Multifunctional microalgae hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery systems, and discloses multifunctional microalgae hydrogel as well as a preparation method and application thereof. According to the multifunctional microalgae hydrogel, natural components are taken as carriers, efficient release of drugs at the far end of the intestinal tract is achieved through pH and an intestinal flora enzyme response mechanism, the retention time of the far end of the intestinal tract is remarkably prolonged in combination with the adhesion characteristic, and the time-space distribution of the drugs is monitored in real time by supporting a fluorescent tracing technology. Meanwhile, by precisely regulating and controlling the homeostasis of intestinal flora, the intestinal barrier and the blood brain barrier are repaired, neuroinflammation, neuronal apoptosis and A beta deposition are remarkably improved, and cognitive impairment is reversed. The technical bottlenecks of poor targeting property and low bioavailability of a traditional oral preparation are broken through, a safe and efficient novel drug delivery strategy is provided for intervening neurodegenerative diseases based on the intestine-brain axis, and the preparation has remarkable clinical transformation potential and application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery systems and relates to a multifunctional microalgae hydrogel and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), the neurodegenerative disease with the highest incidence rate in the world, has become a core challenge threatening social health. AD has senile plaques formed by the deposition of β-amyloid protein (β-amyloid, Aβ) and neurofibrillary tangles caused by hyperphosphorylation of Tau protein as its core pathology, and its clinical manifestations are progressive cognitive decline and behavioral disorders. With the aging of the population, the number of AD patients is growing rapidly, and its death toll will exceed that of cancer, becoming the second largest health threat after cardiovascular disease. AD not only seriously affects the quality of life of patients, but also brings huge care and economic burdens to the patients' families and society. Existing therapies can only temporarily relieve symptoms and cannot block the progression of the disease. Recent studies have found that intestinal flora disorders in AD patients aggravate neuroinflammation and Aβ deposition through the "gut-brain axis", suggesting that targeted intervention of intestinal flora may be a key direction to break through the bottleneck of AD treatment.

[0003] In recent years, Traditional Chinese Medicine (TCM) and its active ingredients have shown great promise in regulating the intestinal microbiota and improving cognitive impairment in AD via the gut-brain axis. Imbalances in the gut microbiota can trigger systemic inflammation, leading to intestinal inflammation and impaired intestinal barrier function. A large number of proinflammatory cytokines enter the bloodstream and cross the blood-brain barrier, triggering central nervous system inflammation and contributing to AD pathology. Through a multi-target synergistic mechanism, TCM and its active ingredients can effectively inhibit the overproliferation of intestinal pathogens and promote the colonization of beneficial bacteria, thereby reducing intestinal inflammation, repairing the intestinal barrier, and blocking the peripheral release of proinflammatory cytokines. This regulatory process not only reduces the permeability of the blood-brain barrier but also inhibits abnormal microglial activation, interrupting the neuroinflammatory cascade, ultimately reducing Aβ deposition and Tau protein pathological damage, and improving cognitive dysfunction. Therefore, TCM and its active ingredients have promising application prospects and enormous development value in treating AD via the gut-brain axis.

[0004] The intestinal flora has a step-by-step colonization characteristic, with about 90% of the flora concentrated in the distal intestine (cecum and colon), while traditional oral Chinese medicine and its active ingredients are easily degraded by gastric acid, absorbed by the small intestine, and blocked by the mucus barrier. Only a small part of the drug can reach the distal intestine. In order to achieve an effective concentration, it is often necessary to significantly increase the dosage, which not only leads to reduced bioavailability, but may also cause side effects such as gastrointestinal irritation. In addition, the interaction with the intestinal flora requires the drug to have a long-term retention ability, while ordinary preparations have a shorter residence time in the distal intestine, making it difficult to continuously regulate the function of the flora. Furthermore, due to the lack of real-time positioning and monitoring methods, it is impossible to accurately understand the distribution and release process of the drug in the gastrointestinal tract, which also limits the regulatory effect of the drug on the intestinal flora.

[0005] Therefore, developing a delivery system that targets the intestinal flora, achieving precise drug release through smart materials, enhancing its adhesion to the intestinal mucosa to prolong the duration of action, and realizing a multifunctional drug delivery system that can monitor the spatiotemporal distribution of drugs in the gastrointestinal tract in real time, has become a breakthrough in improving the efficacy of traditional Chinese medicine and its active ingredients in treating AD by regulating the microbiome-gut-brain axis. Summary of the Invention

[0006] In order to address the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a multifunctional microalgae hydrogel with intestinal flora targeting. The multifunctional microalgae hydrogel has a multilayer structure design, including calcium alginate hydrogel and microalgae (BBR-SP-M) coated in the calcium alginate hydrogel; the microalgae is mucin (Mucin, M) modified drug-loaded platen Spirulina (BBR-SP); the drug is berberine (BBR). The mucin-modified drug-loaded platen microalgae can mimic the components of the intestinal mucus layer and bind to the intestinal epithelial cell MUC2 protein through a disulfide bond to prolong the retention time; at the same time, the calcium alginate hydrogel ionizes and swells under the pH environment of the ileum, and cooperates with the intestinal flora enzyme response degradation to achieve spatiotemporal controlled drug release.

