Oral spirulina delivery hydrogel as well as preparation method and application thereof

Abstract

The invention discloses oral spirulina delivery hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The delivery hydrogel takes spirulina platensis as a raw material, and is added into network-shaped hydrogel formed by xanthan gum / konjac glucomannan / sodium alginate in a combined manner. The method is easy to implement, safe in component and high in encapsulation efficiency. The composite hydrogel has the following effects: after oral administration, the spirulina can be protected to completely pass through the stomach, the intestinal retention is prolonged, and the slow release of the medicine at the focus part is realized; according to the present invention, with the application of the polypeptide, the oxidative stress and the intestinal inflammation can be alleviated, and the expression of the tight junction protein can be significantly recovered so as to enhance the barrier integrity and limit the inflammation leakage, such that the whole body immune homeostasis conversion can be achieved, the good safety and the good compliance can be provided, and the clinical transformation potential as the systemic chronic inflammatory disease adjuvant therapy strategy can be represented; the traditional Chinese medicine composition is especially suitable for diseases with pathological characteristics of intestinal flora imbalance or immune disorder.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oral spirulina delivery hydrogel, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis. The disease is protracted and ultimately leads to irreversible joint damage and functional impairment, severely impacting patients' quality of life. It is estimated that RA affects approximately 1% of the global population. Current clinical treatment primarily relies on disease-modifying antirheumatic drugs (DMARDs), biologics, and immunomodulatory agents such as JAK inhibitors. However, existing treatment regimens generally suffer from inconsistent efficacy, significant side effects, and limited remission rates, necessitating the development of novel adjuvant therapy strategies to improve efficacy and patient adherence.

[0003] In recent years, a growing body of research has shown that impaired intestinal barrier function and gut microbiota dysbiosis play a crucial role in the pathogenesis of rheumatoid arthritis (RA). RA patients often exhibit increased intestinal permeability, downregulation of tight junction proteins (such as ZO-1, Occludin, and Claudin-1), and an increase in inflammatory bacteria in the gut, suggesting that the "gut-joint axis" may be an important source of peripheral immune dysregulation and joint inflammation. Therefore, improving the gut microenvironment and enhancing barrier function holds promise for providing a novel intervention pathway for RA.

[0004] Spirulina is a nutrient-rich cyanobacterial algae, abundant in gamma-linolenic acid, polysaccharides, vitamins, and trace elements. It possesses multiple biological activities, including antioxidant, anti-inflammatory, immunomodulatory, and gut microbiota remodeling effects, and has been listed as a "safe food supplement" by the US FDA. Studies have found that spirulina can promote the proliferation of anti-inflammatory bacteria (such as Muribculaceae, Bacteroides, and Parabacteroides) and inhibit the activity of pathogenic bacteria (such as Desulfovibrio and Enterococcus), thereby improving the intestinal environment and regulating host immune balance.

[0005] However, orally administered spirulina is easily destroyed by gastric acid and digestive enzymes in the gastrointestinal tract, leading to degradation of active substances and low bioavailability, which limits its clinical application. To improve its stability and targeted release effect in the intestine, a delivery system with protective and controlled-release capabilities needs to be constructed.

[0006] Natural polysaccharide-based hydrogels have attracted widespread attention for oral drug delivery due to their excellent biocompatibility and gelling properties. Xanthan gum, konjac glucomannan (KGM), and sodium alginate (SA), as food-grade polysaccharides, possess pH sensitivity and adhesiveness, enabling them to maintain structural stability in the stomach and release active substances in the intestine. Based on this, this invention develops an oral spirulina delivery system (SP-gel) that enhances gel stability through CaCl2 cross-linking, achieving sustained release and localized action of spirulina in the intestine.

