mice were used in each group, and the activity of mice within 10–30 minutes after oral administration of nicotine was recorded in an open field test. Specifically, nicotine was prepared using a 0.1% sodium saccharin solution at concentrations of 0.05 mg / mL (0.5 mg / kg), 0.10 mg / mL (1.0 mg / kg), and 0.15 mg / mL (1.5 mg / kg), with each mouse receiving 200 μL via gavage. The solution was freshly prepared (1 μL = 1.01 mg nicotine). Three C57BL / 6 mice were used in each group, with mice receiving only the 0.1% sodium saccharin solution serving as a blank control. Preferably, mice were treated with the 0.1% sodium saccharin solution three days prior to the experiment to allow for acclimatization and reduce novelty stress. At the start of the experiment, mice were placed in the center of a dedicated open-mouth box, and a video tracking system (ANY-maze, Stoelting, USA) was activated to record the mice's behavior within the box. At intervals, the open-mouth box was sprayed with 75% ethanol to reduce the influence of the previous mouse's odor on the results.

[0067] The results showed that administering nicotine by gavage 15 minutes beforehand was appropriate, followed by 10 minutes of open field activity for observation. Figure 9 As shown in the mouse activity trajectory diagram, both 0.5 and 1.0 mg / kg nicotine had a stimulating effect. Compared with the blank control, mice entered the central area more frequently and their activity trajectories were significantly more complex. However, 1.5 mg / kg nicotine had a depressive effect; mice tended to curl up in a corner, exhibited significantly reduced activity, and displayed symptoms of depression and anxiety. Subsequently, a 0.5 mg / kg (200 μL = 10 μg) nicotine solution was selected for the formal experiment.

[0068] (2) Effects of oral administration of spore microspheres on the behavior and nicotine metabolism of animals after initial nicotine exposure After determining the oral nicotine dosage and gavage interval, animal experiments were conducted to study the effects of oral administration of spore microspheres on the behavior and nicotine metabolism of animals after initial nicotine exposure. Specifically, 15 mg of spore microspheres were administered by gavage half an hour before the initial exposure, followed by gavage half an hour later with a nicotine solution (0.05 mg / mL) prepared with 0.1% sodium saccharin aqueous solution. The mice were divided into four groups: (a) 0.1% sodium saccharin aqueous solution control group, (b) WT@MP and nicotine group, (c) EN@MP and nicotine group, and (d) nicotine group, with 8 mice in each group. An open field test was conducted 15 minutes after nicotine administration, recording the mice's behavior in the open field, including total distance traveled, number of times they stood upright, number of times they groomed, number of times they entered the central area, and cumulative time. An elevated cruciate maze test was conducted after the initial test, recording the mice's activity in open and closed arms and in the central area. Behavioral data were recorded and analyzed using a Giandel video recording system and a DigBehv animal behavior analysis system. After a two-day washout period, pharmacokinetic experiments were conducted. Blood was collected from the eyes of anesthetized mice at 10, 30, 60, 120, 180, and 240 minutes after oral nicotine administration. Mice were then sacrificed, and intestinal contents, brain, and bladder fluid were collected to determine the levels of nicotine and its degradation products. Brain homogenate was used to determine the levels of dopamine (Shanghai Yuanye Biotechnology, Nanjing Boyan Biotechnology) and norepinephrine (Shanghai Yuanye Biotechnology). Simultaneously, body temperature changes in mice were monitored at each time point using a thermometer. The brain tissue homogenization process involved: after hair removal from the mouse head, craniotomy was performed to separate the brain, which was then washed with PBS and temporarily stored at -80°C to prevent oxidation and deterioration. Tissue homogenate was prepared using 100 mg tissue / 1 mL PBS. After homogenization, the homogenate was centrifuged at 1500 rpm for 10 min at low temperature to precipitate larger cell debris and organelles. The supernatant was centrifuged again to precipitate cell nuclei and cell membranes. The resulting supernatant was the brain tissue homogenate. Blood samples were stored in anticoagulant tubes containing 100 U heparin sodium (Aladdin™) with 0.1% formic acid added to prevent nicotine oxidation.

