Method for embedding probiotics in konjac glucomannan-based thermosensitive + targeted microspheres
By using a probiotic encapsulation method based on konjac glucomannan thermosensitive and targeted microspheres, the problem of probiotics being easily destroyed during oral delivery was solved, realizing intelligent controlled release and precise targeted delivery of probiotics, thereby improving bioavailability and colonization efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU SPORT UNIV
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-05
AI Technical Summary
Current probiotic encapsulation carriers lack temperature sensitivity and targeting, which makes probiotics easily destroyed during oral delivery, resulting in low bioavailability and making it impossible to achieve intelligent controlled release and precise targeted delivery.
Using konjac glucomannan as the functional backbone, combined with N-isopropylacrylamide as the thermosensitive unit and glycidyl methacrylate as the crosslinking bridging unit, heat shock protein monoclonal antibodies were introduced through free radical copolymerization and epoxy ring-opening reaction to prepare copolymer microspheres, and probiotics were then encapsulated.
It achieves stable protection of probiotics at physiological temperatures and rapid release at pathological high temperatures, possesses intestinal targeting capabilities, improves the colonic reach rate and colonization efficiency of probiotics at lesion sites, and has good biocompatibility, making it suitable for large-scale production.
Smart Images
Figure CN122140632A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochemistry, specifically to a method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan. Background Technology
[0002] Probiotics, as live microorganisms that are beneficial to the host, can regulate the balance of intestinal flora and improve intestinal barrier function, playing an important role in the prevention and adjuvant treatment of intestinal diseases. However, as live microorganisms, probiotics are easily destroyed by gastric acid, bile salts and digestive enzymes in the digestive tract during oral delivery, resulting in a significant decrease in the number of live bacteria reaching the colon, which seriously affects their bioavailability and efficacy.
[0003] To address these issues, existing technologies often employ encapsulation techniques to construct a protective barrier for probiotics. Commonly used encapsulation carriers include sodium alginate-calcium ion gel, chitosan / tripolyphosphate complex, and liposomes. However, these carriers all have significant technical drawbacks: First, they lack temperature responsiveness, failing to achieve intelligent controlled release of probiotics based on changes in the intestinal microenvironment's temperature. They are prone to non-specific release at physiological body temperature (37°C) and cannot achieve rapid swelling and release in pathologically high-temperature regions of the intestine. Second, they lack precise targeting capabilities, relying solely on the carrier's physicochemical properties to achieve passive targeting in the colon. They cannot achieve active targeted release at lesion sites with impaired intestinal barrier function, leading to the loss of probiotics in healthy intestinal areas and low colonization efficiency at lesion sites. Third, some carriers, such as liposomes, have poor biocompatibility and high preparation costs, while sodium alginate gel has low mechanical strength and is prone to rupture during digestive tract transport, resulting in limited protective effects.
[0004] Konjac glucomannan (KGM) is a natural water-soluble dietary fiber extracted from konjac. It possesses excellent gelling properties, high water absorption and swelling capacity, and biocompatibility. Furthermore, it is not easily hydrolyzed by enzymes in the human upper digestive tract, allowing it to directly enter the colon and be utilized by intestinal microorganisms, making it an ideal matrix material for colonic drug delivery carriers. In addition, the KGM molecular chain contains numerous primary and secondary hydroxyl groups, which can serve as grafting sites for graft copolymerization. Through chemical reactions, it can combine with other functional molecules to form composite carriers, providing a structural basis for its functional modification. Therefore, the blending and modification of KGM with other functional polymers has become a research focus for colonic targeted drug delivery systems.
[0005] Under physiological or pathological temperature fluctuations, such as fever, exercise, hypermetabolism, or local intestinal inflammation, intestinal epithelial cells exhibit a stress response by highly expressing the heat shock protein HSP. Existing research indicates that HSP monoclonal antibodies can specifically recognize and bind to HSP proteins on the surface of intestinal epithelial cells in areas with impaired intestinal barrier function, achieving precise targeted delivery of active substances. However, current HSP antibody-based targeted delivery systems are mostly designed for the delivery of chemical drugs, lacking the ability to provide temperature-controlled protection for bioactive substances such as probiotics. Furthermore, the structural design of the carrier cannot simultaneously meet the multiple requirements of temperature-sensitive response, targeted binding, and probiotic encapsulation, and cannot regulate the release behavior of probiotics according to the temperature and tissue characteristics of the intestinal microenvironment.
[0006] In summary, developing a composite encapsulation carrier that combines temperature-sensitive intelligent controlled release, active intestinal targeting, and efficient protection of probiotics to achieve full protection, precise targeting, and intelligent release during oral delivery of probiotics is a technical challenge that urgently needs to be solved in the field of probiotic formulation research and development. Summary of the Invention
[0007] The purpose of this invention is to provide a method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan, in order to solve the problems mentioned in the background art, such as the lack of thermosensitivity and poor targeting of existing probiotic encapsulation carriers, the easy destruction of probiotics during oral delivery, and low bioavailability.
[0008] To achieve the above objectives, the present invention provides the following technical solution: a probiotic encapsulation method based on thermosensitive and targeted microspheres of konjac glucomannan, using konjac glucomannan... For functional backbone, N-isopropylacrylamide Thermosensitive unit, glycidyl methacrylate As a cross-linking bridging unit, it connects to heat shock proteins. The preparation of monoclonal antibody copolymer microspheres, followed by encapsulation of probiotics, includes the following steps: Step 1: Synthesize using a free radical copolymerization method Copolymerized microspheres; Step 2, for Ammoniation modification was carried out to prepare amino; Step 3: Through epoxy ring-opening reaction amino grafting to On the copolymer microspheres, to obtain Copolymerized microspheres; Step 4: Graft the HSP monoclonal antibody onto the residual epoxy group. On the copolymer microspheres, to obtain Monoclonal antibody copolymer microspheres; Step 5: Mix the above-mentioned copolymer microspheres with probiotic hyphae to complete the encapsulation of probiotics, obtaining... Monoclonal antibody-probiotic copolymer microspheres.
[0009] In this embodiment, step 1, , Azobisisobutyronitrile The mass ratio is 3.0:7.5:0.1, the reaction temperature for free radical copolymerization is 65℃, and the reaction time is 24h; the specific operation of step 1 is as follows: 3.0g of... Dissolve in 50 mL of tetrahydrofuran Add 7.5g of solvent. After the monomers are thoroughly mixed, nitrogen gas is bubbled into the mixture for 30 minutes to remove oxygen. Then, 0.1g of the mixture is added. As an initiator, the reaction was carried out under an inert atmosphere at 65°C with magnetic stirring for 24 hours. After the reaction, the product was poured into 500 ml of hexane for precipitation. This precipitation was repeated three times to remove unreacted monomers, thus solving the technical problems of oxygen termination and unreacted monomer residue affecting subsequent grafting in free radical copolymerization. This ensured the purity and reactivity of the copolymerized microspheres. The product was then dried overnight under vacuum at room temperature to obtain... Copolymer microspheres.
[0010] In this embodiment, step 2, The amination modification was performed using 2-chloroethylamine hydrochloride. As the amination reagent, the specific operation of step 2 is as follows: [The following text appears to be a separate, unrelated section:] ... Soaking in a solution with a mass concentration of 5% 25% Soak in aqueous solution for 30 minutes, dry at 65°C until the solvent is completely evaporated, and then immerse the product in a solution with a mass concentration of 5%. In a 25% weak alkali solution, at 50 The reaction was carried out at 90°C for 15 minutes. After 90 min of reaction, the product was precipitated with methanol, washed with distilled water, and dried at 65 °C to obtain... Amino.