[0007] Studies have shown that this system not only improves the residence time and bioactivity of spirulina in the gastrointestinal tract, but also alleviates RA-related systemic inflammatory responses by increasing tight junction protein expression, reducing oxidative stress levels, and restoring the regulatory T cell (Treg) / Th17 immune balance. Furthermore, its combination with intra-articular injection of hyaluronic acid (HA) significantly reduced synovial hyperplasia and joint damage in mice with collagen-induced arthritis (CIA), demonstrating synergistic anti-inflammatory and immune remodeling effects.

[0008] In addition, 16S rDNA sequencing results showed that SP-gel can reshape the gut microbiota structure, enrich beneficial bacteria such as Muribauculaceae and Bacteroides, and reduce inflammatory bacteria such as Desulfovibrio and Enterococcus, further verifying its mechanism of RA treatment through the "gut-joint axis".

[0009] Based on the above, SP-gel, as a safe, natural, and highly compliant novel oral functional material, has the ability to synergistically regulate the intestinal barrier, flora, and immune system, showing broad application potential in systemic autoimmune diseases such as RA. Summary of the Invention

[0010] The present invention aims to provide a simple, safe, and targeted oral spirulina delivery system, its preparation method, and its application in intestinal inflammation-related diseases.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, an oral spirulina delivery system is provided, the delivery system comprising xanthan gum / konjac glucomannan / sodium alginate (XG / KGM / SA) and Spirulina platensis encapsulated therein, and the preparation method is as follows: Spirulina (SP) samples were added to the above three polysaccharide mixture solution and cross-linked with calcium chloride to form a structurally stable composite hydrogel network (SP-gel).

[0012] In some preferred embodiments, the concentrations of xanthan gum, konjac glucomannan, and sodium alginate in the three polysaccharide mixture solution are all 0.0625% w / v; after the spirulina sample is added to the polysaccharide mixture solution, the final concentration of spirulina is 2 mg / mL. In some preferred embodiments, calcium chloride is used for cross-linking, and the final concentration of calcium chloride is 0.0625 mol / L.

[0013] Secondly, the drug delivery system of this invention is based on a composite hydrogel structure constructed from natural polysaccharides. This structure can effectively encapsulate spirulina, preventing its degradation in the acidic environment of the stomach, protecting its active ingredients, and enabling slow release in the alkaline environment of the intestine. This prolongs the duration of action of spirulina in the digestive tract and increases the effective concentration at the target site. After drug action, it can be naturally excreted from the body through the digestive system, exhibiting good oral bioavailability and biodegradability, and high biosafety. Furthermore, this system possesses multiple biological activities, including antioxidant, anti-inflammatory, and gut microbiota regulation, and has a sound pharmacological basis, making it suitable as a functional adjuvant therapy for chronic immune diseases such as rheumatoid arthritis.

[0014] Furthermore, this invention utilizes the characteristic of spirulina being rich in chlorophyll, which has natural red autofluorescence. It can be used for non-invasive tracing of drug distribution in the gastrointestinal tract under excitation by a specific wavelength light source, realizing in vivo fluorescence imaging after oral administration, and is expected to have both therapeutic and diagnostic functions.

[0015] The drug delivery system described in this invention can be used in combination with hyaluronic acid in rheumatoid arthritis model animals to effectively relieve local joint inflammation, inhibit synovial hyperplasia, and reduce cartilage tissue damage. At the same time, it can improve immune homeostasis and enhance systemic anti-inflammatory response by upregulating the expression of intestinal tight junction protein, reducing intestinal mucosal oxidative stress, regulating the Treg / Th17 cell ratio, increasing IL-10 and decreasing IL-17 levels.

[0016] The beneficial effects of this invention include: The hydrogel system is based on a complex of natural polysaccharides, which is simple to process, uses readily available raw materials, and is inexpensive. It has good swelling and intestinal retention capabilities, and can achieve a balance between structural stability and controlled drug release performance, making it suitable for large-scale production and clinical translation.