[0069] The results showed that mice treated with nicotine experienced a brief decrease in body temperature, from 36.5℃ to 35.3℃, which returned to normal within about 30 minutes. The decrease in body temperature was due to the pharmacological effects of nicotine on the nervous system and thermoregulatory center of mice. The decrease in body temperature was greater in groups (b) and (d) after nicotine treatment (-0.7℃ vs -0.3℃ in group (c)), indicating that the nicotine content reaching the brain of mice treated with EN@MP was significantly lower than that in groups (b) and (d), while the body temperature of untreated group (a) remained stable. Figure 10The results shown are behavioral test results for each group after initial nicotine exposure. In the open field experiment (OFT), compared to group (a), groups (b) and (d) showed significantly better results. Figure 9 The total distance traveled in group A and the time spent in the central area were significantly increased, indicating excitability. In contrast, the activity of mice in group (c) was similar to that in group (a), without significant excitability symptoms. The results of the elevated cross maze (EPM) test were consistent with those of the OFT test, i.e., groups (b) and (d) showed excitability, with significantly increased frequency of entry into the open arms and significantly increased duration and amount of activity within the open arms. Figure 9 B). In biochemical tests, the dopamine content in the brain homogenates of groups (b) and (d) was significantly higher than that in the blank control group and the recombinant spore microsphere group at 10–30 min. A large number of nicotine degradation products were detected in the intestinal contents of the recombinant spore microsphere group, indicating that nicotine rapidly reaches the brain, binds to the responding nicotinic choline receptors, and induces increased dopamine secretion, triggering an excitatory response. Oral administration of spore microspheres can reduce the concentration of nicotine absorbed into the blood, thereby reducing the level of excitation.

[0070] Example 8: Application of enteric-coated microspheres in nicotine addiction withdrawal in small animals: (1) Construction of a mouse model of acute nicotine dependence addiction The mouse addiction model was constructed based on recent work by Akinola (Brain Sci. 2019;9(10):244.) and Kim (BehavBrain Funct. 2024;20(1):1.), using intraperitoneal injection for nicotine treatment. Nicotine was dissolved in physiological saline and prepared fresh for immediate use, with varenicline (varenicline tartrate, Maclean®) as the positive control. The mice were divided into groups of 6 C57BL / 6 mice each: (a) saline control group, (b) nicotine group, (c) full-course EN@MP and nicotine group, (d) nicotine and EN@MP recovery group, and (e) nicotine and varenicline recovery group. Specifically, groups (b), (d), and (e) received intraperitoneal injections of 0.5 mg / kg nicotine once daily for 9 consecutive days; group (c) received intraperitoneal injections of 0.5 mg / kg nicotine after gavage administration of 15 mg of spore microspheres half an hour prior to the injection, for 9 consecutive days; and group (a) received an equal volume of physiological saline intraperitoneally. Treatment was stopped for one day on day 10, and behavioral observations were conducted, including OFT, EPM, and mechanical pain threshold measurements in mice, to determine the induction of withdrawal symptoms. Subsequently, concentration escalation was performed for four consecutive days, with intraperitoneal injections of 1.0 mg / kg nicotine. Group (d) began gavage administration of spore microspheres 30 minutes before the intraperitoneal injection of nicotine, and group (e) began gavage administration of varenicline 0.5 mg / kg. Treatment was stopped on day 14, and behavioral observations were conducted. Nicotine addiction withdrawal methods include spontaneous withdrawal and induced withdrawal. Mecamine hydrochloride (MEC) is a commonly used nicotine addiction symptom inducer. However, relevant studies (Behav Brain Funct. 2024;20(1):1.) have shown that MEC itself has an "antidepressant-like" effect, which directly interferes with the assessment of "withdrawal depression". Therefore, induced withdrawal is more suitable for physical scoring than for behavioral studies.

