Novel multi-dimensional cascade assembled micro-nano intestinal delivery carrier as well as preparation method and application thereof

Abstract

The invention discloses a novel multi-dimensional cascade assembled micro-nano intestinal delivery carrier and a preparation method and application thereof, and relates to the field of biological medicine and functional materials.The preparation method comprises the following steps that S1, phospholipid and a sterol regulator are dissolved in an ethanol solution to form a lipid film, the solution is added for hydration, and the lipid film is obtained; then treating to obtain a monodisperse vesicle suspension; s2, adding the vesicle suspension into the composite solution, and inducing adsorption of an inner shell layer to form intermediate particles; and S3, dropwise adding a cationic electrolyte solution into the intermediate particle suspension, and carrying out interface electrostatic complexing and coordination cross-linking reaction to obtain the delivery carrier. By constructing a cascade self-assembly delivery carrier with a multi-layer core-shell structure, the problem that a large amount of drugs are inactivated due to insufficient shielding in an extreme stomach environment can be solved, so that space-time limited release of the drugs in specific parts of intestinal tracts is realized, and oral bioavailability and targeted therapy efficiency are greatly improved.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and functional materials technology, and in particular to a novel multidimensional cascaded assembly of micro-nano intestinal delivery carriers, their preparation methods, and applications. Background Technology

[0002] The gut is a vital digestive and absorptive organ and an immune barrier, and maintaining its homeostasis is crucial for overall health. Foodborne pathogens pose a significant threat to gut homeostasis, with Listeria monocytogenes, a typical intracellular pathogen, capable of crossing the intestinal mucosal barrier into the bloodstream, causing severe systemic infections and even death, posing a major challenge to public health. Currently, first-line treatment for such infections primarily relies on broad-spectrum antibiotics. However, while antibiotics kill pathogens, they also indiscriminately attack the vast symbiotic microbial community within the gut, leading to drastic changes in gut microbiota structure and disruption of the microecological balance, i.e., intestinal dysbiosis. This dysbiosis not only weakens the gut's inherent colonization resistance, making it easier for pathogens to invade, but may also damage the intestinal epithelial barrier function, exacerbating local and even systemic inflammatory responses, creating a vicious cycle of infection aggravation and barrier damage. Therefore, developing a non-antibiotic intervention strategy that can precisely eliminate pathogens while avoiding harm to beneficial bacteria and actively repairing the damaged barrier has become an urgent research need.

[0003] Nisin is a naturally occurring bacteriocin with broad-spectrum antibacterial activity, particularly effective against various Gram-positive pathogens such as Listeria monocytogenes. Due to its high safety profile, it has been approved for use as a food additive. Studies have shown that this antimicrobial peptide, while inhibiting pathogens, also exhibits the potential to modulate immune responses and alleviate inflammation, making it considered an ideal candidate to replace traditional antibiotics.

[0004] However, developing it into an oral therapeutic faces significant challenges. Nisin is essentially a polypeptide, which, upon passing through the upper digestive tract, encounters the extremely acidic environment of the stomach and degradation by various proteases, leading to a rapid loss of its biological activity and extremely low oral bioavailability. Furthermore, free peptide molecules struggle to target specific sites of infection or injury in the complex intestinal environment, cannot physically repair damaged intestinal barriers after pathogen clearance, and lack the function of regulating dysbiosis. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides a novel multidimensional cascaded assembly micro / nano intestinal delivery carrier, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Firstly, the present invention provides a method for preparing a novel multidimensional cascaded assembly of micro / nano intestinal delivery carriers, comprising the following steps:

[0007] S1. Phospholipids and sterol regulators are dissolved in an ethanol solution, the solvent is removed to form a uniform lipid film, an aqueous solution containing active biomacromolecules and stabilizers is added for hydration, and then ultrasonic treatment is performed under ice bath conditions to obtain a monodisperse vesicle suspension.

[0008] S2. Slowly add the vesicle suspension to the anionic polysaccharide complex solution, stir at room temperature, induce adsorption of the inner shell layer, and form intermediate particles.

[0009] S3. Add a cationic electrolyte solution containing a metal coordination factor dropwise to the intermediate particle suspension. The delivery carrier is obtained through interfacial electrostatic complexation and coordination cross-linking reaction.

[0010] In a preferred embodiment of the present invention, in step S1, the active biomolecule is nisin, and its concentration in the aqueous solution is 20-40 mg / mL; the volume ratio of the lipid film to the aqueous solution is 15 mg: 0.5-2 mL; the stabilizer is one of mannitol, trehalose, or sucrose, and its concentration is 1-5% w / v; the mass ratio of the phospholipid to the sterol regulator is 3-8:1, and the total mass of the phospholipid and sterol regulator to the volume ratio of the ethanol solution is 10-50 mg: 1 mL.