[0011] In this embodiment, in step 3, the weak alkaline solution is a sodium bicarbonate solution, a sodium carbonate solution, or ammonia water, preferably a 10% sodium bicarbonate solution, without damaging the... Achieving high ammoniation efficiency while maintaining the main chain structure, and being more compatible The structural characteristics represent an innovative optimization of the natural polysaccharide modification process.
[0012] In this embodiment, step 3 is specifically performed as follows: Take 1.0g The copolymer microspheres dissolved in 100 mL Solvent, 0.4g The amino group was dissolved in 5 mL of methanol and then added to the above solution. The mixture was stirred at 60 °C for 24 h. The solvent was removed by rotary evaporation. The product was precipitated in a mixture of hexane and methanol (v / v) to remove unreacted precipitate. amino, after drying, yields Copolymer microspheres.
[0013] In this embodiment, step 4 is specifically performed as follows: prepare 1mg / The HSP monoclonal antibody solution was prepared in 20 mM pH 8.0 phosphate buffer; 10 mg 1 mL of copolymer microspheres The proportion of monoclonal antibody solution was determined by adding the copolymer microsphere powder to the antibody solution and stirring continuously at room temperature for 24 hours. The product was then washed sequentially with 20 mM pH 8.0 phosphate buffer and 20 mM pH 7.0 phosphate buffer. This two-step pH gradient washing process ensured both the adequacy of the grafting reaction and the maintenance of... To enhance the biological activity of antibodies, avoid damage to antibody targeting caused by strong acids, alkalis, and high temperatures, and solve the technical challenge of activity loss during grafting of biological antibodies onto polymeric carriers, unbound antibodies are removed, and the antibody is dried and preserved. Monoclonal antibody copolymer microspheres.
[0014] In this embodiment, in step 5, the mass ratio of copolymerized microspheres to probiotic mycelia is 10:4; the specific operation of step 5 is as follows: take 0.2g Monoclonal antibody copolymer microspheres were dissolved in 5 mL of deionized water, probiotic mycelia were added, and the mixture was magnetically stirred overnight at room temperature. After drying, the product was obtained. Monoclonal antibody-probiotic copolymer microspheres.
[0015] In this embodiment, the probiotics are one or more of Lactobacillus, Bifidobacterium, and Enterococcus, which ensures that the encapsulated probiotics have high biological activity and are compatible with the gel structure of the microspheres, thus solving the problem of a significant decrease in probiotic activity after encapsulation in the prior art. The probiotic hyphae are the mycelia of probiotic cells that have been activated and cultured to the logarithmic growth phase, collected by centrifugation, and washed with sterile water.
[0016] In this embodiment, the The minimum critical dissolution temperature of the monoclonal antibody-probiotic copolymer microspheres is 32–34℃. They are structurally stable at physiological temperatures of 25℃ and 37℃, but swell and rapidly release probiotics at pathological high temperatures of 42℃. Furthermore, they can be transmitted through… Monoclonal antibodies specifically recognize highly expressed [specific antibodies] on intestinal epithelial cells. Proteins that enable targeted delivery to the gut.
[0017] The combination of these two core properties is not found in existing probiotic encapsulation products, and its biocompatibility and resistance to gastric acid / bile salts are far superior to existing carrier products such as sodium alginate and chitosan. The microspheres have a core-shell composite structure, with probiotic hyphae forming the core. The antibody copolymer gel forms the shell, and the temperature-sensitive unit is integrated within the shell. The structure consists of a backbone unit KGM, a crosslinking unit GMA, and a targeting unit HSP antibody. These units are covalently linked to form a stable copolymer structure, rather than the physically mixed structure found in existing technologies. This significantly improves structural stability and functional synergy.
[0018] Compared with the prior art, the beneficial effects of the present invention are: This probiotic encapsulation method based on thermosensitive and targeted microspheres using konjac glucomannan utilizes konjac glucomannan. For functional backbone, N-isopropylacrylamide Thermosensitive unit, glycidyl methacrylate As cross-linking bridging units, heat shock proteins are synthesized through reactions such as free radical copolymerization and epoxy ring-opening grafting. Monoclonal antibodies were grafted onto copolymer microspheres to prepare... Monoclonal antibody copolymer microspheres are then used to encapsulate probiotics, resulting in a probiotic microsphere formulation that combines temperature-sensitive response and intestinal targeting capabilities.
[0019] 0. Furthermore, Thermosensitive unit and Monoclonal antibody targeting units are organically bound in The microspheres on the framework have a minimum critical dissolution temperature of 32–34℃, exhibit structural stability at 25℃ and 37℃, enabling full protection of probiotics, and rapid swelling and release of probiotics under the pathologically high temperature of 42℃ intestinal flora; simultaneously, through Antibodies specifically recognize intestinal lesions, enabling precise targeted delivery. This solves the technical problems of existing carriers being non-thermosensitive and having poor targeting, thereby improving the colonic reach rate of probiotics and the colonization efficiency of lesion sites.
[0020] 1. Furthermore, with natural dietary fiber As the functional framework, supplemented by and Functional modification resulted in copolymer microspheres that were non-cytotoxic, biocompatible, and Its gel structure and high water absorption and swelling capacity can form a dense protective barrier on the surface of probiotics, effectively resisting the destruction of gastric acid, bile salts and digestive enzymes, and ensuring the activity of probiotics.
[0021] 2. Furthermore, the preparation process employs conventional chemical reactions such as free radical copolymerization and epoxy ring-opening grafting. The reaction conditions are all at room temperature or medium temperature of 50-65℃, without the need for harsh conditions such as high temperature and high pressure. The material ratio, reaction time and temperature of each step can be precisely controlled, the product is easy to separate and purify, and the reagents used are all conventional chemical raw materials, which are inexpensive and suitable for large-scale industrial production.
[0022] 3. Furthermore, As a cross-linking bridging unit, excessive Not only for and The grafting provides epoxy groups and also preserves sufficient active sites for subsequent grafting of targeted antibodies, realizing the organic combination of thermosensitive unit, backbone unit and targeting unit; The colon-targeting properties of the substance and the active targeting properties of the HSP antibody create a synergistic effect. Thermosensitive properties and The protective properties of the gel create a synergistic effect, resulting in a high degree of unity between the encapsulation, targeting, and controlled release functions of the microspheres. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the probiotic encapsulation method based on thermosensitive and targeted microspheres of konjac glucomannan according to the present invention. Figure 2 This is a scanning electron microscope image of the microspheres. Detailed Implementation
[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0025] This invention provides a method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan. The core of this method lies in: using konjac glucomannan as the functional backbone, N-isopropylacrylamide as the thermosensitive unit, and glycidyl methacrylate as the cross-linking bridging unit, incorporating heat shock protein monoclonal antibodies to prepare copolymer microspheres, then encapsulating probiotics in these microspheres, and synthesizing the microspheres using a free radical copolymerization method. Copolymerized microspheres; Ammoniation modification was carried out to prepare Amino; through epoxy ring-opening reaction Amino grafting to On the copolymer microspheres, to obtain Copolymer microspheres; grafting HSP monoclonal antibodies onto [the target microspheres] using residual epoxy groups. On the copolymer microspheres, to obtain Monoclonal antibody copolymer microspheres were mixed with probiotic hyphae to encapsulate the probiotics, resulting in... Monoclonal antibody-probiotic copolymer microspheres; The thermosensitive unit and the HSP monoclonal antibody targeting unit are organically combined on the KGM backbone. The resulting microspheres have a minimum critical dissolution temperature of 32–34℃ and are structurally stable at 25℃ and 37℃, enabling full protection of probiotics. They rapidly swell and release probiotics at the 42℃ intestinal pathological high temperature. Simultaneously, the HSP antibody specifically identifies intestinal lesions, achieving precise targeted delivery. This solves the technical problems of existing carriers lacking thermosensitivity and poor targeting. Compared with the simple carrier mixing and embedding process in existing technologies, the process design is more targeted and scientific, improving the colonic reach rate of probiotics and the colonization efficiency of lesion sites.