[0017] At the therapeutic level, the spirulina delivered by this system possesses systemic anti-inflammatory and immunomodulatory activities, enhancing intestinal barrier integrity, improving gut microbiota structure, enriching beneficial bacteria (such as Muribacaceae, Bacteroides, and Parabacteroides), and inhibiting pro-inflammatory bacteria (such as Enterococcus and Desulfovibrio), thereby regulating the progression of RA through the gut-immune-joint axis. Furthermore, this delivery system is naturally excreted after metabolism in the body, without the risk of cumulative toxicity or tissue damage. It exhibits excellent biocompatibility and controllable degradation, demonstrating high safety and applicability. In summary, this spirulina-based hydrogel system provides a safe, effective, and patient-compliant oral adjunctive therapy for autoimmune diseases such as RA, and has broad application prospects. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the synthesis of an oral spirulina delivery system; Figure 2 The effects of different concentrations of polysaccharide (0.0156%-2% w / v) and cross-linking agent CaCl2 (0.0156mol / L-2mol / L) on the viscosity of blank gels were investigated. Figure 3 Texture analysis of uncrosslinked blank gel, uncrosslinked spirulina gel, crosslinked blank gel, and crosslinked spirulina gel; Figure 4 The fluorescence signal images of the gastrointestinal tract of mice at different time points after oral administration of the oral spirulina delivery system were obtained using a small animal in vivo imaging system. Figure 5 Scanning electron micrographs showing morphological changes in the digestive tract of mice after oral administration of the spirulina delivery system; Figure 6 A comparative diagram showing the repair effects of oral spirulina delivery system and its components on RA-related intestinal epithelial barrier function damage; Figure 7 A comparative image showing the effects of oral spirulina delivery system (SP-gel) combined with intra-articular injection of hyaluronic acid (HA) on joint lesions and intestinal barrier repair in mice with collagen-induced arthritis (CIA); among them, Figure 7 In the image, 'a' represents a representative photograph of the hind limbs of mice in different treatment groups. Figure 7 b in the figure represents Micro-CT images of bone erosion and joint structure in each group of mice. Figure 7 In the image, c represents the immunofluorescence staining images of the tight junction proteins Claudin-1, Occludin, and ZO-1 in the small intestinal tissue of different groups of mice. Figure 8The effect of oral spirulina delivery system (SP-gel) combined with intra-articular injection of hyaluronic acid (HA) on Tregs (CD4+) in the spleen of mice with collagen-induced arthritis (CIA). + FoxP3 + ) and Th17 cells (CD4) + IL-17A + The impact of ) Figure 9 This is a comparison chart of blood routine (WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelets; HCT, hematocrit) and blood biochemical indicators (ALT, alanine aminotransferase; AST, aspartate aminotransferase; UREA, blood urea; CREA, blood creatinine) test results after 30 days of continuous oral administration of spirulina, blank gel, and spirulina gel. Detailed Implementation

[0019] The present invention will be further described below with reference to the following figures and embodiments, but the present invention is not limited to the following embodiments.

[0020] Example 1. Synthesis of SP-gel, an oral spirulina delivery system.

[0021] Xanthan gum (XG), konjac glucomannan (KGM), and sodium alginate (SA) were weighed and mixed in a mass ratio of 1:1:1, then added to deionized water to adjust the final concentration of each component to 0.0625% (w / v). The resulting mixture was homogenized by ultrasonic stirring until a transparent and homogeneous polysaccharide solution was formed. Subsequently, 10 mg of spirulina was weighed and added to the above polysaccharide solution, mixed well, and dispersed in a system with a total volume of 5 mL to achieve a final SP concentration of 2 mg / mL, thus obtaining a suspension containing SP. A 0.0625 mol / L calcium chloride (CaCl2) solution was added to this suspension, and after pre-reaction at room temperature, it was incubated at room temperature for 12 hours to complete ionic cross-linking, obtaining a structurally stable SP-gel hydrogel. The results are as follows. Figure 1 As shown in the figure. To optimize gel performance, a systematic evaluation was conducted on different polysaccharide concentrations (0.0156%–2%, w / v) and CaCl2 concentrations (0.0156–2 mol / L). Based on a comprehensive analysis of the hydrogel's shape retention and flow properties, a hydrogel with certain flowability and viscosity was selected. The optimal formulation was determined to be a final concentration of 0.0625% (w / v) for all three polysaccharides and a CaCl2 concentration of 0.0625 mol / L. The results are shown in the figure. Figure 2 As shown.