[0007] Another object of the present invention is to provide a method for preparing the multifunctional microalgae hydrogel with intestinal flora targeting.

[0008] Another object of the present invention is to provide the use of the multifunctional microalgae hydrogel with intestinal flora targeting in drugs for treating neurodegenerative diseases.

[0009] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0010] A multifunctional microalgae hydrogel with intestinal flora targeting properties, the gel consisting of a shell structure and a core structure encapsulated within the shell structure; the shell structure is calcium alginate hydrogel, and the core structure is microalgae (BBR-SP-M); the microalgae is mucin (Mucin, M)-modified drug-loaded Spirulina platensis (BBR-SP); the drug is berberine (BBR); the berberine is loaded in the pores and surface of Spirulina platensis, and mucin is modified on the surface of the drug-loaded Spirulina platensis through positive and negative electrical adsorption.

[0011] The preparation method of the multifunctional microalgae hydrogel with intestinal flora targeting comprises the following steps:

[0012] S1. Dissolve berberine in water, add Spirulina platensis, stir, and centrifuge to obtain drug-loaded Spirulina platensis;

[0013] S2. dissolving the drug-loaded Spirulina platensis prepared in step S1 in water, adding mucin, stirring, and centrifuging to obtain mucin-modified drug-loaded Spirulina platensis;

[0014] S3. Dissolve sodium alginate in PBS containing the drug-loaded Spirulina platensis prepared in step S2, add calcium gluconate while vortexing, and after cross-linking, obtain the multifunctional microalgae hydrogel with intestinal flora targeting.

[0015] Preferably, the concentration of berberine in the system in step S1 is 100-1000 μg / ml; more preferably 600 μg / ml.

[0016] Preferably, the stirring time in step S1 is 2 h to 12 h; more preferably 6 h.

[0017] Preferably, the stirring in step S1 is magnetic stirring at a rotation speed of 600 rpm.

[0018] Preferably, the centrifugation condition in step S1 is 2380×g for 10 min.

[0019] Preferably, the concentration of the mucin in the system in step S2 is 0.5-2 mg / ml, and more preferably 1 mg / ml.

[0020] Preferably, the stirring in step S2 is stirring in an ice bath.

[0021] The centrifugation conditions described in step S2 were 2380×g for 5 min.

[0022] Preferably, the concentration of sodium alginate in the system in step S3 is 2 w / v% to 10 w / v%; more preferably, it is 8 w / v%.

[0023] Preferably, the concentration of calcium gluconate in the system in step S3 is 2 w / v% to 4 w / v%, and more preferably 3 w / v%.

[0024] A multifunctional microalgae hydrogel with intestinal flora targeting properties is obtained by the above-mentioned preparation method. The multifunctional microalgae hydrogel has pH-responsive properties and swells and releases drugs in an environment with a pH of ≥7.4.

[0025] The application of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for treating neurodegenerative diseases.

[0026] Furthermore, the neurodegenerative diseases include but are not limited to Alzheimer's disease.

[0027] The application of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for treating Alzheimer's disease based on the microbiome-gut-brain axis.

[0028] The application of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for regulating intestinal flora homeostasis.

[0029] The role of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for improving intestinal inflammation.

[0030] The role of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for repairing intestinal barrier damage and / or blood-brain barrier damage.

[0031] The role of the above-mentioned multifunctional microalgae hydrogel with intestinal flora targeting in the preparation of drugs for reversing cognitive dysfunction.

[0032] Furthermore, the medicine is an oral medicine.

[0033] Furthermore, the effective dose of the drug is 10 mg / kg / day calculated as berberine, and the course of treatment is ≥30 days.

[0034] The present invention constructs a microalgae-based multilayer hydrogel drug delivery system, utilizes the synergistic effect of naturally derived calcium alginate matrix and mucin-modified drug-loaded microalgae, and innovatively develops an intelligent drug delivery system that integrates intestinal distal targeted drug release, enhanced mucosal adhesion, microbial microenvironment response, and dynamic fluorescent tracing. This system uses natural ingredients as carriers and realizes efficient drug release at the distal end of the intestine through a pH-combined intestinal flora enzyme response mechanism. Combined with adhesion properties, it significantly prolongs the distal intestinal retention time and supports real-time monitoring of drug spatiotemporal distribution using fluorescent tracing technology. At the same time, it significantly improves neuroinflammation, neuronal apoptosis, and Aβ deposition, and reverses cognitive dysfunction by precisely regulating intestinal flora homeostasis, repairing the intestinal barrier, and the blood-brain barrier. The present invention breaks through the technical bottleneck of poor targeting and low bioavailability of traditional oral preparations, and provides a safe and efficient new drug delivery strategy for intervening in neurodegenerative diseases based on the "gut-brain axis", with significant clinical translation potential and application value.