[0026] Example 1: To better understand the above technical solution, the following will provide a detailed description of the technical solution in conjunction with the accompanying drawings and specific implementation methods. (Refer to...) Figure 1 As shown in the figure, this is a schematic diagram of a probiotic encapsulation method based on thermosensitive and targeted microspheres of konjac glucomannan. The probiotic encapsulation method based on thermosensitive and targeted microspheres of konjac glucomannan includes the following steps: In step 1, a free radical copolymerization method is used to synthesize... Copolymerized microspheres; Specifically, , Azobisisobutyronitrile The mass ratio is 3.0:7.5:0.1, the reaction temperature for free radical copolymerization is 65℃, and the reaction time is 24h; the specific operation of step 1 is as follows: 3.0g of... Dissolve in 50 mL of tetrahydrofuran Add 7.5g of solvent. After the monomers are thoroughly mixed, nitrogen gas is bubbled into the mixture for 30 minutes to remove oxygen. Then, 0.1g of the mixture is added. As an initiator, the reaction was carried out under an inert atmosphere at 65°C with magnetic stirring for 24 hours. After the reaction, the product was poured into 500 ml of hexane for precipitation. This precipitation was repeated three times to remove unreacted monomers, thus solving the technical problems of oxygen termination and unreacted monomer residue affecting subsequent grafting in free radical copolymerization. This ensured the purity and reactivity of the copolymerized microspheres. The product was then dried overnight under vacuum at room temperature to obtain... Copolymerized microspheres; It should be noted that the origins and selection criteria for each value and process parameter are as follows: 1. Material mass ratio 3.0g: As the core unit of the temperature-sensitive function, this dosage is combined with 50mL The solvent can form a reaction system with a suitable concentration, ensuring both Sufficient dissolution achieves uniform copolymerization, while controlling the particle size of the copolymerized microspheres within the 1-5 μm micrometer range, adapting to the size requirements of subsequent probiotic encapsulation carriers; if Excessive dosage will lead to excessive viscosity of the system, uneven copolymerization reaction, and wide microsphere size distribution; insufficient dosage will prevent the formation of a copolymer skeleton with temperature-sensitive properties, resulting in loss of temperature-sensitive response capability. 7.5g (2.5 times excess): As a crosslinking bridging unit, its side chain contains epoxy active groups, which are used for subsequent grafting. amino and The core sites of monoclonal antibodies, when added in excess, ensure sufficient epoxy groups remain on the surface of the microspheres after copolymerization, meeting the requirements of the two-step grafting reaction; experience has shown that... and At a mass ratio of 2.5:1, the grafting density of epoxy groups on the microsphere surface can reach 1.2 mmol / g, which ensures both subsequent grafting efficiency and avoids [problems caused by] [other factors]. Excessive amounts lead to excessive cross-linking of microspheres and decreased swelling performance; For 0.1g: As an oil-soluble free radical initiator, its decomposition temperature is 50-65℃, which matches the reaction temperature of this product. A dosage of 0.1g can provide an appropriate amount of free radicals, enabling... and The copolymerization rate is moderate, avoiding excessive initiator leading to high free radical concentration, which can cause burst polymerization and microsphere clumping, or insufficient initiator leading to incomplete copolymerization and excessive unreacted monomer residue.
[0027] 2. The solvent is 50 mL : As a nonprotic polar solvent, for , and All three exhibit good solubility, ensuring that they can undergo copolymerization in a homogeneous system. The boiling point is 66℃, close to the reaction temperature of 65℃. The reaction process is mild with minimal volatility, avoiding changes in system concentration due to solvent evaporation. It is miscible with hexane as a subsequent precipitant, which allows the copolymer to precipitate rapidly, facilitating separation and purification. 50 mL 3.0g can be completely dissolved. and 7.5g This is the optimal dosage that balances dissolution effect and reaction efficiency.
[0028] 3. Purge with nitrogen for 30 minutes at a flow rate of 0.5 L / min: In free radical copolymerization, oxygen can capture free radicals in the system, causing the polymerization reaction to terminate. Therefore, thorough deoxygenation is necessary. When the flow rate of high-purity nitrogen is 0.5 L / min, the dissolved oxygen in 50 mL of reaction solution and the oxygen in the space above the reaction flask can be completely replaced within 30 minutes. If the nitrogen purging time is too short or the flow rate is too slow, the deoxygenation will not be thorough, resulting in a low conversion rate of the copolymerization reaction. If the flow rate is too fast, it will cause solvent evaporation and material loss, affecting the concentration of the reaction system.
[0029] 4. Reaction temperature: 65℃: This temperature is... The optimal decomposition temperature, at which point The decomposition rate is moderate, providing a continuous and stable supply of free radicals to ensure a uniform copolymerization reaction; meanwhile, 65℃ effectively promotes... The copolymerization reaction improves the copolymerization conversion rate of the two; if the temperature is below 60℃, The decomposition rate is slow, the free radical concentration is insufficient, and the reaction is incomplete; if the temperature is higher than 70℃, If decomposition is too rapid, the instantaneous concentration of free radicals will be too high, which can easily lead to the formation of microspheres and cause solvent degradation. Increased volatility disrupts the reaction system.
[0030] 5. Reaction time: 24 hours: verified by kinetic experiments. The free radical copolymerization reaction is in a rapid reaction phase in the first 8 hours, the reaction rate slows down after 16 hours, and the copolymerization reaction is basically complete after 24 hours. If the reaction time is insufficient, the copolymerization reaction will be incomplete, the microspheres will have poor structural stability, and there will be a lot of unreacted monomers remaining. If it exceeds 24 hours, it will lead to excessive cross-linking of microspheres, which will reduce the subsequent swelling performance and grafting ability. 24 hours is the optimal time to balance the reaction conversion rate and microsphere performance.
[0031] 6. Precipitate with anhydrous hexane, repeat 3 times: Anhydrous hexane is a non-polar solvent. , and A good solvent, but for The poor solvent properties of the copolymer microspheres allow for rapid precipitation of the copolymer products. Three repeated precipitation cycles are experimentally verified to be the optimal number of cycles. The first precipitation removes most of the unreacted monomers and solvent, while the second and third precipitation cycles further remove trace amounts of unreacted monomers and oligomers adsorbed on the microsphere surface. Testing showed that after three repeated precipitation cycles, the residual amount of unreacted monomers in the microspheres was less than 0.5%, meeting the purity requirements for subsequent grafting reactions. Insufficient precipitation cycles result in residual monomers occupying active sites on the microsphere surface, inhibiting subsequent epoxy ring-opening grafting reactions. Excessive precipitation cycles lead to microsphere loss and reduced yield.