[0011] In a preferred embodiment of the present invention, in step S1, the phospholipid is selected from soybean phospholipid, lecithin, and hydrogenated lecithin; the sterol regulator is cholesterol or phytosterol; and the ultrasonic treatment specifically involves ultrasonic treatment at a power of 50-70 W for 3-10 minutes.

[0012] In a preferred embodiment of the present invention, in step S2, the anionic polysaccharide complex solution comprises 1-2% w / v pectin and 0.2-1.2% w / v oxidized konjac glucomannan, and the volume ratio of the vesicle suspension to the anionic polysaccharide complex solution is 1:2-10; the oxidized konjac glucomannan is prepared by selective oxidation and has an infrared spectrum in the range of 1700-1750 cm⁻¹. -1 It has characteristic absorption peaks within the range.

[0013] In a preferred embodiment of the present invention, in step S2, the temperature of the room temperature stirring is 20-40 ℃, the rotation speed is 100-300 rpm, and the stirring time is 30-90 min.

[0014] In a preferred embodiment of the present invention, in step S3, the cationic electrolyte solution comprises 0.08-0.15 mM zinc acetate and 0.5-2% w / v chitosan solution; the volume ratio of the intermediate particle suspension to the cationic electrolyte solution is 1:1-5.

[0015] In a preferred embodiment of the present invention, in step S3, the conditions for the coordination crosslinking reaction are: pH value of 5.0-6.5, temperature of 25-37 ℃, and time of 1-3 h.

[0016] Secondly, this invention provides a novel multidimensional cascaded assembled micro / nano intestinal delivery carrier, prepared by the method described in any one of the above-mentioned methods. The carrier has a core-shell structure and comprises, from the inside out:

[0017] The core consists of a bilayered vesicle formed by phospholipids and sterol regulators, which encapsulates active biomacromolecules.

[0018] The inner layer, covering the core surface, is a pH-sensitive network layer formed by non-covalent cross-linking of functionalized glucomannan derivatives and pectin;

[0019] The outer layer, covering the surface of the inner layer, is a dense barrier layer formed by the coordination and electrostatic interaction of cationic aminopolysaccharides and polyvalent metal ions, and the outer layer has mucosal adhesion.

[0020] In a preferred embodiment of the present invention, the active biomolecule is nisin; the multivalent metal ion is zinc ion; and the delivery carrier has an average particle size of 200-1200 nm and a surface potential of +15 mV to +35 mV.

[0021] Thirdly, the present invention provides an application of a novel multidimensional cascaded assembly of micro-nano intestinal delivery carriers as described in any one of the above-mentioned methods, for the preparation of drugs for the prevention and / or treatment of intestinal pathogenic bacteria infection, repair of intestinal barrier function and regulation of intestinal microecological balance; wherein the intestinal pathogenic bacteria is Listeria monocytogenes; the drug is administered orally, and the dosage, based on the encapsulated nisin, is 50-100 mg per kilogram of body weight per day.

[0022] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0023] (1) This invention provides a novel multidimensional cascaded assembly micro-nano intestinal delivery carrier, its preparation method and application. By constructing a cascaded self-assembled delivery carrier with a multi-layer core-shell structure, the carrier consists of a lipid core, a polysaccharide hybrid inner layer and a metal coordination outer layer from the inside out. It can respond sequentially to the physiological gradient changes from strong acid to weak base in the gastrointestinal tract. The metal coordination network of the outer layer maintains a dense conformation under the acidic conditions of the stomach and works synergistically with the contracting polysaccharide network of the inner layer to form a physical and chemical double barrier, effectively isolating hydrogen ions and proteases, and effectively ensuring that the encapsulated peptide drugs hardly leak or denature in the stomach and enter the intestine intact. Compared with the single material embedding or simple coating commonly used in existing delivery technologies, it can solve the problem of insufficient shielding in the extreme environment of the stomach, which leads to a large amount of drug inactivation. Furthermore, in the neutral to weakly alkaline environment of the intestine, the outer conformation changes and exhibits mucosal adhesion, while the inner network simultaneously decrosslinks and swells, thereby realizing the spatiotemporally restricted release of drugs in specific parts of the intestine, greatly improving oral bioavailability and targeted therapy efficiency.

[0024] (2) In this invention, the carrier is a food-derived natural polymer. After delivery, the degradation products of the components can continue to exert biological functions. The cationic polysaccharide adheres tightly to the negatively charged intestinal mucosa surface through electrostatic action, which can exert a molecular filling effect. Oxidized konjac polysaccharide and pectin can be used as specific carbon sources by symbiotic microorganisms. After drug release, the carrier components can physically seal the intercellular spaces of epithelial cells that have expanded due to infection, repair the intestinal physical barrier, and selectively promote the proliferation of beneficial bacteria such as lactobacillus. Compared with the defects of the prior art that only focuses on drug delivery and the carrier itself has no therapeutic function, this invention can achieve functional complementarity and synergy between the carrier and the load, thereby realizing a complete intervention chain from pathogen clearance to physical barrier repair to microecological reconstruction, thus providing a comprehensive solution for the full recovery of intestinal homeostasis.