[0035] The present invention has the following advantages and effects compared to the prior art:

[0036] The multifunctional microalgae hydrogel with intestinal flora targeting provided by the present invention protects BBR-SP-M from the influence of the upper gastrointestinal tract; based on the intelligent drug release of the intestinal microenvironment, it can effectively target the intestinal flora; significantly prolong the mucosal retention capacity of the drug in the distal intestinal tract; support fluorescence tracing to achieve dynamic monitoring of drug distribution; effectively regulate the homeostasis of the intestinal flora, inhibit the colonization of intestinal pro-inflammatory bacteria, and promote the colonization of anti-inflammatory bacteria; reduce the level of intestinal inflammation and synergistically restore the intestinal barrier function; reduce the level of peripheral inflammation, thereby restoring the integrity of the blood-brain barrier; reduce the level of neuroinflammation, and reverse neuronal apoptosis and the accumulation of abnormal proteins.

[0037] The matrix materials used in the present invention are all natural ingredients and have excellent biocompatibility and metabolic safety.

[0038] The present invention has the advantages of fewer process steps, simple operation, and fast preparation speed. It can significantly shorten the production cycle, improve production efficiency, is suitable for large-scale continuous production, and has good clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The scanning electron microscope (pseudo-color) images of the multifunctional microalgae hydrogel and its intermediates obtained in Example 1 are as follows: A is Spirulina platensis (SP), B is drug-loaded Spirulina platensis (BBR-SP), C is mucin-modified drug-loaded Spirulina platensis (BBR-SP-M), and D is calcium alginate hydrogel (Gel) encapsulating BBR-SP-M.

[0040] Figure 2A comparison chart of drug release behavior of multifunctional microalgae hydrogels during in vitro simulated gastrointestinal transport.

[0041] Figure 3 Figure 3 is a diagram of the dynamic delivery process of multifunctional microalgae hydrogel in the gastrointestinal tract of mice: A is the degradation process of Gel, and B is the release behavior of BBR revealed by the dual-channel fluorescence co-localization analysis of BBR and SP.

[0042] Figure 4 This is the utility diagram of multifunctional microalgae hydrogel in regulating intestinal flora: where A is α diversity and B is β diversity.

[0043] Figure 5 A diagram showing that multifunctional microalgae hydrogel reduces intestinal inflammation levels; A is the immunofluorescence of CD86, and B is the immunofluorescence of CD206.

[0044] Figure 6 Transmission electron micrograph of multifunctional microalgae hydrogel repairing the intestinal barrier.

[0045] Figure 7 Multifunctional microalgae hydrogel reduces peripheral inflammation levels.

[0046] Figure 8 Transmission electron microscopy image showing multifunctional microalgae hydrogel improving blood-brain barrier permeability.

[0047] Figure 9 This is a pharmacodynamic evaluation diagram of the multifunctional microalgae hydrogel against Alzheimer's disease; A is ASC staining, B is Caspase-1 staining, C is Nissl staining, and D is Aβ 1-42 ELISA test. DETAILED DESCRIPTION

[0048] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0049] The Spirulina platensis (SP) used in the following examples was purchased from Guangyu Biotechnology Co., Ltd., with the product number CDB0201.

[0050] Mucin (M) used in the following examples was purchased from Sigma-Aldrich, catalog number 84082-64-4.

[0051] Example 1: Preparation and characterization of multifunctional microalgae hydrogel

[0052] (1) Preparation of drug-loaded Spirulina platensis

[0053] Weigh 5 mg of SP in pure water, add BBR at a concentration of 600 μg / ml, and then add pure water to a total volume of 10 ml. Stir in a magnetic stirrer at 600 rpm for 6 hours, then centrifuge at 2380 × g for 10 minutes to collect the precipitate. Dilute the supernatant to a certain ratio, and measure the absorbance of BBR using an ultramicrospectrophotometer to calculate the drug loading efficiency.

[0054] (2) Preparation of mucin-modified drug-loaded Spirulina platensis

[0055] The prepared BBR-SP was mixed with 1 mg / ml M, and a certain amount of pure water was added to make the volume 10 ml. The mixture was stirred in an ice bath for 1 h, and the rotation speed was 600 rpm and the centrifugal force was 2380 × g for 5 min. The precipitate was collected as BBR-SP-M.

[0056] (3) Preparation and characterization of multifunctional microalgae hydrogels

[0057] Prepare 8% (w / v) sodium alginate solution and add it to a certain volume of PBS containing the prepared BBR-SP-M in batches, vortexing while adding until the sodium alginate is completely dissolved. Then add 3% (w / v) calcium gluconate while vortexing. After the calcium gluconate is evenly distributed, let it stand at room temperature for 4 hours and observe the gel formation.

[0058] Scanning electron microscopy imaging of the above-formed Gel and its intermediates was performed, and the results were as follows: Figure 1 As shown. SP is spiral in shape, with a diameter of about 50-200μm. The thread-like segments are clearly visible, and the dispersion is good without obvious aggregation. Compared with SP, the surface of BBR-SP is covered with plaque-like substances (as shown by arrows), and the porosity is reduced, but the structure remains intact, indicating that BBR is successfully loaded into the pores and surface of SP. BBR-SP-M forms a smooth and continuous white matte layer on the surface of BBR-SP. The gel matrix (purple-red) of Gel has a smooth and continuous surface, tight cross-linking, uniform skeleton distribution, and no obvious cracks or collapse, indicating that it has good structural stability. BBR-SP-M (green) is clearly visible, embedded and dispersed in the gel matrix, forming a close physical bond with the gel network.