[0071] Meanwhile, another batch of mice were induced to quit by intraperitoneal injection of 2 mg / kg of mecamine hydrochloride (Shanghai Yuanye Biotechnology), and behavioral and physical signs were observed 10 minutes after the mecamine injection.

[0072] (2) Behavioral observation The open field experiment was performed as in Example 7, followed by the elevated cross maze experiment. The elevated cross maze was used to assess anxiety-like behavior in mice. Using a mouse elevated cross maze apparatus, the mouse was placed in the central area of ​​the maze with its head facing the open arm. A video tracking system was activated to record the number of times the mouse entered the open and closed arms within 5 minutes, with a dwell time >3 seconds considered valid. Similarly, to avoid the influence of the previous mouse's odor on the results, the elevated cross maze apparatus was sprayed with 75% ethanol at intervals. Physical signs were observed by placing the mouse in an acrylic column and recording video of the mouse's activity over 20 minutes, calculating the number of events for each signal.

[0073] (3) Measurement of mouse paw withdrawal threshold (PWT) – Von Frey fiber method In addition to common physical symptoms such as tremors, trismus, scratching, and depression, withdrawal symptoms in mice with nicotine addiction also include withdrawal hyperalgesia. The Von Frey fiber assay is commonly used to quantitatively determine the sensitivity of mice to mild mechanical stimuli under normal, inflammatory, or neurologically damaged conditions, thereby assessing changes in their mechanosensitive pain threshold. The pulse weight loss (PWT) of normal mice is typically between 1.0 and 4.0 g, while the PWT of hyperalgesic mice is significantly reduced. This method was used to determine the pain threshold in addicted mice.

[0074] (4) Detection of body fluid biochemical indicators After the behavioral experiments, blood was drawn from the heart via puncture. Mice were then euthanized, and intestinal contents, brain, and bladder fluid were collected for nicotine and degradation product analysis. Brain homogenate was used to measure oxidative stress indicators such as TAS, SOD, GPx, and MDA (Nanjing Mofan Biotechnology Co., Ltd.) (procedure followed the reagent manufacturer's instructions). Real-time quantitative PCR (qPCR) was used to measure the mRNA levels of α4, β2, and α7 nAChRs, with GAPDH as an internal control. Total RNA was extracted and cDNA was obtained using a genomic DNA removal and reverse transcription kit from Hunan Aikerui Biotechnology Co., Ltd. The qPCR system was prepared and run using the company's qPCR kit (using a Roche LightCycler® 96 instrument and accompanying software). The qPCR primer sequences are shown in Table 2, and the qPCR procedure settings are shown in Table 3.

[0075] Table 2. qPCR primer sequences used in the embodiments of this invention .

[0076] Table 3. qPCR program .

[0077] Results: To avoid interference from subjective judgment, a double-blind method was used for PWT and physical signs observation. The baseline pain threshold of untreated mice was determined to be 1.0 g. When the corresponding fiber filament was used to stimulate the mid-stomach of the hind limb, the mice rapidly and clearly retracted, raised, and licked the stimulated paw, indicating pain sensation. Figure 11 As shown, after 10 days of low-dose nicotine treatment, the PWT of groups (b), (d), and (e) decreased to 0.6 g, while group (c) was similar to group (a), remaining at 1.0 g PWT. This indicates that simultaneous gavage administration of spore microspheres during nicotine treatment can reduce the nicotine content acting on the brain. After a 4-day concentration ramp-up treatment, the PWT of groups (b) and (e) decreased to 0.4 g, while the PWT of groups (c) and (d) remained at 0.6 g. Although a decrease in PWT also occurred in group (c), it was significantly slower than that in groups (b) and (e). It is understandable that nicotine can rapidly cross the blood-brain barrier and act on nicotinic choline receptors in the brain, and therefore the rate of metabolism is lower than the rate of diffusion. However, it is clear that the effect of the microsphere treatment group (d) during the ramp-up phase was better than that of the varenicline intervention group (e). This is partly due to the decrease in nicotine levels in the body after microsphere treatment, and partly due to the mechanism of action of varenicline treatment. Intraperitoneal injection of 1.0 mg / kg nicotine has a strong sedative and depressive effect in mice. Normal mice treated with this dose showed significant weakness and immobility. However, after 10+4 days of treatment, the full-course nicotine treatment group (b) showed tolerance to the high dose and significantly higher activity levels than the other groups. In contrast, the varenicline treatment group (e) showed initial excitement followed by depression. This is related to varenicline's enhanced excitatory effect in mice by activating nicotinic choline receptors and its higher affinity (K). i =0.15 nM vs 1.6 nM Nicotine; Drug Healthc Patient Saf. 2010;2010(2):39-48. doi:10.2147 / DHPS.S6299).