[0025] (3) The interfacial complexation locking process mediated by multivalent metal ions in this invention can endow the carrier with extremely high structural stability. This process utilizes zinc ions and coordinating atoms on amino polysaccharide chains to form chelate nodes with spatial topological structures, which can construct a dynamic cross-linked network with covalent bond characteristics on the surface of the carrier. This network can withstand the shear force generated by gastrointestinal peristalsis and inhibit the desorption of inner layer components during long-distance transport. It effectively prevents the sudden release and premature leakage of drugs before reaching the target release site, thereby ensuring the accuracy and reliability of the drug dosage, significantly enhancing the reliability of the carrier in complex physiological environments, making the entire delivery process more controllable, thus laying the foundation for the certainty and reproducibility of the therapeutic effect and enhancing the potential for clinical translation. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a comparison diagram of the infrared spectral characteristics of OKGM and native KGM in Embodiment 1 of the present invention;

[0028] Figure 2 These are SEM images (a) and TEM images (b) of the Nisin nanoparticles (N-Lip) of Example 1 of the present invention;

[0029] Figure 3 This is a potential diagram of Nisin nanoparticles in Example 1 of the present invention;

[0030] Figure 4 This is a particle size distribution diagram of Nisin nanoparticles in Example 1 of the present invention;

[0031] Figure 5 This is a graph showing the release rate of Nisin from Nisin nanoparticles in simulated digestion according to Example 2 of the present invention.

[0032] Figure 6 This is a graph showing the cell viability performance of Nisin nanoparticles in Example 3 of the present invention;

[0033] Figure 7 This is a flowchart of the animal experiment in Embodiment 4 of the present invention;

[0034] Figure 8 This is the FITC transmittance diagram of Embodiment 4 of the present invention;

[0035] Figure 9 This is the Listeria abundance diagram of Example 4 of the present invention;

[0036] Figure 10 This is a graph showing the IFN-γ content in mouse blood in Example 4 of the present invention;

[0037] Figure 11 This is a relative abundance diagram of Lactobacillus johnsonii from Example 4 of the present invention;

[0038] The symbols and their corresponding materials in the figure are as follows: B-Lip is blank liposome; N-Lip is amphiphilic assembly core; N-OP is particles after preliminary modification with pectin and oxidized konjac glucomannan; NPLs is the final Nisin nanoparticles; NLPs-H is the high-dose group; NLPs-L is the low-dose group; free nisin is free nisin; BLPs is the packaging material group (Nisin-free); Amp is the ampicillin group (antibiotic). Detailed Implementation

[0039] 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.

[0040] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0041] Application Overview:

[0042] To overcome the delivery challenges of peptide drugs, existing technologies have attempted to employ strategies such as microencapsulation or liposome encapsulation to improve their stability and targeting. However, these technologies often focus only on the physical protection or sustained release of the active ingredient, resulting in relatively simple carrier structures and insufficiently intelligent responses to environmental changes. They struggle to provide sufficiently strong shielding protection in the acidic environment of the stomach and cannot achieve precise triggered release in specific segments of the intestine.

[0043] More importantly, these delivery systems typically lack biological functions and cannot provide additional synergistic effects beyond drug delivery, such as repairing tight junctions in intestinal epithelial cells or providing selective nutrition to beneficial bacteria to promote their proliferation. Therefore, existing delivery systems are inadequate and have limited effectiveness in addressing complex intestinal homeostasis intervention scenarios requiring synergistic effects of pathogen clearance, physical barrier repair, and microecological reconstruction.

[0044] To address the aforementioned issues, this invention proposes a novel multidimensional cascaded assembly micro / nano intestinal delivery carrier, its preparation method, and its applications. By constructing a cascaded self-assembling micro / nano carrier with a multi-layered core-shell structure, the carrier can sequentially respond to the physiological gradient changes in the gastrointestinal tract from strong acid to weak alkali. In the stomach, the dense outer layer and the contracting inner layer work together to provide strong protection, releasing almost no drugs. After entering the intestine, with the increase in pH and the action of specific enzymes, the outer layer undergoes a conformational change and adheres to the intestinal wall, while the inner network gradually relaxes. Ultimately, nisin is controllably released at the target site. The released antimicrobial peptide can directly kill pathogenic bacteria, while the polysaccharide components after carrier degradation can, on the one hand, physically fill and repair the gaps between damaged epithelial cells like biocement, and on the other hand, selectively promote the growth of beneficial bacteria such as Lactobacillus as a prebiotic. Thus, the three goals of pathogen clearance, barrier repair, and flora regulation are achieved simultaneously, comprehensively intervening in and rebuilding intestinal homeostasis.