[0059] Comparative Example 1: Preparation of drug-loaded Spirulina platensis under different conditions

[0060] 5 mg of SP was weighed and dissolved in pure water. BBR was added at concentrations of 100 μg / ml, 200 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml, and 1000 μg / ml, respectively. Pure water was then added to bring the total volume to 10 ml. The mixture was stirred in a magnetic stirrer at 600 rpm / min for a specified period of time, followed by centrifugation at 2380 × g for 10 minutes. The precipitate was collected. The supernatant was diluted to a specified ratio, and the absorbance of the BBR was measured using an ultramicrospectrophotometer to calculate the drug loading efficiency. The results showed that the drug loading efficiency reached 25% at a concentration of 600 μg / ml, indicating a higher loading of 150 μg of drug. The other conditions showed lower loadings of 37 μg, 70 μg, 103 μg, 107 μg, and 111 μg of drug, respectively. Afterwards, the drug was loaded at a concentration of 600 μg / ml for 2h, 4h, 6h, 8h and 12h, the supernatant was collected to measure the BBR absorbance, and the drug loading rate was calculated. The results showed that the drug loading rate was the highest at 6h, reaching 28.5%. The drug loading rates under other conditions were lower, only 26.5%, 27.4%, 25.0%, 24.2% and 22.8%.

[0061] Comparative Example 2: Preparation of multifunctional microalgae hydrogels under different conditions

[0062] (1) Weigh 5 mg of SP and dissolve it in pure water. Add BBR at a concentration of 600 μg / ml and add pure water to make the total volume 10 ml. Stir in a magnetic stirrer at 600 rpm / min for 6 h and centrifuge at 2380 × g for 10 min to collect the precipitate.

[0063] (2) Preparation of mucin-modified drug-loaded Spirulina platensis

[0064] The prepared BBR-SP was mixed with 1 mg / ml M, and a certain amount of pure water was added to make the volume 10 ml. The mixture was stirred in an ice bath for 1 h, centrifuged, and the precipitate was collected as BBR-SP-M.

[0065] (3) Preparation of multifunctional microalgae hydrogel

[0066] 2%, 4%, 6%, 8%, and 10% (w / v) sodium alginate solutions were prepared and added in batches to a certain volume of PBS containing the prepared BBR-SP-M. The solution was vortexed while adding until the sodium alginate was completely dissolved. 3% (w / v) calcium gluconate was then added while vortexing. After the calcium gluconate was evenly distributed, the solution was left at room temperature for 4 hours and the gel formation was observed. The results showed that the 2% and 4% concentrations failed to form a stable hydrogel. In the bottle inversion test, the hydrogels exhibited fluidity characteristics and collapsed or dripped, indicating that the structural stability under these concentration conditions did not meet the hydrogel formation standard.

[0067] 6%, 8%, and 10% hydrogels were placed in artificial gastric fluid (AGF) containing pepsin (pH 1.2), artificial small intestinal fluid (ASF) (pH 6.8), and artificial colonic fluid (ACF) (pH 7.4) (a total volume of 10 ml). Oral drug release was simulated in a constant temperature water bath shaker. Supernatants were collected at various time points and the BBR release concentration was measured using an ultramicrospectrophotometer. The results showed that the 6% or 10% hydrogels did not exhibit stable release characteristics. Both the 6% and 10% hydrogels exhibited a burst release of approximately 16% and 21% after 1 hour in ACF (pH 7.4), indicating a defect in the stability of the three-dimensional cross-linking network. In contrast, the 8% hydrogel had a more stable release rate, reaching 10% after 1 hour in ACF, and subsequent release was more stable.

[0068] Comparative Example 3:

[0069] The preparation of reference example 1 was different in that the solvent pure water was replaced with anhydrous ethanol or 40% ethanol. The results showed that the drug loading efficiency of SP did not increase after the solvent was changed. At the same time, scanning electron microscopy revealed that the morphology of SP was severely wrinkled.

[0070] Comparative Example 4:

[0071] The preparation of Reference Example 1 differs in that the crosslinking agent is replaced by calcium chloride (CaCl2) from calcium gluconate, and the calcium ion concentration is kept constant. The results show that due to the high solubility of calcium chloride, a gelation reaction is rapidly initiated after addition to the system, forming a dense layer in a short period of time. This dense layer hinders the diffusion of internal calcium ions, thereby affecting the subsequent gelation process, and ultimately resulting in a hydrogel with significant differences in structure and properties.

[0072] Comparative Example 5:

[0073] The preparation was carried out as in Example 1, except that the lyophilized SP was permeabilized with 1% hydrogen chloride. The results showed that the permeabilized SP did not increase the drug loading rate.