[0032] Vacuum drying conditions (25℃, -0.09MPa, 12h): Room temperature vacuum drying avoids damage to the microsphere structure from high temperatures, and a vacuum of -0.09MPa can quickly remove residual hexane and other pollutants from the microspheres. A 12-hour drying time ensures low moisture content in the microspheres, preventing moisture from affecting subsequent grafting reactions; insufficient drying will result in residual solvent interfering with subsequent reactions. The epoxide ring-opening reaction of amino groups reduces grafting efficiency.
[0033] The role of this step in the core technology: It is a fundamental and core step in the preparation of temperature-sensitive targeted microspheres, and the synthesized microspheres... Copolymerized microspheres serve as the core carrier framework for subsequent functionalization modifications, and their preparation quality directly determines the temperature sensitivity, grafting ability, and structural stability of the final probiotic-encapsulated microspheres. Specifically, their role is reflected in the following three aspects: Building the foundation for temperature-sensitive functionality: through and The gathering will The temperature-sensitive properties are introduced into the carrier framework, making the prepared material... The copolymer microspheres exhibit reversible phase transition behavior close to physiological temperature, with a minimum critical dissolution temperature of ≈32–34℃. This lays the structural foundation for the final microspheres to achieve temperature-sensitive controlled-release function that is stable at physiological temperature and expands and releases at pathological high temperature. This is the core feature that distinguishes them from traditional non-temperature-sensitive probiotic encapsulation carriers.
[0034] Provide active grafting sites: After copolymerization, the epoxy groups of its side chains remain on the surface and inside the microspheres, becoming the basis for subsequent grafting. amino and The unique active site of a monoclonal antibody is determined in this step by adding an excess of [a specific ingredient]. It ensures the sufficiency of active sites, providing structural support for the construction of the colon-targeting framework of the vector and the grafting of intestinal active targeting function. It is a key bridge connecting the temperature-sensitive unit with the subsequent targeting unit and framework unit.
[0035] Ensuring carrier structure and purity: Through precise control of copolymerization process parameters and rigorous purification procedures, the resulting... The copolymer microspheres are micron-sized powders with uniform particle size, stable structure and extremely low unreacted monomer residue. This avoids interference from residual monomers in subsequent grafting reactions, ensuring grafting efficiency and uniformity. It also enables the resulting composite microspheres to have good gelling and swelling properties, providing a stable carrier structure for probiotic encapsulation, while avoiding the influence of impurities on probiotic activity.
[0036] In steps 1 and 2 Synergistic effect of grafted amino groups: Step 1, through the design of excessive addition of GMA, pre-emptively reserves sufficient epoxy active sites for this ring-opening reaction, which, in conjunction with step 2... The amino group forms a precise "site-group" match, avoiding low KGM grafting efficiency due to insufficient active sites, ensuring stable grafting of KGM onto the copolymer microspheres, and achieving the organic combination of the natural colon-targeting framework and the temperature-sensitive carrier; step 1 reduces the amount of unreacted monomer residue through three hexane precipitation purifications, avoiding residual... , The monomer enters the system in step 2 and... Amino groups undergo non-specific reactions, preventing The consumption of amino groups reduces grafting efficiency, while avoiding the formation of byproducts from impurities that could affect the structural stability of the microspheres, thus facilitating the ammoniation process in step 2. Grafting provides a high-purity reaction substrate.
[0037] In step 2, for Ammoniation modification was carried out to prepare amino; Specifically, The amination modification was performed using 2-chloroethylamine hydrochloride. As an amination reagent, it is used in a ring-opening process via pre-wetting combined with weak base catalysis. The specific steps in step 2 are as follows: Grafting amino groups onto the hydroxyl sites of the molecular chain. Soaking in a solution with a mass concentration of 5% 25% Soak in aqueous solution for 30 minutes, dry at 65°C until the solvent is completely evaporated, and then immerse the product in a solution with a mass concentration of 5%. In a 25% weak alkali solution, at 50 The reaction was carried out at 90°C for 15 minutes. After 90 min of reaction, the product was precipitated with methanol, washed with distilled water, and dried at 65 °C to obtain... amino; It should be noted that the weighing quantity is... The powder was soaked in a solution with a mass concentration of 5%. In a 25% aqueous solution of 2-chloroethylamine hydrochloride, the mixture was allowed to stand at room temperature for 30 minutes, with gentle stirring every 10 minutes to ensure sufficient contact between KGM and the amination reagent. After soaking, the mixture was transferred to a forced-air drying oven and dried at 65°C until the solvent was completely evaporated, yielding the desired product. Pre-conjugated Intermediate; the intermediate is completely immersed in a solution with a mass concentration of 5%. In a 25% weak alkali solution, at 50 The reaction was stirred for 15 minutes under constant temperature water bath conditions at 90℃. After 90 minutes of reaction, the product was slowly poured into excess anhydrous methanol to precipitate completely. After standing for 2 hours, the supernatant was removed by decantation, and the precipitate was collected. The precipitate was washed repeatedly with deionized distilled water 3-5 times until the washing solution was neutral. The washed product was then dried in a 65°C oven at constant temperature until constant weight, yielding a white powder. Amino.
[0038] The origin and selection basis of each numerical value and process parameter: The amination reagent chosen is 2-chloroethylamine hydrochloride. : It is a small molecule amination reagent; the chloroethyl and amino functional groups in its molecule allow it to react with [agents] under weakly basic conditions. The hydroxyl groups on the molecular chain undergo nucleophilic substitution reactions, achieving highly efficient grafting of amino groups. Furthermore, the grafted amino sites exhibit high activity and can interact with... The epoxy groups of the copolymer microspheres undergo a highly efficient ring-opening reaction; compared with traditional amination reagents such as ethylenediamine and ammonia, The grafted amino sites have low steric hindrance and will not affect the subsequent grafting reaction, and are compatible with... It has high reaction specificity, is not prone to side reactions, and is suitable for adaptation. The optimal amination reagent with the best structural properties; 5% (w / v) of aqueous solution 25%: This concentration range is validated. The high-efficiency range of ammoniation is reached when the concentration is too low. Insufficient molecular content, and The contact probability of hydroxyl groups is low, and the amino grafting density is less than 0.3 mmol / g, which cannot meet the subsequent grafting requirements with epoxy groups; at high concentrations, If the osmotic pressure of the aqueous solution is too high, it will lead to The molecular chain shrinks, which actually reduces the efficiency of amino grafting, and at the same time, excessive... This will increase the difficulty of subsequent purification; 10% is preferred. 20% concentration, at this time The amino grafting density can reach 0.8. 1.2 mmol / g, achieving the optimal balance between grafting efficiency and purification difficulty; Soak at room temperature for 30 minutes: It is a natural polysaccharide, and its molecular chains are prone to aggregation. Room temperature wetting can... The aqueous solution fully penetrates to Inside the aggregate, let Molecules and The hydroxyl groups on the molecular chain are in full contact, laying the foundation for the subsequent amination reaction; the wetting time is less than 30 minutes. Insufficient swelling Uneven contact and uneven distribution of grafting sites; a time longer than 30 minutes does not improve grafting efficiency, but only increases process time. 30 minutes is the optimal time to balance swelling effect and process efficiency. Dry at 65℃ until the solvent is completely evaporated: A drying temperature of 65℃ can quickly remove moisture from the system, allowing... Molecules and The hydroxyl groups are tightly bound together to form a pre-reaction intermediate, which will not cause problems due to excessively high temperatures. Thermal degradation of molecular chains ensures its gelling properties and biocompatibility; if the drying temperature exceeds 80℃, The main chain is prone to breakage, which will significantly reduce the structural stability of the microspheres after subsequent grafting. Maintaining the drying temperature of 65℃ with the drying temperature of subsequent steps can also simplify the requirements for process equipment. 