[0045] To further simplify and make the present invention achieve its objectives and effects, the present invention is described in conjunction with the following specific embodiments, but the present invention is not limited to the scope of the embodiments described herein.

[0046] Example 1:

[0047] A method for preparing a novel multidimensional cascaded assembled micro / nano intestinal delivery carrier includes the following steps:

[0048] S1. Weigh 100 g of konjac glucomannan (KGM) and disperse it in 500 mL of ethanol solution (40% v / v). Adjust the pH to 4.3 at 40 °C. Add a total of 30 mL of 30 wt% H2O2 solution in three portions for selective oxidation. After reacting for 4 h, terminate the reaction with sodium sulfite. Wash the product three times sequentially with 70% v / v and 95% v / v ethanol solutions, and then vacuum dry at 60 °C for 12 h to obtain oxidized konjac glucomannan (OKGM). (Refer to...) Figure 1 As shown, Fourier transform infrared spectroscopy detection revealed that OKGM was at 1730 cm⁻¹. -1 A characteristic absorption peak appeared at the point, and the carbonyl content was determined to be 1.2 g / mmol by titration.

[0049] S2. Soybean lecithin and cholesterol in a mass ratio of 5:1 were dissolved in an ethanol solution. The organic solvent was removed by rotary evaporation at 100 rpm in a 50 ℃ water bath to form a uniform lipid film. A solution containing 30 mg / mL nisin and 3% w / v trehalose was added, and the solution was allowed to stand at 45 ℃ for 2 h for hydration. The suspension was then placed in an ice-water bath and sonicated at 60 W for 5 min to obtain a monodisperse vesicle suspension, yielding a uniform amphiphilic assembly core (N-Lip). The total mass ratio of soybean lecithin and cholesterol to the volume ratio of the ethanol solution was 30 mg:1 mL, and the volume ratio of the lipid film to the aqueous solution was 15 mg:1 mL.

[0050] S3. The vesicle suspension was slowly added dropwise at a rate of 1 drop per second to a composite solution containing 1.5% w / v pectin and 0.5% w / v oxidized konjac glucomannan. The mixture was magnetically stirred at 30 ℃ and 200 rpm for 40 min to induce adsorption of the inner shell layer and form intermediate particles (N-OP). The volume ratio of the vesicle suspension to the composite solution was 1:6.

[0051] S4. Add sodium acetate buffer containing 0.1 mM zinc acetate and 1% w / v chitosan dropwise to the intermediate particle suspension. React at 37 ℃ and pH 5.5 for 2 h to form an outer locking structure. Collect the particles by centrifugation at 4 ℃ and 10000 rpm for 10 min to obtain micro / nano delivery carriers (NLPs) with a three-layer barrier structure. The volume ratio of intermediate particle suspension to sodium acetate buffer is 1:3.

[0052] To verify the successful preparation of Example 1, Figure 2 SEM images of Nisin nanoparticles (N-Lip) from Example 1 are shown below. Figure 2 (a) and TEM image ( Figure 2 (b) shows a core-shell structure under an electron microscope, such as Figure 3 The Nisin nanoparticles, after being encapsulated layer by layer, exhibit an adhesion characteristic with a surface potential of +22.69 mV. Figure 4 As shown, the average particle size of Nisin nanoparticles after the layer-by-layer encapsulation technique is approximately 900 nm.

[0053] Example 2:

[0054] This embodiment is used to verify the sequential release behavior of the delivery vector in Example 1 under simulated gastrointestinal pH environments.

[0055] Experimental Methods: 10 mg of NLPs (nisin lactate) was weighed and placed in a 50 mL centrifuge tube. 20 mL of simulated gastric juice (hydrochloric acid solution containing 0.32% pepsin, pH 2) was added. The centrifuge tube was placed in a 37 ℃ water bath shaker and oscillated at 100 rpm. 5 mL of release medium was collected every 2 hours over 24 hours, and an equal volume of fresh simulated gastric juice was added simultaneously. After filtration through a 0.22 μm filter, the drug concentration was determined using high-performance liquid chromatography (HPLC).

[0056] Release result: such as Figure 5 As shown, in a simulated gastric fluid environment, the cumulative release rate after 2 hours was less than 5%, indicating that the carrier exhibited an extremely low release rate. The results demonstrate that the carrier possesses excellent gastric shielding and intestinal targeted release characteristics.