[0074] Example 2: In vitro simulated gastrointestinal release test of multifunctional microalgae hydrogel

[0075] BBR-SP, BBR-SP-M, and Gel (quantified by SP, SP concentration was 500 μg / ml) of the same concentration were placed in artificial gastric fluid (AGF, pH 1.2) for 2 h, artificial small intestinal fluid (ASF, pH 6.8) for 4 h, and artificial colonic fluid (ACF, pH 7.4) for 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h, with a total volume of 10 ml. The supernatant was taken at each time point, and the BBR release rate was measured to simulate the drug release in simulated gastrointestinal fluid in vitro to determine that the prepared Gel has excellent pH response characteristics.

[0076] The results are as follows Figure 2 As shown in Figure 2, the Gel exhibits significant pH-responsive properties in the release behavior of simulated drugs in gastrointestinal fluid. The BBR release rate of BBR-SP and BBR-SP-M in strongly acidic gastric fluid (pH 1.2) is approximately 20%, because the Gel

[0077] In highly acidic gastric fluid, COOH groups are protonated to form a dense network, preventing BBR-SP-M from diffusing. Consequently, its release rate is only 3%. In weakly acidic intestinal fluid (pH 6.8), the release rate of BBR-SP is approximately 30%, while that of BBR-SP-M is approximately 25%. When exposed to the weakly acidic environment, the gel partially deprotonates its COOH groups, causing slight swelling and a BBR release rate of approximately 7%. In neutral intestinal fluid (pH 7.4), the release rate of BBR-SP reaches approximately 80% after 1 hour, while that of BBR-SP-M is approximately 75%. After 4 hours in ACF, the release rate of BBR reaches 93%, with complete release by 8 hours. After 4 hours in ACF, the release rate of BBR reaches 95%, with complete release by 8 hours. In the neutral colonic fluid (pH 7.4) environment, the -COOH group of the gel is completely ionized. The electrostatic repulsion between the ions causes the gel network structure to swell, forming a diffusion channel for the release of BBR-SP-M. However, BBR is not completely released from BBR-SP-M at 1 hour, so the cumulative release rate is only 10%. However, 90% of BBR is released after 24 hours, and the release is complete after 36 hours. Based on the above results, it can be seen that the gel can release BBR in response to a neutral environment of pH 7.4. Based on this, it is speculated that the prepared gel may be released in the ileum or colon due to its pH-responsive properties. Therefore, in vitro experiments can prove its potential targeting of intestinal flora. In summary, compared with BBR-SP and BBR-SP-M, the gel has a significant protective effect on BBR-SP-M, preventing drug release in the upper gastrointestinal tract (stomach to jejunum). Instead, most of the drug is delivered to the distal intestine and achieves a slow release effect.

[0078] Example 3: Dynamic delivery of multifunctional microalgae hydrogel in the gastrointestinal tract of mice

[0079] (1) Mice were divided into three groups: BBR-SP, BBR-SP-M, and Gel, with three mice in each group. Mice were gavaged with 200 μl of BBR-SP, BBR-SP-M, and Gel (quantified by SP, the dose was 30 mg / kg, and the administration volume was 200 μl for a total of 30 days). Animal experiments were conducted in accordance with the Regulations on Experimental Animal Ethics of Guangzhou University of Chinese Medicine and passed its ethical review (ethics number: 20240410001).

[0080] (2) After a certain period of gavage, the stomach, duodenum, jejunum, ileum, cecum and colon tissues (including contents) of the mice in the Gel group were collected and fixed in 4% glutaraldehyde fixative for more than 4 h. The fixative in the tissues was then discarded and the tissues were washed three times with PBS. They were then fixed with osmium acid fixative for 1-2 h and washed three times with PBS. The tissues were dehydrated with ethanol gradient and dried. The degradation of Gel in various parts of the gastrointestinal tract was observed using a scanning electron microscope.

[0081] (3) The duodenum, jejunum, ileum, and colon of each group of mice were collected and frozen into 20 μm sections. After staining with DAPI for 10 min, the sections were observed under a fluorescence microscope to evaluate drug release and its distribution in the intestinal tissue.

[0082] The results are as follows Figure 3 As shown, the gel maintains an intact morphology and structure from the stomach to the duodenum, with a dense surface. A small amount of BBR-SP-M is exposed outside the gel matrix, and there is no obvious sign of degradation, indicating good stability in the upper gastrointestinal tract. As the gel passes through the jejunum, the pH rises, and the outermost hydrogel begins to swell, developing pores and fissures, resulting in a looser structure. A small amount of BBR-SP-M is exposed, but the majority remains encapsulated within the carrier. By the ileum, the gel fully expands and degrades into fragments, fully exposing the BBR-SP-M. In the cecum and colon, the drug released from the hydrogel interacts with the intestinal flora. The fluorescence of Cy3 (representing BBR) and Cy5 (representing Gel) in BBR-SP and BBR-SP-M do not co-localize starting in the duodenum and jejunum, respectively. This means that BBR in BBR-SP is released from SP in the duodenum, while BBR in BBR-SP-M begins to be released in the jejunum. This means that both release BBR in the upper half of the gastrointestinal tract, and BBR reduces its interaction with intestinal flora through gastrointestinal action or blood circulation. Gel, on the other hand, does not co-localize starting in the ileum, and the released BBR interacts appropriately with the flora in the cecum and colon. Therefore, the delivery process of Gel exhibits a clear spatiotemporal dependence, as evidenced by its stable morphology and structure in the upper gastrointestinal tract, the onset of degradation in the jejunum, and the release of BBR in the ileum. The released BBR is concentrated in the distal intestine to interact with the intestinal flora, indicating that Gel achieves intestinal flora targeting.