5% mass concentration of weak alkaline solution 25%: The core function of a weak base is to provide an alkaline environment for catalysis. hydroxyl and The nucleophilic substitution reaction makes The chloroethyl group was removed, and the amino group was successfully grafted onto the chloroethyl group. Hydroxyl site; at low concentrations, the catalytic activity is insufficient, the reaction rate is slow, and the amination reaction is incomplete; at high concentrations, excessive alkalinity will destroy it. The glycosidic bonds in the molecular chain lead to Degradation, and at the same time, unreacted Self-polymerization occurs, producing byproducts; preferably 10%. A 15% weak alkaline solution provides the best catalytic effect, and there is no... Degradation phenomenon; Reaction temperature 50 90℃: Temperature is the core parameter for controlling the rate of ammoniation reaction. 50℃ is the initial reaction temperature, at which point the weak base begins to act as a catalyst, and the reaction proceeds slowly; as the temperature rises to 70℃... The reaction rate reaches its peak at 80℃, with the highest amino grafting efficiency; temperatures above 90℃ lead to... Molecular chains unwind and may even undergo thermal degradation, while the weak alkaline solution will volatilize violently, disrupting the reaction system; preferably 70 At 80℃, the conversion rate of the ammoniation reaction is optimal within this temperature range, and The structure remains intact; Reaction time 15 90 min: The reaction time is negatively correlated with the reaction temperature. At a low temperature of 50℃, it takes 90 min of reaction to achieve complete amination. 70 At the optimal temperature of 80℃, the reaction can be completed in just 60 minutes, and at a high temperature of 90℃, the reaction equilibrium can be reached in 15 minutes. This time range can be flexibly adjusted according to the actual production temperature conditions to ensure complete amination reaction while avoiding problems caused by excessively long reaction times. It undergoes slow degradation in an alkaline environment, balancing reaction efficiency and product structural integrity; Anhydrous methanol precipitate + washing with distilled water until neutral: Anhydrous methanol is Poor solvents can cause ammoniation to occur. Rapid precipitation, while simultaneously dissolving unreacted substances. The core purpose of washing with distilled water is to remove adsorbed methanol and residual weak base ions from the precipitate. The washing solution should be neutral to avoid interference from residual alkaline substances in the subsequent ring-opening reaction with epoxy groups. Epoxy groups are prone to self-polymerization in an overly alkaline environment. Washing 3-5 times can ensure thorough removal of impurities. The pH of the washing solution should be stable at 6.5-7.5 to meet the system requirements for subsequent reactions. Dry at 65℃ to constant weight: constant weight drying ensures... The water content of the amino group is less than 0.5% to prevent water from entering the subsequent THF / methanol reaction system, reducing solvent solubility, and preventing water from causing... The epoxy groups in the copolymer microspheres are deactivated by hydrolysis; drying to constant weight also ensures that subsequent steps are successful. Precise feeding quality of amino groups improves process repeatability.
[0039] The role of this step in the core technology is to achieve... and The key bridging step in the covalent bonding of copolymerized microspheres is also the process of bringing natural... The colon-targeting properties are integrated into the core components of the temperature-sensitive carrier system, and its preparation... The amino group is the core intermediate connecting the temperature-sensitive unit and the colon-targeting framework, and its core role in the entire technical solution is reflected in four aspects: Introducing active amino sites to achieve covalent grafting: natural The molecular chain contains only hydroxyl functional groups and cannot interact with... The epoxy groups of the copolymer microspheres undergo a specific reaction; this step involves ammoniation modification. Grafting active amino groups onto hydroxyl sites enables... It possesses the structural basis for ring-opening reactions with epoxy groups, thus enabling... Covalent grafting with temperature-sensitive copolymer microspheres, compared to physical mixing, makes the composite carrier structure more stable, preventing component separation during digestive tract transport and ensuring functional continuity. Preserving the core properties of KGM and laying the foundation for colon-targeted therapy: This step employs a mild, weakly alkaline catalysis and a medium-to-low temperature reaction process, achieving amino grafting while fully preserving the [specific properties / characteristics]. It possesses gel properties, high water absorption and swelling capacity, and biocompatibility, and does not damage... Its passive targeting properties in the colon, making it resistant to hydrolysis by upper digestive tract enzymes and allowing direct access to the colon; after grafting After amino groups are grafted onto the copolymer microspheres, this property can be transferred to the final composite carrier, allowing the final microspheres to pass smoothly through the stomach and small intestine and reach the colon, providing a basic guarantee for the colonic delivery of probiotics. Regulating amino grafting density to match subsequent grafting needs: through precise control Concentration, reaction temperature, and time can be flexibly adjusted. The amino grafting density of the amino group, so that it is similar to... The precise matching of epoxy group grafting density in the copolymer microspheres avoids steric hindrance caused by too many amino groups or insufficient grafting caused by too few amino groups, ensuring efficient and uniform subsequent grafting reactions and improving the structural uniformity of the composite carrier.
[0040] Provides gel matrix support for probiotic encapsulation: It is a natural and highly efficient gel material, modified by ammoniation. After amino groups are grafted onto temperature-sensitive microspheres, they interact with... A composite gel framework is formed, which is both thermosensitive and highly swellable, providing a dense and elastic gel network for subsequent probiotic encapsulation. This network can effectively encapsulate probiotic hyphae, resist damage from gastric acid, bile salts and digestive enzymes, and ensure the activity of probiotics during oral delivery. It is one of the core structural foundations for the efficient protection of probiotics.
[0041] Steps 2 and 3 Amino functionalization modification Synergistic cooperation of copolymer microspheres: Step 2 is Introducing amino functional groups, step 3 The surface of the copolymer microspheres contains epoxy functional groups, which can undergo a specific epoxy ring-opening addition reaction to form a stable... The covalent bond, specifically the functional group matching of the amino-epoxy group, is the sole structural basis for the grafting reaction in step 3. Step 2, through process control, ensures the precision of the amino grafting density, achieving an optimal 1:1 match with the epoxy group density of the copolymerized microspheres in step 3, thus improving the conversion rate of the grafting reaction and realizing the efficient combination of the two functional units. Step 2 fully preserves... The colon's passive targeting properties are transferred through covalent grafting in step 3 to... In the copolymerized microspheres, and in the subsequent step 4, the The intestinal active targeting properties of monoclonal antibodies synergistically enable the composite microspheres to possess dual targeting capabilities: passive targeting of the colon and active targeting of the lesion site. If step 2 disrupts... The colon-targeting characteristics cannot be restored after grafting in step 3, and the microspheres will eventually lose their basic ability to deliver to the colon. Therefore, step 2 is an important prerequisite for the subsequent realization of dual-targeting function.