[0057] Example 3:

[0058] This embodiment is used to verify the biocompatibility of the delivery vector in Example 1 with the host cell.

[0059] Experimental Methods: The biocompatibility of the nanocarrier to host cells was evaluated using the CCK-8 assay. RAW264.7 cells and HT-29 cells were respectively inoculated at 1×10⁻⁶. 4 The cells were seeded at a density of cells / well in 96-well plates. After 24 h of incubation, NLPs medium containing gradient concentrations (0-30 µg / mL) was added for further incubation.

[0060] like Figure 6 The results showed that, within the tested concentration range, the survival rates of both cell types were significantly higher than 80%, demonstrating that the vector has excellent biosafety and meets the application requirements of oral drug delivery systems.

[0061] Example 4:

[0062] This embodiment is used to evaluate the in vivo effects of the delivery vector in Example 1 in preventing Listeria monocytogenes infection.

[0063] Experimental model construction and drug administration: such as Figure 7 As shown, C57BL / 6 mice were randomly divided into a control group, a model group, a free Nisin group, and the nanocomposite system (NLPs) group (divided into high and low doses), as well as an ampicillin (Amp) group. During the prevention phase (Day 7 to Day 0), the NLPs group received daily oral gavage of composite particles containing 7.5 mg / mL Nisin. During the infection phase (Day 0), except for the control group, all mice underwent Listeria monocytogenes challenge.

[0064] Evaluation of intestinal physical barrier repair: such as Figure 8As shown, using an in vitro HT-29 cell monolayer model, TNF-α stimulation caused a surge in FITC-glucan permeability to 30.17 ng / mL, while intervention with the NLPs of this invention significantly reduced the permeability to 13.83 ng / mL. This confirms that the physical adhesion between the chitosan component of the carrier's outer layer and the mucosal interface produces a highly efficient biocement sealing effect.

[0065] Pathogen clearance and systemic immune regulation: Bacterial colonization load in the spleen and liver of NLPs group mice 120 h after infection ( Figure 9 The level of IFN-γ in the NLPs group was significantly lower than that in the model group, with an effect equivalent to that of the clinical antibiotic amoxicillin. Simultaneously, the serum IFN-γ level in the NLPs group was significantly lower. Figure 10 This effectively inhibited the cascade outbreak of inflammatory factors.

[0066] Targeted remodeling of the gut microbiota: 16S rRNA sequencing analysis revealed that polysaccharide substrates released from the degradation of NLPs vectors selectively promoted the amplification of Lactobacillus johnsonii. Figure 11 Its relative abundance increased from 10.07% to 24.76%. This niche reconstruction based on the prebiotic effect effectively blocked the recolonization of pathogens and achieved long-term maintenance of intestinal homeostasis.

[0067] To further illustrate the present invention, Example 1 is used as the basis for comparison.

[0068] Comparative Example 1:

[0069] This comparative example is basically the same as Example 1, except that the core vesicles are free of steroid modifiers. The specific steps in S2 are as follows: soybean lecithin is dissolved in an ethanol solution, and the organic solvent is removed by rotary evaporation at 100 rpm in a 50 ℃ water bath to form a uniform lipid film. A solution containing 30 mg / mL nisin and 3% w / v trehalose is added, and the solution is allowed to stand at 45 ℃ for 2 h for hydration. The suspension is then placed in an ice-water bath and ultrasonically treated at 60 W for 5 min to obtain a monodisperse vesicle suspension, thus obtaining the assembled core. The volume ratio of soybean lecithin to ethanol solution is 30 mg: 1 mL, and the volume ratio of lipid film to aqueous solution is 15 mg: 1 mL.

[0070] Comparative Example 2:

[0071] This comparative example is basically the same as Example 1, except that: a single anionic polysaccharide inner layer is used, i.e. step S1 is omitted, and step S3 is specifically as follows: the vesicle suspension is slowly added dropwise at a rate of 1 drop per second to a composite solution containing 1.5% w / v pectin, and the mixture is magnetically stirred at 30 °C and 200 rpm for 40 min to induce adsorption of the inner shell layer and form intermediate particles; wherein, the volume ratio of the vesicle suspension to the composite solution is 1:6.

[0072] Comparative Example 3:

[0073] This comparative example is basically the same as Example 1, except that: there is no metal ion coordination in the outer layer. The specific steps of S4 are as follows: sodium acetate buffer containing 1% w / v chitosan is added dropwise to the intermediate particle suspension, and the mixture is reacted at 37 °C and pH 5.5 for 2 h to form an outer layer locking structure. The particles are collected by centrifugation at 4 °C and 10,000 rpm for 10 min to obtain the micro-nano delivery carrier. The volume ratio of the intermediate particle suspension to the sodium acetate buffer is 1:3.