[0083] Example 4: Effectiveness of multifunctional microalgae hydrogel in regulating intestinal flora

[0084] C57BL / 6J male mice were divided into six groups: ① control group, ② model group, ③ BBR group, ④ BBR-SP group, ② BBR-SP-M group, and ⑥ Gel group. An AD model with intestinal flora disturbance was established by oral administration of aluminum chloride (AlCl3) and intraperitoneal injection of galactose (D-gal) at a dose of 20 mg / kg and 150 mg / kg, respectively, for 2 months. The control group received the same volume of PBS daily by oral and intraperitoneal injection. Two months later, drug administration was resumed. The control and model groups received PBS by oral administration, the BBR group received 200 mg / kg, and the BBR-SP, BBR-SP-M, and Gel groups received 10 mg / kg of BBR, all in a 200 μl volume, for a total of 30 days. After drug administration, feces from each group were collected and placed in sterile EP tubes, snap-frozen in liquid nitrogen, and then transferred to -80°C for storage and sequencing experiments. The forward primer for sequencing was 5′-CCTACGGGRS GCAGCAG-3′; the reverse primer was 5′-GGACTACVVGGG TATCTAATC-3′.

[0085] The results are as follows Figure 4As shown in the figure, compared with the control group, the Chao1 index and ACE index in the model group were significantly decreased, indicating a significant reduction in the species richness of the intestinal microbiota in AD mice. Among the intervention groups, the Chao1 and ACE indices in the BBR, BBR-SP, and BBR-SP-M groups, while slightly improved compared to the model group, remained below the control group. The Chao1 and ACE indices in the Gel group were closest to those in the control group, indicating that the Gel intervention effectively restored the model-induced decrease in species richness, bringing the intestinal microbiota closer to a normal state. Species diversity and richness results showed that the Simpson and Shannon indices in the model group were decreased, indicating a simple and unevenly distributed microbiota structure after modeling. The Simpson and Shannon indices in the Gel group were closest to those in the control group and higher than those in the other intervention groups. This suggests that the Gel treatment not only restored the species richness of AD mice but also maintained a balanced distribution of relative abundance among species within the microbiota, preventing the overgrowth of a single species and thus promoting the stability of the intestinal microbiome. Principal component analysis (PCA) results showed that the centroids of the sample points in the model group were significantly separated from those in the control group, indicating that modeling significantly altered the composition of the intestinal microbiota. The centroids of the samples in the BBR, BBR-SP, and BBR-SP-M groups were somewhat distant from the model group but not from the control group, suggesting that these intervention groups had a modulatory effect on the composition of the intestinal microbiota. However, compared with the Gel group, the centroids of the sample points in the control and Gel groups were closer together, indicating a high degree of similarity in the overall structure and composition of the intestinal microbiota between the two groups. Principal coordinate analysis (PCoA) results showed that the model group samples formed independent clusters, clearly separated from the control and Gel groups, further demonstrating that modeling led to abnormal changes in the intestinal microbiota. Samples in the BBR-SP and BBR-SP-M groups were distributed between the model group and the normal microbiota area, while samples in the BBR, Gel, and control groups clustered closely, with a higher degree of overlap between the Gel group and the control group, indicating that Gel intervention maximized the restoration of the microbiota structure.

[0086] Example 5: Multifunctional microalgae hydrogel reduces intestinal inflammation levels

[0087] Immunofluorescence analysis: Colon tissue was dehydrated in a 30% sucrose solution and then stored in a refrigerator at 4°C. Dehydration was complete when the tissue settled to the bottom of the tube. Next, the cryostat was pre-cooled to -20°C, and the tissue was fixed in OCT embedding medium. The cryostat was then placed in the cryostat and frozen until the OCT appeared white. Sections were cut to 20 μm thick and, using forceps, placed in PBS to thaw the OCT. The slides were washed three times with PBS for 5 minutes each on a shaker. After washing, the slides were blocked with 5% BSA at room temperature for 2 hours. Primary antibody was then added and incubated overnight. The next day, the primary antibody was removed, and the slides were washed three times with PBS for 10 minutes each. Secondary antibody (Cy3-labeled) was then added and incubated at room temperature in the dark for 2 hours. After incubation, the secondary antibody was removed, and the slides were washed three times with PBS. Next, DAPI was incubated in the dark for 10 minutes, followed by mounting and mounting with an anti-fluorescence quencher. Finally, the slides were observed under a microscope. The primary antibodies and their dilution ratios were as follows: CD206 (1:1000), CD86 (1:1000).