[0042] In step 3, the epoxy ring-opening reaction is used to... amino grafting to On the copolymer microspheres, to obtain Copolymerized microspheres; Specifically, the weak alkaline solution is a sodium bicarbonate solution, a sodium carbonate solution, or ammonia water, preferably a 10% sodium bicarbonate solution, which does not damage the alkaline environment. Achieving high ammoniation efficiency while maintaining the main chain structure, and being more compatible The structural characteristics represent an innovative optimization of the natural polysaccharide modification process. The specific steps are as follows: Take 1.0g... The copolymer microspheres dissolved in 100 mL Solvent, 0.4g The amino group was dissolved in 5 mL of methanol and then added to the above solution. The mixture was stirred at 60 °C for 24 h. The solvent was removed by rotary evaporation. The product was precipitated in a mixture of hexane and methanol (v / v) to remove unreacted precipitate. amino, after drying, yields Copolymerized microspheres; It should be noted that 1.0g is weighed. The copolymerized microspheres were placed in a three-necked flask, and 100 mL of tetrahydrofuran was added. Solvent, stir magnetically at room temperature until completely dissolved; separately take 0.4g The amino group was dissolved in 5 mL of anhydrous methanol and ultrasonically dispersed for 5 min until homogeneous. This solution was then slowly added dropwise to the solution in the three-necked flask. The reaction system was placed in a 60 °C water bath and magnetically stirred at 200 r / min for 24 h. After the reaction, most of the solvent was removed by rotary evaporation at 45 °C and -0.08 MPa. The concentrated product was then slowly poured into a mixed precipitant of hexane:methanol = 10:2 (v / v), and allowed to stand for 1 h to allow complete precipitation. The supernatant was then discarded. The precipitate was collected and placed in a vacuum drying oven at 25 °C and -0.09 MPa until constant weight was obtained, yielding a white powder. Copolymer microspheres.
[0043] The origin and selection basis of each numerical value and process parameter: Material ratio: 1.0g copolymer microspheres : 0.4g Amino: Experiments have verified that this ratio, along with the epoxy group density of the copolymerized microspheres from step 1 and step 2... The amino grafting density of the amino group is precisely matched, with a molar ratio of epoxy group to amino group of approximately 1.2:1, ensuring sufficient amino grafting while avoiding... Excessive amino residue improves grafting efficiency; Solvent system, 100mL +5mL methanol: for Methanol is a good solvent and can improve... The solubility of amino groups allows for the formation of a homogeneous reaction system through the combination of the two compounds, avoiding uneven grafting in heterogeneous phases; 100mL 1.0 g of the copolymer microspheres can be completely dissolved, while 0.4 g can be dissolved in just 5 mL of methanol. Amino compounds allow for a balance between solvent usage and reaction efficiency, with no solvent waste. Reaction temperature 60℃, time 24h: 60℃ is the optimal temperature for epoxy ring-opening reaction, which can improve the reaction rate without causing thermal degradation of copolymer microspheres or violent evaporation of solvent; 24h can ensure the complete epoxy ring-opening reaction. If the time is insufficient, the grafting will be inadequate, which will lead to a decrease in the gelation and targeting properties of composite microspheres. The ratio of mixed precipitants is hexane:methanol = 10:2: Its proprietary solvent ratio allows for rapid product precipitation while simultaneously dissolving unreacted solvents. Amino acids offer high precipitation and purification efficiency, and compared to single solvents, they can reduce product loss. Rotary evaporation conditions: 45℃, -0.08MPa: Low-temperature vacuum evaporation can quickly remove solvents while avoiding high-temperature deformation of the grafted composite microsphere structure, thus ensuring stable product performance.
[0044] The core function of this step in terms of core technology is to achieve the covalent fusion of the temperature-sensitive unit and the colon-targeting framework, thereby... Thermosensitive properties and The combination of passive colon targeting and gel properties forms a bifunctional composite carrier framework, laying the structural foundation for subsequent targeted antibody grafting and probiotic encapsulation.
[0045] By retaining the dual-core performance of the carrier, the mild ring-opening reaction conditions ensure that the thermosensitive phase transition properties of the copolymerized microspheres are not lost, and the properties are fully preserved. Its gelling and water-absorbing swelling properties enable the composite carrier to have both probiotic protection and colon delivery capabilities.
[0046] Sufficient active sites are reserved, and the ratio of epoxy groups to amino groups is controlled so that some unreacted epoxy groups are retained on the surface of the microspheres, which is for step 4. Grafting monoclonal antibodies provides a dedicated active site, avoiding site competition.
[0047] By enhancing the structural stability of the carrier and replacing physical mixing with covalent grafting, the composite carrier can resist the erosion of gastric acid and bile salts during transport in the digestive tract, ensuring the full protection of the encapsulated probiotics.
[0048] Steps 3 and 4 are connected Synergistic effect of monoclonal antibodies: Site reservation synergy: This step deliberately preserves some epoxy active sites, which perfectly match the grafting requirements of antibody amino groups in step 4. No additional modification is required, and efficient covalent grafting of the antibody can be achieved directly, ensuring the smooth grafting of targeting function.
[0049] Product performance adaptation: prepared The copolymer microspheres are in powder form with a moderate specific surface area and good dispersibility in phosphate buffer, allowing them to fully contact the antibody solution from step 4 and improve the uniformity of antibody grafting. At the same time, the microspheres are neutral and will not destroy the biological activity of the antibody from step 4.
[0050] Process conditions: The vacuum drying process in this step results in low water content in the microspheres, avoiding interference from moisture with the pH stability of the phosphate buffer in step 4, ensuring that antibody grafting is carried out under the optimal mild conditions of pH 8.0, and preventing antibody denaturation and inactivation.
[0051] Purification process linkage: This step has removed most of the impurities through precipitation, and the resulting composite microspheres have high purity. After entering step 4, only simple buffer washing is needed to remove unbound antibodies, simplifying the purification operation in step 4 and improving process efficiency.
[0052] Functional feature transfer: This step will The colon-targeting properties are incorporated into the composite carrier, and the [product / material] is added in step 4. The active intestinal targeting properties of the antibody form a dual-targeting synergy, laying the functional foundation for the final microspheres to achieve passive delivery to the colon and active recognition of lesion sites.
[0053] In step 4, the HSP monoclonal antibody is grafted onto the residual epoxy group. On the copolymer microspheres, to obtain Monoclonal antibody copolymer microspheres; Specifically, the specific steps are as follows: Prepare 1mg / The HSP monoclonal antibody solution was prepared in 20 mM pH 8.0 phosphate buffer; 10 mg 1 mL of copolymer microspheres The proportion of monoclonal antibody solution was determined by adding the copolymer microsphere powder to the antibody solution and stirring continuously at room temperature for 24 hours. The product was then washed sequentially with 20 mM pH 8.0 phosphate buffer and 20 mM pH 7.0 phosphate buffer. This two-step pH gradient washing process ensured both the adequacy of the grafting reaction and the maintenance of... To enhance antibody bioactivity and prevent damage to antibody targeting by strong acids, alkalis, and high temperatures, this method addresses the technical challenge of activity loss during polymeric carrier grafting of biological antibodies. Unbound antibodies are removed, and the washed product is placed in a vacuum drying oven and dried at 4°C under vacuum until constant weight. It is then sealed and stored in a dry place to obtain the desired product. Monoclonal antibody copolymer microspheres; It should be noted that the origins and selection criteria for each value and process parameter are as follows: Antibody solution concentration 1mg / This concentration is the optimal concentration for antibody grafting. It ensures sufficient binding with the residual epoxy groups on the microspheres, improves the antibody grafting rate, avoids excessive free antibody and increased purification costs due to excessive antibody, and also takes into account the dispersibility of the antibody solution.
[0054] The solvent is 20mM pH 8.0 PBS: 20mM low-concentration PBS can maintain the stability of the system's osmotic pressure and avoid antibody denaturation; pH 8.0 is the optimal pH for the ring-opening reaction of epoxy groups and amino groups, and it is suitable for the physiological activity environment of the antibody without destroying the antibody's spatial structure.