[0022] Example 2. Analysis of the textural properties of different hydrogel systems.

[0023] The mechanical properties of four groups of hydrogel samples were determined using a texture analyzer. The selected samples included: uncrosslinked blank gel (NC-Blank), uncrosslinked spirulina gel (NC-SP), and gel treated with Ca... 2+ Cross-linked blank gel and Ca 2+ Cross-linked spirulina gel (SP-gel, using the optimal formulation determined in Example 1). Test results showed that, compared to the non-cross-linked groups (NC-Blank and NC-SP), the cross-linked groups (Blank-gel and SP-gel) exhibited significant improvements in chewiness and guminess parameters, indicating that Ca... 2+ Mediated ionic crosslinking significantly enhances the mechanical strength of hydrogels. Among these, SP-gel exhibits the most outstanding performance in the aforementioned textural indices, indicating its strongest structural stability. This enhanced performance contributes to improving the hydrogel's shear resistance and intestinal adhesion in the gastrointestinal environment, which is beneficial for its retention in the digestive tract and the sustained release of active substances. This embodiment verifies that the SP-gel possesses excellent mechanical properties and is suitable for constructing hydrogel carrier systems with oral delivery capabilities, as shown in the results. Figure 3 As shown.

[0024] Example 3. Fluorescence imaging capability and in vivo distribution.

[0025] The fluorescence distribution in mice after SP-gel administration via gavage was monitored using a small animal in vivo imaging system to assess its retention capacity in the gastrointestinal tract. The specific method was as follows: 300 μL of SP-gel (SP = 2 mg / mL, Blank-gel = 0.3125 mg / mL) was administered to Balb / c nude mice via gavage. Whole-body fluorescence imaging was performed on the mice at different time points after administration (including initial, several hours, and 24 hours), and changes in fluorescence signal were recorded. The results are as follows: Figure 4 As shown, after gavage administration of SP-gel, the fluorescence signal in the gastrointestinal tract of mice rapidly increased, and a significant signal could still be detected in the intestinal tract 24 hours later, suggesting that SP-gel has good intestinal retention properties. This retention effect may be attributed to the viscosity of the hydrogel system, which helps to prolong its residence time in the intestinal lumen. In addition, the persistent presence of strong fluorescence signal also indicates that SP-gel has the potential to be used as an imaging probe in vivo, and is suitable for constructing an oral delivery system with tracer function.

[0026] Example 4. In vivo degradation.

[0027] To investigate the degradation characteristics of SP-gel in the gastrointestinal tract, 300 μL of SP-gel (SP = 2 mg / mL, Blank-gel = 0.3125 mg / mL) was administered to Balb / c nude mice via gavage. Two hours after administration, the mice were sacrificed, and the contents of the stomach, ileum, cecum, and colon were collected for microscopic morphology observation using scanning electron microscopy (SEM). The results are as follows: Figure 5 As shown, SP-gel maintains its intact helical structure in the stomach; structural rupture begins in the ileum, and it exhibits a distinct fragmented morphology in the cecum and colon. These results indicate that SP-gel exhibits a segmented degradation and region-specific release behavior in the gastrointestinal tract. This progressive degradation mode not only helps prolong the residence time of SP-gel in the gastrointestinal tract but also enables drug release in the lower digestive tract, thereby improving its targeted and sustained release capability during oral delivery. Furthermore, the gradual disintegration of the gel in the distal intestine suggests good in vivo biodegradability, facilitating subsequent excretion and demonstrating high biocompatibility.