[0088] The results are as follows Figure 5 As shown, the CD86 fluorescence intensity in the colon tissue of the model group was significantly increased, indicating increased infiltration of colonic M1 macrophages and enhanced pro-inflammatory responses in the model group. After treatment, the CD86 levels in each group decreased, but the CD86 fluorescence intensity in the Gel group decreased the most compared with the model group and was comparable to that in the control group, indicating that it effectively reduced the level of intestinal inflammation by inhibiting the polarization of macrophage M1. At the same time, CD206 fluorescence detection showed that the expression of M2 anti-inflammatory macrophages in the model group was significantly weakened, while the CD206 intensity in the Gel group increased significantly compared with the model group and was significantly higher than that in the BBR, BBR-SP, and BBR-SP-M groups. This indicates that Gel intervention reduces the expression of colonic M1 macrophages and promotes the transformation of macrophages to M2, inhibiting pro-inflammatory and enhancing anti-inflammatory responses, achieving immune regulation of intestinal inflammation.

[0089] Example 6: Multifunctional microalgae hydrogel repairs intestinal barrier

[0090] The colon tissues of the control group, model group and Gel group were fixed in 4% glutaraldehyde fixative for more than 4 hours, and then the fixative in the colon tissue was poured out. The tissues were washed three times with PBS, fixed with osmium acid fixative for 1-2 hours, washed three times with PBS, dehydrated with ethanol gradient, dried, and the intestinal barrier function of each group was observed by the microscope.

[0091] The results are as follows Figure 6As shown, in the control group, the intestinal villi were neatly arranged, the intestinal epithelial cells were tightly arranged, and the tight junctions between the epithelial cells (red arrows) were numerous and continuous and dense, indicating that the barrier function was intact. In the model group, the villi structure was atrophied, the surface microvilli were sparse, the intestinal epithelial cells had cavities and were not tightly arranged, the tight junctions were few and incomplete, and local areas were broken, indicating that the intestinal barrier permeability was significantly increased. In the Gel group, the villi height and density were close to the control group, and the tight junctions returned to a continuous and dense structure, indicating that the regulation of Gel restored the integrity of the IB.

[0092] Example 7: Multifunctional microalgae hydrogel reduces peripheral inflammation levels

[0093] After deep anesthesia, blood was collected from the orbits of mice in each group. After the blood was allowed to stand for 1 hour, it was centrifuged at 900g for 15 minutes to separate the upper serum layer. Deionized water was used to prepare the corresponding concentrations of capture antibody solution, detection antibody solution, coating solution, washing solution, sample diluent, color development solution, and stop solution. 100μl of capture solution was added to the well plate and stored at 4°C overnight. After blocking with 5% BSA for 2 hours, 40μl of serum was added, followed by 10μl of biomarker antibody and 50μl of enzyme-labeled reagent, and the well plate was incubated at 37°C for 30 minutes. Wash with PBS five times, add 50μl of color development solution to each well, incubate at 37°C in the dark for 10 minutes, add 50μl of stop solution to terminate the reaction, and measure the absorbance at 450nm.

[0094] The results are as follows Figure 7 As shown in the figure, compared with the control group, the serum levels of IL-1β and IL-18 in the model group mice were significantly elevated, indicating a significant increase in peripheral inflammation. After intervention with BBR, BBR-SP, and BBR-SP-M, the levels of IL-1β and IL-18 were significantly reduced, while the IL-1β and IL-18 levels in the Gel group were the lowest and close to those in the control group. This indicates that Gel intervention blocked the levels of peripheral inflammatory factors.

[0095] Example 8: Transmission electron microscopy image of multifunctional microalgae hydrogel improving blood-brain barrier permeability

[0096] The brain tissues of the control group, model group and Gel group were fixed in 4% glutaraldehyde fixative and then photographed under biological transmission electron microscopy.

[0097] The results are as follows Figure 8As shown in the figure, a complete blood-brain barrier structure can be seen in the control group: the vascular endothelial cells have regular morphology, pericytes are closely attached to the vascular wall, astrocytes surround the blood vessels, and the tight junctions (TJs) are dense and obvious, so they can effectively block the penetration of large molecules. No obvious TJs were seen in the model group, indicating that the blood-brain barrier in the model group has undergone pathological changes, resulting in increased BBB permeability. The TEM image of the Gel group showed that the blood-brain barrier structure was intact, and the TJs were restored to continuous and dense connections. The enlarged image shows that the TJs in the Gel group are obvious and orderly. This suggests that Gel intervention increases BBB permeability.

[0098] Example 9: Pharmacodynamic evaluation of multifunctional microalgae hydrogel against Alzheimer's disease

[0099] Immunofluorescence staining: Brain tissue was frozen and sectioned for immunofluorescence staining. The primary antibodies and their dilutions were as follows: ASC (1:1000) and Caspase-1 (1:500).

[0100] Nissl staining: Brains were cut to a desired size, dehydrated in a dehydrator, embedded in paraffin, and sliced ​​into 5 μm thick slices using a paraffin microtome. The slices were dewaxed in xylene and rehydrated in varying degrees of ethanol. The tissue area was circled with an immunohistochemistry pen and incubated in Nissl stain in a 60°C oven for 30 minutes. After incubation, the slices were washed three times with PBS for 5 minutes each, then differentiated with 5% hydrochloric acid-ethanol solution, dehydrated with ethanol, cleared with xylene, and mounted with neutral resin for observation.