[0074] Comparative Example 4:

[0075] This comparative example is basically the same as Example 1, except that the konjac glucomannan in the inner layer is not oxidized, i.e., there is no S1 step. The specific steps of S3 are as follows: the vesicle suspension is slowly added dropwise at a rate of 1 drop per second to a composite solution containing 1.5% w / v pectin and 0.5% w / v natural konjac glucomannan, and the mixture is magnetically stirred at 30 ℃ and 200 rpm for 40 min to induce the adsorption of the inner shell layer and form intermediate particles; wherein, the volume ratio of the vesicle suspension to the composite solution is 1:6.

[0076] Comparative Example 5:

[0077] This comparative example is basically the same as Example 1, except that different metal ions are used for outer layer locking. The specific steps of S4 are as follows: sodium acetate buffer containing 0.1 mmol / L calcium chloride and 1% w / v chitosan is added dropwise to the intermediate particle suspension, and the mixture is reacted at 37 °C and pH 5.5 for 2 h to form an outer layer locking structure. The particles are collected by centrifugation at 4 °C and 10000 rpm for 10 min to obtain a delivery carrier. The volume ratio of the intermediate particle suspension to the sodium acetate buffer is 1:3.

[0078] Performance testing: Using the same experimental methods as in Example 1, the cumulative release rate and FITC permeability of the delivery carriers in Example 1 and Comparative Examples 1-5 were statistically recorded in the gastric fluid environment for 2 hours. The results are shown in Table 1.

[0079] Table 1:

[0080] project Cumulative release rate (%) FITC transmittance (ng / mL) Example 1 4.7 13.83 Comparative Example 1 30.2 28.45 Comparative Example 2 25.6 20.17 Comparative Example 3 40.1 32.89 Comparative Example 4 30.8 25.33 Comparative Example 5 35.5 22.14

[0081] As shown in Table 1, the micro / nano intestinal delivery carrier of Example 1 of this invention exhibits an extremely low cumulative drug release rate in a simulated gastric environment, while the FITC permeability is only 13.83 ng / mL, significantly better than the comparative example. Specifically, the rational ratio of phospholipids and cholesterol in the core lipid bilayer of the carrier of this invention constructs a stable hydrophobic barrier. Cholesterol, as a flow regulator, is embedded between phospholipid molecules, restricting the movement of phosphatidyl chains and enhancing the lipid membrane's resistance to gastric acid. In the inner layer, pectin and oxidized konjac glucomannan form a pH-sensitive network through hydrogen bonds and hydrophobic interactions, undergoing conformational contraction in the acidic environment of the stomach, further blocking the penetration of hydrogen ions and proteases. In the outer layer, zinc acetate and the amino groups of chitosan, and the carboxyl groups of the inner polysaccharide form a dynamic coordination cross-linking network, which can construct a dense physicochemical dual barrier, synergistically achieving zero-leakage protection of the drug in the stomach. Regarding intestinal repair function, the oxidized konjac glucomannan, pectin and chitosan components after carrier degradation can seal the intercellular spaces through molecular filling effect, while acting as prebiotics to promote the proliferation of beneficial bacteria, significantly reduce FITC permeability and repair the intestinal physical barrier.

[0082] A comparison between Example 1 and Comparative Example 1 reveals that Comparative Example 1 did not add cholesterol, a sterol regulator, during the preparation of the core vesicles. Cholesterol, as a key regulatory molecule of the lipid membrane, has a rigid steroidal ring structure that can embed between phospholipid fatty acid chains. By restricting the swinging of phospholipid molecular chains, it increases the order density and mechanical strength of the lipid bilayer, while reducing the permeation of water molecules and ions. In Example 1, the synergistic effect of cholesterol and soybean phospholipids formed a more stable, denser bilayer vesicle structure with a lower leakage rate. In contrast, in Comparative Example 1, the vesicles formed solely by phospholipids exhibited excessive membrane fluidity and a loose structure. Under the extreme acidic environment and enzymatic hydrolysis of simulated gastric juice, membrane integrity was difficult to maintain, leading to premature and substantial leakage of the loaded nisin, with a cumulative release rate as high as 30.2%. Simultaneously, the unstable core also affected the effective assembly and binding between subsequent layers, reducing the overall stability of the carrier structure. Therefore, it performed poorly in the barrier repair model, with the FITC permeability increasing to 28.45 ng / mL.