[0028] Example 5. The repair effect of SP-gel on RA-related intestinal barrier damage.

[0029] A Caco-2 / RAW264.7 co-culture model was established, and epithelial barrier damage was induced by adding fecal supernatant (FSN) from rheumatoid arthritis (RA) model mice. One hour before treatment, SP (20 μg / mL), Blank-gel (3.125 μg / mL), or SP-gel (SP = 20 μg / mL; Blank-gel = 3.125 μg / mL) were added to the upper chamber, respectively. The expression levels of tight junction proteins Occludin, Claudin-1, and ZO-1 were detected by immunofluorescence staining. The results showed that FSN stimulation significantly reduced the expression of these proteins, while the SP-gel treatment group effectively restored their expression, with a repair effect superior to the SP and Blank-gel groups, suggesting that SP-gel has the potential to improve RA-related intestinal barrier damage. Figure 6 .

[0030] Example 6. Effects of oral spirulina delivery system (SP-gel) combined with intra-articular injection of hyaluronic acid (HA) on joint lesions and intestinal barrier repair in mice with collagen-induced arthritis (CIA).

[0031] A collagen-induced arthritis (CIA) model was established using DBA / 1 mice. Treatment was initiated on day 25, with the following methods: (1) RA model group: 300 μL of purified water was administered by gavage daily, and PBS was injected intra-articularly on days 25, 30, 35, 40, and 45 (administration frequency was consistent across all groups); (2) Spirulina hydrogel group: 300 μL of SP-gel (SP = 2 mg / mL, Blank-gel = 0.3125 mg / mL) was administered by gavage, and PBS (10 μL / time) was injected intra-articularly on days 25, 30, 35, 40, and 45; (3) Hyaluronic acid group: 300 μL of purified water was administered by gavage daily, and HA (10 μL / time) was injected intra-articularly on days 25, 30, 35, 40, and 45; (4) Spirulina hydrogel + hyaluronic acid group: 300 μL of PBS was administered orally. SP-gel was injected into the HA joint on days 25, 30, 35, 40 and 45 (10 μL / time), and the treatment cycle continued until day 49. Figure 7 Figure 'a' shows representative images of the hind limbs of mice in each group, demonstrating joint swelling and morphological changes. The results showed that the RA model group had obvious joint swelling and deformity, the spirulina hydrogel group had alleviated joint lesions, and the combination of spirulina hydrogel and hyaluronic acid (spirulina hydrogel + hyaluronic acid group) showed the best joint appearance recovery. Figure 7 In the figure, b is a three-dimensional Micro-CT image of the mouse ankle joint, reflecting the degree of bone destruction and changes in joint morphology. The RA model group showed severe bone destruction and joint structural disorder. Both the spirulina hydrogel group and the hyaluronic acid group showed improvement when treated alone, while the spirulina hydrogel + hyaluronic acid group had the most intact bone structure, clear joint space, and reduced osteophytes. Figure 7 In the image, c represents the immunofluorescence staining image of tight junction proteins Claudin-1, Occludin, and ZO-1 in the ileum tissue, used to assess intestinal barrier integrity. Histone expression in the RA model was significantly decreased, the improvement in the hyaluronic acid group was limited, the Spirulina hydrogel group could restore some expression, and the Spirulina hydrogel + hyaluronic acid group showed the most significant recovery of tight junction protein expression in mice, suggesting that it has a synergistic repair effect on RA-related intestinal barrier dysfunction.

[0032] Example 7. The regulatory effect of oral spirulina delivery system (SP-gel) combined with intra-articular injection of hyaluronic acid (HA) on the ratio of Tregs to Th17 cells in the spleen of mice with collagen-induced arthritis (CIA).