[0101] Brain Aβ 1-42 Concentration: Brain tissue was homogenized completely in a homogenizer, centrifuged at 1500g for 15 minutes, and the supernatant was collected. Aβ was then detected by ELISA. 1-42 concentration.

[0102] The results are as follows Figure 9 As shown, the fluorescence intensity of ASC and Caspase-1 in the brain tissue of the control group was weak and scattered. The fluorescence signal of the model group was significantly enhanced, the ASC fluorescence signal was obviously aggregated, and the number of Caspase-1 positive cells increased and the signal intensity increased, indicating strong neuroinflammation. The fluorescence signal intensity of ASC and Caspase-1 in the Gel group was significantly reduced, which was equivalent to the level of the control group, suggesting that Gel can significantly reduce the level of neuroinflammation. Nissl staining results showed that the hippocampal neurons in the control group were closely arranged, the Nissl bodies were abundant and evenly distributed, and the cell body morphology was intact, while the number of Nissl bodies in the model group was significantly reduced, the neurons were loosely arranged, and some cell bodies were atrophied or dissolved, suggesting that their metabolic function was impaired. The density and distribution pattern of Nissl bodies in the Gel group were close to those of the normal group, the neuronal morphology was restored to its original state, and the cell body was plump. ELISA results showed that the Aβ content in the brains of mice in the model group was significantly increased compared with that in the control group, thus causing severe cognitive impairment. After drug intervention, the Aβ content in each group 1-42The levels showed a significant downward trend, but there was still a gap between them and the control group. The Gel group had the best therapeutic effect. Therefore, Gel has a significant effect in reducing Aβ 1-42 , which improves the cognitive impairment of AD.

[0103] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A multifunctional microalgae hydrogel with intestinal flora targeting, characterized by: The gel consists of a shell structure and a core structure encapsulated in the shell structure; the shell structure is calcium alginate hydrogel, and the core structure is microalgae; the microalgae is mucin-modified drug-loaded Spirulina platensis; the drug is berberine; the berberine is loaded in the pores and surface of Spirulina platensis, and the mucin is modified on the surface of the drug-loaded Spirulina platensis through positive and negative electrical adsorption.

2. The method for preparing the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 1, characterized in that: The steps include: S1. Dissolve berberine in water, add Spirulina platensis, stir, and centrifuge to obtain drug-loaded Spirulina platensis; S2. dissolving the drug-loaded Spirulina platensis prepared in step S1 in water, adding mucin, stirring, and centrifuging to obtain mucin-modified drug-loaded Spirulina platensis; S3. Dissolve sodium alginate in PBS containing the drug-loaded Spirulina platensis prepared in step S2, add calcium gluconate while vortexing, and obtain the multifunctional microalgae hydrogel with intestinal flora targeting after cross-linking.

3. The method for preparing the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 2, characterized in that: The concentration of berberine in the system in step S1 is 100-1000 μg / ml; The concentration of mucin in the system in step S2 is 0.5-2 mg / ml; The concentration of sodium alginate in the system in step S3 is 2 w / v% to 10 w / v%; The concentration of calcium gluconate in the system described in step S3 is 2 w / v% to 4 w / v%.

4. The method for preparing the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 2, characterized in that: The concentration of berberine in the system described in step S1 is 600 μg / ml; The concentration of mucin in the system described in step S2 is 1 mg / ml; The concentration of sodium alginate in the system in step S3 is 8 w / v%; The concentration of calcium gluconate in the system described in step S3 is 3 w / v%.

5. The method for preparing the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 2, characterized in that: The stirring time in step S1 is 2 h to 12 h, further 6 h; The stirring in step S1 is magnetic stirring at a speed of 600 rpm; The centrifugation condition in step S1 is 2380×g for 10 min; The stirring in step S2 is ice bath stirring at a rotation speed of 600 rpm; The centrifugation conditions described in step S2 were 2380×g for 5 min.

6. A multifunctional microalgae hydrogel with intestinal flora targeting, characterized by: The method is obtained by the preparation method described in any one of claims 1 to 5.

7. Use of the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 6 in the preparation of drugs for treating neurodegenerative diseases.

8. The use according to claim 7, characterized in that: The neurodegenerative diseases include Alzheimer's disease.

9. The use of the multifunctional microalgae hydrogel with intestinal flora targeting according to claim 6, characterized in that: The application is at least one of the following applications: (1) Application in the preparation of drugs for treating Alzheimer's disease based on the microbiome-gut-brain axis; (2) Application in the preparation of drugs for regulating the homeostasis of intestinal flora; (3) Role in the preparation of drugs for improving intestinal inflammation; (4) Role in the preparation of drugs for repairing intestinal barrier damage and / or blood-brain barrier damage; (5) Role in the preparation of drugs for reversing cognitive dysfunction.

10. The use according to claim 9, characterized in that: The medicine is an oral medicine; The effective dosage of the drug is 1 mg / kg / day based on berberine, and the course of treatment is ≥30 days.

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