[0083] A comparison between Example 1 and Comparative Example 2 reveals that Comparative Example 2 used only pectin in its inner layer coating, without adding oxidized konjac glucomannan. The inner layer in Example 1 is an interpenetrating network formed by OKGM and pectin through dynamic covalent bonds, hydrogen bonds, and electrostatic interactions. This hybrid network exhibits synergistic pH responsiveness: pectin undergoes carboxylation and shrinkage at low pH and dissociates and swells at high pH; the introduction of OKGM further enhances the network's crosslinking density and mechanical properties, and provides additional swelling drive at specific pH levels. In contrast, the single pectin layer in Comparative Example 2 relies primarily on weak hydrogen bonds between pectin molecular chains and possible calcium ion bridging, resulting in a network structure strength far lower than the hybrid network in Example 1. This weaker inner layer cannot effectively shrink in a gastric acid environment to provide tight protection in conjunction with the outer layer, and is prone to dissociation under mechanical stirring or enzymatic action, leading to weakened inner layer shielding function and premature drug release. Meanwhile, the prebiotic effect provided by the degradation of a single pectin layer is limited, and its selective regulation of gut microbiota is weaker than that of the combination of OKGM and pectin. Therefore, its overall effect on repairing the intestinal barrier and promoting the proliferation of beneficial bacteria is also slightly inferior.

[0084] A comparison between Example 1 and Comparative Example 3 reveals that Comparative Example 3 did not use metal ion coordination factors in its outer layer construction; the outer layer was formed solely through the electrostatic adsorption of the positively charged chitosan particles within the inner layer. In Example 1, Zn... 2+ As a multivalent metal ion, it can form strong coordination bonds with the amino and hydroxyl groups on the chitosan chains. This coordination can construct a three-dimensional, dynamic, but stable cross-linked network between chitosan molecular chains and between chitosan and the surface of the inner layer particles, thereby endowing the outer layer with extremely high mechanical strength and chemical stability. It can effectively resist the shear force generated by gastrointestinal peristalsis and maintain a dense conformation under the strong acid conditions of the stomach. Comparative Example 3 lacks this metal coordination cross-linking. Its outer layer is formed only by relatively weak electrostatic adsorption and physical entanglement, resulting in a loose and unstable structure. Under the simulated acidic environment of gastric juice and the action of proteases, this outer layer is easily damaged, peeled off, or dissolved, losing its protective function. This leads to a cumulative release rate of up to 40.1% of the core drug in the stomach, and the carrier cannot completely reach the intestine to perform its subsequent functions. Therefore, its effect on repairing the intestinal physical barrier is also the worst.

[0085] A comparison between Example 1 and Comparative Example 4 reveals that Comparative Example 4 uses KGM instead of OKGM, and the fundamental difference lies in the functional groups of their molecular structures. OKGM, through selective oxidation, introduces active carbonyl groups into the sugar chains. This not only allows for stronger hydrogen bond interactions with the hydroxyl groups in pectin, but more importantly, it can form pH-sensitive borate ester bonds with the cis-diol structures that may exist on the pectin chains under specific conditions, significantly enhancing the cross-linking strength, stability, and intelligent responsiveness of the inner network. Furthermore, the carbonyl groups of OKGM can also coordinate with zinc ions in the outer solution, strengthening the bonding force between layers. In contrast, the natural KGM used in Comparative Example 4 has molecular chains mainly composed of glucose and mannose linked by β-1,4 glycosidic bonds, rich in hydroxyl groups but lacking highly active carbonyl groups. Therefore, natural KGM can only bond with pectin through weaker non-covalent forces such as hydrogen bonds, resulting in a hybrid network with low cross-linking degree, loose structure, and weak responsiveness. This inner layer does not contract sufficiently in the acidic gastric environment, provides insufficient support to the outer layer, and is easily degraded, resulting in a decrease in its shielding effect. At the same time, its degradation products may be less efficient as prebiotics than functionalized OKGM, affecting the overall barrier repair and microecological regulation effects. Therefore, the FITC permeability increased to 25.33 ng / mL.

[0086] A comparison between Example 1 and Comparative Example 5 shows that: Comparative Example 5 uses calcium chloride (Ca... 2+ ) to replace zinc acetate (Zn 2 + (Ca) acts as an outer metal coordination ion. Although Ca 2+ It can also coordinate with the carboxyl groups and other groups of chitosan, but its coordination ability and the spatial configuration of the resulting coordination compounds are different from those of Zn. 2+ There are significant differences. Zn 2+ Belonging to the transition metal ion family, it possesses empty d orbitals and readily accepts lone pairs of electrons from nitrogen and oxygen atoms in chitosan, forming high-strength coordination complexes with diverse spatial configurations, thereby constructing a dense and stable three-dimensional cross-linked network. Ca... 2+ It is an alkaline earth metal ion, relying mainly on ionic bonds and relatively weak coordination interactions, and the network structure it forms is generally not as good as that of Zn. 2+ The system is stable. Furthermore, Zn 2+ It possesses certain antibacterial activity and wound-healing properties, and can synergize with the therapeutic targets of the carrier. Comparative Example 5 used Ca... 2+ The resulting chitosan outer layer has a lower crosslinking density and mechanical strength than Zn. 2+ The system. In the harsh environment of gastric juice, the outer layer has weak resistance to corrosion and shearing, and cannot provide the robust protection seen in Example 1. Therefore, drug leakage in the stomach is more severe, with a cumulative release rate of 35.5%. Although Ca 2+It may also participate in some biological processes, but its efficiency in building stable delivery barriers is not as good as Zn. 2+ This resulted in the FITC permeability increasing to 22.14 ng / mL.