[0055] The ratio of microspheres to antibody solution is 10 mg: 1 mL: This ratio is precisely matched with the density of residual epoxy groups on the microspheres, achieving uniform antibody grafting and avoiding competition for grafting sites due to an excessive number of microspheres, or waste due to an excessive number of antibodies.
[0056] Reaction at room temperature for 24 hours: Room temperature can maximize the preservation of antibody biological activity and avoid high temperature denaturation; 24 hours ensures that the epoxy ring-opening reaction is fully carried out, achieving stable covalent binding between the antibody and the microspheres.
[0057] pH 8.0 → pH 7.0 gradient washing: First, wash with pH 8.0 PBS to remove unreacted small molecule impurities, then wash with pH 7.0 PBS, which is close to the physiological environment. This not only thoroughly removes free antibodies but also restores the microspheres to neutrality, avoiding alkaline residues from affecting subsequent probiotic encapsulation. At the same time, the mild conditions throughout the process ensure that the antibody targeting activity is not lost.
[0058] 4℃ low-temperature drying: This avoids the destruction of the antibody's spatial structure caused by room temperature / high temperature drying, ensuring that the antibody can still specifically recognize intestinal HSP proteins after grafting.
[0059] The core role of this step in core technologies: Imbuing the carrier with active gut targeting capabilities through grafting Monoclonal antibodies enable the vector to specifically recognize highly expressed antibodies at sites of intestinal lesions. Proteins enable an upgrade from passive targeting of the colon to active targeting of lesion sites, solving the problem of poor targeting of traditional carriers.
[0060] The integration of temperature-sensitive, targeted, and colonic delivery functions enables the composite microspheres to simultaneously possess... Thermosensitive controlled release, passive targeting of the colon The active targeting triple core function of antibodies lays the functional foundation for the precise delivery of probiotics.
[0061] This study addresses the challenge of preserving the activity of grafted biological antibodies by employing mild reaction and washing conditions to achieve covalent grafting of antibodies while retaining their biological activity, thus overcoming the technical bottleneck of grafting bioactive substances onto polymeric carriers.
[0062] To ensure the biocompatibility of the carrier, the composite microspheres grafted with antibodies are non-toxic, have low immunogenicity, and have improved surface hydrophilicity, making them more compatible with the intestinal microenvironment and facilitating the subsequent colonization of probiotics in the gut.
[0063] The synergistic effect of encapsulating probiotics in steps 4 and 5: Synergistic Function: The active targeting characteristics imparted in this step, combined with the temperature-sensitive controlled-release characteristics after encapsulating probiotics in step 5, achieve a precise delivery closed loop of probiotic colon delivery → lesion site identification → pathological high-temperature release, significantly improving the colonization efficiency of probiotics at lesion sites.
[0064] Product performance compatibility: The prepared antibody microspheres are neutral, low-water-content powders with good dispersibility. They can uniformly swell in deionized water in step 5 to form a gel system, which can fully contact the probiotic hyphae and improve the uniformity of encapsulation. At the same time, the antibody structure on the surface of the microspheres will not produce toxicity to the probiotics, thus ensuring the activity of the probiotics.
[0065] Process conditions: This step uses low-temperature drying at 4℃, ensuring the microspheres do not undergo high-temperature denaturation, and is subject to... After washing, there are no impurities left, and no harmful substances will be introduced after entering step 5, avoiding damage to the probiotics and ensuring the number of live probiotics after encapsulation.
[0066] Structural support synergy: The composite microspheres grafted with antibodies still retain and The composite gel framework, with its excellent water absorption, swelling, and gelling properties, provides a dense protective barrier for the encapsulation of probiotics in step 5, effectively resisting the destruction by gastric acid and bile salts, and achieving full protection of probiotics throughout the process.
[0067] Coordination and coordination: The loading capacity of the antibody microsphere gel prepared in this step is precisely matched with the 10:4 polymer-probiotic hyphae mass ratio in step 5, ensuring that the probiotics are uniformly embedded in the gel network. This prevents insufficient embedding due to insufficient gel structure, and also prevents excessive probiotics from damaging the gel structure.
[0068] In step 5, the above-mentioned copolymer microspheres are mixed with probiotic hyphae to complete the encapsulation of probiotics, resulting in... Monoclonal antibody-probiotic copolymer microspheres; Specifically, the mass ratio of copolymerized microspheres to probiotic mycelia is 10:4; the specific operation of step 5 is as follows: take 0.2g Monoclonal antibody copolymer microspheres were dissolved in 5 mL of deionized water, probiotic mycelia were added, and the mixture was magnetically stirred overnight at room temperature. After drying, the product was obtained. Monoclonal antibody-probiotic copolymer microspheres; the probiotics are one or more of Lactobacillus, Bifidobacterium, and Enterococcus, ensuring that the encapsulated probiotics have high biological activity and are compatible with the gel structure of the microspheres, solving the problem of a significant decrease in probiotic activity after encapsulation in the prior art; the probiotic hyphae are the mycelia of probiotic cells that have been activated and cultured to the logarithmic growth phase, collected by centrifugation, and washed with sterile water. The minimum critical dissolution temperature of the monoclonal antibody-probiotic copolymer microspheres is 32–34℃. They are structurally stable at physiological temperatures of 25℃ and 37℃, but swell and rapidly release probiotics at pathological high temperatures of 42℃. Furthermore, they can be transmitted through… Monoclonal antibodies specifically recognize highly expressed [specific antibodies] on intestinal epithelial cells. Proteins, enabling targeted delivery to the gut; It should be noted that the origins and selection criteria for each value and process parameter are as follows: The ratio of microspheres to probiotic hyphae is 10:4: This ratio has been verified to be the optimal encapsulation ratio. The microsphere gel can fully encapsulate the hyphae, ensuring a high encapsulation rate for the probiotics without damaging the gel network structure due to excessive hyphae. At the same time, it avoids insufficient probiotic activity due to excessive microspheres, thus balancing the encapsulation effect and the activity of the formulation.
[0069] 0.2g microspheres with 5mL sterile deionized water: This amount allows the microspheres to fully swell and form a dense and elastic gel system, which perfectly achieves the uniform dispersion and encapsulation of probiotic hyphae. Too little water will result in insufficient swelling of the microspheres, while too much water will make the gel too sparse, both of which will reduce the encapsulation effect.
[0070] Stirring at low speed overnight at room temperature: Room temperature avoids high-temperature inactivation of probiotics, low-speed stirring prevents the hyphae from being damaged by mechanical force, and overnight stirring ensures that the hyphae and gel network are fully combined to achieve uniform encapsulation.
[0071] Freeze-drying process: replaces high-temperature drying, preserves the activity of live probiotics to the greatest extent, and maintains the integrity of the microsphere's gel structure, ensuring that subsequent temperature-sensitive controlled release and targeting functions are not impaired.
[0072] The core role of this step in core technologies: This technology achieves efficient encapsulation and activity protection of probiotics by constructing a physical barrier for probiotics through a gel network to resist damage from gastric acid, bile salts, and digestive enzymes. It solves the problem of significant decrease in probiotic activity after encapsulation in existing technologies and ensures the number of live bacteria in the colon after oral delivery.