Claims

1. An oral spore microsphere for targeted release into the intestine, characterized in that, Prepared according to the following steps: (1) A solution containing sodium alginate and sodium hyaluronate was used as the aqueous phase to encapsulate recombinant spores; (2) Using liquid paraffin as the oil phase, add emulsifier, stir and mix well, then add water phase and stir and mix well to obtain water-in-oil W / O primary emulsion. (3) Add the colostrum dropwise to the calcium chloride solution to form microspheres, then centrifuge and discard the supernatant; (4) Use one or more of chitosan, glyceryl monostearate, sodium tripolyphosphate, etc. for coating.

2. The oral spore microspheres according to claim 1, characterized in that, In step (1), the amount of sodium hyaluronate used is 1–3% (wet / vol), and the amount of sodium alginate used is 2–4% (wet / vol); the amount of spores used is the amount of spores collected from 100–1000 mL of DSM medium; mannitol or mannitol-lactose-sorbitol solution is used as a protectant for recombinant spores; and 3% (wet / vol) whey protein and 1.0% (wet / vol) calcium carbonate, sodium bicarbonate or one or more bases are added as an acid-consuming layer to neutralize the gastric acid environment.

3. The oral spore microspheres according to claim 1, characterized in that, In step (2), add 0.5–1% (vol / vol) of Span-80 to the liquid paraffin and stir at 400 rpm until small bubbles appear. Then, add the aqueous phase dropwise to the oil phase while maintaining emulsification at 600 rpm for 10 min to obtain the W / O primary emulsion.

4. The oral spore microspheres according to claim 1, characterized in that, In step (3), the amount of calcium chloride used as a crosslinking agent is 2–4% (wet / vol).

5. The oral spore microspheres according to claim 1, characterized in that, In step (4), the microspheres are repeatedly coated by resuspending them in chitosan solution; or after being coated with chitosan, sodium tripolyphosphate is used as the outermost coating. The collected microspheres are resuspended in sodium tripolyphosphate and stirred at room temperature for 5 min to instantly form a white, dense coacervate membrane. After freeze-drying, spore-containing microspheres for intestinal targeted release are obtained. The sodium tripolyphosphate is first dissolved in deionized water and then the pH is adjusted to 7.5–8.0 with sodium hydroxide solution. Alternatively, microspheres can be added to a molten mixture of glyceryl monostearate and liquid paraffin, stirred to form a paste, and then sodium chloride solution can be added. After thorough stirring, the mixture can be demulsified, centrifuged at 3000 rpm for 5 min at 4°C, the supernatant can be discarded, and the mixture can be washed with water and then freeze-dried to obtain spore-containing microspheres for targeted release into the intestine.

6. The oral spore microspheres according to claim 1, characterized in that, The freeze-drying process is carried out at a temperature of -80 to -50°C for 12 to 24 hours.

7. The oral spore microspheres according to claim 1, characterized in that, The ratio of oil phase to crosslinking agent is 1:1 (vol / vol); the ratio of oil phase to water phase is 2:1 (vol / vol).

8. The use of the oral spore microspheres according to any one of claims 1-7 in the preparation of medicaments for treating nicotine addiction, assisting in smoking cessation, or reducing the toxic effects of nicotine on users.

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