[0087] The above description is based on the preferred embodiments of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0088] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a novel multidimensional cascaded assembly of micro / nano intestinal delivery carriers, characterized in that, Includes the following steps: S1. Phospholipids and sterol regulators are dissolved in an ethanol solution, the solvent is removed to form a uniform lipid film, an aqueous solution containing active biomacromolecules and stabilizers is added for hydration, and then ultrasonic treatment is performed under ice bath conditions to obtain a monodisperse vesicle suspension. S2. Slowly add the vesicle suspension to the anionic polysaccharide complex solution, stir at room temperature, induce adsorption of the inner shell layer, and form intermediate particles. S3. Add a cationic electrolyte solution containing a metal coordination factor dropwise to the intermediate particle suspension. The delivery carrier is obtained through interfacial electrostatic complexation and coordination cross-linking reaction.

2. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S1, the active biomolecule is nisin, and its concentration in the aqueous solution is 20-40 mg / mL; the volume ratio of the lipid film to the aqueous solution is 15 mg: 0.5-2 mL; the stabilizer is one of mannitol, trehalose, or sucrose, and its concentration is 1-5% w / v; the mass ratio of the phospholipid to the sterol regulator is 3-8:1, and the total mass of the phospholipid and sterol regulator to the volume ratio of the ethanol solution is 10-50 mg: 1 mL.

3. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S1, the phospholipid is selected from soybean phospholipid, lecithin, and hydrogenated lecithin; the sterol regulator is cholesterol or phytosterol; and the ultrasonic treatment specifically involves ultrasonic treatment at a power of 50-70W for 3-10 minutes.

4. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S2, the anionic polysaccharide complex solution contains 1-2% w / v pectin and 0.2-1.2% w / v oxidized konjac glucomannan, and the volume ratio of the vesicle suspension to the anionic polysaccharide complex solution is 1:2-10; the oxidized konjac glucomannan is prepared by selective oxidation and has an infrared spectrum in the range of 1700-1750 cm⁻¹. -1 It has characteristic absorption peaks within the range.

5. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S2, the ambient temperature stirring temperature is 20-40 ℃, the rotation speed is 100-300 rpm, and the stirring time is 30-90 min.

6. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S3, the cationic electrolyte solution contains 0.08-0.15 mM zinc acetate and 0.5-2% w / v chitosan solution; the volume ratio of the intermediate particle suspension to the cationic electrolyte solution is 1:1-5.

7. The novel multidimensional cascaded assembled micro / nano intestinal delivery carrier according to claim 1, its preparation method, and its application, characterized in that: In step S3, the conditions for the coordination crosslinking reaction are: pH value of 5.0-6.5, temperature of 25-37 ℃, and time of 1-3 h.

8. A novel multidimensional cascaded assembly of micro / nano intestinal delivery carriers, characterized in that, The carrier, prepared by any one of claims 1-7, has a core-shell structure and comprises, from the inside out: The core consists of a bilayered vesicle formed by phospholipids and sterol regulators, which encapsulates active biomacromolecules. The inner layer, covering the core surface, is a pH-sensitive network layer formed by non-covalent cross-linking of functionalized glucomannan derivatives and pectin; The outer layer, covering the surface of the inner layer, is a dense barrier layer formed by the coordination and electrostatic interaction of cationic aminopolysaccharides and polyvalent metal ions, and the outer layer has mucosal adhesion.

9. A novel multidimensional cascaded assembly micro / nano intestinal delivery carrier according to claim 8, characterized in that: The active biomolecule is nisin; the multivalent metal ion is zinc ion; the average particle size of the delivery carrier is 200-1200 nm, and the surface potential is +15 mV to +35 mV.

10. The application of a novel multidimensional cascaded assembly of a micro / nano intestinal delivery carrier as described in any one of claims 8-9, characterized in that, This drug is used to prepare a medicine for the prevention and / or treatment of intestinal pathogenic bacteria infection, repair of intestinal barrier function and regulation of intestinal microecological balance; the intestinal pathogenic bacteria is Listeria monocytogenes; the drug is administered orally, and the dosage, calculated based on the encapsulated nisin, is 50-100 mg per kilogram of body weight per day.

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