[0033] This embodiment, based on the same grouping and treatment protocols as in Example 6, further evaluates the impact of each intervention on the systemic immune response, with particular focus on regulatory T cells (Tregs, CD4+). + CD25 + FoxP3 +) and pro-inflammatory Th17 cells (CD4) + IL-17A + The changes in Th17 / Tregs were observed. Mice in each group were sacrificed at the end of day 49, spleen tissue was isolated, single-cell suspensions were prepared, and after erythrocyte lysis, the proportion of T cell subsets was detected by flow cytometry. The results showed that, compared with the RA model group, Spirulina hydrogel intervention significantly downregulated the proportion of Th17 cells (from 0.46% to 0.22%) and upregulated the proportion of Tregs (from 9.47% to 12.0%). The regulatory effect was weaker in the hyaluronic acid group, with the proportion of Th17 cells decreasing to 0.38% and Tregs increasing to 10.3%. In contrast, the Spirulina hydrogel + hyaluronic acid group further reduced the proportion of Th17 cells to 0.13% and significantly increased Tregs to 15.4%, indicating that this combination regimen has a synergistic effect in regulating the Th17 / Treg balance. (Results are referenced from...) Figure 8 .

[0034] Example 8. Oral safety.

[0035] Balb / c mice were administered 300 μL of SP-gel (SP = 2 mg / mL, Blank-gel = 0.3125 mg / mL), 300 μL of Spirulina suspension (SP = 2 mg / mL), and 300 μL of blank gel (Blank-gel = 0.3125 mg / mL) via gavage once daily for 30 consecutive days. After treatment, peripheral blood was collected from the mice for complete blood count and serum biochemical assays. The assays included white blood cell count, red blood cell count, hemoglobin, platelets, ALT, AST, BUN, and CRE. The results are shown below. Figure 9 As shown, after long-term oral administration, the main hematological and biochemical indicators of the SP-gel group mice were within the normal reference range, and there were no obvious toxic reactions, indicating that the oral delivery system has good biosafety under continuous use conditions.

Claims

1. An oral spirulina delivery hydrogel, characterized in that, Comprising: a delivery carrier and spirulina encapsulated in the delivery carrier; the delivery carrier is a mixture of xanthan gum, konjac glucomannan, and sodium alginate.

2. A process for the preparation of oral spirulina delivery hydrogel as claimed in claim 1, wherein, Comprising the following steps: adding spirulina to a mixture of xanthan gum, konjac glucomannan, and sodium alginate so that the final concentration of spirulina is 2 mg / mL, and adding a crosslinking agent to crosslink for 12 hours to form a 3D hydrogel network; wherein the concentration of xanthan gum, konjac glucomannan, and sodium alginate in the mixture of xanthan gum, konjac glucomannan, and sodium alginate is 0.0625% w / v, respectively.

3. The production method according to claim 2, characterized by, The spirulina is spirulina platensis.

4. The production method according to claim 2, characterized by, The mixture of xanthan gum, konjac glucomannan, and sodium alginate is obtained by homogenizing the mixture of each component by ultrasonic stirring.

5. The preparation method according to claim 2, characterized in that, The crosslinking agent is a calcium chloride solution.

6. The production method according to claim 5, wherein The final concentration of calcium chloride is 0.0625 mol / L.

7. Use of the spirulina delivery hydrogel according to claim 1 or of the spirulina delivery hydrogel obtainable by the process according to any one of claims 2 to 6, characterized in that, One or more of the following: (1) preparing a dietary supplement for enhancing intestinal barrier function and / or correcting intestinal flora imbalance; (2) as a natural functional preparation for improving intestinal oxidative stress and mucosal immune function; (3) preparing an oral administration system for adjuvant therapy of rheumatoid arthritis; (4) preparing an adjuvant therapy product for regulating Th17 / Treg immune balance and intervening in the progression of systemic autoimmune diseases; (5) non-invasive tracing for drug distribution in the gastrointestinal tract.

8. Use according to claim 7, characterized in that, The adjuvant therapy of rheumatoid arthritis is specifically oral spirulina delivery hydrogel combined with intra-articular injection of hyaluronic acid.

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