[0073] By integrating all functional units throughout the process, combining temperature-sensitive controlled release, passive colonic targeting, active intestinal targeting, and probiotic activity, an integrated probiotic formulation with protection, targeting, and intelligent release is formed, realizing the final functional implementation of the technical solution.
[0074] To ensure the integrity of the microspheres' temperature-sensitive and targeting functions, a gentle encapsulation process allows the microspheres to retain their original minimum critical dissolution temperature and... Antibody targeting activity ensures precise release under pathological high temperatures in the gut microenvironment.
[0075] To improve the practicality of the formulation, the prepared microspheres are solid powders with good stability and easy storage, and the probiotics are uniformly encapsulated, providing a feasible solution for the subsequent industrial preparation of oral probiotic formulations.
[0076] Coordination and cooperation with each step of the entire process: In conjunction with step 1: the structure built in step 1 The thermosensitive framework is completely transferred to this step, ensuring that the microspheres have a minimum critical dissolution temperature of 32–34°C, which is the core function of achieving physiological temperature stability and pathological high-temperature bacterial release. It is the source support for thermosensitive controlled release.
[0077] In conjunction with steps 2 and 3: Steps 2 and 3 achieve The passive targeting of the colon and the gel properties provide a dense gel embedding framework for this step, which not only ensures efficient encapsulation of probiotics but also allows microspheres to reach the colon directly, laying the foundation for colonic delivery of targeted bacterial release.
[0078] In conjunction with step 4: the grafting in step 4 Monoclonal antibodies endow the microspheres prepared in this step with active intestinal targeting capabilities, and The colon passive targeting forms a dual-target synergy, enabling the precise delivery of probiotics from the colon to the lesion site, avoiding non-specific loss.
[0079] The entire process is adapted and coordinated: the preceding steps all adopt mild reaction, precise purification and low temperature drying processes to ensure the high purity, low impurities and structural integrity of the microspheres. After entering this step, no harmful substances will be introduced to damage the probiotics. Moreover, the microsphere gel loading capacity regulated in the preceding steps is precisely matched with the 10:4 embedding ratio in this step, realizing the linkage and adaptation of process parameters throughout the entire process.
[0080] Full-process functional integration and synergy: This step organically integrates the three core functions of the temperature-sensitive unit, colon-targeting framework, and active targeting antibody built in the previous four steps with the bioactivity of probiotics to form a fully functional integrated formulation. This achieves a closed-loop technology process from carrier design to functional implementation, and ultimately solves the core pain points of the industry such as insufficient protection, poor targeting, and unintelligent release in oral delivery of probiotics.
[0081] The contents not described in detail in this specification are existing technologies known to those skilled in the art.
[0082] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan, characterized in that: Konjac glucomannan For functional backbone, N-isopropylacrylamide Thermosensitive unit, glycidyl methacrylate As a cross-linking bridging unit, it connects to heat shock proteins. The preparation of monoclonal antibody copolymer microspheres, followed by encapsulation of probiotics, includes the following steps: Step 1: Synthesize using a free radical copolymerization method Copolymerized microspheres; Step 2, for Ammoniation modification was carried out to prepare amino; Step 3: Through epoxy ring-opening reaction amino grafting to On the copolymer microspheres, to obtain Copolymerized microspheres; Step 4: Graft the HSP monoclonal antibody onto the residual epoxy group. On the copolymer microspheres, to obtain Monoclonal antibody copolymer microspheres; Step 5: Mix the above-mentioned copolymer microspheres with probiotic hyphae to complete the encapsulation of probiotics, and obtain... Monoclonal antibody-probiotic copolymer microspheres.
2. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 1, characterized in that: Step 1, , Azobisisobutyronitrile The mass ratio is 3.0:7.5:0.1, the reaction temperature for free radical copolymerization is 65℃, and the reaction time is 24h; the specific operation of step 1 is as follows: 3.0g of... Dissolve in 50 mL of tetrahydrofuran Add 7.5g of solvent. After the monomers are thoroughly mixed, nitrogen gas is bubbled into the mixture for 30 minutes to remove oxygen. Then, 0.1g of the mixture is added. As an initiator, the reaction was carried out under an inert atmosphere at 65°C with magnetic stirring for 24 hours. After the reaction was completed, the product was poured into 500 ml of hexane to precipitate. The precipitation was repeated three times to remove unreacted monomers. The product was then dried overnight under vacuum at room temperature to obtain the desired product. Copolymer microspheres.
3. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 2, characterized in that: Step 2, The amination modification was performed using 2-chloroethylamine hydrochloride. As the amination reagent, the specific operation of step 2 is as follows: [The following text appears to be a separate, unrelated section:] ... Soaking in a solution with a mass concentration of 5% 25% Soak in aqueous solution for 30 minutes, dry at 65°C until the solvent is completely evaporated, and then immerse the product in a solution with a mass concentration of 5%. In a 25% weak alkali solution, at 50 The reaction was carried out at 90°C for 15 days. After 90 min of reaction, the product was precipitated with methanol, washed with distilled water, and dried at 65 °C to obtain... Amino.
4. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 3, characterized in that: In step 3, the weak alkaline solution is a sodium bicarbonate solution, a sodium carbonate solution, or ammonia water, preferably a sodium bicarbonate solution with a mass concentration of 10%.
5. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 4, characterized in that: The specific operation of step 3 is as follows: Take 1.0g The copolymer microspheres dissolved in 100 mL Solvent, 0.4g The amino group was dissolved in 5 mL of methanol and then added to the above solution. The mixture was stirred at 60 °C for 24 h. The solvent was removed by rotary evaporation. The product was precipitated in a mixture of hexane and methanol (v / v) to remove unreacted precipitate. amino, after drying, yields Copolymer microspheres.
6. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 5, characterized in that: The specific operation of step 4 is as follows: Prepare 1mg / The HSP monoclonal antibody solution was prepared in 20 mM pH 8.0 phosphate buffer; 10 mg 1 mL of copolymer microspheres To determine the proportion of monoclonal antibody solution, the copolymer microsphere powder was added to the antibody solution, and the reaction was carried out with continuous stirring at room temperature for 24 hours. The product was washed successively with 20 mM pH 8.0 phosphate buffer and 20 mM pH 7.0 phosphate buffer to remove unbound antibody, and then dried and stored. Monoclonal antibody copolymer microspheres.
7. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 6, characterized in that: In step 5, the mass ratio of copolymerized microspheres to probiotic mycelia is 10:4; the specific operation of step 5 is as follows: Take 0.2g Monoclonal antibody copolymer microspheres were dissolved in 5 mL of deionized water, probiotic mycelia were added, and the mixture was magnetically stirred overnight at room temperature. After drying, the product was obtained. Monoclonal antibody-probiotic copolymer microspheres.
8. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 7, characterized in that: The probiotics are one or more of Lactobacillus, Bifidobacterium, and Enterococcus, and the probiotic hyphae are the mycelia of probiotic cells that have been activated and cultured to the logarithmic growth phase, collected by centrifugation, and washed with sterile water.
9. The method for encapsulating probiotics in thermosensitive and targeted microspheres based on konjac glucomannan according to claim 8, characterized in that: The The minimum critical dissolution temperature of the monoclonal antibody-probiotic copolymer microspheres is 32–34℃. They are structurally stable at physiological temperatures of 25℃ and 37℃, but swell and rapidly release probiotics at pathological high temperatures of 42℃. Furthermore, they can be transmitted through… Monoclonal antibodies specifically recognize highly expressed [specific antibodies] on intestinal epithelial cells. Proteins that enable targeted delivery to the gut.